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Human-specific RNA analysis shows uncoupled epithelial-mesenchymal plasticity in circulating and disseminated tumour cells from human breast cancer xenografts

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Abstract

Blood samples, bone marrow, tumours and metastases where possible were collected from SCID mice bearing orthotopic xenografts of the triple-negative MDA-MB-468 cell line or a transplantable ER-positive patient derived xenograft (ED-03), and assessed using human-specific, tandem-nested RT-qPCR for markers relating to detection of circulating (CTCs) and disseminated tumour cells (DTCs), breast cancer clinicopathology, the ‘cancer stem cell’ phenotype, metabolism, hypoxia and epithelial-mesenchymal plasticity (EMP). Increased levels of SNAI1, ILK, NOTCH1, CK20, and PGR, and a decrease/loss of EPCAM in CTCs/DTCs were observed relative to the primary xenograft across both models. Decreased CD24 and EGFR was restricted to the MDA-MB-468 model, while increased TFF1 was seen in the ED-03 model. The major metabolic regulator PPARGC1A, and several hypoxia-related markers (HIF1A, APLN and BNIP3) were significantly elevated in both models. Increased expression of mesenchymal markers including SNAI1 was seen across both models, however CDH1 did not decrease concordantly, and several other epithelial markers were increased, suggesting an uncoupling of EMP to produce an EMP hybrid or partial-EMT. Single cell analysis of ED-03 CTCs, although limited, indicated uncoupling of the EMP axis in single hybrid cells, rather than distinct pools of epithelial or mesenchymal-enriched cells, however dynamic heterogeneity between CTCs/DTCs cannot be ruled out. Reduced CD24 expression was observed in the MDA-MB-468 CTCs, consistent with the ‘breast cancer stem cell’ phenotype, and metastatic deposits in this model mostly resembled the primary xenografts, consistent with the mesenchymal-epithelial transition paradigm.

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Acknowledgements

We gratefully acknowledge Prue Hill and Usha Rani for assessment of pap-stained cytospins; Jason Palmer for generous donation of anti-mitochondrial antibody; Andrzej Januszewski for help with blood collection used in technical experiments; Manisha Shah, Dexing Huang, and Devika Gunasinghe for assisting with xenograft sample processing.

Funding

This research was supported by the National Health and Medical Research Council (Australia, #1027527), the National Breast Cancer Foundation, Australia (EMPathy National Collaborative Research Program, CG-10-04) and St. Vincent’s Hospital Research Endowment Fund—S.C. Dickensen Bequest. We gratefully acknowledge the generous support of the Vermont Cancer Research Fundraising Group, the Phyllis Connor Memorial Trust and the Angior Family Foundation for critical equipment. AT was supported by an Australian Postgraduate Award, AL was supported by an International Research Scholarship from the University of Melbourne and PhD top-up scholarships from the CRC for Cancer Therapeutics (CTx, CTx2), and E.W. Thompson was supported in part by the National Breast Cancer Foundation (CG-10-04; EMPathy). The Translational Research Institute receives support from the Australian Government.

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  1. Anthony Tachtsidis and Anh Viet-Phuong Le have contributed equally to this work.

Authors and Affiliations

  1. University of Melbourne Department of Surgery, St. Vincent’s Hospital, Melbourne, VIC, Australia

    Anthony Tachtsidis, Anh Viet-Phuong Le, Devika Gunasinghe, Mark Waltham & Erik W. Thompson

  2. St. Vincent’s Institute, Melbourne, VIC, Australia

    Anthony Tachtsidis, Tony Blick, Mark Waltham & Erik W. Thompson

  3. Translational Genomics & Epigenomics, Olivia Newton-John Cancer Research Institute, Melbourne, VIC, Australia

    Anh Viet-Phuong Le & Alex Dobrovic

  4. Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia

    Tony Blick, Mark Waltham & Erik W. Thompson

  5. Translational Research Institute, Brisbane, QLD, Australia

    Tony Blick & Erik W. Thompson

  6. The Royal Liverpool and Broadgreen University Hospitals NHS Trust, Prescot Road, Liverpool, UK

    Emma De Sousa

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Tachtsidis, A., Le, A.VP., Blick, T. et al. Human-specific RNA analysis shows uncoupled epithelial-mesenchymal plasticity in circulating and disseminated tumour cells from human breast cancer xenografts. Clin Exp Metastasis 36, 393–409 (2019). https://doi.org/10.1007/s10585-019-09977-y

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