Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 function1,2. 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 perito.......

Representative 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 = 2nd 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.......

Acknowledgements

Eric Ubil was funded, in part, by the American Cancer Society Postdoctoral Fellowship (128770-PF-15-216-01-LIB). The work was supported by a grant from the NIH (R01-CA205398) and a Breast Cancer Research Foundation award (BCRF-18-041) to HSE.

....

Materials

NameCompanyCatalog NumberComments
B16-F10ATCCATCC CRL-6475
cDNA synthesis kitPromegaA3500
DMEM/F12 mediaThermoFisher Scientific- Gibco11320033
Fetal Bovine SerumMilliporeTMS-013-B
J774A.1ATCCATCC TIB-67
Lipopolysaccharides from Escherichia coli O111:B4Sigma-AldrichL5293-2ML
Murine M-CSFProspecCYT-439
Penicillin-Streptomycin (10,000 U/mL)ThermoFisher Scientific- Gibco15140122
Pros1 ELISAMyBioSourceMBS2886720
RAW264.7ATCCATCC TIB-71
Recombinant Mouse IFNγBioLegend575302
Sensimix SYBR Low-ROX kitBiolineQT625-05
Transwell permeable supportsFisher Scientific07-200-170
Trypsin-EDTAThermoFisher Scientific- Gibco25200056

References

  1. Freeman, G. J., et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. Journal of Experimental Medicine. 192 (7), 1027-1034 (2000).
  2. Agata, Y., et al.

Explore More Articles

Tumor secreted Paracrine LigandsMacrophage ActivationCo culturePermeable MembraneImmune SuppressionParacrine SignalingCell to cell ContactMolecular Biological TechniquesPeritoneal MacrophagesTumor CellsDMEMInterferon Gamma

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved