The introduction of checkpoint immunotherapeutic agents in the last decade has revolutionized cancer treatment. Although anti-PD-1, anti-PD-L1 and anti-CTLA4 are promising therapies, many patients fail to respond or relapse due to drug resistance potentially due to redundancy of immune checkpoints. One of the ways to improve the efficacy of this cancer treatment is to target two or even three immune checkpoints. To date, the benefit of combined anti-VISTA/anti-PD-L1 therapy has been confirmed, but no one has investigated the efficacy of blocking these negative immune checkpoints with a bispecific anti-VISTA/anti-PD-L1 antibody.
In this study, the bispecific antibodies (bsAbs) were produced in three formats: symmetric (IgG-HC-scFv), asymmetric (Fab-scFv-Fc(KIH)) and 2 x scFv. The binding and blocking properties of these bispecific antibodies (bsAbs) and their efficacy compared to monotherapy and combination therapy were then determined using endometrial (RL95-2), pancreatic (PANC-1) and breast (BT-20) cancer cell lines.
The bsAbs generated in this study showed weaker binding properties to PD-1 and VISTA in ELISA (EC50) than the parent antibodies (atezolizumab and onvatilimab). Blockade of VISTA/VSIG-3 binding was also weaker with bsAbs compared to onvatilimab, but the ability to block the PD-1/PD-L1 pathway was slightly better than with atezolizumab. The Fc-based bsAbs showed statistically significant higher levels of lysis of endometrial, breast and pancreatic cancer cells. The symmetric bsAbs (IgG-HC-scFv) showed the most promising therapeutic potential. Higher levels of cancer cell lysis were associated with higher levels of pro-inflammatory cytokines. Both the asymmetric and symmetric bsAbs resulted in higher secretion levels of IFN-γ, TNFα and Granzyme B than anti-VISTA, anti-PD-L1 monotherapy and anti-VISTA/anti-PD-L1 combination therapy.
The high level of tumor cell lysis and increased expression of pro-inflammatory cytokines induced by the Fc-based bsAbs suggest a novel approach for the treatment of pancreatic, endometrial and breast cancer.
Copyright © 2025 Bielski, Barczyński, Mikitiuk, Myrcha, Rykała, Boon, Gąsior, Hec-Gałązka, Holak and Sitar.

The identification of affordable and easily accessible indicators to predict overall survival is important for tumor immunotherapy. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which promote tumor immune escape in the tumor microenvironment (TME). This study aimed to determine whether peripheral blood MDSCs could determine their potential as predictors of survival in tumor patients with immunotherapy.
Flow cytometry was used to detect peripheral blood monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic myeloid-derived suppressor cells (G-MDSCs) in 126 patients. Multivariate Cox regression analysis was conducted to examine the associations between peripheral blood MDSCs and patient survival. The receiver operating characteristic (ROC) curve determined the optimal cutoff value for peripheral blood MDSCs and grouped the indicators. The relationship between peripheral blood M-MDSCs and the prognosis and treatment outcome of tumor patients was explored.
The proportion of peripheral blood M-MDSCs was associated with the prognosis of patients with tumors, as were tumor metastasis, the red blood cell count, absolute neutrophil count, absolute monocyte count, and BMI. Multivariate Cox regression analysis revealed that M-MDSCs, absolute lymphocyte value, and tumor metastasis were independent risk factors affecting the prognosis of patients with tumors. Detection of peripheral blood M-MDSCs obtained high sensitivity and specificity for tumor diagnosis. Patients with high M-MDSCs percentage demonstrated reduced survival durations and diminished responses to immunotherapy compared to those with low M-MDSCs percentage.
Peripheral blood M-MDSCs may be used to predict overall survival and immunotherapy efficacy outcomes. This study provides a putative predictive biomarker for clinicians to choose from to predict tumor patients' survival and the selection of receiving immunotherapy regimens.
© 2025. The Author(s).

Context-specific protein-protein interaction (PPI) drive heterogeneity of primary tumor, forming a formidable challenge to effective cancer therapy. However, systematically mapping and modeling these interactions at single-cell resolution across diverse cancer types remains an unmet need. Here, we present Shusi, a large language model-enhanced variational graph auto-encoder model trained on over 75,010 single-cell PPI networks across 23 cancer types, to predict context-specific PPIs. Shusi outperforms existing state-of-the-art methods, as validated through orthogonal experimental evidence. Cancer-specific mutations are significantly enriched in Shusi-predicted networks, offering complementary insights to conventional marker gene-based approaches. Through systematic evaluations, we demonstrate strong associations between Shusi-predicted network topologies, genetic vulnerabilities, and therapeutic sensitivity. Finally, in acute myeloid leukemia (AML), a blood cancer where cell-state heterogeneity drives clinical resistance, Shusi pinpointed JAK2 and SHP1 as actionable vulnerabilities of resistant leukemia subpopulations, as validated experimentally in primary AML. Shusi offers a deep-learning tool for implementing precision medicine based on single-cell protein network architecture.

Transcription factors (TFs) influencing cell fate can be dysregulated in cancer. FLI1 is crucial for hematopoietic stem/progenitor cell (HSPC) function, with STAG2 regulating FLI1 target accessibility. STAG2 depletion enhances HSPC self-renewal, but its role in leukemic transformation is unclear. We uncovered that STAG2 loss maintains FLI1 target accessibility in murine HSPCs and enhances FLI1 binding in NPM1c leukemia. In our Stag2/Npm1c/+ murine model, myeloid-biased HSPCs with increased FLI1 accessibility are reservoirs for transformation, leading to a fully penetrant leukemia. STAG2 deleted NPM1c cell lines exhibit increased chromatin accessibility and chromatin-looping of key stem and leukemia genes including FLI1-target genes CD34 and MEN1. Similarly, enrichment for a CD34+ immunophenotype was observed in co-mutant leukemia patients. STAG2 deficient cells show increased chromatin-bound MENIN and increased sensitivity to MENIN inhibition. Our findings demonstrate that altered chromatin architecture can co-opt oncogenic TF signaling, such as FLI1, as a hallmark of leukemogenesis. Key Findings Loss of STAG2 results in aberrant increased accessibility at FLI1 targets in mouse and human hematopoietic stem and progenitor cells Increased accessibility results in an increased fraction of chromatin-bound FLI1, which overlap with NPM1c targets in STAG2 NPM1c AML cells Stag2 Npm1c co-mutation leads to dysplastic murine AML phenotype arising from myeloid biased progenitors that exhibit increased Fli1 target accessibility In addition to higher chromatin-bound FLI1, co-mutant cells have higher chromatin-bound MENIN, including at the HOXA cluster, rendering cells highly sensitive to MENIN inhibition. Statement of Significance Here, we identify enhanced FLI1 chromatin accessibility as a driver of stemness and leukemic transformation in STAG2 mutant leukemia. Through comprehensive in vivo and in vitro modeling, we demonstrate that altered chromatin architecture can co-opt oncogenic TF activity, like FLI1, to drive divergent leukemia development and therapeutic response.

Chemoresistance is still an important factor affecting the efficacy of treatment in colorectal cancer (CRC) patients. Hypoxia is related to poor prognosis and treatment resistance in cancer. Relevant studies have shown that a hypoxic microenvironment can promote the polarization of M2 macrophages and thus promote tumor development. Previous research has found that bufalin has a wide range of antitumor effects, but whether bufalin can reverse tumor resistance by improving the hypoxic tumor microenvironment is still unclear. In present research, it was found that high expression of SRC-3 in CRC cells under hypoxic conditions promoted the polarization of M2 and caused chemotherapy resistance, while bufalin, a monomeric drug used in Chinese medicine, reduced the level of SRC-3 and HIF-1α, thereby reversing chemoresistance. In addition, overexpression of SRC-3 reduced the hypoxia-mitigating effect of bufalin on CRC cells to promote the polarization of M2. Bufalin also inhibits the polarization of M2 caused by hypoxic CRC cells. Therefore, bufalin has the potential to become a new adjuvant therapy that can be further explored in future studies on its treatment of CRC.