Human Prostate Fibroblasts (HPrF)

Identification of Functional and Diverse Circulating Cancer-Associated Fibroblasts in Metastatic Castration-Naïve Prostate Cancer Patients

Cancer-associated fibroblasts (CAFs) are major drivers of prostate cancer (PCa) progression and metastasis. However, whether they are present in circulation has not been investigated. Booijink et al. determined if there are circulating CAFs (cCAFs) present in the blood of metastatic castration-naïve PCa (mCNPC) patients and characterized phenotype and function.

To isolate circulating cancer-associated fibroblasts (cCAFs), they developed a workflow combining immunofluorescence labeling, flow-activated cell sorting (FACS), and functional characterization (Fig. 1). Fibroblast-activated protein (FAP) was chosen as a CAF surface marker due to its identification on activated CAFs and association with prostate cancer (PCa) prognosis. Human primary prostate fibroblasts (HPrFs) and PCa cells were then spiked into human mononuclear cells (MNCs) to mirror patient samples. 2 × 10⁵ HPrFs (cCAFs) and 2 × 10⁵ LNCaP cells (circulating tumor cells, CTCs) were added to 5 × 10⁷ MNCs. Samples were labeled with antibodies specific for FAP (cCAFs), CD45 (leukocytes), and EpCAM (CTCs). Cells were then sorted by FACS into FAP+ and FAP- fractions for downstream functional or phenotypic characterization, including intracellular staining for fibroblast markers vimentin and collagen-I and analysis of extracellular collagen-I secretion (Fig. 1). They were able to use FACS to successfully isolate FAP+EpCAM- HPrFs (Fig. 2A) and FAP- EpCAM+ LNCaPs. Immunofluorescence imaging showed sorted FAP+EpCAM- HPrFs were positive for collagen-I and vimentin expression. EpCAM+ LNCaPs were negative for both collagen-I and vimentin expression (Fig. 2B). Validation of function utilizing a collagen-I secretion assay showed numerous collagen-I spots present from sorted FAP+EpCAM- HPrFs whereas FAP- EpCAM+ LNCaPs revealed no spots, indicative of collagen-I secretion (Fig. 2B).