Loss of the Y chromosome (LOY) is observed in multiple cancer types, including 10–40% of bladder cancers1,2,3,4,5,6, but its clinical and biological significance is unknown. Here, using genomic and transcriptomic studies, we report that LOY correlates with poor prognoses in patients with bladder cancer. We performed in-depth studies of naturally occurring LOY mutant bladder cancer cells as well as those with targeted deletion of Y chromosome by CRISPR–Cas9. Y-positive (Y+) and Y-negative (Y–) tumours grew similarly in vitro, whereas Y− tumours were more aggressive than Y+ tumours in immune-competent hosts in a T cell-dependent manner. High-dimensional flow cytometric analyses demonstrated that Y− tumours promote striking dysfunction or exhaustion of CD8+ T cells in the tumour microenvironment. These findings were validated using single-nuclei RNA sequencing and spatial proteomic evaluation of human bladder cancers. Of note, compared with Y+ tumours, Y− tumours exhibited an increased response to anti-PD-1 immune checkpoint blockade therapy in both mice and patients with cancer. Together, these results demonstrate that cancer cells with LOY mutations alter T cell function, promoting T cell exhaustion and sensitizing them to PD-1-targeted immunotherapy. This work provides insights into the basic biology of LOY mutation and potential biomarkers for improving cancer immunotherapy.
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This work was supported in part by NIH P01CA278732 and R01CA143971 to D.T. and NIH R01CA262069, R01CA262388 and R01AI077283 to Z.L. J.M.S. and T.D.G. were supported by the Ohio State University Comprehensive Cancer Center’s Tumor Immunology T32 (2T32CA09223-16A1) post-doctoral fellowship award. We thank K. Walsh for providing the LOY gRNA plasmids, and N.-J. Song and B. Riesenberg for development of the ‘all immune phenotyping’ flow antibody panel. We acknowledge resources from the Immune Monitoring and Discovery Platform and the Pelotonia Institute for Immuno-Oncology at OSU Comprehensive Cancer Center (P30CA016058).
Z.L. reports personal consultation fees from Alphamab, HanchorBio, Henlius, Heat Biologic and Ikonisys outside the submitted work. All other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 LOY is associated with a worse clinical outcome for patients with MIBC and NMIBC.
a, Y chromosome genes expressed in normal bladder urothelium that were used to create a Y chromosome gene expression signature. b, Logrank p-values based on stratification by Y chromosome gene expression (normalized FPKM) on TCGA MIBC patient overall survival (OS). Genes resulting in statistically significant OS are plotted in panel c. NE, not expressed. c, Kaplan-Meier plots of OS from TCGA data for males with MIBC and either high or low KDM5D, TBL1Y, UTY (KDM6C), or ZFY expression. d, Kaplan-Meier survival curves stratified by the Y signature score or expression levels for UTY and KDM5D in NMIBC from the E-MTAB-4321 cohort. Survival differences are based on Logrank statistics. e, ChrY gene expression signature scores of TCGA data plotted with respect to extreme downregulation of chromosome Y (EDY, left panel) and Mosaic Alteration Detection for LOY (mLOY, right panel) levels. Statistical significance was determined by Wilcoxon rank-sum test (NoLOY n = 151, LOY n = 90, NoEDY n = 165, EDY n = 76). Boxplots represent the mean with first and third quartile data. Minimum and maximum datapoints are included.
a, Histogram representation of deferentially regulated genes (DEG) from Y+ vs. Y- MB49 RNAseq data per mouse chromosome. b, qRT-PCR analysis of Uty, Kdm5d, Eifs3y, and Ddx3y expression in MB49 clones isolated from the parental MB49 compared to female murine breast cancer (E0771) and bladder cancer cells (NA13), and testis tissue. Curly brackets indicate the clonal lines used to generate the pooled Y+ and Y- MB49 sublines. c, qRT-PCR analysis of Uty and Kdm5d expression in the pooled Y+ and Y- sublines described in a. n = 3 biological replicates. Data are mean ± s.e.m. d, Bar graph of sequencing depth for each chromosome after performing whole exome sequencing (WES) on DNA from the Parental, Y-, and Y+ MB49 cell lines.
a, MB49 Y+ and Y- cells were grown in 0.4% agar for two weeks. Colonies were stained with Nitro-BT and quantified using ImageJ. Average colony number and area were determined from those with a diameter that exceeded 100 µm (n = 4 biological replicates). Data representative of three independent experiments. Statistical significance was determined by two-sided unpaired t-test, P-value = 0.722. Data are mean ± s.e.m. b, In vitro cell proliferation (MTT cell viability) over a 6–8-day time course using three sets of genetically engineered MB49 cells: Y+, Y+ Kdm5d KO, Y+ Uty KO (left panel), Y-, Y- Kdm5d OE, Y- Uty OE (middle panel), and CRISPR-Y-Scr vs. CRISPR-Y-KO (right panel). n = 3 biological replicates. Data are mean ± s.e.m. c, qRT-PCR analysis of Uty expression in MB49 clones isolated from the CRISPR-generated Y-KO and Y-Scr MB49 cell lines. Curly brackets indicate the clonal lines used to generate the pooled Y+ Control and Y- KO MB49 sublines. Representative immunofluorescence images of the CRISPR-generated Y-KO and Y-Scr MB49 cell lines. Scale bar, 150 µM.
a, Volcano plot of DEGs from bulk RNA isolated from Y+ and Y- MB49 tumors grown in male WT mice. Blue (Y+ tumors) and red (Y- tumors) genes correspond to statistically significant (Benjamini-Hochberg method, P < 0.05) genes that have a | >1 log2 | fold-change in expression. b, PCA of DEGs described in a. c, Gene ontology (GO) pathway enrichment score plots of statistically significant gene set enrichment analyses (GSEA) using DEGs from a. NES, normalized enrichment score.
Extended Data Fig. 5 Comprehensive immune phenotyping of tumor-infiltrating leukocytes (TILs) in Y+ and Y- MB49 tumors.
a, UMAPs demonstrating individual spectral flow cytometry analysis of protein marker expression in CD45+ immune cells isolated from Y+ and Y- MB49 tumors grown in WT male mice. b, Heatmap of relative protein expression from immune cells described in a. c, Violin plots of each tumor sample across each cluster from the CD45+ immune cell UMAP (see Fig. 3b). d, Violin plot of PD-1 and PD-L1 mean fluorescence intensity in CD45+ immune cells from Y+ and Y- MB49 tumors. e, Representative dot plots and percentages of CD8+ and CD4+ T cells gated on total CD3+ T cells from CRISPR-Y-Scr (n = 8) and CRISPR-Y-KO (n = 9), MB49 tumors grown for 22 or 17 days, respectively, in male WT mice (left panels). Percentage of CD8+ T cells of total CD3+ T cells per tumor sample (right panel). f, Percentage of CD206+PDL1+ macrophages among total CD11b+F4/80+ macrophages from Control and Y KO MB49 tumors described in e. Statistics were determined using two-sided unpaired t-tests.
Extended Data Fig. 6 GeoMX histological evaluation of infiltrating immune cells in Y- and Y+ MB49 tumors.
a, Table of markers that are functionally categorized for GeoMX evaluation of Y+ and Y- MB49 tumors. b, Representative H&E image (left), immunofluorescence detection of nuclei (blue), cytokeratin (green), CD45+ immune cells (red) (middle), and associated computational digital profiling (right) to quantify markers shown in a. Scale bar, 125 µM. c, Quantification (log2 fold change and P-value) of the markers listed in Y+ versus Y- MB49 tumors (n = 10 tumors per group and three TMA cores per tumor). Data representative of two independent experiments. Statistical significance was determined by two-sided unpaired t-test.
Extended Data Fig. 7 Characterization of tumor-infiltrating CD8+ T cells after PD-1 pathway blockade.
a–c, Relative spectral flow protein expression (a), sample-level violin plots per cluster (b), and heatmap of individual targets per cluster (c) after 200 μg anti-PD-1 or isotype control IgG treatments for 7 days using CD8+ T cells from Y+ and Y- MB49 tumors. d, Representative dot plots and percentages of TOX and/or GZMB-expressing CD8+ T cells from CRISPR-Y-Scr and CRISPR-Y-KO MB49 tumors grown in male WT mice after 200 μg anti-PD-1 or isotype control IgG treatments for 7 days. e–f, Percentage of PD1+TOX+ CD8+ T cells (e) and TOX−CD44+ (top panel) or TOX−ICOS+ (bottom panel) CD8+ T cells (f) from tumor samples described in d. See Fig. 5 for additional method details. Statistical significance was determined by two-sided unpaired t-test. Tests were conducted between isotype controls or between isotype controls and anti-PD1 treatment groups.
a, Heatmap of the indicated pathways and metadata from BC TCGA data. b–c, box plot of tumor neoantigen burden (TNB) per megabase (P = 0.700) (b), and associated pathway enrichment levels (c) from Yhigh and Ylow tumors described in Fig. 1a. Statistical significance was determined by Wilcoxon test (Ylow n = 118 and Yhigh n = 182). Boxplots represent the mean with first and third quartile data. Minimum and maximum datapoints are included.
Normalized enrichment scores of statistically significant GSEA GO pathways using DEGs from Y- vs. Y+ MB49 cell cultures. Purple color denotes DNA repair-related pathways enriched in Y- cells.
Genome instability pathway enrichment scores using RNA-seq data from control and genetically modified MB49 cell lines (Y+ and Y- cells, n = 5 technical replicates. n = 3 for all other cell lines). Two-sided unpaired t-test. Boxplots represent the mean with first and third quartile data. Minimum and maximum datapoints are included.
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Abdel-Hafiz, H.A., Schafer, J.M., Chen, X. et al. Y chromosome loss in cancer drives growth by evasion of adaptive immunity. Nature 619, 624–631 (2023). https://doi.org/10.1038/s41586-023-06234-x
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