Hyperglycaemia, pregnancy outcomes and maternal metabolic disease risk during pregnancy and lactation in a lean gestational diabetes mouse model.

Hyperglycaemia in pregnancy (HIP) is a pregnancy complication characterized by mild to moderate hyperglycaemia that negatively impacts short- and long-term health of mother and child. However, relationships between severity and timing of pregnancy hyperglycaemia and postpartum outcomes have not been systemically investigated. We investigated the impact of hyperglycaemia developing during pregnancy (gestational diabetes mellitus, GDM) or already present pre-mating (pre-gestational diabetes mellitus, PDM) on maternal health and pregnancy outcomes. GDM and PDM were induced in C57BL/6NTac mice by combined 60% high fat diet (HF) and low dose streptozotocin (STZ). Animals were screened for PDM prior to mating, and all underwent an oral glucose tolerance test on gestational day (GD)15. Tissues were collected at GD18 or at postnatal day (PN)15. Among HFSTZ-treated dams, 34% developed PDM and 66% developed GDM, characterized by impaired glucose-induced insulin release and inadequate suppression of endogenous glucose production. No increased adiposity or overt insulin resistance was observed. Furthermore, markers of non-alcoholic fatty liver disease (NAFLD) were significantly increased in PDM at GD18 and were positively correlated with basal glucose levels at GD18 in GDM dams. By PN15, NAFLD markers were also increased in GDM dams. Only PDM affected pregnancy outcomes such as litter size. Our findings indicate that GDM and PDM, resulting in disturbances of maternal glucose homeostasis, increase the risk of postpartum NAFLD development, related to the onset and severity of pregnancy hyperglycaemia. These findings signal a need for earlier monitoring of maternal glycaemia and more rigorous follow-up of maternal health after GDM and PDM pregnancy in humans. KEY POINTS: We studied the impact of high-fat diet/streptozotocin induced hyperglycaemia in pregnancy in mice and found that this impaired glucose tolerance and insulin release. Litter size and embryo survival were compromised by pre-gestational, but not by gestational, diabetes. Despite postpartum recovery from hyperglycaemia in a majority of dams, liver disease markers were further elevated by postnatal day 15. Maternal liver disease markers were associated with the severity of hyperglycaemia at gestational day 18. The association between hyperglycaemic exposure and non-alcoholic fatty liver disease signals a need for more rigorous monitoring and follow-up of maternal glycaemia and health in diabetic pregnancy in humans.

p63 immunoexpression in hair follicles of normal and alopecia X-affected skin of Pomeranian dogs.

BACKGROUND: Alopecia X in Pomeranians is caused by a hair cycle deregulation, associated with downregulation of key regulatory genes of the Wnt and Shh pathways, and stem-cell markers. However, the pathogenesis remains unclear. p63 is an important transcription factor correlated with the aforementioned hair cycle modulating genes. HYPOTHESIS/OBJECTIVES: The aim of this study was to highlight possible changes of p63 immunohistochemical expression within the hair follicles in canine alopecia X compared with normal skin. ANIMALS: Skin biopsies from 19 alopecia X-affected and six control Pomeranians were analysed. MATERIALS AND METHODS: Serial histological sections of skin biopsies harbouring anagen, telogen and kenogen hair follicles were immunohistochemically evaluated for differences in p63 expression in the affected and control samples. RESULTS: Dogs with alopecia X had a significantly decreased immunoexpression of p63 in telogen and kenogen hair follicles. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease of p63 immunoexpression observed in canine alopecia X suggests an involvement of p63 in hair cycle.

E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice.

BACKGROUND AND AIMS: Up-regulation of the E2F-dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F-dependent transcription. They are atypical in that they do not bind to dimerization partner proteins and are not controlled by retinoblastoma protein. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking. APPROACH AND RESULTS: Here, we generated transgenic mice with doxycycline-controlled transcriptional activation of E2f7 and E2f8 and induced their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and, to lesser extent, E2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage, and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity, and cell proliferation in diethylnitrosamine-induced liver tumors. CONCLUSION: Collectively, our findings demonstrate that atypical E2Fs can override cell-cycle entry and progression governed by other E2F family members and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood.

Modeling Phenotypic Heterogeneity of Glycogen Storage Disease Type 1a Liver Disease in Mice by Somatic CRISPR/CRISPR-associated protein 9-Mediated Gene Editing.

BACKGROUND AND AIMS: Patients with glycogen storage disease type 1a (GSD-1a) primarily present with life-threatening hypoglycemia and display severe liver disease characterized by hepatomegaly. Despite strict dietary management, long-term complications still occur, such as liver tumor development. Variations in residual glucose-6-phosphatase (G6PC1) activity likely contribute to phenotypic heterogeneity in biochemical symptoms and complications between patients. However, lack of insight into the relationship between G6PC1 activity and symptoms/complications and poor understanding of the underlying disease mechanisms pose major challenges to provide optimal health care and quality of life for GSD-1a patients. Currently available GSD-1a animal models are not suitable to systematically investigate the relationship between hepatic G6PC activity and phenotypic heterogeneity or the contribution of gene-gene interactions (GGIs) in the liver. APPROACH AND RESULTS: To meet these needs, we generated and characterized a hepatocyte-specific GSD-1a mouse model using somatic CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing. Hepatic G6pc editing reduced hepatic G6PC activity up to 98% and resulted in failure to thrive, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly, hepatic steatosis (HS), and increased liver tumor incidence. This approach was furthermore successful in simultaneously modulating hepatic G6PC and carbohydrate response element-binding protein, a transcription factor that is activated in GSD-1a and protects against HS under these conditions. Importantly, it also allowed for the modeling of a spectrum of GSD-1a phenotypes in terms of hepatic G6PC activity, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly and HS. CONCLUSIONS: In conclusion, we show that somatic CRISPR/Cas9-mediated gene editing allows for the modeling of a spectrum of hepatocyte-borne GSD-1a disease symptoms in mice and to efficiently study GGIs in the liver. This approach opens perspectives for translational research and will likely contribute to personalized treatments for GSD-1a and other genetic liver diseases.

Inhibition of polyploidization in Pten-deficient livers reduces steatosis.

The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver-specific deletion of Pten in mice results in the hyper-activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver-specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten-induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten-deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR-gamma signalling. However, we find no evidence that deregulated genes in Pten-deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten-deficient livers.

PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence-free survival in HCC patients.

Extracellular Vesicles: Novel Opportunities to Understand and Detect Neoplastic Diseases.

With a size range from 30 to 1000 nm, extracellular vesicles (EVs) are one of the smallest cell components able to transport biologically active molecules. They mediate intercellular communications and play a fundamental role in the maintenance of tissue homeostasis and pathogenesis in several types of diseases. In particular, EVs actively contribute to cancer initiation and progression, and there is emerging understanding of their role in creation of the metastatic niche. This fact underlies the recent exponential growth in EV research, which has improved our understanding of their specific roles in disease and their potential applications in diagnosis and therapy. EVs and their biomolecular cargo reflect the state of the diseased donor cells, and can be detected in body fluids and exploited as biomarkers in cancer and other diseases. Relatively few studies have been published on EVs in the veterinary field. This review provides an overview of the features and biology of EVs as well as recent developments in EV research including techniques for isolation and analysis, and will address the way in which the EVs released by diseased tissues can be studied and exploited in the field of veterinary pathology. Uniquely, this review emphasizes the important contribution that pathologists can make to the field of EV research: pathologists can help EV scientists in studying and confirming the role of EVs and their molecular cargo in diseased tissues and as biomarkers in liquid biopsies.

E-Cadherin Expression in Canine Melanocytic Tumors: Histological, Immunohistochemical, and Survival Analysis.

E-cadherin, a glycoprotein involved in cell-cell adhesion, has a pivotal role in epithelial-mesenchymal transition, a process through which neoplastic epithelial cells develop an invasive phenotype. In human cutaneous melanomas, decreased E-cadherin expression is associated with shorter survival and increased Breslow thickness, whereas in the dog its role is poorly understood. Tumor thickness and modified Clark level were recently proposed as useful features to assess canine melanocytic tumors, but no studies investigated their association with E-cadherin expression. We performed immunohistochemistry on 77 formalin-fixed, paraffin-embedded primary canine melanocytic tumors. A 3-tier and a 2-tier classification system for assessing E-cadherin expression were tested, with the latter being more informative for the assessment of canine melanocytic tumors. E-cadherin expression was lower in cutaneous melanomas than melanocytomas, as well as in amelanotic tumors compared to pigmented tumors. In amelanotic melanomas, absent E-cadherin expression was associated with an unfavorable outcome, suggesting a potential use of this marker in defining the prognosis of amelanotic melanomas. E-cadherin expression was lower in tumors with greater tumor thickness and modified Clark level ≥IV, suggesting its possible utility in identifying the most invasive tumors. The expression of E-cadherin in oral melanomas was heterogeneous, but was associated with pigmentation and clinical outcome; thus, E-cadherin evaluation could be advantageous to detect the most aggressive neoplasms. However, cutaneous melanomas without E-cadherin expression frequently had a favorable clinical outcome. Hence, its importance as prognostic factor should be carefully considered depending on the tumor origin.

LED-phototherapy does not induce oxidative DNA damage in hyperbilirubinemic Gunn rats.

BACKGROUND: Phototherapy (PT) is the standard treatment of neonatal unconjugated hyperbilirubinemia. Fluorescent tube (FT)-emitted PT light is known to induce oxidative DNA damage in neonates. Nowadays, however, FTs have largely been replaced by light-emitting diodes (LEDs) for delivering PT. Until now, it is unknown whether LED-PT causes oxidative DNA damage. We aim to determine whether LED-PT induces oxidative DNA damage in hyperbilirubinemic rats. METHODS: Adult Gunn rats, with genetically unconjugated hyperbilirubinemia, received LED-PT in the clinically relevant doses of 10 or 30 µW/cm2/nm. Urine was collected at 0, 24, and 48 h of PT. A group of young Gunn rats received intensive LED-PT of 100 µW/cm2/nm for 24 h. Urine was collected every 8 h and analyzed for the levels of oxidative DNA damage marker 8-hydroxy-2'deoxyguanosine (8-OHdG) and creatinine. DNA damage was evaluated by immunohistochemistry (γH2AX) of skin and spleen samples. RESULTS: LED-PT of 10 and 30 µW/cm2/nm did not affect urinary concentrations of 8-OHdG and creatinine or the 8-OHdG/creatinine ratio. Likewise, intensive LED-PT did not affect the 8-OHdG/creatinine ratio or the number of γH2AX-positive cells in the skin or spleen. CONCLUSIONS: Our results show that LED-PT does not induce oxidative DNA damage in hyperbilirubinemic Gunn rats either at clinically relevant or intensive dosages.

Survivin and Sox9: Potential Stem Cell Markers in Canine Normal, Hyperplastic, and Neoplastic Canine Prostate.

Canine prostatic carcinoma is a relevant model for human prostatic carcinoma. Survivin is proposed as a biomarker of malignancy in human prostatic cancer. Sox9 is a stem cell marker required for prostate development and expressed in several adult tissues. The aims of the present study were to evaluate the patterns and expression levels of 2 putative stem cell markers, survivin and Sox9, in canine benign prostatic hyperplasia (BPH) and prostatic carcinoma to investigate their potential as stem cell markers. Immunohistochemistry with specific antibodies was performed on 3 samples of normal prostate gland, 18 samples of canine BPH, and 16 samples of prostatic carcinoma. The basal cell layer of normal and hyperplastic prostatic lobules had nuclear Sox9 immunolabeling and nuclear and rarely cytoplasmic survivin immunostaining, identifying them as potential stem cell markers. Significantly more frequent survivin and Sox9 expression (≥10% of nuclei) was observed in prostatic carcinoma as compared with BPH. The potential coexpression of survivin with Sox9, androgen receptor, and p63 was also investigated in selected BPH and prostatic carcinoma cases with immunofluorescence, and a partial colocalization was observed. Results indicate that Sox9 and survivin could be considered markers of stemness in canine prostate cells. Given its role in proliferation, cells in the basal cell layer with nuclear survivin expression are likely to be transit-amplifying cells that maintain some stem cell proprieties.

Canine cutaneous melanocytic tumours: significance of ß-catenin and survivin immunohistochemical expression.

BACKGROUND: Recent investigations have highlighted the controversial role of Wnt/ß-catenin pathway activation in human cutaneous melanoma. Survivin has been proposed as a valid prognostic marker for invasive and metastatic melanomas and lymph node melanoma metastasis in human cutaneous melanoma and is a promising therapeutic target. HYPOTHESIS/OBJECTIVES: Our aim was to investigate the immunohistochemical expression of survivin and ß-catenin in canine cutaneous melanocytic tumours, in order to understand their prognostic significance. METHODS: Twenty-one melanocytic tumours (10 melanocytomas and 11 melanomas) were investigated by immunohistochemistry using specific anti-survivin and anti-ß-catenin antibodies. A semi-quantitative method was used to analyse the results; ß-catenin immunolabelling in neoplastic cells was evaluated as cytoplasmic, membranous or nuclear. The number of survivin-positive cells was counted within ~1000 neoplastic cells. Results were related to histopathological features, evaluated in haematoxylin- and eosin-stained slides, and to the clinical data obtained through a telephone survey with referring veterinarians. RESULTS: Despite a low level of expression in the majority of cases, ß-catenin was found to be correlated strongly with malignant behaviour (P < 0.01). An overexpression of nuclear survivin was statistically related to histological features of malignancy, presence of metastasis and death related to melanoma spread (P < 0.01). CONCLUSIONS AND CLINICAL IMPORTANCE: The low nuclear ß-catenin expression, mainly found in metastatic cases, would indicate that ß-catenin activation may have only limited importance in the development or progression of canine cutaneous melanoma. The correlation of nuclear survivin expression with malignancy would indicate that survivin is possibly a useful prognostic marker and therapeutic target in canine melanoma patients.

On the role of survivin as a stem cell biomarker of canine hair follicle and related tumours.

BACKGROUND: Survivin has been identified as one of the most cancer-specific molecules, with a dual function of apoptosis inhibitor and orchestrator of cell division. HYPOTHESIS/OBJECTIVES: Based on our recent results obtained during the study of the role of survivin in epithelial-to-mesenchymal transition, we investigate its potential role in maintenance of stemness in both the normal canine hair follicle and related tumours. METHODS: We performed a simultaneous evaluation, by immunofluorescence, of the expression of survivin and CK15. CK15 was selected as a marker for epidermal and hair follicle stem cells, based on its ability to identify hair follicle stem cells in the normal hair follicle and in canine follicular tumours. In this study, six cases were selected from the cases of hair follicle tumours evaluated in previous studies, based on the highest immunoreactivity for survivin and CK15. Three samples of healthy canine skin were also included as a normal control. RESULTS: A partial co-localization of the molecules was observed in normal hair follicles, as well as in trichoepitheliomas and trichoblastomas. In particular, a different co-expression was observed in relationship to the hair follicle cycle stage. CONCLUSIONS AND CLINICAL IMPORTANCE: These findings suggest that survivin could play an important role in the maintenance of the hair follicle cycle as well as in tumour initiation and maintenance of cancer stem cells.

Cutaneous lesions in pet rabbits following subcutaneous administration of a novel bivalent vaccine against myxomatosis and rabbit haemorrhagic disease.

BACKGROUND: A novel bivalent vaccine to protect against myxomatosis and rabbit haemorrhagic disease is commercially available for pet rabbits. HYPOTHESIS/OBJECTIVES: To describe the appearance of cutaneous lesions arising in pet rabbits positive for myxoma virus (MV) by RT-PCR evaluation shortly after vaccination. ANIMALS: Four pet rabbits presenting with papular, crusting skin lesions ~10 days after vaccination. METHODS: Histological evaluation of formalin-fixed skin biopsies obtained from lesional skin (case 1). Real-time polymerase chain reaction (RT-PCR) evaluation of paraffin-embedded tissue from skin biopsies (case 1) and crusts obtained from the lesion surface (cases 2-4) for myxoma virus are reported as cycle threshold (Ct ) values. RESULTS: Lesions affecting the ear pinna, dorsal aspect of the nose, vulva and/or conjunctiva are reported. Histopathological findings included severe ulcerative, necrotizing dermatitis and intralesional cytoplasmic inclusion bodies in myxoma cells. DNA was amplified from all the paraffin-embedded skin biopsies (Ct  = 34-35) and crusts (Ct  = 20-24). CONCLUSIONS AND CLINICAL IMPORTANCE: Although a wild virus challenge cannot be definitively excluded, veterinarians and pet-owners should be aware that cutaneous lesions have been observed after vaccination with this novel vaccine in low numbers of rabbits.

Computed tomography-guided bone biopsies for evaluation of proliferative vertebral lesions in two boa constrictors (Boa constrictor imperator).

Two boa constrictors (Boa constrictor imperator) presented with paresis of the trunk originating cranial to the cloaca. Radiographs were consistent with proliferative bone lesions involving several vertebrae. Computed tomography (CT) demonstrated the presence of lytic/expansile lesions. Computed tomography-guided biopsies of the lesions were performed without complications. Histology was consistent with bacterial osteomyelitis and osteoarthritis. Gram-negative bacteria (Salmonella sp. and Pseudomonas sp.) were isolated from cultures of the biopsies. Medical treatment with specific antibiotics was attempted for several weeks in both cases without clinical or radiographic improvements. The animals were euthanized, and necropsy confirmed the findings observed upon CT. To the authors' knowledge, this is the first report of the use of CT-guided biopsies to evaluate proliferative vertebral lesions in snakes. In the present report, CT-guided biopsies were easily performed, and both histologic and microbiologic results were consistent with the final diagnosis.

Histomorphological evaluation of thermal injury following palatoplasty performed with CO2 laser or LigaSure device in dogs with brachycephalic obstructive airway syndrome.

The elongated soft palate is an abnormality that characterizes most brachycephalic dogs and contributes to the brachycephalic obstructive airway syndrome (BOAS). Palatoplasty is routinely performed in brachycephalic dogs; several surgical techniques exist. The use of surgical instruments such as monopolar electrocoagulation, CO2 or diode laser, bipolar vessel sealing device and harmonic shears has become routine to reduce the operating time, the intraoperative risk of bleeding and the postoperative oedema. This prospective study aimed to compare the histomorphological effect of a CO2 laser and LigaSure device in palates of dogs undergoing palatoplasty. Twenty owned brachycephalic dogs were included, 10 palatoplasties were performed using CO2 laser and 10 using LigaSure™ device. The dogs were positioned in sternal recumbency. A transoral approach was performed: the elongated soft palate was grasped with Allis forceps and brought rostrally, the palatoplasty was performed using the tonsillar crypts as anatomical landmarks. Surgical specimens were routinely fixed in 10 % formalin. Two sections perpendicular to the surgical margins were trimmed from each sample, paraffin-embedded and stained with hematoxylin and eosin (H&E). Tissue damage induced by the two types of surgical devices was graded (1-4, from minimal to severe) and the depth of thermal injury measured in µm on captured images (using an image analysis program - ImageJ). Mean values and standard deviations (SD) were calculated based on six measurements for each sample. The tissue damage was graded 3.7±0.48 in group LigaSure™ and 2.8±1 in group Laser. The mean depth of thermal injury was 874.94±184.92 µm in the LigaSure™ group and 451,76±137,86 µm in the Laser group. The comparison between the two groups showed significant lower grade and extension of thermal injury in the palate samples obtained with CO2 laser (p<0.05). Additionally, there is a lack of literature that correlates the histological changes with the clinical outcomes of the different palatoplasty methods in brachycephalic dogs. By comparing histological changes and clinical outcomes, we aim to provide valuable insights for optimizing the surgical approach for palatoplasty in brachycephalic dogs, ultimately improving postoperative outcomes for these patients.

The contribution of stem cells to epidermal and hair follicle tumours in the dog.

BACKGROUND: Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog. HYPOTHESIS: Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours. ANIMALS AND METHODS: In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin 15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. RESULTS: In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation.

Epithelial-to-mesenchymal transition: immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of ß-catenin and gain of mesenchymal markers. HYPOSTHESIS/OBJECTIVES: Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. METHODS: Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti-vimentin, -cytokeratin, -survivin and -Hsp72 antibodies. A semi-quantitative method was used to analyse the results, as follows: 0 to <5%; ≥ 5 to <10%; ≥ 10 to <25%; and ≥ 25% of positive cells. Immunofluorescence was performed to investigate survivin-vimentin and survivin-Hsp72 colocalization in selected SCCs. Results - In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin-survivin colocalization. CONCLUSIONS AND CLINICAL IMPORTANCE: A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested.

Extracellular Vesicles in Cancer Drug Resistance: Implications on Melanoma Therapy.

Extracellular vesicles (EVs) are involved in the pathogenesis of neoplastic diseases. Their role in mediating drug resistance has been widely described in several types of cancers, including melanoma. EVs can mediate drug resistance through several different mechanisms, such as drug-sequestration, transfer of pro-survival proteins and RNA, induction of cancer stem cell-like features and interaction with cells of the tumor microenvironment and immune-system. Melanoma is a highly immunogenic tumor originating from the malignant transformation of melanocytes. Several therapeutic strategies currently used in the treatment of melanoma and the combination of BRAF and MEK-inhibitors, as well as immune check-point inhibitors (ICI), have consistently improved the overall survival time of melanoma patients. However, the development of resistance is one of the biggest problems leading to a poor clinical outcome, and EVs can contribute to this. EVs isolated from melanoma cells can contain "sequestered" chemotherapeutic drugs in order to eliminate them, or bioactive molecules (such as miRNA or proteins) that have been proven to play a crucial role in the transmission of resistance to sensitive neoplastic cells. This leads to the hypothesis that EVs could be considered as resistance-mediators in sensitive melanoma cells. These findings are a pivotal starting point for further investigations to better understand EVs' role in drug resistance mechanisms and how to target them. The purpose of this review is to summarize knowledge about EVs in order to develop a deeper understanding of their underlying mechanisms. This could lead to the development of new therapeutic strategies able to bypass EV-mediated drug-resistance in melanoma, such as by the use of combination therapy, including EV release inhibitors.

Cell-Free RNA from Plasma in Patients with Neuroblastoma: Exploring the Technical and Clinical Potential.

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

Immunohistochemical investigation of cell cycle and apoptosis regulators (survivin, ß-catenin, p53, caspase 3) in canine appendicular osteosarcoma.

BACKGROUND: Osteosarcoma (OSA) represents the most common canine primary bone tumour. Despite several pathways have been investigated so far, few molecules have been identified as prognostic tools or potential therapeutic targets, and there is still the need to find out molecular pathways with specific influence over OSA progression to facilitate earlier prognosis and treatment.Aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of a panel of molecules (survivin, ß-catenin, caspase 3 -inactive and active forms- and p53) involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA, and to detect specific relations among them and with histological tumour grade, disease free interval (DFI) and overall survival (OS). RESULTS: Nuclear ß-catenin immunostaining was detected in normal osteoblasts adjacent to the tumour, and in 47% of the cases. Cytoplasmic and/or membranous immunostaining were also observed. Nuclear survivin and p53 positive cells were found in all cases. Moderate/high cytoplasmic ß-catenin expression (≥10% positive cells) was significantly associated with the development of metastasis (P = 0.014); moderate/high nuclear p53 expression (≥10% positive cells) was significantly associated with moderate/high histological grade (P = 0.017) and shorter OS (P = 0.049). Moderate/high nuclear survivin expression (≥15% positive cells) showed a tendency toward a longer OS (P = 0,088). CONCLUSIONS: The present results confirmed p53 as negative prognostic marker, while suggested survivin as a potential positive prognostic indicator, rather than indicative of a poor prognosis. The detection of nuclear ß-catenin immunostaining in normal osteoblasts and the absent/low expression in most of the OSAs, suggested that this pathway could not play a major role in oncogenic transformation of canine osteoblasts. Further studies are needed to confirm these hypotheses.