En trois dimensions modèle de co-culture tumeur stromale-Interaction

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

NOTE: Cette étude a été approuvée par les comités d'éthique appropriés. 1. Culture primaire des fibroblastes pulmonaires humains Collecte de tissu pulmonaire: Obtenir des échantillons humains de tissus pulmonaires de la salle d'opération chirurgicale directement. Prélever environ 1 cm 3 pâtés de maisons de tissus pulmonaires cancéreuses et non-cancéreuses, avec l'échantillon non-cancéreuse recueillies aussi loin de la tumeur que poss…

Representative Results

Cette méthode de co-culture, imitant le micro-environnement de la tumeur, est un outil utile pour étudier les interactions entre les cellules cancéreuses et les fibroblastes incorporés dans des gels de collagène. Dans l'étude précédente, trois paramètres ont été évalués dans ce modèle expérimental: contraction du gel de collagène, l'invasion des cellules cancéreuses et les changements morphologiques. la prolifération des cellules du cancer a également été estimée en utilisant une immunocolo…

Discussion

CAF forment une composante majeure de l'ECM entourant les cellules cancéreuses et non seulement fournir un échafaudage pour la tumeur, mais aussi participer activement au développement de la tumeur ^7. L'accumulation de preuves dénoue l'impact de la CAF ou de leurs molécules apparentées sur le pronostic éventuelle, en soulignant le rôle crucial de la progression tumorale CAF médiation ^8.

Dans l'étude précédente, nous avons utilisé la méthode…

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