Establishment And Characterization Of A Radioresistant Tumor Model Of Human Nasopharyngeal Carcinoma

【Background and objectives】Nasopharyngeal carcinoma （NPC） is an endemic disease in southeastAsia and southeast China. Radiotherapy is the primary treatment for NPCpatients. Concurrent chemoradiotherapy （CCRT） has been considered asthe standered treatment of locally advanced NPC^1-2. Some of the NPCpatients present with local recurrences and distant metastases within 1-2years after treatment due to radioresistance5. The main reason of failure toradiotherapy is radioresistance. At present, numerous studies aimed atidentifying differential genes or differential expressioned proteins（DEPs）associated with cancer radioresistance using high throughput proteomicsmethods between parental cell lines and radioresistant subclones, andulteriorly testified some of the DEPs in vitro^6-9. In vivo research wasseldom reported.Although it was reported that several radioresistant CNE2 subclonecell lines had heen established^6-7, not an animal experiment was reportedso far. As described in our previous research, we also induced aradioresistant NPC subclone cell line named CNE-2R by treating the parental CNE-2 cells with fractioned⁶⁰Co-γray irradiation to a total doseof 6400cGy^8-9. Athymic BALB/c-nu nude mice lack T cells and sufferfrom a lack of cell-mediated immunity, which permits transplantationof tumor cell xenografts. In this present study, we first established aradioresistant tumor model of human NPC by injecting the CNE-2R cellsinto athymic nude mice. Then we irradiated the xenografts withfractionedχ-ray irradiation, and studied its growth characterization.Finally, in order to validate the variations of the DEPs identified by usbefore, we examined the NPM1, nm23-H1 and annexin A3 proteinexpressions in tumor tissues using immunohistochemistry. These arecrucial for understanding the mechanism of the radioresistance andsuppling new biomarkers for clinical use.【Methods】1.CNE-2R cells and CNE-2 cells were subcutaneously injected intohind legs of athymic nude mice, in order to establish a pair of comparabletumor models: a radioresistant tumor model and a raiosensitive tumormodel of human NPC.2.Diameters of subcutaneous xenograft tumors were measured witha caliper as the tumors were palpated, and volumes of the tumors wereregularly calculated, then a growth curve of the two groups of tumors wasdrawn. The tumors-bearing mice received fractionedχ-ray irradiationwhen the tumors reached about 1cm in diameters, 16Gy in 4 fractions delivered over 8 days. Inhibitions of the volumes of the two groups oftumors were compared.3.Tumors were examined by routinely pathological examination, andmorphology of the tumor cells were observed under the light microscope.4.The tumor tissuses were examined by immunohistochemicalstaining for NPM1,nm23-H1 and annexin A3 protein expressions.【Results】1.The CNE-2R xenograft tumor model and the CNE-2 xenografttumor model were established successfully.2.Compared to the NIR-CNE-2 tumors, the NIR-CNE-2R tumorsgrew significantly slowly（P<0.01）. Tumor dobuling times of theNIR-CNE-2R tumors and the NIR-CNE-2 tumors were 4.8 days and 3.9days, respectively. The progression of CNE-2R tumors was not inhibitedby irradiation（P>0.05）, while that of the CNE-2 tumors was inhibited byirradiation（P<0.01）.Tumor dobuling times of the IR-CNE-2R tumors andthe IR-CNE-2 tumors were 6.2 days and 17.1 days, respectively. Thevolume increasing rate of the IR-CNE-2R tumors was higher than that ofthe IR-CNE-2 tumors（P<0.01）.3.A large number of tumor necrosis areas were observed in theNIR-CNE-2R tumors and the NIR-CNE-2 tumors. In the irradiatedtumors, only a few scattered necrotic areas were observed in theIR-CNE-2R tumors. Extensive degeneration and pyknotic cells were observed in tumors after irradiation. Cancer cells were nested, with largenucleus and abundant cytoplasm, round to oval hyperchromatic nuclei.Keratinization is minimal or absent, and the mitotic rate is variable. Thetumor cells possessed the morphological characteristics as human NPCcells, indicating that the comparable tumor models were establishedsuccessfully.4.NPM1 and Annexin A3 protein expressions were significantlylower in the NIR-CNE-2R tumors than in the NIR-CNE-2 tumors（P<0.01）, while Nm23-H1 protein expression was significantly higher（P<0.05）. Annexin A3 protein expression was significantlydown-regulated in the IR-CNE-2R tumors than in the IR-CNE-2 tumors（P<0.01）, while Nm23-H1 protein expression was significantlyup-regulated（P<0.01）. NPM1 protein expression was up-regulated in theIR-CNE-2R tumors compared with the NIR-CNE-2 tumors, although noobvious difference was observed（P>0.05）.【Conclusions】1.The radioresistant tumor model of human NPC（CNE-2R tumor）was established successfully.2.Compared to the NIR-CNE-2 tumors, the NIR-CNE-2R tumorsgrew significantly slowly, and the growth of the IR-CNE-2R tumors wasnot inhibited by irradiation, indicating that the CNE-2R tumors mighthave obtained radioresistance through slowing down growth. 3.NPM1 and Annexin A3 protein expressions were significantlylower in the NIR-CNE-2R tumors than in the NIR-CNE-2 tumors, andNm23-H1 expression was significantly higher, which were inconformance with our previous in vitro study. These results indicated thatNPM1 protein, Annexin A3 protein and Nm23-H1 protein might probablycorelate with radioresistance of NPC. Nevertheless, futher clinicalresearches are needed to conform it.4.NPM1 protein and Nm23-H1 protein expressions wereup-regulated in the IR-CNE-2 tumors than in the IR-CNE-2 tumors,indicating that these two proteins might correlate with DNA repair afterχ-ray iradiation.