Multimodal immunotherapy remodels the tumor microenvironment in hepatocellular carcinoma: Integrative spatial and transcriptomic profiling from a Phase II trial

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-gamma (↑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.