Immunogenic Cell Death Markers Research Articles

In situ endoscopic photodynamic therapy combined with immature DC vaccination induces a robust T cell response against peritoneal carcinomatosis.

Immunogenic cell death (ICD) and ferroptosis have recently emerged as key factors in the anticancer immune response. Among the treatments able to induce ICD and the associated release of danger signals is photodynamic therapy (PDT). Ferroptosis for its part results from lipid peroxidation and is induced by CD8+ T cells to kill nearby cancer cells on IFN-γ production. We aimed to combine the two concepts, that is, to evaluate whether the strong pro-oxidant effects of PDT may promote ferroptosis and antigen release and to develop a procedure for in situ PDT to prepare the soil for highly endocytotic immature dendritic cell (iDC) adoptive transfer. This approach was implemented for managing peritoneal carcinomatosis, a lesion often associated with poor outcomes. We used three-dimensional (3D) heterotypic spheroids made of cancer cells, exposed them to a white light-activated OR141 photosensitizer (PS), and subsequently complexified them by adding iDC and naive lymphocytes. We next used a model of mouse peritoneal carcinomatosis and administered PDT using laparoscopy to locally induce photoactivation using the endoscope light. The immune response following adoptive transfer of iDC was tracked both in vivo and ex vivo using isolated immune cells from in situ vaccinated mice. Cancer cells undergoing PDT-induced cell death significantly increased ICD markers and the infiltration of iDCs in spheroids, relying on ferroptosis. These actions induced the sequential activation of CD8+ and CD4+ T cells as revealed by a significant spheroid 3D structure deterioration and, remarkably, were not recapitulated by conventional ferroptosis inducer RSL3. Using LED light from an endoscope for in situ photoactivation of PS enabled us to apply the vaccination modality in mice with peritoneal tumors. Consecutive intraperitoneal injection of iDCs resulted in delayed tumor growth, increased survival rates, and prevented tumor relapse on rechallenge. CD8+ T cell response was supported by depletion experiments, nodal detection of early activated T cells, and ex vivo T cell-induced cytotoxicity toward spheroids. The combination of in situ PDT locally delivered by an endoscope light and iDC administration induces a durable memory immune response against peritoneal carcinomatosis thereby opening new perspectives for the treatment of a life-threatening condition.

Novel bispecific antibody-drug conjugate targeting PD-L1 and B7-H3 enhances antitumor efficacy and promotes immune-mediated antitumor responses

BackgroundAntibody-drug conjugates (ADCs) offer a promising approach, combining monoclonal antibodies with chemotherapeutic drugs to target cancer cells effectively while minimizing toxicity.MethodsThis study examined the therapeutic efficacy and potential mechanisms of...

Rationally designed catalytic nanoplatform for enhanced chemoimmunotherapy via deploying endogenous plus exogenous copper and remodeling tumor microenvironment

Chemodynamic therapy represents a novel tumor therapeutic modality via triggering catalytic reactions in tumors to yield highly toxic reactive oxygen species (ROS). Nevertheless, low efficiency catalytic ability, potential systemic toxicity and inefficient tumor targeting, have hindered the efficacy of chemodynamic therapy. Herein, a rationally designed catalytic nanoplatform, composed of folate acid conjugated liposomes loaded with copper peroxide (CP) and chloroquine (CQ; a clinical drug) (denoted as CC@LPF), could power maximal tumor cytotoxicity, mechanistically via maneuvering endogenous and exogenous copper for a highly efficient catalytic reaction. Despite a massive autophagosome accumulation elicited by CP-powered autophagic initiation and CQ-induced autolysosomal blockage, the robust ROS, but not aberrant autophagy, underlies the synergistic tumor inhibition. Otherwise, this combined mode also elicits an early onset, above all, long-term high-level existence of immunogenic cell death markers, associated with ROS and aberrant autophagy -triggered endoplasmic reticulum stress. Besides, CC@LPF, with tumor targeting capability and selective tumor cytotoxicity, could elicit intratumor dendritic cells (mainly attributed to CQ) and tumor infiltrating CD8+ T cells, upon combining with PD-L1 therapeutic antibody, further induce significant anti-tumor effect. Collectively, the rationally designed nanoplatform, CC@LPF, could enhance tumor chemoimmunotherapy via deploying endogenous plus exogenous copper and remodeling tumor microenvironment.Graphical abstract

The role of apoptosis, immunogenic cell death, and macrophage polarization in carbon ion radiotherapy for keloids: Targeting the TGF-β1/SMADs signaling pathway

Keloids, characterized by excessive extracellular matrix (ECM) deposition and aberrant fibrous tissue proliferation, present significant therapeutic challenges due to their recalcitrant and recurrent nature. This study explores the efficacy of Carbon Ion Radiotherapy (CIRT) as a novel therapeutic approach for keloids, focusing on its impact on fibroblast proliferation, apoptosis induction, immunogenic cell death (ICD), macrophage polarization, and the TGF-β/SMAD signaling pathway. Utilizing a murine model of keloid formed by subcutaneous injection of zeocin in C57BL/6 mice, we demonstrated that CIRT effectively reduces collagenous fiber synthesis and collagen production in keloid tissues. Further, CIRT was shown to inhibit keloid fibroblast proliferation and to induce apoptosis, as evidenced by increased expression of apoptosis-related proteins and confirmed through flow cytometry and TUNEL assay. Notably, CIRT induced mitochondrial stress, leading to enhanced immunogenicity of cell death, characterized by increased expression of ICD markers and secretion of interferon-γ. Additionally, CIRT promoted a shift from M2 to M1 macrophage polarization, potentially reducing TGF-β release and mitigating ECM deposition. Our findings suggest that CIRT mediates its therapeutic effects through the inhibition of the TGF-β/SMAD signaling pathway, thereby attenuating ECM formation and offering a promising avenue for keloid treatment. This study underscores the potential of CIRT as an innovative strategy for managing keloids, highlighting its multifaceted impact on key cellular processes involved in keloid pathogenesis.

Silymarin suppresses proliferation and PD-L1 expression in colorectal cancer cells and increases inflammatory CD8+ cells in tumor-bearing mice

IntroductionSilymarin as an herbal medicine has shown anticancer effects on tumor cells, while having low toxicity in normal cells. In this study, the effects of Silymarin on proliferation and apoptosis of colorectal cancer cells and its impact on immune response against cancer cells were evaluated in vitro and in vivo. Methods and materialsThe effect of Silymarin on CT-26 and Caco-2 cells proliferation and apoptosis were demonstrated by MTT assay and PI staining. A subcutaneous tumor of colorectal cancer was developed. Silymarin and Doxorubicin were administrated by intravenous injection. qRT-PCR analyses was performed on blood samples and tumor tissues. Spleen tissue was used to evaluate CD8+ T cell immune responses. Histological study was carried out on tumor tissues. ResultsSilymarin showed anti-proliferative effects on CT-26 and Caco-2 cells. The markers of immunogenic cell death (Calreticulin exposure, ATP secretion, and HMGB1 secretion) significantly increased in both cell lines in the presence of silymarin. The expression of genes related to cell proliferation particularly β-Catenin and Cycline D1, and also anti-apoptotic ones such as Bcl-2 significantly reduced in mice treated with Silymarin while the expression of pro-apoptotic Bax increased. The RNA level of PD-L1 decreased in tumor tissues exposed by Silymarin. Moreover, the number of CTLs increased in the spleen of mice treated with Silymarin in comparison with untreated mice. Decreased tumor size and also survival of colorectal cancer cells in Silymarin-treated mice were observed in histological analysis. ConclusionSilymarin treatment showed a suppressive role on colorectal cancer cells almost as much as Doxorubicin. Our study indicated that having a low toxicity profile, cost-effectiveness, and availability of raw materials, plant-derived Silymarin can be a good candidate for further investigation to treat CRC.

The Ability of Clinically Relevant Chemotherapeutics to Induce Immunogenic Cell Death in Squamous Cell Carcinoma.

Immunogenic cell death (ICD) is a crucial mechanism for triggering the adaptive immune response in cancer patients. Damage-associated molecular patterns (DAMPs) are critical factors in the detection of ICD. Chemotherapeutic drugs can cause ICD and the release of DAMPs. The aim of this study was to assess the potential for paclitaxel and platinum-based chemotherapy regimens to induce ICD in squamous cell carcinoma (SCC) cell lines. In addition, we examined the immunostimulatory effects of clinically relevant chemotherapeutic regimens utilized in the treatment of SCC. We screened for differentially expressed ICD markers in the supernatants of three SCC cell lines following treatment with various chemotherapeutic agents. The ICD markers included Adenosine Triphosphate (ATP), Calreticulin (CRT), Annexin A1 (ANXA 1), High Mobility Group Protein B1 (HMGB1), and Heat Shock Protein 70 (HSP70). A vaccination assay was also employed in C57BL/6J mice to validate our in vitro findings. Lastly, the levels of CRT and HMGB1 were evaluated in Serum samples from SCC patients. Addition of the chemotherapy drugs cisplatin (DDP), carboplatin (CBP), nedaplatin (NDP), oxaliplatin (OXA) and docetaxel (DOC) increased the release of ICD markers in two of the SCC cell lines. Furthermore, mice that received vaccinations with cervical cancer cells treated with DDP, CBP, NDP, OXA, or DOC remained tumor-free. Although CBP induced the release of ICD-associated molecules in vitro, it did not prevent tumor growth at the vaccination site in 40% of mice. In addition, both in vitro and in vivo results showed that paclitaxel (TAX) and LBP did not induce ICD in SCC cells. The present findings suggest that chemotherapeutic agents can induce an adjuvant effect leading to the extracellular release of DAMPs. Of the agents tested here, DDP, CBP, NDP, OXA and DOC had the ability to act as inducers of ICD.

The copper (II) complex of salicylate phenanthroline induces immunogenic cell death of colorectal cancer cells through inducing endoplasmic reticulum stress

BackgroundIn our previous study, Cu(sal)phen was found to have anti-tumor effects, yet its precise mechanism remains unknown. Research has shown that dying tumor cells release damage-associated molecular patterns (DAMPs) to promote anti-tumor immune response. Therefore, we have further explored the effects and potential molecular mechanisms of Cu(sal)phen-induced immunogenic cell death (ICD) in colorectal cancer (CRC). MethodsELISA and flow cytometry were used to detect the effects of Cu(sal)phen treatment on ICD markers. The molecular mechanisms of Cu(sal)phen-induced ICD were investigated through the detection of endoplasmic reticulum stress (ERS) and reactive oxygen species (ROS) in vitro using Western blot and flow cytometry. Additionally, a mouse model was constructed to study the effects of Cu(sal)phen on immune cells and anti-tumor-related cytokines in vivo. ResultsCu(sal)phen induced the release of calreticulin (CRT), adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1), the main molecular markers of ICD, by promoting the accumulation of ROS and inducing ERS. Furthermore, Cu(sal)phen promoted the maturation of dendritic cells (DCs) and activation of CD8+T cells, as well as the secretion of interleukin-12 (IL-12) and interferon-γ (IFN-γ), while downregulating transforming growth factor-β (TGF-β) levels, thereby activating the anti-tumor immune response. ConclusionCu(sal)phen has the potential to induce ICD in tumors and activate the adaptive immune response to achieve anti-tumor effects. This makes Cu(sal)phen a promising candidate for the treatment of CRC.

Abstract 6742: PV 10 induces endoplasmic reticulum stress and autophagy, triggering immunogenic cell death and anti-tumor immunity in head and neck squamous cell carcinoma

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) are complex, diverse cancers affecting mucosal linings of the upper aerodigestive tract. Globally, HNSCC is the sixth most common cancer, leading to over 890,000 new cases and more than 450,000 deaths annually. Primary risk factors include human papillomavirus (HPV) infection (HPV-positive HNSCC) and exposure to tobacco and excessive alcohol (HPV-negative HNSCC). Despite multimodal treatments, survival rates remain low due to recurrence and metastasis (R/M HNSCC), creating a need for safe, innovative therapies to reduce tumor burden with minimal adverse events. PV-10 (10% rose bengal sodium) is a novel intratumoral immunotherapeutic agent that has demonstrated promising anti-tumor activity in multiple solid tumor cancer types with few adverse effects in pre-clinical research and clinical trials. However, the role of PV-10 in HNSCCs and its role in enhancing immune response warrants further investigation. Methods: Cell viability was assessed using Alamar blue assay after PV-10 treatment of murine mEER and MTE-RAS HNSCC tumor cell lines. To evaluate the immunomodulatory properties of PV-10, we employed flow cytometry, confocal analysis, and luminescence-based assays. Additionally, we performed a DCFDA assay to measure the reactive oxygen species (ROS) and an Alexa Fluor 488 Annexin V/dead cell apoptosis assay to evaluate apoptosis. To gain insights into the mechanisms underlying PV-10's immunomodulatory effects in cancer cells and tumors from mice, western blotting and multiplex immunohistochemistry (mIHC) were used. Results: Our in vitro findings reveal that PV-10 induces cytotoxicity in both mEER and MTE-RAS cells. Notably, PV-10 promotes a significant increase in ROS, leading to an elevation in late apoptotic cells. Markers of immunogenic cell death (ICD), including a statistically significant increase in the release of damage-associated molecular pattern molecules HMGB1 and ATP, as well as enhanced surface expression of calreticulin, HSP-70, and HSP-90, were observed. At the molecular level, a remarkable activation of endoplasmic reticulum (ER) stress, pro-apoptotic protein, and autophagy markers were observed. Intratumoral PV-10 injection in vivo has shown significant tumor regression in both mEER and MTE-RAS tumors, and a complete response was noticed in some mice, indicating that PV-10 induces potent ICD in both mEER and MTE-RAS tumors. Conclusion: Our collated evidence strongly suggests that PV-10 induces ICD-associated immunogenicity in both mEER and MTE-RAS in vitro and in vivo, fostered by ROS-based ER stress and apoptosis. These findings hold promise for potential therapeutic avenues to manage HNSCC. Citation Format: Sowjanya Thatikonda, Ritu Chaudhary, Yeva Meshkovska, Maria I. Biernacki, Robbert Slebos, Shari P. Thomas, Amelio L. Antonio, Eric A. Wachter, Christine H. Chung. PV 10 induces endoplasmic reticulum stress and autophagy, triggering immunogenic cell death and anti-tumor immunity in head and neck squamous cell carcinoma [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 6742.

Abstract 2622: TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans

Abstract TUB-040 is a novel Antibody-Drug Conjugate (ADC) targeting Napi2b, a highly overexpressed target in ovarian cancer and lung adenocarcinoma. Napi2b is a member of the solute-carrier transporter family with eight transmembrane domains and an antibody-accessible extracellular loop, that regulates sodium-dependent phosphate homeostasis. Ethynylphosphonamidate conjugation chemistry was used to build TUB-040 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting Napi2b using a cleavable linker system. In vitro, TUB-040 is characterized by sub-nanomolar affinity and specific binding to Napi2b, efficient target-mediated internalization and high cytotoxicity towards Napi2b-expressing ovarian and lung cancer cell lines as well as bystander activity against co-cultured target-negative cells. TUB-040 induces DNA damage and apoptosis in a dose-dependent fashion as well as markers of immunogenic cell death. In addition, no unspecific uptake and cytotoxicity in healthy, target-negative human cells could be detected, reducing the risk of target-independent toxicities. Pharmacokinetic analysis showed that - in contrast to maleimide-conjugated ADCs - TUB-040 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation, thus enabling most efficient and continued delivery of Exatecan to the tumor. Across a variety of cell line derived xenograft (CDX) and patient derived xenograft (PDX) models of ovarian and non-small cell lung cancer, including models with low target expression, single dose administration of TUB-040 results in long-lasting tumor growth inhibition with high complete remission rates in vivo. The minimally effective dose (MED) leading to complete remission is 1 mg/kg. Preliminary repeat-dose toxicological assessment of TUB-040 in the pharmacologically relevant species cynomolgus monkey demonstrated that TUB-040 is well tolerated with no signs of lung toxicity and thrombocytopenia. Allometric scaling and PK/PD modelling predicts a human serum half-life greater 10 days and a therapeutic index in humans up to 55. Based on these results, TUB-040, designed with differentiating technology for optimal efficacy and tolerability, is ready for testing in clinical trials. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans [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 2622.

Abstract 5738: Repurposing disulfiram for cancer: A drug delivery and population-based approach

Abstract Purpose: Disulfiram was approved by the FDA for treatment of alcohol abuse in 1951, although for over 40 years there has been evidence supporting disulfiram’s use as an anti-cancer drug. Upon administration, disulfiram is hydrolyzed to form two molecules of DDC, which upon binding Cu forms Cu(DDC)2, the proposed active form against cancer. It was our goal to formulate the water-insoluble Cu(DDC)2 in liposomes to facilitate its repurposing for breast and colon cancers. In addition, we also initiated an ongoing population-based study assessing the protective and treatment effects of disulfiram with cancer. Methods: Cu(DDC)2 liposomes were generated by the thin-film hydration method followed by extrusion, using DSPC and cholesterol (55:45 molar ratio). DDC and CuSO4 were added to the liposomes forming Cu(DDC)2, which was transported inside the liposomes. Anti-tumor activity was evaluated in tumor-bearing mice with or without competent immune systems, and immune system activation was assessed via a cancer vaccination model and the expression of markers of immunogenic cell death (ICD). A study was also initiated for cancer cases and controls in British Columbia (BC Cancer) with at least one prescription of disulfiram from 1996-2022 (PharmaNET), patients treated with immune-modulating therapies (BC Cancer), and outcome of death (Vital Statistics). Results: Liposomes encapsulating Cu(DDC)2 had a size of 100 nm and polydispersity of 0.1, and caused a 40% reduction in tumor burden in a MDA-MB-231 xenograft model. In a syngeneic tumor model of breast cancer the response with immune-competent mice was greater than mice lacking immune function, and a cancer vaccination model and the presence of ATP, HMBG1 and surface-exposed calreticulin indicated the presence of ICD. Conclusions: Cu(DDC)2 was formulated in liposomes facilitating its parenteral administration, and its anti-cancer activity was found to be due in part to activation of the adaptive immune system through ICD. A case-control study is currently being employed to examine the clinical potential of disulfiram, as a protectant and as an adjuvant with immune-activating therapies such as checkpoint inhibitors and anthracyclines to potentially improve efficacy through induction of ICD. Citation Format: Devon Heroux, Ada W. Leung, Roger Gilabert-Oriol, Marcel B. Bally. Repurposing disulfiram for cancer: A drug delivery and population-based approach [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 5738.

Abstract 6578: N17465, a systemically deliverable elastase, attenuates tumorigenesis and stimulates anti-tumor immunity

Abstract Background. Neutrophil elastase (ELANE) - a neutrophil-derived serine protease - kills a wide range of cancer cells without harming non-cancer cells through a unique killing program involving histone H1 and the death domain of CD95. Although ELANE attenuates tumor growth and induces anti-tumor immunity following intratumoral delivery (Cui et al., Cell, 2021), systemic delivery is stymied by the presence of serine protease inhibitors in blood (eg. alpha-1-antitrypsin (A1AT) which rapidly inhibit its enzymatic activity essential for its anti-cancer function. To overcome this challenge, we developed N17465, an enzyme that resists serine protease inhibitors in blood, and tested its effects on tumor growth, both as a monotherapy and in combination with checkpoint inhibitors. Methods. Enzymatic properties: N17465 enzymatic activity was quantified in the +/- of A1AT or plasma in vitro, and in blood following intravenous (I.V) injection into tumor-bearing mice. Enzyme activity assays included a fluorogenic substrate AAPV-AMC and the C-terminal domain of CD95 (amino acids212-335). Anti-cancer properties: For in vitro studies, cells were treated with N17465 and viability was quantified by calcein-AM and immunogenic cell death (ICD) markers. Cancer/non-cancer cells included human/murine cell lines and primary cells from ovarian cancer (OvCa) patients. For in vivo studies, N17465 was injected I.V and effects on tumor growth, immunology and survival were assessed as monotherapy or in combination with a checkpoint inhibitor (anti-CTLA4) in pre-clinical models. Results. N17465 maintained enzymatic activity following incubation with A1AT or mouse/human plasma in vitro, and following I.V injection into CT26 tumor-bearing mice in vivo. Importantly, protecting N17465 from serine protease inhibitors did not interfere with its ability to cleave CD95 (therapeutic target) or selectively kill cancer cells in vitro. Indeed, N17465 induced ICD markers, killed a wide range of murine/human cancer cells and also well tolerated by non-cancer cells. Systemically delivered N17465 produced durable tumor regression in a murine CT26 colon cancer model resulting in ~50% tumor-free mice, induced a favorable immune profile, and synergized with anti-CTLA4. N17465 also exhibited efficacy across a range of xenograft models spanning human colon, prostate, and lung cancer, as well as OvCa patient-derived CDX models. Conclusions. Altogether, our data demonstrate that N17465 selectively kills cancer cells, produces durable responses in mice, induces favorable immunology and synergies with checkpoint inhibitors in pre-clinical models. Its ability to maintain enzymatic activity in blood following systemic delivery, selectively kill a wide range of cancer cells, and stimulate anti-tumor immunity warrants further investigation of this unique therapeutic modality across a wide range of solid tumors. Citation Format: Ravindra Gujar, Chang Cui, Christine Lee, Maria Fumagalli, Nicole Martinez, Hannah Liu, Nicole Grigaitis, Sonia Feau, Lev Becker. N17465, a systemically deliverable elastase, attenuates tumorigenesis and stimulates anti-tumor immunity [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 6578.

Abstract 1366: Dual immune checkpoint inhibition enhances the anti-tumor activity of trastuzumab deruxtecan in preclinical models

Abstract Background Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) composed of an anti-human epidermal growth factor receptor 2 (HER2) antibody, a cleavable tetrapeptide-based linker, and a topoisomerase I inhibitor payload. Recent clinical trials such as BEGONIA have highlighted the potential therapeutic benefit in combining T-DXd with immuno-oncology (IO) agents such as the PD-L1 inhibitor durvalumab. Here, we report preclinical findings demonstrating enhanced anti-tumor activity when T-DXd is combined with dual immune checkpoint inhibition. Methods HER2-expressing human cancer cell lines were treated in vitro with T-DXd and assessed for induction of immunogenic cell death (ICD) markers and expression of NK cell-activating ligands. Human macrophages and T cells were incubated with supernatants from treated cancer cells, and their activation status evaluated via flow cytometry. In vivo, the effects of T-DXd in combination with inhibitors of PD-L1, CTLA-4, and a monovalent bispecific anti-PD-1/TIGIT antibody - a murine surrogate of rilvegostomig - on tumor growth were evaluated in BALB/c mice bearing human HER2-low EMT6 murine mammary tumors. The anti-tumor activity of T-DXd in combination with volrustomig, a monovalent bispecific antibody targeting PD-1 and CTLA-4, was also assessed in hu-CD34+ NSG mice bearing HER2-low Caki-1 renal cell carcinoma tumors. Results In vitro, T-DXd induced ICD via DXd-mediated extracellular release of inflammatory mediators ATP and HMGB1, and cell surface exposure of calreticulin. This was associated with a greater than 2-fold increase in expression of NK cell-activating ligands, and activation of both macrophages and T cells. In immunocompetent mice bearing human HER2 expressing EMT6 tumors, T-DXd treatment drove tumor growth inhibition (53%, P <0.001) that was associated with an increase in tumoral T cells (2.6-fold, P <0.05) and increased CD8+ T cell expression of the immune checkpoints PD-1, TIGIT, and TIM-3. Tumor growth inhibition relative to vehicle-treated mice was enhanced when T-DXd was combined with inhibitors of PD-L1 (108%, P <0.001), PD-L1 plus CTLA-4 (172%, P <0.001), or a bispecific PD-1/TIGIT inhibitor (181%, P <0.001). Pharmacodynamic analysis revealed how the combination of T-DXd and PD-1/TIGIT inhibition increased tumoral NK cells (3.2-fold, P <0.001) and CD8+ T cells (3.8-fold, P <0.001) relative to vehicle. Consistent with findings in syngeneic models, the combination of T-DXd with a bispecific PD-1/CTLA-4 inhibitor also enhanced tumor growth inhibition in a humanised Caki-1 model (121%) compared to T-DXd alone (72%) (P <0.001). Conclusions These data provide insight into the clinical activity observed with T-DXd and provide scientific rationale for combination strategies with novel, bispecific IO agents targeting CTLA-4 or TIGIT in addition to the PD-1/PD-L1 axis. Citation Format: Liam Jenkins, Laura Kazlauskas, Matt Wilson, Scott A. Hammond, Theresa Proia, Jerome Mettetal. Dual immune checkpoint inhibition enhances the anti-tumor activity of trastuzumab deruxtecan in preclinical models [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 1366.

Abstract 5262: MBO enhances anti-tumor immunity in pancreatic ductal adenocarcinoma by inducing immunogenic apoptosis

Abstract Pancreatic ductal adenocarcinoma (PDAC) poses significant therapeutic challenges due to its aggressive nature and limited treatment options. In this study, we explored the anti-cancer potential of MBO, a compound selected through drug repurposing strategy based on its cytotoxic ability and modulation of key survival pathways in PDAC. Our preliminary in vitro experiments demonstrated MBO's potent cytotoxicity against various PDAC cell lines, inhibiting cellular growth and clonogenicity. MBO induced G1 phase cell cycle arrest in a concentration-dependent manner. In the next step, in vivo, studies using xenograft and orthotopic mouse models confirmed MBO's ability to suppress tumor growth and increase CD8+ T cell infiltration. MBO demonstrated a favorable tumor suppressive activity wherein it suppressed tumor growth by approximately 49% and 56% in xenograft and orthotopic models, respectively. A CD8+ T cell depletion experiment revealed partial dependence on CD8+ T cells for MBO's overall anti-tumor efficacy. Further, combining MBO with anti-PD1 therapy exhibited a synergistic effect, enhancing overall T cell infiltration, particularly CD8+ T cells. Our mechanistic studies revealed that MBO induced immunogenic apoptosis, as evidenced by increased expression of immunogenic cell death markers (HSP90 and calreticulin) and apoptotic markers. To further delineate the mechanism, we performed RPPA profiling. RPPA analysis indicated MBO's impact on mitochondrial gene expression and stress response pathways, suggesting its involvement in disrupting cellular survival mechanisms along with stress-induced immunogenic marker expression. Overall, this study demonstrates MBO as a promising anti-cancer agent with the potential to serve as an immune adjuvant. Particularly, in combination with immunotherapies, MBO can serve as a potential option to improve immunotherapeutic response in PDAC tumors providing valuable insights for developing novel therapeutic strategies in PDAC. Citation Format: Shreyas Ramchandra Gaikwad, Sanjay K. Srivastava. MBO enhances anti-tumor immunity in pancreatic ductal adenocarcinoma by inducing immunogenic apoptosis [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 5262.

Comparison of the oncolytic activity of a replication-competent and a replication-deficient herpes simplex virus 1.

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-β mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.

Sterile Water Versus Glycine in Transurethral Resection of Bladder Tumors—Immunogenic and Clinical Implications

Background and objectiveWe compared the oncologic outcomes of patients with non-muscle invasive bladder cancer (NMIBC) who underwent transurethral resection of bladder tumor (TUBRT) using sterile water vs glycine irrigation. The tumoricidal and immunogenic effects of these solutions on urothelial cancer cell lines were investigated. MethodsThe medical records of 530 consecutive patients who underwent TURBT using sterile water or glycine irrigation for NMIBC were reviewed. Recurrence and progression rates were evaluated using time dependent analyses.Bladder cancer cell lines (RT4, T24 and 5637) were treated with glycine and sterile water. Cell viability was evaluated with the XTT assay. Cell membrane calreticulin levels were evaluated with flow cytometry. Extracellular high mobility group box 1 (HMGB1) and heat shock 70 (HSP70) protein levels were evaluated using western blots. Key findings and limitationsAfter propensity score matching each study arm comprised 161 patients. Median follow-up was 13.6 months (IQR 6.2, 24.5). The 2-year recurrence free survival was significantly lower in the sterile water vs glycine group (43% vs 71%, respectively, p<0.0001). Similarly, the 2-years progression free survival was significantly lower in the sterile water vs glycine group (85% vs 94%, respectively, p<0.014). Sterile water treatment resulted in the lowest number of viable cells. Early and late immunogenic cell death markers were markedly elevated in cells treated with glycine. Conclusions and clinical implicationsSterile water compared to glycine irrigation during TURBT for NMIBC was associated with higher recurrence and progression rates. Possible explanation for these findings is the diminished immune response associated with sterile water reflected in a comparatively lesser expression of immune response inducers. Patient summaryWe compared two irrigation fluids in non–muscle-invasive bladder cancer surgery: glycine and sterile water. Glycine outperformed sterile water in cancer recurrence, possibly boosting immunogenicity over sterile water.

Abstract B031: A tale of cancer cell death and immunity

Abstract Patients with tumors that have substantial T cell infiltration benefit from immunotherapy to survive longer. However, several tumor types lack T cells within the tumor and are hence refractory to immunotherapy. Therefore, therapies that render immune-evading cancers susceptible to cytotoxic T cells are highly desirable. We and others have shown that certain types of cancer cell death can kickstart multiple aspects of the anticancer immunity cycle to inflame “cold” tumors. These immunological outcomes were driven by the secretome emitted by a variety of stress and programmed cell death. Many cytotoxic chemoradiotherapies simultaneously activate multiple types of stress and cell death. Hence, it has been a major clinical challenge to identify what specific types of cancer cell death are efficient at eliciting durable anticancer immunity. The overall goal of my lab’s research program is to identify what types of cancer cell death initiate effective anticancer immunity and expand therapeutic approaches to deadly cancers such as pancreatic and brain cancer. Since the start of my lab in September 2020, we have created synthetic models of cell death where inducible expression of cell death executioner proteins activates distinct types of cancer cell death such as apoptosis, necroptosis, and pyroptosis. Using these models, we identified that the secretomes of apoptotic, necroptotic, and pyroptotic tumors display remarkable differences in previously established markers of immunogenic cell death and novel secretomes as identified by mass spectrometry. In follow-up, in vivo studies, we identified differences in the activation and maturation patterns of antigen-presenting cells, such as type I conventional dendritic cells (cDC1). Consistent with the differences in cDC1 functionality we identified differences in the magnitude, diversity, and functionality of effector CD8+ T cells. All these immunological differences contributed to significant differences in the anticancer protective activity of distinct cancer cell death types. During my talk, I will be presenting these findings and discussing how these basic findings can be integrated into translational platforms for the management of non-T cell inflamed pancreatic and brain tumors. Citation Format: Samuel T Workenhe. A tale of cancer cell death and immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B031.

IMMU-42. PP2A-INHIBITION INDUCES STING-TYPE I-MEDIATED IMMUNOLOGIC CELL DEATH IN GBM

Abstract Glioblastoma (GBM) is one of the most lethal forms of brain cancer, and is known to be an aggressive, immunologically cold tumor, resulting in a lack of T-cell infiltration and killing. Thus increasing immune-mediated killing of GBM is a promising therapeutic method. Our group has found that Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase that has been shown to enhance tumor immunogenicity in glioblastoma, does so by inducing immunogenic cell death. We used LB-100, a small molecule inhibitor of the catalytic subunit of PP2A, as a promising drug-therapy. CRISPR-KO of PP2A was generated in murine glioma lines GL261 and SB28. Markers of immunologic cell death, such as calreticulin, gamma-H2AX, and HMGB1 were assessed. In vitro, western blotting of SB28 and GL261 showed significantly increased expression of gamma-H2AX in PP2A-KO compared to WT. Flow-cytometry staining of SB28 and GL261 revealed a dramatic increase in calreticulin expression in PP2A-KO compared to WT. An antibody-based fluorescence assay for HMGB1 also showed dramatic increase in PP2A-KO compared to WT. All experiments were repeated by treating LB-100 for 24 hour to SB28 and GL261 WT and PP2A-KO cells, and revealed a dose-dependent increase in expression of immunogenic cell death markers. In vivo, B6 mice were injected intracranially with SB28 WT or PP2A-deficient tumor and monitored till endpoint. At the endpoint, tumors were harvested, cryosectioned, and stained for calreticulin and gamma-H2AX. Mice injected with PP2A-KO tumor survived longer than WT, and had the highest expression of both calreticulin gamma-H2AX when compared to WT tissue. In conclusion, PP2A-deficiency and inhibition via LB-100 is demonstrated to induce immunogenic cell death in tumors, thus being a promising target for immunotherapy.

Repurposing thioridazine for inducing immunogenic cell death in colorectal cancer via eIF2α/ATF4/CHOP and secretory autophagy pathways

BackgroundColorectal cancer (CRC) is a highly prevalent cancer type with limited targeted therapies available and 5-year survival rate, particularly for late-stage patients. There have been numerous attempts to repurpose drugs to tackle this problem. It has been reported that autophagy inducers could augment the effect of certain chemotherapeutic agents by enhancing immunogenic cell death (ICD).MethodsIn this study, we employed bioinformatics tools to identify thioridazine (THD), an antipsychotic drug, and found that it could induce autophagy and ICD in CRC. Then in vitro and in vivo experiments were performed to further elucidate the molecular mechanism of THD in CRC.ResultsTHD was found to induce endoplasmic reticulum (ER) stress in CRC cells by activating the eIF2α/ATF4/CHOP axis and facilitating the accumulation of secretory autophagosomes, leading to ICD. In addition, THD showed a remarkable ICD-activating effect when combined with oxaliplatin (OXA) to prevent tumor progression in the mouse model.ConclusionsTogether, our findings suggest that the repurposed function of THD in inhibiting CRC involves the upregulation of autophagosomes and ER stress signals, promoting the release of ICD markers, and providing a potential candidate to enhance the clinical outcome for CRC treatment.6ZzBeVddzkUiW38NXX-mrKVideo

Expanding Opportunities in Viral Oncolysis.

Expanding Opportunities in Viral Oncolysis.

Abstract 5038: Evaluation of anti-tumor immunity in response to [177Lu]Lu-PSMA in a mouse model of prostate cancer

Abstract [177Lu]Lu-PSMA improves progression-free and overall survival in metastatic castration resistant prostate cancer. However, the role of immune stimulation by molecularly targeted radionuclide therapy (MRT) is poorly understood. The aim of this study was to evaluate the anti-tumor immune response induced by [177Lu]Lu-PSMA. The markers of immunogenic cell death (ICD), translocation of calreticulin (CRT) to the cell membrane, HMGB1 release and ATP release, were evaluated in the RM1 cell line (PSMA-negative murine prostate carcinoma) and its derivative, RM1-PGLS (stably transduced with human PSMA and SFG-EGFp/Luc), using flow cytometry, ELISA and a luminescence detection assay respectively. The in vivo biodistribution of [177Lu]Lu-PSMA in RM1-PGLS tumor-bearing C57BL/6 mice was investigated using microSPECT-CT imaging, and the in vivo efficacy of [177Lu]Lu-PSMA was tested in RM1-PGLS tumor growth inhibition studies. Vaccination/rechallenge experiments were performed to elucidate whether [177Lu]Lu-PSMA elicits ICD in vivo. The Nanostring Pan Cancer Immune Profiling Platform was used to assess changes in the tumor immune microenvironment (TIME) in response to [177Lu]Lu-PSMA. External radiation (EBRT; 137Cs γ-radiation; 6-10 Gy) was used as a comparator treatment in most experiments. We observed a dose-dependent increase in cell surface CRT following [177Lu]Lu-PSMA and EBRT at 24 and 72 h and of HMGB1 release in response to [177Lu]Lu-PSMA and EBRT at 96 and 144 h. At 24 h following [177Lu]Lu-PSMA (60 MBq) there was a significant increase in ATP release compared to untreated samples. Collectively these data indicate [177Lu]Lu-PSMA causes ICD. A high RM1-PGLS tumor:muscle uptake ratio (210 ± 59) was noted in biodistribution experiments confirming highly specific tumor accumulation of [177Lu]Lu-PSMA. In tumor growth inhibition studies the time for tumors to reach a volume of 400 mm3 was delayed in mice treated with [177Lu]Lu-PSMA (60 MBq) compared to no treatment (30 versus 21 days; P&amp;lt;0.01). We showed that a subcutaneous inoculum of [177Lu]Lu-PSMA (60 MBq)-treated RM1-PGLS cells prevents tumor growth in approximately 50% of cases on rechallenge with viable RM1-PGLS cells one week later. This indicates that [177Lu]Lu-PSMA induces ICD in vivo. We observed a significant reduction in most tumor infiltrating lymphocyte (TIL) types at 2 days after in vivo exposure to [177Lu]Lu-PSMA (80 MBq), followed by an increase in TILs by Day 7. The observed pattern is similar to that after EBRT (6 Gy). There was overlap in the top 10 differentially expressed genes (treatment versus no treatment) for the [177Lu]Lu-PSMA and EBRT groups. The gene showing the greatest level of upregulation at day 7 in [177Lu]Lu-PSMA-treated tumors, IDO1, is known to be involved in immune tolerance and radioresistance. In conclusion, [177Lu]Lu-PSMA induces ICD in vitro and in vivo and modulates the TIME. Citation Format: Gemma Dias, Sarah Able, Irini Skaripa-Koukelli, Rachel Anderson, Gracie Wilson, Katherine A. Vallis. Evaluation of anti-tumor immunity in response to [177Lu]Lu-PSMA in a mouse model of prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5038.