Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNAseq of tumor samples from CT26 syngeneic mouse treated with a PI3Ka/d inhibitor (AZD8835)

ABSTRACT: PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kdelta modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. While PI3K inhibitors as a class and PI3Ka/d specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835 a dual PI3Ka/d inhibitor specifically on the tumor immune microenvironment using syngeneic CT26 mice. CT-26 (5x10^6 cells/mouse) tumor cells were implanted subcutaneously (s.c.) in the left flank of female Balb/c and C57/Bl6 mice, respectively. AZD8835 was dosed at 50mg/kg twice daily in at 2days on/ 5 days off schedule for times indicated in figures or 25mg/kg BID daily. At end of study tumor tissues were then transferred into the gentleMACS C Tube containing RPMI. Tumor samples were processed using the mouse tumor dissociation kit from Miltenyi Biotec. Cells were liberated from tumors for downstream application using a mouse tumor dissociation kit and octodissociator (Miltenyi) according to manufacturer’s instructions. For RNA sequencing, total RNA was extracted using the RNeasy 96 Qiacube HT Kit (Qiagen), quality validated using nanodrop and Quantit RNA Assay Kit (Thermo Fisher), and submitted for TrueSeq Stranded mRNA library preparation, following the manufacturer’s instructions (Illumina). Resulting libraries were sequenced on the HiSeq4000 System.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Anna Coenen-Stass

PROVIDER: E-MTAB-7386 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

PI3Kα/δ inhibition promotes anti-tumor immunity through direct enhancement of effector CD8<sup>+</sup> T-cell activity.

Carnevalli Larissa S LS Sinclair Charles C Taylor Molly A MA Gutierrez Pablo Morentin PM Langdon Sophie S Coenen-Stass Anna M L AML Mooney Lorraine L Hughes Adina A Jarvis Laura L Staniszewska Anna A Crafter Claire C Sidders Ben B Hardaker Elizabeth E Hudson Kevin K Barry Simon T ST

Journal for immunotherapy of cancer 20181227 1

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibi ...[more]

PMID: 30587236

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Project description:We investigated the frequency of CX3CR1+CD8+ T cells in peripheral blood (PB) before and during immune checkpoint inhibitor (ICI) therapy, and delineated the TCR repertoire in peripheral CX3CR1+CD8+ T-cell subsets and CD8+ tumor-infiltrating lymphocytes (TILs) using preclinical models. To understand the clinical utility of CX3CR1 as a circulating T-cell biomarker, we analyzed longitudinal PB samples from non-small cell lung cancer (NSCLC) patients undergoing anti-PD-1 therapy, and evaluated predictive and prognostic value of changes in the frequency of PB CX3CR1+ CD8+ T cells Methods: Female C57BL/6 mice were inoculated subcutaneously with 5 x 10^5 MC38 tumor cells per mouse on the right flank on day 0. When tumor volume reached approximately 50 mm^3, 200 µg of anti-PD-L1 Ab (clone 10F.9G2, BioXcell) and 100 µg of anti-CTLA-4 Ab (clone 9H10, BioXcell) were administered intraperitoneally every 3 days and every other day, respectively. MC38 tumor-bearing mice were treated for 14 days, and spleens and tumors were harvested. [Tumor cells] Tumors were cut into small pieces of 2-4 mm. Single-cell suspensions were obtained by mechanical dispersion consisting of two 30-min incubations at 37°C, 5% CO2 in 5 ml RPMI 1640 (Gibco) and tumor dissociation kit (Miltenyi Biotec) in C Tubes (Miltenyi Biotec) interspersed with three mechanical dispersions on a GentleMACS dissociator (Miltenyi Biotec). The tumor cell suspensions were then filtered through a cell strainer (70 μm; BD Biosciences). An EasySep Mouse CD8a Positive Selection Kit II (STEMCELL Technologies) was used to isolate murine CD8+ tumor-infiltrating lymphocytes (TILs) for TCR sequencing. [Splenocytes] Spleens were homogenized by forcing the tissue through a cell strainer (70 μm; BD Biosciences). Red blood cells in blood and spleen were lysed using ACK Lysis Buffer (Gibco). For TCR sequencing of murine splenic CD8+ T cells, single cell suspensions from freshly isolated splenocytes were stained. CD45+ CD3+ CD8+ T cells were gated, and CD27lo CX3CR1-, CD27hi CX3CR1-, and CX3CR1+ CD8+ T cells were sorted using BD FACSAria II Cell Sorter. DNA from flow-isolated murine splenic CD8+ T cells and CD8+ TILs, and PB CD8+ T cells was extracted using QIAamp DNA Micro Kit (QIAGEN). DNA was quantified using Qubit dsDNA BR Assay (Invitrogen). Amplification and sequencing of TCRβ CDR3 regions was performed using ImmunoSEQ immune profiling system at the survey level (Adaptive Biotechnologies). Sequencing was performed on an Illumina NextSeq system using 150 cycle mid-output kit (Illumina Inc.). Processed data were uploaded to the ImmunoSEQ Platform (Adaptive Biotechnologies) for bioinformatics analysis. Processed data were downloaded and frequencies/counts for TCR clonotypes and diversity were examined by nucleotide sequences after non-productive reads were filtered out. Results: Clonally expanded TCR repertoires of CD8+ TILs are enriched in the peripheral CX3CR1+ subset during ICI therapy. ICI therapy induces high degree of TCR sequence similarity and clonality between CD8+ TILs and the peripheral CX3CR1+ CD8+ T cells.

Dataset Information

RNAseq of tumor samples from CT26 syngeneic mouse treated with a PI3Ka/d inhibitor (AZD8835)

Publications

PI3Kα/δ inhibition promotes anti-tumor immunity through direct enhancement of effector CD8<sup>+</sup> T-cell activity.

Similar Datasets

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