Quantitative screening of the effects of hyper-osmotic stress on cancer cells cultured in 2- or 3-dimensional settings

Abstract

The maintenance of precise cell volume is critical for cell survival. Changes in extracellular osmolarity affect cell volume and may impact various cellular processes such as mitosis, mitochondrial functions, DNA repair as well as cell migration and proliferation. Much of what we know about the mechanisms of cell osmoregulation comes from in vitro two-dimensional (2D) assays that are less physiologically relevant than three-dimensional (3D) in vitro or in vivo settings. Here, we developed a microfluidic model to study the impact of hyper-osmotic stress on the migration, proliferation and ion channel/transporter expression changes of three metastatic cell lines (MDA-MB-231, A549, T24) in 2D versus 3D environments. We observed a global decrease in cell migration and proliferation upon hyper-osmotic stress treatment, with similar responses between 2D and 3D conditions. Specific ion channels/aquaporins are over-expressed in metastatic cells and play a central role during osmo-regulation. Therefore, the effects of hyper-osmotic stress on two transporters, aquaporin 5 (AQP5) and the transient receptor potential cation channel (TRPV4), was investigated. While hyper-osmotic stress had no major impact on the transporters of cells cultured in 2D, cells embedded in collagen gel (3D) decreased their AQP5 expression and exhibited a reduction in intra-cellular translocation of TRPV4. Furthermore, cell dispersion from T24 aggregates embedded in 3D collagen gel decreased with higher levels of hyper-osmotic stress. In conclusion, this study provides evidence on the impact of hyper-osmotic stress on various aspects of metastatic cell progression and highlights the importance of having a 3D cell culture platform in investigating molecular players involved in cancer cell migration.

Osmotic pressure modulates single cell cycle dynamics inducing reversible growth arrest and reactivation of human metastatic cells

“…Since p53 has previously been related to cell cycle delay or apoptosis 32 – 35 , the use of a mutant p53 cell line such as MDA-MB-231 could partially explain the reduced proliferation under hyperosmotic stress. However, new experiments performed with the wild-type p53 MCF7-FUCCI2 cell line, and data from the literature with the wild-type p53 T24 cell line 31 , also show a reduced proliferation under hyperosmotic stress. Nam et al 6 investigated cell cycle progression under 3D mechanical confinement using hydrogels, with an additional osmotic pressure effect, using static single cell EdU proliferation assays.…”

“…This is in agreement with previous observations with prostate cancer cells, where osmotic pressure was linked to a dormant state, but only when seeded at very low (clonogenic) cell density 25 . More recently, Miermont et al 31 also observed this drop in proliferation due to hyperosmotic stress in metastatic cell lines, including MDA-MB-231 breast cancer cells, using bulk cell analyses based only on immunofluorescence staining of proliferation markers. Since p53 has previously been related to cell cycle delay or apoptosis 32 – 35 , the use of a mutant p53 cell line such as MDA-MB-231 could partially explain the reduced proliferation under hyperosmotic stress.…”

“…Recently, a novel biomaterial-based osmotic pressure sensor was developed to possibly fill the gap and enable measurements of osmotic pressure 55 . Osmotic pressure values used in this study were based on those used in previous works 5 , 19 , 25 , 31 , and were below acute hyperosmotic stress values that lead to cell death 56 . It is known that gradients in osmotic pressure and hydrostatic pressure drive fluids out of arterioles and back into the venules 12 – 14 .…”

Tumorigenic epithelial clusters are less sensitive to moderate osmotic stresses due to low amounts of junctional E-cadherin

“…Taken together, these observations have led to the recognition of osmotic pressure as an important parameter in physiological and pathological situations and demonstrate a clear link among cell behavior, cancer and osmotic pressure. However, most previous work used levels of osmotic stress (Racz et al, 2007; Nielsen et al, 2008; Miermont et al, 2019; Bounedjah et al, 2012; Ignatova and Gierasch, 2006) far from the physiological and pathological ranges (Kodama and Mori, 2019; Miermont et al, 2013; Le et al, 2006; Havard et al, 2011; McGrail et al, 2015). Thus, the mechanism by which osmotic stress modulates epithelial cell migration and the precise impact it has on tumorigenic epithelial cells remains poorly understood.…”

Μελέτη Της Επίδρασης Του Ωσμωτικού Στρες Στα Μεσοθηλιακά Κύτταρα Του Υπεζωκότα

“…Στα μεσοθηλιακά κύτταρα έχει φανεί ότι η αναστολή ασβεστιοεξαρτώμενων διαύλων καλίου, που εντοπίζονται στο προπορευόμενο άκρο του μεταναστεύοντος κυττάρου (70) προκαλεί μείωση του ρυθμού μετανάστευσης(116). Σε ότι αφορά τη κυτταρική σειρά σαρκωματοειδούς μεσοθηλιώματος ZL-34 η μείωση της κυτταρικής μετανάστευσης υπό την επίδραση ωσμωτικού στρες μπορεί να εξηγηθεί από το μοντέλο της ωσμωτικής ροής νερού δεδομένου ότι κάτω από τις ίδιες συνθήκες η διαπερατότητα των ZL-34 μειώθηκε(115).Μετά την έκθεση σε υποωσμωτικό στρες ο ρυθμός κυτταρικής μετανάστευσης της μεσοθηλιακής κυτταρικής σειράς MeT-5A και της κυτταρικής σειράς σαρκωματοειδούς μεσοθηλιώματος ZL-34 μειώθηκε, ενώ της κυτταρικής σειράς επιθηλιοειδούς μεσοθηλιώματος παρέμεινε αμετάβλητη. Η επίδραση του υποωσμωτικού στρες έχει ελάχιστα μελετηθεί στα μεσοθηλιακά κύτταρα.…”