Radiation-induced Tumor Cell Death Research Articles

Interleukin-1 receptor associated kinase 1 (IRAK1) is epigenetically activated in luminal epithelial cells in prostate cancer.

The use of immune adjuvants such as toll-like receptor (TLR) agonists reflects a novel strategy in prostate cancer (PCa) therapy. However, interleukin-1 receptor associated kinase 1 (IRAK1), a central effector of TLR signaling, has been shown to be responsible for resistance to radiation-induced tumor cell death. In order to better understand the function and epigenetic regulation of IRAK1 in PCa, we performed in vitro cell culture experiments together with integrative bioinformatic studies using the latest single-cell RNA-sequencing data of human PCa and normal prostate (NOR), and data from The Cancer Genome Atlas. We focused on key effectors of TLR signaling, the Myddosome-complex components IRAK1, IRAK4 and MYD88 (myeloid differentiation primary response 88), and TRAF6 (tumor-necrosis-factor receptor associated factor 6). In PCa, IRAK1-mRNA was specifically enriched in luminal epithelial cells, representing 57% of all cells, whereas IRAK4 and MYD88 were predominantly expressed in leukocytes, and TRAF6, in endothelial cells. Compared to NOR, only IRAK1 was significantly overexpressed in PCa (Benjamini-Hochberg adjusted p<2x10-8), whereas the expression of IRAK4, MYD88, and TRAF6 was unchanged in PCa, and IRAK1-expression was inversely correlated with a specific differentially methylated region (IRAK1-DMR) within a predicted promoter region enriched for H3K27ac (Spearman correlation r<-0.36; Fisher’s test, p<10-10). Transcription factors with high binding affinities in IRAK1-DMR were significantly enriched for canonical pathways associated with viral infection and carcinogenic transformation in the Kyoto Encyclopedia of Gene and Genomes analysis. DU145 cells, exhibiting hypermethylated IRAK1-DMR and low IRAK1-expression, reacted with 4-fold increased IRAK1-expression upon combined treatment with 5-aza-2-deoxycytidine and trichostatin A, and were unresponsive to infection with the uropathogenic Escherichia coli strain UTI89. In contrast, PC3 and LNCaP cells, exhibiting hypomethylated IRAK1-DMR and high endogenous IRAK1-mRNA levels, responded with strong activation of IRAK1-expression to UTI89 infection. In summary, exclusive overexpression of IRAK1 was observed in luminal epithelial cells in PCa, suggesting it has a role in addition to Myddosome-dependent TLR signaling. Our data show that the endogenous epigenetic status of PCa cells within IRAK1-DMR is decisive for IRAK1 expression and should be considered as a predictive marker when selective IRAK1-targeting therapies are considered.

Highlighting the Potential for Chronic Stress to Minimize Therapeutic Responses to Radiotherapy through Increased Immunosuppression and Radiation Resistance.

Simple SummaryStress is an integral part of life and is necessary for proper development and function of every organ. However, there is growing evidence that prolonged activation of the sympathetic stress response stress negatively affects the outcome of many diseases including cancer and impairs the efficacy of widely used therapies. In this review, we specifically focus on the potential mechanisms by which chronic stress could inhibit the efficacy of radiation therapy. We conclude that there is significant evidence for increased suppression of anti-tumor immune responses along with induction of tumor cell survival pathways. Because cancer patients are susceptible to many sources of stress, including stress associated with anxiety and depression, this survey provides a strong rationale for implementing stress-reduction strategies in patients who will be receiving radiation therapy.Ionizing radiation has been used in the treatment of cancer for more than 100 years. While often very effective, there is still a great effort in place to improve the efficacy of radiation therapy for controlling the progression and recurrence of tumors. Recent research has revealed the close interaction between nerves and tumor progression, especially nerves of the autonomic nervous system that are activated by a variety of stressful stimuli including anxiety, pain, sleep loss or depression, each of which is likely to be increased in cancer patients. A growing literature now points to a negative effect of chronic stressful stimuli in tumor progression. In this review article, we present data on the potential for adrenergic stress to influence the efficacy of radiation and in particular, its potential to influence the anti-tumor immune response, and the frequency of an “abscopal effect” or the shrinkage of tumors which are outside an irradiated field. We conclude that chronic stress can be a major impediment to more effective radiation therapy through mechanisms involving immunosuppression and increased resistance to radiation-induced tumor cell death. Overall, these data highlight the potential value of stress reduction strategies to improve the outcome of radiation therapy. At the same time, objective biomarkers that can accurately and objectively reflect the degree of stress in patients over prolonged periods of time, and whether it is influencing immunosuppression and radiation resistance, are also critically needed.

Antitumor, Inhibition of Metastasis and Radiosensitizing Effects of Total Nutrition Formula on Lewis Tumor-Bearing Mice.

Non-small-cell lung cancer (NSCLC) causes high mortality. Radiotherapy is an induction regimen generally applied to patients with NSCLC. In view of therapeutic efficacy, the outcome is not appealing in addition to bringing about unwanted side effects. Total nutrition is a new trend in cancer therapy, which benefits cancer patients under radiotherapy. Male C57BL/6JNarl mice were experimentally divided into five groups: one control group, one T group (borne with Lewis lung carcinoma but no treatment), and three Lewis lung carcinoma-bearing groups administrated with a total nutrition formula (T + TNuF group), a local radiotherapy plus daily 3 Gy in three fractions (T + R group), or a combination TNuF and radiotherapy (T + R + TNuF group). These mice were assessed for their mean tumor volumes, cachectic symptoms and tumor metastasis. TNuF administration significantly suppressed tumor growth and activated apoptotic cell death in NSCLC-bearing mice under radiation. The body-weight gain was increased, while the radiation-induced cachexia was alleviated. Analysis of mechanisms suggests that TNuF downregulates EGFR and VEGF signaling pathways, inhibiting angiogenesis and metastasis. In light of radiation-induced tumor cell death, mitigation of radiation-induced cachexia and inhibition of tumor cell distant metastasis, the combination of TNuF and radiotherapy synergistically downregulates EGFR and VEGF signaling in NSCLC-bearing mice.

Three discipline collaborative radiation therapy (3DCRT) special debate: Equipment development is stifling innovation in radiation oncology.

The field of radiation oncology has recently experienced a period of remarkable transformation of our technical capabilities. These technical and computational advancements have resulted in a tremendous improvement in our ability to precisely and accurately deliver radiation dose. However, this increased emphasis on technical capabilities may be inadvertently diverting attention and funding from scientific developments in other areas, even at a time when exciting new advances are occurring in cancer biology. Much of our recent technical development has been promoted and subsidized by the manufacturers of radiation oncology equipment. The question now becomes “who will encourage and subsidize the research needed to bring our recent biological advances to clinical fruition?” In a recent commentary which inspired this debate, Brown and Adler suggest that we “…are in a golden age of radiation and cancer biology” and that “…the industry's current focus on equipment development alone is undermining significant potential clinical advances in radiation oncology.”3 This is the subject of this month's 3DCRT debate. Arguing for the proposition will be Drs. Leonard Kim, Stephanie Markovina, and Samantha Van Nest. Dr. Leonard Kim has worked on defining microscopic disease target volumes for breast cancer radiotherapy at William Beaumont Hospital and Rutgers Cancer Institute of New Jersey. He is currently Chief Medical Physicist at the MD Anderson Cancer Center at Cooper. Dr. Stephanie Markovina is interested in improving therapeutic response to radiation in cervical cancer and other solid tumors by better understanding molecular mechanisms of radiation induced tumor cell death and survival. She is an Assistant Professor of Radiation Oncology and Cancer Biology at Washington University in St Louis where she specializes primarily in the treatment of patients with gynecologic cancers. Dr. Samantha Van Nest has worked to establish spectroscopic techniques for the personalization of radiation therapy. She is currently a Postdoctoral Associate at Weill Cornell Medicine in New York, where her research investigates the effects of radiation on tumor immunity, with a particular focus on improving our understanding of the abscopal effect. Arguing against the proposition will be Drs. Subarna Eisaman, Lakshmi Santanam, and Julie Sullivan. Dr. Subarna Hamid Eisaman is the clinical director and assistant professor at the University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center Department of Radiation Oncology at the J. Murtha Pavilion in Johnstown, Pennsylvania. She serves as co‐chair of the Radiation Oncology Lung and Lymphoma Via Oncology Pathways Physician Advisory Committee. Her clinical practice includes treatment of breast, GYN, lung, CNS, head and neck, skin and musculoskeletal malignancies. Dr. Lakshmi Santanam is an attending medical physicist at Memorial Sloan Kettering Cancer Center. Her primary interests include motion management and patient safety. She currently serves as vice chair for the AAPM Working Group on RO‐ILS and the Task Group on the Management of Respiratory Motion in Radiation Oncology. Dr. Julie Sullivan is a biologist at the U.S. Food and Drug Administration's (FDA) Center for Devices and Radiological Health. Her scientific interests include the use of radiation‐emitting medical devices in clinical trials and the medical planning for and response to radiological and nuclear incidents.

Increased heat shock protein 70 (Hsp70) serum levels and low NK cell counts after radiotherapy \u2013 potential markers for predicting breast cancer recurrence?

BackgroundBreast cancer is the most common invasive tumor in women worldwide and the second cause of cancer-related deaths. After breast conserving surgery the tumor bed gets irradiated. Radiation-induced tumor cell death has been found to be associated with the release of damage-associated molecular patterns (DAMPs) including free Hsp70 that can stimulate inflammatory immune responses. Therefore, Hsp70 serum levels as well as the composition of lymphocyte subpopulations have been measured in breast cancer patients during therapy and in the follow-up period as potential predictors for clinical outcome.MethodsThe serum of 40 breast cancer patients, who received a breast-conserving surgery and adjuvant radiotherapy (RT) was examined for soluble, free Hsp70 using the R&D Human HSP70 DuoSet and lipHsp70 ELISA. Lymphocyte subpopulations and total lymphocyte counts were analysed by multiparameter flow cytometry in the peripheral blood. Blood samples were collected before (t1), after 30 Gy (t2) and 60 Gy (t3), 6 weeks (t4), 6 months (t5) and 1 year (t6) after RT. Clinical responses were assessed regularly up to 5 years after RT.ResultsPatients who developed a contralateral recurrence or metastases within the first 2 years after RT had significantly higher serum Hsp70 values at the end of RT (t3; p = 0.03) up to 6 weeks after RT (t4; p = 0.007) compared to patients who either remained disease-free or developed a secondary endometrial carcinoma. Clinicopathological parameters such as age, tumor size, grading and TNM-stage of the resected tumors, adjuvant chemotherapy and irradiation dose did not affect serum Hsp70 levels. Elevated free Hsp70 levels might be indicative for a chronic inflammatory response which could support tumor recurrence. Lymphocyte subpopulation analysis revealed lower NK cell counts after RT in recurrence/metastases patients as compared to disease-free patients. In contrast, no significant changes were observed in the proportion of T and B cells.ConclusionLongitudinal elevated serum levels of free Hsp70 up to 6 weeks after RT and dropping NK cell counts might be predictive for an unfavourable prognosis in patients with breast cancer.

Harnessing and Optimizing the Interplay between Immunotherapy and Radiotherapy to Improve Survival Outcomes.

In the past, radiotherapy was primarily used to control local disease, but recent technological advances in accurate, high-dose ionizing radiation (IR) delivery have not only increased local tumor control but in some cases reduced metastatic burden. These "off target" therapeutic effects of IR at nonirradiated tumor sites, also known as abscopal effects, are thought to be mediated by tumor antigen-primed T cells that travel to metastatic sites and promote tumor regression. Similarly, early indications reveal that IR in combination with immune checkpoint inhibitors, such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1), can provide superior therapeutic responses. These observations suggest that local radiotherapy results in altered gene expression, exposure of new antigens, or cell death that can interact with immunotherapy. As such, radiotherapy enhancement of immune responses offers a promising synergy with the potential for substantial clinical benefit. This review focuses on the biology that underlies the mechanisms for the interaction between radiation-induced tumor cell death and enhanced immunologic response. Mol Cancer Res; 16(8); 1209-14. ©2018 AACR.

Abstract 5463: Free fatty acids augment radiation-induced cytotoxicity in cervical cancer cells via mTOR/Akt//PPARg/p53-mediated pathway

Abstract Purposes of the study: Our clinical results show that obese patients with cervical cancer who are treated with definitive chemoradiation have favorable outcomes, and this effect is independent of diabetes or metformin use. PI3K/AKT pathway signaling is decreased in cervical tumors from obese hosts, even in the context of genomic mutations that would otherwise activate PI3K signaling, such as PTEN deletion. The objective of the present study is to determine whether other factors related to obesity, such as circulating free fatty acids, influence radiation responses in vitro. Experimental procedures: Radiation sensitivity experiments were performed in cervical cancer cells with and without PI3K pathway mutations (CaSki (mutant), ME180 (mutant), SiHa (WT). To determine whether combination treatment of free fatty acids (palmitic, oleic and linoleic) plus radiation increases cytotoxicity in cervical cells, we performed cell viability assay, long term colony formation assays (CFA) and morphological examination by vertical microscope. To determine whether the addition of free fatty acids to radiation influences cell survival signaling, a series of Western blots were performed. Summary: We observed that combination treatment of free fatty acids and radiation significantly increased cytotoxicity in all the cervical cancer cell lines tested. Mechanistic investigations revealed that free fatty acids induced potentiation of radiation-induced cytotoxicity through downregulation of p-mTOR, p-Akt and PTEN. Furthermore, we observed that this combination regimen upregulates the expression PPARγ and p53. Conclusions: Free fatty acids potentiated the effect of radiation in PI3K-activated cervical cancer cell lines through mTOR/Akt//PPARg/p53 mediated pathway. Future studies will characterize the mechanism of free fatty acid potentiation of radiation-induced tumor cell death in detail, including the potential contribution of lipid peroxidation and alterations in glucose metabolism. Citation Format: Naoshad Muhammad, Ramachandran Rashmi, Fiona Ruiz, Michael C. Zahner, Julie K. Schwarz. Free fatty acids augment radiation-induced cytotoxicity in cervical cancer cells via mTOR/Akt//PPARg/p53-mediated pathway [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 5463.

PGAM5/FUNDC1/BCL-xL/DRP1 Axis Determines the Mode of Cell Death Induced by Ionizing Radiation in Non–small Cell Lung Cancer

Radiation-induced tumor cell death is widely attributed to multiple distinct mechanisms, mainly including autophagy, apoptosis and necrosis. However, a critical unanswered question is which death pathway a tumor cell chooses in response to various fractionated doses of ionizing radiation (IR). Mitochondrial pathways control both cell apoptosis and necrosis, and play roles in autophagy. The aim of this study was to investigate the role and mechanism of the mitochondrial-dependent cell death regulated by PGAM5/FUNDC1/BCL-xL/DRP1 axis in radiotherapy response of non-small cell lung cancer. Cell death of A549 and Calu-3 NSCLC cell lines induced by various fractionated doses of X-ray radiation (0, 2, 4, 6, 8, 12, 15, 20 Gy/Fraction and 2Gy×9Fraction) was detected by electron microscopy, western blotting, and flow cytometry. Protein interactions were detected by immunofluorescence and immunoprecipitation. Relative low-dose fractionated radiation (LDFRT, <6 Gy /Fraction) were used to induce apoptosis, whereas hypofractionated radiation (HFRT, 6–20 Gy/Fraction) was chosen to induce necrosis in both A549 and Calu-3 cells. Our results demonstrated that LDFRT induced degradation of BCL2-like 1 (BCL2L1), a prominent anti-apoptosis molecule, and released the mitochondrial phosphatase PGAM5 from BCL2L1, which led to its activation and subsequent dephosphorylation of FUN14 domain-containing protein-1 (FUNDC1) at Ser13. The dephosphorylated form of FUNDC1 interacted with light chain 3 (LC3) and induced mitophagy. In addition, peak levels of mitophagy appeared at about 8 hours after 6Gy/Fraction X-ray irradiation in A549, and 4Gy/Fraction X-ray irradiation in Calu-3 cells. Interestingly, the receptor-interacting protein kinase (RIP)1/RIP3 necrosome complex was formed and recruited to the mitochondrial surface at HFRT and then activated PGAM5, dynamin-related protein (Drp1), and mixed lineage kinase domain-like (MLKL) to execute mitochondrial fragmentation and programed cell necrosis. It is worth mentioning that HFRT did not notably induce degradation of BCL2L1 or mitophagy. The PGAM5/FUNDC1/BCL-xL/DRP1 axis might determine mitochondrial fate and the mode of cell death in response to various fractionated doses of X-ray radiation in NSCLC. Targeting these pathways will be useful to understand radiation-induced cell death and may improve the radiotherapeutic effect on human cancers.

Abstract 1649: Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation

Abstract Despite surgery or chemo radiotherapy the five-year survival rate of esophageal carcinoma (eSCC) patients remains around 15% indicating the need for new therapy regimes. Recently the role of the tumor microenvironment has been recognized as an important determinant of radiation responses. Here we investigate the influence of stromal fibroblasts (hF) on radiation induced tumor cell death. To evaluate functional consequences of ionizing radiation hF and eSCC cells were either cultured separately and irradiated (2Gy) or co-cultured after irradiation to investigate radiation dependent tumor-stroma interactions. Subsequently cells were monitored by time-lapse microscopy and cell death was assessed visually as well as by PARP cleavage, live cell staining with Hoechst33342/propidium iodide and quantification of sub-G1 cell cycle. The rate of cell death was barely altered in monocultures. Of note significantly increased cell death of eSCC was observed in co-culture with irradiated hF compared to co-cultures with mock-irradiated fibroblasts (16.10±4.25 fold of control). Microscopic evaluation of co-cultures revealed that hF and cancer cells are connected via tunnelling nanotubes (TNT). Furthermore, TNT formation was found to be dependent on the extracellular matrix component hyaluronan. eSCC cell death was prevented by pharmacological inhibition of TNT formation by cytochalasin B as well as inhibition of hyaluronan by 4-MU. As TNTs are known to mediate intercellular protein or mitochondrial transfer we investigated which stromal proteins are shuttled into eSCC cells by trans-SILAC experiments. For this purpose the proteome of fibroblasts was labeled with heavy amino acids. After co-culturing hF and eSCC were separated by FACS-sorting and labeled stromal proteins were identified in the cancer cells by mass spectrometry. Four consistently shuttled proteins were identified in the cancer cells, which were exclusively transferred after irradiation of both cell lines. Knock-down experiments of these proteins in hF were performed and SHANK1, which is not expressed in eSCC cells, was found to be responsible for the hF induced cell death of eSCC cells in response to radiation. In conclusion, by usage of a novel trans-SILAC approach, we provide evidence that in response to ionizing radiation stromal hF transfer SHANK1 to eSCC cells, thereby inducing cellular death. SHANK1 might therefore serve as potential new pharmacological target to sensitize cancer cells to radiation therapy. Citation Format: Katharina Röck, Alexandra Schütze, Janna Morawitz, Verena Jendrossek, Klaus Pantel, Jens W. Fischer. Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1649.

Abstract C144: LCL161, a SMAC mimetic, induces preferential radiosensitization in human papillomavirus negative head and neck squamous cell carcinoma through induction of apoptosis

Abstract Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetics are a new class of targeted drugs that specifically induce apoptotic cell death and block pro-survival signalling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. The present study was designed to investigate the radiosensitizing effect of a SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization. Material and methods: We examined the correlation between mRNA expression of apoptotic pathway associated molecules and HPV status by using The Cancer Genome Atlas Database (TCGA) in which 279 HNSCC tumors are included. = Protein expression was investigated in 6 HNSCC cell lines (3 HPV[+], 3 HPV[-]), normal oral epithelial cells (NOE) by immunoblotting, and in primary HNSCC tumors by immunohistochemistry (IHC). Clonogenic survival and Alamar Blue assays were carried out in order to explore the potential of LCL161 as a radiosensitizer in HNSCC cell lines. Cell cycle analysis and Annexin-V assays were performed to investigate mechanisms of radiation-induced tumor cell death. Human tumor xenografts were generated to explore the radiosensitization effect of LCL161 on HPV[-] tumors in vivo. Results: TCGA database analysis indicated significantly higher mRNA expression of cIAP1 and RELA, lower expression of CASP3, CASP9, and BCL2 in HPV[-] compared with HPV[+] HNSCC tumors. Consistent with these findings, immunoblotting confirmed that that protein expression of cIAP1 and p65 encoded by RELA were elevated in HPV[-] HNSCC cells.LCL161 displayed minimal single-agent cytotoxicity with IC50 values between 32μM – 95μM in 6 HNSCC cells. LCL161 is a potent inhibitor of cIAPs, and caused downregulation of cIAP1 at nanomolar concentrations in less than 1 hour, while it showed no obvious effect on NFκB, Stat3, Erk, or Akt activation. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-] HNSCC cell lines, but not in HPV[+] HNSCC cells. LCL161 mediated radiosensitisation was associated with enhanced activation of caspases-3, -7, -8, -9, and PARP. Blockage of caspase activation via a pan-caspase inhibitor, z-VAD-fmk, largely abrogated LCL161 radiosensitization. Conclusion: These results suggest that LCL161, a SMAC-mimetic, significantly radiosensitizes a subset of HNSCC tumors, via a mechanism involving caspase activation and apoptosis induction. This agent holds promise for future clinical development as a novel radiosensitizer in the treatment HNSCC HPV[-] tumors. Citation Format: Linlin Yang, Bhavna Kumar, Mitchell Romito, Theodoros N. Teknosa, Amab Chakravarti, Terence M. Williams. LCL161, a SMAC mimetic, induces preferential radiosensitization in human papillomavirus negative head and neck squamous cell carcinoma through induction of apoptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C144.

Abstract A19: Systems biology approach predicts the diagnostic value of T effector: T regulatory cell ratio in clinical response to combined radiation/immunotherapy of high-risk soft tissue sarcoma

Abstract Background: The combination of radiotherapy and immunotherapy for cancer treatment has regained momentum since the recent remarkable clinical success in immune checkpoint strategies. The attraction comes from the concept that radiation-induced tumor cell death will release tumor antigens capable of activating dendritic cells to establish long term adaptive T cell immunity against the tumor. However, such combination trials are difficult to interpret, especially when proper markers are not available that predict clinical response. Using a set of data published on patients with high risk soft tissue sarcoma who were treated with intratumoral administration of autologous dendritic cells and local fractionated external beam radiation, we developed a mathematical model examining the levels of T effector cells and T regulatory cells that were reported in each patient who did or did not demonstrate a clinical response. Materials and Methods: The Phase II clinical trial included 15 Grade 2 high-risk soft tissue sarcoma patients who received radiation/dendritic cell therapy, of which there were 5 responders and 10 non-responders. With the T cell phenotypes that were found in the patients and the accompanying clinical outcome, we designed a mathematical model of tolerogenic and immunogenic tumor subpopulations and their interactions with the host immune system, comprised of T effector and T regulatory cells. Tumor response to the investigational radio-immunotherapy protocol is simulated. The model is calibrated to fit patient-specific pre- and post-treatment response dynamics using computational genetic algorithms. Cell kinetics that separate responder and non-responder cohorts are also categorized. Results: The mathematical model can be calibrated to reproduce patient-specific tumor volume and immune cell number evolution during treatment. Comparison of responder and non-responder cohorts reveals that immune T effector cell recruitment and efficacy are determinants of treatment response. In contrast, tumor growth dynamics were indistinguishable between individual patients and patient cohorts. Increased T effector to T regulatory cell ratio at diagnosis (2.74 in responders vs. 1.88 in non-responders) emerges as a prognostic marker for treatment response. Conclusion: A calibrated quantitative tumor model may help to identify mechanisms that determine treatment response. Current clinical practice predicts treatment outcome predominantly based on tumor characteristics. Our initial work indicates that tumor growth dynamics are indistinguishable between high-risk soft tissue sarcoma patients that respond to radio-immunotherapy and those who do not. Instead, our preliminary studies suggest that the host response to the growing tumor – the quantity and quality of the immune response in particular – may serve exclusively as a prognostic marker. This suggests a departure from the current paradigm to derive prognostic factors from genetic characterization of tumor biopsy samples. Citation Format: Sotiris Prokopiou, Jan Poleszczuk, Mark Robertson-Tessi, Kimberly A. Luddy, Mayer Fishman, Eduardo Moros, Julie Y. Djeu, Heiko Enderling. Systems biology approach predicts the diagnostic value of T effector: T regulatory cell ratio in clinical response to combined radiation/immunotherapy of high-risk soft tissue sarcoma. [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 A19.

Chloroquine Engages the Immune System to Eradicate Irradiated Breast Tumors in Mice

This study used chloroquine to direct radiation-induced tumor cell death pathways to harness the antitumor activity of the immune system. Chloroquine given immediately after tumor irradiation increased the cure rate of MCaK breast cancer in C3H mice. Chloroquine blocked radiation-induced autophagy and drove MCaK cells into a more rapid apoptotic and more immunogenic form of cell death. Chloroquine treatment made irradiated tumor vaccines superior at inducing strong interferon gamma-associated immune responses invivo and protecting mice from further tumor challenge. In vitro, chloroquine slowed antigen uptake and degradation by dendritic cells, although T-cell stimulation was unaffected. This study illustrates a novel approach to improve the efficacy of breast cancer radiation therapy by blocking endosomal pathways, which enhances radiation-induced cell death within the field and drives antitumor immunity to assist therapeutic cure. The study illuminates and merges seemingly disparate concepts regarding the importance of autophagy in cancer therapy.

Radiation as an Immune Modulator

Radiation therapy is currently one of the most widely utilized treatment strategies in the clinical management of cancer. Classically, radiation therapy was developed as an anticancer treatment on the basis of its capacity to induce DNA double strand breaks in exposed cancer cells, ultimately resulting in tumor cell death. Recently, our understanding of radiation effects has expanded widely in terms of the consequences of radiation-induced tumor cell death and the pertinent cells, signaling pathways, and molecular sensors that modify the tumor response to radiation. It is now well accepted that inflammation plays a complex dual role in promoting or inhibiting tumor growth. The capacity of inflammatory responses to alter the tumor response to radiation therapy, and vice versa, is now the subject of intense scientific and clinical investigation. Herein, we review the concepts regarding the immunostimulatory properties of radiation therapy with particular focus on the effects of radiation therapy on the tumor microenvironment.

BlueBerry Isolate, Pterostilbene, Functions as a Potential Anticancer Stem Cell Agent in Suppressing Irradiation-Mediated Enrichment of Hepatoma Stem Cells

For many malignancies, radiation therapy remains the second option only to surgery in terms of its curative potential. However, radiation-induced tumor cell death is limited by a number of factors, including the adverse response of the tumor microenvironment to the treatment and either intrinsic or acquired mechanisms of evasive resistance, and the existence of cancer stem cells (CSCs). In this study, we demonstrated that using different doses of irradiation led to the enrichment of CD133+ Mahlavu cells using flow cytometric method. Subsequently, CD133+ Mahlavu cells enriched by irradiation were characterized for their stemness gene expression, self-renewal, migration/invasion abilities, and radiation resistance. Having established irradiation-enriched CD133+ Mahlavu cells with CSC properties, we evaluated a phytochemical, pterostilbene (PT), found abundantly in blueberries, against irradiation-enriched CSCs. It was shown that PT treatment dose-dependently reduced the enrichment of CD133+ Mahlavu cells upon irradiation; PT treatment also prevented tumor sphere formation, reduced stemness gene expression, and suppressed invasion and migration abilities as well as increasing apoptosis of CD133+ Mahlavu CSCs. Based on our experimental data, pterostilbene could be used to prevent the enrichment of CD133+ hepatoma CSCs and should be considered for future clinical testing as a combined agent for HCC patients.

Radiotherapy Effects on Anti-Tumor Immunity: Implications for Cancer Treatment

Ionizing radiation (IR) is a powerful therapeutic modality for cancer, commonly used for its capacity to kill cancer cells. In this Frontiers Research Topic Book radiotherapy effects are re-visited, from the point of view of the host's immune system (IS). An introductory article from Golden et al. (2012) examines the consequences of the many types of radiation-induced tumor cell death and how these coalesce to generate the key signals that define an immunogenic cell death (ICD). Cancer cells dying by ICD deliver a cascade of signals to the IS that culminates in the generation of anti-tumor T cells by providing a source of antigen for cross-presentation coupled with maturation signals to dendritic cells (DC). The ability of IR to induce an ICD is exploited by novel cancer therapies that have, for instance, shown the benefit of intra-tumoral injection of DC post-radiotherapy in preclinical models. Finkelstein and Fishman (2012) discuss this approach and the emergence of encouraging results from clinical pilot studies. Burnette et al. (2012) provide an overview of the immunological environment existing in tumor-bearing hosts, emphasizing the challenge of overcoming tolerance and immunosuppression to achieve tumor rejection. To overcome this barrier, combinations of IR with specific immunotherapies have been tested by several labs and shown to be effective at eliciting robust anti-tumor immunity. One such strategy, discussed by Mason and Hunter (2012), is the combination of IR with intra-tumoral synthetic oligodeoxynucleotides such as CpG. The preclinical success of this combination was translated to the clinic where it has demonstrated to induce rejection of the irradiated tumor as well as tumors outside the radiation field (abscopal effect). Another strategy, which is in earlier stage of investigation but holds great potential, is nanovectorized radiotherapy discussed by Vanpouille-Box and Hindre (2012). Delivery of radionuclides using nanoparticles has the advantage of providing targeting specificity to the tumor as well as exploiting the intrinsic immunostimulatory properties of nanoparticles. Importantly, IR effects exceed the classical cytocidal properties by also causing phenotypic changes in the fraction of surviving cells, markedly enhancing their susceptibility to T cell-mediated elimination. Kwilas et al. (2012) define these effects of IR as “immunogenic modulation” and illustrate the examples of IR-induced Major Histocompatibility Complex antigens and death receptors, which improve tumor rejection by T cells adoptively transferred or activated by vaccination. However, not all IR-induced modifications of the tumor and its microenvironment favor immune rejection. Chiang et al. (2012) provide novel evidence for accumulation of pro-tumorigenic M2 macrophages in areas of hypoxia present in irradiated tumors. Schaue et al. (2012) discuss the increase of regulatory T cells post-radiotherapy, potentially hindering the development of effective anti-tumor T cell responses. Intriguingly, the dose and fractionation of radiotherapy may play a role in modulating the expansion of effector versus regulatory T cells. This aspect is critically addressed by Demaria and Formenti (2012). Since much of the available preclinical data come from experiments testing single IR doses, further exploration of fractionated regimens is warranted. Overall, the book provides an overview of the available data and evolving concepts in support of a novel use of radiotherapy: that of an immune modulator and optimal partner for immunotherapy. While enthusiasm for the combination of IR and immunotherapy was enhanced by recent anecdotal reports in some cancer patients, much work remains to be done. Hopefully, the book will inspire more investigators to explore this new area, and encourage more discovery of the interaction of IR and immunity.

The convergence of radiation and immunogenic cell death signaling pathways

Ionizing radiation (IR) triggers programmed cell death in tumor cells through a variety of highly regulated processes. Radiation-induced tumor cell death has been studied extensively in vitro and is widely attributed to multiple distinct mechanisms, including apoptosis, necrosis, mitotic catastrophe (MC), autophagy, and senescence, which may occur concurrently. When considering tumor cell death in the context of an organism, an emerging body of evidence suggests there is a reciprocal relationship in which radiation stimulates the immune system, which in turn contributes to tumor cell kill. As a result, traditional measurements of radiation-induced tumor cell death, in vitro, fail to represent the extent of clinically observed responses, including reductions in loco-regional failure rates and improvements in metastases free and overall survival. Hence, understanding the immunological responses to the type of radiation-induced cell death is critical. In this review, the mechanisms of radiation-induced tumor cell death are described, with particular focus on immunogenic cell death (ICD). Strategies combining radiotherapy with specific chemotherapies or immunotherapies capable of inducing a repertoire of cancer specific immunogens might potentiate tumor control not only by enhancing cell kill but also through the induction of a successful anti-tumor vaccination that improves patient survival.

Autocrine regulation of glioblastoma cell-cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and progression of malignant brain tumors. Given the significance of tumor microenvironment in general, and the established role of paracrine VEGF signaling in glioblastoma (GBM) biology in particular, we explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astrocytoma (grade III and IV/GBM)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we show that co-expression of VEGF and VEGF receptors (VEGFRs) occurs commonly in astrocytoma cells. We found VEGF secretion and VEGF-induced biological effects (modulation of cell cycle progression and enhanced viability of glioblastoma cells) to function in an autocrine manner. Morevover, we demonstrated that the autocrine VEGF signaling is mediated via VEGFR2 (KDR), and involves co-activation of the c-Raf/MAPK, PI3K/Akt and PLC/PKC pathways. Blockade of VEGFR2 by the selective inhibitor (SU1498) abrogated the VEGF-mediated enhancement of astrocytoma cell growth and viability under unperturbed culture conditions. In addition, such interference with VEGF-VEGFR2 signaling potentiated the ionizing radiation-induced tumor cell death. In clinical specimens, both VEGFRs and VEGF were co-expressed in astroglial tumor cells, and higher VEGF expression correlated with tumor progression, thereby supporting the relevance of functional VEGF-VEGFR signaling in vivo. Overall, our results are consistent with a potential autocrine role of the VEGF-VEGFR2 (KDR) interplay as a factor contributing to malignant astrocytoma growth and radioresistance, thereby supporting the candidacy of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy.

Arsenic trioxide enhances the therapeutic efficacy of radiation treatment of oral squamous carcinoma while protecting bone

Therapeutic radiation is commonly used in the treatment of squamous cell carcinoma of the oral cavity and pharynx. Despite the proven efficacy of this form of anticancer therapy, high-dose radiation treatment is invariably associated with numerous unwanted side effects. This is particularly true for bone, in which radiation treatment often leads to osteoradionecrosis. The aim of this study was to investigate if treatment with arsenic trioxide (As(2)O(3)) could enhance the antitumor effect of radiotherapy whereas minimizing the destructive effects of radiation on bone. As(2)O(3) treatment induced a dose-dependent (1-20 mumol/L) inhibition of endothelial and tumor cell (OSCC-3 and UM-SCC-74A) survival and significantly enhanced radiation-induced endothelial cell and tumor cell death. In contrast, As(2)O(3) treatment (0.5-7.5 mumol/L) induced the proliferation of osteoblasts and also protected osteoblasts against radiation-induced cell death. Furthermore, As(2)O(3) treatment was able to significantly enhance radiation-induced inhibition of endothelial cell tube formation and tumor cell colony formation. To test the effectiveness of As(2)O(3) and radiation treatment in vivo, we used a severe combined immunodeficiency mouse model that has a bone ossicle and tumor growing side by side subcutaneously. Animals treated with As(2)O(3) and radiation showed a significant inhibition of tumor growth, tumor angiogenesis, and tumor metastasis to the lungs as compared with As(2)O(3) treatment or radiation treatment alone. In contrast, As(2)O(3) treatment protected bone ossicles from radiation-induced bone loss. These results suggest a novel strategy to enhance the therapeutic efficacy of radiation treatment while protecting bone from the adverse effects of therapeutic radiation.

Immunotherapy with Ad-LIGHT enhances radiation induced tumor cell death

Immunotherapy with Ad-LIGHT enhances radiation induced tumor cell death

Radiosensitization of colorectal carcinoma cell lines by histone deacetylase inhibition

BackgroundThe tumor response to preoperative radiotherapy of locally advanced rectal cancer varies greatly, warranting the use of experimental models to assay the efficacy of molecular targeting agents in rectal cancer radiosensitization. Histone deacetylase (HDAC) inhibitors, agents that cause hyperacetylation of histone proteins and thereby remodeling of chromatin structure, may override cell cycle checkpoint responses to DNA damage and amplify radiation-induced tumor cell death.MethodsHuman colorectal carcinoma cell lines were exposed to ionizing radiation and HDAC inhibitors, and cell cycle profiles and regulatory factors, as well as clonogenicity, were analyzed.ResultsIn addition to G2/M phase arrest following irradiation, the cell lines displayed cell cycle responses typical for either intact or defective p53 function (the presence or absence, respectively, of radiation-induced expression of the cell cycle inhibitor p21 and subsequent accumulation of G1 phase cells). In contrast, histone acetylation was associated with complete depletion of the G1 population of cells with functional p53 but accumulation of both G1 and G2/M populations of cells with defective p53. The cellular phenotypes upon HDAC inhibition were consistent with the observed repression of Polo-like kinase-1, a regulatory G2/M phase kinase. Following pre-treatment with HDAC inhibitors currently undergoing clinical investigation, the inhibitory effect of ionizing radiation on clonogenicity was significantly amplified.ConclusionIn these experimental models, HDAC inhibition sensitized the tumor cells to ionizing radiation, which is in accordance with the concept of increased probability of tumor cell death when chromatin structure is modified.