Bing Yike, Wund Zacharie, Abratte Tina, Borlle Lucia, Kang Susie, Southard Teresa, Hume Kelly R
1Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY USA.
2Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY USA.
Cancer Cell Int. 2018 Nov 22;18:192. doi: 10.1186/s12935-018-0690-0. eCollection 2018.
The response of soft tissue sarcomas to cytotoxic chemotherapy is inconsistent. Biomarkers of chemoresistance or chemosensitivity are needed in order to identify appropriate patients for treatment. Given that many chemotherapeutics kill cells through direct DNA interactions, we hypothesized that upregulation of DNA damage response mechanisms would confer resistance to cytotoxic chemotherapy in sarcomas. To study this, we used spontaneously-occurring feline injection-site sarcomas (FISS).
γH2AX and expression were determined in biopsy samples of FISS. γH2AX expression was determined via immunohistochemistry whereas expression was determined via qRT-PCR. Cell lines derived from these sarcoma biopsies were then treated with carboplatin (= 11) or doxorubicin (= 5) and allowed to grow as colonies. Colony forming-ability of cells exposed to chemotherapy was compared to matched, untreated cells and expressed as percent survival relative to controls. ImageJ was used for quantification. A mixed model analysis was performed to determine if an association existed between relative survival of the treated cells and γH2AX or expression in the original tumors. Cell lines were validated via vimentin expression or growth as subcutaneous sarcomas in nude mice.
An association was detected between γH2AX expression and relative survival in cells exposed to carboplatin (= 0.0250). In the 11 FISS tumors evaluated, γH2AX expression ranged from 2.2 to 18.8% (mean, 13.3%). Cells from tumors with γH2AX expression higher than the sample population mean had fourfold greater relative survival after carboplatin exposure than cells from tumors with γH2AX expression less than the mean. There was no association between relative survival after carboplatin exposure and expression (= 0.1608), and there was no association between relative survival after doxorubicin exposure and either γH2AX (= 0.6124) or (= 0.8645) expression. Four cell lines were validated via growth as sarcomas in nude mice. Vimentin expression was confirmed in the other 7 cell lines.
γH2AX expression, but not wild type , may potentially serve as a biomarker of resistance to platinum therapeutics in soft tissue sarcomas. To further investigate this finding, prospective, in vivo studies are indicated in animal models.
软组织肉瘤对细胞毒性化疗的反应并不一致。为了确定适合治疗的患者，需要化疗耐药或化疗敏感性的生物标志物。鉴于许多化疗药物通过直接与DNA相互作用杀死细胞，我们假设DNA损伤反应机制的上调会使肉瘤对细胞毒性化疗产生耐药性。为了研究这一点，我们使用了自发形成的猫注射部位肉瘤（FISS）。
在FISS的活检样本中测定γH2AX和[具体基因]的表达。通过免疫组织化学测定γH2AX的表达，而通过qRT-PCR测定[具体基因]的表达。然后用卡铂（n = 11）或阿霉素（n = 5）处理源自这些肉瘤活检的细胞系，并使其作为集落生长。将暴露于化疗的细胞的集落形成能力与匹配的未处理细胞进行比较，并表示为相对于对照的存活百分比。使用ImageJ进行定量分析。进行混合模型分析以确定处理后细胞的相对存活率与原始肿瘤中γH2AX或[具体基因]表达之间是否存在关联。通过波形蛋白表达或在裸鼠中作为皮下肉瘤生长来验证细胞系。
在暴露于卡铂的细胞中，检测到γH2AX表达与相对存活率之间存在关联（P = 0.0250）。在评估的11个FISS肿瘤中，γH2AX表达范围为2.2%至18.8%（平均值为13.3%）。γH2AX表达高于样本总体平均值的肿瘤细胞在暴露于卡铂后的相对存活率比γH2AX表达低于平均值的肿瘤细胞高四倍。卡铂暴露后的相对存活率与[具体基因]表达之间无关联（P = 0.1608），阿霉素暴露后的相对存活率与γH2AX（P = 0.6124）或[具体基因]（P = 0.8645）表达之间也无关联。通过在裸鼠中作为肉瘤生长验证了4个细胞系。在其他7个细胞系中证实了波形蛋白表达。
γH2AX表达而非野生型[具体基因]可能潜在地作为软组织肉瘤对铂类治疗耐药的生物标志物。为了进一步研究这一发现，需要在动物模型中进行前瞻性的体内研究。
