CD8 T and NK cells mediate killing by delivery of perforin and granzyme B (GZB) stored in lysosome-like granules. We present a flow-cytometry-based protocol combined with a redirected killing assay ...to evaluate granule exocytosis and the cytotoxic potential of human CD8 T cells and NK cells. We describe the assessment of the delivered GZB inside the target cells. We then detail the detection of lysosome membrane protein CD107a exposed on the cell surface of the effector cells upon degranulation.
For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).1
Display omitted
•Flow cytometry protocol to evaluate GZB delivery and activity inside the target cells•Redirected killing assay to assess the full cytotoxic potential of CD8 T cells•Protocol for the evaluation of the ex vivo cytotoxicity of human NK cells
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CD8 T and NK cells mediate killing by delivery of perforin and granzyme B (GZB) stored in lysosome-like granules. We present a flow-cytometry-based protocol combined with a redirected killing assay to evaluate granule exocytosis and the cytotoxic potential of human CD8 T cells and NK cells. We describe the assessment of the delivered GZB inside the target cells. We then detail the detection of lysosome membrane protein CD107a exposed on the cell surface of the effector cells upon degranulation.
Abstract
SARS-CoV-2 is one of the leading causes of mortality worldwide with rising death toll over 5.4 million. The severe presentation of the disease is associated with systemic inflammation, ...activation of the coagulation cascade and immunological dysfunction. Numerous studies implicate endothelial activation and dysfunction as playing an important role; however, the pathways involved are not well-understood. Endothelial injury and damage trigger a mechanism of vascular repair mediated by bone marrow derived endothelial progenitor cells and recently discovered a type of T cells with angiogenic properties. In this study, we hypothesize that SARS-COV-2 alters this pathway. To address this hypothesis, we determined changes in the frequency and phenotype of these two cell types, the circulating endothelial progenitor (EPC) (LIN4−CD45−CD34+) and Tang cells (CXCR4+CD31+) using PBMCs from hospitalized COVID-19 positive (n = 10) and healthy (n = 11) donors. Our results indicate a decrease in the frequency of CD4 and CD8 Tang cells compared with healthy controls (p=0.003; p=0.01). In addition, Tang cells showed a significant increase in the expression of progenitor marker (CD49f) (p<0.001; p=0.016), and no changes were observed in the frequency and phenotype of the LIN4−CD45−CD34+. Moreover, a negative association was observed between Tang cells and plasma levels of the procoagulant PAI-1 biomarker (r=−0.806, p=0.007; r=−0.818, p=0.006), and CD8 Tang cells were inversely correlated with the levels of MMP9 and TNFa, (r=−0.879, p=0.002; r=−0.709, p=0.027). Altogether these data suggest that SARS-CoV-2 infection reduces the frequency of Tang cells and promote activation and may interfere with their vascular repair function.
Abstract
Melanoma is an aggressive and lethal form of cancer with increasing incidence worldwide. In recent years, the FDA approved several promising treatments for malignant melanoma. However, ...treatment often fails due to the survival and regrowth of drug-resistant cells. Studies by our lab and others suggest that Melanoma Initiating Cells (MIC), a CD133+ population that forms xenograft tumors, may represent an intermediate stage in the acquisition of stable drug resistance. This study aims to determine the effect of CD133-expression and drug resistance in metastatic melanoma cells harboring difficult-to-treat mutation signatures by focusing on the two main downstream cascades of MAPK and PI3K/AKT/mTOR. Our results show that triple combination therapy using mebendazole (repurposed anthelmintic BRAF inhibitor), trametinib (MEK inhibitor) and metformin (repurposed anti-diabetic mTOR inhibitor) synergistically reduces cell viability of NRASQ61K melanoma cells when compared with mebendazole, trametinib or metformin alone. Further, the surviving fraction from each monotherapy is highly enriched for CD133+ cells. Thus, the multi-kinase inhibitor trametinib, along with repurposed anthelmintic and anti-diabetic drugs may inhibit MAPK and PI3K/AKT/mTOR pathways, which could potentially be an effective therapy for the resistant subpopulation of melanoma initiating cells.
Citation Format: Maryam AbdusSamad, Anirudh Gaur, Cynthia M. Simbulan-Rosenthal, Edward C. McCarron, Dean S. Rosenthal. Combination therapy with mebendazole, trametinib and metformin eliminates recalcitrant NRASQ61K melanoma cells. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2501.
Most genetic variants associated with type 2 diabetes mellitus (T2DM) have been identified through genome-wide association studies (GWASs) in Europeans. The current study reports a GWAS for ...young-onset T2DM in American Indians. Participants were selected from a longitudinal study conducted in Pima Indians and included 278 cases with diabetes with onset before 25 years of age, 295 nondiabetic controls ≥45 years of age, and 267 siblings of cases or controls. Individuals were genotyped on a ∼1M single nucleotide polymorphism (SNP) array, resulting in 453,654 SNPs with minor allele frequency >0.05. SNPs were analyzed for association in cases and controls, and a family-based association test was conducted. Tag SNPs (n = 311) were selected for 499 SNPs associated with diabetes (P < 0.0005 in case-control analyses or P < 0.0003 in family-based analyses), and these SNPs were genotyped in up to 6,834 additional Pima Indians to assess replication. Rs1861612 in DNER was associated with T2DM (odds ratio = 1.29 per copy of the T allele; P = 6.6 × 10(-8), which represents genome-wide significance accounting for the number of effectively independent SNPs analyzed). Transfection studies in murine pancreatic β-cells suggested that DNER regulates expression of notch signaling pathway genes. These studies implicate DNER as a susceptibility gene for T2DM in American Indians.
Features of melanocortin-4 receptor (MC4R) deficiency have been observed to be more pronounced in childhood. Longitudinal data from a population-based study were used to separate the phenotypic ...effects of MC4R deficiency during childhood and adulthood. The MC4R exon was sequenced in 6,760 individuals of predominantly Pima Indian heritage, and discovered mutations were functionally assessed in vitro. Effects on BMI, height, and slope of BMI change were assessed during childhood (ages 5-20 years) and adulthood (ages 20-45 years). Six mutations affecting MC4R function, including three that may be private to Pima Indians, were found in 159 individuals (2.4%). The slope of BMI increase was greater in individuals carrying an MC4R mutation compared with noncarriers during childhood but not during adulthood. The final adult height obtained was higher in individuals with MC4R deficiency. There was an increased risk for developing type 2 diabetes in individuals with a defective MC4R during childhood and adulthood, but this was only independent of BMI in childhood. The greater rates of body mass accumulation and risk of type 2 diabetes before the age of 20 years in individuals with MC4R deficiency indicate that the effects of these mutations are more apparent during the active growth of childhood.
Abstract
Melanoma is an aggressive and lethal form of cancer, responsible for 9,710 deaths in the US every year, as indicated by the Surveillance Epidemiology and End Results (SEER) program. Although ...early stages are surgically treatable, the advanced or metastatic stages are the most fatal, presumably due to resistance to chemotherapy, radiation or other therapies. Refractoriness of metastatic melanoma is due to self-renewal properties of a subpopulation of resistant cells, which may be the same as what have been termed “melanoma initiating cells” (MIC), many of which have been associated with the expression CD133. CD133 (PROM-1) is a 5-transmembrane-glycoprotein that is a cell surface marker for different cancers including late-stage cutaneous melanoma. By magnetic activated cell sorting, we isolated a CD133-positive population derived from a patient harboring a difficult-to-treat NRASQ61R/BRAFWT profile. The MIC were transfected with CD133-specific small-interfering RNA (siRNAs) to deplete CD133 protein levels in cells. Our results show that knocking down CD133 in NRASQ61R/BRAFWT mutant melanoma renders cells more sensitive to clinically employed-MEK/BRAF inhibitors. Thus, specific gene knockdown or small molecule targeting, along with the multikinase inhibitors, may be a high potency therapeutic of late-stage and recurrent melanoma. Analyses of patient-derived melanomas harboring different mutation signatures are currently in progress to determine if CD133 and MIC play broader roles in melanoma chemoresistance.
Citation Format: Maryam AbdusSamad, Anirudh Gaur, Hengbo Zhou, John L. Zapas, Cynthia M. Simbulan-Rosenthal, Edward C. McCarron, Dean S. Rosenthal. CD133 knockdown sensitizes melanoma to kinase inhibitors. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2015-4224
Abstract
Purpose: High-risk melanoma is a lethal cancer with a high recurrence rates and variable disease-free intervals, due to tumorigenic cell populations that are resistant to treatment. Prominin ...1 (CD133) is a putative stem cell marker in a number of cancers, based on its expression in cells that possess greater tumorigenicity, motility and colony-forming ability in different types of cancer. Furthermore, Prominin 1 is highly expressed in late-stage metastatic melanoma showing its potential as a marker of a resistant cell population. Trametinib and dabrafenib are targeted kinase inhibitors recently approved by the FDA, and now widely used for treatment of melanoma, while the anthelmintic drug mebendazole, a multikinase inhibitor, also exhibits promising anticancer potential. The purpose of this study was to test the relative resistance of CD133+ and CD133- cells to these three agents, as well as to explore potential mechanisms underlying any differences.
Design: Three melanoma cell lines with different kinase mutation profiles were exposed to increasing concentrations of trametinib, dabrafenib, or mebendazole alone, or in combination, either before or after separation into CD133-positive and CD133-negative populations. Cells were then assayed for cell viability and CD133-positivity. Microarray analysis was performed on CD133-positive and CD133-negative cells to explore potential differences in gene expression profiles.
Result: In all parental cell lines, the percentages of the CD133-positive cells increased significantly (P<0.05) after high-dose drug treatment. The CD133-positive, drug resistant cells showed reduced proliferation until after the drug was removed. Correspondingly, the presorted CD133-positive population exhibited significantly higher (P<0.05) IC50s both for single and multidrug treatment. Microarray analysis revealed a significant (P<0.001, >1.5-fold) difference in expression of 265 genes in CD133-positive cells, including putative oncogenes such as POU5F1, NPM1, NES and MYC. Also, 10 of 18 ABC transporter genes were significantly (P<0.05) up-regulated in the CD133-positive population.
Conclusion: Both purified and mixed population-derived CD133 positive cells are more resistant to the three kinase inhibitors. Upregulation of ABC transporter genes and/or oncogenes may contribute to the drug resistance of CD133-positive cells.
Citation Format: Hengbo Zhou, Maryam Abdussamad, Amani Alomari, Anirudh Gaur, Cynthia S. Rosenthal, John L. Zapas, Dean S. Rosenthal. CD133 is associated with resistance of melanoma to multikinase inhibition. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2014-3891
FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are ...also sensitive to MEK inhibition in vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.
A prior genome-wide association study (GWAS) in Pima Indians identified a variant within PFKFB2 (rs17258746) associated with body mass index (BMI). PFKFB2 encodes 6-phosphofructo-2-kinase/fructose ...2,6-bisphosphatase isoform 2, which plays a role in glucose metabolism. To follow-up on the GWAS, tag SNPs across PFKFB2 were genotyped in American Indians (AI) who had longitudinal data on BMI (n = 6839), type 2 diabetes (T2D; n = 7710), diabetic nephropathy (DN; n = 2452), % body fat (n = 555) and insulin secretion (n = 298). Two SNPs were further genotyped in urban AI to assess replication for DN (n = 864). PFKFB2 expression was measured in 201 adipose biopsies using real-time RT-PCR and 61 kidney biopsies using the Affymetrix U133 array. Two SNPs (rs17258746 and rs11120137), which capture the same signal, were associated with maximum BMI in adulthood (β = 1.02 per risk allele, P = 7.3 × 10(-4)), maximum BMI z-score in childhood (β = 0.079, P = 0.03) and % body fat in adulthood (β = 3.4%, P = 3 × 10(-7)). The adiposity-increasing allele correlated with lower PFKFB2 adipose expression (β = 0.81, P = 9.4 × 10(-4)). Lower expression of PFKFB2 further correlated with higher % body fat (r = -0.16, P = 0.02) and BMI (r = -0.17, P = 0.02). This allele was also associated with increased risk for DN in both cohorts of AI odds ratio = 1.64 (1.32-2.02), P = 5.8 × 10(-6), and similarly correlated with lower PFKFB2 expression in kidney glomeruli (β = 0.87, P = 0.03). The same allele was also associated with lower insulin secretion assessed by acute insulin response (β = 0.78, P = 0.03) and 30-min plasma insulin concentrations (β = 0.78, P = 1.1 × 10(-4)). Variation in PFKFB2 appears to reduce PFKFB2 expression in adipose and kidney tissues, and thereby increase risk for adiposity and DN.
Aim/hypothesis
A recent genome-wide trans-ancestry meta-analysis identified seven new loci associated with type 2 diabetes. We assessed the replication of the seven lead single nucleotide ...polymorphisms (SNPs) and evaluated these loci for additional signals in American Indians.
Methods
Seven SNPs were genotyped in 7,710 individuals from a longitudinally studied American Indian population, and associations with type 2 diabetes, BMI and related phenotypes were assessed. Previous genome-wide association study (GWAS) data from these individuals were used to screen for additional type 2 diabetes signals at these loci. A variant independent of the trans-ancestry meta-analysis was identified within
LPP
, and its replication was assessed in an additional 3,106 urban American Indians.
Results
SNP rs6813195 near to
TMEM154
was nominally associated with type 2 diabetes (
p
= 0.01, OR 1.12 95% CI 1.03, 1.22) and adiposity: the type 2 diabetes risk allele was associated with a lower percentage body fat (β = −1.451%,
p
= 4.8 × 10
−4
). Another SNP, rs3130501 near to
POU5F1–TCF19
, was associated with BMI (β = −0.012,
p
= 0.004), type 2 diabetes adjusted for BMI (
p
= 0.02, OR 1.11 95% CI 1.02, 1.22), 2 h glucose concentrations (β = 0.080 mmol/l,
p
= 0.02) and insulin resistance estimated by homeostatic model (β = 0.039,
p
= 0.009). The independent variant identified at the
LPP
locus in our American Indian GWAS for type 2 diabetes was replicated in the additional samples (all American Indian meta-analysis,
p
= 8.9 × 10
−6
, OR 1.29 95% CI 1.15, 1.45).
Conclusions/interpretation
For two of the seven newly identified variants, there was nominal evidence for association with type 2 diabetes and related traits in American Indians. Identification of an independent variant at the
LPP
locus suggests the existence of more than one type 2 diabetes signal at this locus.