Fighting Cancers with Anthrax Lethal Factor
We have evaluated the anti-tumoral potential of LeTx in a subcutaneous xenograft model using three different modes of delivery; intratumoral, intravenous, and intraperitoneal injection. Each mode of delivery has its own strengths and weaknesses. Together they provide a strong indication that LeTx is effective against melanoma in vivo.
Intratumoral Injection
In collaboration with Han-Mo Koo and George Vande Woude, we tested the antitumor activity of LeTx on two different human melanoma xenografts grown in athymic nude mice by intratumoral injection. The strength of this approach is that it is technically easy. Its disadvantage is that it is not clear to what extent the dose is systemically distributed. SK-MEL-28 melanoma cells were injected into nude mice, and after tumors were established, the animals were treated by direct intratumoral injection of LeTx at 2-day intervals. Control tumors were treated with PA alone. Dramatically, all LeTx treated SK-MEL-28 tumor xenografts completely regressed, and the animals remained tumor-free for up to 5 weeks (Fig. 1A). PA alone had no effect on the tumor growth (Fig. 1A). To determine whether the antitumor effect of LeTx is systemic, we used a contralateral xenograft tumor model [63]. MALME-3M melanoma xenograft tumors were established on both the right and left upper dorsal areas of nude mice. At 2-day intervals, we injected LeTx intratumorally only into tumors on the right side. While these tumors completely regressed after six doses and remained undetectable throughout the experiment, the tumors on the left side also regressed (Fig. 1B). Moreover, when the experiment was terminated on day 36, two tumors on the left side had completely regressed, whereas three animals relapsed after initial regression (Fig. 1B). PA alone had no effect on tumor growth (Fig. 1B). A total of 19 doses of LeTx were administered over a 5-week period without obvious adverse effects on the general health or behavior of the animals. These results show that antitumor effects of LeTx are systemic.
Figure 1. In vivo antitumor effects of LeTX on melanoma xenograft tumors. (A) SK-MEL-28 melanoma xenograft tumors at 310 mm3 (day 27) were intratumorally injected with PA alone (PA) as control or with LeTx (n = 7). Five doses were given at 2-day intervals (days 27–35, 1) and then an additional dose 3 days later (day 38, 2). Tumor volume (mm3) is shown with standard deviations. (B) MALME-3M melanoma xenograft tumors were established on both the right and left dorsal upper flank areas of nude mice. When the left-side tumor reached 70 mm3 in size, treatment was initiated by direct intratumoral (IT) injection into the right-side tumors only, with either LeTx or PA alone as control at 2-day intervals (n = 5). When the right-side tumors completely regressed in the LeTx-treated group (day 8), the treatment route was changed to s.c. (SC) injection on the right side of animals (day 10). Individual tumors (small squares) are shown for the left side from day 28 (gray area). Image from Koo, et al. 2002. Proc. Natl. Acad. Sci. U.S.A. 99: 3052–3057.
Intravenous Injection
We also tested the antitumor activity of LeTx on human melanoma xenografts by intravenous injection. The strength of this approach is that the dose is systemically distributed. However, repeated accurate dosing may be technically challenging.
Because LeTx is a binary toxin, we needed to determine the ratio of PA to LF that produces the maximal tumor response. We observed that increasing ratios of PA to LF over the range of 1:1 to 5:1 elicited progressively greater tumor responses. The maximal response was observed at 5:1 and 7:1 ratios (Fig. 2A, p = 0.0178 and 0.0194, respectively, when compared to growth of control LF-only treated tumors). Increased inhibition was not observed at higher ratios (9:1 and 11:1, data not shown). These results indicate that the optimal ratio of PA to LF needed to inhibit tumor growth lies between 3:1 and 5:1 (W/W).
Figure 2: Effects of different ratios (A) and schedules of administration (B) of PA to LF on tumor growth inhibition by intravenously administered LeTx. The Y-axis represents mean tumor volume ± standard error in mm3 at day 14 post-treatment initiation. Mice were treated every Monday, Wednesday and Friday for a total of 6 doses with different ratios of PA to LF (A) and different schedules of administration of PA and LF (B) and at 2 µg LF. Out of the 6 ratios tested (the 9:1 and 11:1 ratios are not shown in this graph) only the 5:1 and 7:1 ratios of PA to LF produced significant tumor growth inhibition as compared to controls (A). LeTx had significant tumor growth inhibition in treated mice as compared to control mice only when PA and LF where administered simultaneously (B). Abi-Habib et al. 2006. Clinical Cancer Res. 12: 7437–7443.
Similarly, we needed to test the effects of the timing of PA and LF administration relative to each other. Only the simultaneous injection of PA and LF produced significant tumor growth inhibition in LeTx-treated mice at day 14 post treatment initiation relative to vehicle-treated mice (p = 0.0015) (Figure 2B). LeTx treatment did not lead to significant tumor growth inhibition when PA and LF injections were separated by one or two hours, starting with either PA or LF (p = 0.062, 0.099, and 0.082, respectively) (Figure 2B). These results indicate that for maximal tumor response PA and LF are best injected simultaneously.
Histologic analysis of Sk-Mel28 xenograft tumors treated with LeTx showed decreased phospho-MAPK (active MAPK) staining (Fig. 3A) that was accompanied by reduced proliferation (Ki-67; Fig. 3B) and increased apoptosis (TUNEL staining; Fig. 3C). Thus, LeTx-treated tumors show immunohistologic evidence of target inhibition and response.
Figure 3. LeTx-treated tumors show histologic evidence of target inhibition and response. Upon completion of the ratio optimization study, tumors were excised and fixed in formalin for immunohistochemical analysis with antibodies against (A) active, phosphorylated ERK (P-ERK), (B) proliferation (Ki-67), and (C) apoptosis (TUNEL) staining. In this image control tumors treated with LF (2 µg) alone are compared to tumors treated with LeTx (10 µg PA, 2 µg LF). Abi-Habib et al. 2006. Clinical Cancer Res. 12: 7437–7443.
Intraperitoneal Injection
Finally, in collaboration with Ralph Abi-Habib and Arthur Frankel, we also tested the antitumor activity of LeTx on human melanoma xenografts by intraperitoneal injection. The strength of this approach is that it is technically easy. However, though the drug is systemically distributed, it is not possible to predict what dose a tumor will receive.
Based on the preceding results, all further experiments were performed using a simultaneous injection of PA and LF at a fixed ratio of 5:1 (w/w). To determine whether LeTx can be administered systemically in a safe and efficacious manner we tested the in vivo potency and selectivity of systemically administered LeTx in a mouse melanoma model. We established the human melanoma xenograft model in anti-NK treated athymic nude mice. Tumors grew subcutaneously in 100% of animals inoculated with 10 million SK-MEL-28 cells and treated with anti-asialo GM1 antibody i.p. at days 4 and 2 prior to tumor inoculation. Tumors reached an average volume of 300 mm3 by day 30 and 750 mm3 at the termination of the experiment at day 60 post tumor inoculation. Intraperitoneal LeTx treatment at a level of cumulative doses near and above the LD10 (see below), including 30, 32, 36, and 40 µg total LF, yielded dramatic and, in some cases, durable responses (Table 1 and Figure 4). In all but the 32 µg total LF dose, all mice were free of measurable tumors at day 30 after tumor inoculation. At the 40 µg total LF dose, mice were still free of measurable tumors at day 60 (p < 0.0001 relative to vehicle-treated mice). At the 30, 32, and 36 µg total LF doses some mice had regrown tumors by day 60, but these doses still produced highly significant tumor growth inhibition as compared to vehicle-treated mice (p = 0.0002, 0.0011, and 0.0001, respectively) (Fig. 4).
Figure 4. LeTx innhibits tumor growth in vivo. SK-MEL-28 melanoma subcutaneous tumor growth (average ± standard error) in athymic nude mice treated i.p. every other day starting at day 1 post-tumor injection with PBS (■), or a 5:1 ratio of PA and LF at the following cumulative doses: 30µg LF (▲), 36µg LF (○), and 40 µg LF (□). The Y-axis represents tumor size in mm3, the X-axis represents days post tumor inoculation (day 0). Treatment ended at day 11 for the 30 µg LF group (6 doses) and at day 15 for the remaining dose groups. All dose groups produced total tumor growth inhibition past day 30. At day 60, only the 40 µg LF group still showed total growth inhibition while the remaining dose groups still produced more than 75% tumor growth inhibition. Abi-Habib et al. 2006. Clinical Cancer Res. 12: 7437–7443.