Loss of CCN1 expression from CAFs does not affect cell proliferation or apoptosis in a syngeneic model of melanoma metastasis. A, PCNA assay in (top) tumor and (bottom) stroma. Cell proliferation was detected using an anti-PCNA antibody. Representative images shown; graphs show the mean percentage of PCNA positive cells in CAFs ± SEM; CCN1f/fn = 7 and CCN1−/−n = 4, Student t test. B, TUNEL assay to detect apoptosis in stroma. Representative images shown; graphs show the mean percentage of TUNEL positive cells in stroma ± SEM, CCN1f/fn = 7 and CCN1−/−n = 4, Student t test.
Funding
Canadian Government | Canadian Institutes of Health Research (CIHR)
Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)
ARTICLE ABSTRACT
Melanoma is the leading cause of skin cancer–related death. As prognosis of patients with melanoma remains problematic, identification of new therapeutic targets remains essential. Matricellular proteins are nonstructural extracellular matrix proteins. They are secreted into the tumor microenvironment to coordinate behavior among different cell types, yet their contribution to melanoma is underinvestigated. Examples of matricellular proteins include those comprising the CCN family. The CCN family member, CCN1, is highly proangiogenic. Herein, we show that, in human patients with melanoma, although found in several tumor cell types, CCN1 is highly expressed by a subset of cancer-associated fibroblasts (CAF) in patients with melanoma and this expression correlates positively with expression of proangiogenic genes and progressive disease/resistance to anti-PD1 checkpoint inhibitors. Consistent with these observations, in a syngeneic C57BL6 mouse model of melanoma, loss of CCN1 expression from Col1A2-Cre-, herein identified as “universal,” fibroblasts, impaired metastasis of subcutaneously injected B16F10 tumor cells to lung, concomitant with disrupted neovascularization and collagen organization. Disruption of the extracellular matrix in the loss of CCN1 was validated using a novel artificial intelligence–based image analysis platform that revealed significantly decreased phenotypic fibrosis and composite morphometric collagen scores. As drug resistance is linked to matrix deposition and neoangiogenesis, these data suggest that CCN1, due to its multifaceted role, may represent a novel therapeutic target for drug-resistant melanoma. Our data further emphasize the essential role that cancer-associated, (universal) Col1A2-Cre-fibroblasts and extracellular matrix remodeling play in coordinating behavior among different cell types within the tumor microenvironment.
In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.
