1130 Investigating macrophage modulation in a murine model of soft tissue sarcoma

Background

Soft tissue sarcomas (STSs) are a heterogeneous group of malignancies that arise from mesodermal tissue. Generally, STSs are treated with morbid surgical resection and radiation therapy.¹ The STS tumour microenvironment (TME) contains an abundance of macrophages² and few lymphocytes.³ In many malignancies, macrophages can be associated tumour progression⁴ and suppression of lymphocytes.⁵ The role of macrophages in STSs needs further investigation. Therefore, the purpose of this study was to modulate macrophages in an immune competent model of STS, and assess the changes in tumour growth and the immune landscape.

Methods

Two models were used to modulate macrophages in an immune competent murine STS model; the Macrophage Fas-Induced Apopotosis (MaFIA) model⁶, and the C-C chemokine receptor-2 Knockout (CCR2 KO) model.⁷ MaFIA mice express a transgene and can be given a pharmacological agent to induce cell death in CSF1R+ cells. The CCR2 KO model shows a deficiency in recruiting monocytes to inflamed tissue. Both models have reduced tumour macrophages in other murine models.^{8, 9} MaFIA and CCR2 KO mice were engrafted with STS cells in the right hindlimb.¹⁰ Tumour volume, macrophages (CD45⁺, CD11b⁺, F4/80⁺, and CD80⁺ or CD206⁺), as well as lymphocytes (CD45⁺, CD3e⁺, and CD4⁺ or CD8⁺) were assessed using flow cytometry.

Results

MaFIA mice showed a ~84% decrease (p<0.01) in tumour volume, whereas the CCR2 KO mice and control mice showed no differences in tumour volume. MaFIA mice showed a significant reduction of CSF1R+ macrophages (53%; p<0.01) and CSF1R+, CD206+ macrophages (54%; p<0.05), but no changes to total macrophages. Interestingly, the CCR2 KO mice showed a significant decrease in tumour macrophages (47%; p<0.01), as well as CD206+ macrophages (41%; p<0.01). The MaFIA model showed no differences in lymphocytes, but the CCR2 KO model showed a significant increase in CD8 T cells (360% increase; p<0.01). Interestingly, CSF1R+ ablative treatment was associated with an increased expression of PD1 in the MaFIA model (211%; p<0.01).

Conclusions

Both CSF1R and CCR2 modulation diminished macrophage subtypes, but CSF1R modulation did not diminish the total macrophage population. However, only the CCR2 KO model improved lymphocyte (CD8 T cell) infiltration, which did not improve disease outcome. Additionally, CSF1R modulation was associated with an increased PD1 expression. Thus, modulating the CCR2 axis influenced the TME but did not change tumour burden. The CSF1R axis diminished STS tumour burden and macrophage subtypes, and could potentially also sensitize STS tumours to anti-PD1 therapy.

References

Cormier JN, Pollock RE. Soft tissue sarcomas. CA Cancer J Clin,2004;54(2):94–109. Sawa-Wejksza K, Kandefer-Szerszen M. Tumor-associated macrophages as target for antitumor therapy. Archivum immunologiae et therapiae experimentalis, 2018;66(2):97–111. Sorbye SW, et al. Prognostic impact of lymphocytes in soft tissue sarcomas. PLoS One 2011;6(1):e14611. Lin Y, Xu J, Lan H, Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. J Hematol Oncol 2019;12(1):76. Peranzoni E, et al. Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti–PD-1 treatment. Proceedings of the National Academy of Sciences 2018;115(17):E4041–E4050. Burnett SH, et al. Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. Journal of Leukocyte Biology 2004;75(4):612–623. Boring L, et al. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in CC chemokine receptor 2 knockout mice. The Journal of Clinical Investigation 1997;100(10):2552–2561. Gabrusiewicz K, et al. Macrophage ablation reduces M2-like populations and jeopardizes tumor growth in a MAFIA-based glioma model. Neoplasia 2015;17(4):374–384. Sanford DE, et al. Inflammatory monocyte mobilization decreases patient survival in pancreatic cancer: a role for targeting the CCL2/CCR2 axis. Clinical Cancer Research 2013;19(13):3404–3415. 10. Hildebrand KM, et al. The Kras G12D; Trp53 fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade. PloS One 2021;16(7):e0253864.

Ethics Approval

Studies involving animals were be conducted according to the Canadian Council on Animal Care guidelines. Ethics approval was obtained from the University of Calgary Health Sciences Animal Care Committee (AC19-0072, 6/25/2021). All mice are housed at the University of Calgary Foothills Campus in a level 2 containment facility.