Hello,
i decided to ask this question, because i am quite confused regard this topic. I've red that in order to obtain TAMs from freshly isolated monocytes, i should first stimulate with MCSF and then with IL-4 and IL-13 (there are also other ways to do this). In other articles, they characterized these cells like M2-SUBTYPE, but not TAMs. So which is the right way to obtain TAMs? I doubt also, about the markers i should use in order to verify afterwards the polarization: one the one hand CD163 it's a typical TAMs marker, on the other hand it is not unregulated after the procedure i mentioned before (MCSF+IL-4). Why? So i should use an other marker like CD206?
I look forward to read your answers.
Thank you very much.
The current view on macrophage M1-M2 phenotypes is that this seems to be an artificial concept. TAMs will have a range of activation states comprised between the extremes of M1 and M2, although it is still assumed that TAMs are a tendentially M2 population.
I would advise you to read Martinez and Gordon, 2014 'The M1 and M2 paradigm of macrophage activation: time for reassessment', it may help clearing your doubts on macrophage polarization.
CD163 and CD206 are typical M2-macrophage type markers, but there are others you can use - CD301, TGF-beta1, CD209 - take a look at Martinz et al, 2006 ' Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Gene Expression'
In a more puristic sense, if you want to obtain tumour-associated macrophages, first you need the tumour itself and then by dissociation of stromal and neoplastic tissue and cell sorting, you may obtain the TAMs.
I hope to have helped, best of luck on your research
Thank you very much . I've already red these articles and i confirm that are very useful. Your approach was very interesting. I will continue reading in order to clear this topic.
Thank you again.
Best regards
M1-M2 compartment is well defined in mouse. In human, M1-M2 macrophages were not fully characterized. M2s are subdivided into M2a (Il4 or IL-13), M2b (IL-10) and M2c (LPS and immune complex). In human CD206 and CD163 is not induced by IL-13/IL-4. I think, you need to induce them with IL-10 and look at CD163.
Hi Meropi
Why not using tumor cells to generate TAMs? We co-cultured monocytes with cancer cells (A549, other cancer cell lines also work) or conditioned medium from cancer cells to polarize these cells. In our hands it worked fine and may resemble better the "natural environment" of TAMs. See: Müller-Quernheim UC, Potthast L, Müller-Quernheim J, Zissel G. Tumor-cell co-culture induced alternative activation of macrophages is modulated by interferons in vitro. J Interferon Cytokine Res 2012: 32(4): 169-177.
The only disadvantage with this model is that we currently do not know which factors released by cancer cells induce this differentiation. However, given the fact that IL-4, IL-13 or IL-10 is not released by cancer cells I am convinced that this model resembles better tha natural situation tan any artificial stimulation.
Hello Gernot,
i've already red your interesting article and i was ready to perform the co-culture this week. Your answer arrived just in time. Strange coincidence! I also think that it is possible to obtain TAMs co-coltivating with tumor cells. Maybe it is difficult for our laboratory this period to check the same markers you chose (CCL18, CXCL10) by ELISA, but i think that i can verify the successful polarization using other markers.
Thank you very much.
Best,
Mary