Cystatin C is glucocorticoid responsive, directs recruitment of Trem2+ macrophages, and predicts failure of cancer immunotherapy

Abstract

Cystatin C (CyC), a secreted cysteine protease inhibitor, has unclear biological functions. Many patients exhibit elevated plasma CyC levels, particularly during glucocorticoid (GC) treatment. This study links GCs with CyC's systemic regulation by utilizing genome-wide association and structural equation modeling to determine CyC production genetics in the UK Biobank. Both CyC production and a polygenic score (PGS) capturing predisposition to CyC production were associated with increased all-cause and cancer-specific mortality. We found that the GC receptor directly targets CyC, leading to GC-responsive CyC secretion in macrophages and cancer cells. CyC-knockout tumors displayed significantly reduced growth and diminished recruitment of TREM2+ macrophages, which have been connected to cancer immunotherapy failure. Furthermore, the CyC-production PGS predicted checkpoint immunotherapy failure in 685 patients with metastatic cancer from combined clinical trial cohorts. In conclusion, CyC may act as a GC effector pathway via TREM2+ macrophage recruitment and may be a potential target for combination cancer immunotherapy.

Keywords: GWAS; PGS; cystatin C; glucocorticoids; immunotherapy; macrophages; renal function.

Graphical abstract

Figure 1

Genomic architecture of cystatin C production (A) Schematic of study plan. The analysis of CyC-production latent trait in UK Biobank (UKB) is leveraged to determine the biological and clinical relevance of CyC. (B) Consort diagram and summary of UKB genome-wide association analysis strategy in the European ancestry population. The software packages utilized for each step are displayed in red. (C) Structural equation model to estimate latent traits of CyC production and renal function. The model schematic, heritability (h²) of eGFR-creatinine and eGFR-CyC, and their genetic correlations derived from LD score regression are shown. Circular arrows refer to variance of each component, and dashed lines refer to covariance between components. RF, renal function. (D) Latent trait effect sizes (CyC production and renal function) for single-nucleotide polymorphisms (SNPs) corresponding to each clumped locus in eGFR-CyC summary. Gene names are annotated per OpenTargets V2G pipeline. (E) Linear model of eGFR-CyC as a function of eGFR-creatinine across all paired blood samples in UKB, including sex as a covariate. The deviation of the eGFR-CyC from the linear fit as indicated by the red arrow is defined as the CyC residual, a surrogate for CyC production. p value refers to Pearson correlation test. (F) Correlation of CyC residual with CyC-production polygenic score (PGS). The continuous PGS has been converted into deciles (1 = lowest, 10 = highest PGS). Only data from the independent validation set (see B) were used. Boxplots show median (central line) with interquartile range (IQR; box) and extrema (whiskers at 1.5× IQR). p value refers to Pearson correlation test.

Figure 2

CyC production is associated with multiple disease states and is prognostic in patients with cancer (A) Multivariate Cox regression to measure effect size for CyC residual on overall survival in UKB. Covariates included age, sex, body mass index (BMI), hemoglobin, C-reactive protein, eGFR-creatinine, and operation status (for cancer-specific subanalysis). Error bars indicate 95% confidence interval. (B) Multivariate Cox regression to measure effect size for CyC-production PGS on subject and parental lifespan in UKB validation cohort. Covariates included PC1-4, recruitment center, genotyping array, year of birth of subject, and sex of subject (if applicable). (C) Phenome-wide association (Cox regression) between CyC-production PGS and 694 time-to-event phenotypes in UKB validation cohort. Covariates included principal components 1–4 (PC1–4), year of birth, and sex. (D and E) Multivariate Cox regression to measure effect size for CyC-production PGS on disease-specific survival for specific cancers in (D) UKB validation cohort (cancers diagnosed since 2000, n = 3,954) and (E) TCGA cohort (n = 4,368). Covariates included PC1–4, age, sex, and a term reflecting whether the patient had curative surgery. Error bars indicate 95% confidence interval; gray squares indicate sample size.

Figure 3

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.

Figure 4

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. (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.

Figure 5

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. Clusters defined by correlation to reference PBMC data, 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.

Figure 6

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).