Thy-1+ Cancer-associated Fibroblasts Adversely Impact Lung Cancer Prognosis

Abstract

Cancer-associated fibroblasts (CAFs) regulate diverse intratumoral biological programs and can promote or inhibit tumorigenesis, but those CAF populations that negatively impact the clinical outcome of lung cancer patients have not been fully elucidated. Because Thy-1 (CD90) marks CAFs that promote tumor cell invasion in a murine model of Kras^G12D-driven lung adenocarcinoma (Kras^LA1), here we postulated that human lung adenocarcinomas containing Thy-1⁺ CAFs have a worse prognosis. We first examined the location of Thy-1⁺ CAFs within human lung adenocarcinomas. Cells that co-express Thy-1 and α-smooth muscle actin (αSMA), a CAF marker, were located on the tumor periphery surrounding collectively invading tumor cells and in perivascular regions. To interrogate a human lung cancer database for the presence of Thy-1⁺ CAFs, we isolated Thy-1⁺ CAFs and normal lung fibroblasts (LFs) from the lungs of Kras^LA1 mice and wild-type littermates, respectively, and performed global proteomic analysis on the murine CAFs and LFs, which identified 425 proteins that were differentially expressed. Used as a probe to identify Thy-1⁺ CAF-enriched tumors in a compendium of 1,586 lung adenocarcinomas, the presence of the 425-gene signature predicted a significantly shorter survival. Thus, Thy-1 marks a CAF population that adversely impacts clinical outcome in human lung cancer.

Figure 1

Detection of cells that express Thy-1 or αSMA in tumor stroma. Images of representative human lung adenocarcinomas with low levels (case 4) or high levels (case 11) of Thy-1 and αSMA. Tissues were subjected to immunohistochemical analysis and counterstained with hematoxylin. Cells that stain positively for Thy-1 or αSMA (brown) are located within stroma surrounding tumor cells (blue). Scale bar, 200 μm.

Figure 2

Positive correlation of Thy-1- and αSMA-expressing cells in tumor stroma. Scatter plot shows the percentages of cells per microscopic field per tumor that stain positively with anti-Thy-1 or anti-αSMA antibodies. Consecutive tissue sections were stained with anti-Thy-1 or anti-αSMA antibodies. Results represent mean values determined from multiple microscopic fields per tumor. Each dot represents a single tumor. P and r values, Pearson’s correlation analysis.

Figure 3

Thy-1-expressing cells localize within the tumor interstitium and on the tumor periphery. Image of human non-small cell lung cancer subjected to immunohistochemical analysis to detect Thy-1. Tissues counterstained with hematoxylin (blue). Cells that stain positively for Thy-1 (brown) are located within interstitial stromal bands (arrows) or at the boundary of tumor and non-tumorous lung tissues (dashed red line), a site of collectively invading tumor cells (asterisks, inset).

Figure 4

Stromal cells that co-express Thy-1 and αSMA. Merged fluorescence micrographs of human lung adenocarcinoma tissue sections co-stained with antibodies that detect Thy-1 or αSMA (pseudocolored green and red, respectively). Nuclei were counterstained with DAPI (pseudocolored blue). Illustrated at higher magnification (inset) are fluorescence and bright field micrographs of a stromal region (A) and a perivascular stromal region (B). The insets contain bright field image of tissues stained with hematoxylin and eosin (top left) and fluorescence micrographs of anti-Thy-1 and -αSMA shown as single channel images (grey scale, top and bottom right) and as merged images (pseudocolored, bottom left). Cells that co-express Thy-1 and αSMA are yellow. Regions of co-localization in Fig. 4A are indicated (ovals). Scale bar, 200 μm.

Figure 5

Quantification of cells that express Thy-1 or αSMA or both. Scatter plot shows the percentages of cells per microscopic field that are Thy-1⁺/αSMA⁻, Thy-1⁻/αSMA⁺, or Thy-1⁺/αSMA⁺. Each dot represents a single microscopic field from the same tumor specimen illustrated in Figs 4 and 5. A total of 13 randomly chosen microscopic fields were analyzed. Mean ± SD (bar and whiskers) from the 13 microscopic fields. P values, two tailed unpaired t test.

Figure 6

Gene Ontology term enrichment in the CAF proteomic signature. The 10 most highly enriched terms among peptides up-regulated in CAFs (top pie chart) or down-regulated in CAFs (bottom pie chart) relative to LFs. Pie segment size is proportional to the gene numbers represented in each term.

Figure 7

αSMA⁺ cells localize within collagenous stroma in a human lung adenocarcinoma. Merged fluorescence micrographs of a human lung adenocarcinoma tissue section co-stained with antibodies that detect type I collagen or αSMA (green and red, respectively). Nuclei were counterstained with DAPI (blue). Zoom (right) of boxed area shows a region of collagenous stroma that contains αSMA⁺ cells and surrounds papillary tumor structures.

Figure 8

The presence of the CAF signature is correlated with a worse prognosis in human lung cancer. Survival analysis of lung adenocarcinoma patients, comparing the differences in risk between tumors, according to degree of manifestation of a CAF proteomic signature (in terms of corresponding mRNA patterns). Kaplan-Meier plot compares top third (“strong manifestation”), bottom third (“weak manifestation”), and middle third (“intermediate”) for a compendium of 12 independent lung adenocarcinoma cohorts. P-values by log-rank test. (B) Table shows correlations by univariate Cox, for each individual array dataset examined, as well as for a “compendium” of all available datasets (featured in Kaplan-Meier plot). Positive beta denotes correlation with worse patient prognosis.

Figure 9

Nuclear YAP in stromal cells. (A) Merged fluorescence micrographs of a Thy-1+ CAF stained with anti-YAP antibodies (red), phalloidin (green), and DAPI (blue). (B) Anti-YAP immunohistochemical analysis of a stromal region within a human lung adenocarcinoma. Within the inset (left), arrows point to stromal cells with nuclei that stain positively (red) or negatively (black) for YAP.