Jia-Ren Lin

The spatial arrangement of immune cells in the tumor microenvironment (TME) varies widely, from dispersed to clustered and tumor-excluded to infiltrating. Multiplexed spatial profiling is an effective means of characterizing tumor-infiltrating lymphocytes and immune complexes such as tertiary lymphoid structures (TLS) in the TME. However, few approaches have been described for objectively parametrizing patterns of immune organization and assessing their association with biological or clinical variables. This makes it difficult to evaluate whether a set of tumors is relatively immunologically cold or hot. In this study, we describe an intuitive set of statistical tools (available in the R package, tlsR) for characterizing lymphocyte patterns in the TME of solid cancers. We applied tlsR to primary prostate cancer, which is often described as immunologically “cold.” Using a cohort of 29 radical prostatectomy specimens stratified into low Gleason grade (LGG; n = 15) and high Gleason grade (HGG; n = 14), we showed that HGG prostate cancer is significantly more infiltrated than LGG prostate cancer, with lymphocytes organized into B or T cell–enriched immune clusters (IC). A subset of these ICs had the B- and T-cell zonation and follicular dendritic cells characteristic of bona fide TLSs. HGGs were also enriched with ICs containing precursors of exhausted T cells and proliferating B cells, and their tumor compartments harbored granzyme B+ cytotoxic T cells in contact with cancer cells. Thus, far from being cold, a subset of HGG prostate cancer has features associated with active immune surveillance, a finding with implications for emerging prostate cancer immunotherapies.

Oxidative stress response (OSR) is suggested as a therapeutic target in triple negative breast cancer (TNBC). However, the cell type specificity and spatial distribution of OSR genes and proteins remain poorly defined. We re-analyzed public datasets, demostrating high OSR gene expression in TNBC tumor cells. We generated a multiplex immunofluorescence dataset of 345 TNBC samples that showed OSR proteins GCLC and NQO1 are differentially expressed across tumor, immune, and stromal cells, and elevated in inflamed regions. We identified a mesenchymal-like tumor cell state characterized by co-expression of GCLC and vimentin (GCLC⁺VIM⁺), with increased proliferation and reduced anastasis-associated signatures, suggesting a vulnerability to chemotherapy. Consistently, TNBC cell lines with higher GCLC expression showed increased sensitivity to cisplatin. Moreover, patients with higher proportions of GCLC⁺VIM⁺ tumor cells had longer overall survival. Our findings indicate that OSR proteins are expressed in spatially and phenotypically distinct tumor microenvironments, and that the GCLC⁺VIM⁺ tumor cell state is associated with favorable outcomes in TNBC.

Mucosal melanoma is a rare, lethal melanoma subtype with distinct genetics and limited benefit from immunotherapy. To define features associated with mucosal melanoma clinical outcomes, we assembled a large, multimodally profiled cohort spanning primary and metastatic disease. Compared with cutaneous melanoma, primary mucosal melanoma showed greater genomic heterogeneity, ploidy and aneuploidy, and a more dedifferentiated, proliferative transcriptional state. Analysis of genomic sequencing of 339 patients with mucosal melanoma identified four mutually exclusive genomic subtypes: CDK4/MDM2/CCND1, SF3B1-mutated, TP53/ATRX, and mucosal melanoma Triple WT, associated with distinct anatomic sites but similar outcomes. In the tumor microenvironment, we observed reduced T cell infiltration and increased CD163 ⁺ tumor-associated macrophages in mucosal melanoma relative to cutaneous melanoma. In matched longitudinal samples, CD163 ⁺ tumor-associated macrophages increased with disease progression and immunotherapy resistance. CD163 ⁺ tumor-associated macrophages engaged non-regulatory T cells through GAL9–TIM-3 interactions, nominating TIM-3 blockade as a therapeutic strategy for mucosal melanoma. These findings define the distinct aggressive biology of mucosal melanoma and highlight potential therapeutic targets and mechanisms of immune resistance.

The RNF43 p.G659fs ( RNF43 ^659mut ) mutation occurs in 8% of colorectal cancers (CRCs) and is enriched in microsatellite instability–high (MSI-H) tumors. Although RNF43 ^659mut promotes tumor growth through phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) activation independent of WNT signaling, its immunologic effects are poorly defined. We show that RNF43 ^659mut shapes an immunosuppressive natural killer (NK) cell landscape in MSI-H CRC. Single-cell RNA sequencing and multiplex immunohistochemistry revealed increased NK cell infiltration in RNF43 ^659mut tumors, yet these cells displayed inhibitory phenotypes. Bulk RNA sequencing and flow cytometry of NK cells cocultured with RNF43 ^659mut isogenic lines and MSI-H patient-derived organoids demonstrated reduced functional NK subsets, decreased activation markers, increased inhibitory receptors, and impaired cytotoxicity. Mechanistically, RNF43 ^659mut -mediated PI3K/AKT activation induced HLA-E expression, suppressing NK cell function. Pharmacological or small interfering RNA–mediated PI3K/AKT inhibition restored NK cell activity and enhanced tumor cell killing. Spatial analysis of MSI-H patient tumors revealed close proximity between NKG2A ⁺ NK cells and HLA-E ⁺ tumor cells, which were themselves adjacent to PI3K/AKT-activated tumor cells, highlighting a localized immunosuppressive niche. These findings uncover an immune evasion mechanism in MSI-H CRC, implicating the PI3K/AKT-HLA-E/NKG2A axis as a promising therapeutic target to overcome immunotherapy resistance.

Given the heterogeneity of HER2-positive breast cancer, reliable biomarkers to guide treatment are needed. We conducted multi-platform biomarker analyses of baseline tumor tissue from the DAPHNe trial of neoadjuvant paclitaxel/trastuzumab/pertuzumab (THP) for HER2-positive early breast cancer to characterize inter- and intra-tumor heterogeneity and to identify molecular predictors of response. A total of 98 patients with stage II-III HER2-positive breast cancer received neoadjuvant THP followed by surgery. Gene expression profiling, spatial protein profiling, and single-cell imaging (cyclic immunofluorescence) were performed on pre-treatment biopsies and a subset of residual disease specimens. Pathologic response was assessed using the residual cancer burden (RCB) score. Among HER2-positive patients included in the biomarker analysis, 34% of patients had node-positive breast cancer, and 66% had hormone receptor (HR)-positive tumors. HR-positive and HR-negative tumors differed significantly across gene expression, protein expression, and single-cell profiling. High ERBB2 gene signature, p53 gene signature, and HER2 protein expression predicted favorable response (RCB 0/1), while ESR1/PGR gene, estrogen receptor (ER) signaling gene signature, and ER alpha protein expression were associated with unfavorable response (RCB 2/3). Single-cell spatial analysis revealed that cancer cells clustered by shared HER2, ER, and PR expression, suggesting local expansion of phenotypically distinct subpopulations. ER-positive cancer cells were associated with lower HLA-A and PD-L1 expression, suggesting a less immunologically active tumor cell state. Baseline HER2 and ER expression are key predictors of response to neoadjuvant HER2-targeted therapy. Single-cell spatial profiling highlights intra-tumoral heterogeneity and suggests ER-driven immune exclusion as a potential mechanism of resistance.

Supplementary Figures provide additional evidence and validation of copy number changes from the ORION-FISH assay.

Table S1 provides the details of antibodies (ORION) and FISH probes used in this manuscript.

Table S2 provides the cohort description.

Detecting chromosomal copy-number alterations together with protein-defined cell states in intact tissue is critical for understanding early clonal evolution and microenvironmental interactions in cancer. We developed ORION-FISH, which integrates high-plex tissue imaging with a morphology-preserving DNA fluorescence in situ hybridization (DNA-FISH) workflow and single-cell registration, yielding measurements concordant with clinical FISH. In high-grade serous ovarian carcinoma (HGSOC), ORION-FISH recapitulated known chromosomal changes while revealing subclonal heterogeneity missed by targeted sequencing. Applied to serous tubal intraepithelial carcinomas, precursors of HGSOC, ORION-FISH identified intermixed epithelial cells with MYC or CCNE1 copy-number gains, as well as concurrent alterations associated with distinct immune microenvironments. In addition, epithelial cells with MYC and CCNE1 copy-number gains were detected in morphologically normal fallopian tube epithelium, along with rare MDM4 increases across epithelial lineages. Together, ORION-FISH provides a framework linking chromosomal copy-number states to protein-defined phenotypes within preserved tissue architecture, enabling context-aware interrogation of early copy-number diversification at single-cell resolution. Significance:We introduce ORION-FISH, a spatially resolved workflow integrating multiplexed protein imaging with DNA-FISH to map genomic alterations within intact tissues. Applying this approach to ovarian cancer precursors reveals early copy-number diversification and associations with the local immune context, providing a foundation for studying how genomic and microenvironmental states coevolve during tumor initiation.

Table S4 provides the statistics summary.