Tumor vascular changes mediated by inhibition of oncogenic signaling

Abstract

Many inhibitors of the epidermal growth factor receptor (EGFR)-RAS-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway are in clinical use or under development for cancer therapy. Here, we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3K, or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion, and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results, as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target, as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor but did respond with vascular alterations to RAS or PI3K inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3K inhibition with decreased tumor hypoxia, increased vascular flow, and morphologic alterations of their vessels, including increased vascular maturity and acquisition of pericyte markers. These changes are similar to the vascular normalization that has been described after the antiangiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.

Figure 1. Tumor hypoxia is reduced after signaling inhibition

Tumors in SCID mice were generated from the HRE-luc SQ20B and the HRE-luc HT1080 cells. When the tumors reached at least 100mm³ in volume, bioluminescent imaging was performed. At the indicated time of treatment with the indicated drugs, bioluminescent imaging was again performed. * indicates p<0.05 by two tailed t-tests compared to controls. a. Representative images from bioluminescent imaging at 10d (L-778,123 (40mg/kg) and Nelfinavir (20mg/kg)) and 14d (Iressa (50mg/kg) and PI-103 (5mg/kg)) to detect luciferase expression in animals bearing SQ20B-luc xenografts. b. Representative images from bioluminescent imaging at 10d treatment as in (a) to detect luciferase expression in animals bearing HT1080-luc xenografts. c. SQ20B xenograft tumor growth measured throughout the time of inhibitor treatment is unaffected by signaling inhibition (p=0.966, ANOVA). d. Immunohistochemistry confirms a reduction in EF5 binding in treated SQ20B tumors from (a).

Figure 2. Signaling inhibition increases tumor blood flow

Tumor blood flow determinations using Doppler ultrasound on xenograft tumors in SCID mice generated from the HRE-luc SQ20B and the HRE-luc HT1080 cells. a. Representative images from 3D power Doppler reconstruction of tumor vascular flow after 14 days treatment with Iressa, L-778,123, PI-103 or Nelfinavir treated as in Figure 1. b. Quantification of Doppler signal:volume ratio shows increases after signaling inhibition in longitudinal studies in tumors after 14 days (Control: p=0.684; Iressa: p=0.006; L778,123: p=0.050; PI-103: p=0.018; Nelfinavir: 0.046. P-values from two tailed t-tests compared to controls) c. Examples of raw contrast kinetics acquired in control (left) and treated (middle, nelfinavir) after bolus i.v. injection of microbubble contrast reagent. Quantification of microbubble velocity in multiple areas within SQ20B tumors (right) in multiple animals demonstrate a significant increase in influx within the tumor parenchyma, (all starred p values <0.001, ANOVA = 0.0002)

Figure 3. Treated tumors demonstrate normalized vascular morphology

SCID mice with SQ20B-GFP tumors were treated with either carrier (50% DMSO, 50% PBS), L778,123 (L, 40mg/kg), Iressa (I, 50mg/kg), Nelfinavir (N) (20mg/kg) or PI-103 (P, 5mg/kg) by daily i.p. injections. CD31-PE and Hoescht were injected iv at 10 min. and 1min. respectively prior to sacrifice. * indicates p<0.05 using two tailed t-tests compared to controls. a. Representative images from a single 0.5μm optical section of tumors showing tumor (green), vascular morphology (red) after indicated treatments. b. 100μm 3D reconstructions of vascular morphology (red) and extra-vascular diffusion (blue). c. Quantitation of perfused vessel density (CD31 staining) and extravascular diffusion (Hoechst staining). d. Calculation of uninterrupted vessel length and tortuosity from the computer generated tracings of vasculature based upon the 3D reconstructions of tumors (for examples see Supplementary Figure 5).

Figure 4. Vascular normalization persists during a 5-14 day timecourse

Xenograft tumors in SCID mice were generated from the SQ20B-GFP and treated with the indicated inhibitors as in Figure1. Each time-point represents a different cohort of mice. a. Top, left: Quantitation of CD31 vascular staining as assessed by confocal microscopy. Middle, left: Quantitation of Hoechst staining as assessed by multiphoton microscopy. Middle, right: Vascular flow determinations based on Doppler ultrasound imaging prior to sacrifice for confocal/multiphoton imaging. Bottom, Left: Uninterrupted tumor vessel length calculated as in Fig 3d for each treatment group at each of three timepoints. Bottom, Right: Tumor vessel tortuosity calculated as in Fig 3d at three timepoints during treatment.

Figure 5. Specific inhibition of PI-3K in a transgenic MMTV Neu model results in vascular normalization

Female mice bearing the MMTV-neu transgene were entered into the experiment when they had developed tumors of 150-200 mm³ in volume. After initial examination, the mice were treated with P-103 (5mg/kg) for 10 days. All p values are from two tailed t-tests compared to controls. * indicates p<0.05. a. Representative images of hypoxia (EF5) staining and 3D reconstruction of Doppler evaluation of breast carcinoma perfusion. Vascular perfusion is increased while hypoxia is reduced. Quantitation of power Doppler signal from images shows significant increase in vascular perfusion after PI-103 treatment. b. At timed intervals prior to sacrifice CD31-PE (10 min.) and dextran (1min.) were injected iv. Tumor vessel morphology was examined as in Figure 3. Quantitation shows an increased in perfused vessel density and an increase in dextran due to extravascular diffusion. Computer generated vascular tracings and further quantitation are shown in Supplemental Figure 6. c. Evaluation and quantitation of vascular maturity by immunohistochemical staining for smooth muscle actin (SMA) and PDGF-β.