Article Figures & Data
Figures
- Figure 1.
Analysis of CAIX expression and activity. A, CAIX expression in HCT116 clones by immunoblot (top) and fluorescence-activated cell-sorting (FACS) analysis (bottom). B, CA9 and CA12 expression in HT29 in normoxic and 0.1% O2 (top). ***, P < 0.001; *, P < 0.05, n = 3, hypoxia. CAIX and CAXII expression in normoxic and 0.1% O2, 72 hours (bottom). C, CA9 expression in U87 shCTL and shCA9 pools in normoxia and 0.1% O2 (hypoxia; 72 hours) measured by (C) quantitative PCR and (D) immunoblot. E, immunoblot of cytoplasmic (C) and membrane (M) fraction of HCT116 clones. F, CO2 hydration rate constant of HCT116 clones at a range of pH ± acetazolomide (ATZ). Immunoblots are representative of 3 blots. Error bars represent SD. FITC, fluorescein isothiocyanate.
- Figure 2.
CAIX increases growth rate in spheroids and xenografts. A, spheroid growth curves of HCT116 (***, P < 0.001, n = 3). B, xenograft growth curves of HCT116 (***, P < 0.001, n = 5). C, spheroid growth curves of HT29 (*, P < 0.05, n = 3). D, xenograft growth curves of HT29 (*, P < 0.05, n = 5). Error bars represent SD.
- Figure 3.
CAIX expression is associated with increased necrosis and apoptosis in spheroid culture. A, representative immunohistochemical images and bar chart of scoring in HCT116 FLCA9 and empty vector (EV) spheroids. CAIX expression, percentage of necrosis, percentage of pimonidazole (PIMO), and caspase-3 staining were significantly different between empty vector and FLCA9 spheroids. B, representative immunohistochemical images and bar charts of scoring of HT29 empty vector and shCA9 spheroids. CAIX expression, percentage of necrosis, percentage of pimonidazole, and cleaved caspase-3 staining were significantly different between empty vector and shCA9. CAIX, pimonidazole, caspase-3, and Ki-67 expression staining are shown in dark brown. Hematoxylin and eosin (H&E), dark pink denotes viable tissue, light pink denotes necrosis. Error bars represent SD. N denotes areas of necrosis. Arrows point to positive staining (***, P < 0.001; **, P < 0.01; *, P < 0.05, n = 5).
- Figure 4.
CAIX expression is associated with increased necrosis in HCT116 xenografts and increased necrosis, apoptosis, and proliferation in HT29 xenografts. A, representative immunohistochemical images of HCT116 empty vector (EV) and FLCA9 xenografts and bar chart of scoring. CAIX expression and proportion of necrosis were significantly different between empty vector and FLCA9. B, representative immunohistochemical images of HT29 empty vector and shCA9 xenografts and bar chart of scoring. CAIX expression, proportion of necrosis, cleaved caspase-3, and Ki-67 expression were significantly different between empty vector and shCA9. CAIX, HIF-1, cleaved caspase-3, Ki-67, and CD34 expression staining is shown in dark brown. H&E, dark pink denotes viable tissue, the light pink stain denotes necrosis. Error bars represent SD. N denotes areas of necrosis. Arrows point to positive staining. Scale bars represent 200 μm. Ki-67 images have a top right × 20 inset image with scale bars representing 100 μm. (***, P < 0.001; *, P < 0.05, n = 5).
- Figure 5.
CAIX knockdown in HT29 reduces Ki-67 positivity in the hypoxic fraction of spheroids and increases CAXII expression in 3D culture and in vivo. A, examples of HT29 empty vector (EV) and shCA9 spheroids stained for Ki-67. Red lines denote the area of central hypoxia. A graph of the percentage of Ki-67 positivity inside the hypoxic core of the HT29 empty vector and shCA9 spheroids. B, analysis of CA9 and CA12 RNA expression in HT29 empty vector and shCA9 spheroids. C, analysis of CA9 and CA12 RNA expression in HT29 empty vector and shCA9 xenografts. D, analysis of CAXII expression by immunohistochemistry with representative images of empty vector and shCA9 spheroids (top) and a bar chart of the scoring (bottom). E, analysis of CAXII expression by immunohistochemistry with representative images of empty vector and shCA9 xenografts (top) and a bar chart of the scoring (bottom). CAXII expression staining is shown in dark brown. N denotes areas of necrosis. Arrows point to positive staining. Scale bars represent 100 μm. (***, P < 0.001; *, P < 0.05, n = 5).
- Figure 6.
CAIX knockdown enhances bevacizumab treatment in xenografts. A, xenograft growth curves of HT29 clones ± bevacizumab treatment. B, xenograft growth curves of HT29 wt ± acetazolamide (ATZ) ± bevacizumab treatment. C, xenograft growth curves of U87 doxycycline (Dox)-inducible shCTL ± Dox and shCA9 − Dox xenografts. D, xenograft growth curves of U87 doxycycline (Dox)-inducible shCA9 ± Dox ± bevacizumab. Arrows denote the start of bevacizumab treatment and/or acetazolamide treatment at 150 mm3 xenograft volume. (***, P < 0.001; **, P < 0.01; *, P < 0.05, n = 5).
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legends 1-4 - (72 KB)
- Supplementary Figure 1 - (1543 KB) - Analysis of the viable tumour volume at the first endpoint of the HCT116 and HT29 xenograft and spheroid experiments revealed that the CAIX expressors had increased viable tissue in HCT116 (A) xenografts (40% more viable tissue at day 14, ***p<0.01, n=10) and (B) spheroids (16% more viable tissue at day 13, **p<0.01, n=5) and HT29 (C) xenografts (34% more viable tissue at day 20, p<0.05, n=5) and (D) spheroids (45% more viable tissue at day 16, *p<0.01, n=5).
- Supplementary Figure 2 - (1056 KB) - Analysis of CA9 RNA expression in U87 xenografts untreated and with chronic Bevacizumab treatment. Bevacizumab treatement increased CA9 expression (***p<0.001, n=5).
- Supplementary Figure 3 - (244 KB) - Bar chart of scoring of immunohistochemistry and representative immunohistochemistry images of HT29 shCA9 and EV xenografts with and without Bevacizumab treatment. CAIX expression (**p<0.01 ***p<0.001, n=5), proportion of necrosis (*p<0.05, n=5) and cleaved caspase 3 expression (*p<0.05, n=5) were significantly different between EV and shCA9. CAIX expression (EV ***p<0.001, n=5), HIF1 (shCA9 *p<0.05, n=5, EV**p<0.01, n=5), cleaved caspase 3 expression (shCA9 **p<0.01, n=5, EV *p<0.05, n=5) and CD34 (shCA9 *p<0.05, n=5, EV *p<0.05, n=5) expression were significantly different between Bevacizumab treated and untreated xenografts. CAIX, HIF1, cleaved caspase 3, KI67 and CD34 expression staining is dark brown. H&E, dark pink denotes viable tissue, light pink denotes necrosis. N denotes areas of necrosis. Arrows point to positive staining. Scale bars represent 200�m on 5x images and 100�m on 20x images.
- Supplementary Figure 4 - (8129 KB) - Bar chart of scoring of immunohistochemistry and representative immunohistochemistry images of HT29 xenografts with and without acetazolomide and/or Bevacizumab treatment. CAIX expression (vehicle (DMSO) *p<0.05, n=5; ATZ **p<0.01, n=5) and CD31 (vehicle (DMSO) *p<0.05, n=5; ATZ **p<0.01, n=5) were significantly different between PBS and Bevacizumab treated xenografts. The amount of necrosis was significantly different between vehicle and ATZ treated xenografts (***P<0.001, n=5). CAIX, and CD31 expression staining is dark brown. H&E, dark pink denotes viable tissue, light pink denotes necrosis. N denotes areas of necrosis. Arrows point to positive staining. Scale bars represent 200�m on 5x images and 100�m on 20x images.
Volume 18, Issue 11
