Manipulating Tumor Associated Macrophages (TAM) in a Mouse Model of B-Cell Non-Hodgkin Lymphoma (NHL)

In vitro stimulation of macrophages can produce classically activated (M1) or alternatively activated (M2) phenotypes. In various solid tumors, tumor associated macrophages (TAM) have been described as approximating an M2 phenotype and are potentially pro-tumoral. We are testing the hypothesis that TAM play an important role in the progression of NHL, and might therefore constitute a rational and effective therapeutic target.

Our aim is to investigate tumor-macrophage interactions by manipulating macrophage numbers and phenotype in a transplantable mouse lymphoma.

We used as a model system B-cell lymphoma arising in Em-myc/bcl-2 transgenic mice, which, when intravenously injected into C57BL/6 mice produces disseminated lymphoma.

Lymphoma growth was assessed by measuring lymph node weight, and cross-sectional tumor area in tissue sections, as well as by flow cytometry. Gene expression changes were determined by real-time PCR in whole lymph nodes or sorted macrophages in control mice or mice with lymphoma, and following interventions to manipulate macrophage populations.

Changes to the cellular composition of the immune microenvironment were assessed by flow cytometry of lymph nodes and blood, and by immunohistochemistry.

The consequences of widespread monocyte and macrophage depletion were first investigated using intravenous injection of the liposome-encapsulated bisphosphonate, sodium clodronate (Liposomal Clodronate, LC).

Intravenous LC successfully depleted macrophages in the bone marrow, lymph nodes, liver and spleen, and correlated with significantly reduced lymphoma mass by weight and flow cytometry, compared to controls (p=0.0058).

Next we examined MaFIA mice (Macrophage Fas Induced Apoptosis, Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) that combine the expression of an enhanced green fluorescent protein on cells with the macrophage-specific csf-1 receptor, with a drug (dimerizer, AP20187) inducible suicide gene leading to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage.

We observed the effects of this non-pharmacological method of macrophage depletion on lymphoma progression.

Lymph node macrophage numbers are not reduced in normal healthy mice treated with the dimerizer, but were significantly reduced in the infiltrated lymph nodes of mice with lymphoma given the dimerizer. This was associated with a reduced lymphoma mass (p=0.044).

Subsequent studies employed adoptive transfer of syngeneic bone marrow derived macrophages (BMDM), in vitro polarized to M1, M2 and model-specific TAM phenotypes; the purpose being to test whether supplementing the TAM population might invigorate lymphoma growth, and whether administering M1 phenotype macrophages might lead to anti-tumor effects.

Adoptive transfer of M1-polarized BMDM attenuated lymphoma growth, and transfer of TAM augmented lymphoma growth (p=0.0003).

Having gathered evidence that reducing TAM numbers might slow lymphoma growth, we explored a novel approach to targeting this macrophage sub-population in lymphoma, with translational potential for human studies. More specific targeting of TAM, and relative sparing of physiological monocytes and resident tissue macrophages, was attempted by targeting the CSF1 receptor with an orally available highly selective small molecule inhibitor. Current literature suggests this might reduce both monocyte trafficking to areas of malignancy, and maturation of monocytes to TAM. This strategy was used against growing lymphomas, resulting in an attenuated TAM population (p=0.0009) and reduced lymphoma mass (p=0.0036), whilst not depleting the circulating monocyte pool.

In vitro cytotoxicity studies confirmed that neither LC, nor the CSF1R inhibitor nor AP20187 dimerizer caused significant direct lymphoma cell death, confirming that the observed in vivo attenuation of lymphoma growth likely resulted from indirect phenomena, such as TAM depletion.

By evaluating a variety of pharmacological, genetic and cellular therapy strategies we have generated a strong body of evidence supporting a causal relationship between TAM abundance and lymphoma progression in this model. These data support the hypothesis that targeting TAM may well provide an attractive therapeutic opportunity to improve outcome in human B-cell lymphomas.

Hagemann:Astra Zeneca and the Medical Research Council: In receipt of a joint studentship with Astra Zeneca and the UK Medical Research Council. Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.