High-LET Proton Irradiation Significantly Alters the Clonogenic and Tumorigenic Potential of Human Breast Cancer Cell Lines In Vitro and In Vivo.

IF 3.3 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Margarita Pustovalova, Rita Mohammad, Yuzhe Wang, Wenyu Xue, Philipp Malakhov, Viktor Nekrasov, Elizaveta Kontareva, Zain Nofal, Vyacheslav Saburov, Dmitry Kolesov, Andreyan Osipov, Sergey Leonov
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Abstract

Background: The implementation of proton beam irradiation (PBI) for breast cancer (BC) treatment is rapidly advancing due to its enhanced target coverage and reduced toxicities to organs at risk. However, the effects of PBI can vary depending on the cell type. This study aimed to explore the effects of PBI on two BC cell lines, MCF7 and MDA-MB-231.

Methods: The relative biological effectiveness (RBE) of PBI was assessed using a clonogenic assay. DNA double-strand break (DSB) repair, epithelial-mesenchymal transition (EMT), and filamentous actin (F-actin) were evaluated using immunofluorescence analysis. The extent of entosis and the senescence-associated β-galactosidase (SA-β-gal) activity were estimated by cytochemistry analysis. The influence of the extracellular matrix was evaluated by cultivating cells in both adherent two-dimensional (2D) environments and within 3D fibrin gels of varying stiffness. The metastatic propensity of cells was investigated using migration tests and the cell encapsulation of carboxylate-modified fluorescent nanoparticles. The comparative tumorigenic potential of cells was investigated using an in vivo model of the chick embryo chorioallantoic membrane (CAM).

Results: PBI demonstrated superior efficacy in eliminating MCF7 and MDA-MB-231 cells with RBE 1.7 and 1.75, respectively. Following PBI, MDA-MB-231 cells exhibited significantly lower clonogenic survival compared to MCF7, which was accompanied by the accumulation of phosphorylated histone H2AX (γH2AX), p53-binding protein 1 (53BP1) and Rad51 foci of DNA DSBs repair proteins. After surviving 7 days post-PBI, MCF7 cells exhibited 2.5-fold higher levels of the senescence phenotype and entosis compared to the MDA-MB-231 offspring. Both PBI-survived cell lines had greater capability for 2D collective migration, but their metastatic potential was significantly reduced. A significant influence of extracellular matrix stiffness on the correlation between F-actin expression in PBI-survived cells-an indicator of cell stiffness-and their ability to uptake nanoparticles, a trait associated with metastatic potential, was observed. PBI-survived MDA-MB-231RP subline exhibited a hybrid EMT phenotype and a 70% reduction in tumor growth in the in vivo model of the chick embryo CAM. In contrast, PBI-survived MCF7RP cells exhibit mesenchymal-to-epithelial transition (MET)-like features, and their in vivo tumor growth increased by 66% compared to parental cells.

Conclusions: PBI triggers various cellular responses in different BC cell lines, influencing tumor growth through mechanisms like DNA damage repair, stress-induced premature senescence (SIPS), and alterations in the stiffness of tumor cell membranes. Our insights into entosis and the effect of extracellular matrix stiffness on metastatic propensity (nanoparticle uptake) enhance the understanding of the role of PBI in BC cells, emphasizing the need for more research to optimize its therapeutic application.

体外和体内高let质子辐照显著改变人乳腺癌细胞系的克隆性和致瘤性潜能
背景:质子束照射(PBI)治疗乳腺癌(BC)的实施正在迅速推进,因为它提高了靶覆盖范围,减少了对危险器官的毒性。然而,PBI的效果可能因细胞类型而异。本研究旨在探讨PBI对两种BC细胞系MCF7和MDA-MB-231的影响。方法:采用克隆测定法评价PBI的相对生物学效果(RBE)。采用免疫荧光法检测DNA双链断裂(DSB)修复、上皮-间质转化(EMT)和丝状肌动蛋白(F-actin)。通过细胞化学分析,测定衰老相关β-半乳糖苷酶(SA-β-gal)活性。通过在贴壁二维(2D)环境和不同硬度的三维纤维蛋白凝胶中培养细胞来评估细胞外基质的影响。通过迁移试验和羧酸修饰的荧光纳米颗粒的细胞包封来研究细胞的转移倾向。采用鸡胚绒毛尿囊膜(CAM)的体内模型,研究了细胞的比较致瘤潜能。结果:PBI对RBE分别为1.7和1.75的MCF7和MDA-MB-231细胞均有较好的杀伤效果。PBI后,MDA-MB-231细胞的克隆存活率明显低于MCF7,这伴随着磷酸化组蛋白H2AX (γH2AX)、p53结合蛋白1 (53BP1)和DNA DSBs修复蛋白Rad51位点的积累。在pbi后存活7天后,MCF7细胞表现出比MDA-MB-231后代高2.5倍的衰老表型和内吞水平。两种pbi存活的细胞系具有更大的二维集体迁移能力,但其转移潜力显着降低。观察到细胞外基质刚度对pbi存活细胞中F-actin表达(细胞刚度指标)与它们摄取纳米颗粒的能力(与转移潜力相关的特性)之间的相关性有显著影响。pbi存活的MDA-MB-231RP亚系在鸡胚CAM体内模型中表现出混合型EMT表型,肿瘤生长减少70%。相比之下,pbi存活的MCF7RP细胞表现出间充质向上皮转化(MET)样特征,与亲本细胞相比,它们的体内肿瘤生长增加了66%。结论:PBI在不同的BC细胞系中触发不同的细胞反应,通过DNA损伤修复、应力诱导的过早衰老(SIPS)和肿瘤细胞膜硬度改变等机制影响肿瘤生长。我们对内吞作用和细胞外基质硬度对转移倾向(纳米颗粒摄取)的影响的见解增强了对PBI在BC细胞中的作用的理解,强调需要更多的研究来优化其治疗应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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