Study On Pluripotent Stem Cells Derived From Tumor Origin And Its Function And Mechanism

Malignant tumor cells share many characteristics with embryonic progenitors including self-renewal and multiple differentiation capacity, which contributes to the conundrum of tumor cell heterogeneity. The differentiation status of primary tumor has long been used to determine the malignant potential of the tumor, i.e., poorly differentiated tumor is often associated with potential for distant metastasis. Induced pluripotent stem cells (iPSCs) technology provides an opportunity to generate cancer-derived stem cells to investigate pathologic process of tumor progression, thereby enabling disease investigation and drag development. However, reprogramming of tumor cells remains a challenge, and the mechanisms of this process are yet to be determined. In this study, using mouse melanoma cell line B16-F10 and mouse pancreatic cancer cell line PanO2, we studied the roles of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, KMOS) and tumor microenvironment in the reprogramming process of tumor cells.We successfully generated the induced pluripotent melanoma cells (B16-iPCCs) using doxycycline-inducible transcription factors delivered by piggyBac (PB) transposon. B16-iPCCs exhibited many features similar to stem cells in terms of morphology and expression of pluripotent genes including Oct4, Sox2, Nanog, Rex-1 and Dax-1. B16-iPCCs could be induced to differentiate into other cellular lineages. Moreover, B16-iPCCs showed enhanced tumorigenicity, increased drug resistance and metastatic potential. B16-iPCCs were epigeneticly modified through demethylation of the promoter region of Nanog and Oct4, and hypermethylation of promoter region of tumor suppressor gene p53. These observations suggested that forced expression of defined transcription factors could reprogram melanoma cells through epigenetic modifications. In addition, the differentiated B16-iPCCs, which were also termed as post B16-iPCCs (P-B16-iPCCs), not only were sensitive to the chemotherapeutic drugs but also showed reduced proliferation rate, soft agar colonies formation and metastasis potential. Our results demonstrated that the PB transposon-based reprogramming method can be used to reprogram tumor cells. The reprogramed tumor cells showed many characteristics of poorly differentiated tumor cells, and can be redifferentiated into other cellular lineages.It is well known that microenvironment plays important roles in both stem cell niche and tumorigenesis. To study the effect of malignant microenvironment on cellular differentiation and phenotypes of iPCCs, we firstly generated reprogrammed mouse pancreatic cancer cells (Pan02-iPCCs) with KMOS transcription factors as described above. Pan02-iPCCs showed pluripotent potential as demonstrated by activation of several pluripotent genes and ability to differentiate into cells with three germ layer properties in vitro. We further evaluated the effect of tumor-conditioned medium, as a mimic of tumor microenvironment, on the cell fate of Pan02-iPCCs-T. The tumor-conditioned medium treated Pan02-iPCCs-T acquired the mesenchymal-like phenotypes and activation of genes associated epithelial-mesenchymal transition (EMT). These data indicate that Pan02-iPCCs-T can be induced to undergo EMT with tumor microenvironment stimuli. Further characterizations of Pan02-iPCCs-T showed many cancer stem cells (CSCs)-like properties including the increased ability to form colonies in soft agar assay, resistance to the chemotherapy reagents, and increased invasion and metastasis potential. In addition, the TGF-β pathway was also activated in Pan02-iPCCs-T.In summary, our findings provide an novel strategy to investigate the molecular mechanisms of cell reprogramming, pluripotency and tumor progression. Althrough fully reprograming of tumor cells remains a major challenge, the induced pluripotent tumor cells we obtained provide valuable model systems for further studies on molecular pathogenesis of tumorigenesis and tumor progression.