Antitumor Effect Of Radiation Research Articles

MEK inhibition enhances the antitumor effect of radiation therapy in NF1-deficient glioblastoma.

Individuals with neurofibromatosis type 1 (NF-1), an autosomal dominant neurogenetic and tumor predisposition syndrome, are susceptible to developing low-grade glioma (LGG) and, less commonly, high-grade glioma (HGG). These gliomas exhibit loss of the neurofibromin gene (NF1), and 10-15% of sporadic HGG have somatic NF1 alterations. Loss of NF1 leads to hyperactive RAS signaling, creating opportunity given the established efficacy of MEK inhibitors (MEKi) in plexiform neurofibromas and some individuals with LGG. We observed that NF1-deficient glioblastoma neurospheres were sensitive to the combination of a MEKi (mirdametinib) with irradiation, as evidenced by synergistic inhibition of cell growth, colony formation, and increased cell death. In contrast, NF1-intact neurospheres were not sensitive to the combination, despite complete ERK pathway inhibition. No neurosphere lines exhibited enhanced sensitivity to temozolomide combined with mirdametinib. Mirdametinib decreased transcription of homologous recombination genes and RAD51 foci, associated with DNA damage repair, in sensitive models. Heterotopic xenograft models displayed synergistic growth inhibition to mirdametinib combined with irradiation in NF1-deficient glioma xenografts, but not those with intact NF1. In sensitive models, benefits were observed at least three weeks beyond the completion of treatment, including sustained phosphor-ERK inhibition on immunoblot and decreased Ki-67 expression. These observations demonstrate synergistic activity between mirdametinib and irradiation in NF1-deficient glioma models and may have clinical implications for patients with gliomas that harbor germline or somatic NF1 alterations.

Abstract 5108: Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas

Abstract Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG) are among the most treatment refractory cancers in children. Despite multimodal therapy, prognosis is dismal with survival of <1 year for DIPG and a 5-year overall survival <10% for other pHGGs. Radiotherapy remains the only standard of care for DIPG that extends the survival by few months. In recent years, immunotherapy is emerging as a potential alternative treatment option. However, several factors have hindered the use of immunotherapy for pHGGs including “cold” immune environment with lack of immune cells infiltration, low tumor mutational burden, and lack of neo-antigen expression that prevents tumor recognition by the immune system. Therefore, novel therapies that activate the immune system while evading the tumor immunosuppression are highly needed. Here, we aim to demonstrate the immunomodulatory anti-tumor effects of THIO (6-thio-2’deoxyguanosine), a nucleoside analog and a telomerase-dependent telomere targeting agent, in DIPG mouse models. Our preclinical in vitro and in vivo data have demonstrated the efficacy of THIO in inducing telomeric damage, G2/M cell cycle arrest, and apoptosis in high-risk medulloblastoma and DIPG. Importantly, we have demonstrated that THIO crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. THIO was shown to induce anti-tumor immunity through micronuclei formation leading to cGAS-STING pathway mediated activation of the innate and adaptive immune response in colon, lung, and hepatocellular carcinoma tumor models. Our preliminary data demonstrate the THIO-dependent induction of micronuclei formation co-localizing with cGAS and leading to STAT1 activation in DIPG cell lines. We have also shown that THIO synergistically sensitizes DIPG cells to ionizing radiation (IR), significantly decreasing cell proliferation. We are currently testing the combination of THIO+IR in an orthotopic mouse model for DIPG. We will present results on the potential of THIO and IR treatments to stimulate anti-tumor immunity through the activation of STING pathway in a syngeneic mouse model of DIPG. These preclinical studies will support the potential use of THIO and IR to treat children with high-risk pediatric brain tumors. Citation Format: Umaru Banlanjo, Shiva Senthil Kumar, Sergei Gryaznov, Rachid Drissi. Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5108.

Injectable dexamethasone-loaded peptide hydrogel for therapy of radiation-induced ototoxicity by regulating the mTOR signaling pathway

Radiation-induced ototoxicity is associated with inflammation response and excessive reactive oxygen species in the cochlea. However, the effectiveness of many drugs in clinical settings is limited due to anatomical barriers in the inner ear and pharmacokinetic instability. To address this issue, we developed an injectable hydrogel called RADA32-HRN-dexamethasone (RHD). The RHD hydrogel possesses self-anti-inflammatory properties and can self-assemble into nanofibrous structures, ensuring controlled and sustained release of dexamethasone in the local region. Flow cytometry analysis revealed that the uptake of FITC-conjugated RHD gel by hair cells increased in a time-dependent manner. Compared to free dexamethasone solutions, dexamethasone-loaded RHD gel achieved a longer and more controlled release profile of dexamethasone. Additionally, RHD gel effectively protected against the inflammatory response, reduced excessive reactive oxygen species production, and reversed the decline in mitochondrial membrane potentials induced by ionizing radiation, leading to attenuation of apoptosis and DNA damage. Moreover, RHD gel promoted the recovery of outer hair cells and partially restored auditory function in mice exposed to ionizing radiation. These findings validated the protective effects of RHD gel against radiation-induced ototoxicity in both cell cultures and animal models. Furthermore, RHD gel enhanced the activity of the mammalian target of rapamycin (mTOR) signaling pathway, which was inhibited by ionizing radiation, thereby promoting the survival of hair cells. Importantly, intratympanic injections of RHD gel exhibited excellent biosafety and do not interfere with the anti-tumor effects of radiotherapy. In summary, our study demonstrates the therapeutic potential of injectable dexamethasone-loaded RHD hydrogel for the treatment of radiation-induced hearing loss by regulating the mTOR signaling pathway.

BRD4 Inhibition Enhances the Antitumor Effects of Radiation Therapy in a Murine Breast Cancer Model.

Bromodomain-containing protein 4 (BRD4) is an intracellular protein that regulates expression of various cellular functions. This study investigated whether BRD4 inhibition can alter the immunomodulatory and antitumor effects of radiation therapy (RT). A murine breast cancer cell line was implanted into BALB/c mice. The dual-tumor model was used to evaluate the abscopal effects of RT. A total of 24 Gy was delivered and BRD4 inhibitor was injected intravenously. Tumor size was measured, and in vivo imaging was performed to evaluate tumor growth. Flow cytometry and immunohistochemistry were performed to examine immunologic changes upon treatment. The combination of BRD4 inhibitor and RT significantly suppressed tumor growth compared to RT alone. BRD4 inhibitor reduced the size of the unirradiated tumor, indicating that it may induce systemic immune responses. The expression of HIF-1α and PD-L1 in the tumor was significantly downregulated by the BRD4 inhibitor. The proportion of M1 tumor-associated macrophages (TAMs) increased, and the proportion of M2 TAMs decreased upon BRD4 inhibition. BRD4 inhibitor expanded CD4+ and CD8+ T cell populations in the tumor microenvironment. Additionally, splenic monocytic myeloid derived suppressor cells, which were increased by RT, were reduced upon the addition of BRD4 inhibitor. Therefore, the addition of BRD4 inhibitor significantly enhanced the systemic antitumor responses of local RT.

Synergistic Antitumor Effect of Combined Radiotherapy and Engineered Salmonella typhimurium in an Intracranial Sarcoma Mouse Model.

Intracranial sarcoma is an uncommon aggressive cancer with a poor prognosis and a high recurrence rate. Although postoperative adjuvant radiotherapy (RT) is the most recommended treatment strategy, it does not significantly improve survival rates. In this study, we used an attenuated Salmonella typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (SLpFlaB) as an immunotherapy to assist with the antitumor effects of RT on intracranial sarcoma. In vitro, the expression of γH2AX and cleaved caspase-3 was analyzed by Western blot. In vivo detection of SLpFlaB colonization time in tumors was measured using an in vivo imaging system (IVIS). Tumor growth delay and elimination were demonstrated in an intracranial mouse model, and the distribution of macrophages, M1 macrophages, and CD8+ cells after treatment was measured using FACS analysis. Our findings in vitro suggest that combination therapy increases S-180 radiosensitivity, the expression of DNA double-strand breaks, and programmed cell death. In vivo, combination treatment causes intracranial sarcoma to be eliminated without tumor recurrence and redistribution of immune cells in the brain, with data showing the enhanced migration and infiltration of CD8+ T cells and macrophages, and an increased proportion of M1 macrophage polarization. Compared to RT alone, the combination therapy enhanced the radiosensitivity of S-180 cells, promoted the recruitment of immune cells at the tumor site, and prevented tumor recurrence. This combination therapy may provide a new strategy for treating intracranial sarcomas.

YTHDF2 inhibition potentiates radiotherapy antitumor efficacy.

RNA N6-methyladenosine (m6A) modification is implicated in cancer progression. However, the impact of m6A on the antitumor effects of radiotherapy and the related mechanisms are unknown. Here we show that ionizing radiation (IR) induces immunosuppressive myeloid-derived suppressor cell (MDSC) expansion and YTHDF2 expression in both murine models and humans. Following IR, loss of Ythdf2 in myeloid cells augments antitumor immunity and overcomes tumor radioresistance by altering MDSC differentiation and inhibiting MDSC infiltration and suppressive function. The remodeling of the landscape of MDSC populations by local IR is reversed by Ythdf2 deficiency. IR-induced YTHDF2 expression relies on NF-κB signaling; YTHDF2 in turn leads to NF-κB activation by directly binding and degrading transcripts encoding negative regulators of NF-κB signaling, resulting in an IR-YTHDF2-NF-κB circuit. Pharmacological inhibition of YTHDF2 overcomes MDSC-induced immunosuppression and improves combined IR and/or anti-PD-L1 treatment. Thus, YTHDF2 is a promising target to improve radiotherapy (RT) and RT/immunotherapy combinations.

Edaravone Exerts Protective Effects on Mice Intestinal Injury without Interfering with the Anti-Tumor Effects of Radiation.

The appropriate dosage of edaravone-a radioprotective agent-and its effect on tumors are unknown. This study evaluated the effects of edaravone on intestinal injuries and tumors in mice induced by whole body X-ray irradiation. Small intestinal mucositis was induced in C3H/HeNSlc mice using a single X-ray dose (15 Gy). Edaravone (15, 30, and 100 mg/kg) was administered 30 min before irradiation to evaluate its protective effect. After 3.5 days, the jejunum was removed and the histological changes were evaluated. Next, C3H/HeNSlc mice with squamous cell carcinoma VII tumors were provided the same single X-ray dose and 100 mg/kg edaravone; further, the tumors were immediately induced after irradiation. The tumor cell viability was detected using an in vivo-in vitro colony formation assay. We found that the intestinal colony-forming ability after irradiation was significantly higher in the 100 mg/kg edaravone group than that in the control group. Moreover, the apoptotic cells in the villi immunohistochemically stained with cleaved caspase-3 were significantly lower in the 100 mg/kg edaravone group than in the control group. We found no radioprotective effects of intraperitoneally inoculated edaravone in both hind legs on squamous cell carcinoma VII tumors. These findings suggest that 100 mg/kg edaravone exerts protective effects on small intestinal injuries without interfering with the antitumor effects of radiation.

Abstract 2812: NL-201, a de novo CD25-independent IL-2Rβγ agonist, synergizes with radiation to generate potent antitumor immunity

Abstract There is a growing interest in combining radiotherapy (RT) with immunotherapeutic agents to improve efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of high-dose recombinant IL-2 is limited by toxicity and the expansion of regulatory T cells (Tregs) through signaling events mediated by IL2Rα (CD25). Here we examined the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway dependency of type I interferon (IFN) response and anti-tumor effect of RT in combination with NL-201, a highly potent and stable CD25-independent IL-2 and IL-15 receptor agonist with enhanced affinity for IL2Rβ and IL2Rγc. In mice implanted with E0771 syngeneic tumors, NL-201 in combination with RT enhanced activation of the STING pathway. This was associated with increased type I IFN production as well as increased activation and number of tumor associated dendritic cells (DCs). In STING (-/-) mice implanted with E0771 tumors, Type I IFN production, tumor-growth inhibition (TGI), and survival advantage was significantly decreased and there were significantly fewer activated CD4+ and CD8+ T-cells. Similarly, CD11c+ knock-out mice implanted with E0771 tumors and treated with the combination exhibited similar TGI, survival, and IFN production compared to vehicle treated mice. Based on these results, we tested the ability of the combination of RT and NL-201 to enhance systemic immunotherapy with an anti-PD-1 checkpoint inhibitor in mice implanted with 4T1 breast tumor cells. Tumor-bearing mice treated with the triple combination exhibited superior TGI and survival, along with increased effector memory T-cell populations as compared to all other treatment groups. These results support our previous findings that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, synergizes with RT, likely via the direct activation of STING. Genetic knockout of STING in tumor-bearing mice abrogated the activity of the NL-201 and RT combination, thus confirming the crucial role of innate immune sensors in the combination treatment’s immune activation and resulting antitumor effect. These results also describe the important role of STING-mediated activation and tumor penetration of DCs, which were crucial for anti-tumor response. Additionally, when combined with anti-PD-1 antibodies, NL-201+RT combination treatment significantly improved TGI of metastatic breast cancers that were previously resistant to checkpoint blockade. This work provides rationale for the continued exploration of RT in combination with CD25-independent IL-2Rβγ agonism as a means to enhance the systemic immune response to treat immunologically challenging tumors. Citation Format: Kristin Huntoon, DaeYong Lee, Xuefeng Li, Yifan Wang, Wen Jiang, Betty Kim. NL-201, a de novo CD25-independent IL-2Rβγ agonist, synergizes with radiation to generate potent antitumor immunity [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 2812.

Abstract 6105: Single high-dose irradiation induced iRhom2 up-regulation promotes macrophage anti-tumor activity through cGAS/STING signaling

Abstract Clinical application of Stereotactic body radiotherapy (SBRT) allows high dose of radiation to be delivered to extracranial target within the body safely, however, high dose per fraction and with fewer fractions(hypofractionation) opened up the radiation oncology field to new questions on variety of dose-fractionation schedules, especially, the immunomodulatory effects of radiotherapy, which changes following the variety of dose-fractionation schedules. In the present study, we excluded the direct antitumor effect of radiation by comparing three different fractionation schedules –8*2Gy, 3*4.5Gy, 1*10Gy, which have the same biologically effective dose (BED). We found the fractionation schedules 1*10Gy had a significant higher antitumor effect, suggesting that a single high-dose induced a more enhanced anti-tumor immunity compared to the conventionally fractionation(8*2Gy) and moderately fractionation (3*4.5Gy). However, in STING deficiency mice, we failed to find a different tumor control effect among the three dose-fractionation schedules, suggesting cGAS/STING signaling was involved in regulating immunomodulatory effects that leading to different anti-tumor immunity. Further mechanism investigation revealed that single high-dose irradiation induced up-regulation of iRhom2 in infiltrated macrophage, and iRhom2 promoted cGAS/STING signaling via maintaining stability of STING and facilitating STING trafficking from ER to perinuclear microsomes. Meanwhile, a positive feedback loop between iRhom2 and TNFα sharpened the signaling responses, and amplified antitumor immunity induced by cGAS-STING-IFN pathway after single high-dose irradiation. Taken together, these results suggest that single high-dose irradiation induced iRhom2 up-regulation promotes macrophage anti-tumor activity through cGAS/STING signaling. Citation Format: Xiaomei Zhao, Shisuo Du, Biao Wang, Zhaochong Zeng. Single high-dose irradiation induced iRhom2 up-regulation promotes macrophage anti-tumor activity through cGAS/STING signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6105.

The mutual relationship between the host immune system and radiotherapy: stimulating the action of immune cells by irradiation.

The effects of irradiation on tumor tissue and the host immune system are interrelated. The antitumor effect of irradiation is attenuated in the immunocompromised hosts. In addition, radiation alone positively and negatively influences the host immune system. The positive effects of radiation are summarized by the ability to help induce and enhance tumor-antigen-specific immune responses. The cancer-immunity cycle is a multistep framework that illustrates how the tumor-antigen-specific immune responses are induced and how the induced antigen-specific immune cells exert their functions in tumor tissues. Irradiation affects each step of this cancer-immunity cycle, primarily in a positive manner. In contrast, radiation also has negative effects on the immune system. The first is that irradiation has the possibility to kill irradiated effector immune cells. The second is that irradiation upregulates immunosuppressive molecules in the tumor microenvironment, whereas the third is that irradiation to the tumor condenses immunosuppressor cells in the tumor microenvironment. When used in conjunction with radiotherapy, immune checkpoint inhibitors can further leverage the positive effects of radiation on the immune system and compensate for the negative effects of irradiation, which supports the rationale for the combination of radiotherapy and immune checkpoint inhibitors. In this review, we summarize the preclinical evidence for the reciprocal effects of radiation exposure and the immune system, and up-front topics of the combination therapy of immune checkpoint inhibitors and radiotherapy.

A Promising Treatment Strategy for Lung Cancer: A Combination of Radiotherapy and Immunotherapy.

Simple SummaryLung cancer is a leading cause of cancer-related deaths worldwide. In the past few decades, radiotherapy has achieved outstanding technical advances and is widely used in the management of lung cancer. The anti-tumor effect of radiotherapy is mainly caused by DNA damage in cancer cells within the irradiated field. In addition, radiotherapy induces anti-tumor immune responses that are essential in cancer control. Recently, immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated protein 4, programmed cell death-1/programmed death-ligand 1, and their inhibitors, have attracted significant attention for overcoming the immunosuppressive conditions in patients with cancer. Furthermore, some studies showed that the combination of immune checkpoint inhibitors and radiotherapy appears promising. In this review, we outlined evidence about the combination of radiotherapy, including particle therapy using protons and carbon ions, with immunotherapy in lung cancer treatment.Lung cancer is a leading cause of cancer-related deaths worldwide despite advances in treatment. In the past few decades, radiotherapy has achieved outstanding technical advances and is being widely used as a definitive, prophylactic, or palliative treatment of patients with lung cancer. The anti-tumor effects of radiotherapy are considered to result in DNA damage in cancer cells. Moreover, recent evidence has demonstrated another advantage of radiotherapy: the induction of anti-tumor immune responses, which play an essential role in cancer control. In contrast, radiotherapy induces an immunosuppressive response. These conflicting reactions after radiotherapy suggest that maximizing immune response to radiotherapy by combining immunotherapy has potential to achieve more effective anti-tumor response than using each alone. Immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated protein 4, programmed cell death-1/programmed death-ligand 1, and their inhibitors, have attracted significant attention for overcoming the immunosuppressive conditions in patients with cancer. Therefore, the combination of immune checkpoint inhibitors and radiotherapy is promising. Emerging preclinical and clinical studies have demonstrated the rationale for these combination strategies. In this review, we outlined evidence suggesting that combination of radiotherapy, including particle therapy using protons and carbon ions, with immunotherapy in lung cancer treatment could be a promising treatment strategy.

Abstract 1394: Poly ADP-ribose polymerase inhibitor promotes the immunoradiotherapeutical efficacy in small cell lung cancer

Abstract This study aimed to investigate the safety and efficacy of radiotherapy combined with poly ADP-ribose polymerase (PARP) inhibitors and immune checkpoint inhibitors in small cell lung cancer (SCLC). Tumor-bearing xenograft mouse model was used to examine the safety and anti-tumor effects of radiotherapy plus niraparib and PD-1 antibodies. SCLC cell lines were used to explore the underlying mechanisms in this combination. PARP inhibitor suppressed SCLC cell proliferation, induced cell apoptosis, and increased cell cycle arrest at G2/M phase. Niraparib treatment augmented radiation-induced double-strand DNA breaks, which subsequently activated the cGAS/STING pathway to increase the expression levels of CXCL10, CCL5 and PD-L1 in SCLC cells. Moreover, the combination of radiation, niraparib and PD-1 antibodies significantly inhibited SCLC tumor growth and prolonged the survival of immunocompetent C57BL/6 mice. The induced CCL5 and CXCL10 activated CD8+ T cells in vivo. Our study indicated that niraparib combined with radiation promoted immune checkpoint inhibitor functions via inducing PD-L1 expression, and activated CD8+ T cells via increasing CXCL10 and CCL5 levels through cGAS/STING pathway. The combination treatment of PARP inhibitors, radiotherapy and immunotherapy were safe in vivo, effectively suppressed SCLC tumor progression, increased survival and decreased the adverse events via lowering the dose of individual treatment. Citation Format: Nannan Zhang, Yanping Gao, Zihang Zeng, Yuan Luo, Xueping Jiang, Jianguo Zhang, Jiali Li, Zhengrong Huang, Yan Gong, Conghua Xie. Poly ADP-ribose polymerase inhibitor promotes the immunoradiotherapeutical efficacy in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1394.

Triple-modality therapy maximizes antitumor immune responses in a mouse model of mesothelioma.

Malignant pleural mesothelioma (MPM) is an intractable disease with an extremely poor prognosis. Our clinical protocol for MPM of subablative radiotherapy (RT) followed by radical surgery achieved better survival compared to other multimodal treatments, but local relapse and metastasis remain a problem. This subablative RT elicits an antitumoral immune response that is limited by the immunosuppressive microenvironment generated by regulatory T (Treg) cells. The antitumor effect of immunotherapy to simultaneously modulate the immune activation and the immune suppression after subablative RT has not been investigated in MPM. Herein, we demonstrated a rationale to combine interleukin-15 (IL-15) superagonist (IL-15SA) and glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) agonist (DTA-1) with subablative RT in mesothelioma. IL-15SA boosted the systemic expansion of specific antitumoral memory CD8+ T cells that were induced by RT in mice. Their effect, however, was limited by the up-regulation and activation of Treg cells in the radiated tumor microenvironment. Hence, selective depletion of intratumoral Treg cells through DTA-1 enhanced the benefit of subablative RT in combination with IL-15SA. The addition of surgical resection of the radiated tumor in combination with IL-15SA and DTA-1 maximized the benefit of RT and was accompanied by a reproducible abscopal response in a concomitant tumor model. These data support the development of clinical trials in MPM to test such treatment options for patients with locally advanced or metastatic tumors.

Enhance the Immune Checkpoint Inhibitors Efficacy with Radiotherapy Induced Immunogenic Cell Death: A Comprehensive Review and Latest Developments.

Simple SummaryEfficient antitumoral immune response is conditioned by a regulated cell death called immunogenic cell death. Many immunologic cell death inducers are currently used in cancer treatment. Among them, radiation therapy is an adaptable, well tolerated and widely used modality of treatment in modern oncology. Moreover, there is growing evidence for synergistic mechanisms between radiotherapy and immune checkpoint inhibitors. Although pre-clinical concepts are numerous, robust clinical evidence is scarce. Radioimmunology is a rapidly evolving discipline with several ongoing clinical trials. In this review, we (i) explain the rationale behind radiotherapy and immune checkpoint-inhibitor association in the light of the most recent knowledge, (ii) provide the results of the latest clinical trials evaluating radiation therapy and immune checkpoint association and (iii) explore the future directions of radioimmunology research.The immunogenic cell death (ICD) is defined as a regulated cell death able to induce an adaptive immunity. It depends on different parameters including sufficient antigenicity, adjuvanticity and favorable microenvironment conditions. Radiation therapy (RT), a pillar of modern cancer treatment, is being used in many tumor types in curative, (neo) adjuvant, as well as metastatic settings. The anti-tumor effects of RT have been traditionally attributed to the mitotic cell death resulting from the DNA damages triggered by the release of reactive oxygen species. Recent evidence suggests that RT may also exert its anti-tumor effect by recruiting tumor-specific immunity. RT is able to induce the release of tumor antigens, to act as an immune adjuvant and thus to synergize with the anti-tumor immunity. The advent of new efficient immunotherapeutic agents, such as immune checkpoint inhibitors (ICI), in multiple tumor types sheds new light on the opportunity of combining RT and ICI. Here, we will describe the biological and radiobiological rationale of the RT-induced ICD. We will then focus on the interest to combine RT and ICI, from bench to bedside, and summarize the clinical data existing with this combination. Finally, RT technical adaptations to optimize the ICD induction will be discussed.

Upregulation of KCNQ1OT1 promotes resistance to stereotactic body radiotherapy in lung adenocarcinoma by inducing ATG5/ATG12-mediated autophagy via miR-372-3p

Stereotactic body radiotherapy (SBRT) has emerged as a standard treatment for non-small-cell lung cancer. However, its therapeutic advantages are limited with the development of SBRT resistance. The SBRT-resistant cell lines (A549/IR and H1975/IR) were established after exposure with hypofractionated irradiation. The differential lncRNAs were screened by microarray assay, then the expression was detected in LUAD tumor tissues and cell lines by qPCR. The influence on radiation response was assessed via in vitro and in vivo assays, and autophagy levels were evaluated by western blot and transmission electron microscopy. Bioinformatics prediction and rescue experiments were used to identify the pathways underlying SBRT resistance. High expression of KCNQ1OT1 was identified in LUAD SBRT-resistant cells and tissues, positively associated with a large tumor, advanced clinical stage, and a lower response rate to concurrent therapy. KCNQ1OT1 depletion significantly resensitized A549/IR and H1975/IR cells to radiation by inhibiting autophagy, which could be attenuated by miR-372-3p knockdown. Furthermore, autophagy-related 5 (ATG5) and autophagy-related 12 (ATG12) were confirmed as direct targets of miR-372-3p. Restoration of either ATG5 or ATG12 abrogated miR-372-3p-mediated autophagy inhibition and radiosensitivity. Our data describe that KCNQ1OT1 is responsible for SBRT resistance in LUAD through induction of ATG5- and ATG12-dependent autophagy via sponging miR-372-3p, which would be a potential strategy to enhance the antitumor effects of radiotherapy in LUAD.

Abstract 2258: CD73 targeting therapy enhances the anti-tumor effect of radiation therapy in rectal cancer

Abstract [Background and Purpose]Extracellular adenosine exerts strong immunosuppressive activity and is considered as an important metabolic immune-checkpoint molecule. CD73 (ecto-5'-nucleotidase) is the rate-limiting enzyme to produce adenosine, and high CD73 expression in tumor tissue is linked to poor prognosis. However, the role of CD73/adenosine signaling in the response against radiation therapy (RT) is less understood. Here, we examined the anti-tumor effects of the combination of CD73 blockade and RT. [Method]LuM1, a highly metastatic subtype of colon26 (murine colon cancer cell) were subcutaneously (sc) inoculated in Balb/c mouse. At day 12,14 and16, 4Gy RT was locally applied selectively to sc tumor with lead shielding of other body. Blocking CD73 mAb (200μg/body) was intraperitoneally administrated at day 12,14,16,19,22 and 25. The mice were sacrificed at day 28 and the growth of irradiated tumor and non-irradiated lung metastasis was evaluated. For evaluating immune function, tumor and spleen were excised at day 18. In addition, expression of CD73 in 64 human rectal cancer after chemoradiotherapy (CRT) was examined with immunohistochemistry and evaluated for the impact on patient outcome. [Result]Flowcytometric analysis showed that LuM1 positively expressed CD73 which was significantly enhanced by 10Gy RT in vitro. Local RT (4Gy x 2) also enhanced the expression of CD73 in LuM1 of sc tumor (MFI: Median=177, 120-234 vs 65.5, 24.3-72.3, n= 5, p=0.0079). Local RT caused significant delay in growth of sc tumor. However, the addition of anti-CD73 mAb further reduced the tumor weight as compared with isotype control (Μ=0.15g, 0.004-0.29g vs 0.34g, 0.1-0.73g, n=8, p=0.0379). Interestingly, the number of metastases in non-irradiated lung was also significantly decreased by anti-CD73mAb (Μ=1, 0-30 vs 12, 1-70, n=8, p=0.040).The percentage of IFNγ (+)/CD4 (+) cells and IFNγ (+)/CD8a (+) cells of splenocytes were increased in RT +Anti-CD73 groups compared to RT +Anti-Isotype group. (Μ=9.95%, 7.71-16.6% vs 3.64%, 1.01-11.7%, n=6, p=0.0022 and Μ=15.5%, 11.9-24.1% vs 5.98%, 1.51-16.6%, n=6, p=0.041).In addition, the percentage of IFNγ (+)/CD4 (+) cells of tumor infiltrating lymphocytes was increased in RT +Anti-CD73 groups compared to RT +Anti-Isotype group. (Μ=12.2%, 9.77-18.2% vs 7.09%, 1.55-9.41%, n=6, p=0.0022).Among the patients who received CRT, 13 patients with high expression level of CD73 both in remnant tumor cells and stroma showed significantly worse recurrence free survival and overall survival than other 51 patients (p=0.0059 and p=0.0077). [Conclusion]RT enhances the tissue expression of CD73, which can provide large amounts of adenosine in irradiated tumor tissue and may seriously suppress the local immune response and impair the response against RT. Our results suggest that functional blockade of CD73 can enhance not only direct but also abscopal effects of RT possibly through T cell activations, which may improve the outcome of the patients with advanced rectal cancer. Citation Format: Hidenori Tsukui, Hisanaga Horie, Akira Saito, Koji Koinuma, Hideyuki Ohsawa, Hironori Yamaguchi, Yasunaru Sakuma, Yoshinori Hosoya, Kotaro Yoshimura, Alan Lefor, Naohiro Sata, Joji Kitayama. CD73 targeting therapy enhances the anti-tumor effect of radiation therapy in rectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2258.

Nuclear Medicine for Optimized Treatment Strategy and Real-Time Therapy Imaging

PET and SPECT provide us with information about biological quantitative values at localized area, even at the deep component of human. They usually have been used for diagnosis, but when combining with radiotherapy, PET and SPECT images would be helpful to make a decision on radiation-dose distribution for treatment because the patient images can be obtained in the similar condition under radiation therapy. In addition, anti-tumor effect of radiation therapy is now precisely controlled for target. Therefore, precise information of tumor, such as extent of tumor and heterogeneity within tumor, would make the treatment more effective, while normal organ functions are preserved. What nuclear medicine can do for the ideal treatment is discussed in this section.

Systemic Immunostimulatory Effects of Radiation Therapy Improves the Outcomes of Patients With Advanced NSCLC Receiving Immunotherapy.

The understanding of localized radiation therapy's immunostimulatory properties combined with its well-known effects on the cell cycle and insights into the immunomodulation mechanisms that occur at the molecular and cellular levels has changed our traditional view of the anticancer effects of ionizing radiation. The potential interactions between the tumor's immune system and radiation therapy have revealed that local radiation has the ability to induce systemic antitumor responses in patients with advanced cancers. The recognition of systemic antitumor effects of radiation therapy has allowed investigators to begin uncovering the integral players in these pathways. Parallel to this, there has been progress in understanding how tumor immunology leads to the development of novel immunotherapies using immune checkpoint blockade therapies in the treatment of advanced cancers. To date there has been limited success in this benefiting only a small fraction of patients. The concept of priming the body's immune system by radiation to make less responsive tumors more responsive to immunotherapy provides an opportunity to explore the use of the combination of radiation therapy and immunotherapy for the treatment of advanced non-small cell lung cancer and other cancers. This article provides an overview of the current state of knowledge of the clinical experience using radiation therapy in combination with immune therapy and discusses the rationale for integrating these 2 modalities in the treatment of advanced non-small cell lung cancer. Available data supports the use of radiation therapy in combination with immunotherapy to achieve improved local and systemic tumor control. Evidence from the early clinical trials has shown that using radiation therapy and immune checkpoint blockade therapies together produces a greater clinical effect than using either modality alone. To maximize the clinical benefit and successful integration of these two modalities as well as optimizing radiation therapy dosing and its schedule, improvement in its field design and the development of reliable predictors of clinical tumor response needs to be established.

Interleukin-2 boosts local and abscopal antitumor effect of radiotherapy combined with anti-PD-1: A translational research from clinical radio-memory effect.

e14244 Background: Preclinical and clinical studies have shown that prior receipt of radiotherapy enhances antitumor immune responses, a phenomenon we call the “radio-memory effect.” However, all of the evidence regarding this effect to date comes from work with anti-PD-1 or anti-PD-L1 inhibitors. Here we explore whether this effect also occurs with other forms of immune therapy, specifically interleukin-2 (IL-2). If so, whether this triple combination therapy could produce synergetic anti-tumor effect. Methods: 1. Clinical work: we retrospectively assessed outcomes in patients with malignant peritoneal effusion (MPE) who had previously received radiotherapy for colon or rectum cancer within 18 months before the intrapleural infusion of IL-2 or cisplatin. All patients received intrapleural infusion of IL-2 or cisplatin, and most had had several cycles of standard chemotherapy for colon or rectum cancer. 2. Translational work: MC38 cell line (5×106) was subcutaneously inoculated into the right lower extremity of C57/BL mice to construct a primary tumor model. Six days later, MC38 cell line (1×106) was subcutaneously inoculated into the left lower limb to construct a second tumor model. The main research is as follows: To explore the optimal radiation dose and fraction for radiotherapy; Whether the addition of IL-2 will further enhance the efficacy of radiotherapy combined with anti-PD-1; To explore the optimal combination model of radiation, anti-PD-1 and IL-2; Whether the addition of IL-2 will increase the abscopal effect produced by radiotherapy and anti-PD-1. Results: 1. We identified 3,747 patients with MPE (median age 66 years [range 24–81)) treated at one of several institutions from August 2009 through May 2016; 412 patients had been treated with IL-2 and 592 with cisplatin and had survived for at least 3 months afterward. 2. Hypofractionated radiotherapy (12Gy × 2) combined with IL-2 is better; IL-2 will enhance the efficacy of radiotherapy combined with anti-PD-1; IL-2 and anti-PD-1 followed by radiotherapy might maximize the anti-tumor effect; IL-2 might increase the abscopal effect produced by radiotherapy combined with anti-PD-1. Conclusions: We speculate that previous radiotherapy could enhance the efficacy of IL-2 and the anti-tumor effect produced by radiotherapy combined with anti-PD-1 could be enhanced by IL-2. The “radio-memory” effect could be beneficial in future studies.

Abstract 1326: Nonprotective autophagy fails to confer resistance to radiation

Abstract Radiation remains a predominant treatment option for advanced, inoperable lung cancer, however, resistance represents a major barrier against effective therapy. The exact mechanisms through which cancer cells exhibit resistance to radiotherapy still remains unresolved. Traditionally, radiation-induced autophagy is considered as a mechanism of resistance which attenuates the antitumor effects of radiotherapy i.e., autophagy plays a cytoprotective role on cell survival. To examine this concept further, we employed H460 non-small cell lung cancer (NSCLC) cells that were either p53 wild-type (p53 +/+) or p53 null (p53 -/-) to study the contribution of autophagy to radiation sensitivity. While autophagy was markedly higher in p53 +/+ cells (determined by p62 degradation, LC3B conversion, and acidic vesicle formation) the p53 +/+ cells were actually more radiosensitive than their counterpart H460 p53 -/- cells, which showed lower levels of radiation-induced autophagy. Despite modest radiosensitization by chloroquine, alternative pharmacological (3-methly adenine) and genetic (ATG5 silencing) inhibition of autophagy failed to radiosensitize either p53 +/+ or p53 -/- cells, indicating that radiation induced autophagy is not cytoprotective in function in these experimental models. Furthermore, secretome analysis (nLC-MS/MS) of p53 +/+ H460 cells showed no significant association with radioresistance-related proteins in comparison with p53 -/- H460 cells. The rate and extent of apoptosis was quite low and similar in the two cells lines, essentially ruling out apoptotic cell death as the basis for differential radiation sensitivity. Finally, Senescence was more pronounced in the p53 +/+ cells compared to the p53 -/- cells, which may contribute to the greater radiation sensitivity in the p53 +/+ cells. However, the most relevant finding in this work was that when autophagy is the nonprotective form, it does not confer inherent radiation resistance in tumor cells. Citation Format: Jingwen Xu, Emmanuel Kenneth Cudjoe, Tareq Saleh, Nipa Patel, Moureq Alotaibi, Yingliang Wu, Santiago Lima, David Gewirtz. Nonprotective autophagy fails to confer resistance to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1326.