Multiplex fluorescent imaging of cancer-associated fibroblasts in colorectal cancer

Abstract

Cancer-associated fibroblasts (CAFs) are a heterogeneous cell population in the tumor stroma and have important prognostic and clinical significance for solid tumors, including colorectal cancer. The identification of CAF presents difficulties due to the lack of a unique diagnostic marker. Detection of CAF by multiplex immunohistochemical staining and assessment of their colocalization. For multiplex IHC staining specimens of 10 colon adenocarcinomas without neoadjuvant treatment were selected. We used «OPAL 7-COLOR MANUAL IHC KIT» (Akoya Biosciences, USA) with five antibodies (FAP, PDGFRβ, CD31, POD, PCK) for staining and Mantra 2 Quantitative Pathology Imaging System (Akoya Biosciences, USA) for evaluation of results. CD31 and CAF markers (FAP, PDGFRβ, POD) are expressed fundamentally in different cells (p<0.0001) in all areas of the tumor (apical, central, invasive margin). Pairs FAP+PDGFRβ in all zones demonstrated significantly higher (p<0.0001) square of tandem staining. It shows that these markers are expressed in the same stromal cells (probably CAF). In pair FAP+POD significant colocalization (p=0.011) was detected only in apical zone. We connect this finding rather with active proliferation of population of young fibroblasts in zones of ulceration and granulations than with CAF. We evaluated co-localization of CAF markers (FAP, PDGFRβ, POD) and endothelial cells (CD31) in different zones of colorectal carcinomas. We showed colocalization of CAF markers for pairs FAP+PDGFRβ in all tumor zones and for pair FAP+POD in apical zone.