Figure 1.
Glucose consumption and lactate production increase under hypoxic conditions.
A and B, Cal33 and OSC19 cells consume more glucose after 48 hours of hypoxic culturing than cells grown in normoxic conditions. C and D, a concomitant rise in lactate production was detected in media from Cal33 and OSC19 cells grown in hypoxic conditions for 48 hours. OSC19 cells are more glycolytic than Cal33 cells. Each dot represents one of four replicate dishes used for each time point and culturing condition. The experiment was performed twice with two replicate dishes. P values indicate a significant difference in metabolite concentrations and were calculated using a two-way ANOVA.
Figure 2.
Cal33 exposure to hypoxia leads to increased expression of HIF-1α, LDH-M, and PDHK1.
Western blot analysis of changes in HIF-1α, LDH-M, and PDHK1 expression in whole cell lysate from Cal33 cultures grown in normoxic (21%), hypoxic (2%), or hypoxic (2%) followed by normoxic (21%) conditions. Cells cultured in 21% oxygen for 16 hours serve as the control for basal protein expression levels. Densitometry values were calculated using ImageJ. Images are representative of duplicate blots.
Figure 3.
Cal33 cells demonstrate greater metabolic flexibility in response to hypoxia than OSC19 cells.
After 16 or 48 hours in 2% (hypoxia) or 21% oxygen (normoxia), 4×104 Cal33 or OSC19 cells were plated in a Seahorse XF24 Extracellular Flux Analyzer plate. The Seahorse Analyzer records rates of OXPHOS & glycolysis via the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of each sample culture. A, basal OCR recorded at the assay start after 16 hour incubation. B, ATP-linked OCR is the difference between the basal OCR and the OCR after the addition of oligomycin to sample cultures (see Figure S1). C, Basal ECAR recorded at the assay start. Three experiments were performed at each time point, with five samples per condition. Boxes represent the interquartile range, horizontal lines indicate the median, the T-bars indicate the range, and individual points are outliers. P values were determined by two-way ANOVA. N = normoxia (21% oxygen), H = hypoxia (2% oxygen). D, Basal OCR recorded at assay start after 48 hour incubation. E, ATP-linked OCR recorded for 48 hour cultures. F, Basal ECAR recorded after 48 hour incubation.
Figure 4.
2-DG uptake in Cal33 xenografts is heterogeneous and is associated with HypoxiSense accumulation.
A, Nude mouse with an ulcerated Cal33 xenograft tumor. B, 3D reconstructions of FMT scans capturing HypoxiSense, AngioSense, and IR800-2-DG signal within the xenograft tumor as shown in A. A manually placed ROI measures the tumor volume. C, Anatomic tumor measurement was calculated from MRI slices. Sagital, axial, and coronal MRI slices of the mouse shown in A and B, with 3D tumor reconstruction. D, two dimensional reconstructions of FMT scans at 2 mm depth within the tumor. E, box plots comparing IR800-2-DG concentration in whole tumors and HypoxiSense-concentrated regions within them (n = 8). Boxes represent the interquartile range, the horizontal line indicates the median, the T-bars indicate the range, and individual points are outliers (p, student t-test). F, Tumors were grouped by the presence or absence of HypoxiSense signal within the tumor (n = 9 for HypoxiSense positive, n = 7 for HypoxiSense negative). The coefficient of variance (CV) for IR800-22-DG concentration within the tumor was calculated (p value was generated using a student's t-test). Each point represents a tumor. Bars indicate the average CV value for the HypoxiSense positive or negative group.
Figure 5.
Heterogeneous staining pattern of HIF-1α, LDH-M, and CAIX expression in xenograft tumors reflects presence of hypoxia.
Representative images of HypoxiSense Cal33 tumor sections were probed for HIF-1α, LDH-M, and CAIX expression by immunohistochemistry (IHC) as described in materials and methods. Gray boxes indicate the region of the tumor shown at 10× magnification. Slides were scanned at 20× on Aperio Imagescope software. Images of whole tumor sections were captured at 20×. Images of regions within tumor slices were captured at 200×. Additional inset of CAIX shows cell-surface staining of this protein in peri-necrotic areas.
Table 1.
HIF-1α, ATP5β and CAIX expression have greater heterogeneity in HypoxiSense-positive tumors as compared to HypoxiSense-negative tumors.
Figure 6.
18F-FDG uptake in human HNSCC tumors is heterogeneous.
A, Non-contrast head CT of patient 2. B, 18F-FDG signal on PET scan. C, Fused PET-CT image with scaled color bar (bq/mL). Yellow ROI represents ROI-E, established by PETEdge. Red ROI represent ROI-C, established by contracting ROI-E by 0.5 cm in all directions. D, Histograms of 18F-FDG signal in ROI-E or ROI-C intensity in each tumor. Binning was set at intervals of 103 mbq/mL. Mean, maximum, minimum, and standard deviation of 18F-FDG signal within each tumor is shown in Table 2. E, Dot plot comparing tumor volume and coefficient of variance (CV) values established by ROI-C for each tumor. The numbers identify the same tumors numbered to the left of the histograms. Tumor volumes and CVs also listed in Table 2.
Table 2.
Tumor characteristics and 18F-FDG-PET values recorded from ROI-C using MIM software.