Monosomy 7 (-7) is occurring as isolated change or in complex karyotypes in 10-20% of myeloid neoplasms with poor prognosis. Although several genes mapping at chromosome 7 have been involved in pathogenetic mechanisms, the -7 molecular landscape is not fully elucidated. Using an epi-transcriptomic approach, new biological insights emerged in monosomy 7. A private 49 stemness gene program was first identified and it included 59.2% of the targets of the homeobox transcription factors, specifically deregulated in -7 by hypermethylated intergenic enhancers. Additionally, 20.4% of the stemness program was determined by the signature generated by IKZF1/7p12.2 deficiency, responsible for upregulation of the CD112 immuno-checkpoint gene. Focusing on CD112, immunohistochemistry typically assigned its expression to bone marrow blasts and myeloid progenitors in monosomy 7. Concomitant increased expression of the inhibitory TIGIT and PVRIG receptors, and decreased expression of the activating DNAM1 receptor, significantly emerged in CD3+, and natural killer (NK) cells from patients with -7. Moreover, receptors deregulation was shown to be induced by CD34+ leukemic cells with CD112 overexpression. Development of an "ex vivo" model, that used cytotoxicity assays with primary cells from AML cases and autologous NK cells after blockade of the TIGIT and PVRIG receptors, allowed us to show enhanced cytolytic activity of NK against leukemic cells with -7. Altogether, these results first disclosed a dysfunctional TIGIT-PVRIG-DNAM1/CD112 axis in myeloid neoplasms with monosomy 7, inspiring the use of inhibitory receptors blockade to exploit the NK autologous reactivity against a still undruggable cytogenetic change of myeloid neoplasms with dismal prognosis.

Large language models (LLMs) have demonstrated impressive capabilities in summarization, reasoning, and content generation, yet their inability to directly interpret large-scale omics data has limited their utility in data-driven hypothesis generation-particularly in mechanism discovery that demands the integration and interpretation of multimodal datasets, heterogeneous models, and deep domain expertise. Conversely, traditional computational algorithms excel at quantitative analysis of omics data but often rely heavily on labor-intensive, expert-driven interpretation to extract biologically meaningful insights. Here, we introduce (cointelligent single-cell spatial cell‒cell communication: iS2C2), a novel cointelligent platform that synergizes mathematically rigorous computational algorithms with the contextual reasoning capabilities of LLMs to automatically generate biologically interpretable hypotheses from single-cell RNA-seq and spatial transcriptomics data. The iS2C2 platform incorporates a transparent and reproducible cell-cell communication analysis pipeline built upon mathematically rigorous algorithms designed to enhance interpretability for integration with LLMs that contextualize algorithmic outputs or predictions using domain-specific knowledge and literature-derived evidence. When applied to Alzheimer's disease and cancer datasets, iS2C2 generated accurate, reproducible, and expert-validated hypotheses, unveiling previously unrecognized signaling pathways and mechanistic insights in disease microenvironments. This cointelligent approach bridges the gap between structured computational analysis and generative reasoning, heralding a paradigm shift toward fully automated, interpretable biological discovery and advancing the frontiers of next-generation precision medicine and systems biology.

Liver metastasis is the primary cause of mortality in colorectal cancer (CRC) patients. To decipher the underlying mechanisms, we performed single-cell RNA sequencing (scRNA-seq) on paired primary colorectal tumors, adjacent tissues and liver metastases from three CRC liver metastasis (CRLM) patients, alongside colorectal tumors and adjacent tissues from three non-metastatic CRC patients. Our analysis revealed a significant enrichment of Enolase 2-expressing (ENO2⁺) cancer cells in CRLM patients compared to their non-metastatic counterparts. Functional characterization, supported by bioinformatics and murine models, demonstrated that ENO2⁺ cancer cells exhibit enhanced epithelial-mesenchymal transition (EMT) and are critical drivers of CRLM. Mechanistically, the ENO2 protein directly binds to macrophage migration inhibitory factor (MIF) within cancer cells, stabilizing MIF by inhibiting its C-terminus of Hsc70-Interacting Protein (CHIP)-mediated ubiquitination and degradation. This ENO2-MIF interaction activates MIF signaling, fostering robust tumor cell-macrophage crosstalk that promotes M2 macrophage polarization, which is validated by spatial transcriptomics showing the colocalization of ENO2⁺ cancer cells and M2 macrophages. Crucially, both organoid and in vivo models confirmed that ENO2 in CRC cells is essential for inducing M2 macrophage polarization via the MIF pathway, thereby facilitating liver metastasis. Knockout of ENO2 significantly suppressed tumor growth and liver metastasis in mouse models. An inhibitor of the ENO2-MIF interaction, pyrithioxin, can effectively reduce the burden of liver metastasis in mice. Collectively, our findings identify ENO2 as a key driver of CRLM by stabilizing MIF to orchestrate M2 macrophage polarization, highlighting the ENO2-MIF axis as a promising therapeutic strategy for CRLM.

BCL-2 has been implicated in prostate cancer (PCa) progression and development of castration-resistant disease (CRPC); however, it remains unclear how the BCL-2- and AR-expressing PCa cell populations evolve across the PCa continuum, how AR molecularly regulates BCL-2 and whether BCL-2 represents a common therapeutic target in heterogeneous CRPC. Here we first show the selective induction of BCL-2 by AR pathway inhibitors (ARPIs). Vectra-based quantitative multiplex immunofluorescence (qmIF) and image mass cytometry (IMC) analyses with single-cell resolution in patient PCa and xenograft models reveal markedly increased BCL-2+ (AR+ or AR-) PCa cells in CRPC. Mechanistically, AR represses BCL-2 transcription through several AR binding sites and ARPIs relieve this repression. Therapeutic studies in cells, organoids and xenografts support BCL-2 as a shared vulnerability across diverse CRPC subtypes. A Phase Ib clinical trial (NCT03751436) combining enzalutamide and BCL-2 inhibitor venetoclax demonstrated reduced circulating tumor cells in responding patients. In summary, by integrating high-content single-cell level imaging analyses with mechanistic studies, extensive preclinical therapeutic experiments and a Phase Ib clinical trial, our studies herein elucidate the AR+/-BCL-2+/- PCa cell subpopulation dynamics and credentials BCL-2 as a vital therapeutic target in heterogeneous CRPC.

Cholangiocarcinoma (CCA) comprises a heterogeneous group of biliary malignant tumors with poor prognosis and limited therapeutic options. Recent studies have highlighted the role of the immune system in the development and progression of intrahepatic CCA (iCCA). In this study, we investigated the role of the scavenger receptor MARCO in iCCA. Employing transcriptomic, spatial proteomic and histological analyses of human samples, MARCO was found on a specific subtype of tumor-associated macrophages linked to immunosuppression and extracellular matrix remodeling within the tumor microenvironment. High MARCO expression in human iCCA tumors correlated with worse overall survival, T cell dysfunction and increased collagen deposition. In line with this, MARCO expression was associated with a TH2-skewed immune response and was increased in macrophages exposed to IL-4 and IL-13. Marco-/- mice were protected against iCCA development, exhibiting reduced tumor burden, fewer innate immune cells related to TH2 responses and attenuated fibrosis. Moreover, Marco-/- mice exhibited lower levels of immunosuppressive markers on macrophages and cytotoxic T cells, resulting in improved overall survival and reduced lung metastases in an orthotopic tumor model. The use of an anti-MARCO antibody further reduced tumor volume in wild-type mice. This study identifies MARCO as a key regulator of immunosuppression, fibrosis and tumor progression in iCCA, and supports its potential as a novel therapeutic target for macrophage-directed immunotherapy.

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy in cancer treatment. Nevertheless, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which have been suggested to cause CAR T-cell fratricide and exhaustion. Whether CMT indeed causes CAR T-cell dysfunction and the molecular mechanisms conferring CMT remain unknown. Using a selective degrader of trogocytosed antigen in CAR T cells, we show that the presence of trogocytosed antigen on the CAR T-cell surface directly causes CAR T-cell fratricide and exhaustion. By performing small molecule screening using a custom high-throughput CMT screening assay, we found that the cysteine protease cathepsin B is essential for CMT and that inhibition of cathepsin B is sufficient to prevent CAR T-cell fratricide and exhaustion, leading to improved long-term in vitro and in vivo CAR T-cell persistence and in vitro antitumor activity. Our data demonstrate that it is feasible to separate CMT from cytotoxic activity, that CAR T-cell persistence, a key factor associated with clinical CAR T-cell efficacy, is directly linked to cathepsin B activity in CAR T cells, and that it is possible to improve CAR T-cell function through selective inhibition of CMT.

Immune-cold tumors fail to respond to immunotherapy due to insufficient lymphocyte infiltration within the tumor tissue. Increasing the objective response rate remains an urgent challenge. Here, we report the development of a monoclonal antibody (6C5) that specifically targets the membrane-proximal epitope of the receptor tyrosine kinase AXL and modulates antitumor immunity. Unlike traditional membrane-distal AXL antibodies, which suppress tumor growth primarily by blocking AXL signaling, 6C5 significantly enhanced innate immune sensing by promoting macrophage-mediated antigen uptake and type I interferon production. These effects further activated dendritic cells and increased CD8+ T-cell infiltration and effector function, thereby reshaping the tumor microenvironment. However, AXL antibody treatment concurrently induced a suppressive subset of PD-1hiFoxp3-CD4+ T cells. These cells displayed a transcriptomic profile resembling that of Tregs, characterized by elevated expression of immune checkpoint molecules (including PD-1 and CTLA4), reduced IL-2 production, and increased IL-2 receptor expression, thereby attenuating antitumor immune responses. The combination of 6C5 with dual immune checkpoint blockade (anti-PD-1 plus anti-CTLA-4) or a PD-1-targeted IL-2 fusion protein therapy mitigated this immunosuppression, resulting in potent tumor regression and durable immune memory. Our findings demonstrate that a membrane-proximal AXL-targeting antibody effectively converts the immune-cold tumor microenvironment, overcoming resistance to both conventional and next-generation immune checkpoint inhibitors.