Overcoming Resistance to Immune Checkpoint Blockade in Liver Cancer with Combination Therapy: Stronger Together?

Primary liver cancer, represented mainly by hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA), is one of the most common and deadliest tumors worldwide. While surgical resection or liver transplantation are the best option in early disease stages, these tumors often present in advanced stages and systemic treatment is required to improve survival time. The emergence of immune checkpoint inhibitor (ICI) therapy has had a positive impact especially on the treatment of advanced cancers, thereby establishing immunotherapy as part of first-line treatment in HCC and CCA. Nevertheless, low response rates reflect on the usually cold or immunosuppressed tumor microenvironment of primary liver cancer. In this review, we aim to summarize mechanisms of resistance leading to tumor immune escape with a special focus on the composition of tumor microenvironment in both HCC and CCA, also reflecting on recent important developments in ICI combination therapy. Furthermore, we discuss how combination of ICIs with established primary liver cancer treatments (e.g. multikinase inhibitors and chemotherapy) as well as more complex combinations with state-of-the-art therapeutic concepts may reshape the tumor microenvironment, leading to higher response rates and long-lasting antitumor immunity for primary liver cancer patients.

Resolving 31 colors on a standard 3-laser full spectrum flow cytometer for immune monitoring of human blood samples.

Immune monitoring of patients on a single-cell level is becoming increasingly important in various diseases. Due to the often very limited availability of human specimens and our increased understanding of the immune systems there is an increasing demand to analyze as many markers as possible simultaneously in one panel. Full spectrum flow cytometry is emerging as a powerful tool for immune monitoring since 5-laser instruments enable characterization of 40 parameters or more in a single sample. Nevertheless, even if only machines with fewer lasers are available, development of novel fluorophore families enables increasing panel sizes. Here, we demonstrate that careful panel design enables the use of 31-color panels on a 3-laser Cytek® Aurora cytometer for analyzing human peripheral blood leukocytes, without the need for custom configuration and using only commercially available fluorochromes. The panel presented here should serve as an example of a 31-fluorochrome combination that can be resolved on a 3-laser full spectrum cytometer and that can be adapted to comprise other (and possibly more) markers of interest depending on the research focus.

Intratumoral dendritic cells and T cells predict survival in gastroenteropancreatic neuroendocrine neoplasms.

Clinical management of gastroenteropancreatic neuroendocrine neoplasms remains challenging. We recently introduced the FMS-like tyrosine kinase 3 ligand (FLT3LG) as a possible biomarker for a proinflammatory tumor microenvironment. Here, we put a spotlight on the quantitative assessment of classical dendritic cells (cDC) and T cells in the context of FLT3LG mRNA levels in a retrospective study on neuroendocrine tumor (NET) G2/G3 and neuroendocrine carcinoma (NEC) of pancreatic and gastric origin. The abundance of cDC and T cells and their relevant subpopulations were determined by immunofluorescent staining and correlated with FLT3LG mRNA levels as well as clinical outcomes. Immune cell counts attested to highly variable infiltration densities. Samples with the presence of cDC or high numbers of T cells exhibited increased FLT3LG expression. Abundance of cDC, defined as HLA-DR+CD11c+ cells with CLEC9a (cDC1) or CD1c (cDC2), as well as T cells correlated with FLT3LG mRNA levels and predicted disease-specific survival. Combining FLT3LG and T cell counts further improved this prediction. Therefore, tumor-infiltrating cDC and T cells are prognostic markers in NET G2/G3 or NEC and FLT3LG mRNA may serve as a simple-to-use biomarker for a quantitative estimate of their abundance, mandating prospective evaluation in the context of immune-targeted therapies.

Temporal expression profiles indicate a primary function for microRNA during the peak of DNA replication after rat partial hepatectomy.

The liver has the unique capacity to regenerate after surgical resection. However, the regulation of liver regeneration is not completely understood. Recent reports indicate an essential role for small noncoding microRNAs (miRNAs) in the regulation of hepatic development, carcinogenesis, and early regeneration. We hypothesized that miRNAs are critically involved in all phases of liver regeneration after partial hepatectomy. We performed miRNA microarray analyses after 70% partial hepatectomy in rats under isoflurane anesthesia at different time points (0 h to 5 days) and after sham laparotomy. Putative targets of differentially expressed miRNAs were determined using a bioinformatic approach. Two-dimensional (2D)-PAGE proteomic analyses and protein identification were performed on specimens at 0 and 24 h after resection. The temporal dynamics of liver regeneration were characterized by 5-bromo- 2-deoxyuridine, proliferating cell nuclear antigen, IL-6, and hepatocyte growth factor. We demonstrate that miRNA expression patterns changed during liver regeneration and that these changes were most evident during the peak of DNA replication at 24 h after resection. Expression of 13 miRNAs was significantly reduced 12-48 h after resection (>25% change), out of which downreguation was confirmed in isolated hepatocytes for 6 miRNAs at 24 h, whereas three miRNAs were significantly upregulated. Proteomic analysis revealed 65 upregulated proteins; among them, 23 represent putative targets of the differentially expressed miRNAs. We provide a temporal miRNA expression and proteomic dataset of the regenerating rat liver, which indicates a primary function for miRNA during the peak of DNA replication. These data will assist further functional studies on the role of miRNAs during liver regeneration.

In Situ Vaccination as a Strategy to Modulate the Immune Microenvironment of Hepatocellular Carcinoma.

Hepatocellular Carcinoma (HCC) is a highly prevalent malignancy that develops in patients with chronic liver diseases and dysregulated systemic and hepatic immunity. The tumor microenvironment (TME) contains tumor-associated macrophages (TAM), cancer-associated fibroblasts (CAF), regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) and is central to mediating immune evasion and resistance to therapy. The interplay between these cells types often leads to insufficient antigen presentation, preventing effective anti-tumor immune responses. In situ vaccines harness the tumor as the source of antigens and implement sequential immunomodulation to generate systemic and lasting antitumor immunity. Thus, in situ vaccines hold the promise to induce a switch from an immunosuppressive environment where HCC cells evade antigen presentation and suppress T cell responses towards an immunostimulatory environment enriched for activated cytotoxic cells. Pivotal steps of in situ vaccination include the induction of immunogenic cell death of tumor cells, a recruitment of antigen-presenting cells with a focus on dendritic cells, their loading and maturation and a subsequent cross-priming of CD8+ T cells to ensure cytotoxic activity against tumor cells. Several in situ vaccine approaches have been suggested, with vaccine regimens including oncolytic viruses, Flt3L, GM-CSF and TLR agonists. Moreover, combinations with checkpoint inhibitors have been suggested in HCC and other tumor entities. This review will give an overview of various in situ vaccine strategies for HCC, highlighting the potentials and pitfalls of in situ vaccines to treat liver cancer.

Independent effects of sham laparotomy and anesthesia on hepatic microRNA expression in rats.

BACKGROUND: Studies on liver regeneration following partial hepatectomy (PH) have identified several microRNAs (miRNAs) that show a regulated expression pattern. These studies involve major surgery to access the liver, which is known to have intrinsic effects on hepatic gene expression and may also affect miRNA screening results. We performed two-third PH or sham laparotomy (SL) in Wistar rats to investigate the effect of both procedures on miRNA expression in liver tissue and corresponding plasma samples by microarray and qRT-PCR analyses. As control groups, non-treated rats and rats undergoing anesthesia only were used. RESULTS: We found that 49 out of 323 miRNAs (15%) were significantly deregulated after PH in liver tissue 12 to 48 hours postoperatively (>20% change), while 45 miRNAs (14%) were deregulated following SL. Out of these miRNAs, 10 miRNAs were similarly deregulated after PH and SL, while one miRNA showed opposite regulation. In plasma, miRNA upregulation was observed for miR-133a and miR-133b following PH and SL, whereas miR-100 and miR-466c were similarly downregulated following anesthesia and surgery. CONCLUSIONS: We show that miRNAs are indeed regulated by sham laparotomy and anesthesia in rats. These findings illustrate the critical need for finding appropriate control groups in experimental surgery.

In vitro evaluation of magnetic resonance imaging contrast agents for labeling human liver cells: implications for clinical translation.

PURPOSE: Magnetic resonance imaging (MRI) is a promising approach for non-invasive monitoring after liver cell transplantation. We compared in vitro labeling of human liver cells with nano-sized (SPIO) and micron-sized iron oxide particles (MPIO). PROCEDURES: The cellular iron load was quantified and phantom studies were performed using 3.0-T MRI. Transferrin receptor and ferritin gene expression, reactive oxygen species (ROS) formation, transaminase leakage, and urea synthesis were investigated over 6 days. RESULTS: Incubation with MPIO produced stronger signal extinctions in MRI at similar iron loads within shorter labeling time. MPIO had no negative effects on the cellular iron homeostasis or cell performance, whereas SPIO caused temporary ROS formation and non-physiologic activation of the iron metabolic pathway. CONCLUSIONS: Our findings suggest that MPIO are suited for clinical translation of strategies for cellular imaging with MRI. Attention should be paid to iron release and oxidative stress caused by biodegradable contrast agents.