Increased expression of cancer-associated fibroblast markers at the invasive front and its association with tumor-stroma ratio in colorectal cancer

Background: The tumor microenvironment has a critical role in regulating cancer cell behavior. Tumors with high stromal content are associated with poor patient outcome. The tumor-stroma ratio (TSR) identifies colorectal cancers (CRC) with poor patient prognosis based on hematoxylin & eosin stained sections. The desmoplastic reaction consists to a great extent of cancer-associated fibroblasts (CAFs) of which different subtypes are known. The aim of this study is to investigate and quantify CAFs present in the tumor stroma of CRC stratified by the TSR to possibly add prognostic significance to the TSR. Methods: The expression of established CAF markers was compared between stroma-low and stroma-high tumors using transcriptomic data of 71 stage I - Ill CRC. Based on literature, fibroblast and stromal markers were selected to perform multiplex immunofluorescent staining on formalin fixed, paraffin-embedded tumor sections of patients diagnosed with stage III colon cancer. Antibodies against the following markers were used: alpha SMA, PDGFR -beta, FAP, FSP1 and the stromal markers CD45 and CD31 as reference. The markers were subsequently quantified in the stroma using the Vectra imaging microscope. Results: The transcriptomic data showed that all CAF markers except one were higher expressed in stroma-high compared to stroma-low tumors. Histologically, stroma-high tumors showed a decreased number of FSP1(+)/CD45(+) cells and a trend of an increased expression of FAP compared to stroma-low tumors. FAP was higher expressed at the invasive part compared to the tumor center in both stroma-high and stroma-low tumors. Conclusions: The increased expression of FAP at the invasive part and in stroma-high tumors might contribute to the invasive behavior of cancer cells. Future functional experiments should investigate the contribution of FAP to cancer cell invasion. Combining the quantity of the stroma as defined by the TSR with the activity level of CAFs using the expression of FAP may result in an expanded stroma-based tool for patient stratification.