In situ tumor arrays reveal early environmental control of cancer immunity [STAMP time course (Day1,3,8) bulk RNAseq ]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE222026 | GEO | 2023/02/17
REPOSITORIES: GEO
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Project description:KPP-GFP (C57Bl/6) cells were implanted into the laser-induced micropores ears of B6.Cg-Foxn1nu/J (C57Bl/6J Jackson background) that were adoptively transferred with 4x10^6 naive CD4tdTomato-positive Tcells (B6 N/J background) the same day. The STAMP microtumors were imaged daily starting at day 3 after tumor cell implantation. On day 8 after tumor cell implantation tumors were classified as either (1) Inflamed, (2) Excluded, (3) Ignored/Desert or (4) Resolved.
2023-02-17 | GSE222027 | GEO
Project description:The immune phenotype of a tumor is a key predictor of its response to immunotherapy1–4. Patients who respond to immune checkpoint blockade generally present with tumors infiltrated by T cells, a phenotype referred to as ‘inflamed’5–7. However, not all inflamed tumors respond to therapy, and even lower response rates occur among patients with tumors that lack T cells (‘desert’) or that spatially exclude T cells to the periphery of the tumor lesion (‘excluded’)8. Despite the importance of these tumor immune phenotypes in patients, little is known about their development, heterogeneity or dynamics due to the technical difficulty of modeling and tracking these features in situ. Here, we introduce STAMP (skin tumor array by microporation), a preclinical approach that combines in vivo high-throughput time-lapse imaging with next generation sequencing of tumor arrays. Using this approach, we follow the early formation of thousands of tumors to show that development of a given immune phenotype is not under strict control of tumor genetics or transcriptional state, but is also influenced by local features of the tumor microenvironment. Only the spatial organization of T cells, specifically early infiltration of T cells recruited by fibroblasts and monocytes into the core of a tumor, was predictive of functional T cell attack and tumor rejection. Evaluating the dynamic immune-history of tumors revealed that early conversion to the immune inflamed phenotype was predictive of therapy-induced or spontaneous tumor regression. Thus, STAMP captures the dynamic and complex relationships of spatial, cellular and molecular components of tumor progression and has the potential to translate therapeutic concepts into successful clinical strategies.
2023-02-17 | GSE222230 | GEO
Project description:In situ tumor arrays reveal early environmental control of cancer immunity [STAMP D8]
| PRJNA917336 | ENA
Project description:Tubulin tyrosine ligase-like family member 5 (TTLL5/STAMP) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamptm/tm) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamptm/tm sperm tails in conjunction with substantial reduction in tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of wt vs. Stamptm/tm males despite the levels of wt STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility. Testes RNA samples for EXON arrays from WT and KO with three biological replications
2013-03-28 | E-GEOD-45546 | biostudies-arrayexpress
Project description:Transcription and translation are intertwined processes where mRNA isoforms are crucial intermediaries. However, methodological limitations in analyzing translation at the mRNA isoform level have impaired our ability to comprehensively establish links between the full-length transcripts and the translatome. This has left gaps in our understanding of critical biological processes, regulatory mechanisms, and disease progression. To address this, we develop an integrated computational and experimental framework called long-read Ribo-STAMP (LR-Ribo-STAMP). LR-Ribo-STAMP capitalizes on advancements in long-read sequencing and RNA-base editing-mediated technologies to simultaneously and scalably profile translation and transcription at both gene and mRNA isoform levels for the first time. In this report, we show agreement between gene-level translation profiles obtained with LR-Ribo-STAMP and those from previously validated short-read Ribo-STAMP data in unperturbed cells. At the mRNA isoform level, we show that LR-Ribo-STAMP successfully profiles translation in unperturbed cells and links mRNA isoforms and regulatory features, such as upstream ORFs (uORFs) and regulatory sequences, to translation measurements. We further demonstrate the method’s effectiveness in profiling disease models by profiling translation at gene and isoform levels in a triple-negative breast cancer cell line under normoxia and hypoxia. Here, we find that LR-Ribo-STAMP effectively delineates orthogonal transcriptional and translation shifts between conditions at gene and isoform levels. At the isoform level, LR-Ribo-STAMP uniquely identifies key regulatory elements and shifts in mRNA isoform transcription that correlate with changes in translational, providing an example of insight that can inform the development of novel therapeutics. Overall, LR-Ribo-STAMP is a significant advancement in translation methods and can have profound implications for basic research and clinical applications.
2024-05-20 | GSE255844 | GEO
Project description:RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
2024-06-13 | GSE240014 | GEO
Project description:RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
2024-06-13 | GSE263870 | GEO
Project description:RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
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2024-06-13 | GSE248461 | GEO
Project description:RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
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