Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Summary

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Abstract

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.

Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

Introduction

Despite significant improvements in cancer immunotherapy over the past decade, our mechanistic understanding of sensitivity and resistance to treatments are still fairly poor¹. It is well-established that tumors display substantial heterogeneity, and that the dynamic interactions of the tumor cells with their microenvironment as well as with the immune cells, impact tumor cell death, invasive behavior and response to treatments that include immunotherapy^1,2,3. As one arm of the adaptive immune system, T-cells execute cell-specific cytotoxicity. The ana....

Protocol

A list and explanation of some frequently used words throughout the protocol can be found in Supplementary File 1.

1. Generation of spheroids

1. Prepare and autoclave 2% agarose in 1x PBS (e.g., 1 g agarose in 50 mL of 1x PBS) and autoclave 35- and 81-microwell rubber molds.
   CAUTION: Avoid using low-melting agarose for generating the agarose casts for IHC processing.
2. Prepare agarose casts
   NOTE: 35-microwell casts are used for invasion, immunofluorescence (IF) and cell isolation assays. 81-microwell casts are used for cytotoxicity, immunohistochemistry (IHC), cell isolation and RNA extraction....

Results

The 3D co-culture model allows for different assays shown in Figure 1A, which can be combined or modified as needed. In our established experimental setup, tumor and T-cells are co-cultured for 2 days followed by initiation of the invasion assay for selection of invasive and/or resistant tumor cells (Figure 1B). On day 4 the quantitation of invasion is performed and “survivor” cells are isolated from the collagen matrix or directly processed for RNA

Discussion

The method presented here describes 3D tumor spheroid generation, which allows co-culture with T-cells, cell-based functional and molecular assays, as well as a variety of monitoring and analysis possibilities using a single device. The major advantage of our approach is that it does not necessitate transfer of the 3D culture to a separate assay and maintains the integrity of the 3D culture throughout the assays.

The workflow presented here can be modified as needed. The incubation times for s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D Petri Dishes	Microtissues Inc	Z764019 & Z764051	referred to as "rubber molds" in the protocols; 81-microwell & 35-microwell molds
8-well Chamber Slides	Lab-Tek	154534
Agarose Type I, low EEO	Sigma-Aldrich	A6013
anti-rabbit-HRP conjugated secondary antibody	Agilent	K4003	ready to use
Collagen Type I, Rat Tail, 100 mg	Millipore	08-115
Collagenase Type 4, 1 g	Worthington	LS004188
DMEM, fetal bovine serum	ThermoFisher	11965092, 16000044	referred to as "cell culture medium" in the protocols
Harris hematoxylin	ThermoFisher	SH30-500D
HistoGel	ThermoFisher	HG-4000-012	referred to as "Hydroxyethyl agarose processing gel" in the protocols
Hoechst	Life Technologies	H1399	1/1000 dilution
Phalloidin 546	Invitrogen	486624	1/200 dilution
rabbit anti-CD8 antibody	Cell Signaling	98941	1/25 dilution
rat anti-keratin 8	DSHB	TROMA-I AB_531826	1/500 dilution
RNeasy Mini Kit	Qiagen	74104	referred to as "RNA extraction kit" in the protocols
RPMI	ThermoFisher	11875093	for T-cell culture medium
Triton X-100	BioRad	1610407	referred to as "Octoxynol" in the protocols
Trizol	ThermoFisher	15596026	referred to as "guanidinium thiocyanate with phenol" in the protocols
Tween 20	Sigma-Aldrich	P1379	referred to as "polysorbate 20" in the protocols
TypLE	ThermoFisher	12604013	referred to as "cell dissociation enzymes solution" in the protocols