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BackgroundIn addition to their established action of synthetic lethality in tumor cells, poly(ADP-ribose) polymerase inhibitors (PARPis) also orchestrate tumor immune microenvironment (TIME) that contributes to suppressing tumor growth. However, it remains not fully understood whether and how PARPis trigger tumor-targeting immune responses.MethodsTo decode the immune responses reshaped by PARPis, we conducted T-cell receptor (TCR) sequencing and immunohistochemical (IHC) analyses of paired clinical specimens before and after niraparib monotherapy obtained from a prospective study, as well as ID8 mouse ovarian tumors. To validate the induction of immunogenic cell death (ICD) by PARPis, we performed immunofluorescence/IHC staining with homologous recombination deficiency tumor cells and patient-derived xenograft tumor tissues, respectively. To substantiate that PARPis elicited tumor cell pyroptosis, we undertook comprehensive assessments of the cellular morphological features, cleavage of gasdermin (GSDM) proteins, and activation of TNF-caspase signaling pathways through genetic downregulation/depletion and selective inhibition. We also evaluated the critical role of pyroptosis in tumor suppression and immune activation following niraparib treatment using a syngeneic mouse model with implanting CRISPR/Cas9 edited Gsdme−/− ID8 tumor cells into C57BL/6 mice.ResultsOur findings revealed that PARPis augmented the proportion of neoantigen-recognized TCR clones and TCR clonal expansion, and induced an inflamed TIME characterized by increased infiltration of both innate and adaptive immune cells. This PARPis-strengthened immune response was associated with the induction of ICD, specifically identified as pyroptosis, which possessed distinctive morphological features and GSDMD/E cleavage. It was validated that the cleavage of GSDMD/E was due to elevated caspase 8 activity downstream of the TNFR1, rather than FAS and TRAIL-R. On PARP inhibition, the NF-κB signaling pathway was activated, leading to increased secretion of TNF-α and subsequent initiation of the TNFR1–caspase 8 cascade. Impeding pyroptosis through the depletion of Gsdme significantly compromised the tumor-suppressing effects of PARP inhibition and undermined the anti-immune response in the syngeneic ID8 mouse model.ConclusionsPARPis induce a specific type of ICD called pyroptosis via TNF–caspase 8–GSDMD/E axis, resulting in an inflamed TIME and augmentation of tumor-targeting immune responses. These findings deepen our understanding of PARPis activities and point toward a promising avenue for synergizing PARPis with immunotherapeutic interventions.Trial registration numberNCT04507841.

BackgroundDespite the remarkable clinical outcomes of epidermal growth factor receptor (EGFR)-targeted therapies in patients with lung cancer, therapeutic resistance eventually develops. This study elucidates the role of galectin-9 (Gal-9), a TIM-3 immune checkpoint ligand, in facilitating tumor immune escape during EGFR tyrosine kinase inhibitor (TKI) therapy, and evaluates the therapeutic potential of combined EGFR-TKI and Gal-9 blockade in preclinical models.MethodsEGFR-TKI-mediated Gal-9 regulation was systematically investigated through multianalysis including RNA-seq transcriptomics, quantitative reverse transcription-PCR, immunoblotting, ELISA, flow cytometry, and immunohistochemical validation across human and murine lung/colorectal cancer cell lines, murine tumor tissues, and paired patient tumor tissues/serum samples. Therapeutic efficacy was evaluated in two syngeneic murine models, with comprehensive immune monitoring of tumor microenvironment (TME), tumor-draining lymph nodes (tdLNs), and splenic compartments. Mechanistic investigations employed CD8+ T-cell/macrophage depletion strategies (anti-CD8α monoclonal antibodies (mAbs)/PLX-3397), type I interferon (IFN-I) pathway inhibition (anti-IFNAR1 mAbs), and lymph node retention approaches (FTY720 administration).ResultsEGFR-TKI treatment significantly induced Gal-9 expression in both tumor cells and host immune cells, particularly myeloid cells. Clinical validation revealed elevated Gal-9 levels in EGFR-TKI-treated patient with lung cancer tumor tissues and serums, correlating with reduced progression-free survival. Mechanistically, EGFR-TKIs triggered DNA damage-potentiated cytosolic double-stranded DNA accumulation and activated tumor-intrinsic STING-IFN-I innate immune pathway that transcriptionally regulated Gal-9 expression. Notably, Gal-9-neutralizing antibodies synergized with EGFR-TKI to markedly inhibit tumor growth in two syngeneic mouse models, including the poorly immunogenic LLC lung tumor model unresponsive to programmed cell death protein-1/programmed death-ligand 1 blockade. The combination therapy remodeled myeloid landscapes toward antigen-presenting phenotypes, promoted dendritic cell accumulation in the tdLN and enhanced CD8+ T response in the TME. Depleting CD8+ T cells or macrophages/monocytes abrogated the therapeutic benefits. Blocking the IFN-I pathway attenuated Gal-9 expression and enhanced the antitumor immunity of afatinib in the LLC tumor model.ConclusionsThese findings identify Gal-9 upregulation as a key mechanism mediating immune evasion and limiting EGFR-TKI efficacy, providing a promising combinational therapeutic strategy of EGFR-TKI and Gal-9 blockade for the treatment of EGFR-driven cancers.

BackgroundApproximately 50% of head and neck squamous cell carcinomas (HNSCC) recur after treatment with curative intent. Immune checkpoint inhibitors are treatment options for recurrent/metastatic HNSCC; however, less than 20% of patients respond. To increase this response rate, it is fundamental to increase our understanding of the spatial tumor immune microenvironment (TIME).MethodsIn total, 53 HNSCC specimens were included. Using a seven-color multiplex immunohistochemistry panel we identified tumor cells, CD163+macrophages, B cells, CD8+T cells, CD4+T helper cells and regulatory T cells (Tregs) in treatment-naive surgical resection specimens (n=29) and biopsies (n=18). To further characterize tumor-infiltrating CD8+T cells, we stained surgical resection specimens (n=12) with a five-color tumor-resident panel including CD103, Ki67, CD8 and pan-cytokeratin. Secretome analysis was performed on matched tumor suspensions (n=11) to measure protein levels.ResultsBased on CD8+T cell infiltrates, we identified four different immunotypes: fully infiltrated, stroma-restricted, immune-excluded, and immune-desert. We found higher cytokine levels in fully infiltrated tumors compared with other immunotypes. While the highest immune infiltrates were observed in the invasive margin for all immune cells, CD163+macrophages and Tregs had the highest tendency to infiltrate the tumor center. Within the tumor center, especially B cells stayed at the tumor stroma, whereas CD163+macrophages, followed by T cells, were more often localized within tumor fields. Also, B cells were found further away from other cells and often formed aggregates while T cells and CD163+macrophages tended to be more closely located to each other. Across resection specimens from various anatomical sites within the head and neck, oral cavity tumors exhibited the highest densities of Tregs. Moreover, the distance from B cells and T cells to tumor cells was shortest in oral cavity squamous cell carcinoma (OCSCC), suggesting more interaction between lymphocytes and tumor cells. Also, the fraction of T cells within 10 µm of CD163+macrophages was lowest in OCSCC, indicating fewer myeloid/T-cell suppressive interactions in OCSCC.ConclusionsWe comprehensively described the TIME of HNSCC using a unique data set of resection specimens. We discovered that the composition, as well as the relative localization of immune cells in the TIME, differed in distinct anatomical sites of the head and neck.

BackgroundHepatocellular carcinoma (HCC) remains a leading cause of cancer-related deaths worldwide, especially in advanced stages where limited treatment options result in poor prognosis. The immunosuppressive tumor immune microenvironment (TIME), characterized by low immune cell infiltration and exhaustion, limits immunotherapy efficacy. To address this, our study investigates the role of C-C motif chemokine ligand 3 (CCL3) in modulating the HCC TIME.MethodsWe analyzed CCL3 expression in human HCC samples from The Cancer Genome Atlas database, focusing on its correlation with inflammatory gene signatures and immune cell infiltration. High-dimensional single-cell RNA sequencing (scRNA-seq), flow cytometry, and multiplex immunofluorescence were used to investigate CCL3’s effects on macrophage function and T cell activation. The biological impact of CCL3 on macrophages was assessed using co-culture systems, confocal imaging, metabolite detection, and inhibition assays. Preclinical HCC models and ex vivo tumor fragment assays further explored how CCL3 modulates immune responses and enhances immune checkpoint blockade efficacy.ResultsOur study shows that CCL3 is suppressed in the tumor microenvironment and positively correlates with immune infiltration and inflammatory responses. Targeted liver delivery of rAAV-Ccl3 reprograms the immune microenvironment in HCC, promoting immune cell recruitment and tertiary lymphoid structure formation, thus suppressing tumor growth via immune engagement. Through scRNA-seq, flow cytometry, and multiplex immunofluorescence, we found that CCL3 enhances macrophage antigen uptake and activates cytotoxic T cells. In vivo and in vitro experiments confirmed that CCL3 facilitates T cell infiltration and upregulates MHC II expression on macrophages, enhancing antigen presentation. The CCL3-CCR5 pathway also boosts macrophage metabolism, increasing lysosomal activity and antigen uptake, thereby strengthening adaptive immune responses and increasing sensitivity to immune checkpoint blockade therapies in preclinical models.ConclusionsThis study highlights the pivotal role of CCL3 in reshaping the TIME and enhancing antitumor immunity in HCC. By promoting immune cell recruitment and enhancing antigen presentation, CCL3 demonstrates significant potential to improve the efficacy of immunotherapy, particularly in combination with immune checkpoint inhibitors. Targeting CCL3 may help to overcome the immunosuppressive TIME in HCC and improve patient outcomes.

BackgroundTumor-associated macrophages (TAMs) are among the most prevalent cells within the tumor microenvironment (TME) of cervical cancer (CC). Although TAMs frequently exhibit an immunosuppressive phenotype, their plasticity enables them as an intriguing reprogrammable target for immunotherapy of CC.MethodsConsensus clustering was employed to delineate immune infiltration patterns in a cohort of 119 patients with CC. Single-cell RNA sequencing, complemented by flow cytometry analysis, was used to characterize hexokinase 3 (HK3)-expressing cell populations. In vivo tumor models were established to assess the functional impact of HK3-expressing cells on the TME, with interventions including Hk3 knockout and CD8+ T-cell depletion. A comprehensive approach involving bulk RNA sequencing, immunoprecipitation assays, confocal microscopy imaging, and in vitro co-culture systems was implemented to elucidate the mechanisms underlying HK3 inhibition-mediated enhancement of antitumor immunity. Furthermore, the therapeutic efficacy of HK3 inhibition, both as a monotherapy and in combination with immunotherapeutic strategies, was systematically evaluated in preclinical tumor models.ResultsWe elucidated a cross-regulation between TAMs and CD8+ T cells, with HK3 serving as a central regulatory node. Upon HK3 expression was upregulated by CD8+ T cells through the IFN-γ-STAT1 signaling axis, TAMs exhibited impaired cross-presentation capacity, which in turn attenuated CD8+ T cell-mediated antitumor immunity. Mechanistically, HK3 physically interacted with mechanistic target of rapamycin (mTOR), promoting nuclear translocation of transcription factor EB (TFEB) and resulting in excessive lysosomal activation and antigen degradation. Moreover, targeting HK3 in combination with immune checkpoint blockade yielded a synergistic effect in enhancing antitumor immunity.ConclusionsTargeting HK3 in TAMs represents a promising therapeutic strategy capable of enhancing antitumor immunity and synergizing with immune checkpoint blockade by restoring efficient antigen cross-presentation.

Resident Golgi protein 73 (GP73) is expressed in many healthy tissues, however overexpression is associated with both viral infections and cancer. As an oncoprotein, GP73 drives tumor progression and plays a fundamental role in immune regulation. A recent publication illustrates a role for GP73 in T-cell antitumor immunity employing GP73 genetically depleted T-cell mouse models. GP73-deficient T-cells were found to detrimentally affect CD8+T cell cytotoxicity and glycolysis primarily due to its interaction with Hypoxia-inducible factor 1α and mTOR levels in hypoxic cells, suggesting a key role for GP73 in T-cell cytotoxicity within the hypoxic tumor microenvironment. This finding opens the door to the potential development of GP73 targeting through ectopic expression of GP73 which was found to restore glycolysis and therefore T-cell cytotoxicity resulting in tumor regression. In addition, GP73 was found to be a potential biomarker to inform clinical treatment of patients undergoing immunotherapy. Could GP73 be the key to establishing a therapeutic strategy for generating improved patient responses to immunotherapy?

BackgroundCombining interleukin-2 (IL-2) agonism with programmed cell death protein 1 (PD-1) checkpoint inhibition has shown synergistic potential in reinvigorating antitumor T cell responses. However, integrating these two mechanisms within a single molecule has been challenging due to competing requirements for PD-1 engagement and IL-2 receptor signaling. ANV600 is a novel bispecific antibody–cytokine fusion protein that targets a non-blocking epitope on PD-1, enabling cis-targeted IL-2Rβγ agonism while preserving combinability with therapeutic PD-1 inhibitors. This design allows for selective expansion of tumor antigen-specific T cells while avoiding the systemic toxicity and regulatory T cell (Treg) expansion associated with conventional IL-2 therapies.MethodsThe PD-1-targeting antibody used in ANV600 was generated by immunization of humanized mice and selected for its ability to bind PD-1 without blocking the binding epitope of PD-1 checkpoint blocking agents. ANV600 was evaluated in multiple syngeneic tumor models using human PD-1 transgenic mice. Tumor-infiltrating lymphocytes were analyzed to assess the selectivity of ANV600 for PD-1+ T cell subsets. Combination studies with pembrolizumab and nivolumab were performed to assess synergy with checkpoint inhibitors.ResultsANV600 significantly inhibited tumor growth as monotherapy across multiple models, including the immune checkpoint-resistant B16F10 melanoma. By targeting PD-1, ANV600 selectively expanded tumor antigen-specific CD8+T cells, particularly progenitor exhausted (Tpex) and cytotoxic exhausted (Tcex) subsets, while sparing Tregs and NK cells. Combination with pembrolizumab and nivolumab resulted in additive effects, consistent with the complementary roles of PD-1 blockade in expanding Tpex cells and IL-2Rβγ signaling in reprogramming Tcex cells. ANV600’s efficacy was dependent on CD8+T cells and primarily driven by tumor-resident T cells, as it remained effective despite blocked lymph node trafficking (FTY720) but was abrogated on CD8+ T cell depletion.ConclusionsANV600 represents a novel approach to delivering IL-2Rβγ agonism specifically to PD-1+ cells while preserving the binding site for PD-1 checkpoint inhibitors. By targeting a non-blocking epitope on PD-1, ANV600 enables the selective expansion of tumor-reactive CD8+ T cells while allowing independent and optimized dosing of both agents. This design ensures combinability with PD-1 inhibitors at clinically relevant doses, including in patients previously treated with checkpoint blockade. These findings support the clinical development of ANV600 as both a monotherapy and a combination therapy in cancer immunotherapy.

The tumor microenvironment (TME) is a complex and ever-changing “rogue organ” composed of its own blood supply, lymphatic and nervous systems, stroma, immune cells and extracellular matrix (ECM). These complex components, utilizing both benign and malignant cells, nurture the harsh, immunosuppressive and nutrient-deficient environment necessary for tumor cell growth, proliferation and phenotypic flexibility and variation. An important aspect of the TME is cellular crosstalk and cell-to-ECM communication. This interaction induces the release of soluble factors responsible for immune evasion and ECM remodeling, which further contribute to therapy resistance. Other aspects are the presence of exosomes contributed by both malignant and benign cells, circulating deregulated microRNAs and TME-specific metabolic patterns which further potentiate the progression and/or resistance to therapy. In addition to biochemical signaling, specific TME characteristics such as the hypoxic environment, metabolic derangements, and abnormal mechanical forces have been implicated in the development of treatment resistance. In this review, we will provide an overview of tumor microenvironmental composition, structure, and features that influence immune suppression and contribute to treatment resistance.

The tumor microenvironment (TME) influences tumor growth, metastatic spread and response to treatment. Often immunosuppression, mediated by the TME, impairs a beneficial response. The complexity of the tumor composition challenges our abilities to design new and more effective therapies. Going forward we will need to ‘manage’ the content and or functionality of the TME to improve treatment outcomes. Currently, several different kinds of treatments are available to patients with cancer: there are the traditional approaches of chemotherapy, radiation and surgery; there are targeted agents that inhibit kinases associated with oncogenic pathways; there are monoclonal antibodies that target surface antigens often delivering toxic payloads or cells and finally there are antibodies and biologics that seek to overcome the immunosuppression caused by elements within the TME. How each of these therapies interact with the TME is currently under intense and widespread investigation. In this review we describe how the TME and its immunosuppressive components can influence both tumor progression and response to treatment focusing on three particular tumor types, classic Hodgkin Lymphoma (cHL), Pancreatic Ductal Adenocarcinoma (PDAC) and Glioblastoma Multiforme (GBM). And, finally, we offer five approaches to manipulate or manage the TME to improve outcomes for cancer patients.