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 response to interferon-gamma (IFNγ), which is expressed by a number of activated immune cells, tumor cells can increase expression of PD-L1 to induce exhaustion of PD-1–expressing activated T cells, thereby preventing them from effectively eradicating tumor cells³. The use of antibodies to block the interaction between PD-L1 and PD-1 is currently used to treat multiple cancer types in humans⁴. In light of this clinical success and others, the identification and targeting of tumor-derived immunosuppressive mechanisms has received increasing attention.

Beyond suppression of adaptive immunity, tumors are also known to secrete factors that suppress the pro-inflammatory responses of innate immune cells. Tumor-derived or tumor-induced secretions, including IL-6, IL-10, VEGF, IL-23, and colony stimulating factor (CSF-1), have been shown to inhibit antitumor responses of natural killer (NK) cells, granulocytes, and dendritic cells in the tumor microenvironment^5,6,7. Tumor cells can also secrete factors that skew the recruitment and differentiation of myeloid-derived cells in the tumor microenvironment to promote suppression of T cell activation^8,9.

One type of innate immune cell that has a profound effect on tumor progression is the macrophage. For many years, the presence of tumor-associated macrophages (TAMs) has been used as a negative prognostic of patient survival¹⁰. The concept that immunosuppressive TAMs dampen immune cell-mediated clearance of tumors was introduced more than 40 years ago¹¹. More recently, it has been shown that the macrophage pro-inflammatory response can be downregulated while a pro-tumor phenotype can be induced in the tumor microenvironment. These immunosuppressive macrophages can contribute to a tolerogenic response, driving tumor progression and resistance to chemo- and immunotherapy¹². Given that macrophages are often one of the most abundant leukocytes with the tumor, restoration of their tumor-specific immune activity represents a potential target for anticancer therapeutics¹³.

While contact-mediated interactions between tumor cells and macrophages can be modeled through direct coculture, the use of permeable membrane supports can elucidate which tumor-secreted factors are immunomodulatory without the potentially confounding influence of tumor-immune cell-cell contact. Using somewhat similar methods, others have demonstrated the potential of identifying secreted factors in microglia/neuronal interactions¹⁴ as well as tumor cells with mesothelial cells¹⁵. We have also successfully used this co-culture technique to characterize the role of a tumor-secreted protein, Pros1, as a suppressor of pro-inflammatory gene expression after the stimulation of peritoneal macrophages with LPS and interferon-gamma¹⁶. Here we describe a straightforward methodology that can be used to interrogate how tumor-secreted factors can affect macrophage activation.

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) of a 0.4 µm polyester membrane insert co-culture 6 well plate (Figure 1A).
   NOTE: The approximate yield of macrophages from each isolation is 1 x 10⁶ cells total, so the average number of cells per well is ~1.5–1.6 x 10⁵ cells in a 6 well plate.
2. Culture the harvested macrophages in Dulbecco’s Modified Eagle Medium (DMEM)/F12, 10% Fetal Bovine Serum (FBS), 1x penicillin/streptomycin, 20 ng/mL macrophage colony stimulating factor (M-CSF) for 3 days at 37 °C, 5% CO₂.
   NOTE: The upper chamber contains 1 mL of culture medium while the lower chamber is filled with 1.5 mL. The medium must be added to each chamber.

2. Coculture of tumor cells with macrophages

1. Prior to use, culture commercially available tumor cells in their respective medium following ATCC-recommended tissue culture methods.
2. Wash adherent tumor cells once with phosphate buffered saline (PBS), add 0.05% trypsin + ethylenediaminetetraacetic acid (EDTA), and incubate at 37 °C until the cells detach. Resuspend the cells in FBS containing medium, quantitate the total number of cells using a hemocytometer or cell counter, and then centrifuge for 5 min at 220 x g to pellet.
3. During centrifugation, aspirate the medium from the upper and lower chambers of the macrophage-containing permeable membrane support plates and replace with fresh medium.
   1. For lower chambers where tumor cells will be plated, fill with 1 mL of medium instead of 1.5 mL to allow for sufficient volume for cell addition.
4. Aspirate medium from pelleted tumor cells and resuspend cells in DMEM/F12 with 10% FBS, 1x penicillin/streptomycin, and 20 ng/mL M-CSF at a concentration of 3 x 10⁵ cells/mL.
5. Add 0.5 mL of 3 x 10⁵ cells/mL tumor cells to the lower chamber of desired wells (Figure 1B).
   NOTE: Cells can be treated immediately.

3. Treatment of co-cultured cells

1. To induce macrophage pro-inflammatory gene expression, treat singly or co-cultured macrophages by adding 100 ng/mL IFNγ and 50 ng/mL LPS.
   1. Vary the duration of treatment times in culture as needed. Macrophage activation occurs within 2 h and some tumor-mediated suppression occurs by 8 h. Co-culture for 24 h yields robust and consistent tumor-derived suppression.
      NOTE: Alternatively, macrophages can be induced to adopt a pro-wound healing phenotype through the addition of factors such as interleukin-10 (IL10), and the effect of the tumor-secreted ligand assessed.
2. As a negative control, culture macrophages singly and leave untreated. As a positive control, treat singly cultured macrophages with 100 ng/mL IFNγ and 50 ng/mL LPS.

4. Downstream analysis of co-cultured cells

1. After desired incubation time has elapsed, isolate the cell lysate or conditioned culture medium as desired, depending on testing needs.
2. To isolate the cell lysate for quantitative polymerase chain reaction (qPCR) analysis, aspirate the media from both chambers of the well and wash once with 2 mL of PBS. Apply RNA lysis buffer to the top chamber containing the macrophages. Gently scrape the membrane to release the cell lysate, and transfer to a collection tube for further processing according to the RNA isolation kit manufacturer’s protocol.

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