Chemotherapeutic Stress Induces Transdifferentiation of Glioblastoma Cells to Endothelial Cells and Promotes Vascular Mimicry.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor affecting adults, with a median survival of approximately 21 months. One key factor underlying the limited efficacy of current treatment modalities is the remarkable plasticity exhibited by GBM cells, which allows them to effectively adapt to changes induced by anticancer therapeutics. Moreover, GBM tumors are highly vascularized with aberrant vessels that complicate the delivery of antitumor agents. Recent research has demonstrated that GBM cells have the ability to transdifferentiate into endothelial cells (ECs), illustrating that GBM cells may use plasticity in concert with vascularization leading to the creation of tumor-derived blood vessels. The mechanism behind this transdifferentiation, however, remains unclear. Here, we show that treatment with temozolomide (TMZ) chemotherapy induces time-dependent expression of markers for glioma stem cells (GSCs) and immature and mature ECs. In addition, GBM tumors growing as orthotopic xenografts in nude mice showed increased expression of GSC and EC markers after TMZ treatment. Ex vivo FACS analysis showed the presence of immature and mature EC populations. Furthermore, immunofluorescence analysis revealed increased tumor-derived vessels in TMZ-recurrent tumors. Overall, this study identifies chemotherapeutic stress as a new driver of transdifferentiation of tumor cells to endothelial cells and highlights cellular plasticity as a key player in therapeutic resistance and tumor recurrence.

Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma.

Emerging evidence reveals enrichment of glioma-initiating cells (GICs) following therapeutic intervention. One factor known to contribute to this enrichment is cellular plasticity-the ability of glioma cells to attain multiple phenotypes. To elucidate the molecular mechanisms governing therapy-induced cellular plasticity, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression analysis (gene microarray analysis) during treatment with standard of care temozolomide (TMZ) chemotherapy. Analysis revealed significant enhancement of open-chromatin marks in known astrocytic enhancers for interleukin-8 (IL-8) loci as well as elevated expression during anti-glioma chemotherapy. The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project data demonstrated that IL-8 transcript expression is negatively correlated with GBM patient survival (p = 0.001) and positively correlated with that of genes associated with the GIC phenotypes, such as KLF4, c-Myc, and HIF2α (p < 0.001). Immunohistochemical analysis of patient samples demonstrated elevated IL-8 expression in about 60% of recurrent GBM tumors relative to matched primary tumors and this expression also positively correlates with time to recurrence. Exposure to IL-8 significantly enhanced the self-renewing capacity of PDX GBM (average threefold, p < 0.0005), as well as increasing the expression of GIC markers in the CXCR2 population. Furthermore, IL-8 knockdown significantly delayed PDX GBM tumor growth in vivo (p < 0.0005). Finally, guided by in silico analysis of TCGA data, we examined the effect of therapy-induced IL-8 expression on the epigenomic landscape of GBM cells and observed increased trimethylation of H3K9 and H3K27. Our results show that autocrine IL-8 alters cellular plasticity and mediates alterations in histone status. These findings suggest that IL-8 signaling participates in regulating GBM adaptation to therapeutic stress and therefore represents a promising target for combination with conventional chemotherapy in order to limit GBM recurrence.

A Dendritic Cell-Targeted Adenoviral Vector Facilitates Adaptive Immune Response Against Human Glioma Antigen (CMV-IE) and Prolongs Survival in a Human Glioma Tumor Model.

Antitumor immunotherapeutic strategies represent an especially promising set of approaches with rapid translational potential considering the dismal clinical context of high-grade gliomas. Dendritic cells (DCs) are the body's most professional antigen-presenting cells, able to recruit and activate T cells to stimulate an adaptive immune response. In this regard, specific loading of tumor-specific antigen onto dendritic cells potentially represents one of the most advanced strategies to achieve effective antitumor immunization. In this study, we developed a DC-specific adenoviral (Ad) vector, named Ad5scFvDEC205FF, targeting the DC surface receptor, DEC205. In vitro analysis shows that 60% of DCs was infected by this vector while the infectivity of other control adenoviral vectors was less than 10%, demonstrating superior infectivity on DCs. Moreover, an average of 14% of DCs were infected by Ad5scFvDEC205FF-GFP, while less than 3% of non-DCs were infected following in vivo administration, demonstrating highly selective in vivo DC infection. Importantly, vaccination with this vehicle expressing human glioma-specific antigen, Ad5scFvDEC205FF-CMV-IE, shows a prolonged survival benefit in GL261CMV-IE-implanted murine glioma models (p < 0.0007). Furthermore, when rechallenged, cancerous cells were completely rejected. In conclusion, our novel, viral-mediated, DC-based immunization approach has the significant therapeutic potential for patients with high-grade gliomas.

Dedifferentiation of Glioma Cells to Glioma Stem-like Cells By Therapeutic Stress-induced HIF Signaling in the Recurrent GBM Model.

Increasing evidence exposes a subpopulation of cancer cells, known as cancer stem cells (CSCs), to be critical for the progression of several human malignancies, including glioblastoma multiforme. CSCs are highly tumorigenic, capable of self-renewal, and resistant to conventional therapies, and thus considered to be one of the key contributors to disease recurrence. To elucidate the poorly understood evolutionary path of tumor recurrence and the role of CSCs in this process, we developed patient-derived xenograft glioblastoma recurrent models induced by anti-glioma chemotherapy, temozolomide. In this model, we observed a significant phenotypic shift towards an undifferentiated population. We confirmed these findings in vitro as sorted CD133-negative populations cultured in differentiation-forcing media were found to acquire CD133 expression following chemotherapy treatment. To investigate this phenotypic switch at the single-cell level, glioma stem cell (GSC)-specific promoter-based reporter systems were engineered to track changes in the GSC population in real time. We observed the active phenotypic and functional switch of single non-stem glioma cells to a stem-like state and that temozolomide therapy significantly increased the rate of single-cell conversions. Importantly, we showed the therapy-induced hypoxia-inducible factors (HIF) 1α and HIF2α play key roles in allowing non-stem glioma cells to acquire stem-like traits, as the expression of both HIFs increase upon temozolomide therapy and knockdown of HIFs expression inhibits the interconversion between non-stem glioma cells and GSCs post-therapy. On the basis of our results, we propose that anti-glioma chemotherapy promotes the accumulation of HIFs in the glioblastoma multiforme cells that induces the formation of therapy-resistant GSCs responsible for recurrence. Mol Cancer Ther; 15(12); 3064-76. ©2016 AACR.

MMP14 as a novel downstream target of VEGFR2 in migratory glioma-tropic neural stem cells.

Neural stem cell (NSC)-based carriers have been presented as promising therapeutic tools for the treatment of infiltrative brain tumors due to their intrinsic tumor homing property. They have demonstrated the ability to migrate towards distant tumor microsatellites and effectively deliver the therapeutic payload, thus significantly improving survival in experimental animal models for brain tumor. Despite such optimistic results, the efficacy of NSC-based anti-cancer therapy has been limited due to the restricted tumor homing ability of NSCs. To examine this issue, we investigated the mechanisms of tumor-tropic migration of an FDA-approved NSC line, HB1.F3.CD, by performing a gene expression analysis. We identified vascular endothelial growth factor-A (VEGFA) and membrane-bound matrix metalloproteinase (MMP14) as molecules whose expression are significantly elevated in migratory NSCs. We observed increased expression of VEGF receptor 2 (VEGFR2) in the focal adhesion complexes of migratory NSCs, with downstream activation of VEGFR2-dependent kinases such as p-PLCγ, p-FAK, and p-Akt, a signaling cascade reported to be required for cellular migration. In an in vivo orthotopic glioma xenograft model, analysis of the migratory trail showed that NSCs maintained expression of VEGFR2 and preferentially migrated within the perivascular space. Knockdown of VEGFR2 via shRNAs led to significant downregulation of MMP14 expression, which resulted in inhibited tumor-tropic migration. Overall, our results suggest, the involvement of VEGFR2-regulated MMP14 in the tumor-tropic migratory behavior of NSCs. Our data warrant investigation of MMP14 as a target for enhancing the migratory properties of NSC carriers and optimizing the delivery of therapeutic payloads to disseminated tumor burdens.