OTME-8. INHIBITORY CD161 RECEPTOR IDENTIFIED IN GLIOMA- INFILTRATING T CELLS BY SINGLE-CELL ANALYSIS OTME-10. INTEGRATED ANALYSIS OF HUMAN GLIOMAS AT THE SINGLE CELL LEVEL IDENTIFIES S100A4 AS A NOVEL IMMUNOTHERAPY TARGET PATHWAY IN GLIOBLASTOMA INVASION

Communication between cancer cells and immune cells is a key deter- minant of the glioblastoma ecosystem and its response to therapies, but remains poorly understood. Here we leveraged single-cell RNA-sequencing (scRNA-seq) of human samples and mouse models, deconvolution analysis of bulk specimen from The Cancer Genome Atlas (TCGA) and functional approaches, to dissect cellular cross-talks in glioblastoma. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal- like (MES-like) states. This effect is mediated, both in vitro and in vivo , by macrophage-derived Oncostatin M (OSM) and its cognate receptor OSMR on glioblastoma cells. We show that MES-like glioblastoma states are also associated with increased expression of a mesenchymal program in macrophages and with increased cytotoxicity of T cells, highlighting extensive alterations of the immune microenvironment with potential therapeutic implications. T-cells are critical effector cells of cancer immunotherapies, but little is known about T-cell gene expression programs in diffuse gliomas. We leveraged single-cell RNA-seq to chart the gene expression and clonal landscape of tumor-infiltrating T-cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. Our analysis revealed subsets of T-cells that expressed several NK-cell receptors, in particular the inhibitory CD161 receptor ( KLRB1 gene). KLRB1 was overexpressed by clonally expanded CD8 T-cells, and larger populations of T-cells expressed CD161 than PD-1. The CLEC2D ligand of CD161 was expressed by malignant cells and myeloid cells, and in-activation of KLRB1 enhanced anti-tumor T-cell function. KLRB1 was also expressed by substantial T-cell populations in multiple other human cancers. CD161 and other NK-cell receptors expressed by T-cells rep- resent opportunities for immunotherapy of diffuse gliomas and other human cancers. Reprograming of cellular metabolism is a hallmark of cancer. The meta- bolic alterations in cancer cells is not only defined by series of genetic muta-tions, but also reflecting the crosstalk between cancer cells and other factors in the microenvironment. Altering metabolism allows cancer cells to overcome unfavorable conditions, to proliferate and invade. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplifica-tion of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. However, the metabolism of high MYC amplified medulloblastoma sub-group remains underexplored. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in different environments – in vitro , in flank xenografts and in orthotopic xenografts. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and the high-MYC medulloblastoma cells in culture. Compared to normal brain, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of nucleotide, hexosamine bio- synthetic pathway (HBP), TCA cycle, and amino acid and glutathione pathways. There was significantly higher glucose up taking and usage in orthotopic xenograft tumor compared to flank xenograft and cells in culture. The data demonstrated that glucose was the main carbon source for the glutamate, glutamine and glutathione synthesis through the TCA cycle. The glutaminase ii pathway was the main pathway utilizing glutamine in MYC-amplified medulloblastoma in vivo. Glutathione was found as the most abundant upregulated metabolite. Glutamine derived glutathione was mainly synthesized through glutamine transaminase K (GTK) enzyme in vivo. In conclusion, we demonstrated that high MYC medulloblastoma adapt to different environments by altering its metabolic pathways des-pite carrying the same genetic mutations. Glutamine antagonists may have therapeutic applications in human patients. Understanding the immune composition of a given tumor is critical to assess its potential responsiveness to cancer immunotherapy. This is especially true for tumors that are intrinsically resistant to immunotherapies, such as GBM. Unfor- tunately, studies on the functional heterogeneity and associated molecular tar-gets of immune-suppressive cells in vivo have been lacking. Here we report an integrated multi-dimensional analysis of the mutational profiles and single-cell transcriptomics of 60,024 glioma and stromal cells from 16 human samples. We identified molecular signatures of seven distinct macrophage subtypes, each with prognostic clinical value. The three inflammatory subtypes showed hallmarks of TNF/NF κ B pathway enrichment and are associated with good outcomes; in contrast, four immunosuppressive subtypes with metabolic pathway hallmarks (oxidative phosphorylation, PI3K/AKT/mTOR, fatty acid metabolism) are associated with poor survival. In addition, we resolved an ongoing controversy in the field regarding the roles of brain resident macrophages, microglia, vs. bone marrow derived macrophages (BMDM) in gliomas. Our data show compelling evidence that microglia are pro-inflammatory and are associated with good survival while BMDMs are mostly immune-suppressive and associated with poor survival. In addition, deciphering immune-suppressive macrophage and Treg molecular signatures enabled us to identify previously unknown immunotherapy targets. In a proof of principle study, we showed that S100A4, a calcium binding protein previously shown to mediate metastasis, was universally upregulated in both innate and adaptive immune suppressor cells, and implantation of gliomas in S100a4-/- host mice significantly extended survival and resulted in pro- inflammatory immune landscape, compared to same glioma cells implanted in B6 control hosts. This functional validation study shows that S100A4 is a highly promising therapeutic target for GBM immunotherapy. cellular invasion, which enables escape from resection and drives inevitable re- currence. Numerous factors have been proposed as the primary driving forces behind GBM’s ability to invade adjacent tissues rapidly, including alterations in the tumor’s cellular metabolism. Though studies have investigated links between GBM’s metabolic profile and its invasive capability, these studies have had two notable limitations. First, while infiltrating GBM cells extending be-yond the tumor edge utilize adaptive cellular machinery to overcome stressors in their microenvironment, these cells at the invasive front have not been the ones sampled in invasive studies, which have used cell lines or banked tumor tissue taken from the readily accessible tumor core. Second, studies of invasion have primarily used two-dimensional (2D) culture systems, which fail to capture the dimensionality, mechanics, and heterogeneity of GBM invasion. To address these limitations, our team has developed two parallel approaches: acquisition of site-directed biopsies from patient GBMs to define regional heterogeneity in invasiveness, and engineering of 3D platforms to study invasion in vitro. Through utilization of these platforms, and by taking advantage of the system-wide, unbiased screens of metabolite profile and gene expression available, our team looks to identify targetable metabolic factors which drive cellular invasion in GBM. Untargeted metabolomics revealed cystathionine to be selectively enriched in the invasive tumor front of both site directed biopsies (fold change 5.8), and 3D organoid models (fold change 14.2). RNA sequencing revealed 7/30(23%) metabolic genes upregulated in the invasive tumor front were involved in cysteine or glutathione metabolism. These re- sults highlight a clear role of the transsulfuration pathway in GBM invasion that our team looks to investigate with further targeted assays. BACKGROUND: The invasive behavior of glioblastoma is considered highly relevant for recurrence. However, the invasion zone is difficult to visu-alize, typically lies outside the resected and irradiated area, and is protected by the blood brain barrier, posing a particular challenge for treatment. We present biological features of invasive growth accompanying tumor pro- gression and invasion based on associated metabolic and transcriptomic changes in patient derived orthotopic xenografts (PDOX) and corres- ponding patients. METHODS: Patients with suspected glioblastoma were enrolled (NCT02904525) and underwent 1 H-MR spectroscopy and imaging ( 1 H-MRS/I, 7T). Tissue obtained at surgery was transplanted orthotopically into immune-compromised mice. Longitudinal follow-up was performed by 1 H-MRS/I (14.1T) on the injected and the contralateral side. The PDOX, the corresponding contralateral side, and the original human tumors under- went RNA-sequencing. RESULTS: The temporal changes of the metabolite profiles characterized the kinetics of invasive growth of PDOX, and were patient specific. Comparison of 1 H-MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel analyses of the patients’ tumors. Associations between the metabolite profiles and the combined transcriptome of the xenografts and the host, re-flected molecular signatures of invasion, comprising extracellular matrix deg- radation and reorganization, growth factor binding, and vascular remodeling. provides insights into the remodeling of the extracellular matrix that is essential for cell-cell communication and regulation of cellular processes. The changes of the structural and biochem- ical properties of the extracellular matrix are of importance for the biological behavior of the tumors and may be subjected to therapeutic targeting. potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs. Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT for- mation upon TGF-b stimulation. To determine downstream signaling me-diators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3. Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling. Glioblastoma multiforme (GBM) cells migrating in physically confined environments are affected by mechanical stress that potentially lead to transcriptomic changes. To simulate those stresses, microfluidic channels were made with micro-patterned polydimethylsiloxane (PDMS) replicating the physical microenvironment of white matter tracts by confining the cells in linear channels similar to the space between axons. We employed a com-bination of microarray transcriptomic profiling and single cell-sequencing analyses to investigate cells undergoing linear confined space migration (LCSM). GBM cells spontaneously migrate through confined spaces along 5x5 mm (height/width) microfluidic channels, 0.5 to 5 mm in length. Our previous studies demonstrated that cells migrating in LCSM are more re- sistant to treatment with temozolomide than the same cells growing in standard monolayer culture (SMC). Cells in confined migration evaluated by microarray-based transcriptomic profiling demonstrated that linear confined migration induces increased expression in pathways involving angio- genesis, cell adhesion, cell motility, DNA damage repair, extracellular matrix structure, HIF1 α , and others. Single cell transcriptomic analysis could iden- tify GBM cells in different migratory states (LCSM vs. SMC), and similar pathways were seen upregulated with additional changes in cholesterol biosynthesis pathways and cell cycle regulation pathways. Trajectory Infer-ence aligned single cells according to changes in migration status and dem- onstrated transcript changes during LCSM were progressive but generally reversible on return to SMC. Pathway analyses showed alterations in the cholesterol biosynthesis pathway and cell cycle regulation in cell clusters of confined migrating cells. Molecular studies confirmed that cholesterol biosynthesis pathway regulatory genes (SQLE, MVD, and HMGCR) are upregulated during LCSM. Expression analysis demonstrated increased G1 phase delay in confined migrating cells (LCSM) confirmed by FUCCI expression analysis. We propose that migration in linear confined spaces like white matter structures produces significant transcriptome changes that produce chemoresistance as a new mechanism for treatment resistance of Glioblastoma.

Reprograming of cellular metabolism is a hallmark of cancer. The metabolic alterations in cancer cells is not only defined by series of genetic mutations, but also reflecting the crosstalk between cancer cells and other factors in the microenvironment. Altering metabolism allows cancer cells to overcome unfavorable conditions, to proliferate and invade. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. However, the metabolism of high MYC amplified medulloblastoma subgroup remains underexplored. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in different environments -in vitro, in flank xenografts and in orthotopic xenografts. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and the high-MYC medulloblastoma cells in culture. Compared to normal brain, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of nucleotide, hexosamine biosynthetic pathway (HBP), TCA cycle, and amino acid and glutathione pathways. There was significantly higher glucose up taking and usage in orthotopic xenograft tumor compared to flank xenograft and cells in culture. The data demonstrated that glucose was the main carbon source for the glutamate, glutamine and glutathione synthesis through the TCA cycle. The glutaminase ii pathway was the main pathway utilizing glutamine in MYC-amplified medulloblastoma in vivo. Glutathione was found as the most abundant upregulated metabolite. Glutamine derived glutathione was mainly synthesized through glutamine transaminase K (GTK) enzyme in vivo. In conclusion, we demonstrated that high MYC medulloblastoma adapt to different environments by altering its metabolic pathways despite carrying the same genetic mutations. Glutamine antagonists may have therapeutic applications in human patients.

OTME-10. INTEGRATED ANALYSIS OF HUMAN GLIOMAS AT THE SINGLE CELL LEVEL IDENTIFIES S100A4 AS A NOVEL IMMUNOTHERAPY TARGET
Nourhan Abdelfattah 1 , Parveen Kumar 2 , Jia-Shiun Leu 1 , William Flynn 2 , David Baskin 1,3 , Kumar Ptichumani 1,3 , Omkar Ijare 1 , Joshy George 2 , Kyuson Yun 1,3 ; 1 Houston Methodist Research Institute, Houston, TX, USA. 2 The Jackson Laboratory, Farmington, CT, USA. 3 Weill Cornell Medical College, New York, NY, USA Understanding the immune composition of a given tumor is critical to assess its potential responsiveness to cancer immunotherapy. This is especially true for tumors that are intrinsically resistant to immunotherapies, such as GBM. Unfortunately, studies on the functional heterogeneity and associated molecular targets of immune-suppressive cells in vivo have been lacking. Here we report an integrated multi-dimensional analysis of the mutational profiles and single-cell transcriptomics of 60,024 glioma and stromal cells from 16 human samples. We identified molecular signatures of seven distinct macrophage subtypes, each with prognostic clinical value. The three inflammatory subtypes showed hallmarks of TNF/NFκB pathway enrichment and are associated with good outcomes; in contrast, four immunosuppressive subtypes with metabolic pathway hallmarks (oxidative phosphorylation, PI3K/AKT/mTOR, fatty acid metabolism) are associated with poor survival. In addition, we resolved an ongoing controversy in the field regarding the roles of brain resident macrophages, microglia, vs. bone marrow derived macrophages (BMDM) in gliomas. Our data show compelling evidence that microglia are pro-inflammatory and are associated with good survival while BMDMs are mostly immune-suppressive and associated with poor survival. In addition, deciphering immune-suppressive macrophage and Treg molecular signatures enabled us to identify previously unknown immunotherapy targets. In a proof of principle study, we showed that S100A4, a calcium binding protein previously shown to mediate metastasis, was universally upregulated in both innate and adaptive immune suppressor cells, and implantation of gliomas in S100a4-/-host mice significantly extended survival and resulted in proinflammatory immune landscape, compared to same glioma cells implanted in B6 control hosts. This functional validation study shows that S100A4 is a highly promising therapeutic target for GBM immunotherapy.

OTME-11. CHARACTERIZING THE IMMUNOLOGIC CONTEXT OF PEDIATRIC BRAIN TUMORS
Robert Galvin, Danielle Maeser, Robert Gruener, R. Stephanie Huang; University of Minnesota, Minneapolis, MN, USA Therapy for pediatric central nervous system (CNS) malignancies can be toxic, and outcomes are suboptimal. Immunotherapy holds promise as a therapeutic avenue, but the poorly understood microenvironment limits its application. The Children's Brain Tumor Network (CBTN) released the Pediatric Brain Tumor Atlas, containing expression profiles of nearly 700 primary CNS tumors. To study the immune microenvironment, a classification from The Cancer Genome Atlas project is applied. High-grade lesions are predominantly lymphocyte deplete (C4, 81%) or immunologically quiet (C5, 11%). Low-grade lesions are more mixed with 46% C4, 21% C5, and a higher proportion of inflammatory subtype (C3, 31%). For survival parameters, adjusting for tumor grade and extent of resection, the hazard ratio is 2.2 (0.78 -6.3), p = 0.13) and 2.4 (0.6 -10.0, p = 0.24) for C4 and C5, respectively. With no events among low-grade tumors, progression-free survival will be another useful metric and released by CBTN in April. Deconvolution of immune cell gene signatures among C4 samples reveals decreased abundance of T cells (OR 0.26, 0.1 -0.5) yet increasing T-cell abundance is associated with decreased survival time in high-grade samples (HR 3.7, 1.4 -10.1). Additionally, there are increased macrophage and decreased microglia signatures among high-grade samples and the C4 and C5 subtypes. It is hypothesized that expression of inhibitory immunomodulators contributes to a pro-tumorigenic microenvironment and represent potential therapeutic targets. In lieu of normal tissue in the data set, differential gene expression experiments between disease states reveals upregulated immunomodulators. Conventional immunomodulators, e.g. PDL1 and CTLA4, are expressed in low-grade samples with C3 subtype, which is abundant in craniopharyngioma. Alternative inhibitory immunomodulators, e.g. KDM1A, EZH2, CD276, are significantly expressed in high-grade samples including diffuse midline glioma. Overall, our analysis contributes to the understanding of the immune microenvironment and identifies potential mechanisms of immune escape among pediatric CNS tumors. ii16 NEURO-ONCOLOGY ADVANCES • JULY 2021 cellular invasion, which enables escape from resection and drives inevitable recurrence. Numerous factors have been proposed as the primary driving forces behind GBM's ability to invade adjacent tissues rapidly, including alterations in the tumor's cellular metabolism. Though studies have investigated links between GBM's metabolic profile and its invasive capability, these studies have had two notable limitations. First, while infiltrating GBM cells extending beyond the tumor edge utilize adaptive cellular machinery to overcome stressors in their microenvironment, these cells at the invasive front have not been the ones sampled in invasive studies, which have used cell lines or banked tumor tissue taken from the readily accessible tumor core. Second, studies of invasion have primarily used two-dimensional (2D) culture systems, which fail to capture the dimensionality, mechanics, and heterogeneity of GBM invasion.
To address these limitations, our team has developed two parallel approaches: acquisition of site-directed biopsies from patient GBMs to define regional heterogeneity in invasiveness, and engineering of 3D platforms to study invasion in vitro. Through utilization of these platforms, and by taking advantage of the system-wide, unbiased screens of metabolite profile and gene expression available, our team looks to identify targetable metabolic factors which drive cellular invasion in GBM. Untargeted metabolomics revealed cystathionine to be selectively enriched in the invasive tumor front of both site directed biopsies (fold change 5.8), and 3D organoid models (fold change 14.2). RNA sequencing revealed 7/30(23%) metabolic genes upregulated in the invasive tumor front were involved in cysteine or glutathione metabolism. These results highlight a clear role of the transsulfuration pathway in GBM invasion that our team looks to investigate with further targeted assays. BACKGROUND: The invasive behavior of glioblastoma is considered highly relevant for recurrence. However, the invasion zone is difficult to visualize, typically lies outside the resected and irradiated area, and is protected by the blood brain barrier, posing a particular challenge for treatment. We present biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes in patient derived orthotopic xenografts (PDOX) and corresponding patients. METHODS: Patients with suspected glioblastoma were enrolled (NCT02904525) and underwent 1 H-MR spectroscopy and imaging ( 1 H-MRS/I, 7T). Tissue obtained at surgery was transplanted orthotopically into immune-compromised mice. Longitudinal follow-up was performed by 1 H-MRS/I (14.1T) on the injected and the contralateral side. The PDOX, the corresponding contralateral side, and the original human tumors underwent RNA-sequencing. RESULTS: The temporal changes of the metabolite profiles characterized the kinetics of invasive growth of PDOX, and were patient specific. Comparison of 1 H-MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multivoxel analyses of the patients' tumors. Associations between the metabolite profiles and the combined transcriptome of the xenografts and the host, reflected molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. CONCLUSION: Integrating metabolic profiles and gene expression of highly invasive PDOX allows in vivo monitoring of progression in the nonenhancing tumor infiltration zone and provides insights into the remodeling of the extracellular matrix that is essential for cell-cell communication and regulation of cellular processes. The changes of the structural and biochemical properties of the extracellular matrix are of importance for the biological behavior of the tumors and may be subjected to therapeutic targeting.

OTME-13. INTEGRATION OF METABOLIC AND TRANSCRIPTIONAL SIGNATURES OF GLIOBLASTOMA INVASION REVEALS EXTRACELLULAR MATRIX REORGANIZATION AND VASCULATURE REMODELING
Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas. By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs. Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3. Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.

OTME-15. PHYSICAL CONFINEMENT INDUCES DIVERSE TRANSCRIPTOMIC CHANGES AND CHEMORESISTANCE IN MIGRATING GLIOBLASTOMA CELLS
James Battiste 1,2 , Anish Babu 1,3 , Rachel Sharp 1,4 , Sydney Scott 1,3 , Ian Dunn 1,2 , Chad Glenn 1,2 , Young-tae Kim 5 , Kenneth Jones 1,4 ; 1 OU Health Science Center, Oklahoma City, OK, USA. 2 Department of Neurosurgery, Oklahoma City, OK, USA. 3 Stephenson Cancer Center, Oklahoma City, OK, USA. 4 Department of Cell Biology, Oklahoma City, OK, USA. 5 UT Arlington, Arlington, TX, USA Glioblastoma multiforme (GBM) cells migrating in physically confined environments are affected by mechanical stress that potentially lead to transcriptomic changes. To simulate those stresses, microfluidic channels were made with micro-patterned polydimethylsiloxane (PDMS) replicating the physical microenvironment of white matter tracts by confining the cells in linear channels similar to the space between axons. We employed a combination of microarray transcriptomic profiling and single cell-sequencing analyses to investigate cells undergoing linear confined space migration (LCSM). GBM cells spontaneously migrate through confined spaces along 5x5 mm (height/width) microfluidic channels, 0.5 to 5 mm in length. Our previous studies demonstrated that cells migrating in LCSM are more resistant to treatment with temozolomide than the same cells growing in standard monolayer culture (SMC). Cells in confined migration evaluated by microarray-based transcriptomic profiling demonstrated that linear confined migration induces increased expression in pathways involving angiogenesis, cell adhesion, cell motility, DNA damage repair, extracellular matrix structure, HIF1α, and others. Single cell transcriptomic analysis could identify GBM cells in different migratory states (LCSM vs. SMC), and similar pathways were seen upregulated with additional changes in cholesterol biosynthesis pathways and cell cycle regulation pathways. Trajectory Inference aligned single cells according to changes in migration status and demonstrated transcript changes during LCSM were progressive but generally reversible on return to SMC. Pathway analyses showed alterations in the cholesterol biosynthesis pathway and cell cycle regulation in cell clusters of confined migrating cells. Molecular studies confirmed that cholesterol biosynthesis pathway regulatory genes (SQLE, MVD, and HMGCR) are upregulated during LCSM. Expression analysis demonstrated increased G1 phase delay in confined migrating cells (LCSM) confirmed by FUCCI expression analysis. We propose that migration in linear confined spaces like white matter structures produces significant transcriptome changes that produce chemoresistance as a new mechanism for treatment resistance of Glioblastoma.