Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer
Daniel Öhlund, Abram Handly-Santana, Giulia Biffi, Ela Elyada, Ana S Almeida, Mariano Ponz-Sarvise, Vincenzo Corbo, Tobiloba E Oni, Stephen A Hearn, Eun Jung Lee, Iok In Christine Chio, Chang-Il Hwang, Hervé Tiriac, Lindsey A Baker, Dannielle D Engle, Christine Feig, Anne Kultti, Mikala Egeblad, Douglas T Fearon, James M Crawford, Hans Clevers, Youngkyu Park, David A Tuveson, Daniel Öhlund, Abram Handly-Santana, Giulia Biffi, Ela Elyada, Ana S Almeida, Mariano Ponz-Sarvise, Vincenzo Corbo, Tobiloba E Oni, Stephen A Hearn, Eun Jung Lee, Iok In Christine Chio, Chang-Il Hwang, Hervé Tiriac, Lindsey A Baker, Dannielle D Engle, Christine Feig, Anne Kultti, Mikala Egeblad, Douglas T Fearon, James M Crawford, Hans Clevers, Youngkyu Park, David A Tuveson
Abstract
Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.
© 2017 Öhlund et al.
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References
- Anders S., and Huber W.. 2010. Differential expression analysis for sequence count data. Genome Biol. 11:R106 10.1186/gb-2010-11-10-r106
- Apte M.V., Haber P.S., Applegate T.L., Norton I.D., McCaughan G.W., Korsten M.A., Pirola R.C., and Wilson J.S.. 1998. Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut. 43:128–133. 10.1136/gut.43.1.128
- Apte M.V., Park S., Phillips P.A., Santucci N., Goldstein D., Kumar R.K., Ramm G.A., Buchler M., Friess H., McCarroll J.A., et al. . 2004. Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells. Pancreas. 29:179–187. 10.1097/00006676-200410000-00002
- Apte M.V., Wilson J.S., Lugea A., and Pandol S.J.. 2013. A starring role for stellate cells in the pancreatic cancer microenvironment. Gastroenterology. 144:1210–1219. 10.1053/j.gastro.2012.11.037
- Arun G., Diermeier S., Akerman M., Chang K.C., Wilkinson J.E., Hearn S., Kim Y., MacLeod A.R., Krainer A.R., Norton L., et al. . 2016. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Genes Dev. 30:34–51. 10.1101/gad.270959.115
- Bachem M.G., Schünemann M., Ramadani M., Siech M., Beger H., Buck A., Zhou S., Schmid-Kotsas A., and Adler G.. 2005. Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology. 128:907–921. 10.1053/j.gastro.2004.12.036
- Bijlsma M.F., and van Laarhoven H.W.. 2015. The conflicting roles of tumor stroma in pancreatic cancer and their contribution to the failure of clinical trials: a systematic review and critical appraisal. Cancer Metastasis Rev. 34:97–114. 10.1007/s10555-014-9541-1
- Boj S.F., Hwang C.I., Baker L.A., Chio I.I., Engle D.D., Corbo V., Jager M., Ponz-Sarvise M., Tiriac H., Spector M.S., et al. . 2015. Organoid models of human and mouse ductal pancreatic cancer. Cell. 160:324–338. 10.1016/j.cell.2014.12.021
- Business Wire 2012 Infinity reports update from phase 2 study of saridegib plus gemcitabine in patients with metastatic pancreatic cancer. .
- Coppé J.P., Desprez P.Y., Krtolica A., and Campisi J.. 2010. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5:99–118. 10.1146/annurev-pathol-121808-102144
- Corcoran R.B., Contino G., Deshpande V., Tzatsos A., Conrad C., Benes C.H., Levy D.E., Settleman J., Engelman J.A., and Bardeesy N.. 2011. STAT3 plays a critical role in KRAS-induced pancreatic tumorigenesis. Cancer Res. 71:5020–5029. 10.1158/0008-5472.CAN-11-0908
- Desmoulière A., Guyot C., and Gabbiani G.. 2004. The stroma reaction myofibroblast: a key player in the control of tumor cell behavior. Int. J. Dev. Biol. 48:509–517. 10.1387/ijdb.041802ad
- Erez N., Truitt M., Olson P., Arron S.T., and Hanahan D.. 2010. Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent Manner. Cancer Cell. 17:135–147. 10.1016/j.ccr.2009.12.041
- Erkan M., Adler G., Apte M.V., Bachem M.G., Buchholz M., Detlefsen S., Esposito I., Friess H., Gress T.M., Habisch H.J., et al. . 2012. StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut. 61:172–178. 10.1136/gutjnl-2011-301220
- Feig C., Jones J.O., Kraman M., Wells R.J., Deonarine A., Chan D.S., Connell C.M., Roberts E.W., Zhao Q., Caballero O.L., et al. . 2013. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc. Natl. Acad. Sci. USA. 110:20212–20217. 10.1073/pnas.1320318110
- Flint T.R., Janowitz T., Connell C.M., Roberts E.W., Denton A.E., Coll A.P., Jodrell D.I., and Fearon D.T.. 2016. Tumor-Induced IL-6 Reprograms Host Metabolism to Suppress Anti-tumor Immunity. Cell Metab. 24:672–684. 10.1016/j.cmet.2016.10.010
- Froeling F.E., Feig C., Chelala C., Dobson R., Mein C.E., Tuveson D.A., Clevers H., Hart I.R., and Kocher H.M.. 2011. Retinoic acid-induced pancreatic stellate cell quiescence reduces paracrine Wnt-β-catenin signaling to slow tumor progression. Gastroenterology. 141:1486–1497: 1497.e1–1497.e14. 10.1053/j.gastro.2011.06.047
- Garrido-Laguna I., Uson M., Rajeshkumar N.V., Tan A.C., de Oliveira E., Karikari C., Villaroel M.C., Salomon A., Taylor G., Sharma R., et al. . 2011. Tumor engraftment in nude mice and enrichment in stroma-related gene pathways predict poor survival and resistance to gemcitabine in patients with pancreatic cancer. Clin. Cancer Res. 17:5793–5800. 10.1158/1078-0432.CCR-11-0341
- Hingorani S.R., Wang L., Multani A.S., Combs C., Deramaudt T.B., Hruban R.H., Rustgi A.K., Chang S., and Tuveson D.A.. 2005. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell. 7:469–483. 10.1016/j.ccr.2005.04.023
- Hingorani S.R., Harris W.P., Beck J.T., Berdov B.A., Wagner S.A., Pshevlotsky E.M., Tjulandin S.A., Gladkov O.A., Holcombe R.F., Korn R., et al. . 2016. Phase Ib Study of PEGylated Recombinant Human Hyaluronidase and Gemcitabine in Patients with Advanced Pancreatic Cancer. Clin. Cancer Res. 22:2848–2854. 10.1158/1078-0432.CCR-15-2010
- Huch M., Bonfanti P., Boj S.F., Sato T., Loomans C.J., van de Wetering M., Sojoodi M., Li V.S., Schuijers J., Gracanin A., et al. . 2013. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32:2708–2721. 10.1038/emboj.2013.204
- Hwang R.F., Moore T., Arumugam T., Ramachandran V., Amos K.D., Rivera A., Ji B., Evans D.B., and Logsdon C.D.. 2008. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res. 68:918–926. 10.1158/0008-5472.CAN-07-5714
- Jacobetz M.A., Chan D.S., Neesse A., Bapiro T.E., Cook N., Frese K.K., Feig C., Nakagawa T., Caldwell M.E., Zecchini H.I., et al. . 2012. Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer. Gut.
- Jesnowski R., Fürst D., Ringel J., Chen Y., Schrödel A., Kleeff J., Kolb A., Schareck W.D., and Löhr M.. 2005. Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine. Lab. Invest. 85:1276–1291. 10.1038/labinvest.3700329
- Kalluri R. 2016. The biology and function of fibroblasts in cancer. Nat. Rev. Cancer. 16:582–598. 10.1038/nrc.2016.73
- Kim E.J., Sahai V., Abel E.V., Griffith K.A., Greenson J.K., Takebe N., Khan G.N., Blau J.L., Craig R., Balis U.G., et al. . 2014. Pilot clinical trial of hedgehog pathway inhibitor GDC-0449 (vismodegib) in combination with gemcitabine in patients with metastatic pancreatic adenocarcinoma. Clin. Cancer Res. 20:5937–5945. 10.1158/1078-0432.CCR-14-1269
- Lee J.J., Perera R.M., Wang H., Wu D.C., Liu X.S., Han S., Fitamant J., Jones P.D., Ghanta K.S., Kawano S., et al. . 2014. Stromal response to Hedgehog signaling restrains pancreatic cancer progression. Proc. Natl. Acad. Sci. USA. 111:E3091–E3100. 10.1073/pnas.1411679111
- Lesina M., Kurkowski M.U., Ludes K., Rose-John S., Treiber M., Klöppel G., Yoshimura A., Reindl W., Sipos B., Akira S., et al. . 2011. Stat3/Socs3 activation by IL-6 transsignaling promotes progression of pancreatic intraepithelial neoplasia and development of pancreatic cancer. Cancer Cell. 19:456–469. 10.1016/j.ccr.2011.03.009
- Mace T.A., Shakya R., Pitarresi J.R., Swanson B., McQuinn C.W., Loftus S., Nordquist E., Cruz-Monserrate Z., Yu L., Young G., et al. . 2016. IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut.:gutjnl-2016-311585 10.1136/gutjnl-2016-311585
- Marusyk A., Tabassum D.P., Altrock P.M., Almendro V., Michor F., and Polyak K.. 2014. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity. Nature. 514:54–58. 10.1038/nature13556
- Moffitt R.A., Marayati R., Flate E.L., Volmar K.E., Loeza S.G., Hoadley K.A., Rashid N.U., Williams L.A., Eaton S.C., Chung A.H., et al. . 2015. Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma. Nat. Genet. 47:1168–1178. 10.1038/ng.3398
- Moir J.A., Mann J., and White S.A.. 2015. The role of pancreatic stellate cells in pancreatic cancer. Surg. Oncol. 24:232–238. 10.1016/j.suronc.2015.05.002
- Nagathihalli N.S., Castellanos J.A., VanSaun M.N., Dai X., Ambrose M., Guo Q., Xiong Y., and Merchant N.B.. 2016. Pancreatic stellate cell secreted IL-6 stimulates STAT3 dependent invasiveness of pancreatic intraepithelial neoplasia and cancer cells. Oncotarget. 7:65982–65992.
- Öhlund D., Elyada E., and Tuveson D.. 2014. Fibroblast heterogeneity in the cancer wound. J. Exp. Med. 211:1503–1523. 10.1084/jem.20140692
- Olive K.P., Jacobetz M.A., Davidson C.J., Gopinathan A., McIntyre D., Honess D., Madhu B., Goldgraben M.A., Caldwell M.E., Allard D., et al. . 2009. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science. 324:1457–1461. 10.1126/science.1171362
- Özdemir B.C., Pentcheva-Hoang T., Carstens J.L., Zheng X., Wu C.C., Simpson T.R., Laklai H., Sugimoto H., Kahlert C., Novitskiy S.V., et al. . 2014. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell. 25:719–734. 10.1016/j.ccr.2014.04.005
- Provenzano P.P., Cuevas C., Chang A.E., Goel V.K., Von Hoff D.D., and Hingorani S.R.. 2012. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell. 21:418–429. 10.1016/j.ccr.2012.01.007
- Putoczki T.L., Thiem S., Loving A., Busuttil R.A., Wilson N.J., Ziegler P.K., Nguyen P.M., Preaudet A., Farid R., Edwards K.M., et al. . 2013. Interleukin-11 is the dominant IL-6 family cytokine during gastrointestinal tumorigenesis and can be targeted therapeutically. Cancer Cell. 24:257–271. 10.1016/j.ccr.2013.06.017
- Quante M., Tu S.P., Tomita H., Gonda T., Wang S.S., Takashi S., Baik G.H., Shibata W., Diprete B., Betz K.S., et al. . 2011. Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth. Cancer Cell. 19:257–272. 10.1016/j.ccr.2011.01.020
- Rhim A.D., Mirek E.T., Aiello N.M., Maitra A., Bailey J.M., McAllister F., Reichert M., Beatty G.L., Rustgi A.K., Vonderheide R.H., et al. . 2012. EMT and dissemination precede pancreatic tumor formation. Cell. 148:349–361. 10.1016/j.cell.2011.11.025
- Rhim A.D., Oberstein P.E., Thomas D.H., Mirek E.T., Palermo C.F., Sastra S.A., Dekleva E.N., Saunders T., Becerra C.P., Tattersall I.W., et al. . 2014. Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer Cell. 25:735–747. 10.1016/j.ccr.2014.04.021
- Sherman M.H., Yu R.T., Engle D.D., Ding N., Atkins A.R., Tiriac H., Collisson E.A., Connor F., Van Dyke T., Kozlov S., et al. . 2014. Vitamin D receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy. Cell. 159:80–93. 10.1016/j.cell.2014.08.007
- Shi J., Wang E., Milazzo J.P., Wang Z., Kinney J.B., and Vakoc C.R.. 2015. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat. Biotechnol. 33:661–667. 10.1038/nbt.3235
- Siegel R.L., Miller K.D., and Jemal A.. 2016. Cancer statistics, 2016. CA Cancer J. Clin. 66:7–30. 10.3322/caac.21332
- Sousa C.M., Biancur D.E., Wang X., Halbrook C.J., Sherman M.H., Zhang L., Kremer D., Hwang R.F., Witkiewicz A.K., Ying H., et al. . 2016. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature. 536:479–483. 10.1038/nature19084
- Srinivas S., Watanabe T., Lin C.S., William C.M., Tanabe Y., Jessell T.M., and Costantini F.. 2001. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1:4 10.1186/1471-213X-1-4
- Straussman R., Morikawa T., Shee K., Barzily-Rokni M., Qian Z.R., Du J., Davis A., Mongare M.M., Gould J., Frederick D.T., et al. . 2012. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 487:500–504. 10.1038/nature11183
- Sugimoto H., Mundel T.M., Kieran M.W., and Kalluri R.. 2006. Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol. Ther. 5:1640–1646. 10.4161/cbt.5.12.3354
- Taga T., and Kishimoto T.. 1997. Gp130 and the interleukin-6 family of cytokines. Annu. Rev. Immunol. 15:797–819. 10.1146/annurev.immunol.15.1.797
- Talar-Wojnarowska R., Gasiorowska A., Smolarz B., Romanowicz-Makowska H., Kulig A., and Malecka-Panas E.. 2009. Clinical significance of interleukin-6 (IL-6) gene polymorphism and IL-6 serum level in pancreatic adenocarcinoma and chronic pancreatitis. Dig. Dis. Sci. 54:683–689. 10.1007/s10620-008-0390-z
- Tian H., Callahan C.A., DuPree K.J., Darbonne W.C., Ahn C.P., Scales S.J., and de Sauvage F.J.. 2009. Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis. Proc. Natl. Acad. Sci. USA. 106:4254–4259. 10.1073/pnas.0813203106
- Trapnell C., Pachter L., and Salzberg S.L.. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 25:1105–1111. 10.1093/bioinformatics/btp120
- Trapnell C., Williams B.A., Pertea G., Mortazavi A., Kwan G., van Baren M.J., Salzberg S.L., Wold B.J., and Pachter L.. 2010. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28:511–515. 10.1038/nbt.1621
- Vonlaufen A., Phillips P.A., Xu Z., Goldstein D., Pirola R.C., Wilson J.S., and Apte M.V.. 2008. Pancreatic stellate cells and pancreatic cancer cells: an unholy alliance. Cancer Res. 68:7707–7710. 10.1158/0008-5472.CAN-08-1132
- Waghray M., Yalamanchili M., Dziubinski M., Zeinali M., Erkkinen M., Yang H., Schradle K.A., Urs S., Pasca Di Magliano M., Welling T.H., et al. . 2016. GM-CSF mediates mesenchymal-epithelial cross-talk in pancreatic cancer. Cancer Discov. 6:886–899. 10.1158/-15-0947
- Xu Z., Vonlaufen A., Phillips P.A., Fiala-Beer E., Zhang X., Yang L., Biankin A.V., Goldstein D., Pirola R.C., Wilson J.S., and Apte M.V.. 2010. Role of pancreatic stellate cells in pancreatic cancer metastasis. Am. J. Pathol. 177:2585–2596. 10.2353/ajpath.2010.090899
- Yuzawa S., Kano M.R., Einama T., and Nishihara H.. 2012. PDGFRβ expression in tumor stroma of pancreatic adenocarcinoma as a reliable prognostic marker. Med. Oncol. 29:2824–2830. 10.1007/s12032-012-0193-0
- Zhang Y., Yan W., Collins M.A., Bednar F., Rakshit S., Zetter B.R., Stanger B.Z., Chung I., Rhim A.D., and di Magliano M.P.. 2013. Interleukin-6 is required for pancreatic cancer progression by promoting MAPK signaling activation and oxidative stress resistance. Cancer Res. 73:6359–6374. 10.1158/0008-5472.CAN-13-1558-T
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