Characterization of cancer stem cells in a syngeneic mouse model

Introduction: Recently, the "cancer stem cell (CSC) hypothesis" has gained significant attention as a possible explanation for treatment resistance and disease relapse. So far, the vast majority of studies about CSCs were performed in immune compromised mouse models. In order to characterize the interactions between CSCs and immune cells in vivo, we decided to study CSCs in a syngeneic immune-competent mouse model.
Materials and methods: The side population (SP) discrimination technique is a commonly used method for the identification of stem cells. We used the DyeCycle Violet (DCV) dye, a recently established alternative for the generally used Hoechst33342 dye. Using this dye we analyzed the SP in different murine and human tumor cell lines and looked at the clonogenic and tumorigenic potential of these cells both in vitro and in vivo.
Results: First we optimized the DCV staining technique using mouse bone marrow cells. The bone marrow SP cells were correlated with hematopoietic stem cells based upon the expression of the surface markers sca-1 and c-kit. Next, we detected a SP in different mouse (B16-F0, 5T33vt) and human (1087-mel, 1088-mel and MCF-7) tumor cell lines. Those tumor SP-cells were then further characterized based on the expression of cell surface markers such as CD133, CD44, sca-1, c-kit, ABCG2, MUC-1, NGFR and CD138. This cell phenotyping revealed that no correlation exists between SP cells and analysed surface markers.
We further investigated the clonogenicity of B16 and 5T33vt cells via an in vitro colony forming assay (CFA). Here, we showed that B16 SP cells displayed an increased advantage to form colonies. Moreover we mimicked the tumor microenvironment in vitro to look at the effect of the environment on B16 SP cells. When cells were maintained under hypoxic conditions for 24 hours followed by reoxygenation for 24 hours an increase in the SP was observed. Furthermore treatment with IL-6 for 5 days also leads to an increased SP.
Finally, we characterized the tumor-growth of B16 SP cells in vivo. For this experiment, B16 cells transduced with a lentiviral vector encoding firefly luciferase were used to follow up tumor growth via bioluminescence imaging. The tumor challenging experiments revealed that B16 SP cells were able to induce tumor formation at a faster rate compared to MP and bulk cells. This increased potential of SP cells to form a tumor is an attribute associated with CSCs. In a last step, we characterized for the first time the effect of CSCs on immune cells in the context of syngeneic mice. Our experiments points out that CSCs could alter the immune system by creating an immunosuppressive tumor microenvironment via the up-regulation of regulatory T cells.
Conclusion: In summary, we show the feasibility for the replacement of the Hoechst 33342 dye by the DCV dye for the SP discrimination technique that aims to identify CSCs. B16 SP cells were shown to be more clonogenic in vitro and possess increased tumor propagating capacity in immune- competent mice. Finally, we provide evidence that CSCs may affect the tumor environment by promoting immunosuppressive mechanisms. In the light of our findings CSCs appear to be a promising target for anti-cancer immunotherapy.