Microsphere cytometry to interrogate microenvironment-dependent cell signaling

Abstract

2018Avhandling for graden philosophiae doctor (ph.d.)Dato for disputas: 06.04.18Trykk:Skipnes Kommunikasjon / Universitetet i Bergen Materialet i denne publikasjonen er omfattet av åndsverkslovens bestemmelser. Tittel:The effects of ageing on microenvironment-contextual epithelial cell signalling Rein Aasland, for valuable insight on academic writing, and for pointing out, as did James, when my words became self-indulgent fun and flowers. Mark LaBarge, for taking me into his lab, and letting me partake in that which concerns absolutely everyone: ageing. To Martha Stampfer for allowing me to poke around in her human cell resources biobank. To James Garbe for being an incredibly sympathetic guy with the most convenient knowledge up his sleeve.I would like to thank my mother Nina-Mamma for never curbing her enthusiasm for tiny biology when I brought home science homework, and my father Ulf for being genuinely impressed with pretty much everything I didincluding when I did it badly. I need to thank my boyfriend, Lars, for meticulous proofreading of the manuscript, and for continuously encouraging me to put my academic whims into writing. I want to point out that Iren Abrahamsen did substantial preliminary work for the microsphere cytometry method. I need to to thank my medical doctor office mates, Gry and Kjersti for valuable insights from the clinical realm where lab rats never go, to remind me who we're doing this for in the first place. Thanks to Sissel for being that steady rock in the corner stall of the lab where PhDs and postdocs fly by. I would like to thank the girls in the lab for taking up the fight against cancer cell Fråtse-Frida, and winning it. Finally, I would like to thank the Cancer Society, VilVite, CCBIO and Forskningsdagene for giving me trust, whether it was to develop flow cytometry protocols or popular science shows, or to interrogate the microenvironment I would like thank just anyone else in the lab and the general world who is interested and willing to contemplate big ideas such as the purpose of being. And at last, every single nerd: nerdy boys and nerdy girls. pathway activation levels in HMEC are attenuated with age, and that the diminished signaling magnitude in HMEC from ageing women correlated with reduced probability of activating oncogene-induced senescence.Our results suggest that attenuated cell signaling response may reduce the likelihood of activating oncogene induced senescence, for cells in ageing women. We hypothesize this is the result of age-related changes to the microenvironment that support age-emergent cellular phenotypes with increased cancer susceptibility.

Microenvironment-contextual cell signaling is attenuated with age

“…In order to measure ECM-mediated cell signaling responses in adherent HMEC, we applied a novel microsphere cytometry technique 13 . Microspheres were coated with E cadherin to mimic cell-cell contact 14 and extracellular matrix proteins (ECM) (collagen 1 (COL1), fibronectin (FN) or laminin (LAM)) to mimic the extracellular matrix ( Fig.…”

“…VARIAN PL-Microspheres SuperCarbonyl White poly(styrene-co-methancrylic acid) 20 μm in diameter (Batch CD185, Agilent Technologies) were coated as described 13 . E-Cadherin (1 µg mL -1 , Sino-Biological Inc. #10204-H08H) was added to the coating suspension..…”

“…Finite life span mammary epithelial cells were obtained from women having undergone reduction mammoplasty, and generously provided from the HMEC biobank 26 . Cells were cultured in M87A complete medium as described 13…”

“…Cultured cells were washed twice with PBS and treated with low concentration trypsin (0.05% trypsin, 0.02% EDTA in PBS). Microsphere adhesion was performed as described 13 . Cell -microsphere incubation time varied between 2-6 hours depending on cell type and choice of ECM coating.…”

“…Pre-treatment (2hr) with MAPK inhibitor PD98059 (20 μM, Cell Signaling #9900) and PI3K inhibitor wortmannin (0.5 μM, Sigma-Aldrich #W1628) was used to set a gate defining the positive population of cells with active ERK or active AKT respectively ( Fig.1 and 4). Cell-microsphere complexes were fixed and permeabilized as described 13 .…”