Etsuko Nakamura

Upregulation of stress-response genes with cell cycle arrest induced by carbon ion irradiation in multiple murine tumors models

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/μm) 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, and Icam1, were upregulated following C-ion irradiation. The expression level of the majority of these genes was similar following γ-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 γ-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 upregulation 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.

Combining carbon ion radiotherapy and local injection of α-galactosylceramide-pulsed dendritic cells inhibits lung metastases in an in vivo murine model.

PURPOSE Our previous report indicated that carbon ion beam irradiation upregulated membrane-associated immunogenic molecules, underlining the potential clinical application of radioimmunotherapy. The antimetastatic efficacy of local combination therapy of carbon ion radiotherapy and immunotherapy was examined by use of an in vivo murine model. METHODS AND MATERIALS Tumors of mouse squamous cell carcinoma (NR-S1) cells inoculated in the legs of C3H/HeSlc mice were locally irradiated with a single 6-Gy dose of carbon ions (290 MeV/nucleon, 6-cm spread-out Bragg peak). Thirty-six hours after irradiation, α-galactosylceramide-pulsed dendritic cells (DCs) were injected into the leg tumor. We investigated the effects on distant lung metastases by counting the numbers of lung tumor colonies, making pathologic observations, and assessing immunohistochemistry. RESULTS The mice with no treatment (control) presented with 168 ± 53.8 metastatic nodules in the lungs, whereas the mice that received the combination therapy of carbon ion irradiation and DCs presented with 2.6 ± 1.9 (P = 0.009) at 2 weeks after irradiation. Immunohistochemistry showed that intracellular adhesion molecule 1, which activates DCs, increased from 6 h to 36 h after irradiation in the local tumors of the carbon ion-irradiated group. The expression of S100A8 in lung tissue, a marker of the lung pre-metastatic phase, was decreased only in the group with a combination of carbon ions and DCs. CONCLUSIONS The combination of carbon ion radiotherapy with the injection of α-galactosylceramide-pulsed DCs into the primary tumor effectively inhibited distant lung metastases.

Villin1, a novel diagnostic marker for cervical adenocarcinoma

The number of new cervical adenocarcinoma (AD) cases has risen slowly, however, its histological similarity to other tumor types and the difficulty of identifying the site of the original tumor makes the diagnosis of cervical AD particularly challenging. We investigated a novel molecular biomarker for cervical AD through the integration of multiple methods of genomic analysis. Tumor samples in discovery set were obtained from 87 patients who underwent radiotherapy, including 31 cervical AD. Microarray analysis and quantitative polymerase chain reaction analysis were performed to screen a candidate diagnostic molecule for cervical AD, and its clinical significance was investigated by immunohistochemical analysis (IHC). We found a difference between biopsy samples of AD and squamous cell carcinoma (SCC) in the expression and genomic copy number of Villin1 (VIL1), which maps to 2q35. IHC revealed 14 VIL1-positive tumors; 13 cervical AD and 1 small cell carcinoma of cervix, while none of SCC or endometrial AD was VIL1-positive. Kaplan-Meier survival curves revealed worse disease-free survival in VIL1-positive tumors. The marker was validated by newly enrolled 65 patients, and VIL1 positive staining showed 52% of sensitivity and 100% of selectivity for cervical AD. In conclusion, we have identified VIL1 as a novel biomarker of cervical AD. VIL1, a major structural component of the brush border cytoskeleton, which was recently found to be an epithelial cell-specific anti-apoptotic protein. Our study suggests the existence of a subtype of cervical tumors which are VIL1 positive with poor radioresponse.

Strain-dependent Damage in Mouse Lung After Carbon Ion Irradiation

PURPOSE To examine whether inherent factors produce differences in lung morbidity in response to carbon ion (C-ion) irradiation, and to identify the molecules that have a key role in strain-dependent adverse effects in the lung. METHODS AND MATERIALS 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 beams (290 MeV/n, in 6 cm spread-out Bragg peak) or with ¹³⁷Cs γ-rays as a reference beam. We performed survival assays and histologic examination of the lung with hematoxylin-eosin and Massons trichrome staining. In addition, we performed immunohistochemical staining for hyaluronic acid (HA), CD44, and Mac3 and assayed for gene expression. RESULTS The survival data in mice showed a between-strain variance after C-ion irradiation with 10 Gy. The median survival time of C3H/He was significantly shortened after C-ion irradiation at the higher dose of 12.5 Gy. Histologic examination revealed early-phase hemorrhagic pneumonitis in C3H/He and late-phase focal fibrotic lesions in C57BL/6J after C-ion irradiation with 10 Gy. Pleural effusion was apparent in C57BL/6J and A/J mice, 168 days after C-ion irradiation with 10 Gy. Microarray analysis of irradiated lung tissue in the three mouse strains identified differential expression changes in growth differentiation factor 15 (Gdf15), which regulates macrophage function, and hyaluronan synthase 1 (Has1), which plays a role in HA metabolism. Immunohistochemistry showed that the number of CD44-positive cells, a surrogate marker for HA accumulation, and Mac3-positive cells, a marker for macrophage infiltration in irradiated lung, varied significantly among the three mouse strains during the early phase. CONCLUSIONS This study demonstrated a strain-dependent differential response in mice to C-ion thoracic irradiation. Our findings identified candidate molecules that could be implicated in the between-strain variance to early hemorrhagic pneumonitis after C-ion irradiation.

Villin1, a diagnostic marker for endometrial adenocarcinoma with high grade nuclear atypia

Villin1 (VIL1) has a role in regulating actin dynamics, cell morphology, anti-apoptotic mechanisms, and epithelial-to-mesenchymal transition. Previously we reported VIL1 as a novel diagnostic marker for cervical adenocarcinoma (AC) with poor radioresponse. This study further investigated the diagnostic role of VIL1 in gynecological tumors especially endometrial AC. We recruited 107 patients with AC (41 tumors in the corpus and 66 tumors in cervix), most of whom treated by total abdominal hysterectomy. Immunohistochemical analysis revealed VIL1-positive tumors in 37% of cases; 10 of 41 corpus tumors and 30 of 66 tumors in the cervix. VIL1-positive tumors were further examined histologically and immunostained for epithelial cell surface marker, EpCAM, and mesenchymal stem cell marker, CD44. Most of these tumors were CD44 negative and EpCAM positive, and the cytoplasmic VIL1 immunoreactivity in endometrial AC was more selective than EpCAM in reflecting histological aggressiveness with high grade nuclear atypia. This study confirmed our previous finding of VIL1 as a diagnostic marker of cervical AC. In addition, VIL1 immunostaining was detected in 25% of endometrial AC cases. These results suggested the existence of an aggressive and VIL1-positive subtype of gynecological tumor.

Change in fibroblast growth factor 2 expression as an early phase radiotherapy-responsive marker in sequential biopsy samples from patients with cervical cancer during fractionated radiotherapy

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.