Chunru Lin

Supplementary Figures and Tables

Long noncoding RNAs (lncRNAs) have emerged as significant regulators of many physiological and pathological processes and play a key role in the onset and progression of a variety of diseases, including cancer. Currently, the functions of the majority of lncRNAs in carcinogenesis are still unclear. Functional characterization of specific lncRNAs is often challenging using the conventional RNA interference method, due to their specific and restricted subcellular localization. Antisense locked nucleic acid (LNA) GapmeRs, which induce RNase H1-mediated RNA cleavage, provide a promising strategy to knock down lncRNAs regardless of their intracellular localization. Here, we describe a protocol for suppressing the lncRNA BIN3 Intronic Transcript 1 (BIN3-IT1) in human breast cancer xenografts using Antisense LNA GapmeRs.

Supplementary Methods file contains extended methods, including the processing and analysis of multiple sequencing datasets.

Supplementary Tables file contains key information and supporting datasets. Table S1 | Clinicopathological features of the 22 MIBC patients. Table S2 | The top 50 most significantly differentially expressed genes between the 15 single cell clusters. Table S3 | GSEA pathway enrichment results for 4 spatial clusters. Table S4 | Clinical characteristics and spatial tumor state composition across samples. Table S5 | Bootstrap-based robustness analysis of spatial tumor state proportions. Table S6 | The top 50 most significantly differentially expressed genes between the 4 spatial clusters.

Supplementary Figures file contains Supplementary Figures S1–S15. Figure S1 | Pathology-guided spatial annotation across 22 MIBC tumors. Figure S2 | Validation of spatial deconvolution accuracy and cell-type compartmentalization. Figure S3 | Additional tumors demonstrating spatial CNV-based subclones and transcriptional divergence. Figure S4 | Cross-platform concordance of CNV profiles inferred from spatial transcriptomics and WES. Figure S5 | Transcriptional and functional features across the luminal-to-basal differentiation continuum. Figure S6 | Single-cell analysis recapitulates the spatial luminal-to-basal differentiation continuum. Figure S7 | Spatial validation of lineage-associated EGFR and FGFR3 expression patterns. Figure S8 | Lineage-associated expression of FGFR3–EGFR signaling and transcriptional regulators across normal tissue, tumor states, and independent cohorts. Figure S9 | Pathology-guided spatial annotation across all MIBC tumors. Figure S10 | Spatial tumor states associate with chemotherapy response and stress-related gene expression. Figure S11 | NECTIN4 expression is enriched in luminal tumors and declines at the tumor-normal boundary. Figure S12 | Subtype-associated genomic alterations and their relationship with basal program activity. Figure S13 | Associations between basal signature scores and clinical or molecular features across MIBC cohorts. Figure S14 | Pathologist-guided TLS annotation confirms spatial transcriptomic predictions. Figure S15 | Lineage-associated chemokine programs and tumor–TLS interactions.

Purpose: SMARCB1-deficient renal medullary carcinoma (RMC) is an ultrarare, lethal malignancy refractory to therapies approved for other renal cell carcinomas, and its rarity makes randomized trials logistically unfeasible. We investigated whether wild-type (WT) epidermal growth factor receptor (EGFR) is a therapeutically actionable dependency in RMC and translated this insight into a deployable clinical regimen. Experimental Design: EGFR expression was assessed by Clinical Laboratory Improvement Amendments–certified immunohistochemistry in RMC tumors and by integrated RNA and chromatin immunoprecipitation sequencing of primary tumors and adjacent kidney. Panitumumab was compared with erlotinib in patient- and cell line–derived xenografts, and its mechanism defined by immunoblotting, confocal co-localization, cell-cycle and apoptosis assays, and natural killer (NK) cell coculture. Panitumumab-based therapy was then evaluated prospectively in 26 patients with RMC across 10 centers on two continents. Results: RMC showed uniformly high membranous EGFR (median H-score 300), with enhancer and promoter reprogramming at the EGFR locus and a ligand switch from EGF to epiregulin and amphiregulin. EGFR expression was uncoupled from SMARCB1 status in vitro. Panitumumab outperformed erlotinib in RMC xenografts, suppressed AKT and ERK1/2 signaling, and routed EGFR to LAMP1-positive lysosomes in vitro; its effect was predominantly cytostatic, with minimal NK cell–mediated cytotoxicity. Clinically, panitumumab-based therapy achieved a 53.9% objective response rate, including 15.4% complete responses, with median progression-free and overall survival rates of 5.8 and 9.5 months, respectively. Conclusions: WT EGFR is a foundational dependency in RMC that is effectively targeted by panitumumab-based therapy. These findings, derived from a nonrandomized registry, provide a biological and clinical rationale for further prospective validation.

Overall survival from treatment initiation with ixazomib plus gemcitabine plus doxorubicin.

Single-cell landscape of TME and epithelial cells from longitudinally collected tumor samples at baseline and during trial therapy.

Cell–cell communication between malignant and TME populations through BMP and WNT signaling.

Probability density functions for the two co-primary endpoints in trial and historical control cohorts.