Table_8_Multi-Omics Characterization of the 4T1 Murine Mammary Gland Tumor Model.XLSX

Background: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. The 4T1 murine mammary cancer cell line is one of the most widely used breast cancer models. Here, we present an integrated map of the genome, transcriptome, and immunome of 4T1.
Results: We found Trp53 (Tp53) and Pik3g to be mutated. Other frequently mutated genes in breast cancer, including Brca1 and Brca2, are not mutated. For cancer related genes, Nav3, Cenpf, Muc5Ac, Mpp7, Gas1, MageD2, Dusp1, Ros, Polr2a, Rragd, Ros1, and Hoxa9 are mutated. Markers for cell proliferation like Top2a, Birc5, and Mki67 are highly expressed, so are markers for metastasis like Msln, Ect2, and Plk1, which are known to be overexpressed in triple-negative breast cancer (TNBC). TNBC markers are, compared to a mammary gland control sample, lower (Esr1), comparably low (Erbb2), or not expressed at all (Pgr). We also found testis cancer antigen Pbk as well as colon/gastrointestinal cancer antigens Gpa33 and Epcam to be highly expressed. Major histocompatibility complex (MHC) class I is expressed, while MHC class II is not. We identified 505 single nucleotide variations (SNVs) and 20 insertions and deletions (indels). Neoantigens derived from 22 SNVs and one deletion elicited CD8+ or CD4+ T cell responses in IFNγ-ELISpot assays. Twelve high-confidence fusion genes were observed. We did not observe significant downregulation of mismatch repair (MMR) genes or SNVs/indels impairing their function, providing evidence for 6-thioguanine resistance. Effects of the integration of the murine mammary tumor virus were observed at the genome and transcriptome level.
Conclusions: 4T1 cells share substantial molecular features with human TNBC. As 4T1 is a common model for metastatic tumors, our data supports the rational design of mode-of-action studies for pre-clinical evaluation of targeted immunotherapies.

背景：肿瘤模型（Tumor models）是解析癌症机制与开发癌症治疗手段的关键载体。4T1小鼠乳腺癌细胞系（4T1 murine mammary cancer cell line）是目前应用最为广泛的乳腺癌模型之一。本研究构建了4T1细胞的基因组、转录组（transcriptome）与免疫组（immunome）整合图谱。结果：本研究检测到Trp53（Tp53）与Pik3g存在突变。乳腺癌中其他常见突变基因，如Brca1与Brca2，并未发生突变。在癌症相关基因中，Nav3、Cenpf、Muc5Ac、Mpp7、Gas1、MageD2、Dusp1、Ros、Polr2a、Rragd、Ros1及Hoxa9存在突变。细胞增殖标志物Top2a、Birc5与Mki67呈高表达；转移标志物Msln、Ect2与Plk1亦呈高表达，上述标志物在三阴性乳腺癌（triple-negative breast cancer, TNBC）中已被证实存在过表达。与乳腺对照样本相比，三阴性乳腺癌标志物Esr1表达水平更低，Erbb2表达水平相对偏低，而Pgr则完全不表达。本研究还发现睾丸癌抗原Pbk以及结直肠/胃肠道癌抗原Gpa33与Epcam呈高表达。主要组织相容性复合体（major histocompatibility complex, MHC）I类分子存在表达，而MHC II类分子则无表达。本研究共鉴定出505个单核苷酸变异（single nucleotide variations, SNVs）与20个插入缺失变异（insertions and deletions, indels）。源自22个SNVs与1个缺失变异的新抗原（neoantigens）在干扰素γ-酶联免疫斑点（IFNγ-ELISpot）实验中可诱导CD8+或CD4+ T细胞应答。本研究共观察到12个高可信度融合基因。未检测到错配修复（mismatch repair, MMR）基因发生显著下调，亦未发现其功能受SNVs/indels影响，这为4T1细胞对6-硫鸟嘌呤产生耐药性提供了实验依据。在基因组与转录组层面均观察到小鼠乳腺肿瘤病毒整合所带来的效应。结论：4T1细胞与人类三阴性乳腺癌具备大量共有的分子特征。鉴于4T1是转移性肿瘤的常用模型，本研究数据可为靶向免疫治疗的临床前药效机制研究的合理设计提供科学支撑。