A novel single-shot therapy for the treatment of breast cancer.

Introduction: Breast cancer is the most prevalent cancer worldwide, with 2.3 million new diagnoses and 685,000 deaths annually. Over the last century, breast cancer treatment has evolved from radical mastectomies to precision immunotherapies, yet the core approach of surgery, radiation, and chemotherapy has remained largely unchanged. Current treatments often require prolonged and recurrent interventions, resulting in significant physical and psychological burdens on patients. We introduce an innovative single-shot therapy using a Drug-Releasing Immune-Activating Polymer (i.e. DRIP) aimed at redefining breast cancer treatment by minimizing systemic toxicity and enhancing tumoricidal activity. The proposed single-shot therapy utilizes ethanol, an anti-neoplastic agent, and ethyl cellulose (i.e. EC) as the scaffold. Ethanol induces necrosis for direct cell kill and ethanol-mediated cell injury releases tumor-associated antigens, alerting the immune system to elicit anti-tumor activity. EC improves injectate localization and reduces off-target leakage of ethanol through in situ gelation. Furthermore, anti-neoplastic drugs can be combined with EC ethanol (i.e. ECE), which upon injection can be sequestered with the ethanol to improve cytotoxic intra-tumoral cell kill while decreasing off-target effects. In addition, we can harness the immunomodulating effects of these drugs. For example, cyclophosphamide decreases the presence of regulatory T cells and doxorubicin increases anti-tumor cytokines IL-10 and TNF-α, which leads to an anti-tumor T cell response. DRIP packages the benefits of cytotoxic cell kill, immunomodulation, and minimally invasive surgery into a single-shot therapy for breast cancer. We evaluated local delivery of doxorubicin in ECE, with or without systemic cyclophosphamide, and systemic delivery of doxorubicin with local ECE.

Materials and

Methods: First, we assessed the effect of ECE on small molecule retention in vitro using a release-into-sink configuration. We prepared 2mL samples containing fluorescent molecular surrogates in anhydrous ethanol with varying concentrations of EC (6%, 10%, 12% w/w). Samples were placed in dialysis tubing within 200mL of water in a beaker on a see-saw rocker (30 osc./min). Serial 2mL water samples were collected from 0 to 360 minutes and analyzed using a spectrophotometer to measure absorbance. For comparison both hydrophobic (fluorescein) and hydrophilic (rhodamine B) molecules were tested. Doxorubicin has a molecular weight of 543 g/mol whereas that of fluorescein and rhodamine B are 332, and 470 g/mol respectively.

Second, we assessed the biodistribution of doxorubicin when administered intratumorally with ECE. Groups of BALB/c mice with 4T1 triple-negative breast tumors were assigned to receive either ECE + local doxorubicin (4mg/kg), ECE + systemic doxorubicin (4mg/kg), or saline. After 4 hours, the mice were euthanized and the heart, lung, liver, kidney, spleen, and tumor were excised. Tissues were minced, homogenized, and then imaged using IVIS.

Third, the efficacy of ECE as a vehicle for intra-tumoral drug delivery was examined in vivo in BALB/c mice with orthotopic 4T1 triple-negative breast tumors. We treated tumors with either saline, 10% w/w ECE with/without a low dose (4mg/kg) of doxorubicin locally, local delivery of doxorubicin (4mg/kg) in saline, systemic delivery of doxorubicin at both low (4mg/kg) and high doses (7.5mg/kg).
Finally, we investigated a combination of ECE ablation with local doxorubicin and systemic cyclophosphamide (200mg/kg).

Results, Conclusions, and Discussions: Higher concentrations of the EC polymer improved the retention of small molecules at the site of polymer phase change (Figure 1). Furthermore, the diffusion of the hydrophobic fluorophore out of the gel was lower than that of the hydrophilic fluorophore (Figure 1). We found a significantly higher doxorubicin fluorescent signal in the tumor for ECE + local doxorubicin at 4 hours post-ablation compared to treatment with either saline or ECE + systemic doxorubicin (Figure 2). Interestingly, we found significantly higher doxorubicin fluorescence in the lungs of mice treated with ECE + systemic delivery compared to saline, indicating significant off-target distribution of systemic doxorubicin (Figure 2). We found that ECE + local doxorubicin was significantly more effective at reducing tumor growth than ablations with the same dose delivered systemically or locally in saline (Figure 3). Additionally, higher systemic doxorubicin doses did not achieve comparable results due to the toxicity of the doxorubicin dose, indicating that the local delivery is not only more effective, but also a safer option. The combination of ECE ablation with local doxorubicin and systemic cyclophosphamide achieved superior results compared to all other treatment groups except ECE + local doxorubicin. Future studies will examine the immunomodulating effects of this drug combination as well as combinations with other anti-neoplastic agents like local cyclophosphamide and paclitaxel.

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