建立三阴乳腺癌干细胞产生的体内模型：小鼠乳腺内诱导的或病人肿瘤移植物中乳腺癌干细胞演变的谱系追踪研究

Triple-negative breast cancer (TNBC) is one of the breast cancer subtype that is very difficult to be cured and is also quite lethal to women worldwide. TNBC tightly connected to breast cancer stem cells (BrCSCs) and some of the BrCSCs are triple-negetive. However, there is almost no clues for where these highly malignant TNBC/BrCSCs originate from, and how these cells progress to TNBC stage.Up to now nothing has been known for the in vivo dynamic conversion of these cells and the corresponding regulatory mechanisms. In our previous investigations, we found that integrin-interacting protein Kindlin-2 could generate TNBC/BrCSCs in vitro by an epigenetic reprogramming process. In the present proposal, we aim to establish a model system for TNBC/BrCSCs in vivo generation and lineage changes by tracing them in the mice mammary glands that are genetically modified by Kindlin-2 transgenic and null mice, in corporaton with the use of Lgr5 knock-in mice.In addition, we will also establish a model with humanized mouse mammary gland by transplantation of patient-derived xenogrant (PDX) from breast cancer patients. Within PDX model that is close to the tumor microenvironment in human body we will trace the TNBC/BrCSCs generation and lineage changes. By means of these models in mouse mammary glands and the transplanted PDX we anticipate to uncover the origin of TNBC/BrCSCs and the commom mechanisms accounting for BrCSCs lineage changes. Therefore, the execution of this research plan will help to set up new systems for anti-TNBC/BrCSCs drug screenings and will formulate new therapeutic strategies for TNBC, which will no doubt be beneficial to the breast cancer patients.

三阴乳腺癌(TNBC)是最难治的乳腺癌亚型之一，严重威胁妇女健康。TNBC与乳腺癌干细胞(BrCSCs)密切相关，部分BrCSCs亚群就是TNBC。然而，病人体内恶性程度最高的TNBC/BrCSCs源自何种细胞，如何进展成为TNBC，其体内动态演变的过程和调控机制尚未知晓。我们前期工作发现Kindlin-2经细胞重编程机制在体外产生了TNBC/BrCSCs。本申请中，我们将综合利用小鼠转基因、基因敲除和敲入的技术，在小鼠体内建立动态追踪Kindlin-2诱导产生TNBC/BrCSCs的谱系变化模型。进一步，我们将在小鼠乳腺内建立人源化乳腺癌的移植物(PDX)，在PDX中以近似人体的环境对Kindlin-2诱导产生的TNBC/BrCSCs进行谱系追踪。研究将揭示乳腺癌中TNBC/BrCSCs的起源和谱系变化规律，有助于抗TNBC/BrCSCs的药物筛选和新临床对策的建立，从而惠及乳腺癌病人。

项目的背景：.在动物体内观察乳腺癌干细胞及乳腺癌干细胞是从何种细胞演变而来？又将演变成什么细胞？乳腺癌的三阴性和乳腺癌干细胞之间是什么关系？这些对乳腺癌的发生机制和治疗对策至关重要。.主要研究内容：.1) 建立乳腺特异性高表达Kindlin-2的转基因小鼠及Kindlin-2 乳腺条件性敲除小鼠；.2) 建立乳腺特异性高表达Kindlin-2转基因MMTV-Kindlin-2-Tg小鼠和MMTV-PyMT 杂交小鼠；.3) 建立Kindlin-2-/- 乳腺条件性敲除小鼠和MMTV-PyMT 杂交小鼠；.4) 在小鼠乳腺内建立人源乳腺癌的移植物(PDX)模型；.5) 在人乳腺癌PDX模型中研究TNBC/BrCSCs的谱系变化规律。.重要结果：.我们构建了MMTV-Kindlin-2转基因小鼠模型，使Kindlin-2在小鼠的乳腺腔上皮细胞中过表达。我们构建了Kindlin-2flox/flox小鼠，使得在小鼠体内各组织器官特异性敲除Kindlin-2基因成为可能。使用MMTV-Cre与K14-Cre工具鼠分别与Kindlin-2flox/flox小鼠杂交，在乳腺腔上皮细胞和肌上皮细胞分别敲除了Kindlin-2基因。乳腺腔上皮细胞特异性高表达Kindlin-2的雌鼠，乳腺导管分支增多，乳腺肿瘤发生率增高。将其与MMTV-PyMT小鼠杂交后，高表达的Kindlin-2促进MMTV-PyMT小鼠乳腺癌向肺发生转移，提示Kindlin-2是一个体内促进癌细胞转移的分子。乳腺腔上皮细胞特异性敲除Kindlin-2的雌鼠，哺乳期乳腺泌乳功能受损，幼鼠发育不良甚至死亡。其与MMTV-PyMT小鼠杂交后，Kindlin-2的缺失抑制了MMTV-PyMT小鼠乳腺肿瘤的生长，肺转移灶降低，提示Kindlin-2是乳腺癌生长和转移必不可少的一个重要分子。Kindlin-2在TNBC BCSC高表达，抑制Kindlin-2能够显著地抑制BCSC形成肿瘤干细胞球的能力，导致乳腺癌肿瘤干细胞CD44+CD24-/low细胞比例降低；抑制Kindlin-2在TNBC PDX模型中抑制肿瘤生长调节乳腺肿瘤亚型转化，即由高恶性basal型向低恶性luminal型转化。