Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Summary

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Abstract

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 µm polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 µm barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Introduction

Recent studies have focused on the ability of cancer cells to avoid detection by immune cells, suppress local immune activation, or to produce a tolerogenic tumor-permissive milieu in the tumor microenvironment. Two broad classes of tumor and immune cell interactions have been described that facilitate these effects: contact-mediated interactions or tumor-secreted ligands. One of the most well-studied and clinically tractable mechanisms of contact-mediated immune inhibition utilized by tumors is the expression of PD-L1, which interacts with PD-1 on T cells to inhibit their activation and function^1,2. In respon....

Protocol

All procedures related to the harvest and use of murine peritoneal macrophages were conducted at the University of North Carolina at Chapel Hill (UNC) and were approved by the UNC Institutional Animal Care and Use Committee (IACUC).

1. Macrophage culture

NOTE: This procedure can utilize primary peritoneal macrophages (described in detail below), bone marrow-derived macrophages, or macrophage cell lines such as J774 (ATCC) or RAW264 (ATCC).

1. Harvest peritoneal macrophages as previously described^16,17 and plate directly into the upper chamber(s) ....

Results

To determine the effect of tumor-secreted ligands on macrophage polarization, the procedure described was utilized. Peritoneal macrophages cultured in the absence of tumor cells were used as negative (untreated = far left) and positive (IFNγ and LPS stimulated = 2^nd from left) controls (Figure 2A). Alternatively, peritoneal macrophages were co-cultured with B16F10 melanoma tumor cells (ATCC) (Figure 1A). Immediately after plating, cells were eith.......

Discussion

The co-culture assay presented here is a modification of previously established assays that allows for the study of tumor-secreted factors on immune cell activation. While cell-cell contact is known to induce changes in immune activity, the ability of tumor-secreted ligands to modulate immune activation is less well understood. We describe a method which, unlike direct co-culture, can be used to interrogate how tumor-derived secreted factors impact immune cell activation without the confounding nature of contact-mediated.......

Materials

Name	Company	Catalog Number	Comments
B16-F10	ATCC	ATCC CRL-6475
cDNA synthesis kit	Promega	A3500
DMEM/F12 media	ThermoFisher Scientific- Gibco	11320033
Fetal Bovine Serum	Millipore	TMS-013-B
J774A.1	ATCC	ATCC TIB-67
Lipopolysaccharides from Escherichia coli O111:B4	Sigma-Aldrich	L5293-2ML
Murine M-CSF	Prospec	CYT-439
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher Scientific- Gibco	15140122
Pros1 ELISA	MyBioSource	MBS2886720
RAW264.7	ATCC	ATCC TIB-71
Recombinant Mouse IFNγ	BioLegend	575302
Sensimix SYBR Low-ROX kit	Bioline	QT625-05
Transwell permeable supports	Fisher Scientific	07-200-170
Trypsin-EDTA	ThermoFisher Scientific- Gibco	25200056