Reversibility of nuclear and 3D Genomic changes in non-cancerous fibroblasts after constricted migration

Metastatic cancer cells and healthy fibroblasts must traverse through tight constrictive spaces to reach secondary sites. We have previously shown that human melanoma (A375) cells that pass through multiple constricted migrations experience stable changes to their nucleus morphology and 3D genome structures and undergo a stable phenotype transition from a low to high migratory state. Here, we investigate whether fibroblasts (BJ-5ta), which are non-cancerous and have an inherent ability to migrate to fulfill roles in wound repair, experience nuclear and 3D genomic changes with constricted migration. While BJ-5ta cells do not get progressively better at migrating with sequential attempts, confocal microscopy analysis of cells stained for Lamin A/C and DAPI after migration shows that these cells do experience nuclear deformations after constricted migration. Unlike the stable changes observed in cancer cells, however, these deformations recovered back to unmigrated levels following proliferation and cell movement and did not induce fibrotic smooth muscle alpha actin expression in these cells. Our study shows that healthy migratory cells are not more robust against alterations caused by constricted migration but can recover from the ones that do arise more readily than cancer cells.

Methods
Constricted Migration Assays:

Transwell filters with 5µm pores (VWR-10769-236) were used. The bottom of each filter was coated with 40µL of 10ug/µL fibronectin (Corning, 354008) for ~30 min. 100,000 BJ-5ta cells in 100µL of fully supplemented media were placed into the top well of the transwell with 500µL of fully supplemented media in the bottom chamber. Cells were allowed to attach overnight. The following day both the top and bottom chamber’s media were replaced with a 4:1 ratio of DMEM and 1x Medium 199, but with 1% BSA instead of 10% FBS. Cells were starved overnight. The following day, the bottom chamber’s media was replaced with a 4:1 ratio of DMEM and Medium 199 supplemented with 20% FBS and 5 μg/mL of FGF (Gibco, PHG0368V).  

For sequential rounds of migration, the migrated cells were seeded into a new well of a 24‐well plate to expand. When cells reached 80–90% confluency, another Transwell migration was performed as previously described. Only the bottom cells were collected and continued to be used for sequential rounds of migration. 

Immunofluorescent Confocal Imaging 

Approximately 50,000 BJ-5ta cells for each condition were seeded into 35‐mm glass bottom dishes (MatTek, P35GC-1.5-10-C). Cells were allowed to attach overnight and then cross‐linked with 4% formaldehyde for 10 min followed by three, 5‐min washes with PBS. After washing, cells were permeabilized with permeabilization buffer (10% goat serum, 0.5% Triton in PBS) for 1 h at room temperature. After the incubation, primary antibody for LaminA/C (Santa Cruz, sc-376248), diluted in antibody dilution buffer (5% Goat serum, 0.25% Triton in PBS), was added and incubated overnight at 4°C. After primary antibody incubation, cells were washed three times with PBS for 5 min for each. Cells were then incubated with secondary antibodies (Alexa Fluor 488, Invitrogen, R37120) per manufacturer's directions (two drops of secondary antibody/1 mL of PBS) for 30 min at room temperature. After secondary antibody incubation, cells were washed three times with PBS and sealed using mounting media with DAPI (Invitrogen, P36962). Slides were treated in the mounting media for 24hr before imaging. Cells were imaged using a Leica Sp8 Confocal microscope equipped with a 63x oil immersion objective. For αSMA experiments, cells were seeded onto 24 well plate coverslips (Fisher Scientific, 50-194-4702) with either no treatment, starvation and FGF spike-in as described earlier, directly after 5um transwell migration, or after migration and allowed to recover for 48hrs. Cells were then fixed as above and stained with an αSMA primary antibody (Invitrogen, MA1-06110) overnight in 4C. Secondary antibody and washes were done as above. Cells were then imaged on a Leica Sp8 Confocal microscope with a 40x water immersion objective. For a positive control for αSMA induction, BJ-5ta cells were treated with 5ng/mL TGFbeta for 48hrs before fixing and staining as described above on 24 well plate sized coverslips.