Seeding and Propagation of Untransformed Mouse Mammary Cells in the Lung

Abstract

The acquisition of metastatic ability by tumor cells is considered a late event in the evolution of malignant tumors. We report that untransformed mouse mammary cells that have been engineered to express the inducible oncogenic transgenes MYC and Kras D12 , or polyoma middle T, and introduced into the systemic circulation of a mouse can bypass transformation at the primary site and develop into metastatic pulmonary lesions upon immediate or delayed oncogene induction. Therefore, previously untransformed mammary cells may establish residence in the lung once they have entered the bloodstream and may assume malignant growth upon oncogene activation. Mammary cells lacking oncogenic transgenes displayed a similar capacity for long-term residence in the lungs but did not form ectopic tumors.Metastatic dissemination of cancer cells, the major cause of cancer mortality, is traditionally viewed as a late-stage event (1), although mammary epithelial cells have been shown to disseminate systemically from early neoplastic lesions in transgenic mice and from ductal carcinoma in situ in women (2). There is ample evidence that the ability of fully transformed tumor cells to metastasize depends on the regulation of developmental programs and external environmental cues (3-11), but to what extent the seeding or growth of tumor cells at the ectopic site is dependent on the initiating transforming event(s) is a subject of debate (12). We have developed a system that separates the process of seeding cells in the lung from the process of malignant growth at an ectopic site by using animals engineered to express potent oncogenes in a doxycycline-dependent mammary-specific manner. After intravenous (IV) injection of marked mammary cells that have different genetic potentials [no oncogenes, or oncogenes that will be expressed only after cells have taken up residence in an ectopic site (the lungs)], normal mammary cells can lodge in the lungs, grow slowly, and become frank metastatic malignancies once potent oncogenes are turned on.We recently described tri-transgenic TetO-MYC;TetO-Kras D12 ;MMTV-rtTA (TOM;TOR; MTB) mice that coordinately express MYC and mutant Kras oncogenes in mammary epithelial cells when fed doxycycline (13). Doxycycline-naïve animals do not express the transgenic oncogenes and have morphologically and functionally normal mammary glands, but they develop diffuse autochthonous tumors within 3 to 4 weeks after doxycycline exposure. Tumors that form because of the expression of these oncogenes display malignant characteristics, such as transplantability and metastasis ( fig. S1) To investigate whether mammary cells from these mice can be induced to form metastasis in the absence of transformation at the primary site, we modified the traditional experimental metastasis assay (14). In the modified approach, instead of IV delivery of tumor cells from doxycycline-treated animals into new recipients, we injected dissociated morphologically normal mammary cells from mature TOM;TOR;MTB animals never exposed to doxycy...

MYC regulation of a “poor-prognosis” metastatic cancer cell state

“…Recent studies suggest that inherited polymorphisms on 8q24 that powerfully modify solid tumor predisposition also influence MYC transcriptional regulation (30). Deregulation of the MYC oncoprotein confers a selective advantage on cancer cells in differing contexts by promoting proliferation, cell survival, differentiation blockade, epithelial to mesenchymal transition, and genetic instability, all of which can contribute to metastasis (27,(31)(32)(33). In fact, a recent report suggested that MYC functions as an indirect regulator of metastasis in a c-RAF-driven mouse model of non-small cell lung cancer by modulating growth, differentiation, and angiogenesis (34).…”

A “latent niche” mechanism for tumor initiation

“…Nonetheless, a latent niche mechanism may act in conjunction with these mechanisms, other tumor-cell-autonomous mechanisms, or both to promote inappropriate self-renewal of neighboring cells. For example, Podsypanina et al recently demonstrated that under experimental conditions normal mammary cells can persist in the lung for extended periods and subsequently become foci for new tumor growth when they are transformed (36). It is conceivable that they are maintained by a latent niche mechanism and that self-renewal or survival signals could predispose such cells to progress to tumors after the acquisition of subsequent transforming mutations (36,37).…”

Premetastatic milieu explained by TLR4 agonist-mediated homeostatic inflammation

“…27 Even in RAGnull mice, normal breast cells can be recruited to the lungs without oncogene activation. 28 Experimental activation of oncogenes after homing results in metastatic colonization. This finding suggests that tumor cells may travel to potential metastatic sites at any stage of the multistep malignant transformation sequence.…”