Immunogenic Cell Death Responses Research Articles

Cu(II) complex that synergistically potentiates cytotoxicity and an antitumor immune response by targeting cellular redox homeostasis

Developing anticancer drugs with low side effects is an ongoing challenge. Immunogenic cell death (ICD) has received extensive attention as a potential synergistic modality for cancer immunotherapy. However, only a limited set of drugs or treatment modalities can trigger an ICD response and none of them have cytotoxic selectivity. This provides an incentive to explore strategies that might provide more effective ICD inducers free of adverse side effects. Here, we report a metal-based complex (Cu-1) that disrupts cellular redox homeostasis and effectively stimulates an antitumor immune response with high cytotoxic specificity. Upon entering tumor cells, this Cu(II) complex enhances the production of intracellular radical oxidative species while concurrently depleting glutathione (GSH). As the result of heightening cellular oxidative stress, Cu-1 gives rise to a relatively high cytotoxicity to cancer cells, whereas normal cells with low levels of GSH are relatively unaffected. The present Cu(II) complex initiates a potent ferroptosis-dependent ICD response and effectively inhibits in vivo tumor growth in an animal model (c57BL/6 mice challenged with colorectal cancer). This study presents a strategy to develop metal-based drugs that could synergistically potentiate cytotoxic selectivity and promote apoptosis-independent ICD responses through perturbations in redox homeostasis.

A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

Near‐Infrared‐II Nanoparticles for Vascular Normalization Combined with Immune Checkpoint Blockade via Photodynamic Immunotherapy Inhibit Uveal Melanoma Growth and Metastasis

AbstractPhotodynamic therapy (PDT) has been widely employed in tumor treatment due to its effectiveness. However, the tumor hypoxic microenvironment which is caused by abnormal vasculature severely limits the efficacy of PDT. Furthermore, the abnormal vasculature has been implicated in the failure of immunotherapy. In this study, a novel nanoparticle denoted as Combo‐NP is introduced, composed of a biodegradable NIR II fluorescent pseudo‐conjugate polymer featuring disulfide bonds within its main chain, designated as TPA‐BD, and the vascular inhibitor Lenvatinib. Combo‐NP exhibits dual functionality by not only inducing cytotoxic reactive oxygen species (ROS) to directly eliminate tumor cells but also eliciting immunogenic cell death (ICD). This ICD response, in turn, initiates a robust cascade of immune reactions, thereby augmenting the generation of cytotoxic T lymphocytes (CTLs). In addition, Combo‐NP addresses the issue of tumor hypoxia by normalizing the tumor vasculature. This normalization process enhances the efficacy of PDT while concurrently fostering increased CTLs infiltration within the tumor microenvironment. These synergistic effects synergize to potentiate the photodynamic‐immunotherapeutic properties of the nanoparticles. Furthermore, when combined with anti‐programmed death‐ligand 1 (PD‐L1), they showcase notable inhibitory effects on tumor metastasis. The findings in this study introduce an innovative nanomedicine strategy aimed at triggering systemic anti‐tumor immune responses for the treatment of Uveal melanoma.

Four Birds with One Stone: A Multifunctional Polypeptide Nanocomposite to Unify Ferroptosis, Nitric Oxide, and Photothermia for Amplifying Antitumor Immunity

AbstractTumor metastasis and relapse mainly results in therapy failure and becomes a big challenge in oncology. Immunogenic cell death (ICD) of tumors mediated immunotherapy (IT) is attracting widely for solving that problem although achieving sufficient ICD and strong immune response is challenging for nanoparticles‐based cancer IT. Herein, a multifunctional polypeptide coordinate nanocomposite that possesses near infrared photothermia (PT) and responsive releases of nitric oxide (NO) and iron ions is constructed, which synergistically kills cancer cells and highly prohibits metastatic 4T1 cells invasion and migration by PT‐boosted NO release and ferroptosis (FT). Remarkably, triple FT‐NO‐PT treatment amplifies the ICD effects and outperforms combo/monotherapy FT‐PT and FT in cancer cells and tumors, which further activates dendritic cells maturation, and primed CD4+T and CD8+T cells immune responses and memory effects, playing four birds with one stone (i.e., FT‐NO‐PT‐IT). The PCSFG‐based FT‐NO‐PT not only fully eradicates 4T1 primary tumors, but also induces strong ICD, immune priming, and memory effects to reject rechallenged 4T1 tumors and inhibit malignant tumor metastasis, demonstrating synergistic amplified ICD effects with strong cell immunities and memory effects by a unified FT‐NO‐PT‐IT.

Marsdenia tenacissima extract induces endoplasmic reticulum stress-associated immunogenic cell death in non-small cell lung cancer cells through targeting AXL

Ethnopharmacological relevanceMarsdenia Tenacissima (Roxb.) Wight et Arn. is a traditional Chinese medicine. Its standardized extract (MTE), with the trade name Xiao-Ai-Ping injection, is widely used for cancer treatment. The pharmacological effects of MTE-inducing cancer cell death have been primarily explored. However, whether MTE triggers tumor endoplasmic reticulum stress (ERS)-associated immunogenic cell death (ICD) is unknown. Aim of the studyTo determine the potential role of endoplasmic reticulum stress in the anti-cancer effects of MTE, and uncover the possible mechanisms of endoplasmic reticulum stress-associated immunogenic cell death induced by MTE. Material and methodsThe anti-tumor effects of MTE on non-small cell lung cancer (NSCLC) were examined through CCK-8 and wound healing assay. Network pharmacology analysis and RNA-sequencing (RNA seq) were performed to confirm the biological changes of NSCLCs after MTE treatment. Western blot, qRT-PCR, reactive oxygen species (ROS) assay, and mitochondrial membrane potential (MMP) assay were used to explore the occurrence of endoplasmic reticulum stress. Immunogenic cell death-related markers were tested by ELISA and ATP release assay. Salubrinal was used to inhibit the endoplasmic reticulum stress response. SiRNA and bemcentinib (R428) were used to impede the function of AXL. AXL phosphorylation was regained by recombinant human Gas6 protein (rhGas6). The effects of MTE on endoplasmic reticulum stress and immunogenic cell death response were also proved in vivo. The AXL inhibiting compound in MTE was explored by molecular docking and confirmed by Western blot. ResultsMTE inhibited cell viability and migration of PC-9 and H1975 cells. Enrichment analysis identified that differential genes after MTE treatment were significantly enriched in endoplasmic reticulum stress-related biological processes. MTE decreased mitochondrial membrane potential (MMP) and increased ROS production. Meanwhile, endoplasmic reticulum stress-related proteins (ATF6, GRP-78, ATF4, XBP1s, and CHOP) and immunogenic cell death-related markers (ATP, HMGB1) were upregulated, and the AXL phosphorylation level was suppressed after MTE treatment. However, when salubrinal (an endoplasmic reticulum stress inhibitor) and MTE were co-treated cells, the inhibitory effects of MTE on PC-9 and H1975 cells were impaired. Importantly, inhibition of AXL expression or activity also promotes the expression of endoplasmic reticulum stress and immunogenic cell death-related markers. Mechanistically, MTE induced endoplasmic reticulum stress and immunogenic cell death by suppressing AXL activity, and these effects were attenuated when AXL activity recovered. Moreover, MTE significantly increased the expression of endoplasmic reticulum stress-related markers in LLC tumor-bearing mouse tumor tissues and plasma levels of ATP and HMGB1. Molecular docking illustrated that kaempferol has the strongest binding energy with AXL and suppresses AXL phosphorylation. ConclusionMTE induces endoplasmic reticulum stress-associated immunogenic cell death in NSCLC cells. The anti-tumor effects of MTE are dependent upon endoplasmic reticulum stress. MTE triggers endoplasmic reticulum stress-associated immunogenic cell death by inhibiting AXL activity. Kaempferol is an active component that inhibits AXL activity in MTE. The present research revealed the role of AXL in regulating endoplasmic reticulum stress and enriched the anti-tumor mechanisms of MTE. Moreover, kaempferol may be considered a novel AXL inhibitor.

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Abstract 2633: Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer

Abstract Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric 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 2633.

A Cascade-Targeted Enzyme-Instructed Peptide Self-Assembly Strategy for Cancer Immunotherapy through Boosting Immunogenic Cell Death.

Immunogenic cell death (ICD) approaches by encumbering mitochondrial functions provide great promise for the treatment of malignant tumors, but these kinds of ICD strategies are still in their infancy. Here, one multifunctional drug-loaded, cascade-targeted, and enzyme-instructed self-assembling peptide nanomedicine (Comp. 4) for ICD-based cancer therapy is constructed. Comp. 4 consists of 1) lonidamine (LND) that specifically interferes with mitochondrial functions; 2) a programmed death ligand 1 (PD-L1) binding peptide sequence (NTYYEDQG) and a mitochondria-specific motif (triphenylphosphonium, TPP) that can sequentially control the cell membrane and mitochondria targeting capacities, respectively; and 3) a -GD FD FpD Y- assembly core to in situ organize peptide assemblies responsive to alkaline phosphatase (ALP). Comp. 4 demonstrates noticeable structural and morphological transformations in the presence of ALP and produces peptide assemblies in mouse colon cancer cells (CT26) with high expressions of both ALP and PD-L1. Moreover, the presence of PD-L1- and mitochondria-specific motifs can assist Comp. 4 for effective endocytosis and endosomal escape, forming peptide assemblies and delivering LND into mitochondria. Consequently, Comp. 4 shows superior capacities to in vivo induce abundant mitochondrial oxidative stress, provoke robust ICD responses, and produce an immunogenic tumor microenvironment, successfully inhibiting CT26 tumor growth by eliciting a systemic ICD-based antitumor immunity.

Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.

Research progress in inducing immunogenic cell death of tumor cells.

Immunogenic cell death (ICD) is a regulated cell death (RCD) pathway. In response to physical and chemical signals, tumor cells activate specific signaling pathways that stimulate stress responses in the endoplasmic reticulum (ER) and expose damage-associated molecular patterns (DAMPs), which promote antitumor immune responses. As a result, the tumor microenvironment is altered, and many tumor cells are killed. The ICD response in tumor cells requires inducers. These inducers can be from different sources and contribute to the development of the ICD either indirectly or directly. The combination of ICD inducers with other tumor treatments further enhances the immune response in tumor cells, and more tumor cells are killed; however, it also produces side effects of varying severity. New induction methods based on nanotechnology improve the antitumor ability and significantly reduces side effects because they can target tumor cells precisely. In this review, we introduce the characteristics and mechanisms of ICD responses in tumor cells and the DAMPs associated with ICD responses, summarize the current methods of inducing ICD response in tumor cells in five distinct categories: chemical sources, physical sources, pathogenic sources, combination therapies, and innovative therapies. At the same time, we introduce the limitations of current ICD inducers and make a summary of the use of ICD responses in clinical trials. Finally, we provide an outlook on the future of ICD inducer development and provide some constructive suggestions.

Aptamer-conjugated nano-liposome for immunogenic chemotherapy with reversal of immunosuppression.

Cancer cells have various immune evasion mechanisms that resist the immune cells by reprogramming the tumor microenvironment (TME), such as programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase-1 (IDO1) overexpression. One of the approaches to restore antitumor immune response by T-cells is through induction of immunogenic cell death (ICD). Thus, drug carrier containing IDO1 siRNA and ICD inducer would be effective anticancer regimen to modulate the immunosuppressive TME by reversing the IDO1-mediated immunosuppression in a synergistic combination with ICD induction. However, numerous nanocarrier platforms for co-delivery of multiple drugs mostly depend on the enhanced permeation and retention (EPR), which is insufficient to achieve selectivity in tumor sites harboring various types of cells. We designed a targeted drug delivery system using nano-sized liposomes functionalized with anti-CD44 and anti-PD-L1 DNA aptamers, which target breast cancer cells and inhibit PD-1/PD-L1 interaction between cancer cells and T-cells. To reverse immunosuppressive TME and reactivate immune response, cancer-targeting nano-liposomes were prepared to contain immunogenic cell death inducer (Doxorubicin, DOX) and IDO1 siRNA, namely Aptm[DOX/IDO1]. The Aptm[DOX/IDO1] specifically delivered the loaded DOX and IDO1 siRNA into target breast cancer cells through aptamer-mediated endocytosis. Cancer-targeted DOX/IDO1 siRNA delivery enhanced ICD and suppressed IDO1 expression with significantly high toxicity in cancer cells. We demonstrated that Aptm[DOX/IDO1] could achieve synergistic antitumor effects by facilitating ICD response and simultaneous reversal of the immunosuppressive TME with IDO1 knockdown in the subcutaneous breast cancer model mice, thus reducing tumor size. These antitumor effects were exerted with intratumoral infiltration of CD8+ cytotoxic T lymphocyte as well as attenuation of regulatory T-cell recruitment in the tumor sites. We further proved that our Aptm[DOX/IDO1] strategy significantly reduced tumor metastasis in tumor-xenograft mice through a synergistic combination of cancer cell-targeted ICD induction and reversal of the IDO1-mediated immunosuppressive TME. Our nanocarrier platform based on cationic liposomes containing DOX and IDO1 siRNA, which are conjugated with two DNA aptamers targeting the cancer cell surface, accomplished synergistic chemoimmunotherapy through tumor-specific immune modulation into immune-favorable TME in vivo.

Liposomal Delivery of Mitoxantrone and a Cholesteryl Indoximod Prodrug Provides Effective Chemo-immunotherapy in Multiple Solid Tumors.

We developed a custom-designed liposome carrier for codelivery of a potent immunogenic cell death (ICD) stimulus plus an inhibitor of the indoleamine 2,3-dioxygenase (IDO-1) pathway to establish a chemo-immunotherapy approach for solid tumors in syngeneic mice. The carrier was constructed by remote import of the anthraquinone chemotherapeutic agent, mitoxantrone (MTO), into the liposomes, which were further endowed with a cholesterol-conjugated indoximod (IND) prodrug in the lipid bilayer. For proof-of-principle testing, we used IV injection of the MTO/IND liposome in a CT26 colon cancer model to demonstrate the generation of a robust immune response, characterized by the appearance of ICD markers (CRT and HMGB-1) as well as evidence of cytotoxic cancer cell death, mediated by perforin and granzyme B. Noteworthy, the cytotoxic effects involved natural killer (NK) cell, which suggests a different type of ICD response. The immunotherapy response was significantly augmented by codelivery of the IND prodrug, which induced additional CRT expression, reduced number of Foxp3+ Treg, and increased perforin release, in addition to extending animal survival beyond the effect of an MTO-only liposome. The outcome reflects the improved pharmacokinetics of MTO delivery to the cancer site by the carrier. In light of the success in the CT26 model, we also assessed the platform efficacy in further breast cancer (EMT6 and 4T1) and renal cancer (RENCA) models, which overexpress IDO-1. Encapsulated MTO delivery was highly effective for inducing chemo-immunotherapy responses, with NK participation, in all tumor models. Moreover, the growth inhibitory effect of MTO was enhanced by IND codelivery in EMT6 and 4T1 tumors. All considered, our data support the use of encapsulated MTO delivery for chemo-immunotherapy, with the possibility to boost the immune response by codelivery of an IDO-1 pathway inhibitor.

Paclitaxel/sunitinib-loaded micelles promote an antitumor response in vitro through synergistic immunogenic cell death for triple-negative breast cancer

Chemotherapy-induced immunogenic cell death (ICD) may offer a strategy to improve the effect of the therapeutic treatment of triple-negative breast cancer (TNBC) by eliciting broad antitumor immunity. However, chemotherapy shows a limited therapeutic effect because of multi-drug resistance and the immunosuppressive tumor microenvironment (TME) of TNBC. The unique pharmacological actions of sunitinib (SUN) indicate its possible synergies with paclitaxel (PTX) to enhance chemo-immunotherapy for TNBC. Here, we prepared a co-delivery platform composed of poly(styrene-co-maleic anhydride) (SMA) via a self-assembly process for a combination of PTX and SUN, which was able to induce a higher synergistic ICD. The nanomicellar delivery of PTX and SUN loaded at an optimal ratio of 1:5 (PTX:SUN) presented the characteristics of an appropriate particle size, long-term stability, and time sequence release which synergistically promoted the apoptosis of MDA-MB-231 tumor cells. Moreover, we demonstrated that the combination of PTX and SUN could significantly induce a synergistic effect because it promoted an ICD response, improved tumor immunogenicity, and regulated immunosuppressive factors in the TME. Overall, PTX and SUN with synergistic effects entrapped in a self-assembly nano-delivery system could offer the potential for clinical applicationof a combination chemo-immunotherapy strategy to improve the effect of the therapeutic treatment of TNBC.

Immunogenic cell death by neoadjuvant oxaliplatin and radiation protects against metastatic failure in high-risk rectal cancer

ObjectiveHigh rates of systemic failure in locally advanced rectal cancer call for a rational use of conventional therapies to foster tumor-defeating immunity.MethodsWe analyzed the high-mobility group box-1 (HMGB1) protein, a measure of immunogenic cell death (ICD), in plasma sampled from 50 patients at the time of diagnosis and following 4 weeks of induction chemotherapy and 5 weeks of sequential chemoradiotherapy, both neoadjuvant modalities containing oxaliplatin. The patients had the residual tumor resected and were followed for long-term outcome.ResultsPatients who met the main study end point—freedom from distant recurrence—showed a significant rise in HMGB1 during the induction chemotherapy and consolidation over the chemoradiotherapy. The higher the ICD increase, the lower was the metastatic failure risk (hazard ratio 0.26, 95% confidence interval 0.11–0.62, P = 0.002). However, patients who received the full-planned oxaliplatin dose of the chemoradiotherapy regimen had poorer metastasis-free survival (P = 0.020) than those who had the oxaliplatin dose reduced to avert breach of the radiation delivery, which is critical to maintain efficient tumor cell kill and in the present case, probably also protected the ongoing radiation-dependent ICD response from systemic oxaliplatin toxicity.ConclusionThe findings indicated that full-dose induction oxaliplatin followed by an adapted oxaliplatin dose that was compliant with full-intensity radiation caused induction and maintenance of ICD and as a result, durable disease-free outcome for a patient population prone to metastatic progression.

Transformable Nanoparticle‐Enabled Synergistic Elicitation and Promotion of Immunogenic Cell Death for Triple‐Negative Breast Cancer Immunotherapy

AbstractAlthough cisplatin‐based neoadjuvant chemotherapy is an efficient therapy approach for triple‐negative breast cancer (TNBC), it has dismal prognosis and modestly improved survival benefit. Here, a synergistic immunotherapy of TNBC premised on the elicitation and promotion of immunogenic cell death (ICD) response, through a transformable nanoparticle‐enabled approach for contemporaneous delivery of cisplatin, adjudin, and WKYMVm is reported. The nanoparticles can sequentially respond to matrix metalloproteinases‐2, pH, and glutathione to achieve structural transformation with the advantages of optimal size change, efficient drug delivery, and well‐controlled release. Cisplatin and adjudin can synergistically amplify reactive oxygen species (ROS) cascade and eventually increase the formation of hydrogen peroxide and downstream highly toxic ROS like •OH, which can elicit ICD response by mechanisms of endoplasmic reticulum stress, apoptotic cell death, and autophagy. WKYMVm can further promote anti‐TNBC immunity by activation of formyl peptide receptor 1 to build stable interactions between dendritic cells and dying cancer cells. Thus, the nanoparticles achieve significant primary tumor regression and pulmonary metastasis inhibition as well as a remarkable survival benefit, with boosting of the innate and adaptive anti‐TNBC immunity.

Ecto-Calreticulin is essential for an efficient immunogenic cell death stimulation in mouse melanoma.

Skin melanoma remains one of the most aggressive and difficult to treat human malignancy, with an increasing incidence every year. Although surgical resection represents the best therapeutic approach, this is only feasible in cases of early diagnosis. Furthermore, the established malignancy is resistant to all therapeutic strategies employed so far, resulting in an unacceptable patient survival rate. Although the immune-mediated therapeutic approaches, based on anti-PD1 or anti-CTLA4, are very promising and under clinical trial experimentation, they could conceal not yet fully emerged pitfalls such as the development of autoimmune diseases. Therefore, alternative therapeutic approaches are still under investigation, such as the immunogenic cell death (ICD) process. Here we show that the lack of calreticulin translocation onto mouse melanoma cell membrane prevents the stimulation of an effective ICD response in vivo.

Abstract 4006: Oxaliplatin-induced immune factors and long-term disease control in rectal cancer - a reflection of immunogenic tumor cell death

Abstract Purpose: In contemporary cancer therapy, the ability of the immune system to recognize and destroy tumor cells is increasingly acknowledged. Following cytotoxic damage of tumor cells, the priming of tumor-targeting T lymphocytes via presentation of shed tumor antigens by dendritic cells may ultimately result in systemic anti-tumor activity, a concept referred to as immunogenic cell death (ICD). Recent preclinical studies have highlighted oxaliplatin as an ICD-inducing agent. In a prospective study (NCT00278694) of curatively intended treatment of locally advanced rectal cancer (LARC), oxaliplatin-containing induction neoadjuvant chemotherapy (NACT; two cycles of the Nordic FLOX regimen) was administered prior to chemoradiotherapy (CRT) and surgery. Within the spectrum of possible ICD responses to NACT, we analyzed circulating osteoprotegerin (OPG) and fms-related tyrosine kinase 3 ligand (FLT3LG), both of which are factors implicated in activation of the antigen-specific interaction between dendritic cells and cytotoxic T lymphocytes. Experimental procedures: Immunoassay measurements of OPG and FLT3LG were performed in serum samples collected from study patients at baseline and following the induction NACT. Estimated 5-year progression-free survival (PFS) and overall survival (OS) were assessed using the log-rank test. Results: Fifty-six patients (38 T2-3 cases and 18 T4 cases) with median follow-up time of 65 months were included in the analysis. During follow-up, 18 patients (nine in each of the T2-3 and T4 groups) experienced metastatic progression as a PFS event. For the entire study population, significant increases in serum levels of both OPG and FLT3LG (from median baseline values of 46.1 pg/ml and 66.7 pg/ml to median post-NACT values of 53.6 pg/ml and 140 pg/ml, respectively) were observed. Patients were separated into cases with (n = 37) or without (n = 19) increase in circulating OPG levels and more (n = 30) or less (n = 26) than two-fold increase in circulating FLT3LG levels. When grouped together, patients with the designated increases in both factors (n = 25) or in either of OPG or FLT3LG values (n = 21) during NACT had significantly better PFS rates than the remaining cases (n = 10) (86%, 72%, and 20%, respectively; p < 0.001). Corresponding OS rates were 100%, 88%, and 60% (p < 0.001), suggesting that even patients who experienced metastatic progression had reasonably good OS. Conclusion: Increase in circulating OPG and FLT3LG following oxaliplatin-containing induction chemotherapy was associated with favorable long-term outcome in LARC patients given curatively intended neoadjuvant treatment consisting of CRT and surgery. These immune effectors may have mediated systemic anti-tumor immunity invoked by ICD-inducing oxaliplatin effects. Citation Format: Sebastian Meltzer, Erta Kalanxhi, Svein Dueland, Kjersti Flatmark, Kathrine Røe Redalen, Anne Hansen Ree. Oxaliplatin-induced immune factors and long-term disease control in rectal cancer - a reflection of immunogenic tumor cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4006.

Cancer-Associated Fibroblasts from Lung Tumors Maintain Their Immunosuppressive Abilities after High-Dose Irradiation

Accumulating evidence supports the notion that high-dose (>5 Gy) radiotherapy (RT) regimens are triggering stronger pro-immunogenic effects than standard low-dose (2 Gy) regimens. However, the effects of RT on certain immunoregulatory elements in tumors remain unexplored. In this study, we have investigated the effects of high-dose radiotherapy (HD-RT) on the immunomodulating functions of cancer-associated fibroblasts (CAFs). Primary CAF cultures were established from lung cancer specimens derived from patients diagnosed for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immunomodulation in experiments with peripheral blood mononuclear cells from random, healthy donors. Regulation of lymphocytes behavior was checked by lymphocyte proliferation assays, lymphocyte migration assays, and T-cell cytokine production. Additionally, CAF-secreted immunoregulatory factors were studied by multiplex protein arrays, ELISAs, and by LC-MS/MS proteomics. In all functional assays, we observed a powerful immunosuppressive effect exerted by CAF-conditioned medium on activated T-cells (p > 0.001), and this effect was sustained after a single radiation dose of 18 Gy. Relevant immunosuppressive molecules such as prostaglandin E2, interleukin-6, and -10, or transforming growth factor-β were found in CAF-conditioned medium, but their secretion was unchanged after irradiation. Finally, immunogenic cell death responses in CAFs were studied by exploring the release of high motility group box-1 and ATP. Both alarmins remained undetectable before and after irradiation. In conclusion, CAFs play a powerful immunosuppressive effect over activated T-cells, and this effect remains unchanged after HD-RT. Importantly, CAFs do not switch on immunogenic cell death responses after exposure to HD-RT.