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.

This Transwell co-culture method is used to study paracrine signaling used by tumor cells to suppress the immune response. This method can be used to discover novel secreted ligands, as well as the mechanistic underpinnings of their immune suppressive effects. We have previously used this method to show how tumor-secreted factors can dampen macrophage pro-inflammatory gene transcription.

We believe this tool has wide-ranging implications in the study of tumor-derived immune suppression. One of the main advantages of this technique is that it can be used to study paracrine signaling between tumor cells and immune cells without the potentially confounding variable of cell-to-cell contact. This platform is also amenable to a variety of molecular biological techniques for downstream analysis.

After harvesting peritoneal macrophages, plate them directly into the upper chambers of a 0.4 micrometer polyester membrane insert co-culture six-well plate. Then added supplemented DMEM to the well such that the top chamber contains one milliliter and the bottom chamber contains 1.5 milliliters. Incubate for three days at 37 degrees Celsius with 5%carbon dioxide.

First, culture commercially available tumor cells in their respective medium, following ATCC recommended tissue culture methods. Next, wash the adherent tumor cells once with PBS. Add 0.05%trypsin in EDTA and incubate at 37 degrees Celsius until the cells detach.

Then re-suspend the cells in media containing FBS. Use a hemocytometer or cell counter to quantitate the total number of cells and centrifuge at 220 times G for five minutes to pellet. During centrifugation, aspirate the medium from the upper and lower chambers of the macrophage containing permeable membrane support plates and replace with fresh medium.

For the lower chambers where tumor cells will be plated, fill with one milliliter of medium instead of 1.5 milliliters to leave sufficient volume for cell addition. When the centrifugation is complete, aspirate the medium from the pelleted tumor cells and re-suspend the cells in supplemented DMEM at a concentration of 300, 000 cells per milliliter. Add 0.5 milliliters of this cell suspension to the lower chamber of the desired wells.

To induce macrophage pro-inflammatory gene expression, treat singly or co-cultured macrophages by adding interferon gamma at a concentration of 100 nanograms per milliliter and LPS at a concentration of 50 nanograms per milliliter. Vary the duration of treatment times in culture as needed. Macrophage activation occurs within two hours, and some tumor-mediated suppression occurs by eight hours.

Co-culture for 24 hours yields robust and consistent tumor-derived suppression. As a negative control, culture macrophages singly and leave untreated. As a positive control, treat singly-cultured macrophages with interferon gamma and LPS at the same concentrations used for the samples.

After the desired incubation time has elapsed, isolate the cell lysate or condition culture medium as desired depending on testing needs. To isolate the cell lysate for quantitative polymerase chain reaction analysis, aspirate the medium from both chambers of the well and wash once with two milliliters of PBS. Apply RNA lysis buffer to the top chamber containing the macrophages.

After this, gently scrape the membrane to release the cell lysate and transfer it to a collection tube for further processing according to the RNA isolation kit manufacturer's protocol. The co-culture method described here involves the culture of macrophages and tumor cells without physical contact. Peritoneal macrophages cultured in the absence of tumor cells are used as negative and positive controls.

Peritoneal macrophages co-cultured in the presence of B16F10 tumor cells but without activating stimuli do not increase expression of pro-inflammatory associated genes. This implies that tumor-secreted ligands are either not sufficient to induce pro-inflammatory gene expression by themselves, or if there is immune activation by tumor secretions, paracrine ligands are sufficient to suppress it to its native levels. This co-culture method illustrates that when macrophages polarized by interferon gamma and LPS are cultured in the presence of tumor cells, suppression of inflammation-associated gene expression was reduced by as much as 60%A comparable level of macrophage pro-inflammatory gene suppression is observed when the murine macrophage cell line J774 is substituted for peritoneal macrophages.

Previous work has identified protein S as a tumor-secreted factor that can inhibit macrophage activation, so the permeable membrane support co-culture model is used in conjunction with a lysA to assay the concentration of protein S in conditioned media after 24 hours. In conditioned media from interferon gamma and LPS-treated B16F10 melanoma cells, protein S is expressed in a concentration of approximately 475 nanograms per milliliter. Peritoneal macrophages treated in the same conditions expressed protein S at approximately 61 nanograms per milliliter.

Interestingly, when co-cultured, the concentration of protein S in the interferon gamma and the LPS-treated well was approximately 86 nanograms per milliliter. This suggests that either macrophages ingest tumor-secreted protein S or that the amount of protein S secreted by B16F10 cells is substantially decreased when in the presence of macrophages. These results highlight profound changes of macrophage activation and paracrine signaling when macrophages are co-cultured with tumor cells.

The Transwell co-culture method can answer a variety of questions pertaining to paracrine signaling. Western blot analysis could be conducted to determine how tumor-secreted proteins alter various signaling pathways in macrophages.