Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer. METHODS: Murine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRß), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed. RESULTS: We have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5-6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRß, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRß+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance. CONCLUSION: We demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.

Enteroantigen (eAg)-binding B lymphocytes in the mouse - phenotype, distribution, function and eAg-specific antibody secretion.

Studies reporting beneficial effects of B lymphocytes in autoimmune diseases have been accumulating and a regulatory role for certain B cell subsets is hence getting more and more recognition. Recently, B cells were shown to exhibit a regulatory effect in a T cell transfer model of colitis. Here, B cells exposed to enteroantigen (eAg) ex vivo abrogated the colitogenic effect if co-transplanted with Treg-depleted (CD4+CD25-) T cells into severe combined immune deficiency (SCID) mice. These data may imply a role for B cells that bind eAg (eAg+ B cells) in the immunopathology of colitis. Here, we report the detection of a subset of eAg+ B cells, including both B2 and B1 lineages, and show that these cells are present in all peripheral lymphoid organs of the mouse including the peritoneal cavity. eAg+ B cells are far more efficient as eAg-presenting cells than unfractionated splenocytes or eAg- B cells in causing proliferation of eAg-specific T cells. In comparison with eAg- B cells, eAg+ B cells secrete a significant amount of IL-10 in vitro, suggesting an anti-inflammatory potential. Compared with wild-type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme-specific B cells show inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19(+) B cells by eAg and hybridization with hypoxanthine-aminopterin-thymidine (HAT) sensitive ×63 lymphoma cells followed by cloning at limiting dilution conditions, around 10% of the hybridoma cells secrete eAg-specific antibodies.