Name: studying the effects of tumor-secreted paracrine ligands on macrophage activation using co-culture with permeable membrane supports
Uploaded: 2019-11-28T13:30:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports at JoVE.com

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.

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).
<ol>
	<li>Harvest peritoneal macrophages as previously described16,17 and plate directly into the upper chamber(s) of a 0.4 &#181;m 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&#8211;1.6 x 105 cells in a 6 well plate.</li>
	<li>Culture the harvested macrophages in Dulbecco&#8217;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 &#176;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.</li>
</ol>
2. Coculture of tumor cells with macrophages
<ol>
	<li>Prior to use, culture commercially available tumor cells in their respective medium following ATCC-recommended tissue culture methods.</li>
	<li>Wash adherent tumor cells once with phosphate buffered saline (PBS), add 0.05% trypsin + ethylenediaminetetraacetic acid (EDTA), and incubate at 37 &#176;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.</li>
	<li>During centrifugation, aspirate the medium from the upper and lower chambers of the macrophage-containing permeable membrane support plates and replace with fresh medium.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Treatment of co-cultured cells
<ol>
	<li>To induce macrophage pro-inflammatory gene expression, treat singly or co-cultured macrophages by adding 100 ng/mL IFN&#947; and 50 ng/mL LPS.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>As a negative control, culture macrophages singly and leave untreated. As a positive control, treat singly cultured macrophages with 100 ng/mL IFN&#947; and 50 ng/mL LPS.</li>
</ol>
4. Downstream analysis of co-cultured cells
<ol>
	<li>After desired incubation time has elapsed, isolate the cell lysate or conditioned culture medium as desired, depending on testing needs.</li>
	<li>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&#8217;s protocol.</li>
</ol>

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The authors have nothing to disclose.

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 signaling. Given the potential clinical importance of tumor-secreted ligands in immunosuppression, this method offers an easy to use tool to study these mechanisms in ways not addressed by direct co-culture.
Essentially, the co-culture method described here involves the culture of macrophages (primary cells: resident, bone marrow-derived, or a macrophage cell line) with tumor cells (cell line or primary cells) without physical contact. It is critical that the macrophages be cultured on a 0.4 &#181;m pore size polyester membrane insert to allow free permeability of tumor-secreted ligands while preventing possible macrophage cell migration through the filter. It is also important to add the described amount of culture medium to the upper and lower chambers to ensure proper cell coverage. Experimental design, such as inclusion of one cell type only controls, is another key factor when planning co-culture experiments. Several other factors, described in more detail later, can also have effects on the results of the assay and it is important to keep each in mind during study design.
Two useful modifications to the outlined protocol to consider are the duration of co-culture or the relative ratio of tumor cells to macrophages. In the method described, macrophages and tumor cells are plated at roughly equal concentrations. An important point to consider is the relative proportion of macrophages to tumor cells that naturally occurs in the tumor microenvironment. To better mimic the tumor microenvironment, the relative proportion of macrophages could be altered to mirror what is found within the tumor in vivo, though preliminary work may be necessary to establish the correct ratio.
Another critical point, and possible limitation of the system, is that treatments applied to stimulate one cell type in the assay may have unexpected or unintended effects on the other cell type. As described here, addition of LPS and IFN&#947; is intended to stimulate pro-inflammatory gene expression in macrophages. However, in Ubil et al. we showed that IFN&#947; also induces the expression and secretion of immunosuppressive Pros116, and others have shown the effects of LPS on tumor cells18. It is therefore essential to include the appropriate controls to monitor potential off-target or unintended effects on the other cell type to verify experimental outcomes. One method by which this may be achieved is by treating individual cell types with the agents of interest and monitoring effects using standard molecular biology assays.
When designing permeable membrane support experiments, it is also important to consider possible diffusion gradients of secreted ligands. The relative rate of ligand secretion, the duration of co-culture and whether the culture plate is maintained in a stationary position can all have effects on the results. In addition, it is possible for some secreted ligands to adhere to the surface of culture plates.
While the representative results shown are characteristic of this system, the degree of pro-inflammatory gene suppression observed when co-culturing other tumor lines may vary dramatically. In Ubil et al., we show that some human tumor lines can almost completely suppress the pro-inflammatory gene expression of a human macrophage cell line16. Conversely, other tumor cell types or cell lines may vary substantially in their immunosuppressive capabilities. The cause for these variances is unclear but is an area for further study.
Permeable membrane support co-culture is a robust methodology that can be easily modified to address a variety of questions and can be adapted to a range of molecular biology readouts including qRT-PCR, Western blot, and ELISA. The system can be used to interrogate individual gene functions, such as ligands or receptors, when genetic alterations like those from gene-deleted mice or CRISPR edits are made to either macrophages or the tumor cells. The system is also amenable to the study of pharmacological activators or inhibitors and their effects on paracrine signaling. Also, while not discussed here, the system can be used to study the effects of immune activation on tumor cell gene expression.
This method has been used successfully in the discovery and characterization of a novel function for an immunosuppressive, tumor-secreted ligand. This robust tool can be utilized to interrogate the much broader subset of non-contact tumor/immune interactions in the hopes of discovering new therapeutic targets.

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 response to interferon-gamma (IFN&#947;), which is expressed by a number of activated immune cells, tumor cells can increase expression of PD-L1 to induce exhaustion of PD-1&#8211;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 microenvironment5,6,7. 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 activation8,9.
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.

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

studying the effects of tumor-secreted paracrine ligands on macrophage activation using co-culture with permeable membrane supports

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 &#181;m 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 &#181;m 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.

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 (IFN&#947; 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 either treated with IFN&#947; and LPS or left untreated. After 24 h of culture, macrophages were harvested, RNA prepared, and qRT-PCR performed to measure the expression of pro-inflammatory genes. Using the co-culture system described here, we show that peritoneal macrophages co-cultured in the presence of B16F10 tumor cells, but without activating stimuli (LPS + IFN&#947;), did not increase expression of pro-inflammatory&#8211;associated genes (Figure 2A, B16F10 membrane/untreated). This implies that tumor-secreted ligands are 1) not sufficient by themselves to induce pro-inflammatory gene expression or 2) if there is immune activation by tumor secretions, paracrine ligands are sufficient to suppress it to naive levels. This co-culture method illustrates that when macrophages polarized by IFN&#947; and LPS are cultured in the presence of tumor cells, suppression of inflammation-associated gene expression was reduced by as much as 60% (Figure 2A, B16F10 Membrane/IFN&#947;+LPS). A comparable level of macrophage pro-inflammatory gene suppression was observed when the murine macrophage cell line J774 was substituted for peritoneal macrophages (Figure 2B).
Our previous work identified Pros1 as a tumor-secreted factor that can inhibit macrophage activation16. Using the permeable membrane support co-culture model in conjunction with ELISA, we assayed the concentration of Pros1 in conditioned medium after 24 h. We observed that in conditioned medium from IFN&#947; and LPS treated B16F10 melanoma cells Pros1 was expressed at 475 ng/mL &#177; 120 ng/mL (Figure 3). Peritoneal macrophages treated in the same conditions expressed Pros1 at 61 ng/mL &#177; 5 ng/mL (Figure 3). Interestingly, when co-cultured, the Pros1 within the IFN&#947; and LPS treated well was 86 ng/mL &#177; 15 ng/mL. This suggests that 1) macrophages consume tumor-secreted Pros1 or 2) the amount of Pros1 secreted by B16F10 cells is substantially decreased when in the presence of macrophages. Results from both Figure 2 and Figure 3 highlight profound changes of macrophage activation and paracrine signaling when macrophages are co-cultured with tumor cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60453/60453fig1.jpg" /> 
Figure 1. Schematic for permeable membrane support co-culture of tumor cells with macrophages. Positive and negative treatment controls can be applied to singly cultured macrophage wells (A). To determine the effects of tumor-secreted signals on macrophage activation, macrophages are cultured in the upper chamber of permeable membrane support co-culture plates and tumor cells cultured in the lower chamber (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60453/60453fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60453/60453fig2.jpg" /> 
Figure 2. Tumor paracrine signals suppress macrophage pro-inflammatory polarization. Macrophage expression of inflammation-associated genes was assayed by qRT-PCR in untreated or IFN&#947; and LPS stimulated macrophages in the presence or absence of tumor cells. Expression of pro-inflammatory genes was decreased in peritoneal macrophages (A) or J774 macrophage cell line (B) when cultured in the presence of tumor cells separated by a permeable membrane support so that the effect was transmitted by a paracrine soluble ligand. *p&#160;&#60; 0.05 relative to untreated,&#160;&#8224;p&#160;&#60; 0.05 relative to IFN&#947; and LPS stimulated. Data are mean &#177; SEM;&#160;p&#160;values calculated by two-tailed Student&#8217;s&#160;t&#160;test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60453/60453fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60453/60453fig3.jpg" /> 
Figure 3. Coculture of tumor cells and macrophages leads to changes in the amount of tumor-secreted paracrine ligands found in conditioned medium. ELISA was used to determine the concentration of Pros1 in tumor alone, macrophage alone, or co-cultured conditioned medium after 24 h. Co-culture leads to a decrease in the amount of Pros1 relative to tumor cell only controls. *p&#160;&#60; 0.05 relative to untreated. p values calculated by two-tailed Student&#8217;s t test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60453/60453fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports at JoVE.com

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

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.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

studying-effects-tumor-secreted-paracrine-ligands-on-macrophage

Nanyang Technological University

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JoVE Journal

Cancer Research

7.4K Views.  University of North Carolina at Chapel Hill. Deze Transwell co-cultuur methode wordt gebruikt om paracrine signalering gebruikt door tumorcellen om de immuunrespons te onderdrukken bestuderen. Deze methode kan worden gebruikt om nieuwe afgescheiden liganden te ontdekken, evenals de mechanistische onderbouwing van hun immuunonderdrukkende effecten. We hebben deze methode eerder gebruikt om te laten zien hoe tumor-gesecreteerde factoren macrofaag pro-inflammatoire gentranscriptie kunnen dempen.Wij geloven dat deze tool heeft brede ...

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Medicine

Examining Macrophage-Tumor Spheroid Interactions in a 3D Collagen Matrix Setup

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Neuroscience

Matrix-Based Tumor Invasion Model Using Microglia Glioblastoma Co-Culture

Source: Coniglio, S., et al.&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53990">Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion.</a> J. Vis. Exp. (2016)
In this video, glioblastoma and microglia cells are mixed with a polymer matrix solution and transferred to a porous chamber insert where a gel forms, embedding the cells. Microglia stimulate the migration of glioblastoma cells through the porous insert, mimicking tumor cell invasion into surrounding tissue, Cells are then fixed and prepared for further analysis.

Source: Coniglio, S., et al.&#160;Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion. J. Vis. Exp. (2016)
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Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

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