Abstract
BackgroundCancer-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.MethodsMurine 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 PDGFRβ), 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.ResultsWe 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, PDGFRβ, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRβ+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance.ConclusionWe 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.
Highlights
Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment
Which CAFs are tumour-promoting CAFs, and how can we distinguish these from the tumour-restraining CAFs? While studies reporting on tumour-suppressing functions of CAFs are clearly the minority, they highlight the need for gaining more knowledge regarding CAF subpopulations with potentially opposite functions, if targeting of CAFs is to be a successful add-on to cancer treatment
Unbiased detection of CAFs through negative selection strategy We carried out three independent biological repeats of the orthotopic breast tumour model shown in Fig. 1a, implanting either 5 × 105 4T1 or 4T07 cells into the mammary fat pad of the Alpha-smooth muscle actin (αSMA)-red fluorescent protein (RFP) BALB/c mice, analysing a total of 128 tumours and 12 healthy mammary fad pads
Summary
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. Cancer-associated fibroblasts (CAFs) contribute to a plethora of pro-tumorigenic functions, such as supporting cancer stem cells, providing protection against chemotherapy, and creating an immunosuppressive tumour microenvironment (TME) [1]. This has sparked great interest in targeting CAFs to treat various types of solid cancers [2]. While studies reporting on tumour-suppressing functions of CAFs are clearly the minority, they highlight the need for gaining more knowledge regarding CAF subpopulations with potentially opposite functions, if targeting of CAFs is to be a successful add-on to cancer treatment Which CAFs are tumour-promoting CAFs, and how can we distinguish these from the tumour-restraining CAFs? While studies reporting on tumour-suppressing functions of CAFs are clearly the minority, they highlight the need for gaining more knowledge regarding CAF subpopulations with potentially opposite functions, if targeting of CAFs is to be a successful add-on to cancer treatment
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