Irradiated Tumor Cells Research Articles

Alpha Particles Induce Autophagy in Multiple Myeloma Cells.

Radiation emitted by the radionuclides in radioimmunotherapy (RIT) approaches induce direct killing of the targeted cells as well as indirect killing through the bystander effect. Our research group is dedicated to the development of α-RIT, i.e., RIT using α-particles especially for the treatment of multiple myeloma (MM). γ-irradiation and β-irradiation have been shown to trigger apoptosis in tumor cells. Cell death mode induced by (213)Bi α-irradiation appears more controversial. We therefore decided to investigate the effects of (213)Bi on MM cell radiobiology, notably cell death mechanisms as well as tumor cell immunogenicity after irradiation. Murine 5T33 and human LP-1 MM cell lines were used to study the effects of such α-particles. We first examined the effects of (213)Bi on proliferation rate, double-strand DNA breaks, cell cycle, and cell death. Then, we investigated autophagy after (213)Bi irradiation. Finally, a coculture of dendritic cells (DCs) with irradiated tumor cells or their culture media was performed to test whether it would induce DC activation. We showed that (213)Bi induces DNA double-strand breaks, cell cycle arrest, and autophagy in both cell lines, but we detected only slight levels of early apoptosis within the 120 h following irradiation in 5T33 and LP-1. Inhibition of autophagy prevented (213)Bi-induced inhibition of proliferation in LP-1 suggesting that this mechanism is involved in cell death after irradiation. We then assessed the immunogenicity of irradiated cells and found that irradiated LP-1 can activate DC through the secretion of soluble factor(s); however, no increase in membrane or extracellular expression of danger-associated molecular patterns was observed after irradiation. This study demonstrates that (213)Bi induces mainly necrosis in MM cells, low levels of apoptosis, and autophagy that might be involved in tumor cell death.

Abstract IA03: Turning a tumor into a vaccine factory: In situ vaccination for low-grade lymphoma

Abstract Background: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients' non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. Methods: Flt3L – the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of: intratumoral Flt3L administration to increase DC within the tumorlow-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, andintratumoral poly-ICLC administration to activate tumor antigen-loaded DC. Results: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for >6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced >10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Conclusions: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study. Research supported by: Damon Runyon Cancer Research Foundation Clinical Investigator Award Lymphoma Research Foundation Clinical Research Mentorship Program Citation Format: Thomas Marron, Nina Bhardwaj, Elizabeth Crowley, Tibor Keler, Thomas Davis, Andres Salazar, Joshua Brody. Turning a tumor into a vaccine factory: In situ vaccination for low-grade lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA03.

Abstract A17: The role of radiation-induced TGF-β in the generation of MDSCs

Abstract Myeloid derived suppressor cells (MDSCs) are a subset of myeloid precursors that are only present in pathological situations such as pathogen infection and cancer. Very little is known about the regulation of this cells as well as the regulation of its immunosuppressive function. We are interested in whether MDSC abundance is regulated by tumor cells secreted factors as a means to evade anti-tumor immune responses and how this might be affected by radiation therapy. Our hypothesis is that radiation therapy activation of TGFβ, which has a prominent role in the induction and maintenance of MDSC, promotes MDSC in the tumor microenvironment, thereby limiting tumor control. To test this we first established co-cultures of tumor cell lines and freshly isolated PBMCs from normal donors. MDSC are rarely in healthy donor mono-cultures (less than 1%). However, co-culture with either MDA-MB231 breast cancer cell line or with U118-MG glioma cancer cells resulted in a significant enrichment in the population of MDSCs (up to 12%). We then isolated cells based on CD33 expression for functional characterization. Compared to CD33- cells, the CD33+ population expresses significantly higher levels of ARG1, PDL1, IL10 and TGFB1, which are markers of active MDSC. This model will be modified by co-culture with irradiated tumor cell lines in conjunction with TGFβ inhibitors in ongoing studies. We can postulate that the combination of radiotherapy and TGFβ inhibitors, which are nowadays in clinical trials for different cancer types, could have important therapeutic benefits. Citation Format: Alba Gonzalez-Junca, Ilenia Pelicciotta, Kyla Elizabeth Discroll, Mary Helen Barcellos-Hoff. The role of radiation-induced TGF-β in the generation of MDSCs. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A17.

531 MVX-ONCO-1: First in man, Phase I clinical trial combining encapsulation cell technology and irradiated autologous tumor cells for personalized cell-based immunotherapy. Safety, feasibility and clinical outcome results

Reference EPFL-CONF-214198View record in Web of Science Record created on 2015-12-02, modified on 2016-08-09

Abstract 993: The relationship between autophagy, senescence, and DNA repair

Abstract Radiotherapy is used along with other modalities such as surgery, chemotherapy, and immunotherapy to either shrink tumors before surgery or kill surviving tumor cells post surgery. Although radiation (and chemotherapy) induce different modes of cell death, recurrence of cancer may occur. It has been shown that DNA damage can induce cells to undergo both autophagy and senescence where both responses can function either as a pro-survival mechanism or in promoting loss of self-renewal capacity in the tumor cell. The relationship between autophagy, senescence, and the DNA repair system has not been well-characterized. The primary aims of this work were to understand whether autophagy and senescence might be permissive for DNA repair, thereby allowing the tumor cells to overcome the persistent DNA damage and recover using HCT116 WT and HCT116 Ligase IV (-/-) human colon carcinoma cells. Irradiation caused HCT116 Ligase IV (-/-) cells to lose their capacity to proliferate followed by delayed cell death, whereas HCT116 WT cells showed proliferative recovery 72 hours post-treatment, consistent with deficiencies in DNA repair in the Ligase IV (-/-) cells. Irradiation of both cell lines resulted in enlargement in cell size and accumulation of acidic vacuoles, indicative of the induction of autophagy. Inhibition of autophagy by Bafilomycin and Choroquine resulted in radio-sensitization in HCT116 WT cells but not in HCT116 Ligase IV (-/-) cells, indicating that radiation-induced autophagy in HCT116 WT cells is likely to be cytoprotective. Both repair competent HCT116 colon carcinoma cells and Ligase IV deficient cells demonstrated significant increases in senescent populations upon irradiation based on β-galactosidase staining and enlargement of treated cells. The PARP inhibitor, Olaparib, enhanced the efficacy of radiation through interference with DNA repair and the promotion of both autophagy and senescence but not apoptosis. Taken together, it appears that both autophagy and senescence may facilitate DNA repair during exposure to genotoxic stress, and that autophagy and senescence are closely linked responses in irradiated tumor cells. Citation Format: Moureq R. Alotaibi, Lawrence Povirk, Daivd Gewirtz. The relationship between autophagy, senescence, and DNA repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2015-993

Abstract SY39-03: Radiation-mediated DNA sensing pathway for immunity and tumor regression

Abstract It is thought that ablative radiotherapy (RT), used for rapid control tumor growth, induces genotoxic stress and mitosis crisis leading to prolonged dormancy of irradiated tumor at local and distal site in most patients. We have unexpectedly observed that initial reduction of tumor burden following ablative RT also depends largely on type I IFN for CTL. Targeting tumor with IFN can control tumor growth through initiating a coordinated innate and adaptive immune attack against tumor cells. However, the mechanism for radiation-mediated type I IFN induction remains unclear. Here, we demonstrated that STING, but not MYD88, was required for type I IFN-dependent antitumor effects of radiation. STING in dendritic cells (DCs) controlled radiation-mediated IFN-β induction and was activated by irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) mediated DCs sensing of irradiated-tumor cells. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-β treatment rescued cGAS/STING-deficient immune responses. Accordingly, enhancing STING signaling by cGAMP administration promoted antitumor efficacy of radiation. Our results reveal that the molecular mechanism of radiation-mediated antitumor immunity depends on a proper cytosolic DNA-sensing pathway, pointing towards a new understanding of radiation and host interactions. Furthermore, we uncover a new strategy to improve radiotherapy by cGAMP treatment. RT induced IFN can also upregulate PD-L1 that suppress T cell-mediated damage and develop radiation resistance for relapse over time. Anti-PDL-1 antibody can greatly reduce radiation resistance leading to complete tumor regression. Furthermore, our study challenges the rationale for current radio/chemotherapy strategies and highlights the importance of immune activation in preventing tumor relapse. Citation Format: Yang-xin Fu. Radiation-mediated DNA sensing pathway for immunity and tumor regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY39-03. doi:10.1158/1538-7445.AM2015-SY39-03

Abstract 4140: Role of nitric oxide in invasiveness of tumor cells irradiated with carbon-ion beams

Abstract We previously reported that carbon-ion (C-ion) irradiation suppresses the invasiveness of several pancreatic cancer cell lines such as MIAPaCa-2, BxPC-3, and AsPC-1; however, we also observed that C-ion irradiation enhanced PANC-1 invasion. Most invading PANC-1 cells were nitric oxide (NO)-producing cells; the NOS-NO-PI3K-AKT pathway was activated in these cells. In addition, the actin-binding AKT substrate, Girdin, which modulates the actin organization and has important role in AKT-dependent cell motility, was activated and co-localized with p-AKT at the lamellipodia. C-ion irradiation increased the NO-producing cell population, thereby enhancing C-ion irradiation-enhanced PANC-1 invasion. In contrast to PANC-1 cells, NO levels as well as the number of NO-producing cells were decreased in C-ion irradiated MIAPaCa-2, indicated that the NO reduction might cause the suppression of MIAPaCa-2 invasion. The aim of this study is to clarify the role of NO in altered invasiveness of C-ion irradiated tumor cells. Treatment of PANC-1 with NO donor, DETA/NO, enhanced PANC-1 invasion. Interestingly, DETA/NO also increased MIAPaCa-2 invasion, indicated that NO contributes to the enhancement of MIAPaCa-2 invasiveness. The reduced invasiveness of C-ion irradiated MIAPaCa-2 was slightly but significantly recovered by the treatment with DETA/NO, but the recovered levels were still much less than the invasiveness of non-irradiated MIAPaCa-2. Thus, the reduction of NO levels involves in the decreased invasiveness of C-ion irradiated MIAPaCa-2, but there are additional factor regulating the invasiveness of cells irradiated with C-ion beams. Indeed, C-ion irradiation reduced GTP-Rac1 and GTP-RhoA expression, the active form of Rac1 and RhoA, which are known as two master regulators of cell motility. The reduction of GTP-Rac1 or GTP-RhoA was recovered by the treatment of proteasome inhibitor, indicated that those proteins were undergo degradation via the ubiquitin-proteasome pathway. So far, IAPs, Inhibitors of Apoptosis Proteins, and HACE1, HECT-domain containing E3 ubiquitin-ligase, were reported as direct E3 ubiquitin ligase of Rac1. Of those, XIAP was selectively induced and was co-precipitated with GTP-Rac1 in C-ion irradiated MIAPaCa-2. In conclusion, NO has a significant role in enhancing invasive potential of both PANC-1 and MIAPaCa-2 cells. The alteration of NO levels upon C-ion irradiation modulates the invasiveness of irradiated cells. Also, reductions of GTP-Rac1 and GTP-RhoA have the additional effects on the C-ion reduced MIAPaCa-2 invasion. Several studies have reported that NO modulates ubiquitin-proteasomal degradation of proteins. The effect of NO on the GTP-Rac1 and GTP-RhoA degradation remain unknown, and further researches are needed to clarify whether the NO reduction affects to the GTP-Rac1 or GTP-RhoA degradation observed in C-ion irradiated MIAPaCa-2. Citation Format: Mayumi Fujita, Kaori Imadome, Yoshimi Shoji, Robert Cheng, Aparna H. Kesarwala, David A. Wink, Takashi Imai. Role of nitric oxide in invasiveness of tumor cells irradiated with carbon-ion beams. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4140. doi:10.1158/1538-7445.AM2015-4140

Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells--A Key Role for Heat-Shock Protein 70 and Receptor CD91.

Immune responses contribute to the success of radiotherapy of solid tumors; however, the mechanism of triggering CD8(+) T-cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8(+) T-cell stimulation. We established a cross-presentation model in which DCs present a naturally expressed oncofetal tumor antigen (5T4) from irradiated DU145 prostate cancer cells to 5T4-specific T cells. The aim was to establish which immunogenic signals are important in radiation-induced cross-presentation. Radiation (12 Gy) caused G2-M cell-cycle arrest and cell death, increased cellular 5T4 levels, high-mobility protein group-B1 (HMGB1) release, and surface calreticulin and heat-shock protein-70 (Hsp70) expression in DU145 cells. DCs phagocytosed irradiated tumor cells efficiently, followed by upregulation of CD86 on phagocytic DCs. CD8(+) 5T4-specific T cells, stimulated with these DCs, proliferated and produced IFNγ. Inhibition of HMGB1 or the TRIF/MyD88 pathway only had a partial effect on T-cell stimulation. Unlike previous investigators, we found no evidence that DCs carrying Asp299Gly Toll-like receptor-4 (TLR4) single-nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, pretreatment of tumor cells with Hsp70 inhibitors resulted in a highly statistically significant and robust prevention of antigen cross-presentation and CD86 upregulation on DCs cocultured with irradiated tumor cells. Blocking the Hsp70 receptor CD91 also abolished cross-presentation. Together, the results from our study demonstrate that irradiation induces immunologically relevant changes in tumor cells, which can trigger CD8(+) T-cell responses via a predominantly Hsp70-dependent antigen cross-presentation process.

Increased HMGB1 and cleaved caspase-3 stimulate the proliferation of tumor cells and are correlated with the poor prognosis in colorectal cancer.

BackgroundDying tumor cells after irradiation could promote the proliferation of living tumor cells might cause tumor relapse and treatment failure. Our previous study showed that activated caspase-3 after irradiation probably participates in tumor repopulation. In this study, we investigated whether high mobility group box 1(HMGB1) is also involved in tumor repopulation.MethodsColorectal tumor cells were irradiated. The cleaved caspase-3 (CC3) in irradiated tumor cells and HMGB1 in the supernatant of irradiated tumor cells were detected by Western blot. A large number of irradiated colorectal tumor cells (feeder cells) were then co-cultured with a small number of luciferase-labeled living colorectal tumor cells (reporter cells) and proliferation of reporter cells was measured by bioluminescence imaging. The CC3 and HMGB1 protein expression in colorectal tumor and peritumoral tissues were detected by immunohistochemistry and their correlation with prognosis were analyzed.ResultsThe irradiated colorectal tumor cells underwent apoptosis and necrosis and produced CC3 in tumor cells and HMGB1 in the supernatant of cultured cells. The increased expression of secretory HMGB1 correlated with CC3 level and proliferating cell nuclear antigen (PCNA) after irradiation in vitro. The irradiated dying cells remarkably stimulated living tumor cell proliferation. Interestedly, immunohistochemistry staining showed that positive HMGB1, CC3, and Ki67 expression were significantly higher in colorectal tumor tissues than in peritumoral tissues (p <0.01). The Kaplan-Meier survival analysis revealed that high HMGB1, CC3, and Ki67 levels were significantly associated with poor prognosis (p <0.05, p <0.01). Multivariate analysis using Cox proportional hazards model showed that TNM staging and HMGB1 were independent prognostic factors in patients with colorectal cancer (CRC) (p <0.01, p <0.001).ConclusionBoth apoptotic and necrotic cells could stimulate proliferation of living tumor cells, and the increased expression of CC3 and HMGB1 in tumor cells could be new markers for poor prognosis in colorectal cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0166-1) contains supplementary material, which is available to authorized users.

Phase III multicenter trial of eltrapuldencel-T: Autologous dendritic cells loaded with irradiated autologous tumor cells (DC-TC) in granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with metastatic melanoma (INTUS trial).

TPS9082 Background: Melanomas harbor non-synonymous mutations that can result in unique, immunogenic tumor associated antigens (TAA). Recognition of TAA can be induced or enhanced by vaccination. T...

Enhanced protective immunity derived from dendritic cells with phagocytosis of CD40 ligand transgene-engineered apoptotic tumor cells via increased dendritic cell maturation.

Dendritic cells (DCs) play a pivotal role in regulating CD8+ cytotoxic T-lymphocyte (CTL) responses. Currently, DC vaccines have been used in experimental animal models and clinical trials for evaluation of antitumor immunity. However, their efficacy is limited, warranting the improvement of DC-based cancer vaccines. CD40 ligand (CD40L) stimulates DC activation and maturation via CD40-CD40L interaction. We demonstrated that DCs that had phagocytized apoptotic tumor cells induced antitumor immunity. We generated CD40L-expressing (EG7-CD40L) and the control (EG7-Null) EG7 tumor cells by transfection of EG7 tumor cells with CD40L-expressing adenoviral vector AdVCD40L and the control vector AdV(pLpA), respectively. We also generated DC vaccines (DC-EG7/CD40L and the control DC-EG7/Null) using DCs with phagocytosis of irradiated EG7-CD40L and EG7-Null tumor cells, and assessed their phenotype and immunogenicity by flow cytometry and animal studies in C57BL/6 mice. We demonstrate that an irradiation of 9000-rad induced Annexin V-expressing cell apoptosis in most (~75%) tumor cells, and provide evidence for phagocytosis of apoptotic tumor cells by flow cytometry and confocal microscopy. The DC-EG7/CD40L cells showed higher expression of DC maturation markers (Ia(b), CD40, CD80, and CD86) and peptide/major histocompatibility complex I than the control DC-EG7/Null cells. In addition, DC-EG7/CD40L vaccine stimulates more efficient (0.97%) tumor-specific CTL responses than DC-EG7/Null cells (0.31%). Furthermore, 80% (4/5) of mice immunized with DC-EG7/CD40L vaccine become tumor-free after EG7 tumor cell challenge, whereas DC-EG7/Null vaccine only delays immunized mouse death. Dendritic cells that have phagocytized CD40L-expressing apoptotic tumor cells appear to offer new strategies in DC cancer vaccines.

Radiotherapy combined with immune checkpoint blockade immunotherapy: Achievements and challenges

To date, several kinds of immunomodulating monoclonal antibodies (mAbs) have been applied in clinical trials, such as anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) mAb and anti-programmed death-1 (anti-PD-1) mAb. With the recent success of cancer immunotherapy, especially the checkpoint inhibitors, the renewed interest in immunotherapy as a treatment modality has gained extensive attention. The irradiated tumor cell death can enhance antitumor immunity by inducing antigen expression on tumor cells and activating lymphocytes. Radiotherapy (RT) combined with immunotherapy has revealed promising outcomes in various animal models. However, this new paradigm is often considered as a medical spectacle without a unifying model, and its mechanisms have yet to be elucidated. The purpose of this review is to investigate previously published studies of radiotherapy combined with checkpoint blockades by the following aspects: exploring the potential mechanisms; identifying the most beneficial dose, fraction and target site for RT; finding an appropriate time window to combine these two treatments; and discussing the toxicity and suitable treatment evaluating criteria.

Unusual immunogenicity of live cancer cells (TUM6P.957)

Abstract The ability of irradiated tumor cells to elicit tumor immunity has been widely established. Using two murine tumor models, we observe that injection of about 20,000 live tumor cells fails to develop tumors in recipient mice. (About ten times that dose leads to a successful tumor implantation.) However, mice injected with this sub-tumorigenic dose of cells become resistant to a subsequent lethal challenge. Remarkably, as many as 20 million (M) irradiated tumor cells (i.e. 1,000 times the sub-tumorigenic dose) are required to elicit resistance to tumor challenge. We assumed that the live cells in the sub-tumorigenic dose multiply in vivo and generate a large immunizing dose, before they are rejected. Indeed, imaging in vivo of the mice challenged with the sub-tumorigenic dose revealed that the tumor cells multiply at the site of challenge ~100 fold to ~2M cells over a week followed by a rapid decline. Thus the total antigen load in a sub-tumorigenic live inoculum (2M cells) is still only about 10% of the antigenic load in an irradiated cell inoculum (20M cells). While we explore the mechanisms of the unusual immunogenicity of live cells, we presently conclude (i) that higher immunogenicity is not a direct function of antigen load, and (ii) live cells cross-prime the naïve T cell response in a way that is distinctly different from, and more efficient than irradiated cells.

Enhanced dendritic cell-based immunotherapy using low-dose cyclophosphamide and CD25-targeted antibody for transplanted Lewis lung carcinoma cells.

Regulatory T cells (Tregs) is one of the main obstacles to the success of cancer immunotherapy. The effect of dendritic cell (DC)-based immunotherapy can be attenuated by immune suppressive functions of Tregs. We used a CD25-targeted antibody and low-dose cyclophosphamide (CTX) as immunomodulators to increase the antitumor effect of intratumoral injection of immature DCs into the irradiated tumor cells (IR/iDC). CTX or CD25-targeted antibody alone showed a significant reduction in the number of Tregs within the tumor microenvironment. When they are combined with IR/iDC, the number of Tregs was further reduced. Although IR/IDC showed strong antitumor effects such as reduction in tumor growth, increase in Th1 immune response, and improvement of survival, the therapeutic effect was further improved by combining treatments with immunomodulators. CTX and CD25-targeted antibody showed no significant difference in tumor growth when combined with IR/iDC, but CTX further increased the number of interferon (IFN)-γ-secreting T cells, cytotoxicity, and survival rate. Although irradiation induced depletion of T lymphocytes, administration of DCs recovered this depletion. Particularly, the lymphocytes were more significantly increased when CTX and IR/iDC were combined. Low-dose CTX has already been used as an immunomodulator in clinical trials, and it offers several advantages, including convenience, low-cost, and familiarity to clinicians. However, CD25-targeted antibody cannot only deplete Tregs, but also may affect IL-2-dependent effector T lymphocytes. Therefore, CTX is an effective means to inhibit Tregs, and an effective immunomodulatory agent for multimodality therapy such as combination treatment of conventional cancer therapy and immunotherapy.

Quantitative proteomic analysis of single or fractionated radiation-induced proteins in human breast cancer MDA-MB-231 cells.

BackgroundRadiotherapy is widely used to treat cancer alone or in combination with surgery, chemotherapy, and immunotherapy. However, damage to normal tissues and radioresistance of tumor cells are major obstacles to successful radiotherapy. Furthermore, the immune network around tumors appears to be connected to tumor progression and recurrence.MethodsWe investigated the cytosolic proteins produced by irradiated tumor cells by using a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture. MDA-MB-231 breast cancer cells were treated with a single or fractionated 10 Gray dose of 137Cs γ-radiation, which was selected based on cell viability.ResultsRadiation-induced proteins were differentially expressed based on the fractionated times of radiation and were involved in multiple biological functions, including energy metabolism and cytoskeleton organization. We identified 46 proteins increased by at least 1.3-fold, and high ranks were determined for cathepsin D, gelsolin, arginino-succinate synthase 1, peroxiredoxin 5, and C-type mannose receptor 2.ConclusionThese results suggest that a number of tumor-derived factors upregulated by γ-radiation are promising targets for modulation of the immune response during radiation treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-3701-5-2) contains supplementary material, which is available to authorized users.

Phase III randomized, double-blinded, placebo-controlled multicenter trial of Eltrapuldencel-T: autologous dendritic cells loaded with irradiated autologous tumor cells (DC-TC) in GM-CSF in patients with metastatic melanoma

Phase III randomized, double-blinded, placebo-controlled multicenter trial of Eltrapuldencel-T: autologous dendritic cells loaded with irradiated autologous tumor cells (DC-TC) in GM-CSF in patients with metastatic melanoma

Combining radiation therapy with immunotherapy: clinical translation

Ionizing radiation induces immunogenic cell death of tumors, an effect likely to contribute to the success associated with radiotherapy of cancer [1]. Recent discovery suggests that radiotherapy can be applied as a powerful adjuvant to immunotherapy and, in fact, can contribute to convert the irradiated tumor into an in situ vaccine, resulting in specific immunity against metastases [2]. Preclinical models of syngeneic tumors have reliably predicted clinical success, in distinct tumor settings and immunotherapy/radiation combinations [3-5]. As a first proof of principle trial, we translated the preclinical evidence of a successful combination with Flt3 ligand and RT [6] to a protocol of GM-CSF and RT, and demonstrated out of field objective responses in 27% of patients with multiple metastases of solid tumors, defined as an abscopal effect [7]. Parallel mechanistic studies in the lab in the syngeneic 4T1 mouse model of metastatic breast cancer demonstrated at intratravital microscopy that RT with anti-CTLA-4 increased the arrest of T cells in contact with tumor cells. The latter required interaction of NKG2D on CD8+ T cells with its ligand retinoic acid early inducible-1 (Rae-1) on the tumor cells, up-regulated by RT. Blocking NKG2D-Rae-1 interactions increased markedly the motility of anti-CTLA-4 treated T cells inhibiting their contact with irradiated tumor cells, and abrogated immune-mediated tumor rejection, suggesting a critical role of radiation-induced NKG2D ligands for the antitumor effects of anti-CTLA-4 [8]. In humans, a similar block of NKG2D is mediated by soluble MICA (sMICA), which is released by some tumors and reaches high levels in the serum [9]. Dranoff et al reported that in some patients sMICA levels dropped after initiation of Ipilimumab, due to the generation of anti-sMICA antibodies that led to its clearance [10]. Decreased levels of sMICA were associated with increased expression of NKG2D in T and NK cells, and corresponded to response to treatment. Anti-sMICA antibodies and sMICA levels can be measured in serum with ELISA by using recombinant MICA protein and anti-human MICA monoclonal antibodies [10]. Since RT is known to upregulate MICA on the surface of tumor cells [11] biopsies of tumors before and after radiotherapy and Ipilimumab could also be tested for expression of MICA.The preclinical success of the combination of anti-CTLA-4 antibody and RT was mirrored by abscopal responses in metastatic melanoma and NSCLC patients irradiated to one lesion, during Ipilimumab. This evidence inspired our current trial testing radiotherapy with CTLA-4 blockade in metastatic melanoma. In this study patients with newly diagnosed metastatic melanoma eligible to first line Ipilimumab are randomly assigned to Ipilimumab alone or Ipilimumab and radiotherapy to one metastatic lesion. Preliminary results in seven patients demonstrate feasibility of the combination, without additive toxicity. The novel role of radiotherapy as a powerful adjuvant to immunotherapy warrants more research to define the optimal immunotherapy/RT combinations: currently 35 trials of RT+immunotherapy are ongoing in USA.

OX40 costimulation acts synergistically with GVAX boosting the elimination of tumor cells in a murine melanoma model

Event Abstract Back to Event OX40 costimulation acts synergistically with GVAX boosting the elimination of tumor cells in a murine melanoma model Andrea J. Manrique Rincón1, 2, Camila Marques Beraldo1, Jessica Marcelino Toscaro1, Anna C. Pereira Vieira De Carvalho1 and Marcio C. Bajgelman1, 2* 1 National Center For Research in Energy and Materials – CNPEM, Brazilian Biosciences National Laboratory– LNBio , Brazil 2 University of Campinas, Medical School, Brazil GM-CSF-expressing tumor cell-based vaccine (GVAX) induces a potent antitumor response and is considered as a gold standard to compare efficacy of new strategies that are being developed to produce specific anti-tumor immunity. Although GVAX has surprising results in animal models, there is no comparable success in the clinic. This observation can be associated to the complexity of the immune system and low immunogenicity of tumor cells that may be protected by immunosuppressive mechanisms which mediate activation and proliferation of effectors lymphocytes. The costimulatory TNFSF receptors can modulate phenotypic properties of T cell, reducing the activation threshold, inducing a greater level of proliferation and preventing cell death after activation. In this work, we evaluate the anti tumor effect of modified B16F10 irradiated tumor cells expressing TNFRSF4, also know as OX40 ligand (CD134), which receptor is expressed on the surface of activated T cells and play a role in the maintenance of the immune response.We have performed in vivo assays in C57BL/6 mice that were challenged with wild type B16 F10 tumor cells and treated with modified B16F10 irradiated cells. We compare tumor progression in 4 groups of mice divided according to the modified B16 F10 they received (A) PBS, (B) GVAX, (C) OX40L and (D) combination of GVAX and OX40L. Our data suggests that OX40L induces the same reduction in tumor mass as the gold standard GVAX, and the combination of the two approaches provides a synergistic effect, enhancing the elimination of tumor cells. Acknowledgements The authors acknowledge to the funding agency FAPESP. Process Number 2013/02041-6. Keywords: Immunotherapy, Tumor cell vaccine, Melanoma, GVAX, OX40 Ligand Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Tumor immunology Citation: Manrique Rincón AJ, Marques Beraldo C, Marcelino Toscaro J, Pereira Vieira De Carvalho AC and Bajgelman MC (2015). OX40 costimulation acts synergistically with GVAX boosting the elimination of tumor cells in a murine melanoma model. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00067 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 May 2015; Published Online: 14 Sep 2015. * Correspondence: PhD. Marcio C Bajgelman, National Center For Research in Energy and Materials – CNPEM, Brazilian Biosciences National Laboratory– LNBio, CAMPINAS, Sao Paulo, 13083-970, Brazil, marcio.bajgelman@lnbio.cnpem.br Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Andrea J Manrique Rincón Camila Marques Beraldo Jessica Marcelino Toscaro Anna C Pereira Vieira De Carvalho Marcio C Bajgelman Google Andrea J Manrique Rincón Camila Marques Beraldo Jessica Marcelino Toscaro Anna C Pereira Vieira De Carvalho Marcio C Bajgelman Google Scholar Andrea J Manrique Rincón Camila Marques Beraldo Jessica Marcelino Toscaro Anna C Pereira Vieira De Carvalho Marcio C Bajgelman PubMed Andrea J Manrique Rincón Camila Marques Beraldo Jessica Marcelino Toscaro Anna C Pereira Vieira De Carvalho Marcio C Bajgelman Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of Irradiated Tumor Cell Lysate Vaccine and Low Doses of Gamma Irradiation in Tumor Regression

Role of Irradiated Tumor Cell Lysate Vaccine and Low Doses of Gamma Irradiation in Tumor Regression

Immunity to the vacuolar ATPase complex accessory unit ATP6S1 in patients with malignant melanoma.

The augmentation of high-titer antibodies to ATP6S1 is associated with favorable clinical outcomes in patients who received vaccination with autologous, irradiated tumor cells engineered to secrete GM-CSF and allogeneic bone marrow transplantation. Cellular immune responses to ATP6S1 are unknown. To define its role as an immune target, examination of cellular responses to ATP6S1 and immunity related to current therapies such as checkpoint blockade is needed. We used an overlapping peptide library representing the full-length ATP6S1 protein to screen for cellular responses from the peripheral blood of patients with stage III and IV melanoma. Reactive peptide pools were used to determine the individual peptide activity and epitopes. Recombinant ATP6S1 protein was used in an ELISA to assess potential correlation with humoral immune responses and changes in immunity related to CTLA-4 blockade with ipilimumab in these patients. We observed a broad array of CD4(+) and CD8(+) cellular responses against ATP6S1, including the identification of several MHC class I and II ATP6S1 epitopes. The generation of specific CD4(+) and cytotoxic T cells revealed potent functional capability elicited by ipilimumab treatment in patients with metastatic melanoma, which revealed potent functional capability, including cytokine production, proliferation responsiveness to melanoma cell lines, and tumor-cell killing. Furthermore, the augmented humoral immune responses to ATP6S1 as a function of ipilimumab treatment were associated with beneficial clinical outcomes. These results support the continued development of ATP6S1 as a biomarker and therapeutic target.