Interrogation and modulation of the immunosuppressive effects of TAMs using in vitro cultures.
The tumour microenvironment (TME) supports the development of pro-tumoral macrophages. These Tumour Associated Macrophages (TAMs) resemble M2-macrophages and are characterised by an immune-suppressive phenotype. In the TME, TAMs can induce proliferation and survival of tumour cells, facilitate angiogenesis, and suppress anti-tumour immune responses via expression of co-inhibitory molecules such as PD-L1 and cytokines such as IL-10 or TGF-β1. Therefore, TAMs are a highly attractive target for innovative cancer immunotherapies. Understanding whether candidate immunotherapies can reverse TAM-mediated immune suppression or reprogram TAMs to a pro-inflammatory phenotype is key to the development of effective cancer immunotherapy.
Aquila has a suite of customisable human in vitro and ex vivo macrophage assays that can be tailored and used to build a data package to suit your requirements. Our expertise allows you to test your novel compound activity upon TAM-like macrophage generation, phenotype and suppressive function, selecting the most effective compounds for further investigation using patient-derived immune cells.
Whatever your stage of drug development, we can tailor our assays to meet your needs. From generation of myeloid cell-derived macrophages, we offer both monoculture (M1 or M2 macrophage assays) and co-culture macrophage assays, through to immunophenotyping and functional assays to confirm drug effectiveness or identify indirect toxic effects, using in vitro generated and/or ex vivo derived macrophages from tumour samples and ascites fluid.
Phenotyping Macrophages and Repolarisation of M2 Macrophages
Our macrophage assays can be used to screen libraries of small molecules or biologics. Human PBMC-derived monoctyes differentiated with M-CSF are skewed towards a CD163+ M2 phenotype, whereas GM-CSF lead to differentiation of CD163lo M1 macrophages.
IL-10 is believed to be a key component of TAM-driven T cell suppression. M2 macrophages produce high levels of IL-10, and little IL-12 in response to LPS (M1 macrophages show the opposite) (Figure 1).
Figure 1. Macrophage Gene Expression.
Small molecule inhibitors can re-polarise M2 macrophages, rendering them potentially more immuno-stimulatory (high IL-12, low IL-10) (Figure 2).
Figure 2. Repolarising M2 Macrophages with a Small Molecule Drug.
Macrophage Suppression Assay
Aquila offer a tailorable Macrophage Suppression Assay for screening molecules or biologics in their ability to reverse macrophage suppression, an important mechanism of action for a potential immunotherapy cancer treatment (Figure 3).
Figure 3. Macrophage Suppression Assay.
The tumour microenvironment drives macrophages to acquire a pro-tumoural phenotype (M2). Our assay screens compounds for their potential to switch or re-polarise macrophages to an anti-tumour (M1) state, which – in turn – reduces the immune suppression of T cells.
We can measure expression of M2 markers and/or inhibitory molecules using flow cytometry and qPCR. We can measure cytokine release by ELISA or multiplex. Further assay readouts include monocyte to macrophage differential rate, viability and apoptosis (Figure 4).
Figure 4. Reversal of Macrophage Suppression.
Ex Vivo TAM Assays
Understanding the role of pro-tumoral macrophages within the TME is vital to immuno-oncology research. TAMs are immunosuppressive, resembling M2, rather than the more proinflammatory M1 phenotype.
Aquila can investigate the effect of candidate therapeutics on macrophages generated in vitro or derived from tumour samples and ascites fluid.
We can investigate the surface chemistry of TAMs to determine optimal pre-clinical in vitro models for large scale analyses. We also offer readouts measuring clinically relevant changes in immune function including proliferation, gene expression and production of immunomodulatory cytokines (Figure 5).
Figure 5. Immunophenotyping human CD14+ cells: different macrophage subsets express different markers.
Tumour derived immune cells can show IFN-γlo/IL-10hi responses when stimulated with anti-CD3. This response profile can be reversed when cultured with macrophage-targeting small molecule inhibitors (Figure 6).
Figure 6. Induction of IFN-γ and inhibition of IL-10 production in tumour associated immune cell cultures using a small molecule drug.
A Case Study on p38 MAP Kinase Inhibition
IL-10 is believed to be a key component of TAM-driven T cell suppression. M2 macrophages produce high levels of IL-10 and little IL-12 in response to LPS (M1 macrophages show the opposite). p38 MAP Kinase signalling is a key mediator of IL-10 production by macrophages and is therefore an attractive therapeutic target. The p38 inhibitor LY2228820 reduced the ability of M2-polarised human macrophages to produce IL-10 in response to TLR4 ligation (Figure 7).
Figure 7. The p38 inhibitor LY2228820 reduced the ability of M2-polarised human macrophages to produce IL-10 in response to TLR4 ligation.
M2 macrophages potently suppress IFN-γ production by anti-CD3 stimulated PBMC, and this suppression can be partially reversed by neutralisation of IL-10. LY2228820 was also able to reverse macrophage suppression in this culture (Figure 8).
Figure 8. Macrophage Suppression Assay
Aquila has access to a range of fresh clinical material. Addition of LY2228820 to cultures of tumour material from ovarian carcinoma (tumour and ascites) and renal carcinoma was able to inhibit TLR4-driven IL-10 production (Figure 9).
Figure 9. Clinical Samples: IL-10 and IL-12p40 expression.
This case study highlights how Aquila can build a package of consistent data, using immune cells from both healthy donors and the clinic, to support your drug discovery and development program.
Get in touch to discuss your macrophage study requirements and how Aquila can support you to build a stronger data package and speed up your route to clinic.