Hematogenous metastasis is a multi-step process involving complex tumor-stroma interactions [1
]. Most of the current efforts of metastasis research focus on the identification of genes and signaling pathways that mediate metastasis with an emphasis on the organ-specific functions of the molecular mediators [1
]. These efforts strived to understand the biochemical compatibility of tumor and stroma during metastasis and have successfully extend the concept of the “seed” and “soil” hypothesis into molecular levels [28
]. However, another potentially important aspect of the “seed” and “soil” hypothesis, the biophysical compatibility between tumor cells and target organs, has been largely overlooked in the modern era of metastasis research. Systemic dissemination of tumor cell is not a random process. Instead, physical properties of tumor cells can influence the efficiency of the arrest and seeding in secondary organs. In the present study, by investigating the organ-specific metastasis behaviors of hypertetraploid cells and their isogenic hyperdiploid counterparts, we found enlarged cell size confers a quantitative advantage when lodging at organs with continuous vasculature (i.e. lung and brain), but not in organ that have fenestrated capillaries (bone). This result is in line with a recent study suggesting that organ-specific vascular structures accounted for the selective advantages of TGFβ-induced Angptl4 to promote lung metastasis but not bone metastasis [29
]. The correlation between larger cell size with increased efficiency of metastatic seeding was also observed in a recent study using intravital real-time imaging of lung metastasis seeding and progression [30
]. However, we could not rule other possible mechanisms that could also account for the increased lung and brain metastasis by LM2-hyper cells. For example, hypertetraploid cells may be more resistant to rapid apoptosis after initial arrest compared with hyperdiploid cells in lung and brain, but not in bone, although the mechanism of organ specificity of apoptosis behavior changes by ploidy duplication is elusive.
It is obvious that enlarged cell size by ploidy duplication is not necessary for metastasis to occur; for example, LM2-modal is already highly metastatic to lung. BM1-modal is essentially non-metastatic to lung [16
]. If enlarged cell size were sufficient to confer metastasis ability to lung, we would expect BM1-hyper became lung-metastatic. Our preliminary experiments indicated that BM1-hyper, similar to BM1-modal, was still not able to form lung metastasis (data not shown). This finding indicates that the cell size increase as a consequence of ploidy shift is by itself not sufficient to enable lung metastasis formation. Taken together, the increased cell size only serves as a biophysical modulator of the metastasis ability. Gene expression pattern and biochemical activity of tumor cells still play a dominant role in determining the metastatic potential and organ specificity of malignant tumor cells. Indeed, the most likely reason for the inability of BM1-modal and BM1-hyper cells to colonize the lung is the lack of essential genes for lung metastasis, such as ANGPTL4, EREG and COX2
The enlarged cell size is a consequence of ploidy duplication, as observed in our study. Ploidy duplication, or polyploidy in general, has profound influence during evolution [9
] as well as cancer progression. Tetraploidy caused by cykokinesis failure promoted tumorigenesis [9
] and was proposed as an intermediate stage for cancer aneuploidy during the early stages of tumorigenesis [9
]. It should be noted that MDA-MB-231 is already aneuploid before any experimental handling. The ploidy shift studied here was the transition from hyperdiploidy (one type of aneuploidy) to hypertetraploidy (another type of aneuploidy). Ploidy duplication may lead to chromosome reshuffling and massive instability. However, our observation indicated that the hypertetraploid descendants were genetically stable, consistent with our previously reported finding that tumor-tumor cell fusion in MDA-MB-231 generated stable hybrids [19
]. The genetic stability is possibly linked to the ability of supernumerary centrosomes to cluster into two functional mitotic spindles in advanced malignant cells [19
]. The similarity of genomic stability between hypertetraploid cells and cells derived from fusion of MDA-MB-231 cells [19
] raises the possibility that cell fusion may initiate ploidy duplication. Alternatively, endoreduplication as the result of cytokinesis failure or mitotic slippage can also lead to chromosome doubling [31
]. During the course of our study, we observed that not all MDA-MB-231 derived cells were able to spontaneously progress to higher ploidy after extensive period of cell culture. Another MDA-MB-231 subline, SCP2, did not progress to higher ploidy when cultured for nearly one year. Since both SCP2 and LM2 undergo spontaneous cell fusion at a low frequency [19
], yet only LM2 could undergo massive ploidy duplication, endoreduplication instead of cell fusion is more likely to be the cause of ploidy shift in our current experimental system. Nevertheless, the exact cellular and molecular mechanism of ploidy duplication remains obscure and should be further investigated.
Overall, results from this study suggest that metastasis is not simply a metastasis gene-driven event, but also a process subjected to influence by the biophysical properties of the constantly evolving tumor cells. Importantly, our study provided a direct mechanistic explanation for the previous clinical observations that hypertetraploidy and enlarged nuclear size are linked to poor survival and metastasis [5
]. In addition, the model system established in our study may become a useful platform for functional studies of putative molecular mediators of ploidy duplication, which could be explored as potential new targets for prophylactic cancer therapy to prevent metastasis. Further efforts should investigate the generality of these findings in breast as well as other types of cancer and explore the clinical association of hyperploidy with organ-specific metastases.