Brain tumors remain among the most aggressive cancers due to their ability to adapt to microenvironmental stress to sustain malignancy and resist therapy. We propose Drosophila lethal(3) tumorous brain [l(3)tb] mutant, which develops rapidly expanding brain tumors, as a genetically tractable in vivo model to study stress-adaptive tumor growth. The progressive brain enlargement in l(3)tb/l(3)tb larvae is accompanied by robust tumour size-dependent induction of Hsp70, a molecular chaperone linked to poor glioma prognosis. These findings position the l(3)tb model as a powerful platform for evaluation of stress tolerance in brain tumours.
","acknowledgements":"We thank ISLS, BHU for the Confocal microscope facility.
","authors":[{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","writing_originalDraft"],"email":"abhijit.biswas10@bhu.ac.in","firstName":"Abhijit ","lastName":"Biswas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","fundingAcquisition","project","resources","supervision","validation","writing_reviewEditing"],"email":"devanjan@bhu.ac.in","firstName":"Devanjan","lastName":"Sinha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0001-5060-2075"}],"awards":[],"conflictsOfInterest":"The authors declare no conflicts of interest.
","dataTable":null,"extendedData":[],"funding":"Indian Council of Medical Research Ad-hoc grant (5/13/87/2020/NCD-III), BHU-Institute of Eminence grant (R/Dev/IoE/Incentive/2021-22/32452) to D.S. CSIR doctoral fellowship to A.B.
","image":{"url":"https://portal.micropublication.org/uploads/6887c1c747a107ae74694d665624739a.jpg"},"imageCaption":"(A) Morphological difference between larval progeny coming from Wild type (Oregon R+) and l(3)tb heterozygous parents, respectively. (B) Wild type larval brain after 6 days of egg laying (AEL) and (C-G) Larval brain size of delayed 3rd instar l(3)tb homozygous larvae in the indicated time point, showing gradual increase in brain size. (H) Tumourous brain of l(3)tb homozygous 3rd instar larvae (12 days AEL) [n=12] showing significant differences in optic lobe size compared to wild type [n=19] and l(3)tb homozygous 6 days larvae (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents optic lobe size. (I-J) Tumourous brain of l(3)tb homozygous 3rd instar larvae after 12 days of egg laying (AEL) [n=12], showing significant differences in Hsp70 intensity and distribution, compared to wild type [n=19] and l(3)tb homozygous 6 days larvae after egg laying (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents Hsp70 intensity (I) and % of Hsp70 expressed area (J), in each optic lobe respectively. (K-M) Confocal projection of wild type (K), l(3)tb 6 days (L) and l(3)tb- 12 days (M) homozygous larval brain showing Hsp70 staining in optic lobe area, scale bar: 50 μm. (O) Dot plot showing correlation of Hsp70 distribution with whole DAPI area of optic lobe of different genotypes [n=12, 9, 11 accordingly] where X-axis and Y-axis represents DAPI+ area and Hsp70+ area, respectively. Unpaired Student’s t-test, not significant (ns)p > 0.05, ***p < 0.001, **p < 0.01, *p < 0.05.
","imageTitle":"Temporal expression of stress inducible Hsp70 in developing l(3)tb brain tumour of Drosophila melanogaster
","methods":"Immunostaining: GFP+ clone bearing wing imaginal discs were dissected out in 1X PBS from the larvae of appropriate age and fixed in 4% formaldehyde for 20min. After fixation, wing discs were rinsed thrice with 0.1% PBST (0.1% Triton X-100 in 1X PBS) for 15 min each. After rinsing, discs were kept in blocking solution (10% fetal calf serum, 0.1% Bovine serum albumin, 0.1% Triton X-100, 0.02% Thiomersal and 0.1% sodium deoxycholate, in 1XPBS) for 1 hr. After that, discs were incubated with desired primary antibody: rat anti-Hsp70(7Fb 1:200, Sigma) for overnight at 4°C, following that discs were rinsed thrice with 0.1% PBST for 15min each and kept in a blocking solution for 1 hr. Thereafter, those discs were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 (1:200, Molecular Probes, USA), for 2hr at room temperature. After rinsing those discs thrice with 0.1% PBST, counterstained with 6-diamidino-2-phenylindole dihydrochloride (DAPI,1μg/ml, Thermo Fisher Scientific, Cat# D1306) and finally, mounted in an anti-fade mounting media 1,4-Diazabicyclo [2.2.2] octane (DABCO), Sigma, Cat# D27802, 2.5% DABCO in 70% glycerol made in 1X PBS). All the immunostained disc images were taken in Zeiss LSM 900 confocal microscopy with Zeiss ZEN4.3 blue edition software using Apo 20X (0.8 NA) and 63X (1.4 NA) oil immersion objectives. Images were assembled in Adobe Photoshop (2012). Quantitative analysis of acquire images were performed by using Fiji. Statistical analysis was done in Graph Pad Prism 8.4.2 by applying Unpaired Students’ t- Test.
Live Imaging of whole larvae: Photographs of larvae and adults were taken by using Axio Cam Zeiss camera mounted on Nikon SMZ800N stereo binocular microscope.
","reagents":"Reagents
a. Fly Stocks:
1. l3tb/l3tb or l3tb/TM3, Ser
2.Oregon R+
b. Chemicals:
1.Hsp70 antibody (7Fb, Sigma)
","patternDescription":"Description
Primary brain tumours are characterized by severe cellular and molecular heterogeneity that are associated with aggressive disease progression, therapeutic resistance and recurrence (Nicholson and Fine, 2021; Qazi et al., 2017). Traditional in vitro cell culture systems, including both two-dimensional and three-dimensional cultures, are intrinsically limited because they fail to incorporate the necessary host-tumour environment. Brain tumour progression is dictated by intricate, non-cell autonomous signaling originating from the surrounding glia, vasculature, and immune cells (Pasqualini et al., 2020). Models that cannot incorporate this crucial microenvironment, risk overlooking the therapeutic targets related to the complex dynamics of tumour-host interaction.
Among the different adaptive features followed by rapidly proliferating tumours, stress adaptation has recently been appreciated to be one of the major drivers of tumour growth (Hong et al., 2021; Seiler et al., 2020). Brain tumors, particularly gliomas and glioblastomas, possess a variety of stress adaptive mechanisms that help them survive, proliferate, and resist therapy in an otherwise hostile micro-environment (Combs et al., 2016; Graner et al., 2007). These mechanisms act across metabolic, genetic, and signaling pathways to maintain homeostasis under oxidative, hypoxic, and therapeutic stress conditions (Li et al., 2023). Although therapy-induced stress result in accumulation of misfolded proteins that subsequently triggers apoptosis, activation of chaperones such as Grp78 induce the unfolded protein response that allows the cell to manage the misfolded proteins and increase therapy tolerance(Kusaczuk et al., 2024; Liu et al., 2020). This requires a need to develop suitable models to study the factors responsible for development of stress tolerance in brain tumours.
The common fruit fly, Drosophila melanogaster serves to overcome the limitations of conventional brain tumour models since it exhibits rapid life cycle and production of large number of offsprings that can allow high-throughput screening of large drug libraries(Rudrapatna et al., 2012). This simultaneously generates information on drug bio-availability, toxicity parameters and host-tumour interactions, allowing researchers to rapidly test compounds in the complex in vivo context(Munnik et al., 2022). Further, the exceptional molecular conservation of the fly system with humans allows precise engineering of brain tumours that closely mimic the human disease. For example, glioma model has been developed to mimic the human glioblastomas, by constitutive co-activation of EGFR and PI3K pathways specifically in glial cells by using tissue specific Gal4 drivers (Read et al., 2009). In a P-element mutagenesis screen, a specific gene mutation called as the l(3)tb (lethal tumorous brain) was isolated that is characterized by excess accumulation of neuroblasts due to their uncontrolled growth, leading to larval and pupal lethality. The brain of l(3)tb homozygous larvae gradually increases in size with extended larval periods till death. Hence, the mutation was initially named as lethal (3) tumorous brain [l(3)tb]. Larvae homozygous for l(3)tb mutant, along with the presence of tumourous brain, show 12-13 days extended larval life, overgrowth in leg, wing and eye-antennal discs (Kunar and Roy, 2021; Mishra et al., 2020). These defects were rescued by introducing a functional copy of DCP2 in the mutant background (Mishra et al., 2020). DCP2 is an evolutionarily conserved mRNA decapping enzyme encoded by DCP2 gene present at 73A1 region in left arm of Drosophila chromosome 3 (Kunar and Roy, 2021). It belongs to the NUDIX family of pyro-phosphatases and was first identified in yeasts (Dunckley and Parker, 1999). DCP2 along with DCP1 cleaves the 5’ methyl guanosine cap of mRNA and is involved in cell cycle regulation and DNA repair(She et al., 2008). DCP2 has been found to be upregulated in lung cancer and gliomas where it promotes cell proliferation, invasion, suppression of apoptosis and tumour immune cell infiltration (Watson et al., 2008).
In human cancers, Hsp70 is commonly overexpressed and are integral to mitigating stress, promoting cell survival and preventing cell death. Hsp70 positive tumours are more aggressive and resistant to therapy (Liu et al., 2021; Murphy, 2013). In case of brain tumours, the intensity and localization of Hsp70 within the cell correlate strongly with tumor malignancy (Babi et al., 2022). High-grade gliomas exhibit a significantly greater frequency of Hsp70 overexpression in both the nucleus and cytosol, indicating the elevated requirement for chaperone-mediated survival capacity (Graner et al., 2007; Lobinger et al., 2021). Further, high levels of extracellular Hsp70, released by the necrotic cells in large sized tumours, are associated with an unfavorable prognosis and overall survival (Shevtsov et al., 2024). Hence, Hsp70 serves as an important diagnostic biomarker and therapeutic target for many brain tumours.
Therefore, we sought to determine whether the l(3)tb homozygous mutant of Drosophila, which develops tumourous brains, could serve as a tractable model to study the development of Hsp70-mediated brain tumours. In our study the mutant flies showed a phenotype similar to previous reports (Mishra et al., 2020). Homozygous l(3)tb homozygous mutant larvae show developmental delay and most of the larvae die before pupation. Only a few of the larvae enter pupal stage and eventually die (Fig. 1A). During the early stages of larval growth, the brain size of l(3)tb homozygous mutant larvae was found to be smaller than that of age-matched controls, indicating a developmental delay (Fig. 1B-C) and during the prolonged larval period, the brain size exponentially increased, ultimately leading to larval lethality (Fig. 1D-G). At the later stages of development, the size of l(3)tb/l(3)tb brain were approximately 3-fold larger than that of control (Fig. 1H). To further assess the dependency of these aggressively growing brain tumours on Hsp70, we performed immunostaining using an anti-Hsp70 antibody. Control brains from third instar wild type larvae showed minimal Hsp70 expression which was distributed only in a few cells of brain at the left and right optic lobe region (Fig. 1I, J and K). A similar pattern of minimalistic Hsp70 staining had been proposed in non-heat-shocked larval brain samples (Krebs and Feder, 1997). Similar to wild type condition, third instar l(3)tb/l(3)tb larvae during initial stages of development, exhibited Hsp70 expression in a few cells of the optic lobes (Fig. 1L). However, during later stages, these Hsp70-positive regions expanded progressively along with tumour growth, eventually encompassing most of the optic lobe area (Fig. 1M-N). Quantitative analysis revealed an approximately 15-fold increase in Hsp70 expression in l(3)tb/l(3)tb tumourous brains compared to wild-type controls (Fig. 1I). Similarly, the area of optic lobe covered by Hsp70 expressing cells increased to ~35% in 12-day old tumour (Fig. 1J). Further, a direct association was observed between the brain size, quantified as DAPI positive area, and the area covered with Hsp70 expressing cells (Fig.1O). These observations indicated a strong correlation between temporal induction of Hsp70 and brain tumour progression.
In conclusion, the l(3)tb mutant of Drosophila offers a unique opportunity to model the interplay between oncogenic drivers and stress-adaptive chaperones in brain tumours. By recapitulating both the genetic and micro-environmental dimensions of tumour biology, this system provides a powerful and cost-effective platform for mechanistic discovery and therapeutic exploration on the role of Hsp70 in aggressive brain tumours. Most importantly, our findings indicate a direct link between Hsp70 expression and tumour progression in the l(3)tb brain tumour model, underscoring the promise of this conserved chaperone as a diagnostic and therapeutic target in stress-adapted malignant brain tumours. By labelling individual cells in the brain, it will be of interest to see which cell type express Hsp70 whose proliferation ultimately results in expanded growth of brain size. This work highlights the possible development of l(3)tb/l(3)tb Drosophila brain tumour model to study stress-tolerance in brain tumours with potential translational outcomes.
","references":[{"reference":"Babi, A., K. Menlibayeva, T. Bex, A. Doskaliev, S. Akshulakov, and M. Shevtsov. 2022. Targeting Heat Shock Proteins in Malignant Brain Tumors: From Basic Research to Clinical Trials. Cancers (Basel). 14.
","pubmedId":"","doi":""},{"reference":"Combs, S.E., T.E. Schmid, P. Vaupel, and G. Multhoff. 2016. Stress Response Leading to Resistance in Glioblastoma-The Need for Innovative Radiotherapy (iRT) Concepts. Cancers (Basel). 8.
","pubmedId":"","doi":""},{"reference":"Dunckley, T., and R. Parker. 1999. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 18:5411-5422.
","pubmedId":"","doi":""},{"reference":"Graner, M.W., R.I. Cumming, and D.D. Bigner. 2007. The heat shock response and chaperones/heat shock proteins in brain tumors: surface expression, release, and possible immune consequences. J Neurosci. 27:11214-11227.
","pubmedId":"","doi":""},{"reference":"Hong, H., M. Ji, and D. Lai. 2021. Chronic Stress Effects on Tumor: Pathway and Mechanism. Front Oncol. 11:738252.
","pubmedId":"","doi":""},{"reference":"Krebs, R.A., and M.E. Feder. 1997. Tissue-specific variation in Hsp70 expression and thermal damage in Drosophila melanogaster larvae. J Exp Biol. 200:2007-2015.
","pubmedId":"","doi":""},{"reference":"Kunar, R., and J.K. Roy. 2021. The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms. Cell Tissue Res. 386:261-280.
","pubmedId":"","doi":""},{"reference":"Kusaczuk, M., E.T. Ambel, M. Naumowicz, and G. Velasco. 2024. Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma. Biochim Biophys Acta Rev Cancer. 1879:189054.
","pubmedId":"","doi":""},{"reference":"Li, S., C. Wang, J. Chen, Y. Lan, W. Zhang, Z. Kang, Y. Zheng, R. Zhang, J. Yu, and W. Li. 2023. Signaling pathways in brain tumors and therapeutic interventions. Signal Transduct Target Ther. 8:8.
","pubmedId":"","doi":""},{"reference":"Liu, H., Z. Li, Q. Li, C. Jia, N. Zhang, Y. Qu, and D. Hu. 2021. HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. J Bioenerg Biomembr. 53:405-413.
","pubmedId":"","doi":""},{"reference":"Liu, K., K. Tsung, and F.J. Attenello. 2020. Characterizing Cell Stress and GRP78 in Glioma to Enhance Tumor Treatment. Front Oncol. 10:608911.
","pubmedId":"","doi":""},{"reference":"Lobinger, D., J. Gempt, W. Sievert, M. Barz, S. Schmitt, H.T. Nguyen, S. Stangl, C. Werner, F. Wang, Z. Wu, H. Fan, H. Zanth, M. Shevtsov, M. Pilz, I. Riederer, M. Schwab, J. Schlegel, and G. Multhoff. 2021. Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients. Front Mol Biosci. 8:669366.
","pubmedId":"","doi":""},{"reference":"Mishra, R., R. Kunar, L. Mandal, D.P. Alone, S. Chandrasekharan, A.K. Tiwari, M.G. Tapadia, A. Mukherjee, and J.K. Roy. 2020. A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumor Suppressor in Drosophila melanogaster. G3 (Bethesda). 10:2601-2618.
","pubmedId":"","doi":""},{"reference":"Munnik, C., M.P. Xaba, S.T. Malindisa, B.L. Russell, and S.A. Sooklal. 2022. Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies. Front Genet. 13:949241.
","pubmedId":"","doi":""},{"reference":"Murphy, M.E. 2013. The HSP70 family and cancer. Carcinogenesis. 34:1181-1188.
","pubmedId":"","doi":""},{"reference":"Nicholson, J.G., and H.A. Fine. 2021. Diffuse Glioma Heterogeneity and Its Therapeutic Implications. Cancer Discov. 11:575-590.
","pubmedId":"","doi":""},{"reference":"Pasqualini, C., T. Kozaki, M. Bruschi, T.H.H. Nguyen, V. Minard-Colin, D. Castel, J. Grill, and F. Ginhoux. 2020. Modeling the Interaction between the Microenvironment and Tumor Cells in Brain Tumors. Neuron. 108:1025-1044.
","pubmedId":"","doi":""},{"reference":"Qazi, M.A., P. Vora, C. Venugopal, S.S. Sidhu, J. Moffat, C. Swanton, and S.K. Singh. 2017. Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma. Ann Oncol. 28:1448-1456.
","pubmedId":"","doi":""},{"reference":"Read, R.D., W.K. Cavenee, F.B. Furnari, and J.B. Thomas. 2009. A drosophila model for EGFR-Ras and PI3K-dependent human glioma. PLoS Genet. 5:e1000374.
","pubmedId":"","doi":""},{"reference":"Rudrapatna, V.A., R.L. Cagan, and T.K. Das. 2012. Drosophila cancer models. Dev Dyn. 241:107-118.
","pubmedId":"","doi":""},{"reference":"Seiler, A., A.K. Sood, J. Jenewein, and C.P. Fagundes. 2020. Can stress promote the pathophysiology of brain metastases? A critical review of biobehavioral mechanisms. Brain Behav Immun. 87:860-880.
","pubmedId":"","doi":""},{"reference":"She, M., C.J. Decker, D.I. Svergun, A. Round, N. Chen, D. Muhlrad, R. Parker, and H. Song. 2008. Structural basis of dcp2 recognition and activation by dcp1. Mol Cell. 29:337-349.
","pubmedId":"","doi":""},{"reference":"Shevtsov, M., D. Bobkov, N. Yudintceva, R. Likhomanova, A. Kim, E. Fedorov, V. Fedorov, N. Mikhailova, E. Oganesyan, S. Shabelnikov, O. Rozanov, T. Garaev, N. Aksenov, A. Shatrova, A. Ten, A. Nechaeva, D. Goncharova, R. Ziganshin, A. Lukacheva, D. Sitovskaya, A. Ulitin, E. Pitkin, K. Samochernykh, E. Shlyakhto, and S.E. Combs. 2024. Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors. Cancer Res Commun. 4:2025-2044.
","pubmedId":"","doi":""},{"reference":"Watson, P.M., S.W. Miller, M. Fraig, D.J. Cole, D.K. Watson, and A.M. Boylan. 2008. CaSm (LSm-1) overexpression in lung cancer and mesothelioma is required for transformed phenotypes. Am J Respir Cell Mol Biol. 38:671-678
","pubmedId":"","doi":""}],"title":"The Drosophila l(3)tb mutant as a model to study Hsp70-mediated brain tumor progression
","reviews":[{"reviewer":{"displayName":"Sa Kan Yoo"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":"1776361069075"}]},{"id":"7a4b4603-dec8-4993-ba25-3f3c4dcc98f5","decision":"revise","abstract":"Brain tumors remain among the most aggressive cancers due to their ability to adapt to microenvironmental stress to sustain malignancy and resist therapy. We propose Drosophila lethal(3) tumorous brain [l(3)tb] mutant, which develops rapidly expanding brain tumors, as a genetically tractable in vivo model to study stress-adaptive tumor growth. The progressive brain enlargement in l(3)tb/l(3)tb larvae is accompanied by robust tumor size-dependent induction of Hsp70, a molecular chaperone linked to poor glioma prognosis. These findings position the l(3)tb model as a powerful platform for evaluation of stress tolerance in brain tumors.
","acknowledgements":"We thank ISLS, BHU for the Confocal microscope facility.
","authors":[{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","writing_originalDraft"],"email":"abhijit.biswas10@bhu.ac.in","firstName":"Abhijit ","lastName":"Biswas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","fundingAcquisition","project","resources","supervision","validation","writing_reviewEditing"],"email":"devanjan@bhu.ac.in","firstName":"Devanjan","lastName":"Sinha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0001-5060-2075"}],"awards":[],"conflictsOfInterest":"The authors declare no conflicts of interest.
","dataTable":{"url":null},"extendedData":[],"funding":"Indian Council of Medical Research Ad-hoc grant (5/13/87/2020/NCD-III), BHU-Institute of Eminence grant (R/Dev/IoE/Incentive/2021-22/32452) to D.S. CSIR doctoral fellowship to A.B.
","image":{"url":"https://portal.micropublication.org/uploads/6887c1c747a107ae74694d665624739a.jpg"},"imageCaption":"(A) Morphological difference between larval progeny coming from Wild type (Oregon R+) and l(3)tb heterozygous parents, respectively. (B) Wild type larval brain after 6 days of egg laying (AEL) and (C-G) Larval brain size of delayed 3rd instar l(3)tb homozygous larvae in the indicated time point, showing gradual increase in brain size. (H) Tumourous brain of l(3)tb homozygous 3rd instar larvae (12 days AEL) [n=12] showing significant differences in optic lobe size compared to wild type [n=19] and l(3)tb homozygous 6 days larvae (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents optic lobe size. (I-J) Tumourous brain of l(3)tb homozygous 3rd instar larvae after 12 days of egg laying (AEL) [n=12], showing significant differences in Hsp70 intensity and distribution, compared to wild type [n=19] and l(3)tb homozygous 6 days larvae after egg laying (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents Hsp70 intensity (I) and % of Hsp70 expressed area (J), in each optic lobe respectively. Statistical analyses was performed by one-way ANOVA followed by Tukey's post-hoc multiple-comparison test; ****P<0.0001, **p < 0.01, *p < 0.05, not significant (ns)p > 0.05. (K-M) Confocal projection of wild type (K), l(3)tb 6 days (L) and l(3)tb- 12 days (M) homozygous larval brain showing Hsp70 staining in optic lobe area, scale bar: 50 μm. (O) Dot plot showing relationship of Hsp70 expression with size of optic lobe (demarcated from the DAPI+ area) of different genotypes [n=12, 9, 11 accordingly] where X-axis and Y-axis represents DAPI+ area and Hsp70+ area, respectively. Statistical significance of enhanced Hsp70 expression (WT vs l(3)tb brain) in the optic lobe area was determined through unpaired t-test, ***P (two=tailed)<0.001.
","imageTitle":"Temporal expression of stress inducible Hsp70 in developing l(3)tb brain tumour of Drosophila melanogaster
","methods":"Immunostaining: GFP+ clone bearing wing imaginal discs were dissected out in 1X PBS from the larvae of appropriate age and fixed in 4% formaldehyde for 20min. After fixation, wing discs were rinsed thrice with 0.1% PBST (0.1% Triton X-100 in 1X PBS) for 15 min each. After rinsing, discs were kept in blocking solution (10% fetal calf serum, 0.1% Bovine serum albumin, 0.1% Triton X-100, 0.02% Thiomersal and 0.1% sodium deoxycholate, in 1XPBS) for 1 hr. After that, discs were incubated with desired primary antibody: rat anti-Hsp70(7Fb 1:200, Sigma) for overnight at 4°C, following that discs were rinsed thrice with 0.1% PBST for 15min each and kept in a blocking solution for 1 hr. Thereafter, those discs were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 (1:200, Molecular Probes, USA), for 2hr at room temperature. After rinsing those discs thrice with 0.1% PBST, counterstained with 6-diamidino-2-phenylindole dihydrochloride (DAPI,1μg/ml, Thermo Fisher Scientific, Cat# D1306) and finally, mounted in an anti-fade mounting media 1,4-Diazabicyclo [2.2.2] octane (DABCO), Sigma, Cat# D27802, 2.5% DABCO in 70% glycerol made in 1X PBS). All the immunostained disc images were taken in Zeiss LSM 780 confocal microscopy with Zeiss ZEN2.3 SP1 Black edition software using Apo 20X (0.8 NA) and 63X (1.4 NA) oil immersion objectives. Images were assembled in Adobe Photoshop (2012). Quantitative analysis of acquire images were performed by using Fiji. Statistical analysis was done in Graph Pad Prism 8.4.2 as mentioned in the figure legends.
Imaging of larvae adults and larval brain: Photographs of larvae and adults were taken by using Axio Cam Zeiss camera mounted on Nikon SMZ800N stereo binocular microscope. Photographs of larval brain was taken using Nikon DS-Fi2 camera mounted on Nikon Eclipse 90i microscope.
","reagents":"Reagents
a. Fly Stocks:
1. l3tb/l3tb or l3tb/TM3, Ser (Mishra et al., 2020), where l3tb/TM6b, Tb flies used by the authors were rebalanced using TM3, Ser balancer to generate l3tb/TM3, Ser flies.
2.Oregon R+
b. Chemicals:
1.Hsp70 antibody (7Fb, Sigma)
","patternDescription":"Description
Primary brain tumors are characterized by severe cellular and molecular heterogeneity that are associated with aggressive disease progression, therapeutic resistance and recurrence (Nicholson and Fine, 2021; Qazi et al., 2017). Traditional in vitro cell culture systems, including both two-dimensional and three-dimensional cultures, are intrinsically limited because they fail to incorporate the necessary host-tumor environment. Brain tumor progression is dictated by intricate, non-cell autonomous signaling originating from the surrounding glia, vasculature, and immune cells (Pasqualini et al., 2020). Models that cannot incorporate this crucial microenvironment, risk overlooking the therapeutic targets related to the complex dynamics of tumor-host interaction.
Among the different adaptive features followed by rapidly proliferating tumors, stress adaptation has recently been appreciated to be one of the major drivers of tumor growth (Hong et al., 2021; Seiler et al., 2020). Brain tumors, particularly gliomas and glioblastomas, possess a variety of stress adaptive mechanisms that help them survive, proliferate, and resist therapy in an otherwise hostile micro-environment (Combs et al., 2016; Graner et al., 2007). These mechanisms act across metabolic, genetic, and signaling pathways to maintain homeostasis under oxidative, hypoxic, and therapeutic stress conditions (Li et al., 2023). Although therapy-induced stress result in accumulation of misfolded proteins that subsequently triggers apoptosis, activation of chaperones such as Grp78 induce the unfolded protein response that allows the cell to manage the misfolded proteins and increase therapy tolerance(Kusaczuk et al., 2024; Liu et al., 2020). This requires a need to develop suitable models to study the factors responsible for development of stress tolerance in brain tumors.
The common fruit fly, Drosophila melanogaster serves to overcome the limitations of conventional brain tumor models since it exhibits rapid life cycle and production of large number of offsprings that can allow high-throughput screening of large drug libraries(Rudrapatna et al., 2012). This simultaneously generates information on drug bio-availability, toxicity parameters and host-tumor interactions, allowing researchers to rapidly test compounds in the complex in vivo context(Munnik et al., 2022). Further, the exceptional molecular conservation of the fly system with humans allows precise engineering of brain tumors that closely mimic the human disease. For example, glioma model has been developed to mimic the human glioblastomas, by constitutive co-activation of EGFR and PI3K pathways specifically in glial cells by using tissue specific Gal4 drivers (Read et al., 2009). In a P-element mutagenesis screen, a specific gene mutation called as the l(3)tb (lethal tumorous brain) was isolated that is characterized by excess accumulation of neuroblasts due to their uncontrolled growth, leading to larval and pupal lethality (Mishra et al., 2020). The brain of l(3)tb homozygous larvae gradually increases in size with extended larval periods till death. Hence, the mutation was initially named as lethal (3) tumorous brain [l(3)tb]. Larvae homozygous for l(3)tb mutant, along with the presence of tumourous brain, show 12-13 days extended larval life, overgrowth in leg, wing and eye-antennal discs (Kunar and Roy, 2021; Mishra et al., 2020). These defects were rescued by introducing a functional copy of DCP2 in the mutant background (Mishra et al., 2020). DCP2 is an evolutionarily conserved mRNA decapping enzyme encoded by DCP2 gene present at 73A1 region in left arm of Drosophila chromosome 3 (Kunar and Roy, 2021). It belongs to the NUDIX family of pyro-phosphatases and was first identified in yeasts (Dunckley and Parker, 1999). DCP2 along with DCP1 cleaves the 5’ methyl guanosine cap of mRNA and is involved in cell cycle regulation and DNA repair(She et al., 2008). DCP2 has been found to be upregulated in lung cancer and gliomas where it promotes cell proliferation, invasion, suppression of apoptosis and tumor immune cell infiltration (Watson et al., 2008).
In human cancers, Hsp70 is commonly overexpressed and are integral to mitigating stress, promoting cell survival and preventing cell death. Hsp70 positive tumors are more aggressive and resistant to therapy (Liu et al., 2021; Murphy, 2013). In case of brain tumors, the intensity and localization of Hsp70 within the cell correlate strongly with tumor malignancy (Babi et al., 2022). High-grade gliomas exhibit a significantly greater frequency of Hsp70 overexpression in both the nucleus and cytosol, indicating the elevated requirement for chaperone-mediated survival capacity (Graner et al., 2007; Lobinger et al., 2021). Further, high levels of extracellular Hsp70, released by the necrotic cells in large sized tumors, are associated with an unfavorable prognosis and overall survival (Shevtsov et al., 2024). Hence, Hsp70 serves as an important diagnostic biomarker and therapeutic target for many brain tumors.
Therefore, we sought to determine whether the l(3)tb homozygous mutant of Drosophila, which develops tumourous brains, could serve as a tractable model to study the development of Hsp70-mediated brain tumors. In our study the mutant flies showed a phenotype similar to previous reports (Mishra et al., 2020). Homozygous l(3)tb homozygous mutant larvae show developmental delay and most of the larvae die before pupation. Only a few of the larvae enter pupal stage and eventually die (Fig. 1A). During the early stages of larval growth, the brain size of l(3)tb homozygous mutant larvae was found to be smaller than that of age-matched controls, indicating a developmental delay (Fig. 1B-C) and during the prolonged larval period, the brain size exponentially increased, ultimately leading to larval lethality (Fig. 1D-G). At the later stages of development, the size of l(3)tb/l(3)tb brain were approximately 3-fold larger than that of control (Fig. 1H). To further assess the induction of Hsp70-mediated stress-response in these aggressively growing brain tumors, we performed immunostaining using an anti-Hsp70 antibody. Control brains from third instar wild type larvae showed minimal stress-inducible Hsp70 expression which was distributed only in a few cells of brain at the left and right optic lobe region (Fig. 1I, J and K). A similar pattern of minimalistic Hsp70 staining had been proposed in non-heat-shocked larval brain samples (Krebs and Feder, 1997). Similar to wild type condition, third instar l(3)tb/l(3)tb larvae during initial stages of development, exhibited Hsp70 expression in a few cells of the optic lobes (Fig. 1L). However, during later stages, these Hsp70-positive regions expanded progressively along with tumor growth, eventually encompassing most of the optic lobe area (Fig. 1M-N). Quantitative analysis revealed an approximately 15-fold increase in Hsp70 expression in l(3)tb/l(3)tb tumourous brains compared to wild-type controls (Fig. 1I). Similarly, the area of optic lobe covered by Hsp70 expressing cells increased to ~35% in 12-day old tumor (Fig. 1J). Further, a direct association was observed between the brain size, quantified as DAPI positive area, and the area covered with Hsp70 expressing cells (Fig.1O). These observations indicated a strong correlation between temporal induction of Hsp70 and brain tumor progression.
In conclusion, the l(3)tb mutant of Drosophila offers a unique opportunity to model the interplay between oncogenic drivers and stress-adaptive chaperones in brain tumors. By recapitulating both the genetic and micro-environmental dimensions of tumor biology, this system provides a powerful and cost-effective platform for mechanistic discovery and therapeutic exploration on the role of Hsp70 in aggressive brain tumors. Most importantly, our findings indicate a direct link between Hsp70 expression and tumor progression in the l(3)tb brain tumor model, underscoring the promise of this conserved chaperone as a diagnostic and therapeutic target in stress-adapted malignant brain tumors. By labelling individual cells in the brain, it will be of interest to see which cell type express Hsp70 whose proliferation ultimately results in expanded growth of brain size. This work highlights the possible development of l(3)tb/l(3)tb Drosophila brain tumor model to study stress-tolerance in brain tumors with potential translational outcomes.
","references":[{"reference":"Babi, A., K. Menlibayeva, T. Bex, A. Doskaliev, S. Akshulakov, and M. Shevtsov. 2022. Targeting Heat Shock Proteins in Malignant Brain Tumors: From Basic Research to Clinical Trials. Cancers (Basel). 14.
","pubmedId":"","doi":""},{"reference":"Combs, S.E., T.E. Schmid, P. Vaupel, and G. Multhoff. 2016. Stress Response Leading to Resistance in Glioblastoma-The Need for Innovative Radiotherapy (iRT) Concepts. Cancers (Basel). 8.
","pubmedId":"","doi":""},{"reference":"Dunckley, T., and R. Parker. 1999. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 18:5411-5422.
","pubmedId":"","doi":""},{"reference":"Graner, M.W., R.I. Cumming, and D.D. Bigner. 2007. The heat shock response and chaperones/heat shock proteins in brain tumors: surface expression, release, and possible immune consequences. J Neurosci. 27:11214-11227.
","pubmedId":"","doi":""},{"reference":"Hong, H., M. Ji, and D. Lai. 2021. Chronic Stress Effects on Tumor: Pathway and Mechanism. Front Oncol. 11:738252.
","pubmedId":"","doi":""},{"reference":"Krebs, R.A., and M.E. Feder. 1997. Tissue-specific variation in Hsp70 expression and thermal damage in Drosophila melanogaster larvae. J Exp Biol. 200:2007-2015.
","pubmedId":"","doi":""},{"reference":"Kunar, R., and J.K. Roy. 2021. The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms. Cell Tissue Res. 386:261-280.
","pubmedId":"","doi":""},{"reference":"Kusaczuk, M., E.T. Ambel, M. Naumowicz, and G. Velasco. 2024. Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma. Biochim Biophys Acta Rev Cancer. 1879:189054.
","pubmedId":"","doi":""},{"reference":"Li, S., C. Wang, J. Chen, Y. Lan, W. Zhang, Z. Kang, Y. Zheng, R. Zhang, J. Yu, and W. Li. 2023. Signaling pathways in brain tumors and therapeutic interventions. Signal Transduct Target Ther. 8:8.
","pubmedId":"","doi":""},{"reference":"Liu, H., Z. Li, Q. Li, C. Jia, N. Zhang, Y. Qu, and D. Hu. 2021. HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. J Bioenerg Biomembr. 53:405-413.
","pubmedId":"","doi":""},{"reference":"Liu, K., K. Tsung, and F.J. Attenello. 2020. Characterizing Cell Stress and GRP78 in Glioma to Enhance Tumor Treatment. Front Oncol. 10:608911.
","pubmedId":"","doi":""},{"reference":"Lobinger, D., J. Gempt, W. Sievert, M. Barz, S. Schmitt, H.T. Nguyen, S. Stangl, C. Werner, F. Wang, Z. Wu, H. Fan, H. Zanth, M. Shevtsov, M. Pilz, I. Riederer, M. Schwab, J. Schlegel, and G. Multhoff. 2021. Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients. Front Mol Biosci. 8:669366.
","pubmedId":"","doi":""},{"reference":"Mishra, R., R. Kunar, L. Mandal, D.P. Alone, S. Chandrasekharan, A.K. Tiwari, M.G. Tapadia, A. Mukherjee, and J.K. Roy. 2020. A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumor Suppressor in Drosophila melanogaster. G3 (Bethesda). 10:2601-2618.
","pubmedId":"","doi":""},{"reference":"Munnik, C., M.P. Xaba, S.T. Malindisa, B.L. Russell, and S.A. Sooklal. 2022. Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies. Front Genet. 13:949241.
","pubmedId":"","doi":""},{"reference":"Murphy, M.E. 2013. The HSP70 family and cancer. Carcinogenesis. 34:1181-1188.
","pubmedId":"","doi":""},{"reference":"Nicholson, J.G., and H.A. Fine. 2021. Diffuse Glioma Heterogeneity and Its Therapeutic Implications. Cancer Discov. 11:575-590.
","pubmedId":"","doi":""},{"reference":"Pasqualini, C., T. Kozaki, M. Bruschi, T.H.H. Nguyen, V. Minard-Colin, D. Castel, J. Grill, and F. Ginhoux. 2020. Modeling the Interaction between the Microenvironment and Tumor Cells in Brain Tumors. Neuron. 108:1025-1044.
","pubmedId":"","doi":""},{"reference":"Qazi, M.A., P. Vora, C. Venugopal, S.S. Sidhu, J. Moffat, C. Swanton, and S.K. Singh. 2017. Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma. Ann Oncol. 28:1448-1456.
","pubmedId":"","doi":""},{"reference":"Read, R.D., W.K. Cavenee, F.B. Furnari, and J.B. Thomas. 2009. A drosophila model for EGFR-Ras and PI3K-dependent human glioma. PLoS Genet. 5:e1000374.
","pubmedId":"","doi":""},{"reference":"Rudrapatna, V.A., R.L. Cagan, and T.K. Das. 2012. Drosophila cancer models. Dev Dyn. 241:107-118.
","pubmedId":"","doi":""},{"reference":"Seiler, A., A.K. Sood, J. Jenewein, and C.P. Fagundes. 2020. Can stress promote the pathophysiology of brain metastases? A critical review of biobehavioral mechanisms. Brain Behav Immun. 87:860-880.
","pubmedId":"","doi":""},{"reference":"She, M., C.J. Decker, D.I. Svergun, A. Round, N. Chen, D. Muhlrad, R. Parker, and H. Song. 2008. Structural basis of dcp2 recognition and activation by dcp1. Mol Cell. 29:337-349.
","pubmedId":"","doi":""},{"reference":"Shevtsov, M., D. Bobkov, N. Yudintceva, R. Likhomanova, A. Kim, E. Fedorov, V. Fedorov, N. Mikhailova, E. Oganesyan, S. Shabelnikov, O. Rozanov, T. Garaev, N. Aksenov, A. Shatrova, A. Ten, A. Nechaeva, D. Goncharova, R. Ziganshin, A. Lukacheva, D. Sitovskaya, A. Ulitin, E. Pitkin, K. Samochernykh, E. Shlyakhto, and S.E. Combs. 2024. Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors. Cancer Res Commun. 4:2025-2044.
","pubmedId":"","doi":""},{"reference":"Watson, P.M., S.W. Miller, M. Fraig, D.J. Cole, D.K. Watson, and A.M. Boylan. 2008. CaSm (LSm-1) overexpression in lung cancer and mesothelioma is required for transformed phenotypes. Am J Respir Cell Mol Biol. 38:671-678
","pubmedId":"","doi":""}],"title":"The Drosophila l(3)tb mutant as a model to study stress-inducible Hsp70-expressing brain tumor
","reviews":[{"reviewer":{"displayName":"Sa Kan Yoo"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":null}]},{"id":"0aa19041-817a-4bf7-91fb-f5149f6f433c","decision":"accept","abstract":"Brain tumors remain among the most aggressive cancers due to their ability to adapt to microenvironmental stress to sustain malignancy and resist therapy. We propose Drosophila lethal(3) tumorous brain [l(3)tb] mutant, which develops rapidly expanding brain tumors, as a genetically tractable in vivo model to study stress-adaptive tumor growth. The progressive brain enlargement in l(3)tb/l(3)tb larvae is accompanied by robust tumor size-dependent induction of Hsp70, a molecular chaperone linked to poor glioma prognosis. These findings position the l(3)tb model as a powerful platform for evaluation of stress tolerance in brain tumors.
","acknowledgements":"We thank ISLS, BHU for the Confocal microscope facility.
","authors":[{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","writing_originalDraft"],"email":"abhijit.biswas10@bhu.ac.in","firstName":"Abhijit ","lastName":"Biswas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","fundingAcquisition","project","resources","supervision","validation","writing_reviewEditing"],"email":"devanjan@bhu.ac.in","firstName":"Devanjan","lastName":"Sinha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0001-5060-2075"}],"awards":[],"conflictsOfInterest":"The authors declare no conflicts of interest.
","dataTable":{"url":null},"extendedData":[],"funding":"Indian Council of Medical Research Ad-hoc grant (5/13/87/2020/NCD-III), BHU-Institute of Eminence grant (R/Dev/IoE/Incentive/2021-22/32452) to D.S. CSIR doctoral fellowship to A.B.
","image":{"url":"https://portal.micropublication.org/uploads/6887c1c747a107ae74694d665624739a.jpg"},"imageCaption":"(A) Morphological difference between larval progeny coming from Wild type (Oregon R+) and l(3)tb heterozygous parents, respectively. (B) Wild type larval brain after 6 days of egg laying (AEL) and (C-G) Larval brain size of delayed 3rd instar l(3)tb homozygous larvae in the indicated time point, showing gradual increase in brain size. (H) Tumourous brain of l(3)tb homozygous 3rd instar larvae (12 days AEL) [n=12] showing significant differences in optic lobe size compared to wild type [n=19] and l(3)tb homozygous 6 days larvae (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents optic lobe size. (I-J) Tumourous brain of l(3)tb homozygous 3rd instar larvae after 12 days of egg laying (AEL) [n=12], showing significant differences in Hsp70 intensity and distribution, compared to wild type [n=19] and l(3)tb homozygous 6 days larvae after egg laying (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents Hsp70 intensity (I) and % of Hsp70 expressed area (J), in each optic lobe respectively. Statistical analyses was performed by one-way ANOVA followed by Tukey's post-hoc multiple-comparison test; ****P<0.0001, **p < 0.01, *p < 0.05, not significant (ns)p > 0.05. (K-M) Confocal projection of wild type (K), l(3)tb 6 days (L) and l(3)tb- 12 days (M) homozygous larval brain showing Hsp70 staining in optic lobe area, scale bar: 50 μm. (O) Dot plot showing relationship of Hsp70 expression with size of optic lobe (demarcated from the DAPI+ area) of different genotypes [n=12, 9, 11 accordingly] where X-axis and Y-axis represents DAPI+ area and Hsp70+ area, respectively. Statistical significance of enhanced Hsp70 expression (WT vs l(3)tb brain) in the optic lobe area was determined through unpaired Student's t-test, where 6 days old WT brain were individually compared with either 6 days old l(3)tb brain or 12 days old l(3)tb brain. ***P (two-tailed)<0.001, **P(two-tailed)<0.01.
","imageTitle":"Temporal expression of stress inducible Hsp70 in developing l(3)tb brain tumour of Drosophila melanogaster
","methods":"Immunostaining: GFP+ clone bearing wing imaginal discs were dissected out in 1X PBS from the larvae of appropriate age and fixed in 4% formaldehyde for 20min. After fixation, wing discs were rinsed thrice with 0.1% PBST (0.1% Triton X-100 in 1X PBS) for 15 min each. After rinsing, discs were kept in blocking solution (10% fetal calf serum, 0.1% Bovine serum albumin, 0.1% Triton X-100, 0.02% Thiomersal and 0.1% sodium deoxycholate, in 1XPBS) for 1 hr. After that, discs were incubated with desired primary antibody: rat anti-Hsp70(7Fb 1:200, Sigma) for overnight at 4°C, following that discs were rinsed thrice with 0.1% PBST for 15min each and kept in a blocking solution for 1 hr. Thereafter, those discs were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 (1:200, Molecular Probes, USA), for 2hr at room temperature. After rinsing those discs thrice with 0.1% PBST, counterstained with 6-diamidino-2-phenylindole dihydrochloride (DAPI,1μg/ml, Thermo Fisher Scientific, Cat# D1306) and finally, mounted in an anti-fade mounting media 1,4-Diazabicyclo [2.2.2] octane (DABCO), Sigma, Cat# D27802, 2.5% DABCO in 70% glycerol made in 1X PBS). All the immunostained disc images were taken in Zeiss LSM 780 confocal microscopy with Zeiss ZEN2.3 SP1 Black edition software using Apo 20X (0.8 NA) and 63X (1.4 NA) oil immersion objectives. Images were assembled in Adobe Photoshop (2012). Quantitative analysis of acquire images were performed by using Fiji. Statistical analysis was done in Graph Pad Prism 8.4.2 as mentioned in the figure legends.
Imaging of larvae adults and larval brain: Photographs of larvae and adults were taken by using Axio Cam Zeiss camera mounted on Nikon SMZ800N stereo binocular microscope. Photographs of larval brain was taken using Nikon DS-Fi2 camera mounted on Nikon Eclipse 90i microscope.
","reagents":"Reagents
a. Fly Stocks:
1. l3tb/l3tb or l3tb/TM3, Ser (Mishra et al., 2020), where l3tb/TM6b, Tb flies used by the authors were rebalanced using TM3, Ser balancer to generate l3tb/TM3, Ser flies.
2.Oregon R+
b. Chemicals:
1.Hsp70 antibody (7Fb, Sigma)
","patternDescription":"Description
Primary brain tumors are characterized by severe cellular and molecular heterogeneity that are associated with aggressive disease progression, therapeutic resistance and recurrence (Nicholson and Fine, 2021; Qazi et al., 2017). Traditional in vitro cell culture systems, including both two-dimensional and three-dimensional cultures, are intrinsically limited because they fail to incorporate the necessary host-tumor environment. Brain tumor progression is dictated by intricate, non-cell autonomous signaling originating from the surrounding glia, vasculature, and immune cells (Pasqualini et al., 2020). Models that cannot incorporate this crucial microenvironment, risk overlooking the therapeutic targets related to the complex dynamics of tumor-host interaction.
Among the different adaptive features followed by rapidly proliferating tumors, stress adaptation has recently been appreciated to be one of the major drivers of tumor growth (Hong et al., 2021; Seiler et al., 2020). Brain tumors, particularly gliomas and glioblastomas, possess a variety of stress adaptive mechanisms that help them survive, proliferate, and resist therapy in an otherwise hostile micro-environment (Combs et al., 2016; Graner et al., 2007). These mechanisms act across metabolic, genetic, and signaling pathways to maintain homeostasis under oxidative, hypoxic, and therapeutic stress conditions (Li et al., 2023). Although therapy-induced stress result in accumulation of misfolded proteins that subsequently triggers apoptosis, activation of chaperones such as Grp78 induce the unfolded protein response that allows the cell to manage the misfolded proteins and increase therapy tolerance(Kusaczuk et al., 2024; Liu et al., 2020). This requires a need to develop suitable models to study the factors responsible for development of stress tolerance in brain tumors.
The common fruit fly, Drosophila melanogaster serves to overcome the limitations of conventional brain tumor models since it exhibits rapid life cycle and production of large number of offsprings that can allow high-throughput screening of large drug libraries(Rudrapatna et al., 2012). This simultaneously generates information on drug bio-availability, toxicity parameters and host-tumor interactions, allowing researchers to rapidly test compounds in the complex in vivo context(Munnik et al., 2022). Further, the exceptional molecular conservation of the fly system with humans allows precise engineering of brain tumors that closely mimic the human disease. For example, glioma model has been developed to mimic the human glioblastomas, by constitutive co-activation of EGFR and PI3K pathways specifically in glial cells by using tissue specific Gal4 drivers (Read et al., 2009). In a P-element mutagenesis screen, a specific gene mutation called as the l(3)tb (lethal tumorous brain) was isolated that is characterized by excess accumulation of neuroblasts due to their uncontrolled growth, leading to larval and pupal lethality (Mishra et al., 2020). The brain of l(3)tb homozygous larvae gradually increases in size with extended larval periods till death. Hence, the mutation was initially named as lethal (3) tumorous brain [l(3)tb]. Larvae homozygous for l(3)tb mutant, along with the presence of tumourous brain, show 12-13 days extended larval life, overgrowth in leg, wing and eye-antennal discs (Kunar and Roy, 2021; Mishra et al., 2020). These defects were rescued by introducing a functional copy of DCP2 in the mutant background (Mishra et al., 2020). DCP2 is an evolutionarily conserved mRNA decapping enzyme encoded by DCP2 gene present at 73A1 region in left arm of Drosophila chromosome 3 (Kunar and Roy, 2021). It belongs to the NUDIX family of pyro-phosphatases and was first identified in yeasts (Dunckley and Parker, 1999). DCP2 along with DCP1 cleaves the 5’ methyl guanosine cap of mRNA and is involved in cell cycle regulation and DNA repair(She et al., 2008). DCP2 has been found to be upregulated in lung cancer and gliomas where it promotes cell proliferation, invasion, suppression of apoptosis and tumor immune cell infiltration (Watson et al., 2008).
In human cancers, Hsp70 is commonly overexpressed and are integral to mitigating stress, promoting cell survival and preventing cell death. Hsp70 positive tumors are more aggressive and resistant to therapy (Liu et al., 2021; Murphy, 2013). In case of brain tumors, the intensity and localization of Hsp70 within the cell correlate strongly with tumor malignancy (Babi et al., 2022). High-grade gliomas exhibit a significantly greater frequency of Hsp70 overexpression in both the nucleus and cytosol, indicating the elevated requirement for chaperone-mediated survival capacity (Graner et al., 2007; Lobinger et al., 2021). Further, high levels of extracellular Hsp70, released by the necrotic cells in large sized tumors, are associated with an unfavorable prognosis and overall survival (Shevtsov et al., 2024). Hence, Hsp70 serves as an important diagnostic biomarker and therapeutic target for many brain tumors.
Therefore, we sought to determine whether the l(3)tb homozygous mutant of Drosophila, which develops tumourous brains, could serve as a tractable model to study the development of Hsp70-mediated brain tumors. In our study the mutant flies showed a phenotype similar to previous reports (Mishra et al., 2020). Homozygous l(3)tb homozygous mutant larvae show developmental delay and most of the larvae die before pupation. Only a few of the larvae enter pupal stage and eventually die (Fig. 1A). During the early stages of larval growth, the brain size of l(3)tb homozygous mutant larvae was found to be smaller than that of age-matched controls, indicating a developmental delay (Fig. 1B-C) and during the prolonged larval period, the brain size exponentially increased, ultimately leading to larval lethality (Fig. 1D-G). At the later stages of development, the size of l(3)tb/l(3)tb brain were approximately 3-fold larger than that of control (Fig. 1H). To further assess the induction of Hsp70-mediated stress-response in these aggressively growing brain tumors, we performed immunostaining using an anti-Hsp70 antibody. Control brains from third instar wild type larvae showed minimal stress-inducible Hsp70 expression which was distributed only in a few cells of brain at the left and right optic lobe region (Fig. 1I, J and K). A similar pattern of minimalistic Hsp70 staining had been proposed in non-heat-shocked larval brain samples (Krebs and Feder, 1997). Similar to wild type condition, third instar l(3)tb/l(3)tb larvae during initial stages of development, exhibited Hsp70 expression in a few cells of the optic lobes (Fig. 1L). However, during later stages, these Hsp70-positive regions expanded progressively along with tumor growth, eventually encompassing most of the optic lobe area (Fig. 1M-N). Quantitative analysis revealed an approximately 15-fold increase in Hsp70 expression in l(3)tb/l(3)tb tumourous brains compared to wild-type controls (Fig. 1I). Similarly, the area of optic lobe covered by Hsp70 expressing cells increased to ~35% in 12-day old tumor (Fig. 1J). Further, a direct association was observed between the brain size, quantified as DAPI positive area, and the area covered with Hsp70 expressing cells (Fig.1O). These observations indicated a strong correlation between temporal induction of Hsp70 and brain tumor progression.
In conclusion, the l(3)tb mutant of Drosophila offers a unique opportunity to model the interplay between oncogenic drivers and stress-adaptive chaperones in brain tumors. By recapitulating both the genetic and micro-environmental dimensions of tumor biology, this system provides a powerful and cost-effective platform for mechanistic discovery and therapeutic exploration on the role of Hsp70 in aggressive brain tumors. Most importantly, our findings indicate a direct link between Hsp70 expression and tumor progression in the l(3)tb brain tumor model, underscoring the promise of this conserved chaperone as a diagnostic and therapeutic target in stress-adapted malignant brain tumors. By labelling individual cells in the brain, it will be of interest to see which cell type express Hsp70 whose proliferation ultimately results in expanded growth of brain size. This work highlights the possible development of l(3)tb/l(3)tb Drosophila brain tumor model to study stress-tolerance in brain tumors with potential translational outcomes.
","references":[{"reference":"Babi, A., K. Menlibayeva, T. Bex, A. Doskaliev, S. Akshulakov, and M. Shevtsov. 2022. Targeting Heat Shock Proteins in Malignant Brain Tumors: From Basic Research to Clinical Trials. Cancers (Basel). 14.
","pubmedId":"","doi":""},{"reference":"Combs, S.E., T.E. Schmid, P. Vaupel, and G. Multhoff. 2016. Stress Response Leading to Resistance in Glioblastoma-The Need for Innovative Radiotherapy (iRT) Concepts. Cancers (Basel). 8.
","pubmedId":"","doi":""},{"reference":"Dunckley, T., and R. Parker. 1999. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 18:5411-5422.
","pubmedId":"","doi":""},{"reference":"Graner, M.W., R.I. Cumming, and D.D. Bigner. 2007. The heat shock response and chaperones/heat shock proteins in brain tumors: surface expression, release, and possible immune consequences. J Neurosci. 27:11214-11227.
","pubmedId":"","doi":""},{"reference":"Hong, H., M. Ji, and D. Lai. 2021. Chronic Stress Effects on Tumor: Pathway and Mechanism. Front Oncol. 11:738252.
","pubmedId":"","doi":""},{"reference":"Krebs, R.A., and M.E. Feder. 1997. Tissue-specific variation in Hsp70 expression and thermal damage in Drosophila melanogaster larvae. J Exp Biol. 200:2007-2015.
","pubmedId":"","doi":""},{"reference":"Kunar, R., and J.K. Roy. 2021. The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms. Cell Tissue Res. 386:261-280.
","pubmedId":"","doi":""},{"reference":"Kusaczuk, M., E.T. Ambel, M. Naumowicz, and G. Velasco. 2024. Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma. Biochim Biophys Acta Rev Cancer. 1879:189054.
","pubmedId":"","doi":""},{"reference":"Li, S., C. Wang, J. Chen, Y. Lan, W. Zhang, Z. Kang, Y. Zheng, R. Zhang, J. Yu, and W. Li. 2023. Signaling pathways in brain tumors and therapeutic interventions. Signal Transduct Target Ther. 8:8.
","pubmedId":"","doi":""},{"reference":"Liu, H., Z. Li, Q. Li, C. Jia, N. Zhang, Y. Qu, and D. Hu. 2021. HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. J Bioenerg Biomembr. 53:405-413.
","pubmedId":"","doi":""},{"reference":"Liu, K., K. Tsung, and F.J. Attenello. 2020. Characterizing Cell Stress and GRP78 in Glioma to Enhance Tumor Treatment. Front Oncol. 10:608911.
","pubmedId":"","doi":""},{"reference":"Lobinger, D., J. Gempt, W. Sievert, M. Barz, S. Schmitt, H.T. Nguyen, S. Stangl, C. Werner, F. Wang, Z. Wu, H. Fan, H. Zanth, M. Shevtsov, M. Pilz, I. Riederer, M. Schwab, J. Schlegel, and G. Multhoff. 2021. Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients. Front Mol Biosci. 8:669366.
","pubmedId":"","doi":""},{"reference":"Mishra, R., R. Kunar, L. Mandal, D.P. Alone, S. Chandrasekharan, A.K. Tiwari, M.G. Tapadia, A. Mukherjee, and J.K. Roy. 2020. A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumor Suppressor in Drosophila melanogaster. G3 (Bethesda). 10:2601-2618.
","pubmedId":"","doi":""},{"reference":"Munnik, C., M.P. Xaba, S.T. Malindisa, B.L. Russell, and S.A. Sooklal. 2022. Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies. Front Genet. 13:949241.
","pubmedId":"","doi":""},{"reference":"Murphy, M.E. 2013. The HSP70 family and cancer. Carcinogenesis. 34:1181-1188.
","pubmedId":"","doi":""},{"reference":"Nicholson, J.G., and H.A. Fine. 2021. Diffuse Glioma Heterogeneity and Its Therapeutic Implications. Cancer Discov. 11:575-590.
","pubmedId":"","doi":""},{"reference":"Pasqualini, C., T. Kozaki, M. Bruschi, T.H.H. Nguyen, V. Minard-Colin, D. Castel, J. Grill, and F. Ginhoux. 2020. Modeling the Interaction between the Microenvironment and Tumor Cells in Brain Tumors. Neuron. 108:1025-1044.
","pubmedId":"","doi":""},{"reference":"Qazi, M.A., P. Vora, C. Venugopal, S.S. Sidhu, J. Moffat, C. Swanton, and S.K. Singh. 2017. Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma. Ann Oncol. 28:1448-1456.
","pubmedId":"","doi":""},{"reference":"Read, R.D., W.K. Cavenee, F.B. Furnari, and J.B. Thomas. 2009. A drosophila model for EGFR-Ras and PI3K-dependent human glioma. PLoS Genet. 5:e1000374.
","pubmedId":"","doi":""},{"reference":"Rudrapatna, V.A., R.L. Cagan, and T.K. Das. 2012. Drosophila cancer models. Dev Dyn. 241:107-118.
","pubmedId":"","doi":""},{"reference":"Seiler, A., A.K. Sood, J. Jenewein, and C.P. Fagundes. 2020. Can stress promote the pathophysiology of brain metastases? A critical review of biobehavioral mechanisms. Brain Behav Immun. 87:860-880.
","pubmedId":"","doi":""},{"reference":"She, M., C.J. Decker, D.I. Svergun, A. Round, N. Chen, D. Muhlrad, R. Parker, and H. Song. 2008. Structural basis of dcp2 recognition and activation by dcp1. Mol Cell. 29:337-349.
","pubmedId":"","doi":""},{"reference":"Shevtsov, M., D. Bobkov, N. Yudintceva, R. Likhomanova, A. Kim, E. Fedorov, V. Fedorov, N. Mikhailova, E. Oganesyan, S. Shabelnikov, O. Rozanov, T. Garaev, N. Aksenov, A. Shatrova, A. Ten, A. Nechaeva, D. Goncharova, R. Ziganshin, A. Lukacheva, D. Sitovskaya, A. Ulitin, E. Pitkin, K. Samochernykh, E. Shlyakhto, and S.E. Combs. 2024. Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors. Cancer Res Commun. 4:2025-2044.
","pubmedId":"","doi":""},{"reference":"Watson, P.M., S.W. Miller, M. Fraig, D.J. Cole, D.K. Watson, and A.M. Boylan. 2008. CaSm (LSm-1) overexpression in lung cancer and mesothelioma is required for transformed phenotypes. Am J Respir Cell Mol Biol. 38:671-678
","pubmedId":"","doi":""}],"title":"The Drosophila l(3)tb mutant as a model to study stress-inducible Hsp70-expressing brain tumor
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":null}]},{"id":"02833ab9-377b-4c14-ad20-d4a5a628ce90","decision":"edit","abstract":"Brain tumors remain among the most aggressive cancers due to their ability to adapt to microenvironmental stress to sustain malignancy and resist therapy. We propose Drosophila lethal(3) tumorous brain [l(3)tb] mutant, which develops rapidly expanding brain tumors, as a genetically tractable in vivo model to study stress-adaptive tumor growth. The progressive brain enlargement in l(3)tb/l(3)tb larvae is accompanied by robust tumor size-dependent induction of Hsp70, a molecular chaperone linked to poor glioma prognosis. These findings position the l(3)tb model as a powerful platform for evaluation of stress tolerance in brain tumors.
","acknowledgements":"We thank ISLS, BHU for the Confocal microscope facility.
","authors":[{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","writing_originalDraft"],"email":"abhijit.biswas10@bhu.ac.in","firstName":"Abhijit ","lastName":"Biswas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","fundingAcquisition","project","resources","supervision","validation","writing_reviewEditing"],"email":"devanjan@bhu.ac.in","firstName":"Devanjan","lastName":"Sinha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0001-5060-2075"}],"awards":[],"conflictsOfInterest":"The authors declare no conflicts of interest.
","dataTable":{"url":null},"extendedData":[],"funding":"Indian Council of Medical Research Ad-hoc grant (5/13/87/2020/NCD-III), BHU-Institute of Eminence grant (R/Dev/IoE/Incentive/2021-22/32452) to D.S. CSIR doctoral fellowship to A.B.
","image":{"url":"https://portal.micropublication.org/uploads/c65a8f1c210337cd359e117bb4248e23.jpg"},"imageCaption":"(A) Morphological difference between larval progeny coming from Wild type (Oregon R+) and l(3)tb heterozygous parents, respectively. (B) Wild type larval brain after 6 days of egg laying (AEL) and (C-G) Larval brain size of delayed 3rd instar l(3)tb homozygous larvae in the indicated time point, showing gradual increase in brain size. (H) Tumourous brain of l(3)tb homozygous 3rd instar larvae (12 days AEL) [n=12] showing significant differences in optic lobe size compared to wild type [n=19] and l(3)tb homozygous 6 days larvae (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents optic lobe size. (I-J) Tumourous brain of l(3)tb homozygous 3rd instar larvae after 12 days of egg laying (AEL) [n=12], showing significant differences in Hsp70 intensity and distribution, compared to wild type [n=19] and l(3)tb homozygous 6 days larvae after egg laying (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents Hsp70 intensity (I) and % of Hsp70 expressed area (J), in each optic lobe respectively. Statistical analyses was performed by one-way ANOVA followed by Tukey's post-hoc multiple-comparison test; ****P<0.0001, **p < 0.01, *p < 0.05, not significant (ns)p > 0.05. (K-M) Confocal projection of wild type (K), l(3)tb 6 days (L) and l(3)tb- 12 days (M) homozygous larval brain showing Hsp70 staining in optic lobe area, scale bar: 50 μm. (O) Dot plot showing relationship of Hsp70 expression with size of optic lobe (demarcated from the DAPI+ area) of different genotypes [n=12, 9, 11 accordingly] where X-axis and Y-axis represents DAPI+ area and Hsp70+ area, respectively. Statistical significance of enhanced Hsp70 expression (WT vs l(3)tb brain) in the optic lobe area was determined through unpaired Student's t-test, where 6 days old WT brain were individually compared with either 6 days old l(3)tb brain or 12 days old l(3)tb brain. ***P (two-tailed)<0.001, **P(two-tailed)<0.01.
","imageTitle":"Temporal expression of stress inducible Hsp70 in developing l(3)tb brain tumour of Drosophila melanogaster
","methods":"Immunostaining: Larval brains were dissected out in 1X PBS from the larvae of appropriate age and fixed in 4% formaldehyde for 20min. After fixation, brains were rinsed thrice with 0.1% PBST (0.1% Triton X-100 in 1X PBS) for 15 min each. After rinsing, brains were kept in blocking solution (10% fetal calf serum, 0.1% Bovine serum albumin, 0.1% Triton X-100, 0.02% Thiomersal and 0.1% sodium deoxycholate, in 1X PBS) for 1 hr. After that, brains were incubated with desired primary antibody: rat anti-Hsp70 (7Fb 1:200, Sigma) for overnight at 4°C, following that brains were rinsed thrice with 0.1% PBST for 15 min each and kept in a blocking solution for 1 hr. Thereafter, those brains were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 (1:200, ThermoFisher Scientific, USA), for 2 hr at room temperature. After rinsing those brains thrice with 0.1% PBST, counterstained with 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg/ml, Thermo Fisher Scientific, Cat# D1306) and finally, mounted in an anti-fade mounting media 1,4-Diazabicyclo [2.2.2] octane (DABCO), Sigma, Cat# D27802, 2.5% DABCO in 70% glycerol made in 1X PBS). All the immunostained brain images were taken in Zeiss LSM 780 confocal microscopy with Zeiss ZEN2.3 SP1 Black edition software using Apo 20X (0.8 NA) and 63X (1.4 NA) oil immersion objectives. Images were assembled in Adobe Photoshop (2012). Quantitative analysis of acquire images were performed by using Fiji (ImageJ). Statistical analysis was done in GraphPad Prism 8.4.2 as mentioned in the figure legends.
Imaging of larvae adults and larval brain: Photographs of larvae and adults were taken by using Axio Cam Zeiss camera mounted on Nikon SMZ800N stereo binocular microscope. Photographs of larval brain was taken using Nikon DS-Fi2 camera mounted on Nikon Eclipse 90i microscope.
","reagents":"Reagents
a. Fly Stocks:
1. l3tb/l3tb or l3tb/TM3, Ser (Mishra et al., 2020), where l3tb/TM6b, Tb flies used by the authors were rebalanced using TM3, Ser balancer to generate l3tb/TM3, Ser flies.
2. Oregon R+ (Wild-type strain of Drosophila melanogaster)
b. Chemicals:
Name of reagent | Source | Catalog No |
Hsp70 antibody (clone 7Fb) | Sigma | SAB5200204 |
DAPI | Thermo Fisher Scientific | D1306 |
DABCO | Sigma | D27802 |
Anti-rat secondary antibody conjugated with Alexa Flour 488 | Thermo Fisher Scientific | A-11006 |
Description
Primary brain tumors are characterized by severe cellular and molecular heterogeneity that are associated with aggressive disease progression, therapeutic resistance and recurrence (Nicholson and Fine, 2021; Qazi et al., 2017). Traditional in vitro cell culture systems, including both two-dimensional and three-dimensional cultures, are intrinsically limited because they fail to incorporate the necessary host-tumor environment. Brain tumor progression is dictated by intricate, non-cell autonomous signaling originating from the surrounding glia, vasculature, and immune cells (Pasqualini et al., 2020). Models that cannot incorporate this crucial microenvironment, risk overlooking the therapeutic targets related to the complex dynamics of tumor-host interaction.
Among the different adaptive features followed by rapidly proliferating tumors, stress adaptation has recently been appreciated to be one of the major drivers of tumor growth (Hong et al., 2021; Seiler et al., 2020). Brain tumors, particularly gliomas and glioblastomas, possess a variety of stress adaptive mechanisms that help them survive, proliferate, and resist therapy in an otherwise hostile micro-environment (Combs et al., 2016; Graner et al., 2007). These mechanisms act across metabolic, genetic, and signaling pathways to maintain homeostasis under oxidative, hypoxic, and therapeutic stress conditions (Li et al., 2023). Although therapy-induced stress result in accumulation of misfolded proteins that subsequently triggers apoptosis, activation of chaperones such as Grp78 induce the unfolded protein response that allows the cell to manage the misfolded proteins and increase therapy tolerance (Kusaczuk et al., 2024; Liu et al., 2020). This requires a need to develop suitable models to study the factors responsible for development of stress tolerance in brain tumors.
The common fruit fly, Drosophila melanogaster serves to overcome the limitations of conventional brain tumor models since it exhibits rapid life cycle and production of large number of offsprings that can allow high-throughput screening of large drug libraries (Rudrapatna et al., 2012). This simultaneously generates information on drug bio-availability, toxicity parameters and host-tumor interactions, allowing researchers to rapidly test compounds in the complex in vivo context (Munnik et al., 2022). Further, the exceptional molecular conservation of the fly system with humans allows precise engineering of brain tumors that closely mimic the human disease. For example, glioma model has been developed to mimic the human glioblastomas, by constitutive co-activation of EGFR and PI3K pathways specifically in glial cells by using tissue specific Gal4 drivers (Read et al., 2009). In a P-element mutagenesis screen, a specific gene mutation called as the l(3)tb (lethal tumorous brain) was isolated that is characterized by excess accumulation of neuroblasts due to their uncontrolled growth, leading to larval and pupal lethality (Mishra et al., 2020). The brain of l(3)tb homozygous larvae gradually increases in size with extended larval periods till death. Hence, the mutation was initially named as lethal (3) tumorous brain [l(3)tb]. Larvae homozygous for l(3)tb mutant, along with the presence of tumourous brain, show 12-13 days extended larval life, overgrowth in leg, wing and eye-antennal discs (Kunar and Roy, 2021; Mishra et al., 2020). These defects were rescued by introducing a functional copy of DCP2 in the mutant background (Mishra et al., 2020). DCP2 is an evolutionarily conserved mRNA decapping enzyme encoded by DCP2 gene present at 73A1 region in left arm of Drosophila chromosome 3 (Kunar and Roy, 2021). It belongs to the NUDIX family of pyro-phosphatases and was first identified in yeasts (Dunckley and Parker, 1999). DCP2 along with DCP1 cleaves the 5’ methyl guanosine cap of mRNA and is involved in cell cycle regulation and DNA repair (She et al., 2008). DCP2 has been found to be upregulated in lung cancer and gliomas where it promotes cell proliferation, invasion, suppression of apoptosis and tumor immune cell infiltration (Watson et al., 2008).
In human cancers, Hsp70 is commonly overexpressed and are integral to mitigating stress, promoting cell survival and preventing cell death. Hsp70 positive tumors are more aggressive and resistant to therapy (Liu et al., 2021; Murphy, 2013). In case of brain tumors, the intensity and localization of Hsp70 within the cell correlate strongly with tumor malignancy (Babi et al., 2022). High-grade gliomas exhibit a significantly greater frequency of Hsp70 overexpression in both the nucleus and cytosol, indicating the elevated requirement for chaperone-mediated survival capacity (Graner et al., 2007; Lobinger et al., 2021). Further, high levels of extracellular Hsp70, released by the necrotic cells in large sized tumors, are associated with an unfavorable prognosis and overall survival (Shevtsov et al., 2024). Hence, Hsp70 serves as an important diagnostic biomarker and therapeutic target for many brain tumors.
Therefore, we sought to determine whether the l(3)tb homozygous mutant of Drosophila, which develops tumourous brains, could serve as a tractable model to study the development of Hsp70-mediated brain tumors. In our study the mutant flies showed a phenotype similar to previous reports (Mishra et al., 2020). Homozygous l(3)tb homozygous mutant larvae show developmental delay and most of the larvae die before pupation. Only a few of the larvae enter pupal stage and eventually die (Fig. 1A). During the early stages of larval growth, the brain size of l(3)tb homozygous mutant larvae was found to be smaller than that of age-matched controls, indicating a developmental delay (Fig. 1B-C) and during the prolonged larval period, the brain size exponentially increased, ultimately leading to larval lethality (Fig. 1D-G). At the later stages of development, the size of l(3)tb/l(3)tb brain were approximately 3-fold larger than that of control (Fig. 1H). To further assess the induction of Hsp70-mediated stress-response in these aggressively growing brain tumors, we performed immunostaining using an anti-Hsp70 antibody. Control brains from third instar wild type larvae showed minimal stress-inducible Hsp70 expression which was distributed only in a few cells of brain at the left and right optic lobe region (Fig. 1I, J and K). A similar pattern of minimalistic Hsp70 staining had been proposed in non-heat-shocked larval brain samples (Krebs and Feder, 1997). Similar to wild type condition, third instar l(3)tb/l(3)tb larvae during initial stages of development, exhibited Hsp70 expression in a few cells of the optic lobes (Fig. 1L). However, during later stages, these Hsp70-positive regions expanded progressively along with tumor growth, eventually encompassing most of the optic lobe area (Fig. 1M-N). Quantitative analysis revealed an approximately 15-fold increase in Hsp70 expression in l(3)tb/l(3)tb tumourous brains compared to wild-type controls (Fig. 1I). Similarly, the area of optic lobe covered by Hsp70 expressing cells increased to ~35% in 12-day old tumor (Fig. 1J). Further, a direct association was observed between the brain size, quantified as DAPI positive area, and the area covered with Hsp70 expressing cells (Fig.1O). These observations indicated a strong correlation between temporal induction of Hsp70 and brain tumor progression.
In conclusion, the l(3)tb mutant of Drosophila offers a unique opportunity to model the interplay between oncogenic drivers and stress-adaptive chaperones in brain tumors. By recapitulating both the genetic and micro-environmental dimensions of tumor biology, this system provides a powerful and cost-effective platform for mechanistic discovery and therapeutic exploration on the role of Hsp70 in aggressive brain tumors. Most importantly, our findings indicate a direct link between Hsp70 expression and tumor progression in the l(3)tb brain tumor model, underscoring the promise of this conserved chaperone as a diagnostic and therapeutic target in stress-adapted malignant brain tumors. By labelling individual cells in the brain, it will be of interest to see which cell type express Hsp70 whose proliferation ultimately results in expanded growth of brain size. This work highlights the possible development of l(3)tb/l(3)tb Drosophila brain tumor model to study stress-tolerance in brain tumors with potential translational outcomes.
","references":[{"reference":"Babi A, Menlibayeva K, Bex T, Doskaliev A, Akshulakov S, Shevtsov M. 2022. Targeting Heat Shock Proteins in Malignant Brain Tumors: From Basic Research to Clinical Trials. Cancers 14: 5435.
","pubmedId":"","doi":"10.3390/cancers14215435"},{"reference":"Combs S, Schmid T, Vaupel P, Multhoff G. 2016. Stress Response Leading to Resistance in Glioblastoma—The Need for Innovative Radiotherapy (iRT) Concepts. Cancers 8: 15.
","pubmedId":"","doi":"10.3390/cancers8010015"},{"reference":"Dunckley T, Parker R. 1999. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. The EMBO Journal 18: 5411-5422.
","pubmedId":"","doi":"10.1093/emboj/18.19.5411"},{"reference":"Graner MW, Cumming RI, Bigner DD. 2007. The Heat Shock Response and Chaperones/Heat Shock Proteins in Brain Tumors: Surface Expression, Release, and Possible Immune Consequences. The Journal of Neuroscience 27: 11214-11227.
","pubmedId":"","doi":"10.1523/JNEUROSCI.3588-07.2007"},{"reference":"Hong H, Ji M, Lai D. 2021. Chronic Stress Effects on Tumor: Pathway and Mechanism. Frontiers in Oncology 11: 10.3389/fonc.2021.738252.
","pubmedId":"","doi":"10.3389/fonc.2021.738252"},{"reference":"Krebs RA, Feder ME. 1997. Tissue-Specific Variation In Hsp70 Expression and Thermal Damage in Drosophila Melanogaster Larvae. Journal of Experimental Biology 200: 2007-2015.
","pubmedId":"","doi":"10.1242/jeb.200.14.2007"},{"reference":"Kunar R, Roy JK. 2021. The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila—insights from expression paradigms. Cell and Tissue Research 386: 261-280.
","pubmedId":"","doi":"10.1007/s00441-021-03503-x"},{"reference":"Kusaczuk M, Ambel ET, Naumowicz M, Velasco G. 2024. Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1879: 189054.
","pubmedId":"","doi":"10.1016/j.bbcan.2023.189054"},{"reference":"Li S, Wang C, Chen J, Lan Y, Zhang W, Kang Z, et al., Li. 2023. Signaling pathways in brain tumors and therapeutic interventions. Signal Transduction and Targeted Therapy 8: 10.1038/s41392-022-01260-z.
","pubmedId":"","doi":"10.1038/s41392-022-01260-z"},{"reference":"Liu H, Li Z, Li Q, Jia C, Zhang N, Qu Y, Hu D. 2021. HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. Journal of Bioenergetics and Biomembranes 53: 405-413.
","pubmedId":"","doi":"10.1007/s10863-021-09904-5"},{"reference":"Liu K, Tsung K, Attenello FJ. 2020. Characterizing Cell Stress and GRP78 in Glioma to Enhance Tumor Treatment. Frontiers in Oncology 10: 10.3389/fonc.2020.608911.
","pubmedId":"","doi":"10.3389/fonc.2020.608911"},{"reference":"Lobinger D, Gempt J, Sievert W, Barz M, Schmitt S, Nguyen HT, et al., Multhoff. 2021. Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.669366.
","pubmedId":"","doi":"10.3389/fmolb.2021.669366"},{"reference":"Mishra R, Kunar R, Mandal L, Alone DP, Chandrasekharan S, Tiwari AK, et al., Roy. 2020. A Forward Genetic Approach to Mapping a\n P\n -Element Second Site Mutation Identifies\n DCP2\n as a Novel Tumor Suppressor in\n Drosophila melanogaster. G3 Genes|Genomes|Genetics 10: 2601-2618.
","pubmedId":"","doi":"10.1534/g3.120.401501"},{"reference":"Munnik C, Xaba MP, Malindisa ST, Russell BL, Sooklal SA. 2022. Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies. Frontiers in Genetics 13: 10.3389/fgene.2022.949241.
","pubmedId":"","doi":"10.3389/fgene.2022.949241"},{"reference":"Murphy ME. 2013. The HSP70 family and cancer. Carcinogenesis 34: 1181-1188.
","pubmedId":"","doi":"10.1093/carcin/bgt111"},{"reference":"Nicholson JG, Fine HA. 2021. Diffuse Glioma Heterogeneity and Its Therapeutic Implications. Cancer Discovery 11: 575-590.
","pubmedId":"","doi":"10.1158/2159-8290.CD-20-1474"},{"reference":"Pasqualini C, Kozaki T, Bruschi M, Nguyen THH, Minard-Colin Vr, Castel D, Grill J, Ginhoux F. 2020. Modeling the Interaction between the Microenvironment and Tumor Cells in Brain Tumors. Neuron 108: 1025-1044.
","pubmedId":"","doi":"10.1016/j.neuron.2020.09.018"},{"reference":"Qazi MA, Vora P, Venugopal C, Sidhu SS, Moffat J, Swanton C, Singh SK. 2017. Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma. Annals of Oncology 28: 1448-1456.
","pubmedId":"","doi":"10.1093/annonc/mdx169"},{"reference":"Read RD, Cavenee WK, Furnari FB, Thomas JB. 2009. A Drosophila Model for EGFR-Ras and PI3K-Dependent Human Glioma. PLoS Genetics 5: e1000374.
","pubmedId":"","doi":"10.1371/journal.pgen.1000374"},{"reference":"Rudrapatna VA, Cagan RL, Das TK. 2011. Drosophila cancer models. Developmental Dynamics 241: 107-118.
","pubmedId":"","doi":"10.1002/dvdy.22771"},{"reference":"Seiler A, Sood AK, Jenewein J, Fagundes CP. 2020. Can stress promote the pathophysiology of brain metastases? A critical review of biobehavioral mechanisms. Brain, Behavior, and Immunity 87: 860-880.
","pubmedId":"","doi":"10.1016/j.bbi.2019.12.013"},{"reference":"She M, Decker CJ, Svergun DI, Round A, Chen N, Muhlrad D, Parker R, Song H. 2008. Structural Basis of Dcp2 Recognition and Activation by Dcp1. Molecular Cell 29: 337-349.
","pubmedId":"","doi":"10.1016/j.molcel.2008.01.002"},{"reference":"Shevtsov M, Bobkov D, Yudintceva N, Likhomanova R, Kim A, Fedorov E, et al., Combs. 2024. Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors. Cancer Research Communications 4: 2025-2044.
","pubmedId":"","doi":"10.1158/2767-9764.CRC-24-0094"},{"reference":"Watson PM, Miller SW, Fraig M, Cole DJ, Watson DK, Boylan AM. 2008. CaSm (LSm-1) Overexpression in Lung Cancer and Mesothelioma Is Required for Transformed Phenotypes. American Journal of Respiratory Cell and Molecular Biology 38: 671-678.
","pubmedId":"","doi":"10.1165/rcmb.2007-0205OC"}],"title":"The Drosophila l(3)tb mutant as a model to study stress-inducible Hsp70-expressing brain tumor
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":null}]},{"id":"2c71d5c6-2e91-4990-b82d-3c73ddacc06b","decision":"edit","abstract":"Brain tumors remain among the most aggressive cancers due to their ability to adapt to microenvironmental stress to sustain malignancy and resist therapy. We propose Drosophila lethal(3) tumorous brain [l(3)tb] mutant, which develops rapidly expanding brain tumors, as a genetically tractable in vivo model to study stress-adaptive tumor growth. The progressive brain enlargement in l(3)tb/l(3)tb larvae is accompanied by robust tumor size-dependent induction of Hsp70, a molecular chaperone linked to poor glioma prognosis. These findings position the l(3)tb model as a powerful platform for evaluation of stress tolerance in brain tumors.
","acknowledgements":"We thank ISLS, BHU for the Confocal microscope facility.
","authors":[{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","writing_originalDraft"],"email":"abhijit.biswas10@bhu.ac.in","firstName":"Abhijit ","lastName":"Biswas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","fundingAcquisition","project","resources","supervision","validation","writing_reviewEditing"],"email":"devanjan@bhu.ac.in","firstName":"Devanjan","lastName":"Sinha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0001-5060-2075"}],"awards":[],"conflictsOfInterest":"The authors declare no conflicts of interest.
","dataTable":{"url":null},"extendedData":[],"funding":"Indian Council of Medical Research Ad-hoc grant (5/13/87/2020/NCD-III), BHU-Institute of Eminence grant (R/Dev/IoE/Incentive/2021-22/32452) to D.S. CSIR doctoral fellowship to A.B.
","image":{"url":"https://portal.micropublication.org/uploads/c65a8f1c210337cd359e117bb4248e23.jpg"},"imageCaption":"(A) Morphological difference between larval progeny coming from Wild type (Oregon R+) and l(3)tb heterozygous parents, respectively. (B) Wild type larval brain after 6 days of egg laying (AEL) and (C-G) Larval brain size of delayed 3rd instar l(3)tb homozygous larvae in the indicated time point, showing gradual increase in brain size. (H) Tumourous brain of l(3)tb homozygous 3rd instar larvae (12 days AEL) [n=12] showing significant differences in optic lobe size compared to wild type [n=19] and l(3)tb homozygous 6 days larvae (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents optic lobe size. (I-J) Tumourous brain of l(3)tb homozygous 3rd instar larvae after 12 days of egg laying (AEL) [n=12], showing significant differences in Hsp70 intensity and distribution, compared to wild type [n=19] and l(3)tb homozygous 6 days larvae after egg laying (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents Hsp70 intensity (I) and % of Hsp70 expressed area (J), in each optic lobe respectively. Statistical analyses was performed by one-way ANOVA followed by Tukey's post-hoc multiple-comparison test; ****P<0.0001, **p < 0.01, *p < 0.05, not significant (ns)p > 0.05. (K-M) Confocal projection of wild type (K), l(3)tb 6 days (L) and l(3)tb- 12 days (M) homozygous larval brain showing Hsp70 staining in optic lobe area, scale bar: 50 μm. (O) Dot plot showing relationship of Hsp70 expression with size of optic lobe (demarcated from the DAPI+ area) of different genotypes [n=12, 9, 11 accordingly] where X-axis and Y-axis represents DAPI+ area and Hsp70+ area, respectively. Statistical significance of enhanced Hsp70 expression (WT vs l(3)tb brain) in the optic lobe area was determined through unpaired Student's t-test, where 6 days old WT brain were individually compared with either 6 days old l(3)tb brain or 12 days old l(3)tb brain. ***P (two-tailed)<0.001, **P(two-tailed)<0.01.
","imageTitle":"Temporal expression of stress inducible Hsp70 in developing l(3)tb brain tumour of Drosophila melanogaster
","methods":"Immunostaining: Larval brains were dissected out in 1X PBS from the larvae of appropriate age and fixed in 4% formaldehyde for 20min. After fixation, brains were rinsed thrice with 0.1% PBST (0.1% Triton X-100 in 1X PBS) for 15 min each. After rinsing, brains were kept in blocking solution (10% fetal calf serum, 0.1% Bovine serum albumin, 0.1% Triton X-100, 0.02% Thiomersal and 0.1% sodium deoxycholate, in 1X PBS) for 1 hr. After that, brains were incubated with desired primary antibody: rat anti-Hsp70 (7Fb 1:200, Sigma) for overnight at 4°C, following that brains were rinsed thrice with 0.1% PBST for 15 min each and kept in a blocking solution for 1 hr. Thereafter, those brains were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 (1:200, ThermoFisher Scientific, USA), for 2 hr at room temperature. After rinsing those brains thrice with 0.1% PBST, counterstained with 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg/ml, Thermo Fisher Scientific, Cat# D1306) and finally, mounted in an anti-fade mounting media 1,4-Diazabicyclo [2.2.2] octane (DABCO), Sigma, Cat# D27802, 2.5% DABCO in 70% glycerol made in 1X PBS). All the immunostained brain images were taken in Zeiss LSM 780 confocal microscopy with Zeiss ZEN2.3 SP1 Black edition software using Apo 20X (0.8 NA) and 63X (1.4 NA) oil immersion objectives. Images were assembled in Adobe Photoshop (2012). Quantitative analysis of acquire images were performed by using Fiji (ImageJ). Statistical analysis was done in GraphPad Prism 8.4.2 as mentioned in the figure legends.
Imaging of larvae adults and larval brain: Photographs of larvae and adults were taken by using Axio Cam Zeiss camera mounted on Nikon SMZ800N stereo binocular microscope. Photographs of larval brain was taken using Nikon DS-Fi2 camera mounted on Nikon Eclipse 90i microscope.
","reagents":"Reagents
a. Fly Stocks:
1. l3tb/l3tb or l3tb/TM3, Ser (Mishra et al., 2020), where l3tb/TM6b, Tb flies used by the authors were rebalanced using TM3, Ser balancer to generate l3tb/TM3, Ser flies.
2. Oregon R+ (Wild-type strain of Drosophila melanogaster)
b. Chemicals:
Name of reagent | Source | Catalog No |
Hsp70 antibody (clone 7Fb) | Sigma | SAB5200204 |
DAPI | Thermo Fisher Scientific | D1306 |
DABCO | Sigma | D27802 |
Anti-rat secondary antibody conjugated with Alexa Flour 488 | Thermo Fisher Scientific | A-11006 |
Description
Primary brain tumors are characterized by severe cellular and molecular heterogeneity that are associated with aggressive disease progression, therapeutic resistance and recurrence (Nicholson and Fine, 2021; Qazi et al., 2017). Traditional in vitro cell culture systems, including both two-dimensional and three-dimensional cultures, are intrinsically limited because they fail to incorporate the necessary host-tumor environment. Brain tumor progression is dictated by intricate, non-cell autonomous signaling originating from the surrounding glia, vasculature, and immune cells (Pasqualini et al., 2020). Models that cannot incorporate this crucial microenvironment, risk overlooking the therapeutic targets related to the complex dynamics of tumor-host interaction.
Among the different adaptive features followed by rapidly proliferating tumors, stress adaptation has recently been appreciated to be one of the major drivers of tumor growth (Hong et al., 2021; Seiler et al., 2020). Brain tumors, particularly gliomas and glioblastomas, possess a variety of stress adaptive mechanisms that help them survive, proliferate, and resist therapy in an otherwise hostile micro-environment (Combs et al., 2016; Graner et al., 2007). These mechanisms act across metabolic, genetic, and signaling pathways to maintain homeostasis under oxidative, hypoxic, and therapeutic stress conditions (Li et al., 2023). Although therapy-induced stress result in accumulation of misfolded proteins that subsequently triggers apoptosis, activation of chaperones such as Grp78 induce the unfolded protein response that allows the cell to manage the misfolded proteins and increase therapy tolerance (Kusaczuk et al., 2024; Liu et al., 2020). This requires a need to develop suitable models to study the factors responsible for development of stress tolerance in brain tumors.
The common fruit fly, Drosophila melanogaster serves to overcome the limitations of conventional brain tumor models since it exhibits rapid life cycle and production of large number of offsprings that can allow high-throughput screening of large drug libraries (Rudrapatna et al., 2012). This simultaneously generates information on drug bio-availability, toxicity parameters and host-tumor interactions, allowing researchers to rapidly test compounds in the complex in vivo context (Munnik et al., 2022). Further, the exceptional molecular conservation of the fly system with humans allows precise engineering of brain tumors that closely mimic the human disease. For example, glioma model has been developed to mimic the human glioblastomas, by constitutive co-activation of EGFR and PI3K pathways specifically in glial cells by using tissue specific Gal4 drivers (Read et al., 2009). In a P-element mutagenesis screen, a specific gene mutation called as the l(3)tb (lethal tumorous brain) was isolated that is characterized by excess accumulation of neuroblasts due to their uncontrolled growth, leading to larval and pupal lethality (Mishra et al., 2020). The brain of l(3)tb homozygous larvae gradually increases in size with extended larval periods till death. Hence, the mutation was initially named as lethal (3) tumorous brain [l(3)tb]. Larvae homozygous for l(3)tb mutant, along with the presence of tumourous brain, show 12-13 days extended larval life, overgrowth in leg, wing and eye-antennal discs (Kunar and Roy, 2021; Mishra et al., 2020). These defects were rescued by introducing a functional copy of DCP2 in the mutant background (Mishra et al., 2020). DCP2 is an evolutionarily conserved mRNA decapping enzyme encoded by DCP2 gene present at 73A1 region in left arm of Drosophila chromosome 3 (Kunar and Roy, 2021). It belongs to the NUDIX family of pyro-phosphatases and was first identified in yeasts (Dunckley and Parker, 1999). DCP2 along with DCP1 cleaves the 5’ methyl guanosine cap of mRNA and is involved in cell cycle regulation and DNA repair (She et al., 2008). DCP2 has been found to be upregulated in lung cancer and gliomas where it promotes cell proliferation, invasion, suppression of apoptosis and tumor immune cell infiltration (Watson et al., 2008).
In human cancers, Hsp70 is commonly overexpressed and are integral to mitigating stress, promoting cell survival and preventing cell death. Hsp70 positive tumors are more aggressive and resistant to therapy (Liu et al., 2021; Murphy, 2013). In case of brain tumors, the intensity and localization of Hsp70 within the cell correlate strongly with tumor malignancy (Babi et al., 2022). High-grade gliomas exhibit a significantly greater frequency of Hsp70 overexpression in both the nucleus and cytosol, indicating the elevated requirement for chaperone-mediated survival capacity (Graner et al., 2007; Lobinger et al., 2021). Further, high levels of extracellular Hsp70, released by the necrotic cells in large sized tumors, are associated with an unfavorable prognosis and overall survival (Shevtsov et al., 2024). Hence, Hsp70 serves as an important diagnostic biomarker and therapeutic target for many brain tumors.
Therefore, we sought to determine whether the l(3)tb homozygous mutant of Drosophila, which develops tumourous brains, could serve as a tractable model to study the development of Hsp70-mediated brain tumors. In our study the mutant flies showed a phenotype similar to previous reports (Mishra et al., 2020). Homozygous l(3)tb homozygous mutant larvae show developmental delay and most of the larvae die before pupation. Only a few of the larvae enter pupal stage and eventually die (Fig. 1A). During the early stages of larval growth, the brain size of l(3)tb homozygous mutant larvae was found to be smaller than that of age-matched controls, indicating a developmental delay (Fig. 1B-C) and during the prolonged larval period, the brain size exponentially increased, ultimately leading to larval lethality (Fig. 1D-G). At the later stages of development, the size of l(3)tb/l(3)tb brain were approximately 3-fold larger than that of control (Fig. 1H). To further assess the induction of Hsp70-mediated stress-response in these aggressively growing brain tumors, we performed immunostaining using an anti-Hsp70 antibody. Control brains from third instar wild type larvae showed minimal stress-inducible Hsp70 expression which was distributed only in a few cells of brain at the left and right optic lobe region (Fig. 1I, J and K). A similar pattern of minimalistic Hsp70 staining had been proposed in non-heat-shocked larval brain samples (Krebs and Feder, 1997). Similar to wild type condition, third instar l(3)tb/l(3)tb larvae during initial stages of development, exhibited Hsp70 expression in a few cells of the optic lobes (Fig. 1L). However, during later stages, these Hsp70-positive regions expanded progressively along with tumor growth, eventually encompassing most of the optic lobe area (Fig. 1M-N). Quantitative analysis revealed an approximately 15-fold increase in Hsp70 expression in l(3)tb/l(3)tb tumourous brains compared to wild-type controls (Fig. 1I). Similarly, the area of optic lobe covered by Hsp70 expressing cells increased to ~35% in 12-day old tumor (Fig. 1J). Further, a direct association was observed between the brain size, quantified as DAPI positive area, and the area covered with Hsp70 expressing cells (Fig.1O). These observations indicated a strong correlation between temporal induction of Hsp70 and brain tumor progression.
In conclusion, the l(3)tb mutant of Drosophila offers a unique opportunity to model the interplay between oncogenic drivers and stress-adaptive chaperones in brain tumors. By recapitulating both the genetic and micro-environmental dimensions of tumor biology, this system provides a powerful and cost-effective platform for mechanistic discovery and therapeutic exploration on the role of Hsp70 in aggressive brain tumors. Most importantly, our findings indicate a direct link between Hsp70 expression and tumor progression in the l(3)tb brain tumor model, underscoring the promise of this conserved chaperone as a diagnostic and therapeutic target in stress-adapted malignant brain tumors. By labelling individual cells in the brain, it will be of interest to see which cell type express Hsp70 whose proliferation ultimately results in expanded growth of brain size. This work highlights the possible development of l(3)tb/l(3)tb Drosophila brain tumor model to study stress-tolerance in brain tumors with potential translational outcomes.
","references":[{"reference":"Babi A, Menlibayeva K, Bex T, Doskaliev A, Akshulakov S, Shevtsov M. 2022. Targeting Heat Shock Proteins in Malignant Brain Tumors: From Basic Research to Clinical Trials. Cancers 14: 5435.
","pubmedId":"","doi":"10.3390/cancers14215435"},{"reference":"Combs S, Schmid T, Vaupel P, Multhoff G. 2016. Stress Response Leading to Resistance in Glioblastoma—The Need for Innovative Radiotherapy (iRT) Concepts. Cancers 8: 15.
","pubmedId":"","doi":"10.3390/cancers8010015"},{"reference":"Dunckley T, Parker R. 1999. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. The EMBO Journal 18: 5411-5422.
","pubmedId":"","doi":"10.1093/emboj/18.19.5411"},{"reference":"Graner MW, Cumming RI, Bigner DD. 2007. The Heat Shock Response and Chaperones/Heat Shock Proteins in Brain Tumors: Surface Expression, Release, and Possible Immune Consequences. The Journal of Neuroscience 27: 11214-11227.
","pubmedId":"","doi":"10.1523/JNEUROSCI.3588-07.2007"},{"reference":"Hong H, Ji M, Lai D. 2021. Chronic Stress Effects on Tumor: Pathway and Mechanism. Frontiers in Oncology 11: 10.3389/fonc.2021.738252.
","pubmedId":"","doi":"10.3389/fonc.2021.738252"},{"reference":"Krebs RA, Feder ME. 1997. Tissue-Specific Variation In Hsp70 Expression and Thermal Damage in Drosophila Melanogaster Larvae. Journal of Experimental Biology 200: 2007-2015.
","pubmedId":"","doi":"10.1242/jeb.200.14.2007"},{"reference":"Kunar R, Roy JK. 2021. The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila—insights from expression paradigms. Cell and Tissue Research 386: 261-280.
","pubmedId":"","doi":"10.1007/s00441-021-03503-x"},{"reference":"Kusaczuk M, Ambel ET, Naumowicz M, Velasco G. 2024. Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1879: 189054.
","pubmedId":"","doi":"10.1016/j.bbcan.2023.189054"},{"reference":"Li S, Wang C, Chen J, Lan Y, Zhang W, Kang Z, et al., Li. 2023. Signaling pathways in brain tumors and therapeutic interventions. Signal Transduction and Targeted Therapy 8: 10.1038/s41392-022-01260-z.
","pubmedId":"","doi":"10.1038/s41392-022-01260-z"},{"reference":"Liu H, Li Z, Li Q, Jia C, Zhang N, Qu Y, Hu D. 2021. HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. Journal of Bioenergetics and Biomembranes 53: 405-413.
","pubmedId":"","doi":"10.1007/s10863-021-09904-5"},{"reference":"Liu K, Tsung K, Attenello FJ. 2020. Characterizing Cell Stress and GRP78 in Glioma to Enhance Tumor Treatment. Frontiers in Oncology 10: 10.3389/fonc.2020.608911.
","pubmedId":"","doi":"10.3389/fonc.2020.608911"},{"reference":"Lobinger D, Gempt J, Sievert W, Barz M, Schmitt S, Nguyen HT, et al., Multhoff. 2021. Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.669366.
","pubmedId":"","doi":"10.3389/fmolb.2021.669366"},{"reference":"Mishra R, Kunar R, Mandal L, Alone DP, Chandrasekharan S, Tiwari AK, et al., Roy. 2020. A Forward Genetic Approach to Mapping a\n P\n -Element Second Site Mutation Identifies\n DCP2\n as a Novel Tumor Suppressor in\n Drosophila melanogaster. G3 Genes|Genomes|Genetics 10: 2601-2618.
","pubmedId":"","doi":"10.1534/g3.120.401501"},{"reference":"Munnik C, Xaba MP, Malindisa ST, Russell BL, Sooklal SA. 2022. Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies. Frontiers in Genetics 13: 10.3389/fgene.2022.949241.
","pubmedId":"","doi":"10.3389/fgene.2022.949241"},{"reference":"Murphy ME. 2013. The HSP70 family and cancer. Carcinogenesis 34: 1181-1188.
","pubmedId":"","doi":"10.1093/carcin/bgt111"},{"reference":"Nicholson JG, Fine HA. 2021. Diffuse Glioma Heterogeneity and Its Therapeutic Implications. Cancer Discovery 11: 575-590.
","pubmedId":"","doi":"10.1158/2159-8290.CD-20-1474"},{"reference":"Pasqualini C, Kozaki T, Bruschi M, Nguyen THH, Minard-Colin Vr, Castel D, Grill J, Ginhoux F. 2020. Modeling the Interaction between the Microenvironment and Tumor Cells in Brain Tumors. Neuron 108: 1025-1044.
","pubmedId":"","doi":"10.1016/j.neuron.2020.09.018"},{"reference":"Qazi MA, Vora P, Venugopal C, Sidhu SS, Moffat J, Swanton C, Singh SK. 2017. Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma. Annals of Oncology 28: 1448-1456.
","pubmedId":"","doi":"10.1093/annonc/mdx169"},{"reference":"Read RD, Cavenee WK, Furnari FB, Thomas JB. 2009. A Drosophila Model for EGFR-Ras and PI3K-Dependent Human Glioma. PLoS Genetics 5: e1000374.
","pubmedId":"","doi":"10.1371/journal.pgen.1000374"},{"reference":"Rudrapatna VA, Cagan RL, Das TK. 2011. Drosophila cancer models. Developmental Dynamics 241: 107-118.
","pubmedId":"","doi":"10.1002/dvdy.22771"},{"reference":"Seiler A, Sood AK, Jenewein J, Fagundes CP. 2020. Can stress promote the pathophysiology of brain metastases? A critical review of biobehavioral mechanisms. Brain, Behavior, and Immunity 87: 860-880.
","pubmedId":"","doi":"10.1016/j.bbi.2019.12.013"},{"reference":"She M, Decker CJ, Svergun DI, Round A, Chen N, Muhlrad D, Parker R, Song H. 2008. Structural Basis of Dcp2 Recognition and Activation by Dcp1. Molecular Cell 29: 337-349.
","pubmedId":"","doi":"10.1016/j.molcel.2008.01.002"},{"reference":"Shevtsov M, Bobkov D, Yudintceva N, Likhomanova R, Kim A, Fedorov E, et al., Combs. 2024. Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors. Cancer Research Communications 4: 2025-2044.
","pubmedId":"","doi":"10.1158/2767-9764.CRC-24-0094"},{"reference":"Watson PM, Miller SW, Fraig M, Cole DJ, Watson DK, Boylan AM. 2008. CaSm (LSm-1) Overexpression in Lung Cancer and Mesothelioma Is Required for Transformed Phenotypes. American Journal of Respiratory Cell and Molecular Biology 38: 671-678.
","pubmedId":"","doi":"10.1165/rcmb.2007-0205OC"}],"title":"The Drosophila l(3)tb mutant as a model to study stress-inducible Hsp70-expressing brain tumor
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Steven Marygold"},"openAcknowledgement":false,"submitted":null}]},{"id":"ad0937f5-e916-4a22-ae22-f12558b29d1e","decision":"publish","abstract":"Brain tumors remain among the most aggressive cancers due to their ability to adapt to microenvironmental stress to sustain malignancy and resist therapy. We propose Drosophila lethal(3) tumorous brain [l(3)tb] mutant, which develops rapidly expanding brain tumors, as a genetically tractable in vivo model to study stress-adaptive tumor growth. The progressive brain enlargement in l(3)tb/l(3)tb larvae is accompanied by robust tumor size-dependent induction of Hsp70, a molecular chaperone linked to poor glioma prognosis. These findings position the l(3)tb model as a powerful platform for evaluation of stress tolerance in brain tumors.
","acknowledgements":"We thank ISLS, BHU for the Confocal microscope facility.
","authors":[{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","dataCuration","formalAnalysis","methodology","investigation","writing_originalDraft"],"email":"abhijit.biswas10@bhu.ac.in","firstName":"Abhijit ","lastName":"Biswas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Banaras Hindu University, Varanasi, UP, IN"],"departments":["Department of Zoology"],"credit":["conceptualization","fundingAcquisition","project","resources","supervision","validation","writing_reviewEditing"],"email":"devanjan@bhu.ac.in","firstName":"Devanjan","lastName":"Sinha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0001-5060-2075"}],"awards":[],"conflictsOfInterest":"The authors declare no conflicts of interest.
","dataTable":{"url":null},"extendedData":[],"funding":"Indian Council of Medical Research Ad-hoc grant (5/13/87/2020/NCD-III), BHU-Institute of Eminence grant (R/Dev/IoE/Incentive/2021-22/32452) to D.S. CSIR doctoral fellowship to A.B.
","image":{"url":"https://portal.micropublication.org/uploads/c65a8f1c210337cd359e117bb4248e23.jpg"},"imageCaption":"(A) Morphological difference between larval progeny coming from Wild type (Oregon R+) and l(3)tb heterozygous parents, respectively. (B) Wild type larval brain after 6 days of egg laying (AEL) and (C-G) Larval brain size of delayed 3rd instar l(3)tb homozygous larvae in the indicated time point, showing gradual increase in brain size. (H) Tumourous brain of l(3)tb homozygous 3rd instar larvae (12 days AEL) [n=12] showing significant differences in optic lobe size compared to wild type [n=19] and l(3)tb homozygous 6 days larvae (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents optic lobe size. (I-J) Tumourous brain of l(3)tb homozygous 3rd instar larvae after 12 days of egg laying (AEL) [n=12], showing significant differences in Hsp70 intensity and distribution, compared to wild type [n=19] and l(3)tb homozygous 6 days larvae after egg laying (6 days AEL) [n=12]. X-axis represents larval genotypes with their age and Y-axis represents Hsp70 intensity (I) and % of Hsp70 expressed area (J), in each optic lobe respectively. Statistical analyses was performed by one-way ANOVA followed by Tukey's post-hoc multiple-comparison test; ****P<0.0001, **p < 0.01, *p < 0.05, not significant (ns)p > 0.05. (K-M) Confocal projection of wild type (K), l(3)tb 6 days (L) and l(3)tb- 12 days (M) homozygous larval brain showing Hsp70 staining in optic lobe area, scale bar: 50 μm. (O) Dot plot showing relationship of Hsp70 expression with size of optic lobe (demarcated from the DAPI+ area) of different genotypes [n=12, 9, 11 accordingly] where X-axis and Y-axis represents DAPI+ area and Hsp70+ area, respectively. Statistical significance of enhanced Hsp70 expression (WT vs l(3)tb brain) in the optic lobe area was determined through unpaired Student's t-test, where 6 days old WT brain were individually compared with either 6 days old l(3)tb brain or 12 days old l(3)tb brain. ***P (two-tailed)<0.001, **P(two-tailed)<0.01.
","imageTitle":"Temporal expression of stress inducible Hsp70 in developing l(3)tb brain tumour of Drosophila melanogaster
","methods":"Immunostaining: Larval brains were dissected out in 1X PBS from the larvae of appropriate age and fixed in 4% formaldehyde for 20min. After fixation, brains were rinsed thrice with 0.1% PBST (0.1% Triton X-100 in 1X PBS) for 15 min each. After rinsing, brains were kept in blocking solution (10% fetal calf serum, 0.1% Bovine serum albumin, 0.1% Triton X-100, 0.02% Thiomersal and 0.1% sodium deoxycholate, in 1X PBS) for 1 hr. After that, brains were incubated with desired primary antibody: rat anti-Hsp70 (7Fb 1:200, Sigma) for overnight at 4°C, following that brains were rinsed thrice with 0.1% PBST for 15 min each and kept in a blocking solution for 1 hr. Thereafter, those brains were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 (1:200, ThermoFisher Scientific, USA), for 2 hr at room temperature. After rinsing those brains thrice with 0.1% PBST, counterstained with 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg/ml, Thermo Fisher Scientific, Cat# D1306) and finally, mounted in an anti-fade mounting media 1,4-Diazabicyclo [2.2.2] octane (DABCO), Sigma, Cat# D27802, 2.5% DABCO in 70% glycerol made in 1X PBS). All the immunostained brain images were taken in Zeiss LSM 780 confocal microscopy with Zeiss ZEN2.3 SP1 Black edition software using Apo 20X (0.8 NA) and 63X (1.4 NA) oil immersion objectives. Images were assembled in Adobe Photoshop (2012). Quantitative analysis of acquire images were performed by using Fiji (ImageJ). Statistical analysis was done in GraphPad Prism 8.4.2 as mentioned in the figure legends.
Imaging of larvae adults and larval brain: Photographs of larvae and adults were taken by using Axio Cam Zeiss camera mounted on Nikon SMZ800N stereo binocular microscope. Photographs of larval brain was taken using Nikon DS-Fi2 camera mounted on Nikon Eclipse 90i microscope.
","reagents":"a. Fly Stocks:
1. l3tb/l3tb or l3tb/TM3, Ser (Mishra et al., 2020), where l3tb/TM6b, Tb flies used by the authors were rebalanced using TM3, Ser balancer to generate l3tb/TM3, Ser flies.
2. Oregon R+ (Wild-type strain of Drosophila melanogaster)
b. Chemicals:
Name of reagent | Source | Catalog No |
Hsp70 antibody (clone 7Fb) | Sigma | SAB5200204 |
DAPI | Thermo Fisher Scientific | D1306 |
DABCO | Sigma | D27802 |
Anti-rat secondary antibody conjugated with Alexa Flour 488 | Thermo Fisher Scientific | A-11006 |
Primary brain tumors are characterized by severe cellular and molecular heterogeneity that are associated with aggressive disease progression, therapeutic resistance and recurrence (Nicholson and Fine, 2021; Qazi et al., 2017). Traditional in vitro cell culture systems, including both two-dimensional and three-dimensional cultures, are intrinsically limited because they fail to incorporate the necessary host-tumor environment. Brain tumor progression is dictated by intricate, non-cell autonomous signaling originating from the surrounding glia, vasculature, and immune cells (Pasqualini et al., 2020). Models that cannot incorporate this crucial microenvironment, risk overlooking the therapeutic targets related to the complex dynamics of tumor-host interaction.
Among the different adaptive features followed by rapidly proliferating tumors, stress adaptation has recently been appreciated to be one of the major drivers of tumor growth (Hong et al., 2021; Seiler et al., 2020). Brain tumors, particularly gliomas and glioblastomas, possess a variety of stress adaptive mechanisms that help them survive, proliferate, and resist therapy in an otherwise hostile micro-environment (Combs et al., 2016; Graner et al., 2007). These mechanisms act across metabolic, genetic, and signaling pathways to maintain homeostasis under oxidative, hypoxic, and therapeutic stress conditions (Li et al., 2023). Although therapy-induced stress result in accumulation of misfolded proteins that subsequently triggers apoptosis, activation of chaperones such as Grp78 induce the unfolded protein response that allows the cell to manage the misfolded proteins and increase therapy tolerance (Kusaczuk et al., 2024; Liu et al., 2020). This requires a need to develop suitable models to study the factors responsible for development of stress tolerance in brain tumors.
The common fruit fly, Drosophila melanogaster serves to overcome the limitations of conventional brain tumor models since it exhibits rapid life cycle and production of large number of offsprings that can allow high-throughput screening of large drug libraries (Rudrapatna et al., 2012). This simultaneously generates information on drug bio-availability, toxicity parameters and host-tumor interactions, allowing researchers to rapidly test compounds in the complex in vivo context (Munnik et al., 2022). Further, the exceptional molecular conservation of the fly system with humans allows precise engineering of brain tumors that closely mimic the human disease. For example, glioma model has been developed to mimic the human glioblastomas, by constitutive co-activation of EGFR and PI3K pathways specifically in glial cells by using tissue specific Gal4 drivers (Read et al., 2009). In a P-element mutagenesis screen, a specific gene mutation called as the l(3)tb (lethal tumorous brain) was isolated that is characterized by excess accumulation of neuroblasts due to their uncontrolled growth, leading to larval and pupal lethality (Mishra et al., 2020). The brain of l(3)tb homozygous larvae gradually increases in size with extended larval periods till death. Hence, the mutation was initially named as lethal (3) tumorous brain [l(3)tb]. Larvae homozygous for l(3)tb mutant, along with the presence of tumourous brain, show 12-13 days extended larval life, overgrowth in leg, wing and eye-antennal discs (Kunar and Roy, 2021; Mishra et al., 2020). These defects were rescued by introducing a functional copy of DCP2 in the mutant background (Mishra et al., 2020). DCP2 is an evolutionarily conserved mRNA decapping enzyme encoded by DCP2 gene present at 73A1 region in left arm of Drosophila chromosome 3 (Kunar and Roy, 2021). It belongs to the NUDIX family of pyro-phosphatases and was first identified in yeasts (Dunckley and Parker, 1999). DCP2 along with DCP1 cleaves the 5’ methyl guanosine cap of mRNA and is involved in cell cycle regulation and DNA repair (She et al., 2008). DCP2 has been found to be upregulated in lung cancer and gliomas where it promotes cell proliferation, invasion, suppression of apoptosis and tumor immune cell infiltration (Watson et al., 2008).
In human cancers, Hsp70 is commonly overexpressed and are integral to mitigating stress, promoting cell survival and preventing cell death. Hsp70 positive tumors are more aggressive and resistant to therapy (Liu et al., 2021; Murphy, 2013). In case of brain tumors, the intensity and localization of Hsp70 within the cell correlate strongly with tumor malignancy (Babi et al., 2022). High-grade gliomas exhibit a significantly greater frequency of Hsp70 overexpression in both the nucleus and cytosol, indicating the elevated requirement for chaperone-mediated survival capacity (Graner et al., 2007; Lobinger et al., 2021). Further, high levels of extracellular Hsp70, released by the necrotic cells in large sized tumors, are associated with an unfavorable prognosis and overall survival (Shevtsov et al., 2024). Hence, Hsp70 serves as an important diagnostic biomarker and therapeutic target for many brain tumors.
Therefore, we sought to determine whether the l(3)tb homozygous mutant of Drosophila, which develops tumourous brains, could serve as a tractable model to study the development of Hsp70-mediated brain tumors. In our study the mutant flies showed a phenotype similar to previous reports (Mishra et al., 2020). Homozygous l(3)tb homozygous mutant larvae show developmental delay and most of the larvae die before pupation. Only a few of the larvae enter pupal stage and eventually die (Fig. 1A). During the early stages of larval growth, the brain size of l(3)tb homozygous mutant larvae was found to be smaller than that of age-matched controls, indicating a developmental delay (Fig. 1B-C) and during the prolonged larval period, the brain size exponentially increased, ultimately leading to larval lethality (Fig. 1D-G). At the later stages of development, the size of l(3)tb/l(3)tb brain were approximately 3-fold larger than that of control (Fig. 1H). To further assess the induction of Hsp70-mediated stress-response in these aggressively growing brain tumors, we performed immunostaining using an anti-Hsp70 antibody. Control brains from third instar wild type larvae showed minimal stress-inducible Hsp70 expression which was distributed only in a few cells of brain at the left and right optic lobe region (Fig. 1I, J and K). A similar pattern of minimalistic Hsp70 staining had been proposed in non-heat-shocked larval brain samples (Krebs and Feder, 1997). Similar to wild type condition, third instar l(3)tb/l(3)tb larvae during initial stages of development, exhibited Hsp70 expression in a few cells of the optic lobes (Fig. 1L). However, during later stages, these Hsp70-positive regions expanded progressively along with tumor growth, eventually encompassing most of the optic lobe area (Fig. 1M-N). Quantitative analysis revealed an approximately 15-fold increase in Hsp70 expression in l(3)tb/l(3)tb tumourous brains compared to wild-type controls (Fig. 1I). Similarly, the area of optic lobe covered by Hsp70 expressing cells increased to ~35% in 12-day old tumor (Fig. 1J). Further, a direct association was observed between the brain size, quantified as DAPI positive area, and the area covered with Hsp70 expressing cells (Fig.1O). These observations indicated a strong correlation between temporal induction of Hsp70 and brain tumor progression.
In conclusion, the l(3)tb mutant of Drosophila offers a unique opportunity to model the interplay between oncogenic drivers and stress-adaptive chaperones in brain tumors. By recapitulating both the genetic and micro-environmental dimensions of tumor biology, this system provides a powerful and cost-effective platform for mechanistic discovery and therapeutic exploration on the role of Hsp70 in aggressive brain tumors. Most importantly, our findings indicate a direct link between Hsp70 expression and tumor progression in the l(3)tb brain tumor model, underscoring the promise of this conserved chaperone as a diagnostic and therapeutic target in stress-adapted malignant brain tumors. By labelling individual cells in the brain, it will be of interest to see which cell type express Hsp70 whose proliferation ultimately results in expanded growth of brain size. This work highlights the possible development of l(3)tb/l(3)tb Drosophila brain tumor model to study stress-tolerance in brain tumors with potential translational outcomes.
","references":[{"reference":"Babi A, Menlibayeva K, Bex T, Doskaliev A, Akshulakov S, Shevtsov M. 2022. Targeting Heat Shock Proteins in Malignant Brain Tumors: From Basic Research to Clinical Trials. Cancers 14: 5435.
","pubmedId":"","doi":"10.3390/cancers14215435"},{"reference":"Combs S, Schmid T, Vaupel P, Multhoff G. 2016. Stress Response Leading to Resistance in Glioblastoma—The Need for Innovative Radiotherapy (iRT) Concepts. Cancers 8: 15.
","pubmedId":"","doi":"10.3390/cancers8010015"},{"reference":"Dunckley T, Parker R. 1999. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. The EMBO Journal 18: 5411-5422.
","pubmedId":"","doi":"10.1093/emboj/18.19.5411"},{"reference":"Graner MW, Cumming RI, Bigner DD. 2007. The Heat Shock Response and Chaperones/Heat Shock Proteins in Brain Tumors: Surface Expression, Release, and Possible Immune Consequences. The Journal of Neuroscience 27: 11214-11227.
","pubmedId":"","doi":"10.1523/JNEUROSCI.3588-07.2007"},{"reference":"Hong H, Ji M, Lai D. 2021. Chronic Stress Effects on Tumor: Pathway and Mechanism. Frontiers in Oncology 11: 10.3389/fonc.2021.738252.
","pubmedId":"","doi":"10.3389/fonc.2021.738252"},{"reference":"Krebs RA, Feder ME. 1997. Tissue-Specific Variation In Hsp70 Expression and Thermal Damage in Drosophila Melanogaster Larvae. Journal of Experimental Biology 200: 2007-2015.
","pubmedId":"","doi":"10.1242/jeb.200.14.2007"},{"reference":"Kunar R, Roy JK. 2021. The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila—insights from expression paradigms. Cell and Tissue Research 386: 261-280.
","pubmedId":"","doi":"10.1007/s00441-021-03503-x"},{"reference":"Kusaczuk M, Ambel ET, Naumowicz M, Velasco G. 2024. Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1879: 189054.
","pubmedId":"","doi":"10.1016/j.bbcan.2023.189054"},{"reference":"Li S, Wang C, Chen J, Lan Y, Zhang W, Kang Z, et al., Li. 2023. Signaling pathways in brain tumors and therapeutic interventions. Signal Transduction and Targeted Therapy 8: 10.1038/s41392-022-01260-z.
","pubmedId":"","doi":"10.1038/s41392-022-01260-z"},{"reference":"Liu H, Li Z, Li Q, Jia C, Zhang N, Qu Y, Hu D. 2021. HSP70 inhibition suppressed glioma cell viability during hypoxia/reoxygenation by inhibiting the ERK1/2 and PI3K/AKT signaling pathways. Journal of Bioenergetics and Biomembranes 53: 405-413.
","pubmedId":"","doi":"10.1007/s10863-021-09904-5"},{"reference":"Liu K, Tsung K, Attenello FJ. 2020. Characterizing Cell Stress and GRP78 in Glioma to Enhance Tumor Treatment. Frontiers in Oncology 10: 10.3389/fonc.2020.608911.
","pubmedId":"","doi":"10.3389/fonc.2020.608911"},{"reference":"Lobinger D, Gempt J, Sievert W, Barz M, Schmitt S, Nguyen HT, et al., Multhoff. 2021. Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.669366.
","pubmedId":"","doi":"10.3389/fmolb.2021.669366"},{"reference":"Mishra R, Kunar R, Mandal L, Alone DP, Chandrasekharan S, Tiwari AK, et al., Roy. 2020. A Forward Genetic Approach to Mapping a\n P\n -Element Second Site Mutation Identifies\n DCP2\n as a Novel Tumor Suppressor in\n Drosophila melanogaster. G3 Genes|Genomes|Genetics 10: 2601-2618.
","pubmedId":"","doi":"10.1534/g3.120.401501"},{"reference":"Munnik C, Xaba MP, Malindisa ST, Russell BL, Sooklal SA. 2022. Drosophila melanogaster: A platform for anticancer drug discovery and personalized therapies. Frontiers in Genetics 13: 10.3389/fgene.2022.949241.
","pubmedId":"","doi":"10.3389/fgene.2022.949241"},{"reference":"Murphy ME. 2013. The HSP70 family and cancer. Carcinogenesis 34: 1181-1188.
","pubmedId":"","doi":"10.1093/carcin/bgt111"},{"reference":"Nicholson JG, Fine HA. 2021. Diffuse Glioma Heterogeneity and Its Therapeutic Implications. Cancer Discovery 11: 575-590.
","pubmedId":"","doi":"10.1158/2159-8290.CD-20-1474"},{"reference":"Pasqualini C, Kozaki T, Bruschi M, Nguyen THH, Minard-Colin Vr, Castel D, Grill J, Ginhoux F. 2020. Modeling the Interaction between the Microenvironment and Tumor Cells in Brain Tumors. Neuron 108: 1025-1044.
","pubmedId":"","doi":"10.1016/j.neuron.2020.09.018"},{"reference":"Qazi MA, Vora P, Venugopal C, Sidhu SS, Moffat J, Swanton C, Singh SK. 2017. Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma. Annals of Oncology 28: 1448-1456.
","pubmedId":"","doi":"10.1093/annonc/mdx169"},{"reference":"Read RD, Cavenee WK, Furnari FB, Thomas JB. 2009. A Drosophila Model for EGFR-Ras and PI3K-Dependent Human Glioma. PLoS Genetics 5: e1000374.
","pubmedId":"","doi":"10.1371/journal.pgen.1000374"},{"reference":"Rudrapatna VA, Cagan RL, Das TK. 2011. Drosophila cancer models. Developmental Dynamics 241: 107-118.
","pubmedId":"","doi":"10.1002/dvdy.22771"},{"reference":"Seiler A, Sood AK, Jenewein J, Fagundes CP. 2020. Can stress promote the pathophysiology of brain metastases? A critical review of biobehavioral mechanisms. Brain, Behavior, and Immunity 87: 860-880.
","pubmedId":"","doi":"10.1016/j.bbi.2019.12.013"},{"reference":"She M, Decker CJ, Svergun DI, Round A, Chen N, Muhlrad D, Parker R, Song H. 2008. Structural Basis of Dcp2 Recognition and Activation by Dcp1. Molecular Cell 29: 337-349.
","pubmedId":"","doi":"10.1016/j.molcel.2008.01.002"},{"reference":"Shevtsov M, Bobkov D, Yudintceva N, Likhomanova R, Kim A, Fedorov E, et al., Combs. 2024. Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors. Cancer Research Communications 4: 2025-2044.
","pubmedId":"","doi":"10.1158/2767-9764.CRC-24-0094"},{"reference":"Watson PM, Miller SW, Fraig M, Cole DJ, Watson DK, Boylan AM. 2008. CaSm (LSm-1) Overexpression in Lung Cancer and Mesothelioma Is Required for Transformed Phenotypes. American Journal of Respiratory Cell and Molecular Biology 38: 671-678.
","pubmedId":"","doi":"10.1165/rcmb.2007-0205OC"}],"title":"The Drosophila l(3)tb mutant as a model to study stress-inducible Hsp70-expressing brain tumor
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