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Project description:The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA and TCR sequencing to detect and characterize "tumor-matching" (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared with nonmatching T cells in blood and were less exhausted than matching cells in tumors. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome, we identified candidate cell surface markers for TM cells in mice and patients and validated NKG2D, CD39, and CX3CR1 in mice. These data show that the TCR can be used to identify tumor-relevant cells for characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells.
| S-EPMC7933992 | biostudies-literature
Project description:Single-cell analyses identify circulating anti-tumor CD8 T cells and markers for their enrichment
| PRJNA668141 | ENA
Project description:Tobacco smoke exposure contributes to the global burden of communicable and chronic diseases. To identify immune cells affected by smoking, we use single-cell RNA sequencing on peripheral blood from smokers and nonsmokers. Transcriptomes reveal a subpopulation of FCGR3A (CD16)-expressing Natural Killer (NK)-like CD8 T lymphocytes that increase in smokers. Mass cytometry confirms elevated CD16+ CD8 T cells in smokers. Inferred as highly differentiated by pseudotime analysis, NK-like CD8 T cells express markers characteristic of effector memory re-expressing CD45RA T (TEMRA) cells. Indicative of immune aging, smokers' CD8 T cells are biased toward differentiated cells and smokers have fewer naïve cells than nonsmokers. DNA methylation-based models show that smoking dose is associated with accelerated aging and decreased telomere length, a biomarker of T cell senescence. Immune aging accompanies T cell senescence, which can ultimately lead to impaired immune function. This suggests a role for smoking-induced, senescence-associated immune dysregulation in smoking-mediated pathologies.
| S-EPMC7644053 | biostudies-literature
Project description:Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.
| S-EPMC4770429 | biostudies-literature
Project description:Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.
| S-EPMC2935889 | biostudies-literature
Project description:Cancer-related mortality of solid tumors remains the major cause of death worldwide. Circulating tumor DNA (ctDNA) released from cancer cells harbors specific somatic mutations. Sequencing ctDNA opens opportunities to non-invasive population screening and lays foundations for personalized therapy. In this study, two commercially available platforms, Roche's Avenio ctDNA Expanded panel and QIAgen's QIAseq Human Comprehensive Cancer panel were compared for (1) panel coverage of clinically relevant variants; (2) target enrichment specificity and sequencing performance; (3) the sensitivity; (4) concordance and (5) sequencing coverage using the same human blood sample with ultra-deep next-generation sequencing. Our finding suggests that Avenio detected somatic mutations in common cancers in over 70% of patients while QIAseq covered nearly 90% with a higher average number of variants per patient (Avenio: 3; QIAseq: 8 variants per patient). Both panels demonstrated similar on-target rate and percentage of reads mapped. However, Avenio had more uniform sequencing coverage across regions with different GC content. Avenio had a higher sensitivity and concordance compared with QIAseq at the same sequencing depth. This study identifies a unique niche for the application of each of the panel and allows the scientific community to make an informed decision on the technologies to meet research or application needs.
| S-EPMC7057974 | biostudies-literature
Project description:Human blood contains cell-free DNA (cfDNA), with circulating tumor-derived DNAs (ctDNAs) widely used in cancer diagnosis and treatment. However, it is still difficult to efficiently and accurately identify and distinguish specific ctDNAs from normal cfDNA in cancer patient blood samples. In this study, ctDNA fragment length distribution analysis showed that ctDNA fragments are frequently shorter than the normal cfDNAs, which is consistent with previous findings. Interestingly, the ctDNA fragment length was found to be partially associated with the mutant allele frequency, with a low mutant allele frequency (< ~0.6%) associated with a longer ctDNA fragment length when compared to normal cfDNAs. The findings of this study contribute to improving the detection of low-frequency tumor mutations.
| S-EPMC7059766 | biostudies-literature
Project description:BackgroundCirculating cell-free DNA (cfDNA) detection for non-invasive diagnosis requires higher sensitivity and accuracy due to the low circulating tumor DNA (ctDNA) content. Many methods have been developed to improve detection of ctDNA, including ultra-deep sequencing or enrichment of shorter cfDNA fragments, such as those in the range of 90-150 bp.MethodsHere, we developed a method for single-stranded DNA (ssDNA) library preparation with a large proportion of magnetic beads to enrich the shorter cfDNA fragments. We aimed to determine if this could increase the ctDNA content and thus improve the sensitivity of ctDNA detection by testing the method in blood samples from patients with advanced cancers (non-small cell lung cancers, esophageal squamous cell carcinoma, cholangiocarcinoma, colorectal cancer and liver cancer).ResultsThis method was able to obtain shorter cfDNA both in commercial cfDNA references and real world clinical cfDNA samples. Plasmid simulation experiments showed that using a large proportion of magnetic beads to construct the library could obtain more ctDNA derived from shorter-fragment plasmids, which could significantly improve the detection of ctDNA especially in the low-variant allele frequency sample. In real-world clinical samples, this method may be able to increase the opportunity to obtain alteration reads from short fragments, which was important to low frequency detection.ConclusionsThe ssDNA library preparation with large proportion of magnetic beads could increase the opportunity to obtain alteration reads from short fragments, which is crucial for low variant allele frequency detection.
| S-EPMC8044499 | biostudies-literature
Project description:CD8+ T cells are crucial to establish antitumor immunity, and their high infiltration associates with favorable prognoses. However, several CD8+ T cell subpopulations in the tumor microenvironment may play different roles in prognosis, progression, and immunotherapy. Here, we analyzed prior published single-cell RNA-sequencing (scRNA-seq) melanoma data to explore the heterogeneity of CD8+ T cell subpopulations and identified 7 major subpopulations. We found that high infiltration of exhausted CD8+ T cell subpopulation 2 would contribute to unfavorable prognoses. In contrast, a large proportion of naive/memory cells and cytotoxic CD8+ T cell subpopulation 3 would lead to favorable prognoses. Notably, the proportion of the cytotoxic CD8+ T cell subpopulation 3 would decrease in later-stage melanoma samples, while that of the exhausted CD8+ T cell subpopulation 2 would increase. We also found that high abnormal activities of metabolic pathways existed in exhausted CD8+ T cell subpopulation 1. Significantly, immunosuppressive checkpoints PD-1 and CTLA-4 signaling pathways were upregulated in exhausted CD8+ T cell subpopulations. In addition, a dynamic transcript landscape of immune checkpoints among different subpopulations was also depicted in this study. Moreover, we identified three overexpressed genes (PMEL, TYRP1, and EDNRB) that were significantly correlated to poor prognoses and only expressed in exhausted CD8+ T cell subpopulation 2. Importantly, they showed the highest expression in melanoma samples compared to other tumors. In general, we characterized the CD8+ T cell subpopulations in melanoma and identified that not only genes of immunosuppressive checkpoints but also PMEL, TYRP1, and EDNRB could serve as potential targets for melanoma therapy.
| S-EPMC7851490 | biostudies-literature
Project description:Effective capture and analysis of a single circulating tumor cell (CTC) is instrumental for early diagnosis and personalized therapy of tumors. However, due to their extremely low abundance and susceptibility to interference from other cells, high-throughput isolation, enrichment, and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge. Herein, we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation, CTC enrichment, and single-cell immunoblotting (ieSCI). The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting. The ieSCI-chip successfully identified a subgroup of apoptosis-negative (Bax-negative) cells, which traditional bulk analysis did not detect, from cisplatin-treated cells. Furthermore, we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition (EMT) analysis. The expression level of a tumor cell marker (EpCAM) can be directly determined in isolated CTCs at the single-cell level, and the therapeutic response to anticancer drugs can be simultaneously monitored. Therefore, the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.
| S-EPMC8807661 | biostudies-literature