Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis-associated adhesion of ovarian cancer cells

Abstract

Background: IL-17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation.

Methods: Flow-cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL-17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses.

Results: We identified Th17 cells as the main population of IL-17A- and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL-17A and its receptor subunit IL-17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL-17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro-inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL-17A and TNF synergistically induced the Th17-promoting cytokines IL-6 and IL-1β as well as the Th17-attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor-promoting role of Th17 cells in OC.

Conclusions: Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL-17A/TNF-mediated induction of mesothelial-mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.

Keywords

mesothelial cells / metastasis / ovarian carcinoma / Th17 cells

References

[1] Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72:7-33.

[2] Torre LA, Trabert B, DeSantis CE, et al. Ovarian cancer statistics, 2018. CA Cancer J Clin. 2018;68:284-296.

[3] Kipps E, Tan DS, Kaye SB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer. 2013;13:273-282.

[4] Worzfeld T, Pogge von Strandmann E, Huber M, et al. The Unique Molecular and Cellular Microenvironment of Ovarian Cancer. Front Oncol. 2017;7:24.

[5] Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010;177:1053-1064.

[6] Nieman KM, Kenny HA, Penicka CV, et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med. 2011;17:1498-1503.

[7] Pogge von Strandmann E, Reinartz S, Wager U, Muller R. Tumor-Host Cell Interactions in Ovarian Cancer: Pathways to Therapy Failure. Trends Cancer. 2017;3:137-148.

[8] Sommerfeld L, Finkernagel F, Jansen JM, et al. The multicellular signalling network of ovarian cancer metastases. Clin Transl Med. 2021;11:e633.

[9] Sodek KL, Murphy KJ, Brown TJ, Ringuette MJ. Cell-cell and cell-matrix dynamics in intraperitoneal cancer metastasis. Cancer Metastasis Rev. 2012;31:397-414.

[10] Iwanicki MP, Davidowitz RA, et al. Ovarian cancer spheroids use myosin-generated force to clear the mesothelium. Cancer Discov. 2011;1:144-157.

[11] Kenny HA, Chiang CY, White EA, et al. Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion. J Clin Invest. 2014;124:4614-4628.

[12] Heath RM, Jayne DG, O'Leary R, Morrison EE, Guillou PJ. Tumour-induced apoptosis in human mesothelial cells: a mechanism of peritoneal invasion by Fas Ligand/Fas interaction. Br J Cancer. 2004;90:1437-1442.

[13] Steitz AM, Schroder C, Knuth I, et al. TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion. iScience. 2023;26:108401.

[14] Bajwa P, Kordylewicz K, Bilecz A, et al. Cancer-associated mesothelial cell-derived ANGPTL4 and STC1 promote the early steps of ovarian cancer metastasis. JCI Insight. 2023;8:e163019.

[15] Rynne-Vidal A, Au-Yeung CL, Jiménez-Heffernan JA, et al. Mesothelial-to-mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer. J Pathol. 2017;242:140-151.

[16] Slack-Davis JK, Atkins KA, Harrer C, Hershey ED, Conaway M. Vascular cell adhesion molecule-1 is a regulator of ovarian cancer peritoneal metastasis. Cancer Res. 2009;69:1469-1476.

[17] Kulbe H, Thompson R, Wilson JL, et al. The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in epithelial ovarian cancer cells. Cancer Res. 2007;67:585-592.

[18] Wang W, Wu J, Mukherjee A, et al. Lysophosphatidic acid induces tumor necrosis factor-alpha to regulate a pro-inflammatory cytokine network in ovarian cancer. FASEB J. 2020;34:13935-13948.

[19] Charles KA, Kulbe H, Soper R, et al. The tumor-promoting actions of TNF-alpha involve TNFR1 and IL-17 in ovarian cancer in mice and humans. J Clin Invest. 2009;119:3011-3023.

[20] van Loo G, Bertrand MJM. Death by TNF: a road to inflammation. Nat Rev Immunol. 2023;23:289-303.

[21] Mills KHG. IL-17 and IL-17-producing cells in protection versus pathology. Nat Rev Immunol. 2023;23:38-54.

[22] Zhao J, Chen X, Herjan T, Li X. The role of interleukin-17 in tumor development and progression. J Exp Med. 2020;217:e20190297.

[23] Picard FSR, Lutz V, Brichkina A, et al. IL-17A-producing CD8(+) T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts. Gut. 2023;72:1510-1522.

[24] Salazar Y, Zheng X, Brunn D, et al. Microenvironmental Th9 and Th17 lymphocytes induce metastatic spreading in lung cancer. J Clin Invest. 2020;130:3560-3575.

[25] Zhang Y, Zoltan M, Riquelme E, et al. Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells. Gastroenterology. 2018;155:210-223. e213.

[26] Xiang T, Long H, He L, et al. Interleukin-17 produced by tumor microenvironment promotes self-renewal of CD133+ cancer stem-like cells in ovarian cancer. Oncogene. 2015;34:165-176.

[27] Kryczek I, Banerjee M, Cheng P, et al. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood. 2009;114:1141-1149.

[28] Bilska M, Pawlowska A, Zakrzewska E, et al. Th17 Cells and IL-17 As Novel Immune Targets in Ovarian Cancer Therapy. J Oncol. 2020;2020:8797683.

[29] Rei M, Goncalves-Sousa N, Lança T, et al. Murine CD27(-) Vgamma6(+) gammadelta T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages. Proc Natl Acad Sci U S A. 2014;111:E3562-E3570.

[30] Zhu Q, Wu X, Wang X. Differential distribution of tumor-associated macrophages and Treg/Th17 cells in the progression of malignant and benign epithelial ovarian tumors. Oncol Lett. 2017;13:159-166.

[31] Foord E, Arruda LCM, Gaballa A, Klynning C, Uhlin M. Characterization of ascites- and tumor-infiltrating gammadelta T cells reveals distinct repertoires and a beneficial role in ovarian cancer. Sci Transl Med. 2021;13:eabb0192.

[32] Griffin GK, Newton G, Tarrio ML, et al. IL-17 and TNF-alpha sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation. J Immunol. 2012;188:6287-6299.

[33] Noack M, Beringer A, Miossec P. Additive or Synergistic Interactions Between IL-17A or IL-17F and TNF or IL-1beta Depend on the Cell Type. Front Immunol. 2019;10:1726.

[34] Witowski J, Pawlaczyk K, Breborowicz A, et al. IL-17 stimulates intraperitoneal neutrophil infiltration through the release of GRO alpha chemokine from mesothelial cells. J Immunol. 2000;165:5814-5821.

[35] Wang CQF, Akalu YT, Suarez-Farinas M, et al. IL-17 and TNF synergistically modulate cytokine expression while suppressing melanogenesis: potential relevance to psoriasis. J Invest Dermatol. 2013;133:2741-2752.

[36] Kouri VP, Olkkonen J, Nurmi K, et al. IL-17A and TNF synergistically drive expression of proinflammatory mediators in synovial fibroblasts via IkappaBzeta-dependent induction of ELF3. Rheumatology (Oxford). 2023;62:872-885.

[37] Ince TA, Sousa AD, Jones MA, et al. Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours. Nat Commun. 2015;6:7419.

[38] Reinartz S, Lieber S, Pesek J, et al. Cell type-selective pathways and clinical associations of lysophosphatidic acid biosynthesis and signaling in the ovarian cancer microenvironment. Mol Oncol. 2019;13:185-201.

[39] Yang WL, Roland IH, Godwin AK, Xu XX. Loss of TNF-alpha-regulated COX-2 expression in ovarian cancer cells. Oncogene. 2005;24:7991-8002.

[40] Khan SM, Funk HM, Thiolloy S, et al. In vitro metastatic colonization of human ovarian cancer cells to the omentum. Clin Exp Metastasis. 2010;27:185-196.

[41] Sivakumar S, Lieber S, Librizzi D, et al. Basal cell adhesion molecule promotes metastasis-associated processes in ovarian cancer. Clin Transl Med. 2023;13:e1176.

[42] Grayson WL, Zhao F, Bunnell B, Ma T. Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun. 2007;358:948-953.

[43] Gerber SA, Rybalko VY, Bigelow CE, et al. Preferential attachment of peritoneal tumor metastases to omental immune aggregates and possible role of a unique vascular microenvironment in metastatic survival and growth. Am J Pathol. 2006;169:1739-1752.

[44] Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15-21.

[45] Booeshaghi AS, Chen X, Pachter L. A machine-readable specification for genomics assays. bioRxiv 2023.

[46] Chen X, Roelli P, Hereñú D, Höjer P, Stuart T. 2023.

[47] Wolf FA, Angerer P, Theis FJ. SCANPY: large-scale single-cell gene expression data analysis. Genome Biology. 2018;19:15.

[48] McInnes L, Healy J, Melville J. Umap: Uniform manifold approximation and projection for dimension reduction. 2018.

[49] Amir el AD, Davis KL, Tadmor MD, et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol. 2013;31:545-552.

[50] van der Maaten LJP, Hinton GE. Visualizing High. Dimensional Data Using t-SNE. J Mach Learn Res. 2008;9:26.

[51] Blondel VD, Guillaume J-L, Lambiotte R, Lefebvre E. Fast unfolding of communities in large networks. J Stat Mech Theory Exp. 2008;2008:P10008.

[52] Cao Y, Wang X, Peng G. SCSA: A Cell Type Annotation Tool for Single-Cell RNA-seq Data. Front Genet. 2020;11:490.

[53] Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139-140.

[54] Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2015;1:417-425.

[55] Győrffy B. Discovery and ranking of the most robust prognostic biomarkers in serous ovarian cancer. Geroscience. 2023;45:1889-1898.

[56] Finkernagel F, Reinartz S, Schuldner M, et al. Dual-platform affinity proteomics identifies links between the recurrence of ovarian carcinoma and proteins released into the tumor microenvironment. Theranostics. 2019;9:6601-6617.

[57] Huber M, Lohoff M. IRF4 at the crossroads of effector T-cell fate decision. Eur J Immunol. 2014;44:1886-1895.

[58] Yáñez-Mó M, Lara-Pezzi E, Selgas R, et al. Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells. N Engl J Med. 2003;348:403-413.

[59] van Baal JO, Van de Vijver KK, Nieuwland R, et al. The histophysiology and pathophysiology of the peritoneum. Tissue Cell. 2017;49:95-105.

[60] Kastelein AW, Vos LMC, de Jong KH, et al. Embryology, anatomy, physiology and pathophysiology of the peritoneum and the peritoneal vasculature. Semin Cell Dev Biol. 2019;92:27-36.

[61] Yang J, Antin P, Berx G, et al. Guidelines and definitions for research on epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2020;21:341-352.

[62] Kuol N, Stojanovska L, Apostolopoulos V, Nurgali K. Role of the nervous system in cancer metastasis. J Exp Clin Cancer Res. 2018;37:5.

[63] Slominski RM, Raman C, Chen JY, Slominski AT. How cancer hijacks the body's homeostasis through the neuroendocrine system. Trends Neurosci. 2023;46:263-275.

[64] Polette M, Mestdagt M, Bindels S, et al. Beta-catenin and ZO-1: shuttle molecules involved in tumor invasion-associated epithelial-mesenchymal transition processes. Cells Tissues Organs. 2007;185:61-65.

[65] Assarsson E, Lundberg M, Holmquist G, et al. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One 2014;9:e95192.

[66] Wik L, Nordberg N, Broberg J, et al. Proximity Extension Assay in Combination with Next-Generation Sequencing for High-throughput Proteome-wide Analysis. Mol Cell Proteomics. 2021;20:100168.

[67] Hellström I, Raycraft J, Hayden-Ledbetter M, et al. The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma. Cancer Res. 2003;63:3695-3700.

[68] Graumann J, Finkernagel F, Reinartz S, et al. Multi-platform Affinity Proteomics Identify Proteins Linked to Metastasis and Immune Suppression in Ovarian Cancer Plasma. Front Oncol. 2019;9:1150.

[69] Luckel C, Picard FSR, Huber M. Tc17 biology and function: Novel concepts. Eur J Immunol. 2020;50:1257-1267.

[70] Revu S, Wu J, Henkel M, et al. IL-23 and IL-1beta Drive Human Th17 Cell Differentiation and Metabolic Reprogramming in Absence of CD28 Costimulation. Cell Rep. 2018;22:2642-2653.

[71] Sallusto F, Zielinski CE, Lanzavecchia A. Human Th17 subsets. Eur J Immunol. 2012;42:2215-2220.

[72] Miyahara Y, Odunsi K, Chen W, Peng G, Matsuzaki J, Wang RF. Generation and regulation of human CD4+ IL-17-producing T cells in ovarian cancer. Proc Natl Acad Sci U S A. 2008;105:15505-15510.

[73] Chen X, Zhang X, Xu R, et al. Implication of IL-17 producing abetaT and gammadeltaT cells in patients with ovarian cancer. Hum Immunol. 2020;81:244-248.

[74] Lee W, Ko SY, Mohamed MS, et al. Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum. J Exp Med. 2019;216:176-194.

[75] Singel KL, Emmons TR, Khan ANH, et al. Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment. JCI Insight. 2019;4:e122311.

[76] Cannon MJ, Goyne HE, Stone PJ, et al. Modulation of p38 MAPK signaling enhances dendritic cell activation of human CD4+ Th17 responses to ovarian tumor antigen. Cancer Immunol Immunother. 2013;62:839-849.

[77] Lan C, Huang X, Lin S, et al. High density of IL-17-producing cells is associated with improved prognosis for advanced epithelial ovarian cancer. Cell Tissue Res. 2013;352:351-359.

[78] Block MS, Dietz AB, Gustafson MP, et al. Th17-inducing autologous dendritic cell vaccination promotes antigen-specific cellular and humoral immunity in ovarian cancer patients. Nat Commun. 2020;11:5173.

[79] Sandoval P, Jimenez-Heffernan JA, Rynne-Vidal Á, et al. Carcinoma-associated fibroblasts derive from mesothelial cells via mesothelial-to-mesenchymal transition in peritoneal metastasis. J Pathol. 2013;231:517-531.

[80] Davidowitz RA, Selfors LM, Iwanicki MP, et al. Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance. J Clin Invest. 2014;124:2611-2625.

[81] Mogi K, Yoshihara M, Iyoshi S, et al. Ovarian Cancer-Associated Mesothelial Cells: Transdifferentiation to Minions of Cancer and Orchestrate Developing Peritoneal Dissemination. Cancers (Basel). 2021;13:1352.

[82] Lopez-Cabrera M. Mesenchymal Conversion of Mesothelial Cells Is a Key Event in the Pathophysiology of the Peritoneum during Peritoneal Dialysis. Adv Med. 2014;2014:473134.

[83] Casey RC, Oegema TR, Skubitz KM, Pambuccian SE, Grindle SM, Skubitz AP. Cell membrane glycosylation mediates the adhesion, migration, and invasion of ovarian carcinoma cells. Clin Exp Metastasis. 2003;20:143-152.

[84] Gardner MJ, Catterall JB, Jones LM, Turner GA. Human ovarian tumour cells can bind hyaluronic acid via membrane CD44: a possible step in peritoneal metastasis. Clin Exp Metastasis. 1996;14:325-334.

[85] Lessan K, Aguiar DJ, Oegema T, Siebenson L, Skubitz AP. CD44 and beta1 integrin mediate ovarian carcinoma cell adhesion to peritoneal mesothelial cells. Am J Pathol. 1999;154:1525-1537.

[86] Jones LM, Gardner MJ, Catterall JB, Turner GA. Hyaluronic acid secreted by mesothelial cells: a natural barrier to ovarian cancer cell adhesion. Clin Exp Metastasis. 1995;13:373-380.

[87] Clark R, Krishnan V, Schoof M, et al. Milky spots promote ovarian cancer metastatic colonization of peritoneal adipose in experimental models. Am J Pathol. 2013;183:576-591.

[88] Liu J, Geng X, Li Y. Milky spots: omental functional units and hotbeds for peritoneal cancer metastasis. Tumour Biol. 2016;37:5715-5726.

[89] Kenny HA, Nieman KM, Mitra AK, Lengyel E. The first line of intra-abdominal metastatic attack: breaching the mesothelial cell layer. Cancer Discov. 2011;1:100-102.

[90] Aboobacker S, Kurn H, Al Aboud AM. Secukinumab. StatPearls. 2023.

[91] Rossi JF, Lu ZY, Jourdan M, Klein B. Interleukin-6 as a therapeutic target. Clin Cancer Res. 2015;21:1248-1257.

[92] Maniati E, Berlato C, Gopinathan G, et al. Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment. Cell Rep. 2020;30:525-540. e527.

[93] Kim O, Park EY, Klinkebiel DL, et al. In vivo modeling of metastatic human high-grade serous ovarian cancer in mice. PLoS Genet. 2020;16:e1008808.

[94] Iyer S, Zhang S, Yucel S, et al. Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy. Cancer Discov. 2021;11:384-407.

RIGHTS & PERMISSIONS

2024 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.