Dying Tumor Cells Research Articles

Drug-loaded microbubble delivery system to enhance PD-L1 blockade immunotherapy with remodeling immune microenvironment

BackgroundAlthough programmed cell death protein 1 (PD-1)/ programmed cell death-ligand protein 1 (PD-L1) checkpoint blockade immunotherapy demonstrates great promise in cancer treatment, poor infiltration of T cells resulted from tumor immunosuppressive microenvironment (TIME) and insufficient accumulation of anti-PD-L1 (αPD-L1) in tumor sites diminish the immune response. Herein, we reported a drug-loaded microbubble delivery system to overcome these obstacles and enhance PD-L1 blockade immunotherapy.MethodsDocetaxel (DTX) and imiquimod (R837)-loaded microbubbles (RD@MBs) were synthesized via a typical rotary evaporation method combined with mechanical oscillation. The targeted release of drugs was achieved by using the directional "bursting" capability of ultrasound-targeted microbubble destruction (UTMD) technology. The antitumor immune response by RD@MBs combining αPD-L1 were evaluated on 4T1 and CT26 tumor models.ResultsThe dying tumor cells induced by DTX release tumor-associated antigens (TAAs), together with R837, promoted the activation, proliferation and recruitment of T cells. Besides, UTMD technology and DTX enhanced the accumulation of αPD-L1 in tumor sites. Moreover, RD@MBs remolded TIME, including the polarization of M2-phenotype tumor-associated macrophages (TAMs) to M1-phenotype, and reduction of myeloid-derived suppressor cells (MDSCs). The RD@MBs + αPD-L1 synergistic therapy not only effectively inhibited the growth of primary tumors, but also significantly inhibited the mimic distant tumors as well as lung metastases.ConclusionPD-L1 blockade immunotherapy was enhanced by RD@MBs delivery system.

Dual-Cascade Activatable Nanopotentiators Reshaping Adenosine Metabolism for Sono-Chemodynamic-Immunotherapy of Deep Tumors.

Immunotherapy is an attractive treatment strategy for cancer, while its efficiency and safety need to be improved. A dual-cascade activatable nanopotentiator for sonodynamic therapy (SDT) and chemodynamic therapy (CDT)-cooperated immunotherapy of deep tumors via reshaping adenosine metabolism is herein reported. This nanopotentiator (NPMCA ) is constructed through crosslinking adenosine deaminase (ADA) with chlorin e6 (Ce6)-conjugated manganese dioxide (MnO2 ) nanoparticles via a reactive oxygen species (ROS)-cleavable linker. In the tumor microenvironment with ultrasound (US) irradiation, NPMCA mediates CDT and SDT concurrently in deep tumors covered with 2-cm tissues to produce abundant ROS, which results in dual-cascade scissoring of ROS-cleavable linkers to activate ADA within NCMCA to block adenosine metabolism. Moreover, immunogenic cell death (ICD) of dying tumor cells and upregulation of the stimulator of interferon genes (STING) is triggered by the generated ROS and Mn2+ from NPMCA , respectively, leading to activation of antitumor immune response. The potency of immune response is further reinforced by reducing the accumulation of adenosine in tumor microenvironment by the activated ADA. As a result, NPMCA enables CDT and SDT-cooperated immunotherapy, showing an obviously improved therapeutic efficacy to inhibit the growths of bilateral tumors, in which the primary tumors are covered with 2-cm tissues.

The therapeutic mavericks: Potent immunomodulating chaperones capable of treating human diseases.

Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti-inflammatory functions respectively. CRT induces pro-inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti-inflammatory cytokines, inhibits DC maturation and heightens T-regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an 'eat-me' signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long-term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease-relevant immune responses leading to a beneficial clinical outcome.

XBP1‐elicited environment by chemotherapy potentiates repopulation of tongue cancer cells by enhancing miR‐22/lncRNA/KAT6B‐dependent NF‐κB signalling

Tumour repopulation initiated by residual tumour cells in response to cytotoxic therapy has been described clinically and biologically, but the mechanisms are unclear. Here, we aimed to investigate the mechanisms for the tumour-promoting effect in dying cells and for tumour repopulation in surviving tongue cancer cells. Tumour repopulation in vitro and in vivo was represented by luciferase activities. The differentially expressed cytokines in the conditioned medium (CM) were identified using a cytokine array. Gain or loss of function was investigated using inhibitors, neutralising antibodies, shRNAs and ectopic overexpression strategies. We found that dying tumour cells undergoing cytotoxic therapy increase the growth of living tongue cancer cells in vitro and in vivo. Dying tumour cells create amphiregulin (AREG)- and basicfibroblast growth factor (bFGF)-based extracellular environments via cytotoxic treatment-induced endoplasmic reticulum stress. This environment stimulates growth by activating lysine acetyltransferase 6B (KAT6B)-dependent nuclear factor-kappa B (NF-κB) signalling in living tumour cells. As direct targets of NF-κB, miR-22 targets KAT6B to repress its expression, but long noncoding RNAs (lncRNAs) (XLOC_003973 and XLOC_010383) counter the effect of miR-22 to enhance KAT6B expression. Moreover, we detected increased AREG and bFGF protein levels in the blood of tongue cancer patients with X-box binding protein-1 (XBP1) activation in tumours under cytotoxic therapy and found that XBP1 activation is associated with poor prognosis of patients. We also detected activation of miR-22/lncRNA/KAT6B/NF-κB signalling in recurrent cancers compared to paired primary tongue cancers. We identified the molecular mechanisms of cell death-induced tumour repopulation in tongue cancer. Such insights provide new avenues to identify predictive biomarkers and effective strategies to address cancer progression.

EGFR inhibition in EGFR-mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) elicit potent cell cycle arrest in EGFR-mutant non-small-cell lung cancer (NSCLC) cells. However, little is known about the mechanisms through which these drugs alter the tumor phenotype that contributes to the immune escape of EGFR-mutant cells. Using EGFR-mutant NSCLC cell lines and tissue samples from patients, we investigated the changes in immune checkpoints expressed in tumor cells following EGFR inhibition. Subsequently, we also analyzed the role of soluble factors from the dying tumor cells in the activation of immune signaling pathways involved in therapy resistance. Upon EGFR-TKI treatment, we found that EGFR-mutant cells upregulated the expression of innate immune checkpoint CD24 in vitro. We then analyzed biopsy samples from six patients who developed resistance to a first-generation EGFR-TKI without the acquired T790M mutation. Immunohistochemistry revealed that levels of tumor CD24 expression were increased upon treatment compared with those from pre-treatment samples. Monocyte-derived macrophages facilitated antibody-dependent cellular phagocytosis when EGFR-TKI-treated EGFR-mutant cells were incubated with anti-CD24 antibodies in vitro, suggesting that CD24 may be a therapeutical target for EGFR-mutant lung cancer. Moreover, EGFR inhibition accelerated the release of cell-free DNA (cfDNA) from dying tumor cells, which activated the type I interferon signaling pathways in human THP-1 monocytes in a stimulator of interferon genes-dependent manner. Our study indicates that EGFR inhibition in EGFR-mutant NSCLC cells fosters a tumor microenvironment associated with immune escape. Thus, CD24 targeted therapy and cfDNA monitoring may contribute to improved treatment outcomes in patients with EGFR-mutant NSCLC.

Combination of magnetic hyperthermia and immunomodulators to drive complete tumor regression of poorly immunogenic melanoma.

Hyperthermia using magnetic nanoparticles enables tumor-specific heating and can destroy tumor tissues. This approach works as in situ vaccination with tumor antigens released from dying tumor cells. However, in situ vaccination caused by magnetic hyperthermia is often insufficient to induce complete regression of poorly immunogenic tumors surrounded by an immunosuppressive microenvironment. In this study, we explored a novel strategy for immunotherapy using magnetic hyperthermia to regress poorly immunogenic melanoma. Magnetic hyperthermia induced tumor cell death in a B16-F10 melanoma mouse model. After hyperthermia treatment, we found elevated levels of HMGB1, which is known to be released from dying cells to promote inflammation, and the proinflammatory cytokine TNF-α was increased in serum of the mice. Systemic administration of glycyrrhizin, an HMGB1 inhibitor, reduced the levels of TNF-α in serum and successfully delayed the regrowth of tumors after magnetic hyperthermia. To achieve complete tumor regression, TLR9 activation by intratumor injection of CpG was combined with systemic administration of anti-PD-1 antibody and anti-CTLA-4 antibody. The combination therapy of magnetic hyperthermia at 46°C with the immunomodulators (glycyrrhizin+CpG+anti-PD-1+anti-CTLA-4) achieved complete tumor regression in 80% of growing 5-mm B16-F10 tumors. These findings have important implications for the development of novel cancer immunotherapy using magnetic hyperthermia for poorly immunogenic tumors.

Tumor cell surface modification with immuno-amplified nanoparticles to enhance cancer immunotherapy

The process of cancer immunogenic cell death (ICD) provides adjuvanticity and antigenicity from dying tumor cells, thereby stimulating host immune system and promoting antitumor immunity. However, due to the immune evasion of tumor cells and the immunosuppressive tumor microenvironment formed in the process of cancer progression, it is far from satisfactory in the efficacy of the cancer treatments based on ICD. Herein, we report an immuno-amplified nanoparticle (IANP) that can modify mannose onto the tumor cell surface while delivering ICD-inducing drug doxorubicin (DOX) into the tumor cytoplasm. IANP consists of a DOX-loaded polymer core encapsulated within a mannose modified, fusogenic liposome. After reaching tumor cells, IANP achieved to transfer the mannose groups onto the surface of tumor cells through membrane fusion, and simultaneously transport the polymer core into tumor cells for DOX delivery. With this unique ability, IANP triggered the ICD of tumor cells and facilitated the activation of dendritic cells (DCs) via the mannose-C-type lectin receptors (CLRs) interaction, leading to the enhanced immunogenic effects of chemotherapy-induced tumor cell death. As a result, intratumoral injection of IANP achieved to trigger ICD of tumor cells and enhance the anti-tumor immune responses, thereby suppressing the tumor growth effectively. This work demonstrated a potential strategy towards the development of novel ICD-based cancer immunotherapies.

Expanding cross-presenting dendritic cells enhances oncolytic virotherapy and is critical for long-term anti-tumor immunity

Immunotherapies directly enhancing anti-tumor CD8+ T cell responses have yielded measurable but limited success, highlighting the need for alternatives. Anti-tumor T cell responses critically depend on antigen presenting dendritic cells (DC), and enhancing mobilization, antigen loading and activation of these cells represent an attractive possibility to potentiate T cell based therapies. Here we show that expansion of DCs by Flt3L administration impacts in situ vaccination with oncolytic Newcastle Disease Virus (NDV). Mechanistically, NDV activates DCs and sensitizes them to dying tumor cells through upregulation of dead-cell receptors and synergizes with Flt3L to promote anti-tumor CD8+ T cell cross-priming. In vivo, Flt3L-NDV in situ vaccination induces parallel amplification of virus- and tumor-specific T cells, including CD8+ T cells reactive to newly-described neoepitopes, promoting long-term tumor control. Cross-presenting conventional Type 1 DCs are indispensable for the anti-tumor, but not anti-viral, T cell response, and type I IFN-dependent CD4+ Th1 effector cells contribute to optimal anti-tumor immunity. These data demonstrate that mobilizing DCs to increase tumor antigen cross-presentation improves oncolytic virotherapy and that neoepitope-specific T cells can be induced without individualized, ex vivo manufactured vaccines.

Establishing Immunocompetent Leukemia Models to Investigate the Impact of CAR T Cells on the Immune Microenvironment and Bone Marrow Niche

Background Chimeric antigen receptor (CAR) T cells need to overcome limited expansion and persistence within the highly immunosuppressive tumor microenvironment (TME) to successfully eliminate acute myeloid leukemia (AML) in the clinic. CD123 has been identified as a promising target for AML because of its high expression on leukemia stem cells and AML blasts, with a clear increased differential expression when compared to healthy hematopoietic progenitors (HPCs). To date, preclinical evaluation of CD123-CAR T cells have used human xenograft models, which lack a functional immune system. This cross-species evaluation of CAR T cells inadequately addresses interactions between CAR T cells and the immune microenvironment, a limitation of many CAR T cell therapy studies. We have generated immunocompetent murine leukemia models to evaluate murine CD123 (mCD123)-specific CAR T cells and thus investigate the impact of the AML TME on CAR T cell dysfunction. Methods and Results We used biopanning of a murine phage display library on recombinant mCD123 to select a panel of mCD123-specific single chain variable fragments (scFvs). 4 scFvs, encoding 'top binders', were subcloned into a MSGV1 retroviral vector encoding a CD28.z-CAR to generate 4 mCD123-CARs. Second-generation mCD123-CAR T cells were generated by retroviral transduction of activated splenocytes. Only one mCD123-CAR construct was expressed on the cell surface as judged by flow cytometric analysis using F(ab')2 (55.8±11.5% N=7) or recombinant mCD123 (59.0±12.1% N=6) for detection, and T cells expressing this CAR were selected for further analysis. To test the antigen specificity of mCD123-CAR T cells, we expressed full-length mCD123 on 2 murine leukemia cell lines: C1498, a spontaneous myelomonocytic AML cell line, and a Arf-null, BCR-ABL1 preB-ALL. In addition, we analyzed a panel of virally induced NUP98::KDM5A AML generated by retroviral transduction of murine HPCs and determined all samples naturally expressed mCD123 antigen at different densities (range 38-77% N=4). We co-cultured mCD123-CAR in the presence of mCD123+ leukemia cell lines, with mB7-H3-CAR, SP6-CAR, or non-transduced T cells serving as controls. mCD123-CAR T cells recognized CD123+ targets, as evidenced by significantly increased cytokine secretion and cytolytic activity in comparison to controls (N=6, p<0.01). Antitumor activity was maintained in repeat stimulation assays for up to 5 stimulations. To determine in vivo toxicity of mCD123-CAR T cells, non-tumor bearing C57BL/6 mice were given 200 mg/kg cyclophosphamide (Cy) and one effector T cell infusion generated from transgenic luciferase mice (N=5) and monitored by bioluminescence imaging for 10 days. Bone marrow and spleen were harvested 10 days after injection for flow cytometry evaluation and colony formation unit (CFU) readouts. We detected no significant differences in toxicities between mCD123-CAR and control T cells. We next evaluated the anti-leukemia activity of mCD123-CAR T cells. C57BL/6 mice were injected with preB-ALLArf-/CD123+ cells on day 0 and received 100 mg/kg of Cy on day 2. On day 4, they were injected with 10x106 mCD123-CAR T cells. Mice receiving tumor only, tumor + Cy, or SP6-CAR served as controls. mCD123-CAR T cells had significant anti-leukemia activity resulting in a prolonged survival advantage in comparison to controls (N=5 per group, p<0.001). We focused on delineating the TME in our established models and preliminary analysis of comprehensive flow cytometry and single cell transcriptomic data on bone marrow. Our findings revealed that the TME of our established immune competent leukemia CAR T cell therapy models recapitulate human disease, having an increase in dying tumor cells within the bone marrow of mice treated with mCD123-CAR, with a number of other populations (e.g. dendritic cells, regulatory T cells) approximating the tumor-only microenvironment profile. Conclusions We have developed functional mCD123-CAR T cells targeting AML and ALL and have established immunocompetent models to test them. mCD123-CAR T cells demonstrated potent anti-leukemic activity in vitro and have anti-leukemia activity with minimal to no toxicity in vivo. These findings will provide insight into the impact of the AML TME on CAR T cell functionality, and inform future strategies to improve CAR T cells in the immunosuppressive TME.

A mitochondria-targeted molecular phototheranostic platform for NIR-II imaging-guided synergistic photothermal/photodynamic/immune therapy

Phototherapy is a conducive and non-invasive strategy for cancer therapy under light irradiation. Inspiringly, fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) holds a great promise for imaging-guided phototherapy with deep penetration and high spatiotemporal resolution. However, most phototherapeutics still face great challenges, including complicated synthesis of agents, potential biotoxicity and unsatisfied therapeutic outcomes. Herein, a near-infrared laser triggered molecular photosensitizer FEPT, modified with triphenylphosphine PEGylation (PEG2000-TPP), is developed for NIR-II imaging-guided mitochondria-targeting synergistic photothermal therapy (PTT)/photodynamic therapy (PDT)/immune therapy (IMT). The mitochondria-targeting photosensitizer FEPT can produce reactive oxygen species (ROS) and hyperpyrexia upon 808 nm laser irradiation, resulting in mitochondrial dysfunction and photo-induced apoptosis via caspase-3 pathway. Phototherapy-induced hyperthermia or ROS triggers the release of immunogenic intracellular substrates from dying tumor cells, thereby promoting the activation of antitumor immunity. Herein, this work provides a practicable strategy to develop a molecular phototheranostic platform for imaging-guided cancer therapy via mitochondria-targeting.Graphical

Efferocytosis drives myeloid NLRP3 dependent inflammasome signaling secretion of IL-1β to promote tumor growth

Caspase-1 signaling in myeloid suppressor cells can promote T-cell independent cancer progression, but the regulation of inflammasome signaling within the highly heterogeneous myeloid population in the tumor milieu remains elusive. To resolve this complexity, single cell transcriptomic profile of Head and Neck Squamous Cell Carcinoma (HNSCC) identified distinct inflammasome-associated genes within specific clusters of tumor-infiltrating myeloid cells. Among these myeloid cells, the sensor protein, NLRP3, and downstream effector IL-1β transcripts were enriched in discreet monocytic and macrophage subtypes in the TME. We showed that deletion of NLRP3, but not AIM2, phenocopied caspase-1/IL-1β dependent tumor progression in vivo. Paradoxically, we found myeloid-intrinsic caspase-1 signaling increased myeloid survival contrary to what would be predicted from the canonical pyroptotic function of caspase-1. This myeloid NLRP3/IL-1β signaling axis promotion of tumor growth was found to be gasdermin D independent. Mechanistically, we found that phagocyte-mediated efferocytosis of dying tumor cells in the TME directly activated NLRP3-dependent inflammasome signaling to drive IL-1β secretion. Subsequently we showed that NLRP3-mediated IL-1β production drives tumor growth in vivo. Dynamic RNA velocity analysis showed a robust directional flow from efferocytosis gene-set high macrophages to an inflammasome gene-set high macrophage population. We provide a novel efferocytosis-dependent inflammasome signaling pathway which mediates homeostatic tumor cell apoptosis that characterizes chronic inflammation-induced malignancy.

Multi-Omics Analysis Reveals the Unexpected Immune Regulatory Effects of Arsenene Nanosheets in Tumor Microenvironment.

Arsenene, a two-dimensional (2D) monoelemental layered nanosheet composed of arsenic, was recently reported to feature outstanding anticancer activities. However, the specific biological mechanism of action remains unknown. In this work, we extensively analyzed the mechanism of arsenene in vivo and in vitro and discovered the unexpected immune regulatory capability of arsenene for the first time. Analysis of cell phenotypes in tumor microenvironment by single-cell RNA sequencing revealed that arsenene remodeled the tumor microenvironment by recruiting a high proportion of anticancer immune cells to eliminate the tumor. Mechanistically, arsenene significantly activated T cell receptor signaling pathways to produce antitumor immune cells while inhibiting DNA replication and TCA cycle pathways of tumor cells in vivo. Further proteomic analysis on tumor cells revealed that arsenene induced reactive oxygen species production and oxidative stress damage by targeting thioredoxin TXNL1. The overloaded reactive oxygen species (ROS) further triggered endoplasmic reticulum stress responses to release damage-associated molecular patterns (DAMPs) and "eat-me" signals from dying tumor cells, leading to the activation of antigen-presenting processes to induce the subsequent effector tumor-specific CD8+ T cell immune responses. This unexpected discovery indicated for the first time that 2D inorganic nanomaterials could effectively activate direct anticancer immune responses, suggesting arsenene as a promising candidate nanomedicine for future cancer immunotherapy.

Combined Chemo-Immuno-Photothermal Therapy for Effective Cancer Treatment via an All-in-One and One-for-All Nanoplatform.

Tumor metastasis and recurrence are recognized to be the main causes of failure in cancer treatment. To address these issues, an "all in one" and "one for all" nanoplatform was established for combined "chemo-immuno-photothermal" therapy with the expectation to improve the antitumor efficacy. Herein, Docetaxel (DTX, a chemo-agent) and cynomorium songaricum polysaccharide (CSP, an immunomodulator) were loaded into zein nanoparticles coated by a green tea polyphenols/iron coordination complex (GTP/FeIII, a photothermal agent). From the result, the obtained nanoplatform denoted as DTX-loaded Zein/CSP-GTP/FeIII NPs was spherical in morphology with an average particle size of 274 nm, and achieved pH-responsive drug release. Moreover, the nanoplatform exhibited excellent photothermal effect both in vitro and in vivo. It was also observed that the nanoparticles could be effectively up take by tumor cells and inhibited their migration. From the results of the in vivo experiment, this nanoplatform could completely eliminate the primary tumors, prevent tumor relapses on LLC (Lewis lung cancer) tumor models, and significantly inhibit metastasis on 4T1 (murine breast cancer) tumor models. The underlying mechanism was also explored. It was discovered that this nanoplatform could induce a strong ICD effect and promote the release of damage-associated molecular patterns (DAMPs) including CRT, ATP, and HMGB1 by the dying tumor cells. And the CSP could assist the DAMPs in inducing the maturation of dendritic cells (DCs) and facilitate the intratumoral infiltration of T lymphocytes to clear up the residual or disseminated tumor cells. In summary, this study demonstrated that the DTX-loaded Zein/CSP-GTP/FeIII is a promising nanoplatform to completely inhibit tumor metastasis and recurrence.

Self-Assembled Immunostimulatory Tetrahedral Framework Nucleic Acid Vehicles for Tumor Chemo-immunotherapy.

Some chemotherapeutic agents, such as anthracyclines and oxaliplatin, can induce immunogenic cell death (ICD) with additional immune responses against cancer. However, ICD-based immunotherapy is limited by the nonspecific distribution of drugs and various side effects. Here, an immunostimulatory self-assembled tetrahedral framework nucleic acid (tFNA) vehicle was constructed to potentiate the chemo-immunotherapy, in which doxorubicin (DOX) acted as a chemotherapeutic agent and an ICD-inducer. Meanwhile, the immunostimulatory CpG-tFNA was employed as a nanocarrier to deliver DOX and an adjuvant to enhance the immunotherapy. Damage-associated molecular patterns (DAMPs) generated by DOX from dying tumor cells, such as calreticulin (CRT), high mobility group protein 1(HMGB1), and adenosine triphosphate (ATP), can activate dendritic cells (DCs) and trigger an immunological response. Afterward, CpG-tFNA with immunostimulatory properties works to boost the DOX-induced immunotherapy. Consequently, CpG-tFNA/DOX showed excellent antitumor effects and immunological activation, including CD8+ T cell proliferation and antitumor cytokine TNF-α and IFN-γ secretion. Moreover, chemo-immunotherapy can also be enhanced synergistically when coadministered with PD-L1. In conclusion, CpG-tFNA/DOX promotes the ICD-associated chemo-immunotherapy and strengthens the connection between traditional chemotherapy and immunotherapy, representing a novel strategy for clinical application. Moreover, the concept of ICD-related immunotherapy can also be extended to other treatments such as radiotherapy which can induce immunogenic cell death as well.

Autophagy activation by dietary piceatannol enhances the efficacy of immunogenic chemotherapy.

Immunogenic cell death (ICD) promotes the immune antitumor response via releasing damage-associated molecular patterns (DAMPs) from dying tumor cells. The induction of autophagy improves the efficacy of multiple immunogenic chemotherapies. Here, we show that piceatannol, a dietary phenolic compound that is widely distributed in multiple fruits and vegetables such as grapes, blueberries, and mushrooms, induces autophagy and enhances oxaliplatin (OXA)-induced anticancer immune response. Specifically, piceatannol enhanced OXA-induced release of DAMPs, several key hallmarks of ICD including ATP release, cell surface exposure of calreticulin, and high-mobility group box 1 (HMGB1) release. Mechanistically, piceatannol promoted autophagy via activating TFEB/TFE3, two key transcription factors of the autophagy-lysosome pathway, and inhibiting autophagy attenuated piceatannol plus OXA-induced ATP release. Furthermore, piceatannol induced endoplasmic reticulum stress, which is critical for its role in enhancing OXA-induced cell surface exposure of calreticulin, another key hallmark of ICD. Consistently, the combination of piceatannol with OXA promoted the anticancer effects in immunocompetent mice. Taken together, our results indicate the importance and great potential of dietary piceatannol in cancer immunotherapy. Therefore, piceatannol may be used as an ICD enhancer that improves the efficacy of chemotherapeutics such as OXA in cancer treatment with minimized toxicity.

Biodegradable hydrogen peroxide nanogenerator for controllable cancer immunotherapy via modulating cell death pathway from apoptosis to pyroptosis

Damage-associated molecular patterns (DAMPs) generated by dying tumor cells can react with host immune systems to activate autonomous immune responses. However, there are still huge challenges in achieving precise release of DAMPs for controllable cancer immunotherapy. As we all know, apoptosis is a process by which cells automatically end their lives determined by genes, and generally does not cause the strong release of DAMPs and immune responses. However, pyroptosis, as an emerging unique pathway of programmed cell death, can trigger a strong inflammatory response by generating large amount of DAMPs. Based on this, we prepared ZnO2 as the donator of H2O2 to trigger pyroptosis and apoptosis by using high concentration of ZnO2 (HZnO2) and low concentration of ZnO2 (LZnO2) respectively, and further compared the different immune activation effects between these two cell death pathways. For the first time, we found HZnO2 can induce pyroptosis effectively and its immune activation effect is better than that of apoptosis induced by LZnO2. Therefore, this nanoplatform provides a new pyroptosis inducer and a new insight for understanding the relationship between cell death pathway and immune responses.

Integrating chromatin accessibility states in the design of targeted sequencing panels for liquid biopsy

Dying tumor cells shed DNA fragments into the circulation that are known as circulating tumor DNA (ctDNA). Liquid biopsy tests aim to detect cancer using known markers, including genetic alterations and epigenetic profiles of ctDNA. Despite various advantages, the major limitation remains the low fraction of tumor-originating DNA fragments in a high background of normal blood-cell originating fragments in the cell-free DNA (cfDNA) pool in plasma. Deep targeted sequencing of cfDNA allows for enrichment of fragments in known cancer marker-associated regions of the genome, thus increasing the chances of detecting the low fraction variant harboring fragments. Most targeted sequencing panels are designed to include known recurrent mutations or methylation markers of cancer. Here, we propose the integration of cancer-specific chromatin accessibility states into panel designs for liquid biopsy. Using machine learning approaches, we first identify accessible and inaccessible chromatin regions specific to each major human cancer type. We then introduce a score that quantifies local chromatin accessibility in tumor relative to blood cells and show that this metric can be useful for prioritizing marker regions with higher chances of being detected in cfDNA for inclusion in future panel designs.

Abstract 5504: Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to restrain acquired resistance from antigen-loss-variant tumor cells

Abstract Adoptive cell therapy (ACT) with tumor-specific T cells expressing T-cell-receptors (TCR) or chimeric antigen receptors (CAR) has not mediated sustained responses in solid tumors. Several factors limit the efficacy of ACT in solid tumors and lead to primary resistance and acquired resistance (refers to recurrence after the initial response). These include but not limited to the insufficient proliferation of T cells, the low capacity of T cells to penetrate stroma-rich solid tumors, and heterogeneity of antigen expression in solid tumors, which allows the escape of T cell attack by tumor cells when becoming antigen-loss-variant cancer cell (ALV). Hence, it is urgent to develop novel ACT strategies to eradicate solid tumors and prevent acquired resistance. We have previously reported (Yong Lu, Cancer Cell, 2018) that tumor-specific IL-9-producing CD4+ Th9 cells represent a novel T cell paradigm for ACT - they are less exhausted, fully cytolytic, and hyperproliferative. This hyperproliferation of Th9 cells is driven by a unique Th9 master transcriptional factor Pu.1 mediated Pu.1-Traf6-NFκB signaling to ensure Th9 cells persist as long as “stem cell-like” T cells, which is not presented in other known CD4+ T cells subsets. In our most recent study, we demonstrate that murine and human Th9 cells, but not Th1/Tc1 or Th17 cells, expressing tumor-specific T-cell-receptors (TCR) or chimeric antigen receptors (CAR), eradicate advanced tumors that contain ALVs. This unprecedented antitumor capacity of Th9 cells is attributed to both enhanced direct tumor cell killing and bystander antitumor effects promoted by intratumor release of IFNα/β. Mechanistically, tumor-specific Th9 cells increase the intratumor accumulation of extracellular ATP (eATP, released from dying tumor cells), because of a unique feature of Th9 cells that lack the expression of ATP degrading ectoenzyme CD39. Intratumor enrichment of eATP promotes the monocyte infiltration and stimulates their production of IFNα/β by inducing eATP-endogenous retrovirus-TLR3/MAVS pathway activation. These results identify tumor-specific Th9 cells as a unique T cell subset endowed with the unprecedented capacity to eliminate ALVs for curative responses. Citation Format: Yong Lu. Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to restrain acquired resistance from antigen-loss-variant tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5504.

Abstract 242: TCTP is a target for cancer immunotherapy modulating myeloid-derived suppressor cells

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogenous group of myeloid cells that are highly suppressive to antitumor lymphocyte function and trafficking to the tumor. The accumulation of MDSCs in the tumor immune microenvironment (TIME) makes immunosuppressive TIME and progressive tumor. In fact, despite notable clinical outcomes in cancer immunotherapy, many patients are refractory or relapsed to immune checkpoint inhibitor therapy, wherein obstacles imposed by TIME contribute to a great extent. Here, we show that translationally controlled tumor protein (TCTP) released by dying tumor cells is an immunomodulator crucial to full-blown MDSC accumulation in the TIME in mouse models. We provide evidence that extracellular TCTP mediates recruitment of the polymorphonuclear MDSC (PMN-MDSC) population in the TIME, which is Toll-like receptor-2 (TLR2) dependent. For human translation, we confirmed that human TCTP binds directly to human TLR2 extracellular domain and stimulates human PBMC to produce multiple cytokines. To explore therapeutic applicability, we generated anti-TCTP antibodies for TCTP inhibition. The antibodies successfully neutralized TCTP in molecular and cellular level. The antibodies also suppressed PMN-MDSC accumulation and tumor growth. The combination of anti-TCTP antibody and immune checkpoint inhibitor showed synergistic effect on tumor growth inhibition. In conclusion, extracellular TCTP is an immunomodulator recruiting PMN-MDSCs to TIME. When TCTP is removed or neutralized, PMN-MDSC counts in TIME decreased. Anti-TCTP therapy may offer a new immunotherapy strategy. Citation Format: Jeong-Hyun Ryou, Gwanghee Lee, Tadatsugu Taniguchi, Hideyuki Yanai, Sho Hangai. TCTP is a target for cancer immunotherapy modulating myeloid-derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 242.

Abstract 1286: Oral administration of poly gamma glutamic acid significantly enhances the antitumor immune response of doxorubicin in a murine cancer model through regulating tumor microenvironment

Abstract Immunogenic cell death (ICD) is a type of cancer cell death triggered by certain chemotherapeutic drugs, oncolytic viruses, and other therapies. In particular, doxorubicin (DOX), one of the chemo-drugs, is well known to induce ICD, which involves the activation of the immune system against cancer in immunocompetent hosts. ICD comprises the release of damage-associated molecular patterns (DAMPs) from dying tumor cells that result in the activation of tumor-specific immune responses, thus eliciting long-term efficacy of anticancer drugs by combining direct cancer cell killing and antitumor immunity. However, the antitumor immunity can be achieved by overcoming the immunosuppressive tumor microenvironment (TME). Most current treatment strategies harnessing the tumor microenvironment focus on T cell-immune response, either by promoting activating signals or suppressing inhibitory ones.We here present a novel approach to cope with the inhibitory TME, combinatory treatment of DOX with poly gamma glutamic acid (γ-PGA). PGA, a safe edible biomaterial secreted by B. subtillis, has been evaluated in the clinical trials with the patients diagnosed as cervical intraepithelial neoplasia 1 (CIN 1). From the study using murine cancer models, including MC38, we found the combinatory treatment with γ-PGA restored the downregulation of NK cell activity caused by DOX, enhanced ICD and T cell responses, and reduced proinflammatory cytokines in the tumor tissues. Taken together, co-administration of γ-PGA exhibited synergistic anti-cancer effects by favoring the immunosuppressive TME in the combinatory treatment with conventional chemo-drugs. Citation Format: Joungwoo Choi, Solmin Jung, Yena Oh, Jaepyeong Jang, Yeondong Cho, Do young Lee, Youngcheol Park, Kwangil Jeong. Oral administration of poly gamma glutamic acid significantly enhances the antitumor immune response of doxorubicin in a murine cancer model through regulating tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1286.