Longitudinal imaging studies of tumor microenvironment in mice treated with the mTOR inhibitor rapamycin

Abstract

Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3)) and measurements of tumor pO(2) and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2) levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2)<10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

Figure 1. Effect of rapamycin on SCC tumor growth and mTOR signaling pathway.

(A) Tumor sizes of the SCC tumors in the mice leg treated with vehicle (control, •), 5 mg/kg bw/day (▴), and 10 mg/kg bw/day (▪) rapamycin. (B) Western blot analysis of S6 protein expression and the abundance of its phosphorylated form in SCC tumor cells treated with rapamycin (100 nM). (C, D) Immunostaining of pS6 in SCC xenograft of control and rapamycin treated (10 mg/kg bw/day, 2 days).

Figure 2. Anatomy, pO₂, and blood volume images of SCC tumor.

T₂-weighted anatomical image (top) of a SCC tumor-bearing mouse, and the corresponding pO₂ maps (middle) and blood volume images (bottom) measured by EPRI and MRI. The adjacent center six slices of the 3D images were displayed, and the every slice has 2 mm thickness.

Figure 3. Effect of rapamycin treatments on tumor pO₂ and blood volume.

Anatomy, pO₂, and blood volume images of SCC tumor in mice leg treated with vehicle (A) and 10 mg/kg bw/day rapamycin (B). The center slice of each 3D image is displayed. Treatment was initiated 8 days after tumor implantation (Day 0). The images of day 0 were obtained before beginning of the treatments. (C, D) Frequency histograms of pO₂ and blood volume in the SCC tumors of (A) and (B). The values indicate median pO₂ and blood volume in the tumor region.

Figure 4. Changes in tumor oxygenation and blood volume.

Median pO₂ values (A), percentage of hypoxic fraction (B), and mean blood volume (C) in the control and rapamycin treated SCC tumors. The values are average of 5 or 6 mice and error bars represent standard deviations. Oxygen delivery per unit blood volume (D) was calculated by dividing tumor pO₂ by blood volume. * p<0.05 as compared with control, ** p<0.01 as compared with control, † p<0.05 as compared with rapamycin day 8, †† p<0.005 as compared with rapamycin day 8.

Figure 5. Immunohistochemical analysis of CD31 and αSMA in SCC xenograft.

(A) Representative images of control and rapamycin treated SCC xenograft. Green is CD31, red is αSMA, and blue is DAPI. (B) Percentage of CD31 and αSMA in the SCC tumor of control (n = 3) and rapamycin treated (n = 4, 10 mg/kg bw/day, 2 days) mice.

Figure 6. Effect of combination of rapamycin and X-irradiation on tumor growth.

Growth kinetics of SCC tumors implanted in mice leg, treated with vehicle (control, •), X-irradiation (5 Gy/day, 3 days, □), rapamycin (10 mg/kg/day, 5 days, ▴), and rapamycin and X-irradiation (◊).