Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Summary

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Abstract

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Introduction

Solid tumors are composed of heterogeneous cell populations which are guided by the tumor microenvironment (TME), however, the origin of most of the cells has yet to be discovered. Mainly stromal and immune cells (fibroblasts, endothelial cells, monocytes, macrophages, dendritic cells, B cells, T cells, and their subsets) reflect the tumor heterogeneity in lung, breast, renal, and other solid cancers^1,2,3. Understanding the origin of each subtype and their trans-differentiation potential is of utmost need for developing advanced therapies against these cancers. The analysis o....

Protocol

1. Cell culture

1. Culture human lung adenocarcinoma cell line A549, and human monocytic cell line THP-1 in RPMI1640 media supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified chamber with 5% CO₂.
2. Culture MRC-5 human lung fibroblast cells in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37 °C in a humidified chamber with 5% CO₂.

2. Preparation of multicellular tumor spheroids using A549 lung adenocarcinoma cell line, MRC5 fibroblasts, and THP-1 monocytes

NOTE: The multice....

Results

Figure 1 shows the development of multicellular tumor spheroids using three different cell populations-A549 (lung adenocarcinoma), MRC-5 (fibroblasts), and THP-1 (monocytes)-by the hanging drop method as observed on day 7 and day 10 under the microscope. On day 7, spheroids were compact and rigid with a 260 ± 5.3 µm diameter, and on day 10, spheroids were 480 ± 7.5 µm in diameter (Figure 1A upper panel, Figure 1B-D).......

Discussion

The present study introduces the development of multicellular tumor spheroids comprising tumor cells, stromal cell population (i.e., fibroblasts), and immune cell population (i.e., monocytes) using a modified hanging drop method. Fibroblasts and monocytes/macrophages are among the most significant populations that constitute the tumor microenvironment (TME), and their presence is often linked with poor patient prognosis¹⁶. When present in the TME, fibroblasts transform, exhibiting a specific cance.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies
APC anti-human α-SMA	R&D systems	Cat# IC1420A
Anti-fibroblast microbeads	Miltenyi Biotec	Cat# 130-050-601
Cell lines
A549 lung adenocarcinoma cells	NCCS Pune	-
MRC-5 fetal lung fibroblasts	ATCC	CCL-171
THP-1 Human monocytes	NCCS Pune	-
Chemicals
BSA	Himedia	Cat# 9048-46-8
2,6-dichloroindophenol (DCPIP)	SRL	Cat# 55287
Calcein-AM	Thermo Fisher Scientific	Cat# C3099
DAPI	Thermo Fisher Scientific	Cat# D1306
DCFDA	Sigma	Cat# D6883
DMEM	Gibco	Cat# 11995073
DPBS	Gibco	Cat# 14190-144
EDTA	Thermo fisher scientific	Cat# 17892
EGTA	SRL	Cat# 62858
EZcoun Lactate Dehydrogenase Cell Assay Kit	HiMedia	Cat# CCK036
FBS	Gibco	Cat# 10082147
Halt Protease and Phosphatase Inhibitor Cocktail (100X)	Thermo Fisher Scientific	Cat# 87786
HEPES	Thermo Fisher Scientific	Cat# 15630080
Horse heart Cytochrome c	SRL	Cat# 81551
Image-iT Red hypoxia reagent	Thermo Fisher Scientific	Cat# H10498
JC-1 Dye	Thermo Fisher Scientific	Cat# T3168
KCl	Merck	Cat# P9541
MgCl2	Merck	Cat# M8266
MOPS	Thermo Fisher Scientific	Cat# 69824
Nacl	Sigma-Aldrich	Cat# S9888
NADH MB Grade	SRL	Cat# 54941
NP-40	Thermo Fisher Scientific	Cat# 85124
Penicillin/Streptomycin	Gibco	Cat# 15140122
Phenazine methosulfate (PMS)	SRL	Cat# 55782
Propidium iodide	Thermo fisher scientific	Cat# P1304MP
RPMI 1640	Gibco	Cat# 11875093
Single Cell Lysis Kit	Thermo Fisher Scientific	Cat# 4458235
Sodium ascorbate	Merck	Cat# A7631
Sodium cyanide	Sigma	Cat# 205222
Sodium Deoxycholate	Thermo Fisher Scientific	Cat# 89904
Sodium dodecyl sulphate	Sigma-Aldrich	Cat# L3771
Sodium succinate hexahydrate	SRL	Cat# 36313
Sucrose	Sigma	Cat# S0389
SuperScript VILO cDNA synthesis kit	Thermo Fisher Scientific	Cat# 11754-050
Triton X-100	Sigma	Cat# T8787
Trypsin 0.25% EDTA	Gibco	Cat# 25200072
Universal SYBR Green Supermix	BIO-RAD	Cat# 172-5124
Plasticware
MACS LS Columns	Miltenyi Biotec	Cat# 130-042-401
Equipment
Countess II FL Automated Cell Counter	Thermo Fisher Scientific	Cat# AMQAF1000
EVOS XL core imaging system	Thermo Fisher Scientific	Serial Number F0518-1727-0191
LAS X software	Leica Microsystems
Leica fluorescent inverted microscope	s	DMi8 automated S/N 424150)
Midi MACS separator	Miltenyi Biotec	Cat# 130-042-302