Antitumoral immunity induced by gel ethanol ablation to treat unresectable colorectal cancer metastases in the liver

Introduction: Colorectal cancer is the second leading cause of cancer-related mortality worldwide, and this burden is expected to double by 2040. Approximately half of all colorectal patients develop liver metastases during the course of the disease. While hepatic resection is a curative approach, many of these patients are not eligible for surgery due to severe extrahepatic disease and a lack of adequate postoperative liver volume or subsequent hepatic functionality. Because of these limitations, ablative therapies, in which tumors are locally destroyed, offer a strong alternative treatment option. Ethanol ablation is one such ablative technique, where pure ethanol is directly administered into tumors to induce necrosis. The ethanol, however, tends to leak from the injection site, which reduces efficacy and causes adverse effects. To reduce leakage, we added a polymer ethyl cellulose (EC) to ethanol, which allows for the formation of a gel upon injection into tissue. Our novel formulation of EC-ethanol was initially tested in a preclinical model of carcinoma; all tumors completely regressed after 7 days. While our initial studies show promise, key translational questions remain that will inform how EC-ethanol could be part of a therapeutic strategy to treat colorectal liver metastasis. Recent evidence suggests that some ablative therapies can activate antitumoral immunity against tumor antigens and can be used to fortify the impact of immunotherapies. The immune responses following EC-ethanol ablation, however, remain unclear. Thus, the goal is this work seeks to characterize the immune effects following EC-ethanol ablation in an immunogenic mouse model of colorectal disease.

Materials and

Methods: The study protocol was approved by the National Institutes of Health Clinical Center IACUC in compliance with the U.S. Animal Welfare Regulations (DRD23-04). BALB/c mice were inoculated by subcutaneous flank injection with 1x10^6 CT26 colorectal carcinoma cells suspended in 100 µL Hank’s Buffered Saline Solution. Whole blood was collected shortly afterwards via submandibular bleed, with the serum extracted and cryopreserved. Flank tumors were measured with calipers every 2-3 days. Once tumors reached a volume of 250 mm^3, mice were divided randomly into EC-ethanol ablation cohorts and a sham control group. For the EC-ethanol cohorts, 60 µL of 6% EC-ethanol (w/w) was intratumorally infused with a 27-gauge hypodermic needle at 30 mL/hr infusion rate, all controlled by a programmable syringe pump. Sham procedures were performed by insertion of the needle without drug administration, for the same duration as the treatment cohorts. Six hours after the procedure, blood and serum was collected. After 3 and 7 days, the mice were euthanized by CO2 asphyxiation, and blood was collected via terminal cardiac puncture, and serum extracted. All serum specimens were analyzed via an electrochemiluminescence assay for detecting pro-inflammatory cytokines. All tumors were collected and fixed in 10% neutral-buffered formalin before processing for paraffin-embedded sectioning. 5 µm thick sequential sections were obtained from each tumor and mounted onto glass slides, where histologic examination via hematoxylin & eosin (H&E) staining and immunohistochemistry (IHC) staining for immune markers were performed. All image analyses were performed by a certified veterinary pathologist.

Results, Conclusions, and Discussions: Representative H&E and IHC stains of CD8a and PD-1 for EC-ethanol- and sham-treated tumors are shown in Figure 1. The EC-ethanol group experienced an increase in CD8a+ cytotoxic T-cells (8.3% positive cells) and PD-1 expression (8.7% positive cells) within the ablation zone 3 days post-treatment, compared to that of the sham treatment (3.6% for CD8a and 7.3% for PD-1). Notably, many of the positive-stained cells were situated in the periphery margins of the ablation zone, particularly for CD8a. There was no significant difference in the presence of CD4+ helper T-cells, FOXP3 regulatory T-cells, and F4/80 macrophages within the tumor area.
Select pro-inflammatory serum cytokine levels of the mice are shown in Figure 2. Four distinct time points are described: 1) baseline (0 hours), 2) 6 hours, 3) 3 days, and 4) 7 days post-treatment. For the EC-ethanol group, interleukin-6 (IL-6) experienced significant elevation in expression at 6 hours (38-fold) and 3 days (22-fold) before falling back to baseline levels at day 7; the sham treatment group only saw a modest increase at 3 days (16-fold). Keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO) for the EC-ethanol group experienced similar trends in increased expression at 6 hours and 3 days (8-fold and 5-fold, respectively) before falling back to baseline levels. All other cytokines tested (IFN-γ, TNF-α, IL-1β. IL-2, IL-12p70, IL-5, IL-4, IL-10) did not exhibit significant differences between the two treatment groups.
In conclusion, EC-ethanol treatment of colorectal tumors saw between 5- and 38-fold increase in pro-inflammatory cytokines in serum and mass accumulation of cytotoxic T-cells within the margins of the ablation zone, compared to the sham control, indicating ample antitumoral activity induced by EC-ethanol within the first three days post-treatment. Future studies will implement a bilateral murine flank tumor model to study the strength of the antitumor effects observed in this study to potentially generate abscopal effects.

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