scholarly journals 82 Single-cell RNA sequencing and CITE-Seq analysis of bladder cancer patient urine with matched tumor and peripheral blood suggests urine as a window into the tumor immune microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A90-A90
Author(s):  
Michelle Tran ◽  
Adam Farkas ◽  
Kristin Beaumont ◽  
Timothy O’Donnell ◽  
Reza Mehrazin ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patient ◽  
Single Cell ◽  
Cancer Patients ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Single Cells ◽  
Bladder Cancer Patient ◽  
Immune Microenvironment ◽  
Tumor Immune Microenvironment

BackgroundFDA-approved immunotherapies for early and advanced stage bladder cancer have response rates of 15–65% in bladder cancer, suggesting that tumor-associated resistance mechanisms undermine their efficacy. Accordingly, there is an unmet need to identify accessible biomarkers that predict response. Urine, which is in direct contact with urothelial tumors, represents an easily accessible patient material that may reflect cellular and/or genetic signatures related to immune resistance. It has been demonstrated that urine from bladder cancer patients contains not only tumor cells, which are routinely assessed by clinical urinalyses, but also immune cells that previous studies suggest may reflect the tumor microenvironment (TME).1 However, the concordance between cells in the urine and those in bladder tumors is unknown., Here, we characterized patient urine in an unbiased fashion by performing the first single-cell RNA sequencing (scRNAseq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on matched bladder cancer patient urine, tumor, and peripheral blood.MethodsMatched tumor tissue, urine, and peripheral blood were collected from bladder cancer patients (n=7) during surgery; either trans-urethral resection of bladder tumor or cystectomy. All three tissues were processed to single-cell suspensions and sequenced using the 10X Genomics platform (scRNAseq: 17 samples, CITE-seq: 3 samples). These sequencing approaches permitted quantification of both transcriptomic and surface protein expression of 54,469 cells total.2 3 Analysis was performed using Seurat, Enrichr, and Monocle packages and platforms.4 5 6Results scRNAseq of urine from bladder cancer patients revealed several immune populations including CD4+ and CD8+ T cells, Treg cells, NK cells, B cells, neutrophils, dendritic cells, monocytes, and macrophages in addition to non-hematopoietic lineages including bladder epithelial cells, neuronal cells, prostate epithelial cells, fibroblasts, myofibroblasts, and endothelial cells. The composition and transcriptional profiles of urine immune cells were more similar to TME immune cells than to peripheral blood immune cells. Urine immune cells expressed gene signatures associated with hypoxia, anergy, pro-inflammation, and glucose deprivation that were more similar to tumor immune cells than those in the peripheral blood.ConclusionsOur work represents the first scRNAseq and CITEseq profiling of cancer patient urine. Our study suggests several viable immune cells shed in bladder cancer patient urine that look more transcriptionally and phenotypically similar to the TME than peripheral blood cells. This important finding has several implications for future research and clinical applications as urine can be sampled non-invasively in scenarios when tumor resection may not be feasible.ReferencesWong YNS, Joshi K, Khetrapal P, et al. Urine-derived lymphocytes as a non-invasive measure of the bladder tumor immune microenvironment. Journal of Experimental Medicine. 2018; 215:2748–59.Zheng GXY, Terry JM, Belgrader P, et al. Massively parallel digital transcriptional profiling of single cells. Nature Communications 2017; 8.Stoeckius M, Hafemeister C, Stephenson W, et al. Simultaneous epitope and transcriptome measurement in single cells. Nature Methods 2017;14, 865–68.Butler A, Hoffman P, Smibert, P, et al. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nature Biotechnology 2018; 36: 411–20.Xie Z, Bailey A, Kuleshov MV, et al. Gene set knowledge discovery with Enrichr. Current Protocols 2021.Trapnell C, Cacchiarelli D, Grimsby J, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nature Biotechnology 2014; 32: 381–6.Ethics ApprovalThe study was approved by Mount Sinai Institution’s Ethics Board, approval number 10–1180. Participants gave informed consent before taking part in the study.

Can Intratumoral neutrophil lymphocyte ratio (NLR) be a prognostic biomarker in breast cancer patients?

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e12573-e12573
Author(s):  
Yoshihisa Tokumaru ◽  
Masanori Oshi ◽  
Vijayashree Murthy ◽  
Eriko Katsuta ◽  
Nobuhisa Matsuhashi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Cancer Patients ◽  
Immune Cells ◽  
Breast Cancer Patients ◽  
Immune Microenvironment ◽  
High Group ◽  
Anti Cancer ◽  
Tumor Immune Microenvironment ◽  
Er Positive

e12573 Background: In breast cancer patients, it is well known that the elevation of neutrophil lymphocyte ratio (NLR) in the blood are reported to associate with poor prognosis based on the notion that neutrophils represent pro-cancer, and lymphocytes represent anti-cancer immune cells. Tumor immune microenvironment has been demonstrated to play critical roles in the outcome of breast cancer patients. However, there is scarce evidence on the clinical relevance of intratumoral NLR in breast cancer patients. In the current study, we hypothesized that intratumoral NLR high tumors are associated with worse survival particularly in TNBC that is known to have high immune cell infiltration. Methods: A total of 1904 breast cancer patients’ data from METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) and analyzed. NLR was calculated by the gene expressions of CD66b (CEACAM8) and CD8 (CD8A). NLR high and low were divided by the median. Overall Survival (OS) and Disease-Free Survival were calculated utilizing Kaplan Meier method between intratumoral NLR high and low groups. xCell algorithm was used to analyze the infiltrated immune cells within the tumor immune microenvironment as we have previously published. Results: Intratumoral NLR high group was associated with worse OS in whole, ER-positive/HER2-negative, and triple negative (TN) subtypes, in agreement with the previous studies. TN subtype alone demonstrated worse DFS of NLR high group. Surprisingly, gene set enrichment analysis (GSEA) demonstrated no gene set enrichment to NLR high group, which implicates that there is no distinctive mechanism that associate with worse survival. Whereas, immune response-related gene sets significantly enriched to NLR low group in TN subtype. This enrichment was consistent in ER-positive/HER2-negative. Compared with ER-positive/HER2-negative subtype, anti-cancer immune cells such as CD4+ T cells, CD8+ T cells, M1 macrophage, and helper T helper type 1 cells were significantly infiltrated in TN patients (p < 0.001 for all genes), where M2 macrophages and neutrophils were less and regulatory T cells and T helper type 2 cells were more infiltrated in TN subtype. Furthermore, intratumoral NLR was significantly lower in TN compared with ER-positive/HER2-negative subtype (p < 0.001). These results suggest that intratumoral NLR low group is associated with better survival due to favorable tumor immune microenvironment in TN subtype rather than NLR high group has worse survival. Conclusions: Intratumoral NLR low tumor demonstrated more favorable OS and more favorable DFS in TN patients. Intratumoral NLR low breast cancer was associated with enhanced immune response and higher infiltration of anti-cancer immune cells were observed in TN subtype compared to ER-positive/HER2-negative which may contribute to the favorable outcome of in TN breast cancer.

scholarly journals Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer

2021 ◽  
Author(s):  
Inga-Maria Launonen ◽  
Nuppu Lyytikäinen ◽  
Julia Casado ◽  
Ella Anttila ◽  
Angéla Szabó ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Single Cell ◽  
Single Cells ◽  
High Grade ◽  
Prognostic Role ◽  
Immune Microenvironment ◽  
Proteomic Data ◽  
Tumor Immune Microenvironment ◽  
Cellular Phenotypes

Abstract The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generated spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes (HRwt). We identified a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we found an opposing prognostic role of a proliferative tumor-cell phenotypic subpopulation in the HR-genotypes, which associated with enhanced spatial tumor-immune interactions by the CD8+ and CD4+T-cells in BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the premise to improve immunotherapeutic strategies and patient stratification in HGSC.

Clinical relevance of miR-143 expression in ER-positive breast cancer patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e12574-e12574
Author(s):  
Yoshihisa Tokumaru ◽  
Masanori Oshi ◽  
Eriko Katsuta ◽  
Nobuhisa Matsuhashi ◽  
Manabu Futamura ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Immune Cells ◽  
High Expression ◽  
Breast Cancers ◽  
Breast Cancer Patients ◽  
Immune Microenvironment ◽  
Anti Cancer ◽  
Tumor Immune Microenvironment ◽  
Er Positive

e12574 Background: MicroRNA-143(miR-143) is a well-known tumor suppressive microRNA in various malignancies, including breast cancer. Recently, the tumor immune microenvironment has been reported to associate with progression of breast cancers. However, the association with the tumor immune microenvironment and miR-143 in breast cancers remains ambiguous. Given these backgrounds, we hypothesized that high expression of miR-143 is associated with favorable effect to the tumor immune microenvironment which leads to better survival of ER positive breast cancer patients. Methods: Two major publicly available breast cancer cohorts were used for this study. A total of 753 patients from The Cancer Genome Atlas (TCGA) and total of 1283 patients from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) were used. Results: We defined the higher quartile of miR-143 expression levels as high and the remainder as low expression groups. There was no significant difference in patient clinicopathlogical features between two groups. Gene set enrichment analysis (GSEA) revealed that miR-143 high expression tumors enriched Helper T cell type 1 (Th1) related gene sets indicating the upregulation of anti-cancer immune cells. Also, the cell composition of anti-cancer immune cells, such as Th1 and Macrophage M1 were higher with miR-143 high tumors (p < 0.001 and p < 0.01 respectively) in whole group. On the contrary, pro-cancer immune cells such as Th2 and M1 were lower with miR-143 high tumors (p < 0.01 and p < 0.001 respectively) in whole group. Interestingly, among the subtypes, we found that ER positive subgroup followed this trend of high infiltration rate of anti-cancer immune cells and low infiltration rate of pro-cancer immune cells. Furthermore, only ER positive subgroup demonstrated the survival benefit with miR-143 high expression tumors. Conclusions: We demonstrated that high expression of miR-143 in ER breast cancer associate with favorable tumor immune microenvironment, upregulation of the anti-cancer immune cells and suppression of the pro-cancer immune cells, and associate with better survival of the breast cancer patients.

scholarly journals Pseudogene HLA-DRB6 Regulates Immune Microenvironment of Cutaneous Melanoma by miR-338/CXCL10 Axis

2022 ◽  
Author(s):  
Jianmin Ren ◽  
Jinglu Yu ◽  
Yang Shi ◽  
Inam Ullah Khan ◽  
Jiansheng Huang
Keyword(s):  
Single Cell ◽  
Immune Cells ◽  
Cutaneous Melanoma ◽  
Target Genes ◽  
Mrna Levels ◽  
Sequencing Data ◽  
Immune Microenvironment ◽  
Potential Biomarker ◽  
Tumor Immune Microenvironment ◽  
The Relationship

Abstract Background: The relationship between the pseudogene and tumor immune microenvironment in cutaneous melanoma is unclear. In this study, we analyzed the role of the pseudogene HLA-DRB6 and its effect on the tumor immune microenvironment in skin cutaneous melanoma (SKCM) using bioinformatics tools. Method: The GEPIA database was used to analyze the expression of HLA-DRB6 and CXCL10 mRNA in tumor tissues. The TIMER database was used to analyze the relationship between mRNA levels and the infiltration of immune cells. The enrichment of HLA-DRB6 and CXCL10 in melanoma tissues was analyzed by single cell portal. The binding sites of HLA-DRB6 with its target genes was predicted via starBase database. The gene expression profiling and clinical data from GEO database (GSE94873) was used to verify the potential of CXCL10 as a biomarker. Result: The expression of HLA-DRB6 in SKCM tumor is higher than in normal tissues, and patients with high HLA-DRB6 expression had a better prognosis (P<0.05). Furthermore, HLA-DRB6 is positively correlated with the infiltration of immune cells such as B cells, CD4+ T, and CD8+ T lymphocytes, and the expression of immune checkpoint molecules such as PD-1, PD-L1, and CTLA-4. Single cell transcriptome sequencing data showed that HLA-DRB6 is mainly enriched in macrophages and had the highest correlation with CXCL10 than other chemokines (cor=0.66, P<0.0001). In addition, we found that CXCL10 can be used as a potential biomarker for predicting responsiveness and survival rate in SKCM patients who treated with Tremelimumab (a human anti-CTLA-4 antibody). Conclusion: In the microenvironment of SKCM, HLA-DRB6 is mainly enriched in macrophages and regulates the expression of CXCL10 through the ceRNA mechanism. Furthermore, the CXCL10 in peripheral blood can be used as a biomarker to predict the responsiveness and the prognosis for patients treated with tremelimumab.

Urine cell flow cytometry analysis of PD-L1 expression and DNA content for bladder cancer.

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 466-466
Author(s):  
Pin-I Chen ◽  
Alice (Xiaoyang) Wang ◽  
Mustafa Deebajah ◽  
Shaheen Alanee ◽  
Bruce Kendrick Patterson
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Bladder Cancer ◽  
Epithelial Cells ◽  
Cancer Patient ◽  
Cancer Patients ◽  
Dna Content ◽  
Urine Samples ◽  
Normal Donor ◽  
Bladder Cancer Patient

466 Background: Bladder cancer is the fifth most common cancer in the United States. PD-1/PD-L1, a pathway used by cancer cells to evade immune response, correlates with bladder cancer severity and has emerged as a target in bladder cancer treatment. Chromosomal instability is also a prominent feature associated with the development of bladder cancer. A method to unbiasedly analyze PD-L1 expression and DNA content in cells from urine samples will help us better understand bladder cancer. Methods: To evaluate the PD-L1 expression and DNA content, we developed a 4-color flow assay. Cells in urine samples were pelleted, fixed/permeabilized (in incellMAX, IncellDx Inc.) and stained with antibodies against pan-cytokeratin (CK), CD45, PD-L1 and a cell cycle dye. The stained samples were analyzed by a flow cytometer alongside stained control cells. Results: Fifty bladder cancer patient and 15 normal donor urine samples were collected and tested with this assay. We could distinguish epithelial cells (pan-CK+) and white blood cells (WBCs, CD45+) in urine samples and obtain PD-L1 expression and DNA content information simultaneously from these cell populations. The patient samples showed a significantly higher percentage of WBCs with substantial PD-L1 expression. The percentage of PD-L1 positive epithelial cells was not distinguishable between normal donor and patient samples. However, increased post G1 epithelial cells ( > 5%) were observed in a majority of bladder cancer patients, with around 25% of samples showing a DNA index above 1.05. In addition, a comparison of urine collection fixatives showed that incellMAX-fixed samples had the best single cell recovery and DNA content measurement, as shown by lower cell cycle dye staining variability (lower rCV). Statistically significant differences were found between cancer patients and normal samples. Conclusions: We developed a flow cytometry-based method to investigate PD-L1 and DNA content simultaneously in cells from urine samples. Comparing urine samples from bladder cancer patients and normal yielded statistically significant differences that could provide valuable information for bladder cancer patient management.

scholarly journals Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer

2021 ◽  
Author(s):  
Inga-Maria Launonen ◽  
Nuppu Lyytikäinen ◽  
Julia Casado ◽  
Ella Anttila ◽  
Angéla Szabó ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Single Cell ◽  
Spatial Data ◽  
Single Cells ◽  
High Grade ◽  
Spatial Interactions ◽  
Immune Microenvironment ◽  
Tumor Immune Microenvironment ◽  
Cellular Phenotypes

Abstract The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis on 112 tumor cores we generated single-cell spatial data for 21 markers in 124,623 single cells from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and 13 tumors without any alterations in HR genes (HRwt). We identified a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we found an opposing prognostic role of a proliferative tumor-cell phenotypic subpopulation in the HR-genotypes, which associated with enhanced spatial interactions in the tumor-immune cellular communities. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the premise to improve immunotherapeutic strategies and patient stratification in HGSC.

scholarly journals Uranium concentration variation dependency on gender correlated with age of bladder cancer patient

2019 ◽  
Vol 10 (3) ◽  
pp. 1730-1734
Author(s):  
Aseel S. Mahmood ◽  
Omar Sh. Shafeq ◽  
Mohamed S. Shafiq
Keyword(s):  
Bladder Cancer ◽  
Cancer Patient ◽  
Cancer Patients ◽  
Uranium Concentration ◽  
Bladder Cancer Patient ◽  
Age Group ◽  
Average Period ◽  
Age And Gender ◽  
Males And Females ◽  
And Gender

Human health was and still the most important problem and objective of all most researches. Finding out what causes in the decadence of healthiness of Iraqi population is our tendency in the present work, Uranium causing cancer that is affected by a correlation between age and gender of bladder cancer patients is studied in the present work. Mean of Uranium concentration (Uc) decreased with increasing age for all age group without dependency on gender. While, there is a wide dispersion in Mean Uc excretion between males and females, due to the effect of correlated gender with age, where female Mean Uc is maximum at age 50-69 year (2.355 µg/L), and it's higher than male Mean Uc (2.022 µg/L) in this age stage because of menopause, also average period of illness and the percentage of patients are affected by correlated gender with age. So that factor of gender correlation with age affects in the calculation of background levels and radiation exposure and causing bladder cancer incidence.

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