Triple negative breast tumors contain heterogeneous cancer cells expressing distinct KRAS-dependent collective and disseminative invasion programs

Inter-patient and intra-tumoral heterogeneity complicate the identification of predictive biomarkers and effective treatments for basal triple negative breast cancer (b-TNBC). Invasion is the initiating event in metastasis and can occur by both collective and single-cell mechanisms. We cultured primary organoids from a b-TNBC genetically engineered mouse model in 3D collagen gels to characterize their invasive behavior. We observed that organoids from the same tumor presented different phenotypes that we classified as non-invasive, collective and disseminative. To identify molecular regulators driving these invasive phenotypes, we developed a workflow to isolate individual organoids from the collagen gels based on invasive morphology and perform RNA sequencing. We next tested the requirement of differentially regulated genes for invasion using shRNA knock-down. Strikingly, KRAS was required for both collective and disseminative phenotypes. We then performed a drug screen targeting signaling nodes upstream and downstream of KRAS. We found that inhibition of EGFR, MAPK/ERK, or PI3K/AKT signaling reduced invasion. Of these, ERK inhibition was striking for its ability to potently inhibit collective invasion and dissemination. We conclude that different cancer cells in the same b-TNBC tumor can express different metastatic molecular programs and identified KRAS and ERK as essential regulators of collective and single cell dissemination. Overall design: C3(1)-TAg organoids were plated in 3D fibrillar collagen I for 3 days. We classified organoids based on their invasive phenotype (non-invasive, invasive (collective) and disseminative. Organoids were then removed from the gels with sharp forceps and were kept on dry ice. Trizol (Invitrogen) was then added and RNA extracted. Three independent experiments were conducted.

患者间及瘤内异质性给基底样三阴性乳腺癌（basal triple negative breast cancer, b-TNBC）的预测性生物标志物识别与有效治疗方案开发带来了极大挑战。侵袭是肿瘤转移的起始事件，可通过集体侵袭与单细胞侵袭两种机制发生。我们从基因工程改造的基底样三阴性乳腺癌小鼠模型中分离原代类器官，并在三维胶原凝胶中进行培养，以表征其侵袭行为。我们观察到同一肿瘤来源的类器官呈现出不同表型，将其分为非侵袭型、集体侵袭型与播散型三类。为筛选调控这些侵袭表型的分子调控因子，我们建立了一套基于侵袭形态学从胶原凝胶中分离单个类器官的流程，并开展RNA测序。随后我们利用短发夹RNA（short hairpin RNA, shRNA）敲降技术，检测差异表达基因对侵袭过程的必要性。令人惊喜的是，KRAS对集体侵袭型与播散型表型均为必需。随后我们针对KRAS上下游的信号节点开展了药物筛选，发现抑制EGFR、MAPK/ERK或PI3K/AKT信号通路均可降低侵袭能力。其中，ERK抑制剂展现出极强的集体侵袭与播散抑制效果。综上，同一基底样三阴性乳腺癌肿瘤内的不同癌细胞可表达不同的转移分子程序，我们同时确定KRAS与ERK是集体侵袭及单细胞播散的核心调控因子。

实验整体设计：将C3(1)-TAg类器官接种于三维纤维状I型胶原中培养3天。我们根据侵袭表型将类器官分为非侵袭型、侵袭（集体侵袭）型与播散型三类。随后用尖头镊子从凝胶中分离类器官，并将样品置于干冰中保存。随后加入Trizol（Invitrogen）试剂提取RNA。本实验共开展3次独立重复实验。