Metronomic  chemotherapy  offsets  HIFα  induction  upon maximum-tolerated dose in metastatic cancers (2020)
Figure 2

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 (LDMCTX + LDMCPB vs vehicle).

B: HIF-1α levels in HT29 liver metastatic nodules.

Left: vehicle- treated controls.

Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB).

Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB).

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 LDMCTX + MTDCPB vs vehicle or P= 0.0123 LDMCTX + LDMCPB vs vehicle; #, P= 0.0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli 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 50th (red line), 25th and 75th 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.