InVivoMAb mouse IgG2c isotype control, anti-dengue virus

Spatial omics has advanced our understanding of tissue-level biology, yet tools to systematically link gene functional perturbations to spatial phenotypes and signaling pathways remain limited. To address this, we developed spatial CRISPR screen sequencing (SPAC-seq), a high-throughput spatial CRISPR screen platform, and TARDIS (target prioritization toolkit for perturbation data in spatial omics), a statistical spatial perturbation analysis toolkit. Using SPAC-seq and TARDIS, we linked gene perturbations to spatial phenotypes and pathways, uncovering how Icam1 loss in tumor cells promotes metastasis via immune suppression and macrophage polarization. In CD8+ T cells, we revealed Cd44's role in regulating spatial phenotypes by interacting with Spp1 on macrophages. We also demonstrated the model of the transcription factor-chemokine receptor axis coupling cell states with chemotaxis. SPAC-seq and TARDIS provide an effective framework to study spatially resolved functional genomics and pathways across diverse biological and disease contexts.

Resistance to antiangiogenics is a major challenge in cancer therapy. These agents can either normalize or exacerbate tumor vascular abnormality and hypoxia. The mechanisms of resistance remain unclear in the latter setting. By integrating data from mouse models and clinical trials, we showed that hypoxia-inducing anti-VEGF therapy upregulated programmed cell death ligand 1 (PD-L1), yet failed to sensitize tumors to PD-L1 blockade. Mechanistically, early hypoxic stress triggered epithelial osteopontin (SPP1) production, which recruited monocytes and skewed macrophages toward M2 states, suppressing T cell cytotoxicity. Pharmacological SPP1 depletion impeded the development of hypoxia, reduced M2 infiltration, restored T cell activity, and enabled synergy between antiangiogenics and anti-PD-L1. Genetic dissection - tumor-epithelial Spp1-KO grafts and bone marrow chimeras generated by lethal irradiation and reconstitution with Spp1-/- or WT hematopoietic donors - showed that myeloid SPP1 contributed only marginally compared with epithelial SPP1. These findings identified SPP1 as a central mediator of resistance to hypoxia-inducing antiangiogenics, contributed to a comprehensive model of antiangiogenic resistance, and supported SPP1-targeted strategies to personalize immunotherapy and antiangiogenic therapy according to tumor hypoxia.

Inflammation elicited by rAAV vectors continues to present a critical challenge for the long-term efficacy and safety of gene therapy in the eye. Preclinical models of gene therapy-associated uveitis (GTAU) show that despite the resolution of early acute inflammatory response, persistent subclinical inflammation remains. Here, we employ the GTAU model in Cx3cr1CreER:R26-tdTomato+/- mice to reveal that intravitreal rAAV2 administration elicits sustained microglial dysregulation and retention of CD3+ T cells extending to 50 days post-injection. Deploying pharmacologic and genetic approaches, we define the absolute requirement for microglia and T cells to mediate rAAV2-induced inflammation. Targeted depletion confirmed that microglia-independent mechanisms initiate GTAU, while elimination of lymphocytes prevented both inflammation and microglial activation. Systematic evaluation of therapeutic strategies reveals identified inhibition of T cell recruitment via sphingosine-1-phosphate receptor modulation, but not B cell depletion, as an effective steroid-sparing strategy to prevent both acute and long-term subclinical inflammation. Collectively, our findings challenge the paradigm of microglia-driven ocular inflammation and support the utility of targeted T cell immunomodulation strategies to control GTAU and maintain long-term ocular homeostasis.