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Journal of Experimental & Clinical Cancer Research

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Open Access 01-12-2021 | Breast Cancer | Research

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

Authors: Freja Albjerg Venning, Kamilla Westarp Zornhagen, Lena Wullkopf, Jonas Sjölund, Carmen Rodriguez-Cupello, Pontus Kjellman, Mikkel Morsing, Morteza Chalabi Hajkarim, Kyoung Jae Won, Janine Terra Erler, Chris Denis Madsen

Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2021

Abstract

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 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.

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, 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.

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.

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Title: Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer
Authors: Freja Albjerg Venning
Kamilla Westarp Zornhagen
Lena Wullkopf
Jonas Sjölund
Carmen Rodriguez-Cupello
Pontus Kjellman
Mikkel Morsing
Morteza Chalabi Hajkarim
Kyoung Jae Won
Janine Terra Erler
Chris Denis Madsen
Publication date: 01-12-2021
Publisher: BioMed Central
Keywords: Breast Cancer
Breast Cancer
Published in: Journal of Experimental & Clinical Cancer Research / Issue 1/2021
Electronic ISSN: 1756-9966
DOI: https://doi.org/10.1186/s13046-021-01944-4

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