STEM-26. BLOOD-TUMOR BARRIER IS COMPOSED OF MECHANOSENSING TUMOR CELLS THAT MASK THERAPEUTIC VULNERABILITY

Abstract Two major obstacles in brain cancer treatment are the blood-tumor barrier (BTB), which restricts delivery of most therapeutic agents, and the quiescent brain tumor-initiating cells (BTICs), which evade cell cycle-targeting chemotherapy. Mechanosensation, the transduction of mechanical cues into cellular signaling, underlies physiological processes such as touch, pain, proprioception, hearing, respiration, epithelial homeostasis, and vascular and lymphatic development. We report that medulloblastoma (MB) BTICs are mechanosensing, a property conferred by force-activated ion channel Piezo2. In contrast to the prevailing view that astrocytes function as a physical barrier in BTB, BTICs project endfeet to ensheathe capillaries. MB develops a tissue stiffness gradient as a function of distance to capillaries. Piezo2 senses substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion at BTIC growth cones, which allow BTICs to mechanically interact with their substrate and sequester β-Catenin to prevent WNT/β-Catenin signaling in BTICs. Our single cell analysis uncovers a two-branched BTIC trajectory that progresses from a deep quiescent state to two cycling states. Tumor cell-specific Piezo2 knockout reverses the off-on WNT/β-Catenin signaling states in BTICs and endothelial cells, collapses the BTB, reduces quiescence depth of BTICs, and markedly enhances MB response to chemotherapy. Our study reveals that BTICs co-opt astrocytic mechanism to contribute to the BTB and provides the first evidence that BTB depends on mechanochemical signaling to mask tumor chemosensitivity. Targeting Piezo2 addresses BTB and BTIC properties that underlie therapy failures in brain cancer.

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DDRE-33. MELATONIN AS A MASTER METABOLIC SWITCH FOR GLIOBLASTOMA

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab024.055 ◽

2021 ◽

Vol 3 (Supplement_1) ◽

pp. i13-i14

Author(s):

Beatriz Irene Fernandez-Gil ◽

Carla Vazquez-Ramos ◽

Alexandra Bechtle ◽

Paola Suarez-Meade ◽

Neda Qosja ◽

...

Keyword(s):

Tumor Growth ◽

Brain Cancer ◽

Cell Metabolism ◽

Metabolic Reprogramming ◽

Tumor Initiating Cells ◽

Metabolic Switch ◽

Powerful Approach ◽

Internal Ph ◽

Brain Tumor Initiating Cells

Abstract Glioblastoma (GBM) is the most common form of malignant primary brain cancer in adults with a median survival of only 15 months. Therefore, new therapies to suppress malignant brain cancer are needed. Brain Tumor Initiating Cells (BTICs) are a GBM subpopulation of cells with a highly glycolytic profile that are thought to be responsible of the resistance of GBM to treatments. Metabolic reprogramming allows tumor cells to survive in unsupportive microenvironments. Manipulating tumor metabolism to counteract GBM resistance arises as a powerful approach with minimum effects in normal counterparts. At pharmacological concentrations, melatonin displays oncostatic properties. This is thought to be due to an increase in mitochondrial oxidative phosphorylation through the effects of melatonin in mitochondria, key organelle in metabolic homeostasis. We hypothesize that melatonin could alter BTIC metabolism, by inducing an anti-Warburg effect and as consequence, melatonin will decrease the viability of GBM cells and tumor growth. We found that treatment of GBM cell lines with 3mM melatonin significantly altered tumor cell metabolism. We observed that melatonin downregulated the lactate symporter MCT4 (p<0.002), inducing a significant intracellular accumulation of lactate (p<0.002) while decreasing it in the extracellular media (p<0.001). This was followed by a decrease in the internal pH (p<0.002). These effects were compensated by an increase in the oxygen consumption rate (OCR) followed by decay that leaded to an increase in ROS production (p<0.001). All these changes result in a depletion of cellular ATP (p<0.001) and eventually drove to a decrease in the proliferation (p<0.001) and cell death (p<0.001). When applied in vivo we observed a significant reduction in the tumor growth (p<0.001), volume (p<0.002) and weight (p<0.002), as well as a drop in the proliferation marker ki67 (p<0.001) and a fibrosis increase in treated tumors. These results position melatonin as a strong therapeutic candidate for GBM therapy.

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ETS-Domain Transcription Factor Elk-1 Regulates Stemness Genes in Brain Tumors and CD133+ BrainTumor-Initiating Cells

Journal of Personalized Medicine ◽

10.3390/jpm11020125 ◽

2021 ◽

Vol 11 (2) ◽

pp. 125

Author(s):

Melis Savasan Sogut ◽

Chitra Venugopal ◽

Basak Kandemir ◽

Ugur Dag ◽

Sujeivan Mahendram ◽

...

Keyword(s):

Transcription Factor ◽

Brain Tumor ◽

Brain Tumors ◽

System Development ◽

Nervous System Development ◽

Stem Cell Population ◽

Tumor Initiating Cells ◽

Specific Domain ◽

Brain Tumor Initiating Cells ◽

Ets Domain

Elk-1, a member of the ternary complex factors (TCFs) within the ETS (E26 transformation-specific) domain superfamily, is a transcription factor implicated in neuroprotection, neurodegeneration, and brain tumor proliferation. Except for known targets, c-fos and egr-1, few targets of Elk-1 have been identified. Interestingly, SMN, SOD1, and PSEN1 promoters were shown to be regulated by Elk-1. On the other hand, Elk-1 was shown to regulate the CD133 gene, which is highly expressed in brain-tumor-initiating cells (BTICs) and used as a marker for separating this cancer stem cell population. In this study, we have carried out microarray analysis in SH-SY5Y cells overexpressing Elk-1-VP16, which has revealed a large number of genes significantly regulated by Elk-1 that function in nervous system development, embryonic development, pluripotency, apoptosis, survival, and proliferation. Among these, we have shown that genes related to pluripotency, such as Sox2, Nanog, and Oct4, were indeed regulated by Elk-1, and in the context of brain tumors, we further showed that Elk-1 overexpression in CD133+ BTIC population results in the upregulation of these genes. When Elk-1 expression is silenced, the expression of these stemness genes is decreased. We propose that Elk-1 is a transcription factor upstream of these genes, regulating the self-renewal of CD133+ BTICs.

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CS-14 * NEUROTROPHIN RECEPTORS TrkB AND TrkC ARE REQUIRED FOR SURVIVAL OF BRAIN TUMOR INITIATING CELLS

Neuro-Oncology ◽

10.1093/neuonc/nou242.14 ◽

2014 ◽

Vol 16 (suppl 5) ◽

pp. v54-v54

Author(s):

R. Kenchappa ◽

S. Lawn ◽

N. Krishna ◽

X. Qu ◽

D. Fenstermacher ◽

...

Keyword(s):

Brain Tumor ◽

Neurotrophin Receptors ◽

Tumor Initiating Cells ◽

Brain Tumor Initiating Cells

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Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1701911114 ◽

2017 ◽

Vol 114 (30) ◽

pp. E6147-E6156 ◽

Cited By ~ 53

Author(s):

Dou Yu ◽

Omar F. Khan ◽

Mario L. Suvà ◽

Biqin Dong ◽

Wojciech K. Panek ◽

...

Keyword(s):

Brain Tumor ◽

Therapeutic Potential ◽

Therapy Resistance ◽

Tumor Initiating Cells ◽

Convection Enhanced Delivery ◽

Individual Gene ◽

Brain Tumor Initiating Cells ◽

Novel Therapeutic Strategies

Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood–brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

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Microglia induces Gas1 expression in human brain tumor-initiating cells to reduce tumorigenecity

Scientific Reports ◽

10.1038/s41598-018-33306-0 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 4

Author(s):

Susobhan Sarkar ◽

Candice C. Poon ◽

Reza Mirzaei ◽

Khalil S. Rawji ◽

Walter Hader ◽

...

Keyword(s):

Brain Tumor ◽

Human Brain ◽

Tumor Initiating Cells ◽

Human Brain Tumor ◽

Brain Tumor Initiating Cells

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miR-146 connects stem cell identity with metabolism and pharmacological resistance in breast cancer

The Journal of Cell Biology ◽

10.1083/jcb.202009053 ◽

2021 ◽

Vol 220 (5) ◽

Author(s):

Chiara Tordonato ◽

Matteo Jacopo Marzi ◽

Giovanni Giangreco ◽

Stefano Freddi ◽

Paola Bonetti ◽

...

Keyword(s):

Breast Cancer ◽

Breast Cancer Subtype ◽

Transcriptional Analysis ◽

Quiescent State ◽

Tumor Initiating Cells ◽

Physiological Relevance ◽

Basal Like Breast Cancer ◽

Xenograft Tumor Growth

Although ectopic overexpression of miRNAs can influence mammary normal and cancer stem cells (SCs/CSCs), their physiological relevance remains uncertain. Here, we show that miR-146 is relevant for SC/CSC activity. MiR-146a/b expression is high in SCs/CSCs from human/mouse primary mammary tissues, correlates with the basal-like breast cancer subtype, which typically has a high CSC content, and specifically distinguishes cells with SC/CSC identity. Loss of miR-146 reduces SC/CSC self-renewal in vitro and compromises patient-derived xenograft tumor growth in vivo, decreasing the number of tumor-initiating cells, thus supporting its pro-oncogenic function. Transcriptional analysis in mammary SC-like cells revealed that miR-146 has pleiotropic effects, reducing adaptive response mechanisms and activating the exit from quiescent state, through a complex network of finely regulated miRNA targets related to quiescence, transcription, and one-carbon pool metabolism. Consistent with these findings, SCs/CSCs display innate resistance to anti-folate chemotherapies either in vitro or in vivo that can be reversed by miR-146 depletion, unmasking a “hidden vulnerability” exploitable for the development of anti-CSC therapies.

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The pro-tumorigenic effects of metabolic alterations in glioblastoma including brain tumor initiating cells

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer ◽

10.1016/j.bbcan.2018.01.004 ◽

2018 ◽

Vol 1869 (2) ◽

pp. 175-188 ◽

Cited By ~ 32

Author(s):

Catherine J. Libby ◽

Anh Nhat Tran ◽

Sarah E. Scott ◽

Corinne Griguer ◽

Anita B. Hjelmeland

Keyword(s):

Brain Tumor ◽

Tumor Initiating Cells ◽

Metabolic Alterations ◽

Brain Tumor Initiating Cells

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Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712363115 ◽

2018 ◽

Vol 115 (22) ◽

pp. E5086-E5095 ◽

Cited By ~ 33

Author(s):

Liang Xu ◽

Ye Chen ◽

Anand Mayakonda ◽

Lynnette Koh ◽

Yuk Kien Chong ◽

...

Keyword(s):

Brain Tumor ◽

Acquired Resistance ◽

Transcriptional Network ◽

Next Generation ◽

Transcriptional Program ◽

Tumor Initiating Cells ◽

Self Renewal ◽

Brain Tumor Initiating Cells ◽

Bet Protein

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.

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TMOD-21. A NOVEL IN-VITRO METHOD TO MODEL MACROPHAGES IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.882 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi220-vi220

Author(s):

Hasan Alrefai ◽

Andee Beierle ◽

Lauren Nassour ◽

Nicholas Eustace ◽

Zeel Patel ◽

...

Keyword(s):

Cell Types ◽

In Vitro Models ◽

Tumor Initiating Cells ◽

Serum Free ◽

Brain Tumor Initiating Cells ◽

Multiple Cell ◽

Anti Inflammatory ◽

Free Media ◽

Inflammatory Macrophages

Abstract BACKGROUND The GBM tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemoresistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Others have addressed this problem by employing 3D bioprinting to incorporate astrocytes and macrophages in an extracellular matrix; however, they used serum-containing media and classically polarized anti-inflammatory macrophages. Serum has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, highlighting the importance of excluding it from these models. Additionally, tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. METHODS THP-1 monocytes and normal human astrocytes (NHAs) were transitioned into serum-free HL-1 and neurobasal-based media, respectively. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft(PDX) lines, using a transwell insert. CD206 expression was used to validate polarization and a cytokine array was used to characterize the cells. RESULTS There was no difference in proliferation rates at 72 hours for THP-1 monocytes grown in serum-free HL-1 media compared to serum-containing RPMI 1640 (p > 0.95). Macrophages polarized via transwell inserts expressed the lymphocyte chemoattractant protein, CCL2, whereas resting(M0), pro-inflammatory(M1), and anti-inflammatory(M2) macrophages did not. Additionally, these macrophages expressed more CXCL1 and IL-1ß relative to M1 macrophages. We have also demonstrated a method to maintain a tri-culture model of GBM PDX cells, NHAs, and TAMs in a serum-free media that supports the growth/maintenance of all cell types. CONCLUSIONS We have demonstrated a novel method by which we can polarize macrophages towards a tumor-supportive phenotype that differs in cytokine expression from traditionally polarized macrophages. This higher-fidelity method of modeling TAMs in GBM can aid in the development of targeted therapeutics that may one day enter the clinic in hopes of improving outcomes in GBM.

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