Browsing by Subject "Tumor ablation"
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Item Open Access Designing a Low-Cost Cancer Therapeutic with Ethanol Ablation and Immunomodulation(2021) Nief, Corrine AudreyBreast cancer outcomes globally are dependent on access to advanced operating room technology and radiation therapy facilities. In low-income countries, 90% of patients cannot access either radiation or surgery due to a lack of infrastructure, medical specialists, and funds. Therefore, there is a dire need for effective, resource-appropriate technology to improve cancer care in the absence of radiation and surgery, particularly for breast cancer which is the most common cancer in women globally.Breast cancer is a disease with a significant disease burden in both low- and high-resource settings. In both settings, breast cancer is fundamentally treated based on the degree of spread. Non-metastatic, focal tumors are treated with "local" therapy with or without additional "locoregional" therapy based on the degree of local invasiveness. When invasive tumors are found, treatment must include "systemic" immuno- or chemo-therapy as there is a presumed presence of circulating tumor cells. Some treatments, like radiation, occasionally incite an "abscopal effect" whereby tumor death in situ exposes tumor-associated antigens (TAAs), eliciting systemic, immune-mediated destruction of distant tumors; however, this mechanism remains elusive. An alternative "local" cancer therapy, ablation, involves focal destruction of tissue using a small instrument delivered under the skin with image guidance. Various ablation modalities (cryotherapy and radiofrequency ablation) have been observed producing the aforementioned abscopal effect because the necrotic/apoptotic tumor milieu remains in situ, activating tumor-specific cytotoxic T cells. Ablation with ethanol is particularly suited for low-resource settings as it can be performed with only a needle and syringe and may be guided with minimal imaging (ultrasound). Ablation with ethanol has been extensively used for hepatocellular carcinomas, and even though it is fast and effective, the injection of liquid ethanol into a dense tumor is difficult to control. Currently, ethanol ablation often requires multiple treatment sessions for residual or recurrent tumors. Here I utilized the phase-changing formulation of ethanol and the polymer ethylcellulose to increase coverage of a target ablation zone and produce a greater anti-tumor response. Previously, our lab has shown that ethyl cellulose-ethanol (ECE) ablation could more efficiently ablate superficial hamster cheek-pouch tumors than pure ethanol. However, a treatment strategy for breast cancer in low-HDI settings must address invasive disease which previous work with ECE had yet to address. In low-HDI settings, where there is less access to diagnostic services, many patients present with advanced disease. Even in high-HDI settings, current treatment options fall short for patients with recurrent or metastatic breast cancer. The goal of the dissertation was to develop a low-cost, easily accessible method for treating invasive breast cancers. To achieve this goal, I attempted to produce a reliable abscopal response using ECE ablation and other easily accessible drugs. I first optimized ECE ablation for use in a mouse breast cancer model, finding the maximum tolerable dose, and optimizing target tissue necrosis by modulating ethylcellulose concentration. I then characterized the local and systemic immune response to ECE ablation in several tumor models to identify a strategy for improving anti-tumor responses. To enhance the likelihood of an abscopal effect after ECE, I then utilized cyclophosphamide (CP) and buffer therapy to reverse tumor microenvironment (TME) hostility. Oral sodium bicarbonate buffer therapy (bicarb) reduces tumor acidosis and has been shown to increase cytotoxic T lymphocyte (CTL) infiltration into tumors and decrease CTL anergy. CP, a widely accessible chemotherapy, has immunomodulatory effects when used at low, non-curative doses, specifically depleting pro-tumor regulatory T cells. I demonstrated that an anti-tumor response after ECE ablation is more likely in a tumor primed with sodium bicarbonate and low-dose CP. I will refer to this combination treatment as ECE + CP + bicarb. To optimize the treatment and demonstrate efficacy, small animal tumor models were utilized to determine in vivo anti-cancer responses. Both non-metastatic and metastatic models were utilized to determine both the local and systemic response to the new therapeutic ECE + CP + bicarb to understand for which types of breast cancer this therapy was appropriate. Aim 1) Maximize breast tumor necrosis using ethanol ECE injections. First, I optimized ECE delivery to increase target tissue necrosis while minimizing adverse events and tumor growth. I used various dosing schedules to determine the maximum tolerable ECE dose in murine 67NR flank tumors, which is 6 mL/kg or 150 µL for a 25 g mouse. The concentration of ethylcellulose in ECE was modulated to determine the role of the phase-changing polymer on the target tissue ablation. I found that 6% ethylcellulose produced the most tumor necrosis and injectate retention at the injection site, thus 6% ECE was selected as the optimal concentration for these non-metastatic 67NR tumors. I also demonstrated that compared to ethanol alone, ECE improves the ablation zone's compactness and decreases local adverse events due to ethanol leakage. Using Raman spectroscopy through ex vivo tissue, I found that ECE slows ethanol diffusion through 67NR tumors compared to pure ethanol alone. Finally, I demonstrated that ECE improves long-term survival compared to an injection of the same volume of pure ethanol in murine tumors. While I developed a method of ECE that able to reduce primary tumor growth in a non-metastatic model, the local and systemic effect of ECE was still unknown. Aim 2) To characterize the local and distant immune response to ECE. To develop a therapy capable of treating invasive breast cancer, our goal was to create a systemic anti-tumor immune response initiated by tumor ablation. However, the immune response to ECE ablation had yet to be characterized. By comparing an injection of ECE to an injection of the same volume of saline in a mouse tumor model, the effect of ECE could be monitored. In this aim I demonstrated that ECE increases tumor-infiltrating lymphocytes in several models, including chemically-induced and cell-line derived tumors. Additionally, in mice lacking CD8+ T cells, the anti-tumor response of ECE was significantly reduced when compared to immunocompetent mice, suggesting reliance on CD8+ T cell immunity. In the metastatic 4T1 model, ECE increased splenic populations of activated CD8+ T cells and decreased the number of splenic CD11b+Ly6G+Ly6C+ neutrophils. Finally, I discovered that after a single ECE injection, the number of metastases were decreased compared to saline injections and standard of care treatment: surgical excision. Local ECE ablation was found to produce local and systemic immunomodulation favoring an anti-tumor immune phenotype; however, most primary tumors never completely regressed. Therefore, the readily-accessible, low-cost agents CP and bicarb were implemented to further enhance the anti-tumor immune response following ECE ablation. Aim 3) Enhancing ECE with readily-accessible, low-cost immunomodulatory agents. In Aim 2 the immune response to ECE ablation was characterized, however, it was not strong enough to cure animals with invasive TNBC. I hypothesized that ECE ablation was insufficient to cure malignant TNBC due to the highly immunosuppressive TME. Two methods for reducing TME immunosuppression were employed: low-dose CP and oral bicarb therapy. A single low-dose of CP was utilized to deplete Tregs before ablation. Bicarb was ingested by mice for the duration of the study to decrease tumor acidosis and increase the infiltration of anti-tumor T cells into the TME. TNBC cell lines with a range of natural immunogenicity were utilized to test the efficacy of ECE + CP + bicarb including 4T1, 67NR and EO771. The combination of ECE + CP + bicarb eradicated a majority of tumors, eliminating primary tumors and metastatic disease for most animals. Furthermore, the anti-tumor response was found to have a CD8+ T cell-dependent manner in EO771 tumors. In all three cell lines, mice cured with ECE + CP + Bicarb experienced a reduced tumor growth rate when re-challenged with a tumor. When surgery was used instead of ECE ablation, the antimetastatic effect was reduced implying that the in situ necrosis left by ECE ablation is crucial for the systemic anti-tumor response. In summary, I successfully created a novel anti-cancer therapeutic using ECE + CP + bicarb that is effective in aggressive TNBC tumors. The work in these Aims laid a foundation for the use of ECE ablation in breast tumors. A safe and effective dosing strategy was identified in small animal models, as well as methods for boosting the anti-tumor response to ECE ablation. An anti-tumor response to ECE ablation was identified along with the antimetastatic properties of local ECE ablation. These findings provoke many new research questions about the interplay of acidosis, wound healing, inflammation, and necrosis in the TIME and how they affect systemic disease progression. ECE ablation still requires much more investigation to reach the ultimate goal of impacting patient outcomes. For example, the mechanism for the anti-tumor and anti-metastatic response has yet to be fully elucidated. The work here suggests that CD8 T cells are implicated in the therapeutic response; however, the impact of ECE ablation on other crucial players in the TIME (myeloid cell populations, tumor metabolism, hypoxia, and the extracellular matrix) are largely unknown. Additionally, since the therapeutic power of ECE + CP + bicarb does not rely on specific tumor biomarkers, ECE + CP + bicarb could be effective in other tumor types. Specifically, we are interested in using ECE for cervical cancer which disproportionately affects low-HDI settings resulting in significant mortality globally. Another strategic use of the immunomodulatory effect of ECE is in combination with immunotherapies. ECE ablation induces a local inflammatory response and releases necrotic tumor debris that may increase the strength of the response to checkpoint inhibitors. Future research is needed to assess these new combinations.
Item Open Access Development of an Injectable Ablative Therapy for Resource-Limited Settings: Applications in Tumor Ablation(2020) Morhard, RobertAlthough 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.