Ethanol ablation reliably achieves an anti-metastatic response after modulating tumor acidity and regulatory T cells

Abstract

<h3>Objectives:</h3> Ethylcellulose (ECE) injections are a low-cost, accessible ablation method for patients with localized tumors. In order to increase the immunogenicity of the tumor microenvironment in triple-negative breast cancers, cyclophosphamide (cyclo) and sodium bicarbonate (bicarb) were used to prime the immune system prior to ECE injections in pre-clinical models of cancer. Cyclo specifically depletes pro-tumor regulatory T cells when once at a low-dose of 200 mg/kg (human equivalent dose of 16 mg/kg). Daily oral bicarb has been shown to reduce tumor pH and enhance infiltration of cytotoxic T cells into the tumor. <h3>Methods:</h3> Immunocompetent BALB/c mice were injected with orthotopic triple-negative (P53-null) breast cancer cell lines (4T1, 67NR). Tumor-bearing mice were treated with combinations of ECE, cyclo, and bicarb. On day 7 after tumor inoculation bicarb at 200 mM was added to the drinking water, on day 9 cyclo at 200 mg/kg IP was given, and on day 10 ECE ablation was performed with an intratumoral injection of 150 µL. Tumor growth rates, overall survival, and metastatic burden after treatment were quantified (n=5-7). To quantify metastatic disease, lungs were harvested at day 60 or earlier if the humane tumor burden limit of 2 cm³ was exceeded. H&E was used to quantify the metastatic burden in whole lung sections. <h3>Results:</h3> In both 4T1 and 67NR pre-clinical models, the combination of ECE+cyclo+bicarb significantly improved survival (P=0.005, P=0.01) and reduced metastases (P=0.02, P=0.003). In the 4T1 tumor model (Fig. 1A), tumors responded following the initial inflammatory response from ablation when primed with cyclo+bicarb resulting in increased overall survival (Fig. 1B). More mice were without evidence of disease at day 60 after receiving ECE+cyclo+bicarb (5/7) than sham+cyclo+bicarb (0/6), ECE alone (0/5), and sham alone (0/6). 4T1 is highly metastatic, shown in representative whole lung H&E (Fig. 1C); however, the addition of ECE to cyclo+bicarb significantly decreased the metastatic burden at death (Fig. 1C, P<0.05). The combination of ECE+cyclo+bicarb resulted in significantly increased survival compared to all other combinations in both 67NR and 4T1 tumor models (P<0.05). <h3>Conclusions:</h3> The addition of cyclo+bicarb to ECE produced a potent systemic anti-tumor response reducing the size of the primary tumors and metastases <i>in vivo.</i>