EXTH-59. IMMORTALIZATION OF MOUSE BONE MARROW-DERIVED MACROPHAGES FOR BRAIN TUMOR THERAPY DEVELOPMENT

Abstract

Abstract BACKGROUND Macrophages can cross blood-brain-barrier (BBB) and infiltrate brain parenchyma with malignancies such as glioma. Recently, many efforts have been made to develop macrophage-based cancer therapy, including CAR–macrophages. However, lack of proliferation in differentiated macrophages hinders their utility. We sought to ameliorate this problem by immortalizing macrophages to assist potential therapy development. Bone marrow derived macrophages (BMDMs) were harvested from C57Bl/6 mice and transduced with mouse telomerase reverse transcriptase (mTert) gene via retrovirus. Following puromycin selection, morphology, proliferation, lineage marker expression and phagocytosis were measured by microscopy, flow cytometry and IncuCyte assays. RESULTS Through overexpression of mTert, we have successfully immortalized mouse BMDMs, termed as iBMDMs. Morphologically, iBMDMs largely maintained features of primary BMDMs. The established iBMDMs proliferated for over 20 subcultures without obvious reduction in growth. Proliferation of iBMDMs (doubling time, dt=52h) was similar to IC-21(dt=53h), an established mouse macrophage line immortalized by SV40-T antigen, and slower than Raw264.7 (dt=23h), an immortalized mouse macrophage line driven by murine leukemia virus. Furthermore, iBMDMs can grow without macrophage colony-stimulating factor (M-CSF) (dt=54h). Macrophage lineage markers CD11B and F4/80 expression levels were higher in primary BMDMs (CD11B+=62.8±0.53%, F4/80+=54.9±0.38%, CD11B+F4/80+=52.7±0.41%, n=2 for all flow results) and iBMDMs (CD11B+=39.1±5.29%, F4/80+=26.4±7.93%, CD11B+F4/80+=24.6±5.95%) than in IC-21 (CD11B+=10.7±0.37%, F4/80+=19.4±1.88%, CD11B+F4/80+=9.13±0.37%) and Raw264.7 (CD11B+=34.3±5.32%, F4/80+=14.1±4.40%, CD11B+F4/80+=8.33±4.19%) cells lines. Lastly, we evaluated the phagocytic ability of iBMDMs through IncuCyte assays. Similar to primary BMDMs, we observed a concentration dependent increase in red fluorescence, which indicates the phagocytosis of E. coli derived bioparticles, in iBMDMs with and without M-CSF. CONCLUSIONS We immortalized bone marrow-derived macrophages of C57Bl/6 mouse origin with competent immune function via mTert transduction. These cells can be used in preclinical immunotherapy development for glioma treatment. Ongoing work is focused on assessing their ability to penetrate BBB and infiltrate brain tumor in a mouse glioma model.