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The glutathione S-transferase Gstt1 drives survival and dissemination in metastases

Nature Cell Biology volume 26, pages 975–990 (2024)Cite this article

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Abstract

Identifying the adaptive mechanisms of metastatic cancer cells remains an elusive question in the treatment of metastatic disease, particularly in pancreatic cancer (pancreatic adenocarcinoma, PDA). A loss-of-function shRNA targeted screen in metastatic-derived cells identified Gstt1, a member of the glutathione S-transferase superfamily, as uniquely required for dissemination and metastasis, but dispensable for primary tumour growth. Gstt1 is expressed in latent disseminated tumour cells (DTCs), is retained within a subpopulation of slow-cycling cells within existing metastases, and its inhibition leads to complete regression of macrometastatic tumours. This distinct Gstt1high population is highly metastatic and retains slow-cycling phenotypes, epithelial–mesenchymal transition features and DTC characteristics compared to the Gstt1low population. Mechanistic studies indicate that in this subset of cancer cells, Gstt1 maintains metastases by binding and glutathione-modifying intracellular fibronectin, in turn promoting its secretion and deposition into the metastatic microenvironment. We identified Gstt1 as a mediator of metastasis, highlighting the importance of heterogeneity and its influence on the metastatic tumour microenvironment.

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Fig. 1: A shRNA screen targeting metastatic genes identifies Gstt1 as a regulator of metastasis.
Fig. 2: Gstt1 is required for metastatic maintenance and dissemination.
Fig. 3: Gstt1 is expressed in latent DTCs and in a subpopulation of slow-cycling macrometastatic cells.
Fig. 4: Gstt1high cells represent a slow-cycling, aggressive metastatic subpopulation and retain features of latent DTCs.
Fig. 5: Gstt1 interacts with and glutathione-modifies fibronectin to enhance metastasis.
Fig. 6: Glutathione availability dictates Gstt1 expression to enhance fibronectin deposition and promote metastasis.

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Data availability

All RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE232199. There is no restriction on data availability. Previously published data that were re-analysed here can be accessed through the Human Metastatic Cancer Database (HMCDB) and are available under accession code GSE63124. Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD051110 (https://www.ebi.ac.uk/pride/). Human PDA survival data were derived from KMPlot (https://kmplot.com/analysis/). The dataset derived from this resource that supports the findings of this study is available at https://doi.org/10.1038/s41598-021-84787-5. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

In-house codes were previously used in the published works. Appropriate references to the original works are provided.

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Acknowledgements

We thank all the members of the Mostoslavsky laboratory for critical discussions throughout the years and editing of the manuscript, especially T. L. Clarke. We would also like to thank N. Bardeesy for experimental discussions and critical evaluation of the manuscript, and S. Martin and J. Ju for technical confocal expertise and experimental discussions. R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work is supported by NIH grants (R01CA235412 and R01GM128448) and a Krantz Breakthrough Award to R.M., and an ACS Postdoctoral Fellowship and NIH grant (K99/R00CA252600-01) as well as the Maryland Department of Health’s Cigarette Restitution Fund Program and the National Cancer Institute–Cancer Center Support Grant (CCSG) (P30CA134274) to C.M.F. We also thank the members of the HSCI-CRM Flow Cytometry Facility at Massachusetts General Hospital, R. Bronson at the Rodent Histopathology Core at Harvard Medical School, the NextGen Sequencing Core at Massachusetts General Hospital, Center for Innovative Biomedical Resources at the University of Maryland School of Medicine and Greenebaum Comprehensive Cancer Center, the Massachusetts General Hospital Translational Imaging Core for their technical expertise.

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Author notes

  1. Ruben Boon

    Present address: Galapagos NV, 2800 Mechelen, Belgium

  2. These authors contributed equally: Hyo Min Cho, Ruben Boon.

Authors and Affiliations

  1. The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA

    Christina M. Ferrer, Hyo Min Cho, Ruben Boon, Tiziano Bernasocchi, Elizabeth R. Haggerty, Irene Mitsiades, Daniel E. McLoughlin, Esther Rheinbay, Dejan Juric & Raul Mostoslavsky

  2. The Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Christina M. Ferrer, Hyo Min Cho, Tiziano Bernasocchi & Raul Mostoslavsky

  3. University of Maryland School of Medicine and the Greenebaum Comprehensive Cancer Center, Baltimore, MD, USA

    Christina M. Ferrer

  4. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Lai Ping Wong, Murat Cetinbas & Ruslan Sadreyev

  5. Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA

    Gregory R. Wojtkiewicz

  6. Termeer Center for Targeted Therapies, Massachusetts General Hospital, Boston, MA, USA

    Daniel E. McLoughlin & Dejan Juric

  7. University of Vermont Larner College of Medicine, Burlington, VT, USA

    Reem Aboushousha, Hend Abdelhamid & Yvonne M. W. Janssen-Heininger

  8. Fred Hutchison Cancer Research Center, Seattle, WA, USA

    Sita Kugel

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Contributions

C.M.F. conducted experiments, wrote the manuscript, and designed and interpreted most experiments. R.B., H.M.C., T.B. and E.R.H. conducted and assisted with experiments. L.P.W., M.C. and R.S. provided all the computational analysis for transcriptomic experiments. G.R.W. performed mouse BLI. D.E.M. and D.J. provided access to rapid autopsy samples and clinical expertise. S.K. generated the genetically engineered pancreatic mouse models. E.R. and I.M. evaluated pancreatic cancer datasets for bioinformatic analysis of gene expression and survival. Y.M.J.-H., R.A. and H.A. performed and interpreted in vitro FN-SSG experiments. R.M. designed experiments, interpreted the data, and wrote and edited the manuscript.

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Correspondence to Christina M. Ferrer or Raul Mostoslavsky.

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Extended data

Extended Data Fig. 1 Functional validation of shRNA screen hits including Gstt1 in metastatic cells.

(A) Top 17 shRNA screen hits (minus GSTT1, n = 16) were analyzed for impact on overall survival in pancreatic cancer (KMPlot) based on mean expression of selected genes (N = 177 samples). A long-rank test was used to determine significance (*P = 0.0369). (B) Schematic depicting PDAC mouse models used for obtaining YFP/GFP+ primary and metastatic cells. Created with Biorender.com. (C) Representative fluorescence-activated cell sorting (FACS) strategy to obtain YFP/GFP+ metastatic cells. (D) Expression of Top 17 hits in primary matched GFP+ sorted cells represented as CPM values. Genes significantly differentially expressed between metastases (n = 5) and primary tumors (n = 4) from both mouse models displayed with red asterisk. Two-sided t-test was used to determine statistical significance between primary tumors and metastases (Nr1h3, *P = 0.0220, Gstm1, *P = 0.0496, Itih4, *P = 0.050). (E) qRT-PCR expression analysis of top 6 gene targets in primary (n = 3) and matched metastatic (n = 3) PDAC cell lines. Data represented as metastatic cell line mRNA expression relative to primary tumor-derived cells, s.e.m. Two-way ANOVA was used to determine statistical significance between groups (**P = 0.0065). (F) qRT-PCR for individual shRNA gene knockdowns. Data represents gene expression in a single cell line with n = 2 technical replicates. (G) Individual metastatic cell lines (n = 3) were generated to express each shRNA once and subjected to soft agar assay in (G) for n = 3 replicates. Brightfield images of soft agar colonies from liver metastatic cell lines using individual shRNAs validating top 6 screen hits. (H) Soft agar growth using two independent shRNAs per each gene target. Gene knockdown validation was performed once with three technical replicates for each gene. Data are represented as percent growth relative to NT Control, s.e.m. Two-sided t-test was used to determine statistical significance between groups (*P = 0.0264, **P = 0.0031, **P = 0.0083, **P = 0.0033, **P = 0.0093, *P = 0.0199, *P = 0.0480). (I) Western blot depicting PDAC-derived primary and metastatic cell lines stably expressing control or two independent Gstt1 shRNAs. (J) Soft agar assay growth in matched primary and metastatic cell lines. Representative bright-field image (2.5X). (K) Quantification of soft agar growth. Data are represented as number of colonies per well relative to control. The experiment was performed in triplicate with three technical replicates each. Data are represented as mean s.d. Two-sided t-test was used to determine statistical significance between groups (****P < 0.0001). (L) 2D growth curve in metastatic cell lines. The experiment was performed in triplicate with three technical replicates each. Data are represented as mean s.e.m. Two-sided t-test was used to determine statistical significance between groups.

Source data

Extended Data Fig. 2 Gstt1 expression and catalytic activity is required for spontaneous and experimental metastases in BC and PDAC mouse models.

(A) Liver metastatic (PDAC) cell lines stably expressing indicated constructs were analyzed by western blot for Gstt1 expression 3 days post-doxycycline (relates to cells injected in Fig. 1I). (B) Diagram depicting experimental schematic for data in Fig. 1I. Created with Biorender.com. (C) Schematic depicting experimental metastasis experiment. Mice were sacrificed when the first group demonstrated clinical evidence of metastatic burden and lungs were harvested and analyzed via H&E. Created with Biorender.com. (D) Cells injected in Fig S2C were analyzed by western blot for Gstt1 expression. (E) Representative H&E Images from lungs from each condition, taken at endpoint. (F) Lung metastatic burden was evaluated using H&E and represented as total number of macrometastases per lung tissue. Data are represented as mean s.e.m. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance of all groups compared to ipCW-Gstt1 WT +Dox group (****P < 0.0001). (G) Western blot depicting BC-derived primary and lung metastatic cell lines stably expressing pooled Gstt1 shRNAs. (H) Metastatic-derived lung cell line from the 4T1 BC mouse model stably expressing control or pooled shGstt1 were injected retro-orbitally to generate experimental lung metastases (n = 5 or n = 8 mice per group). Mice were sacrificed when the first group demonstrated clinical evidence of metastatic burden. Created with Biorender.com. (I) In vivo validation of Gstt1 knockdown demonstrates reduced metastatic burden in BC experimental metastasis model. Data represented as mean s.e.m. Two-sided t-test was used to determine statistical significance between groups (*P = 0.0216). (J) Primary-derived cell lines from the 4T1 BC mouse model stably expressing pooled shGstt1 were injected orthotopically into the mammary fat pad, and mice were monitored for primary tumor size and the formation of end-stage metastases. Primary tumors were measured over time, metastases were quantified at endpoint. Created with Biorender.com. (K) Primary tumor growth curves of orthotopically injected 4T1 cells into the mammary fat pad (n = 3 mice per group). Data are represented as mean s.e.m. for each timepoint. Two-way ANOVA with a post-hoc Holm-Sidak’s multiple comparisons test. P-values taken at each timepoint. (L) Quantification of spontaneous metastases derived from 4T1 orthotopic injections in the mammary fat pad (n = 3 mice per group). Data are represented as mean s.e.m. Two-sided t-test was used to determine statistical significance between groups (*P = 0.020). (M) Representative H&E images from spontaneous lung metastases in (L). (N) Immunofluorescence staining of Gstt1 and Cytokeratin 19 in mouse PDAC primary tumor, liver and lung metastases. (O) Quantification of immunofluorescence staining of Gstt1 populations in all CK19+ primary and metastatic lesions. Data represented as percent of CK19+ cells per metastatic lesion. Quantification represents the average of 6 fields from a minimum of 5 individual metastatic lesions from n = 3 independent mice. Data are represented as mean s.d. Two-sided t-test was used to determine statistical significance between groups (****P < 0.0001). (P) Representative immunofluorescence images of GSTT1 and Cytokeratin 19 in PDA-derived primary matched liver metastases from 3 independent rapid autopsy patients. (Q) Western blot analysis of GSTT1 levels in multiple primary and matched metastatic tissues derived from two rapid autopsy patients with pancreatic cancer. (R) Western blot analysis of GSTT1 levels in two metastatic derived human cell lines expressing control and two individual short hairpins targeting GSTT1. (S) Quantification of soft agar colony growth in liver metastasis-derived pancreatic cancer cell lines expressing either control or short hairpins targeting GSTT1. Data represent average of three technical replicates from three independent experiments. Data are represented as mean s.e.m. Two-sided t-test was used to determine statistical significance between control and each individual shRNA for each cell line (****P < 0.0001).

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Extended Data Fig. 3 Gstt1 is dispensable for primary tumor growth and regulates metastatic dissemination after primary tumor resection.

(A) Western blot depicting Gstt1 and Tubulin protein levels in 4T1 cells expressing indicated constructs. (B) Schematic depicting effect of Gstt1 on spontaneous metastases in an orthotopic resected BC model. Created with Biorender.com. (C) Tumor size of surgically removed primary tumors. 1 iCas9 mouse was excluded from analysis due to delayed primary tumor growth. Data are represented as mean s.d. Two-sided t-test was used to determine statistical significance between groups (n.s.). (D) Representative bioluminescent image of metastatic burden 3 weeks post-surgery. iCas9 mouse #3 (middle) imaged prior to PT removal. (E) Measurement of lung photon flux (p/s) over time post-surgical removal of the primary tumor. Each set of data points represents an individual mouse. (F) H&E quantification of spontaneous lung metastases post-surgery, harvested at endpoint. Data are represented as mean s.e.m. Two-sided t-test was used to determine statistical significance between groups (*P = 0.0399). (G) Schematic depicting effect of Gstt1 re-expression on spontaneous metastases in an orthotopic resected BC model. Created with Biorender.com. (H) Western blot depicting Gstt1 and Tubulin protein levels in primary-derived 4T1 cells expressing indicated constructs. (I) Representative bioluminescent image of primary tumors 3 weeks after tumor cell injection. (J) Bioluminescence imaging of weekly primary tumor growth prior to surgical removal. Values are represented as mean s.d. Two-way ANOVA with a post-hoc Geisser-Greenhouse multiple comparisons test was used to determine statistical significance between groups. P-values taken at final endpoint (**P = 0.0040). (K) Representative bioluminescent image of end-point metastatic burden 4 weeks post-surgery. (L) Measurement of lung photon flux (p/s) over time, taken at endpoint. Data are represented as mean s.d. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance between groups (***P = 0.003). (M) Experiment in (G) was repeated without BLI for post-mortem metastatic tissue analysis. H&E quantification of spontaneous lung metastases post-surgery, taken at endpoint. Data points derived from a combination of both experiments (NT Control -Dox, n = 4 mice; shGstt1 -Dox, n = 8 mice; shGstt1 +Dox, n = 7 mice). Data are represented as mean s.d. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance between groups (****P < 0.0001). (N) H&E quantification of the number of spontaneous macrometastases discovered outside the lung per tissue. Data are represented as mean s.d. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance between groups (****P < 0.0001). (O) H&E quantification of number of tissues outside the lung presenting with macrometastatic tumors. Data are represented as mean s.d. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance between groups (***P = 0.0007).

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Extended Data Fig. 4 Gstt1 is dispensable for primary tumor growth and required for dissemination of latent DTCs in an orthotopic model of PDAC.

(A) PDAC-derived primary tumor cells expressing either Control or shGstt1 were orthotopically injected into the pancreas (1 × 104 cells, n = 3 mice per group). Mice were euthanized when primary tumor burden resulted in poor body condition (abdominal ascites, hunched posture, lethargy, ~5 weeks), primary tumor size was measured, and liver and lung tissues analyzed for DTC content. Created with Biorender.com. (B) Measurement of primary tumor volume, post-euthanasia. Data are represented as mean s.e.m. A two-sided t-test was used to determine statistical significance between groups (P = 0.5609, not-significant). (C) Representative image of H&E stained orthotopic primary tumors from each group. (D) Immunofluorescence staining of orthotopically-derived disseminated tumor cells (DTCs) using Cytokeratin 19. Inset panel demonstrates a magnified image of CK19-positive DTCs. (E) Quantification of single DTCs ~5 weeks post-orthotopic injection, representing the average of 10 fields (20X field) from a minimum of n = 3 mice per group. Data are represented as mean s.d. two-sided t-test was used to determine statistical significance between groups (*P = 0.0384). (F) Immunofluorescence staining of Cytokeratin 19, Gstt1 and PCNA in spontaneous primary tumors and DTCs from orthotopic derived lung and liver tissues.

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Extended Data Fig. 5 Gstt1 is required for and enhances dissemination of Gstt1High/PCNALow latent DTCs.

(A) Immunofluorescence staining of experimental disseminated tumor cells (DTCs) using 2-weeks post-injection. DTCs were identified using Cytokeratin-19 as a tumor cell marker and co-stained with PCNA. (B) Quantification of the percent of PCNA+ and PCNA- CK19+ DTCs from (A). Quantification represents the average of 10 fields from a minimum of n = 3 mice per group (20X field). Data are represented as mean s.d. 2-way ANOVA was used to determine statistical significance between groups (all PCNA- vs PCNA+ groups, ****P < 0.0001). (C) Experimental disseminated tumor cells (DTCs) from (A) were stained with CK19 and Gstt1. Arrows indicate double-positive DTCs. (D) Flow cytometry analysis of lung metastatic cell populations 0, 2, and 7-days post-incubation with membrane dye CM-DiI. Data represented as % of CM-Dil+ and CM-Dil- cells relative to the total live cell population. Quantification represents n = 3 independent sorting experiments in one cell line. Data are represented as mean s.e.m. A two-sided t-test was used to determine statistical significance between groups (***P = 0.009, *P = 0.0465, *P = 0.0433).

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Extended Data Fig. 6 Gstt1 enhances EMT signaling and promotes anchorage-independent growth and metastasis in a subset of PDAC tumor cells.

(A) Representative gating strategy of GFP+ metastatic mCherry populations isolated from lungs of SCID mice. Upper left part of image created with Biorender.com. (B) Principal component analysis (PCA) of mCherryhigh (n = 3) and mCherrylow (n = 3) cell populations based on identified differentially expressed genes (DEGs) (>2FC, FDR 0.05). Each paired sample set indicates sorted populations from n = 3 independent biological replicates and n = 3 independent sorting experiments. (C) Gene set enrichment analysis (GSEA) finds hallmark enrichment of ‘Epithelial-to-Mesenchymal Transition’, ‘TGF-Beta Signaling’ and ‘Angiogenesis’ pathways in the mCherryhigh population. (D) mCherrylow sorted lung metastatic cell lines stably expressing ipCW, ipCW-Gstt1 wild-type and ipCW-Gstt1 R234G were treated with doxycycline for 5 days and subsequently subjected to soft agar growth assay. (E) ImageJ quantification of soft agar colony growth (averaged optical density per well) in all four conditions expressed as relative to mCherrylow. Data represents n = 3 independent sorting experiments with n = 3 soft agar replicates each. Data are represented as mean s.e.m. A two-sided t-test was used to determine statistical significance between groups (*P = 0.0335, *P = 0.0496, P = 0.2465 (ns)). (F) Western blot depicting Gstt1 levels in ipCW, ipCW-Gstt1 wild-type and ipCW-Gstt1-R234G overexpressing mCherrylow cells used in experiment (E). (G) mCherryhigh and mCherrylow populations were sorted and injected via tail vein into SCID mice (n = 10 cells, n = 102 cells, n = 103 cells) to generate experimental metastases to the lung. Groups were sacrificed when the first mouse in each group displayed clinical evidence of metastasis. Quantification of metastatic burden in H&E-stained slides, n = 4 or n = 5 mice per group (as indicated). Data are represented as mean s.e.m. A two-sided t-test was used to determine statistical significance between groups (*P = 0.0202, *P = 0.0315). (H) Representative image of H&E stained mCherry lungs from mice injected with 102 cells per group.

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Extended Data Fig. 7 The Gstt1High macrometastatic subpopulation regulates cell cycle progression and shares dissemination gene signatures with latent DTCs.

(A) A panel of metastatic cell lines (independently derived from PDAC liver and lung metastases) expressing either Control or shGstt1 were subjected to RNA-Seq. DAVID biological pathway analysis on Control vs shGstt1 differentially expressed gene signatures (95 UP, 217 DN) (>2FC, FDR 0.01) (GO_TERMs). Pathways were ranked by p-value(Log10) using an unpaired, two-sided t-test with post-hoc Bonferroni correction. (B) Expression (Log2CPM) of ‘Cell Cycle’ GO_TERM genes enriched in Control vs shGstt1 from (A) (>2FC, FDR 0.01). (C) Metastatic-derived PDAC cells stably expressing control, shGstt1 #1 or shGstt1 #2 were grown for 5 days, fixed, and analyzed for cell cycle stages using propidium iodide and flow cytometry. Cell cycle stages were analyzed using FlowJo. Data represents n = 3 independent experiments. Data are represented as mean s.d. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance between groups (*P = 0.0247). (D) Dissemination gene panel identified by Hosseini et al, 2018 was analyzed in mCherry populations and DTCs. Data represented as genes commonly enriched in mCherryhigh cells and DTC populations compared to mCherrylow. Data are represented as mean s.e.m. Two-way ANOVA with a post-hoc Tukey’s multiple comparisons test was used to determine statistical significance between groups (Log2CPM, ****P < 0.0001). (E) Dissemination gene panel (n = 5 genes) identified in Hosseini et al, 2018 from (D) were analyzed for impact on overall survival in pancreatic cancer (KMPlot). Data represents overall survival (OS) in N = 177 patient dataset based on mean expression of selected genes. A long-rank test was used to determine significance (*P = 0.0022). (F) Proliferation genes commonly downregulated in mCherryhigh cells and DTC populations compared to mCherrylow. Data are represented as mean s.e.m. Two-way ANOVA with a post-hoc Tukey’s multiple comparisons test was used to determine statistical significance between groups (Log2CPM, ****P < 0.0001).

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Extended Data Fig. 8 Gstt1 interacts with and glutathione-modifies Fibronectin protein in a subset of metastatic cells.

(A) Lung metastatic cell lines (PDAC) stably expressing wild-type ipCW-Gstt1 were treated with doxycycline for 5 days, lysed and subjected to immunoprecipitation using a Gstt1 antibody (whole cell lysate). Pull downs were analyzed for interactors and enriched peptides using unbiased mass spectrometry (M/S) for data in main Fig. 5A. Asterisks denote IgG band. (B) Gstt1 pull downs in liver metastatic cells were blotted for validation of the interaction with Plectin. (C) Log2CPM expression values for FN1 gene from mCherryHigh vs mCherryLow RNA-Seq from Fig4. Data are represented as mean s.d. FN1 was absent from the mCherryHigh vs mCherryLow DEG list (using FDR 0.05 cutoff) and therefore considered not differentially expressed between populations. Data represent sorting from n = 3 independent experiments. (D) Gene set enrichment analysis (GSEA) for ‘Integrin Cell Surface Interactions’ pathways in the mCherryhigh population. (E) Confocal imaging of immunofluorescence staining of Gstt1, Fibronectin and Cytokeratin 19 (pancreatic cell marker) in mouse CK19lowGstt1high and CK19highGstt1low liver metastatic lesions. A minimum of 5 metastatic lesions, across 3 independent mice were analyzed for co-staining. (F) Confocal imaging of immunofluorescence staining of Gstt1, Fibronectin and Cytokeratin 19 (pancreatic cell marker) in mouse pancreatic primary tumors. (G) Lung metastatic cell lines (PDAC) stably expressing ipCW, ipCW-Gstt1 wild-type and ipCW-Gstt1 R234G were treated with doxycycline for 5 days. Cells were lysed and subjected to immunoprecipitation using a Fibronectin antibody (whole cell lysate). Pull downs were blotted for FN-SSG (GSH antibody), Fibronectin and Gstt1. (Right panel) Input controls. (H) Lung metastatic cell lines (PDAC) stably expressing ipCW, ipCW-Gstt1 wild-type and ipCW-Gstt1 R234G were treated with doxycycline for 5 days. Cells were plated on glass chamber slides, fixed and stained with indicated antibodies. (I) Quantification of GSH and Fibronectin double positive cells. Quantification represents n = 3 biological replicates and an average of n = 5 fields of view per replicate (20X mag). Data are represented as mean s.d. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance (*P = 0.0203). (J) Western blot from cells plated in (I).

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Extended Data Fig. 9 Gstt1 mediates anchorage-independent growth, EMT, and Fibronectin ECM deposition through the regulation of intracellular Fibronectin in metastatic cells.

(A) Sorted mCherryhigh and mCherrylow populations were stably transduced with shFN1 and subjected to soft agar growth assay. Representative images of soft agar wells. (B) Quantification of soft agar colony growth in all four conditions expressed as relative to mCherrylow. Data represents n = 3 independent sorting experiments with n = 3 soft agar replicates each. Data are represented as mean s.e.m. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance (***P = 0.0007). (C) Western blot analysis of lysates from cells plated in (A) and (B). (D) Liver metastatic (PDAC) cell lines stably expressing non-targeting Control, shGstt1 or ipCW-Gstt1 in the presence of control shRNA or shRNA targeting FN1 were plated and conditioned media was collected 5 days after dH2O or doxycycline treatment (where indicated). Conditioned media was subjected to an ELISA assay to measure extracellular Fibronectin deposition. Fibronectin levels were normalized to cellular protein content. Data is normalized to Control. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons (compared to NT Control) was performed to determine statistical significance (**P = 0.002). Data represents a minimum of n = 3 independent experiments (except for ipCW -Dox sample, n = 2) with n = 2–4 technical replicates each. (E) Conditioned media from (D) was normalized to cellular protein content and subjected to western blotting for Fibronectin protein.

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Extended Data Fig. 10 Gstt1 expression responds to glutathione availability to modulate cellular ROS in metastatic cells.

(A) Metastatic cells were grown for 48 hours under suspension conditions +/− 100 uM NAC. Quantification of %GFP+ cells using CellROXGreen analyzed by flow cytometry. Data are represented as mean s.d. Data represents n = 2 independent experiments. (B) Cells from CellROX experiments in Fig. 6A, B and Supp Fig. 9A were subjected to RNA extraction and qRT-PCR for Gstt1. For attachment and suspension NT Control results, data are represented as mean s.e.m. Two-way ANOVA was used statistical significance between groups (*P = 0.0115) and represent a minimum of 3 independent experiments. For suspension Gstt1 KD conditions, data represent n = 2 independent experiments. (C) Gstt1Low mouse PDAC-derived metastatic cells were treated with either dH2O control or (100 uM) NAC under 2D attachment or low-attachment ‘suspension’ conditions for 48 hours. Cell lysates were subjected to western blotting for the indicated antibodies.

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Ferrer, C.M., Cho, H.M., Boon, R. et al. The glutathione S-transferase Gstt1 drives survival and dissemination in metastases. Nat Cell Biol 26, 975–990 (2024). https://doi.org/10.1038/s41556-024-01426-7

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