Radioresistance Research Articles

P1.03-34 The lncRNA NEAT1 Promotes Radioresistance via the MiR-491-5p/CAPG Axis in NSCLC

Long noncoding RNAs (lncRNAs) have been implicated in various biological processes and pathological conditions in cancer. However, the exact roles of LncRNA NEAT1 and its underlying mechanisms in radio resistance of non-small cell lung cancer (NSCLC) remain largely unclear. The expression of LncRNA NEAT1 was measured in NSCLC tissues and cell lines by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The radio sensitivity of NSCLC cells including H358, H226, HCC827, H1975, H1395 and H23 were detected by colony formation. Lentivirus-mediated short hairpin RNAs were used to knock down NEAT1 expression in H358 cells. Furthermore, the role of NEAT1 on tumor cell biological behavior and radio resistance were explored through MTT, colony formation, transwell migration, and invasion assays in vitro. Luciferase reporter assay was used to verify interaction between miR-491-5p and NEAT1, CAPG. The potential mechanism of LncRNA NEAT1 was identified by Western blot. Additionally, the association between the survival time and miR-491-5p expression in lung adenocarcinoma patients were evaluated based on the TCGA data. NEAT1 was highly expressed in NSCLC tissues and cell lines. NEAT1 was up-expressed in radiosensitive NSCLC cells and low-expressed in radioresistant NSCLC cells. Conversely, miR-491-5P was low-expressed in radiosensitive NSCLC cells and up-expressed in radioresistant NSCLC cells. In addition, we revealed a reciprocal repression between NEAT1 and miR-491-5P. CAPG was identified as a down-stream target of miR-491-5P. Further experiments revealed that lncRNA NEAT1 silencing inhibited cell proliferation, invasion and radio resistance in vitro. Overexpression of CAPG rescued the effects of NEAT1 downregulation on proliferation, invasion and radio resistance. In addition, mechanistic analysis showed that lncRNA NEAT1 upregulated the miR-491-5p-targeted gene CAPG through acting as a competitive “sponge” of miR-491-5p. By cox regression analysis, a tendency towards a survival benefit in patients with high miR-491 expression was observed in 430 lung adenocarcinoma patients of the TCGA database. Our findings suggest that NEAT1 regulated proliferation, invasion and radio resistance by modulating the miR-491-5p/CAPG axis in NSCLC.

Prognostic impact of glioblastoma stem cell markers OLIG2 and CCND2.

AimsGlioblastoma (GBM) is the most common and lethal malignant brain tumor in adults. Glioma stem cells (GSCs) are implicated in this poor prognosis and in radio(chemo‐)resistance. We have previously demonstrated that among potentially highly specific GSC markers oligodendrocyte lineage transcription factor 2 (OLIG2) appears to be the most specific and cyclin D2 (CCND2) the only one related to cell cycle regulation. The purpose of this work was to investigate the clinical significance and the evolution of OLIG2 and CCND2 protein expression in GBM.Methods and resultsImmunohistochemical expression analysis of Olig2 and Ccnd2 was carried out on a cohort of human paired GBM samples comparing initial resections with local recurrent tumors after radiation therapy (RT) alone or radio‐chemotherapy with temozolomide (RT‐TMZ). Uni‐ and multivariate logistic regression analysis revealed that significant risk factors predicting early mortality (<12 months) are: subtotal surgery for recurrence, time to recurrence <6 months, Ccnd2 nuclear expression at initial surgery ≥30%, and Olig2 nuclear expression <30% at second surgery after RT alone and RT‐TMZ.ConclusionsWe demonstrated that patients for whom nuclear expression of Olig2 becomes low (<30%) after adjuvant treatments have a significantly shorter time to recurrence and survival reflecting most probably a proneural to mesenchymal transition of the GSCs population. We also highlighted the fact that at initial surgery, high nuclear expression (≥30%) of CCND2, a G1/S regulator specific of GSCs, has a prognostic value and is associated with early mortality (<12 months).

Radiosensitivity Index Predicts for Locoregional Control with Preoperative Radiation in Soft Tissue Sarcomas

Sarcomas are a heterogeneous group of malignancies composed of a wide variety of histopathologic features and response to radiation (RT). We have previously developed and validated a genome-based gene expression signature to measure tumor intrinsic radiosensitivity, called the radiosensitivity index (RSI). We hypothesize that comparing the RSI based on histology may show differences in radiosensitivity, which would translate to a difference in locoregional control (LRC) in sarcomas treated with preoperative RT. A cohort of 113 resected sarcoma samples were identified from our prospective observational protocol. RSI was analyzed as previously described, with histologies defined as radiosensitive (RS) or radioresistance (RR) based on values ≤ and >0.55, respectively. A cohort of 123 patients treated between 11/1987 and 12/2015 with preoperative RT was assessed for clinical correlation. We evaluated locoregional control (LRC), which was estimated via Kaplan-Meier method and compared via log-rank. Cox proportional hazard regression method was utilized for univariate(UVA) and multivariate analyses(MVA). RSI sarcoma samples showed a median RSI of 0.55 (0.09-1.01), with variation by sarcoma subtypes, categorized as either RS angiosarcoma (0.44, range 0.20-0.64 n=14) and leiomyosarcoma(0.52, range 0.09-0.70, n=53) or RR(non-myxoid liposarcoma(0.60, range 0.25-1.01, n=49)). With a median follow up of 50.6 months and age of 57 years (28-89 years), a cohort of 123 sarcoma patients were identified. The RS group consisted of 29 leiomyosarcoma and 19 angiosarcoma patients, whereas the RR cohort included 75 non-myxoid liposarcoma patients. There was an improvement in the 4-year LRC in the RS cohort (95% vs. 79.3%, p=0.021), without a significant difference in 4-year OS (63.8% vs 74.5%, p=0.121), when compared to the RR cohort. RS histology was associated with improved LRC on UVA (HR 0.21 95%CI 0.05-0.91 p=0.037) and trended towards significance on MVA (HR 0.208 9 95%CI 0.035-1.236 p=0.084). In this analysis we demonstrate a significant difference in LRC between RR and RS histologies which translated to a clinically significant difference in treatment outcomes based on radiosensitivity. Risk-adjusted genomic-adjusted radiation dosing may have a role in optimizing locoregional control in this challenging disease.

Utilizing the Radiosensitivity Index (RSI) to Predict Pelvic Failure in Endometrial Cancer Treated with Adjuvant Radiotherapy

Various adjuvant approaches are utilized in the management of endometrial cancer based on surgical pathology and institutional preference. The radiosensitivity index (RSI) is a previously validated multi-gene expression index that estimates tumor radiosensitivity. In this study, we evaluate RSI as a genomic predictor for pelvic failure (PF) in patients treated with adjuvant radiotherapy. A total of 204 consecutively treated patients with endometrial cancer were identified from our IRB-approved institutional tissue biorepository. All patients underwent hysterectomy with bilateral salpingo-oophorectomy with the majority undergoing lymph node dissection (n=181; 88%) between 01/99 and 04/11 and followed until 01/19. Gene expression was from Affymetrix Hu-RSTA-2a520709 (Affymetrix; Santa Clara, CA). The RSI 10-gene signature was calculated for each sample using the previously published algorithm. Radiophenotype was determined by dichotomization of RSI at the previously identified cutpoint of 0.375; ≥0.375 = radioresistant (RR) and <0.375 = radiosensitive (RS). Time to event analysis was performed with Kaplan-Meier estimates and the log-rank test. Associations between radiophenotype and outcomes were explored with univariable (UVA) and multivariable (MVA) Cox regression. The threshold for statistical significance in all tests was set at p<0.05. Median follow-up was 38.5 months (range: 0.2-216). The median RSI was 0.42 (range: 0.11-0.70). There were no significant differences in RS and RR patients in age (p=0.99), serosa and/or adnexa involvement (p=0.37), vaginal and/or parametrial involvement (p=0.48), cervical stromal invasion (p=0.59), receipt of adjuvant chemotherapy (p=0.12), and node involvement (p=0.78). A total of 83 (41%) patients were treated with adjuvant radiotherapy with vaginal brachytherapy (n=19; 23%), pelvic radiotherapy (n=26; 31%), or both (n=38; 46%). In patients treated with radiation, RR patients were more likely to undergo PF (3 year pelvic control 84% vs 100%; p=0.02) with worse PF free survival (PFFS) (3 year PFFS 65% vs. 89%; p=0.04). However, since RSI is a radiation specific signature it did not predict PF (p=0.86) or PFFS (p=0.57) in patients not treated with radiation. Factors found to predict PF on UVA included grade 3/1-2 (HR 3.8; 95% CI 1.2-14.8, p=0.03), serosa and/or adnexa involvement (5.9; 95% CI 1.6-20.2, p=0.008), lymph node involvement (4.9; 95% CI 1.5-18.8, 0.009), and RR/RS (7.7; 95% CI 1.5-140.9, 0.01). On MVA, factors that continued to predict for PF included RR/RS (10.7; 95% CI 1.9-202.4, p=0.004), lymph node involvement (4.1; 95% CI 1.2-16.3, p=0.03), and serosa and/or adnexa involvement (4.3; 95% CI 1.2-16.4, p=0.03). RSI was found to be a significant predictor of PF in patients treated with adjuvant radiotherapy on MVA. We propose RSI may be a method to predict which patients are most likely to fail in the pelvis and candidates for treatment escalation in the adjuvant setting.

Hypoxia-Induced Autophagy Promotes EGFR Loss in Specific Cell Contexts, Which Leads to Cell Death and Enhanced Radiosensitivity

Treatment failure through radio resistance of tumors is associated with activation of the epidermal growth factor receptor (EGFR). Tumor cell proliferation, DNA-repair, hypoxia and metastases-formation are four mechanisms in which EGFR signalling has an important role. However, the effect of hypoxia on EGFR expression is still controversial. In this study, we evaluated the in vitro and in vivo effects of hypoxia on EGFR and its mechanism, as well as its relationship with radiosensitivity. Treatment failure through radio resistance of tumors is associated with activation of the epidermal growth factor receptor (EGFR). Tumor cell proliferation, DNA-repair, hypoxia and metastases-formation are four mechanisms in which EGFR signalling has an important role. However, the effect of hypoxia on EGFR expression is still controversial. In this study, we evaluated the in vitro and in vivo effects of hypoxia on EGFR and its mechanism, as well as its relationship with radio sensitivity. In a panel of cancer cells with low or high cell density under hypoxia, viable cell counting assay was used to calculate the percentage of cell death. Western blot was used to detect the levels of proteins involved in EGFR signaling and autophagy. The localization of EGFR and autophagy-related molecules was observed by confocal microscopy. The xenograft model in nude mice was used to elucidate the in vivo correlation of hypoxia with EGFR and p62. Colony formation assay was performed to assess the radiosensitivity. In this study, we demonstrated that hypoxia enhanced EGFR expression and sustained cell survival in SiHa, CAL 27 and A549 cells at both low and high cell densities, while in MCF-7, MDA-MB-231 and HeLa cells, EGFR and cell survival were regulated by hypoxic treatment in a cell-density dependent manner: upregulated at low cell density and down regulated at high cell density. In MCF-7 and HeLa xenografts in nude mice, EGFR expression varied inversely with the pimonidazole level that was used as an indicator of hypoxia, accordant with the effect of hypoxia at high cell density in vitro. Hypoxia induced more remarkable cell autophagy at high cell density than at low cell density. Autophagy inhibitor 3MA, rather than proteasome inhibitor MG132 inhibited hypoxia-mediated EGFR loss and shifted cell death to cell survival in HeLa cells. The MCF7 cells’ sensitivity to ionizing radiation (IR) under hypoxia was also conditional on the cell densities when the hypoxia treatment was introduced, inversely associated with the expression levels of EGFR. Hypoxia can decrease EGFR expression in some cell lines by enhancing autophagy at high cell density, leading to cell death and hypersensitivity to radiotherapy. This study may help understand how hypoxia influences EGFR expression and radiosensitivity.

MiR-129-5p enhances the radiosensitivity of thyroid myeloid cell MZ-CRC-1 by inhibiting HMGB1

Objective To explore whether MiR-129-5p participates in radiosensitivity of medullary thyroid cell MZ-CRC-1 by inhibiting the gene expression of high mobility group protein B1 (HMGB1). Methods The radioresistant cell line MZ-CRC-1/R was established from MZ-CRC-1. Cell survival fraction was analyzed by colony formation assay. The expressions of miR-129-5p in MZ-CRC-1 and MZ-CRC-1/R cells were detected by qRT-PCR. Cell viability was determined by MTT assay. Cell apoptosis was measured by flow cytometry. Dual-luciferase reporter assay was performed to confirm the relationship between miR-129-5p and HMGB1. Besides, the protein expressions of HMGB1 and p-AKt were evaluated by western blot. Results Compared with that of MZ-CRC-1 cells, the survival fraction of MZ-CRC-1/R cells was significantly increased (t=3.038, 4.330, 4.885, 4.568, P<0.05), the cell viability of MZ-CRC-1/R cells was also increased (t=3.637, 7.734, 11.896, 14.522, P<0.05), and the expression of miR-129-5p(0.26±0.03) was significantly decreased in MZ-CRC-1/R cells(1.00±0.06) (t=19.107, P<0.05). Compared with miR-NC-inhibitor group, cell viability was promoted and cell apoptosis was blocked in the miR-129-5p-inhibitor group (t=5.156, 6.005, 9.649, 8.659, P<0.05). Moreover, miR-129-5p mimic suppressed cell viability and enhanced cell apoptosis after irradiation (t=3.118, 5.034, 6.005, 7.488, 6.362, P<0.05). Overexpression of miR-129-5p inhibited the protein expressions of HMGB1 and p-AKt (t=9.325, 10.614, P<0.05). In addition, HMGB1 depletion rescued cell apoptosis that was reduced by miR-129-5p inhibitor in MZ-CRC-1 cells (t=6.700, P<0.05), while HMGB1 overexpression attenuated the effect of miR-129-5p upregulation on MZ-CRC-1/R cells (t=7.073, P<0.05). Conclusions miR-129-5p increased the radiosensitivity of medullary thyroid-like cell MZ-CRC-1 by inhibiting HMGB1. Key words: Medullary thyroid carcinoma cells; miR-129-5p; HMGB1; Radiosensitivity

Abstract LB-280: FLASH: A novel paradigm changing tumor irradiation platform that enhances therapeutic ratio by reducing normal tissue toxicity and activating immune pathways

Abstract Summary: A novel irradiation platform using a proton beam leads to an improved therapeutic window through sparing of normal tissue toxicity. Preliminary experiments suggest involvement of immune pathways in mediating this effect. Abstract: Radiation therapy is administered to nearly half of all the cancer patients. The beneficial effect of this treatment is limited by the radio resistance of the cancer cells and by the adverse effects of radiation on the surrounding normal tissues. Strategies that would reduce normal tissue toxicity while maintaining potent tumor cell killing would enhance the therapeutic ratio and make radiotherapy more effective. Recent publications have shown that delivering radiation at an ultra-high dose rate ≥40Gy/s (ie. FLASH) vs conventional dose rate of 0.03-0.05Gy/s (CONV), significantly reduces the normal tissue toxicity in lung and skin while still being as effective in killing tumor cells (Favaudon et al Sci Trans Med 2014). These studies were done using experimental radiotherapy equipment able to deliver electrons in that dose rate range in preclinical models. However, translation of this approach into the clinic using electron beams may be challenging. In this work, we show for the first time, reduced normal tissue toxicity in pre-clinical mouse models using proton FLASH on a clinical device capable of translation to humans. The study divided C57BL/6 mice into groups (n=6 to 20) which were sham-irradiated or exposed to a single-dose of 15, 17.5 and 20 Gy Conventional (1Gy/sec protons) or FLASH (40 Gy/sec) protons through whole thorax irradiation, and then sampled at 8, 16, 24, and 34 weeks post-irradiation for evaluation of complications and histopathological analysis of lung fibrosis. We observed up to a 30% reduction in lung fibrosis, as well as reduced incidence of skin dermatitis and improved overall survival in FLASH- vs conventionally-treated mice. Evaluation of the molecular mechanism underlying the FLASH effect was performed through genome wide microarray analysis. This revealed that the major pathways differentially regulated between the two treatments included DNA damage and repair, inflammation and immune modulation. Dendritic cell maturation, PKC signaling in lymphocytes, TH1 pathway modulation and calcium-induced T lymphocyte apoptosis were elevated after CONV radiation and decreased after FLASH treatment. Further analysis of these pathways is ongoing. Together these results suggest that FLASH treatment using clinical proton beams might allow for sparing of both early and late tissue toxicity after radiation and this phenomenon involves pathways related to differential DNA damage response and immune modulation. Citation Format: Swati Girdhani, Eric Abel, Alexander Katsis, Andrew Rodriquez, Shilpa Senapati, Angel KuVillanueva, Isabel L. Jackson, John Eley, Zeljko Vujaskovic, Renate Parry. FLASH: A novel paradigm changing tumor irradiation platform that enhances therapeutic ratio by reducing normal tissue toxicity and activating immune pathways [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 LB-280.

The hypermethylation of the CDKN2A and CHFR promoter region is a key regulatory mechanism of CDKN2A and CHFR expression in esophageal squamous cell carcinoma

Background: The aim of this study was to evaluate the correlation between CDKN2A and CHFR expression and the methylation status of the CpG island in the promoter region of esophageal squamous cell carcinoma (ESCC) cell lines, radioresistant ESCC cell lines and ESCC clinical tissue specimens. Methods: To determine whether the methylation of CHFR and CDKN2A occurs in ESCC and enhanced in radioresistant ESCC cell lines, the DNA methylation of the following was analyzed by pyrosequencing and quantitative real-time polymerase chain reaction (PCR): (I) 28 ESCC tissues; (II) the ESCC cell lines KYSE-150 and KYSE-180; and (III) the radioresistant ESCC cell lines KYSE-150-radioresistance and KYSE-180-radioresistance. Results: The methylation of CDKN2A and CHFR was found to be enhanced in the radioresistant ESCC cell lines KYSE-150-radioresistance and KYSE-180-radioresistance (P CDKN2A and CHFR was found to decrease correspondingly (P CDKN2A and CHFR was found to be higher in the ESCC tissue and paracarcinoma tissue than in the adjacent benign tissue. The overall survival rates for ESCC patients were significantly correlated with the levels of CDKN2A and CHFR expression (P Conclusions: Our findings suggest that the hypermethylation of the CDKN2A and CHFR promoter region is a key regulatory mechanism of CDKN2A and CHFR expression in ESCC. Hypermethylated CDKN2A and CHFR are potential biomarkers for predicting the radioresistance of ESCC.

Mir-361-5p enhances radiosensitivity of osteosarcoma cells by directly downregulating FOXM1

Objective To investigate the effect of microRNA361-5p on radiosensitivity of osteosarcoma cells and its downstream regulatory mechanisms. Methods The radioresistant osteosarcoma cell line HOS-R was constructed and the expression of microRNA-361-5p in HOS and HOS-R cells was detected by RT-qPCR.The survival rate and apoptosis rate were detected by clone formation assay and flow cytometry in HOS-R cells treated with up-regulated or down-regulated of miR-361-5p, or FOXM1 depletion. Dual fluorescent luciferase reporter and western blot assays were used to measure the relationship between miR-361-5p and FOXM1.he effects of miR-361-5p on radiosensitivity of osteosarcoma cells were determined by cloning formation assay and flow cytometry. Results RT-qPCR results showed that miR-361-5p was low expressed in HOS-R cells. Colony formation and flow cytometry assays demonstrated that overexpression of miR-361-5p significantly decreased the survival rate and increased the apoptosis rate of HOS-R cells. In contrast, FOXM1 downregulation inhibited cell survival rate and induced apoptosis. Moreover, miR-361-5p negatively regulated FOXM1 expression. Besides, FOXM1 overexpression attenuated the miR-361-5p upregulation-mediated promotion of radiosensitivity in HOS-R cells. Conclusion miR-361-5p was involved in the radiosensitivity of osteosarcoma cells by inhibiting FOXM1. Key words: Osteosarcoma cell line; Gene expression regulation; Radiosensitivity

Integrating radiosensitive genes improves prediction of radiosensitivity or radioresistance in patients with oesophageal cancer.

Oesophageal cancer is a serious disease worldwide. In China, the incidence of esophageal cancer was reported to be ~478,000 in 2015. In the same year, the incidence of esophageal cancer in the United States was ~16,910. Radiotherapy serves as an important tool in the treatment of oesophageal cancer, and although radiation therapy has progressed over time, the prognosis of the majority of patients with oesophageal cancer remains poor. Additionally, the sensitivity of patients with oesophageal cancer to radiotherapy and chemotherapy is not yet clear. Although there are a number of studies on the radiosensitivity of oesophageal cancer cell lines, the vastly different results from different cell lines make them unreliable to use as a guide in clinical practice. Therefore, a common radiosensitive gene signature may provide more reliable results, and using different combinations of common gene signatures to predict the outcome of patients with oesophageal cancer may generate a unique gene signature in oesophageal cancer. In the present study, the radiosensitive index and prognostic index were calculated to predict clinical outcomes. The prognostic index of a 41-gene signature combination is the largest combination of gene signatures used for classifying oesophageal cancer patients into radiosensitive (RS) and radioresistance (RR) groups, to the best of our knowledge, and this gene signature was more effective in patients classified as having Stage III oesophageal cancer. Furthermore, four genes (carbonyl reductase 1, serine/threonine kinase PAK2, ras-related protein Rab 13 and twinfilin-1) may be sufficient to classify patients into either RS or RR. Subsequent to gene enrichment analysis, the cell communication pathway was significantly different between RS and RR groups in oesophageal cancer. These results may provide useful insights in improving radiotherapy strategies in clinical decisions.

Understanding the mechanism underlying the acquisition of radioresistance in human prostate cancer cells.

Acquisition of radioresistance (RR) has been reported during cancer treatment with fractionated irradiation. However, RR is poorly understood in the prognosis of radiotherapy. Although radiotherapy is important in the treatment of prostate cancer (PCa), acquisition of RR has been reported in PCa with an increased number of cancer stem cells (CSCs), neuroendocrine differentiation (NED) and epithelial-mesenchymal transition. However, to the best of our knowledge, the mechanism underlying RR acquisition during fractionated irradiation remains unclear. In the present study, human PCa cell lines were subjected to fractionated irradiation according to a fixed schedule as follows: Irradiation (IR)1, 2 Gy/day with a total of 20 Gy; IR2, 4 Gy/day with a total of 20 Gy; and IR3, 4 Gy/day with a total of 56 Gy. The expression of cluster of differentiation (CD)44, a CSC marker, was identified to be increased by fractionated irradiation, particularly in DU145 cells. The expression levels of CD133 and CD138 were increased compared with those in parental cells following a single irradiation or multiple irradiations; however, the expression levels decreased with subsequent irradiation. RR was evidently acquired by exposure to 56 Gy radiation, which resulted in increased expression of the NED markers CD133 and CD138, and increased mRNA expression levels of the pluripotency-associated genes octamer-binding transcription factor 4 and Nanog homeobox. These data indicate that radiation-induced CSCs emerge due to the exposure of cells to fractionated irradiation. In addition, the consequent increase in the expression of NED markers is possibly induced by the increased expression of pluripotency-associated genes. Therefore, it can be suggested that cancer cells acquire RR due to increased expression of pluripotency-associated genes following exposure to fractionated irradiation.

Multi-cellular tumor spheroids formation of colorectal cancer cells on Gelatin/PLCL and Collagen/PLCL nanofibrous scaffolds

In recent years, more attention has been drawn to development of three-dimensional in-vitro tumor models, Multi Cellular Tumor Spheroids (MCTS), due to their similarity to in vivo tumors models. Interaction of tumor cells with the extracellular matrix (ECM) has an important role in stimulating microenvironmental signaling and MCTS formation. Recently, a number of scaffolds based on natural-nano pattering have been proposed which can superbly mimic the topographical and biochemical features of ECM. In this study, we investigated whether the natural-synthetic polymer nanofibers can promote the three-dimensional (3D) MCTS formation of HT29 colorectal cancer cells in compare to synthetic nanofibers. Nanofibers were fabricated by blending of collagen (Col) and gelatin (Gel) with poly (L-lactide co-ε-caprolactone) (PLCL) polymer, separately. Generally, nanofibers exhibited proper structural properties in term of morphology, hydrophilic nature and mechanical integrity. The results revealed that HT29 colorectal cancer cells can form 3D spheroids with uniform morphology and smooth surface on both Col/PLCL and Gel/PLCL nanofibers while the spheroids were unstable and irregular in shape on PLCL nanofibers. In addition, the cells were dispersed on non-coated plates as confluent cell monolayer. There were no significant differences between the number and diameter of MCTSs on both Col/PLCL and Gel/PLCL nanofibers. On the other hand, the radio resistance of cells on Col/PLCL and Gel/PLCL nanofibers was higher compared with either PLCL nanofibers or non-coated plates. In conclusion, the results showed that scaffolds provided by Col/PLCL and Gel/PLCL nanofibers can mimic the properties of ECM in case of in vitro MCTS formation so they can be suggested to use in tumor drug screening studies.

Abstract P3-12-24: Tumor-secreted predictive biomarkers of response to radiotherapy in breast cancer

Abstract Background:In breast cancer (BC), radiotherapy (RT) is used adjuvantly to prevent recurrence and also in the palliative setting. Clinical signs of RT response are often not apparent for several weeks post-treatment and we currently lack tools to predict or monitor tumor response to RT early during treatment. The aim was to identify tumor-secreted biomarkers whose release reflects response to RT, which could be monitored during treatment in the blood or intratumorally by an implantable biosensor, currently under development within the Implantable Microsystems for Personalised Anti-Cancer Therapy (IMPACT) program. Methods: A series of experiments assessed the effect of different radiation doses (2-10Gy) on 3 human BC cell lines – MDA-MB-231 (ER-), MCF-7 (ER+) and HBL-100 (ER-) –, 1 canine breast cancer and 2 sheep lung cancer lines. Culture media was collected from each dose experiment at a range of post-radiation time-points (1-24 hours). Proteins were isolated from collected media for secretome mass spectrometry (MS) analysis. A subset of treatment/time conditions were repeated in the same BC cell lines and radioresistant (RR) derivatives from which RNA was extracted and analysed using Lexogen QuantSeq for whole-genome transcriptomics.In-lab candidate biomarker validation was carried out using immuhistochemistry (IHC), immunofluorescence (IF) and western blotting (WB) using validated antibodies. Levels of candidate biomarkers were also assessed in normal and untreated BC tissues using IHC. ELISA-based methods are currently under investigation for detection of the lead candidate biomarkers in the blood of large animal cancer models treated with RT. Results: Biomarker discovery using the MS data revealed 4 promising candidates: EIF3G, SEC24C, YBX3 and TK1. These are released from BC and animal cancer cells sensitive to radiation in a dose-dependent manner 24 hours after treatment. Analysis of the transcriptomic data showed an 8-fold higher expression of the genes encoding the 4 candidates in the radio-sensitive parental cell lines compared to the RR cell lines. IF and WB confirmed lower intracellular expression of the 4 proteins in RR cells compared to the parental lines. WB of collected culture media confirmed release of each of the 4 candidates 24 hours after a 2Gy dose of radiation in only the parental lines. GAPDH was not found in these media samples, demonstrating that protein release was not due to cell lysis. Conclusions: · We have identified 4 promising biomarkers which are released from cancer cells sensitive to RT and not released from RR derivatives. · All 4 candidates are released 24 hours after a 2Gy radiation dose, which fits with the current clinical dosing schedule where radiation is administered at 24 hour intervals. Ongoing work will elucidate if these biomarkers can be reliably detected in blood or intratumorally using implantable biosensors. · There are currently no validated predictive tools to monitor RT response during treatment. If successfully validated, these biomarkers could have a clinical role in personalising RT dosing schedules and durations for solid tumors in the neoadjuvant and palliative setting, thus optimising treatment and preventing the administration of ineffective RT and its associated side effects. Citation Format: Meehan J, Gray M, Turnbull AK, Martinez-Perez C, Bonello M, Ward C, Langdon SP, McLaughlin S, MacLennan M, Dixon JM, Wills J, Quinn N, Finich AJ, von Kriegsheim A, Cameron D, Kunkler IH, Murray A, Argyle D. Tumor-secreted predictive biomarkers of response to radiotherapy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-12-24.

Evaluation of radiation-induced genotoxicity on hu-man melanoma cells (SK-MEL-37) by flow cytometry

Micronucleus assay is a test used to evaluate genotoxic damage in cells, which can be caused by various factors, like ionizing radiation. Interactions between radiation energies and DNA can cause breakage, leading to use chromosomal mutations or loss of genetic material, important events that could be induced in solid tumors to mitigate its expansion within human body. Melanoma has been described as a tumor with increased radio resistance. This work evaluated micronuclei percentages (%MN) in human melanoma cells (SK-MEL-37), irradiated by gamma radiation, with doses between 0 and 16Gy. Cell suspensions were irradiated in PBS by a 60Co source in doses between 0 and 16Gy, and incubated by 48h. Then cell membranes were lysed in the presence of SYTOX Green and EMA dyes, preserving nuclear membranes. Using this method, EMA-stained nuclei could be discriminated as those derived from dead cells, and SYTOX nuclei and micronuclei could be quantified. Micronuclei percentages were found to be proportional to dose, (R2 = 0.997). Only the highest dose (16Gy) could induce statistically significant increase of MN (p&lt;0.0001), although cultures irradiated by 4, 8 and 16Gy showed significant increase of dead cell fractions. Calculation of the nuclei-to-beads ratio showed that 8 and 16Gy could reduce melanoma cell proliferation. Results showed that although cell death and loss of proliferative capacity could be observed on cultures irradiated at lower doses, genotoxic damage could be induced only on a higher dose. Resistance to radiation-induced genotoxicity could explain a relatively high radio resistance of melanoma tumors.

Abstract B168: A Radiosensitivity gene signature and PD-L1 status predict clinical outcome of patients with glioblastoma multiforme in The Cancer Genome Atlas Dataset

Abstract Background: A combination of radiotherapy and immune checkpoint blockade, such as programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1) blockade, is being actively tested in clinical trial settings. Here, we tried to identify a subset of patients that could potentially benefit from this strategy using The Cancer Genome Atlas (TCGA) dataset for glioblastoma (GBM). Methods: A total of 399 cases were clustered into radiosensitive (RS) versus radioresistant (RR) groups based on radiosensitivity gene signature and were further stratified into PD-L1 high versus PD-L1 low groups according to the median expression of CD274 mRNA. Differential and integrated analyses with expression and methylation data were performed. CIBERSORT was used to enumerate the immune repertoire that resulted from transcriptome profiles. Results: We identified a subset of GBM patients, the “PD-L1 high-RR group” that had a worse clinical outcome compared to the other groups. In this group, differentially expressed genes (DEG) were highly enriched for an immune response and mapped into activation of PI3K-AKT and MAPK signaling pathways. Through integration of DEG and differentially methylated regions, kinase MAP3K8, which is involved in T-cell receptor signaling, was found to be upregulated, while BAI1, a factor that inhibits angiogenesis, was silenced. CIBERSORT showed that a higher infiltration of the immune repertoire, which included M2 macrophages and regulatory T-cells, contributed to an immunosuppressive tumor microenvironment. Conclusion: Looking at the results collectively, the “PD-L1-high-RR group” could potentially benefit from radiotherapy combined with PD-1/PD-L1 blockade and angiogenesis inhibition. Citation Format: Inah Kim, Bum Sup Jang. A Radiosensitivity gene signature and PD-L1 status predict clinical outcome of patients with glioblastoma multiforme in The Cancer Genome Atlas Dataset [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B168.

MiR-590-5p Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Radiation by Blocking Autophagy via Targeting ATG3

Background: Resistance against radio therapy is growing concern in treating patients of pancreatic cancer (PC). Improving our depth with regards to mechanisms involved in radio resistance could help in developing strategies for increasing patient's response to this therapy. Here we investigated the role of micro RNAs (miRs) and specifically miR-590-5p in radio-resistance of PC cells. Methods: We successfully developed radio resistant PC cell lines in our lab and subjected them to microarray analysis to compare the levels of miRs with their original parental cell lines. We then transfected the PC cells using miR mimics or inhibitor and performed clonogenic survival assay to study the effects on radiation sensitivity. We also studied the effect of miR-590-5p on autophagy using electron microscopy and Immunoblotting analysis. Luciferase assay was done for identifying mRNA targets of miR-590-5p. Results: The radio resistant PC cells exhibited decreased expression of miR-590-5p with elevated autophagy against the parental cells. The over expression of miR-590-5p inhibited the radiation mediated autophagy and the miR-590-5p inhibitor encouraged autophagy in PC cells. The up-regulation of miR-590-5p enhanced the sensitivity of PC cells towards radiation. We confirmed that ATG-3 as a favorable target of miR-590-5p and was unregulated in radio resistant cells, we also found that ATG-3 was associated with autophagy. MiR-590-5p inhibited radiation-mediated autophagy and enhanced radio sensitivity of PC cells. The results suggested an inverse correlation for miR-590-5p and autophagy in PC tissues. Conclusions: The expression of miR-590-5p in PC cells is associated with elevated levels of autophagy along with ATG-3, which leads to radio resistance; miR-590-5p can be employed to enhance the radio sensitivity of PC cells. Funding Statement: The project was self financed. Declaration of Interests: The authors have no conflict of interest. Ethics Approval Statement: All the patients were educated about the study and informed consent forms were signed before the study. The study was approved from institutional review of research committee at The Second Xiangya Hosipital of Central South University, Changsha, Hunan, China

Research progress on radioresistance and sensitization of tumor cells in three-dimensional culture system

Three-dimensional cell culture is a novel type of in vitro culture method. This system can mimic the microenvironment of cell growth in vivo, where cells exhibit similar state and function to that in the in vivo environment. Currently, three-dimensional culture system has been widely applied in tissue engineering and tumor cell biology fields, etc. Radiotherapy is an important treatment of cancer. Radioresistance of tumor cells is the main cause of treatment failure, tumor recurrence and metastasis. Tumor cells can exhibit the resistant characteristics in the three-dimensional culture system, similar to those of tumor cells in vivo. Therefore, three-dimensional culture system can be adopted to investigate the radioresistance and underlying mechanism of tumor cells and identify the key factors regulating the radioresistance of tumor cells, which plays a pivotal role in enhancing the radioresistance and improving the effect of radiotherapy. This article will review recent research progress in the radioresistance and sensitization of tumor cells under three-dimensional culture system, aiming to provide reference for relevant investigations. Key words: Three-dimensional culture; Tumor cell; Radioresistance; Radiosensitization; Matrix material

Increased PD-L1 expression in radioresistant HNSCC cell lines after irradiation affects cell proliferation due to inactivation of GSK-3beta.

At present, targeting PD-1/PD-L1 axis for immune checkpoint inhibition has improved treatment of various tumor entities, including head and neck squamous cell carcinoma (HNSCC). However, one part of the patient cohort still shows little improvement or even hyperprogression. We established three radioresistant (RR) and three radiosensitive (RS) HNSCC cell lines. RR cells showed prolonged survival as well as delayed and diminished apoptosis after irradiation with vimentin expression but no E-cadherin expression, whereas RS cell lines died early and exhibited early apoptosis after irradiation and high vimentin expression. Here, we present results demonstrating differential basal PD-L1 gene and protein expression in RR and RS HNSCC cell lines. Moreover, we observed a radiation dose dependent increase of total PD-L1 protein expression in RR cell lines up to 96h after irradiation compared to non-irradiated (non-IRR) cells. We found a significant GSK-3beta phosphorylation, resulting in an inactivation, after irradiation of RR cell lines. Co-immunoprecipitation experiments revealed decreased interaction of GSK-3beta with PD-L1 in non-IRR compared to irradiated (IRR) RR cells leading to PD-L1 stabilization in RR cells. PD-L1 knockdown in RR cells showed a strong decrease in cell survival. In summary, our results suggest an irradiation dependent increase in basal PD-L1 expression in RR HNSCC cell lines via GSK-3beta inactivation.

Translational Research: A Future Strategy for Managing Squamous Cell Carcinoma of the Head and Neck?

Squamous Cell Carcinoma of the Head and Neck (SCCHN) are neoplasms arising from the epithelium of the first aero-digestive tract. They are very heterogeneous both clinically and biologically. Classic and well acknowledged risk factors are alcohol and tobacco consumption and other forms of smokeless tobacco assumption, although lately the incidence of Human Papilloma Virus (HPV)-related SCCHN is rapidly increasing. HPV-related tumors are very different from their alcohol and tobacco-associated counterpart, as they show strong chemo and radio sensitivity and thus can often be treated with conservative treatment strategies. Moreover, peculiar biologic features characterize HPV-related tumors, such as wild type TP53, low expression of Epidermal Growth Factor Receptor (EGFR), wild type CCND1 and high expression of P16. In contrast, alcohol and tobacco related SCCHN show opposite features, together with higher number of chromosomal and genetic abnormalities, conferring them chemo and radio resistance. We have performed a narrative review of the PubMed database with the aim to study the mutational landscape of SCCHN. Several lines of evidence support the existence of at least two genetically different types of SCCHN, one virus-related and the other alcohol and/or tobacco-related, characterized by both clinical and biological opposite features. Virus related SCCHN are very chemo and radiosensitive, so suitable for organ preserving strategy, which in the near future may be induction chemotherapy followed by association of chemotherapy and underpowered radiotherapy. Alcohol and tobacco related SCCHN are themselves strongly heterogeneous and can be divided in different entities on the basis of the "Driver" genetic aberration, responsible for carcinogenesis. The most frequently mutated genes in alcohol and tobacco-related SCCHN are TP53, NOTCH1, CCND1, CDKN2A, EGFR and PI3KCA. Virus-related SCCHN can be managed with chemo-radiotherapy. Alcohol and tobacco-related tumors should be further characterized on the basis of their "Driver Mutations" in order to select effective targeted therapies.

PD-1 and PD-L1 Expression Predicts Radiosensitivity and Clinical Outcomes in Head and Neck Cancer and is Associated with HPV Infection.

Objectives: PD-1 and PD-L1 overexpression in malignant tumors in response to radiotherapy is correlated with a poor prognosis. Human papilloma virus (HPV) infection impacts intrinsic radiosensitivity of head and neck cancers (HNCs). Herein, this study aims to determine PD-1/PD-L1 expression differences in tumors with different HPV statuses and their prognostic value in patients with different radiosensitivity gene signatures to define the characteristics of patients who will benefit from radiotherapy combined with anti-PD-1/PD-L1 therapy.Material and methods: According to the identified gene signature related to radiosensitivity, 517 patients from the TCGA HNSCC cohort were selected and divided into the radioresistant (RR) group and radiosensitive (RS) group using a K-mean clustering algorithm. All data analyses were conducted using SPSS and GraphPad Prism.Results: PD-L1 expression is upregulated in tumor tissue (unpaired t test, P=0.0363; paired t test, P=0.0584) compared with normal tissue. PD-L1 was positively correlated with PD-1 expression (P<0.0001). The HPV/p16-positive group was significantly high PD-1 expression (P<0.0001). PD-L1 expression (P=0.0005) and PD-1 expression (P<0.0001) were significantly increased in the RS group compared with that in the RR group. In the patients who were treated with radiotherapy, the PD-1-high group was associated with better recurrence-free survival (RFS) (HR, 0.4892; 95% CI, 0.2357-1.015; P=0.023). Within the RR group, high PD-L1 expression was associated with reduced overall survival (OS) (HR, 2.196; 95% CI, 1.081-4.46; P=0.0108) compared with low PD-L1 expression. In the RR group, HPV/p16-negative patients with high PD-L1 expression exhibited reduced OS (HPV: HR, 2.334; 95% CI, 0.7828-6.961; P=0.0313; p16: HR,2.486; 95% CI, 0.8559-7.219; P=0.0192) compared with that of patients with low PD-L1 expression. In the PD-L1-high group, RR patients exhibited reduced OS (HR, 0.4858; 95% CI, 0.2136-1.105; P=0.0189) and RFS (HR, 0.4371; 95% CI, 0.1421-1.345; P=0.0231) compared with that of RS patients.Conclusion: Our findings demonstrated that high PD-1/PD-L1 expression was strongly related to radiosensitivity, and high PD-1 expression was significantly associated with HPV/p16-positive HNCs. Patients in the radioresistant group and patients in the HPV/p16-negative group with a radioresistant gene signature could benefit from the combination of radiotherapy and anti-PD-1/PD-L1 therapy.