Supplementary Figure 2: iNANIVID microenvironments and hypoxia responses in vitro and in vivo. | Nature Cell Biology

Supplementary Figure 2: iNANIVID microenvironments and hypoxia responses in vitro and in vivo.

From: Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments

Supplementary Figure 2

(A) Representative intra-vital images of mCherry and GFP signal of MDA-231 HIF reporter cells grown on the CAM for 3 days in indicated distances of PBS- or Hi-NANIVIDs. Scale bar 10 μm; n = 3 tumors. (B) Fluorescence was measured: D = distal, M = middle distance, P = proximal to NANIVID. (C) Quantification of overall (all distances) mCherry signal in PBS- and Hi-NANIVID influenced MDA-231 HIF tumors. Bars show mean + SEM of n = 3 tumors per group, >180 cell cluster; one tailed Student’s t test. (D) Red mCherry signal at indicated distances after 3 days in vivo. D = distal, M = middle distance, P = proximal to iNANIVID (see Supplemental Figure 2B). Bars show mean + SD of n = 3 tumors, >60 cell clusters each distance. (E) T-HEp3 tumors in CAMs implanted with DFOM or PBS iNANAVIDs harvested at day 3 (D3) and day 6 (D6) and stained for HIF1α; scale bar 10 μm. Graph: average HIF1α fluorescence intensity (red) was quantified using MetaMorph. Each dot one cell. Bars show mean + SD; n = 400 cells, 2 tumors per treatment; Mann-Whitney test. (F) Representative images of IHC staining for DEC2, NR2F1 and Glut1 in 6 days PBS- or Hi-NANIVID treated T-HEp3 CAM tumors. White arrows: negative, black arrows positive cells; n = 2. (G) Fold change of expression in positive cells from PBS- to Hi-NANIVID treated CAM tumors; bars indicate mean + SEM. See Fig 2E + F for 3 day treated tumors. Each point one HPF. Scale bar 10 μm, n = 11 HPF of 2 tumors; Mann-Whitney test. (H) T-HEp3 tumors 6 days on Hi-iNANAVID CAMs were harvested and cultured for 24 hours with either PBS (n = 4) or 90 μM DFOM (n = 3), 120 cells per condition. Cells were stained for GLUT1. Representative images shown, scale bar 50 μm and 10 μm. Average fluorescence intensity per cell (green, GLUT1) was quantified using MetaMorph. Each dot one cell. Bars show mean + SD. Note re-induced GLUT1 expression in cells that had downregulated the response to DFOM in vivo (see Fig S2F + G). Mann-Whitney test. (I) Representative images of IF staining for phospho-Rb (pRb) and cleaved Caspase3 (Cl-C3) of 3 days PBS- or Hi-NANIVID treated T-HEp3 CAM tumors. Scale bar 50 μm; n = 3 tumors. For quantification see Fig 2G + H.

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