Fig 1: CyC directs recruitment of TREM2+ macrophages and promotes failure of cancer immunotherapy(A) Tumor growth curves (mean and standard error of the mean) for single-flank sgScrambled (n = 8) and CST3-/- (CST3 knockout [KO], n = 8) tumors; 100,000 cells were inoculated in right flank (cohort A).(B) Tumor growth curves (mean and standard error of the mean) for biflank paired sgScrambled (n = 5) and CST3-/- (n = 5) tumors; 50,000 cells were inoculated in both flanks (cohort C). Mice received three doses of anti-PD-L1 antibody. p values refer to paired two-sided t tests.(C and D) Proliferation index (proportion of Ki67+ cells/total cells) (C) and proportion (D) of non-epithelial cells in histological sections from paired biflank sgScrambled and CST3-/- tumors (pooled cohorts C and D). p values refer to paired two-sided t tests.(E) Uniform manifold approximation and projection (UMAP) of 14,416 cells, annotated with cell type, from 4 tumor samples (2 sgScrambled, 2 CST3-/-).(F) Proportion of Trem2+ macrophages in sgScrambled and CST3-/- tumors; p value is adjusted p value from linear model of logit-transformed proportions.(G) Number of Trem2+ cells per mm2 from digital image analysis of Trem2 immunohistochemistry in paired biflank sections from sgScrambled and CST3-/- tumors. p value refers to paired two-sided t test.(H) Multivariate (Cox and logistic) regression of Z scored CyC-production PGS against immuno-oncology biomarkers (progression-free survival [PFS], overall survival [OS], durable clinical benefit [DCB]) in meta-analysis of European patients (n = 685) treated with checkpoint immunotherapy (anti-CTLA4 or anti-PD1/PD-L1). Sample sizes for each clinical endpoint were n = 342, 685, and 670, respectively. In each model, covariates included PC1–4, sex, and primary cancer. Error bars reflect 95% confidence interval. Lower hazard ratios (survival, Cox regression) or higher odds ratios (durable clinical benefit, logistic regression) reflect better therapeutic outcomes (annotated with purple arrow).(I) Sensitivity analysis indicating odds ratio and 95% confidence interval for DCB in each cancer type. p values refer to two-sided t tests unless otherwise stated. *p < 0.05; **p < 0.01, ***p < 0.001.Boxplots show median (central line) with interquartile range (IQR; box) and extrema (whiskers at 1.5× IQR).
Fig 2: CyC production is dynamically regulated in disease states(A and B) Significant (p < 0.05) changes in paired (A) CST3 gene expression (reverse transcription PCR) and (B) extracellular CyC concentration in PMA-treated human THP-1 cells (macrophage-like) normalized to cellular protein content during 0- to 18-h DEX (100 nM) treatment. There are 6 biological replicates per group.(C) Change in normalized extracellular CyC concentration in macrophage-like THP-1 cells after 18-h treatment with DEX (100 nM) or VEH control.(D and E) Creatinine-normalized plasma CyC (C2 ratio) at specific time points with sufficient data in hospitalized COVID-19 patients treated with DEX or standard of care (control [CTRL]) as part of cohorts based in (D) Calgary, Canada, and (E) Charité Hospital, Germany. Day 1 in the Calgary, Canada, cohort refers to a time window of 72 h after admission to the ICU. Error bars indicate standard error of the mean.(F) Single-cell CST3 gene expression in each cell cluster in melanoma tumors (n = 12) from Jerby-Anon et al.64 Clusters defined by correlation to reference PBMC data,60 with unclassified cells that exhibit detectable clonal copy-number variation classified as tumors.(G) Plasma CyC concentration in BALBc mice after inoculation with colon-26 (C26) tumor cells. Cachexia is defined by >15% body weight loss, and pre-cachexia refers to 14 days after tumor inoculation; tumor bearing refers to day 7 after tumor inoculation.(H) Significant positive correlation between plasma corticosterone and plasma CyC during tumor progression in C26 model.(I) Extracellular CyC concentrations in C26 cells normalized to cellular protein content after 0-, 6-, 12-, 18-, or 24-h treatment with 100 nM DEX. Each time point comprises at least 4 biological replicates. p values refer to two-sided t tests. VEH, vehicle; DEX, dexamethasone.
Fig 3: CyC is predominantly produced by myeloid cells in health(A) Tissue-specific expression quantitative trait score (eQTS) analysis to identify tissues with significant correlation (Spearman coefficient) between CyC-production PGS and tissue-specific CST3 gene expression in GTEx cohort. p values are uncorrected, as each correlation test is performed in a non-overlapping set of tissue-specific samples.(B and C) Distribution of normalized single-cell CST3 expression (log-transcripts per million [TPM]) in cell clusters isolated from (B) spleen and (C) peripheral blood mononuclear cells (PBMCs). Clusters defined by correlation to reference PBMC data.60(D) Mean CST3 gene expression (log-TPM) in each cell cluster from multiple tissue-specific single-cell RNA sequencing projects, harmonized by Human Protein Atlas. The top cell cluster and tissue-specific macrophage cell type (if not top cluster) by tissue is annotated.(E) Two-sample Mendelian randomization using blood-specific cis-eQTLs for CST3 (eQTLGen) as exposure and CyC-production latent trait GWAS as outcome. Error bars correspond to standard errors, and point color refers to linkage with top cis-eQTL.(F and G) Non-significant (p > 0.05) changes in (F) CST3 gene expression (reverse transcription PCR) during 0- to 18-h DEX (100 nM) treatment and (G) extracellular CyC concentration in human THP-1 cells (monocyte-like) normalized to cellular protein content after 18-h treatment with DEX (100 nM) or VEH control.(H and I) Each condition comprises 10 biological replicates. Plasma CyC concentration in healthy (H) BALB/cJ and (I) C57BL/6J mice treated with VEH or 20 mg/kg DEX.(J) Creatinine-normalized plasma CyC (C2 ratio) in patients with primary adrenal insufficiency treated with placebo (VEH) or hydrocortisone (CORT) in a crossover experimental medicine study. The administered intranveous (i.v.) CORT dose was 0.03 mg/kg/h between 12 and 7 a.m. (the time point of sampling), achieving near-physiological GC exposure.(G–J) p values refer to two-sided t tests. VEH, vehicle; DEX, dexamethasone.
Fig 4: CyC is a glucocorticoid response gene in vitro(A) Co-localization of summary statistics for CyC-production from UKB and plasma cortisol from CORNET Consortium at SERPINA1/6 locus. rs2749527 variant is highlighted in red.(B) Trans-eQTL analysis examining association between genetic instrument rs2749527 and CST3 gene expression in visceral adipose fat (VAF) in STARNET cohort.(C) Cis-eQTL association between rs2749527 and SERPINA6 (encodes cortisol-binding globulin) in liver in STARNET cohort. p values for additive and recessive models are shown. See Figure S3 for replication analysis in GTEx.(D) Gene set enrichment analysis (MAGMA) across CyC-production summary statistics (UKB) for steroid signaling-related gene sets.(E) Functional genomics in A549 cell line (ENCODE project) treated with 100 nM dexamethasone for 0 min to 12 h. ChIP-seq (for glucocorticoid receptor/NR3C1) and ATAC-seq (at 0 h) at CST3 locus identifies a glucocorticoid-responsive and accessible distal enhancer element.(F and G) Time course of (F) GR recruitment (at distal enhancer) and (G) CST3 gene expression (log-CPM) following dexamethasone (DEX) treatment in A549 cells (ENCODE project). Trendline and shaded 95% confidence interval correspond to regression of gene expression as a function of log-time.(H and I) Extracellular CyC concentration in (H) A549 cells and (I) HeLa cells normalized to cellular protein content after 18-h treatment with 100 nM DEX or vehicle (VEH) control. Each condition comprises at least 5 biological replicates; horizontal bars indicate mean extracellular CyC. p values correspond to two-sided t tests. See Figure S3 for timecourse.(B and C) Boxplots show median (central line) with IQR (box) and extrema (whiskers at 1.5× IQR). Outliers beyond 1.5× IQR are shown as dots. SH, steroid hormone; SP, signaling pathway; VEH, vehicle; DEX, dexamethasone.
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