Distribution Characteristics and Species Diversity of Bacteria in Hepatocellular Carcinoma Tissues

This study was to explore the differences in the distribution and species diversity of bacteria between hepatocellular carcinoma (HCC) tissues and normal liver tissues. 28 HCC patients treated with surgery were selected as the research objects (HCC group), and 19 healthy volunteers with normal physical examinations were included in the control group (Normal group). The tumor specimens were obtained by intraoperative and biopsy puncture, and a 16S ribosomal ribonucleic acid (rRNA) library was constructed. Based on the sequencing data obtained by the IlluminaHi Seq sequencing platform, the differences of bacteria in the liver tissues of the HCC group and the Normal group were analyzed at the level of phyla, family, and genus. The Ace, Chao1, and Shannon of the two groups were compared. The results showed that IlluminaHi Seq sequencing obtained a total of 11,714,659 valid sequences, with an average of 131,625 sequences per sample. The proportions of Bacteroidetes, Firmicutes, and Proteobacteria in HCC group and Normal group were 48.75% versus 34.16%, 37.44% versus 18.02%, and 10.85% versus 39.26%, respectively. The Bacteroidaceae, Prevotellaceae, Lachnospiraceae, and Ruminococcaceae accounted for 22.49%, 20.62%, 16.54%, and 19.93% in Normal group; while those in the HCC tissues accounted for 26.83%, 14.22%, 11.14%, and 13.18%, respectively. The dominant bacteria at the genus level in HCC group and Normal group were Bacteroides and Prevotella-9, with the proportions of 24.19% versus 26.04% and 14.19% versus 8.44%, respectively. The difference in operational taxonomic unit (OTU) numbers of HCC and Normal group were compared and analyzed, which were 1,266 and 1,082, respectively, and the number of common OTU in the two tissues was 875. The Ace in HCC tissue and normal liver tissue were 1063.8±66.79 and 1003.6±52.19, respectively. The Ace in HCC tissue was greater than that in normal liver tissue (P < 0.05). The Chao1 and Shannon in HCC tissue were 1022.9±67.74 and 5.4269±0.3608, respectively; while those in normal liver tissue were 1003.6±66.79 and 5.2842±0.9714, respectively. The Chao1 and Shannon in HCC tissues were much higher than those in Normal group (P < 0.05). It showed that there was no difference in the types of bacterial species in HCC tissues, but the proportions of their flora at the level of phyla, family, and genus changed greatly, which may be related to the occurrence of HCC. This study could provide a reference for the diagnosis and treatment of HCC.

Single-cell RNA sequencing reveals the heterogeneity of infiltrating immune cell profiles in the hepatic cystic echinococcosis microenvironment

Human cystic echinococcosis, caused by the larval stage of echinococcus granulus sensu lato , has been reported a near-cosmopolitan zoonotic disease. Various infiltrating immune cells gather around the lesion and produce lesion microenvironment, however cellular composition and heterogeneity in hepatic cystic echinococcosis lesion microenvironment are incompletely understood. Here, 81,865 immune cells isolated from peripheral blood, peri-lesion liver tissue, and adjacent normal liver tissue from four cystic echinococcosis patients were profiled using single-cell RNA sequencing. We identified 23 discrete cell populations, and found distinct differences in infiltrating immune cells between tissue environments. Despite the significant similarity between peri-lesion and adjacent normal liver tissue-resident immune cells, the cellular proportions of innate lymphocyte 2 and plasmacytoid dendritic cells were higher in peri-lesion liver tissue. Interestingly, the immunosuppressive gene NFKBIA was up-regulated in these cells. Seven subsets of CD4 + T cell populations were found, and there were more Treg-CD4 + T and Th2-CD4 + T cells in peri-lesion tissue than those in adjacent normal tissue. There was close contact between CD4 + T cells and ILC2 and pDCs cells, which caused up-regulation of genes related to positive immune activity in adjacent normal liver tissue. However, expression of genes related to immunosuppression, especially the immune inhibitory checkpoint gene NKG2A/HLA-E, was obviously higher in peri-lesion tissue, suggesting that cellular interaction resulted in an inhibitory microenvironment in the CE lesion. This work offers new insights into the transcriptional heterogeneity of infiltrating immune cells in hepatic cystic echinococcosis lesion microenvironment at single-cell level, and provides potential target signatures for diagnosis and immunotherapies.

Tumor heterogeneity for differentiation between liver tumors and normal liver tissue in 18F-FDG PET/CT

Aim Malignancies show higher spatial heterogeneity than normal tissue. We investigated, if textural parameters from FDG PET describing the heterogeneity function as tool to differentiate between tumor and normal liver tissue. Methods FDG PET/CT scans of 80 patients with liver metastases and 80 patients with results negative upper abdominal organs were analyzed. Metastases and normal liver tissue were analyzed drawing up to three VOIs with a diameter of 25 mm in healthy liver tissue of the tumoral affected and results negative liver, whilst up to 3 metastases per patient were delineated. Within these VOIs 30 different textural parameters were calculated as well as SUV. The parameters were compared in terms of intra-patient and inter-patient variability (2-sided t test). ROC analysis was performed to analyze predictive power and cut-off values. Results 28 textural parameters differentiated healthy and pathological tissue (p < 0.05) with high sensitivity and specificity. SUV showed ability to differentiate but with a lower significance. 15 textural parameters as well as SUV showed a significant variation between healthy tissues out of tumour infested and negative livers. Mean intra- and inter-patient variability of metastases were found comparable or lower for 6 of the textural features than the ones of SUV. They also showed good values of mean intra- and inter-patient variability of VOIs drawn in liver tissue of patients with metastases and of results negative ones. Conclusion Heterogeneity parameters assessed in FDG PET are promising to classify tissue and differentiate malignant lesions usable for more personalized treatment planning, therapy response evaluation and precise delineation of tumors for target volume determination as part of radiation therapy planning.

Effect of Intrahepatic Arterial Delivery of Sorafenib on Normal Liver Tissue of Rabbit: An Experimental Study

Objective: To assess the safety, feasibility and eluting efficiency of intrahepatic arterial delivery of sorafenib on normal liver tissue of rabbit. Methods: 24 New Zealand rabbits were randomly divided into three groups: group Ⅰ (Lipiodol-sorafenid), group Ⅱ (Lipiodol) and group Ⅲ (Sorafenib). Group Ⅰ and Ⅱ were treated by transcatheter selective hepatic arterial embolization with emulsions of lipiodol and sorafenib or with only lipiodol, while group Ⅲ was given hepatic arterial infusion with sorafenib. Sorafenib concentration in plasma was determined by HPLC (high performance liquid chromatography) in 0 min, 20 min, 1h, 2h, 4h, 8h, 16h, 32h and 48h respectively. The breathing rate, heart rate, rectal temperature and body weight were measured, as well the blood routine test and the function of liver, kidney, and heart. Two animals of each group were respectively killed in the 3rd day, 1st, 3rd and 6th week after treatment. Histopathologic study was done to liver, heart, kinney, lung, brain, gall bladder and intestine. Result: ① The peak sorafenib concentration (Cmax)and AUC(Area under curve) in plasma in groupⅠwas 2.46±0.101μg/ml and 945.72 ± 52.3 μg/mL.min respectively, while in group Ⅲ which was 3.78±0.180 ug/ml and 546.98±21.1μg/mL.min. Compared with groupⅢ, the Cmax and AUC of groupⅠhad a significant statistics difference (p<0.05). ② The breathing rate, heart rate, rectal temperature and AST/ALT,WBC,NEU% of group Ⅰand groupⅢhas a significant statistics difference(p<0.05) in the 3rd day. ③CK ,CK-MB, DB, Cr，BUN,RBC,PLT in plasma did not change in all group.④Local necrosis was seen in group Ⅰand group Ⅱin the 3rd day and 1st week, but they did not seem to be different. Group Ⅲ showed no necrosis. Granulation tissue with bile duty, portal vein and microfossils hyperplasia were seen in local necrosis area in the 3rd week. No pathological changes were found in brain, heart, kidney, intestine and gallbladder. Conclusion: TAE with emulsions of lipiodol and sorafenib is feasible, safe and has some slow-release effect.

Elasticity Characterization of Liver Cancers Using Shear Wave Ultrasound Elastography: Comparison Between Hepatocellular Carcinoma and Liver Metastasis

Purpose: This was a feasibility study of shear wave ultrasound elastography for characterization of liver tumors and to compare the tissue elasticity values of hepatocellular carcinoma (HCC), liver metastases, and normal liver tissues. Methods: Forty-one patients and 30 healthy volunteers were recruited and categorized into HCC, liver metastasis, and control groups based on their computed tomography and sonographic examinations. Elasticity values of different groups were compared statistically. Results: Mean (standard deviation) elasticity values for HCC, liver metastasis, and normal liver tissue were 51.45 (14.96), 49.89 (13.82), and 6.63 (1.65) kilopascal, respectively. Statistically significant differences were found between the elasticity values of HCC and liver metastasis with normal liver tissue. Based on the receiver operating characteristics analysis, 18.25 kilopascal may differentiate the malignant focal liver lesions from the normal liver tissue with both sensitivity and specificity of 100%. Conclusion: Shear wave ultrasound elastography may be able to differentiate HCC and liver metastasis from normal liver tissue based on the tissue elasticity values.

Effect of Rho/ROCK pathway inhibition on metastasis-free and overall survival in biomarker selected, orthotopic, patient-derived models of pancreatic cancer.

e15759 Background: PC is a highly lethal disease which metastasises early. The sequencing efforts of the Australian Pancreatic Cancer Genome Initiative (APGI) identified ROCK-1 as a target of interest. The Rho/ROCK pathway is essential in cellular movement, metastasis, vasculogenesis and stromal signaling. Fasudil (F) is a ROCK inhibitor which has been shown to be safe for human use. Using unique, patient derived models, we have previously shown that F plus gemcitabine (G) does not reduce cellular proliferation in vitro but reduces tumour size and improves overall survival (OS) in subcutaneous patient-derived xenografts (PDX). However, PDXs are critisised for not fully recapitulating the human condition. Here we aimed to generate a patient-derived orthotopic xenograft (O-PDX) which could be used to assess the effects of F + G on metastasis formation and OS. Methods: One patient-derived cell line (PDCL) was labled with firefly luciferase. This was injected into the pancreas of NOD/Shi- scid/IL-2Rγnull (NSG) mice to generate the O-PDX model. The O-PDX was imaged weekly with the IVIS imaging system. The O-PDXs were treated with saline control (S) (n = 8), gemcitabine (G) (n = 10) fasudil (F) (n = 6) or gemcitabine + fasudil (GF) (n = 10). Mice were culled once radiographic or clinical evidence of ascites was detected. Metastasis free (MFS) and OS were recorded. Vessel quantification was performed via CD-31 staining. Results: Metastases seen (spleen, liver, lung, ovary) on imaging were confirmed using GFP stains on immunohistochemistry. F prolonged MFS but not OS when compared with S. GF significantly prolonged MFS (33.5 versus 43 days; p = 0.0339) and OS (51.5 versus 64 days; p = 0.0035). CD-31 staining showed that treatment with F resulted in increased vascularity in the liver metastases and normal liver tissue but not in the pancreas tumour. Conclusions: The addition of Fasudil to gemcitabine improves MFS and OS in an orthotopic model of PC and is a promising new therapeutic option. The increase in vascularity of the metastases and normal liver tissue suggest that changes in vascularity are important and may result in improved delivery of gemcitabine.

Accuracy of real-time shear wave elastography in the assessment of normal liver tissue in the guinea pig (cavia porcellus)

Shear wave elastography is a novel technique enabling real-time measurement of the elasticity of liver tissue. The color map is superimposed on the classic ultrasound image of the assessed tissue, which enables a precise evaluation of the stiffness of the liver tissue. The aim of the study was to assess the stiffness of normal liver tissue in the guinea pig using shear wave elastography. The study was carried out on 36 guinea pigs using the SuperSonic Imagine Aixplorer scanner, and a 1 to 6 MH convex SC6-1 transducer. An ultrasound guided Try-Cut liver core needle biopsy was carried out in all the studied animals and the collected samples were examined to exclude pathological lesions. The mean liver tissue stiffness ranged from 0.89 to 5.40 kPa. We found that shear wave elastography is an easy, non-invasive technique that can be used to assess the stiffness of liver tissue. The obtained results can be used in future studies to assess the types and changes of liver tissue in the course of various types of liver disease.