biostudies-arrayexpressKlas HatjeMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15144We used transgenic mice challenged subcutaneously with Panc02-H7-Fluc tumors to compare the efficacy of tobemstomig with nivolumab and relatlimab-like monotherapies, as well as their combination, and further investigated their mechanism of action. We used a murinized version of tobemstomig, nivolumab and relatlimab-like anti-LAG-3 to prevent the formation of anti-drug antibodies and to prolong the treatment to 3 weeks. We performed single cell RNA sequencing on the tumor samples collected at day 28. We found both qualitative and quantitative differences between vehicle, mu-tobemstomig, mu-nivolumab monotherapy and in combination with mu-relatlimab-like anti-LAG-3.biostudies-arrayexpressNucleic Acid Extraction - After thawing a batch of samples, the cells were stained with a mix of FACS and oligo-labelled antibodies and sorted for CD3+T cells before performing scRNA-seq. Prior to sorting, the cells were washed with PBS, and cell number and viability were evaluated. Approximately 1 million cells per sample were resuspended in PBS and incubated with a Fc blocking reagent. The mix of FACS and oligo-labelled antibodies was added, and after incubation and washing, the cells were resuspended for sorting. The cell number and viability of the sorted cells were determined, and a total of 10,000 viable cells per sample were loaded into the 10x Genomics Chromium Connect Instrument.Sample Collection - Mice were sacrificed, and tumor tissue was isolated. The tumor tissue was disrupted and then digested in an enzyme mix containing RPMI with 10 mg/ml DNAse and 0.25 mg/ml Liberase for 30 minutes at 37°C. The resulting tissue mix was filtered to obtain a single-cell suspension. Lymphocytes were mechanically isolated from draining lymph nodes, filtered, and resuspended to a single-cell suspension. The resulting single-cell suspensions from tumors and lymph nodes were stored in liquid nitrogen in pZerve freezing medium.Sequencing - The resulting libraries were sequenced in an Illumina NovaSeq6000 sequencer according to 10x Genomics recommendations. The sequencing parameters were R1 = 26, i7 = 10, i5 = 10, and R2 = 90. The targeted sequencing depth was approximately 20,000 reads per cell for the GEX library and 5,000 reads per cell for both the TCR and feature barcoding libraries.Library Construction - cDNA and library preparation was performed according to the manufacturer’s indications using the 10x Genomics Chromium Next GEM Automated Single Cell 5’ Reagent Kits v2 with TCR and feature barcoding.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Normalization was performed using count depth scaling to 10,000 total counts per cell, resulting in the cp10k (counts per 10,000) unit. Count values were then log-transformed using the natural logarithm: ln(cp10k + 1).UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsMus musculusKlas HatjefalseSingle cell RNA-seq of Panc02-H7-Fluc model with vehicle tobemstomig, nivolumab and relatlimab-like treatmentWe used transgenic mice challenged subcutaneously with Panc02-H7-Fluc tumors to compare the efficacy of tobemstomig with nivolumab and relatlimab-like monotherapies, as well as their combination, and further investigated their mechanism of action. We used a murinized version of tobemstomig, nivolumab and relatlimab-like anti-LAG-3 to prevent the formation of anti-drug antibodies and to prolong the treatment to 3 weeks. We performed single cell RNA sequencing on the tumor samples collected at day 28. We found both qualitative and quantitative differences between vehicle, mu-tobemstomig, mu-nivolumab monotherapy and in combination with mu-relatlimab-like anti-LAG-3.2025-11-27T00:00:00Z2025-11-27T16:10:40.581Z2025-05-19T09:52:55.98ZE-MTAB-15144ERP172709EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184