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Purpose: Our previous report indicated that carbon ion beam irradiation upregulated immunogenic, underlining the potential clinical application of radioimmunotherapy. The antimetastatic efficacy of a local combination therapy of carbon-ion radiotherapy and immunotherapy was examined using in vivo murine model. Methods and Materials: Tumors of the mouse squamous cell carcinoma (NR-S1) cells inoculated in the leg of C3H/HeMsNrs mice were locally irradiated with a single 6 Gy dosage of carbon ions (290 MeV/nucleon, 6 cm spread-out Bragg peak). Thirty-six hours after irradiation, alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) were injected into the leg tumor. The effects on distant lung metastases were investigated using pathological observations and immunohistochemistry. Results: The mice in the control group, which were not irradiated and not injected with alpha-GalCerpulsed DCs, had 168 +/- 53.8 metastatic nodules in their lungs at 2 weeks. The lung metastases were significantly decreased in the mouse which received the combination therapy, and the mean number of lung metastatic nodules was 2.6 +/- 1.9 (P = 0.009). In addition, the size of each lung metastasis was significantly decreased in compared to the control group (P = 0.009). Immunohistochemistry showed that ICAM-1, which activates DCs, was highly expressed on the local tumors of the carbon ionirradiated group. The expression of S100A8 in lung tissue, a marker of the lung pre-metastatic phase, was decreased in the combination therapy group. Conclusion: The combination of carbon ion radiotherapy with the injection of alpha-GalCer-pulsed DCs into the primary tumors significantly decreased the number of lung metastases.

BACKGROUND: The authors previously demonstrated that fibroblast growth factor 2 (FGF2) expression levels in tumor cells (FGF2-T) may be an indicator of the efficacy of radiotherapy in patients with cervical cancer (CC). In the current study, this finding was extended in newly enrolled patients and was investigated further in stromal FGF2 (FGF2-S) expression. METHODS: Sixty-nine patients with CC were recruited as a validation set for the immunohistochemical detection of FGF2-T from biopsy samples that were taken before (pretreatment) or 1 week after the initiation of radiotherapy (midtreatment). The authors also investigated the expression of FGF2 in tumor FGF2-S and investigated vascular endothelial growth factor (VEGF), and cluster of differentiation 31 (CD31) (also called platelet endothelial cell adhesion molecule) in these patients and in an additional 35 patients from a previous study. RESULTS: FGF2 expression was detected in tumor cells from all patients and in stromal cells from 87% of patients. FGF2-T was significantly higher in midtreatment samples (P = .0002), and a high ratio of midtreatment/pretreatment FGF2-T was related significantly to a better prognosis (P = .025). Increased VEGF expression after the initiation of radiotherapy was related significantly to positive FGF2-S in pretreatment samples (P = .035); however, it was not related to prognosis or microvessel density detected by CD31 expression. CONCLUSIONS: Radiation causes a response in tumor cells and adjacent normal cells and changes the extracellular matrix environment. In this study, the authors confirmed their previous findings and demonstrated that changes in FGF2-T expression may be used as a marker to monitor the effectiveness of radiotherapy in patients with CC. These findings should improve patient selection for molecular targeted therapies, such as cytokine inhibitors, after standard-of-care treatment. Cancer 2010. © 2010 American Cancer Society.

Cancer cells often develop drug resistance. In cisplatin-resistant HeLa cisRcells, fibroblast growth factor 13 (FGF13/FHF2) gene and protein expression was strongly upregulated, and intracellular platinum concentrations were kept low. When the FGF13 expression was suppressed, both the cells' resistance to platinum drugs and their ability to keep intracellular platinum low were abolished. Overexpression of FGF13 in parent cells led to greater resistance to cisplatin and reductions in the intracellular platinum concentration. These cisplatin-resistant cells also showed increased resistance to copper. In preoperative cervical cancer biopsy samples from poor prognoses patients after cisplatin chemoradiotherapy, FGF13-positive cells were detected more abundantly than in the biopsy samples from patients with good prognoses. These results suggest that FGF13 plays a pivotal role in mediating resistance to platinum drugs, possibly via a mechanism shared by platinum and copper. Our results point to FGF13 as a novel target and useful prognostic guide for cancer therapy.

The aim of this study is to establish carbon ion beam (C-ion) resistant cancer cells and to investigate the difference in cellular characteristics between C-ion resistant and X-ray resistant cancer cells. The X-ray resistant cancer cell was already established using mouse squamous cell carcinoma cell line NRS1. The cells were repeatedly irradiated with a total dose 60 Gy of 200 kVp X-ray, and this repetitive X-ray irradiation generated the X-ray resistant cancer cell NRS1-X60. Surprisingly the result from colony formation assay after irradiation of 290 MeV/n C-ion showed that NRS1-X60 cells also acquired significant C-ion resistance, Although C-ion irradiation is known to be more effective for killing cells of X-ray resistant tumor. The survival fractions of NRS1-X60 cells at 10 Gy of X-ray or 5 Gy of C-ion is approximately 6.3- or 6.7-folds increased compared with that in NRS1 cells, respectively. DNA repair potential in NRS1-X60 cells, which is represented by the disappearance of g-H2AX foci at one day after X-ray or C-ion irradiation, is promoted by approximately 2.3-fold compared with that in NRS1 cells for both irradiations. Currently, the NRS1 cells were repeatedly irradiated with a total dose 30 Gy of C-ion, and we established NRS1-C30 cells. We will report the difference in X-ray, C-ion sensitivity, and DNA repair potential between NRS1-X60, NRS1-C30, and NRS1., Heavy Ion in Therapy and Space Radiation Symposium 2013 (HITSRS2013)

Radiation therapy has played an important role for treatment of patients diagnosed with cancer. Metastasis, radioresistance and adverse reactions are the most crucial issues for radiotherapy to overcome. Identification of molecular markers that predict prognosis and/or risk for normal tissues within the irradiated field may help to improve currently available therapies as well as to develop novel therapies. \nWe have identified several molecular markers for predicting prognosis by using clinical materials, such as biopsies, from patients before/after radiotherapy, including carbon ion radiotherapy. However, it is not possible to obtain biopsies from inaccessible tumors such as those that are targets for carbon ion radiotherapy. To resolve these issues, we have taken blood samples in addition to clinical samples before/after radiation to find novel molecular markers. \nIn this symposium, I would like to present our research project to identify molecular markers for carbon ion radiotherapy by using the next-generation sequencer at Research Institute for Radiation Biology and Medicine, Hiroshima University., 3rd International symposium Biological Effects of Low Dose Radiation

Purpose: Hyaluronan (HA), a polysaccharide, is synthesized by a hyaluronan synthase (HAS). HA interacts with cells in dynamic processes such as embryogenesis and oncogenesis through interactions with several types of surface receptors like CD44 that play a role in regulating important signaling pathways, including the PI3K pathway. To identify predictive markers for radiotherapy effectiveness in cervical cancer, we have been analyzing the expression and genetic variation of some key genes such as FGF2, CD44, and HAS1. This study sought to determine the association between polymorphisms in CD44 and HAS1 genes with radiotherapy effectiveness in patients with cervical cancer. Materials and Methods: The study population comprised 121 cervical cancer patients who were treated with pelvic radiotherapy (the median dose of external beam = 50.6 Gy [range, 45.0 - 60.6 Gy] and median dose of brachytherapy = 24.0 Gy [range, 19.0 - 36.5 Gy]). The patients were defined as good (n = 82, mean age = 61 years [range, 32-83 years]) or poor (n = 39, mean age = 54 years [36-82 years]) responders on the basis of their 2-year disease-free survival. Genomic DNA was obtained from their blood samples and was genotyped using 19 SNPs in CD44 and 4 SNPs in HAS1. The association between each SNP and prognosis was evaluated by multivariate logistic regression analysis conditioned on the data pertaining to smoking, histological classification (squamous cell carcinoma/adenocarcinoma), and the International Federation of Gynecology and Obstetrics (FIGO) staging (I, II, III, IVA) of the tumors. Results: In logistic regression analysis conditioned on FIGO staging and smoking habits (the other factors did not contribute to the final model), P-values of 0.0056 was obtained for the SNP, T-2016C in CD44 (OR = 2.49, 95% CI = 1.31 - 4.76). The variant was located in the 5' upstream region of the gene, suggesting that patients with the variant contribute to produce different amounts of CD44. Conclusions: Thus, radiotherapy effectiveness in patients with cervical cancer may be predicted, at least in part, by the genotype of CD44. Because of the limited sample size used in this study, further replication studies and functional analyses are required to confirm the results of the association analysis. However, the present data may be useful for analyzing the mechanisms underlying interaction between HA and CD44 and the subsequent cervical cancer-associated signaling., CSH Asia / ICMS (The International Cancer Microenvironment Society) Joint Conference on Tumor Microenvironment

[Objectives] The carbon-ion (C-ion) radiotherapy (RT) is an advanced RT using positively-charged carbon-ion particles. Due to the biological properties and excellent dose distribution, the C-ion RT is effective in resistance tumor to the conventional RT. However, metastasis control is one of the important issues in C-ion RT. Therefore, we have examined the possibility of control of metastasis by combined therapy with C-ion. In this study, we focused on a combined therapy of C-ion irradiation and dendritic cell (DC) immunotherapy and examined the effects of this treatment in the mouse lung metastatic models. [Methods] The mouse carcinoma cell lines (NR-S1, LLC, LM8) inoculated into the legs of C3H/He or C57BL/6J mice were irradiated with a single dosage of carbon-ion (6Gy or 2 Gy, 290 MeV/nucleon, 6 cm SOBP). At 36 hours after irradiation, DCs were injected into the mice. The numbers of lung metastasis were evaluated using pathological observations. [Results] We established the mouse metastasis model using squamous carcinoma cell line NR-S1, lung cancer cell line, LLC, and osteosarcoma cell line, LM8. Under conditions that there are no significant effects by the treatments on the growth of transplanted tumors, the number of lung metastasis was significantly decreased by the combined therapy. [Conclusions] Our present results demonstrated that combined therapy of C-ion irradiation and DC immunotherapy was effective in repression of lung metastasis against these tumors., The 2nd Japan-China Symposium on Cancer Research

The aim of this study was to investigate whether inherent factors produce variance of lung morbidity in response to carbon-ion (C-ion) irradiation, and to identify the molecules that have a key role in strain dependent lung adverse effects. Three strains of female mice (C3H/He Slc, C57BL/6J Jms Slc and A/J Jms Slc) were locally irradiated in the thorax with either C-ion beam (290 MeV/n, in 6 cm SOBP) or with 137Cs gamma-rays as a reference beam. We performed survival assays, histological examinations of the lung with hematoxylin-eosin staining, Masson s trichrome staining, immunohistochemical staining for CD44 and Mac3, and gene expression analyses. Intra-alveolar hemorrhage was assessed by Berlin blue staining and measurement of the iron concentration of bronchoalveolar lavage fluid. Survival data presented a mouse strain-variance after C-ion irradiation at 10 Gy. The histological examination revealed early phase hemorrhagic pneumonitis in C 3! H/He mice and late phase pulmonary fibrosis in C57BL/6J mice after C-ion irradiation at 10 Gy. Microarray analysis of irradiated lung tissue in three mouse strains identified differential expression changes of the growth differentiation factor 15 gene (Gdf15), that regulates inflammatory and apoptotic pathways and macrophage function and for the hyaluronan synthase 1 gene (Has1), that plays a role in hyaluronan metabolism. Immunohistochemical analysis showed that the ratio of CD44-positive cells as surrogate marker for hyaluronan accumulation and Mac3-positive cells as maker for inflammation and macrophage infiltration in irradiated lung varied significantly among three mouse strains. This study demonstrated the strain-dependent differential response in mice to high-LET C-ion irradiation. Our findings identified possible molecules relating to the strain-variance of lung toxicity after C-ion irradiation., 14th International Congress of Radiation Research(ICRR’2011)

It is well known that a cyclic cascade of inflammatory cytokines, together with the activation of macrophages, is initiated very early after irradiation for development of lung fibrosis in a late phase. Interleukin-6 (IL-6), one of inflammatory cytokines, is involved in development of lung fibrosis. Although IL-6 is induced by radiation, the role of IL-6 in lung fibrosis caused by radiation was unclear. In this study, we evaluated a role of IL-6 in the late-phase inflammatory response and subsequent fibrotic changes after carbon-ion (c-ion) irradiation using wild-type (WT) and IL-6 knock out (IL-6 KO) mice. The mice underwent thoracic irradiation with 10 Gy of c-ion beam or sham-irradiation and were examined by histology. At 24 weeks after irradiation, the infiltration of macrophages, detected by positive immunohistological staining with Mac3 antibody, was observed in alveolar spaces both in WT and IL-6 KO mice. The thickening of bronchiolar and alveolar walls exhibited in WT mice, but not KO mice, and fibrotic changes, detected by Masson-Trichrome staining, were observed only in the lungs of WT mice, while it was attenuated in IL-6 KO mice. These results indicated that IL-6 plays an important role for fibrotic changes of alveolar wall after irradiation., The 53ｒd Annual Meeting of The Japan Radiation Research Society

Propose: Irradiation of carbon-ion (C-ion) leads to the formation of severe DNA damage without dependency on the cell cycle phase and oxygen status. However, the molecular mechanisms underlying the therapeutic response to C-ion are not fully understood. In this study, we investigated the molecular pathways induced by C-ion radiotherapy using sequentially taken biopsy samples of cervical cancers to understand the biological mechanism specific to C-ion radiotherapy. Methods: Biopsy specimens were obtained from 60 patients with cervical cancer, before and during fractionated C-ion radiotherapy. C-ion treatment was initiated with a fraction dose of 2.2 GyE and total dose was 71.2- 74.4 GyE. A second biopsy was taken 1 day after the start of radiation. Transcriptome analysis was performed using single-color oligo-microarrays consisting of 44 K human sequences for biopsy samples from 22 patients. Expression values of genes before and during radiotherapy were compared and the statistical analysis was used with the Wilcoxon signed rank test (p < 0.001). The changes of gene expression were confirmed by Real-time PCR analysis, and further immunohistological analysis was performed to confirm the expression and localization of target proteins using newly enrolled 38 patients. Results: Two hundred and twenty genes were identified as C-ion responded genes (CRGs) in cervical cancers under our microarray-analysis conditions. We found three new CRGs in addition to the previously reported ionizing radiation-induced genes such as CDKN1A, BAX, Fas, and DDB2. The p53-target genes such as MDM2, TRIAP1 and GADD45 were also responded to C-ion in the cervical cancers. Comparison of the expression profiles of the human cervical cancers with those of the experimental murine tumors (Imadome et al. Cancer Biol. Ther. 7: 208 – 217, 2008) showed that the genes categorized into the cell-communication and/or the stress-response were commonly involved in both the expression profiles after C-ion irradiation. Conclusions: Genes responded to C-ion irradiation in human cervical cancers were identified. These genes were mainly involved in cell communication or stress response pathways. Our findings may lead to a better understanding the molecular mechanisms underlying the therapeutic response to C-ion., PTCOG 49 (49th Annual Meeting of the Particle Therapy Co-Operative Group)

Purpose Tumor microenvironment possesses extreamly important role for tumor progression and metastasis. Cytokines have autocrine and paracrine functions, and they are also secreted by normal and cancerous cells. Herewith we investigated an indicator for the efficacy of radiotherapy in cervical cancers (CC) using microarray analysis and immunohistochemical analysis. Patients and methods One hundred and four patients with CC were recruited and divided into two groups (research set: n =35, and validation set: n =69). Microarray analysis was performed in research set and further immunohistochemical analysis (IHA) was performed for all patients to detect candidate radioresponsive markers using pre-radiotherapy and mid-radiotherapy biopsy samples, which were taken one week after initiation of radiotherapy. Results FGF2 in tumor cells (FGF2–T) significantly increased in midtreatment samples compared with pretreatment samples in research set of patients, and the ratio change of FGF2-T was significantly related with better prognosis. This evidence was confirmed in validation set. Next using all 104 patimets we found IHA positive FGF2 in stromal cells (FGF2-S) in 85 patients, and the radiotherapy-induced increase of FGF-S in 23 patients. Though positive FGF2-S in pretreatment samples was significantly related with increased expression change of VEGF, it was not related with poor prognosis. Conclusion Radiation causes severing the normal or cancerous associations with adjacent cells and changes the extracellular matrix environment. Therefore, we need to investigate not only pretreatment status of tumors, but also modified tumor structures during fractionated radiotherapy. In this study, we found FGF2-T expression change as a monitoring marker for the effectiveness of radiotherapy, and found the relationship between FGF2-S in pretreatment status and VEGF expression change in a subgroup of patients., 5th International Conference on Tumor Microenvironment: Progression, Therapy & Prevention

We previously reported radiotherapy-responsive genes, which were revealed by comprehensive transcriptome analysis using the microarray technology for sequential biopsies for cervical cancer patients with radiotherapy (RT). We found several tens of RT-responsive genes, including well-known radiation-responsive apoptosis/cell cycle-related genes, such as cdkn1a/p21, CAD, and Bax, and several tens of genes categorized into extracellular matrix (ECM), such as HPSE and CD44. In addition, several cytokines were also included. In this study, we investigated the possibility that some of those molecules, which were changed their expressions by RT, might be new biomarkers for the effectiveness of RT. Sequential biopsy samples were immunohistochemically analyzed before and during RT in cervical cancer patients, and the prognostic value of such changes of 15 candidate molecules for disease failure after RT were evaluated. \nPatients and Methods: Biopsy specimens were obtained from 91 patients with cervical cancers before (pretreatment) and 1 week after initiation (midtreatment) of radiotherapy. which were in RT-responsive genes or related with those genes, were performed to detect protein expression using an automated streptavidin-biotin immunoperoxidase staining system. Positive area/numbers of cells proportion (%) of immunostaining were analyzed using an image analysis system or the positive staining appearance was scored by grading system. Patients were defined as good or poor responders based on their two-year disease-free survival. Aberrant genomic change, human papillomavirus infection, and p53 status in tumor were also evaluated. \nResults: Protein expression of cdkn1a/p21, Bax, p53 and FGF2 in midtreatment samples (mid) was significantly higher than in pretreatment samples (pre). Protein expression of P-cadherin, ICAD, S100, p73 and VEGF in mid was significantly lower than in pre. Discontinuity of laminin staining pattern in mid was significantly higher than in pre. The ratio change (mid versus pre) of FGF2 expression in poor responders was significantly lower than that in good responders (P < 0.05). The number of cases with discontinuity of laminin staining pattern at pre was significantly higher in the poor responders (P < 0.05). \nConclusions: Using biopsy specimens from pretreatment and midtreatment cervical cancers, we revealed significant changes in expression of several proteins during fractionated radiotherapy. We also found that some of these ratio changes were significantly associated with prognosis. These molecular features in sequential biopsy samples might help us to identify patients at high risk of disease failure after radiotherapy and to provide tool for personalized radiotherapy., European Society for Therapeutic Radiology and Oncology Annual Congress(ESTRO27)

Objective: To elucidate the in vivo biological effects induced by carbon-ion irradiation using comprehensive expression analysis. Materials and Methods: We examined gene expression changes after carbon-ion (C-ion) irradiation (290 MeV/m, SOBP 6 cm middle, 50 kev/m) with a single dose of 30 Gy in four mouse tumors (NR-S1, SCCVII, NFSa, and #8520) transplanted into the hind legs of C3H/HeNrs mice, using 44K single-color oligo-microarrays at 6 hours (h), 1 day, and 3 days after irradiation. Gamma rays of 30 Gy and 50 Gy were used as a reference beam. Identification of C-ion-responsive genes was based on a false discovery rate of < 5% using the Wilcoxon test (P < 0.001) and the Benjamini-Hochberg correction. Results: In all tumors, the level of expression of several tens of genes, including Ccl3, Ccng1, Cd80, Cdkn1a, Cxcl2, IL7r, Lrdd, Mgmt, Mmp8, and Polk, was significantly altered 6 h and day 1 following C-ion irradiation. At day 3, several hundred genes, many of which are also classified as stress-response or cell-communication genes, including Tnfrsf5, Ikbke, Ifi202b, and Icam1, were upregulated following C-ion irradiation. The expression level of the majority of these genes was similar following -ray treatment, although the change was not as extensive and intertumor variance was apparent. Several genes, including Ikbke, Serpina3n, and Saa3, responded differentially following C-ion irradiation than after -ray irradiation. Pathological investigation and immunohistochemical analysis of Cdkn1a revealed cell cycle arrest with mitotic catastrophe in tumors irradiated by C-ions. Conclusions: This study revealed significant C-ion induced up-regulation of stress-responsive and cell-communication genes common to different tumor types. These findings provide evidence for the efficacy of this modality for the treatment of local tumors., 第66回日本癌学会学術総会

Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas: NR-S1 (radioresistant) and SCCVII (radiosensitive), after irradiation with 137-Cs gamma rays at doses of 30, 50 and 70 Gy or 290 MeV/u carbon ions at a dose of 30 Gy. Potential genes related to radiosensitivity were selected by comparing the expression values before and after irradiation (with both gamma rays and carbon ions) using a filter for at least 1.5-fold changes. Furthermore, candidate genes which had significantly different ratio values between the two tumors (P < 0.05) were detected by unpaired Student's t-tests. Subsequent analysis by quantitative reverse-transcription polymerase chain reaction (RT-PCR) confirmed our microarray data. Protein expression and function were examined by immunohistochemical studies. Results: Four genes, Efna1, Sprr1a, Srgap3 and Xrra1, were selected as potential genes related to radioresistance after gamma and carbon ion irradiation. RT-PCR confirmed that Efna1 was induced in radioresistant NR-S1. Efna1, a proangiogenic factor, was expressed in the cytoplasm of tumor cells and significant increases in microvascular density were observed in the radioresistant NR-S1. Conclusions: We found that Efna1 may be a potential molecule related to radioresistance in murine tumors., The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncology

Objective: To identify changes in gene expression related to the concurrent use of platinum compounds with radiotherapy, in the treatment of cervical cancer. Patients and Methods: Biopsy specimens were obtained from 53 patients with the uterine carcinoma, before and during fractionated radiotherapy. Twenty-five patients were treated with radiotherapy (RT) alone, while 28 received the same radiotherapy plus concomitant chemotherapy with cisplatin (CRT). Changes in gene expression induced by treatment were investigated using CodeLink single-color oligo-microarrays consisting of 44K human sequences. Paraffin-embedded samples were used to examine apoptosis and the expression of protein related with treatment-responsive genes. Changes in mRNA expression were assessed for these genes by real-time reverse transcriptase-polymerase chain reaction. Results: We found several tens of genes, including CDKN1A, BAX, TNFSF8, RRM2B, and FGF2, responded to CRT using microarray analysis, and suggested CRT activated TNF-mediated cell death pathway and extra-cellular matrix pathway related with cell death pathway. Apoptotic cells were significantly increased in both groups. In immunohistochemical study, CRT significantly increased the numbers of cases with diffusely distributed CDKN1A-positive cells (P < 0.002). Protein expression of ICAD was recognized weakly in nucleus of tumor cells in pre-treatment samples and decreased significantly after CRT (P < 0.05). BAK1 increased its intensity in half of cases at mid-treatment. The expression changes of FGF2 were revealed to be significantly different between good responders and poor responders (P < 0.05). Conclusions: CRT produced a homogenous pattern of changes in expression of known radiation-responsive genes. Our data suggest that concurrent use of cisplatin produced a radiosensitizing effect in most of these cervical cancer patients. Microarray analysis appeared a useful tool to get a comprehensive overview of the changes in gene expression after irradiation. In addition, transcriptional profiling of sequential biopsies after irradiation would improve our understanding of the biological effectiveness of radiotherapy, and provide information on potential targets for adjuvant therapy., The 13th International Congress of Radiation Research

Developments in radiation technology have lead to the clinical improvement of Carbon ion (C-ion) irradiation with well-localized energy distributions (high LET). While the development of high LET has a number of advantages over the more traditional radiation technology for the treatment of cancer, the mechanisms underlying the superior biological effectiveness of C-ion irradiation is not fully understood. Several studies have investigated the biological mechanism of C-ion irradiation in vitro. The main disadvantage of these in vitro studies includes significant differences in the gene expression profile between cell lines in vitro and the same cell lines growing in vivo and the effect of other factors related to the cellular microenvironment on the gene expression profile. Previous studies suggest that more appropriate in vivo tumor models allowing translation of findings to the clinical setting are required. We used microarray technology to examine the fate of multiple C-ion-irradiated tumors in vivo to gain a comprehensive overview of the changes in gene expression induced by C-ion irradiation. Four murine tumors were irradiated in vivo with C-ions at a single dose, and gamma-rays were used as a reference beam. \nObjective: To elucidate the in vivo biological effects induced by carbon-ion irradiation using comprehensive expression analysis. Materials and Methods: We examined gene expression changes after C-ion irradiation (290 MeV/m, SOBP 6 cm middle) with a single dose of 30 Gy in four mouse tumors (NR-S1, SCCVII, NFSa, and #8520) transplanted into the hind legs of C3H/HeNrs mice, using 44K single-color oligo-microarrays at 6 hours (h), 1 day, and 3 days after irradiation. Gamma-rays of 30 Gy and 50 Gy were used as a reference beam. Identification of C-ion-responsive genes was based on a false discovery rate of < 5% using the Wilcoxon test (P < 0.001) and the Benjamini-Hochberg correction. Results: The first finding of this study was that C-ion irradiation causes significant changes in the expression of genes that are already known to be related to radiation-induced tumor regression. A hypothetical C-ion-induced pathway indicated that a well known radiation-induced mechanism of tumor regression was similarly activated by C-ion irradiation. Radiation-induced apoptosis related genes, including Casp4, Bcl2a1b, and TNF family were also listed as C-ion responsive genes. Second, at 6 h and 1 day following C-ion irradiation, several stress-responsive genes or cell communication-related genes were also upregulated. In all tumors, the level of expression of several tens of genes, including Ccl3, Ccng1, Cd80, Cdkn1a, Cxcl2, IL7r, Lrdd, Mgmt, Mmp8, and Polk, was significantly altered 6 h and day 1 following C-ion irradiation. Third, at day 3, several hundred genes, many of which are also classified as stress-response or cell-communication genes, including Tnfrsf5, Ikbke, and Icam1, were upregulated following C-ion irradiation. The expression level of the majority of these genes was similar following gamma-ray treatment, although the change was not as extensive and intertumor variance was apparent. Several genes, including Ikbke, Serpina3n, and Saa3, responded differentially following C-ion irradiation than after gamma-ray irradiation. Pathological investigation and immunohistochemical analysis of Cdkn1a revealed cell cycle arrest with mitotic catastrophe in tumors irradiated by C-ions. \nConclusions: This study revealed significant C-ion induced up-regulation of stress-responsive and cell-communication genes common to different tumor types. These findings provide evidence for the efficacy of this modality for the treatment of local tumors. In addition, antimetastatic effects of local C-ion irradiation in murine model and evaluation of C-ion responsive genes in clinical biopsy samples taken from cervical cancer patients would be presented., NIRS International Workshop on Particle Radiation Science