Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction

Abstract

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.

Keywords: doxorubicin tumor entrapment; retargeted tissue factor; vascular infarction; vascular targeting.

Figure 1. Fluorescence- and HPLC-based quantification of doxorubicin content in tumor tissue

Doxorubicin was injected at a dose of 5 mg/kg bw i.v. and the intratumoral concentration was determined over a time of up to 240 hours by spectrofluorometric analysis (A) 1 h n = 3, 2 h n = 4, 6 h n = 2, 65 h n = 2, 120 h n = 5 and 240 h n = 2) and up to 120 hours by HPLC (B) 1 h n = 3, 2 h n = 2, 4 h n = 3, 6 h n = 2, 65 h n = 3 and 120 h n = 3). The maximum fluorescence signal (tissue C_max) was obtained at 6 hours (t_max) after injection with a following slow monophasic decrease of intratumoral doxorubicin concentrations and remaining intratumoral levels still detectable after 240 hours. tTF-NGR (1mg/kg bw i.v.) or PEGylated tTF-NGR (5 mg/kg bw i.v.) injection 4 hours before doxorubicin yielded a decreased uptake of doxorubicin into tumor tissue in comparison to a fast opposite sequence (C). P-values showed significance in uptake blockade (p = 0.0079, **, Mann-Whitney test). Furthermore, tumor fluorescence microscopy photographs (D) visualized the decrease in autofluorescence of intratumoral doxorubicin injected after tTF-NGR (D, b) over the opposite sequence injecting tTF-NGR after doxorubicin (D, a). AU, arbitrary units. All data are presented in means ± SEM.

Figure 2. Fluorescence-based quantification of doxorubicin wash-out kinetics upon combinatorial application of doxorubicin and tTF-NGR or randomly PEGylated tTF-NGR, respectively

The HT1080 tumor-bearing mice were injected with a 1st application of tTF-NGR or PEGylated tTF-NGR 6 hours after doxorubicin injection at intratumoral C_max of doxorubicin, and vascular occlusion was upheld by repeated application of tTF-NGR or randomly PEGylated tTF-NGR, respectively, over time. This combinatorial protocol significantly retarded wash-out times of doxorubicin from the tumor with prolonged high intratumoral drug levels in the tumor tissue after 65 and 120 hours upon doxorubicin - tTF-NGR sequences as compared to control sequences of doxorubicin - saline (p = 0.0043 at 65 h, p = 0.0095 at 120 h; **Mann-Whitney test). Non-PEGylated tTF-NGR had a more pronounced effect than randomly PEGylated tTF-NGR. Data are presented in means ± SEM. AU, arbitrary units.

Figure 3. Fluorescence-based quantification of doxorubicin content in normal lung tissue

For details see Figures 1 and 2. Identical time points following doxorubicin - tTF-NGR sequences were taken for comparative studies on normal organ tissues. In normal tissues such as lung no entrapment of doxorubicin by tTF-NGR could be shown, indicating that doxorubicin organ toxicity should not be altered by this combination with tTF-NGR. AU, arbitrary units. Data are shown as means ± SEM.

Figure 4. Fluorescence-based quantification of apoptosis in tumor and organ tissues in situ

Petri dish with tumors (2–3 weeks after cell inoculation) or organs 98 hours post-injection of Annexin-Vivo 750 and 120 hours after start of treatment (A) color-coded fluorescence reflectance images: (a) doxorubicin 5 mg/kg bw (day 0); (b) tTF-NGR 1 mg/kg bw (day 0, 2, 5); (c) doxorubicin 5 mg/kg bw (day 0) followed by tTF-NGR 1 mg/kg bw (6 h after doxorubicin, and days 2, 5); (d) doxorubicin 5 mg/kg bw followed by PEGylated tTF-NGR 5 mg/kg bw (6 h after doxorubicin, and days 2, 5). High fluorescence values are observed in the tumors (A, b-d). Semi-quantitative data showed significant higher levels of tumor cell apoptosis in situ in mice, which received the doxorubicin - tTF-NGR than in mice receiving a doxorubicin - saline control sequence (A) p = 0.0381, *, Mann-Whitney test), whereas doxorubicin - PEGylated tTF-NGR vs. doxorubicin (p = 0.114) and doxorubicin vs. doxorubicin – tTF-NGR (p = 0.610) showed a trend in favour of the combination, but no significant tTF-NGR differences in fluorescence signal. (B) The liver and at lower levels the heart showed signs of in situ apoptosis in mice injected with tTF-NGR and doxorubicin - tTF-NGR (B, a-d, liver), but there was no quantitative difference between tTF-NGR alone or the combination of doxorubicin - tTF-NGR, respectively (B, right panel). CAVE: The absolute values cannot be compared between the single organs and tumors, since different organ sizes interfere (e.g. liver size bigger than tumor size). AU, arbitrary units. All data are presented in means ± SEM.

Figure 5. Doxorubicin-induced phosphatidylserine (PS) externalization on HUVEC enhances the procoagulatory activity of tTF-NGR

APC Annexin V stains early apoptotic cells, which display PS on their surface, TOPRO3 iodide stains necrotic cells (A and B). The amount of PS on the outer surface of HUVECs correlates with the dosage of and incubation time with doxorubicin. Upon 16 hours treatment with 2 μM doxorubicin, the fraction of PS positive cells rises from 14% (non-treated cells) to 47%, whereas the number of necrotic cells only increases moderately from 7% to 13% (A). For the chart in (B), the percentage of stained cells in R1 has been reduced by the fraction of unstained cells in R1 (not shown). Doxorubicin concentrations of 2 μM and 20 μM display significant differences to controls for all incubation times (2 μM: p_8h = 0.0007, p_16h = 0.0180, p_24h = 0.0030 (**); 20 μM: p_8h = 0.0022, p_16h = 0.0198, p_24h = 0,0028 (**); two-tailed t-test). For these concentrations, 24 h incubation leads to significantly (p < 0.05, *) elevated PS exposure when compared to 8 hours. Lower concentrations do not show efficacy in terms of augmenting PS levels on the outer cell membrane (B). Data are shown in means ± SEM. (C) After 16 h treatment with 2 μM doxorubicin, HUVECs showed significantly improved ability to activate FX in the presence of tTF-NGR (p = 0.0015, **). This effect is due to additional PS on their surface and not to nonspecifc effects following necrosis, since TOPRO3 iodide staining was below 10% and since the increased coagulability could be completely reverted by blocking surface PS by Annexin V binding to PS as shown in the right bars (−doxorubicin, +annexin; +doxorubicin, +annexin, p = 0.00003; ***). Data are represented in means ± SEM (one outlier has been excluded from analysis).

Figure 6. Doxorubicin-induced phosphatidylserine (PS) expression on HT1080 fibrosarcoma cells enhances the procoagulatory activity of tTF-NGR

For experimental details see Figure 5. HT1080 fibrosarcoma cells are more susceptible to doxorubicin treatment than HUVECs. Dosages of 0.2 μM doxorubicin yield considerably higher apoptosis levels than controls (0.2 μM: p = 0.0559-0.4482; 2 μM: p_8h = 0.1045, _16h = 0.0073, p_24h = 0.0260 (*); 20 μM: p_8h = 0.0981, p_16h = 0.008, p_24h = 0.0096 (**); (A and B). With similar time and dose dependency as in HUVECs PS externalization exceeds HUVECs by up to 100% whereas necrosis (TOPRO3) remained low. If necessary, different gates for APC Annexin V and TOPRO3-stained cells were set to account for distinct staining behaviour. (C) As a result, Factor X activation assays show a proportionally increased procoagulatory effect of HT1080 cells upon doxorubicin treatment (p = 0.0264, *), which is completely reversible upon PS inhibition by Annexin V (p = 0.0268, *). All values are shown in means ± SEM.

Figure 7. Doxorubicin-induced phosphatidylserine (PS) expression on M21-melanoma cells enhances the procoagulatory activity of tTF-NGR

For experimental details see Figure 5. Experiments with M21-melanoma cells showed a clear trend for doxorubicin to enhance PS externalization on the cell surface (A and B) with a higher degree of simultaneous necrosis than in HUVECs (2 μM: p_8h = 0.2411, p_16h = 0.2040, p_24h = 0.0325 (*); 20 μM: p_8h = 0.8166, p_16h = 0.0013, p_24h = 0.0006 (***)). (C) Doxorubicin treatment enhanced the procoagulatory effect of tTF-NGR in the presence of doxorubicin-treated M21 cells, which is entirely reversible upon PS inhibition by Annexin V. Adherent and suspension cells were considered for analysis collectively. Asterisks denote statistical significance (p < 0.05, *); all values are presented in means + SEM.

Figure 8. In vivo therapeutic activity against tumor xenografts of combinatorial application of doxorubicin - tTF-NGR or - TMS(PEG)₁₂ tTF-NGR

Drugs were applied intravenously at the doses and time points indicated. Data are presented as means + SEM. Asterisks indicate statistical significance for the groups as indicated (p < 0.05, *; p < 0.001, **; p < 0.0001, ***; Mann-Whitney test). (A) Therapeutic activity of doxorubicin (5 mg/kg bw, see arrow; n = 13), tTF-NGR at optimal dose (1.5 mg/kg bw, daily application, see 5 arrows; n = 16), or the combination of both drugs (n = 14) compared to the PBS control (n = 15) in a human M21 melanoma xenograft model. Three experiments with CD-1- and BALB/c-nude mice were combined. Start of treatment: 35 days after tumor implantation (CD-1), and 52 days (BALB/c), respectively. Tumor volumes are presented as percentage (start of the therapy = 100%; max. tumor size included into the evaluations was 1.2 cm³ at the start of the therapy). In comparison to the PBS control, the therapies with doxorubicin, combination of doxorubicin and tTF-NGR, and tTF-NGR alone revealed highly significant decrease in tumor volumes on day 7. Comparison of the combination therapy versus doxorubicin alone indicates statistical significance (p = 0.026), while the comparison with tTF-NGR alone reveals a non-significant trend in favor of the combination (p = 0.533). (B) Therapeutic activity of doxorubicin (5 mg/kg bw, see arrow; n = 5), tTF-NGR (1 mg/kg bw; n = 5) or TMS(PEG)₁₂ tTF-NGR (5 mg/kg bw; n = 4), respectively (both applied every other day, see 4 arrows), or the combination of both drugs (each n = 4) at identical doses in a HT1080-human fibrosarcoma xenograft model. In comparison to the PBS control, the therapies with doxorubicin, doxorubicin followed by tTF-NGR, and tTF-NGR alone revealed a significant decrease in tumor volume on day 21 (p = 0.0317, p = 0.0286, p = 0.0259, respectively); comparison of the combination schedule ‘doxorubicin followed by tTF-NGR’ versus doxorubicin alone shows a non-significant trend in favor of the combination (p = 0.105), while comparison of ‘doxorubicin followed by tTF-NGR’ versus tTF-NGR alone reveals statistical significance (p = 0.0489).

Figure 9. Tumor macroscopic appearance and proliferation as shown by Ki-67 staining

(A) Appearance of subcutaneous HT1080 tumor after treatment with doxorubicin (5 mg/kg bw, i.v.) followed by tTF-NGR (1 mg/kg bw, i.v.). During the time of treatment (5 days), the tumor collapsed with signs of necrosis. (B) Immunofluorescence staining of tumor sections (mean n = 2 per group) with anti-Ki67 antibody (green), indicating the proliferation rate in control mice after 21 days (a), in doxorubicin - tTF-NGR treated mice after 7 days (b), in doxorubicin treated mice after 21 days (c), in tTF-NGR treated mice after 21 days (d) and in doxorubicin - tTF-NGR treated mice after 21 days (e). Nuclei were stained with DAPI (blue). The measured binary area fraction (4 sections of each tumor) showed significant decrease of tumor cell proliferative activity in the doxorubicin - tTF-NGR sequence (b) on day 7 over saline control (a; **p = 0.008, Mann-Whitney test). After 21 days proliferative capacity of the doxorubicin monotherapy tumors recovered completely (c), whereas pronounced anti-proliferative effects of the doxorubicin - tTF-NGR sequence (e) over single drugs (c, d) was retained, however, without reaching statistically significant differences comparing the combination vs. doxorubicin (p = 0.133) or tTF-NGR (p = 0.600) alone.