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Gene expression profiling of 10 tumor samples from a GBM patient

ABSTRACT: Hypothetically the intratumor genomic heterogeneity has the potential to foster the tumor-infiltrating lymphocytes (TILs) diversity or activity, but no one has directly connected them by simultaneously investigating somatic mutations, TILs diversity and immune response activity. We have performed whole-exome sequencing, immune repertoire sequencing and gene expression profiling analysis simultaneously on ten spatially separated tumor samples obtained from two tumor bulks (the primary tumor and its metastatic offspring) of a glioblastoma multiforme (GBM) patient as well as his peripheral blood. We found that although the multi-region samples from one tumor shared more common mutations than those from different ones, the TIL populations did not. The TILs repertoire diversity was not significantly correlated with and the number of nonsynonymous mutations (Spearman's rank correlation, p = 0.4614), but it was highly related to the local immune status as it showed a significant positive correlation both with the local immune cytolytic activity and its down regulating factors. Furthermore, the tumor region having the highest TILs repertoire diversity was founded to show simultaneous local immune activation and suppression. A 20-gene signature was found to represent such contradictory local immune status. 50 GBM patients were stratified by the expression pattern of this signature in their tumors. Survival analysis suggested most of GBM patients (~80%) might be suffered from the simultaneous local immune activation and suppression status, which was unfavorable to patients’ prognoses (log-rank test, p = 0.012). This finding was validated when the similar analysis applied to the existing TCGA GBM dataset (log-rank test, p = 0.005). Through integration analysis of multi-omics data, we provided new insight into the complex relationship among the characteristics of TILs repertoire, somatic mutation pattern, and immune status.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83301 | GEO | 2017/06/13

SECONDARY ACCESSION(S): PRJNA325525

REPOSITORIES: GEO

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Project description:The interaction of neoplastic cells with their tumor microenvironment (TME) is required for cancer progression. However, in vitro cancer models, including recent in vitro 3-dimensional (3D) organoid cultures of primary human tumors, are typically comprised exclusively of neoplastic epithelium, with stromal and/or immune interactions requiring artificial reconstitution. As relevant to cancer immunotherapy, the unified co-culture of primary tumor epithelia with their endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has remained particularly elusive. Here, we used a single 3D air-liquid interface (ALI) methodology to successfully propagate 3D Patient-Derived Tumor Organoids (PDOs) as primary tumor epithelia together with native immune and myofibroblast stromal compartments without reconstitution. Derived from >100 diverse human surgically resected tumor samples or from murine tumors in syngeneic immunocompetent mice, PDOs preserved cancer histologic subtypes and mutational spectrum with endogenous T, B, NK cells and macrophages integrally embedded amidst the tumor epithelium. PDO-based TILs accurately recapitulated the T cell receptor (TCR) spectrum of the original tumors, as determined by a robust droplet-based immune profiling solution that links gene expression and immune repertoire in single cells. Anti-PD-1 or anti-PD-L1 treatment of organoids from murine tumors from syngeneic immunocompetent hosts induced activation and cytolytic activity of tumor antigen-specific TILs, indicating successful PDO modeling of immune checkpoint blockade (ICB) and anti-tumor immunity. Crucially, the anti-PD-1 antibody nivolumab activated TILs in PDOs from human lung, renal and melanoma clinical tumor resections. The organoid-based propagation of primary tumor epithelium en bloc with its endogenous immune stroma should facilitate mechanistic investigation of TME-specific local tumor immunity, with applications for functional testing of individualized patient immunotherapy responses.

Project description:Background: Anti-tumor immunity is highly heterogeneous between individuals; however, underlying mechanisms remain elusive, despite their potential to improve personalized cancer immunotherapy. Head and neck squamous cell carcinomas (HNSCCs) vary significantly in immune infiltration and therapeutic responses between patients, demanding a mouse model with appropriate heterogeneity to investigate mechanistic differences. Methods: We developed a unique HNSCC mouse model to investigate underlying mechanisms of heterogeneous anti-tumor immunity. This model system may provide a better control for tumor-intrinsic and host-genetic variables, thereby uncovering the contribution of the adaptive immunity to tumor eradication. We employed single-cell T-cell receptor (TCR) sequencing coupled with single-cell RNA sequencing to identify the difference in TCR repertoire of CD8 tumor infiltrating lymphocytes (TILs) and the unique activation states linked with different TCR clonotypes. Results: We discovered that genetically identical wild-type recipient mice responded heterogeneously to the same SCC tumors orthotopically transplanted into the buccal mucosa. While tumors initially grew in 100% of recipients and most developed aggressive tumors, ~25% of recipients reproducibly eradicated tumors without intervention. Heterogeneous anti-tumor responses were dependent on CD8 T cells. Consistently, CD8 TILs in regressing tumors were significantly increased and more activated. Single-cell TCR-sequencing revealed that CD8 TILs from both growing and regressing tumors displayed evidence of clonal expansion compared with splenic controls. However, top TCR clonotypes and TCR specificity groups appear to be mutually exclusive between regressing and growing TILs. Furthermore, many TCRa/TCRb sequences only occur in one recipient. By coupling single-cell transcriptomic analysis with unique TCR clonotypes, we found that top TCR clonotypes clustered in distinct activation states in regressing vs. growing TILs. Intriguingly, the few TCR clonotypes shared between regressors and progressors differed greatly in their activation states, suggesting a more dominant influence from tumor microenvironment than TCR itself on T cell activation status. Conclusions: We reveal that intrinsic differences in the TCR repertoire of TILs and their different transcriptional trajectories may underlie the heterogeneous anti-tumor immune responses in different hosts. We suggest that anti-tumor immune responses are highly individualized and different hosts employ different TCR specificities against the same tumors, which may have important implications for developing personalized cancer immunotherapy.

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Gene expression profiling of 10 tumor samples from a GBM patient

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