Single-cell proteomics defines the cellular heterogeneity of localized prostate cancer

Abstract

Localized prostate cancer exhibits multiple genomic alterations and heterogeneity at the proteomic level. Single-cell technologies capture important cell-to-cell variability responsible for heterogeneity in biomarker expression that may be overlooked when molecular alterations are based on bulk tissue samples. This study aims to identify prognostic biomarkers and describe the heterogeneity of prostate cancer and the associated microenvironment by simultaneously quantifying 36 proteins using single-cell mass cytometry analysis of over 1.6 million cells from 58 men with localized prostate cancer. We perform this task, using a high-dimensional clustering pipeline named Franken to describe subpopulations of immune, stromal, and prostate cells, including changes occurring in tumor tissues and high-grade disease that provide insights into the coordinated progression of prostate cancer. Our results further indicate that men with localized disease already harbor rare subpopulations that typically occur in castration-resistant and metastatic disease.

Keywords: cellular heterogeneity; prostate cancer; single-cell proteomics.

Graphical abstract

Figure 1

Schematic of method for characterization of primary prostate cancer tissue using mass cytometry (A) The patient cohort consisted of 58 primary prostate cancer cases. For 16 patients, tumor and adjacent benign prostatic tissue (ABPT) samples were available. The remaining samples were from randomly selected regions from a prostatectomy without tumor assessment. Samples were analyzed by mass cytometry, and data were analyzed using Franken. (B) Markers used to categorize prostate epithelial cells as luminal, basal, or transitional, and markers used to identify tumor cells, cells from the microenvironment, and functional features, such as proliferation, apoptosis, or hypoxia.

Figure 2

Prostate cancer samples have similar overlapping phenotypic profiles (A) Heatmap of scaled mean signal of marker expression in 55 Franken clusters; numbers are colored according to metaclusters resulting from hierarchical clustering merging (using Pearson correlation dissimilarities) of Franken clusters. Bar plot below the heatmap corresponds to the number of cells found in each cluster. (B) UMAP map of 23,200 (400 per patient) cells colored by cellular metacluster as indicated in (A). (C) UMAP map of 23,200 (400 per patient) cells colored by patient. (D and E) Boxplots of frequencies of the main cell types across all 58 samples from tumor, ABPT, and RPT. Significant changes were seen between tumor and ABPT in the proportion of granulocytes (two-sided Wilcoxon signed rank paired test, p = 0.008). N = 17. (E) Boxplots of frequencies of the main cell types in samples from all 58 patients in our cohort stratified by intermediate- and high-grade tumors. Changes in luminal and T cell compartments are significant according to a two-sided Mann-Whitney-Wilcoxon test (p = 0.028 and 0.014, respectively). Intermediate N = 46 and high grade N = 12.

Figure 3

Stratification of samples reveals prostate tissue changes associated with tumor and advanced disease (A) UMAP of 23,200 cells (400 per patient) colored by expression of indicated marker. (B) Representative tissue sample stained for CD3 from a tissue microarray generated from prostate samples from the same cohort analyzed by mass cytometry. (C) Densities of T cells as determined by CD3 staining (p = 0.042). (D) Representative tissue sample stained for CD15 from a tissue microarray generated from prostate samples from the same cohort analyzed by mass cytometry. (E) Densities of granulocytes as determined by CD15 staining (p = 0.058). Scale bar, 50 μm. (F) Proportion of T cell metaclusters in ABPT and tumor samples across patients with paired samples (p = 0.066, 0.169, 0.023, 0.002, and 0.332 for TC01–05, respectively). N = 17. (G) Summary table of clusters that were significant when comparing ABPT and tumor samples (N = 17 for both groups). Metaclusters enriched in ABPT are colored in blue, while those enriched in tumor samples are colored in red. Comparison between intermediate- and high-grade patient samples (for combined tumor/ABPT; intermediate N = 46 and high grade N = 12). Metaclusters enriched in patients with high-grade disease are colored in dark red. Only significant relationships are colored, and remaining comparisons are shown in gray. (H) Proportion of macrophage metaclusters in ABPT and tumor samples across patients with paired samples (p = 0.095, 0.169, 0.515, 0.0004, and 0.014 for MA01–05, respectively). N = 17. In panels (F) and (H), dots are colored by disease severity (intermediate versus high grade). In all boxplots, boxes illustrate the IQR (25th to 75th percentile), the median is shown as the middle band, and the whiskers extend to 1.5 times the IQR from the top (or bottom) of the box to the furthest datum within that distance. Statistical testing between dependent paired tumor and ABPT samples was done using a Wilcoxon signed rank paired-sample statistical tests (two-sided). Independent intermediate- and high-grade samples were tested using a two-sided Wilcoxon rank-sum test. Number of patients in each group is indicated by N.

Figure 4

Characterization of epithelial tumor clusters and patient groups (A) Bar indicating which metaclusters were significantly enriched between three pairs of conditions: (top bar) high- and intermediate-grade samples, irrespective of tumor status; (middle bar) tumor versus ABPT; (bottom bar) intermediate- and high-grade tumor regions only. All significant nominal p values are indicated given their level of significance for the figure, and those still significant after Bonferroni correction are indicated by a dark black outline. p values for all tested hypotheses are given in Table S1. (B) Pairwise Tanimoto similarity of intermediate- (I) and high-grade (H) tumor (T) and benign tumor-adjacent (TA) samples for metaclusters in the microenvironment and epithelium. Color intensity and the size of the circle are proportional to the Tanimoto similarity. (C) Correlation of metaclusters across 17 tumor patient samples. Correlations in the paired adjacent benign tissue that were lost in tumor are indicated by an L in the correlation plot, while correlations that were gained are indicated by a G. Only Spearman correlations with a significance level <0.05 are shown to exclude spurious correlations. Metacluster labels are colored to reflect cell types as in Figure 2A. (D) UMAP projections of 23,200 cells (400 cells per patient) colored by expression of indicated epithelial and prostate-specific markers. Maximum signal (=1) is shown in gray. (E) CD15 and p63 co-stained showing CD15 expression in epithelial cells from patients with acinar (left) and with ductal (middle) carcinoma as well as absence of CD15 in normal glands (right) showing basal cell layer expressing p63. Scale, 25 μm. (F) Number of patients with cells belonging to a specific metacluster. Colors and labels matched to panels (A) and (C). (G) Grouped patient samples represented by proportion of metaclusters. Colors in bar plot reflect those on panels (A) and (C).