Macrophage Assays

Myeloid precursors can differentiate into multiple cell types, one of which is the macrophage. Macrophage exhibit multiple phenotypes, ranging from inflammatory (M1) through a spectrum of phenotypes to anti-inflammatory cells (M2). In the TME a broader set of tumor-associated macrophage (TAM) can be found. TAM and myeloid-derived suppressor cells (MDSC) exhibit overlapping features and play a key role in promoting tumor growth and metastasis. TAM populations are often induced by the TME to become anti-inflammatory, creating a regulatory environment and supporting tumour growth through production of pro-angiogenic factors such as VEGF. A key aim in generating an effective anti-tumour immune response is therefore to mobilise these populations of myeloid cells by driving them to become more pro-inflammatory in phenotype and cytokine production, to present antigen to T cells and to enhance their ability to phagocytose tumour cells.

Director of Science, Rhiannon Jenkinson, discusses the advantages of using the innate immune system, such as macrophage, to target tumor growth.

Charles River have developed a range of in vitro macrophage assays to model the role of myeloid-derived cells in the TME. These assays provide an understanding of whether the suppressive mechanisms of this cell type can be overcome by immuno-modulators.

Charles Rivers’ Macrophage Assays include:

  • Macrophage differentiation and polarization (M1/M2)
  • Tumor associated macrophage (TAM)
  • Phagocytosis (flow cytometry/IncuCyte®)
  • Antigen presentation and cytokine production
  • Receptor internalization and trafficking (confocal microscopy)
  • MDSC (Myeloid-Derived Suppressor Cells) suppression assay

 

CD14+ monocytes can be differentiated into macrophage or TAM, with a range of characteristics by exposure to human tumor cell supernatants, or to MDSC-like cells, following exposure to GM-CSF and IL-6. Polarisation under different conditions drives macrophage with a range of inflammatory (M1) or anti-inflammatory (M2) phenotypes.
Figure 1: CD14+ monocytes isolated from healthy donors can be differentiated into macrophage or TAM, with a range of characteristics by exposure to human tumor cell supernatants, or to MDSC-like cells, following exposure to GM-CSF and IL-6. Polarisation under different conditions drives macrophage with a range of inflammatory (M1) or anti-inflammatory (M2) phenotypes.

 

Polarisation of macrophage and the ability of a therapeutic to inhibit or drive polarisation into a particular phenotype can be assessed by flow cytometric analysis of a panel of surface markers associated with the different polarisation states.
Figure 2: Polarisation of macrophage and the ability of a therapeutic to inhibit or drive polarisation into a particular phenotype can be assessed by flow cytometric analysis of a panel of surface markers associated with the different polarisation states. In the context of driving anti-tumour responses the aim is often to cause a switch from polarisation to a Tam or anti-inflammatory phenotype to a cell type with more inflammatory characteristics.

 

Novel therapeutics can be tested for their ability to switch macrophage cytokine production from a regulatory TAM or M2 profile to production of cytokines associated with inflammation.
Figure 3: Novel therapeutics can be tested for their ability to switch macrophage cytokine production from a regulatory TAM or M2 profile to production of cytokines associated with inflammation. TAM typically have an anti-inflammatory/ pro-angiogenic phenotype, producing high levels of IL-10 and VEGF and low levels of IL-6 and IL-12. Multiplex analysis of a panel of cytokines provides a signature phenotype for macrophage polarised under different conditions. Therapeutics can be added during monocyte to macrophage differentiation or during polarization.

 

Novel compounds can be tested for their ability to enhance phagocytosis in flow cytometric, confocal or IncuCyte based assays.
Figure 4: Novel compounds can be tested for their ability to enhance phagocytosis in flow cytometric, confocal or IncuCyte based assays.

a. Fluorescently labelled CD47 expressing Raji or DLD-1 tumour cells were co-cultured with monocyte derived macrophage in the presence of anti-CD47 antibody which blocks the tumour ‘don’t eat me signal’ or opsonising antibodies. Anti-CD47 or opsonising antibody enhanced macrophage phagocytosis of tumour cells.
b. Confocal imaging of fluorescently labelled particle uptake and trafficking to measure the ability of a therapeutic to enhance uptake and internalization.

Macrophage Cell Based Assay Frequently Asked Questions (FAQs)

  • When can my therapeutic be added to the cultures?

    If the aim is to examine if your therapeutic alters monocyte to macrophage differentiation and the subsequent ability of macrophage to respond to polarising signals your therapeutic can be added during monocyte to macrophage differentiation. It is then removed before polarising signals are provided to the cells If you wish to examine if your therapeutic inhibits or alters responses to polarising signals then it would be added during polarisation to the M1/M2/TAM phenotypes and the effect on polarisation in terms of surface markers and cytokine production and phagocytosis measured.

  • What does the flow cytometry panel of antibodies show?

    This panel of antibodies includes detection of expression of surface markers which are required for antigen presentation and co-stimulation and therefore indicates how well cells may act as antigen presenting cells. Other markers are associated with responses to chemokines and cytokines and phagocytosis of certain targets, they are indicative that the cells have been programed into a certain phenotype which will influence the functional phenotype of these cells.

  • Which cell types can be used as targets in the phagocytosis assay?

    Any tumour cell type can be used in the phagocytosis assay, the choice of cell will impact on the positive controls (does the cell line express CD47? Is there an appropriate opsonising antibody which could be used?) . The percentage uptake and kinetics will be dependent on the target cell and typically needs optimising for each cell type used.