Hepatitis C virus (HCV) exists as a heterogenous quasispecies, but the phenotypic consequences of viral variability are widely unexplored. Here we identify a replication enhancing domain (ReED) in non-structural protein 5A conferring high replication fitness to clinical isolates. Accumulation of mutations in the ReED mediates high genome replication capacity. In a cohort of liver transplant patients, high replicator variants are exclusively found in individuals with severe disease outcome, suggesting that high viral replication fitness is associated with increased viral pathogenesis. Analysis of large sequence cohorts reveals that overall only 10% of viral genomes show genetic signatures of high replicators, which are enriched in recipients of liver transplantations, patients developing hepatocellular carcinoma and in HIV coinfected individuals. Overall, our data suggests that low replication fitness is a hallmark of HCV, contributing to establishment of persistence, whereas high replicators appear to have an advantage under conditions of immune suppression, thereby enforcing pathogenesis.

The investigation of hepatocarcinogenesis is a major field of interest in oncology research and rodent models are commonly used to unravel the pathophysiology of onset and progression of hepatocellular carcinoma. HCC is a highly vascularized tumor and vascular remodeling is one of the hallmarks of tumor progression. To date, only a few detailed data exist about the vasculature and vascular remodeling in rodent models used for hepatocarcinogenesis. In this study, the vasculature of HCC and the preneoplastic foci of alteration (FCA) of different mouse models with varying genetic backgrounds were comprehensively characterized by using immunohistochemistry (CD31, Collagen IV, αSMA, Desmin and LYVE1) and RNA in situ hybridization (VEGF-A). Computational image analysis was performed to evaluate selected parameters including microvessel density, pericyte coverage, vessel size, intratumoral vessel distribution and architecture using the Aperio ImageScope and Definiens software programs. HCC presented with a significantly lower number of vessels, but larger vessel size and increased coverage, leading to a higher degree of maturation, whereas FCA lesions presented with a higher microvessel density and a higher amount of smaller but more immature vessels. Our results clearly demonstrate that vascular remodeling is present and crucial in early stages of experimental hepatocarcinogenesis. In addition, our detailed characterization provides a strong basis for further angiogenesis studies in these experimental models.

Objectives: This paper reports the preclinical evaluation of stable tumor-specific gold nanoparticles (AuNPs) activated by neutron irradiation as a therapeutic option for the treatment of cancers characterized by high tumor angiogenesis. Methods: A selection of promising AuNPs with high avidity to α_vβ₃-expressing glioma (U-87 MG) cells (IC₅₀ = 82–104 nM) were chosen with different surface loading of Arg-Gly-Asp (RGD) peptides as tumor targeting vectors for integrin α_vβ₃, a target which is overexpressed in tissues displaying high tumor angiogenesis. Three different [¹⁹⁸Au]AuNPs were evaluated applying three injection methods, intravenous (i.v.), intraperitoneal (i.p.), and intratumoral (i.t.), each in a group of six U-87 MG xenograft–bearing mice (54 female athymic nude mice in total). Their biodistribution and tumor accumulation was assessed by in vivo imaging within 1–7 days after injection and 7 days after injection by ex vivo measurement. Results: The developed [¹⁹⁸Au]AuNPs exhibited suboptimal biodistribution by i.v. application (accumulation pattern tail > liver > spleen, no significant tumor accumulation) and by i.p. application (accumulation pattern spleen >> liver > pancreas, slight tumor accumulation of <0.3 %ID/g). However, an acceptable biodistribution by i.t. application was observed (5.5 %ID/g in liver, 4.9 %ID/g in spleen, and 3.0 %ID/g in tumor). Conclusions: Despite the very promising in vitro results, the in vivo evaluation suggests that the [¹⁹⁸Au]AuNPs represent a platform for the development of restricted therapeutic strategies.

Flow cytometry of T cells in tdLNs and the TME

Lymphatic PD-L1 regulates proliferating CD44+ CD62L+ PD-1+ CD8+ T cells within YUMMER1.7-draining LNs. A, Study design overview. PD-L1LECKO mice were treated with tamoxifen for five consecutive days before being inoculated intradermally with YUMMER1.7 melanoma cells (indicated by the arrow). On day 7, 8, and 9, mice received FTY720 intraperitoneally (IP) before being sacrificed on day 10. B and C, Count per LN or mg of tumor for CD3+ T cells, CD4+ T cells, CD8+ T cells, and Ki67+ CD62L+ CD44+ PD-1+ CD8+ T cells in the tdLNs (B) and primary tumors (C) of the YUMMER1.7 melanoma model under the indicated treatment. N = 3–5 mice/group. *, P < 0.05; **, P < 0.01; one-way ANOVA with a Šídák post test. D, Schematic representation of the proposed mode of action. In Cre-mice (top), PD-L1 expressed by LECs in tdLNs inhibits activation, proliferation, or LN egress of early activated CD8+ T cells, limiting their migration and accumulation within the TME. In Cre+ mice lacking lymphatic PD-L1 expression, early activated CD8+ T cell output of tdLNs is enhanced, resulting in increased infiltration of the TME. BV, blood vessel; LV, lymphatic vessel.

Lymphatic PD-L1 deletion reduces PD-1+ Th1 cells in the YUMMER1.7 primary tumor specifically. A, UMAP with unsupervised clustering of CD4+ T cells in the tdLNs and individual UMAP for each melanoma model and condition. B, Heatmap depicting mean marker expression across all clusters. C, Heatmap of the cluster frequencies of CD4+ T cells in the tdLNs under the indicated treatment. D, UMAP with unsupervised clustering of CD4+ T cells in the TME and individual UMAP for each melanoma model and condition. E, Heatmap depicting mean marker expression across all clusters. F, Heatmap of the cluster frequencies of CD4+ T cells in the TME under the indicated treatment. *, P < 0.05; Student t test. G, Frequency of PD-1+ CD44+ Foxp3− CD4+ T cells (cluster 7) in the TME under the indicated conditions determined by classic flow cytometry gating. N = 4–7 mice/group. *, P < 0.05; Student t test. APO, apotracker.