Project description:Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a known anti-tumor immunity marker in myeloid cell subpopulations. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II LGGs. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

Project description:Therapeutic screening of potential anticancer agents relies on representative cancer models. In vitro cell culture models have been long questioned to be representative for human malignant glioma. Therefore, in the present study genomic profiles of both short-term (2 weeks; n=8) and long-term (6 and 12 weeks; n=3) primary cell cultures and spheroid cultures were compared with their parental malignant gliomas. Cancer genomic profiles were obtained by 6400 BAC array comparative genomic hybridization. In 7 out of 8 short-term primary cell cultures, the genomic profiles clustered further from their parental tumors than the spheroid cultures. In 4 out of 8 samples, the changes were substantial and included chromosomal regions associated with prognosis and therapeutic response. The average correlation coefficients between parental tumor profiles and spheroid profiles was 0.89 (range: 0.79 to 0.97), whereas those between parental tumors and cell cultures was 0.62 (range: 0.10 to 0.96). In 2 out of 3 primary cell cultures progressive genetic changes appeared at 6 and 12 weeks after initial preservation, whereas the spheroid cultures were genetically stable throughout. It is concluded that the cancer genomic profile of primary cell cultures from malignant glioma is inconsistent with the parental tumorâs profile already after 2 weeks with subsequent progressive genetic changes. Because malignant glioma spheroids are genetically stable, biological characteristics of the parental tumor are better reflected. This indicates that the spheroid model is better for therapeutic screening. Keywords: comparative genomic hybridization Two in vitro culture models (primary cell culture and organotypic spheroid culture) and their parental tumor were compared in whole-genome DNA copy number profile. Malignant glioma surgical specimens from 8 patients were divided in parental tumor (T), primary cell culture (C) and organotypic spheroid culture (S). After 2 weeks, genomic DNA was extracted from culture harvests. For 3 of 8 surgical specimens (patient 55, 58, 60) cultures were extended to 6 and 12 weeks to determine DNA copy number changes in time. Primary cell culture harvests at 2, 6 and 12 weeks were named C1, C2, C3 and organotypic spheroid cultures harvests at 2, 6 and 12 weeks were named S1, S2, S3.

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Modeling Malignant Progression in Glioma

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