Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET

Abstract

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[(18)F]fluoro-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC(50) values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 microM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.

Figure 1

Everolimus decreases FDG uptake by murine melanoma B16/BL6 metastases. Murine B16/BL6 cells were injected intradermally in the ears of C57BL/6 mice, and after 2 weeks, the lymph node metastases were studied by FDG-PET as described in the Materials and Methods section, immediately before and 2 days after treatment with everolimus (10 mg/kg p.o.). (A) Representative experiment showing horizontal head/thorax images of mice with lymph node (red arrow) and primary ear tumors (blue arrow) before and after treatment with everolimus. (B) Individual SUVs for tumors (n = 10 per treatment group) and the mean ± SEM fractional effect of treatment, where ***P < .001. (C) Mean ± SEM fractional changes for the effect of vehicle or everolimus on rTBVol, n = 19 per group, and BFI, n = 14 per group.

Figure 2

Everolimus decreases FDG uptake by human H596 lung tumor xenografts. Human tumors were created by s.c. implantation of viable H596 tumor tissue in Harlan athymic mice. (A) For efficacy, mice were treated with vehicle or with everolimus (10 mg/kg p.o.) for 21 days. Results show mean ± SEM. (B and C) A separate cohort of tumor-bearing mice was studied by FDG-PET as described in the Materials and Methods section, immediately before and 2 days after treatment with everolimus. Results show the individual SUVs for tumors (n = 10 per treatment group) and the mean ± SEM fractional effect of treatment, where *P < .05, **P <.01. (D) Histology and IHC of ablated tumors on day 2 after completion of the second PET scan.

Figure 3

Everolimus inhibits growth of insensitive human tumors but does not affect FDG uptake. Human HCT116 (colon) and KB31 (cervical) were created by s.c. injection of cells in Harlan athymic mice. In all cases, mice were treated daily p.o. with vehicle or everolimus (10 mg/kg). (A and B) Efficacy in HCT116 tumors for 1 week (A) or 2 weeks (B) showing the mean ± SEM with the associated T/C_TVol at the end point. (C and D) FDG-PET in HCT116 tumors in the respective experiment showing the mean fractional change for each treatment compared with day 0 (C) or the individual tumor SUV at two different time points (D). (E and F) Efficacy in KB31 tumors with the fractional change in FDG-PET compared with day 0 for each treatment, where *P < .05, ***P < .001.

Figure 4

Everolimus decreases FLT uptake by human H596 lung tumor xenografts. Human tumors were created by s.c. implantation of viable H596 tumor tissue in Harlan athymic mice. Tumors were studied by FLT-PET as described in the Materials and Methods section, immediately before and 2 days after treatment with everolimus (10 mg/kg p.o.). (A) Representative experiment showing horizontal whole-body images of mice with tumors (blue arrow) before and after treatment with everolimus; red arrow indicates the bladder. (B) Individual SUVs for tumors (n = 6 per treatment group), and the mean ± SEM fractional effect of treatment, where **P < .01. (C) Histology and IHC of ablated tumors on day 2 after completion of the second PET scan.

Figure 5

Everolimus does not affect FLT uptake by human HCT116 colon tumor xenografts. Human HCT116 tumors were created by s.c. injection of cells in Harlan athymic mice. (A and B) Tumors were studied by FLT-PET as described in the Materials and Methods section, immediately before (day 0) and on days 3 and 10 after treatment with vehicle or everolimus (10 mg/kg p.o.). Results show (n = 6 per treatment group) the individual SUVs for tumors (A) and the mean ± SEM fractional effect of treatment (B). (C) Histology and IHC of ablated tumors on day 10 after completion of the second PET scan.

Figure 6

Patupilone inhibits growth and FDG uptake by rat mammary BN472 tumors. Rat BN472 tumors were created by transplantation of viable tissue into the mammary fat pad and, after 2 weeks, were treated once with vehicle or patupilone (0.7 mg/kg i.v.). Results show the mean ± SEM for (A) efficacy (n = 12 per group) and (B) fractional change in FDG uptake compared with day 0 (n = 11 per group), where *P < .05, **P < .01, ***P < .001. (C) Pearson correlation between the fractional change in FDG uptake and the change in tumor volume (T_Vol) at the end point of day 6 compared with day 0.

Figure 7

Effects of NVP-AAL881 on rat mammary BN472 tumors. Rat BN472 tumors were created by transplantation of viable tissue into the mammary fat pad and, after 2 weeks, were treated twice daily with vehicle or NVP-AAL881 (15 mg/kg p.o.). Results show the mean ± SEM (n = 6 per group) for (A) efficacy and (B) fractional change in FDG uptake compared with day 0, where ***P < .001. (C) A separate cohort of treated rats showing the relative Sinerem concentration (compared with day 0) in each tumor determined on day 3 and day 7 after treatment initiation. Rats were injected i.v. with Sinerem (8 µl/kg) 24 hours before measurements as described in the Materials and Methods section.

Figure 8

Effects of PTK/ZK on rat mammary BN472 tumors. Rat BN472 tumors were created by transplantation of viable tissue into the mammary fat pad and after 2 weeks were treated daily with vehicle or PTK/ZK (200 mg/kg p.o.). Results show the mean ± SEM (n = 6 per group) for (A) efficacy and (B) fractional change in FDG uptake compared with day 0. On day 9, the PET tracer FMISO was injected i.v. (150 µl), and after 90 minutes, images were made of the tumor and muscle as described in the Materials and Methods section. (C) Results show the mean ± SEM ratio of tumor-muscle where hypoxic conditions are defined as a TMRR > 1.4. (D) Tumors were ablated, and IHC was performed for the percent of cells positive for caspase 3.