Development of an Injectable Ablative Therapy for Resource-Limited Settings: Applications in Tumor Ablation

Although two-thirds of the global cancer mortality burden is predicted to occur in low- and middle-income countries (LMICs), citizens of these countries have disproportionately less access to resources and facilities to provide effective care. Surgery, radiation therapy, and chemotherapy form the foundation of effective cancer care in high-income countries (HICs), but these modalities are largely unavailable in LMICs. Stemming from this disparity, long-term cancer survival rates are lower, and the mortality-to-incidence ratio is higher in LMICs. With limited healthcare spending and a large portion of expenditures out-of-pocket, non-communicable diseases such as cancer lead to financial catastrophe for millions of families annually and are a barrier to global development. To expand global access to cancer care and buttress the anti-cancer capabilities of overextended healthcare systems in LMICs, it is necessary to develop a therapy compatible with the constraints imposed by resource-limited settings.

To accomplish this goal, the work presented here describes a low-cost injectable ablative therapy suitable for widespread use in LMICs. This therapy is a modification of an existing technique entailing intratumoral injection of ethanol to induce necrosis of malignant cells (termed “ethanol ablation”) utilized to reduce tumor volume with either curative or palliative intent. Modifications are based on analysis of the mechanics of the injection process and entail the incorporation of the water-insoluble, ethanol-soluble polymer ethyl cellulose and reduction of the infusion rate and volume. Ethanol ablation is one of the original forms of tumor ablation, treatments in which the tumor microenvironment is altered via chemical or thermal means to destroy malignant tissue, and has achieved widespread clinical success in HICs. It is appealing for use in LMICs because it is low-cost, portable, electricity-independent, and minimally invasive. However, injected ethanol is highly pressurized and forms cracks within tissue leading to excessive leakage and an unpredictable distribution of injected ethanol, poor tumor coverage, and damage to adjacent organs. With the recognition of pressure-induced crack formation as a source of leakage, reducing the infusion rate and volume will improve localization. Further, the incorporation of ethyl cellulose is likely to reduce leakage because it forms a gel upon exposure to the aqueous tissue environment and reduces the permeability of fractured tissue. These innovations are poised to improve upon ethanol ablation while retaining its suitability for use in resource-limited settings.

Three specific aims were proposed to establish crack formation as a limiting factor for efficacy of ethanol ablation, characterize this novel tumor ablation technique and develop a framework for tailoring treatment protocols to specific lesion types and sizes. The first aim described the rheological properties of ethyl cellulose-ethanol and the gelling behavior upon exposure to water and found that reducing the infusion rate and incorporating ethyl cellulose decreased leakage in tissue-mimicking surrogates and improved ablative efficacy in chemically induced squamous cell carcinoma tumors in the hamster oral cavity. The viscosity of ethyl cellulose-ethanol solutions increases with the ethyl cellulose concentration, which has been found to improve localization of injected solutions. Further, as expected from a water-insoluble polymer, gel formation increases with higher ethyl cellulose concentrations and higher water-to-ethanol ratios as well. These findings motivate the use of higher ethyl cellulose concentrations and low infusion volumes, and indicate that gel forms upon injection as water diffuses into and ethanol diffuses away from the injection site.

Tissue-mimicking surrogates composed of agarose were utilized because they are transparent and poroelastic. This makes visualization of injected ethanol feasible in a material that replicates the dynamics of tissue’s mechanical response to infusion. In these surrogates, ethyl cellulose was demonstrated to reduce leakage and increase the distribution volume of injected ethanol, but only at moderate infusion rates. At infusion rates typically used in conventional ethanol ablation (approximately 100 mL/hr), excessive leakage was observed for pure ethanol and ethyl cellulose-ethanol alike. This result, taken in context with the established linear relationship between infusion pressure and rate, suggests that reducing the infusion rate is necessary to localize injected ethanol in addition to incorporating ethyl cellulose.

To demonstrate proof-of-concept of improved therapeutic efficacy, chemically induced oral squamous cell carcinoma tumors in the hamster oral cavity were utilized as they are similar to human primary tumors. Further, since they protrude from the surface of the oral cavity and injected fluid is not confined by adjacent tissue, they are susceptible to leakage and more difficult to treat. To evaluate conventional ethanol ablation in this model, high-rate (100 mL/hr) infusions were performed with an infusion volume 4x greater than the tumor volume. This protocol led to regression of only 4 of 13 treated tumors. However, with the reduction of the infusion rate to 10 mL/hr and infusion volume to a quarter of tumor volume, and the incorporation ethyl cellulose, 7 of 7 tumors regressed completely. In the absence of ethyl cellulose, reduction of infusion rate and volume led to regression of 0 of 5 tumors.

With the characterization of ethyl cellulose-ethanol and demonstration of proof-of-concept in Aim 1, the objective of Aim 2 was to investigate the role of infusion pressure in the mechanics of crack formation, as well as of ethyl cellulose in preventing leakage. Pressure-induced crack formation has been described to occur at a material-inherent critical pressure dictated by the fracture toughness and elasticity and can be quantified as the maximum pressure achieved during the infusion of air. In this aim, transparent tissue-mimicking surrogates were fabricated to match the critical pressure of ex vivo swine liver. To determine the relevance of the critical pressure, infusions were performed with two contrast agents dissolved in ethanol– one smaller than the surrogate pore size (fluorescein) and one larger (graphite). When the agarose pore structure was unfractured, only fluorescein was visible. After it was fractured, both contrast agents were visible. Using this system, fracture was observed to occur at the critical pressure and a modified technique to detect fractures via infusion pressure was established. While previous studies have demonstrated that fracture can be observed during the infusion, this is only possible with low-viscosity fluids unlike ethyl cellulose-ethanol. In these studies, it was demonstrated that unfractured agarose retains an elevated post-infusion pressure, but fractured agarose allows the pressure to dissipate rapidly. This result allows for non-invasive detection of crack formation in tissue during infusion of viscous fluids.

In ex vivo swine liver, as was the case in tissue-mimicking surrogates, crack formation was detected when the critical pressure was exceeded and increased leakage. In these studies, the injected ethanol distribution was determined by adding fluorescein to the injection solution, freezing tissue after the infusion, sectioning it, and imaging with a fluorescent microscope. Since the infusion pressure increases with rate and volume, this finding motivates the use of low rates and volumes when possible to improve localization. For low-volume infusions in which the pressure remained below the critical pressure, there was minimal leakage. While leakage, and the infusion pressure, increased with infusion rate (from 1 to 10 mL/hr) for pure ethanol, it did not increase for 6% ethyl cellulose-ethanol. The gel formation behavior of ethyl cellulose reduces leakage in the presence of infusion-induced cracks.

Having established proof-of-concept of ethyl cellulose-ethanol and its mechanism of action in localizing injected ethanol, the focus of Aim 3 was to characterize computed tomography (CT) imaging as rapid, non-destructive method to visualize injected ethanol, optimize the ethyl cellulose concentration, and investigate the relationship between the injected ethanol distribution and resultant extent of induced necrosis. Since ethanol is less attenuating of x-rays than water or tissue, it is readily visible with CT imaging. However, the accuracy of extraction of ethanol concentration from CT imaging has not yet been established. Utilizing ethanol-water mixtures as in vitro surrogates, the random and systematic components of measurement error were quantified, with the combined error defined as the root sum square of both components. The random error component arises from the variance of the radiodensity of a solution of fixed concentration. The systematic error component was quantified as the difference between the predicted and true radiodensity of ethanol-water mixtures, with the predicted value determined by a linear two-point calibration equation with pure water and ethanol at the extremes. The total measurement error was 13.4% with both components contributing approximately equal amounts. This error is low enough to confidently delineate between treated and untreated tissue.

Having established the utility of CT imaging to quantify the ethanol distribution volume, the ethyl cellulose concentration was optimized in ex vivo rat liver tissue submerged in buffer over a wider range of concentrations than has been feasible in previous models. The optimal ethyl cellulose concentration was defined as the formulation that maximized the volume of tissue infiltrated with a cytotoxic (> 20%) ethanol concentration. In these studies, 12% ethyl cellulose maximized the ethanol distribution volume by 8-fold in comparison to pure ethanol. It also led to the most spherical distributions as defined by the aspect ratio quantified as the ratio of the radius of gyration to the effective radius. These results were confirmed in in vivo rat liver in which 12% ethyl cellulose-ethanol yielded a distribution volume 3-times greater than pure ethanol.

In addition to improving localization of injected ethanol, 12% ethyl cellulose increased the extent of induced necrosis by 6-times in comparison to pure ethanol. Necrosis was quantified by excising treated tissue 24 hours post-ablation, cryopreserving, sectioning, and staining it with NADH-diaphorase. There was an approximate one-to-one equivalence of the ethanol distribution volume with the necrotic volume for 12% ethyl cellulose-ethanol. This validates the concentration-based thresholding strategy utilized to determine the ethanol distribution volume and confirms the utility of CT imaging. CT imaging is particularly appealing to assess the morphology of the ablative extent as three-dimensional reconstruction of the ablative extent from pathology is challenging. The equivalence between the distribution volume visualized with CT imaging and necrotic volume determined via pathology motivates further use of CT imaging in optimization of the ablation parameters. Pure ethanol had a necrotic volume of nearly half of the injected ethanol volume. While the comparison of this relationship between pure ethanol and 12% ethyl cellulose-ethanol was not statistically significant, it is indicative of prolonged exposure time achieved by ethyl cellulose that may be caused by delayed vascular clearance in vivo. This aim establishes CT imaging with concentration-based thresholding as a non-destructive, high-throughput method to optimize ablation parameters and tailor treatment to specific lesion types and sizes.

In conclusion, the objective of this work was to establish ethyl cellulose-ethanol ablation as an effective tumor ablation technique suitable for use in resource-limited settings with the goal of expanding global access to cancer treatment. In pursuit of this goal, aim 1 assessed the rheological and gelling behavior of ethyl cellulose-ethanol, established improved localization, and demonstrated proof-of-concept in treatment of chemically induced oral tumors. Aim 2 investigated the relationship between crack formation and infusion pressure, adapted an established model to detect crack formation by demonstrating that post-infusion pressure dissipation is characteristic of fractured tissue, and found that ethyl cellulose decreases leakage when cracks do form. Finally, aim 3 characterized the ethanol concentration measurement accuracy of CT imaging, optimized the ethyl cellulose concentration, and investigated the relationship between ethanol distribution volume and the resultant extent of induced necrosis. Ultimately, this work demonstrates that ethyl cellulose reduces leakage associated with ethanol ablation, improves therapeutic efficacy, and establishes a methodology for further optimization and to tailor treatment for specific applications.