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Heterotypic clustering of circulating tumor cells and circulating cancer-associated fibroblasts facilitates breast cancer metastasis

Breast Cancer Research and Treatment volume 189, pages 63–80 (2021)Cite this article

Abstract

Background

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.

Methods

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.

Results

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.

Conclusion

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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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CAF:

Cancer-associated fibroblast

TME:

Tumor microenvironment

BC:

Breast cancer

CTC:

Circulating tumor cell

cCSC:

Circulating cancer stem cell-like cell

cCAF:

Circulating cancer-associated fibroblast

MSC:

Mesenchymal stem cell

B-FGF:

Basic fibroblast growth factor

FAP:

Fibroblast activation protein

EMT:

Epithelial to mesenchymal transition

ECM:

Extracellular matrix

CSC:

Cancer stem cell

FBS:

Fetal bovine serum

NSG:

NOD-scid-gamma

IHC:

Immunohistochemistry

IF:

Immunofluorescence

MMTV PYMT:

FVB/N-Tg (MMTV-PyVT)634Mul/Jc

CK7:

Cytokeratin 7

ER:

Estrogen receptor

PR:

Progesterone receptor

AR:

Androgen receptor

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Acknowledgements

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.

Funding

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).

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Authors and Affiliations

  1. Sheila and David Fuente Graduate Program in Cancer Biology, Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL, USA

    Utsav Sharma

  2. Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, DC, USA

    Utsav Sharma, Kelsie Medina-Saenz, Philip C. Miller & Marc E. Lippman

  3. Department of Laboratory Medicine and Pathology, Masonic Cancer Center, University of Minnesota, 2231 6th St. SE Minneapolis, Minneapolis, MN, 55455, USA

    Benjamin Troness, Angela Spartz & Dorraya El-Ashry

  4. Miami Cancer Institute, Baptist Health South Florida, Miami, FL, USA

    Ana Sandoval-Leon

  5. Department of Pathology, The University of Tennessee Health Science Center, Memphis, TN, USA

    Deanna N. Parke & Tiffany N. Seagroves

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  1. Utsav Sharma
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Contributions

US, KM-S, PCM, MEL, and DE-A contributed to conception and design.US, KM-S, PCM, BT, AS, and DE-A contributed to development of methodology. US, KM-S, PCM, BT, AS, AS-L, DNP, TNS, and DE-A were involved in acquisition of data (animal work, image analysis, patient sample navigation, and processing, etc.). US, KM-S, PCM, BT, AS, MEL, and DE-A were involved in analysis and interpretation of data. US, KM-S, PCM, MEL, and DE-A writing and/or revision of manuscript. US, KM-S, PCM, BT, AS, DNP, TNS, MEL, and DE-A contributed to review of manuscript.

Corresponding author

Correspondence to Dorraya El-Ashry.

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The authors declare that they have no competing interests.

Ethical approval

This study was approved by the Institutional Review Board at the University of Miami (protocol numbers 20130312 and 20160356).

Consent to participate

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

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Keywords

  • Breast Cancer
  • Metastasis
  • Tumor microenvironment
  • Circulating tumor cells
  • Cancer-associated fibroblasts
  • Breast cancer stem cells
  • Animal models of cancer