Generation and characterisation of two D2A1 mammary cancer sublines to model spontaneous and experimental metastasis in a syngeneic BALB/c host

In vivo and in vitro analyses revealed distinct and shared characteristics of the metastatic D2A1-m1 and D2A1-m2 sublines. In particular, D2A1-m1 cells are more aggressive in experimental metastasis assays, while D2A1-m2 cells are more efficient at disseminating from the primary tumour in spontaneous metastasis assays. Surprisingly, classical metastasis-associated in vitro phenotypes such as enhanced proliferation, migration and invasion are reduced in the sublines compared to the parental cell line. Further, evasion of immune control cannot fully explain their enhanced metastatic properties. By contrast, both sublines show increased resistance to apoptosis when cultured in non-adherent conditions and, for the D2A1-m2 subline, increased 3D tumour spheroid growth. Moreover, the enhanced spontaneous metastatic phenotype of the D2A1-m2 sublines is associated with the ability to recruit an activated tumour stroma and promote directional migration of fibroblasts. Gene expression profiling revealed that the two metastatic sublines are distinct but more closely related to each other than the parental D2A1 cells.

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