Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the fourth-leading cause of all cancer-related deaths around the world. Liver transplantation, surgery, and local ablation are curative therapies for early-stage HCC. However, post-treatment outcomes can vary based on histopathologic stage. Poorly-differentiated HCC are associated with higher rates of tumor progression and lower overall survival compared to well-differentiated HCC after therapy. In this study, we aimed to characterize the cancer stem cell (CSC) profile of histopathologically-proven well and poorly-differentiated HCCs in an in-vitro environment. We characterized the stem-like profile of each type of HCC based on their surface markers and susceptibility to NK cell-mediated cytotoxicity.Methods: Flow cytometry was used to quantify differential expression of MHC-class I, CD54, and CD44 between well- and poorly-differentiated HCCs. Primary untreated NK cells, IL-2 stimulated primary NK cells, and supercharged (sNK) cell-mediated cytotoxicity was assessed against well- and poorly-differentiated HCCs. IFN-γ supernatant from each respective NK cell experimental arm was also used to induce differentiation of HCCs. Finally, we characterized the temporal NK effector cell cytotoxicity using real-time quantitative analysis of imaging and impedance (eSight study).Results: Poorly-differentiated HCCs demonstrated low surface expression of MHC-class I and CD54, and high expression of CD44. Treatment of NK cells secreted IFN-γ or IFN-γ cytokine induced differentiation in HCCs. Poorly-differentiated HCCs in comparison to well-differentiated HCC were more susceptible to NK cell-mediated cytotoxicity in primary NK cells, IL-2 stimulated primary NK cells, and sNK cells. sNK cells induced significantly higher cytotoxicity against well-differentiated HCCs in comparison to untreated or IL-2-stimulated primary NK cells. These findings were recapitulated with real-time quantitative imaging analysis.Conclusions: Poorly-differentiated HCCs were found to have surface marker patterns of CSCs, making them highly susceptible to NK cell-based immunotherapy. NK-cell based therapy can potentially be leveraged as a neoadjuvant or adjuvant therapy in poorly-differentiated HCCs. Supercharged NK cells, which can be rapidly expanded to therapeutic levels, are uniquely capable of lysing both poorly- and well-differentiated HCCs. This finding suggests that sNK cells not only exhibit enhanced features against NK cells’ targets but also are capable of activating T cells to induce cytotoxicity against well-differentiated HCCs with high expression of MHC class I.

Natural killer (NK) cells are innate lymphoid cells that exhibit high levels of cytotoxicity against NK-specific targets. NK cells also produce various cytokines, and interact with T cells, B cells, and dendritic cells to effectively serve as frontliners of the innate immune system. Produce various cytokines, and interact with T cells, B cells, and dendritic cells to effectively serve as frontliners of the innate immune system. Moreover, NK cells constitute the second most common immune cell in the liver. These properties have drawn significant attention towards leveraging NK cells in treating liver cancer, especially hepatocellular carcinoma (HCC), which accounts for 75% of all primary liver cancer and is the fourth leading cause of cancer-related death worldwide. Notable anti-cancer functions of NK cells against HCC include activating antibody-dependent cell cytotoxicity (ADCC), facilitating Gasdermin E-mediated pyroptosis of HCC cells, and initiating an antitumor response via the cGAS-STING signaling pathway. In this review, we describe how these mechanisms work in the context of HCC. We will then discuss the existing preclinical and clinical studies that leverage NK cell activity to create single and combined immunotherapies.

The goal of this study was to optimize a lung-tuned monopole antenna to deliver microwave energy at 2.45 GHz into a novel ventilator-controlled ex vivo lung model. An analytic\approach was utilized to create an optimized monopole antenna that was impedance-matched to aerated lung tissue. This lung-tuned antenna was then fabricated using a copper 0.085” semi-rigid copper coaxial cable. For validation, the lung-tuned antenna was inserted centrally into lobes of a ex vivo porcine lung that was fully inflated to physiologically appropriate volumes. Microwave ablations were then created at 50 and 100 W for 1 minute and 5 minutes. Reflected power, cross sectional ablation sizes and spherical shape of the lung-tuned antenna were compared against a liver-tuned antenna in the ventilator-controlled ex vivo lung tissue. The study showed that the lung-tuned antennas delivered energy significantly more efficiently, with less reflected power, compared to the conventionally-used liver-tuned antennas at 50 W at 1 minute (11.8 ${\pm }$ 3.0 vs 16.3 ${\pm }$ 3.1 W; p value = 0.03) and 5 minutes (16.2 ${\pm }$ 2.8 vs 19.4 ${\pm }$ 2.9 W; p value = 0.04), although this was only true using 100 W at the 1 min time point (29.0 ${\pm }$ 3.5 vs 38.0 ${\pm }$ 5.3 W; p value = 0.02). While overall ablation zone sizes were comparable between the two types of antenna, the lung-tuned antenna did create a significantly more spherical ablation zone compared to the liver-tuned antenna at the 1 minute, 50 W setting (aspect ratio: 0.43 ${\pm }$ 0.07 vs 0.38 ${\pm }$ 0.04; p value = 0.04). In both antenna groups, there was a significant rise in the ablation zone aspect ratio between 1 and 5 minutes, indicating that higher power and time settings can increase the spherical shape of ablation zones when using tuned antennas. Adapting this combined analytic and parametric approach to antenna design can be implemented in adaptive tissue-tuning for real-time microwave ablation optimization in lung tissue.