Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Summary

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Abstract

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 µm intervals, covering a depth of 90 µm in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Introduction

Invasion is a process in which neoplastic cells migrate through the extracellular matrix into the surrounding tissue^1,2. As the tumor progresses, neoplastic cells are exposed to increasingly complex microenvironments. The tumor microenvironment includes various components of the extracellular matrix and non-neoplastic cell types. Stromal cells, including fibroblasts and resident macrophages, remodel the microenvironment by secreting extracellular matrix components and growth factors, and cytokines, which in turn influence neoplastic cell functions³. Stromal and infiltrating leukocyte ce....

Protocol

The use of primary human cells was approved by the Institutional Human Research Ethics Committee (CAAE 57895822.0.0000.5416) of the School of Dentistry at Araraquara - UNESP. The details of the reagents and the equipment used in the study are listed in the Table of Materials.

1. Preparation of cells for 3D cell culture

NOTE: All steps in this section must be performed within a laminar flow hood.

1. Obtain single-cell suspen.......

Discussion

Three-dimensional spheroid cultures are a powerful approach to investigate various aspects of cell biology. In this study, we report a protocol for evaluating neoplastic cell invasion using heterospheroids that mimic the tumor microenvironment in a multilayered and more dynamic approach. This method allows for the assessment of co-localization and patterns of invasion in the presence of non-neoplastic stromal cells.

There are some critical steps that warrant consideration. First, it is importa.......

Materials

Name	Company	Catalog Number	Comments
96 Well Bioprinting Kit	Greiner Bio-one	655840	Spheroid drive magnetic field. Holding drive. 96 well microplate, cell-repellent surface
CellTrace CFSE Cell Proliferation Kit	Invitrogen, ThermoFisher Scientific	C34554
CellTracker Red CMTPX	Invitrogen, ThermoFisher Scientific	C34552
CellTracker Violet BMQC Dye	Invitrogen, Thermo Fisher	C10094
DMEM	Gibco, ThermoFisher Scientific	31600-034
DMEM/F12	Gibco, ThermoFisher Scientific	12400-024
Extracellular matrix with Myogel	doi: 10.1186/s12885-015-1944-z		Mix composed of myogel (2.4 mg/mL), collagen (0.8 mg/mL) and non-supplemented medium of neoplastic cell
FBS	Gibco, ThermoFisher Scientific	12657-029
Hydrocortisone	Sigma	H088
ImageJ	National Institutes of Health		Software
Nanoshuttle-PL	Greiner Bio-one	657841	Magnetic nanoparticles
Needle 23 G	Medix	AHMD004	25 x 0.60 mm (23 G x 1)
PBS pH 7.2 (1x)	Gibco, ThermoFisher Scientific	2012-027
Penicillin-Streptomycin (5,000 U/mL)	Gibco, ThermoFisher Scientific	15070063
Pipet Tips	Axygen, Corning Inc.	TF-200-L-R-S	Pipet tip with barrier (filter), 200 µL Low Retention Filter, short, maximum recovery
RPMI	Gibco, ThermoFisher Scientific	31800-022
TransWell 24 well	Costar, Corning Inc.	3422	Transwell Permeable Supports 6.5 mm Insert, 24 well plate; 0.8 µm Polycarbonate Membrane
Zen 3.4 (blue edition)	Zeiss Group		Software