LGG-05. SINGLE-CELL RNA SEQUENCING OF PEDIATRIC LOW-GRADE GLIOMAS REVEALS INTRATUMORAL HETEROGENEITY

Abstract INTRODUCTION The glioblastoma microenvironment contains immune cells, particularly well-defined macrophage populations; however, intratumoral neutrophils and their effects on GBM biology are under-characterized. While tumor-associated neutrophils (TANs) were initially thought as passive bystanders due to their short-lived nature, current investigation of TANs in other cancer types revealed distinct pro-tumoral roles. Therefore, we sought to characterize TANs in the glioblastoma microenvironment and define their oncologic effects. METHODS Following informed consent, patient-derived GBM samples were collected for flow cytometry of TANs, which were then used to produce conditioned media (CM) for in Vitro studies on tumor cell proliferation and ELISA quantification of TAN-secreted factors. Single-cell RNA sequencing was performed on TANs to identify pro-tumoral factors. RESULTS Flow cytometric analysis (CD11b+/CD15+ /CD66b+) indicated higher percentages of TAN-infiltration to glioblastoma compared to low-grade gliomas (1.76% [n = 13] vs 0.33% [n = 6], P = .03). Using the Transwell migration assay with glioblastoma tumor CM, we found recruitment of circulating neutrophils to tumor sites is mediated by leukotriene-B4 and that this chemoattraction can be blocked with an LtB4 receptor antagonist, LY293111. We then performed single-cell RNA sequencing of isolated TANs and identified upregulation of osteopontin. Osteopontin is linked to GBM stem cell-like phenotype maintenance, thus, we decided to investigate osteopontin as a possible driver of pro-tumoral signaling. Osteopontin concentration was significantly higher in TAN CM than in patient-matched peripheral blood neutrophil CM (3.2 ng/mL [n = 3] vs 0.02 ng/mL [n = 3], P < .05). In Vitro, TAN CM led to significantly increased GBM cell proliferation and increased stem marker expression (Nanog, Oct4, Sox2) when incubated with neurospheres from an established GBM line. Pro-tumoral effects were lost in presence of osteopontin-neutralizing antibodies. CONCLUSION This study elucidates a possible mechanism of neutrophil-mediated pro-tumoral signaling. We found that neutrophils are recruited to tumor sites and play a biologically relevant role in GBM cellular proliferation and maintenance of a stem cell phenotype via osteopontin secretion.

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Abstract 4899: Investigating mutation co-operativity in early tumorigenesis of low-grade serous ovarian carcinoma with organoid model system and single-cell RNA sequencing

10.1158/1538-7445.am2020-4899 ◽

2020 ◽

Author(s):

Joyce Yu Han Zhang ◽

Dawn Cochrane ◽

Kieran Campbell ◽

Minh Bui ◽

Germain Ho ◽

...

Keyword(s):

Ovarian Carcinoma ◽

Single Cell ◽

Rna Sequencing ◽

Model System ◽

Low Grade ◽

Serous Ovarian Carcinoma ◽

Single Cell Rna Sequencing

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182 Single-cell RNA-sequencing reveals SCC intratumoral heterogeneity

Journal of Investigative Dermatology ◽

10.1016/j.jid.2018.03.187 ◽

2018 ◽

Vol 138 (5) ◽

pp. S31

Author(s):

A. Ji ◽

A. Rubin ◽

S. Hollmig ◽

S. Aasi ◽

P. Khavari

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Intratumoral Heterogeneity ◽

Single Cell Rna Sequencing

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Single cell RNA sequencing of upper tract urothelial carcinoma to reveal significant heterogeneity of the tumor and immune microenvironment.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.6_suppl.484 ◽

2021 ◽

Vol 39 (6_suppl) ◽

pp. 484-484

Author(s):

Peter Reisz ◽

Andrew Tracey ◽

Fengshen Kuo ◽

Jasmine Thomas ◽

Timothy Nguyen Clinton ◽

...

Keyword(s):

Urothelial Carcinoma ◽

Single Cell ◽

Rna Sequencing ◽

Immune Cell ◽

Marker Genes ◽

Low Grade ◽

Significant Heterogeneity ◽

High Grade ◽

Single Cell Rna Sequencing ◽

Tissue Specimens

484 Background: Upper tract urothelial carcinoma (UTUC) comprises 5-10% of urothelial malignancies but demonstrates unique clinical and molecular characteristics compared to urothelial carcinoma of the bladder. Prior investigations have used bulk profiling of tumor tissue to identify molecular subtypes, classifying the majority of UTUC as luminal and T-cell depleted. However, bulk sequencing does not allow for analysis of the significant heterogeneity known to be present in urothelial tumors. Single-cell RNA sequencing (scRNA-seq) allows examination of intra-tumoral heterogeneity, clonality, and the complex interactions of the immune tumor microenvironment (TME). We sought to apply this technology to better characterize UTUC and the TME. Methods: Single cell RNA sequencing (scRNA-seq) was performed on nine UTUC tissue specimens from six different patients collected fresh via ureteroscopic biopsy using an established institutional process and the 10X Genomics platform. Sequencing reads were normalized and analyzed using R/Seurat package. We assessed the composition of each tumor specimen with known marker genes for molecular subtypes (luminal, basal, squamous, EMT, and claudin-low). We then assessed the composition of immune cells in each specimen using known marker genes. We compared high- and low-grade specimens by subtype composition and immune cell infiltrates. Results: Lineage density analyses demonstrate the intra- and inter-tumoral heterogeneity of the nine endoscopic samples analyzed by molecular subtype composition. There is higher expression of luminal and claudin-low subtypes across all samples. The high-grade samples have higher expression of squamous markers. There is significant heterogeneity of immune cell infiltrates in seven specimens (two specimens were excluded due to low CD45+ cell counts). There is higher macrophage infiltration in high-grade samples, which was the only significant difference (Wilcoxon two-sided p-value = 0.05). Conclusions: This is the first known study using scRNA-seq expression analysis to characterize the notable heterogeneity of high and low-grade UTUC and the associated TME. Lineage density analysis demonstrates high luminal gene expression across samples, which has been demonstrated on prior bulk sequencing studies. The immune TME is also heterogeneous, with notable increased infiltration of macrophages in high-grade disease. There are unique limitations to performing and analyzing scRNA-seq of fresh UTUC tissue specimens, thus data should be interpreted cautiously. However, this study demonstrates the marked heterogeneity of UTUC tumors and frames our current approaches to bulk molecular subtyping of urothelial cancers and immune deconvolution. Further high-resolution studies are needed to characterize UTUC and inform bulk-sequencing efforts.

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