Tumour-reprogrammed stromal BCAT1 fuels branched-chain ketoacid dependency in stromal-rich PDAC tumours

Abstract

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-β-SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells.

Extended Data Fig. 1. Transcriptomic analysis of BCAA metabolic genes in PDAC tumors

a. Expression of BCAT1 healthy tissue samples from the GTEx database (Brain, n=2642; Prostate, n=245; Testis, n=361; Pancreas, n=328; Ovary, n=180). b. Expression of genes involved in BCAA metabolism in samples from GSE21501 (n=132). Tumor samples with dominant epithelial markers and dominant fibroblast markers are deconvolved to compare expression of metabolic genes between pancreatic cancer cells and stromal cells in the TME. ROBO1 is a marker for validation that has been found to be expressed in stromal cells but not in cancer cells in independent studies. c. Expression of genes involved in BCAA metabolism in samples from GSE36924 (n=91). d. Expression of genes involved in BCAA metabolism in samples from GSE62165 (n=118). Samples with dominant epithelial markers and dominant fibroblast markers are deconvolved to compare expression of metabolic genes between epithelial cells and stromal cells (a-d). e. Gene expression of BCAA pathway genes and ROBO1 in paired epithelial and stromal compartments obtained by laser microdissection (GSE 93326, n=63 paired samples). Violin plot represents all data points in each group (a-d). Boxplot limits represent median and interquartile range (IQR), and notches represent 1.5*IQR (a-e). Data analyzed using multiple, two-tailed, unpaired, Student’s t-test (a-d); multiple, two-tailed, paired, Student’s t-test (e).

Extended Data Fig. 2. Characterization of BCAA metabolism

a. Representative IHC staining image comparing BCAT1 expression between stromal and tumor compartments. Experiments were repeated independently three times with similar results. b. Representative IF images showing protein expression of stromal αSMA, BCAT1 and Vimentin from paired healthy and PDAC tissue. Experiments were repeated independently three times with similar results. Experiments were repeated independently twice with similar results. c. Absolute cell numbers of PDAC CAFs were determined in the presence or absence of BCAA. n = 3 biologically independent samples. d. Absolute cell numbers of PDAC cell were determined in the presence or absence of BCAAs. n = 3 biologically independent samples. Data are presented as mean ± s.d.

Extended Data Fig. 3. PDAC cells are BCAT2 dependent for growth

a. Fluorescence microscopy images merged with brightfield images comparing growth of GFP-labeled Mia Paca-2 and Panc-1 cells in contact co-cultures with CAFs or NOFs under BCAA deprivation. Experiments were repeated independently three times with similar results. b. Relative growth rates of Mia Paca-2 cells co-cultured with CAFs or NOFs at different seeding ratios under BCAA deprivation. n = 3 biologically independent samples. c. Relative growth rates of AsPC1 and BxPC-3 cells co-cultured with CAFs or NOFs under BCAA deprivation. n = 3 biologically independent samples. d. Relative growth rates of Mia Paca-2 cells in various concentrations of BCAAs or BCKAs. n = 6 biologically independent samples. e. Model for the rescue of proliferation in BCAT2 KD cancer cells by BCKAs released from CAFs under BCAA deprivation. f. Relative growth rates of Mia Paca-2 and Panc-1 cells co-cultured with ATG-5/7 knockdown CAFs. n = 3 biologically independent samples. g. Relative growth rates of Mia Paca-2 and Patu 8988t cells cocultured with CAFs treated with autophagy inhibitors (chloroquine, Bafilomycin A1 and LY294002) under BCAA deprivation. n = 3 biologically independent samples. *P < 0.0001. Data are presented as mean ± s.d. Two-tailed, unpaired, Student’s t-test (c).

Extended Data Fig. 4. BCKDH complex is essential for PDAC cells growth and cell biosynthesis.

a. Relative proliferation rates of Mia Paca-2, Panc-1 and Patu 8988t cells expressing control shRNA or two independent shRNAs to DBT. n = 8 biologically independent samples. b. Colony-formation assay of DBT knockdown pancreatic cell lines. n = 3 biologically independent samples. c. Relative growth rates of Patu 8988t, Mia Paca-2 cells, and CAFs treated with BCKDK inhibitor, 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2). n = 3 biologically independent samples. d. Relative growth rates of MiaPaca-2 and Patu 8988t cells under BCAA deprivation and low glucose and low glutamine conditions after supplementation with BCKAs. n = 3 biologically independent samples. e. Schematic for the loss of rescue in DBT knockdown cancer cells by BCKAs released from CAFs under BCAA deprivation. f. Colocalization of Mitotracker and RexMito fluorescence in Mia Paca-2 cells. Mitotracker (red), RexMito (green), and DAPI (blue). Experiments were repeated independently three times with similar results. g. Substrate-specific oxygen consumption rate (OCR) in permeabilized pancreatic cancer cells. n = 4 biologically independent samples. h. OCR of Panc-1 cells after BCAT2 and DBT knockdown. n = 18 biologically independent samples. i. Substrate-specific OCR of BCAT2 knockdown pancreatic cancer cells. n = 4 biologically independent samples. j. Substrate-specific of DBT knockdown pancreatic cancer cells. n = 4 biologically independent samples. *P < 0.0001. Data are presented as mean ± s.d. One-way ANOVA with Tukey’s post hoc comparison (a,j); two-way ANOVA with Dunnett’s multiple comparison test (i,j).

Extended Data Fig. 5. CAFs have upregulated collagen uptake under BCAA deprivation.

a. BCAT activity in CAFs treated with Gabapentin measured by spectrophotometric assay. n = 6 biologically independent samples. b. Growth rate of Panc-1 cancer cells with Gabapentin, BCKAs, and CAF coculture under BCAA deprivation c. The effect of knockdown of BCAT1 in CAFs on CAF growth rates. n = 4 biologically independent samples. d. Uptake of DQ-Collagen by CAFs after 24 h measured using confocal imaging. Experiments were repeated independently three times with similar results. e. Uptake of DQ-Collagen by PDAC cell lines and CAFs after 24 h measured using confocal imaging. Experiments were repeated independently three times with similar results. f. Flow cytometry assay of MRC2 expression in PDAC cell lines. Experiments were repeated independently three times with similar results. g. Flow cytometry assay of MRC2 expression in CAFs. Experiments were repeated independently three times with similar results. *P < 0.0001. Data are presented as mean ± s.d. One-way ANOVA with Tukey’s post hoc comparison (b).

Extended Data Fig. 6. CAFs uptake collagen through the proteasome.

a. Uptake of DQ-Collagen by CAFs transfected with siControl or siuPARP measured using confocal imaging after 24 h. Experiments were repeated independently three times with similar results. b. CAFs are cultured with ¹³C-BCAAs for 12 h prior to inducing BCAA deprivation. Spent media and cells are collected after 6, 12, 24, and 48 h under deprivation. Media samples are analyzed for secreted BCKAs using LC-QTOF and intracellular samples are analyzed for BCAAs using GC-MS. c. Intracellular BCAA levels measured after 6, 12, 24 and 48 h under BCAA deprivation. Mole percent enrichment of intracellular BCAAs measured after 6, 12, 24, and 48 h under BCAA deprivation. n = 3 biologically independent samples. d. Influence of TGF-β and BCAA deprivation on the proteasome activity in CAFs (n=6). e. Relative growth rates of Mia Paca-2 and Panc-1 cells cocultured with CAFs treated with MG-132 under BCAA deprivation conditions. n = 8 biologically independent samples. f. Mass isotopomer distribution of BCAAs after acid hydrolysis of decellularized ECM proteins produced by CAFs cultured with ¹³C-BCAAs. n = 3 biologically independent samples. g. Fractional enrichment of amino acids after acid hydrolysis of decellularized ECM proteins produced by CAFs cultured with ¹³C-BCAAs. n = 3 biologically independent samples. Data are presented as mean ± s.d.

Extended Data Fig. 7. Stromal BCAT1 is regulated by cancer-cell derived TGF-β.

a. BCAT2 expression in NOFs treated with pancreatic cancer cell conditioned media (CM). n = 8 biologically independent samples. b. α-smooth muscle actin, (α-SMA) expression in NOFs cultured with pancreatic cancer cell-CM over 4 weeks. n = 8 biologically independent samples. c. Fibroblast specific protein (FSP1) expression in NOFs cultured with pancreatic cancer cell-CM over 4 weeks. n = 8 biologically independent samples. d. Podoplanin (PDPN) expression in NOFs cultured with pancreatic cancer cell-CM over 4 weeks. n = 8 biologically independent samples. e. BCAT2, α-SMA, FSP-1 and PDPN expression in MSCs treated with pancreatic cancer cell CM. n = 6 biologically independent samples. f. Expression of BCAA related genes in CAFs treated with pancreatic cancer cell-CM. n = 8 biologically independent samples. g. BCAT2 expression in CAFs treated with TGF-β and BCAT1 expression in cancer cells treated with TGF-β. n = 8 biologically independent samples. h. BCAT2 and α-SMA expression in NOFs cultured with pancreatic cancer cell CM in presence of anti-TGFB1 antibodies or isotype antibodies for 3 weeks. n = 8 biologically independent samples. Data are presented as mean ± s.d.

Extended Data Fig. 8. Cancer cells regulate stromal BCAT1 through SMAD5.

a. Representative images from IF analysis of BCAT1 and α-SMA expression in NOFs cultured with pancreatic cancer cell CM in presence of anti-TGFB1 antibodies or isotype antibodies for 3 weeks. Experiments were repeated independently twice with similar results. b. BCAT1 and BCAT2 expression in control and integrin α_vβ₅ KO CAFs cultured with pancreatic cancer cell CM for 3 weeks. n = 4 biologically independent samples. c. ELISA for TGF-β secretion levels from CAFs and PDAC cell lines. n = 8 biologically independent samples. d. SMAD2 expression in NOFs treated with pancreatic cancer cell CM. n = 8 biologically independent samples. e. SMAD3 expression in NOFs treated with pancreatic cancer cell CM. n = 8 biologically independent samples. f. SMAD4 expression in NOFs treated with pancreatic cancer cell CM. n = 8 biologically independent samples. g. SMAD5 binding motif. h. ChIP assays performed with control IgG and anti-SMAD4 antibodies in CAFs treated with PBS control or TGF-β. n = 6 biologically independent samples. *P < 0.0001. Data are presented as mean ± s.d. Multiple, two-tailed, unpaired, Student’s t-test (b); one-way ANOVA with Tukey’s post hoc comparison (c).

Extended Data Fig. 9. Validation of stromal BCAT1 and PDAC DBT in patient-derived CTCs.

a. Representative images of CTCs separated by Labyrinth. Cells are stained with DAPI (blue), cytokeratin (red), CD45 (green) and Vimentin (pink). Experiments were repeated independently three times with similar results. b. The influence of BCAAs and BCKAs on the growth of CTCs. n = 8 biologically independent samples. c. Extracellular concentration of BCKAs secreted by CAFs in monoculture and cocultured with CTCs over 6, 12, 24, and 48 h. n = 3 biologically independent samples. d. Extracellular concentration of BCKAs secreted by CAFs in monoculture or cocultured with CTCs, and CTCs in monoculture for 48 h. n = 4 biologically independent samples. e. Relative growth rate of PDAC cell lines and CTC lines under BCAA deprivation but supplemented with αKG, malate, succinate, acetate, citrate, NEAA mixture, or a combination in BCAA-deprived media. n = 8 biologically independent samples. f. Schematic of the protocol used to generate CTC derived organoid with CAF secreted ECM. g. Representative images from a CTC-derived organoid. Cytokeratin is shown in green and the nuclei stained with DAPI are shown in blue. Experiments were repeated independently three times with similar results. h. Representative FACS data of Pan-Cytokeratin positive tumor cells in CTC derived organoids. Experiments were repeated independently three times with similar results. i. Representative images of CTC derived organoids. Cells are stained with DAPI (blue), cytokeratin (red), CD45 (green) and Vimentin (Pink). Experiments were repeated independently twice with similar results. *P < 0.0001. Data are presented as mean ± s.d. Multiple, two-tailed, unpaired, Student’s t-test (b).

Extended Data Fig. 10. Validation of stromal BCAT1 and PDAC DBT in patient-derived tissue slices.

a. Representative Live Dead assay of tissue slice at Day 0 and Day 14. Live cells fluoresce bright green, whereas dead cells fluoresce red. Positive controls were fixed by methanol. Experiments were repeated independently three times with similar results. b. Efficiency of BCAT1 and DBT siRNAs in the human PDAC tissue slices. Expression of BCAT1, BCAT2, DBT, BCKDHA and BCKDHB in the human PDAC tissue slices treated with BCAT1 and DBT siRNAs. n = 6 biologically independent samples. Data are presented as mean ± s.d.

Fig. 1. Characterization of BCAA metabolism in CAFs and cancer cells.

a. BCAA transaminases (BCAT1/2), deaminate BCAAs to branched chain α-ketoacids (BCKAs), α-ketoisovalerate (KIV), α-keto-β-methylbutyrate (KMV), and α-ketoisocaproate (KIC). Then the mitochondrial BCKA dehydrogenase (BCKDH) complex consisting of three catalytic components, α-ketoacid dehydrogenase (E1), dihydrolipoyltransacylase (E2), and dihydrolipoamide dehydrogenase (E3) irreversibly oxidizes BCKAs. b. Immunoblots of BCAT1, BCAT2 and DBT expression in CAFs and pancreatic cancer cell lines. HSP90 and Vinculin used as loading control. Experiments were repeated independently three times with similar results. c. Relative BCAT1/2 mRNA expression in CAFs and PDAC lines, normalized to gene expression in CAF1. n = 4 biologically independent samples. d. Relative BCKDHA, BCKDHB, and DBT mRNA expression determined by qRT–PCR in CAFs and pancreatic cancer cell lines. Expression normalized to gene expression in CAF1. n = 4 biologically independent samples. e. Expression of genes in BCAA metabolism in samples from TCGA PDAC dataset (n=179). Violin plot represents all samples in each group. f. t-SNE clustering of single-cell gene expression of PDAC tumor cells (n=1352 single cells from N=2 patient samples). g. BCAT1 is predominantly expressed in single cells identified as CAFs, while BCAT2 is primarily expressed in single cells identified as PDAC cells. h. Single-cell gene expression of BCAA metabolic genes from N=24 PDAC tumor samples (n=41986 single cells) and N=11 healthy pancreatic tissue samples (n=15544 single cells) by t-SNE-clustered cell-types. i. BCAT1 gene expression in paired epithelial and stromal compartments obtained by laser microdissection of human PDAC tumors (GSE93326, n=63 paired samples). j. Representative IHC staining comparing BCAT1 expression between stromal and tumor compartments. Experiments were repeated independently three times with similar results. k. Newly synthesized BCKA flux determined by ¹³C-BCAA tracing in PDAC cells and CAFs. n = 3 biologically independent samples. l. Relative proliferation rates of Mia Paca-2, Panc-1 and Patu 8988t pancreatic cancer cells or CAFs under BCAA deprivation. n = 4 biologically independent samples. *P < 0.0001. Data are presented as mean ± s.d. One-way ANOVA with Tukey’s post hoc comparison (a); two-tailed, paired, Student’s t-test (i); multiple, two-tailed, unpaired, Student’s t-test (e, k). Boxplot limits represent median and interquartile range (IQR), and notches represent 1.5*IQR (e,i).

Fig. 2. PDAC cells are BCAT2 dependent for growth and respiration.

a. Fluorescence microscopy images comparing growth of GFP-labeled Mia Paca-2 and Panc-1 cells in contact co-cultures with CAFs or NOFs under BCAA deprivation. Experiments were repeated independently three times with similar results. b. Relative proliferation rates of Mia Paca-2, Patu 8988t and Panc-1 pancreatic cancer cells under BCAA deprivation. n = 3 biologically independent samples. c. BCKA secretion by CAFs estimated by measuring extracellular concentration of BCKAs, KIC and KMV, at 6, 12, 24, and 48h by LC-MS. n = 3 biologically independent samples. d. Fate of ¹³C-BCKAs in PDAC cells elucidated by measuring mole percent enrichment (MPE) of TCA cycle intermediates that represent BCKA oxidation, and of intracellular BCAAs and BCAAs from acid-hydrolyzed proteins that represent de novo protein synthesis. n = 7 biologically independent samples for intracellular metabolites and n = 4 biologically independent samples for protein hydrolyzed metabolites. e. Relative proliferation rates of Mia Paca-2, Panc-1, and Patu 8988t pancreatic cancer cells with BCAT2 knockdown by shRNA. n = 4 biologically independent samples. f. Relative proliferation rates of Panc-1 and Patu 8988t pancreatic cancer cells with BCAT2 knockdown by CRISPR. n = 4 biologically independent samples. g. Mole percent enrichment (MPE) of BCAAs in hydrolyzed protein obtained from BCAT2 knockdown Mia Paca-2 cells cultured with ¹³C-BCKA. n = 4 biologically independent samples. h. FACS analysis of GFP-labeled Mia Paca-2 cells detected with Alexa 647-labeled antibodies to puromycin (puro-A647). n = 3 biologically independent samples. i. Representative images of SUnSET assay of Mia Paca-2 cells cultured in the indicated medium for 48h. Whole-cell lysates were subjected to western blotting with puromycin antibody. Experiments were repeated independently three times with similar results. j. CAF cocultures rescue the loss of growth in BCAT2-knockdown PDAC cells. n = 6 biologically independent samples. Data are presented as mean ± s.d. *P < 0.0001. Two-way ANOVA with Dunnett’s multiple comparison test (b,e,f,j); multiple, two-tailed, unpaired, Student’s t-test (d,g); One-way ANOVA with Tukey’s post hoc comparison (h).

Fig. 3. BCKDH complex is essential for PDAC cell growth and cell biosynthesis.

a. Absolute cell numbers of PDAC cells expressing control shRNA or two independent shRNAs to DBT. n = 3 biologically independent samples. b. Colony-formation assay of DBT knockdown PDAC cell lines. n = 3 biologically independent samples. c. BCAA deprivation in the cancer monoculture can be rescued by BCKAs. n = 4 biologically independent samples. d. BCKA has no influence on CAF proliferation. n = 4 biologically independent samples. e. Relative proliferation rates of DBT knockdown cells in BCAA depleted media under BCKA replete conditions. n = 8 biologically independent samples. f. Relative proliferation rates of DBT knockdown cells co-cultured with CAFs. n = 3 biologically independent samples. g-h. NADH/NAD⁺ ratio measured using confocal florescence imaging of Mia Paca-2 cells in BCAA depleted media under BCKA replete conditions. n = 5 biologically independent samples. Experiments were repeated independently three times with similar results. i. NADH/NAD⁺ ratio measured using confocal florescence imaging of Mia Paca-2 cells transfected with siControl or siDBT. n = 5 biologically independent samples. j. NADH/NAD⁺ ratio measured using confocal florescence imaging of Mia Paca-2 cells in complete media with 4mM αKG added BCKAs n = 4 biologically independent samples. k. EdU uptake was measured in Mia Paca-2 cells in the presence of αKG and/or BCKAs after 1 day. n = 3 biologically independent samples. l. Substrate-specific oxygen consumption rate (OCR) in permeabilized pancreatic cancer cells measured using Seahorse Analyzer. n = 6 biologically independent samples. m. NADH/NAD⁺ ratio measured using confocal florescence imaging of Mia Paca-2 cells transfected with siControl or siBCAT2 in complete media or BCAA depleted media under BCKA replete conditions. n = 6 biologically independent samples. n. Substrate-specific OCR in permeabilized cells. n = 4 biologically independent samples. o. OCR measurements in DBT and BCAT2 knockdown cells. n = 6 biologically independent samples. p. Substrate-specific OCR measurements of BCAT2 knockdown cells. n = 4 biologically independent samples. q. Substrate-specific OCR measurement of DBT knockdown cells. n = 4 biologically independent samples. Data are presented as mean ± s.d. *P < 0.0001. Multiple, two-tailed, unpaired, Student’s t-test (b);two-way ANOVA with Dunnett’s multiple comparison test (c,d,e,f,l,p,q); One-way ANOVA with Tukey’s post hoc comparison (h,I,j,k,m,n).

Fig. 4. BCAT1 regulates stromal cells’ synthesis of ketoacids.

a. BCKA secretion by CAFs treated with 10mM Gabapentin. n = 3 biologically independent samples. b. Effect of 10mM Gabapentin on CAF-mediated rescue of MiaPaca-2 growth rate under BCAA deprived conditions. n = 6 biologically independent samples. c. Effect of BCAT1 and BCAT2 knockdown in CAFs using shRNA-BCAT1 and shRNA-BCAT2, respectively on CAF-mediated rescue of cell growth rate under BCAA deprivation. n = 3 biologically independent samples. d. Relative proliferation rates of Mia Paca-2 cells cocultured with CAFs and Collagen or 10mM Gabapentin under BCAA deprivation. n = 6 biologically independent samples. e. FACS analysis of GFP-labeled Mia Paca-2 cells detected with puromycin antibodies in the co-culture system with 10mM Gabapentin. n = 6 biologically independent samples. f. Effect of 10mM Gabapentin on NADH/NAD⁺ ratio of cancer cells cocultured with CAFs. n = 6 biologically independent samples. g. Uptake of DQ-Collagen by CAFs assessed using confocal imaging after 24 h. Experiments were repeated independently three times with similar results. h. Proteasome activity in CAFs treated with TGF-β and under BCAA deprivation. n = 6 biologically independent samples. i. Colocalization of collagen and proteasome analyzed by immunofluorescence against 20S proteasome and FITC-collagen. Experiments were repeated independently three times with similar results. j. Relative proliferation rates of Mia Paca-2 pancreatic cancer cells cocultured with CAFs in combination with Collagen or Delanzomib under BCAA deprivation. n = 6 biologically independent samples. k. BCKA secretion by CAFs treated with Delanzomib. n = 3 biologically independent samples. l. Schematic of the protocol used to synthesize ECM labeled with ¹³C-BCAAs and secretion of ¹³C-BCKAs after culturing BCAA-deprived CAFs in ECM labeled with ¹³C-BCAAs. m. Scanning electron microscopy image of CAF-derived 3-D matrices. Experiments were repeated independently two times with similar results. n. Fractional enrichment of BCAAs after acid hydrolysis of decellularized ECM proteins produced by CAFs cultured with ¹³C-BCAAs. n = 3 biologically independent samples. o. Fractional enrichment of BCKAs secreted by CAFs after 48h of being cultured under BCAA deprivation on ECM labeled with ¹³C-BCAAs. n = 3 biologically independent samples. Data are presented as mean ± s.d. *P < 0.0001. Multiple, two-tailed, unpaired, Student’s t-test (a,k); two-tailed, unpaired, Student’s t-test (e,f); two-way ANOVA with Dunnett’s multiple comparison test (b,c,d,h,j).

Fig. 5. Cancer cells regulate BCAT1 in stromal cells through TGF-β.

a. Effect of pancreatic cancer cell conditioned media (CM) on BCAT1 expression in NOFs over 4 weeks. n = 8 biologically independent samples. b. Effect of pancreatic cancer cell CM on BCAT1 expression in primary MSCs over 4 weeks. n = 6 biologically independent samples. c. Effect of pancreatic cancer cell CM on BCAT1 expression in various CAFs. n = 6 biologically independent samples. d. Growth rate of pancreatic cancer cells cultured with activated NOFs under BCAA deprivation. n = 6 biologically independent samples. e. BCAT1 mRNA expression measured in CAFs after 2 days of treatment with TGF-β. n = 6 biologically independent samples. f. BCAT1 expression in NOFs cultured with pancreatic cancer cell-CM in the presence of neutralizing anti-TGFβ1 antibodies or isotype antibodies for 3 weeks. n = 8 biologically independent samples. g. Effect of pancreatic cancer cell CM on SMAD5 expression in NOFs over 4 weeks. n = 6 biologically independent samples. h. ChIP assays performed with control IgG and anti-SMAD5 antibodies in CAFs treated with PBS control or TGF-β. n = 4 biologically independent samples. i. Transient transfection assays in CAFs with the reporter plasmid containing BCAT1 promoter. n = 8 biologically independent samples. j. Representative IHC staining image comparing SMAD5 expression between stromal and tumor compartments. Experiments were repeated independently three times with similar results. k. mRNA expression of BCAT1 in CAFs treated with siRNAs targeting SMAD4 or SMAD5. n = 6 biologically independent samples. l. Immunoblots showing BCAT1 protein expression in CAFs treated with control siRNA and SMAD5 siRNA. Experiments were repeated independently three times with similar results. m. TGF-β secreted by cancer cells regulates BCAT1 expression in CAFs by activating SMAD5, which binds to the BCAT1 promoter. Data are presented as mean ± s.d. *P < 0.0001. One-way ANOVA with Tukey’s post hoc comparison (a,b,f,g,k); multiple, two-tailed, unpaired, Student’s t-test (c,e); two-way ANOVA with Dunnett’s multiple comparison test (d,h,i).

Fig. 6. Validation of stromal BCAT1 and PDAC DBT in patient-derived CTCs and tissue slices.

a. CTCs are isolated from the blood of PDAC patients using the microfluidics-based Labyrinth™ Chip. Isolated CTCs are purified to generate CTC cell-lines used for downstream analyses. b. Heatmap of gene expression of BCAT1, BCAT2, BCKDHA and DBT measured by qRT-PCR from Day 0 CTCs isolated from PDAC patients, CAF1 cells, and cells isolated from a healthy subject. n = 7 biologically independent samples. c. Relative BCAT1, BCAT2, BCKDHA, BCKDHB, and DBT mRNA expression determined by qRT-PCR in CAFs and patient-derived CTCs. n = 4 biologically independent samples. d. Immunoblots of BCAT1 and BCAT2 expression in CAFs and Patient-derived CTCs. HSP90 is used as loading control. Experiments were repeated independently three times with similar results. e. KIC concentration in spent media from CAFs in monoculture or cocultured with CTC line. n = 3 biologically independent samples. *,p=0.0001,**,p=0.0008,***,p=0.0093. f. Representative images from CTC derived organoid. Cytokeratin is shown in green and the nuclei stained with DAPI are shown in blue. Experiments were repeated independently three times with similar results. g. EdU staining and SUnSET assay on Pan Cytokeratin+ tumor cells in the CTC derived organoid cultured with CAFs treated with vehicle or 10mM Gabapentin. n = 6 biologically independent samples. h. Schematic of human PDAC tissue slice culture. Freshly biopsied tumor is embedded in agarose and sliced into 200 μm thick slices using a vibrating microtome. Slices are cultured for downstream metabolic and functional analyses. i. Percentage PCNA-positive and Ki67-positive in Pan Cytokeratin+ tumor cells identified using IF. n = 6 biologically independent samples. j. Fractional enrichment of BCAAs of human PDAC tissue slices cultured with ¹³C-BCKAs. n = 5 individual tissue samples from distinct patients. Violin plot represents entire range of values, lines at median, 10-90 percentiles. k. Representative images from SUnSET IF analysis of PDAC tissue treated with vehicle or 10mM Gabapentin. n = 3 biologically independent samples. Data are presented as mean ± s.d. except j. *P < 0.0001 except e. Multiple, two-tailed, unpaired, Student’s t-test (e); two-tailed, unpaired, Student’s t-test (g,k); one-way ANOVA with Tukey’s post hoc comparison (I,j).