Cancer-associated fibroblasts (CAFs) are activated fibroblasts in the cancer stroma and are at the leading edge of many solid tumors, including breast, colon, and melanoma.[1]–[5] CAFs are the most prominent cell type within the tumor stroma of many cancers and an important player in the cancer-microenvironment which consists of a dynamic mixture of fibroblasts, monocytes/macrophages, endothelial cells, lymphocytes, and granulocytes.[2], [6] CAFs drive tumor progression by directly stimulating tumor cell proliferation through the secretion of various growth factors and cytokines such as hepatocyte growth factor, transforming growth factor-β, stromal cell-derived factor 1, and interleukin-6 as well as by remodeling the cancer microenvironment through deposition of extracellular matrix and recruitment of other players such as various inflammatory cells and endothelial cells.[1]–[3] Furthermore, activated CAFs contribute to cancer progression by inducing angiogenesis.[1]–[3] During cancer progression, CAFs contribute to invasive growth of cancer by secreting several proteases such as matrix metalloproteinase or cathepsins and inducing the epithelial to mesenchymal transition (EMT) [1]–[3].

Although fibroblasts are widely distributed and easily recognizable due to their fusiform or spindle-like shape, fibroblasts remain poorly defined in molecular terms and there is no known specific and reliable molecular fibroblast markers.[4] Therefore, the identity of CAF is also poorly understood and CAF marker with absolute specificity has not been identified. There are several well-established indicators of CAF such as smooth muscle actin (SMA), fibroblast stimulating protein-1 (FSP-1), platelet-derived growth factor α (PDGFRα), and PDGFRβ.[4] However, none of them are both exclusive to CAFs and present in all CAFs.[4] Instead, each of these CAF markers is estimated to represent a certain and different phenotype of CAFs. In addition, fibroblasts are highly heterogeneous and fibroblasts from different anatomical sites have considerably different expression profile [7].

In general, CAFs have been known to have distinct morphology characterized as large and plump cells distinguished from normal fibroblasts which are thin, wavy, and small spindle cells.[4], [6], [8] However, we have preliminarily found that some ESCC had stromal fibroblasts having histology of relatively normal fibroblast, while some had distinct morphology of CAF.

The classification by histological morphology of individual CAF and its clinical relevance have not been studied, although some previous reports showed that histological categorization of stromal fibrosis was correlated with clinical outcome of colon cancer [9], breast cancer [10], and lung cancer [11].

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors, with a 5-year survival rate of only 10%.[12] CAF have seldom been discussed in ESCC despite its significance in cancer progression. Only very few studies have investigated the biological role of the tumor stroma including angiogenesis in ESCC.[13]–[18] Because fibroblasts in different parts of the body are intrinsically different, the result of CAF study in cancer of different organs cannot be directly applied to ESCC.

Therefore, we studied clinical relevance of CAF in ESCC by histological classification according to individual cell morphology and immunohistochemical studies of five CAF markers such as FAP, SMA, FSP-1, PDGFRα, PDGFRβ. In addition, we assessed the association between CAF and other cancer-microenvironment related factors such as microvessel density (MVD) and tumor associated macrophage (TAM) infiltration or EMT phenotype.

In this study, we first examined the expression pattern and clinical significance of CAF markers as well as the histological categories and other cancer-microenvironments in ESCC. Histologically immature CAF, defined as a large, plump spindle-shaped morphology, was associated with increased MVD, marked TAM and complete EMT phenotype. It was also a strong independent prognostic factor. Five CAF markers were heterogeneously expressed in individual case, but the total number of highly-expressed CAF markers was larger in immature CAF type than in mature one.

Fibroblasts seem to be the most versatile of connective-tissue cells, displaying a remarkable capacity to differentiate into other members of the family such as fat cells, cartilage cells, and bone cells. “Mature” fibroblasts with a lesser capacity for transformation may, for example, exist side by side with “immature” fibroblasts (often called mesenchymal cells) that can develop into a variety of mature cell types. Generally, immature fibroblasts are known to show typically a large plump and euchromatic nucleus with one or two nucleoli and rough endoplasmic reticulum and prominent Golgi apparatus on ultrastructural findings. In this study, we classified CAF of ESCC as a mature and immature type on the basis of histological features. As a result, immature type was observed in 55.2% of ESCC and was associated with adverse clinical outcome. Also it was associated with increased MVD and the complete EMT phenotype, which are known to be the mechanism of cancer progression.[1]–[6], [22] In addition, we noted the TAM as indicated by CD68 expression was significantly more frequent in ESCC with immature type CAF. Generally TAM is regarded as the M2 phenotype of macrophages that promote angiogenesis, growth, invasion, migration, and metastasis.[23]–[25] Infiltrating TAMs are correlated with poor prognosis in breast cancer.[23] In agreement with this report, enhanced infiltration of TAM in ESCC was also correlated with adverse clinical outcome. However, further study is required to determine more specific relationship between CAF phenotype and M2 phenotype of TAM in ESCC. These results suggest that our histological classification of stromal fibroblast is reliable and clinically relevant despite the lack of detailed understanding of its molecular mechanism.

We chose SMA, FSP-1, FAP, PDGFRα, and PDGFRβ as CAF markers based on previous studies.[1], [2], [4] This is the first study to investigate the expression and significance of SMA, FSP-1, FAP, PDGFRα, and PDGFRβ in ESCC as far as we know. Except SMA, which is widely used in daily practice, the remaining four antibodies are relatively new and have been used mostly for research purposes. Therefore we attempted to validate these four antibodies and found that all four antibodies were specific in both Western blotting and immunohistochemistry in formalin fixed and paraffin embedded tissue (Figure S2).

Our results revealed that SMA expression in ESCC stromal fibroblasts was associated with larger size, advanced T stage, lymph node metastasis, and poor prognosis. Despite the difference in organs and cancer types, this result is highly consistent with previous studies in which patients with abundant stromal myofibroblasts expressing SMA showed a poorer prognosis in oral, colorectal and breast cancers.[26]–[29] Our results also showed that patients expressing FSP1 were older and had shorter survival rates, which was also consistent with previous studies performed on breast cancer.[30] PDGFRβ expression was associated with poorly differentiated tumors (p = 0.010) but was not associated with prognosis. PDGFRα expression in CAF is an essential factor in the progression of lung cancer [31] and this result was partly consistent with our result. The patients with FAP expression showed slightly reduced OS (p = 0.070) and FAP expression was associated with more frequent death (p = 0.030). These results are partly in line with previous studies performed in pancreatic and colorectal cancers.[32], [33] Regarding the prognostic value of PDGFRβ, there is some disagreement between our result and those of previous two studies which remarked the association of stromal PDGFRβ expression and poor prognosis in prostatic and pancreatic cancer, respectively.[34], [35] This discrepancy may be attributable to different cancer types or organs. Our results demonstrated that some of individual CAF markers were significant prognostic predictors of ESCC. However, the expression pattern of CAF markers was highly heterogeneous in every individual case, reflecting the heterogeneity of CAF population. Considering the diversity of cellular origin of CAF and immense heterogeneity of CAF phenotypes, additional CAF markers with a good specificity are required [2].

Because of heterogeneity of CAF phenotype, the evaluation of CAF markers by tissue microarray (TMA) may have a potential limitation compared with evaluation by whole block-based immunostaining. However, we found that CAFs were evenly distributed irrespective of heterogeneity of CAF phenotype in more than half of ESCC cases (Figure S3A–B) in preliminary study examining expression of FSP1, a representative CAF marker, in the whole blocks of 10 ESCC cases. In the remaining cases, CAF distribution showed regional concentration around diverse location, such as invading front, surface necrosis or muscle layer (Figure S3C–E), without a specific correlation with anatomical location or depth of invasion. And CAF’s “hot spot” intensely crowded with CAFs and stromal cells was easily recognized on H&E section. To minimize selection bias, we used large-sized tissue core (2.0 mm in diameter) for TMA construction and each tissue core was selected to contain at least one or more CAF’s “hot spot”.

In ESCCs, recently several reports have shown the clinical significance and the role of tumor stroma including CAF in cancer progression. Wang et al. reported that the tumor-stroma ratio determined by microscopic evaluation was an independent predictor of survival in ESCC.[13] Liu et al. showed that the densities of myofibroblasts, lymphocytes, macrophages and microvessels were increased characteristically in the tumor stroma of ESCC and were usually associated with lymph node involvement.[14] Zhang et al. compared gene expression profiling of tumor fibroblasts from ESCC to that of normal fibroblasts and found that genes associated with cell proliferation, the extracellular matrix, and the immune response are differentially expressed.[16] These results are generally in line with our results in that the presence of activated CAFs was associated with an unfavorable outcome of ESCC. However, this is the first study that has evaluated the exact clinicopathological relevance of each CAF markers using a large cohort of patients with ESCC. In addition, this is the first study to analyze stromal fibroblasts in ESCC based on histology and show its prognostic effect in a large cohort of patients with ESCC.

In conclusion, our results demonstrate that histological classification of CAF is a powerful prognostic predictor for ESCC and is associated with increased MVD and TAM as well as complete EMT phenotype. Our results also suggest that CAF markers have potentials to be diagnostic and therapeutic targets in ESCC.