PA-L1/LF efficacy in orthotopic BHT-101 and DRO xenografts. Mice bearing 5-day old BHT-101 or DRO orthotopic tumors were treated with either 30 μg PA-L1/10 μg LF in 500 μl PBS or PBS alone 3 times a week for 2 weeks. At day 21, mice were euthanized and tumor volume was determined. Error bars represent SEM. (A) PBS-treated BHT-101 and DRO tumors averaged 285 and 200 mm³ while PA-L1/LF-treated tumors were 3.7 and 4 mm³, respectively. (B) Representative mice from each tumor type and treatment group are shown. Tumors are indicated by white arrows. *unpaired t test p value <.05.

PA-L1 and PA/LF sensitivity of BHT-101 and DRO cell lines. The anaplastic thyroid carcinoma cell line BHT-101 and the cell line DRO were treated with serial dilutions of PA-L1 or PA keeping LF at a concentration of 5.5 nM and the incorporation of [³H]-thymidine was measured after 48 h. Data is expressed as a percent of the untreated cells [³H]-thymidine incorporation. Sensitivity was arbitrarily set at an IC₅₀ <100 pmol/L with <10% of control [³H]-thymidine incorporation at 10 nM PA-L1/PA in combination with 5.5 nM LF. (A) BHT-101 (■) was extremely sensitive to PA-L1/LF (IC₅₀ 20 pmol/L with 8.7% [³H]-thymidine incorporation at 10 nM PA-L1/5.5 nM LF) while the cell line DRO (▲) was relatively resistant to PA-L1/LF (IC₅₀ 1255 pmol/L with 18.3% [³H]-thymidine incorporation at 10 nM PA-L1/5.5 nM LF). (B) The PA-L1/LF sensitive BHT-101 (■) was also sensitive to PA/LF (IC₅₀ 18 pmol/L with 9.7% [³H]-thymidine incorporation at 10 nM PA/5.5 nM LF) (Fig. 1B). In comparison, DRO (▲) exhibited an 18.6-fold enhanced resistance to PA/LF (IC₅₀ 334 pmol/L with 1.5% [³H]-thymidine incorporation at 10 nM PA/5.5 nM LF).

PA-L1/LF inhibits tumor cell proliferation and vascularization. (A) PBS and PA-L1/LF-treated BHT-101 and DRO tumor cell Ki-67 expression was compared. PBS-treated BHT-101 tumors were found to have 40% tumor cells positive for Ki-67 while tumors treated with PA-L1/LF were reduced to 21.4%. PBS-treated DRO tumors were 56.6% Ki-67 positive while PA-L1/LF treatment was found to reduce DRO Ki-67 staining to 19.1%. (B) Representative Ki-67 immunohistochemical stains are shown from PBS or PA-L1/LF-treated BHT-101 and DRO tumors. PBS-treated tumors demonstrated uniform Ki-67 staining while PA-L1/LF-treated xenografts had Ki-67 positive cells on the periphery of the tumor mass. (C) Tumor vascularization was determined via CD31 staining. PBS-treated BHT-101 and DRO tumors exhibited a mean vascularization of 35.1 and 27.1 vessels per field of view (0.6mm²), respectively. PA-L1/LF treatment induced an 83.8 and 81.2% decrease to an average of 5.7 and 5.1 vessels per 0.6 mm² in BHT-101 and DRO tumors. (D) Vessels of PBS-treated BHT-101 and DRO xenografts were long, tortuous, and disorganized in nature with observable lumens (inset). In contrast, PA-L1/LF-treated xenografts exhibited poor tumor neovascularization. Vessels that were present were found to be short in length, extremely under developed, and had negligible lumen diameters. Error bars represent SEM. * unpaired t test p value <.05. Scale = 100 μm, total magnification 100x.

PA-L1/LF treatment significantly improves long term survival of mice bearing ATC orthotopic xenografts. Long-term survival of mice bearing BHT-101 and DRO orthotopic tumors was determined. (A) Mice bearing BHT-101 (black solid line) and DRO (black dashed line) receiving PBS alone showed rapid disease progression with no survivors remaining past day 32 in either cell line. PA-L1/LF treatment improved the long term survival of mice with BHT-101 (gray solid line) and DRO (gray dashed line) tumors 1.59 and 1.52-fold improvement in mean survival from 27.5 to 43.8 days and 23.7 days 36.1 days, respectively. (B) Mean survival of vehicle-treated mice with BHT-101 (black solid line) and DRO (black dashed line) xenografts was 23.6 and 24.3 days. Daily therapy with 60 mg/kg sorafenib for 30 days increased mean survival 1.82 and 1.46-fold to 43 and 35.7 days for BHT-101 (gray solid line) and DRO (gray dashed line) xenografts, respectively. One BHT-101 xenograft mouse treated with sorafenib demonstrated survival to 55 days. (C) Improvements in long term survival by PA-L1/LF and sorafenib were subsequently compared. Long term survival of mice bearing BHT-101 (light gray bars) and DRO (dark gray bars) tumors treated with PA-L1/LF were significantly improved from mice treated with PBS as well as mice treated with sorafenib versus vehicle alone. However, differences in long term survival between sorafenib and PA-L1/LF were not significantly different. Error bars represent SEM.* unpaired t test p value <.05.

PA-L1/LF treatment exhibits dramatic anti-tumor activity in late stage disease. Mice bearing well-established BHT-101 or DRO tumors were treated with either 1, 2, or 4 doses of 30 μg PA-L1/10 μg LF. Animals receiving multiple doses were treated every other day. After the last scheduled dose, the animal was euthanized and tumor responses were compared to animals not receiving therapeutic intervention. H&E staining demonstrates extensive necrosis in both BHT-101 and DRO tumors (A). Scale = 20 μm, total magnification 400x. (B) BHT-101 (light gray bars) necrosis increased to 43.5% with a single dose of PA-L1/LF while DRO (dark gray bars) necrosis increased 41.5%. Both tumors exhibited ≥98% necrotic area with two doses of 30 μg PA-L1/10 μg LF. (C) BHT-101 (light gray bars) and DRO (dark gray bars) tumors exhibited a corresponding 13.9 and 37.9% decrease of Ki-67 expression, respectively. Two doses of 30 μg PA-L1/10 μg LF induced essentially 100% senescence of tumor cell proliferation and no significant change with 4 PA-L1/LF doses. (D) Representative DRO tumors from each dose number are shown. Error bars represent SEM. *unpaired t test p value <.05. Scale = 100 μm, total magnification 100x

PA-L1 activation, LF internalization, and ERK1/2 inhibition in BHT-101 and DRO cell lines. (A) BHT-101 and DRO cells were treated with either 90 nM LF-β-Lac alone or in combination with 26 nM PA/PA-L1. Cells were subsequently loaded with the β-Lactamase enzyme substrate, the membrane-permeable fluorogenic dye CCF2/AM, and subjected to flow cytometry analysis. Intact CCF2/AM emits fluorescence at 520 nm (green) due to intramolecular fluorescence resonance transfer between 7-hydroxycoumarin and flourescin, while LF-β-Lac-mediated CCF2/AM hydrolysis will cause an emission at 447 nm (blue) from the liberated donor coumarin. Tumor cell PA/PA-L1 activation and subsequent LF-β-Lac internalization will result in blue light emission, while cells that do not internalize LF will emit green fluorescence. Error bars represent SEM. BHT-101 cells were 72 and 97% positive for blue fluorescence when treated with either 26 nM PA or PA-L1 in combination with 90 nM LF-β-Lac, respectively. PA/LF-β-Lac-treated DRO cells were ≥98% positive for intracellular LF-β-Lac activity while cells treated with PA-L1/LF-β-Lac had negligible blue fluorescence. (B) PA and PA-L1/LF-mediated MEK1 and MEK2 cleavage was determined. Treatment of BHT-101 cells with 10 nM PA/PA-L1 in combination with 5.5 nM LF for 16 h exhibited ≥75 and 74% MEK1 cleavage and ≥95 and 96% MEK2 cleavage. DRO cells treated with PA/LF exhibited complete MEK1/2 cleavage while PA-L1/LF treatment induced a 57 and 31.8% reduction in MEK1 and MEK2, respectively. Treatment with 10 μM sorafenib did not reduce total MEK1/2 levels. (C) Pretreated serum starved BHT-101 and DRO cells were exposed to 30 ng/ml EGF for 15 min in order to determine ERK1/2 activation levels. The multi-kinase inhibitor 10 μM sorafenib was included as phospho-ERK1/2 positive control BHT-101 cells treated with 10 μM sorafenib or 10 nM PA/PA-L1 in combination with 5.5 nM LF reduced ERK1/2 phosphorylation by ≥ 99%. sorafenib and PA-L1/LF-treated DRO cells resulted in a modest 27 and 25.1% decrease in ERK1/2 phosphorylation, respectively. Complete ERK1/2 inhibition was observed with 10 nM PA/5.5 nM LF.