Blood 2002;99(10):3838C43. a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN? population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGF signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide-dependent manner whereas FAP+PDPN? pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location. INTRODUCTION Cancer-associated fibroblasts (CAFs) are the predominant non-hematopoietic stromal cell type in tumors, and their abundance often correlates with poor prognosis (1C4). Several roles have been CD163L1 ascribed to CAFs, including production of tumor mitogenic factors, deposition of extracellular matrix (ECM), stimulation of angiogenesis(5C7) and even immune cell trafficking and activation(5).. Targeting fibroblast-activation protein (FAP) to eradicate CAFs has demonstrated that a reduction in fibroblasts decreased collagen content and tumor burden(8C12), improving the effectiveness of immunotherapy(13,14). Similarly, abrogation of CXCL12 produced by FAP+ cells synergized with PD-L1 blockade to control the growth of pancreatic cancer in mice (13). Whether CAFs suppress the function of tumor-infiltrating T cells through direct or indirect mechanisms remains unclear. One of the factors that has limited our understanding of how CAFs modulate anti-tumor immunity is a lack of specific markers to identify CAFs. FAP, for example, is expressed by some tumor-infiltrating immune cells (14,15) and is also expressed in lymph nodes (LNs)(16,17). Similarly, expression of alpha-smooth muscle actin (SMA), another putative fibroblast-specific marker (18) (19,20), is detected in other stromal cells. Another limitation is represented by a dearth of techniques to freshly isolate low abundance stromal cells with high viability and reproducibility from tumors. Furthermore, the potential heterogeneity within CAFs adds complexity to the issue of fibroblast identification (21) (22). We developed a protocol to isolate mouse and human stromal cells from tumors, which enabled the identification of two FAP+ stromal subsets differentiated by the expression of the glycoprotein podoplanin (PDPN). FAP+PDPN+ cells expressed canonical fibroblast genes and exhibited some resemblance to fibroblasts of LNs, including the ability to construct a reticular network of fibers and secrete chemokines(23,24), which engenders interactions of tumor-infiltrating lymphocytes and stromal cells. The PDPN? subset of FAP+ cells was identified as cancer-associated pericytes (PDPN? CAPs), as confirmed by their localization around vasculature. Functionally, PDPN+ CAFs suppressed T cell proliferation through nitric oxide (NO) production, whereas PDPN? CAPs had no effect on T cell proliferation. Taken together, our study highlights heterogeneity within FAP+ tumor mesenchymal cells, identifying nitric oxide as a stromal mediator of immunosuppression. MATERIALS AND METHODS Mice and tumor models. (Fig. 2E), as also depicted in skin fibroblasts and LN FRCs, underscoring their common lineage. FAP+PDPN? stroma exhibited pronounced expression of genes previously attributed to pericytes, including (5,34)(Fig. 2F). In addition, their expression pattern resembled that of primary pericytes from LNs (IAPs, integrin alpha7+ pericytes) (35), strengthening the argument that PDPN? tumor stromal cells represent a population of perivascular cells in tumors. Further analysis PF-05231023 of signature genes associated with different LN resident stromal cells also revealed similarities between PDPN+ tumor stromal cells and LN FRCs, including expression of and exhibited membrane ruffles (Supplementary Fig. S1F), characteristic of contractile cells. Additionally, confocal microscopic analysis of 4T1 cryosections revealed perivascular localization of FAP+ PDPN? stromal cells, as well as expression of integrin 7, a marker of LN pericytes (Fig. 3D). Differential expression of fibroblast- and pericyte-specific proteins among FAP+ tumor stroma was confirmed by flow cytometry. Expression of CD140 and cadherin-11, both fibroblast markers, was restricted to PDPN+ tumor stroma, whereas integrin 7 was only expressed by the PDPN? subset (Fig. 3E). In addition, these two subsets both expressed SMA and CD140. These two populations of FAP+ mesenchymal cells were also observed in primary human breast carcinomas (Fig. 3F and Supplementary Fig. S1G). Similar to murine tumor stromal cells, both of these FAP+ populations in human tumors also expressed SMA and CD140 (Supplementary Fig. S1H). Only the subset expressing both FAP and PF-05231023 PDPN exhibited fibroblastic markers such as CD140 (Fig. PF-05231023 3G) and CD44 (Supplementary Fig. S1H). Together, these results demonstrate that FAP is expressed not only by PDPN+ CD140+ cadherin-11+ CAFs (hereafter.