The Combination of the Tyrosine Kinase Receptor Inhibitor SU6668 with Paclitaxel Affects Ascites Formation and Tumor Spread in Ovarian Carcinoma Xenografts Growing Orthotopically

Animals.

Female NCr-mu/mu mice were obtained from the animal production colony of the National Cancer Institute (NCI-BTB-DTP), Frederick Cancer Research and Development Center (Frederick, MD). Mice were used at 6-to-8 weeks of age and had a mean body weight of 21 ± 2 g. The mice were housed in filtered-air laminar-flow cabinets and were manipulated using aseptic procedures.

Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (Decreto Legislativo No. 116, Gazzetta Ufficiale (G.U.), Suppl. 40, Feb. 18, 1992; Circolare No. 8, G.U., July, 1994) and international laws and policies (European Economic Community Council Directive 86/609, Official Journal Legislation 358. 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996).

HOC Xenografts.

HOC79 and HOC22 xenografts were established from patient ascites and maintained i.p. in nude mice as described previously (36 , 37) . These xenografts grow in the peritoneal cavity of nude mice producing ascites and solid lesions on abdominal organs, primarily the diaphragm, omentum, and pancreas. Both HOC xenograft models secrete high levels of VEGF in ascites.

Dose Finding and Drug Treatment.

SU6668 {(Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid} was provided by SUGEN Inc. (South San Francisco, CA). SU6668 was formulated as 50 mg/ml in an aqueous-base cremophor vehicle [16.92% 1 n sodium hydroxide solution, 24.6% cremophor EL (polyoxyethylated castor oil), 1.56% benzyl alcohol, 35.14% PEG 400, 16.79% deionized water] and was stored in the dark at −20°C in ready-to-use aliquots. Each aliquot was thawed at the beginning of the cycle and kept at 4°C for the full-cycle treatment. SU6668 administered p.o. has shown dose-dependent inhibition of xenograft models, with 200 mg/kg being the most, nontoxic, efficacious dose (34) . Accordingly, dose-finding studies on our model of HOC 79 survival showed significant activity at doses of 200 mg/kg, but only a modest effect with 50 mg/kg. On this basis, the dose of 200 mg/kg (100 μl p.o.), daily in cycles of five injections per week was chosen for our studies.

Paclitaxel (provided by Indena S.p.A., Milan, Italy) was dissolved in 50% cremophor EL and 50% ethanol and was further diluted in 5% glucose; it was injected i.v. at the dose of 6 mg/kg every other day or 20 mg/kg once a week, as specified in the “Results.” The vehicles (defined as “vehicle-SU6668” and “vehicle-paclitaxel”) were administered by the same schedule and route as were the active compounds.

For the combination studies, the doses and schedules of paclitaxel were chosen on the basis of dose-finding studies on the 1A9 xenograft model (38) transplanted s.c. into nude mice. In this model, paclitaxel at different doses and schedules was administered continuously until tumor reached 1.5 g. Twenty mg/kg (one-third of the MTD) weekly and 6 mg/kg (one-tenth of the MTD) every other day in combination with SU6668 (200 mg/kg) were selected as the most active schedules administrable for a prolonged period with no signs of toxicity.

Evaluation of Treatment.

HOC22 and HOC79 xenografts were injected i.p. as a cell suspension into 10–12 nude mice (10 × 10⁶ cells/animal) for each experimental group. When indicated, four additional mice per group were given injections to determine tumor burden at the beginning or end of the treatment period (see “Necropsy… ” below). Treatment was started 7 days after tumor transplantation unless otherwise specified and continued for 3, 5, or 7 weeks as detailed in the “Results.” Mice were monitored twice a week for body weight loss and tumor formation in the peritoneal cavity (abdominal distension) and were euthanized when they became moribund, the day of death being considered the limit of survival. At autopsy, the peritoneal cavity was macroscopically examined to ascertain the presence of tumor. Results are plotted as the percentage survival against days after tumor transplant. The %ILS was calculated as 100 × [(median survival day of treated group − median survival day of control group)/median survival day of control group]. Differences in survival were analyzed by the log-rank test (7) . Data are representative of two different experiments.

Necropsy and Evaluation of Tumor Burden.

At the beginning and end of the treatment period, four nude mice per group were euthanized by carbon dioxide inhalation and were necropsied to establish the tumor burden. Ascites was harvested and centrifuged, and the volume of fluid was recorded by subtracting the pellet volume. The number of cells in the pellet (representative of tumor burden) was determined for each mouse (7) . The cell suspension was spun in a cytocentrifuge, and the cytology samples were air-dried and were stained with May-Grünwald Giemsa.

The pancreas, omentum, and diaphragm were collected for histology, fixed in 10% neutral buffered formalin, and were routinely processed for paraffin embedding. Five-μm thick sections were stained with H&E and examined. Tumor deposits were classified as nodes (when composed of >100 tumor cells), nodules (10–100 tumor cells), and small nodules (<10 tumor cells). The involvement of each organ was scored as “severe” (grade 3) if one or more tumor nodes were detected, “moderate” (score 2) if more than 10 small nodules/nodules were detected, “mild” (score 1) if fewer than 10 small nodules/nodules were detected, and “absent” (score 0) if no tumor deposits were detected.

Growth Factor Levels in Ascites.

Ascites, harvested as above, was processed as described in detail elsewhere (39) . Briefly, the supernatant after centrifugation was frozen and maintained at −80°C until analysis. VEGF, PDGF, and FGF-2 were measured using a commercial ELISA kit (Human, Quantikine; R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The sensitivity of the assay was 9 pg/ml, 1.7 pg/ml, and 3 pg/ml for the three growth factors, respectively. Each sample was analyzed in duplicate.

Response of HOC Xenograft to SU6668.

Two HOC xenografts, HOC22 and HOC79, were implanted in the peritoneal cavity of female nude mice, and treatment with SU6668 started on day 7 when tumor involvement of the omentum and pancreas, with ascites was already present (7) . Three weekly cycles of SU6668 increased the survival time for mice bearing HOC79 (%ILS = 23% and 29%, in two independent experiments) and HOC22 (%ILS = 67% and 81% in two independent experiments). The HOC79 xenograft, showing a low response to SU6668 as a single-agent treatment, was chosen for additional studies, described as follows.

Analysis of the tumor burden at the beginning and end of treatment (days 7 and 25) of representative mice bearing HOC79 (four per group) showed that SU6668 limited tumor growth in the peritoneal cavity (Fig. 1)⇓ . The ascitic fluid (Fig. 1A)⇓ in mice treated with SU6668 accumulated less as treatment proceeded (2.2 ± 0.04 ml at the beginning of treatment on day 7, and 3 ± 0.05 ml at the end of treatment on day 25) compared with vehicle-treated mice (6,6 ± 2 ml on day 25). The tumor burden in the ascites (43 × 10⁶ ± 20 cells, on day 7) was inhibited in mice receiving SU6668 (92 × 10⁶ ± 9 cells), compared with vehicle (165 × 10⁶ ± 31 cells) on day 25 (Fig. 1B)⇓ . Accordingly, VEGF levels paralleled tumor growth in vehicle-treated mice (1,450 pg ± 350 on day 7, and 10,145 ± 1,939 pg on day 25) and were lower in SU6668-treated mice (6,574 ± 3,300 pg at the end of the treatment, day 25; Fig. 1C⇓ ). PDGF levels in vehicle-treated mice were 8.2 ± 4.6 pg on day 7, and 73 ± 30 pg on day 25, and were lower in SU6668-treated mice (32.3 ± 16 pg at the end of the treatment, day 25; Fig. 1D⇓ ). No significant levels of FGF-2 were detected in the ascitic fluid. The tumor burden, the level of growth factors, and the survival time of mice that were given the oral vehicle (vehicle-SU6668) were no different from untreated mice (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Effect of SU6668 on ascites formation and tumor burden in ovarian carcinoma xenografts. HOC79 was transplanted i.p. in nude mice and treatments started 7 days later. Mice were treated with SU6668 (200 mg/kg p.o.) and vehicle daily (every day for 5 days) for three cycles (days 7–11, 14–18, 21–25). At the beginning (untreated, day 7) and at the end of the treatment (day 25), four animals per group were sacrificed to estimate the tumor burden as volume of ascitic fluid (A), number of cells in the ascites (B), VEGF levels (C), and PDGF level (D). Data are the mean ± SD (n = 4). P ≤ 0.005 (a), P ≤ 0.01 (b), P ≤ 0.001 (c), P ≤ 0.05 (d) versus untreated (day 7); and P ≤ 0.01 (e), P ≤ 0.05 (f) versus vehicle (day 25). Differences were analyzed by the nonparametric Mann-Whitney test (significance, P ≤ 0.05).

Response to SU6668 Depends on the Treatment Schedule.

HOC79 is a fast-growing tumor that causes hemorrhagic ascites and tumor deposits to the omentum, pancreas, and diaphragm (see results in Table 1⇓ ). The antitumor activity of SU6668 was tested against early-stage (beginning of treatment 3 days after tumor implantation) and advanced-stage tumor (beginning of treatment 21 days after tumor implantation; Fig. 2A⇓ ). Cytohistological results showed that, 3 days after HOC79 injection, all of the mice presented tumor cells in the peritoneal lavage and microscopic tumor deposit in the omentum and pancreas. Treatment with SU6668 that was started at this time significantly improved the survival of mice bearing HOC79 (MST, 45 days; range, 40–49 days; %ILS, 45%) compared with vehicle-treated mice (MST, 31 days; range, 27–33 days). In contrast, treatment when the tumor was advanced did not affect the course of the tumor (MST, 35 days; range, 29–47 days; %ILS, 9%) compared with mice receiving vehicle at the same time (MST, 32 days; range, 30–34 days). In this group, only a few mice were able to complete the three cycles of treatment, and at necropsy, all of the mice had the same tumor burden as vehicle-treated mice.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

The effect of SU6668 depends on the schedule of treatment. HOC79 was transplanted i.p. into nude mice. A, mice received SU6668 (200 mg/kg p.o.) and the vehicle daily, every day for 5 days, for three cycles starting 3 days (days 3–7, 10–14, 17–21) or 21 days (days 21–25, 28–32, 35–39) after tumor transplantation (n = 8). P < 0.001, treated at day 3, versus vehicle. B, mice received SU6668 (200 mg/kg p.o.) and the vehicle daily, every day for 5 days, for three cycles (days 3–7, 10–14, 17–21) or 7 cycles (days 3–7, 10–14, 17–21, 23–28, 31–35, 38–42, 45–49; n = 10–12). P < 0.001, treated versus vehicle (both schedules).

Fig. 2B⇓ shows that the prolonged treatment, i.e., for seven cycles, further improved the survival of mice bearing HOC79 (MST, 53 days; range, 52–61 days; %ILS, 81%) compared with mice treated for only three cycles (MST, 45 days; range, 40–49 days; %ILS, 45%). Vehicle-treated mice had a MST of 29 days (range, 28–31 days) and 31 days (range, 27–33 days) after 7 and 3 cycles, respectively, of treatment (Fig. 2B)⇓ . SU6668, 200 mg/kg/injection, was well tolerated for the prolonged treatment, with no obvious side effects. However, in this case also, all of the animals had progressive disease, and their death by tumor was confirmed at necropsy.

Effect of SU6668 in Combination with Paclitaxel on HOC79 Xenografts.

To see whether the response to the compound could be improved, we tested it in combination with paclitaxel (Fig. 3)⇓ . HOC79 is a xenograft only partially responsive to paclitaxel, and tumors progress under paclitaxel treatment (37) . Five-week therapy with paclitaxel increased the survival of mice (MST, 92 days; range, 66–140 days; %ILS, 206%) compared with vehicle-treated mice (MST, 30 days; range, 27–34 days). In this study, the 5-week treatment with SU6668 also significantly increased the survival of mice (MST, 50 days; range, 42–50 days; %ILS, 66%). Paclitaxel in combination with SU6668 significantly prolonged the survival of mice bearing HOC79 (MST, 154 days; range, 140–192 days; %ILS, 413%), and it was significantly more potent than paclitaxel as a single agent. The combination with paclitaxel did not cause significant body weight loss. Moreover, clinical signs, gross lesions, and histological hepatic findings, indicative of toxic effect, were not observed in animals necropsied at the end of the treatment.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Effect of SU6668 in combination with paclitaxel on the survival of mice bearing HOC79. HOC79 was transplanted i.p. in nude mice, and treatments started 7 days later. Mice were treated with SU6668, paclitaxel, SU6668 + paclitaxel, or vehicles. Treatment schedules were as follows: SU6668 200 mg/kg p.o. (every day for 5 days) for five cycles (days 7–11, 14–18, 21–25, 28–32, 35–39); paclitaxel 20 mg/kg i.v. (every 7 days for 5 doses: day 7, 14, 21, 28, 35; n = 10). P < 0.001, treated groups (paclitaxel and SU6668, each alone) versus vehicle; P < 0.01, combination versus paclitaxel. Differences in survival were analyzed by the log-rank test.

Effect of Different Schedules of Paclitaxel in Combination with SU6668 on the Survival and Tumor Burden in Mice Bearing HOC79 Xenografts.

HOC79, implanted i.p., formed tumors in all of the mice, with a median survival of 42 days (range, 37–43 days; Fig. 4⇓ ). To define the optimal treatment condition of paclitaxel in combination with SU6668, two different schedules of paclitaxel 20 mg/kg (optimal dose) every 7 days for 3 doses and 6 mg/kg (minimal active dose) every 2 days for 10 doses, both for a total of 60 mg/kg (MTD) were used (Fig. 4⇓ ; Table 1⇓ ). HOC79 was sensitive to both schedules, 20 mg/kg, every 7 days for 3 doses being more active (MST, 82 days; range, 71–138 days; %ILS, 95%) than 6 mg/kg every 2 days for 10 doses (MST, 65 days; range, 57–86 days; %ILS, 54%). SU6668 alone, given for only three weeks, although less active than paclitaxel, also increased the survival of nude mice (MST, 52 days; range, 48–55 days; %ILS, 23%). Combined, the two drugs significantly prolonged survival of mice bearing HOC79 (MST, 80 days; range, 72–97 days; %ILS, 90%, and MST, 127 days; range, 83–152 days; %ILS, 200%, with 6 and 20 mg/kg/dose, respectively). As shown in Fig. 4⇓ , both paclitaxel schedules significantly boosted the antitumor effect of SU6668. Interestingly, the same increase in survival was obtained with the high doses of paclitaxel (20 mg/kg/dose) given as a single agent (%ILS, 95%) and with low doses of paclitaxel (6 mg/kg/dose) but combined with SU6668 (%ILS, 90%).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Effect of different schedule of paclitaxel with SU6668 on the survival of mice bearing HOC79. HOC79 was transplanted i.p. into nude mice, and treatments started 7 days later. Mice were treated with SU6668, paclitaxel, SU6668 + paclitaxel or vehicles (n = 10). Paclitaxel doses were 6 mg/kg i.v. (PTX 6; every 2 days for 10 doses: day 7, 9, 11, 13, 15, 17, 19, 21, 23, 25) and 20 mg/kg i.v. (PTX 20; every 7 days for 3 doses: day 7, 14, 21). SU6668 dose was 200 mg/kg p.o. (every day for 5 days) for three weekly cycles: days 7–11, 14–18, 21–25). Results were shown as day of survival (median with range) and %ILS (ILS %). Differences in survival were analyzed by the log-rank test. a, P < 0.001 versus vehicle; b, P < 0.001 versus SU6668; c, P < 0.05 versus paclitaxel.

To investigate the effects of these combination therapies on the growth of HOC79, we did an interim necropsy of four mice per group at the end of treatment (day 26). Results are summarized in Table 1⇓ . Vehicle-treated mice had more ascitic fluid (median, 5.8 ml) and more tumor cells (median, 397 × 10⁶ cells) than mice treated with either SU6668 (ascites 2.1 ml and cells 90 × 10⁶) or paclitaxel alone (6 and 20 mg/kg/injection: ascites 2.6 and 2.3 ml, respectively, and cells 67 × 10⁶ and 13 × 10⁶, respectively; Table 1⇓ ). The blockade of ascitic fluid accumulation in the groups given the combination of paclitaxel with SU6668 was paralleled by a decrease in tumor cells. Only a few cells were detectable in the peritoneal lavage from mice treated with the combination at 6 mg/kg/dose (18 × 10⁶), and almost no cells were detectable in those treated with the combination at 20 mg/kg (Table 1)⇓ . The level of VEGF and PDGF in ascites paralleled the tumor burden (data not shown). Ascites and organs were collected for cytological and histopathological analysis (see description that follows and Table 1⇓ ).

Cytological and Histological Analysis.

In cytological specimens from untreated mice, the neoplastic cells formed clusters, often in a papillary configuration. Single, usually larger neoplastic, cells could also be seen. In all cases, there were few inflammatory cells. Similar findings were observed in samples from mice treated with SU6668 alone. In animals treated with paclitaxel alone and with the combination of paclitaxel plus SU6668, the neoplastic cells were more anaplastic, showing marked anisokaryosis and frequent, atypical, mitotic figures.

On day 26 at the end of treatment, vehicle-treated mice had widespread tumor implants on the omentum, pancreas, and diaphragm (Fig. 5)⇓ . No metastases were detected to organs outside the peritoneal cavity, and the tumor did not invade the host tissue to which they were adherent. The pattern of tumor dissemination did not change along with the treatments. Animals treated with SU6668 alone had metastatic deposits similar to untreated controls (Table 1)⇓ . A dose-schedule-related effect was observed, with greater activity in animals given paclitaxel 20 mg/kg for 3 injections than in those given 6 mg/kg for 10 injections. However, in animals treated with the combination of paclitaxel plus SU6668, the reduction of metastatic deposits occurred with both schedules. The reduction of tumor involvement in mice treated with the high dose of paclitaxel and with the combination of paclitaxel plus SU6668 was particularly evident in the diaphragm and was also evident in the diaphragm when the low dose of paclitaxel was used (Table 1⇓ and Fig. 5⇓ ). These findings suggest that the combination treatment affects tumor dissemination in the peritoneal cavity.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

Hystopathology of tumor deposits in the peritoneal cavity of mice bearing HOC79. Example of organ involvement of mice bearing HOC79 treated with the combination of SU6668 and paclitaxel. A and C, untreated; B and D, treated with SU6668 and paclitaxel. For details, see Table 1⇓ . H&E, ×100. A, large tumor mass surrounded by densely packed lymphocytes is present in the omental tissue of untreated mice. B, two small tumor nodules surrounded by densely packed lymphocytes are present in the omental tissue of mice treated with paclitaxel plus SU6668. C, diffuse papillary growth of the tumor along the peritoneal surface of the diaphragm of untreated mice. D, a small tumor nodule surrounded by lymphocytes is present on the peritoneal surface of treated mice.