Project description:Given that TREX1-deficient tumor cells showed a growth delay in immunocompetent but not immunodeficient hosts, we characterize the consequences of CT26 tumor-intrinsic TREX1 loss on the host immune system by performing single-cell RNA sequencing on intra-tumoral immune cells sorted from control and TREX1 KO CT26 tumors.
Project description:The DNA exonuclease TREX1 degrades endogenous cytosolic DNA. Cytosolic DNA triggers the cGAS/STING pathway which increases type I interferon. To investigate the physiological significance of TREX1 loss on in vivo tumor growth, we implanted control and TREX1-deficient CT26 tumor cells into immunocompetent BALB/c hosts.Tumor cells were collected 7 days after tumors reached around 200mm3.
Project description:We hypothesized that the immune microenvironment of the bone marrow influences the progression of myeloma outgrowth in the 5TGM1 transfer model of multiple myeloma. Therefore we sorted bone marrow T, B, NK, neutrophils, and monocytes/macrophages from control and tumor-bearing C57Bl/6 and KaLwRij mice.
Project description:By generating a paired single cell RNA-sequencing database of the tumor niche from 10 newly diagnosed MM patients, we created a unique dataset allowing the in-depth analyses of stromal-immune interactions within the tumor microenvironment. Using this database, we identified the presence of inflammatory stromal fibroblasts in the bone marrow of Myeloma patients.The stromal inflammation was associated with NF-κB signaling, and sources of IL-1β or TNFα were specific immune subsets previously shown to be altered in MM, suggesting the presence of an immune cell-mediated feed-forward loop of bone marrow inflammation in MM. By tracking inflammatory signatures over time in individual patients undergoing first-line treatment using bulk RNA sequencing, we show that bone marrow inflammation is not reverted by successful anti-tumor therapy (see related accession number), suggesting a role for stromal fibroblasts and bone marrow inflammation in disease persistence or relapse.
Project description:Neutrophils are the most abundant nucleated cell type in the bone marrow. A pro-tumor bias in this cell type may have implications for bone-marrow residing malignancies, such as multiple myeloma. Here, we generated single cell transcriptomic overviews of the entire myeloid compartment, including the entire neutrophilic lineage, of the bone marrow of 6 newly diagnosed myeloma patients, 5 treated myeloma patients and 4 non-cancer controls. We find dat mature neutrophils in myeloma patients, both newly diagnosed and treated, have an activated and pro-inflammatory phenotype, accompanied by increased transcription of pro-inflammatory cytokines, such as IL-1β, and myeloma cell survival factors, such as BCMA-ligand BAFF/TNFSF13B. Moreover, inflammatory stromal cells can activate naive neutrophils to acquire an inflammatory phenotype as is seen in patients. Previously, we have shown that inflammatory stromal cells characterized the bone marrow of newly diagnosed myeloma patients. Here, we generate single cell RNA sequencing dataset of non-hematopoietic bone marrow cells of patients after induction treatment, high-dose melphalan, stem cell transplantation and consolidation treatment. We show that this intensive treatment reduced, but did not normalize, stromal inflammation.
Project description:We performed 10x single cell RNA and VDJ sequencing on sorted T cells (7AAD- Calcein blue+ CD45+ THY1.1- TCRb+) in an orthotopic EMT6 tumor model 7 days after treatment initiation in four experimental groups: 1) Control 2) aPD-L1 3) aTGFb 4) aPD-L1 and aTGFb.
Project description:Langerhans cells (LC) in skin help initiate the immune response to locally presented antigens. We performed high-resolution single-cell RNA-sequencing (scRNAseq) analysis for antigen presenting cells including LC in normal mouse skin, and in mouse skin expressing the human papillomavirus (HPV) 16 E7 oncogene. Ear skin was collected from normal and trangenic mice. Dissociated CD45+ cells were processed for scRNA-seq using the 10X Genomics Chromium 3' gene expression kit (v2).
Project description:The dataset includes the raw FASTQ files generated from the 10x Genomics Single Cell 3’ v3.1 experiments. For samples processed with TotalSeq Hashtag reagents, the corresponding hashtag oligonucleotide and condition annotations are provided as separate .csv files. The processed matrices contain filtered readcounts for each cell barcode and, where multiplexing was used, the demultiplexed sample assignments derived from the hashtag data as .csv files. Synovial biopsies were taken using 14G Precisa Needle (HS Hospital Service, Italy) in ultrasound-guided protocol, and digested as described previously (Alivernini et al, 2020). Live CD45+ cells from the synovial tissue (ST) of healthy donors (n=3), patients with active RA (n=5) and patients in sustained remission (n=2) were sorted for single cell RNA (scRNA) or CITE (scCITE) sequencing using the 10x Genomics platform. Maximum 20,000 immune cells from tissue were sorted into Protein LoBind 1.5 ml Eppendorf tube containing 300 μl of RPMI media with 10% of FSC. Cells were loaded onto a Chromium Controller (10X Genomics) for single-cell partitioning, followed by library preparation using Single-Cell 3’ Reagent Kits v3.1. For 3 healthy ST and 4 ST from active RA (Figure S1), Totalseq Hashtag were used (Biolegend #394601, #394603, #394605, #394607, #394609) to combine 2 samples per run, and TotalSeq™-A Human Universal Cocktail (V1.0) was used to collect protein expression (CITEseq) data together with transcriptome data. Single-cell libraries were sequenced on the Illumina HiSeq 4000 system to a minimum depth of 50k reads/cell.