Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer

Abstract

The tumor microenvironment strongly influences cancer development, progression, and metastasis. The role of carcinoma-associated fibroblasts (CAFs) in these processes and their clinical impact has not been studied systematically in non-small cell lung carcinoma (NSCLC). We established primary cultures of CAFs and matched normal fibroblasts (NFs) from 15 resected NSCLC. We demonstrate that CAFs have greater ability than NFs to enhance the tumorigenicity of lung cancer cell lines. Microarray gene-expression analysis of the 15 matched CAF and NF cell lines identified 46 differentially expressed genes, encoding for proteins that are significantly enriched for extracellular proteins regulated by the TGF-β signaling pathway. We have identified a subset of 11 genes (13 probe sets) that formed a prognostic gene-expression signature, which was validated in multiple independent NSCLC microarray datasets. Functional annotation using protein-protein interaction analyses of these and published cancer stroma-associated gene-expression changes revealed prominent involvement of the focal adhesion and MAPK signaling pathways. Fourteen (30%) of the 46 genes also were differentially expressed in laser-capture-microdissected corresponding primary tumor stroma compared with the matched normal lung. Six of these 14 genes could be induced by TGF-β1 in NF. The results establish the prognostic impact of CAF-associated gene-expression changes in NSCLC patients.

Fig. 1.

Characterization of CAF and lung NF. (A) Representative H&E-stained sections of normal lung and a lung adenocarcinoma with prominent desmoplasia (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 represents means ± SD of eight replicate samples. (C Left) In contrast to the parent cell lines, NF 094^YFPhTERT and CAF 094^YFPhTERT 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. (Scale bar: 40 μm.) (D) Matrigel invasion ability of H460 and A549 cell lines was enhanced by coculture with four pairs of primary CAF. Significance was tested with the Mann–Whitney test. (E) Gelatin zymography shows activation of MMP-2 when tumor cells were cocultured with CAF compared with coculturing with NF. The bands show the lytic zones. (F) Both primary (CAF 094) and immortalized (CAF 094^YFPhTERT) cells enhanced the in vivo tumorigenicity of both A549 and H460 cells in SCID mice (mean ± SEs with eight mice in each group).

Fig. 2.

Differentially expressed genes in 15 paired CAF vs. NF and in corresponding NSCLC stroma vs. normal lung. A total of 46 differentially expressed genes [22 up-regulated (blue sidebar) and 24 down-regulated (yellow sidebar)] were identified by using paired SAM analysis (absolute fold change > 2; q < 0.1). Heatmap plot of scaled gene-expression levels (mean centered for each gene and SD set to 1). Columns represent paired samples of CAF and NF. Rows are genes with mean fold change between CAFs and NFs (in parentheses). Asterisks denote 14 differentially expressed genes (6 up- and 8 down-regulated) overlapping with NSCLC stroma vs. normal lung analysis.

Fig. 3.

Prognostic significance of CAF-associated gene-expression signature in multiple independent cohorts of NSCLC patients. (A) The 11-gene (13-probe set) signature is prognostic in 218 patients randomly assigned to training set in the DCC lung adenocarcinoma study. (B–D) By using the fixed algorithm, the prognostic value of the signature was tested on the microarray data of the remaining 218 DCC patients (assigned as testing set; B), NSCLC patients from Duke (C), and NSCLC patients from SKKU (D). Patients were classified as low-risk and high-risk groups by the signature. Significant differences in survival outcome between these two groups were observed in both the training and testing sets; 5-y overall survival was used for the DCC and Duke patients, and disease-free survival was used for SKKU patients.

Fig. 4.

Proteins encoding the CAF and tumor stroma-associated genes from seven different epithelial tumors share common involvement in the MAPK signaling pathway and focal adhesion. CAF/tumor stroma characteristic gene signatures from seven studies were mapped to PPIs. In the resulting PPI network, 55 proteins interacting with multiple NSCLC stroma and CAF proteins identified in this study (NSCLC; top) and additional tumor stroma/CAF studies from breast cancer, cholangiocarcinoma (CCA), and basal cell carcinoma (BCC) show significant enrichment in focal adhesion and the MAPK pathway (P = 1.7 × 10⁻⁴ and P = 7.7 × 10⁻⁴, respectively). Shapes (nodes) represent proteins, and lines (edges) indicate physical PPIs, colored based on annotation to KEGG focal adhesion (hsa04510; yellow). PPI network was visualized with NAViGaTOR 2.1.14; indirectly linked proteins and interactions are faded-out to reduce image complexity.