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    Cancer Technologies

    (G-242) Establishment of a series of lung-metastatic breast cancer lines using a fully immunocompetent mouse model

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row G - Poster # 242
    Introduction:: In vivo systems to model breast cancer metastasis are often performed using mice lacking a complete immune system, due to the difficulty of achieving reliable and replicable metastasis in models such as the FvB mouse, where immune clearing of tumor grafts is common. Establishment of syngeneic models of metastasis using primary tumor cell isolation are a useful tool to address this, but careful validation of methods and characterization of the primary cell line are necessary. Here we have developed a model of tumor metastasis which allows for study of the differences in the tumor microenvironment, vascularization, immune landscape, and physiological cues associated with the primary tumor and metastatic niche. By investigating the morphology and behavior of cancer cells isolated from metastatic nodules after repeated reimplantation to the mammary fat pad, conclusions can be drawn about the relationship between cell phenotype and metastatic potential in an immune-competent model of cancer metastasis.

    Materials and Methods:: FvB (PyMT-/-) mice were implanted with a 1mm3 tumor graft from a FVB/N-Tg(MMTV-PyVT) mouse displaying spontaneous mammary carcinoma. After 6 weeks, metastatic nodules to the lung were counted, and 1mm3 lung tumors were reimplanted into the mammary fat pad for a subsequent generation of mice exhibiting primary tumor development and metastasis. This process was repeated twice to yield 3 generations of PyMT cancer cell lines generated from dissociated nodules after metastasis to lung. Multi-cell suspensions were then assayed using flow cytometry and fluorescent staining of FAP to confirm the presence of fibroblasts still present after tumor dissociation, and fibroblast de-enrichment was performed using CD90 magnetic bead sorting to establish a primary metastatic cancer cell line.

    Results, Conclusions, and Discussions::

    Results: By implanting FVB/N-Tg(MMTV-PyVT) tumor grafts into FVB mice of the same lineage, we can robustly observe metastatic nodules in the lung 8 weeks after implantation (Fig 1A). We report an orthotopic engraftment of 100%, with a metastatic rate of 100% in mice that reached endpoints. Serial tumor implantation and harvesting of lung nodules can be performed, and phenotypic differences in the metastatic potential of embedded tumor cells can be observed after each reimplantation (Fig 1B,C). Mesenchymal status of cells cultured on glass was assayed by using fluorescent staining to measure the relative ratio of vimentin and E-cadherin expression. Finally, presence of fibroblasts in the primary cell suspension was confirmed through staining for FAP (Fig 1D,E). Fibroblasts were de-enriched from the cell line using bead sorting for the CD90 protein, and post-sorting cytometry was performed to confirm that fibroblasts were no longer present (Fig 1F,G). Cell lines may be established for study in vitro on the changes to cell morphology, migratory potential, and proliferative ability which may confer cells the increased metastatic ability observed in vivo. 




    Conclusions: Primary tumor volume was significantly increased in tumors following round 3 of metastatic nodule reimplantation. In addition, an increase in metastatic burden was seen in lungs from mice in the Generation 3 cohort when compared to Generation 1. The FvB mouse strain is fully immunocompetent and generating metastasis in this model is not trivial; further investigation into the differences between generations of cells undergoing graft implantation may lend insights into the adaptive ability of cells to gain metastatic potential in this mouse model.




    Discussion: While studies of subcutaneous or intra-glandular mammary tumor injections yield high engraftment rates, these tumors rarely metastasize, which limits the clinical relevance of many mouse studies that cannot contend with the immunological factors which have a major impact on therapeutic efficacy1. In addition, these methods are often incompletely described in the literature, leading to an inability to compare apples to apples where studies involving novel cancer therapeutics are concerned. This study provides a methodology for observing metastasis in the FVB mouse, and provides a future avenue for immunotherapy studies. 




    Acknowledgements (Optional): : T32 DK101003

    References (Optional): : 1. Grzelak CA, Goddard ET, Lederer EE, et al. Elimination of fluorescent protein immunogenicity permits modeling of metastasis in immune-competent settings. Cancer Cell. 2022;40(1):1-2. doi:10.1016/j.ccell.2021.11.004