Chimeric antigen receptor (CAR) macrophage therapy shows significant potential for solid tumors owing to the intrinsic tumor infiltration and phagocytic capacity of macrophages. However, its clinical translation is limited by macrophage phenotypic plasticity within the immunosuppressive tumor microenvironment and the complexity of ex vivo cell manufacturing. It is essential to develop techniques that enable macrophages to be activated specifically by antigens while sustaining their proinflammatory activity in vivo. Here, we report a mannose-modified lipid nanoparticle (LNP) platform for the co-delivery of CAR-encoding messenger RNA (mRNA) and the Toll-like receptor (TLR) 7/8 agonist resiquimod (R848), enabling in situ generation of proinflammatory CAR macrophages. In vitro, we assessed macrophage-preferential uptake, CAR expression efficiency, TLR7/8 agonist-mediated macrophage polarization, and immune activation. In vivo efficacy was assessed in syngeneic and humanized mouse models of triple-negative breast cancer, including postoperative recurrence and lung metastasis models. Systemic administration of M-LNP/CAR+R848 induced robust CAR expression in tumor-associated macrophages and promoted sustained M1 polarization. Engineered macrophages exhibited enhanced antigen-specific phagocytic activity and tumor cell clearance, and promoted CD8+ T cell proliferation and NK cell infiltration, thus coordinating innate and adaptive immune responses. Functional macrophage depletion experiments demonstrated that tumor control was dependent on macrophages. In vivo treatment significantly reduced the growth of primary tumors, prevented postoperative recurrence, and prolonged survival in mice with lung metastases in both syngeneic and humanized models. Our findings demonstrate that M-LNPs enabling co-delivery of mRNA and an innate immune agonist enable in situ generation of proinflammatory CAR macrophages and induce durable antitumor immunity. This controllable and non-integrative strategy allows tunable immune activation, provides a flexible platform for CAR macrophage-based immunotherapy in triple-negative breast cancer.
Circulating tumor antigens (ctA; tumor markers) are blood-based proteins that can offer prognostic value in non-small cell lung cancer (NSCLC) and may serve as potential early surrogates for survival. Given the significantly reduced testing time and cost of ctA compared with circulating tumor DNA (ctDNA), we further explored the utility of ctA using samples collected from over 2,300 patients participating in five clinical trials (IMpower130, 131, 132, 150, and 110). We analyzed a panel of six ctA (CA125 (cancer antigen 125), CEA (carcinoembryonic antigen), Cyfra21-1 (cytokeratin 19 fragment 21-1; CYFRA), NSE (neuron-specific enolase), SCC (squamous cell carcinoma antigen), and ProGRP (progastrin-releasing peptide)) and CRP (C-reactive protein) from the serum of patients with metastatic NSCLC in these trials, which investigated combinations of atezolizumab (anti-programmed death-ligand 1)±bevacizumab±chemotherapy. Previous work showed that an optimized cut-off using two ctA or a machine learning (ML) model of ctDNA features, both taken at 6 weeks, can stratify patients with stable disease (SD) for survival risk in IMpower150. Building on this approach, we applied an ML model combining ctA features at baseline and at 6 weeks, trained across a much larger aggregate dataset from multiple clinical studies. Previous findings from ctA analysis of IMpower150 were confirmed and found to be applicable to several other trials analyzed in this study. We found that ML model predictions provided similar prognostic performance (c-index of 0.73 and 0.71 in squamous and non-squamous test datasets, respectively), with CYFRA being the top feature for both histologies. While ctA demonstrated limited potential in differentiating treatment effects to inform early drug development, deriving an optimal prediction cut-off for 1-year overall survival (OS) showed that ctA model predictions could effectively stratify patients by radiographic response with 61% sensitivity and 78% specificity, adding significant prognostic value to radiographic imaging. Patients with partial response (PR), progressive disease (PD), or stable disease (SD) at either 6 weeks of treatment or best confirmed overall response could be separated into low-risk or high-risk groups for OS.
Local tumor progression and metastasis substantially compromise the therapeutic efficacy of thermal ablation in liver cancer. Although the inflammatory microenvironment contributes, the specific role of neutrophils after ablation remains unclear. This study aimed to clarify the roles of neutrophil extracellular traps (NETs) after thermal ablation and evaluate the translational potential of NETs-targeted strategies. The adverse prognostic impact of thermal ablation-induced neutrophil and inflammatory changes in liver cancer was retrospectively evaluated. Circulating NETs were quantified by ELISA, and their association with local tumor progression was analyzed. An orthotopic liver cancer mouse model and patient-derived neutrophils elucidated the spatiotemporal dynamics and mechanisms of ablation-induced NETs, and pharmacologic inhibition was used to investigate NET formation, function, and translational potential. Thermal ablation rapidly activates neutrophils, elicits a sustained local inflammatory response and NET formation, which is closely associated with poor prognosis in patients with liver cancer. Depletion of neutrophils, inhibition of NET formation, or degradation of thermal ablation-induced NETs markedly suppressed post-ablation tumor progression. Mechanistically, tumor cell-intrinsic reactive oxygen species generated under heat stress contributed to nuclear factor kappa-B (NF-κB) activation, promoting p65 binding to theC-X-C motif chemokine ligand 1 (CXCL1) promoter and transcriptional upregulation of CXCL1. This process drove CXCL1-C-X-C motif chemokine receptor 2 (CXCR2) signaling and led to the formation of a characteristic band-like NETs-enriched zone at the peri-ablational margin. In turn, NETs activated toll-like receptor (TLR)9-dependent NF-κB,mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 3 (STAT3) signaling in heat-stressed cancer cells, reinforcing a pro-inflammatory feedback loop. Concurrently, NETs impaired CD8+ T-cell responses and fostered an immunosuppressive microenvironment. Together, this coordinated pro-inflammatory and immunosuppressive effect promotes liver cancer progression following thermal ablation, which can be effectively reversed by dual blockade of NF-κB and CXCR2. Our research uncovers a novel mechanism underlying tumor-promoting inflammation after thermal ablation, highlighting the critical role of NETs as mediators through TLR9 activation of downstream inflammatory pathways and the significant potential of NETs-targeted strategies in combination with thermal ablation.
Why are clonal neoantigens paradoxically rare in established tumors? Why does tumor-associated antigen (TAA) burden inversely correlate with T-cell infiltration? Why do damage-associated molecular pattern (DAMP)/danger signal-inducing interventions (messenger RNA (mRNA) vaccines, stereotactic body radiotherapy (SBRT), immunogenic chemotherapy) enhance checkpoint blockade activity in "cold" tumors? We propose the "DEA Hypothesis" to resolve these paradoxes: durable antitumor immunity for established tumors requires three convergent signals-(1) Disinhibition of T cells through checkpoint blockade, (2) Enhanced recognition via the presentation of clonal neoantigens, and crucially, (3) Alerting the host through release of DAMPs/danger signal(s). We frame cancer as a subversion of an ancient "Guardian" system-the emergency hematopoietic response that evolved to orchestrate immune defense following epithelial breach or major trauma. Tumors hijack this system by promoting restorative hematopoiesis at the expense of immunity and masquerading as chronic wounds. This framework explains key observations: the paucity of clonal neoantigens reflects successful immunoediting over years of tumor evolution, not failed presentation. TAAs persist in cold tumors because insufficient DAMPs limit the "Decision to Attack"; and mRNA vaccines, recently SBRT, and immunogenic chemotherapy succeed in cold tumors by providing the missing Alert. For cold tumors, rational immunotherapy must extend beyond checkpoint blockade to reconstitute the Alert signal-not merely releasing the brakes but providing the ignition to reawaken the ancient Guardian.Graphic abstract: The DEA hypothesis proposes that effective cancer immunotherapy requires three convergent signals: Disinhibition (checkpoint blockade), Enhanced recognition (clonal neoantigens), and Alerting the host (DAMP release). Panel I depicts immune decision points in tumor-draining lymph nodes. Panel II shows mutation-driven DAMP generation via the Poly(ADP-Ribose) Polymerase 1 - High Mobility Group Box 1 (PARP1-HMGB1) axis. Panel III illustrates the three-signal model converging on type 1 conventional dendritic cells activation. Panel IV demonstrates how immunoediting shapes stage-specific therapeutic strategies for cold, warm, and hot tumors.
Resistance to anti-programmed cell death protein-1 (PD-1) treatment in gastric cancer (GC) is closely associated with an immunosuppressive tumor microenvironment. However, the role of neutrophils in resistance to anti-PD-1 therapy remains unclear. Single-cell RNA sequencing was performed on tumor samples from patients with advanced GC receiving anti-PD-1 therapy to identify neutrophil subsets associated with neutrophil extracellular traps (NETs). Multilevel experimental validation was conducted using multiomics analysis, flow cytometry, multiplex immunofluorescence, and in vitro co-culture. Therapeutic strategies targeting NETs and CD8+ T-cell exhaustion were evaluated in a mouse model of YTN16 tumors. We identified a NETs-associated neutrophil subset enriched in patients with GC resistant to anti-PD-1 treatment. This subset was marked by CD177, and it exhibited a high potential for NETs release. Peripheral blood NETs levels and CD177+ neutrophil ratios in patients with GC act as markers for evaluating the efficacy of PD-1 inhibitors. Furthermore, transforming growth factor-β1 (TGF-β1), which was highly expressed in GC and spatially colocalized with CD177+ neutrophils, might induce neutrophils to release NETs via the Smad3-NFE2 axis. NETs promoted CD8+ T cell exhaustion by activating the MEK/ERK-c-Fos/JunB axis, as evidenced by increased PD-1/TIM3 expression and reduced interferon-gamma/tumor necrosis factor-alpha secretion. In vivo experiments confirmed that targeted inhibition of NETs formation using DNase I or TGF-β1 inhibitors significantly suppressed tumor growth and CD8+ T cell exhaustion. Notably, the MEK inhibitor trametinib reversed the immunosuppressive microenvironment associated with CD8+ T cell exhaustion and synergistically enhanced the antitumor efficacy with anti-PD-1 therapy. TGF-β1 drives CD177+ neutrophils to release NETs, which induce CD8+ T cell exhaustion via the ERK-c-Fos-JunB pathway, thereby mediating resistance to anti-PD-1 treatment in GC. Furthermore, targeting NETs formation and combining trametinib with PD-1 inhibitors can significantly reverse CD8+ T cell exhaustion, exert synergistic antitumor effects, and offer a potential therapeutic strategy for overcoming resistance to anti-PD-1 therapy in GC.
Deficient DNA mismatch repair/microsatellite instability-high (dMMR/MSI-H) cancers are very sensitive to immune checkpoint inhibitors (ICIs), yet their use is frequently complicated by immune-related adverse events (irAEs). Our study analyzed the impact of grade ≥3 irAEs (immune-related severe adverse events (irSAEs)) on survival in patients with dMMR/MSI-H digestive cancers treated with ICIs. We conducted an international, multicenter, ambispective study involving 1,175 patients from 34 centers. The primary endpoint was the correlation between the occurrence of irSAEs and progression-free survival (PFS). Secondary endpoints included factors associated with irSAEs, correlation between irSAEs and overall response rate (ORR) and overall survival (OS). Prespecified landmark and time-dependent survival models were used to account for the time to irSAE occurrence. Among 1,175 patients treated with ICIs for digestive cancers, 49.1% were female, median age was 66.9 years (IQR 53.7-77.3) and 82.9% had colorectal cancer. Overall, 382 patients (32.5%) had an irAE, including 117 (10.0%) irSAEs. Median time to irSAEs occurrence was 3.78 months (IQR 1.97-7.62). The most frequent irSAEs were gastrointestinal (3.4%), hepatic (1.8%) and dermatologic (1.0%). The only factor associated with irSAEs was the use of an ICI combination versus monotherapy (20.0% vs 11.4%, p=0.03). Among patients with irSAEs, 71.5% received oral and 29.8% intravenous corticosteroids, and 15.7% immunosuppressive agents. Overall, in 26.9% of patients, ICIs were resumed following irSAEs, with an irAE recurrence rate of 57.1%. irSAE was associated with a better ORR (48.6% vs 34.6%, p<0.0001). The multivariable Cox model revealed that irSAE as a time-dependent variable was not associated with better PFS (HR=1.280 (95% CI 0.898 to 1.824), p=0.172) or OS (HR=1.043 (95% CI 0.712 to 1.528), p=0.828). Likewise, landmark analysis at 3 months and 6 months also showed that irSAEs were not associated with better PFS or OS. The occurrence of irSAE was not independently associated with better survival in patients with dMMR/MSI-H digestive cancers treated with ICIs.
Immune checkpoint blockade is minimally active in unselected castration-resistant prostate cancer (CRPC) and does not reproducibly yield durable decreases in prostate-specific antigen (PSA) levels. The Quick Efficacy Seeking Trial was designed to employ a combination of agents to initiate an immune response (with BN-Brachyury vaccine), potentiate that response (with nogapendekin-alfa inbakicept (NAI), an interleukin (IL)-15 receptor superagonist), and reduce or eliminate immunosuppressive entities in the tumor microenvironment (with bintrafusp alfa, a dual inhibitor of programmed death-ligand 1 and transforming growth factor beta). Epacadostat (an indoleamine 2,3-dioxygenase (IDO) inhibitor) was also employed in one cohort to reduce immune suppression induced by IDO's conversion of tryptophan to kynurenine. Patients with CRPC enrolled sequentially to receive vaccine + bintrafusp alfa (Arm 2.1), vaccine + bintrafusp alfa + NAI (Arm 2.2), and vaccine + bintrafusp alfa + NAI + epacadostat (Arm 2.3), with the primary objective to determine response rate. Adverse events in Arms 2.1 and 2.2 were manageable and consistent with the safety profiles of each agent individually, and notable for five individuals developing isolated adrenocorticotropic hormone deficiency. Arm 2.3 was closed early due to skin toxicity. Sustained declines in PSA were seen in 1/13 (8%) patients in Arm 2.1, 7/24 (29%) patients in Arm 2.2, including six with proficient mismatch repair/microsatellite stable tumors, and 0/6 (0%) patients in Arm 2.3. Analyses of peripheral immune profiles provided evidence of a multifaceted antitumor immune response, including IL-15 receptor superagonist NAI-dependent expansion and activation of natural killer cells and CD8+ T cells, increased effector-to-suppressor immune cell ratios, and induction of cytotoxic immune gene programs. NCT03493945.
Cancer-associated mesothelial cells (CAMCs) are key modulators of the ovarian tumor microenvironment, contributing to tumor growth and immune evasion. Mesothelial cells (MCs) maintain peritoneal homeostasis and immune surveillance and represent the first point of contact during abdominal dissemination of ovarian cancers. Yet, their role in ovarian tumor immunity remains poorly understood. Lineage tracing, 3D models, and spatial transcriptomic profiling were used to characterize CAMC origin, localization, and phenotypic transitions during ovarian cancer progression. Multiplex cytokine panels were used to define the cytokine profiles associated with MC transformation into CAMCs. Functional studies were conducted in syngeneic ovarian cancer mouse models to assess the impact of CAMCs on tumor growth and response to immunotherapy. In parallel, CAMC-driven changes in immune cell phenotype and functional state within the tumor microenvironment were characterized. We demonstrate that CAMCs originate from peritoneal MCs, populate the tumor surface, and progressively infiltrate the tumor core while undergoing a phenotypic transition toward a fibroblast-like phenotype. We characterize the function of an unrecognized CAMC signature marked by SERPINB2+ expression and a combination of markers absent in normal MCs. CAMCSerpinb2+ cells have reduced expression of pro-inflammatory cytokines (IL-2, IL-7, IL-12, IL-15) and increased expression of IL-10, TGFβ1, and CCL17 compared with normal MCs. Functionally, the presence of CAMCSerpinb2+ cells correlates with accelerated tumor growth, reduced CD4+ T and B cell infiltration, an expanded Treg population, and ultimately resistance to combination immunotherapy in a syngeneic mouse model of ovarian cancer. These findings identify CAMCs as central regulators of immune suppression in ovarian cancer and reveal a distinct SERPINB2+ immunosuppressive CAMC state associated with tumor progression and immunotherapy resistance. Targeting CAMCs may represent a promising therapeutic strategy to restore antitumor immunity and improve responses to current ovarian cancer treatments and immunotherapies.
Programmed death receptor 1 (PD-1) blockade produces high response rates in resectable and locally advanced cutaneous squamous cell carcinoma (CSCC), but how many doses are needed and whether surgery or radiation after immunotherapy (consolidation) adds benefit in deep responders (patients with substantial clinical responses) remain unclear. Observational inference is challenging because treatment decisions are response-guided, doses accumulate over time, and treatment selection depends on patient characteristics that also affect outcomes. We conducted a retrospective cohort study of 189 patients with resectable, borderline-resectable, locally advanced, or limited metastatic CSCC treated with immune checkpoint inhibitors as part of management (2019-2025). We summarized responses, treatment discontinuation patterns, and time-to-event outcomes. To evaluate dose-response relationships, we fit Bayesian regression models adjusting for prespecified baseline confounders. Directed acyclic graphs were used to formalize causal assumptions and identify minimally sufficient adjustment sets. For event-free survival (EFS), we used a prespecified landmark analysis among patients receiving ≥2 doses to reduce guarantee-time bias. Objective response occurred in 119/189 (63%), including 52/189 (27.5%) complete responses. Nearly half of patients received ≤2 doses (92/189, 48.7%). Among 50 complete responders managed without surgery, 1 recurrence was observed over a median follow-up of 25.4 months. Dose-response models showed a consistent but modest positive association between additional doses and response: in a linear model, each additional dose was associated with ~1.09 fold higher odds of response (posterior probability >95%). Flexible threshold models suggested early concentration of benefit, strongest at 2 versus 1 dose (93.6% posterior probability of benefit), with persistent uncertainty in effect magnitude (66% probability of ≥6 percentage-point absolute increase). In the EFS landmark modeling cohort (n=177), receipt of ≥3 versus 2 doses showed a 93% posterior probability of reduced event risk, but the 89% credible interval spanned the null (HR 0.61-1.01), indicating substantial uncertainty regarding incremental downstream benefit. In real-world CSCC, durable disease control frequently occurred after limited immunotherapy exposure, and clinical responders often did well without routine surgical consolidation. Although additional doses may modestly improve outcomes, observed gains appear concentrated early with uncertain incremental value beyond two doses. These patterns support conceptualizing treatment as frontline immunotherapy with response-guided subsequent treatment rather than fixed-duration neoadjuvant therapy.
Primary liver cancer, predominantly hepatocellular carcinoma (HCC), has limited therapeutic options. While mutation-derived neoantigen vaccine holds promise, its success is hindered by low antigen availability. This study explores transcriptome-derived neoantigens (neoantigen-encoding tumor-specific transcripts, neoTSTs) in HCC, characterizing their features, generation mechanisms, and therapeutic potential. We developed a computational pipeline integrating STAR/StringTie-based transcript assembly with multiexon/single-exon reference datasets (23,972 human control samples) for tumor-specific transcripts (TSTs) identification. A custom sliding-window algorithm compared TST-encoded peptides against UniProt, with neoTSTs predicted using netMHCPan. This framework was applied to 1,013 patients with liver cancer. NeoTSTs were validated through proteomics, immunopeptidomics, and HLA-transgenic models. Multiomics analyses characterized splicing patterns, transposable elements, and transcription factor regulation. Single-cell RNA-seq and Hep53.4 murine models assessed tumor coverage and immunotherapeutic efficacy. We analyzed RNA-seq data from 1,013 patients with liver cancer and constructed a multilayered reference dataset. Using a customized pipeline, we identified an average of 60 neoTSTs per patient, significantly surpassing mutation-derived neoantigens (neoMuts). NeoTSTs exhibited higher population frequencies, with 73.1% providing multiple epitopes, and were validated through mass spectrometry and HLA transgenic mouse models. Mechanistically, neoTSTs were generated via retained introns, transposable element activation, HNF4A-regulated alternative promoters, and de novo transmembrane domain generation. Single-cell analysis revealed neoTSTs cover >75% of tumor cells and identified antigen-presenting cancer-associated fibroblasts that enriched in immunotherapy responders and amplified CD4+ T-cell responses. In murine HCC models, neoTST vaccination outperformed neoMuts, inducing dual major histocompatibility complex-I/II activation and significant tumor growth inhibition. NeoTSTs represent a superior neoantigen source in HCC, compensating for the limitations of mutation-derived targets. The remarkable abundance and patient-to-patient sharedness of neoTSTs underscore their dual potential: (1) as personalized immunotherapeutic targets, and (2) as broadly applicable antigens for low-TMB tumors. These findings provide a transformative framework for expanding treatment options in HCC immunotherapy.
Metabolic competition and nutrient restriction in the tumor microenvironment (TME) shape the immune infiltrate in tumors and subsequently tumor immunity. In this study we used the transgenic melanoma mouse model tg(Grm1)EPv, which spontaneously develops melanoma due to the ectopic expression of the metabotropic glutamate receptor 1 (Grm1) in melanocytes to investigate if aberrant glutamate metabolism drives tumor formation and affects immune cell function. We performed liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomic analyses and RNA sequencing on tumor-free and tumor-bearing tg(Grm1)EPv tissues to characterize metabolic alterations associated with tumor progression. Flow cytometry was used to examine changes in immune cell subsets within the TME. To assess the functional relevance of glutamate metabolism, we inhibited glutathione metabolism using L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase that depletes cellular glutathione levels. LC-MS/MS-based metabolomic analyses and RNA sequencing revealed changes in glutamate and glutamine metabolism, a glycolytic shift (Warburg effect), and reduced ATP levels in advanced tumors compared with tumor-free tissue, suggesting respiratory chain dysfunction. These metabolic changes in the TME are advantageous for the tumor cells and unfavorable for immune cells, such as dendritic cells (DC). Indeed, flow cytometry analysis of myeloid subsets during tumor progression showed a decline in tumor-infiltrating conventional type 2 DC and macrophages, alongside an increase in neutrophil and monocyte populations in advanced lesions. Interference with glutamate metabolism using BSO induced immunogenic cell death, namely ferroptosis, in an tg(Grm1)EPv-derived cell line in vitro. Therefore, we evaluated the combination of this inhibitor with immunotherapy as a promising new approach for the treatment of tumors in the tg(Grm1)EPv mouse model. We observed that tumor growth could be delayed in vivo when BSO was combined with a therapy regimen boosting DC numbers and activation. This inhibition of tumor growth was supported by the infiltration of activated T cells. Overall, our findings provide novel insights into the importance of combining metabolic intervention with immunotherapy for the treatment of patients with melanoma, particularly those bearing glutamate pathway-active or immunologically cold tumors. This knowledge can drive the design of novel therapeutic strategies for patients with cancer.
Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty (SB) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying Doxycycline (Dox)-inducible SB mutagenesis system (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8+ T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8+ T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. Under chronic stimulation, SB-mutagenized CD8+ T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2, known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.
Fecal microbiota transplantation (FMT) has shown promise in overcoming resistance to immune checkpoint inhibitors (ICIs) in early-phase cancer trials. We investigated the safety, feasibility and efficacy of FMT from ICI responders to patients with advanced cancers progressing on ICIs. This was a single-arm phase IIa basket trial (MITRIC; NCT05286294) including patients with ICI-refractory cancer. Long-term ICI responders were used as FMT donors. Patients received FMTs in combination with ICIs; two FMT administrations (by colonoscopy) were scheduled before the first radiological evaluation after 6 weeks, and up to three later FMTs were allowed (by enema). Co-primary endpoints were the evaluation of FMT-related adverse events and objective response rate. Feasibility, clinical benefit rate, progression-free survival (PFS), overall survival (OS), implant engraftment, immune response and biomarkers were among the secondary objectives. The study enrolled 12 patients with melanoma (n=9), head and neck squamous cell carcinoma (HNSCC; n=1), renal cell carcinoma (n=1) or microsatellite instability-high pancreatic cancer (n=1). FMT was well tolerated, whereas immune-related toxicity occurred in 6/12 patients. All patients received the first FMT; 10/12 patients also underwent the second FMT. No objective responses were observed, while 5/12 patients recorded stable disease. Clinical benefit per-protocol (stable disease >6 months) was achieved in a patient with melanoma, who had regression of some lesions and remains alive after 33 months without further systemic treatment. Mixed responses with regression of some lesions were observed in another melanoma patient, and in a patient with HNSCC. The median PFS was 1.5 months, and median OS was 10.1 months. Sequencing of fecal samples indicated engraftment after the first FMT in most patients. Mass cytometry analysis of peripheral blood cells suggested that an activated and differentiated T-cell signature was associated with improved PFS and OS, while a naïve T-cell phenotype and a myeloid-dominant environment were unfavorable. CD14+ monocytes and serum interleukin-8 increased at group level over time. FMT in combination with ICIs was safe and feasible in patients with advanced cancers, but with limited clinical activity. Further studies are required to clarify the potential benefit of FMT, identify the appropriate patient population and define criteria for donor selection. NCT05286294.
Although chromosome 14q deletion (14q-) is present in up to 40% of clear cell renal cell carcinoma (ccRCC) cases, its immunologic and molecular impact remains unclear. Because TRAF3 and NFΚBIA, key regulators of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, are located on 14q, we hypothesize that 14q- in ccRCC promotes increased inflammation and unique determinants of immune checkpoint blockade (ICB) response. We collected data from 825 patients with ccRCC from publicly available sources, our own independent retrospective study, and the Oncology Research Information Exchange Network. These data include bulk RNA-sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), and whole exome sequencing (WES). We also include reverse phase protein array from DepMap cell lines (n=277). Next, we performed multiplex immunofluorescence for spatial analysis of immune cells on 23 patient slides with paired WES and ICB response. Finally, progression-free survival, 14q- status (assessed in bulk RNA-seq or whole exome sequencing), and kidney inflammatory marker EDNRB expression were assessed in two independent ICB immunotherapy trials. 14q- status is associated with increased NF-κB-target transcription and p65 phosphorylation. Tumor scRNA-seq revealed increased expression of CCL20, an NF-κB target and lymphotactic protein. Simultaneously, single-cell and bulk RNA-seq demonstrated higher effector CD8+T cell infiltration in 14q- tumors compared with controls. We investigated myeloid panel mIF images from 14q- tumors and found dendritic cells (DCs) significantly aggregate with tumor cells in ICB responders compared with non-responders. Cell-cell communication analysis on DCs and tumor cells from a 14q- responder revealed tumor-derived EDN1 is uniquely interacting with EDNRB on DCs. In two ICB trials, 14q- combined with elevated EDNRB expression correlated with significantly improved progression-free survival. This is the first evidence that 14q- in ccRCC is associated with an antitumor inflammatory phenotype, possibly driven by aberrant NF-κB activation. Furthermore, 14q- with high EDNRB expression (EDNRB+) may serve as a predictive biomarker for ICB response in ccRCC. Finally, our results indicate patients with 14q-/EDNRB- may respond poorly to immunotherapy and new treatment options should be explored for this cohort.
Cancer immunotherapies have significantly improved treatment efficacy and patient survival by exploiting antigen-specific T cells to eliminate cancer cells. However, current approaches for identifying and isolating antigen-specific T cells typically require prior knowledge of target antigens, limiting discovery, and reducing the ability to consistently detect rare tumor-reactive T cells. We therefore sought to develop an unbiased platform for the identification and enrichment of antigen-specific T cells using naturally processed and presented tumor antigens. We developed ATTACH (Assessment of T cells Tethered to Antigen Class I Histocompatibility), a microfluidic platform that applies controlled shear stress and leverages tumor cells as a natural source of endogenous major histocompatibility complex (MHC)-peptide complexes to isolate antigen-specific T cells based on MHC/peptide binding avidity. ATTACH was evaluated in both human and mouse systems for its ability to enrich rare tumor-reactive T-cell populations and deplete bystander virus-specific T cells. ATTACH resulted in up to a 10-fold enrichment of antigen-specific T cells across both human and mouse systems, enabling the isolation of clonotypes present at frequencies as low as 0.1%. In addition to enriching rare tumor-reactive T cells, ATTACH efficiently depleted virus-specific bystander T cells. ATTACH provides a streamlined and unbiased approach for the rapid identification and isolation of antigen-specific T cells, and may facilitate the optimization of cellular therapies for the treatment of solid tumors.
Metastatic castration-resistant prostate cancer (mCRPC) is among the leading causes of cancer-related mortality in men worldwide. Treatment options for mCRPC typically include chemotherapy and androgen receptor pathway inhibitors. Immune checkpoint inhibitors (ICIs) have demonstrated a limited effect in mCRPC. We hypothesized that the addition of stereotactic body radiation therapy (SBRT) could enhance immune responses and improve treatment outcomes. Patients with mCRPC who had received at least two prior lines of therapy were randomized 1:1 to receive SBRT with nivolumab and ipilimumab (arm A) or nivolumab and ipilimumab (arm B). The dual primary endpoints in the study were prostate-specific antigen (PSA) response rate and objective response rate (ORR). Secondary endpoints included overall survival (OS), PSA, radiologic progression-free survival, and toxicity. We enrolled 91 patients, and 81 patients received at least one treatment with ICIs and were eligible for evaluation of the efficacy and safety endpoints. Of the evaluable patient population, the confirmed PSA response rate was 21.6% (95% CI 9.8% to 38.2%) in arm A, and 20.5% (95% CI 9.8% to 35.3%) in arm B. ORR was 16.7% (95% CI 4.7% to 37.4%) and 22.2% (95% CI 10.1% to 39.2%) in arms A and B, respectively. Median OS was 10.2 months (95% CI 7.1 to 15.2) in arm A and 9.2 months (95% CI 7.1 to 14.4) in arm B. Treatment-related adverse events grade 3-4 were observed in 27 patients (33.3%). The addition of SBRT to nivolumab and ipilimumab did not improve outcomes; however, a fraction of the patients had a response to the treatment combination of nivolumab and ipilimumab. Explorative translational research is needed to identify possible biomarkers of response to immunotherapy with ICIs in mCRPC. European Union Clinical Trial Registry (https://www.clinicaltrialsregister.eu) EudraCT number: 2018-003461-34 and on https://clinicaltrials.gov (NCT05655715).
The tumor microenvironment, particularly the tumor stroma, plays a critical role in tumor progression, immune evasion, and therapeutic resistance. However, its interaction with the immune landscape in rectal cancer (RC) remains incompletely understood. This study aimed to comprehensively characterize the stromal-immune ecosystem associated with the tumor stroma ratio (TSR) in RC and to evaluate its clinical and therapeutic relevance. We analyzed a multicenter cohort of 498 patients with treatment-naïve RC in whom TSR was assessed on H&E-stained sections. Integrative multi-omics analyses were performed, including bulk RNA sequencing (n=118) and single-cell RNA/T-cell receptor (TCR) sequencing (n=10). Key findings were validated by immunohistochemistry (n=114) and multiplex immunofluorescence (n=20). Survival analyses and statistical comparisons were conducted to evaluate clinical associations and treatment responses. High TSR was an independent predictor of unfavorable disease-free survival and cancer-specific survival and was associated with aggressive clinicopathological features. Single-cell analyses revealed that TSR-high tumors exhibited a profoundly immunosuppressive microenvironment, characterized by clonally expanded terminally exhausted CD8+ T cells (CD8+ Tex-CXCL13) and activated CD4+ regulatory T cells (CD4+ Treg-TNFRSF4). Two LRRC15+ cancer-associated fibroblast (CAF) subsets (mCAF-CTHRC1 and mCAF-FAP) were enriched in TSR-high tumors. Among them, mCAF-CTHRC1 was associated with increased Treg abundance and activation features, with predicted interactions with CD4+ Treg-TNFRSF4 cells through the LGALS9-CD44 signaling axis. In addition, SPP1-expressing monocytes (Mon-SPP1) and malignant epithelial cells were prominent in TSR-high tumors and showed a predicted SPP1-CD44 interaction with T-cell subsets, suggesting potential involvement in immunosuppressive stromal-immune interactions. In patients receiving neoadjuvant therapy, pretreatment TSR-low tumors showed improved pathological response and survival outcomes compared with TSR-high tumors. In the neoadjuvant chemoradiotherapy plus immunotherapy cohort, TSR-low tumors were associated with a significantly higher major pathological response rate, whereas pathological complete response showed a non-significant trend in the same direction. High TSR identifies a clinically aggressive subtype of RC characterized by a profoundly immunosuppressive stromal-immune ecosystem enriched for exhausted T cells, immunosuppressive CAF programs, and SPP1-associated stromal-myeloid interactions. These findings highlight LGALS9-, LRRC15-, and SPP1-related stromal-immune pathways as candidate stromal-immune therapeutic vulnerabilities that warrant further mechanistic and preclinical validation.
TIGIT (T cell immunoreceptor with Ig and ITIM domains) has emerged as a key exhaustion marker of intratumoral natural killer (NK) cells, but results from clinical trials with TIGIT blockade have been largely negative. Recent data have suggested that a subset of TIGIT-expressing NK cells can show increased functionality. We hypothesized that there are differences in function between peripheral and intratumoral TIGIT-expressing NK cells in patients with sarcoma and preclinical sarcoma models, which undermine the efficacy of systemic TIGIT blockade. We sought to investigate differences in TIGIT+ NK cells using systemic versus intratumoral TIGIT-blocking strategies. Peripheral and intratumoral NK cells were analyzed from human patients and mice with osteosarcoma (OSA) and soft tissue sarcoma (STS). NK phenotype and function were evaluated using flow cytometry, immunohistochemistry, live-cell imaging, and RNA sequencing. Mouse antimouse-IgG1 TIGIT blockade was delivered systemically or intratumorally in flank models of OSA (K7M2) and STS (MCA-205). Clinical and genomic data were evaluated using Caris CODEai. Expression of the TIGIT ligand CD155 on myeloid and tumor cells was evaluated as a marker of TIGIT function. Using multiple readouts, TIGIT+ NK cells from the spleen of tumor-bearing mice or peripheral blood of patients with STS and OSA showed increased functionality compared with TIGIT- NK, while TIGIT+ NK cells from the sarcoma tumor microenvironment (TME) of mice and humans were dysfunctional, with upregulated senescence and inhibitory gene pathways. Systemic TIGIT blockade reinvigorated intratumoral NK cell function but inhibited peripheral NK cells, while intratumoral administration of TIGIT blockade significantly delayed tumor growth and prolonged survival with partial reversal of NK dysfunction in the TME. Clinical and genomic data demonstrated that more NK cell infiltration in human STS was prognostic of improved overall survival only when intratumoral CD155 expression was low, indicating that elevated CD155 expression is correlated with greater NK cell dysfunction. We identified a cross-species role of TIGIT expression dependent on location, where peripheral TIGIT+ NK cells showed evidence of enhanced functionality, while intratumoral TIGIT+ NK cells were dysfunctional. These differences impacted antitumor effects of TIGIT blockade, suggesting that intratumoral delivery of TIGIT blockade may be a novel translational strategy in high-risk bone and soft tissue sarcomas.
Conventional antibody discovery approaches that do not account for enrichment-driven biases, such as epitope immunogenicity, PCR amplification bias, or protein expression efficiency, may result in under-representation of rare yet functionally relevant clones, necessitating labor-intensive in vitro screening to identify agonistic antibodies among a large number of dominant clones. Thus, efficient screening methods for agonistic antibodies are urgently needed. OX40 is a promising target for cancer immunotherapy due to its role in enhancing T-cell activation and survival. However, effective anti-OX40 agonistic antibodies have not yet been developed. We developed a novel screening strategy that involves the selection of nanobody clone pools enriched by biopanning against gp34-engaged and non-engaged OX40-expressing cells, next-generation sequencing, and computational clustering and subtraction analysis to identify clones recognizing the ligand-receptor interface. Representative nanobody clones underwent in vitro validation, including epitope mapping, binding affinity measurements, and functional assessments. Furthermore, we engineered the selected nanobody to enhance its in vivo efficacy. We also performed structural analysis of the nanobody-OX40 complex. Our epitope-directed approach efficiently identified nanobody clones recognizing functionally relevant epitopes distinct from dominant immunogenic regions. Notably, clone Nb479 demonstrated robust agonistic activity, closely mimicking the natural ligand gp34 with extensive OX40-binding interactions. Trimerization of Nb479 facilitated potent OX40 activation without the need for a cross-linking scaffold. Conjugation of the Nb479 trimer with an anti-serum albumin nanobody exhibited significantly improved pharmacokinetics in vivo and enhanced antitumor activity in a mouse model treated with CD19 chimeric antigen receptor T cells. This study presents an innovative epitope-directed approach that greatly accelerates the discovery of functionally potent agonistic nanobodies by effectively circumventing enrichment-driven epitope bias. Our approach and engineered multivalent anti-OX40 nanobody offer a powerful platform to advance immunotherapeutic strategies for cancer treatment.
CD161+CD8+ T cells, a cytotoxic T cell subset, remain poorly defined in terms of their clinical significance and functional role in esophageal squamous cell carcinoma (ESCC), which has limited the advancement of precision immunotherapy strategies. We integrated a multicenter cohort of ESCC patients who underwent either surgery or neoadjuvant therapy (chemotherapy or chemoimmunotherapy). Multiplex immunofluorescence staining, survival analyses, and assessment of major pathological response (MPR) and pathological complete response (pCR) were performed to elucidate the clinical relevance of CD161+CD8+ T cell infiltration. The functional state and cellular interactions of this subset were characterized using single-cell RNA sequencing. A CT-based radiomics model was also developed for non-invasive prediction. High intratumoral infiltration of CD161+CD8+ T cells was identified as an independent prognostic factor for prolonged overall survival and disease-free survival (both p<0.01) and appeared to correlate with a higher MPR and pCR rate following neoadjuvant therapy. This subset was enriched in treatment responders, exhibited transcriptional features associated with cytotoxicity and activation, and showed gene expression profiles suggestive of potential interactions with tumor-associated macrophages, possibly involving the TNFRSF9/TNFSF9 (4-1BB/4-1BBL) signaling axis. A radiomics model built on the XGBoost algorithm accurately predicted the infiltration level of this subset (area under the curve=0.889), and predicted high infiltration was correlated with favorable pathological response. We identify CD161+CD8+ T cells as a pivotal prognostic and predictive biomarker in ESCC. This subset is associated with a putative 4-1BB/TNFSF9-mediated interaction network involving macrophages, correlating with a coordinated anti-tumor immune microenvironment. The CT-based radiomics model could provide a noninvasive means for assessing immune phenotypes, with potential applicability in patient stratification and treatment selection.