Verticillin A-Loaded Surgical Buttresses Prevent Local Pancreatic Cancer Recurrence in a Murine Model

Abstract

The fungal metabolite verticillin A is a potent and selective histone methyltransferase inhibitor. It regulates apoptosis, the cell cycle, and stress response, and displays potent activity in the suppression of tumor cell growth in several different in vivo models. Verticillin A sensitizes pancreatic cancer cells to anti-PD-1 immunotherapy by regulating PD-L1 expression. However, as with many natural products, delivery and systemic toxicity are challenges that must be overcome to advance their use as a chemotherapeutic. To both reduce systemic toxicity and improve delivery, we report a verticillin A-loaded surgical buttress, which is well-tolerated at a dose as high as 40 mg/kg. In contrast, free verticillin A administered systemically results in toxicity at a dose of 3 mg/kg. The verticillin A-loaded buttress suppresses tumor recurrence in vivo in a safe and dose-dependent manner against a highly aggressive and metastatic model of pancreatic cancer.

Keywords: drug-loaded surgical buttress; histone methyltransferase; local drug delivery; pancreatic cancer; prolonged drug release; verticillin A.

Figure 1

(A) Schematic of the preparation and composition of a verticillin A-loaded surgical buttress. (B) Illustration of pancreatic cancer treatment using a verticillin A-loaded surgical buttress to prevent local recurrence of the disease.

Figure 2

Kinetics of verticillin A release. (A) Daily release rate of verticillin A normalized by the surface area of the loaded buttress and plotted as the amount (i.e., μg) released/cm²/day (Dashed line) and percent released/cm²/day (solid line). (B) Cumulative release profile of verticillin A-loaded buttress over 90 days plotted as the mass of verticillin A (μg, left side) and percent (%, right side) of total loaded verticillin A in the buttress (i.e., 1200 μg). (C) Structure of verticillin A (C₃₀H₂₈N₆O₆S₄). For plots (A) and (B), each time point represents three experimental replicates. Error bars represent the standard deviation.

Figure 3

In vitro cytotoxicity of verticillin A, either alone (A, B) or after release from a verticillin A-loaded surgical buttresses (C, D), against human (A, C) and murine (B, D) pancreatic cancer cells (Miapaca2 and Panc02-H7, respectively). (A) IC₅₀ value of verticillin A against human pancreatic cancer cells (Miapaca-2). (B) IC₅₀ value of verticillin A against murine pancreatic cancer cells (Panc02-H7). (C) Long-term cytotoxicity of verticillin A after release from 1200 and 100 μg verticillin A-loaded buttresses against human pancreatic cancer cells (Miapaca2) over 10 weeks. (D) Long-term cytotoxicity of verticillin A after release from 1200 and 100 μg verticillin A-loaded buttresses against murine pancreatic cancer cells (Panc02-H7) over 10 weeks. Note, in both panels (C) and (D), the unloaded buttress represents the buttress shown in the diagram at the right of Figure 1A, where no test compound was loaded. Table S3 provides more details about how verticillin A was loaded onto each buttress.

Figure 4

Maximum tolerable dose (MTD) study of systemically administered verticillin A vs dose-escalation study of verticillin A-loaded surgical buttresses using nontumor bearing mice. (A, B) Percent of animal’s weight change and survival plotted over 20 days after intraperitoneal injection of verticillin A in a dose range of 0–5 mg/kg. (C, D) Percent of animal’s weight change and survival over 30 days after subcutaneous implantation of verticillin A-loaded surgical buttresses with a dose range of 0–800 μg (i.e., 0 to 40 mg/kg). In all panels, n represents the number of mice per treatment group.

Figure 5

In vivo efficacy study of verticillin A-loaded surgical buttresses. (A) Percent of freedom from local tumor recurrence over 14 days after subcutaneous implantation of 100 μg and 800 μg verticillin A-loaded buttresses (i.e., equivalent to 5 and 40 mg/kg, respectively). (B) Images of the recurred tumors among the treatment groups after 14 days. (C) Percent of tumor recurrence among the treatment groups. (D) Percent of animals’ weight change among the treatment groups over 14 days. In panel (A), n represents the number of mice per treatment group. Note, the color coding in panels (C) and (D) follows the key shown in panel (A).

Figure 6

Histological analysis of harvested liver and kidney tissue of mice treated with gemcitabine or verticillin A (polymer and IP administration). All tissue samples were comparable to the control (unloaded polymer-coated buttresses), except for the liver tissue of mice treated with verticillin via IP (bottom right). The lesions in the tissues (indicated with red arrows) are indicative of hepatotoxicity.