Heterogeneity of carcinoma-associated fibroblasts and REV-ERB-induced phenotype molding in pancreatic ductal adenocarcinoma

Abstract

Tumor-stroma crosstalk is essential for PDAC formation. Therefore, the stroma, and specifically the carcinoma-associated fibroblasts (CAFs), are the target of therapeutic alternatives to treat highly desmoplastic tumors such as PDAC. Lately, many CAFs-targeted therapies have been published. However, some of these strategies have not been completely satisfactory results may be because of the presence of CAFs heterogeneity. Therefore, the objectives of this thesis are to identify specific biomarkers of CAFs subpopulations to describe specific signatures to each one. And, with this information, to develop strategies to reprogram protumoral CAFs towards a less supportive subpopulation. Firstly, we isolated CAFs from human PDAC samples. These CAFs were used to perform in vitro cultures without or with pancreatic tumor cells. The transcriptional analysis of these cultures revealed the presence of two different CAFs signatures: the mono-culture and the co-culture. The first one obtained from the culture of CAFs without tumor cells and the second one from the culture of CAFs with tumor cells. Specifically, mono-cultured CAFs show a myofibroblast-like phenotype, while de co-cultured CAFs present an enrichment in inflammatory processes, proliferation and lipid biosynthesis. In addition, these signatures correlated with others described in different patients’ datasets. Afterwards, we used independent PDAC patients’ datasets to study the prognostic value or our signatures. Our signatures, their signaling pathways or even specific markers of such processes were associated with a prognostic value. In particular, patients with high expression of the mono-cultured CAFs signature, their corresponding pathways or, biomarkers, correlated with a better prognosis. However, patients pathways or, biomarkers, correlated with a better prognosis. However, patients with high expression of co-cultured CAFs signature were associated with a worse prognosis. Secondly, we performed a transcriptomic characterization of all CAFs isolated from human PDAC tumor samples and we defined 3 CAFs subpopulations with different expression patterns and functional features: the myoCAFs, the lipoCAFs, and the ecmCAFs. Result that confirmed the CAFs heterogeneity in PDAC. We also used our different CAFs subtypes to evaluate the effects of the tumorstroma crosstalk by some functional assays. The results suggested that paracrine communication between CAFs and tumor cells, was not able to induce functional changes in tumor cells. However, the transcriptional analysis of CAFs co-cultured with tumor cells, in which there is contact between different cell types, confirmed that the presence of tumor cells induced the expression of activated-fibroblast genes in CAFs. Finally, we evaluated the CAFs’ subtypes biomarkers spatial distribution on human PDAC tissue samples. This validation confirmed the coexistence of different subpopulation of CAFs in the same tumor. And, at the same time, it confirmed the existence 2 main type of PDAC tumors, ones characterized by a high myofibroblast content (aSMAÓ/FAPÔ) and, others with a low myofibroblast content (aSMAÔ/FAPÓ). We could also correlate the distribution of these biomarkers with the tumor histology. Poorly differentiated tumors, without well-defined glandular structure, usually showed a low expression of myofibroblast markers. In this and other previous works haven been described that CAFs undergo transcriptomic and metabolomic modifications in response to tumor signals. Many of these changes are modulated by nuclear receptors and transcription factors. Therefore, in this thesis CAFs were treated with REV-ERB drugs. The REV-ERB is a nuclear receptor that acts as a repressor of many processes up- or downregulated in our CAFs signatures. The intention of this treatment was to modulate CAFs towards a less tumor-supportive state. Specifically, the treatment of CAFs with SR9009, a REV-ERB agonist, caused a dedifferentiation state in CAFs. The dedifferentiation state was characterized by an increased capacity to store intracellular lipids, a reduced lipid metabolism and a reduced expression of the classic fibroblast-activation biomarkers. These events have been described as a properly of pancreatic stellate cells (PSCs), a cell type considered CAFs’ precursors. Thus, this study offers new therapeutic opportunities for PDAC and other highly desmoplastic tumors treatment through modulation of CAFs toward less activated stages.