Induction Of Immunogenic Cell Death Research Articles

Targeting immunogenic cell stress and death for cancer therapy.

Immunogenic cell death (ICD), which results from insufficient cellular adaptation to specific stressors, occupies a central position in the development of novel anticancer treatments. Several therapeutic strategies to elicit ICD - either as standalone approaches or as means to convert immunologically cold tumours that are insensitive to immunotherapy into hot and immunotherapy-sensitive lesions - are being actively pursued. However, the development of ICD-inducing treatments is hindered by various obstacles. Some of these relate to the intrinsic complexity of cancer cell biology, whereas others arise from the use of conventional therapeutic strategies that were developed according to immune-agnostic principles. Moreover, current discovery platforms for the development of novel ICD inducers suffer from limitations that must be addressed to improve bench-to-bedside translational efforts. An improved appreciation of the conceptual difference between key factors that discriminate distinct forms of cell death will assist the design of clinically viable ICD inducers.

Dual molecule targeting HDAC6 leads to intratumoral CD4+ cytotoxic lymphocytes recruitment through MHC-II upregulation on lung cancer cells

BackgroundDespite the current therapeutic treatments including surgery, chemotherapy, radiotherapy and more recently immunotherapy, the mortality rate of lung cancer stays high. Regarding lung cancer, epigenetic modifications altering cell cycle, angiogenesis...

Abstract LB116: Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors

Abstract In solid cancers, an immunosuppressive tumor microenvironment (TME) restrains immunotherapy. Induction of immunogenic cell death in tumor cells and simultaneously programming the TME from immunosuppressive to inflammatory employing dsRNA agonists for endosomal Toll-like receptor 3 (TLR3) represents an appealing in situ immunomodulation strategy. However, uptake of dsRNA by tumor cells is limited and systemic administration is linked to adverse side effects. Here we describe an exceptional therapeutically effect of systemically delivered TLR3 agonist RIBOXXOL using a recently developed antibody-guided nanoparticle-system designated “Rapid Inducer of Cellular Inflammation and Apoptosis” (RICIA) for targeting epidermal growth factor variant III (EGFRvIII) on tumor cells. We suggest that systemically applied monotherapy with RICIA might be effective in experimental solid tumors. This includes, (i) preferential enrichment of the immune-stimulatory RIBOXXOL and (ii) subsequent induction of a type-I interferon response in tumor cells expressing TLR3, and (iii) synergistic inflammation of the TME through TLR3 agonist thereby expediting anti-tumor immune responses. The effects of RICIA-treatment were comprehensively assessed in vitro and in murine syngeneic epidermal growth factor receptor variant III (EGFRvIII)-positive SMAvIII gliomas. Our in vitro studies confirm uptake of RICIA by EGFRvIII-mediated endocytosis in human and murine glioma cells and furthermore demonstrate TLR3-triggered type-I interferon release and mRNA signatures indicative of an innate immune response against virus. We show in the SMAvIII tumor model that monotherapy with picomolar amounts of RICIA induces regression of subcutaneous (s.c.) tumors leading to increase in survival time and results in complete tumor clearance in a significant fraction of mice accompanied by immune memory when compared to RICIAs equipped with an unspecific control antibody. Mechanistically, RICIA treatment reshapes the immunosuppressive tumor environment by mimicking viral infection leading to increased expression of the interferon beta (IFN-β) gene (Ifnb1), by subsequent induction of an inflammatory state characterized by RNA-expression signatures indicating induction of apoptosis, interleukin 1beta (IL-1β) signaling, and programming immunosuppressive M2 TAMs to activated pro-inflammatory M1 TAMs. In addition, a single stereotactic injection of RICIA into established intracranial glioma significantly improves mean survival time of treated mice and is able to induce complete tumor clearance thus showing therapeutic potency of RICIA in a relevant pre-clinical setting for glioma. Thus, we show that targeted activation of TLR3 represents a promising therapeutic strategy for immune-modulation of tumors with the prospect to elicit long-lasting anti-tumor immune responses. Citation Format: Achim Temme, Alexander Hagstotz, Isabell Schau, Michael Seifert, Duran Sürün, Stefan Zimmermann, Frank Buchholz, Luise Rupp, Rebekka Wehner, Marc Schmitz, Gabriele Schackert, Ilker Y. Eyüpoglu. Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB116.

Tumor microenvironment reprogramming combined with immunogenic enhancement by nanoemulsions potentiates immunotherapy

The combination of immune checkpoint inhibitors and immunogenic cell death (ICD) inducers has become a promising strategy for the treatment of various cancers. However, its efficacy remains unmet because of the dense stroma and defective vasculatures in the tumor microenvironment (TME) that restricts the intratumoral infiltration of cytotoxic T lymphocytes (CTLs). Herein, cancer-associated fibroblasts (CAFs)-targeted nanoemulsions are tailored to combine the ICD induction and the TME reprogramming to sensitize checkpoint blockade immunotherapy. Melittin, as an ICD inducer and an antifibrotic agent, is efficiently encapsulated into the nanoemulsion accompanied by a nitric oxide donor to improve its bioavailability and tumor targeting. The nanoemulsions exhibited dual functionality by directly inducing direct cancer cell death and enhancing the tumoral immunogenicity, while also synergistically reprogramming the TME through reversing the activated CAFs, decreasing collagen deposition and restoring tumor vessels. Consequently, these nanemulsions successfully facilitated the CTLs infiltration and suppressing the recruitment of immunosuppressive cells. A combination of AE-MGNPs and anti-CTLA-4 antibody greatly elicited a striking level of antitumor T-cell response to suppress tumor growth in CAFs-rich colorectal tumor models. Our work emphasized the integration of the ICD induction with simultaneous modulation of the TME to enhance the sensitivity of patients to checkpoint blockade immunotherapy.

Enhancing cancer immunotherapy with photodynamic therapy and nanoparticle: making tumor microenvironment hotter to make immunotherapeutic work better

Cancer immunotherapy has made tremendous advancements in treating various malignancies. The biggest hurdle to successful immunotherapy would be the immunosuppressive tumor microenvironment (TME) and low immunogenicity of cancer cells. To make immunotherapy successful, the ‘cold’ TME must be converted to ‘hot’ immunostimulatory status to activate residual host immune responses. To this end, the immunosuppressive equilibrium in TME should be broken, and immunogenic cancer cell death ought to be induced to stimulate tumor-killing immune cells appropriately. Photodynamic therapy (PDT) is an efficient way of inducing immunogenic cell death (ICD) of cancer cells and disrupting immune-restrictive tumor tissues. PDT would trigger a chain reaction that would make the TME ‘hot’ and have ICD-induced tumor antigens presented to immune cells. In principle, the strategic combination of PDT and immunotherapy would synergize to enhance therapeutic outcomes in many intractable tumors. Novel technologies employing nanocarriers were developed to deliver photosensitizers and immunotherapeutic to TME efficiently. New-generation nanomedicines have been developed for PDT immunotherapy in recent years, which will accelerate clinical applications.

Abstract CT035: Proof-of-concept and preliminary efficacy of triple IAP blockade to maximize immunogenic cell death and induce efficient adaptive immune response: First report on the ASTEROID phase 1 trial

Abstract Background: Dying cells play an important role in CD8+ T cell-mediated immunity. While apoptosis is mainly immunologically silent, necroptosis is immunogenic and can more efficiently initiate an adaptive immune response that could be augmented by immune checkpoint inhibition (ICI). Inhibitor of Apoptosis (IAP) proteins are key gatekeepers of how a cell dies, and frequently overexpressed in cancer. ASTX660 (tolinapant) is a triple blocker of cIAP1/2 and XIAP, sensitizing cancer cells to death ligands through ripoptosome formation, activating ‘danger’ co-stimulatory signals and dendritic cell (DC) maturation required to prime a potent antitumor response. Methods: We assessed the safety, pharmacodynamic (PD), and clinical activity of 7 days of oral ASTX660 given prior to pembrolizumab 200 mg (Q3W), in patients (pts) refractory to standard therapy. TITE-CRM was used to explore ASTX660 doses 90-180 mg. Serial paired blood and tumor samples were analyzed to interrogate the effect of ASTX660 on cell death, tumor microenvironment and host immune system. Results: 22 pts have been enrolled; median age 55 years (range 38-81); male:female 5:17; all ECOG PS ≤1; median 4.5 prior therapies. Tumor types included breast, head and neck (HN), hepatocellular, cervical, pancreatic and cholangiocarcinoma. Combination ASTX660-pembrolizumab is currently recruiting at highest dose 180 mg without any dose limiting toxicities (DLT) seen within the 6-week DLT observation period. Most common adverse events (AE) were G1-2 rash, nausea, and asymptomatic increases in amylase, lipase and transaminases; all reversible and easily manageable. Four iRECIST confirmed partial responses were seen in 10 efficacy evaluable pts treated at biologically active dose levels 120 and 150 mg (Overall response rate 40%), including 2 pts with ER+ HER2- breast cancer, 1 cervical adenocarcinoma pt (exceptional response following initial pseudoprogression) and a HN cancer pt refractory to ICI monotherapy. 3/4 responders are ongoing. Efficacy data from pts treated at highest dose level 180 mg is pending. FACS of paired blood samples from responders shows a significant rise in %CD57+/CD3+ cytotoxic effector T cells [mean 11.90% (pre-) vs 14.25% (post-ASTX660); p=.0176]. These changes were not seen in the non-responders group. Full PD from paired blood and tumour sampling will be shared at the meeting. Conclusion: ASTX660-pembrolizumab is is well-tolerated with durable antitumor responses in pts with immunologically cold tumors, providing the first clinical proof-of-concept of immunogenic cell death induction leading to effective adaptive immune response when combined with ICI. Recommended Phase 2 dose optimization and expansion cohorts in immunologically cold cancers are ongoing. Citation Format: Crescens Tiu, Wing Yau, Victoria Sanchez-Perez, Giovanni Codacci-Pisanelli, Ching Leung, Eden S. Andrews, Bristi Basu, Anna Zachariou, Toby Prout, Mona Parmar, Alison Turner, Xiaoran Lai, Zhulin Yin, Karen E. Swales, Syed Haider, Thomas Lund, Diego C. Dos Reis, Tatiany L. Silveira, Alan S. Dunlop, Ann-Marie Baker, Bora Gurel, Ana Ferreira, Claudia Bertan, Nina Tunariu, Edward W. Johnston, Alistair Ring, Tencho Tenev, Tomoko Smyth, Simone Jueliger, Udai Banerji, Johann S. de Bono, Alec Paschalis, Adam Sharp, Anna Minchom, Christina Yap, Pascal Meier, Juanita S. Lopez. Proof-of-concept and preliminary efficacy of triple IAP blockade to maximize immunogenic cell death and induce efficient adaptive immune response: First report on the ASTEROID phase 1 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT035.

Celastrol Elicits Antitumor Effects through Inducing Immunogenic Cell Death and Downregulating PD-L1 in ccRCC.

Targeting immunogenic cell death (ICD) is considered a promising therapeutic strategy for cancer. However, the commonly identified ICD inducers promote the expression of programmed cell death ligand 1 (PD-L1) in tumor cells, thus aiding them to evade the recognition and killing by the immune system. Therefore, the finding of novel ICD inducers to avoid enhanced PD-L1 expression is of vital significance for cancer therapy. Celastrol (CeT), a triterpene isolated from Tripterygium wilfordii Hook. F induces various forms of cell death to exert anti-cancer effects, which may make celastrol an attractive candidate as an inducer of ICD. In the present study, bioinformatics analysis was combined with experimental validation to explore the underlying mechanism by which CeT induces ICD and regulates PD-L1 expression in clear cell renal cell carcinoma (ccRCC). The results showed that EGFR, IKBKB, PRKCQ and MAPK1 were the crucial targets for CeT-induced ICD, and only MAPK1 was an independent prognostic factor for the overall survival (OS) of ccRCC patients. In addition, CeT triggered autophagy and up-regulated the expressions of HMGB1 and CRT to induce ICD in 786-O cells in vitro. Importantly, CeT can down-regulate PD-L1 expression through activating autophagy. At the molecular level, CeT suppressed PD-L1 via the inhibition of MAPK1 expression. Immunologically, the core target of celastrol, MAPK1, was tightly correlated with CD8+ T cells and CD4+ T cells in ccRCC. These findings indicate that CeT not only induces ICD but also suppresses PD-L1 by down-regulating MAPK1 expression, which will provide an attractive strategy for ccRCC immunotherapy.

Pyroptosis activates conventional type I dendritic cells to mediate the priming of highly functional anticancer T cells

BackgroundInitiation of antitumor immunity is reliant on the stimulation of dendritic cells (DCs) to present tumor antigens to naïve T cells and generate effector T cells that can kill cancer...

Endoplasmic reticulum-targeted delivery of celastrol and PD-L1 siRNA for reinforcing immunogenic cell death and potentiating cancer immunotherapy

The prospect of employing chemoimmunotherapy targeted towards the endoplasmic reticulum (ER) presents an opportunity to amplify the synergistic effects of chemotherapy and immunotherapy. In this study, we initially validated celastrol (CEL) as an inducer of immunogenic cell death (ICD) by promoting ER stress and autophagy in colorectal cancer (CRC) cells. Subsequently, an ER-targeted strategy was posited, involving the codelivery of CEL with PD-L1 small interfering RNAs (siRNA) using KDEL peptide-modified exosomes derived from milk (KME), to enhance chemoimmunotherapy outcomes. Our findings demonstrate the efficient transportation of KME to the ER via the Golgi-to-ER pathway. Compared to their non-targeting counterparts, KME exhibited a significant augmentation of the CEL-induced ICD effect. Additionally, it facilitated the release of danger signaling molecules (DAMPs), thereby stimulating the antigen-presenting function of dendritic cells and promoting the infiltration of T cells into the tumor. Concurrently, the ER-targeted delivery of PD-L1 siRNA resulted in the downregulation of both intracellular and membrane PD-L1 protein expression, consequently fostering the proliferation and activity of CD8+ T cells. Ultimately, the ER-targeted formulation exhibited enhanced anti-tumor efficacy and provoked anti-tumor immune responses against orthotopic colorectal tumors in vivo. Collectively, a robust ER-targeted delivery strategy provides an encouraging approach for achieving potent cancer chemoimmunotherapy.

Advancements in Stimulus-Responsive Co-Delivery Nanocarriers for Enhanced Cancer Immunotherapy.

Cancer immunotherapy has emerged as a novel therapeutic approach against tumors, with immune checkpoint inhibitors (ICIs) making significant clinical practice. The traditional ICIs, PD-1 and PD-L1, augment the cytotoxic function of T cells through the inhibition of tumor immune evasion pathways, ultimately leading to the initiation of an antitumor immune response. However, the clinical implementation of ICIs encounters obstacles stemming from the existence of an immunosuppressive tumor microenvironment and inadequate infiltration of CD8+T cells. Considerable attention has been directed towards advancing immunogenic cell death (ICD) as a potential solution to counteract tumor cell infiltration and the immunosuppressive tumor microenvironment. This approach holds promise in transforming "cold" tumors into "hot" tumors that exhibit responsiveness to antitumor. By combining ICD with ICIs, a synergistic immune response against tumors can be achieved. However, the combination of ICD inducers and PD-1/PD-L1 inhibitors is hindered by issues such as poor targeting and uncontrolled drug release. An advantageous solution presented by stimulus-responsive nanocarrier is integrating the physicochemical properties of ICD inducers and PD-1/PD-L1 inhibitors, facilitating precise delivery to specific tissues for optimal combination therapy. Moreover, these nanocarriers leverage the distinct features of the tumor microenvironment to accomplish controlled drug release and regulate the kinetics of drug delivery. This article aims to investigate the advancement of stimulus-responsive co-delivery nanocarriers utilizing ICD and PD-1/PD-L1 inhibitors. Special focus is dedicated to exploring the advantages and recent advancements of this system in enabling the combination of ICIs and ICD inducers. The molecular mechanisms of ICD and ICIs are concisely summarized. In conclusion, we examine the potential research prospects and challenges that could greatly enhance immunotherapeutic approaches for cancer treatment.

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

Copper-Doped Luteolin Carbon Dots with Antitumor Properties Based on the Induction of Immunogenic Cell Death

Copper-Doped Luteolin Carbon Dots with Antitumor Properties Based on the Induction of Immunogenic Cell Death

Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma

Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant anti-tumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABARAP is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in high-risk MM patients. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent anti-tumor T cell response. Low GABARAP was independently associated with shorter MM patient survival and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, like bortezomib, with an autophagy inducer, like rapamycin, may improve patient outcomes in MM, where low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Enzyme Core Spherical Nucleic Acid That Enables Enhanced Cuproptosis and Antitumor Immune Response through Alleviating Tumor Hypoxia.

Cuproptosis, a copper-dependent cell death process, has been confirmed to further activate the immune response and mediate the immune resistance. However, hypoxic tumor microenvironment hampers cuproptosis sensitivity and suppresses the body's antitumor immune response. Herein, we have successfully immobilized and functionalized catalase (CAT) with long single-stranded DNA containing polyvalent CpG sequences through rolling circle amplification (RCA) techniques, obtaining an enzyme-cored spherical nucleic acid nanoplatform (CAT-ecSNA-Cu) to deliver copper ions for cuproptosis. The presence of long-stranded DNA-protected CAT enhances mitochondrial respiration by catalyzing the conversion of H2O2 to O2, thereby sensitizing cuproptosis. Meanwhile, increased tumor oxygenation suppresses the expression of the hypoxia-inducible factor-1 (HIF-1) protein, resulting in the alleviation of the immunosuppressive tumor microenvironment. Of note, cuproptosis induces immunogenic cell death (ICD), which facilitates dendritic cell (DC) maturation and enhances antigen presentation through polyCpG-supported Toll-like receptor 9 (TLR9) activation. Furthermore, cuproptosis-induced PD-L1 upregulation in tumor cells complements checkpoint blockers (αPD-L1), enhancing antitumor immunity. The strategy of enhancing cuproptosis-mediated antitumor immune responses by alleviating hypoxia effectively promotes the activation and proliferation of effector T cells, ultimately leading to long-term immunity against cancer.

Mechanisms of triggering antitumor immunity: from photodynamic effects to immunogenic cell death

The development of cancer immunotherapies provides hope to millions of patients for better clinical outcomes after tumor treatment. Thus, investigating the fundamental mechanisms of antitumor immunity activation is an urgent task. Numerous studies have outlined the effect of immunogenic cell death (ICD) on cancer cells, and this outcome is both sophisticated and simple. Different stimuli can cause ICD; however, photodynamic exposure has been proven to be an effective inducer of programed death on par with radiotherapy. The link between triggering a photodynamic response in cancer cells and triggering ICD has been poorly described in experimental works. The question of which molecular cascades are activated after photodynamic therapy (PDT) irradiation and the way damage-associated molecular patterns (DAMPs) are released is intriguing. Much is known about reactive oxygen species generation and endoplasmic reticulum stress but little about the Golgi apparatus. Photosensitizers of different types can exert different effects, including completely nonimmunogenic ones.&#x0D; This review describes the cascades that link the induction of cell death by photodynamic exposure and the immunogenic pattern of DAMP release. The photosensitizers that have shown potential as ICD inducers and the different pathways of programed death that occur during PDT exposure are also discussed.

HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness. Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

Immunoantitumor Activity and Oxygenation Effect Based on Iron-Copper-Doped Folic Acid Carbon Dots.

Cancer metastasis and recurrence are closely associated with immunosuppression and a hypoxic tumor microenvironment. Chemodynamic therapy (CDT) and photothermodynamic therapy (PTT) have been shown to induce immunogenic cell death (ICD), effectively inhibiting cancer metastasis and recurrence when combined with immune adjuvants. However, the limited efficacy of Fenton's reaction and suboptimal photothermal effect present significant challenges for successfully inducing ICD through CDT and PTT. This paper described the synthesis and immunoantitumor activity of the novel iron-copper-doped folic acid carbon dots (CFCFB). Copper-doped folic acid carbon dots (Cu-FACDs) were initially synthesized via a hydrothermal method, using folic acid and copper gluconate as precursors. Subsequently, the nanoparticles CFCFB were obtained through cross-linking and self-assembly of Cu-FACDs with ferrocene dicarboxylic acid (FeDA) and 3-bromopyruvic acid (3BP). The catalytic effect of carbon dots in CFCFB enhanced the activity of the Fenton reaction, thereby promoting CDT-induced ICD and increasing the intracellular oxygen concentration. Additionally, 3BP inhibited cellular respiration, further amplifying the oxygen concentration. The photothermal conversion efficiency of CFCFB reached 55.8%, which significantly enhanced its antitumor efficacy through photothermal therapy. Immunofluorescence assay revealed that treatment with CFCFB led to an increased expression of ICD markers, including calreticulin (CRT) and ATP, as well as extracellular release of HMGB-1, indicating the induction of ICD by CFCFB. Moreover, the observed downregulation of ARG1 expression indicates a transition in the tumor microenvironment from an immunosuppressive state to an antitumor state following treatment with CFCFB. The upregulation of IL-2 and CD8 expression facilitated the differentiation of effector T cells, resulting in an augmented population of CD8+ T cells, thereby indicating the activation of systemic immune response.

A Comprehensive Strategy Based on High Clinical Translational Nanosystem for Programmable Immunotherapy of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBCs), known as an immunologically cold tumor, is difficult to completely eliminate with existing monotherapies, let alone metastasis and recurrence. It is urgent to design a rational combination of multiple therapies to programmatically reconstitute tumor microenvironment (TME) and reverse the immune "cold" into "hot" inflammatory tumors to improve the therapeutic effect. Hence, in this work, a multifunctional nanosystem (FeSH NPs) that integrates metal-polyphenol coordination complex as a photothermal agent and polyphenol, salvianolic acid B (SAB) as immunomodulator is designed and fabricated for synergistic photothermal-immunotherapy of TNBCs combined with anti-PD-L1 antibody. Guided by photothermal/photoacoustic dual-mode imaging, photothermal therapy (PTT) caused by FeSH NPs induces immunogenic cell death (ICD) under 808nm laser irradiation. Subsequently, the loaded SAB is released with the addition of deferoxamine mesylate (DFO) to remodel TME, specifically TGF-β inhibition and PD-L1 upregulation, and eliminate the primary tumors. The combination of PTT and TME reprogramming by FeSH NPs further synergizes with anti-PD-L1 antibody to eradicate recurrence and inhibit metastasis of TNBCs concurrently. Given the biosafety of FeSH NPs throughout the lifecycle, this work provides a protocol with high clinical translational promise for comprehensive programmed therapeutics of immunologically cold tumors TNBCs. This article is protected by copyright. All rights reserved.

Abstract 6649: Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with few therapeutic options. Tumor Treating Fields (TTFields) are low-intensity electric fields that disrupt cellular processes critical for cancer cell division and tumor progression and are approved for the treatment of glioblastoma and pleural mesothelioma. A phase 3 clinical trial is in progress to test the efficacy and safety of TTFields concomitant with standard-of-care gemcitabine and nab-paclitaxel (GnP) as front-line treatment of locally advanced PDAC. TTFields have been shown to induce immunogenic cell death (ICD) in cancer cells and elicit a systemic anti-cancer immune response. Accordingly, the benefit of applying TTFields together with immune checkpoint inhibitors (ICI) was demonstrated in a phase 3 study in patients with non-small cell lung carcinoma. This preclinical study investigated TTFields-induced immune responses in PDAC. Methods: An orthotopic PDAC tumor model was established by inoculating 4 × 105 Panc02 pancreatic cancer cells with stable luciferase expression into the pancreas tail of immune-competent C57BL/6 mice. The mice were randomized into four groups following bioluminescence detection of tumor volume: control, TTFields (150 kHz, 10 days), GnP (50 mg/kg for each drug, i.p. injected once a week for a total of two administrations), and GnP + TTFields. At the end of treatment, tumor volume was measured using caliper, and blood was collected for spectral flow cytometry-based immunophenotyping and ELISA testing for the ICD marker HMGB-1. In vitro, Panc02 cells were treated for 72h with TTFields (150 kHz, 1.62 V/cm RMS) and examined by flow cytometry for apoptosis and ATP secretion (using quinacrine staining) and surface exposure of calreticulin. Results: TTFields markedly reduced the tumor size in Panc02 tumor-bearing mice compared to control mice. Analysis of peripheral blood effector (CD62L-CD44+) and central (CD62L+CD44+) memory T cells demonstrated the induction of circulating effector-memory cytotoxic CD8+ T cells. Additionally, serum HMGB-1 concentrations were significantly elevated in mice treated with TTFields compared to control mice, implying the involvement of ICD in the response to TTFields. In vitro, TTFields application induced apoptosis of Panc02 cells and increased ICD, as indicated by increased surface exposure of calreticulin and ATP depletion. When TTFields were used in Panc02 tumor bearing mice concomitantly with GnP, a substantial reduction in tumor size was detected, and a complete tumor regression was observed in a number of mice. Conclusions: Our results suggest that TTFields may enhance the effects of standard-of-care chemotherapy for the treatment of PDAC. Furthermore, TTFields were shown to induce ICD and activate an antitumor immune response in PDAC, supporting future studies evaluating the efficacy of TTFields together with ICI in PDAC. Citation Format: Tal Kan, Tharwat Haj Khalil, Yiftah Barsheshet, Tali Voloshin, Avital Vorontsov, Boris Brant, Lilach Koren, Roni Blatt, Shay Cahal, Efrat Zemer-Tov, Rom Paz, Anat Klein-Goldberg, Catherine Tempel Brami, Sara Jacobovitch, Alexandra Volodin, Bella Koltun, Cfir David, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6649.

Abstract 2377: HER2-ADC efficacy is rooted in its induction of immunogenic cell death with FcGR activation and a systemic bystander effect

Abstract Background: T-DXd is an antibody-drug conjugate (ADC) targeting HER2 that has demonstrated superior clinical efficacy over previous HER2-targeting agent Trastuzumab emtansine (T-DM1). Notably, T-Dxd is clinically effective in breast cancers (BC) with a range of HER2 expression. While its purported mechanism of action (MOA) involves its cleavable linker leading to bystander killing, it remains unclear how this potential MOA accounts for efficacy in HER2-low cancers or the role FCGR-mediated signaling and immune induction plays in its clinical efficacy. Method: In vitro studies utilized co-culture, internalization, FCGR activation, ADCP, flow cytometry, and gene expression by QRT-PCR and ELISA assays. Assessments of tumor antigen-specific T cell stimulation involved JEDI T cells. In vivo studies were conducted using in vivo implantation of HER2+ and HER2- cells, PK assessments of T-Dxd/DM1 and therapeutic effects determined using an endogenous HER2 transgenic mouse model that measured HER2-specific adaptive immune responses, in combination with immune checkpoint blockade anti-CD47. Results: T-Dxd elicited a systemic bystander effect that was only partially dependent on HER2 expression, mediated by specific cathepsins in the extracellular TME. Notably, T-Dxd cytotoxicity induced immunogenic cell death (ICD), while also retaining the ability to engage FCGRs to promote ADCP and innate immune activation. ICD was mediated by secretion of specific Dxd driven DAMPs (HMGB1 and eATP), resulting in TLR4/STING activation. ICD and FCGR activation along with antigen ADCP together elicited myeloid cell activation (CD80, CD40) and antigen presentation, resulting in effective expansion of tumor antigen-specific T cells responses, demonstrated by co-culture assays using EGFP/HER2+ BC cells and CD8+ JEDI T cells. T cell activation required TLR4 and STING pathways while in vivo, T-Dxd anti-tumor responses associated with augmented HER2-specific T and B cell responses. However, T-Dxd also induced CD47 to limit phagocytosis. But, the use of CD47/SIRPa blocking antibodies dramatically enhanced CD8+ T cell expansion in vitro and anti-tumor efficacy in vivo upon T-Dxd treatment. Conclusion: Our study demonstrates that T-Dxd is cleavable in HER2 negative BC in vivo, which permits more widespread Dxd activity against HER2 low BC. Critically, Dxd elicits ICD, resulting in innate immune stimulation of myeloid cells, which when combined with FCGR activation and ADCP, leads to a striking induction of tumor-specific adaptive immunity. This combination was unique to T-Dxd, which may underlie its clinical superior efficacy. However, T-Dxd activity also elicits suppressive CD47 expression which could be negated using CD47/SIRPA blockade. We found that this combination dramatically enhanced T-Dxd resulted therapeutic efficacy, thus supporting clinical exploration of these combinations. Citation Format: Li-Chung Tsao, Xingru Ma, John S. Wang, Timothy N. Trotter, Tao Wang, Jun-Ping Wei, Gang-jun Lei, Jason McBane, Ping Fan, Ivan Spasojevic, Zachary C. Hartman. HER2-ADC efficacy is rooted in its induction of immunogenic cell death with FcGR activation and a systemic bystander effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2377.