Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mus), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mu-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a selfadvantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. STEM CELLS 2016;34:1151-1162 SIGNIFICANCE STATEMENTA specialized microenvironment called niche that maintains cancer stem cells (CSCs) has been studied as a promising target of cancer therapy. However, as recapitulating the niche by conventional approaches is challenging due to its biological and physical complexity, some important aspects of CSC biology might remain unelucidated. We here provide the first demonstration of the utility of synthetic polymer as a niche mimic for GSCs. Our approach also reveals an important finding, that is, self-organization of the GSC niche, which appends a new aspect to the current concept of CSC regulation and could help development therapeutic strategies for cancer eradication.

Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism.

Cancer stem cells (CSCs) are maintained under special microenvironment called niche, and elucidation and targeting of the CSC niche will be a feasible strategy for cancer eradication. Tumor-associated macrophages (TAMs) are known to be involved in cancer progression and thus can be a component of CSC niche. Although TAMs are known to play multiple roles in tumor progression, involvement of CSCs in TAM development fully remains to be elucidated. Using rat C6 glioma side population (SP) cells as a model of glioma CSCs, we here show that CSCs induce the TAM development by promoting survival and differentiation of bone marrow-derived monocytes. CSC-induced macrophages can be separated into two distinct subsets of cells, CD11c low and CD11c high cells. Interestingly, only the CD11c high subset of cells have protumoral activity, as shown by intracranial transplantation into immune-deficient mice together with CSCs. These CD11c high macrophages were observed in the tumor formed by co-transplantation with CSCs. Furthermore, CSCs produced GM-CSF and anti-GM-CSF antibody inhibited CSC-induced TAM development. In conclusion, CSCs have the ability to selfcreate their own niche involving TAMs through CSC-derived GM-CSF, which can thus be a therapeutic target in view of CSC niche disruption.

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