Tredimensjonal Co-kultur Modell for Tumor-stromal Interaksjon

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

MERK: Denne studien ble godkjent av de aktuelle etiske komiteer. 1. Primær Culture of Human lungefibroblaster Samling av lungevev: Skaff menneskelige lunge vevsprøver fra kirurgisk operasjonsstuen direkte. Samle omtrent 1 cm 3 blokker fra cancerøs og ikke-cancerøs lungevev, med det ikke-kreft prøve samlet så langt borte fra tumor som mulig. Suspen prøvene i Dulbeccos modifiserte Eagles medium (DMEM) supplert med 100 enheter / ml penicillin, 100…

Representative Results

Dette co-kultur metode, etterligne svulstens mikromiljø, er et nyttig verktøy for å undersøke samspillet mellom kreftceller og fibroblaster innebygd i kollagen gels. I forrige undersøkelse ble tre parametere evaluert i denne eksperimentelle modellen: kollagen gel sammentrekning, kreftcelle invasjon og morfologisk endring. Kreftcelle spredning ble også beregnet ved hjelp av Ki67 immunofarging 4. Lungevev Prøver ble samlet fra kreft og ikke-kreft seksjoner av en resektert lungelapp (figur 1A).</s…

Discussion

Kafeer utgjør en stor del av ECM rundt kreftceller og ikke bare gi et stillas for svulsten, men også delta aktivt i svulst utvikling ^7. Akkumulere bevis avslører virkningen av kafeer eller deres nærstående molekyler på eventuell prognose, fremhever de kritiske roller CAF-mediert tumorprogresjon ^8.

I forrige undersøkelse, benyttet vi utvekst metode for å isolere lunge kafeer ^fire. I dette eksperimentet, er opprettholdelsen av vev seksjonen adhesjon mot…

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