Blood vessel organoids (BVOs) derived from human pluripotent stem cells have emerged as a novel system to understand human vascular development, model disorders, and develop regenerative therapies. However, it is unclear which molecular states constitute BVOs and how cells differentiate and self-organize within BVOs in vitro and after transplantation. Here we reconstruct BVO development over a time course using single-cell transcriptomics. We observe progenitor states that bifurcate into endothelial and mural fates, and find that BVOs do not acquire definitive arterio-venous endothelial identities in vitro . Chromatin accessibility profiling identifies gene regulatory network (GRN) features associated with endothelial and mural fate decisions, and transcriptome-coupled lineage recording reveals multipotent progenitor states within BVOs. We perform single-cell genetic perturbations within mosaic BVOs to dissect the impact of transcription factor (TF) and receptor depletion on cell differentiation, and highlight multiple TFs including MECOM and ETV2 as strong-effect regulators of human BVO development. We show that manipulation of VEGF and Notch signaling pathways alters BVO morphogenesis and endothelial GRNs, and induces arteriovenous-like state differentiation. We analyze matured BVOs after transplantation using scRNA-seq, and observe matured endothelium with clear arteriovenous specification. We also observe off-target cell fates with bone and adipocyte features, suggesting multipotent states reside within the BVOs in vitro that expand and diversify in less restrictive conditions. Finally, we map vascular disease associated genes to BVO cell states to highlight the potential of BVOs for disease modeling. Altogether, our data and analyses provide the first comprehensive cell state atlas of BVO development and illuminate both the power and limitation of BVOs for translational research.

Accurately predicting whether a new molecule will be safe and effective in humans remains a fundamental challenge in biomedical research, requiring models that can reliably inform clinical outcomes. In oncology, drug attrition rates remain disproportionately high compared to other therapeutic areas, largely due to the poor translatability of conventional preclinical models that fail to capture the complexity of human immune-tumor-stroma interactions (1, 2). Here, we describe human vascularized organoids (VascO), formed through self-assembly of an immune cell perfused capillary network and stromal compartment, interfacing with patient-derived colorectal cancer organoids that enable the combined safety and efficacy assessment of tumor-targeted immunotherapies. VascO generates functional and integrated vascular networks within tumor organoids that sustain organoid growth and recapitulate the morphology and functionality of tumor vasculature. Single-cell transcriptomics reveals that the tumor organoids-educated VascO niche reprograms otherwise healthy fibroblasts into canonical cancer-associated fibroblast states, adopting my-ofibroblastic or inflammatory signatures, and faithfully mirroring donor-specific tumor cues. In parallel, VascO tumor vessels display an expanded tip-cell program, aberrant morphology, and VEGF/ANG2-driven permeability, recapitulating defining hallmarks of tumor vasculature. Immune perfusion with a tumor-targeted T cell bispecific antibody induces dynamic immune trafficking, infiltration and engagement of effector cells with the tumor tissue, leading to its effective killing. A protease-activated variant restricts T cell activation to the tumor microenvironment, retaining potent tumoricidal activity while sparing donor-matched healthy colon organoids, and thereby improving the therapeutic index. VascO thus models a patient-specific, tumor-educated microenvironment that is amenable to assessing the combined safety and efficacy of new drug candidates and provides a scalable platform to improve the preclinical assessment of innovative cancer immunotherapies.

Purpose: Immune checkpoint inhibitors (CPI) have revolutionized cancer therapy, yet many patients derive limited benefit because of poor immune infiltration within the tumor microenvironment (TME). The presence of tertiary lymphoid structures (TLS) and high endothelial venules (HEV) correlates with improved immunotherapy responses. This study evaluated whether selective activation of lymphotoxin β receptor (LTBR) signaling, targeted to fibroblast activation protein (FAP)-expressing tumor stroma, could remodel the TME to enhance immune cell infiltration and potentiate immunotherapy. Experimental Design: FAP-LTBR was engineered as a novel tumor-targeted LTBR agonist and was characterized for its binding, activation, and immunomodulatory properties in vitro and in vivo. Functional effects were assessed using primary human endothelial cells, three-dimensional (3D) microfluidic vascular models, and multiple murine tumor models. Spatial transcriptomics and 3D immunophenotyping elucidated TME remodeling. Therapeutic efficacy was tested as monotherapy and in combination with CPIs or T-cell engagers. Results: FAP-LTBR selectively activated endothelial cells and induced chemokine secretion in a FAP-dependent manner, enhancing T-cell adhesion and extravasation in vitro. In murine models, FAP-LTBR promoted HEV differentiation, TLS-like immune aggregates, and broad tumor inflammation marked by increased B- and T-cell infiltration, including stem-like TCF1+ CD8+ T cells. FAP-LTBR synergized with CPIs and T-cell engagers to induce durable tumor regression, with superior CD8+ T-cell recruitment and redistribution into tumor cores. Conclusions: FAP-LTBR represents a first-in-class tumor-targeted LTBR agonist that remodels the TME, promoting HEV differentiation, immune cell infiltration, and the formation of organized lymphoid aggregates, which collectively enhance immunotherapy efficacy in preclinical models.

Changes in abundance of various cell populations in tumors upon treatment with FAP-LTBR.