MRes in Biosciences dissertation - supplementary moviesStudent ID: 23218258Glioblastoma Multiforme (GBM) is a highly aggressive and lethal form of brain cancer with no curative treatment, largely due to the limited understanding of its motility regulation. One type of motility, chemotaxis, is often presented in the field as a primary mechanism promoting GBM migration. However, it fails to address GBM migration in a complex physiological setting. Thus, it is imperative to investigate alternative model(s) that encompass non-chemotactic mechanisms in better reflecting GBM cell migration behaviours. This study aims to establish an in vitro system to understand how GBM cells interact with one another and their environment, orchestrating their rapid spread. Migration assays were performed under live cell microscopy imaging to observe migratory behaviour of GBM cells using (i) one and multiple cultured primary E2 GBM spheroid(s), (ii) different coating materials, and (iii) conditioned media from migration assay (CM-M) and cell culture (CM-C). A novel pump assay was also developed to determine the presence and role of diffusible molecule(s) influencing GBM cells’ migration under varying media flow rates and types. Complex self-generated chemotaxis mechanisms were identified as potential drivers of GBM cells’ motility. Contact inhibition of locomotion was observed as a dominant steering mechanism in some instances. The secretion of large quantities of extracellular vesicles (EVs) and formation of a self-generated pH gradient were additional drivers likely steering GBM migration. In the pump assay, flow rate did not significantly affect GBM cell motility; however, CM-M pumped in resulted in lamellipodia collapse of migratory cells, revealing possible secreted by-product(s) in CM-M involved in modulating GBM migration. In conclusion, this in vitro system suggests that GBM migration is governed by complex chemotactic models and multiple mechanisms simultaneously, reflecting the disease’s complexity. These insights could aid in developing more effective therapeutic strategies to impede GBM’s rapid invasion.Supplementary Movie 1 (SM1). Migration assay of a spheroid with non-migratory cells that have lamellipodia formed on dish coated using Laminin Coating Protocol for Cell Culture by Sigma Aldrich at 10X magnification under brightfield microscopy. Scale bar = 100μm.Supplementary Movie 2 (SM2). Migration assay of a spheroid on laminin coated dish using Laminin Coating Protocol for neurospheres by Sigma Aldrich at 10X magnification. Scale bar = 100μm.Supplementary Movie 3 (SM3). Manual tracking of randomly selected cels out from spheroid using ImageJ to compare difference in migratory speed migratory away from spheroid in migration assay based on SM2. Scale bar = 100μm.Supplementary Movie 4 (SM4). Laminin-based migration assay of multiple different sized spheroids at 10X magnification using brightfield microscopy channel. Scale bar = 100μm.Supplementary Movie 5 (SM5). Goat fibronectin-based migration assay at 10X magnification. Scale bar = 100μm.Supplementary Movie (SM6). Human fibronectin-based migration assay at 10X magnification. Scale bar = 100μm.Supplementary Movie 7 (SM7). Laminin-based migration assay using brightfield microscopy at 10X magnification without CO2 supply and HEPES buffer added. Scale bar = 100μm.Supplementary Movie 8 (SM8). Secretion of EVs observed under darkfield microscopy at 10X magnification of migration assay without CO2 supply and HEPES buffer added. Scale bar = 100μm.Supplementary Movie 9 (SM9). Secretion of EVs accumulated on one side of dish observed under darkfield microscopy at 5x magnification of migration assay without CO2 supply and HEPES buffer added. Scale bar = 100μm.Supplementary Movie 10 (SM10). Laminin-based migration assay with two spheroids exhibiting contact inhibition of locomotion when cells collide at 10X magnification. Scale bar = 100μm.Supplementary Movie 11 (SM11). Laminin-based migration assay using CM-M. Scale bar = 100μm.Supplementary Movie 12 (SM12). Laminin-based migration assay using CM-C. Scale bar = 100μm.Supplementary Movie 13 (SM13). Pump assay with regular fresh media pumped in on laminin coated dish. Rate used to pump media in was 3ml/h and rate media was withdrawn from the assay was 2.9ml/h. Pumps activated at approximately 2h and 5 min and stopped around 3h 15min. Scale bar = 100μm.Supplementary Movie 14 (SM14). Pump assay with laminin-based CM-M pumped in on laminin coated dish. Rate used to pump media in was 3ml/h and rate media was withdrawn from the assay was 2.9ml/h. Pumps activated at approximately 3h 30min and stopped around 4h 30min. Scale bar = 100μm.Supplementary Movie 15 (SM15). Pump assay with laminin-based CM-C pumped in on laminin coated dish. Rate used to pump media in was 3ml/h and rate media was withdrawn from the assay was 2.9ml/h. Pumps activated at approximately 2h 20min and stopped around 3h 50min. Scale bar = 100μm.Supplementary Movie 16 (SM16). Pump assay with (human) fibronectin regular media pumped in on human fibronectin coated dish. Rates used for media flow were 1.0ml/h pumped in and 0.9ml/h pumped out. Pumps activated at approximately 1h 50min and stopped around 2h 55min. Scale bar = 100μm. Supplementary Movie 17 (SM17). Pump assay with (goat) fibronectin regular media pumped in on goat fibronectin coated dish. Rates used for media flow were 0.7ml/h pumped in and 0.6ml/h pumped out. Pumps were activated at approximately 3h 20min and stopped around 5h 15min. Scale bar = 100μm.Supplementary Movie 18 (SM18). Pump assay with (goat) fibronectin CM-M pumped in on goat fibronectin coated dish. Rates used for media flow were 1.0ml/h pumped in and 0.9ml/h pumped out. Pumps were activated at approximately 2h 15 min and stopped around 3h 25min. Scale bar = 100μm.