Identifying the intercellular networks regulating estrogen receptor expression with a high definition single cell printer

 描述：这项提议的长期目标是通过结合液滴微流体技术、机器人自动化和显微技术的进步来重新发明活细胞打印。这项提议的直接科学目标是通过使用原代或有限寿命的人乳腺上皮细胞构建组合细胞相互作用阵列来重建原位微环境。具体地说，我们将应用多细胞相互作用阵列来扰乱导致雌激素受体表达异质性模式的细胞网络。雌激素受体(ER)是人类乳腺生长的关键调节因子，是人类最常见的乳腺癌所必需的。然而，值得注意的是，雌激素受体只在人类乳腺中的一种细胞类型-腔上皮细胞中表达。更值得注意的是，受体以二元方式表达，在正常组织中只有5%-20%的腔上皮细胞表达。关于ER在管腔内表达的调节机制，我们知之甚少，但现有证据表明，微环境中的多种其他类型的细胞是至关重要的。我们将使用拟议的高清晰度单细胞打印机来探索原代人类细胞类型、可溶性因子和细胞外基质成分的大量组合，以确定维持乳腺ER表达所需的最小细胞间回路。我们假设，管腔人群中ER的表达是由细胞与肌上皮细胞的细胞间接触以及涉及成纤维细胞和至少一个负责稳定雌激素受体表达的其他基质成分的旁分泌回路共同调节的。具体目标1：建立和测试原型单细胞表型分析工具。在这个目标中，我们将建立一个原型平台，能够组装和成像数以万计的具有精确定义的成分的三维多细胞凝聚体。首先，我们将设计和构建能够将含有单细胞、基质成分或生长因子的微滴输送到目标表面的微流体。接下来，我们将构建光学检测硬件，编写控制软件，并集成系统。最后，我们将设计和构建能够将含有单细胞、基质成分或生长因子的微滴输送到目标表面的微流体。接下来，我们将构建光学检测硬件，编写控制软件，并集成系统。最后，我们将 将使用培养的原代人类乳腺上皮细胞进行对照实验，以建立该系统组装和概括多细胞相互作用的能力。(ADAM)“特定目标2：从培养的原始人类乳房组织中组装异型2D和3D组织微阵列，以识别调节雌激素受体表达的多细胞电路。我们将使用人类乳房早期传代的原代成纤维细胞、内皮细胞、腔上皮细胞和肌上皮细胞来确定维持或增强腔人口中ER表达所需的最低限度的细胞相互作用。这些细胞将与Matrigel以及参与ER功能的生长因子和激素的混合物结合成微观组织。每个打印的组织将结合10个打印的液滴，跨越细胞、基质成分和可溶性因子的组合。在细胞Arras固定、染色和高含量成像后，将在培养48小时后测量ER表达，以确定细胞和延伸或增强ER表达的因素的组合。

 DESCRIPTION: The long-term objective of this proposal is to reinvent live-cell printing by combining advances in droplet microfluidics, robotic automation, and microscopy techniques. The immediate scientific goal of this proposal is to recreate the in situ microenvironment by building combinatorial cellular interaction arrays using primary or limited life-span human mammary epithelial cells. Specifically, we will apply the multicellular interaction arrays to disset the cellular network that contributes to heterogeneous patterns of estrogen receptor expression. Estrogen receptor (ER) is the key regulator of human mammary gland growth and is necessary for the most prevalent forms of human breast cancer. Remarkably, however, the estrogen receptor is only expressed in one cell type in the human mammary gland - luminal epithelial cells. More remarkably still, the receptor is expressed in a binary fashion, and only in 5-20% of luminal epithelial cells in a normal tissue. Little is known about the mechanisms through which ER expression is regulated within the luminal population, but available evidence suggests that multiple other cell types in the microenvironment are critical. We will use the proposed high definition single cell printer to explore a large combination of primary human cell types, soluble factors, and ECM components to identify the minimal intercellular circuit necessary to sustain ER expression in the mammary gland. We hypothesize that ER expression in the luminal population is regulated by a combination of cell-cell contact with myoepithelial cells and a paracrine circuit involving fibroblasts and at least one other stromal component responsible for stabilizing estrogen receptor expression. " Specific Aim 1: Build and test the prototype single cel phenotype analysis tool. In this aim, we will build a prototype platform capable of assembling and imaging tens of thousands of 3D multicellular agglomerates with precisely defined compositions. First, we will design and construct microfluidics capable of delivering picodroplets containing single cells, matrix components, or growth factors to a target surface. Next, we will build the optical detection hardware, write control software, and integrate the system. Finally, we will perform control experiments using cultured primary human mammary epithelial cells to establish the capability of the system to assemble and recapitulate multicellular interactions. (Adam) " Specific Aim 2: Assemble heterotypic 2D and 3D tissue microarrays from cultured primary human breast tissue to identify the multicellular circuit regulating estrogen receptor expression. We will use early passage primary fibroblasts, endothelial cells, luminal epithelial and myoepithelial cells from the human breast to identify the minimal cellular interactions necessary to sustain or augment ER expression in the luminal population. These cells will be combined into microtissues with Matrigel and a cocktail of growth factors and hormones involved in ER function. Each printed tissue will combine 10 printed droplets spanning a combination of cells, matrix components, and soluble factors. ER expression will be measured after 48 hours in culture following fixing, staining, and high-content imaging of the cellular arras to identify combinations of cells and factors extending or enhancing ER expression.