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

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.

STEM-04. PD-1 INDEPENDENT OF PD-L1 LIGATION PROMOTES GLIOBLASTOMA GROWTH THROUGH THE NFkB PATHWAY

Brain tumor-initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that a proportion of human and murine BTICs expressed programmed cell death protein (PD-1). Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation, and self-renewal of BTICs. Mechanistically, site-directed mutagenesis, RNA sequencing and pharmacological inhibitors implicated SHP-2-mediated activation of NFkB downstream of PD-1 in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require PD-L1 ligation; thus, the therapeutic blocking antibodies inhibiting PD-1/PD-L1 interaction which failed in glioblastoma trials could not overcome the growth advantage of PD-1 in BTICs. Finally, mice with intracranial Pdcd1 over- or underexpressing BTICs had shorter or longer survival, respectively, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a non-immune resistance mechanism of GBM patients to PD-1 or PD-L1 blocking therapies.

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

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.

A NR2E1-Interacting Peptide of LSD1 Inhibits the Proliferation of Brain Tumor Initiating Cells

The recurrence of malignant brain tumor, like glioblastoma, is often due to the existence of brain tumor initiating cells (BTICs) with stem cell properties. As the mortality ratio of brain tumor relapsed patients is very high and lack of efficient therapies, there is an urgent need to develop novel therapeutic methods targeting BTICs. NR2E1 (TLX), an orphan nuclear receptor, is critical for the self-renewal of BTICs. In this study, we found that NR2E1 recruits LSD1, a lysine demethylase, to demethylate mono- and di-methylated histone 3 Lys4 (H3K4me/me2) at the Pten promoters and repress its expression, thereby promoting BTIC proliferation. Using Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) method, we identified four LSD1 peptides that may interact with NR2E1. One of the peptides that locates at the LSD1 SWIRM domain strongly inhibited BTIC proliferation by promoting Pten expression through interfering NR2E1 and LSD1 function. Furthermore, overexpression of this peptide in human BTIC can inhibit its formation of brain tumor. Hence, this peptide exhibits an interesting potential for therapeutic intervention in malignant brain tumors in future.

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

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.

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

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.

An Arf/Rab cascade controls the growth and invasiveness of glioblastoma

Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.