Aurora kinase A inhibition plus Tumor Treating Fields suppress glioma cell proliferation in a cilium-independent manner.

Tumor Treating Fields (TTFields) extend the survival of glioblastoma (GBM) patients by interfering with a broad range of tumor cellular processes. Among these, TTFields disrupt primary cilia stability on GBM cells. Here we asked if concomitant treatment of TTFields with other agents that interfere with GBM ciliogenesis further suppress GBM cell proliferation in vitro. Aurora kinase A (AURKA) promotes both cilia disassembly and GBM growth. Inhibitors of AURKA, such as Alisertib, inhibit cilia disassembly and increase ciliary frequency in various cell types. However, we found that Alisertib treatment significantly reduced GBM cilia frequency in gliomaspheres across multiple patient derived cell lines, and in patient biopsies treated ex vivo. This effect appeared glioma cell-specific as it did not reduce normal neuronal or glial cilia frequencies. Alisertib-mediated depletion of glioma cilia appears specific to AURKA and not AURKB inhibition, and attributable in part to autophagy pathway activation. Treatment of two different GBM patient-derived cell lines with TTFields and Alisertib resulted in a significant reduction in cell proliferation compared to either treatment alone. However, this effect was not cilia-dependent as the combined treatment reduced proliferation in cilia-depleted cell lines lacking, ARL13B, or U87MG cells which are naturally devoid of ARL13B+ cilia. Thus, Alisertib-mediated effects on glioma cilia may be a useful biomarker of drug efficacy within tumor tissue. Considering Alisertib can cross the blood brain barrier and inhibit intracranial growth, our data warrant future studies to explore whether concomitant Alisertib and TTFields exposure prolongs survival of brain tumor-bearing animals in vivo.

KR158 spheres harboring slow-cycling cells recapitulate GBM features in an immunocompetent system.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment-resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor-microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM-pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

Comment on Mahajan, S.; Schmidt, M.H.H. Distinct Lineage of Slow-Cycling Cells Amidst the Prevailing Heterogeneity in Glioblastoma. Cancers 2023, 15, 3843.

We greatly appreciate the interest, careful reading, and appraisal by Mahajan and Schmidt [...].

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

Cilia at the Crossroads of Tumor Treating Fields and Chemotherapy.

Glioblastoma (GBM), the most common and lethal primary brain tumor in adults, requires multi-treatment intervention which unfortunately barely shifts the needle in overall survival. The treatment options after diagnosis and surgical resection (if possible) include irradiation, temozolomide (TMZ) chemotherapy, and now tumor treating fields (TTFields). TTFields are electric fields delivered locoregionally to the head/tumor via a wearable medical device (Optune®). Overall, the concomitant treatment of TTFields and TMZ target tumor cells but spare normal cell types in the brain. Here, we examine whether primary cilia, microtubule-based "antennas" found on both normal brain cells and GBM cells, play specific roles in sensitizing tumor cells to treatment. We discuss evidence supporting GBM cilia being exploited by tumor cells to promote their growth and treatment resistance. We review how primary cilia on normal brain and GBM cells are affected by GBM treatments as monotherapy or concomitant modalities. We also focus on latest findings indicating a differential regulation of GBM ciliogenesis by TTFields and TMZ. Future studies await arrival of intracranial TTFields models to determine if GBM cilia carry a prognostic capacity.

Heterogeneity of glioblastoma stem cells in the context of the immune microenvironment and geospatial organization.

Glioblastoma (GBM) is an extremely aggressive and incurable primary brain tumor with a 10-year survival of just 0.71%. Cancer stem cells (CSCs) are thought to seed GBM's inevitable recurrence by evading standard of care treatment, which combines surgical resection, radiotherapy, and chemotherapy, contributing to this grim prognosis. Effective targeting of CSCs could result in insights into GBM treatment resistance and development of novel treatment paradigms. There is a major ongoing effort to characterize CSCs, understand their interactions with the tumor microenvironment, and identify ways to eliminate them. This review discusses the diversity of CSC lineages present in GBM and how this glioma stem cell (GSC) mosaicism drives global intratumoral heterogeneity constituted by complex and spatially distinct local microenvironments. We review how a tumor's diverse CSC populations orchestrate and interact with the environment, especially the immune landscape. We also discuss how to map this intricate GBM ecosystem through the lens of metabolism and immunology to find vulnerabilities and new ways to disrupt the equilibrium of the system to achieve improved disease outcome.

Tumor Treating Fields Suppression of Ciliogenesis Enhances Temozolomide Toxicity.

Tumor Treating Fields (TTFields) are low-intensity, alternating intermediate-frequency (200 kHz) electrical fields that extend survival of glioblastoma patients receiving maintenance temozolomide (TMZ) chemotherapy. How TTFields exert efficacy on cancer over normal cells or interact with TMZ is unclear. Primary cilia are microtubule-based organelles triggered by extracellular ligands, mechanical and electrical field stimulation and are capable of promoting cancer growth and TMZ chemoresistance. We found in both low- and high-grade patient glioma cell lines that TTFields ablated cilia within 24 h. Halting TTFields treatment led to recovered frequencies of elongated cilia. Cilia on normal primary astrocytes, neurons, and multiciliated/ependymal cells were less affected by TTFields. The TTFields-mediated loss of glioma cilia was partially rescued by chloroquine pretreatment, suggesting the effect is in part due to autophagy activation. We also observed death of ciliated cells during TTFields by live imaging. Notably, TMZ and TTFields have opposing effects on glioma ciliogenesis. TMZ-induced stimulation of ciliogenesis in both adherent cells and gliomaspheres was blocked by TTFields. Surprisingly, the inhibitory effects of TTFields and TMZ on tumor cell recurrence are linked to the relative timing of TMZ exposure to TTFields and ARL13B+ cilia. Finally, TTFields disrupted cilia in patient tumors treated ex vivo. Our findings suggest that the efficacy of TTFields may depend on the degree of tumor ciliogenesis and relative timing of TMZ treatment.

Slow-Cycling Cells in Glioblastoma: A Specific Population in the Cellular Mosaic of Cancer Stem Cells.

Glioblastoma (GBM) exhibits populations of cells that drive tumorigenesis, treatment resistance, and disease progression. Cells with such properties have been described to express specific surface and intracellular markers or exhibit specific functional states, including being slow-cycling or quiescent with the ability to generate proliferative progenies. In GBM, each of these cellular fractions was shown to harbor cardinal features of cancer stem cells (CSCs). In this study, we focus on the comparison of these cells and present evidence of great phenotypic and functional heterogeneity in brain cancer cell populations with stemness properties, especially between slow-cycling cells (SCCs) and cells phenotypically defined based on the expression of markers commonly used to enrich for CSCs. Here, we present an integrative analysis of the heterogeneity present in GBM cancer stem cell populations using a combination of approaches including flow cytometry, bulk RNA sequencing, and single cell transcriptomics completed with functional assays. We demonstrated that SCCs exhibit a diverse range of expression levels of canonical CSC markers. Importantly, the property of being slow-cycling and the expression of these markers were not mutually inclusive. We interrogated a single-cell RNA sequencing dataset and defined a group of cells as SCCs based on the highest score of a specific metabolic signature. Multiple CSC groups were determined based on the highest expression level of CD133, SOX2, PTPRZ1, ITGB8, or CD44. Each group, composed of 22 cells, showed limited cellular overlap, with SCCs representing a unique population with none of the 22 cells being included in the other groups. We also found transcriptomic distinctions between populations, which correlated with clinicopathological features of GBM. Patients with strong SCC signature score were associated with shorter survival and clustered within the mesenchymal molecular subtype. Cellular diversity amongst these populations was also demonstrated functionally, as illustrated by the heterogenous response to the chemotherapeutic agent temozolomide. In conclusion, our study supports the cancer stem cell mosaicism model, with slow-cycling cells representing critical elements harboring key features of disseminating cells.

Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2+/CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner.

Glioblastoma (GBM) is the most common and malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized in part by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive, hematopoietic cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a potent subset of myeloid cells, expressing monocytic (M)-MDSC markers, distinguished by dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate into the TME. This study evaluated the T cell suppressive function and migratory properties of CCR2+/CX3CR1+ MDSCs. Bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Recombinant and glioma-derived CCL2 and CCL7 induce the migration of CCR2+/CX3CR1+ MDSCs with similar efficacy. KR158B-CCL2 and -CCL7 knockdown murine gliomas contain equivalent percentages of CCR2+/CX3CR1+ MDSCs compared to KR158B gliomas. Combined neutralization of CCL2 and CCL7 completely blocks CCR2-expressing cell migration to KR158B cell conditioned media. CCR2+/CX3CR1+ cells are also reduced within KR158B gliomas upon combination targeting of CCL2 and CCL7. High levels of CCL2 and CCL7 are also associated with negative prognostic outcomes in GBM patients. These data provide a more comprehensive understanding of the function of CCR2+/CX3CR1+ MDSCs and the role of CCL2 and CCL7 in the recruitment of these immune suppressive cells and further support the significance of targeting this chemokine axis in GBM.

Metabolomics Monitoring of Treatment Response to Brain Tumor Immunotherapy.

Immunotherapy has revolutionized care for many solid tissue malignancies, and is being investigated for efficacy in the treatment of malignant brain tumors. Identifying a non-invasive monitoring technique such as metabolomics monitoring to predict patient response to immunotherapy has the potential to simplify treatment decision-making and to ensure therapy is tailored based on early patient response. Metabolomic analysis of peripheral immune response is feasible due to large metabolic shifts that immune cells undergo when activated. The utility of this approach is under investigation. In this review, we discuss the metabolic changes induced during activation of an immune response, and the role of metabolic profiling to monitor immune responses in the context of immunotherapy for malignant brain tumors. This review provides original insights into how metabolomics monitoring could have an important impact in the field of tumor immunotherapy if achievable.

HDAC6 Signaling at Primary Cilia Promotes Proliferation and Restricts Differentiation of Glioma Cells.

Histone deacetylase 6 (HDAC6) is an emerging therapeutic target that is overexpressed in glioblastoma when compared to other HDACs. HDAC6 catalyzes the deacetylation of alpha-tubulin and mediates the disassembly of primary cilia, a process required for cell cycle progression. HDAC6 inhibition disrupts glioma proliferation, but whether this effect is dependent on tumor cell primary cilia is unknown. We found that HDAC6 inhibitors ACY-1215 (1215) and ACY-738 (738) inhibited the proliferation of multiple patient-derived and mouse glioma cells. While both inhibitors triggered rapid increases in acetylated alpha-tubulin (aaTub) in the cytosol and led to increased frequencies of primary cilia, they unexpectedly reduced the levels of aaTub in the cilia. To test whether the antiproliferative effects of HDAC6 inhibitors are dependent on tumor cell cilia, we generated patient-derived glioma lines devoid of cilia through depletion of ciliogenesis genes ARL13B or KIF3A. At low concentrations, 1215 or 738 did not decrease the proliferation of cilia-depleted cells. Moreover, the differentiation of glioma cells that was induced by HDAC6 inhibition did not occur after the inhibition of cilia formation. These data suggest HDAC6 signaling at primary cilia promotes the proliferation of glioma cells by restricting their ability to differentiate. Surprisingly, overexpressing HDAC6 did not reduce cilia length or the frequency of ciliated glioma cells, suggesting other factors are required to control HDAC6-mediated cilia disassembly in glioma cells. Collectively, our findings suggest that HDAC6 promotes the proliferation of glioma cells through primary cilia.

Metabolic heterogeneity and adaptability in brain tumors.

The metabolic complexity and flexibility commonly observed in brain tumors, especially glioblastoma, is fundamental for their development and progression. The ability of tumor cells to modify their genetic landscape and adapt metabolically, subverts therapeutic efficacy, and inevitably instigates therapeutic resistance. To overcome these challenges and develop effective therapeutic strategies targeting essential metabolic processes, it is necessary to identify the mechanisms underlying heterogeneity and define metabolic preferences and liabilities of malignant cells. In this review, we will discuss metabolic diversity in brain cancer and highlight the role of cancer stem cells in regulating metabolic heterogeneity. We will also highlight potential therapeutic modalities targeting metabolic vulnerabilities and examine how intercellular metabolic signaling can shape the tumor microenvironment.

Adult immuno-oncology: using past failures to inform the future.

In oncology, "immunotherapy" is a broad term encompassing multiple means of utilizing the patient's immune system to combat malignancy. Prominent among these are immune checkpoint inhibitors, cellular therapies including chimeric antigen receptor T-cell therapy, vaccines, and oncolytic viruses. Immunotherapy for glioblastoma (GBM) has had mixed results in early trials. In this context, the past, present, and future of immune oncology for the treatment of GBM was discussed by clinical, research, and thought leaders as well as patient advocates at the first annual Remission Summit in 2019. The goal was to use current knowledge (published and unpublished) to identify possible causes of treatment failures and the best strategies to advance immunotherapy as a treatment modality for patients with GBM. The discussion focuses on past failures, current limitations, failure analyses, and proposed best practices moving forward.

Dysregulation of Glutamate Transport Enhances Treg Function That Promotes VEGF Blockade Resistance in Glioblastoma.

Anti-VEGF therapy prolongs recurrence-free survival in patients with glioblastoma but does not improve overall survival. To address this discrepancy, we investigated immunologic resistance mechanisms to anti-VEGF therapy in glioma models. A screening of immune-associated alterations in tumors after anti-VEGF treatment revealed a dose-dependent upregulation of regulatory T-cell (Treg) signature genes. Enhanced numbers of Tregs were observed in spleens of tumor-bearing mice and later in tumors after anti-VEGF treatment. Elimination of Tregs with CD25 blockade before anti-VEGF treatment restored IFNγ production from CD8+ T cells and improved antitumor response from anti-VEGF therapy. The treated tumors overexpressed the glutamate/cystine antiporter SLC7A11/xCT that led to elevated extracellular glutamate in these tumors. Glutamate promoted Treg proliferation, activation, suppressive function, and metabotropic glutamate receptor 1 (mGlutR1) expression. We propose that VEGF blockade coupled with glioma-derived glutamate induces systemic and intratumoral immunosuppression by promoting Treg overrepresentation and function, which can be pre-emptively overcome through Treg depletion for enhanced antitumor effects. SIGNIFICANCE: Resistance to VEGF therapy in glioblastoma is driven by upregulation of Tregs, combined blockade of VEGF, and Tregs may provide an additive antitumor effect for treating glioblastoma.

CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors.

Chimeric antigen receptor (CAR) T-cell therapy targeting solid tumors has stagnated as a result of tumor heterogeneity, immunosuppressive microenvironments, and inadequate intratumoral T cell trafficking and persistence. Early (≤3 days) intratumoral presentation of CAR T cells post-treatment is a superior predictor of survival than peripheral persistence. Therefore, we have co-opted IL-8 release from tumors to enhance intratumoral T-cell trafficking through a CAR design for maximal antitumor activity in solid tumors. Here, we demonstrate that IL-8 receptor, CXCR1 or CXCR2, modified CARs markedly enhance migration and persistence of T cells in the tumor, which induce complete tumor regression and long-lasting immunologic memory in pre-clinical models of aggressive tumors such as glioblastoma, ovarian and pancreatic cancer.

Infiltrative and drug-resistant slow-cycling cells support metabolic heterogeneity in glioblastoma.

Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast-cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow-cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose-deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7-related metabolic pathways is a viable therapeutic strategy.

Personalized Tumor RNA Loaded Lipid-Nanoparticles Prime the Systemic and Intratumoral Milieu for Response to Cancer Immunotherapy.

Translation of nanoparticles (NPs) into human clinical trials for patients with refractory cancers has lagged due to unknown biologic reactivities of novel NP designs. To overcome these limitations, simple well-characterized mRNA lipid-NPs have been developed as cancer immunotherapeutic vaccines. While the preponderance of RNA lipid-NPs encoding for tumor-associated antigens or neoepitopes have been designed to target lymphoid organs, they remain encumbered by the profound intratumoral and systemic immunosuppression that may stymie an activated T cell response. Herein, we show that systemic localization of untargeted tumor RNA (derived from whole transcriptome) encapsulated in lipid-NPs, with excess positive charge, primes the peripheral and intratumoral milieu for response to immunotherapy. In immunologically resistant tumor models, these RNA-NPs activate the preponderance of systemic and intratumoral myeloid cells (characterized by coexpression of PD-L1 and CD86). Addition of immune checkpoint inhibitors (ICIs) (to animals primed with RNA-NPs) augments peripheral/intratumoral PD-1+CD8+ cells and mediates synergistic antitumor efficacy in settings where ICIs alone do not confer therapeutic benefit. These synergistic effects are mediated by type I interferon released from plasmacytoid dendritic cells (pDCs). In translational studies, personalized mRNA-NPs were safe and active in a client-owned canine with a spontaneous malignant glioma. In summary, we demonstrate widespread immune activation from tumor loaded RNA-NPs concomitant with inducible PD-L1 expression that can be therapeutically exploited. While immunotherapy remains effective for only a subset of cancer patients, combination therapy with systemic immunomodulating RNA-NPs may broaden its therapeutic potency.

Scalable Culturing of Primary Human Glioblastoma Tumor-Initiating Cells with a Cell-Friendly Culture System.

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 108 cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.

Using Carboxy Fluorescein Succinimidyl Ester (CFSE) to Identify Quiescent Glioblastoma Stem-Like Cells.

Tumor resistance to conventional therapies is a major challenge toward the eradication of cancer, a life-threatening disease. This resistance mainly results from tumor heterogeneity and more specifically from the existence of "stem-like" cells that remain in a quiescent state for long periods of time and thus escape commonly used anti-cancer drugs resulting in treatment failure. Therefore, targeting this subpopulation would present a viable strategy to overcome tumor burden. This daunting task requires a deep and thorough understanding of the biology of the quiescent stem-cell population, their interaction with tumor microenvironments, and mechanisms used to sustain themselves despite aggressive therapies. In this chapter, we describe detailed technical procedures for the isolation of quiescent or infrequently dividing stem-like cells in cultured glioblastoma tumor cells using carboxy fluorescein succinimidyl ester (CFSE) staining and flow cytometric analysis. Quiescent glioblastoma cells with stem-like features are characterized and subsequently isolated based on their ability to retain the CFSE labeling.