Metastasis blood test by flow cytometry: in vivo cancer spheroids and the role of hypoxia.

Cancer hypoxia correlates with therapeutic resistance and metastasis, suggesting that hypoxic adaptation is a critical survival advantage for cancer stem cells (CSCs). Hypoxic metabolism, however, may be a disadvantage in aerobic circulation as the extremely low incidence of metastasis-compared to the high circulating tumor-cell numbers (CTCs)-appears to suggest. As rare metastatic CSCs still survive, we searched for a mechanism that protects them from oxygen in circulation. CSCs form multicellular spheroids in vitro from virtually all cancers tested. We asked, therefore, whether cancers also form spheroids in vivo and whether circulating spheroids play a role in metastasis. We used metabolic, apoptotic and hypoxia assays, we measured aerobic barriers and calculated hypoxia vs. spheroid-size correlations. We detected metabolic/oxidative stress in spheroids, we found correlation between stem cell presence and hypoxia and we showed that the size of hypoxic spheroids is compatible with circulation. To detect spheroids in patients, we worked out a new light-scatter flow cytometry blood test and assayed 67 metastatic and control cases. We found in vivo spheroids with positive stem cell markers in cancer blood and they showed exclusive correlation with metastasis. In conclusion, our data suggest that metastatic success depends on CSC-association with in vivo spheroids. We propose that the mechanism involves a portable "micro-niche" in spheroids that may support CSC-survival/adaptation in circulation. The new assay may establish a potential early marker of metastatic progression.

Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel.

The molecular mechanisms conferring resistance to docetaxel in prostate cancer patients remain partially understood. We generated docetaxel resistant derivatives of the androgen independent prostate cancer cell lines PC-3 and DU-145. Docetaxel rapidly induces DU-145 cell death via apoptosis and the drug resistant cells were produced by periodically exposing proliferating DU-145 cultures to small doses of docetaxel. In PC-3 cells docetaxel induces delayed cell death via mitotic catastrophe evident by profound multinucleation and formation of giant cells. Mononucleated progeny of the giant PC-3 cells shows significant resistance to docetaxel. Gene expression profiling of these docetaxel resistant PC-3 cells revealed sets of docetaxel inducible and constitutively expressed genes associated with major cancer pathways. A contradictory overlap with DU-145 docetaxel resistant cells was also found. Analyses suggested significant changes associated with apoptotic function, DNA repair, cell growth, survival and proliferation, metabolism, maintenance of cytoskeleton and extracellular matrix formation. These cellular processes often contribute to drug resistance and our study identified a set of genes managing this phenotype. Additional analyses of the drug resistant PC-3 cells using shRNA constructs determined direct relevance of Cyclin G2 to docetaxel resistance as well as prevention of multinucleation, whereas the knockdown of upregulated CYP1B1 showed no effect on either of these processes. Downregulated GBP1 was explored by ectopic overexpression and even though GBP1 has a potential to mediate resistance to docetaxel, it was not utilized in PC-3 cells. The results suggest complex combination of gene expression pattern changes that enables resistance to docetaxel while preventing death via multinucleation.