对于肿瘤基质相互作用的三维共培养模式

Summary

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Abstract

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Introduction

Cancer tissue can be perceived as a type of organ, which evolves through close interactions between the cancer and the tumor stromal microenvironment, composed of cancer-associated fibroblasts (CAFs), immune cells, tumor vessels and the extracellular matrix (ECM). CAFs are the major source of soluble factors (cytokines, growth factors and chemokines) that exert mitogenic, pro-migratory and pro-invasive effects on cancer cells. They also stimulate tumor vessel formation and recruit precursor cells, such as bone marrow-derived cells (BMDC). Activated CAFs are involved in the production and remodeling of the ECM, thereby promoting the growth and spread of cancer cells¹. CAFs also provide a niche that facilitates tumor cell colonization and metastasis and are capable of conferring stem cell phenotypes onto neighboring cancer cells. Pathological observations suggest that stromal reactions or fibrotic changes in cancer tissues are indicative of a poor prognosis. Recent studies have also demonstrated that tumor stromal features, such as the gene signature, can predict patient prognosis. Furthermore, CAF-derived factors can modulate sensitivity to chemotherapy, highlighting the role of CAFs in determining drug sensitivity and resistance².

As CAFs play a multifaceted role in the promotion of tumor progression through signaling pathways that mediate interactions between CAFs and different cell types within the tumor microenvironment, they have attracted increasing attention as novel targets for cancer therapies. The heterogeneity of the cell populations within the cancer microenvironment presents an obstacle for targeting CAFs. Several markers for CAFs have been proposed, such as α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific proten-1 (FSP-1: also called S100A4); however, these molecular markers are not specific for distinguishing CAFs from other cells present in non-cancerous tissues³. Therefore, further studies are needed to obtain more knowledge about the specific properties of CAFs. To this end, it is informative to characterize primary cultured CAFs compared with patient-matched normal fibroblasts.

Recently, analyses on patient-derived CAFs have been reported in several cancer types, revealing unique gene expression patterns and cell behaviors compared with fibroblasts derived from non-cancerous tissues. Using isolated CAFs from human lung cancer tissues, we developed a three-dimensional co-culture method, enabling us to evaluate the properties of lung CAFs. In this model, we investigated the effects of the CAFs on lung cancer cell invasion, proliferation and collagen gel contraction, which experimentally recapitulated the tumor-promoting roles of lung CAFs⁴.

Protocol

注：此研究是通过适当的伦理委员会。 人肺成纤维细胞1.原代培养收集肺组织： 得到直接从外科手术室人肺组织样本。收集来自癌性和非癌肺组织约1cm 3块，以收集作为远离肿瘤尽可能的非癌性样品。 暂停在Dulbecco改良的Eagle培养基（DMEM）中补充有100单位/ ml青霉素，100微克/毫升链霉素和0.25微克/毫升两性霉素B（无血清培养基）的样品。保持在无?…

Representative Results

该共培养方法，模仿肿瘤微环境，是一种有用的工具，调查癌细胞和嵌入在胶原凝胶的成纤维细胞之间的相互作用。胶原凝胶收缩，癌细胞的侵袭和形态变化：在以前的研究中，三个参数在这个实验模型进行了评价。还预计使用Ki67的免疫染色4癌细胞增殖。肺组织样品从一个切除肺叶（ 图1A）的癌性和非癌的部分收集。样品切成小片，并在DMEM中培养在补充有10％FBS的（ ?…

Discussion

的CAF形成周围癌细胞ECM的一个重要组成部分，而不是仅提供一个支架的肿瘤，但也积极参与肿瘤发展^7。越来越多的证据揭开CAF或与其相关的分子上的最终预后的影响，突出CAF介导的肿瘤进展⁸的关键作用。

在以前的研究中，我们采用的方法生长隔离肺的CAF ^4。在这个实验中，组织切片的粘附维持到餐具表面是关键的。细胞不能从组织切片漂浮在培养基?…

Materials

Name	Company	Catalog Number
DMEM	Sigma-Aldrich	D5796
FBS	GIBCO	10437
Collagen type IA	Nitta gelatin Inc.	CELL-1A
Reconstitution buffer	Nitta gelatin Inc.
Cover slip	NUNC	174934
Silicone grease	Dow Corning Toray	High vacuum grease
Dispase I	WAKO	386-02271
6-well plate	BD Falcon	353046
Cell strainer (70 μm)	BD Falcon	352350