Perylene Tetracarboxylic Dianhydride Carbon Dots Deplete Glutathione to Induce Immunogenic Cell Death in Tumor Cells.

SY03-01: The desirable death of the cancer cell: Immunogenic cell death for optimal chemotherapy

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

Even with the recent advancements in cancer immunotherapy, treatments are still associated with debilitating side effects and unacceptable fail rates. Induction of immunogenic cell death (ICD) in tumors is a promising approach to cancer treatment that may overcome these deficiencies. Cells undergoing ICD pathways enhance the interactions between cancerous cells and immune cells of the patient, resulting in the generation of anti-cancer immunity. The goal of this therapy relies on the engagement and reestablishment of the patient's natural immune processes to target and eliminate cancerous cells systemically. The main objective of this research was to determine if non-thermal plasma could be used to elicit immunogenic cancer cell death for cancer immunotherapy. My hypothesis was that plasma induces immunogenic cancer cell death through oxidative stress pathways, followed by development of a specific anti-tumor immune response. This was tested by investigating the interactions between plasma and multiple cancerous cells in vitro and validating anti-tumor immune responses in vivo. Following plasma treatment, two surrogate ICD markers, secreted adenosine triphosphate (ATP) and surface exposed calreticulin (ecto-CRT), were emitted from all three cancerous cell lines tested: A549 lung carcinoma cell line, CNE-1 radiation-resistant nasopharyngeal cell line and CT26 colorectal cancer cell line. When these cells were co-cultured with macrophages, cells of the innate immune system, the tumoricidal activity of macrophages was enhanced, thus demonstrating the immunostimulatory activity of cells undergoing ICD. The underlying mechanisms of plasma-induced ICD were also evaluated. When plasma is generated, four major components are produced: electromagnetic fields, ultraviolet radiation, and charged and neutral reactive species. Of these, we determined that plasma-generated charged and short-lived reactive oxygen species (ROS) were the major effectors of ICD. Following plasma treatment, ROS immediately increased. When chemical attenuators of ROS were used, intracellular ROS was abrogated and emission of ICD markers were attenuated. This strongly suggests that plasma-induced ICD is associated with increased intracellular ROS. The gold-standard approach to evaluating whether a stimulus can elicit genuine ICD relies on a vaccination assay. CT26 colorectal cancer cells were treated at ICD-inducing regimes of plasma and injected into syngeneic Balb/c mice. One week later, mice were challenged with live CT26 cancer cells. Tumor progression was moderated in animals immunized with plasma-treated CT26 cells. Altogether, these provide strong evidence that plasma regimes can be adapted for a new application: ICD induction. Next, a study was conducted to test the potential of plasma to induce ICD in tumors in animals. Plasma treatment of subcutaneous tumors in mice elicited the emission of ecto-CRT and high mobility group box 1 (HMGB1), another marker of ICD, in the tumor and also recruited CD11c+ and CD45+ immune cells locally. This was followed by development of cancer-specific splenic T cells, indicating that a systemic anti-tumor response was elicited from localized plasma treatment of the tumor. Overall, this work demonstrates the development of non-thermal plasma as a novel method of inducing immunogenic cell death for cancer immunotherapy. The obtained results further our understanding of plasma-cellular interaction mechanisms and highlight the potential for clinical translation.

The deep and inner beds of solid tumors lack lymphocytic infiltration and are subjected to various immune escape mechanisms. Reversing immunosuppression deep within the tumor is vital in clinical cancer therapy, however it remains a huge challenge. In this work, we have demonstrated the use of a second window near-infrared (NIR(II)) photothermal treatment to trigger more homogeneous and deeper immunogenic cancer cell death in solid tumors, thereby eliciting both innate and adaptive immune responses for tumor control and metastasis prevention. Specifically, photothermal transducers with similar components, structures, and photothermal conversion efficiencies, but different absorptions in red light, NIR(I), and NIR(II) biowindows, were constructed by controlling the self-assembly of gold nanoparticles on fluidic liposomes. In vitro, photothermal treatments induced immunogenic cell death (ICD) that were accompanied by the release of damage-associated molecular patterns (DAMPs) regardless of the wavelength of incident lasers. In vivo, NIR(II) light resulted in a more homogeneous release and distribution of DAMPs in the deeper parts of the tumors. With the induction of ICD, NIR(II) photothermal therapy simultaneously triggered both innate and adaptive immune responses and enabled efficient tumor control with 5/8 of the mice remaining tumor-free in the cancer vaccination assay. Additionally, the NIR(II) photothermal treatment in combination with checkpoint blockade therapy exerted long-term tumor control over both primary and distant tumors. Finally, using systemically administered two-dimensional polypyrrole nanosheets as a NIR(II) transducer, we achieved striking therapeutic effects against whole-body tumor metastasis via a synergistic photothermal-immunological response.

Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models. Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively. Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect. Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody. Citation Format: Ruslan Novosiadly, David Schaer, Nelusha Amaladas, Erik Rasmussen, Zhao Hai Lu, Andreas Sonyi, Carmine Carpenito, Yanxia Li, Shuang Luo, Andrew Capen, Catalina Meyer, Xiaodong Huang, Jason Manro, Gregory Donoho, Thompson Doman, Gerald Hall, Sandaruwan Geeganage, Michael Kalos. Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4549.

Biology-Oriented Design Strategies of AIE Theranostic Probes

BackgroundWe have been developing a non-thermal, drug-free tumor therapy called Nano-Pulse Stimulation (NPS) that delivers ultrashort electric pulses to tumor cells which eliminates the tumor and inhibits secondary tumor growth. We hypothesized that the mechanism for inhibiting secondary tumor growth involves stimulating an adaptive immune response via an immunogenic form of apoptosis, commonly known as immunogenic cell death (ICD). ICD is characterized by the emission of danger-associated molecular patterns (DAMPs) that serve to recruit immune cells to the site of the tumor. Here we present evidence that NPS stimulates both caspase 3/7 activation indicative of apoptosis, as well as the emission of three critical DAMPs: ecto-calreticulin (CRT), ATP and HMGB1.MethodsAfter treating three separate cancer cell lines (MCA205, McA-RH7777, Jurkat E6-1) with NPS, cells were incubated at 37 °C. Cell-culture supernatants were collected after three-hours to measure for activated caspases 3/7 and after 24 h to measure CRT, ATP and HMGB1 levels. We measured the changes in caspase-3 activation with Caspase-Glo® by Promega, ecto-CRT with anti-CRT antibody and flow cytometry, ATP by luciferase light generation and HMGB1 by ELISA.ResultsThe initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm2. Reducing this current inhibits cell death. Activated caspase-3 increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h post treatment. This increase is non-linear and peaks at 15–20 J/mL for all field strengths. 10 and 30 kV/cm fields exhibited the lowest response and the 12 and 15 kV/cm fields stimulated the largest amount of caspase activation. We measured the three DAMPs 24 h after treatment. The expression of cell surface CRT increased in an energy-dependent manner in the NPS treated samples. Expression levels reached or exceeded the expression levels in the majority of the anthracycline-treated samples at energies between 25 and 50 J/mL. Similar to the caspase response at 3 h, secreted ATP peaked at 15 J/mL and then rapidly declined at 25 J/mL. HMGB1 release increased as treatment energy increased and reached levels comparable to the anthracycline-treated groups between 10 and 25 J/mL.ConclusionNano-Pulse Stimulation treatment at specific energies was able to trigger the emission of three key DAMPs at levels comparable to Doxorubicin and Mitoxantrone, two known inducers of immunogenic cell death (ICD). Therefore NPS is a physical modality that can trigger immunogenic cell death in tumor cells.

Recent data suggest that therapy-induced immunogenic cancer cell death can stimulate a therapeutic anti-cancer immune response that then contributes to the control (or even to the elimination) of residual tumor cells. Indeed, some chemotherapeutic agents (e.g. X Rays, oxaliplatin and anthracyclines) can induce immunogenic apoptosis, while others cannot (e.g. cisplatin, mitomycin C and etoposide) [Casares, 2005; Obeid, 2007; Apetoh, 2007; Obeid, 2007; Ghiringhelli, 2009]. Thus far, we know that the immunogenicity of tumor cell death is closely linked to the surface exposure of calreticuline (CRT), the secretion of ATP and the release of HMGB1 that bind to CRT receptor, P2RX7 and TLR4 on host dendritic cells respectively. Chemotherapeutic agents that fail to induce CRT exposure or HMGB1 release are unable to induce immunogenic cell death. Our published data indicate that two loss-of-function alleles (that affect toll-like receptor-4 [TLR4] and purinergic P2 receptor X7 [P2RX7] of 12 and 30% of Caucasians, respectively) reduce the efficacy of conventional anticancer therapies, for instance in anthracyline-treated breast carcinomas and in oxaliplatin-treated colon cancers. We have determined novel defects in the emission or perception of immunogenic cell death signals negatively affecting the therapeutic response of human cancers that will be presented. Mouse preclinical models allowed us to unravel novel cellular and molecular pathways involved in the immunogenicity of cell death. These data will be reviewed. Based on this concept, which remains to be proven, one can formulate four predictions: First, therapeutic regimens that are intrinsically unable to elicit immunogenic death should be less efficient than therapies that can trigger immunogenic cell death. Second, cancers that intrinsically lack the capacity to emit immunogenic signals during tumor cell death should fail to induce a specific immune response and have a bad prognosis. Third, immune defects that weaken the response against dying tumor cells, including defects in the perception of immunogenic signals, should worsen the prognosis of cancer and compromise the therapeutic response. Fourth, the immunogenic cell death induced by chemotherapy should influence the composition and the architecture of the immune infiltrate present in tumors, which in turn should affect the therapeutic response. Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN2-1

SummaryProtons are highly related to cell viability during physiological and pathological processes. Developing new probes to monitor the pH variation could be extremely helpful to understand the viability of cells and the cell death study. Carbonized polymer dots (CPDs) are superior biocompatible and have been widely applied in bioimaging field. Herein, a new type of extreme-pH suitable CPDs was prepared from citric acid and o-phenylenediamine (CA/oPD-CPDs). Due to the co-existence of hydrophilic and hydrophobic groups, CA/oPD-CPDs tend to aggregate in neutral condition with a dramatic decrease of fluorescence, but disperse well in both acidic and alkaline conditions with brighter emission. This specialty enables them to selectively illuminate lysosomes in cells. Moreover, CA/oPD-CPDs in the cytoplasm could serve as a sustained probe to record intracellular pH variation during apoptosis. Furthermore, CA/oPD-CPDs present a continuous fluorescence increase upon 2-h laser irradiation in living cells, underscoring this imaging system for long-term biological recording.

We have previously shown that nearly half of mesothelioma patients have tumors with low autophagy and that these patients have a significantly worse outcome than those with high autophagy. We hypothesized that autophagy may be beneficial by facilitating immunogenic cell death (ICD) of tumor cells following chemotherapy. An important hallmark of ICD is that death of tumor cells is preceded or accompanied by the release of damage-associated molecular pattern molecules (DAMPs), which then can stimulate an antitumor immune response. Therefore, we measured how autophagy affected the release of three major DAMPs: high mobility group box 1 (HMGB1), ATP, and calreticulin following chemotherapy. We found that autophagy in three-dimensional (3D) models with low autophagy at baseline could be upregulated with the cell-permeant Tat-BECN1 peptide and confirmed that autophagy in 3D models with high autophagy at baseline could be inhibited with MRT 68921 or ATG7 RNAi, as we have previously shown. In in vitro 3D spheroids, we found that, when autophagy was high or upregulated, DAMPs were released following chemotherapy; however, when autophagy was low or inhibited, DAMPs release was significantly impaired. Similarly, in ex vivo tumors, when autophagy was high or upregulated, HMGB1 was released following chemotherapy but, when autophagy was low, HMGB1 release was not seen. We conclude that autophagy can be upregulated in at least some tumors with low autophagy and that upregulation of autophagy can restore the release of DAMPs following chemotherapy. Autophagy may be necessary for ICD in this tumor.

An emerging concept of ¡§a key‐lock paradigm¡ between the immunogenic cell death and dendrite cell (DC) is recently proposed. Immunogenic cell death is characterized by immunogenic factors (HMGB1, calreticulin, heat shock proteins), which are helpful for the maturation, antigen uptake and presentation of DC and serve as powerful immunological adjuvants. Whether some specific phytocompounds claimed to have anti‐tumor effects can deliver the immunogenic signals and keep expending the tumor‐recognition factors is a challenging question. To address this possibility, we first determined the expression profiles of immunogenic factors in tumor cells lysate (TCL) obtained from B16 cells treated with test phytocompounds and chemotherapeutic drugs at varying concentrations. Shikonin can induced tumor cell death and specific immunogenic factors expression in TCL in a dose‐dependent manner. The cytotoxic effects of splenocytes on target cancer cells were much higher in TCL‐shikonin‐DC vaccinated mice than in TCL‐DMSO‐DC vaccinated mice. TCL‐shikonin‐DC vaccination can substantially decrease the tumor growth and maintain over 70% of survival rate of tumor‐bearing mice after 35 days of tumor cell injection. In conclusion, shikonin is shown here to sensitize tumor cells to the lytic effects of DC‐activated immune effector cells, which may warrant future evaluation as adjuvant of tumor lysate‐pulsed DC vaccine.

SY28-03: Targeting tetraploidy for cancer therapy by pharmacological and immunological strategies.

BackgroundIt is well accepted that the immune system efficiently contributes to positive outcomes of chemotherapeutic cancer treatment by activating immunogenic cell death (ICD). However, only a limited number of ICD-inducing compounds are well characterized at present; therefore, identification of novel ICD inducers is urgently needed for cancer drug discovery, and the need is becoming increasingly urgent.MethodsHerein, we assessed the antitumour activity of bullatacin by MTS assay and apoptosis assay. ICD biomarkers, such as calreticulin (CRT), high-mobility group protein B1 (HMGB-1), heat shock protein (HSP)70, HSP90 and ATP, were assessed by Western blotting, ELISA and flow cytometry. Western blot and qPCR assays were performed to explore the underlying mechanisms of bullatacin-induced ICD. Flow cytometry was used to detect macrophage phagocytosis.ResultsFirst, bullatacin induced apoptosis in both SW480 cells and HT-29 cells in a time-dependent manner at 10 nM, as assessed by flow cytometry. Moreover, Western blot and flow cytometry assays showed that CRT and HSP90 (biomarkers of early ICD) significantly accumulated on the cell membrane surface after approximately 6 h of treatment with bullatacin. In addition, ELISAs and Western blot assays showed that the second set of hallmarks required for ICD (HMGB1, HSP70 and HSP90) were released in the conditioned media of both SW480 and HT-29 cells after 36 h of treatment. Furthermore, qPCR and Western blot assays indicated that bullatacin triggered ICD via activation of the endoplasmic reticulum stress (ERS) signalling pathway. Finally, bullatacin promoted macrophage phagocytosis.ConclusionThis study documents that bullatacin, a novel ICD inducer, triggers immunogenic tumour cell death by activating ERS even at a relatively low concentration in vitro.

BackgroundTisotumab vedotin (TV) is an investigational antibody-drug conjugate composed of a tissue factor (TF)-directed human monoclonal antibody covalently linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable...

3076 Background: Recent studies have identified molecular events characteristic of immunogenic cell death. These include surface exposure of calreticulin, HSP70 and HSP90, release of intranuclear HMGB1 and secretion of ATP from dying cells. Several chemotherapeutic agents, including anthracyclins, oxaliplatin and bortezomib, and hypericin-based photodynamic therapy have been described to induce the immunogenic cell death in human tumor cells. We investigated the potential of high hydrostatic pressure (HHP) to induce immunogenic cell death in human tumor cells. Methods: Prostate and ovarian cancer cell lines and primary tumor cells were treated by HHP and we analyzed the kinetics of the expression of immunogenic cell death markers. HHP killed tumor cells expressing immunogenic cell death markers were tested for their ability to activate dendritic cells (DCs), to induce tumor specific T cells and regulatory T cells. Results: HHP induced rapid expression of HSP70, HSP90 and calreticulin on the cell surface of all tested cell lines and primary tumor cells. HHP also induced release of HMGB1 and ATP from treated cells. The kinetics of expression was similar to doxorubicin, HHP, however, induced 1.5-2 fold higher expression of HSP70, HSP90 and calreticulin. The interaction of DCs with HHP-treated tumor cells led to the faster rate of phagocytosis, significant upregulation of CD83, CD86 and HLA-DR and release of IL-6, IL-12p70 and TNFα. The ability of HHP-killed tumor cells to promote DCs maturation was cell contact dependent. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor-specific CD4+ and CD8+IFN-g-producing T cells even in the absence of additional maturation stimulus. DCs pulsed with HHP treated tumor cells also induced the lowest number of regulatory T cells among the tested conditions. Cells treated by HHP can by cryopreserved in liquid nitrogen and retain their immunogenic properties upon thawing thus allowing for their convenient use in the manufacturing of cancer immunotherapy products. Conclusions: High hydrostatic pressure is a reliable and very potent inducer of immunogenic cell death in the wide range of human tumor cell lines and primary tumor cells.