Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF–cCAF homotypic clusters, and in heterotypic clusters with CTCs.
In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I–IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells.
We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is involved in heterotypic clustering of these cells.
We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Circulating tumor cell
Circulating cancer stem cell-like cell
Circulating cancer-associated fibroblast
Mesenchymal stem cell
Basic fibroblast growth factor
Fibroblast activation protein
Epithelial to mesenchymal transition
Cancer stem cell
Fetal bovine serum
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The authors thank the University of Miami Sylvester Cancer Center IVIS imaging core and University Imaging Center at the University of Minnesota for expert assistance with in vivo imaging and data analysis. They thank the University of Miami Clinical Research Services and S. Khadaran for navigating patient recruitment and sample acquisition. They thank L Machlin, T Yeasky, D Koyuncu, BJ Wasserlauf, SM Bare, and S Copley for technical assistance.
This work was supported in part by Breast Cancer Research Foundation BCRF-19-099 (ME Lippman), Prevent Cancer Foundation (D El-Ashry), and Sylvester Comprehensive Cancer Center, University of Miami and Masonic Cancer Center, University of Minnesota institutional funds (D El-Ashry).
Conflict of interests
The authors declare that they have no competing interests.
This study was approved by the Institutional Review Board at the University of Miami (protocol numbers 20130312 and 20160356).
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All study participants provided written informed consent prior to study enrollment. Animal work was done in accordance with approved protocols from the Institutional Animal Care and Use Committee at the University of Miami and the University of Minnesota.
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Sharma, U., Medina-Saenz, K., Miller, P.C. et al. Heterotypic clustering of circulating tumor cells and circulating cancer-associated fibroblasts facilitates breast cancer metastasis. Breast Cancer Res Treat 189, 63–80 (2021). https://doi.org/10.1007/s10549-021-06299-0
- Breast Cancer
- Tumor microenvironment
- Circulating tumor cells
- Cancer-associated fibroblasts
- Breast cancer stem cells
- Animal models of cancer