Deconvolution of Cell Type-Specific Drug Responses in Human Tumor Tissue with Single-Cell RNA-seq
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE148842 | GEO | 2020/04/18
REPOSITORIES: GEO
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Project description:Purpose: Authentic preclinical models of renal cell carcinoma (RCC) are lacking. We aimed to establish and characterize primary RCC cultures and demonstrate the feasibility of evaluating drug responses in vitro and in vivo. Materials and Methods: Previously published methodology, with minor modifications, was used to establish, cryopreserve, and serially passage RCC cells from nephrectomy and tumorgraft specimens. Cells were characterized for immuno- and molecular phenotype by immunochemistry, DNA sequencing and gene expression profiling. Tumorigenic potential was evaluated by implanting cells under the renal capsule of immunocompromised mice. The ability to monitor xenograft growth by magnetic resonance imaging (MRI) was investigated. Responses to a tyrosine kinase inhibitor (TKI) and an mTOR inhibitor were measured. Results: Primary cultures were successfully established from 11 clear cell and 1 chromophobe RCC, cryopreserved and serially passaged. Retention of immuno- and molecular phenotypes was demonstrated. Cultured cells formed xenografts in mice that could be measured by MRI. Patient-specific responses to drugs were observed in vitro and response to an TKI was confirmed in vivo. Conclusions: Our study is the first to show the derivation of primary cultures from RCC tumorgrafts, and to demonstrate the ability of primary RCC cultures to generate xenografts in mice. Our results suggest the feasibility of establishing large, well-annotated banks of RCC primary cultures for high-throughput drug screening in vitro and validation in vivo, providing a versatile platform together with xenografts and patient-derived precision-cut tissue slice tumorgrafts we developed previously for precilinical studies of RCC. Microarray analyses were performed to compare the gene expression profile of one of the primary cultures (case 7) to its parental tumor and tissue slice tumorgrafts in the study. Tissue slice grafts and parent tumors were preserved in Allprotect tissue reagent (Qiagen, Valencia, CA) at -20°C prior to RNA extraction using an AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Valencia, CA). The quality of RNA was determined using an Agilent 2100 Bioanalyzer (Agilent Biotechnologies, Santa Clara, CA). Microarray hybridization was performed using Illumina Human HT-12 v4 Beadchips (Illumina Inc., San Diego, CA) according to the manufacturerâ??s directions. Expression data was rank invariant normalized using BeadStudio software (Illumina Inc.).
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Project description:In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic1, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model2,3 to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.
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Project description:Purpose: The aim of this study was to create a patient-derived slice model by combining cryopreservation technique with precision-cut slice culture method and explore its effectivity of predicting anti-cancer drug sensitivity in vitro. Methods: We prepared 0.3 mm thick tissue slices by a microtome and maintain its cell viability by cryopreservation technique. Slices were cultured individually in the presence or absence of regorafenib (REG) for 72 hours. Alterations in morphology and gene expression were assessed by histological and genetic analysis. Overall viability was also analyzed in tissue slices by CCK-8 quantification assay and fluorescent staining. Tissue morphology and cell viability could be evaluated to quantify drug effects. Results: Histological and genetic analysis showed that no significant alterations in morphology and gene expression were induced by vitrification‑based cryopreservation. The viability of warmed HCC tissues was up to 90% of the fresh tissues. The viability and proliferation could be retained for at least four days in filter culture system. The positive drug responses in precision-cut slice culture in vitro were evaluated by tissue morphology and cell viability. Conclusion: In summary, the successful application of precision-cut HCC slice culture combining cryopreservation technique in a systematic drug screen demonstrates the feasibility and utility of slice culture method for drug response.
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