InVivoMAb anti-mouse PD-1 (CD279)

The clinical efficacy of immune checkpoint inhibitors (ICIs) in colorectal cancer (CRC) remains limited. Modulation of the glucagon-like peptide-1 receptor (GLP-1R) may enhance T-cell-mediated antitumor responses. The present study aimed to evaluate the antitumor effects of the GLP-1R antagonist Exendin 9-39 (Exe-9) combined with anti-programmed cell death protein-1 (PD-1) treatment in preclinical CRC models. Using in vitro co-culture assays, ELISA and in vivo murine models, alongside immunohistochemical and molecular analyses of clinical samples, HT-29 and MC38-OVA colon cancer cell lines were co-cultured in vitro with activated T cells in the presence of Exe-9. In vivo, male BALB/c mice were injected with MC38 to establish a CRC model and nude mice were used to assess T-cell dependency. To evaluate this synergistic effect, BALB/c mice with CRC were treated with Exe-9, anti-PD-1 or a combination. Additionally, clinical CRC samples were analyzed to assess the association of GLP-1R expression with the immunotherapy response. Exe-9 significantly enhanced T-cell-mediated cytotoxicity in CRC cell lines and reduced tumor growth in immunocompetent CRC mice; however, this effect was not observed in nude mice. Furthermore, combination therapy with the GLP-1R antagonist and anti-PD-1 yielded an improved antitumor effect compared with either treatment alone, and high GLP-1R ex2pression in clinical samples correlated with poor ICI response. These findings suggest that GLP-1R antagonism potentiates T-cell-mediated antitumor immunity and may provide a promising adjunctive therapeutic strategy for patients with CRC when combined with ICIs in the future.

Although programmed cell death-1 (PD-1) inhibitors have shown promising and durable responses in patients with several types of cancer, many patients show resistance to PD-1 inhibitors. Recent evidence has demonstrated that immunosuppressive cells are induced in tumor microenvironment and inhibit the anti-tumor effects of anti-PD-1 monoclonal antibody (αPD-1 mAb). To investigate whether nintedanib-a multi-tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor, fibroblast growth factor receptor, and platelet-derived growth factor receptor-suppresses immunosuppressive cells and enhances the anti-tumor effects of αPD-1 mAb in preclinical models, flowcytometry, immunohistochemistry, and RNA sequencing of tumor-tissue, tumor-draining lymph nodes, spleens were conducted. RNA sequencing of murine tumor tissues revealed that nintedanib decreased the gene signatures related to myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs). Flow cytometry showed that nintedanib significantly decreased the MDSC and CAF percentage in tumor-bearing hosts and increased IFN-ϒ+CD4+ and CD8+ T cells infiltrating into tumors. Immunohistochemical analysis demonstrated that nintedanib treatment significantly increased the number of CD8+ T cells in the internal area of the tumor. Adding nintedanib to anti-PD-1 mAb therapy significantly inhibited in vivo tumor progression. These results indicate that nintedanib suppresses MDSCs and CAFs by inhibiting VEGFR-, PDGFR-, and FGFR-mediated signaling, thereby increasing effector T-cell infiltration into tumors and enhancing the anti-tumor effects of αPD-1 mAb therapy.

Adoptive transfer of T lymphocytes specific for neoantigens can elicit immunity against solid tumors in patients. However, how these antigens affect T-cell function, effector differentiation, and persistence remains unclear. We examined how an identical CD8+ T-cell product was shaped by melanoma expressing either a low-avidity self/tumor-associated antigen or high-avidity neoantigen and kinetically profiled T-cell differentiation in these two contexts across host tissues. High-avidity neoantigen expression was sufficient to activate naïve CD8+ T cells-leading to robust tumor regression and long-term protective immunity upon tumor rechallenge. Mechanistically, transferred naïve CD8+ T cells reacting to high-avidity neoantigen exhibited enhanced cytokine production, heightened effector function, and sustained persistence compared with the low-avidity wild-type tumors. Antitumor activity to these high-avidity tumors was preserved even in the absence of functional host T and B lymphocytes, and early lymph node (LN) trafficking was found to be essential for adoptive T-cell therapy efficacy. Expanded effector or stem memory T cells were compared with the naïve pmel-1 T-cell product. Stem memory (but not effector memory) cells exhibited similar antitumor efficacy and LN trafficking patterns to the naïve cells in mice with high-avidity neoantigen-expressing tumors. These findings highlight how differential tumor antigens shape divergent cellular fate and uncover a critical role of T-cell trafficking in LNs in shaping high-avidity neoantigen-specific responses.

Glioblastoma multiforme (GBM), a highly invasive brain tumor, is severely restricted in T-cell infiltration and anti-tumor activity due to its immunosuppressive microenvironment. However, commonly used preclinical GBM mouse models cannot fully recapitulate the refractoriness of human GBM or effectively distinguish therapeutic efficacy. In this study, we evaluated the efficacy and mechanisms of therapies based on the novel sesquiterpene lactone small-molecule compound, ACT001, using the refractory G422TN-GBM mouse model. ACT001 alone exerted evident anti-G422TN-GBM effects in vivo and in vitro, but it only slightly prolonged animal survival. ACT001 combined with concurrent radiotherapy and temozolomide (RT/TMZ) exerted synergistic effects by suppressing tumor progression and extending animal survival. Importantly, the RT/TMZ/ACT001 regimen could achieve cure (long-term survival, >100 d, 26.7%) and immune cure (passing the tumor-rechallenge assay, >100 d, 12.5%) in G422TN mice. However, combining the anti-PD-1 antibody (αPD-1) with RT/TMZ/ACT001 did not further improve survival. Mechanistically, RT/TMZ/ACT001 substantially activated the tumor necrosis factor (TNF) pathway, inducing tumor cells and stromal cells in the microenvironment to express the chemokine C-X-C motif chemokine 10 (CXCL10), thereby promoting T-cell infiltration, especially CD8+ T cell, into the tumor site. Pharmacological inhibition of the TNF signaling pathway with R-7050 completely abolished the synergistic efficacy of RT/TMZ/ACT001. Taken together, our results demonstrate that ACT001 combined with RT/TMZ can overcome the immunosuppressive barrier of GBM to achieve immune cure in GBM via TNF-CXCL10-CD8+ signaling, strongly suggesting the priority of combining ACT001 with RT/TMZ rather than with αPD-1 in clinical trials.

Mechanical forces are emerging physical cues that regulate biochemical signals of immune cells for antitumor immunity. Owing to the lack of precise tools to impose intracellular forces, little is known about whether and how organelle-level forces trigger mechanotransduction for antitumor immunity. Here, we developed a magneto-mechanical force-triggered lysosomal membrane permeabilization (MagLMP) strategy to induce durable macrophage repolarization for in vivo applications. Self-assembled magnetic nanomotors are driven by rotational magnetic fields, facilitating dynamic damage to the lysosomal membrane by a finely tuned torque-induced vortex. Intriguingly, galectin 9 (Gal9) was found to be critical for sensing cyclic MagLMP, which dynamically activated AMP-activated protein kinase (AMPK), enhanced activation of nuclear factor kappa B (NF-κB), and induced metabolic alterations for sustained M1-like macrophage repolarization, followed by mounting of antitumor immunity. Through single-cell RNA sequencing of tumor tissues, as well as macrophage depletion-reconstitution models involving intratumoral transfer of Gal9-KO bone marrow-derived macrophages (BMDMs) and AMPK shRNA-transduced Gal9-KO BMDMs, we confirmed the Gal9-AMPK-NF-κB axis as the essential pathway by which MagLMP functions in antitumor therapy. In a mouse model of lung adenocarcinoma in situ, overall survival was extended after intravenous administration of nanomotors followed by cyclic MagLMP, and one third of mice survived for more than 300 days. Together, these results demonstrate an intracellular mechanical strategy that can dynamically manipulate innate immune responses in vivo, providing a tool for durable immunotherapy through organelle mechanotransduction.

Host immunity status and hypoxia are the hallmarks of radiosensitivity. Induction of anti-PD-1 immunotherapy demonstrates promise in locally advanced tumor radiotherapy, but whether anti-PD-1 immunotherapy improves radiosensitivity is unclear.

Portal vein tumor thrombus (PVTT) is among the most lethal complications of hepatocellular carcinoma (HCC), yet its molecular mechanisms and immune features remain poorly characterized. To address this gap, we performed a comprehensive multi-omics analysis of 99 specimens from 47 patients, integrating nCounter profiling, single-cell RNA sequencing, digital spatial profiling, and proteomics to construct the first spatial map of the PVTT microenvironment. These analyses revealed marked intratumoral heterogeneity and enrichment of myofibroblast-like cancer-associated fibroblasts (myCAFs) arising through a macrophage-to-myofibroblast transition. Nidogen-1 (NID1) was identified as a stromal driver of immune barriers, highly expressed in PVTT cores and associated with impaired antitumor immunity. Guided by these mechanistic insights, we repurposed acarbose, a Food and Drug Administration-approved drug, to inhibit the NID1 axis. Functional assays demonstrated that acarbose disrupted myCAF-mediated immune barriers, suppressed PVTT progression, and synergized with anti-programmed death-1 (anti-PD-1) therapy in preclinical models. Furthermore, analysis of an independent clinical cohort of 810 HCC patients revealed a substantially lower incidence of PVTT among those receiving acarbose, underscoring its translational potential. Collectively, these findings establish the immune-stromal landscape of PVTT, uncover NID1-driven stromal remodeling as a mechanism of immune evasion, and highlight drug repurposing as an immediately actionable strategy to improve outcomes in HCC with PVTT.

Background: Immune checkpoint inhibitors are ineffective in the majority of colorectal cancers (CRCs) that are microsatellite stable. However, the underlying reasons for their unresponsiveness and mechanisms of immune evasion are poorly understood. In the present study, we aimed to elucidate the mechanisms underlying the immune evasion driven by CRC cells. Methods: We performed single-cell RNA sequencing of tumor tissues from 30 CRC patients and syngeneic mice implanted with transformation-related protein 53 (Trp53) knockout CT26 cells. Gene expression and correlations of individual tumor microenvironment (TME) components were analyzed, and their functional significance was investigated using syngeneic mouse models and cell line co-culture experiments. Results: CCAAT enhancer-binding protein beta (CEBPB) expression was increased in tumor protein 53 (TP53)-mutated CRCs. We confirmed that wild-type TP53 negatively regulated CEBPB expression in CRC cell lines. CEBPB expression was associated with decreased intratumoral T cell infiltration and negatively impacted survival in CRC patients. In the intercellular correlation analysis of gene expression, tumor epithelial cell CEBPB expression was significantly correlated with cytotoxic T-lymphocyte associated protein 4 (CTLA4) expression in T cells, especially in regulatory and exhausted T cells. Cebpb overexpression promoted tumor growth in the immunocompetent syngeneic mouse models, which was accompanied by increased CTLA-4 expression in tumor-infiltrating CD4+ T cells. In vitro co-culture experiments also showed that tumor cell CEBPB overexpression increased CTLA4 in T cells. Conclusions: Tumor cell CEBPB expression, up-regulated by TP53 mutation, can increase CTLA4 expression in T cells and negatively affect patient outcomes. These findings suggested a central role of tumor cell CEBPB in shaping an immunosuppressive TME.

Although peritumoural visceral adipose tissue (tVAT) is anatomically close to tumours such as colorectal cancer, the immune landscape of this tissue and its functional contribution to tumour immunity remain poorly defined. Here, we performed single-cell RNA analysis on the tVAT from patients with colorectal cancer to map its immune landscape and observed that tVAT exhibited a highly immune-infiltrated microenvironment enriched with lymphocytes, especially tumour-specific CD8⁺ T cells. Mechanistically, tVAT competes with the tumour for these immunocytes by activating the CXCL12-CXCR4 axis to promote tumour immune escape. Moreover, tumour-derived factors induce an adipose-mesenchymal transformation process where the adipose stromal cells trans-differentiated into adipose-derived cancer-associated fibroblasts, which secrete large amounts of CXCL12 in tVAT. Clinically, targeting adipose-tumour interaction substantially enhances diagnostic and therapeutic efficacy of anti-PD-1 therapy. These findings offer an understanding of the dynamic crosstalk between tVAT and tumour immune escape, highlighting the tVAT as a potential target for cancer immunotherapy.

Background: CC chemokines orchestrate intercellular communication and modulate tumor microenvironment. This study investigates the role of C-C motif chemokine ligand 24 (CCL24) in immune regulation in colorectal cancer (CRC). Methods: CCL24 expression and its clinical relevance in CRC were analyzed via bioinformatics and tissue microarrays. Genetic knockout of CCL24, or antibody-mediated inhibition of CCL24 was performed in AOM/DSS-induced mouse CRC models. CCL24 knockout (CCL24ko) CRC cells were co-cultured with macrophages or CD8+ T cells. Mouse MC38 CRC cells with CCL24ko were implanted into C57BL/6 mice to generate subcutaneous or metastasis models. Molecular docking was conducted to identify potential pharmacological inhibitors of CCL24. Results: CCL24 is abundantly expressed in CRC tissues and linked to T cell dysfunction and unfavorable patient survival. Inhibition or knockout of CCL24 suppressed AOM/DSS-induced colorectal tumorigenesis in mice, reduced the population of tumor-associated macrophages (TAMs), and increased CD8+ T cell numbers. While the morphology of CCL24ko cells showed minimal changes in vitro, their tumorigenic ability was reduced in immunocompetent but not in immunodeficient mice. CCL24 did not directly alter CD8+ T cell populations; instead, CCL24+ tumor cells recruited CCR3+ TAMs, which promote immunosuppression by promoting nuclear translocation of YAP1, a key transcription factor of the Hippo pathway. Gracillin, a natural compound, was identified as a CCL24 inhibitor and synergized with 5-fluorouracil and programmed cell death 1 monoclonal antibody therapies in allograft-bearing mice. Conclusion: CCL24 facilitates recruitment of CCR3+ TAMs, enhancing the immunosuppressive TME in CRC. Targeting CCL24 with agents like gracillin represents a promising therapeutic strategy.

Glioblastoma (GBM) is the most common primary brain tumor with an overall survival under 21 months. Despite extensive research effort, patient outcomes have improved minimally over the past several decades. The Inhibitor of Apoptosis (IAP) proteins are critical survival factors implicated in both immune regulation and gliomagenesis. Small molecule IAP antagonists called SMAC mimetic compounds (SMCs) are under investigation as cancer therapeutics across multiple malignancies, including GBM. SMCs induce GBM cell death in the presence of inflammatory cytokines, synergize with immune checkpoint inhibitors (ICI), and induce death of microglia and macrophages. Although SMCs show significant efficacy in murine models, complete eradication is not achieved. Here, we aimed to understand the limitations of SMCs in murine GBM and identify strategies to enhance efficacy of combination treatment with ICIs with the goal of informing future translational efforts.

Discovering more targets is of great importance for developing alternative interventions for tumor therapy. The roles of transmembrane protein 175 (TMEM175) in neurodegeneration diseases have been reported, however its functions in tumor immune surveillance are not known. We show that TMEM175 conditional knockout in macrophages inhibits the tumor growth and metastasis through promoting the anti-tumor immunity in the tumor microenvironment (TME), including elevated M1-like polarization, reduced M2-like polarization, and facilitated recruitment and activation of T cells and nature killer cells (NKs). The anti-tumor immunity is abrogated by caspase-1 inhibitor VX-765, anti-IL-1β, and anti-IL-18. Tmem175-/- bone marrow-derived macrophages (BMDMs) show enhanced tumor antigen cross-presentation that is further strengthened by IL-1β and IL-18. NLRP3 is robustly elicited in Tmem175-/- BMDMs by the tumor cell debris through lysosomal permeabilization and cathepsin B leakage. Finally, Tmem175-/- mice are more responsive to anti-PD-1. Our works implies TMEM175 to be a potential target for immunotherapy.

Discovering more targets is of great importance for developing alternative interventions for tumor therapy. The roles of transmembrane protein 175 (TMEM175) in neurodegeneration diseases have been reported, however its functions in tumor immune surveillance are not known. We show that TMEM175 conditional knockout in macrophages inhibits the tumor growth and metastasis through promoting the anti-tumor immunity in the tumor microenvironment (TME), including elevated M1-like polarization, reduced M2-like polarization, and facilitated recruitment and activation of T cells and nature killer cells (NKs). The anti-tumor immunity is abrogated by caspase-1 inhibitor VX-765, anti-IL-1β, and anti-IL-18. Tmem175-/- bone marrow-derived macrophages (BMDMs) show enhanced tumor antigen cross-presentation that is further strengthened by IL-1β and IL-18. NLRP3 is robustly elicited in Tmem175-/- BMDMs by the tumor cell debris through lysosomal permeabilization and cathepsin B leakage. Finally, Tmem175-/- mice are more responsive to anti-PD-1. Our works implies TMEM175 to be a potential target for immunotherapy.

Background: Lung cancer remains a major global health burden. RNA-binding proteins (RBPs) play crucial roles in post-transcriptional gene regulation, and their dysregulation is frequently implicated in tumorigenesis. The present study aimed to elucidate the molecular network governed by the highly expressed RBP TIMELESS in lung adenocarcinoma (LUAD) and determine its mechanistic role in LUAD progression. Methods: The Cancer Genome Atlas-LUAD, Gene Expression Omnibus, and single-cell RNA sequencing datasets were analyzed to identify aberrantly expressed RBP genes. The RBP gene TIMELESS exhibited the most significant effect on LUAD cell death and was selected for further study. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation sequencing and RNA sequencing were employed to identify ferroptosis-related targets directly bound by TIMELESS. Molecular mechanisms underlying the TIMELESS-mediated regulation of ferroptosis in LUAD were investigated via immunoprecipitation-mass spectrometry, glutathione S-transferase pull-down, immunofluorescence-fluorescence in situ hybridization, RNA immunoprecipitation, poly(A)-tail, and RNA stability assays. In an orthotopic lung cancer mouse model treated with erastin (a ferroptosis inducer) and programmed cell death protein 1 (PD-1) blockade, the role of TIMELESS in therapeutic response was assessed via flow cytometry and multiplex immunofluorescence (mIF). Infiltrating immune cells in LUAD were analyzed by tissue microarrays (TMAs) via mIF. Results: TIMELESS significantly affected LUAD cell proliferation and death, and TIMELESS knockdown significantly enriched RNA-binding and ferroptosis pathways. Transferrin (TF) was identified as a direct TIMELESS target governing ferroptosis. TIMELESS was revealed to bind Ccr4-Not transcription complex subunit 3 (CNOT3) to promote TF mRNA degradation. TIMELESS depletion combined with erastin and PD-1 blockade enhances efficacy, prolongs survival, increases T cell and M1 macrophage infiltration, and reduces M2 macrophage infiltration. Further, high TIMELESS expression was inversely correlated with ferroptosis marker 4-hydroxynonenal but positively correlated with programmed cell death ligand 1 (PD-L1), reduced T cell and M1 macrophage infiltration, and increased M2 macrophage infiltration. Conclusions: TIMELESS recruits CNOT3 to accelerate TF mRNA degradation, thereby suppressing ferroptosis and promoting LUAD growth. These findings suggest that the TIMELESS/TF regulatory axis may be a promising therapeutic target for LUAD.

Colorectal cancer (CRC) is a leading cause of cancer-related death and remains a significant global health challenge. Cancer vaccines have emerged as a promising immunotherapy for long-term tumor control. While Listeria monocytogenes (Lm)-based intravenous vaccines can generate tumor-reactive CD8 T cells, clinical trial success has been limited. Here, we sought to determine whether in vivo targeting of gastrointestinal tissues with foodborne delivery of Lm-based cancer vaccines controlled tumor growth in murine models of CRC.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited treatment options due to the absence of hormone receptors and HER2 amplification. Immune checkpoint blockade, particularly targeting PD-1/PD-L1, has emerged as a promising therapeutic strategy. However, the response rate of TNBC patients to this monotherapy remains low. This study explores the systemic effect of PD-1 blockade on the immune and hematopoietic systems in 4T1 TNBC mice and demonstrates its limited efficacy in reducing the tumor burden and changing the number of tumor-infiltrating immune cells. However, PD-1 blockade increases systemic immune activity, as demonstrated by increased T cells and DCs in the peripheral blood, which may be associated with inflammatory side effects of this treatment. In addition, PD-1 blockade does not rescue the hematopoietic damage caused by TNBC, highlighting a limitation in long-term response. Furthermore, PD-1 blockade in tumor-free mice leads to an increase in hematopoietic stem/progenitor cells, suggesting that PD-1 blockade may yield better benefits post-tumor resection.

Hepatocellular carcinoma (HCC) is the fastest growing cause of cancer-related mortality and there are limited therapies1. Although endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are implicated in HCC, the involvement of the UPR transducer ATF6α remains unclear2. Here we demonstrate the function of ATF6α as an ER-stress-inducing tumour driver and metabolic master regulator restricting cancer immunosurveillance for HCC, in contrast to its well-characterized role as an adaptive response to ER stress3. ATF6α activation in human HCC is significantly correlated with an aggressive tumour phenotype, characterized by reduced patient survival, enhanced tumour progression and local immunosuppression. Hepatocyte-specific ATF6α activation in mice induced progressive hepatitis with ER stress, immunosuppression and hepatocyte proliferation. Concomitantly, activated ATF6α increased glycolysis and directly repressed the gluconeogenic enzyme FBP1 by binding to gene regulatory elements. Restoring FBP1 expression limited ATF6α-activation-related pathologies. Prolonged ATF6α activation in hepatocytes triggered hepatocarcinogenesis, intratumoural T cell infiltration and nutrient-deprived immune exhaustion. Immune checkpoint blockade (ICB)4 restored immunosurveillance and reduced HCC. Consistently, patients with HCC who achieved a complete response to immunotherapy displayed significantly increased ATF6α activation compared with those with a weaker response. Targeting Atf6 through germline ablation, hepatocyte-specific ablation or therapeutic hepatocyte delivery of antisense oligonucleotides dampened HCC in preclinical liver cancer models. Thus, prolonged ATF6α activation drives ER stress, leading to glycolysis-dependent immunosuppression in liver cancer and sensitizing to ICB. Our findings suggest that persistently activated ATF6α is a tumour driver, a potential stratification marker for ICB response and a therapeutic target for HCC.

Lung cancer, primarily non-small cell lung cancer (NSCLC), causes the highest cancer-related mortality. Although PD-1/PD-L1 inhibitors have improved survival in advanced NSCLC, they can cause immune-related adverse events. Cordyceps sinensis (C. sinensis), a traditional Chinese medicine used for tonifying the lung and kidney and enhancing immune function, has shown therapeutic promise in combination with anti-PD-1 therapy for NSCLC. This study aimed to explore the anti-tumor effect of wild C. sinensis combined with anti-mouse PD-1 in the treatment of Lewis lung adenocarcinoma (LLC) and to elucidate the underlying pharmacodynamic mechanism. LLC mouse model was established via inoculation with LLC cells, followed by treatment with anti-mouse PD-1, C. sinensis, or their combination. The tumor volume, weight, and histological changes of LLC mice were evaluated. The proportions of tumor-infiltrating immune cells in blood and tumors were evaluated by flow cytometry, immunohistochemistry, and immunofluorescence. The underlying mechanisms of the combination of C. sinensis and anti-mouse PD-1 therapy in LLC mice were investigated using an integrated transcriptomics and metabolomics analysis. Treatment with anti-mouse PD-1, C. sinensis, or their combination significantly reduced tumor volume and weight, and attenuated the histopathological changes of LLC mice tumors. Among which, medium-dose C. sinensis combination exhibited significant improvements. Furthermore, the combination of C. sinensis and anti-mouse PD-1 significantly increased the proportion of CD8+ T cells and decreased the abundance of Tregs and PMN-MDSCs. Integrated transcriptomics and metabolomics analysis revealed that the combination of C. sinensis and anti-mouse PD-1 can enhance anti-tumor immunity in LLC mice by acting on key immune-related genes, including DGKA, PLA2G7, AMPD1, ATP8B4, and BST1, thereby modulating glycerophospholipid metabolism, the TCA cycle, purine metabolism, and nicotinate-nicotinamide metabolism. Wild C. sinensis combined with anti-mouse PD-1 therapy exerts therapeutic effects against LLC by targeting immune-related genes, modulating associated pathways, increasing the proportion of CD8+ T cells, and reducing the infiltration of Tregs and PMN-MDSCs, thereby suppressing tumor growth and inhibiting LLC progression. Further research and clinical studies are needed to validate and expand upon these promising findings.

Background: Lung metastasis is a leading cause of breast cancer (BC)-related mortality, driven by the immunosuppressive traits of the metastatic tumor microenvironment. However, the mechanisms underlying cell-cell crosstalk in shaping immune evasion within the metastatic niche remain poorly defined. Neutrophil extracellular traps (NETs) and their associated proteins, such as cathelicidin, have emerged as key mediators of metastatic regulation in cancer. Here, we aimed to decipher the interaction between a neutrophil subset characterized by high expression of lymphocyte antigen 6 complex locus g (Ly6ghigh) and cluster of differentiation 8-positive T lymphocytes (CD8+ T cells), mediated via cathelicidin embedded in NETs, as well as their synergistic mechanism and cooperative role in promoting lung metastasis of BC. Methods: We characterized neutrophil heterogeneity and functional dynamics by performing single-cell RNA sequencing and flow cytometry on lung tissues derived from murine models of BC lung metastasis. We utilized cathelicidin-related antimicrobial peptide (Cramp) knockout mice to dissect the role of cathelicidin in NETs. The spatial colocalization of apoptotic CD8+ T cells and NETs was analyzed using multiplex immunofluorescence, and the molecular interactions were probed by protein binding assays. Results: Neutrophils in the lung metastatic niche were classified into 2 subsets based on the Ly6g expression: Ly6ghigh and Ly6glow neutrophils. Ly6glow neutrophils, which were recruited in the macrometastatic stage, exhibited myeloid-derived suppressor cell-like characteristics. Notably, Ly6ghigh neutrophils induced CD8+ T cell apoptosis through NET formation, with apoptotic CD8+ T cells spatially clustered within NET-rich areas. Mechanistically, NET-derived cathelicidin (Cramp in mice) directly bound to mitochondrial adenine nucleotide translocator 1 (Ant1) in CD8+ T cells, triggering conformational changes and complex formation with voltage-dependent anion channel 1 (Vdac1). These events resulted in the opening of the mitochondrial permeability transition pore and loss of mitochondrial membrane potential. Conclusions: Our study demonstrates that Ly6ghigh neutrophils play a critical role in immunosuppression and immune evasion through NET-induced apoptosis of CD8+ T cells. These findings underscore the importance of NETs and cathelicidin in BC lung metastasis, suggesting their potential as therapeutic targets in restoring antitumor immunity and in preventing metastatic progression.

Background: Osteosarcoma remains largely refractory to immune checkpoint inhibitor (ICI) monotherapy, and strategies to modulate the tumor immune microenvironment are being actively explored. Mild hyperthermia has been reported to influence antitumor immune responses; however, its impact in combination with PD-1 blockade in osteosarcoma has not been well characterized. Methods: Murine LM8 osteosarcoma cells were subjected to mild thermal stimulation, and changes in PD-L1 expression were evaluated. LM8-bearing mice were treated with mild hyperthermia, anti-PD-1 antibody, or their combination. Tumor growth, lung metastasis, and survival were assessed. Tumor-infiltrating immune cells were profiled using single-cell RNA sequencing to descriptively characterize immune-associated transcriptional features under each treatment condition. Results: Mild thermal stimulation (42 °C, 30 min) increased PD-L1 expression in LM8 cells in vitro. In vivo, combination therapy significantly suppressed primary tumor growth compared with control (χ2 = 29.75, p = 1.6 × 10-6) and reduced lung metastasis burden, with a significant decrease in metastatic nodules (p < 0.01). Kaplan-Meier analysis demonstrated a significant survival benefit in the combination group (log-rank p < 0.001). Single-cell RNA sequencing revealed an increased proportion of CD8+ T cells with reduced exhaustion-associated gene expression and a shift toward pro-inflammatory (M1-like) macrophage transcriptional profiles. Conclusions: PD-1 blockade combined with mild hyperthermia was associated with enhanced antitumor efficacy and immune-associated transcriptional remodeling in a murine osteosarcoma model, supporting further preclinical evaluation of this combination strategy.