Background/Objectives: Cervical cancer (CC) is a significant global health challenge, particularly in low- and middle-income countries (LMICs), where limited access to human papillomavirus (HPV) vaccination and effective CC screening results in a majority of cases and fatalities among women. Moreover, existing vaccines do not target HPV-independent cancers. Current screening methods are expensive and time-consuming, with a limited emphasis on CC protein biomarkers. Therefore, we aimed to validate critical markers that allow the development of affordable point-of-care screening tests for resource-limited settings. Methods: This study first optimized a cell lysis and protein extraction protocol for CC cell lines and clinical cervical swabs. Subsequently, four proteins—topoisomerase II alpha (TOP2A), minichromosome maintenance complex component 2 (MCM2), valosin-containing protein (VCP), and cyclin-dependent kinase inhibitor 2A (p16INK4a)—were quantified in the resulting lysates using enzyme-linked immunosorbent assays, as well as in cervical tumors and squamous intraepithelial lesions (SILs) using immunohistochemistry for further validation. Results: Acetone precipitation allowed for efficient cell isolation, and radioimmunoprecipitation assay buffer yielded the highest protein recovery. VCP and p16INK4a were overexpressed across all cancer cell lines compared to primary cells. All four biomarkers were overexpressed in high-grade SIL (HSIL) swab specimens and tumor samples, including CC subtypes, G1–G3 tumor grades, and HSILs. Lastly, we showed that the proteins could accurately classify swabs and tissue specimens into clinically relevant groups. Conclusions: The quantitative analysis of these biomarkers, along with the subsequent sensitive and specific clinical classification, highlights their potential application in SIL early detection and CC prevention, particularly in LMICs.
**背景/目的：** 宫颈癌是全球范围内一项重大的健康挑战，在中低收入国家尤为突出。这些国家由于人乳头瘤病毒疫苗接种和有效宫颈癌筛查的可及性有限，导致女性病例和死亡人数占绝大多数。此外，现有疫苗并不针对非HPV依赖型癌症。目前的筛查方法昂贵且耗时，对宫颈癌蛋白质生物标志物的关注有限。因此，本研究旨在验证关键标志物，以便为资源有限地区开发可负担的即时筛查检测方法。 **方法：** 本研究首先优化了适用于宫颈癌细胞系和临床宫颈拭子的细胞裂解及蛋白质提取方案。随后，使用酶联免疫吸附测定法对所得裂解液中的四种蛋白质——拓扑异构酶IIα、微小染色体维持蛋白复合体组分2、含缬酪肽蛋白和细胞周期蛋白依赖性激酶抑制剂2A——进行了定量分析，并进一步使用免疫组织化学法在宫颈肿瘤和鳞状上皮内病变组织中进行验证。 **结果：** 丙酮沉淀法可实现高效的细胞分离，而放射免疫沉淀测定缓冲液可获得最高的蛋白质回收率。与正常原代细胞相比，含缬酪肽蛋白和细胞周期蛋白依赖性激酶抑制剂2A在所有癌细胞系中均过表达。所有四种生物标志物在高级别鳞状上皮内病变拭子样本和肿瘤样本（包括宫颈癌亚型、G1-G3级肿瘤以及高级别鳞状上皮内病变）中均过表达。最后，我们证明这些蛋白质能够准确地将拭子和组织样本分类到临床相关组别中。 **结论：** 对这些生物标志物的定量分析，以及随后进行的灵敏且特异的临床分类，突显了它们在鳞状上皮内病变早期检测和宫颈癌预防（尤其是在中低收入国家）中的潜在应用价值。
肿瘤(癌症)患者之家