Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time-of-day dependent changes of proliferation and DNA damage in HCC. Using transgenic c-myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ-H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev-erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ-H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time-ofday dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.

The circadian system is an endogenous timekeeping system that synchronizes physiology and behavior with the 24 h solar day. Mice with total deletion of the core circadian clock gene Bmal1 show circadian arrhythmicity, cognitive deficits, and accelerated age-dependent decline in adult neurogenesis as a consequence of increased oxidative stress. However, it is not yet known if the impaired adult neurogenesis is due to circadian disruption or to loss of the Bmal1 gene function. Therefore, we investigated oxidative stress and adult neurogenesis of the two principle neurogenic niches, the hippocampal subgranular zone and the subventricular zone in mice with a forebrain specific deletion of Bmal1 (Bmal1 fKO), which show regular circadian rhythmicity. Moreover, we analyzed the morphology of the olfactory bulb, as well as olfactory function in Bmal1 fKO mice. In Bmal1 fKO mice, oxidative stress was increased in subregions of the hippocampus and the olfactory bulb but not in the neurogenic niches. Consistently, adult neurogenesis was not affected in Bmal1 fKO mice. Although Reelin expression in the olfactory bulb was higher in Bmal1 fKO mice as compared to wildtype mice (Bmal1 WT), the olfactory function was not affected. Taken together, the targeted deletion of Bmal1 in mouse forebrain neurons is associated with a regional increase in oxidative stress and increased Reelin expression in the olfactory bulb but does not affect adult neurogenesis or olfactory function.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

No abstract

In the brain, generation of new neurons throughout life, the adult neurogenesis, has important functional aspects. Adult neurogenesis is a multistep process covering proliferation of neuronal stem/precursor cells (NPCs), migration of neuroblasts, differentiation into mature neurons, and integration into pre-existing neuronal networks. Moreover, neurogenesis is heterogeneous among mammals depending on species and age. 1 Under normal physiological conditions, adult neurogenesis takes place in specific regions of the adult mouse brain, known as "neurogenic niches,"

novel 3D flow-perfusion bioreactor system as an ex vivo model for NE cancer and for personalized therapeutic testing. Materials/Methods: Polydimethylsiloxane bioreactors were designed to house 3D extracellular matrix (ECM) made of bovine collagen I and Matrigel in various ratios to determine the optimal composition. TT (RFP/ Luc) cell line and freshly collected tumor cell suspensions originating from mouse or patients were suspended in the ECM. The bioreactors were connected to a peristaltic pump to circulate media through the NE cancer surrogates. Growth was determined by non-invasive fluorescence and bioluminescence imaging using the IVIS Lumina system. H&E stained histologic sections were prepared for morphologic analysis by a pathologist. Finally, the growth of the surrogates were assessed after 6gy radiation and after perfusion with potential drug candidate Thailandepsin-A (TDP-A). Results: The optimal ECM composition for NE cancer cell growth was determined to be 50% collagen I and 50% Matrigel. Linear growth of cell line (RFP) surrogates were observed up to 14 days (RFP 7.1 fold increase at 14 days compared to 0 days). Similarly, imaging of the human NE tumor surrogates with near infrared dye showed an increased signal from days 3 to 9 (1.4 fold increase). Histologic sections showed epithelial clustering characteristic of NE cancers. Surrogates originating from subcutaneous xenograft were used for therapeutic testing. While the bioluminescence signal of surrogate perfused with DMSO control increased 2.2 fold in 2 days, perfusion with 25nM TDP-A lead to 1.2 fold increase and 6gy radiation showed no growth after 2 days. Conclusion: We demonstrated that the 3D surrogates system can be used to simulate physiologically relevant stromal context for NE cancer cells and grow patient derived tumors for therapeutic testing. The surrogates were compatible with standard histological techniques. Further testing should be done with additional patient tumors, various radiation dose regimens, FDA approved drugs and drug candidates to test cytotoxic and anti-proliferative effects.

Chronic liver diseases including hepatocellular carcinoma (HCC) create a state of chronic inflammation that affects the brain via the liver–brain axis leading to an alteration of neurotransmission and cognition. However, little is known about the effects of HCC on the hippocampus, the key brain region for learning and memory. Moreover, radiotherapy used to treat HCC has severe side effects that impair patients’ life quality. Thus, designing optimal strategies, such as chronotherapy, to enhance the efficacy and reduce the side effects of HCC treatment is critically important. We addressed the effects of HCC and the timed administration of radiotherapy in mice on the expression of pro-inflammatory cytokines, clock genes, markers for glial activation, oxidative stress, neuronal activity and proliferation in the hippocampal neurogenic niche. Our data showed that HCC induced the upregulation of genes encoding for pro-inflammatory cytokines, altered clock gene expressions and reduced proliferation in the hippocampus. Radiotherapy, in particular when applied during the light/inactive phase enhanced all these effects in addition to glial activation, increased oxidative stress, decreased neuronal activity and increased levels of phospho(p)-ERK. Our results suggested an interaction of the circadian molecular clockwork and the brain’s innate immune system as key players in liver–brain crosstalk in HCC and that radiotherapy when applied during the light/inactive phase induced the most profound alterations in the hippocampus.