Figure 2. LDM chemotherapy selectively offsets HIF‐1α levels in colon cancer metastases to the liver.

• A
  Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size. Left: Histogram of pooled cross‐sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. A decreasing linear trend for median metastatic diameter was observed (left to right, slope = −143.2, P = 0.043 by post‐test for trend). Right: Dichotomized metastatic size at median diameter of vehicle‐treated tumors. Liver nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ² (df = 5) = 6.80; P = 0.2361 (not significant) for overall effects on size; χ² (df = 1) = 4.71; P = 0.0299 (LDM^CTX + LDM^CPB versus vehicle).
• B
  HIF‐1α levels in HT29 liver metastatic nodules. Left: Vehicle‐treated controls. Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDM^CTX + LDM^CPB). Middle/right: LDM cyclophosphamide + MTD capecitabine (LDM^CTX + MTD^CPB). Right: Automatic quantification of the effect of monotherapies or doublet regimens on HIF‐1α⁺ areas in individual metastatic nodules. F _2,13 = 4.796 and P = 0.028 for overall treatment by Brown–Forsythe ANOVA; *P = 0.038 LDM^CTX + MTD^CPB versus vehicle or P = 0.0123 LDM^CTX + LDM^CPB versus vehicle; ^# P = 0.0098 LDM^CTX + LDM^CPB versus LDM^CTX + MTD^CPB by Benjamini, Krieger, and Yekutieli post hoc test.
• C
  Correlation between metastatic and peri‐metastatic HIF‐1α levels in the liver. Left: Nodule immunostaining and parenchymal HIF‐1α expression. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F _1,57 = 150.7 and P < 0.0001; slope ≠ 0 by F‐test. Right: Correlation between intra‐metastatic and peri‐metastatic parenchymatous ring in the liver. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F _1,54 = 55.2 and P < 0.0001; slope ≠ 0 by F‐test.
• D
  Correlation between metastatic and peri‐metastatic HIF‐2α levels in the liver. Left: Nodule immunostaining and parenchymal HIF‐2α expression. Right: Correlation between intra‐metastatic and peri‐metastatic parenchymatous ring in the liver. Each point represents median values for each nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F _1,54 = 55.2 and P < 0.0001; slope ≠ 0 by F‐test. n, number of nodules.

Data information: Violin plots present 50^th (red line), 25^th, and 75^th percentiles (blue line); numbers in brackets indicate number of nodules (n) or animals (mice). L, low‐dose metronomic; M, maximum‐tolerated dose; ns, not significant; Met, metastasis; r, correlation coefficient. Dashed green lines encircle the histological limit between metastatic nodules and their surrounding normal liver parenchyma. Insets show high‐magnification images of regions marked with asterisks (*). Blue frames in C and D indicate consecutive sections from the same liver metastasis, stained for HIF‐1α or HIF‐2α, respectively.