Glioma stem cell (GSC)-derived autoschizis-like products confer GSC niche properties involving M1-like tumor-associated macrophages.

Spontaneous necrosis is a defining feature of glioblastomas (GBMs), the most malignant glioma. Despite its strong correlations with poor prognosis, it remains unclear whether necrosis could be a possible cause or mere consequence of glioma progression. Here we isolated a particular fraction of necrotic products spontaneously arising from glioma cells, morphologically and biochemically defined as autoschizis-like products (ALPs). When administered to granulocyte macrophage colony-stimulating factor (GM-CSF)-primed bone marrow-derived macrophage/dendritic cells (Mφ/DCs), ALPs were found to be specifically engulfed by Mφs expressing a tumor-associated macrophage (TAM) marker CD204. ALPs from glioma stem cells (GSCs) had higher activity for the TAM development than those from non-GSCs. Of note, expression of the Il12b gene encoding a common subunit of IL-12/23 was upregulated in ALPs-educated Mφs. Furthermore, IL-12 protein evidently enhanced the sphere-forming activity of GBM patient-derived cells, although interestingly IL-12 is generally recognized as an antitumoral M1-Mφ marker. Finally, in silico analysis of The Cancer Genome Atlas (TCGA) transcriptome data of primary and recurrent GBMs revealed that higher expression of these IL-12 family genes was well correlated with more infiltration of M1-type TAMs and closely associated with poorer prognosis in recurrent GBMs. Our results highlight a role of necrosis in GSC-driven self-beneficial niche construction and glioma progression, providing important clues for developing new therapeutic strategies against gliomas.

Induction of protumoral CD11c(high) macrophages by glioma cancer stem cells through GM-CSF.

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 self-create their own niche involving TAMs through CSC-derived GM-CSF, which can thus be a therapeutic target in view of CSC niche disruption.

A Synthetic Polymer Scaffold Reveals the Self-Maintenance Strategies of Rat Glioma Stem Cells by Organization of the Advantageous Niche.

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 (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-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 self-advantageous 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.

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

BACKGROUND: Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. METHODS: The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. RESULTS: SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable. CONCLUSION: Inhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

A niche-mimicking polymer hydrogel-based approach to identify molecular targets for tackling human pancreatic cancer stem cells.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is one of the most fatal human cancers, but effective therapies remain to be established. Cancer stem cells (CSCs) are highly resistant to anti-cancer drugs and a deeper understanding of their microenvironmental niche has been considered important to provide understanding and solutions to cancer eradication. However, as the CSC niche is composed of a wide variety of biological and physicochemical factors, the development of multidisciplinary tools that recapitulate their complex features is indispensable. Synthetic polymers have been studied as attractive biomaterials due to their tunable biofunctionalities, while hydrogelation technique further renders upon them a diversity of physical properties, making them an attractive tool for analysis of the CSC niche. METHODS: To develop innovative materials that recapitulate the CSC niche in pancreatic cancers, we performed polymer microarray analysis to identify niche-mimicking scaffolds that preferentially supported the growth of CSCs. The niche-mimicking activity of the identified polymers was further optimized by polyethylene glycol (PEG)-based hydrogelation. To reveal the biological mechanisms behind the activity of the optimized hydrogels towards CSCs, proteins binding onto the hydrogel were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and the potential therapeutic targets were validated by looking at gene expression and patients' outcome in the TCGA database. RESULTS: PA531, a heteropolymer composed of 2-methoxyethyl methacrylate (MEMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA) (5.5:4.5) that specifically supports the growth and maintenance of CSCs was identified by polymer microarray screening using the human PAAD cell line KLM1. The polymer PA531 was converted into five hydrogels (PA531-HG1 to HG5) and developed to give an optimized scaffold with the highest CSC niche-mimicking activities. From this polymer that recapitulated CSC binding and control, the proteins fetuin-B and angiotensinogen were identified as candidate target molecules with clinical significance due to the correlation between gene expression levels and prognosis in PAAD patients and the proteins associated with the niche-mimicking polymer. CONCLUSION: This study screened for biofunctional polymers suitable for recapitulation of the pancreatic CSC niche and one hydrogel with high niche-mimicking abilities was successfully fabricated. Two soluble factors with clinical significance were identified as potential candidates for biomarkers and therapeutic targets in pancreatic cancers. Such a biomaterial-based approach could be a new platform in drug discovery and therapy development against CSCs, via targeting of their niche.

Cancer Stem Cell-Associated Immune Microenvironment in Recurrent Glioblastomas.

Glioblastoma multiforme (GBM) is the most incurable tumor (due to the difficulty in complete surgical resection and the resistance to conventional chemo/radiotherapies) that displays a high relapse frequency. Cancer stem cells (CSCs) have been considered as a promising target responsible for therapy resistance and cancer recurrence. CSCs are known to organize a self-advantageous microenvironment (niche) for their maintenance and expansion. Therefore, understanding how the microenvironment is reconstructed by the remaining CSCs after conventional treatments and how it eventually causes recurrence should be essential to inhibit cancer recurrence. However, the number of studies focusing on recurrence is limited, particularly those related to tumor immune microenvironment, while numerous data have been obtained from primary resected samples. Here, we summarize recent investigations on the immune microenvironment from the viewpoint of recurrent GBM (rGBM). Based on the recurrence-associated immune cell composition reported so far, we will discuss how CSCs manipulate host immunity and create the special microenvironment for themselves to regrow. An integrated understanding of the interactions between CSCs and host immune cells at the recurrent phase will lead us to develop innovative therapies and diagnoses to achieve GBM eradication.

Bacteria break barrier to promote metastasis.

The intestinal microbiota promote colorectal cancer, but their role in metastasis is poorly defined. In this issue of Cancer Cell, Bertocchi et al. report that intratumoral bacteria disrupt the gut vascular barrier, causing bacterial dissemination to the liver and the formation of a premetastatic niche, favoring recruitment of metastatic cells.

Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation.

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.