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Cancer Technologies

Cancer Technologies - Poster Session C

Poster H6 - Investigating the Influence of Lactate and Macrophage Inhibitory Factor on Macrophage Migration

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Alterations in cellular metabolism is a common hallmark in cancer progression. Even though metabolic reprogramming plays a critical role, the role of inflammatory mediators in the regulation of metabolic reprogramming is currently not understood. Among these mediators is macrophage inhibitory factor (MIF). MIF is overexpressed in solid tumors due to the presence of hypoxia inducible factor (HIF), which results in the promotion of macrophage migration and has been implicated as a regulator of inflammation, demonstrating various effects on both non-immune and immune cells. However, it is poorly understood how this overexpression of MIF affects metabolic rates of the migrating macrophages. We aim to better understand this by exploring the relationship between MIF and lactate, a byproduct of glycolysis, to understand how MIF concentrations can alter metabolite concentrations in migrating macrophages (1-3). In this study, we use a multicellular spheroid/transwell platform to investigate changes in macrophage migration, in addition to a conventional metabolic intermediate detection assay to investigate 1) how MIF inhibition using ISO-1 and 2) hypoxic conditions modulate lactate secretion and macrophage migration towards a 3D simulated solid tumor model. We hypothesize that MIF inhibition will decrease lactate secretion and macrophage migration, regardless of whether it was exposed to normoxic or low hypoxic (1%) conditions, which could signify changes in the glycolytic metabolism. By investigating macrophage migration patterns and their metabolic states, we aim to unravel the crosstalk between MIF signaling and macrophage behavior (functional and metabolic patterns).

Materials and

Methods:  RAW 264.7 (ATCC© TIB-71) murine macrophages and CT26 (ATCC© CRL-2638) murine colorectal carcinoma cells were cultured in RPMI-1640 culture medium supplemented with 1% gentamicin and 10% FBS. We brought RAW 264.7 cells to a concentration of 1 x 106 cells/mL and CT26 to a concentration of 2 x 106 cells/mL before creating a single cell suspension. After suspension creation, we deposited 20 µL hanging drops on an inverted 100 mL petri dish lid, then flipped them on top of the bottom of the petri dish filled with 10 mL of phosphate buffered saline (PBS). We placed dishes on an orbital shaker set to 70 RPM in a 37°C incubator for three days before being transferred to a suspension culture for an additional seven days (Figure 1). After the transfer, a transwell migration platform was created. Wells were randomly placed into two treatment groups: 1) No treatment (control) and 2) MIF inhibition (ISO-1, abcam (ab142140). First, we transferred 600 µL of spheroids and then media (control) or 100 µg/mL of ISO-1to the lower well. In the upper well, we added RAW 264.7 macrophages at a concentration of 200,000 cells/mL. The transwell platform was transferred to a normoxic incubator (21% O2, 5% CO2) or a hypoxic chamber (1% O2, 5% CO2) for 24 hours (Figure 2). We measured migration rates in normoxic samples using DAPI staining. A Mouse Lactate ELISA Kit (MyBioSource) was used to quantify lactate in lower well concentrations according to manufacturer’s instructions.

Results, Conclusions, and Discussions: We first examined the differences lactate concentrations from the transwell and spheroid samples from our transwell/spheroid platform to quantify how macrophages’ rates of glycolysis are altered in the absence of MIF. For the normoxic control and normoxic inhibitor the mean lactate concentrations were 0.10 mM and 0.16 mM respectively with no significant difference found between the groups (p=0.63). For the hypoxic control and hypoxic inhibitor, the average lactate concentrations were found to be 0.25 mM and 0.28 mM, respectively with no significant difference found (p=0.86). Between the normoxic and hypoxic inhibitors there was no significant difference (p= 0.51) however between the normoxic and hypoxic control groups, a significant difference was observed (p=0.04). Based on these results, the inhibition of MIF with ISO-1, regardless of normoxic or hypoxic exposure, showed higher lactate concentrations (Figure 2). It was also observed that when the platform was exposed to hypoxia, lactate levels were higher than under normoxic conditions.

Next, we investigated macrophage migration towards our 3D spheroid model under normoxic conditions with and without exposure to ISO-1 (Figure 3). It was found that the inhibition of MIF using ISO-1 showed major significant differences compared to no inhibition (p = 0.0009). Taken together, after 24 hours of normoxic culture conditions, MIF inhibition increased lactate production and macrophage migration, which has been proven in other studies (4). Future work includes investigating macrophage migration after exposure to low hypoxia (1% O2) conditions and investigating the changes in macrophage polarization during migration.

Acknowledgements (Optional):  

References:



1. Macrophage migration inhibitory factor (MIF): A multifaceted cytokine regulated by genetic and physiological strategies - ScienceDirect. https://www.sciencedirect.com/science/article/pii/S0163725821002266.

2. Hompland, T., Fjeldbo, C. S. & Lyng, H. Tumor Hypoxia as a Barrier in Cancer Therapy: Why Levels Matter. Cancers 13, 499 (2021).

3. Choi, S. Y. C., Collins, C. C., Gout, P. W. & Wang, Y. Cancer-generated lactic acid: a regulatory, immunosuppressive metabolite? J. Pathol. 230, 350–355 (2013).

4. Tao, H., Zhong, X., Zeng, A., & Song, L. (2023). Unveiling the veil of lactate in tumor-associated macrophages: a successful strategy for immunometabolic therapy. Frontiers in immunology, 14, 1208870. https://doi.org/10.3389/fimmu.2023.1208870