Effect of p53 Status on Tumor Response to Antiangiogenic Therapy Joanne L. Yu, Janusz W. Rak, Brenda L. Coomber, Daniel J. Hicklin, and Robert S. Kerbel

Supplementary Material

Experimental details for Fig. 1 Treatment of p53^+/+ and p53^-/- tumors was initiated at an approximate volume of 100 mm³, which occurred on day 21 for p53^-/- tumors (control, 106.0 ± 34.8 mm³; treatment, 106.4 ± 30.1 mm³), and day 23 for p53^+/+ tumors (control, 106.7 ± 47.7 mm³; treatment, 107.4 ± 46.4 mm³). This slightly longer latency of 2-3 days was consistently observed with p53^+/+ tumors, although control p53^+/+ and p53^-/- tumors expanded at similar rates thereafter. Antiangiogenic ("metronomic") scheduling of vinblastine was as previously described, at 1/10th to 1/20th the maximum tolerated dose, administered twice weekly, to target the tumor vasculature (1).

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Supplemental Figure 1. Relationship between hypoxia and apoptosis in p53^+/+ and p53^-/- tumors. EF5 staining (A, D, G; Cy5, red), TUNEL staining (B, E, H; fluorescein, green) and dual staining (C, F, I) of the same tumor cryosections visualized by confocal microscopy. (A-C) p53^+/+ tumor, (D-F) p53^-/- tumor, (G-I) p53^-/- tumor section pre-treated with 20 U/ml DNase as a positive control for labeling of DNA fragmentation. Scale bar in (A) indicates 100 m for (A) through (I). Mice bearing p53^+/+ or p53^-/- tumors were injected intravenously with 0.25 ml of 10 mM EF5 prepared in 0.9% saline, 3 hours before tumor excision. 10 m tumor cryosections were cut, fixed in 4% paraformaldehyde, and apoptosis detected first using the TUNEL method according to manufacturer's instructions (Boehringer-Mannheim). After blocking overnight, slides were stained for 4 hours with the Cy5-conjugated ELK3-51 antibody to identify areas of tumor hypoxia (2). Dual-labeled images were visualized using a LSM 510 confocal microscope (Zeiss).

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Supplemental Figure 2. Disruption of p21 does not influence vascular dependence or response to anti-angiogenic therapy. (A) The relative percentage of p21^-/- cells is similar in perivascular (Hoechst-bright) and distal (Hoechst-dim) areas of mixed tumors. Tumors were established from 1:1 mixtures of p21^+/+ and p21^-/- cells and sorted based on Hoechst fluorescence as described. Cells displaying the 5% highest and lowest Hoechst fluorescence intensities were collected, genomic DNA extracted, and assayed for p21 status. The PCR assay involved amplification of p53 exon 6 as before (present in both p21^+/+ and p21^-/- genomic DNA), but rather than p53 exon 2, a region of the hygromycin resistance cassette used in targeted deletion of one of the p21 alleles in the p21^-/- cells was amplified (5'-GTG TCA CGT TGC AAG ACC TG-3' and 5'-AGC ATC AGC TCA TCG AGA GC-3'). Thus, the relative intensity of the hygromycin PCR product vs. the exon 6 product for each DNA sample would be proportional to the relative percentage of p21^-/- cells in each population of sorted cells. The average relative percentage of p21^-/- cells was 45.7 ± 4.5% in the proximal (Hoechst-bright) tumor cell samples, comparable to 42.9 ± 4.3% in cells collected from the distal, more hypoxic tumor regions. (B) Mice bearing tumors established by subcutaneous injection of 5x10⁶ parental HCT116 (p21^+/+) and p21^-/- cells (3) were treated with DC101/Vbl or saline, as described for the p53 experiment, starting at a tumor size of 100 mm³. Tumor volume (mm³) was estimated from caliper measurements using the standard formula: (length ( width²)/2. (C) Growth of p21^+/+ and p21^-/- tumors is inhibited with similar kinetics. After 14 days of therapy, the volume of treated p21^+/+ tumors decreased to 27.9 ± 3.2% that of untreated controls, comparable to the decrease in volume of treated p21^-/- tumors to 25.3 ± 3.4% that of controls. All error bars represent SD.

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References for supplemental material

1. G. Klement et al., J. Clin. Invest. 105, R15 (2000).

2. E.M. Lord, L. Harwell, C.J. Koch, Cancer Res. 53, 5721 (1993).

3. T. Waldman, K.W. Kinzler, B. Vogelstein, Cancer Res. 55, 5187 (1995).