Development of an immunosuppressed orthotopic hepatocellular carcinoma rat model for the evaluation of chemo- and radioembolization therapies.

Hepatocellular carcinoma (HCC) is widely known to be chemo-resistant and presents with significant liver disease resulting in low tolerability to systemic chemotherapy. As a counter measure, more targeted therapies such as trans-arterial chemoembolization (TACE) and trans-arterial radioembolization (TARE) have been developed. To further optimize these therapies, animal models are critical in elucidating the molecular events in disease progression and test new treatment options. The present study focuses on the development of a hepatoma bearing rat model. N1S1 rat hepatoma cells were transfected by a lentiviral method and injected into the liver of Sprague Dawley (SD) and Rowett Nude (RNU) rats. Longitudinal tumor growth was observed by bioluminescence imaging (BLI) and liver/tumor histology. In both models, tumors were visible within 4 days post cell inoculation. Tumor take rates were 52 % and 73 % for male and female SD rats, respectively, and 100 % for male RNU rats. By day 12 and 15 post inoculation, we recorded complete tumor regression in male and female SD rats. Liver histology showed advanced fibrosis in the tumor regressed SD rats, whilst RNU rats exhibited the characteristic sheet pattern of Novikoff tumor with mild liver fibrosis. Increased CD3 and TUNEL staining observed in SD rat livers may be key factors for tumor regression. Our data reveal that the immunocompetent SD rats are not recommended as a model for therapeutic investigations. The immunosuppressed RNU rats, however, are characterized by consistent and reliable tumor growth and thus a desirable model for future therapeutic investigations.

A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma.

MYCN amplification (MNA) is a defining feature of high-risk neuroblastoma (NB) and predicts poor prognosis. However, whether genes within or in close proximity to the MYCN amplicon also contribute to MNA+ NB remains poorly understood. Here, we identify that GREB1, a transcription factor encoding gene neighboring the MYCN locus, is frequently coexpressed with MYCN and promotes cell survival in MNA+ NB. GREB1 controls gene expression independently of MYCN, among which we uncover myosin 1B (MYO1B) as being highly expressed in MNA+ NB and, using a chick chorioallantoic membrane (CAM) model, as a crucial regulator of invasion and metastasis. Global secretome and proteome profiling further delineates MYO1B in regulating secretome reprogramming in MNA+ NB cells, and the cytokine MIF as an important pro-invasive and pro-metastatic mediator of MYO1B activity. Together, we have identified a putative GREB1-MYO1B-MIF axis as an unconventional mechanism promoting aggressive behavior in MNA+ NB and independently of MYCN.

Development of an in vivo system to model breast cancer metastatic organotropism and evaluate treatment response using the chick embryo.

Metastatic lesions produced through the process of systemic tumor cell dissemination and growth at distant sites are challenging to treat and the primary cause of patient mortality. Developing in vivo models of metastasis with utility in evaluating molecular targets and therapeutics in a timely manner would expedite the path to therapeutic discovery. Here, we evaluated breast cancer metastasis and metastatic organotropism using the chick embryo. We developed a method to evaluate metastasis using the MDA231 cell line. Then, using cell lines with demonstrated tropism for the bone, brain, and lung, we evaluated organotropism. Rapid and robust organ-specific metastasis was modeled in the chick embryo and, importantly, recapitulated metastatic organotropism congruent to what has been demonstrated in mice. Treatment response in the metastatic setting was also evaluated and quantified. This work establishes the chick embryo as a model for studies aimed at understanding organotropism and therapeutic response in the metastatic setting.

Longitudinal bioluminescence imaging to monitor breast tumor growth and treatment response using the chick chorioallantoic membrane model.

The development of successful treatment regimens for breast cancer requires strong pre-clinical data generated in physiologically relevant pre-clinical models. Here we report the development of the chick embryo chorioallantoic membrane (CAM) model to study tumor growth and angiogenesis using breast cancer cell lines. MDA-MB-231 and MCF7 tumor cell lines were engrafted onto the chick embryo CAM to study tumor growth and treatment response. Tumor growth was evaluated through bioluminescence imaging and a significant increase in tumor size and vascularization was found over a 9-day period. We then evaluated the impact of anti-angiogenic drugs, axitinib and bevacizumab, on tumor growth and angiogenesis. Drug treatment significantly reduced tumor vascularization and size. Overall, our findings demonstrate that the chick embryo CAM is a clinically relevant model to monitor therapeutic response in breast cancer and can be used as a platform for drug screening to evaluate not only gross changes in tumor burden but physiological processes such as angiogenesis.

Anti-Inflammatory Activity of the Wild Mushroom, Echinodontium tinctorium, in RAW264.7 Macrophage Cells and Mouse Microcirculation.

The aim of this study was to investigate the anti-inflammatory activity of a previously un-studied wild mushroom, Echinodontium tinctorium, collected from the forests of north-central British Columbia. The lipopolysaccharide (LPS)-induced RAW264.7 macrophage model was used to study the in vitro anti-inflammatory activity. The crude alkaline extract demonstrated potent anti-inflammatory activity, and was further purified using a "bio-activity-guided-purification" approach. The size-exclusion and ion-exchange chromatography yielded a water-soluble anti-inflammatory polysaccharide (AIPetinc). AIPetinc has an average molecular weight of 5 kDa, and is a heteroglucan composed of mainly glucose (88.6%) with a small amount of galactose (4.0%), mannose (4.4%), fucose (0.7%), and xylose (2.3%). In in vivo settings, AIPetinc restored the histamine-induced inflammatory event in mouse gluteus maximus muscle, thus confirming its anti-inflammatory activity in an animal model. This study constitutes the first report on the bioactivity of Echinodontium tinctorium, and highlights the potential medicinal benefits of fungi from the wild forests of northern British Columbia. Furthermore, it also reiterates the need to explore natural resources for alternative treatment to modern world diseases.

Inonotus obliquus attenuates histamine-induced microvascular inflammation.

Cell-to-cell communication is a key element of microvascular blood flow control, including rapidly carrying signals through the vascular endothelium in response to local stimuli. This cell-to-cell communication is negatively impacted during inflammation through the disruption of junctional integrity. Such disruption is associated with promoting the onset of cardiovascular diseases as a result of altered microvascular blood flow regulation. Therefore, understanding the mechanisms how inflammation drives microvascular dysfunction and compounds that mitigate such inflammation and dysfunction are of great interest for development. As such we aimed to investigate extracts of mushrooms as potential novel compounds. Using intravital microscopy, the medicinal mushroom, Inonotus obliquus was observed, to attenuate histamine-induced inflammation conducted vasodilation in second-order arterioles in the gluteus maximus muscle of C57BL/6 mice. Mast cell activation by C48/80 similarly disrupted endothelial junctions and conducted vasodilation but only histamine was blocked by the histamine antagonist, pyrilamine not C48/80 suggesting the importance of mast cell activation. Data presented here supports that histamine induced inflammation is a major disruptor of junctional integrity, and highlights the important anti-inflammatory properties of Inonotus obliquus focusing future assessment of mast cells as putative target for Inonotus obliquus.

Invadopodia are chemosensing protrusions that guide cancer cell extravasation to promote brain tropism in metastasis.

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

Growth-Inhibitory and Immunomodulatory Activities of Wild Mushrooms from North-Central British Columbia (Canada).

Wild mushrooms, especially from North America, have not been systematically explored for their medicinal properties. Here we report screening for the growth-inhibitory and immunomodulatory activities of 12 species collected from multiple locations in north-central British Columbia, Canada. Mushrooms were characterized using morphology and DNA sequencing, followed by chemical extraction into 4 fractions using 80% ethanol, 50% methanol, water, and 5% sodium hydroxide. Growth-inhibitory, immunostimulatory, and anti-inflammatory activities of 5 mushrooms (Leucocybe connata, Trichaptum abietinum, Hydnellum sp., Gyromitra esculenta, and Hericium coralloides) are reported here, to our knowledge for the first time. Growth-inhibitory effects were assessed using the cytotoxic MTT assay. Immunostimulatory activity was assessed by tumor necrosis factor-α production in Raw 264.7 macrophages, whereas anti-inflammatory activity was assessed based on the inhibition of lipopolysaccharide-induced tumor necrosis factor-α production. The ethanol and aqueous extracts of Hydnellum sp. were potent growth inhibitors, with a half-maximal inhibitory concentration of 0.6 mg/mL. All 5 fungi displayed strong immunostimulatory activity, whereas only L. connata and T. abietinum showed strong anti-inflammatory activity. For the 7 other fungi investigated, which included well-known medicinal species such as Inonotus obliquus, Phellinus igniarius, and Ganoderma applanatum, the remarkable similarities in the biological activities reported here, and by others for specimens collected elsewhere, suggest that mushrooms can produce similar metabolites regardless of their habitat or ecosystem. This is to our knowledge the first study to explore wild mushrooms from British Columbia for biological activities that are relevant to cancer, and the results provide an initial framework for the selection of mushroom species with the potential for discovery of novel anticancer compounds.