Development of 4T1 breast cancer mouse model system for preclinical carbonic anhydrase IX studies.

IF 2.8 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Zane Kalniņa, Ilva Liekniņa, Svetlana Koteloviča, Ramona Petrovska, Gediminas Žvinys, Agne Petrosiute, Asta Zubrienė, Matīss Toms Laugalis, Vendija Skeltona, Juris Jansons, Madara Kreishmane, Edita Čapkauskaitė, Daumantas Matulis, Kaspars Tārs
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引用次数: 0

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, for which targeted treatment is currently lacking. Carbonic anhydrase IX (CAIX) is a known cancer target due to its selective overexpression in hypoxia, a hallmark of many solid cancers including TNBC. This study aimed to develop a robust murine TNBC cell line 4T1-based model system that could be used in the comprehensive preclinical evaluation of targeting CAIX. The model is based on the original 4T1 breast cancer cell line and two genetically edited versions of it-one with biallelic CRISPR/Cas9-mediated Car9 inactivation and another with constitutively expressed Car9, thus ensuring negative and positive controls for CAIX production in the model system, respectively. The generated cell lines were validated for CAIX production and characterised functionally in vitro and in vivo after orthotopic implantation in syngeneic BALB/c mice. Results demonstrated significantly reduced primary tumour growth and metastatic progression rates in animals with CAIX-deficient tumours, while the CAIX-expressing tumours had vascularised phenotypes with prominent central areas of coagulative necrosis. The differential CAIX expression levels in the model were preserved during tumour growth in syngeneic mice, as verified by in vivo imaging using a novel high-affinity CAIX-specific near-infrared (NIR) fluorescent imaging probe, GZ22-4. Constitutive overexpression of autologous CAIX did not elicit specific autoantibody responses in vivo, demonstrating the suitability of this model for evaluating the efficacy of anti-CAIX vaccination as a therapeutic strategy. The in vivo study was repeated as an independent experiment and demonstrated good robustness of the developed model.

用于临床前碳酸酐酶IX研究的4T1乳腺癌小鼠模型系统的建立。
三阴性乳腺癌(TNBC)是最具侵袭性的乳腺癌类型,目前缺乏靶向治疗。碳酸酐酶IX (CAIX)是已知的癌症靶点,因为它在缺氧中选择性过表达,这是包括TNBC在内的许多实体癌症的标志。本研究旨在建立一个强大的基于小鼠TNBC细胞系4t1的模型系统,可用于靶向CAIX的临床前综合评估。该模型基于原始的4T1乳腺癌细胞系和两个基因编辑版本-一个具有双等位基因CRISPR/ cas9介导的Car9失活,另一个具有组成性表达的Car9,从而分别确保了模型系统中CAIX产生的阴性和阳性对照。在同基因BALB/c小鼠原位植入后,对所生成的细胞系进行了体外和体内CAIX生产验证和功能表征。结果显示,在患有caix缺陷肿瘤的动物中,原发性肿瘤生长和转移进展率显著降低,而表达caix的肿瘤具有血管化表型,中心区域有明显的凝固性坏死。采用新型高亲和CAIX特异性近红外(NIR)荧光成像探针GZ22-4进行体内成像证实,在同基因小鼠的肿瘤生长过程中,模型中CAIX的差异表达水平得以保留。在体内,自体CAIX的组成性过表达不会引起特异性的自身抗体反应,这证明了该模型用于评估抗CAIX疫苗接种作为治疗策略的有效性的适用性。体内研究作为独立实验重复进行,表明所建立的模型具有良好的鲁棒性。
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来源期刊
FEBS Open Bio
FEBS Open Bio BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
5.10
自引率
0.00%
发文量
173
审稿时长
10 weeks
期刊介绍: FEBS Open Bio is an online-only open access journal for the rapid publication of research articles in molecular and cellular life sciences in both health and disease. The journal''s peer review process focuses on the technical soundness of papers, leaving the assessment of their impact and importance to the scientific community. FEBS Open Bio is owned by the Federation of European Biochemical Societies (FEBS), a not-for-profit organization, and is published on behalf of FEBS by FEBS Press and Wiley. Any income from the journal will be used to support scientists through fellowships, courses, travel grants, prizes and other FEBS initiatives.
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