GSM8302400: PAM007, CD8 T cells, scTCRseq; Homo sapiens; OTHER - SRA

SRX24781436: GSM8302400: PAM007, CD8 T cells, scTCRseq; Homo sapiens; OTHER
1 ILLUMINA (NextSeq 550) run: 34M spots, 4.9G bases, 1.8Gb downloads

External Id: GSM8302400_r1

Submitted by: Immuno-Oncology, Obstetrics&Gynecology, University Medical Center Groningen

Study: Neoadjuvant immune checkpoint blockade in women with mismatch repair deficient endometrial cancer: a phase I study

PRJNA1119430 • SRP511345 • All experiments • All runs

show Abstracthide Abstract

Neoadjuvant immune checkpoint blockade (ICB) has shown unprecedented activity in mismatch repair deficient (MMRd) colorectal cancers, but its effectiveness in MMRd endometrial cancer (EC) remains unknown. In this investigator-driven, phase I, feasibility study (NCT04262089), 10 women with MMRd EC of any grade, planned for primary surgery, received two cycles of neoadjuvant pembrolizumab (200 mg IV) every three weeks. All patients completed treatment without severe toxicity. A partial radiologic response was observed in 3/10 patients, 5/10 patients had stable disease and 2/10 patients were non-evaluable on Magnetic Resonance Imaging (MRI). Pathologic response was observed in 5/10 patients, with 2 patients showing a major pathologic response. No patient achieved a complete pathologic response. At median duration of follow-up of 22.5 months, two non-responders experienced disease recurrence. Monoclonal T cell expansion significantly correlated with treatment response. Tumour-draining lymph nodes displayed clonal overlap with intra-tumoural T cell expansion. All pre-specified endpoints were reached. Neoadjuvant ICB with pembrolizumab proved safe and induced pathologic, radiologic, and immunologic responses in MMRd EC, warranting further exploration of extended neoadjuvant treatment. Overall design: Intratumoral T cells of 3 patients following neoadjuvant treatment were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or abscence of CD45, CD3, CD4, CD8, CD19, CD14, CD56, EpCAM, CXCR5 and PD1 and analyzed using scRNAseq and scTCRseq.

Sample: PAM007, CD8 T cells, scTCRseq

SAMN41656133 • SRS21499365 • All experiments • All runs

Organism: Homo sapiens

Library:

Name: GSM8302400

Instrument: NextSeq 550

Strategy: OTHER

Source: OTHER

Selection: other

Layout: PAIRED

Construction protocol: Tumour digests were thawed in FCS and resuspended in RPMI + 10% FCS. After 1.5h incubation at 37°C/5% CO2, samples were stained and incubated with a CXCR5 PerCP-Cy5.5 antibody for an additional 0.5h at 37°C. After a total of 2h recovery at 37°C/5% CO2, samples were centrifuged and resuspended in 100 µl PBS + 2% FCS. Samples were incubated for 30-45 minutes on ice (in dark) with the following antibodies: CD45 BV605, CD8 APC Cy7, CD4 PE, CD19 BV421, PD1 APC, CD3 PE-Cy7 and the dump channel antibodies CD14 FITC, CD56 FITC and EpCAM FITC. 3 µl of each antibody was used per 1x10e6 cells. Samples were washed twice with PBS + 2% FCS and filtered using a 35 µm strainer (Falcon) before sorting on a Beckman Coulter MoFlo Astrios cytometer. UltraComp eBeads (Thermo Fisher Scientific) were used as compensation controls. Before sorting, Propidium Iodide was added to exclude dead cells. Sorting was done in 1.5 mL Low Bind DNA tubes (Eppendorf) in 150 µl PBS 0.04 % BSA. CD45+ CD3+ CD19- CD8+ and CD4+ cells were sorted. Sorted cells were centrifuged and resuspended at a concentration of 727 cells/µl (total 16.5 µl) for PAM-007 or 2121 cells/µl (total 16.5 µl) for PAM-010 and PAM-011, in PBS 0.04% BSA. Library was performed according to manufacturer's protocol (10x genomics): 'chromium next GEM single cell 5' reagents kit v2 (dual index)'. For the cDNA amplification step, 13 cycli were done. For each sample, both the V(D)J library as well as the gene expression (GEX) library was constructed. All cDNA quality control and quantification steps were done with the Qubit 4 Fluorometer (Thermo Fisher) and the Agilent 4200 Tapestation (with D5000 and high sensitivity D5000 screen tapes). For the sequencing, V(D)J and GEX libraries were pooled at a 1:4 molar ratio, with 2 GEX libraries and 2 V(D)J libraries in one pool. 1.5 pM of this superpool was sequenced on a NextSeq 500 (Illumina inc., San Diego, CA, USA) A 150 bp NextSeq high-output reagent kit was used for the sequencing (paired end).

Runs: 1 run, 34M spots, 4.9G bases, 1.8Gb

Run	# of Spots	# of Bases	Size	Published
SRR29263787	33,998,421	4.9G	1.8Gb	2024-07-19

ID:: 33106774

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