Browsing by Author "Nief, Corrine Audrey"

Breast 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.