Abstract Background: Spatial imaging outputs continue to grow in scale and complexity. While brightfield IHC and H&E remain the qualitative gold standard for antibody-based assessment, mIF offers quantitative protein measurement on a single slide. However, challenges such as non-specific binding, imaging artifacts, and variability across sites and operators limit confidence in mIF reproducibility. A quantitative, robust method is needed to assess concordance between IHC and mIF stains. Methods: Using a pan-cancer dataset with a 4-plex mIF panel and matched IHC sections from consecutive slides, we first co-registered images into a shared coordinate space with Valis, applying global rigid and non-rigid transformations from feature matches. IHC stain channels were isolated via stain-matrix-based deconvolution. A tissue mask was generated on the mIF image using Otsu thresholding and morphology operations and then projected onto the IHC slide.Tissue was divided into tiles whose size accounted for section-to-section distance, registration error, and biological variability. Within each tile, random windows were sampled to perform two tests: (1) identify whether the tile contains high stain intensity and (2) determine whether the corresponding IHC and mIF tiles exhibit statistically concordant staining. This approach yields both a DICE score for high-stain region overlap and a stain concordance metric capturing agreement across high- and low-stain regions. Tile-level results and heatmaps are visualized in SpaceIQ™. Results: Concordance between mIF and IHC varied substantially across markers, with CD8 showing the highest and FoxP3 the lowest agreement, a trend consistent across samples. Concordance heatmaps also revealed strong spatial effects, with some tissue regions highly concordant and others clearly discordant. Expert visual review matched these quantitative findings. Conclusions: This segmentation-free framework identifies substantial marker- and region-specific variation in concordance between mIF and IHC staining. Because the method is marker-agnostic and compensates for registration error and inter-section biological differences, it provides a generalized, quantitative approach for evaluating agreement between paired mIF and IHC slides across platforms. Citation Format: Brian Falkenstein, Raymond Yan, A. Burak Tosun, S. Chakra Chennubhotla, Filippo Pullara. Robust segmentation-free stain quality concordance metrics in the SpaceIQ™ multi-omic analysis platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 715.

Abstract Background: Cancer remains a major global health challenge. While standard-of-care (SoC) screenings (e.g., colonoscopy, mammography) improve early detection and survival, limitations persist: many cancer types lack screening guidelines, existing methods are single-cancer early detection (SCED) tests, and invasive protocols with low adherence lead to inefficiencies. Multi-cancer early detection (MCED) tests using liquid biopsy technologies provide a potential solution by screening for multiple cancers, including those not targeted by current SoC, within a single test. From a health economic perspective, supplementing SoC with MCED could enhance cost-effectiveness by broadening detection and reducing missed diagnoses. Methods: This modeling study evaluated two strategies: 1) integrating MCED testing into SoC screenings, and 2) optimizing colorectal cancer screening with a two-step process (FIT followed by colonoscopy), further enhanced by MCED. U.S. SoC screening data from 2021 for five common cancers (breast, cervical, colorectal, lung, prostate), U.S. Census 2020 statistics, and real-world screening cohort parameters informed screening population overlap, cancer incidence, and detection rates for both SoC screenings and MCED. Results: Among 75.6 million individuals screened in 2021, SoC alone detected 193,554 cancer cases at a total cost of $43.2 billion ($223,034 per detected case). Integrating SeekIn's two-step MCED test (OnceSeek followed by SeekInCare, $190 per person) raised total cost to $57.5 billion but increased detected cases to 539,909, nearly three times SoC alone, while halving the cost per case to $106,556. In contrast, using GRAIL’s Galleri MCED ($949 per person) increased detected cases further, with an additional 297,141 cases, but escalated total costs to $114.9 billion. Notably, optimizing the colorectal screening protocol (FIT followed by colonoscopy) reduced SoC spending by $22.1 billion, covering all participants' TwoStep MCED costs and achieving a surplus of $7.7 billion. This strategy yielded 531,556 detected cases (2.7x SoC) and reduced per-case cost to $66,637 (29.9% of SoC). Conclusion: Integrating cost-effective MCED tests, such as TwoStep test, into current SoC screenings can substantially improve cancer detection, lower per-case identification costs, and keep overall expenditures manageable. Targeted optimization, exemplified by a FIT-first colorectal approach, maximizes cost savings and amplifies the benefits of adding MCED tests to established national screening programs, all without requiring an increase in the overall budget. Collectively, these findings highlight a unique opportunity: with thoughtful integration, MCED can reshape population health by delivering timely, accessible, and affordable screening that saves more lives. Citation Format: Mao Mao, Shuaipeng Geng, Shiyong Li, Yinyin Chang, Shujia Hao. Cost-effectiveness analysis of integrating multi-cancer early detection into current cancer screenings in the United States [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5104.

Abstract Perineural invasion (PNI)—the infiltration of tumor cells within, around, or through nerve fibers—is a hallmark of aggressive tumor biology and an independent predictor of poor prognosis in head and neck squamous cell carcinoma (HNSCC). Despite its high prevalence, reported in up to 90% of cases, the molecular and spatial mechanisms enabling PNI remain poorly characterized. Here, we apply spatial transcriptomics to delineate the cellular, molecular, and microenvironmental features that drive PNI and its associated immune contexture. Spatial transcriptomic datasets (GSE300147, GSE252265, and GSE281978) were processed using standardized pipelines for quality control, normalization, and unsupervised clustering (UMAP). Cell populations were annotated based on canonical markers, identifying distinct epithelial, neural, Schwann, immune, and axon guidance-related clusters. Composite module scores were calculated to quantify PNI activity, neuronal and Schwann cell identity, axon guidance signaling, and immune infiltration. Correlation analyses and Gene Ontology (GO) enrichment were used to define key biological pathways associated with spatially resolved PNI phenotypes. UMAP visualization revealed spatially distinct neural-enriched tumor clusters co-localizing with Schwann and axon guidance signatures, indicating a neural-like invasive niche. Correlation analysis demonstrated strong associations between PNI and neuronal (r = 0.89) and axon guidance (r = 0.65) programs, with moderate correlation to Schwann cell activity (r = 0.49). In contrast, PNI exhibited weak negative associations with T-cell (r = -0.26) and macrophage (r = -0.12) scores, consistent with an immune-excluded perineural microenvironment. GO enrichment further supported these trends. PNI-high regions showed activation of pathways involved in chemokine-mediated signaling, cellular adhesion, and fluid and lipid transport—processes that may facilitate neural infiltration and axon-tumor communication. PNI-low regions, by contrast, were enriched for immune-related functions including T-cell and B-cell receptor signaling, interleukin-2 production, and tumor necrosis factor regulation, consistent with active antitumor immunity. Collectively, these data reveal that PNI progression coincides with the emergence of a neural-dominant, immune-silent niche enriched for chemotactic and axon guidance signals. Together, these findings define the spatial neuroimmune landscape of PNI in HNSCC, implicating reciprocal nerve-tumor signaling and localized immune evasion as synergistic drivers of invasion. By integrating spatial resolution with transcriptomic profiling, this study provides mechanistic insight into how cancer cells remodel the perineural microenvironment to support invasion and immune escape. Citation Format: Riya Chhabra, Alfred Kao, Suravi Bajaj, Reena Ding, Daniel John, Wei Tse Li, Jessica Y. Wang-Rodriguez, Weg M. Ongkeko. Spatial neuroimmune crosstalk driving perineural invasion in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5559.

Abstract Cold tumors evade immunotherapy through limiting T cell priming inside draining LN or infiltration into TME. Here, we have developed sequential strategies to overcome the limitation. Firstly, we have developed new mRNA vaccine expressing membrane cytokines with shared or mutated antigens to more and better prime tumor specific T cells (TST) without toxicity. Secondly, we generated tetramer forms of NGR that fusing into Fc-pro-IL2 that selectively targeted CD13 enriched on TME for more IL-2 to rejuvenate dysfunctional T cells. Unexpectedly, cis-delivery of IL-2 on CD13 on tumor vessels can bridge the cross-talk between tumor vessels and TST. Activated T cells can remodel tumor vessels to allow more infiltration. To sustain their anti-tumor activities, we have sequentially delivered tumor-activating cytokines (pro-IL2) guided by anti-PD-1 antibody (anti-PD-1-pro-IL2) or radiation-activating TLR agonists to help DC-T cell interaction inside TME to sustain T-cell effector function. Together, we have sequential strategies converting cold to hot tumors and then simultaneously reinvigorates TIL to overcome the limitations of current immunotherapies. Citation Format: Yang-Xin Fu, . The Immune strategies to convert cold to hot tumors and overcome resistances [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4245.

Abstract Advances in long read sequencing technologies have significantly improved accuracy and throughput, making them useful for clinical applications and personalized medicine. Ultra-long DNA molecules resulting from high quality extractions, if left intact, are generally inefficiently converted into libraries and sequenced on long-read platforms. Therefore, fragmentation is strongly recommended for high-throughput long read sequencing. DNA fragmentation upstream of long read sequencing typically also results in comparable fragment sizes across samples and experiments with higher and consistent N50 lengths as well as improved sequencing yields compared to ultra-long DNA. Current methods for DNA fragmentation include mechanical shearing and enzymatic fragmentation. Mechanical shearing (e.g., Covaris® g-TUBE and Megaruptor®) is the gold standard method for DNA fragmentation upstream of long read library preparation. Drawbacks to these methods include the use of expensive consumables and instruments that are not automation-friendly and result in sample loss. In contrast, enzymatic DNA fragmentation does not require expensive instruments and is automation friendly, however existing fragmentation parameters may not be optimal for long read sequencing. To address these constraints, we developed a novel enzymatic fragmentation solution, NEBNext UltraShear® Long Read (UltraShear LR), that is quick, tunable, and automation friendly. Enzymatic fragmentation is time-dependent and can be used to generate a wide-range of DNA fragment sizes (2 to 30 kb) suitable for different applications and sequencing platforms. UltraShear LR is robust across a wide range of gDNA input amounts (250 to 5,000 ng) as well as different gDNA samples and species (e.g., animal, plant and human). Here we demonstrate that UltraShear LR fragmentation generates high quality libraries with tunable read lengths by sequencing on Oxford Nanopore™ Technologies and PacBio® platforms. UltraShear LR libraries retain base modifications (including CpG methylation) and identify more CpGs than mechanically sheared libraries at the same read count. Additionally, we applied the Twist cancer hotspot long read sequencing capture panel using matched normal and tumor samples to identify copy number variations and perform variant calling, demonstrating the applicability of UltraShear LR for clinically relevant samples. The UltraShear LR system generates time-dependent DNA fragment sizes that are reproducible in a robust and cost-effective manner. UltraShear LR overcomes many limitations of mechanical shearing methods by simplifying sample processing, increasing throughput, and preserving base modifications. These advantages collectively improve usability and data quality in long read sequencing library preparation, making the approach well-suited for clinical applications and personalized medicine. Citation Format: Keerthana Krishnan, Brittany S. Sexton, Matt Angel, Karen McKay, Jonathan Sanford, Ruby Moulton, Louise Williams, Bradley W. Langhorst, Pingfang Liu, V.K. Chaithanya Ponnaluri. NEBNext UltraShear® Long Read: Enzymatic DNA fragmentation for long read sequencing of clinically relevant samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 285.

Abstract Purpose: This study evaluated the clinical outcomes and toxicity of hypo-fractionated prostate radiotherapy combined with elective pelvic nodal irradiation and androgen deprivation therapy (ADT) in patients with unfavorable intermediate-risk, high-risk, and very high-risk localized prostate cancer. Methods: A retrospective analysis was conducted on 152 consecutively treated patients from 2014 to 2024. After excluding 21 deaths unrelated to prostate cancer, 131 patients formed the study cohort. All patients received volumetric modulated arc therapy (VMAT) with daily cone-beam CT image guidance. Treatment consisted of prostate hypo-fractionation delivered with a simultaneous integrated boost and pelvic nodal irradiation to 46-50 Gy in 20-25 fractions. ADT was administered for 6-24 months according to disease risk. Patients were followed for biochemical relapse-free survival (bRFS), disease-free survival (DFS), overall survival (OS), and genitourinary (GU) and gastrointestinal (GI) toxicity. Results: With a median follow-up of 55 months, the cohort demonstrated a DFS rate of 93.8% and an OS rate of 97.7%. Eight patients experienced progression, including five metastatic deaths and three biochemical failures. Cumulative grade III or higher GU toxicity was 3.8%, including one grade IV event; no grade III or higher GI toxicity occurred. Toxicity remained low despite pelvic treatment, likely attributable to modern VMAT planning, daily image guidance, and consistent bowel and bladder preparation. Conclusions: Hypo-fractionated prostate radiotherapy with elective pelvic nodal coverage and ADT produced excellent long-term disease control with minimal severe toxicity in high-risk and very high-risk prostate cancer. These findings support the safety and effectiveness of elective pelvic nodal irradiation when delivered with contemporary image-guided radiotherapy techniques and reinforce its potential role in the modern management of high-risk disease. Citation Format: Eashwer Reddy, Affan Ahmad Ansari, Inamul Haque, Dunia Khaled, John Park. Hypofractionated prostate and pelvic nodal radiotherapy with androgen deprivation therapy for high-risk prostate cancer: A retrospective analysis of oncologic outcomes and toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7369.

Abstract Background: Recurrent glioblastoma (rGBM) remains refractory to immunotherapy due to sparse and suppressed T cell infiltration. We recently reported that survival correlated with immune activation signatures in rGBM patients receiving the oncolytic HSV-1 (oHSV) rQNestin34.5v.2 (CAN-3110). Here, we provide in-situ and molecular evidence that a single oncolytic virus injection can induce durable, tumor-reactive T cell immunity in rGBM. Methods: We integrated highly multiplexed spatial proteomics (CODEX), spatial transcriptomics (Xenium) with custom probes for viral, immune and TCR targets, and bulk TCR-sequencing on paired pre- and post-treatment specimens from a phase 1 clinical trial (NCT03152318). Results: T cell densities strongly increased after treatment, with deep infiltration into viable tumor regions persisting up to two years after a single intratumoral oHSV injection. Cytotoxic GZMB+ T cells were located in close proximity with cleaved-caspase 3+ apoptotic tumor cells, and shorter T cell-tumor distance correlated with longer-progression free survival, demonstrating ongoing anti-tumor immunity. Spatial transcriptomics identified CD8+ T cells states expressing early TCR activation (NR4A1, CD69) and tissue residency (ZNF683 [HOBIT], ITGAE [CD103]) programs enriched in the tumor bed while stem-like T cells localized within lymphoid aggregates. Bulk and spatial TCR analyses revealed in-situ expansion of pre-existing tumoral T cell clones whose amplification correlated with survival. Expanded clones featured tissue resident phenotypes and were positioned closer to tumor cells than non-expanded T cells. Viral remnants were limited to necrotic regions and did not co-localize with T cells, suggesting persistent tumor recognition rather than viral antigen. Conclusion: These results provide in-situ evidence that a single intratumoral oncolytic virus injection can amplify pre-existing T cells clones and induce sustained T cell mediated tumor cytotoxicity even after virus clearance. This suggests that oncolytic virotherapy is as potent T cell activating strategy in rGBM. Citation Format: Maxime Meylan, Ye Tian, Lijian Wu, Alexander L. Ling, Daniel Kovarsky, Graham L. Barlow, Linh D. Nguyen, Jason Pyrdol, Lucas Westphal, Michel Julius, Nicolas L. Gonzalez Castro, Sydney D. Dumont, Andres Santos, Itay Tirosh, Mario L. Suva, E Antonio Chiocca, Kai W. Wucherpfennig. Pre-existing T cells drive durable anti-tumor immunity after oncolytic virus therapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7743.

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype with high relapse rates and limited therapies, emphasizing the need for mechanism-based strategies. EZH2, the catalytic subunit of PRC2, drives TNBC proliferation, metastasis, and chemoresistance, while dopamine D1 receptor (D1R) activation induces apoptosis, autophagy, and invasion inhibition. Immunohistochemistry confirmed strong EZH2 expression and elevated H3K27me3, but catalytic inhibition alone is insufficient due to non-enzymatic functions. We hypothesized that dual targeting of EZH2 and D1R would synergistically suppress TNBC by disrupting the EZH2-D1R axis. Computational modeling using a full-length AlphaFold EZH2 structure and three ligands (GSK126, A77636, SKF38393) identified canonical and cryptic binding pockets via PocketMiner and Fpocket, revealing the SET domain as the primary high-affinity site. DiffDock docking showed stable, high-affinity binding of GSK126, while A77636 and SKF38393 displayed dynamic, less-specific interactions. Molecular dynamics simulations over 100 ns confirmed GSK126’s stability and persistent contacts at key residues (624, 684, 686), providing mechanistic insight. Immunoprecipitation validated an EZH2-D1R interaction, and D1R knockdown reduced combination efficacy. Experimentally, combination therapy reduced 2D cell viability (CI = 0.24). In droplet-based tumor spheroids (TSIMS), GSK126 + A77636 decreased spheroid diameter by 62% and induced necrosis without apoptosis, with confocal imaging showing EZH2 degradation, H3K27me3 loss, and cytoskeletal disruption. EZH2 knockout cells failed to form spheroids. In a vascularized SynTumor organ-on-chip model, the combination reduced circulating tumor cells by 50% within 96 hours, demonstrating suppression of metastatic spread. Together, these silico-guided and experimental findings establish a translationally actionable dual-target strategy that synergistically inhibits TNBC growth and metastasis. (Supported by DOD: W81XWH2010065, Eswar Shankar.). Citation Format: Francesco Drago, Libere Ndacayisaba, Xilal Y. Rima, Gautam Sarathy, Deborah Ramsey, Gwen Fewell, Senthil Saravanamuthu, Sanjay Gupta, Giovanni Nigita, Eduardo Reátegu, Pierre Giglio, Christian Rolfo, Eswar Shankar. Structure-guided dual targeting of EZH2 and dopamine D1 receptor suppresses TNBC growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1488.

Abstract Background: Prostate cancer (PCa) remains a highly lethal disease due to the rapid emergence of neuroendocrine (NE)-like variants from adenocarcinoma. It is predicted that de novo NEPC will emerge from 17% of localized PCa, while treatment-related NEPC will account for 20% of advanced PCa. Clinically, NEPC patients frequently harbor visceral metastasis (62%, P<0.001) compared with CRPC patients (24%), with poor overall survival of 12-17 months. Thus, exploring the unknown mechanism underlying cell-state transition from adeno to NEPC will help identify new targets to overcome lineage plasticity and improve PCa patient survival. Methods: We developed a novel indolent and metastatic PCa mouse model by overexpressing cMyc and knocking out Pten with/without Mutant p53 (R172H). Global transcriptional profiling was performed in indolent and aggressive mice and ASPORIN (ASPN) knockdown (KD) to identify differential gene expression, biological, and pathway analyses. ASPN ectopic overexpression (OE) and KD clones confirmed ASPN biological function and on-target proteins/genes using RT-PCR and western blot analyses. ASPN in vitro function was analyzed in the Incucyte® live imaging system, colony growth assay, and proliferation assays. Results: The novel Ptenfl/fl; Hi-Myc; Trp53R172H/+; Rosa-26; PB-Cre4+ (PCTPLuc) mouse exhibits phenotypic resemblance to PCa visceral metastasis to the lung, liver, inguinal lymph node, and intestine with poor survival as compared with age-matched indolent Ptenfl/fl; Hi-Myc; Rosa-26Luc; PB-Cre4+ (PCPLuc). Unbiased RNA-Seq analysis of PCa adenocarcinoma tissues from PCTP mice revealed a significant association between unique extracellular matrix (ECM) protein clusters and liver and lung metastasis. Asporin (ASPN) emerged as the top differentially expressed ECM protein among the top 25 genes. Consistently, ASPN knockdown and overexpression were directly associated with PCa cellular phenotypes of proliferation and colony growth. Mechanistically, ASPN modulation impacts pERK, CyclinD3, EMT proteins, and a neuroendocrine-like phenotype, as well as cell differentiation-related transcriptomes such as cellular retinoic acid-binding protein 2 (CRABP2), along with Chromogranin A (CHGA), NeuroD1, and INSM1. Using a publicly available TCGA database, we observed a significant overexpression of ASPN and CRABP2 in PCa tissues (N=497) relative to normal (N=52) (P<0.001). Finally, ASPN ectopic overexpression in immortalized RWPE-1 cells confirmed an aggressive in vitro phenotypes. Conclusion: For the first time, we explored the influence of ASPN/CRABP2 and other signaling involved in luminal epithelial to neuroendocrine differentiation and validated CRABP2 as a novel target to prevent NEPC transdifferentiation. Our findings support ASPN’s new role, mechanism(s) and as a target for PCa and other visceral metastasis disease. Citation Format: Parthasarathy Seshacharyulu, Shobhit Lall, Sushanta Halder, Sakthivel Muniyan, Zahraa Wajih Alsafwani, Ramakanth Chirravuri-Venkata, Moorthy P. Ponnusamy, Surinder K. Batra. Novel ASPORIN/CRABP2 axis drives cell-state plasticity from adenocarcinoma to neuroendocrine-like prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5573.

Abstract Histological examination of follicular lymphoma (FL) biopsies remains the cornerstone for diagnosis and staging of FL. Single cell sequencing approaches, while transcriptomically rich, require tissue dissociation and therefore lose the native spatial context that may underpin FL progression and transformation to a high-grade lymphoma, typically diffuse large B cell lymphoma (DLBCL). By single cell spatial profiling, we aimed to characterize the spatiotemporal heterogeneity and interplay between malignant B cells and the tumor microenvironment (TME) during transformation from FL to DLBCL histology. Single cell spatial transcriptomics (Nanostring CosMx) and proteomics (Cell DIVE) were performed on FFPE tissue cores of 14 tFL patients with paired FL (tFL-FL) and DLBCL (tFL-DLBCL) timepoints, and 13 FL patients without evidence of progression or transformation (non-tFL; >6y of follow-up). Spatial omics confirmed malignant B cell dominated follicular structures in non-tFL and tFL-FL while absent in tFL-DLBCL, consistent with pathologic classification. Follicles were then delineated (defining intra-follicular, peri-follicular and extra-follicular regions) and enriched proliferation and exhaustion features were observed in intra-follicular regions compared to extra-follicular counterparts. Moreover, differential spatial interaction patterns were observed, amongst which Galectin-9 crosstalk components were identified as being differentially expressed and exhaustion-related pathways were more prevalent in intra-follicular and peri-follicular regions in non-tFL and tFL-FL timepoints. Macrophage phenotypes also markedly shifted across spatial regions with progression: intrafollicular FOLR2+ and IL4I1+ macrophages present in non-tFL progressively diminished, while CD163+, SPP1+, and NLRP3+ macrophages became increasingly enriched in extra-follicular regions in tFL-DLBCL. Cell neighborhood analysis showed differential abundance among non-tFL, tFL-FL, and tFL-DLBCL timepoints. For B-cell-predominant cell neighborhoods (CNs), the germinal center (GC) score was higher in non-tFL than tFL-FL, whereas tFL-FL and tFL-DLBCL shared similar GC score levels. Within CNs of similar cell type compositions, T cells were more exhausted and macrophages were more polarized towards anti-inflammatory phenotypes in the transformed state. Subsequent single cell proteomics validated the differential follicular Galectin-9 signaling and macrophage-related gene expression patterns among spatial regions. In summary, we delineated the spatial landscape of both indolent and transformed FL. We identified a spatiotemporal shift in TME composition, spatial interactions, and cell neighborhoods. This study provides novel insights into the spatial lymphoma architecture associated with FL transformation, contributing to a refined disease evolution model. Citation Format: Shaocheng Wu, Eric Lee, Anne-Sophie Fratzscher, Andrew Lytle, Spencer D. Martin, David G. Huntsman, David W. Scott, Christian Steidl, Andrew Roth. Single cell spatial multi-omic characterization of the tumor microenvironment in transformed follicular lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6198.