InVivoMAb rat IgG2a isotype control, anti-trinitrophenol

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.

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.

Tumour-induced mechanisms of immune evasion hinder immune response to cancer, particularly in melanoma. mRNA translation, by ensuring accurate protein synthesis, regulates cancer phenotypes and immune response, but the underlying mechanisms remain unclear. Here, we reveal how O-sialoglycoprotein endopeptidase (OSGEP), catalysing the tRNA modification N6-threonylcarbamoyladenosine (t6A), drives protein homeostasis in cancer cells to maintain T-cell exclusion and prevent anti-tumour immune response. t6A-deficient melanoma cells disrupt efficient cytoplasmic translation of ANN codons (trinucleotides with A in the first position and N = any nucleotide), causing specific protein aggregation and the formation of integrated stress response-dependent stress granules. We discovered that OSGEP loss triggers melanoma regression by relocating RIG-I to stress granules, leading to its pathway activation. As a result, T-cells are recruited to the tumour site and orchestrate an anti-tumour immune response. Finally, an OSGEP-driven gene signature in melanoma patients is associated with T-cell infiltration and improved overall survival. Together, our findings position t6A tRNA modification as a promising therapeutic target for melanoma treatment.

Most colorectal cancers (CRCs) are mismatch repair-proficient (pMMR) and microsatellite stable (MSS), and they respond poorly to immune checkpoint inhibitors (ICIs). Radiotherapy (RT) can promote antitumor immunity but may also trigger adaptive immune suppression through checkpoint upregulation, providing a rationale for combination therapies.

Radiation therapy (RT) is recognized for its ability to induce DNA damage within cancer cells, leading to cancer cell death and promotion of anti-tumor immune responses. However, this beneficial effect is often counterbalanced by the presence of suppressive Tregs. Although factors such as RT-induced transforming growth factor β (TGF-β) can contribute to increased Treg accumulation within the tumor, the dynamics of Treg movement, and recruitment in the post-RT tumor microenvironment are not fully understood. Our study examined Treg migration following RT, revealing that RT disrupts Treg migration to the tumor-draining lymph node (TdLN) and alters their phenotype. Combining RT with anti-CCR8 therapy, which selectively depletes Tregs within the tumor, significantly reduced tumor burden, and increased survival in preclinical models. This combination also proved effective against distant and unirradiated tumors. Additionally, efficacy of combination therapy was CD8 T cell dependent. These findings highlight the potential of combining RT with Treg-targeting therapies to enhance anti-tumor immunity.

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.

Metastatic tumor cell dissemination is the leading cause of cancer-related deaths. Clustered circulating tumor cells (CTCs) possess higher metastatic potential than single CTCs. Epithelial adherens junction (AJ) proteins typically mediate stable cell-cell interactions; however, these proteins are frequently lost in highly aggressive triple-negative breast cancers (TNBCs), raising the question of how CTCs from such tumors cluster. Here we show that the extracellular matrix (ECM) component hyaluronan (HA) mediates AJ-independent CTC clustering in TNBCs. HA is necessary and sufficient to drive clustering of tumor cells expressing its receptor CD44. Mechanistically, HA initiates contact between neighboring cells through actin-based membrane protrusions. As cells are pulled closer, these initial interactions expand to membrane-membrane contact and are subsequently stabilized by desmosomes. CTC-derived HA also acts as a docking platform to promote heterotypic cluster formation by recruiting non-CTCs, including immune cells. Thus, this ECM-receptor interaction enables CTC clustering and survival under shear stress, enhancing TNBC metastasis.

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.

Clonal hematopoiesis (CH) driven by JAK2V617F is known to accelerate atherosclerosis through inflammasome activation and release of interleukin (IL)-1β and -18; yet, the specific contribution of IL-18 has remained unclear. In this study, we demonstrate that antibody inhibition of IL-18 in JAK2V617F CH mice increases plaque collagen but paradoxically promotes both early lesion growth and advanced necrotic core formation. Mechanistically, IL-18 blockade reverses absent in melanoma 2 inflammasome activation but shifts cell death toward apoptosis, and together with impaired efferocytosis, results in greater necrosis. These events are coordinated by reduced interferon gamma signaling, which enhances collagen deposition while decreasing expression of efferocytotic genes. Our findings challenge the prevailing notion that IL-18 inhibition stabilizes atherosclerotic plaques and provide new mechanistic insight into the interplay among inflammasome biology, adaptive immunity, and plaque stability.

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.

Immunosuppression and metastasis are critical hallmarks of breast cancer, often linked to poor patient outcomes. The secreted cytokine chitinase-3 like 1 (CHI3L11) is frequently overexpressed in breast cancer samples and promotes an immunosuppressed tumor microenvironment. Notably, CHI3L1 expression is elevated in metastatic patient samples when compared to the matched primary breast tumor. To investigate its role in breast cancer metastasis, we generated an inducible Genetically Engineered Mouse Model (GEMM) that overexpresses CHI3L1 in the mammary epithelium. Ectopic expression of CHI3L1 in the Polyomavirus Middle T (PyMT) mouse model of breast cancer suppressed anti-tumor immune responses, accelerated mammary tumor onset and enhanced lung metastasis. Mechanistically, elevated CHI3L1 expression in the mammary epithelium enhanced neutrophil recruitment, which subsequently degraded the extracellular matrix and increased the number of circulating tumor cells. These findings reveal a key mechanism driving metastatic dissemination and argue that therapeutically targeting Chi3l1 could enhance anti-tumor immunity and suppress metastasis.

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.

The expression level of PD-L1 is one of the most widely used predictive markers of immune checkpoint blockade (ICB) efficacy in the clinic, suggesting the importance of regulating PD-L1 expression. However, no published reports have addressed the systematic exploration of the regulation of immune checkpoint molecules from the perspective of mutual exclusion (ME) in gene expression. The ME analysis, based on gene plasticity, provides a novel perspective on the intergenic regulatory paradigm. Here, multiple negative regulators of PD-L1 expression are identified, and dual-specificity phosphatase 9 (DUSP9) is selected for intensive study. DUSP9 negatively regulates PD-L1 expression in multiple tumor cells, and mechanistically, DUSP9 dephosphorylates STAT3 to mediate the inhibitory role. In syngeneic tumor models, the combination of DUSP9 targeting and PD-1 antibody can enhance therapeutic sensitivity. The clinical data demonstrated that elevated DUSP9 expression is correlated with diminished PD-1/PD-L1 antibody response rates. Consequently, DUSP9 emerges as a promising target for enhancing treatment response in combination with PD-1 antibody, and functions as a potential marker for predicting the efficacy of tumor immunotherapy. This research demonstrates an efficient method for identifying negative regulators of highly plastic genes (HPGs), which can predict immunotherapy responses and identify new targets for combination therapy with ICB.

Thimerosal is a mercury-containing preservative widely used in vaccines. This study aimed to investigate its potential antitumor effects and mechanisms in solid malignancies, particularly colorectal cancer (CRC) and melanoma.

Insufficient infiltration of CD8+ T cells in the tumor microenvironment (TME) critically restricts antitumor immunity and cancer immunotherapy efficacy. The purpose of this study was to identify novel tumor cell-intrinsic regulators of T-cell infiltration and to elucidate their mechanisms of action.

Decorin (DCN) predominantly produced by fibroblasts is a small leucine-rich proteoglycan with tumor-suppressive property. However, whether DCN has a role in shaping the tumor immune microenvironment remains elusive.

Microsatellite stable (MSS) colorectal cancer (CRC) is often considered a "cold" tumor with limited response to programmed death-1 (PD-1) antibody monotherapy. The mechanisms underlying its intrinsic resistance to immunotherapy remain unclear. Here, we show that cathepsin D (CTSD) is highly expressed in MSS CRC and contributes significantly to immunotherapy resistance. Mechanistically, CTSD, acting as a protease, interacts with the α2 domain of the major histocompatibility complex (MHC) class I via the light chain of its catalytic domain, promoting MHC class I degradation through lysosomal pathways and impairing its recycling to the cell surface. This mechanism shields tumor cells from cytotoxic T-cell-mediated killing and facilitates immune evasion. Notably, genetic deletion or pharmacological inhibition of CTSD using pepstatin A prevents immune escape and enhances anti-PD-1 efficacy. These findings identify CTSD as a key mediator of immune evasion in MSS CRC and support the development of a combination therapy comprising CTSD inhibition and anti-PD-1 immunotherapy.

The dual challenges of limited therapeutic options due to de novo or acquired resistance and psychological distress in patients with melanoma necessitate innovative treatment strategies. Here, we identify paroxetine hydrochloride (PH), a Food and Drug Administration (FDA)-approved antidepressant, as an alternative therapeutic for BRAFV600E-mutated melanoma, including BRAFi/MEKi-resistant cases. Furthermore, our findings reveal that PH acts as an unrecognized inducer of pyroptosis. By triggering pyroptosis, PH remodels the tumor-permissive microenvironment in recurrent melanoma to potentiate anti-PD-1 therapy while maintaining a favorable safety profile. Mechanistically, PH impedes 5-hydroxytryptamine (5-HT) reuptake, leading to epigenetic reprogramming by reducing histone serotonylation (H3Q5ser) at the promoters of DNA repair genes. Impaired DNA damage repair pathways in turn trigger genome instability, proteostasis imbalance, and subsequent endoplasmic reticulum stress, ultimately inducing pyroptosis. Our findings uncover the underlying mechanism by which 5-HT drives melanoma progression and highlight PH as a promising candidate with multiple clinical potentials for treating melanoma.

DF6215 is a rationally engineered interleukin-2 (IL-2) Fc-fusion protein developed to overcome efficacy and safety limitations of traditional IL-2 cancer immunotherapy. Unlike non-alpha (non-α) IL-2 variants that eliminate CD25 binding and underperform clinically, DF6215 retains moderate IL-2 receptor α (IL-2Rα) affinity while enhancing IL-2Rβγ signaling and extending the half-life via an engineered immunoglobulin (Ig)G1 Fc domain. This design preferentially expands cytotoxic CD8+ T cells and natural killer cells over regulatory T cells, resulting in favorable effector-to-regulatory cell ratios, enhanced immune activation, and robust tumor regression in mouse models. In poorly immunogenic tumors, DF6215 synergized with PD-1 blockade to achieve durable responses without added toxicity. Cynomolgus monkey studies confirm DF6215's pharmacodynamics and favorable safety profile, with no signs of vascular leak syndrome or cytokine release syndrome. These findings position DF6215 as a differentiated IL-2 capable of modulating the tumor microenvironment and achieving potent anti-tumor immunity with improved tolerability, supporting its advancement into clinical trials for solid tumors.