TY - JOUR

T1 - Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer

AU - Venning, Freja Albjerg

AU - Zornhagen, Kamilla Westarp

AU - Wullkopf, Lena

AU - Sjölund, Jonas

AU - Rodriguez-Cupello, Carmen

AU - Kjellman, Pontus

AU - Morsing, Mikkel

AU - Hajkarim, Morteza Chalabi

AU - Won, Kyoung Jae

AU - Erler, Janine Terra

AU - Madsen, Chris Denis

N1 - Funding Information: This work was supported by the Danish Council for Independent Research YDUN grant (1084181001, F.A.V.), Dansk Kræftforsknings Fond (F.A.V.), the European Research Council (ERC-2015-CoG-682881-MATRICAN; L.W., J.T.E.), the Novo Nordisk Foundation Hallas Møller Stipend (C.D.M., J.T.E.), the Novo Nordisk Foundation (NNF17CC0027852, K.J.W.), the Ragnar Söderberg Foundation, Sweden (N91/15, C.D.M.), Swedish Cancer Society (CAN 2016/783, 19 0632 Pj, and 190007, C.D.M), Swedish Research Council (2017–03389 and 2020–02088, C.D.M.), Ollie and Elof Ericsson's Foundation (2017, C.D.M.), Crafoord Foundation (20171049 and 20190798, C.D.M.), and Åke Wiberg Foundation, Sweden (M16–0120 and M17–0235, C.D.M.). Open Access funding provided by Lund University. Publisher Copyright: © 2021, The Author(s).

PY - 2021/5/20

Y1 - 2021/5/20

N2 - 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.

AB - 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.

KW - Breast cancer progression

KW - CAF heterogeneity

KW - CAF subpopulations

KW - Cancer-associated fibroblast (CAF)

KW - Flow cytometry analysis

U2 - 10.1186/s13046-021-01944-4

DO - 10.1186/s13046-021-01944-4

M3 - Journal article

C2 - 34016130

AN - SCOPUS:85106562096

VL - 40

JO - Journal of Experimental and Clinical Cancer Research (Online)

JF - Journal of Experimental and Clinical Cancer Research (Online)

SN - 1756-9966

IS - 1

M1 - 175

ER -