Immune checkpoint inhibitors, such as anti-PD-1/PD-L1, are a novel class of inhibitors that function as a tumor suppressing factor via modulation of immune cell-tumor cell interaction. To date, PD-1/PD-L1 inhibitors have been approved for the treatment of specific types of tumors and obtained good clinical efficacy. However, patients with osteosarcoma showed poor response to anti-PD-1/PD-L1 therapy, the mechanism of which is not well understood. In this study, we found that osteosarcoma tissues were heavily infiltrated by myeloid-derived suppressor cells (MDSCs) which could inhibit cytotoxicity T cell (CTL) expansion. Further study revealed that the vast majority of tumor-infiltrating MDSCs were CXCR4 positive and could migrate toward an SDF-1 gradient. The binding of SDF-1 to its receptor CXCR4 results in the activation of downstream AKT pathway that mediates reduced apoptosis of MDSCs. We also demonstrated that AMD3100, a CXCR4 antagonist, has a synergistic effect with anti-PD-1 antibody in tumor treatment in a murine model of osteosarcoma. These findings provide the basis for establishing CXCR4 antagonist and PD-1/PD-L1 inhibitors co-administration as a novel therapeutic regimen for patients with osteosarcoma and hold great promise for improving the therapeutic effect of osteosarcoma.

Background: Chimeric antigen receptor macrophage (CAR-M) therapy is a novel cancer immunotherapy approach that integrates CAR structure and macrophage functions. CAR-M therapy has shown unique and impressive antitumor effects in immunotherapy for solid tumors. However, the polarization state of macrophages can affect the antitumor effect of CAR-M. We hypothesized that the antitumor activity of CAR-Ms may be further improved after inducing M1-type polarization. Methods: In this report, we constructed a novel HER2-targeting CAR-M, which was composed of humanized anti-HER2 scFv, CD28 hinge region and FcγRI transmembrane domain and intracellular domain. Phagocytosis, tumor-killing capacities, and cytokine release of CAR-Ms were detected with or without M1-polarization pretreatment. Several syngeneic tumor models were used to monitor the in vivo antitumor activity of M1-polarized CAR-Ms. Results: After polarization with LPS combined with interferon-γ in vitro, we found that the phagocytic and tumor-killing capacities of CAR-Ms against target cells were significantly enhanced. The expression of costimulatory molecules and proinflammatory cytokines was also significantly increased after polarization. By establishing several syngeneic tumor models in vivo, we also demonstrated that infusing polarized M1-type CAR-Ms could effectively suppress tumor progression and prolong the survival of tumor-bearing mice with enhanced cytotoxicity. Conclusions: We demonstrated that our novel CAR-M can effectively eliminate HER2-positive tumor cells both in vitro and in vivo, and M1 polarization significantly enhanced the antitumor ability of CAR-M, resulting in a stronger therapeutic effect in solid cancer immunotherapy.

Background: Hydroxysafflor Yellow A (HSYA), which is one of the most important active ingredients of the Chinese herb Carthamus tinctorius L, is widely used in the treatment of cerebrovascular and cardiovascular diseases. However, the potential protective effect of HSYA in spinal cord ischemia/reperfusion (I/R) injury is still unknown. Methods: Thirty-nine rabbits were randomly divided into three groups: sham group, I/R group and HSYA group. All animals were sacrificed after neurological evaluation with modified Tarlov criteria at the 48th hour after reperfusion, and the spinal cord segments (L4-6) were harvested for histopathological examination, biochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Results: Neurological outcomes in HSYA group were slightly improved compared with those in I/R group. Histopathological analysis revealed that HSYA treatment attenuated I/R induced necrosis in spinal cords. Similarly, alleviated oxidative stress was indicated by decreased malondialdehyde (MDA) level and increased superoxide dismutase (SOD) activity after HSYA treatment. Moreover, as seen from TUNEL results, HSYA also protected neurons from I/R-induced apoptosis in rabbits. Conclusions: These findings suggest that HSYA may protect spinal cords from I/R injury by alleviating oxidative stress and reducing neuronal apoptosis in rabbits.

The efficacy of immune checkpoint blockade (ICB) therapy depends on sufficient infiltration and activation of primed tumor-specific cytotoxic T lymphocytes (CTLs) in the tumor microenvironment. However, many tumor types, including osteosarcoma, mainly display immune-desert or immune-excluded phenotypes, which are characterized by a lack of tumor-infiltrating lymphocytes and a poor response to ICB monotherapy. Thus, novel therapeutic strategies are urgently needed to surmount these obstacles. In this study, we found that the expression of the c-Myc oncogene is negatively correlated with the T cell infiltration rate in osteosarcoma. Pharmacological inhibition of c-Myc with JQ-1 significantly reduced tumor burden and improved overall survival in an immunocompetent syngeneic murine model of osteosarcoma (K7M2). A mechanistic study revealed that JQ-1 administration dramatically reprogrammed the tumor immune microenvironment (TIME) within K7M2 tumors. On the one hand, JQ-1 can promote T cell trafficking into tumors by increasing the expression and secretion of T cell-recruiting chemokines. On the other hand, JQ-1 is capable of facilitating crosstalk between antigen-presenting dendritic cells and T cells through the CD40/CD40L costimulatory pathway, leading to activation of tumor-specific CTLs. Combined treatment with anti-PD-1 antibody and JQ-1 resulted in more pronounced tumor regression than either monotherapy, showing an obvious synergistic effect. These findings uncover for the first time that c-Myc inhibition can promote T cell infiltration and activation in osteosarcoma in multiple ways, delivering a one-two punch for modulating TIME. The present work also provides the basis for establishing c-Myc inhibitor and ICB coadministration as a novel therapeutic regimen for patients with osteosarcoma.

Notch signaling plays a vital role in tumorigenicity and tumor progression by regulating proliferation, invasion, and the tumor microenvironment. Previous research by our group indicated that Notch ligand Delta-like 1 (Dll1) is involved in angiogenesis in melanoma, and we noticed that it took a longer time to trypsinize Dll1-expressing B16 melanoma cells than the control cells. In this article, we extended our study to investigate the effects of Dll1 on tumor cell adhesion and metastasis. Dll1 overexpression activated Notch signaling in B16 tumor cells and significantly enhanced the adhering capacity of B16 tumor cells both in vitro and in vivo. B16-Dll1 cells also had a higher metastatic potential than their counterpart in the mouse model of lung metastasis. Along with increased Dll1 expression, N-cadherin, but not E-cadherin, was upregulated in B16-Dll1 cells. These data suggested that Notch ligand Dll1 may enhance the adhesion and metastasis of melanoma cells by upregulation of N-cadherin.

Background: Hepatocellular carcinoma (HCC) immunotherapy is limited by antigenic heterogeneity and an immunosuppressive microenvironment. This study engineered chimeric antigen receptor macrophages (CAR-Ms) targeting stress-inducible NKG2D ligands (NKG2DLs), broadly overexpressed in HCC, to enhance phagocytic clearance and remodel immunity. Methods: NKG2DL expression in HCC and association with survival were analyzed. CAR-Ms were constructed by fusing the NKG2D extracellular domain to FcγRI signaling. In vitro assays assessed phagocytosis, cytokine secretion, signaling, and T cell interactions. Therapeutic efficacy was evaluated in immunocompetent mice bearing subcutaneous, orthotopic, or metastatic HCC models, with or without anti-PD-L1. Tumor progression, immunity, and survival were analyzed via bioluminescence imaging, flow cytometry, histopathology, and serum biochemistry. Statistics analyses were performed using t-tests, ANOVA, and log-rank tests. Results: NKG2DLs were significantly upregulated in human HCC and correlated with poor prognosis. CAR-Ms selectively engulfed NKG2DL⁺ tumor cells, polarized to an M1 phenotype, and activated PI3K-AKT and cGAS-STING pathways, driving phagocytosis and pro-inflammatory cytokines secretion. They enhanced T cell chemokines (Cxcl10, Ccl5) and antigen presentation, boosting T cell recruitment and activation in vitro. In subcutaneous models, CAR-Ms suppressed tumor growth, reprogrammed tumor-associated myeloid cells toward M1, and induced durable immune memory (100% tumor rejection upon rechallenge), with T cell activation. In orthotopic models, CAR-M monotherapy induced complete regression by week 5 and 100% survival, with elevated CD8⁺ T cells and CAR-M specifically homing to liver tumors. CAR-Ms suppressed metastasis in peritoneal/pulmonary models. Combining CAR-Ms with PD-L1 blockade accelerated tumor clearance and survival versus monotherapies, enhancing T cell cytotoxicity. Safety assessments showed no significant organ toxicity based on histopathology and serum biochemistry. Conclusions: NKG2D-directed CAR-Ms eliminate HCC through integrated innate phagocytosis, adaptive immune activation, and myeloid reprogramming, overcoming key therapeutic barriers. Combination with anti-PD-L1 enhances therapeutic efficacy by leveraging innate-adaptive crosstalk, providing a promising approach for HCC immunotherapy.