Irradiated Tumor Cells Research Articles

Oxidized mitochondrial DNA sensing by STING signaling promotes the antitumor effect of an irradiated immunogenic cancer cell vaccine

Exposure to ionizing radiation, a physical treatment that inactivates live tumor cells, has been extensively applied to enhance the antitumor responses induced by cancer cell vaccines in both animal research and human clinical trials. However, the mechanisms by which irradiated cells function as immunogenic tumor vaccines and induce effective antitumor responses have not been fully explored. Here, we demonstrate that oxidized mitochondrial DNA (mtDNA) and stimulator of interferon genes (STING) signaling play a key roles in the enhanced antitumor effect achieved with an irradiated tumor cell vaccine. Elevations in ROS and oxidized mtDNA 8-OHG content could be induced in irradiated tumor cells. Oxidized mtDNA derived from irradiated tumor cells gained access to the cytosol of dendritic cells (DCs). Oxidized mtDNA, as a DAMP or adjuvant, activated the STING-TBK1-IRF3-IFN-β pathway in DCs, which subsequently cross-presented irradiated tumor cell-derived antigens to CD8+ T cells and elicited antitumor immunity. The results of our study provide insight into the mechanism by which an irradiated cell vaccine mediates antitumor immunity, which may have implications for new strategies to improve the efficacy of irradiated vaccines.

Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-presenting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9-/- tumors abolishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effectively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.

Irradiated tumor cell-derived microparticles mediate tumor eradication via cell killing and immune reprogramming.

Radiotherapy (RT) is routinely used in cancer treatment, but expansion of its clinical indications remains challenging. The mechanism underlying the radiation-induced bystander effect (RIBE) is not understood and not therapeutically exploited. We suggest that the RIBE is predominantly mediated by irradiated tumor cell-released microparticles (RT-MPs), which induce broad antitumor effects and cause immunogenic death mainly through ferroptosis. Using a mouse model of malignant pleural effusion (MPE), we demonstrated that RT-MPs polarized microenvironmental M2 tumor-associated macrophages (M2-TAMs) to M1-TAMs and modulated antitumor interactions between TAMs and tumor cells. Following internalization of RT-MPs, TAMs displayed increased programmed cell death ligand 1 (PD-L1) expression, enhancing follow-up combined anti-PD-1 therapy that confers an ablative effect against MPE and cisplatin-resistant MPE mouse models. Immunological memory effects were induced.

Orphan designation: Two allogeneic irradiated pancreatic tumour cell lines, Treatment of pancreatic cancer

Orphan designation: Two allogeneic irradiated pancreatic tumour cell lines, Treatment of pancreatic cancer

Radiation-induced small extracellular vesicles as "carriages" promote tumor antigen release and trigger antitumor immunity.

Rationale: Accumulating evidence supports the importance of radiation therapy in the induction of antitumor immunity. Small extracellular vesicles (sEVs) play essential roles in tumor antigen loading and delivery. However, the role of sEVs in radiation-induced antitumor immunity remains unclear. It is therefore important to determine the role and regulatory mechanisms of sEVs in radiation-induced immunity.Methods: Tumor cells were irradiated (8 Gy), and sEVs were purified via ultracentrifugation. Primary tumor and experimental lung metastasis models were established in mice to evaluate antitumor immunity triggered by immunization with sEVs. Proteomic and bioinformatic analyses were performed to identify altered cargos in sEVs induced by radiation. Peptides derived from up-regulated proteins in sEVs were designed and synthesized as vaccines according to major histocompatibility complex (MHC) I binding and immunogenicity.Results: Here, we demonstrated that sEVs derived from irradiated tumor cells could trigger antitumor immunity against primary tumor and experimental lung metastasis by enhancing CD8+ and CD4+ T cell infiltration. Radiation may also enrich sEVs with tumor antigens and heat-shock proteins. Furthermore, CUB domain-containing protein 1 (CDCP1) derived from radiation-induced sEVs was identified as a novel tumor-associated antigen and developed as a peptide vaccine that may generate antitumor immune responses.Conclusions: Our results demonstrate that the use of sEVs secreted by irradiated tumor cells constitutes an efficient approach for tumor antigen delivery and presentation and highlight the role of sEVs in radiation-triggered antitumor immunity.

Non Habitual Immunotherapy by Using Irradiated Whole Tumor Cells

Non Habitual Immunotherapy by Using Irradiated Whole Tumor Cells

Virus specific tolerance enhanced efficacy of cancer immuno-virotherapy

BackgroundActivation of the immune system to fight cancer is a major goal in immunology and oncology. Although cancer treatment using oncolytic viruses shows promising results, virus mediated oncolysis induces a weak anti-tumor immune response. Upon application of viruses, immune responses against the virus play a significant role in limiting tumor virotherapy. Although suppression of host immunity increases the efficacy of virotherapy against the tumor, but inhibits anti-tumor immune responses. Induction of viral specific tolerance before viral replication may cause the virus to efficiently replicate in tumor cells without affecting the immune responses against tumor antigens. Investigation of the combined strategy of virotherapy and immunotherapy using irradiated tumor cells along with IL-2 and interferon-alpha in virus specific tolerant mice was the goal of this study. Materials and methodsFor tolerance induction, the newborn mice were injected with vesicular stomatitis virus (VSV) subcutaneously. After injection of TC-1 tumor cells to adult tolerant mice and formation of a tumor, irradiated TC-1 cells along with IL-2 and Interferon-alpha expression plasmid were injected twice in mice and followed by virotherapy. Size of tumors and CTL activity against the virus and tumor cells were measured. ResultThe results showed increased efficacy of virotherapy in combination with immune-stimulators and tumor cells injection in tolerant mice compared to normal mice. ConclusionSpecific tolerance against the oncolytic virus enhances the efficacy of virotherapy both in monotherapy and in combination with immunotherapy.

Irradiated lactic acid-stimulated tumour cells promote the antitumour immunity as a therapeutic vaccine.

Low pH and lactate accumulation are characteristic features of the tumour microenvironment. With our study finding that high concentrations of lactic acid can inhibit tumour growth, it is conceivable that the administration of lactic acid could promote antitumour immunity in the context of a tumour vaccine. To test this concept, we studied the antitumour effect of irradiated tumour cells stimulated with lactic acid in mouse xenograft models to potentially improve whole-cell tumour vaccines. In this study, we found that the effects of lactic acid-stimulated tumour cells on dendritic cells (DCs) included enhancing phagocytic function and stimulating maturation and aggregation. Moreover, lactic acid could potentiate the immunogenicity of an irradiated whole-tumour cell vaccine and thus inhibit tumour growth. We further verified that the antitumour immune response was mediated by CD8+ T cells. In addition, interferon-γ-expressing CD4+ and CD8+ T cell numbers increased in the spleen and lymph nodes of mice immunized with the irradiated lactic acid-stimulated cells. Furthermore, changes in the tumour microenvironment were also observed in this study. Our findings may be helpful for developing a new strategy for cancer therapy.

Necroptosis regulates tumor repopulation after radiotherapy via RIP1/RIP3/MLKL/JNK/IL8 pathway

BackgroundTumor cell repopulation after radiotherapy is a major cause for the tumor radioresistance and recurrence. This study aims to investigate the underlying mechanism of tumor repopulation after radiotherapy, with focus on whether and how necroptosis takes part in this process.MethodsNecroptosis after irradiation were examined in vitro and in vivo. And the growth-promoting effect of necroptotic cells was investigated by chemical inhibitors or shRNA against necroptosis associated proteins and genes in in vitro and in vivo tumor repopulation models. Downstream relevance factors of necroptosis were identified by western blot and chemiluminescent immunoassays. Finally, the immunohistochemistry staining of identified necroptosis association growth stimulation factor was conducted in human colorectal tumor specimens to verify the relationship with clinical outcome.ResultsRadiation-induced necroptosis depended on activation of RIP1/RIP3/MLKL pathway, and the evidence in vitro and in vivo demonstrated that the inhibition of necroptosis attenuated growth-stimulating effects of irradiated tumor cells on living tumor reporter cells. The JNK/IL-8 were identified as downstream molecules of pMLKL during necroptosis, and inhibition of JNK, IL-8 or IL-8 receptor significantly reduced tumor repopulation after radiotherapy. Moreover, the high expression of IL-8 was associated with poor clinical prognosis in colorectal cancer patients.ConclusionsNecroptosis associated tumor repopulation after radiotherapy depended on activation of RIP1/RIP3/MLKL/JNK/IL-8 pathway. This novel pathway provided new insight into understanding the mechanism of tumor radioresistance and repopulation, and MLKL/JNK/IL-8 could be developed as promising targets for blocking tumor repopulation to enhance the efficacy of colorectal cancer radiotherapy.

Effect of Ionizing Radiation on Human EA.hy926 Endothelial Cells under Inflammatory Conditions and Their Interactions with A549 Tumour Cells.

Purpose Most tumours are characterized by an inflammatory microenvironment, and correlations between inflammation and cancer progression have been shown. Endothelial cells (ECs), as part of the tumour microenvironment, play a crucial role in inflammatory processes as well as in angiogenesis and could be critical targets of cancer therapy like irradiation. Therefore, in the present study we investigated the effect of ionizing radiation on endothelial cells under inflammatory conditions and their interactions with tumour cells. Methods Nonactivated and TNF-α treatment-activated human EC EA.hy926 were irradiated with doses between 0.1 Gy and 6 Gy with a linear accelerator. Using a multiplex assay, the accumulation of various chemokines (IL-8, MCP-1, E-selectin, and P-selectin) and soluble adhesion molecules (sICAM-1 and VCAM-1) as well as protein values of the vascular endothelial growth factor (VEGF) was measured in the supernatant at different time points. The adhesion capability of irradiated and nonirradiated A549 tumour cells to EA.hy926 cells was measured using flow cytometry, and the migration of tumour cells was investigated with a scratch motility assay. Results In contrast to unirradiated cells, IR of ECs resulted in a modified release of chemokines IL-8 and MCP-1 as well as the adhesion molecules sICAM-1 and VCAM-1 in the EC, whereas concentrations of E-selectin and P-selectin as well as VEGF were not influenced. IR always affected the adhesion capability of tumour cells to ECs with the effect dependent on the IR-treated cell type. TNF-α treatment generally increased adhesion ability of the tumour cells. Tumour cell migration was clearly inhibited after IR. This inhibitory effect was eliminated for radiation doses from 0.5 to 2 Gy when, additionally, an inflammatory environment was predominant. Conclusions Our results support past findings suggesting that ECs, as part of the inflammatory microenvironment of tumours, are important regulators of the actual tumour response to radiation therapy.

Syngeneic B16-F1 cells are more efficient than allogeneic Cloudman cells as antigen source in DC-based vaccination in the B16-F1 murine melanoma model

A major obstacle to obtaining relevant results in cancer vaccination has been the lack of identification of immunogenic antigens. Dendritic cell (DC)-based cancer vaccines used preventively may afford protection against tumor inoculation, but the effect of antigen choice on anti-tumor protection is not clear. When using irradiated syngeneic tumor cells to load DCs, tumor self-antigens are provided, including tumor-associated antigens (TAAs) and neoantigens generated by tumor mutations. On the other hand, allogeneic tumor cells could only supply shared TAAs. To assess the advantages of each source in protective vaccination, we analyzed in C57BL/6 mice the effect of loading DCs with irradiated syngeneic B16-F1 or allogeneic Cloudman melanoma cells; both cell lines were characterized by whole exome sequencing and RNAseq. Tumor cell components from the two irradiated cell lines were efficiently internalized by DCs, and transported to MHC-class II positive tubulovesicular compartments (MIICs). DCs loaded with allogeneic irradiated Cloudman cells (DC-ApoNecALLO) induced a partially effective anti-melanoma protection, although Cloudman and B16-F1 cells share the expression of melanocyte differentiation antigens (MDAs), cancer-testis antigens (CTAs) and other TAAs. DCs loaded with syngeneic B16-F1 cells (DC-ApoNecSYN) established a more potent and long-lasting protection and induced a humoral anti-B16F1 response, thus suggesting that neoepitopes are needed for inducing long-lasting protection.

Abstract 1357: Serum levels of programmed cell death molecule-1 (PD-1) as a biomarker in metastatic melanoma patients randomized for treatment with autologous dendritic cell or tumor cell vaccines

Abstract The purpose of this study was to analyze PD-1 serum levels, in patients with metastatic melanoma who were enrolled in a randomized phase 2 trial, to determine: [1] if baseline PD-1 serum levels were prognostic for overall survival (OS), [2] if baseline PD-1 levels were predictive for survival benefit in the specific treatment arms, [3] if PD-1 levels changed between week-0 and week-4 by treatment, and [4] if changes in PD-1 levels were predictive of survival benefit overall and by treatment. Patients with metastatic melanoma were enrolled in a randomized phase 2 clinical trial comparing treatment with autologous dendritic cell vaccines (DCV) presenting autologous tumor antigens (ATA) derived from irradiated cancer cells from short-term autologous cell lines, compared to tumor cell vaccines (TCV) consisting of irradiated tumor cells [Dillman et al. J ImmunoTher Cancer 2018]. Vaccines were injected s.c. weekly for three weeks, then at weeks 8, 12, 16, 20, and 24. OS was longer for patients treated with DCV (median 43 vs 20 months; 70% reduction in risk of death). Cryopreserved serum samples from week-0 and week-4 for 39/42 patients were analyzed using a quantitative, multiplex enzyme-linked immunosorbent assay (Raybiotech, Inc., Norcross, GA). The patients for whom PD-1 levels were missing were two TCV-treated subjects who had rapidly progressive disease and had no week-4 samples, and one DCV-treated subject who survived more than 5-years, but rescinded permission to test his blood. Baseline serum PD-1 levels ranged from 0 to 511 ng/ml. After three weekly vaccinations, median serum levels of PD-1 did not change between week-0 and week-4 (1.51 and 1.58 ng/ml), nor did mean serum levels change (52.5 and 52.6 ng/ml). For all 39 patients, baseline PD1 levels were not prognostic for OS. DCV was associated with longer OS than TCV both in patients whose baseline PD1 was <1.2 ng/ml and > 1.2 ng/ml. Among DCV-treated subjects, baseline levels <50 ng/ml were associated with OS > 3.5 years in 9/13 compared to 0/4 for those with levels > 50 ng/ml (p=0.029), but there was no difference in the TCV arm (5/19 vs 1/3, p=1.00). Overall, PD1 levels decreased after 3 DCV injections, but increased after 3 TCV injections. DCV-treated subjects who had a decrease in PD-1 were more likely to survive 4 years (6/9 vs 1/8, p=0.05) but this difference was not seen in patients treated with TCV (0/6 vs 6/16, p=0.289). CONCLUSONS: In these metastatic melanoma patients, baseline serum PD1 levels were not prognostic for survival, but were predictive of survival benefit in patients treated with DCV, as was a decrease in PD1 level after 3 weekly DCV vaccinations. PD1 levels should be prospectively evaluated in larger studies to see if they may be useful as a companion diagnostic and/or surrogate marker for efficacy. Citation Format: Robert O. Dillman, Gabriel I. Nistor, Aleksandra J. Poole, Andrew N. Cornforth. Serum levels of programmed cell death molecule-1 (PD-1) as a biomarker in metastatic melanoma patients randomized for treatment with autologous dendritic cell or tumor cell vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1357.

Tumor Cells Modified with Newcastle Disease Virus Expressing IL-24 as a Cancer Vaccine

Interleukin-24 (IL-24) is a promising agent for cancer immunotherapy that induces apoptosis of tumor cells and enhances T cell activation and function. In order to improve the antitumor activity induced by Newcastle disease virus (NDV)-modified tumor vaccine, we generated a recombinant NDV expressing IL-24 using reverse genetics. Irradiated tumor cells infected with LX/IL-24 showed stable IL-24 expression. The cytotoxicity assay showed that LX/IL-24-infected murine melanoma cells significantly enhanced the antitumor immune response in vitro. Then, the antitumor effects of virus-infected tumor cells were examined in the murine tumor models. LX/IL-24-infected tumor cells exhibited strong antitumor effects both in prophylaxis and therapeutic models. LX/IL-24-infected tumor cells increased infiltration of CD4+ T cells and CD8+ T cells in tumor sites, and the antitumor activity of the tumor vaccine modified with LX/IL-24 was dependent on CD8+ T cells. Taken together, our data well illustrates that LX/IL-24-modified tumor cells are a promising agent for cancer immunotherapy.

Survival by stage and tumor measurability in metastatic melanoma patients treated with autologous dendritic cell tumor cell vaccines.

2637 Background: Survival of cancer patients is greatly affected by stage and tumor burden. The purpose of this study was to determine survival for melanoma patients who were treated with patient specific vaccines in the context of prospective clinical trials, by cohorts defined by stage and tumor measurability. Methods: Metastatic melanoma patients were treated with autologous dendritic cells loaded with antigens from irradiated cells from short-term autologous tumor cell lines (DCV). All patients had a metastatic melanoma lesion surgically resected, from which a tumor cell line was established. Irradiated tumor cells (ITC) were incubated with autologous dendritic cells (DC) to produce the DCV, which were injected s.c. in 500 micrograms GM-CSF weekly x 3 weeks, then monthly for 5 months. Data was pooled for DCV-treated patients enrolled in either of two phase II trials: one single-arm (NCT00948480), one randomized (NCT00436930). Patients were assigned to one of three cohorts based on their most advanced stage of disease prior to treatment, and whether they had measurable disease at the time of treatment. Survival was determined per Kaplan and Meier. Results: The final therapeutic products consisted of autologous DC with non-phagocytosed ITC making up 0% to 20% of cells in the final product. There were 45 men and 27 women. Median age was 52 years (range 17 to 83). Tumor sources were 37-lymph node, 20-viscera, and 15-soft tissue. No patients were lost to follow up; all surviving patients were followed 5 years. Toxicity was minimal. Median overall survival (OS) for all 72 patients was 49.4 mos; 5-year OS 46%. There was no correlation between survival and the number of DC or ITC in the first three injections. Patients with recurrent stage 3 disease that had not recurred (n=18) had a 72% 5-year OS; patients with non-measurable stage 4 (n=30) had a 53% 5-year OS. Patients with measurable stage 4 (n=18) had received an average of four prior therapies. They had a median OS of 18.5 months, and 2-year OS of 46%. Conclusions: This patient-specific DCV was associated with encouraging survival in all three clinical subsets. Because of its mechanism of action and absence of toxicity, it should be evaluated further. Clinical trial information: NCT00948480, NCT00436930.

Unbiased functional identification and therapeutic targeting of T cell neoantigens in a spontaneous murine squamous cell carcinoma

Abstract The comparative resistance of some cancers including head and neck squamous cell carcinoma (HNSCC) to checkpoint blockade is speculated to derive from the low frequency of expressed somatic mutations targeted by T cells as neoantigens (NeoAg). SCC VII, a spontaneously arising murine squamous carcinoma resembling human HNSCC in several key features, is likewise poorly immunogenic as irradiated tumor cells alone fail to induce protective immunity in syngeneic hosts. Justifying use of this model to identify NeoAgs, we confirm activated CD4+ and CD8+ T cells are detectable and essential for vaccine efficacy of SCC VII and polyI:C co-administration. Whole-exome sequencing tumor versus normal genome identified 39 nonsynonymous missense mutations that were synthesized into 81 representative 20-mers. NeoAg-specific CD4+ T cell IFN-γ responses were found against mutations of Pik3ca, Ctnnd1, and Otud5 while both CD4+ and CD8+ T cells produced IFN-γ when stimulated by a single Cltc mutation containing overlapping minimal epitopes during in vitro recall assays. NeoAgs eliciting CD4+ T cell responses alone were not protective in vivo. However, the more immunogenic Cltc NeoAg prophylactically protected hosts from tumor challenge and caused regression of established tumors as an immunotherapy. Anti-PD-1 combinatorial blockade resulted in synergistic tumor rejection by boosting Cltc-specific responses and increasing response diversification via epitope spreading. These data show that a functional NeoAg identification platform can be used to select immunotherapeutically relevant targets and filtration of neoepitopes that co-prime both CD4+ and CD8+ T cell responses is superior for practical intervention of poorly immunogenic tumors.

The immune mediated role of extracellular HMGB1 in a heterotopic model of bladder cancer radioresistance

Radical cystectomy (RC) together with bilateral pelvic lymph node dissection remains the standard treatment for muscle invasive bladder cancer (MIBC). However, radiation-based treatments such as tri-modal therapy (TMT) involving maximally performed transurethral resection of bladder tumor (TURBT), radiotherapy (XRT), and a chemosensitizer represent an attractive, less invasive alternative. Nevertheless, 25–30% of MIBC patients will experience local recurrence after TMT and half will develop metastasis. Radioresistance of tumor cells could potentially be one of the causes for local recurrence post treatment. High mobility group box-1 (HMGB1) was shown to play a role in bladder cancer radioresistance through its intracellular functions in promoting DNA damage repair and autophagy. Recently, HMGB1 was found to be passively released from irradiated tumor cells. However, less is known about the involvement of extracellular HMGB1 in impairing radiation response and its exact role in modulating the tumor immune microenvironment after XRT. We identified a novel mechanism of bladder cancer radioresistance mediated by the immunological functions of HMGB1. The combination of radiation plus extracellular HMGB1 inhibition markedly improved the radiation response of tumors and resulted in marked changes in the immune landscape. Moreover, combining radiation and HMGB1 inhibition significantly impaired tumor infiltrating MDSCs and TAMs -but not Tregs- and shifted the overall tumor immune balance towards anti-tumoral response. We conclude that extracellular HMGB1 is involved in bladder cancer radioresistance through promoting pro-tumor immune mechanisms.

Cyclooxygenase-2-Mediated Up-Regulation of Mitochondrial Transcription Factor A Mitigates the Radio-Sensitivity of Cancer Cells.

Mitochondrial transcription factor A (TFAM) regulates mitochondrial biogenesis, and it is a candidate target for sensitizing tumor during therapy. Previous studies identified that increased TFAM expression conferred tumor cells resistance to ionizing radiation. However, the mechanisms on how TFAM are regulated in irradiated tumor cells remain to be explored. In this research, we demonstrated the contribution of cyclooxygenase-2 (COX-2) to enhancing TFAM expression in irradiated tumor cells. Our results showed TFAM was concomitantly up-regulated with COX-2 in irradiated tumor cells. Inhibition of COX-2 by NS-398 blocked radiation-induced expression of TFAM, and prostaglandin E2 (PGE2) treatment stimulated TFAM expression. We next provided evidence that DRP1-mediated mitochondrial fragmentation was a reason for TFAM up-regulation in irradiated cells, by using small interfering RNA (siRNA) and selective inhibitor-targeted DRP1. Furthermore, we proved that p38-MAPK-connected COX-2, and DRP1-mediated TFAM up-regulation. Enhanced phosphorylation of p38 in irradiated tumor cells promoted DRP1 expression, mitochondrial fragmentation, and TFAM expression. NS-398 treatment inhibited radiation-induced p38 phosphorylation, while PGE2 stimulated the activation of p38. The results put forward a mechanism where COX-2 stimulates TFAM expression via p38-mediated DRP1/mitochondrial fragmentation signaling in irradiated tumor cells, which may be of value in understanding how to sensitize cancer cells during radiotherapy.

Th17 responses to autologous dendritic cell vaccine.

132 Background: Dendritic cells (DC) loaded with autologous tumor antigens in vitro, can induce a Th17 phenotype preceding a Th1 type response. We examined this in patients with metastatic melanoma who had been treated in a randomized trial with either DC loaded with autologous tumor antigens (ATA) from self-renewing tumor cells (DCV) or irradiated tumor cells (TCV). Methods: Blood samples were obtained at baseline, one week before three weekly vaccinations (week-0), and at week-4, (one week after the third vaccination). They were analyzed for growth factors, tumor markers and biomarkers using a quantitative, multiplex ELISA. Monocytes, lymphocytes, loaded and unloaded DC were analyzed for cytokine production and intercellular responses using mixed lymphocyte co-cultures (MLC). Results: When compared to baseline or the TCV arm, DCV-treated patients showed reductions in tumor markers, inflammation and angiogenesis in parallel with Th17 pathway activation, IL12 p70 heterodimer expression was detected in monocytes and DC only after antigen loading. The IL12 p40 fraction was present after differentiation and was enhanced after antigen loading. Similarly, the p19 component of IL23 was expressed before DC loading and later enhanced. MLC showed induction of Th17, Th1 and CD8 subpopulations. In the presence of antigen-loaded DCs, the FOXp3 cells were reduced in number, an effect that was canceled by neutralizing the IL12p40 subunit or by the adding tumor cells alone in the MLC. Conclusions: DC produced ex vivo can activate multiple cytotoxic pathways depending on IL12 subunits. Expression of IL23p19 and the shared IL12p40 fraction are responsible for Th1 priming (IL12 incomplete) and Th17 induction (IL23 complete), determinants of the reactive phenotype of naïve DCs. IL12 p70 heterodimer expression after DC loading enables a targeted Th1 type response while neutralizing the IL12 subunit p40, enhances Foxp3 positive T-regs. DC vaccines produced ex vivo could induce a Th1/Th17 cytotoxic response against tumors. In the absence of antigens, however, The Th17 phenotype can become tolerizing in the microenvironment. The findings are important for applications in settings in which tumor infiltration with Th17 correlates with survival, such as in advanced ovarian cancer. Clinical trial information: NCT00436930.

Autologous tumor cell vaccination combined with systemic CpG-B and IFN-\u03b1 promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial

In this study the toxicity and efficacy of an irradiated autologous tumor cell vaccine (ATV) co-injected with a class-B CpG oligodeoxynucleotide (CpG-B) and GM-CSF, followed by systemic CpG-B and IFN-α administration, were examined in patients with metastatic renal cell carcinoma (mRCC). A single-arm Phase II trial was conducted, in which patients with mRCC were intradermally injected with a minimum of three whole-cell vaccines containing 0.7–1.3 × 107 irradiated autologous tumor cells (ATC), admixed with 1 mg CpG-B and 100 µg GM-CSF, followed by bi-weekly s.c. injections with 8 mg CpG-B and s.c. injections with 6 MU IFN-α three times per week. Fifteen patients were treated according to the protocol. Treatment was well tolerated. Objective clinical responses occurred in three patients, including one long-term complete response. Disease stabilization occurred in another three patients. Positive delayed type hypersensitivity (DTH) responses to ATC were absent before treatment but present in 13 out of 15 patients during treatment. Immune monitoring revealed activation of plasmacytoid dendritic cells, non-classical monocytes and up-regulation of both PD-1 and CTLA4 on effector T cells upon treatment. Moreover, a pre-existing ex vivo IFN-γ response to ATC was associated with clinical response. ATV combined with systemic CpG-B and IFN-α is tolerable, safe, immunogenic and able to elicit anti-tumor responses in patients with mRCC. Immune activation and treatment-induced up-regulation of PD-1 and CTLA4 on circulating T cells further suggest an added benefit of combining this approach with immune checkpoint blockade [added]

Radiotherapy-induced immunogenic effects in bladder cancer.

418 Background: Many patients with bladder cancer (BC) undergo radiotherapy (RT) during the course of their treatment. There is emerging evidence that RT can cause immune stimulatory changes within the tumour microenvironment (TME), potentially contributing to its efficacy. We aimed to determine if RT induces immunogenic changes in murine BC cell lines, and develop a pre-clinical model of BC with a TME reflective of de novo tumours in order to test this premise in vivo. Methods: Immunogenic effects of RT were determined using murine vaccination studies with irradiated tumour cells. RT-induced immuno-phenotypic changes in surface antigen expression on tumour cells were ascertained using flow cytometry. An orthotopic BC model was established using MBT2 cells instilled intravesically in C3H/Hen mice, and resulting tumours monitored with ultrasound (US). We used immunohistochemical (IHC) staining to determine the immune contexture of the TME within developing orthotopic tumours. Results: C57BL/6 mice inoculated with irradiated MB49 cells demonstrated improved survival compared to control mice after subsequent rechallenge with viable tumour cells. This effect was not seen in C3H mice implanted with irradiated MBT2 cells. RT led to upregulation of immune stimulatory molecules CD80, MHC I, and Fas on MB49 but not MBT2 cells. Tumours developed in 80% of mice following catheter implant, and visible on US 3-4 weeks after instillation. Profiling of the TME with IHC demonstrated that tumours contained few CD8+ T-cells, but high numbers of myeloid cells. Conclusions: RT induces immune stimulatory effects on murine BC cells, including upregulation of several surface proteins. In future work, we will determine the effects of RT on the TME in the orthotopic model, and correlate these with the expression of various immunogenic cell surface proteins. This may lead to the discovery of a biomarker to predict which patients with BC would benefit from combination of an immunomodulatory agent with RT.