Fig. 4. HIF1α regulates metastasis via promotion of hybrid EMT state and metabolic plasticity.

a Athymic female mice (n = 10) bearing PyMT1/GPx2 KD tumours were treated post onset with daily i.p injection of vehicle (DMSO) or 10 μg/kg of echinomycin/DMSO for 21 days starting at 64 mm³ tumour volume. Representative tumours from both groups are shown (left boxes). Mice then underwent survival surgery to remove primary tumour and were left untreated for 1 week to recover. Treatment was then resumed daily for one more week as above. Four weeks later, mouse lungs were removed (second boxes), sectioned, H&E-stained and scanned; scans of whole lung lobes (third boxes) from both groups are shown (third boxes). b The number of lung foci from H&E stained sections are shown; Mean ± SEM; ****p < 0.0001. c GPx2 KD tumours from 3 independent mice that were treated with vehicle or echinomycin were immunoblotted for SLUG, VIM, pAMPK, GLUT1 vs ACTIN. d, e KRT8/KRT14 (d) or VIM and E-CAD (e) double-immunostained PyMT1/GPx2KD tumour sections treated with either or echinomycin from 3 mice each are shown. f Co-staining for four markers consisting of KRT8 (yellow), KRT14 (red), GLUT1 (blue), and pAMPK (green) shows individual and overlay staining. Robust quadruple staining was observed in GPx2KD tumours especially in Area 1 and also in part of Area 3, but not in Area 2 which was only KRT8 + /pAMPK+ (right panels). By contrast, control PyMT1 tumours were strongly KRT8 + /pAMPK+ (left panels). Staining was done in 3 mice (2 tumours each) in 3 sections from each tumour. g, h Bar graphs display baseline OCR and ECAR for GPx2 KD tumour cells treated with 5 nM echinomycin vs DMSO; normalised results are shown as Mean ± SEM; *p < 0.05, ****p < 0.0001.