Hypoxia downregulates MHC class I expression <i>in vitro</i> in 3D but not in 2D culture systems and requires the deeper hypoxia achieved in the 3D system.

Abstract

<p><b>(A-C):</b> MCA205 tumor cells were cultured as 2D monolayers <b>(A)</b> or as 3D spheroids <b>(B,C)</b> and cultured under 21% O₂ or 1% O₂ for 48h. Levels of MHC class I expression was determined using flow cytometry. Representative histograms <b>(B)</b> and associated quantification and statistics <b>(C)</b> of 4 independent experiments are shown. The significance of differences was analyzed by the Student’s t-test (two-sided); p = 0.013 (C). Grey filled: Unstained control; Red: Hypoxia; Blue: Normoxia. MFI: mean fluorescence intensity. Inset: 40X magnification of MCA205 grown at 1% O₂ in 2D culture <b>(A)</b> or in 3D culture <b>(B)</b>. Error bars indicate SD. <b>(D)</b> Representative flow cytometry histograms of Hypoxyprobe-1 (HP) indicating significantly increased levels of hypoxia in MCA205 cells grown under 1% oxygen for 48h as 3D spheroids (Red histogram) as compared with 2D monolayers (Blue histogram). n = 4. <b>(E)</b> Contour plots representing intensity of hypoxia within MCA205 cultures grown as 2D monolayers show 98% of the population was intermediately hypoxic. <b>(F)</b> MCA205 cells grown as 3D spheroids show two distinct populations of intermediately hypoxic (56%; HP MFI = 309) and severely hypoxic (39.5%; HP MFI: 1412) regions. <b>(G, H)</b> Gating on the 2 distinct hypoxic populations in the spheroid revealed inverse correlation between MHC class I and hypoxia levels. Less hypoxic cells had significantly higher percentage of MHC class I positive cells <b>(G)</b> and more hypoxic regions had lower percentage of MHC class I positive cells <b>(H)</b>. <b>(D-H)</b> Representative data of 4 independent experiments.</p