Methods and computational tools to study eukaryotic cell migration in vitro.

Cellular movement is essential for many vital biological functions where it plays a pivotal role both at the single cell level, such as during division or differentiation, and at the macroscopic level within tissues, where coordinated migration is crucial for proper morphogenesis. It also has an impact on various pathological processes, one for all, cancer spreading. Cell migration is a complex phenomenon and diverse experimental methods have been developed aimed at dissecting and analysing its distinct facets independently. In parallel, corresponding analytical procedures and tools have been devised to gain deep insight and interpret experimental results. Here we review established experimental techniques designed to investigate specific aspects of cell migration and present a broad collection of historical as well as cutting-edge computational tools used in quantitative analysis of cell motion.

A Preliminary Evaluation of the Prognostic Role of HER-2 and HER-3 Immunohistochemical Expression in Canine Melanomas.

Canine melanoma is a malignant and aggressive neoplasm showing clinical, histological, and molecular features similar to the human counterpart. In human medicine, epidermal growth factor receptors (EGFRs) have already been suggested as prognostic markers and potential therapeutic targets in cutaneous melanoma. The aim of this study was to evaluate the expression of HER-2 and HER-3 in canine melanomas by immunohistochemistry and correlate their expression to the clinicopathological parameters of the examined tumors. Thirty-seven canine melanoma samples were recruited. Data regarding signalment and clinical parameters were also collected. The population was composed of 18 cutaneous, 16 oral/mucosal, and three digital/foot pad melanomas. Histopathological investigations were carried out to analyze histological type, ulceration, and mitotic count. On each sample, immunohistochemistry was performed using an anti-Melan-A or anti-Melanoma antigen, i.e., anti-HER-2 and anti-HER-3 antibodies. HER-2 and HER-3 positivity were classified using already established scoring criteria and a statistical analysis was carried out. The results highlighted that HER-2 expression was observed in 48.6% of the samples and HER-3 expression in 18.9%. The highest HER 2 score (3+) was recorded in 16.2% of the samples, while the coexpression of the two receptors was detected in 13.5% of the samples. A statistically significant association (p < 0.05) was observed between the expression of HER-2 and HER-3 and the presence of ulceration in oromucosal tumors. This work confirms the expression of HER-2 and HER-3 in canine melanomas and suggests a putative association with negative prognostic parameters. Further studies are necessary to strengthen these data by increasing the samples size and combining pathological examinations with molecular biology in the investigation of EGFR family receptors.

N2FXm, a method for joint nuclear and cytoplasmic volume measurements, unravels the osmo-mechanical regulation of nuclear volume in mammalian cells.

In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, current methods to measure cytoplasmic and nuclear volumes, including confocal 3D reconstruction, have limitations, such as relying on two-dimensional projections or poor vertical resolution. Here, to overcome these limitations, we describe a method, N2FXm, to jointly measure cytoplasmic and nuclear volumes in single cultured adhering human cells, in real time, and across cell cycles. We find that this method accurately provides joint size over dynamic measurements and at different time resolutions. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we show that N2FXm can give relevant insights on how mechanical forces exerted by the cytoskeleton on the nuclear envelope can affect the growth of nucleus volume by biasing nuclear import. Our method, by allowing for accurate joint nuclear and cytoplasmic volume dynamic measurements at different time resolutions, highlights the non-constancy of the nucleus/cytoplasm ratio along the cell cycle.

A planar polarized MYO6-DOCK7-RAC1 axis promotes tissue fluidification in mammary epithelia.

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.

Cancerogenic parasites in veterinary medicine: a narrative literature review.

Parasite infection is one of the many environmental factors that can significantly contribute to carcinogenesis and is already known to be associated with a variety of malignancies in both human and veterinary medicine. However, the actual number of cancerogenic parasites and their relationship to tumor development is far from being fully understood, especially in veterinary medicine. Thus, the aim of this review is to investigate parasite-related cancers in domestic and wild animals and their burden in veterinary oncology. Spontaneous neoplasia with ascertained or putative parasite etiology in domestic and wild animals will be reviewed, and the multifarious mechanisms of protozoan and metazoan cancer induction will be discussed.

Exploring the link between viruses and cancer in companion animals: a comprehensive and comparative analysis.

Currently, it is estimated that 15% of human neoplasms globally are caused by infectious agents, with new evidence emerging continuously. Multiple agents have been implicated in various forms of neoplasia, with viruses as the most frequent. In recent years, investigation on viral mechanisms underlying tumoral transformation in cancer development and progression are in the spotlight, both in human and veterinary oncology. Oncogenic viruses in veterinary medicine are of primary importance not only as original pathogens of pets, but also in the view of pets as models of human malignancies. Hence, this work will provide an overview of the main oncogenic viruses of companion animals, with brief notes of comparative medicine.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

Confirmation of the Prognostic Value of Foxp3+ Cells in Canine Mammary Tumors.

Foxp3+ cell counts were evaluated by immunohistochemistry in 59 canine mammary tumors, 20 adenomas, and 39 carcinomas in three different compartments: intratumoral, within the adjacent stroma, and in the distant stroma. Foxp3+ lymphocyte counts were compared with histotype, grading, presence of lymphatic invasion, immunohistochemical expression of estrogen and progesterone receptors, expression of c-erbB-2, and the overall survival (OS). Our findings confirmed that Foxp3+ cells were significantly higher in canine mammary carcinomas compared to adenomas. A significantly higher number of Foxp3+ cells were detected in grade III carcinomas compared to grade II carcinomas, as well as in tumors with lymphatic invasion and loss of ER-expression. Finally, a high number of Foxp3+ cells was associated with poor prognosis. In conclusion, our findings highlighted the association of Foxp3+ lymphocytes with negative clinicopathological features and shorter overall survival (OS), thus confirming the role of Tregs as a negative prognostic marker in canine mammary carcinomas.

Preliminary Assessment of Tumor-Associated Tissue Eosinophilia (TATE) in Canine Mast Cell Tumors: Prevalence and Prognostic Relevance and Its Association with Neoangiogenesis.

Mast cell tumor (MCT) is the most common malignant skin tumor in dogs. In order to gain more information on the prognostic markers in MCT, the role of the eosinophil granulocytes infiltrates was investigated and assessed by the evaluation of tumor-associated tissue eosinophilia (TATE) in 87 canine cutaneous MCTs. In human medicine, high TATE are often described in highly angiogenic tumors: we therefore assessed the vascular endothelial growth factor (VEGF) expression in neoplastic mast cells. TATE and VEGF expression were compared between themselves, with histological grading, immunohistochemical expression of KIT and Ki-67, and with the recurrence. We found a statistically significant correlation between TATE and Patnaik grading (p = 0.041), Kiupel grading (p = 0.022), immunohistochemical KIT expression (p = 0.015), and tumor recurrence (p = 0.000). No associations were observed with Ki-67 and VEGF expression. This is the first evaluation of TATE and its prognostic value in canine MCTs in veterinary oncology. This study suggest that this investigation could be an important source of information for this tumor and for other neoplasms.

Skeletal muscle dissemination in a dog with T-cell lymphoma.

A 5-year-old spayed female American Staffordshire was referred for weakness, reluctance to move and distension of the abdomen. Three weeks before, the dog underwent surgery for excision of a nodular mass suspected to be a non-epitheliotropic cutaneous T-cell lymphoma (NE-CTCL). Computed tomography revealed heterogeneous enhancing mesenteric masses and nodular lesions of soft tissue density, and infiltration of the abdominal muscular wall. Moreover, a pattern of diffuse muscle nodules in the skeletal muscles was visible, with lesions showing homogenous, heterogeneous or ring enhancement. Necrosis was histologically observed and these lesions were infiltrated by CD3-positive and CD20-, CD79a- and Iba1-negative neoplastic lymphocytes. On the basis of the immunopathological features metastatic NE-CTCL was suspected. Skeletal muscle metastasis has been rarely reported in small animals and this case report further confirms that this possibility should be considered in dogs with lymphoma.

Immunohistochemical Characterization of Immune System Cells in Lymphoid Organs from Roe and Fallow Deer.

Roe and Fallow deer are common wild ruminants widely distributed in Italy. Infectious diseases of these species can potentially pose health risks to domestic animals and humans. However, few studies have been conducted in which immune system cells in these species were phenotyped. The aims of this study were to determine the cross-reactivity of a wide anti-human panel of commercial antibodies on formalin-fixed and paraffin-embedded (FFPE) samples and to describe the distribution of roe and fallow deer main immune cell subsets in the lymph nodes and spleen. Twenty retromandibular lymph nodes (RLNs) and spleen samples were collected from 10 roe deer and 10 fallow deer and were tested by a panel of 12 commercial anti-human antibodies. The CD79a, CD20, CD3, Iba-1, MAC387, and AM-3K antibodies were successfully labeled cells in cervine tissue, while the Foxp3 and the CD68 did not show suitable immunostaining. This study supplies the first immunohistochemical description of immune cell subpopulations in non-pathological spleen and RLNs from roe and fallow deer and provides an easily repeatable manual IHC protocol to immunolocalize cervine B-, T-cells, and macrophages subsets in FFPE tissue samples.

Toxoplasmosis in Captive Ring-Tailed Lemurs (Lemur catta).

Toxoplasma gondii is one of the most common protozoan parasites and is widely present in all warm-blooded animals. Although clinical disease is uncommon, some species, including ring-tailed lemurs (Lemur catta), have been found to develop acute and lethal toxoplasmosis. The aim of this study was to describe the pathologic, immunohistochemical, serological, and molecular findings of an outbreak of fatal toxoplasmosis in three captive ring-tailed lemurs in Central Italy in 2009. The animals died acutely within few days. The necropsy was immediately performed; necrotic lesions in the spleen, liver, and kidney, as well as interstitial pneumonia, were found histologically. All animals had high titers of anti-T. gondii-specific antibodies (1:1280 IgM and 1:640 IgG) according to a modified agglutination test (MAT) and immunohistochemistry showed scattered tachyzoites in the target organs. Diagnosis was confirmed by PCR and clonal type II was identified. In addition, the seven co-habiting lemurs were seronegative. This paper reports the first outbreak of acute disseminated toxoplasmosis in captive ring-tailed lemurs in Italy. These findings confirm the high susceptibility of this endangered species to toxoplasma infection, which may be considered a further threat to captive population viability.

Occurrence of Bacterial and Protozoan Pathogens in Red Foxes (Vulpes vulpes) in Central Italy.

Most surveys of pathogens in red foxes (Vulpes vulpes) have focused on particular agents. The aim of this study was to verify, with bacteriological and molecular analyses, the occurrence of the main bacterial and protozoan pathogens that are able to infect canids, in red foxes regularly hunted in Central Italy. Spleen, brain, kidney and fecal samples from red foxes were submitted to bacteriological and/or molecular analyses to detect Salmonella spp., Yersinia spp., Listeria monocytogenes, Brucella spp., Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Leptospira spp., Neospora caninum, Hepatozoon canis, Babesia spp. and microsporidia. Two (9.1%) strains of Yersinia enterocolitica biotype 1 and 2 (9.1%) of Yersinia frederiksenii were isolated from 22 fecal samples. Among the 22 spleen samples, seven (31.8%) were PCR-positive for H. canis and 3 (13.6%) for Babesia vulpes. Kidneys from two (2.9%) foxes, among 71 tested, were PCR-positive for L. interrogans. Even though the analyses were carried out on a small number of animals, the results suggested that red foxes from the selected geographic area may act as reservoirs of some investigated pathogens.

Skin Photodamage Lesions in a Bilateral Feline Auricular Primary Fibrosarcoma.

As with human species, recent studies also suggest a photoinduced etiopathology for non-epithelial cutaneous tumors in feline species. We report a recent case of a ten-year-old male cat with a white-hair coat and mesenchymal neoplasms of both auricles. Cytology, complete blood count (CBC), serum biochemistry and imaging examinations were performed. After surgery, the samples underwent routinary histopathology and were additionally stained with orcein. A routine analysis yielded values within a normal range and the imaging examination showed no abnormalities, suggesting that the bilateral presentation of neoplasms was primary rather than metastatic. The cytology was inconclusive, but, through histopathology, two well-differentiated fibrosarcomas were diagnosed and histopathological changes related to chronic UV exposure (such as epidermal hyperplasia, stratification disorders, keratinocyte dysplasia and an accumulation of elastotic material) were documented in the skin adjacent to the lesions. An orcein stain succeeded in highlighting elastosis. The elastic fibers lost their regular structure and orientation and appeared to be fragmented, wavy to branched and knotted. A morphometric analysis showed that the amount of elastotic material in the dermis close to the tumors was more than double compared with the more distant areas. Elastosis is considered to be a hallmark of photodamage; thus, an involvement of UV rays in the carcinogenic process of the tumors may be suspected.

The Italian Network of Laboratories for Veterinary Oncology (NILOV) 2.0: Improving Knowledge on Canine Tumours.

Advances in tumour research are crucial, and comparative oncology can improve the knowledge in several ways. Dogs are not only models of specific naturally occurring tumours but can also be sentinels of environmental exposures to carcinogens, as they share the same environment with their owners. The purpose of this work was to describe the data collected by The Italian Network of Laboratories for Veterinary Oncology in the first 9 years of activity (2013-2021) and to evaluate their potential epidemiological significance. Frequencies of tumour topographies and main morphologies in dogs were described, analysed and compared, calculating age-adjusted proportional morbidity ratios and considering several risk factors (breed, sex, period and region of residence). These observations allowed us to highlight differences not only in morphology and topography of some tumours but also to formulate hypotheses on the potential role of some risk factors, e.g., neutering/spaying or geographical location. In our opinion, the results of this case series confirm the importance of initiating and consolidating animal cancer registration initiatives that would facilitate the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumours in dogs from a comparative perspective.

Virulence Genes of Pathogenic Escherichia coli in Wild Red Foxes (Vulpes vulpes).

Different pathotypes of Escherichia coli can cause severe diseases in animals and humans. Wildlife may contribute to the circulation of pathogenic pathotypes, including enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC). This study analyzed 109 DNA samples previously extracted from fecal specimens collected from red foxes (Vulpes vulpes) to detect E. coli virulence genes eaeA, hlyA, stx1, and stx2, that characterize the EPEC, STEC, and EHEC strains. Thirty-one (28.4%) samples were positive for at least one investigated virulence gene: eaeA gene was detected in 21 (19.2%) samples, hlyA in 10 (9.1%), stx1 in 6 (5.5%), and stx2 in 4 (3.6%). Nine DNA samples resulted positive for two or three virulence genes: five (4.6%) samples were positive for eaeA and hlyA genes, two (1.8%) for eaeA and stx1, one (0.9%) for hlyA and stx1, one (0.9%) for eaeA, hlyA and stx2. Red foxes seem to be involved in the epidemiology of these infections and their role could be relevant because they may be source of pathogenic E. coli for other wild animals, as well as domestic animals and humans.

Presence of a mouse mammary tumour virus-like in feline lymphomas: a preliminary study.

The mouse mammary tumour virus (MMTV) is implicated in the aetiology of murine mammary carcinomas and a variant of it, the type B leukemogenic virus, can cause murine thymic lymphomas. Interestingly, a MMTV-like virus is suspected to be involved in human breast cancer and feline mammary carcinomas. However, to date, no cases of MMTV-like sequence amplifications have been described in lymphoid neoplasms in veterinary literature. The aim of this study was to investigate the presence of env nucleotide sequences and protein 14 (p14) of a MMTV-like virus in fifty-three feline lymphoma samples. Our results show that MMTV-like sequences were detected in 5/53 tumours (9.4%): three gastrointestinal lymphomas (one B-type diffuse large, one B-type small non-cleaved, and one T-type diffuse mixed lymphoma); and two nasal lymphomas (one B-type diffuse small cleaved lymphoma and one B-type diffuse mixed lymphoma). P14 expression was detected in the cytoplasm, and rarely in nuclei, exclusively of neoplastic cells from PCR-positive tumours. The correlation between the presence of the MMTV-env like sequences (MMTVels) and p14 antigen was statistically significant in nasal lymphomas. All cats with MMTVels-positive lymphoma had a history of contact with the outdoor environment and/or catteries, and two deceased subjects shared their environment with cats that also died of lymphoma. In conclusion, this study succeeds in demonstrating the presence of MMTVels and p14 in feline lymphomas. The characterization of the immunophenotype of MMTVels-positive lymphomas could contribute to the understanding of a possible role of a MMTV-like virus in feline tumour aetiology. The significant association between the presence of the viral sequences in lymphoid tumours and their nasal localization, together with the data collected through supplementary anamnesis, should be further analysed in order to understand the epidemiology of the virus.

Mouse Mammary Tumor Virus (MMTV) and MMTV-like Viruses: An In-depth Look at a Controversial Issue.

Since its discovery as a milk factor, mouse mammary tumor virus (MMTV) has been shown to cause mammary carcinoma and lymphoma in mice. MMTV infection depends upon a viral superantigen (sag)-induced immune response and exploits the immune system to establish infection in mammary epithelial cells when they actively divide. Simultaneously, it avoids immune responses, causing tumors through insertional mutagenesis and clonal expansion. Early studies identified antigens and sequences belonging to a virus homologous to MMTV in human samples. Several pieces of evidence fulfill a criterion for a possible causal role for the MMTV-like virus in human breast cancer (BC), though the controversy about whether this virus was linked to BC has raged for over 40 years in the literature. In this review, the most important issues related to MMTV, from its discovery to the present days, are retraced to fully explore such a controversial issue. Furthermore, the hypothesis of an MMTV-like virus raised the question of a potential zoonotic mouse-man transmission. Several studies investigate the role of an MMTV-like virus in companion animals, suggesting their possible role as mediators. Finally, the possibility of an MMTV-like virus as a cause of human BC opens a new era for prevention and therapy.

The transcription factor PREP1(PKNOX1) regulates nuclear stiffness, the expression of LINC complex proteins and mechanotransduction.

Mechanosignaling, initiated by extracellular forces and propagated through the intracellular cytoskeletal network, triggers signaling cascades employed in processes as embryogenesis, tissue maintenance and disease development. While signal transduction by transcription factors occurs downstream of cellular mechanosensing, little is known about the cell intrinsic mechanisms that can regulate mechanosignaling. Here we show that transcription factor PREP1 (PKNOX1) regulates the stiffness of the nucleus, the expression of LINC complex proteins and mechanotransduction of YAP-TAZ. PREP1 depletion upsets the nuclear membrane protein stoichiometry and renders nuclei soft. Intriguingly, these cells display fortified actomyosin network with bigger focal adhesion complexes resulting in greater traction forces at the substratum. Despite the high traction, YAP-TAZ translocation is impaired indicating disrupted mechanotransduction. Our data demonstrate mechanosignaling upstream of YAP-TAZ and suggest the existence of a transcriptional mechanism actively regulating nuclear membrane homeostasis and signal transduction through the active engagement/disengagement of the cell from the extracellular matrix.

Molecular and Pathological Detection of Hepatitis E Virus in Roe Deer (Capreolus capreolus) and Fallow Deer (Dama dama) in Central Italy.

Hepatitis E virus (HEV) is a common causative agent of acute hepatitis in the world, with a serious public health burden in both developing and industrialized countries. Cervids, along with wild boars and lagomorphs, are the main wild hosts of HEV in Europe and constitute a documented source of infection for humans. The aim of this study was to evaluate the presence of HEV in roe deer (Capreolus capreolus) and fallow deer (Dama dama) living in Tuscany, Central Italy. Liver samples from 48 roe deer and 60 fallow deer were collected from carcasses during the hunting seasons. Following the results obtained from molecular and histopathologic studies, 5/48 (10.4%) roe deer and 1/60 (1.7%) fallow deer liver samples were positive for the presence of HEV RNA. All PCR-positive livers were also IHC-positive for viral antigen presence, associated with degenerative and inflammatory lesions with predominantly CD3+ cellular infiltrates. This study represents the first identification in Italy of HEV RNA in roe and fallow deer and the first study in literature describing liver alterations associated with HEV infection in cervids. These results demonstrate that HEV is present in wild cervid populations in Italy and confirm the potential zoonotic role of these species.