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

Albjerg Venning, Freja ; Zornhagen, Kamilla Westarp ; Wullkopf, Lena ; Sjölund, Jonas LU ; Rodriguez-Cupello, Carmen LU orcid ; Kjellman, Pontus LU ; Morsing, Mikkel LU ; Hajkarim, Morteza Chalabi ; Won, Kyoung Jae and Erler, Janine Terra , et al. (2021) In Journal of Experimental and Clinical Cancer Research 40(1). p.1-1
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... (More)
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. (Less)
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Experimental and Clinical Cancer Research
volume
40
issue
1
article number
175
pages
21 pages
publisher
BioMed Central (BMC)
external identifiers
  • pmid:34016130
  • scopus:85106562096
ISSN
1756-9966
DOI
10.1186/s13046-021-01944-4
language
English
LU publication?
yes
id
426249b7-3b66-4dd5-9956-454ae81e5a2f
alternative location
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138934/pdf/13046_2021_Article_1944.pdf
date added to LUP
2021-05-26 10:14:47
date last changed
2022-04-27 02:03:29
@article{426249b7-3b66-4dd5-9956-454ae81e5a2f,
  abstract     = {{Background<br/>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.<br/><br/>Methods<br/>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.<br/><br/>Results<br/>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.<br/><br/>Conclusion<br/>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.}},
  author       = {{Albjerg Venning, Freja and Zornhagen, Kamilla Westarp and Wullkopf, Lena and Sjölund, Jonas and Rodriguez-Cupello, Carmen and Kjellman, Pontus and Morsing, Mikkel and Hajkarim, Morteza Chalabi and Won, Kyoung Jae and Erler, Janine Terra and Madsen, Chris Denis}},
  issn         = {{1756-9966}},
  language     = {{eng}},
  month        = {{05}},
  number       = {{1}},
  pages        = {{1--1}},
  publisher    = {{BioMed Central (BMC)}},
  series       = {{Journal of Experimental and Clinical Cancer Research}},
  title        = {{Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer}},
  url          = {{http://dx.doi.org/10.1186/s13046-021-01944-4}},
  doi          = {{10.1186/s13046-021-01944-4}},
  volume       = {{40}},
  year         = {{2021}},
}