10X genomics single cell RNA sequencing of tumor sections from tumor bearing KrasG12D/+Lkb1fl/fl(KL) mice after Ad-cre injection for 10 weeks
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ABSTRACT: 10X genomics single cell RNA sequencing of tumor sections from tumor bearing KrasG12D/+Lkb1fl/fl(KL) mice after Ad-cre injection for 10 weeks
Project description:LKB1 deficiency is widely acknowledged to induce immune-desert tumors in the non small cell lung cancer. Dissecting the "cold" tumor microenvironment (TME) would promote a better understanding of mechnism of the immune evasion triggered by LKB1 loss, thereby facilitating to seek therapeutic targets. Here, we exploited single-cell RNA sequencing to show the immune landscape of genetically engineered mouse model (GEMM) bearing a conditional activating mutation of endogenous Kras (KrasLSL-G12D/+) with or without Lkb1 conditional knockout (Lkb1fl/fl). The heterogeneity of TME and celler communication were analyzed.
Project description:Tumor-infiltrating cells comprise many celltypes, including monocytes, macrophages, dendritic cells, neutrophils, stromal cells and epithelial cells, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which may promote or limit tumor outgrowth, but remain poorly understood. Here, we used single-cell RNA sequencing to map cell heterogeneity in non-small cell lung cancer mouse model. We uncovered 11 cell types in tumor sections, most of which were reproducibly found across tumor bearing mice. we obtained a series of genes which expression levels were very low, such as cytokines and chemokines. It is very helpful for us to understand the signal cascade and ligands-receptors interaction between different cell types.
Project description:Comparative gene expression profiling analysis of RNA-seq data in CD45- lung tumor cells from tumor-burdened lungs of KrasLSL-G12D/+Lkb1fl/fl and KrasLSL-G12D/+Lkb1fl/flUsp25-/- mice 10 weeks after they were intranasally injected with Ad-Cre.
Project description:Ribosome profiling on sections taken from a kidney tumor For two tumors: 2 sections of normal tissue and 4 samples of tumor tissue, for another two tumors 1 section of normal tissue and 1 section of tumor tissue
Project description:5000 target cells per sample where sequenced from 2 gastric tumor samples and 1 sample of adjacent gastric mucosa Twist2Cre;Lkb1fl/+;RosaRTdtomato+/- mice. Drop-seq 10X Genomics (10X v3 library kit) was used for single cell barcoding and library preparation. Samples were sequenced with Novaseq6000.
Project description:mRNA transcriptome sequencing of tumor bearing lungs from KrasLSL-G12D/+Lkb1fl/fl, KrasLSL-G12D/+Lkb1fl/flIl1f9-/-(KL9) and KrasLSL-G12D/+Lkb1fl/flIl1f5-/-(KL5) or KrasLSL-G12D/+Tp53fl/fl, KrasLSL-G12D/+Tp53fl/flIl1f9-/-(KP9) and KrasLSL-G12D/+Tp53fl/flIl1f5-/-(KP5) after 10 weeks Ad-cre injection Tumor-burdened lungs from KL/KL5/KL9 mice and KP/KP5/KP9 mice were perfused through alveolar lavage and cardiac lavage with PBS, dry it quickly, and then homogenized in 2 ml of TRIzol (Invitrogen). Total RNAs were prepared and the quality of RNAs was determined by agarose gel electrophoresis and spectrophotometer analysis. Poly(A) mRNA was subsequently purified from 10μg total RNA using NEBNext Oligo d(T)25 Magnetic Beads Isolation Module. First-strand complementary DNA was synthesized with NEBNext RNA First-Strand Synthesis Module. NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module was used for the synthesis of the complementary strand of first-strand cDNA. The resulting double-stranded DNA was purified and Vazyme TruePrep DNA Library Prep kit V2 was used to prepare libraries followed by sequencing on an Illumina Hiseq X Ten platform with 150-bp paired-end reads strategy (Novogene). Quality control of mRNA-seq data was performed by using Fatsqc (v0.11.9) and low-quality bases were trimmed by Trim_galore (0.6.4_dev). All RNA-seq data were mapped to the mouse genome (Mus_musculus_Ensemble_94) by Hisat2 (v.2.0.5) and allowed a maximum of two mismatches per read. Gene expression level was calculated by FeatureCounts (v.2.0.0) with default parameters and normalized by FPKM (Fragments Per Kilobase of exon model per Million mapped fragments).
Project description:Rhabdomyosarcoma is a pediatric malignancy thought to arise from the uncontrolled proliferation of myogenic cells. Here, we have generated models of rhabdomyosarcoma in the zebrafish by inducing oncogenic KRASG12D expression at different stages during muscle development. Several zebrafish promoters were used including the cdh15 and rag2 promoters that drive gene expression in early muscle progenitors, and the mylz2 promoter that expresses in differentiating myoblasts. The tumors that developed differed in their ability to recapitulate normal myogenesis. cdh15:KRASG12D and rag2:KRASG12D fish developed tumors that displayed an inability to fully undergo muscle differentiation by histologic appearance and gene expression analyses. In contrast, mylz2:KRASG12D tumors more closely resembled mature skeletal muscle and were most similar to well-differentiated human rhabdomyosarcoma by gene expression. mylz2:KRASG12D fish showed significantly improved survival compared to cdh15:KRASG12D and rag2:KRASG12D fish. Tumor-propagating activity was enriched in myf5-expressing cell populations within all of the tumor types. Our results demonstrate that oncogene expression at different stages during muscle development has profound effects on the ability of tumor cells to recapitulate normal myogenesis, altering the tumorigenic capability of these cells. 32 samples total: 7 WT muscle, 9 mylz2-KRAS, 9 cdh15-KRAS, and 7 rag2-KRAS tumors