Search Results (32)

Osteosarcoma (OSA) is the most common primary bone malignancy in dogs, characterized by aggressive growth and high metastatic potential. Despite advances in treatment, the prognosis for affected animals remains poor, mainly due to metastatic disease. Metastasis is a complex process that involves forming new blood vessels in the primary tumor (angiogenesis), intravasation, the transport of cancer cells to other locations, extravasation, and the growth of cancer cells in the secondary site. Gold nanoparticles (AuNPs), due to their unique physicochemical properties, are considered promising tools in cancer therapy, both as drug delivery systems and potential anti-metastatic agents. Previously, it has been demonstrated that 500 µg/mL glutathione-stabilized gold nanoparticles (Au-GSH NPs) inhibit cancer cell extravasation—one of the steps of the metastatic cascade. This study aimed to evaluate the anti-metastatic properties of Au-GSH NPs through their influence on OSA cell migration, proliferation, and colony formation in vitro, as well as their antiangiogenic properties on the chick embryo chorioallantoic (CAM) model. Additionally, we investigated whether these effects are associated with changes in alpha-2-macroglobulin (A2M) expression, as it was previously demonstrated to play an essential role in the metastatic cascade. Au-GSH NPs significantly inhibited migration and colony formation in canine osteosarcoma cells (from OSCA-8, OSCA-32, and D-17 cell lines) at 200 µg/mL concentrations. Interestingly, at 500 µg/mL, Au-GSH NPs inhibited angiogenesis on the CAM model and cancer cell migration, but fewer colonies were formed. These results may be directly related to the higher efficiency of Au-GSH NPs uptake by OSA cells at the dose of 200 μg/mL than at the dose of 500 μg/mL, as demonstrated using Microwave Plasma Atomic Emission Spectroscopy (MP-AES). Moreover, this is the first study that demonstrates a significant increase in A2M expression in cancer cells after Au-GSH NPs treatment. This study provides new insight into the potential use of Au-GSH NPs as anti-metastatic agents in canine osteosarcoma, indicating that their anti-metastatic properties may be related to A2M. However, further in vitro and in vivo studies are needed to explore the molecular mechanism underlying these effects and to evaluate the clinical relevance of AuNPs in veterinary oncology. Full article

Previously, we reported that mammalian cells, specifically human dermal fibroblasts (HDFs), could be transdifferentiated by lactic acid bacteria (LAB). Later, we observed that HDFs incorporated LAB-derived ribosomes, forming the ribosome-induced cell clusters (RICs) and transdifferentiating into cells derived from all three germ layers. Based on this insight, we hypothesized that incorporating ribosomes into non-mammalian cells could reveal the universality of this mechanism and open the door to commercial applications. Our current study demonstrates that ribosome incorporation can transdifferentiate chick primary muscle-derived cells (CMCs) into adipocytes, osteoblasts, and chondrocytes. Furthermore, the culture medium supernatant from ribosome-incorporated CMCs was found to significantly enhance CMC’s proliferation. RNA-seq analysis revealed that RICs-CMC exhibit increased expression of genes related to multi-lineage cell growth. In addition, we developed a novel technological shift in meat production—the “CulNet System”—which replicates organ interactions within mechanical systems for cell-cultured meat production. While significant efforts are still required to implement this technology in a cost-effective manner, we believe that combining the “CulNet System” with ribosome-incorporated multipotent cells that have prolonged culture capability could substantially improve the scalability and cost-effectiveness of cultured chicken meat production. This report highlights a promising approach for cell-culture-based meat production, offering a sustainable alternative to traditional methods. Full article

Uveal melanoma (UM) is the most common intraocular tumor in adults, and nearly 50% of patients develop metastatic disease with a high mortality rate. Therefore, the development of relevant preclinical in vivo models that accurately recapitulate the metastatic cascade is crucial. We exploited the chick embryo chorioallantoic membrane (CAM) xenograft model to quantify both experimental and spontaneous metastasis by qPCR analysis. Our study found that the transplanted UM cells spread predominantly and early in the liver, reflecting the primary site of metastasis in patients. Visible signs of pigmented metastasis were observed in the eyes, liver, and distal CAM. Lung metastases occurred rarely and brain metastases progressed more slowly. However, UM cell types of different origins and genetic profiles caused an individual spectrum of organ metastases. Metastasis to multiple organs, including the liver, was often associated with risk factors such as high proliferation rate, hyperpigmentation, and epithelioid cell type. The severity of liver metastasis was related to the hepatic metastatic origin and chromosome 8 abnormalities rather than monosomy 3 and BAP1 deficiency. The presented CAM xenograft model may prove useful to study the metastatic potential of patients or to test individualized therapeutic options for metastasis in different organs. Full article

This study aimed to investigate the interactions between corn particle size (PS) and conditioning temperature (CT) on the performance, carcass traits, intestinal morphology, and immune responses in broilers fed a corn-soybean meal-based diet. A total of 360 one-day-old male broiler chicks (Ross 308) were randomly allocated into six dietary treatments in a 2 × 3 factorial arrangement, consisting of two corn PS (finely ground with geometric mean diameter (GMD) of 357 µm (PS_F) vs. coarsely ground corn with GMD of 737 µm (PS_C), and three CT [unconditioned (CT_U), conditioned at 75 °C (CT₇₅) and 90 °C (CT₉₀)]. Birds were accommodated in 30 pens with five replicates and 12 chicks per each pen. There was no interaction between corn PS and CT on the growth performance and immune response of broilers at any growth phases. However, during the starter (0–10 days) period, the average daily weight gain (ADWG) and feed conversion ratio (FCR) of PS_F-fed birds were significantly improved compared to those fed PS_C (p < 0.05). During the starter (0–10 days) and grower (11–24 days) periods, increasing the conditioning temperature of corn increased the ADWG, while in the starter phase only the CT₇₅ caused a lower FCR (p < 0.05). Broilers fed PS_F corn showed the lowest FCR during the finisher (25–42 days) period compared to those fed PS_C (p < 0.05). Conditioning corn at 75 °C reduced FCR during the finisher (25–42 days) period compared to the birds fed CT_U and CT₉₀ corn (p < 0.05). In whole experimental periods (1–42 days), PS_F and CT₇₅ treatment increased the ADWG compared to the PS_C and CT_U (p < 0.05). The CT₇₅ treatment improved primary total anti-sheep red blood cell (SRBCs) titer (IgT) and IgM and secondary IgT and IgG responses compared to the other experimental groups (CT_U and CT₉₀) (p < 0.05). No significant PS × CT interaction was found on the Newcastle disease (ND) antibody titer of broiler chickens (p > 0.05). Feeding CT₇₅ corn reduced duodenum and jejunum relative lengths compared to the birds fed diets containing CT_U corn. Significant PS × CT interactions (p < 0.05) were observed for villus height, villus height to crypt depth, crypt depth, muscle thickness, and absorption surface area of the jejunum. The highest carcass yield was observed in the PS_F-CT₇₅ group (p < 0.05). In conclusion, the use of finely ground corn (PSF) conditioned at 75 °C (CT75) was beneficial to growth performance, development of the digestive tract, jejunum histomorphometry and the immune responses of broilers. Full article

In ovo injection of the Marek’s disease (MD) vaccine (MDV) has been widely practiced in commercial US hatcheries. However, the MDV is very sensitive and may not be compatible with some nutrients when administered together by in ovo injection. When individually administered by in ovo injection, L-Ascorbic acid (L-AA) and 25-hydroxyvitamin D₃ (25OHD₃) have previously exhibited very promising results on the post-hatch physiological and immunological characteristics of broilers subjected to stressful commercial conditions. However, the compatibility of the MDV with these vitamins has not been previously explored. Their compatibility must first be established before their combined administration by in ovo injection can be considered. Therefore, the objective in this study was to determine the compatibility of the MDV with various levels of 25OHD₃ or L-AA. The treatments employed were MDV-alone, MDV in combination with 0.6 (low) or 2.4 (high) μg doses of 25OHD₃, or MDV in combination with 1.2 (low) or 12 (high) mg doses of L-AA. The live and dead ratio of primary chick embryo fibroblast cells infected by the MD virus (CEF-MDV) in each treatment was determined every 30 min for 2 h. The L-AA at both the low and high doses resulted in a 70% death of CEF-MDV within 1 h, but either dose of the 25OHD₃ exhibited only an approximate 5% lower CEF-MDV survival as compared to those in the MDV-alone treatment. Therefore, it is suggested that the two designated doses of 25OHD₃ have the potential to be effectively combined with the MDV for subsequent administration by in ovo injection. Full article

Canine osteosarcoma (OS) is an aggressive bone tumor with high metastatic potential and poor prognosis, mainly due to metastatic disease. Nanomedicine-based agents can be used to improve both primary and metastatic tumor treatment. Recently, gold nanoparticles were shown to inhibit different stages of the metastatic cascade in various human cancers. Here, we assessed the potential inhibitory effect of the glutathione-stabilized gold nanoparticles (Au-GSH NPs) on canine OS cells extravasation, utilizing the ex ovo chick embryo chorioallantoic membrane (CAM) model. The calculation of cells extravasation rates was performed using wide-field fluorescent microscopy. Transmission electron microscopy and Microwave Plasma Atomic Emission Spectroscopy revealed Au-GSH NPs absorption by OS cells. We demonstrated that Au-GSH NPs are non-toxic and significantly inhibit canine OS cells extravasation rates, regardless of their aggressiveness phenotype. The results indicate that Au-GSH NPs can act as a possible anti metastatic agent for OS treatment. Furthermore, the implemented CAM model may be used as a valuable preclinical platform in veterinary medicine, such as testing anti-metastatic agents. Full article

The S2 subunit serves a crucial role in infectious bronchitis virus (IBV) infection, particularly in facilitating membrane fusion. Using reverse genetic techniques, mutant strains of the S2 locus exhibited substantially different syncytium-forming abilities in chick embryonic kidney cells. To determine the precise formation mechanism of syncytium, we demonstrated the co-ordinated role of Abl2 and its mediated cytoskeletal regulatory pathway within the S2 subunit. Using a combination of fluorescence quantification, RNA silencing, and protein profiling techniques, the functional role of S2 subunits in IBV-infected cells was exhaustively determined. Our findings imply that Abl2 is not the primary cytoskeletal regulator, the viral S2 component is involved in indirect regulation, and the three different viral strains activate various cytoskeletal regulatory pathways through Abl2. CRK, CRKL, ABI1, NCKAP1, and ENAH also play a role in cytoskeleton regulation. Our research provides a point of reference for the development of an intracellular regulatory network for the S2 subunit and a foundation for the rational design of antiviral drug targets against Abl2. Full article

Resection margin adequacy plays a critical role in the local control of sarcomas. Fluorescence-guided surgery has increased complete resection rates and local recurrence-free survival in several oncological disciplines. The purpose of this study was to determine whether sarcomas exhibit sufficient tumor fluorescence (photodynamic diagnosis (PDD)) after administration of 5-aminolevulinic acid (5-ALA) and whether photodynamic therapy (PDT) has an impact on tumor vitality in vivo. Sixteen primary cell cultures were derived from patient samples of 12 different sarcoma subtypes and transplanted onto the chorio-allantoic membrane (CAM) of chick embryos to generate 3-dimensional cell-derived xenografts (CDXs). After treatment with 5-ALA, the CDXs were incubated for another 4 h. Subsequently accumulated protoporphyrin IX (PPIX) was excited by blue light and the intensity of tumor fluorescence was analyzed. A subset of CDXs was exposed to red light and morphological changes of both CAMs and tumors were documented. Twenty-four hours after PDT, the tumors were excised and examined histologically. High rates of cell-derived engraftments on the CAM were achieved in all sarcoma subtypes and an intense PPIX fluorescence was observed. PDT of CDXs resulted in a disruption of tumor-feeding vessels and 52.4% of CDXs presented as regressive after PDT treatment, whereas control CDXs remained vital in all cases. Therefore, 5-ALA mediated PDD and PDT appear to be promising tools in defining sarcoma resection margins (PDD) and adjuvant treatment of the tumor bed (PDT). Full article

Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess drug targets using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, potentially offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies. Full article

Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy. Full article

Unlike in many mammals, poultry testes are found in the abdominal cavity where they develop and perform spermatogenesis at high body temperature. Scarce reports among current publications detail the growth of testes and ST morphometry among juvenile chicks. Therefore, this study aims to investigate changes in components occurring in Gallus domesticus testes, by assessing the GSI and morphologically and histologically evaluating the testes and ST morphometry from 1-wk- to 4-mo-old. Right and left testes were collected from 70 healthy chickens divided into seven age-related groups (n = 10) and then immersed into the alcoholic acetate formalin (AAF) fixative solution. Hematoxylin- and eosin-stained tissues were used for microscopic observations. The findings revealed that both testes exhibited smooth features from 1-wk-old to 1-mo-old, and thereafter showed a consistent increase in vascularization until 4-mo-old. Histologically, both testes exhibited unclear ST, with ST apoptotic resorption observed in the 1-wk-old chicks. Until 1-mo-old, ST formed and few spermatogonia differentiated into primary spermatocytes, with all spermatogenic cells observed at 3-mo-old, i.e., sexual maturity. These findings suggest that both testes develop in analogy, and their sizes including increases in length and diameter are related to the spermatogenic activity in the ST. Subsequently, ST resorption by apoptosis is assumed to participate in the physiological mechanism regulating germ cells (GC). Finally, the GSI tended to increase with growth. Full article

Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS. Full article

Retinoblastoma (RB) is a primary intraocular malignancy in childhood. Relapses may develop and cause secondary cancers during later development. This study was set up to identify optimal cell culture conditions for RB cell growth in vitro and to optimize tumor growth in an in vivo model. RB cell lines (Y79 and WERI-Rb1) were cultivated under three different in vitro conditions and apoptosis, proliferation and cell growth, as well as expression profiles of two epithelial-mesenchymal transition (EMT) markers, were analyzed. EMT gene expression profiles were not generally changed, whereas apoptosis levels, tumor cell proliferation, and in vitro growth were significantly influenced by different cell culture conditions. In order to optimize the time-limited chick chorioallantoic membrane (CAM) assay, we investigated two different time points of tumor cell inoculation (embryonic development day EDD8 and EDD10) as well as three different cell concentrations. We showed that inoculation at EDD8 led to decreased tumor formation and chicken viability, whereas different cell concentrations did not change size and weight of developing tumors. Our findings demonstrate that medium conditions in vitro as well as the starting point for CAM inoculation in ovo significantly influence the experimental outcome of investigations using RB cell lines. Full article

The combination of Resminostat (HDACi) and Ruxolitinib (JAKi) exerted cytotoxic effects and inhibited proliferation of CTCL cell lines (MyLa, SeAx) in previously published work. A xenograft tumor formation was produced by implanting the MyLa or SeAx cells on top of the chick embryo chorioallantoic membrane (CAM). The CAM assay protocol was developed to monitor the metastatic properties of CTCL cells and the effects of Resminostat and/or Ruxolitinib in vivo. In the spontaneous CAM assays, Resminostat and Ruxolitinib treatment inhibited the cell proliferation (p < 0.001) of MyLa and SeAx, and induced cell apoptosis (p < 0.005, p < 0.001, respectively). Although monotherapies reduced the size of primary tumors in the metastasis CAM assay, the drug combination exhibited a significant inhibition of primary tumor size (p < 0.0001). Furthermore, the combined treatment inhibited the intravasation of MyLa (p < 0.005) and SeAx cells (p < 0.0001) in the organs, as well as their extravasation to the liver (p < 0.0001) and lung (p < 0.0001). The drug combination also exerted a stronger inhibitory effect in migration (p < 0.0001) rather in invasion (p < 0.005) of both MyLa and SeAx cells. It further reduced p-p38, p-ERK, p-AKT, and p-STAT in MyLa cells, while it decreased p-ERK and p-STAT in SeAx cells in CAM tumors. Our data demonstrated that the CAM assay could be employed as a preclinical in vivo model in CTCL for pharmacological testing. In agreement with previous in vitro data, the combination of Resminostat and Ruxolitinib was shown to exert antitumor effects in CTCL in vivo. Full article

Adenoviral gizzard erosion is an emerging disease with negative impact on health and production of chickens. In this study, we compared in vitro and in vivo characteristics of a fowl adenovirus serotype 1 (FAdV-1), attenuated by 53 consecutive passages in primary chicken embryo liver (CEL) cell cultures (11/7127-AT), with the virulent strain (11/7127-VT). Whole genome analysis revealed near-complete sequence identity between the strains. However, a length polymorphism in a non-coding adenine repeat sequence (11/7127-AT: 11 instead of 9) immediately downstream of the hexon open reading frame was revealed. One-step growth kinetics showed delayed multiplication of 11/7127-AT together with significantly lower titers in cell culture (up to 4 log₁₀ difference), indicating reduced replication efficiency in vitro. In vivo pathogenicity and immunogenicity were determined in day-old specific pathogen-free layer chicks inoculated orally with the respective viruses. In contrast to birds infected with 11/7127-VT, birds infected with 11/7127-AT did not exhibit body weight loss or severe pathological lesions in the gizzard. Virus detection rates, viral load in organs and virus excretion were significantly lower in birds inoculated with 11/7127-AT. Throughout the experimental period, these birds did not develop measurable neutralizing antibodies, prevalent in birds in response to 11/7127-VT infection. Differences in pathogenicity between the virulent FAdV-1 and the attenuated strain could not be correlated to prominently discriminate genomic features. We conclude that differential in vitro growth profiles indicate that attenuation is linked to modulation of viral replication during interaction of the virus with the host cells. Thus, hosts would be unable to prevent the rapid replication of virulent FAdV leading to severe tissue damage, a phenomenon broadly applicable to further FAdV serotypes, considering the substantial intra-serotype virulence differences of FAdVs and the variation of diseases. Full article