Villasboas, Jose Caetano

Abstract Background: Hepatocellular carcinoma (HCC) is largely refractory to immune checkpoint blockade due to immune exclusion, stromal barriers, and aberrant vasculature. We evaluated whether multimodal therapy - external beam radiotherapy (EBRT) for antigen release, intratumoral dendritic cell (DC) vaccination for antigen presentation, PD-L1 blockade for immune activation, and VEGF inhibition for vascular normalization - could convert “cold” HCC tumors into sustained immune-inflamed states. Methods: Serial biopsies (baseline, post-EBRT, end-of-treatment [EOT], and progression [PD] when available) from three patients enrolled in an ongoing phase II trial (NCT03942328) underwent multiplex immunofluorescence, spatial neighborhood analysis, and transcriptomic profiling. Longitudinal changes in cellular composition, immune-stromal architecture, spatial contact networks, and pathway activity were compared. Results: Baseline tumor microenvironments (TMEs) were strongly immune-excluded, dominated by tumor (PanCK+60-70%) and stroma (SMA+/Vimentin+25-30%) with sparse immune infiltration (<10-15%). Post-therapy, immune complexity and spatial connectivity increased 2-3-fold in all patients. Patient 1 showed robust cold-to-hot conversion with CD8+(3x), CD4+(2.2x), and DC (2.3x) expansion, increased CD8-DC interactions (2.5x), and reduced tumor fraction (-40%). Transcriptomics showed marked upregulation of IFN-γ (↑3.4x), HLA-DRA (↑2.7x), and reductions in VEGFR2 (↓50%) and HIF1A (↓40%), consistent with vascular normalization. Patient 2 exhibited stromal/vascular remodeling and improved T-cell infiltration but restricted immune diversity, with CD4+/CD8+ dyads (↑2.2x) and B/NK cells(↓ 60%). Neighborhood diversity rose (3→7 communities) and immune-tumor proximity improved (distance ↓ 35%). Patient 3 showed transient inflaming with CD8+(↑3x), DC(↑2x), and new CD4-CD20/CD4-CD56 interactions, followed by relapse marked by stromal/myeloid re-expansion (SMA+↑1.8x, CD14+↑2.5x), angiogenic resurgence (VEGFR2/CD34+↑2.2x), contraction of immune networks, and genomic shift from activating CTNNB1 mutation loss to ATM gain. DCs accumulated but localized mainly to DC-DC or DC-myeloid niches, indicating non-productive antigen presentation. Conclusions: Multimodal EBRT+DC+PD-L1/VEGF blockade induces four-sided synergistic TME reprogramming - antigen release, immune priming, checkpoint release, and vascular normalization - promoting cold-to-hot conversion in HCC. Durable inflaming correlates with sustained T-cell/DC expansion and vascular remodeling, whereas relapse reflects stromal/myeloid reinforcement and genomic adaptation. These findings define mechanistic trajectories of TME remodeling and highlight actionable barriers to sustained immunotherapy response in HCC. Citation Format: Lionel Aurelien Kankeu Fonkoua, Christopher L. Hallemeier, Basim Salem, Arina Tkachuk, Kaitlynn McCay, Panwen Wang, Ying Li, Thomas D. Atwell, Krishan R. Jethwa, Caitlin Conboy, Nguyen H. Tran, Leslie A. Washburn, Melody Wu, Chen Wu, Andre De Menezes Silva Corraes, Rondell P. Graham, Jose Caetano Villasboas, Kevin Regan, Zuoyi Shao, Rayaan Kamal, Nathan R. Foster, Haidong Dong, Sean Park, Yi Lin, Lewis R. Roberts. Multimodal immunotherapy remodels the tumor microenvironment in hepatocellular carcinoma: Integrative spatial and transcriptomic profiling from a Phase II trial [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 7935.

Abstract Chimeric antigen receptor (CAR) T-cell therapy achieves high response rates in relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, yet only 40% of patients achieve durable remission. Identifying biological programs linked to long-term response remains critical. As metabolic fitness underlies T-cell persistence and effector function, we profiled metabolic pathways associated with clinical outcomes following commercial CD19 CAR T-cell therapy. Single-cell RNA sequencing of peripheral blood mononuclear cells collected at baseline (BL), peak CAR-T expansion (PK), and one-month post-infusion (M1) was analyzed using CellRanger v7.0.1, immunopipe. Seurat v4.3.0 was applied for unsupervised clustering to delineate cell subsets based on top differentially expressed genes. Patients were categorized as durable complete remission (CR ≥6 months; n = 16), primary refractory (PD1; n = 4), or relapse after initial response (PD2; n = 12). Gene-set enrichment analysis defined metabolic pathway activity across T-cell, monocyte, dendritic cell (DC), and natural killer (NK) subsets. Oxidative phosphorylation (OXPHOS) emerged as the dominant metabolic program distinguishing clinical outcomes. At BL and PK, OXPHOS was consistently enriched in PD1 relative to CR across T-cell, monocyte, and DC subsets, suggesting early oxidative activation in non-responders. By M1, this pattern inverted, with higher OXPHOS activity in CR. PD2 largely paralleled PD1, but several subsets including CD8 T central memory, classical monocytes TGFβ, intermediate monocytes CD38, monocytic myeloid-derived suppressor cell (mMDSC) HIF1A, and mMDSC SIRPA, showed OXPHOS enrichment in CR at BL and/or PK that persisted through M1. In contrast, DC and NK subsets exhibited the opposite pattern: OXPHOS was enriched in CR at PK (conventional DC 2 (cDC2), plasmacytoid DC (pDC), NK, proliferating NK), but shifted toward enrichment in PD2 at M1 (cDC2, NK, NK CD56bright). Glycolysis (GLY) followed a similar trajectory in monocytes and NK cells, with enrichment in PD1/PD2 at BL and PK, followed by enrichment in CR at M1. In T-cells, PD1 maintained GLY enrichment from BL through PK, with no significant differences observed at M1. The inositol-phosphate metabolism pathway showed a more static pattern and was consistently enriched in multiple PD2 effector and memory T-cell subsets without reversal at M1. Taken together, these data reveal distinct, lineage-specific metabolic signatures that differentiate durable remission from early and late progression. Dynamic OXPHOS and GLY programming, characterized by lower activity early and enhanced activity at M1 in CR, may reflect adaptive metabolic programming that supports sustained antitumor immunity. Immune-metabolic profiling may therefore serve as a biomarker of response and highlight actionable metabolic pathways to enhance CAR T-cell durability. Citation Format: Melinda S.Y. Tan, Panwen Wang, Patrizia Mondello, Jacqueline Turner, Andre de Menezes Silva Corraes, Chen Wu, Zuoyi Shao, Kevin Regan, Ma Audrey, Arushi Khurana, Nora N. Benanni, Yucai Wang, Paul Hampel, Jonas Paludo, Saad J. Kenderian, Urshila Durani, Patrick B. Johnston, Jose Caetano Villasboas, Stephen M. Ansell, Ying Li, Haidong Dong, Hu Zeng, Yi Lin. Metabolic pathway signatures defining response to CD19 CAR T-Cell therapy in aggressive B-cell non-hodgkin 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 3278.

Abstract Introduction: Therapeutic outcomes in B-cell non-Hodgkin’s lymphoma (B-NHL) have substantially improved with the introduction of chimeric antigen receptor T-cell (CAR-T) therapy. Yet, persistent immune dysfunction and underlying metabolic dysregulation remain major obstacles to achieving durable clinical responses in B-NHL. We hypothesize that mitochondrial pathways play a critical role in mediating anti-tumor immunity during CAR-T therapy. Methods: Peripheral blood mononuclear cells were collected from healthy controls (CNTRL, n=5) and patients with advanced stage lymphoma (LYM) who received FDA approved CAR-T (n=32). Samples were collected before lymphodepletion (BL), at peak CAR-T expansion (PK) and one-month post-infusion (M1). Single-cell RNA sequencing was used to interrogate differential mitochondrial gene expression. Samples were obtained from LYM patients with complete remission for >6 months (CR), primary refractory (PD1), or relapsed (PD2) disease. Results: Compared to CNTRLs, LYM patients exhibited reduced expression of cytochrome c oxidase. At BL, CR patients demonstrated broader mitochondrial differential gene expression across all T-cell subsets relative to PD1 and PD2 and showed higher expression of MT-ATP-6, MT-ATP-8, and MT-CO3. Following CAR-T infusion, global mitochondrial gene expression increased across patients corresponding with expansion of CD8 effector T cells (Tem tumor circulating) and activated peripheral memory T cells (Tpm). Certain mitochondrial genes, such as MT-CO1, were durably expressed across LYM patients at BL, PK, and M1 in peripheral and central memory T cells (Tpm, Tcm). At PK, cytochrome c oxidase and ATP synthase genes were upregulated in CR Tpm and Tcm. At M1, MT-ATP-6, MT-ATP-8, MT-CO3 were significantly upregulated in CD8 memory precursor effector T-cells (MPECs) of CR patients compared to both PD1 and PD2. While CR and PD2 patients appeared clinically responsive at M1, CR samples maintained significantly higher MT-ATP-8 expression across T cell subsets (CD8 MPECS, Tpm, Tcm) compared to PD2 (p values 0.008; <0.0001; <0.0001). Conclusions: We report a temporal shift in immunometabolism with CAR-T therapy in LYM. Clinical responders exhibited sustained mitochondrial activity characterized by persistent expression of ATP synthase in CD8 MPECs and cytochrome c oxidase in Tpm and Tcm. In sum, mitochondrial programming may distinguish effective versus dysfunctional T cells and identify targets for therapeutic development. Citation Format: Jacqueline Turner, Panwen Wang, Patrizia Mondello, Melinda Tan, Christoph Schaefers, Chen Wu, Andre de Menezes Silva Corraes, Kevin Regan, Zuoyi Shao, Ma Audrey, Arushi Khurana, Nora N. Benanni, Yucai Wang, Paul Hampel, Saad J. Kenderian, Jonas Paludo, Urshila Durani, Patrick B. Johnston, Jose Caetano Villasboas, Stephen M. Ansell, Haidong Dong, Ying Li, Zeng Hu, Yi Lin. Mitochondrial transcriptome profiles associated with clinical responses to CAR-T therapy in aggressive 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 1213.

Abstract BACKGROUND Diffuse large B-cell lymphoma (DLBCL) is a high-grade malignancy and the most common subtype of lymphoma worldwide. Standard therapy with anthracycline-based chemotherapy and rituximab is able to cure about two thirds of patients but a significant proportion will develop relapsed/refractory disease. Salvage treatment options in this setting are limited and only effective in a minority of patients. Most of these patients will eventually die from progressive disease, underscoring the pressing clinical need for the development of new treatment strategies. Checkpoint blockade therapy has demonstrated efficacy in a variety of cancers and involves modulating the host's immune response towards an effective anti-tumoral attack. Trials using programmed cell death protein 1 (PD-1) blockers in advanced hematological malignancies demonstrated remarkable activity in patients with Hodgkin lymphoma. Unfortunately, results in DLBCL were marginal and short lived. The mechanisms underlying the failure of immune checkpoint blockade in DLBCL are not known. To better understand mechanisms of immune evasion we utilized mass cytometry to the study of immune subsets in the tumor microenvironment (TME) of DLBCL. METHODS Viably cryopreserved cell suspensions created from clinical specimens of patients with DLBCL were obtained from our biorepository. Specimens were stained using a cocktail of metal-tagged antibodies recognizing over 32 surface proteins and acquired on the mass cytometer (CyTOF2™, Fluidigm). Control tissue consisted of reactive lymph node (LN) tissue, tonsil and spleen, obtained from patients without diagnosis of cancer. Analysis was performed using Cytobank (www.cytobank.com). Institutional Review Board approved the conduct of this study. RESULTS Thirteen patient samples were available for analysis (7 DLBCL, 6 controls). DLBCL samples originated from LN (4), spleen (2), and adrenal gland (1). Control tissue consisted of tonsil (3), reactive LN (2), and normal spleen (1). Global analysis of CD45+ cells showed that T-cells were more commonly found in the TME of DLBCL than controls (DLBCL 47.2% vs. 24.5% in controls; Figure 1). The difference persisted when the analysis was restricted to LN specimens (50.2% in DLBCL vs. 25.1% in normal LN). On both DLBCL and control samples the majority of T-cells were CD4+ (61.1% in DLBCL; 75.3% in control) compared to CD8+ T-cells (33.3% in DLBCL; 20% in control). DLBCL samples had increased proportion of regulatory T-cells (Treg) compared to controls (16.9% vs. 6.5%), even after stratifying for tissue of origin (Figure 2). Multiparametric analysis of cytotoxic (CD8+) T-cell subsets revealed that majority of CD8+ T-cells in DLBCL has an effector memory (Tem) phenotype (81.9%) compared to tissue-matched control (43%). PD-1 expression was present in the majority of CD8+ T-cells and mapped to the Effector Memory and Effector subsets. The majority of PD-1 positive cells co-expressed CD27 (>80%). One of the samples (DLBCL arising in the spleen from CLL background) also demonstrated bright PD-1 expression in B-cells (Figure 3). CONCLUSIONS Mass cytometry reveals that the tumor microenvironment of DLBCL is enriched with CD4+ T-cells including a higher proportion of Treg compared to tissue-matched control. We also demonstrated that most cytotoxic T-cells in the TME of DLBCL have an effector memory phenotype and express other markers of activation and prior antigenic exposure. Co-expression of PD-1 and CD27 was common in CD8+ T-cells, providing rationale for studies of combination immunotherapy. PD-1 expression in B-cells of DLBCL arising from CLL background deserves further investigation. Figure 1 Proportion of major immune subsets found in the TME of DLBCL (blue) and control tissue (red). Figure 1. Proportion of major immune subsets found in the TME of DLBCL (blue) and control tissue (red). Figure 2 Proportion of regulatory T-cells (Treg) found in the TME of DLBCL (blue) compared to control (red). Bars represent standard error. Figure 2. Proportion of regulatory T-cells (Treg) found in the TME of DLBCL (blue) compared to control (red). Bars represent standard error. Figure 3 viSNE map of CD45+ cells from a patient with DLBCL arising from a CLL background. Each unit on the dot plot represents a cell. Their coordinates on the map are functions of their phenotypic distance. Major subsets were identified using unbiased clustering and annotated (A). Events were color coded according to the expression of representative markers (B-G). A heat map (H) displays median intensity of expression of a comprehensive set of markers included in the panel for each immune subset. Figure 3. viSNE map of CD45+ cells from a patient with DLBCL arising from a CLL background. Each unit on the dot plot represents a cell. Their coordinates on the map are functions of their phenotypic distance. Major subsets were identified using unbiased clustering and annotated (A). Events were color coded according to the expression of representative markers (B-G). A heat map (H) displays median intensity of expression of a comprehensive set of markers included in the panel for each immune subset. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.