Immunogenic Cell Death Markers Research Articles

Cisplatin and oxaliplatin induce similar immunogenic changes in preclinical models of head and neck cancer

ObjectivesPrior studies suggest that oxaliplatin is unique among platinum chemotherapy drugs in its ability to enhance anti-tumor immunity, but the immune mechanisms of different platinum chemotherapy drugs have not been previously compared in preclinical models of head and neck squamous cell carcinoma (HNSCC). Materials and methodsHuman HNSCC cell lines were treated with cisplatin or oxaliplatin, then assessed for markers associated with immunogenic cell death (ICD) and antigen processing. A syngeneic mouse model of oral cancer was then used to compare the effects of cisplatin vs. oxaliplatin, alone or in combination with anti-PD-1 immunotherapy, on tumor growth and survival. A subset of spleens and tumors were analyzed for ICD markers and immune cell infiltrates by flow cytometry. ResultsCisplatin and oxaliplatin both increased cell surface levels of calreticulin, HSP70, MHC class I and PD-L1 in multiple cell lines. Inoculation of immunocompetent mice with cells killed in vitro by either drug resulted in failure of subsequently-injected live tumor cells to establish and grow in a small proportion of animals. Systemic cisplatin and oxaliplatin induced similar tumor growth delay when combined with anti-PD-1 therapy. ConclusionsTreatment of HNSCC cells with platinum chemotherapy appears to induce some features of anti-tumor immunity, which may be enhanced by anti-PD-1 therapy. Cisplatin, the standard drug for HNSCC, appears to affect anti-tumor immunity in a similar fashion to oxaliplatin in these preclinical models.

PD-1 Blockade Following Isolated Limb Perfusion with Vaccinia Virus Prevents Local and Distant Relapse of Soft-tissue Sarcoma.

The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma.Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines. PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines. Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation.

STAT3 Contributes To Oncolytic Newcastle Disease Virus-Induced Immunogenic Cell Death in Melanoma Cells.

Background: Oncolytic viruses (OVs) are emerging as potent inducers of immunogenic cell death (ICD), releasing danger-associated molecular patterns (DAMPs) that induce potent anticancer immunity. Oncolytic Newcastle disease virus (NDV) has been shown to educe ICD in both glioma and lung cancer cells. The objective of this study is to investigate whether oncolytic NDV induces ICD in melanoma cells and how it is regulated.Methods: Various time points were actuated to check the expression and release of ICD markers induced by NDV strain, NDV/FMW in melanoma cell lines. The expression and release of ICD markers induced by oncolytic NDV strain, NDV/FMW, in melanoma cell lines at various time points were determined. Surface-exposed calreticulin (CRT) was inspected by confocal imaging. The supernatants of NDV/FMW infected cells were collected and concentrated for the determination of ATP secretion by ELISA, HMGB1, and HSP70/90 expression by immunoblot (IB) analysis. Pharmacological inhibition of apoptosis, autophagy, necroptosis, ER Stress, and STAT3 (signal transducer and activator of transcription 3) was achieved by treatment with small molecule inhibitors. Melanoma cell lines stably depleted of STAT3 were established with lentiviral constructs. Supernatants from NDV-infected cells were intratumorally injected to mice bearing melanoma cells-derived tumors.Results: Oncolytic NDV induced CRT exposure, the release of HMGB1 and HSP70/90 as well as secretion of ATP in melanoma cells. Inhibition of apoptosis, autophagy, necroptosis or ER stress attenuated NDV/FMW-induced release of HMGB1 and HSP70/90. Moreover, NDV/FMW-induced ICD markers in melanoma cells were also suppressed by either treatment with a STAT3 inhibitor or shRNA-mediated depletion of STAT3. Of translational importance, treatment of mice bearing melanoma cells-derived tumors with supernatants from NDV/FMW-infected cells significantly inhibited tumor growth.Conclusions: Our data authenticate that oncolytic NDV/FMW might be a potent inducer of ICD in melanoma cells, which is amalgamated with several forms of cell death. We also show that STAT3 plays a role in NDV/FMW-induced ICD in melanoma cells. Together, our data highlight oncolytic NDV as propitious for cancer therapeutics by stimulatingan anti-melanoma immune response.

PC-8 Novel chemoimmunotherapy combinations for pancreatic cancer

Introduction: Pancreatic cancer (PC) is a devastating malignancy. It is usually diagnosed at an incurable stage and has the poorest of prognoses of any cancer. Despite its relatively low incidence, it is the fourth leading cause of cancer-related death. A few chemotherapies have shown modest efficacy but are associated with significant toxicities and development of tumour resistance. For these reasons the median survival after diagnosis is about 4.6 months, 18% of patients survive one year and less than 5% survive for 5 years. Without improvement in PC therapy it may soon become the second leading cause of cancer related deaths. Immunotherapies are beginning to show efficacy as first line therapies for a number of cancers but have yet to prove effective against PC. This is due to the dense desmoplastic stroma and immune suppressive microenvironment characteristic of advanced disease. Thus novel chemo-immunotherapeutic combinations will likely be required to target the tumour and its microenvironment to create the conditions in which immunotherapies, such as checkpoint inhibition, will be effective. The data presented here details the development of combination chemotherapy capable of killing tumour cells whilst increasing the immunogenicity of the tumour. Results: Combinations based upon the chemotherapeutic drug Gemcitabine were studied. Gemcitabine was combined with Zolodronic acid, Pomalidomide, Oxaliplatin and mycobacterial adjuvants. These combinations demonstrated increased cytotoxicity of pancreatic cell lines compared to single agent treatment. Gemcitabine increased markers of immune recognition on these cell lines independently of its cytotoxic properties whilst Oxaliplatin increased markers of immunogenic cell death and uptake of tumour cells into dendritic cells. Combination treatment was able to enhance dendritic cell maturation and the activation of CD8+ T-cells, measured as increases in the expression of IFN-γ. Discussion: Taken together the data presented here shows that gemcitabine based combinations exhibit superior in vitro cytotoxicity and immunogenic properties compared to single agent therapy. Each of these agents has previously been paired with gemcitabine for treatment of pancreatic cancer, demonstrating low toxicity and potential clinical efficacy. This supports their development as 3 or 4 agent combinations. Further research using pre-clinical pancreatic cancer models is planned including the use of checkpoint inhibition in combination with Chemoimmunotherapy.

Naphthyl quinoxaline thymidine conjugate is a potent anticancer agent post UVA activation and elicits marked inhibition of tumor growth through vaccination

Anticancer anthracyclines are cytotoxic drugs that can induce antitumor immune response as a secondary effect through immunogenic cell death (ICD) mechanism. However, the immunogenic potency is quite limited, possibly due to that these chemotherapeutic agents are not specifically developed as ICD inducers. Thus, new drug entities through studies focusing on enhanced ICD induction would significantly promote antitumor immune response in the vaccination application. We report here a naphthyl quinoxaline thymidine conjugate as a new class of cytotoxic compounds that effectively induced in vivo antitumor activity through the vaccination application. Synthesized naphthyl quinoxaline conjugates were weak fluorescent thymidine analog yet exhibited a pronounced anticancer activity in the low nanomolar range post UVA activation. The potent activity of naphthyl conjugate was able to induce the marked detection of ICD markers including ATP and HMGB1 extracellular and calreticulin intracellularly at 2 h post UVA activation. Most importantly, mice vaccinated with cells treated with naphthyl conjugate plus UVA exhibited complete tumor growth inhibition in the tumor challenge study, and the induced immunogenic inhibition was much more effective than that of mitoxantrone anthracycline drug. All these results demonstrate the high potential of naphthyl quinoxaline conjugate for the cancer cell vaccine against tumor.

CRGD target liposome delivery system promoted immunogenic cell death through enhanced anticancer potency of a thymidine conjugate under UVA activation as a cancer vaccine

Conventional chemotherapeutic and photodynamic therapy have recently been shown to also elicit immune response against cancer through the immunogenic cell death mechanism, which can be potentially translated into effective cancer vaccines. However, there are few studies on the potential of nanodelivery system to promote the immunogenic cell death as a cancer vaccine. We reported here that cRGD target liposome delivery system was capable to promote the immunogenic cell death through enhanced potency of a thymidine conjugate post UVA activation as a cancer vaccine. Liposomes and cRGD target liposomes were found to significantly increase the cellular accumulation of the thymidine conjugate and subsequently translated into enhanced cytotoxic potency after UVA activation. More importantly, cRGD target liposomes of the thymidine conjugate markedly promoted the early detection of immunogenic cell death markers including ATP, HMGB1 and calreticulin. Subsequent in vivo vaccination−challenge study confirmed effective tumor growth inhibition by the cRGD liposomal thymidine conjugate and UVA treated cancer cells as the cancer vaccine. In addition, liposomes and cRGD target liposomes alone did not shown any induction of the immunogenic cell death markers, revealing the adjuvant nature of the nanodelivery system.

Abstract A142: Anti-mesothelin immunotoxins induce markers of immunogenic cell death and when injected locally into AE17M mesothelioma tumors enhance the effect of CTLA-4 blockade

Abstract Background: SS1P and LMB-100 are anti-mesothelin immunotoxins composed of a targeting antibody fragment genetically fused to a truncated pseudomonas exotoxin A. We have previously repotrted a synergistic antitumor effect when SS1P or LMB-100 were injected locally into tumors of murine breast cancer in combination with antibodies that block the immune check point CTLA-4. In this study our goal is to explore if this treatment can be applied in a murine mesothelioma model. Method: The mouse mesothelioma AE17M cell line, expressing human mesothelin, was treated in culture with anti-mesothelin immunotoxins. We collected data on the cytotoxic activity and ability to induce markers of immunogenic cell death. In vivo effects were performed on AE17M tumors growing in C57/Bl6 mice. Results: AE17M cells are sensitive to both SS1P and LMB-100 in culture, but when injected IV, the immunotoxins did not affect AE17M tumor growth rate. To overcome obstacles in drug penetration we injected immunotoxins directly into AE17M tumors and found that they only increased the survival of mice from 16 days to 20 days. In addition, anti-CTLA-4 monotherapy failed to control AE17M tumor growth rate in most mice. But when locally injected SS1P or LMB-100 was combined with i.p. anti-CTLA-4, tumor regressions occurred and the median survival increased to 80 days or longer. In search for possible mechanisms enabling immunotoxins to contribute to anti-CTLA-4 activity, we examined their ability to induce markers of immunogenic cell death. We found that both LMB-100 and SS1P increased release of ATP from AE17M cells. In addition, LMB-100 induced calreticulin expression on the surface of both AE17M cells and KLM-1 cells. These results point to possible pathways allowing immunotoxins to promote anti-tumor immunity and sensitize tumors for anti-CTLA-4 effect. Conclusion: Our findings in the AE17M model provide additional support for the use of local immunotoxins in combination with anti-CTLA-4. Citation Format: Yasmin Leshem, Emily King, Yoram Reiter, Ira Pastan. Anti-mesothelin immunotoxins induce markers of immunogenic cell death and when injected locally into AE17M mesothelioma tumors enhance the effect of CTLA-4 blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A142.

Physical plasma-treated saline promotes an immunogenic phenotype in CT26 colon cancer cells in vitro and in vivo

Metastatic colorectal cancer is the fourth most common cause of cancer death. Current options in palliation such as hyperthermic intraperitoneal chemotherapy (HIPEC) present severe side effects. Recent research efforts suggested the therapeutic use of oxidant-enriched liquid using cold physical plasma. To investigate a clinically accepted treatment regimen, we assessed the antitumor capacity of plasma-treated saline solution. In response to such liquid, CT26 murine colon cancer cells were readily oxidized and showed cell growth with subsequent apoptosis, cell cycle arrest, and upregulation of immunogenic cell death (ICD) markers in vitro. This was accompanied by marked morphological changes with re-arrangement of actin fibers and reduced motility. Induction of an epithelial-to-mesenchymal transition phenotype was not observed. Key results were confirmed in MC38 colon and PDA6606 pancreatic cancer cells. Compared to plasma-treated saline, hydrogen peroxide was inferiorly toxic in 3D tumor spheroids but of similar efficacy in 2D models. In vivo, plasma-treated saline decreased tumor burden in Balb/C mice. This was concomitant with elevated numbers of intratumoral macrophages and increased T cell activation following incubation with CT26 cells ex vivo. Being a potential adjuvant for HIPEC therapy, our results suggest oxidizing saline solutions to inactivate colon cancer cells while potentially stimulating antitumor immune responses.

Combination of chemotherapy and physical plasma elicits melanoma cell death via upregulation of SLC22A16

Malignant melanoma is an aggressive cancer that develops drug resistance leading to poor prognosis. Efficient delivery of chemotherapeutic drugs to the tumor tissue remains a major challenge in treatment regimens. Using murine (B16) and human (SK-MEL-28) melanoma cells, we investigated traditional cytotoxic agents in combination with cold physical plasma-derived oxidants. We report synergistic cytotoxicity of doxorubicin and epirubicin, and additive toxicity of oxaliplatin with plasma exposure in coefficient of drug interaction analysis. The combination treatment led to an increased DNA damage response (increased phosphorylation of ATM, γ-H2AX foci, and micronuclei formation). There was also an enhanced secretion of immunogenic cell death markers ATP and CXCL10 in cell culture supernatants following combination treatment. The observed synergistic effects in tumor cells was due to enhanced intracellular doxorubicin accumulation via upregulation of the organic cationic transporter SLC22A16 by plasma treatment. The doxorubicin uptake was reversed by pretreating cells with antioxidants or calcium influx inhibitor BTP2. Endoribonuclease-prepared siRNAs (esiRNA)-mediated knockdown of SLC22A16 inhibited the additive cytotoxic effect in tumor cells. SK-MEL 28 and THP-1 monocytes co-culture led to greater THP-1 cell migration and SK-MEL-28 cytotoxicity when compared with controls. Taken together, we propose pro-oxidant treatment modalities to sensitize chemoresistant melanoma cells towards subsequent chemotherapy, which may serve as therapeutic strategy in combination treatment in oncology.

SS1P Immunotoxin Induces Markers of Immunogenic Cell Death and Enhances the Effect of the CTLA-4 Blockade in AE17M Mouse Mesothelioma Tumors.

SS1P is an anti-mesothelin immunotoxin composed of a targeting antibody fragment genetically fused to a truncated fragment of Pseudomonas exotoxin A. Delayed responses reported in mesothelioma patients receiving SS1P suggest that anti-tumor immunity is induced. The goal of this study is to evaluate if SS1P therapy renders mesothelioma tumors more sensitive to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) immune checkpoint blockade. We evaluated the ability of SS1P to induce adenosine triphosphate (ATP) secretion and calreticulin expression on the surface of AE17M mouse mesothelioma cells. Both properties are associated with immunogenic cell death. Furthermore, we treated these tumors with intra-tumoral SS1P and systemic CTLA-4. We found that SS1P increased the release of ATP from AE17M cells in a dose and time-dependent manner. In addition, SS1P induced calreticulin expression on the surface of AE17M cells. These results suggest that SS1P promotes immunogenic cell death and could sensitize tumors to anti-CTLA-4 based therapy. In mouse studies, we found that the combination of anti-CTLA-4 with intra-tumoral SS1P induced complete regressions in most mice and provided a statistically significant survival benefit compared to monotherapy. The surviving mice were protected from tumor re-challenge, indicating the development of anti-tumor immunity. These findings support the use of intra-tumoral SS1P in combination with anti-CTLA-4.

Hmox1 Upregulation Is a Mutual Marker in Human Tumor Cells Exposed to Physical Plasma-Derived Oxidants.

Increasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 (nrf2) target heme oxygenase 1 (hmox1) was upregulated in all cell lines 4 h post PTM-treatment. nrf2 was not changed, but its baseline expression inversely and significantly correlated with hmox1 expression after exposure to PTM. Besides awarding hmox1 a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.

Abstract 2757: Antibody-drug conjugate payloads induce markers of immunogenic cell death in cancer cells

Abstract Activating the immunologic destruction of cancer cells is a clinically validated therapeutic approach. Immunogenic cell death (ICD) is a reported mechanism of activating the immune system wherein anti-cancer treatments induce specific tumor cell markers, followed by dendritic cell activation and T-cell recruitment. Antibody-drug conjugates (ADCs) are targeted biotherapeutics which deliver cytotoxins to cancer cells. Our hypothesis is that ADCs also may have immune-modulating properties and that the payloads delivered by ADCs may induce ICD. We developed three assays to evaluate ICD in cultured tumor cell lines: (1) HMGB1 extracellular protein levels, (2) cell surface calreticulin translocation, (3) and ICD-induced dendritic cell activation (ICD-DCA). First, cytototoxity was assessed for all compounds, then ICD studies were conducted at dose-multiples relative to each compound's anti-proliferative IC50. Next, cells were treated with various chemotherapeutic agents and extracellular HMGB1 protein detected by ELISA. However, HMGB1 levels strongly correlated with cytotoxicity, and did not differ among treatments. We next assessed calreticulin (CRT), an ER-resident protein reportedly translocated to the cell surface upon ICD. We evaluated the specificity of commercial antibodies to detect CRT in human (A549) or murine (EMT6) cell lines. Cells were incubated with CRT siRNA specific for murine or human sequences. Flow cytometry (for cell surface CRT) and immunoblots (for total CRT) confirmed that a commonly used CRT antibody detects CRT in murine cells, but not human cells. Murine EMT6, 4T1, B16F10, or CT26 cells were then treated with compounds of varied mechanisms-of-action. Our assays confirm previous reports that anthracycline chemotherapeutics (e.g., doxorubicin) induce CRT surface translocation. We also discovered that analogs of both the calicheamicin and cyclopropylpyrroloindolone (CPI) DNA inhibitor class of ADC payloads robustly induced CRT surface translocation in several murine cancer lines within 24 hr, even at relatively low subtoxic doses. Some DNA inhibitors did not induce CRT. Microtubule inhibitor auristatins and mertansine (DM1) induced CRT translocation, but at high cytotoxic doses and after 48 hr incubation. Next, tumor cells were treated with compounds, washed, then co-cultured with murine bone-marrow derived dendritic cells (BMDC). The DNA inhibitors which induced CRT on tumor cells also increased levels of CD86 activation marker and cytokine release from BMDC in these co-cultures. Hence, we have demonstrated that calreticulin, a reported marker of ICD, is activated in cultured murine tumor cells by unconjugated payloads typically delivered by ADCs. These compounds can also induce DC activation in tumor:BMDC co-cultures. These data suggest the potential for immune system activation using cytotoxic ADC payloads. Citation Format: Xingzhi Tan, My-Hanh Lam, Shoba Ragunathan, Keziban Unsal-Kacmaz, Frank Loganzo. Antibody-drug conjugate payloads induce markers of immunogenic cell death in cancer cells [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 2757.

PO-360 Vaccinia virus delivered by isolated limb perfusion combines with PD-1 blockade to prevent local and distant relapse in soft-tissue sarcoma

IntroductionSystemic relapse is the major cause of mortality in extremity soft tissue sarcoma (ESTS). Traditional systemic therapies for metastatic sarcoma have limited efficacy, as do novel agents such as immune checkpoint inhibitors (ICI). Oncolytic virotherapy is capable of improving the efficacy of ICI in some non-sarcomatous pathology. In a rat model of ESTS, we have previously found isolated limb perfusion (ILP) to be ideally suited to delivering a vaccinia virus (GLV-1h68), alongside melphalan and tumour necrosis factor-α (TNFα). Using this model, we sought to investigate the effects of GLV-1h68 delivered by ILP on the tumour microenvironment and determine whether viral ILP can sensitise sarcomas to subsequent immune checkpoint blockade.Material and methodsIn vivo experiments were performed in Brown Norway rats bearing BN175 sarcoma in accordance with a Home Office animal license. Therapeutics included GLV-1h68, a PD-1 inhibitor (J43), melphalan and TNFα.Results and discussionsPD-1 inhibition had limited in vivo monotherapy efficacy in BN175 sarcoma. Pre-treatment with GLV-1h68 delivered by ILP prior to PD-1 blockade markedly improved therapy, with complete tumour regression in a third of animals. Without further treatment, resistant disease rapidly evolved leading to both local and distant relapse. However, when performed as a neoadjuvant treatment prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented local and distant relapse in all animals. Treatment with both GLV-1h68 and PD-1 blockade were found to be necessary for durable cure. In vitro, GLV-1h68 induced ATP, calreticulin and HMGB1, markers of immunogenic cell death. In vivo, viral ILP and PD-1 blockade significantly increased the number of intra-tumoural CD4+ and CD8+ effector cells, with an increased proportion of these cells expressing activation markers. Viral ILP and PD-1 blockade also altered the topography of intra-tumoural immune invasion; significantly increasing CD8+ cells within the tumour parenchyma relative to the invasive margin. The accumulation of effector cells within regional lymph nodes, located outside of the perfusion field, was also noted indicative of at least a locoregional, and perhaps, systemic immune-priming.ConclusionViral ILP and PD-1 blockade combine to prevent local and distant relapse in an animal model of high-grade ESTS. These data provide a strong rationale for clinical translation of this neoadjuvant combination immunotherapy.

Abstract 267: Nonsteroidal anti-inflammatory drugs induce ER stress- and BID-dependent immunogenic cell death to suppress colorectal tumorigenesis

Abstract Prevention or early detection represents a key approach for reducing the mortality and morbidity of CRC. Use of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with decreased risk of CRC in numerous epidemiological studies, clinical trials, and animal model studies. However, the mechanism by which NSAIDs suppress colorectal tumorigenesis has remained unclear. We previously demonstrated that the chemopreventive effect of NSAIDs is mediated by a synthetic lethal interaction between death receptor signaling and loss of the gatekeeper APC tumor suppressor, which is dependent on the BH3-only Bcl-2 family member BID. In this study, we found a critical role of endoplasmic reticulum (ER) stress upstream of death receptor signaling and BID activation. Elevated levels of ER stress and cell death markers were detected in advanced adenomas from patients taking NSAIDs including aspirin. Inhibiting ER stress using a pharmacological approach abolished the apoptotic effect of NSAIDs in CRC cells and normal colonic epithelial cells with APC loss and also suppressed the chemopreventive activity of the NSAID sulindac in APCMin/+ mice. Importantly, our results unveiled a critical role of ER stress and BID-dependent cell death in triggering immune-mediated elimination of nascent cells that lose APC. Markers of immunogenic cell death (ICD), including plasma membrane translocation of calreticulin and phagocytosis by dendritic cells, were suppressed in cells with BID knockout or ER stress inhibition. BID knockout or ER stress inhibition also reduced tumor infiltrating lymphocytes (TILs) in sulindac-treated APCMin/+ mice. Collectively, our results suggest that NSAIDs suppress colorectal tumorigenesis by enhancing immunosurveillance through ER stress and BID-dependent ICD. These results provide novel insight into the chemopreventive mechanism of NSAIDs, which may help design more effective chemopreventive strategies and agents. Citation Format: Rochelle Elayne Fletcher, Brian Leibowitz, Yijun Wang, Fernando Concha-Benavente, Robert Ferris, Robert Schoen, Jian Yu, Lin Zhang. Nonsteroidal anti-inflammatory drugs induce ER stress- and BID-dependent immunogenic cell death to suppress colorectal tumorigenesis [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 267.

Abstract 2744: Telomelysin-induced immunogenic cell death synergizes with anti-PD-1 antibody in non-immunogenic gastrointestinal tumors

Abstract Background: Telomelysin (OBP-301) is a telomerase-specific oncolytic adenovirus, in which the telomerase promoter controls its replication and induction of cell death in human tumor cells. A FDA-approved phase I study has confirmed the safety and biological activity of intratumoral administration of Telomelysin in patients with advanced solid tumors in the US. Anti-programmed death-1 antibody (PD-1 Ab) has dramatically improved clinical outcomes for patients with melanoma or lung cancer; however, the clinical benefit is limited to small population of patients with certain degree of tumor infiltrating lymphocytes (TILs). Therefore, more effective strategy to enhance the immunogenicity of non-immunogenic tumors is needed. Here, we show the potential of our oncolytic adenovirus as an immunogenic agent sensitizing non-immunogenic gastrointestinal tumors to PD-1 Ab. Methods: A murine colon cancer cell line CT26 (BALB/c) and a murine pancreatic cancer cell line PAN02 (C57BL/6) were used in this study. An OBP-301 variant (OBP-502) that expresses the RGD mutant fiber to infect murine tumor cells via interaction with integrin was used as an oncolytic adenovirus. PD-1 Ab (clone 4H2) was obtained from Ono Pharmaceutical Co. Ltd. As immunogenic cell death markers, extracellular ATP and high mobility group box 1 (HMGB1) levels were measured following OBP-502 infection. In vivo subcutaneous tumor models, CD8-positive TILs, vaccination efficiency of OBP-502-treated cells and combination therapy of OBP-502 with PD-1 Ab were evaluated. Results: OBP-502 efficinetly killed CT26 and PAN02 cells, and also significantly increased extracellular ATP and HMGB1 on both cell lines in vitro, suggesting that OBP-502-induced cell death might be immunogenic. Indeed, in the CT26 and PAN02 subcutaneous tumor models, OBP-502 induced massive accumulation of TILs compared to control. Vaccination of mice with OBP-502-infected CT26 cells rejected tumors in three of 10 vaccinated mice (30%) and also significantly suppressed the growth of remaining seven CT26 tumors. The combination therapy of OBP-502 and PD-1 Ab significantly suppressed the growth of CT26 subcutaneous tumors and successfully eradicated tumors in four of 12 mice (33%) while either single treatment failed to eradicate in most mice. When these 4 tumor-free mice treated with the combination therapy were re-challenged by inoculation of CT26 cells, two of 4 mice (50%) remained tumor-free. Similar antitumor effects of the combination therapy were observed in the PAN02 tumor model.H.E staining of major organs harvested after each treatment demonstrated no serious systemic toxicity that PD-1 Ab has been reported to cause sometimes in clinical practice. Conclusion: Our telomerase-specific oncolytic adenovirus has the potential to activate antitumor immunity, leading to sensitization of non-immunogenic gastrointestinal tumors to PD-1 Ab. Citation Format: Nobuhiko Kanaya, Shinji Kuroda, Toshiaki Morihiro, Yoshihiko Kakiuchi, Tetsushi Kubota, Satoru Kakiuchi, Masahiko Nishizaki, Yasuo Urata, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Telomelysin-induced immunogenic cell death synergizes with anti-PD-1 antibody in non-immunogenic gastrointestinal tumors [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 2744.

PO-413 Immune modulating properties of cyclophosphamide synergise with immunotherapy in preclinical models of neuroblastoma

IntroductionNeuroblastoma (NB) is one of the most common childhood cancers accounting for 15% of paediatric cancer deaths. Current therapies, which include chemotherapy, are highly toxic with significant treatment related mortality. There is little scope for further intensification therefore alternatives strategies such as immunotherapy, are a priority for improving patient outcomes. Classically, chemotherapy is regarded as immunosuppressive but recent work has highlighted that some may illicit an immunogenic form of cell death. This work sets out to identify the immunomodulating effects of cyclophosphamide (CPM) on tumour infiltrating immune cells and whether these properties can synergise with immunomodulatory antibodies in preclinical models of NB.Material and methodsThe effects of CPM and doxorubicin on tumour cells were investigated in vitro by analyses of immunogenic cell death (ICD) markers. Two different in vivo subcutaneous murine NB models (NXS2 +NB9464D), were treated i.p with different doses of CPM. Tissues were harvested and detailed immunophenotyping performed. Combination of CPM and anti-PD-1 therapy was investigated in both models using tumour growth and survival as end points. Combination therapy was also assessed in TH-MYCN mice which develop spontaneous neuroblastoma.Results and discussionsChemotherapy application in vitro led to an increase in expression of the ICD markers, ecto-calreticulin and Hsp-70. CPM was found to have numerous immune modulating activities in vivo, including the reduction of intratumoral Treg cells, even at low doses (20 mg/kg), in both NB models. Combination therapy was shown to increase CD8+ and CD4+ percentages within tumours, along with an increase CD4+ effector and memory cell proportions. Anti-PD-1 therapy synergised with CPM to improve median survival and slow tumour growth. Metronomic dosing of CPM and anti-PD-1 antibody led to tumour regression in TH-MYCN tumour bearing mice.ConclusionThis work supports combining low dose CPM to enhance immunomodulatory antibody therapy, to generate therapeutic anti-neuroblastoma immunity. Ongoing work is focused on elucidating the mechanism of action of the CPM and anti-PD-1 combination therapy, along with identifying the optimal chemotherapy and antibody dosing strategies.

Photothermal Therapy Generates a Thermal Window of Immunogenic Cell Death in Neuroblastoma

A thermal "window" of immunogenic cell death (ICD) elicited by nanoparticle-based photothermal therapy (PTT) in an animal model of neuroblastoma is described. In studies using Prussian blue nanoparticles to administer photothermal therapy (PBNP-PTT) to established localized tumors in the neuroblastoma model, it is observed that PBNP-PTT conforms to the "more is better" paradigm, wherein higher doses of PBNP-PTT generates higher cell/local heating and thereby more cell death, and consequently improved animal survival. However, in vitro analysis of the biochemical correlates of ICD (ATP, high-motility group box 1, and calreticulin) elicited by PBNP-PTT demonstrates that PBNP-PTT triggers a thermal window of ICD. ICD markers are highly expressed within an optimal temperature (thermal dose) window of PBNP-PTT (63.3-66.4°C) as compared with higher (83.0-83.5°C) and lower PBNP-PTT (50.7-52.7°C) temperatures, which both yield lower expression. Subsequent vaccination studies in the neuroblastoma model confirm the in vitro findings, wherein PBNP-PTT administered within the optimal temperature window results in long-term survival (33.3% at 100d) compared with PBNP-PTT administered within the higher (0%) and lower (20%) temperature ranges, and controls (0%). The findings demonstrate a tunable immune response to heat generated by PBNP-PTT, which should be critically engaged in the administration of PTT for maximizing its therapeutic benefits.

Combination of Vaccine-Strain Measles and Mumps Viruses Enhances Oncolytic Activity against Human Solid Malignancies

ABSTRACTOncolytic measles and mumps viruses (MeV, MuV) have a potential for anti-cancer treatment. We examined the anti-tumor activity of MeV, MuV, and MeV-MuV combination (MM) against human solid malignancies (HSM). MeV, MuV, and MM targeted and significantly killed various cancer cell lines of HSM but not normal cells. MM demonstrated a greater anti-tumor effect and prolonged survival in a human prostate cancer xenograft tumor model compared to MeV and MuV. MeV, MuV, and MM significantly induced the expression of immunogenic cell death markers and enhanced spleen-infiltrating immune cells. In conclusion, MM combination significantly improves the treatment of human solid malignancies.

Natural Killer T-cell Immunotherapy in Combination with Chemotherapy-Induced Immunogenic Cell Death Targets Metastatic Breast Cancer.

Natural killer T (NKT) cells are glycolipid-reactive lymphocytes that promote cancer control. In previous studies, NKT-cell activation improved survival and antitumor immunity in a postsurgical mouse model of metastatic breast cancer. Herein, we investigated whether NKT-cell activation could be combined with chemotherapeutic agents to augment therapeutic outcomes. Gemcitabine and cyclophosphamide analogues enhanced the potential immunogenicity of 4T1 mammary carcinoma cells by increasing the expression of antigen-presenting molecules (MHC-I, MHC-II, and CD1d) and promoting exposure or release of immunogenic cell death markers (calreticulin, HMGB1, and ATP). In 4T1 primary tumor and postsurgical metastasis models, BALB/c mice were treated with cyclophosphamide or gemcitabine. NKT cells were then activated by transfer of dendritic cells loaded with the glycolipid antigen α-galactosylceramide (α-GalCer). Chemotherapeutic treatments did not impact NKT-cell activation but enhanced recruitment into primary tumors. Cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cell monotherapies decreased tumor growth in the primary tumor model and reduced metastatic burden and prolonged survival in the metastasis model. Combining chemotherapeutics with NKT-cell activation therapy significantly enhanced survival, with surviving mice exhibiting attenuated tumor growth following a second tumor challenge. The frequency of myeloid-derived suppressor cells was reduced by gemcitabine, cyclophosphamide, or α-GalCer-loaded dendritic cell treatments; cyclophosphamide also reduced the frequency of regulatory T cells. Individual treatments increased immune cell activation, cytokine polarization, and cytotoxic responses, although these readouts were not enhanced further by combining therapies. These findings demonstrate that NKT-cell activation therapy can be combined with gemcitabine or cyclophosphamide to target tumor burden and enhance protection against tumor recurrence. Cancer Immunol Res; 5(12); 1086-97. ©2017 AACR.

123 - Induction of Immunogenic Cell Death upon Combinatorial Therapy with Cold Physical Plasma and Chemotherapeutic Agents in Melanoma

Introduction Chemotherapeutic agents remain as the essential regimen against malignant melanoma. Variety of combination therapies have been employed to sensitize tumor cells, circumvent drug resistance, and reduce systemic toxicity. However, there is still need for innovative combinational approaches to resolve these difficulties. In the current study, we investigated the potential synergism of cold physical plasma in conjunction with chemotherapeutic agents and identified mechanisms of synergistic immunogenic cell death (ICD) in tumor cells in vitro Methods In this study, we pretreated murine B16F10 and human SK-Mel 28 melanoma cells with increasing concentrations of Doxorubicin, Epirubicin, Oxaliplatin, Vorinostat and SAHA for 24h. This was followed by 30 secs exposure cold physical plasma effluent of an atmospheric pressure argon plasma jet (kINPen). After 6-hour incubation, IC50 was determined using alamar blue assay and compared with single treatment modalities. Cell culture supernatants were used to quantify markers of immunogenic cell death (CART, ATP, HMGB1, CXCL10) by ELISA, immunofluorescence and flow cytometry. Total cell counts, intracellular superoxide levels (DHE), mitochondria membrane potential (TMRE), cell membrane integrity (sytox green) and cellular morphology were quantitatively assessed using live cell and high throughput confocal imaging (Operatta; Perkin Elmer). Co culture and transwell migration assays was performed on THP1 monocytes and RAW 264.7 macrophages. For SK-Mel 28 tumor cells, 3D spheroid models were also investigated. Results Combination therapy of chemotherapeutic agents and cold physical plasma significantly decreased the IC50 values in melanoma cell lines in comparison to single treatment modalities in 2D and 3D culture conditions. The tumor cell killing was mediated by elevation of superoxide and loss of mitochondrial membrane potential. Co culturing SK-Mel 28 cells with THP1 monocytes or RAW macrophages with B16F10 following combination therapy led to pronounced tumor cell killing. ICD markers such as ATP, HMGB1, CART, and CXCL10 were induced in both single treatment conditions however were significantly elevated upon combination therapy demonstrating a synergistic effect. Transwell migration assay revealed migration of THP1 monocytes and RAW 264.7 macrophages following incubation with conditioned medium. In this study, we have demonstrated that cold physical plasma is a valuable tool to induce ICD in tumors pretreated with conventional chemotherapeutic agents.