Inducing a double hit to tumors using intratumoral drug delivery with local ablation

Introduction:: Intratumoral drug administration is of great interest to clinicians because it allows for delivery of higher doses of drugs at the tumor site than systemic delivery while simultaneously reducing systemic toxicity. A major limitation of direct injection of chemotherapeutic drugs is tumors’ high interstitial pressure, leading to leakage of the drug away from the target site, reducing the concentration of the drug within the tumor limiting efficacy, and exposing healthy tissue to the drug, potentially leading to dangerous local side effects. To reduce leakage outside of the tumor, we have developed a novel ablation system consisting of ethyl cellulose and ethanol (ECE). ECE has two unique characteristics compared to other drug loading systems. First, ethyl cellulose, which is generally recognized as safe (GRAS) by the FDA, is soluble in ethanol but insoluble in water, creating an ethanol-rich gel when injected into tissue. The high viscosity of the gel prevents leakage away from the target site and maintains a high concentration of ethanol within the tumor. Secondly, incorporation of ethanol disrupts the cell membrane, permeabilizing the cells inducing necrosis. Therefore, ECE can be loaded with chemotherapeutic drugs to generate a double hit to tumors: necrotic cell death from ECE and apoptotic cell death from chemotherapy. Our objective was to demonstrate the safety and efficacy of chemotherapy-loaded ECE through a biodistribution study looking at chemotherapy concentrations in the heart, kidney, liver, lung, spleen, and tumor, as well as a long term survival studies in a murine model of breast cancer.


Materials and Methods:: To demonstrate that chemotherapy-loaded ECE is safe and reduces systemic distribution of chemotherapy, we performed an in vivo biodistribution study. 4T1-Luc murine breast tumors were established in 5-6-week-old female BALB/c mice. Mice were randomized to one of three groups (n=10 mice per group): ECE + local doxorubicin, ECE + systemic doxorubicin, or saline injection (no ECE) alone. Tissue (heart, kidney, liver, lung, spleen, and tumor) was collected 4 hours after treatment and homogenized for analysis with IVIS imaging to quantify fluorescence from doxorubicin. Four-hours was chosen as doxorubicin has a rapid uptake in tissue (uptake half-life of 5 minutes according to the FDA) and long elimination half-life (20-48 hours). Fluorescence results were compared using an analysis of variance (ANOVA) with Tukey HSD post-hoc testing.



Improved survival was demonstrated using the same 4T1-Luc murine breast tumor model as the biodistribution study. Mice were randomized to one of eight groups (n=~10 mice per group): saline injection (no ECE) alone, saline with local doxorubicin, or saline with systemic doxorubicin at 2 doses; ECE injection alone, ECE with local doxorubicin, or ECE with systemic doxorubicin at 2 doses. Treatment began when tumors were palpable and ~50 µL in volume (day 10 of study). Tumor volume was measured every 3 days following treatment until a humane endpoint or tumor burden was reached. Humane endpoints that occurred before tumor volume were recorded as adverse events. Overall survival was compared using a Log-rank test.


Results, Conclusions, and Discussions:: Results



The time course for the biodistribution study demonstrating the safety of chemotherapy-loaded ECE is shown in Figure 1A. Figure 1B shows representative IVIS images of the homogenized tumor, spleen, kidney, liver, lung, and heart for each treatment group 4 hours after treatment. Figure 1C shows quantification of the doxorubicin fluorescence intensity. There were no significant differences in doxorubicin fluorescence within the heart, kidney, liver, and spleen; however, there was a significant increase in doxorubicin fluorescence within the lungs from mice treated with ECE + systemic doxorubicin compared to saline control. Further, there was a significant increase in doxorubicin fluorescence within the tumors of mice treated with ECE + local doxorubicin compared to ECE + systemic doxorubicin and saline control. There was no significant difference in doxorubicin fluorescence within the tumors of mice treated with ECE + systemic doxorubicin and saline control.



Long term tumor growth and survival were examined in BALB/c mice with orthotopic 4T1-Luc tumors according to the timeline in Figure 1D. Figures 1E and 1F show the tumor growth curves and overall survival for control and treated mice. ECE + local doxorubicin achieved the longest tumor growth delay and overall survival. Overall survival for ECE + local doxorubicin was significantly longer than treatment with the same doxorubicin dose delivered systemically or locally. Statistics for comparisons with saline and ECE + local doxorubicin are shown to the right of the legend, with significant differences seen between groups with different letters. Mice receiving systemic doxorubicin had higher toxicity resulting in early euthanasia, further corroborating the biodistribution study demonstrating that the local delivery is not only more effective, but also safer.



Conclusions

Chemotherapy-loaded ECE reduces systemic chemotherapeutic distribution and increases chemotherapy concentration within the tumor, which translates into delayed tumor growth and improved overall survival.



Discussions

Cornerstone cancer treatments like surgery and radiation therapy are often not accessible in countries with lower human development indices (HDI), however, chemotherapies like doxorubicin are on the WHO essential medicines list and are often available. ECE loaded with chemotherapeutics has potential to be an effective treatment for accessible cancers, particularly in lower HDI countries.


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