Many epithelial cancers orchestrate prognostically significant intratumoral tertiary lymphoid structures (TLSs), yet the mechanisms behind their antitumor activity remain unclear. Here we show that intratumoral TLS in ∼14% of human high-grade serous ovarian cancer (HGSOC) harbor highly oligoclonal B cells expressing IgA or IgG, which are not found in distant tumor regions. By engineering a recombinant antibody with the dominant B cell receptor (BCR) sequence from a mature ovarian cancer TLS, we inhibited the growth of the corresponding autologous tumor in vivo by targeting the extracellular domain of tumor-promoting GPR85. Additionally, tumor regions surrounding TLS exhibited an inflammatory environment that restricts tumor growth. TLS therefore exert anti-tumor immune pressure through the local production of isotype-switched anti-tumor antibodies and by promoting antitumor inflammation. Our results provide mechanistic insight into the elusive nature of antitumor immunity associated with TLS and underscore the importance of coordinated cellular and humoral responses in human cancer.

The groundbreaking SWOG S1801 and NADINA trials herald the use of neoadjuvant immune checkpoint blockade (NICB) as the standard of care for stage III-IV melanomas. The shift from radiographic to pathologic response scoring offers an unprecedented window of opportunity to interrogate ‘on-treatment’ biospecimens for the immune mechanisms of response and resistance at curative-intent surgeries. To date, single-cell/bulk RNA studies have demonstrated that TCF7+ stem-like CD8+ T cells and tertiary lymphoid structures (TLS) are positive predictors of ICB response. However, how these immune cells organize and communicate within the spatial context of the neoadjuvant tumor microenvironment remains poorly understood. We assembled a retrospective cohort of 68 patients with stage 3 melanoma: 35 NICB (Ipilimumab-nivolumab, nivolumab-relatlimab, nivolumab) (14 complete response, 5 partial response, 14 non-response) and 33 treatment-naive (TxN). We dissected the spatial molecular architecture of all tumor beds (n= 68) and 10 uninvolved LNs (5 post-PD-1/5 TxN) using integrative multiplexed error-robust fluorescence in situ hybridization (MERFISH, 305 genes), Vectra multiplex IF and RNAscope multiomics in 38 whole slides and 3 TMAs (77 cores). We also performed single-cell-FFPE-seq using matched blocks (n=12) as an orthogonal benchmark for the MERFISH data. To analyze these high-dimensional datasets, we built a computational framework that includes 1) cell-typing with scFFPEseq validation, 2) novel spatial quantification methods to compute receptor-ligand (R-L) interactions that account for cell-cell distance and chemical signaling, 3) a graph-based clustering that quantifies germinal centers (GCs)/TLS, B-cell follicles and other spatial topography, and 4) MERFISH/ scRNA imputation via generative AI. Here, we showed that the quantity and size of spatially resolved GC/TLS and B-cell follicles surrounded by TCF7+ stem-like CD4+ and CD8 + T-cells, plasma cells, Tregs and myeloids are associated with positive response to neoadjuvant ICB. Our R-L analysis further revealed the critical importance of CXCR4-CXCL12 between TCF7+ T-cells and M2-myeloids and CCR7-CCL19/21 between TCF7+ T-cells in organizing the immune hubs in NICB response. Lastly, we incorporated scFFPEseq and MERFISH data into a unified latent embedding by environmental variational inference (ENVI) allowing us to impute melanoma phylogenetics, geographically accentuated hypoxic foci, and immune hubs within the spatial MERFISH images. We leveraged cutting-edge spatial -omics technologies and novel computational methods to resolve the immunologic hallmarks of NICB response in metastatic melanoma. We believe our approach will usher in a new paradigm for the investigation of other cancer biospecimens in the new era of standard of care neoadjuvant immunotherapy. Zichao Liu, Xiaofei Song, Jodi Balasi, Wei-Shen Chen, Jiang He, Justin He, Jonathan Nguyen, Carlos Moran-Segura, Joseph Johnson, Chaomei Zhang, Jane Messina, Zena Sayegh, Nan Sun, Douglas Marchion, Sean Yoder, Vernon K. Sondak, Jeffrey H. Chuang, Pei-Ling Chen. Spatially resolved immunologic hallmarks of response to neoadjuvant immune checkpoint blockade in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5809.

AbstractPurpose: Therapeutic efficacy of KRASG12C(OFF) inhibitors (KRASG12Ci) in KRASG12C-mutant non–small cell lung cancer (NSCLC) varies widely. The activation status of RAS signaling in tumors with KRASG12C mutation remains unclear, as its ability to cycle between the active GTP-bound and inactive GDP-bound states may influence downstream pathway activation and therapeutic responses. We hypothesized that the interaction between RAS and its downstream effector RAF in tumors may serve as indicators of RAS activity, rendering NSCLC tumors with a high degree of RAS engagement and downstream effects more responsive to KRASG12Ci compared with tumors with lower RAS–RAF interactions. Experimental Design: We developed a method for measuring in situ RAS binding to RAF in cancer samples using proximity ligation assays (PLA) designed to detect panRAS–CRAF interactions. Results: The panRAS–CRAF PLA signal correlated with levels of both RAS-GTP and phosphorylated ERK protein, suggesting that this assay can effectively assess active RAS signaling. We found that elevated panRAS–CRAF PLA signals were associated with increased sensitivity to KRASG12Ci in KRASG12C-mutant NSCLC cell lines, xenograft models, and patient samples. Applying a similar PLA approach to measure the interactions between EGFR and its adapter protein growth factor receptor–bound protein 2 as a surrogate for EGFR activity, we found no relationship between EGFR activity and response to KRASG12Ci in the same samples. Conclusions: Our study highlights the importance of evaluating in situ RAS–RAF interactions as a potential predictive biomarker for identifying patients with NSCLC most likely to benefit from KRASG12Ci. The PLA developed for quantifying these interactions represents a valuable tool for guiding treatment strategies.

9562 Background: The groundbreaking neoadjuvant ICB clinical trials have established beyond doubt that ICB before surgery will become the new standard of care for metastatic melanoma. Importantly, the shift from radiographic to pathologic response scoring offers an unprecedented window of opportunity to interrogate a goldmine of ‘on-treatment’ biospecimens. To date, single-cell profiling and region-based spatial transcriptomics (ST) have highlighted the importance of tertiary lymphoid structures (TLS) and stem-like T-cells as positive predictors of ICB response. However, the molecular mechanisms by which tumor infiltrating lymphocytes communicate and organize multicellular immune hubs within the spatial context of the tumor ecosystem remains poorly understood. Methods: To identify robust biomarkers of response to neoadjuvant ICB, we assembled a cohort of 58 stage III melanoma patients treated with neoadjuvant ICB [24 ipilimumab-nivolumab (IPI-NIVO), 21 NIVO-relatlimab (RELA), 13 PD1 mono] and deployed transformative technologies and computational methods, including single-cell FFPE sequencing, multiplexed FISH single-cell ST (MERFISH 305 genes), digital pathology, 3D open-top light-sheet imaging and AI-based computational pathology, to decipher the complex neoadjuvant ICB tumor ecosystem in response to therapy. We also developed 2 critical computational tools, SCIRA (Spatial Cellular Interaction and Receptor Activation), to compute receptor-ligand (R-L) interactions in whole slide images, and GC-SCAN (graph-based spatial clustering against noise), a graph-based algorithm that quantifies locally clustered structures from spatial -omics data. Results: Our results showed that the quantity and size of hyper-expanded germinal center/TLS, with increased GC: non-GC B-cell ratio, plasma cells and spatially resolved stem-like T-cells are strongly associated with response. IPI-NIVO elicited significantly stronger GC proliferation compared to NIVO-RELA and PD1 monotherapy, suggesting anti-CTLA4 can robustly induce germinal center/TLS proliferation. SCIRA spatial R-L analyses revealed the critical chemokine R-L interactions that organize the GC- and T-cell zones in response to therapy. Lastly, 3D light-sheet imaging revealed remarkable morphologic heterogeneity in 3D, with interconnected GC-TLS networks that are indicative of long-range molecular gradients. Conclusions: Our investigations herein have provided a comprehensive characterization of the immune architectures, cellular communications and 3D large-scale morphologic organizations of the TME that drive response to neoadjuvant ICB therapy. We believe the results of this study will enable the development of robust predictive biomarkers to guide the design of next generation combination ICB therapies in the clinical trial setting for melanoma and other cancer types.

Cancers evolve in a dynamic ecosystem. Thus, characterizing cancer's ecological dynamics is crucial to understanding cancer evolution and can lead to discovering novel biomarkers to predict disease progression. Ductal carcinoma in situ (DCIS) is an early-stage breast cancer characterized by abnormal epithelial cell growth confined within the milk ducts. In this study, we show that ecological analysis of hypoxia and acidosis biomarkers can significantly improve prediction of DCIS upstaging. First, we developed a novel eco-evolutionary designed approach to define habitats in the tumor intraductal microenvironment based on oxygen diffusion distance. Then, we identified cancer cells with metabolic phenotypes attributed to their habitats, including CA9 for hypoxia responding phenotype, and LAMP2b for acid adapted phenotype. Traditionally these markers have shown limited predictive capabilities for DCIS progression, if any. However, when analyzed from an ecological perspective, their power to differentiate between non-upstaged and upstaged DCIS increased significantly. Second, we discovered distinct niches with spatial patterns of these biomarkers and used the distribution of such niches to predict patient upstaging. The niches were characterized by pattern analysis of both cellular and spatial features. With a 5-fold validation on the biopsy cohort, we trained a random forest classifier to achieve the area under curve (AUC) of 0.74. Our results affirm the importance of tumor ecological features in eco-evolutionary-designed approaches for novel biomarkers discovery.

Melanoma incidence and mortality rates are historically higher for men than women. Although emerging studies have highlighted tumorigenic roles for the male sex hormone androgen and its receptor (AR) in melanoma, cellular and molecular mechanisms underlying these sex-associated discrepancies are poorly defined. Here, we delineate a previously undisclosed mechanism by which androgen-activated AR transcriptionally upregulates fucosyltransferase 4 ( FUT4 ) expression, which drives melanoma invasiveness by interfering with adherens junctions (AJs). Global phosphoproteomic and fucoproteomic profiling, coupled with in vitro and in vivo functional validation, further reveal that AR-induced FUT4 fucosylates L1 cell adhesion molecule (L1CAM), which is required for FUT4-increased metastatic capacity. Tumor microarray and gene expression analyses demonstrate that AR-FUT4-L1CAM-AJs signaling correlates with pathological staging in melanoma patients. By delineating key androgen-triggered signaling that enhances metastatic aggressiveness, our findings help explain sex-associated clinical outcome disparities and highlight AR/FUT4 and its effectors as potential prognostic biomarkers and therapeutic targets in melanoma.

A hybrid off-lattice agent-based model has been developed to reconstruct the tumor tissue oxygenation landscape based on histology images and simulated interactions between vasculature and cells with microenvironment metabolites. Here, we performed a robustness sensitivity analysis of that model's physical and computational parameters. We found that changes in the domain boundary conditions, the initial conditions, and the Michaelis constant are negligible and, thus, do not affect the model outputs. The model is also not sensitive to small perturbations of the vascular influx or the maximum consumption rate of oxygen. However, the model is sensitive to large perturbations of these parameters and changes in the tissue boundary condition, emphasizing an imperative aim to measure these parameters experimentally.

A specific splicing isoform of RNASET2 is associated with worse oncologic outcomes in clear cell renal cell carcinoma (ccRCC). However, the interplay between wild-type RNASET2 and its splice variant and how this might contribute to the pathogenesis of ccRCC remains poorly understood. We sought to better understand the relationship of RNASET2 in the pathogenesis of ccRCC and the interplay with a pathogenic splicing isoform (RNASET2-SV) and the tumor immune microenvironment. Using data from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium, we correlated clinical variables to RNASET2 expression and the presence of a specific RNASET2-SV. Immunohistochemical staining with matched RNA sequencing of ccRCC patients was then utilized to understand the spatial relationships of RNASET2 with immune cells. Finally, in vitro studies were performed to demonstrate the oncogenic role of RNASET2 and highlight its potential mechanisms. RNASET2 gene expression is associated with higher grade tumors and worse overall survival in The Cancer Genome Atlas cohort. The presence of the RNASET2-SV was associated with increased expression of the wild-type RNASET2 protein and epigenetic modifications of the gene. Immunohistochemical staining revealed increased intracellular accumulation of RNASET2 in patients with increased RNA expression of RNASET2-SV. In vitro experiments reveal that this accumulation results in increased cell proliferation, potentially from altered metabolic pathways. RNASET2 exhibits a tumor-promoting role in the pathogenesis of ccRCC that is increased in the presence of a specific RNASET2-SV and associated with changes in the cellular localization of the protein.

Abstract Hypoxia is common in many solid tumors, including bladder cancer (BC). Preclinical murine models that mimic relevant molecular and phenotypical characteristics of clinical BC were used to analyze the immune cell composition within the hypoxic tumor microenvironment. The experimental protocols involve intravesical installations, adoptively transferred T cells, and chemotherapy and were designed to recreate therapeutic procedures used in a recently open feasibility clinical trial of intravesical adoptive cell therapy (ACT) for BC patients. We analyzed 18 murine BC tissues from a treatment protocol that included intravesical installment of MB49-OVA tumor cells on day 0, gemcitabine treatment on day 10, and/or intravesical transfer of OT-I transgenic T cells on day 14, in addition to untreated controls and normal bladder tissues. The OT-I cells mimic the T cells used in clinical ACT. The collected tissues were sliced, and consecutive sections were stained for vasculature, tumor, and immune cells: CD8+, CD4+, and myeloid-derived suppressor cells (MDSCs). Subsequently, these histology images were co-registered, segmented, and preprocessed to identify the tumor boundaries, select viable vasculature, resolve cell overlaps, and annotate bladder cavities. A multi-cell off-lattice hybrid agent-based model that combines discrete cells and vasculature with continuous oxygen kinetics was developed to recreate the differential oxygenation in the tumor microenvironment. In all tissues, the hypoxic microenvironment was more prevalent in the tumor regions than in the nontumor regions due to differences between the irregular tumor vasculature vs. normal vasculature. The T cell and MDSC infiltration was increased in the tumors treated with gemcitabine (as mono- or combination therapy). Despite the significant hypoxia level in these tumors, only small proportions of CD8+s and CD4+s were hypoxic, as the majority resided in the well-oxygenated portions of the tumor. High infiltration of CD8+ cells was observed in tumors treated with OT-I cells compared to the controls. However, only a small proportion were hypoxic, and most CD8+s and MDSCs were well-oxygenated. The CD8+s were located far from the intravesical source, with some cells near the vasculature. Hypoxic native CD8+s in untreated tissues were observed inside the tumors, and most well-oxygenated CD8+s were near the tumor borders. The CD8+s also resided in the well-oxygenated regions in the normal tissues. Our reconstructed tissue oxygenation based on histology images revealed that the hypoxic tumor microenvironment hinders T cell accumulation in treated and untreated tumors. Such quantitative analyses may encourage designing more effective hypoxia-mediated therapies. Citation Format: Awino Maureiq E. Ojwang, Anjun Hu, McKenzie Williams, Sarah Bazargan, Joseph O. Johnson, Shari Pilon-Thomas, Katarzyna A. Rejniak. A histological data-driven analysis of the hypoxic microenvironment of preclinical murine bladder tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B024.

There is a critical unmet need for safe and efficacious neoadjuvant treatment for cisplatin-ineligible patients with muscle invasive bladder cancer. We launched a Phase 1b study using the combination of intravesical cretostimogene grenadenorepvec (oncolytic serotype 5 adenovirus encoding granulocyte-macrophage colony stimulating factor) with systemic nivolumab in cisplatin-ineligible patients with cT2-4aN0-1M0 muscle invasive bladder cancer. The primary objective was to measure safety and the secondary objective to assess the antitumor efficacy of the combination. No dose limiting toxicity was encountered in 21 patients enrolled and treated. Combination treatment achieved a pathologic complete response rate of 42.1%, which was associated with baseline free E2F activity and tumor mutational burden. Although T cell infiltration was broadly induced following intravesical oncolytic virotherapy, the formation and maturation of tertiary lymphoid structures was specifically associated with complete response, emphasizing the importance of adaptive humoral immune responses. Together, these results highlight the potential of this combination regimen to enhance therapeutic efficacy in cisplatin-ineligible patients with muscle invasive bladder cancer, warranting additional study as a neoadjuvant therapeutic option. ClinicalTrials.gov identifier: NCT04610671.

Abstract Sotorasib demonstrated efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. There is therefore a clear need for a predictive biomarker to guide treatment decisions. Proximity ligation assay (PLA) is an immunofluorescent-based approach for analysis of endogenous protein-protein interactions, in which the signal can be detected only when two target proteins are within 40 nm. This method measures drug-targetable signaling-associated protein complexes in cells and tissues. KRAS activity has recently been shown to be related to the sensitivity to sotorasib. We therefore conducted a preclinical study to evaluate whether RAS activation inferred by PLA might predict response to KRASG12C inhibitors. We first examined panRAS:CRAF PLA reaction for a KRASG12C-mutant H358 NSCLC cell line, by comparing a complete PLA reaction with both panRAS and CRAF antibodies and an incomplete PLA reaction in the absence of either panRAS or CRAF antibodies. We observed high panRAS:CRAF PLA intensity in H358 with both antibodies, which was abrogated in the absence of either panRAS or CRAF antibodies. We next evaluated the specificity of the PLA reaction by RNA interference. Transfection with sipanRAS or siCRAF resulted in a reduction in panRAS:CRAF PLA signal. We then performed panRAS:CRAF PLA with a panel of KRAS G12C-mutant NSCLC cell lines. The intensity of panRAS:CRAF PLA signal varied between these cell lines, with H358 and LU65 having higher and H1792 and LU99 having lower intensity of panRAS:CRAF PLA signal. To further understand the RAS-RAF interaction, we evaluated a CRAF-RAS binding domain pulldown assay for the cell lines. Different levels of CRAF-bound panRAS were observed in the cell lines, with H358 and LU65 having higher and H1792 and LU99 having lower level of CRAF-bound panRAS. The intensity of panRAS:CRAF PLA strongly correlated with CRAF-bound panRAS (r = 0.70, P = 0.037), suggesting that panRAS:CRAF PLA works sufficiently well to detect the RAS-RAF complex. We investigated the antitumor efficacy of AMG510 in vitro and found that a heterogeneous response to AMG510 was observed in the cell lines, with the 50% inhibitory concentration values of AMG510 for H358 and LU65 below 30 nmol/L and those for H1792, and LU99 above 10 μmol/L. The intensity of panRAS:CRAF PLA was strongly associated with the sensitivity of the G12C-mutated cells to AMG510 (r = 0.74, P = 0.01). We next examined sections of FFPE tumors from NSCLC cell line-derived xenograft models. The panRAS:CRAF PLA signal was higher in mice with sotorasib-sensitive H358 than with resistant H1792 (P = 0.02). Our results suggest that RAS:RAF PLA may have values as a predictive marker to identify which patients can obtain the greatest benefit from sotorasib. Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Joseph Johnson, Eric B. Haura. RAS:RAF proximity ligation assay may predict response to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A001.

Sotorasib demonstrated efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. There is therefore a clear need for a predictive biomarker to guide treatment decisions. Proximity ligation assay (PLA) is an immunofluorescent-based approach for analysis of endogenous protein-protein interactions, in which the signal can be detected only when two target proteins are within 40 nm. This method measures drug-targetable signaling-associated protein complexes in cells and tissues. KRAS activity has recently been shown to be related to the sensitivity to sotorasib. We therefore conducted a preclinical study to evaluate whether RAS activation inferred by PLA might predict response to KRASG12C inhibitors. We first examined panRAS:CRAF PLA reaction for a KRASG12C-mutant H358 NSCLC cell line, by comparing a complete PLA reaction with both panRAS and CRAF antibodies and an incomplete PLA reaction in the absence of either panRAS or CRAF antibodies. We observed high panRAS:CRAF PLA intensity in H358 with both antibodies, which was abrogated in the absence of either panRAS or CRAF antibodies. We next evaluated the specificity of the PLA reaction by RNA interference. Transfection with sipanRAS or siCRAF resulted in a reduction in panRAS:CRAF PLA signal. We then performed panRAS:CRAF PLA with a panel of KRAS G12C-mutant NSCLC cell lines. The intensity of panRAS:CRAF PLA signal varied between these cell lines, with H358 and LU65 having higher and H1792 and LU99 having lower intensity of panRAS:CRAF PLA signal. To further understand the RAS-RAF interaction, we evaluated a CRAF-RAS binding domain pulldown assay for the cell lines. Different levels of CRAF-bound panRAS were observed in the cell lines, with H358 and LU65 having higher and H1792 and LU99 having lower level of CRAF-bound panRAS. The intensity of panRAS:CRAF PLA strongly correlated with CRAF-bound panRAS (r = 0.70, P = 0.037), suggesting that panRAS:CRAF PLA works sufficiently well to detect the RAS-RAF complex. We investigated the antitumor efficacy of AMG510 in vitro and found that a heterogeneous response to AMG510 was observed in the cell lines, with the 50% inhibitory concentration values of AMG510 for H358 and LU65 below 30 nmol/L and those for H1792, and LU99 above 10 μmol/L. The intensity of panRAS:CRAF PLA was strongly associated with the sensitivity of the G12C-mutated cells to AMG510 (r = 0.74, P = 0.01). We next examined sections of FFPE tumors from NSCLC cell line-derived xenograft models. The panRAS:CRAF PLA signal was higher in mice with sotorasib-sensitive H358 than with resistant H1792 (P = 0.02). Our results suggest that RAS:RAF PLA may have values as a predictive marker to identify which patients can obtain the greatest benefit from sotorasib. Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Joseph Johnson, Eric B. Haura. RAS:RAF proximity ligation assay may predict response to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A001.

Dimeric IgA (dIgA) can move through cells via the IgA/IgM polymeric immunoglobulin receptor (PIGR), which is expressed mainly on mucosal epithelia. Here, we studied the ability of dIgA to target commonly mutated cytoplasmic oncodrivers. Mutation-specific dIgA, but not IgG, neutralized KRASG12D within ovarian carcinoma cells and expelled this oncodriver from tumor cells. dIgA binding changed endosomal trafficking of KRASG12D from accumulation in recycling endosomes to aggregation in the early/late endosomes through which dIgA transcytoses. dIgA targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of KRASG12D-mutated ovarian and lung carcinomas in a manner dependent on CD8+ T cells. dIgA specific for IDH1R132H reduced colon cancer growth, demonstrating effective targeting of a cytoplasmic oncodriver not associated with surface receptors. dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers.

Supplementary Figures 1-4 from Oncogenic B-RafV600E Induces Spindle Abnormalities, Supernumerary Centrosomes, and Aneuploidy in Human Melanocytic Cells

Supplemental Materials Methods with additional references.