宫颈癌仍然是全世界女性面临的重大健康挑战，对撒哈拉以南非洲等发展中地区的影响尤为严重。利用开发合适的临床前模型来研究细胞增殖、分化和基因表达的最新进展，我们使用 RNA 测序来比较在 3D 和 2D 培养系统中生长的 SiHa 宫颈癌细胞的转录组谱。 3D 培养物的通路分析揭示了免疫激活、血管生成和组织重塑通路的上调。细胞因子、趋化因子、基质金属蛋白酶和立即早期基因的高表达表明 3D 培养物比 2D 单层培养物更好地复制肿瘤微环境。 HPV 基因表达分析进一步证明，与 2D 培养物相比，3D 培养物中 HPV16 E1、E2、E6 和 E7 基因的表达水平更高。此外，通过使用一组线性模型，我们鉴定出 3D 培养中与 2D 培养条件相比有 79 个显着差异表达的基因，且与 HPV16 病毒基因效应无关。随后，我们在 SiHa 细胞系和新开发的患者来源的宫颈癌细胞系中在蛋白质水平上验证了其中 5 个基因。此外，相关性分析发现 26 个人类基因在 2D 和 3D 培养条件下与病毒基因呈正相关。通过 qRT-PCR 在我们的患者衍生细胞系中验证了前 5 个 3D 与 2D 差异表达和 HPV 相关基因。总而言之，这些发现表明 3D 培养提供了卓越的模型系统来探索免疫逃避、癌症进展和抗病毒治疗的机制。版权所有 © 2024 Kumar、Iden、Tsaih、Schmidt、Ojesina 和 Rader。

Cervical cancer remains a significant health challenge for women worldwide, with a disproportionate impact on developing regions like sub-Saharan Africa. Taking advantage of recent advancements in developing suitable preclinical models to study cell proliferation, differentiation, and gene expression, we used RNA sequencing to compare the transcriptomic profiles of SiHa cervical cancer cells grown in 3D versus 2D culture systems. Pathway analysis of 3D cultures revealed upregulation of immune activation, angiogenesis, and tissue remodeling pathways. The high expression of cytokines, chemokines, matrix metalloproteinases, and immediate early genes, suggests that 3D cultures replicate the tumor microenvironment better than 2D monolayer cultures. HPV gene expression analysis further demonstrated higher expression levels of HPV16 E1, E2, E6, and E7 genes in 3D versus 2D cultures. Further, by using a set of linear models, we identified 79 significantly differentially expressed genes in 3D culture compared to 2D culture conditions, independent of HPV16 viral gene effects. We subsequently validated five of these genes at the protein level in both the SiHa cell line and a newly developed, patient-derived cervical cancer cell line. In addition, correlation analysis identified 26 human genes positively correlated with viral genes across 2D and 3D culture conditions. The top five 3D versus 2D differentially expressed and HPV-correlated genes were validated via qRT-PCR in our patient derived cell line. Altogether, these findings suggest that 3D cultures provide superior model systems to explore mechanisms of immune evasion, cancer progression and antiviral therapeutics.Copyright © 2024 Kumar, Iden, Tsaih, Schmidt, Ojesina and Rader.