Migration State-Dependent Traction Force Generation and Energy Regulation in Cancer Cells

Introduction: : Invasion and migration of cancer cells to the surrounding tissue is an important step in metastasis, which is the primary cause of cancer-associated death. Cancer cells are also known to have an altered and more dynamic metabolic program compared to normal cells, which influences their metastatic potential [1,2]. Recent evidence further suggests a direct connection between the bioenergetics and the migration of cancer cells [3,4]. For example, increased cellular ATP:ADP ratio and oxidative phosphorylation have been positively correlated with migratory capability [5] and protrusive activities of breast cancer cells [3]. During the metastatic cascade, cancer cells experience different microenvironments and may exhibit different migration states and metabolic adaptations to the local environment [3,5]. However, how the migration states and the corresponding energy utilization of cancer cells influence cancer progression is not clear. Faster cancer cell migration does not necessarily correlate with higher metastatic potential. In fact, in breast cancer, weakly migratory cells were found to be more metastatic than highly migratory cells in vivo [6]. Hence, it is important to understand the behaviors of cancer cells when they are actively migrating or not actively migrating, to develop targeted therapeutics to prevent metastasis. Here, we engineered the microenvironment to either permit or restrict cancer cell migration in 2D and 3D, and investigated how the migration state regulates cellular bioenergetics and traction force generation, providing insight into cancer cell behavior during the metastatic cascade.

Materials and

Methods: : To allow for free cell migration on 2D surfaces, MDA-MB-231 human breast cancer cells were seeded on fibronectin-coated glass. To prevent cells from migrating freely and create a non-migrating state, the cells were seeded on fibronectin-coated micropatterns with a teardrop shape mimicking the size and shape of a migrating cell, as previously described [3] (Fig. 1A-B). To measure cellular contractility, cells were seeded on patterned or un-patterned polyacrylamide elastic hydrogels, and traction force microscopy (TFM) was used to measure the total traction force generated by each cell with the ImageJ Plugins, PIV, and FTTC [7,8]. To prevent free cell migration in 3D, cells seeded in collagen gels were treated with 25 µM GM6001, a broad-spectrum matrix metalloproteinase inhibitor, to prevent them from degrading the collagen matrix. Control cells were not treated to permit free migration. To evaluate the bioenergetics of cells in different migration states, the PerecevalHR probe was expressed in the cells to measure intracellular ATP:ADP ratio, and the Peredox probe was expressed to measure intracellular NADH:NAD+ ratio, as previously described [2]. Additionally, the fluorescent dyes TMRM and 2-NBDG were used to measure mitochondrial membrane potential (MMP) and glucose uptake of the cells, respectively. Cells were imaged using an LSM900 confocal microscope at 12 and 24 hours post-seeding for 2D and 3D cells, respectively.

Results, Conclusions, and Discussions::

Results: Non-migrating cells were found to exhibit an increased ATP:ADP ratio and MMP (Fig. 1C-E) than migrating cells in 2D, consistent with previous reports [3]. Non-migrating cells also had increased glucose uptake (Fig. 1F). Similar results were observed for migrating and non-migrating cells in 3D (Fig. 1C-F). These results indicate that cells may upregulate their energy production and level when prevented from migrating freely. Intriguingly, a significant difference between migrating and non-migrating cells for cellular NADH:NAD+ ratio was only found in 3D, not 2D (Fig. 1G). As NADH:NAD+ ratio reflects cellular redox balance, this indicates cells migrating in 2D and 3D may have differential reliance on oxidative phosphorylation or glycolysis as their main energy production pathway. As non-migrating fibroblasts were previously found to generate stronger traction forces than migrating cells [9], we hypothesized that non-migrating cancer cells may upregulate their energy level to support a strong traction generation. To test this hypothesis, we measured traction forces in our 2D cells. Surprisingly, our preliminary results revealed greater total force in migrating cells compared to non-migrating cells (Fig. 1H-I), opposite to previous findings [9].
Discussions: Highly motile cells were previously reported to exhibit a higher ATP:ADP ratio than weakly motile cells in 3D matrices [5]. This does not contradict our results, as the weakly motile population in that study may represent cells inherently incapable of migration, whereas our non-migrating cells retain migratory potential but are physically restricted from moving. To further validate our findings, we will apply additional methods to inhibit cell migration in 3D. Additionally, we observed an opposite trend in traction force generation between migrating and non-migrating cancer cells and fibroblasts, suggesting that cancer cells may utilize a distinct energy pathway to generate force compared to normal cells. We plan to investigate this further.

Conclusions: Together, we found that cancer cells temporarily restricted from migration become metabolically hyperactive. While the underlying mechanisms and consequences of this upregulation remain under investigation, this novel finding offers important insight into how a cell’s migratory state may influence cancer progression.

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