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

Abstract

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.Malignant cells can enter circulation very early from most cancers (circulating tumor cells, CTCs), 1 yet metastases do not occur until much later (e.g., in pancreatic cancer).2 Enumeration of circulating cancer cells is part of the diagnostic arsenal, but not considered a direct marker of metastasis. Part of the reason is that the majority of cells in most cancers are not capable of seeding metastatic growth.3 Metastatic dissemination is the function of cancer stem cells (CSC), but the overwhelming majority of CTCs do not show stem cell features. 4 An assay to detect circulating CSCs, therefore, could serve as a marker for metastatic predisposition, 5 but it is not available in the oncology practice today.To develop such an assay we took advantage of a unique feature of CSCs, spheroid formation. Spheroids are small multicellular organoids formed in vitro by cells from most cancers. Spheroids are stabilized by cellular contacts that may also play a role in differentiation (e.g., the P19 model).6 With most cancers only a subset of cells can form spheroids and these cells share features with CSCs. 7,8 The same has been demonstrated with cell lines from a variety of tissues. 9 The almost universal potential for spheroids suggests that it may be a basic feature of CSCs akin to clonogenicity or growth in soft agar.We reasoned that if in vitro spheroid formation reflects an important biological function, it may also occur in vivo. To investigate thi...

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