38 Spatial immune profiling of human glioblastoma tissue reveals the presence of aggregated lymphoid niches in the tumor microenvironment

BackgroundGlioblastomas (GBM) account for 60% of adult primary brain tumors. With few advances in therapeutics, median overall survival remains 15-months post-diagnosis. Immunotherapies may provide therapeutic benefit in GBM patients; however, no predictive immune features currently inform therapeutic stratification in GBM. We have shown that, independently of known prognosticators, radiographic tumor contact with the lateral ventricle (C-GBM) correlates with 7-months worse survival prognosis compared to patients with ventricle non-contacting GBM (NC-GBM). This study sought to characterize the GBM immune microenvironment and identify targetable mechanisms of immunosuppression correlating with worse outcomes in C-GBM.MethodsTwelve patients presented with pathologically confirmed primary, IDH wildtype C-GBM and thirteen with NC-GBM. Multiplex immunohistochemistry (mxIHC) was performed on formalin-fixed paraffin embedded (FFPE) tissue for each patient interrogating 8 predictive immune markers (CD3, CD4, CD8, FOXP3, CD68, IBA1, PD-1, and PD-L1). Machine learning tools characterized tumor-infiltrating immune populations and identified biomarkers correlating with C-GBM and patient survival. K-means clustering identified immunological neighborhoods within the tissue and a log odds ratio was used to quantify the likelihood of cell-cell interactions in the tissue.ResultsC-GBM tumors were enriched in monocyte-derived macrophages (MDM) compared to NC-GBM (19 ± 8% vs. 6 ± 2%; p<0.001) and depleted in lymphocytes (2.9 ± 1% vs. 7.6 ± 2%; p<0.001) and tissue-resident microglia (1.8 ± 0.3% vs. 7 ± 3%; p<0.001). Further, T cells in C-GBM co-expressed the checkpoint receptors PD-1, suggesting T cell exhaustion in the C-GBM tumor microenvironment. K-means clustering identified 10 immunological niches prevalent in GBM tissue. Macrophage-tumor niches were most common niche in the tissue accounting for 17.93% of all niches, followed by T cell-microglia-tumor niches (17.72%). Conversely, tumor-tumor niches were the least prevalent, accounting for only 2.51% of niches. Within niches, T cell-T cell interactions occurred more frequently than expected by random chance (log odds ratio = 0.90) whereas T cell-macrophage interactions occurred less frequently than expected by random chance (log odds ratio = -1.61). Pathological assessment of the tissue confirmed the presence of lymphoid aggregates in regions of myeloid exclusion in the tissue.ConclusionsThese findings suggest that factors within the periventricular space may influence antitumor immunity within GBM, and have identified clinically targetable immune biomarkers in glioblastoma. The prevalence of T cell niches in GBM tumors suggests the establishment tertiary lymphoid aggregates may be targetable to improve patient outcomes. Lastly, radiologic assessment of lateral ventricle contact by standard-of-care MRI may guide clinical trial design for immunotherapies in neuro-oncology.AcknowledgementsThis study was funded by NIH/NCI grant K00 CA212447 and supported by the Translational Pathology Shared Resource at Vanderbilt University (P30 CA068485).Ethics ApprovalPrimary glioblastoma tumors obtained in accordance with the Declaration of Helsinki and with institutional IRB approval (#131870) along with patient written informed consent.

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IMMU-11. SELECTIVE ENRICHMENT OF SUPPRESSED IMMUNE CELLS IN THE TUMOR MICROENVIRONMENT CORRELATES WITH ESTABLISHMENT OF DISTINCT IMMUNOLOGIC NICHES IN HUMAN GLIOBLASTOMAS CONTACTING THE LATERAL VENTRICLE

Neuro-Oncology ◽

10.1093/neuonc/noab196.370 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi94-vi94

Author(s):

Todd Bartkowiak ◽

Asa Brockman ◽

Bret Mobley ◽

Akshitkumar Mistry ◽

Sierra Barone ◽

...

Keyword(s):

T Cell ◽

Lateral Ventricle ◽

Odds Ratio ◽

Clinical Trial Design ◽

Standard Of Care ◽

Therapeutic Benefit ◽

Primary Glioblastoma ◽

Selective Enrichment ◽

Immune Biomarkers ◽

Log Odds

Abstract Glioblastomas (GBM) account for 60% of adult primary brain tumors. With few advances in therapeutics, median overall survival remains 15-months post-diagnosis. Immunotherapies may provide therapeutic benefit in GBM patients; however, no predictive immune features currently inform therapeutic stratification in GBM. We have shown that, independently of known prognosticators, radiographic tumor contact with the lateral ventricle (C-GBM) correlates with 7-months worse prognosis compared to patients with ventricle non-contacting GBM (NC-GBM). This study sought to characterize the GBM immune microenvironment and identify targetable mechanisms of immunosuppression correlating with worse outcomes in C-GBM. Primary glioblastoma specimens were resected in accordance with the Declaration of Helsinki (IRB #131870). Twelve patients presented with C-GBM and thirteen with NC-GBM. Machine learning tools applied to mass cytometry data characterized tumor-infiltrating immune populations and identified biomarkers correlating with C-GBM and patient survival. C-GBM tumors were enriched in blood-derived macrophages compared to NC-GBM (19 ± 8% vs. 6 ± 2%; p< 0.001) and depleted in lymphocytes (2.9 ± 1% vs. 7.6 ± 2%; p< 0.001) and tissue-resident microglia (1.8 ± 0.3% vs. 7 ± 3%; p< 0.001). Further, T cells in C-GBM co-expressed the checkpoint receptors PD-1 and TIGIT, suggesting acute T cell exhaustion. Multiplex immunohistochemistry (mxIHC) on matched FFPE tissue provided spatial context to risk-stratifying immune populations, and defined structured immunological niches within the TME. Macrophage-tumor niches were most common (36%), followed by T cell-microglia-tumor niches (26%). Within niches, T cell-T cell interactions were more prevalent (log odds ratio = 0.90) whereas T cell-macrophage interactions were less prevalent (log odds ratio = -1.61). These findings suggest that factors within the periventricular space may influence antitumor immunity within tumors, and identify clinically targetable immune biomarkers in glioblastoma. Notably, radiologic assessment of lateral ventricle contact by standard-of-care MRI may guide clinical trial design for immunotherapies in neuro-oncology.

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The Tim3-galectin-9 interactions in the tumor microenvironment of nasopharyngeal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.2629 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 2629-2629

Author(s):

Dora Lai Wan Kwong ◽

Ngar-Woon Kam ◽

Wai Chun Tse ◽

Sing Hei Lok ◽

George Tsao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Tumor Cells ◽

Cell Lines ◽

Nasopharyngeal Cancer ◽

Cell Interactions ◽

Cell Contact ◽

Potential Marker ◽

Significant Difference

2629 Background: The complex cell interactions within the tumor microenvironment (TME) have become a crucial point in cancer research. Yet, the cell interactions might not only depend on the frequency of immune cells, but also on the inter-individual distances as cells might interact via soluble factors and/or cell-cell contact. Accordingly, the mapping of TME has recently gained importance. The aim of this study is to investigate the alternations between galectin-9 (G9) and its natural immunosuppressive receptor, T cell immunoglobulin and mucin domain 3 (Tim3) in nasopharyngeal cancer (NPC). Methods: Using multiplexed quantitative immunofluorescence, we measured the levels of G9 and Tim3 in 95 NPC patients cancerous and 8 normal specimens in tissue microarray format. Cell densities and cell-to-cell distances were quantified. The interaction between G9-expressing tumor cell lines and T cells were also studied. Results: G9-expressing tumor cells were detected in all NPC cases and were significantly higher than normal tissue. Elevated G9 was associated with shorter overall survival (OS: 89% vs 70.5% at 7 years, p: 0.019). Incremental percentages of Tim3+ cells were shown in top 10% cases strongly positive for G9-expressing tumor cells. The number of Tim3+ cells was calculated at 15µm intervals from the nearest G9-expressing tumor cells, of which a significant difference of Tim3+ cells was observed at the 0-15µm distance from G9-expressing cell in cancerous compared to normal tissues. Epithelial short distances were associated with a unfavourable prognosis. Observed short distance were hypothesized to represent Tim3+ cells actively interacting with G9-expressing tumor cells. Accordingly, In vitro cocultured of G9-ovexpressing NPC cell lines induced Tim3 expression on T cells which suppressed the T-cell mediate cytotoxicity on tumor cells. Conclusions: Our findings indicate a specific preexisting profile of Tim3+ and G9-expressing tumor cells and demonstrated that Tim3+ cells were mainly found intratumorally within 15µm of a NPC cell. The relevance of Tim3+ and G9+ distances reflect a potential marker of their functional interaction. Our results could have important implications for clinical therapeutic strategies. Since high G9 expression have poorer OS, they would deserve a different therapeutic strategy.[Table: see text]

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Single-Cell Transcriptomics Reveals the Complexity of the Tumor Microenvironment of Treatment-Naive Osteosarcoma

Frontiers in Oncology ◽

10.3389/fonc.2021.709210 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yun Liu ◽

Wenyu Feng ◽

Yan Dai ◽

Mengying Bao ◽

Zhenchao Yuan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Single Cell ◽

Cell Types ◽

Survival Prognosis ◽

Accurate Treatment ◽

Depth Study ◽

Treatment Naïve

Osteosarcoma (OS), which occurs most commonly in adolescents, is associated with a high degree of malignancy and poor prognosis. In order to develop an accurate treatment for OS, a deeper understanding of its complex tumor microenvironment (TME) is required. In the present study, tissues were isolated from six patients with OS, and then subjected to single-cell RNA sequencing (scRNA-seq) using a 10× Genomics platform. Multiplex immunofluorescence staining was subsequently used to validate the subsets identified by scRNA-seq. ScRNA-seq of six patients with OS was performed prior to neoadjuvant chemotherapy, and data were obtained on 29,278 cells. A total of nine major cell types were identified, and the single-cell transcriptional map of OS was subsequently revealed. Identified osteoblastic OS cells were divided into five subsets, and the subsets of those osteoblastic OS cells with significant prognostic correlation were determined using a deconvolution algorithm. Thereby, different transcription patterns in the cellular subtypes of osteoblastic OS cells were reported, and key transcription factors associated with survival prognosis were identified. Furthermore, the regulation of osteolysis by osteoblastic OS cells via receptor activator of nuclear factor kappa-B ligand was revealed. Furthermore, the role of osteoblastic OS cells in regulating angiogenesis through vascular endothelial growth factor-A was revealed. C3_TXNIP+ macrophages and C5_IFIT1+ macrophages were found to regulate regulatory T cells and participate in CD8+ T cell exhaustion, illustrating the possibility of immunotherapy that could target CD8+ T cells and macrophages. Our findings here show that the role of C1_osteoblastic OS cells in OS is to promote osteolysis and angiogenesis, and this is associated with survival prognosis. In addition, T cell depletion is an important feature of OS. More importantly, the present study provided a valuable resource for the in-depth study of the heterogeneity of the OS TME.

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