A cell-delivered and cell-activated SN38-dextran prodrug increases survival in a murine disseminated pancreatic cancer model

Abstract

Enzyme-activated prodrugs have been investigated and sought after as highly specific, low-side-effect treatments, especially for cancer therapy. Unfortunately, excellent targets for enzyme-activated therapy are rare. Here a system based on cell delivery that can carry both a prodrug and an activating enzyme to the cancer site is demonstrated. Raw264.7 cells (mouse monocyte/macrophage-like cells, Mo/Ma) are engineered to express intracellular rabbit carboxylesterase (InCE), which is a potent activator of the prodrug irinotecan to SN38. InCE expression is regulated by the TetOn® system, which silences the gene unless a tetracycline, such as doxycycline, is present. Concurrently, an irinotecan-like prodrug, which is conjugated to dextran and can be loaded into the cytoplasm of Mo/Ma, is synthesized. To test the system, a murine pancreatic cancer model is generated by intraperitoneal (i.p.) injection of Pan02 cells. Engineered Mo/Ma are loaded with the prodrug and are injected i.p. Two days later, doxycycline was given i.p. to activate InCE, which activated the prodrug. A survival study demonstrates that this system significantly increased survival in a murine pancreatic cancer model. Thus, for the first time, a prodrug/activating enzyme system, which is self-contained within tumor-homing cells and can prolong the life of i.p. pancreatic tumor bearing mice, is demonstrated.

Figure 1. SN38 loading of monocyte-like cells

A+B. Monocyte-like cells were incubated overnight with 10 mg/mL of SN38-dextran. The medium was then removed and cells were washed with PBS and fresh medium was added. A. bright field and B. fluorescence with DAPI filter were taken at 20X magnification. C+D. Control monocyte-like cells were incubated without dextran overnight. The medium was then removed and cess were washed with PBS and fresh medium was added. C. bright field and D. fluorescence with DAPI filter were taken at 20X magnification. Scale bars are 100 mm.

Figure 2. Toxicity of SN38-dextran prodrug

Cells were incubated overnight at varying concentrations of SN38-dextran. In the morning the medium was removed, the cells were washed in PBS, and fresh medium was added. An MTT assay was then run to determine cell viability.

Figure 3. Monocyte-like cells home to tumor tissue but not healthy tissue

Monocyte-like cells, treated with PKH26 were injected i.p. to Pan02 tumor bearing mice. A: dark field fluorescence from pancreatic tumor tissue three days after monocyte-like cells injection, magnification 10x. B: H&E staining of serial sections of the tissue above. C-F: dark field fluorescence from healthy tissue three days after monocyte-like cell injection, magnification 20x, C. Lung; D. Pancreas; E. Kidney; F. Liver. Blue is DAPI nuclear staining and orange is PKH26 fluorescence (monocyte-like cells). All scale bars are 100 mm.

Figure 4. Duration to clinical symptoms (survival)

Mice were treated as described and were monitored every twelve hours using the body condition scoring system. Mice were euthanized when they scored 2 or less and the day/time was recorded (n=5 or 6 for each group). P<0.05 for SN38 treatment versus all other groups.

Figure 5. Model of developed system

(1) DS monocyte-like cells were loaded with SN38-Dextran prodrug and injected intraperitoneally into mice bearing Pan02 tumors. The monocyte-like cells specifically infiltrated the tumors. (2) Three days after injecting monocyte-like cells, doxycycline was injected intraperitoneally. The doxycycline disables the tet repressor, activating the InCE which cleaves the prodrug into active SN38. The SN38 is then released from the cell and taken up by nearby cancer cells.

Figure 6. Synthesis of SN38-dextran prodrug

200 mg of dextran (1, M_w=70 kDa) was reacted with succinic anhydride (2) in the presence of lithium chloride to give succinic acid-modified dextran (3). (3) was then reacted with SN38 after activation with EDC and 4-DMAP to give SN38-linked dextran (4).