Abstract P2-02-08: Slit2 induced anti-tumor activity may be mediated through metabolism driven immunomodulation
Authors: Kaul K , Ahirwar DK , Benej M , Denko N , Ganju R 
Published: May 2019

Metabolism of immune cells plays an important role in regulating tumor growth by modulating the anti-tumor M1 phenotype or pro-tumor M2 phenotype in macrophages. However, the role of bone marrow derived macrophages (BMDM) and their metabolic profile in promoting tumor growth is unknown. Slit2 is an anti-tumor molecule that is suppressed in breast cancer; however, the mechanism by which Slit2 mediates its function is not fully elucidated. We hypothesize that Slit2 mediated metabolic reprogramming of BMDMs favors anti-tumor M1 phenotype in these macrophages, which in turn reduces tumor growth.

Here we assessed cellular metabolism in BMDMs from the MMTV-PyMT mouse, a model showing spontaneous tumor development. Shortly, age matched female mice with palpable tumors and control mice without tumors were treated with recombinant Slit2 (Slit2) or PBS intraperitoneally every third for 2 weeks (n=4 mice per group). Tumor volume was measured in each mouse before and at the end of treatment. Next, mice were euthanized and bone marrow was flushed from both the tibia and femor for culturing in vitro in the presence of macrophage chemotactic factor rich conditioned media. Rate of glycolysis was described based on extracellular acidification rate (ECAR) as measured by the Seahorse Bioanalyzer® under glycostress conditions. Lactate dehydrogenase (LDH) activity, which is linked with breast cancer progression and pro-tumor macrophage phenotype in experimental models, was also assayed using a commercially available kit. Finally, to elucidate potential pathways involved in Slit2 induced metabolic change, we assessed the expression of several proteins and factors involved in cellular metabolism.

Firstly, we observed that PyMT mice treated with Slit2 showed lower tumor volume compared to mice treated with PBS confirming Slit2 anti-tumor activity. Hematoxylin & Eosin staining of tumor sections from these mice also showed better tissue structure, with a higher cytoplasm to nuclear ratio in Slit2 treated PyMT mice compared to PBS treated mice. Furthermore, BMDMs from PyMT mice show lower aerobic glycolysis with higher lactate dehydrogenase activity (LDH) compared to control mice. Moreover, treatment with Slit2 appeared to lower LDH activity and trended to increase glycolysis in the BMDMs isolated from Slit2 treated PyMT compared to PBS treated PyMT.

Expression analyses using quantitative PCR showed a 2-4 fold decrease in PGC-1α and CPT-2 in Slit2 treated BMDMs, indicating a reduction in fatty acid oxidation in these cells. This coupled with a 3 fold increase in IL-6 expression, and 2-3 fold decrease in arginase and IL-10 expression in tumor tissue suggest a potential shift from pro-tumor M2 to anti-tumor M1 phenotype. In spite of these preliminary trends, changes in metabolism and associated signals may be clearer in isolated, enriched populations of macrophages alone. Nevertheless, our findings suggest that Slit2 reduces tumor growth by affecting immune cell metabolism. Furthermore, these studies provide novel evidence of the potential immunomodulatory effects of Slit2 on macrophages. This may lead to development of Slit2 as a novel non-invasive therapeutic strategy against highly aggressive and metastatic cancers associated with high mortality and low quality of life.

Citation Format: Kaul K, Ahirwar DK, Benej M, Denko N, Ganju R. Slit2 induced anti-tumor activity may be mediated through metabolism driven immunomodulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-08.