Development Of Clinically Relevant Models Of Oligo-And Polymetastatic Progression And In Vitro Characterization Of The Effect Of Mir-200c On Biological Features Of Oligometastatic Cells

Background : Metastases are the leading cause of cancer related mortality. Oligometastasis( es) is charactered by a limited number of metastasis(had limited organ involvement and had less than 5 discrete metastatic foci). In contrast, the polymetastasis is shown more than 5 metastatic foci in tissue, or involved multiple anatomic sites. Oligometastasis is a form of stable metastatic dissemination that presents the window for curative treatment. About 25% of oligometastatic patients, treated with metastasis-directed therapies, could achieve long-term survival. Treatment failure is attributed to the progression from the oligometastatic state to the widespread polymetastasis. We recently showed that a 20-fold higher miRNA- 200 c expression, found in patients progressed clinically from the oligometastatic state to polymetastases, could be used to separate stable oligometastatic patients from those who underwent polymetastatic progression. In this project, we set out to begin to define the role of miR-200 c in promoting oligo- to polymetastastic progression from the different level of molecules,cells and tumors. These observations suggest that miR-200 c may play an important role in regulating oligo- to polymetastatic progression. Purpose : To develop reproducible and clinically relevant MDA-MB-435-based pre-clinical animal models for oligometastatic and polymetastatic progression. And to explore the role of miR-200 c in promoting oligo- to polymetastastic progression. Method: Green fluorescent protein(GFP) labeled MDA-MB-435(435-P) cancer cells developed a high incidence of spontaneous lung metastases, We utilized the high lung colonization efficiency feature of this human tumor model to develop stable MDA-MB-435-GFP xenograft models of oligometastatic and polymetastatic progression, the whole body macroscopic metastases were evaluated during the course of 10~12 weeks after tail vein injection of 2 x 106 cancer cells. We observed a significant difference in miR-200 c expression between oligometastatic and polymetastatic MDA-435 cells. So we further developed an oligometastatic cell line that overexpress miR-200 c by lentivirus infection(MDA-435-MU-200c). At the same time, negative control group(infected with empty plasmid LV3NC) and positive control group(polymetastastic cell lines) were established to verify the effect of characterization for oligometastases by overpressing miR-200 c in vitro. Results:1、According to the definition of clinical oligometastasis state,we establish stable xenograft models of oligometastatic and polymetastatic progression and oligometastatic and polymetastatic lung derivative cell lines. 2 、 we have successfully developed the oligometastatic cell line that overexpressed miR-200 c.The efficiency of virus infection was checked by FCM and Real Time-PCR. 3、The results of agar colony assay show that clonogenic formation rate of infected cells was similar to positive control and higher than negative control. The difference is statistically significant.4、The results of CCK-8 experiment show that miRNA-200 c overexpression had no significant effect on cell proliferation as determined by the doubling time.5 、The results of transwell migration assay and invasion assay show that miR-200 c overexpression enhanced migration ability and invasion ability of oligometastatic cells. The difference is statistically significant(P<0.05).Conclusion:1、we first developed oligometastasis(es) and polymetastases models of the MDA-MB-435 human tumor in nude mice and obtained oligometastatic and polymetastatic lung derivative cell lines that exhibited stable oligo- and polymetastatic phenotypes during serial passages in vivo.2、In vitro, oligometastatic cells present the characteristics of polymetastatic cells after overexpressing miR-200 c. These observations collectively show the ability of miR-200 c in promoting metastatic cancer cells to acquire more severe metastatic behaviors in vitro, a function that is consistent with its elevated expression during oligmetastatic to polymetastatic progression.3 、 The effect of miR-200 c in survival efficiency and migration(invasion) ability of tumor cells show that miR-200 c may be a new therapeutic target for preventing oligo- to polymetastatic progression. The definitive role of miRNA-200 c in promoting oligo- to polymetastatic progression requires further in vivo characterization and confirmation which are currently underway.