Prognostic gene-expression signature of carcinoma- associated fibroblasts in non-small cell lung cancer Roya Navaba,1, Dan Strumpfa,1, Bizhan Bandarchia,1, Chang-Qi Zhua,1, Melania Pintiliea, Varune Rohan Ramnarinea, Emin Ibrahimova, Nikolina Radulovicha, Lisa Leunga, Malgorzata Barczyka,b, Devang Panchala, Christine Toa, James J. Yuna, Sandy Dera, Frances A. Shepherda,c, Igor Jurisicaa,d,e, and Ming-Sound Tsaoa,e,f,2 aThe Campbell Family Institute for Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada M5G 2M9; bDepartment of Biomedicine, Jonas Lies vei, N-5009 Bergen, Norway; Departments of cMedicine, dComputer Science, and eMedical Biophysics, and fLaboratory of Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5A 2N4 Edited* by Tak Wah Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved March 10, 2011 (received for review September 28, 2010) The tumor microenvironment strongly influences cancer develop- Results ment, progression, and metastasis. The role of carcinoma-associated Cultured CAFs Display Features of Myofibroblasts. By using a study fibroblasts (CAFs) in these processes and their clinical impact has protocol approved by the Institutional Research Ethics Board, not been studied systematically in non-small cell lung carcinoma CAFs and NFs were cultured from 15 surgically resected primary (NSCLC). We established primary cultures of CAFs and matched nor- NSCLCs, and the histologically confirmed normal lung tissue was mal fibroblasts (NFs) from 15 resected NSCLC. We demonstrate that obtained from the same lobe (Table S1A). Both the primary CAFs have greater ability than NFs to enhance the tumorigenicity of cultured CAFs and tumor stromal fibroblasts expressed α-smooth lung cancer cell lines. Microarray gene-expression analysis of the 15 muscle actin (α-SMA) (Fig. 1A), a marker of myofibroblasts (7). matched CAF and NF cell lines identified 46 differentially expressed These primary CAFs and NFs could be maintained in culture for genes, encoding for proteins that are significantly enriched for ex- up to 20 population doublings. Primary fibroblasts preserved their tracellular proteins regulated by the TGF-β signaling pathway. We ability to induce collagen gel contraction (8), which was greater have identified a subset of 11 genes (13 probe sets) that formed with CAFs than NFs (Fig. 1B). These results showed that CAFs a prognostic gene-expression signature, which was validated in can maintain the phenotypic properties of myofibroblasts even in multiple independent NSCLC microarray datasets. Functional anno- the absence of continuing interaction with carcinoma cells. tation using protein–protein interaction analyses of these and pub- To facilitate further the in vitro and in vivo studies using CAFs lished cancer stroma-associated gene-expression changes revealed and NFs, one pair of primary cultured CAFs and NFs was prominent involvement of the focal adhesion and MAPK signaling immortalized with lentivirus expressing human telomerase YFPhTERT pathways. Fourteen (30%) of the 46 genes also were differentially (hTERT). The resulting immortalized CAF 094 and expressed in laser-capture–microdissected corresponding primary NF 094YFPhTERT cell lines showed loss of senescence (Fig. 1C). tumor stroma compared with the matched normal lung. Six of these 14 genes could be induced by TGF-β1 in NF. The results CAFs Enhance Invasion and Tumorigenicity of NSCLC Cells. By using establish the prognostic impact of CAF-associated gene-expression the coculture Matrigel invasion assay, CAFs increased the in- changes in NSCLC patients. vasiveness of both NCI-H460 and A549 NSCLC cells compared with NFs (Fig. 1D and Fig. S1A). This effect was not attributable B integrin α11 | NAViGaTOR | adenocarcinoma | extracellular matrix to an effect on proliferation of the tumor cells (Fig. S1 ). We also observed that the invading tumor cells appeared elongated and fibroblast-like only when they were cocultured with CAFs but not ung cancer is the leading cause of cancer deaths worldwide C (1). Non-small cell lung carcinoma (NSCLC) accounts for with NFs (Fig. S1 ). A similar alteration of tumor cell appear- L ance was observed when they were exposed to conditioned media 85% of all lung cancers. One of the most consistent histological D features of cancer cell invasion is the appearance of desmoplasia: of CAFs but not NFs (Fig. S1 ). By using gelatin zymography, an stromal changes characterized by the activation of stromal active lytic band (68 kDa) of matrix metalloproteinase 2 (MMP-2) fibroblasts into carcinoma-associated fibroblasts (CAFs), in- was observed in CAFs but not in NFs cocultured with tumor cells creased matrix protein disposition, new blood vessel formation, (Fig. 1E), suggesting a role for MMP-2 activation in CAF-induced and immune cell infiltration (2, 3). These changes have been enhancement of tumor cell invasion. reported to promote tumor cell growth, invasion, metastases, and Subcutaneous coimplantation of A549 lung adenocarcinoma resistance to treatment as well as to mediate immune reaction cells with CAF 094 into severe combined immunodeficient against tumor cells (2, 4, 5). Published prognostic gene signatures (SCID) mice significantly enhanced tumor growth compared for NSCLC have included genes encoding for ECM proteins with controls (Fig. 1F). A similar effect was observed with the such as collagens 1A1, 1A2, and 9; glypican 3; intercellular ad- immortalized CAF 094YFPhTERT (Fig. 1F). These findings were hesion molecule 1 (ICAM-1); laminin B1; selectin L; selectin P; validated further with the NCI-H460 cell line (Fig. 1F). and secreted protein acidic and rich in cysteine (SPARC) (3, 5). Furthermore, stromal elements, including PDGF-C, VEGF-A, IL-8, endothelin-1 (EDN1), osteopontin (SPP1), and the che- Author contributions: R.N., D.S., and M.-S.T. designed research; R.N., D.S., B.B., E.I., N.R., mokine CXCL1, recently have been suggested as potential ther- L.L., M.B., D.P., and J.J.Y. performed research; R.N., D.S., C.-Q.Z., M.P., V.R.R., C.T., I.J., and apeutic targets in skin and breast cancers (4, 6). M.-S.T. analyzed data; and R.N., D.S., B.B., C.-Q.Z., M.P., N.R., S.D., F.A.S., I.J., and M.-S.T. To gain greater insight into the gene-expression characteristics in wrote the paper. CAFs and tumor stroma of NSCLC and more direct evidence for The authors declare no conflict of interest. their clinical role, we conducted microarray analyses on paired CAF and normal fibroblasts (NFs) cultured from 15 resected NSCLC *This Direct Submission article had a prearranged editor. specimens and their corresponding laser-capture–microdissected Data deposition: The data reported in this paper have been deposited in the Gene Ex- (LCM) tumor stroma and histologically normal lung parenchyma. pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE22874). We identified a NSCLC stromal prognostic signature that could be 1R.N., D.S., B.B., and C.-Q.Z. contributed equally to this work. validated in multiple independent published expression datasets of 2To whom correspondence should be addressed. E-mail: [email protected]. primary NSCLCs. The results establish the clinical relevance of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stromal gene-expression changes in lung cancer. 1073/pnas.1014506108/-/DCSupplemental. 7160–7165 | PNAS | April 26, 2011 | vol. 108 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1014506108 Downloaded by guest on September 29, 2021 A C Normal Tumor Primary hTERT 100 +CAF 094YFPhTERT +NF 094YFPhTERT +NF 094 75 +CAF 094 H&E NF 094 50 25 SMA α Population Doubling (PD) 0 CAF 094 0 25 50 75 100 Time (Days) NF CAF D H460 A549 800 p= 0.0286 400 p= 0.0025 700 600 p= 0.0286 300 p= 0.0286 500 p= 0.0022 Cytokeratin AE1/AE3 400 200 300 P<0.0001 p= 0.0286 200 p= 0.0286 100 --------------------------------------------------- 100 --------------------------------------------------- 0 0 Vimentin CAF549 CAF520 CAF089 CAF094 CAF549 CAF520 CAF089 CAF094 Invasion (% of Control NF) Invasion (% of Control NF) E H460 A549 H460 A549 H460 A549 SMA H460 A549 NF 520 CAF 520 NF 089CAF 089NF 089CAF 089NF 520CAF 520NF 520CAF 520NF 094CAF 094NF 094CAF 094 α 72 kDa pro MMP-2 68 kDa MMP-2 B 100 NF 094 F A549 A549 CAF 094 3 80 3 +CAF 094 YFPhTERT +CAF 094 +NF 094 YFPhTERT 60 +NF 094 Control P<0.00053 P<0.0032 Control 40 P=0.01 20 % of initial gel area Tumor Volume (cm ) Volume Tumor 0 (cm ) Volume Tumor 025811 Time (h) Days Days H460 H460 100 NF 549 3 CAF 549 3 YFPhTERT 80 +CAF 094 +CAF 094 YFPhTERT +NF 094 +NF 094 60 Control P<0.0001 Control P<0.0001 40 P=0.001 P<0.0001 20 P=0.001 Tumor Volume (cm ) Volume Tumor % of initial gel area 0 (cm ) Volume Tumor 025811 Time (h) Days Days Fig. 1. Characterization of CAF and lung NF. (A) Representative H&E-stained sections of normal lung and a lung adenocarcinoma with prominent des- moplasia (DS) and showing strong staining of the tumor stromal fibroblasts (S) but not tumor cells (T) for α-SMA. Both CAF and NF stain positive for vimentin but negative for cytokeratin (AE1–AE3 antibody). (Scale bar: 20 μm.) (B) Time-dependent collagen gel contraction induced by NF and CAF. Each point rep- resents means ± SD of eight replicate samples. (CLeft) In contrast to the parent cell lines, NF 094YFPhTERT and CAF 094YFPhTERT continue to proliferate beyond 10–20 population doublings. (Right) The senescence-associated acidic β-galactosidase enzyme activity (blue staining) was detected in primary fibroblasts that failed to continue doubling but not in hTERT-immortalized cells.