Effectiveness of combined modality radiotherapy of orthotopic human squamous cell carcinomas in Nu/Nu mice using cetuximab, tirapazamine and MnSOD-plasmid liposome gene therapy

Abstract

Hypoxic regions limit the radiocontrollability of head and neck carcinomas. Whether or not combinations of plasmid/liposome mediated overexpression of normal tissue protective manganese superoxide dismutase (MnSOD), cetuximab (C225), and the hypoxic cytotoxin tirapazamine (TPZ) enhanced radiotherapeutic effects was tested in a CAL-33 orthotopic mouse cheek tumor model. The tumor volume continued to increase in the control (untreated) mice, with a ninefold increase by 10 days when the tumors exceeded 2 cm(3). The mice receiving 14 Gy only showed reduced tumor growth to 3.1+/-0.1 fold at day 10. The mice receiving MnSOD-PL, C225, TPZ plus 14 Gy had the best outcome with 0.7+/-0.1 fold increase in tumor volume by 10 days (p=0.015) compared to irradiation only. The addition of MnSOD-PL, TPZ, and C225 to irradiation optimized the therapeutic ratio for the local control of hypoxic region-containing CAL-33 orthotopic tumors.

Figure 1

Effect of MnSOD-PL transfection on radiosensitivity of CAL-33 cells. MnSOD-overexpression by MnSOD-PL transfection increases radiosensitivity by CAL-33 cells in vitro. D₀ for CAL-33 1.75±0.02 Gy, D₀ for CAL-33-MnSOD=1.17±0.08 Gy; difference is significant at p=0.0018.

Figure 2

Nitric oxide (NO) production measured spectrophotometrically in human CAL-33 cell pellets following irradiation.

Figure 3

Effect of TPZ on radiation killing of CAL-33 cells in vitro under normoxic (A) and hypoxic conditions (B). CAL-33 cells or MnSOD-PL transfected CAL-33 cells were either (A) incubated in TPZ 10 μM for 1 hour and irradiation survival curves performed under normal oxygen conditions, or B) held for 18 hours at 3% oxygen prior to TPZ treatment and irradiation.

Figure 4

Orthotopic human CAL-33 tumor in the cheek of a nu/nu mouse. Microscopic histopathology of tumor at day 10 after injection of 1×10⁶ cells into the cheek (hematoxylin and eosin). Open arrow shows tumor. Closed arrow identified blood vessel, infiltration with tumor cells. A=×100, B=enlargement of A ×300.

Figure 5

PET Imaging of CAL-33 tumors in nu/nu mice ([¹⁸F]-FDG vs. [¹⁸F]-FMISO). Representative summed microPET images taken 40–90 min post-injection of radiotracer, with arrows indicating the location of the tumor. Representative time-activity curves of tumor uptake in mice bearing tumors following injection of [¹⁸F]-FDG and [¹⁸F]-FMISO. Data are expressed in terms of standardized uptake values (SUV).

Figure 6

Micro-PET scan showing the effect of MnSOD plus 18 Gy on CAL-33 orthotopic tumors in the cheek of athymic nude mice. Ten days after injection of 1×10⁶ CAL-33 cells into each cheek, the mice were injected with MnSOD-PL 100 μg/100 μl (mouse on left) or with blank-PL (mouse on right) then irradiated with 18 Gy to the head and neck. The next day mice received i.v. FMISO (200 uCi) (A) and were scanned. Twnty-four hours later the mice were reimaged after FDG injection (B). Mouse on right in A displays a big hypoxic tumor as identified by the uptake of FMISO (yellow area – arrow); in the corresponding FDG image (B) the tumor shows reduced FDG uptake in the same region suggesting an area of necrosis or hypoxia. Mouse (left) that received MnSOD-PL shows reduced hypoxic area.

Figure 7

Micro-PET scans showing the effect of TPZ and MnSOD-PL, plus C225, on CAL-33 orthotopic tumors in cheek of 14 Gy-irradiated nude mice. Mice were injected with FMISO before scanning. Mouse 1 is a nonirradiated control, mouse 2 was treated with TPZ (60 mg/kg) 10 min before 14 Gy, and mouse 3 with MnSOD-PL intraorally, C225, and TPZ before irradiation.

Figure 8

Increased radiocontrollability in orthotopic oral cavity CAL-33 squamous cell carcinoma tumor-bearing nu/nu mice by tirapazamine (TPZ), cetuximab (C225), and MnSOD-PL administration. Groups of ten adult nu/nu female mice were injected in the cheek pouch with 1×10⁶ CAL-33 cells. The treatments were administered at 10 days (0.3 cm diameter) and tumor volume monitored. Control: no treatment; MnSOD-PL (100 μl containing 100 μg plasmid) intraorally, TPZ (60 mg/kg) intraperitoneally in 0.1 ml, + C225 100 μl containing 10 mg/kg intraperitoneally; treated mice received either 14 Gy (A) or 18 Gy (B) to the oral cavity.