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Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Despite their identification in some cancers, pro-tumoral cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of primary glioblastoma cultures yielded cells with CAF morphology, CAF transcriptomic profile, and mesenchymal lineage in single-cell RNA-seq. Glioblastoma CAFs were attracted to glioblastoma stem cells (GSCs) and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF-β and TGF-β as mediators of GSC effects on CAFs, and osteopontin and hepatocyte growth factor as mediators of CAF-induced GSC enrichment. Glioblastoma CAFs also induced M2 macrophage polarization by producing the EDA fibronectin variant. Glioblastoma CAFs were enriched in the subventricular zone which houses neural stem cells that produce GSCs. Including CAFs in GSC-derived xenografts induced in vivo growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Cancer-associated fibroblasts (CAFs) constitute a key component of the tumor microenvironment. While pro-tumoral CAFs have been identified in some cancers, CAFs had been presumed absent in glioblastoma given the lack of brain fibroblasts. We found that serial trypsinization of primary glioblastoma cultures yields cells that morphologically resemble fibroblasts and transcriptomically resemble CAFs as shown by bulk RNA-seq and single-cell RNA-seq. Moreover, Single-cell RNA-seq from patient GBMs showed a mesenchymal lineage for CAFs. We demonstrate that Glioblastoma CAFs are chemotactically attracted to glioblastoma stem cells (GSCs) and CAFs enriched GSCs. To identify CAF/GSC interaction mediators, we created a resource of inferred crosstalk by mapping the expression of receptors to their cognate ligands/agonists. This analysis suggested PDGF-b and TGF-b as mediators of GSC recruitment and proliferation of CAFs, and osteopontin and hepatocyte growth factor (HGF) as mediators of CAF-induced GSC enrichment, hypothesis confirmed by blocking antibodies. Glioblastoma CAFs also induce hypertrophied vessels and M2 macrophage polarization, the latter through unique CAF production of the EDA fibronectin variant which binds macrophage toll-like receptor 4 (TLR4) in a targetable manner. Glioblastoma CAFs were enriched in the subventricular zone which houses the neural stem cells that produce GSCs. Depleting CAFs in GSC-derived xenografts slowed their in vivo growth. These findings are among the first to identify glioblastoma CAFs and reveal their involvement with GSCs, making them an intriguing target.

Among clinical analyses, elevated neutrophil-lymphocyte ratio has been correlated with poor outcomes of glioblastoma patients independent of other prognostic factors. Additionally, our flow cytometric studies of primary patient samples found neutrophil percentage to be significantly higher in higher-grade glioma versus lower-grade glioma. Tumor-associated neutrophils (TANs) comprise less than 2% of the glioblastoma microenvironment. While TANs were initially considered passive bystanders due to their short-lived nature, investigation of TANs in other cancers revealed distinct pro-tumoral roles. Therefore, we transcriptomically characterized glioblastoma TANs and defined their oncologic effects. Transcriptomic analysis of patient-matched TANs versus peripheral blood neutrophils revealed that functionally quiescent circulating neutrophils infiltrate IDH1-wild type glioblastoma via leukotriene B4 chemoattraction, where tumor cells morphologically and transcriptomically activate them to become TANs. Single-cell RNA-sequencing of patient-matched TANs and peripheral blood neutrophils revealed a subset of tumor-activated neutrophils which adopt a pro-tumoral secretory phenotype, marked by activation of the IL-17 signaling pathway and high osteopontin production. Using immunofluorescence stains of primary patient glioblastoma sections, we demonstrated that activated, myeloperoxidase-positive TANs reside in the perivascular niche of glioblastoma in close proximity to glioblastoma stem-like cells (GSCs) and CD31-positive endothelial cells. Further analysis in culture demonstrated that TAN-secreted osteopontin drives the formation, self-renewal, and proliferation of GSC-containing neurospheres. These results were validated using a syngeneic stem cell-derived IDH1-wild type murine glioblastoma model in vivo. Thus, while TANs are rare in glioblastoma, their enrichment in the glioblastoma perivascular niche uniquely positions them to support the GSCs that are crucial to therapeutic resistance of GBM.

Glioblastoma is a primary malignant brain tumor with a median survival under two years. The poor prognosis glioblastoma caries is largely due to cellular invasion, which enables escape from resection and drives inevitable recurrence. While numerous factors have been proposed as the primary driving forces behind glioblastoma’s ability to invade adjacent tissues rapidly, little attention has been paid to the alterations in tumor cell metabolism needed for tumor cells to thrive in isolation in the peritumoral white matter. To improve on biased 2D cell culture studies, we defined the links between glioblastoma metabolism in invasion using unbiased CRISPR screens and metabolomics performed in biomimetic 3D hydrogels and regional biopsies of patient glioblastomas. Through these platforms, we identified targetable metabolic factors which drive cellular invasion in glioblastoma. Metabolomics revealed cystathionine to be selectively enriched in the invasive tumor front of both site-directed biopsies (6-fold change), and 3D organoid models (14-fold change). 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 glioblastoma invasion, revealing a targetable alteration unique to invading glioblastoma cells.

Glioblastoma (GBM) is a primary malignant brain tumor with a median survival under two years. The poor prognosis GBM caries is largely due to 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 its cellular metabolism. Though studies have investigated links between GBM’s metabolic profile and its invasive capabilities, these studies have had two notable limitations. First, while infiltrating GBM cells utilize adaptive cellular machinery to overcome stressors in their microenvironment, the cells at the invasive tumor front have rarely been sampled in previous studies, which have primarily used banked 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 developed two complementary approaches: acquisition of site-directed biopsies from patient GBMs to define regional heterogeneity in invasiveness, and engineering of three dimensional (3D) platforms to study invasion in culture. Through utilization of these platforms, and by taking advantage of the system-wide, unbiased screens of metabolite profile and gene expression available, our team looked to accomplish the goal of identifying targetable metabolic factors which drive cellular invasion in GBM. Pilot RNA-Sequencing data revealed 87 of the top 250 (35%) genes preferentially expressed in the tumor invasive edge, and 30 of the top 250 (12%) genes preferentially expressed in the tumor core were involved in cellular metabolism. KEGG pathways analysis demonstrated enrichment of glycolytic, pentose phosphate, and response to amino acid starvation pathways at the tumor invasive edge. These preliminary studies demonstrate a distinct metabolic phenotype in invasive GBM cells which will be further explored with system wide screens.