OPEN Circulating tumor cell-derived lines exhibit an amoeboid mode of migration

OPEN Circulating tumor cell-derived lines exhibit an amoeboid mode of migration

Epithelial-to-mesenchymal transition has been described as a critical step in the metastatic process. Recent studies have detected circulating tumor cells in hybrid EMT-states in the blood of cancer patients, however fully epithelial CTC states were also detected, challenging the EMT dogma. Besides invasion through EMT, the amoeboid migration mode has been proposed as an alternative migration mechanism for CTCs, but functional evidence is lacking. In this study, we aimed to investigate the migration mode retained in established CTC lines using in vitro and in vivo assays as well as patient samples. We found that established CTCs are strictly epithelial and yet develop blebbing-amoeboid movement in three-D environments, including individual as well as amoeboid-collective migration modes. Thus, clinically isolated CTCs from epithelial cancer patients retain a range of invasion strategies during metastasis, including amoeboid dissemination.

The vast majority of cancer related deaths result from distant organ metastasis. Colorectal cancer is one of the most frequent cancers with almost two million new colorectal cancer cases diagnosed and approximately one million caused deaths in twenty twenty worldwide. The overall five-year survival stands at sixty-five percent. However, for patients with localized stage colorectal cancer, it reaches ninety-one percent, dropping sharply to fourteen percent when the tumor has metastasized. Deciphering mechanisms involved in the metastatic process has become urgently needed to propose alternative therapeutic strategies. One of the least understood stages of the metastatic process is the transition from the primary site to organ colonization, mediated by circulation of tumor cells within the bloodstream which may retain or not features acquired in the primary tumor microenvironment. Circulating tumor cell counting has been proposed as a promising biomarker in many cancers including colorectal cancer, with EpCAM positive cell enumeration as an FDA-approved prognostic marker. However, CTC phenotypic characterization has been initiated with the ability to amplify these cells in culture. In this context, we have been the first to amplify several CTC lines from the blood of patients with metastatic colorectal cancer.

Epithelial to mesenchymal transition as a mandatory step in the metastatic process is still controversial. In fact, epithelial, mesenchymal and hybrid phenotypes have been detected in CTCs isolated from patients with different tumor types. Mesenchymal single cells use integrins and metalloprotease-dependent degradation of the extracellular matrix to invade the surrounding stroma and get access to vessels for dissemination. Distinct from cells with the mesenchymal phenotype, amoeboid migration, also used by immune cells, is another type of tumor cell migration. It relies on changes in cell shape and changes in actin architecture to squeeze between the extracellular matrix but does not depend on integrins and proteolytic degradation of extracellular matrix. Concomitantly with the downregulation of metalloprotease and integrin expression, upregulation of the Rho/ROCK signaling pathway leads to mesenchymal-amoeboid transition. However, epithelial cancer cells are not required to undergo EMT to acquire an amoeboid migration, they can also undergo epithelial-amoeboid transition.

Using different CTC lines previously established from metastatic colorectal cancer patients, we questioned their mode of migration. As a dominant shared characteristic, colorectal cancer-CRC-CTCs maintained a strong epithelial phenotype. Many studies have speculated that CTC could use an amoeboid mode of migration, but because of the lack of relevant models no phenotypical proof has been described. Using standard colorectal cancer cell lines, primary samples, and CTC lines established in our laboratory, our colleagues were the first to show that cancer cells can migrate collectively in an amoeboid-like manner. In the present work, we exploit the unique model of CTC lines to further test whether cell individualization depends on amoeboid or rather EMT-related strategies. Using in vitro and in vivo models as well as ex vivo patient tumor samples, we show that CTCs employ both individual and collective amoeboid modes of migration, similar to macrophages or other immune populations, supporting a concept of diversity of cancer invasion strategies.

Results CTCs display a phenotypic plastic signature and express epithelial markers

To understand which migration mode is used by CTCs, we interrogate the transcriptomes of the CTCs derived from patients forty-four (CTC forty-four) and forty-five (CTC forty-five) and compare it to the cell line established from the primary tumor of patient forty-four (CPP forty-four). The three cell lines were scored against Epithelial, Mesenchymal, TGF-beta-induced EMT and Mesenchymal Amoeboid Transition signatures using sIngscore. Results highlighted first the contrast between the high Epithelial feature of CTCs and the strong mesenchymal/EMT characteristics of matching primary tumor cells established from patient forty-four. The second interesting observation made by comparing the expression profile of the three cell lines for the MAT features is that CTCs may have acquired an amoeboid program instead of retaining the mesenchymal program to migrate and circulate.

To go deeper in the phenotypic characterization of the patient derived CTCs, we compared the expression of epithelial and mesenchymal markers at the mRNA and protein level in two circulating tumor cell lines (CTC forty-four and CTC forty-five) with a primary tumor cell line (CPP forty-four) and a liver metastasis tumor cell line (CPP forty-five). CTC forty-five and CPP forty-five have been established from the same patient but the first line is from the blood and the second is from a liver metastasis. Fig. one B shows that CTC lines express high levels of the epithelial marker E-cadherin mRNA but not the mesenchymal markers, Vimentin and Zeb-one. Conversely, cell lines from primary tumor and liver metastasis (CPP forty-four and CPP forty-five) exhibit higher mRNA expression level for the mesenchymal genes, especially in the case of CPP forty-five. In particular, CPP forty-five has a higher Vimentin expression suggesting to be further committed in the EMT process compared to CPP forty-four. At the protein level, Fig. one C confirms the epithelial phenotype of the two CTC lines and the mesenchymal phenotypes of the corresponding CPP lines. We previously characterized the strong metastatic initiating potential of CTC forty-four and CTC forty-five but the total absence of mesenchymal phenotype in both lines was unexpected according to the metastatic cascade. Taken together, these results suggest an epithelial migration independent of mesenchymal trait but probably by using an amoeboid migration mode. To validate this Epithelial-amoeboid phenotype, we stained both CTC lines and the corresponding CPP lines with the epithelial marker EpCAM and the phosphorylated form of myosin light chain two (pMLC two), specific for amoeboid phenotype. The phosphorylation and membrane localization of MLC two is visible only for the two CTC lines and not for the corresponding CPP lines (Fig. one D). Altogether these results suggest that CTCs derived from patient blood samples have a strong epithelial phenotype.