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.

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.

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.

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.

Auricular Non-Epithelial Tumors with Solar Elastosis in Cats: A Possible UV-Induced Pathogenesis.

The photoinduced etiopathology of actinic keratosis and squamous cell carcinoma in feline species is well known. This etiology has also been reported for non-epithelial cutaneous tumors in other species. To date, no cases of auricular non-epithelial cutaneous neoplasms erased in a contest of actinic keratosis in cats have been reported. The aim of this study was to describe feline auricular non-epithelial cutaneous neoplasms associated with typical UV-induced cutaneous lesions and solar elastosis. The study was conducted on five feline cases diagnosed with auricular non-epithelial cutaneous tumors (two fibrosarcomas, one mixosarcoma, one epithelioid melanoma and one hemangiosarcoma), selected from the Tumor Registry of the Department of Veterinary Sciences of the University of Pisa (1998-2018). Ten and six feline auricular biopsies of normal skin and skin with actinic keratosis, respectively, were used as controls. Orcein stain was used to investigate solar elastosis. Histological changes related to chronic solar irradiation were documented in the skin adjacent to the neoplastic lesions in the five cats. Considering the anatomical localization and the results of histopathology, this study suggests that non-epithelial cutaneous neoplasms may have a UV-induced etiopathogenesis in the feline species.

Pathological Features and Molecular Phenotype of MMTV Like-Positive Feline Mammary Carcinomas.

In the last few years MMTV-like nucleotide sequences were detected in some feline and canine mammary tumours. Due to the confirmed role of cats in the epidemiology of the MMTV-like virus, the aim of this study was to investigate the main pathological features of positive feline mammary carcinomas (FMCs). Twenty-four FMCs were collected at the University of Bologna, submitted to laser microdissection and analysed by nested fluorescence-PCR using primer sets specific for MMTV env sequence. For immunohistochemistry, an antibody against MMTV protein 14 (p14) was used. MMTV-like sequences were detected in three out of 24 FMCs (12.5%), one tubular carcinoma, one tubulopapillary carcinoma and one ductal carcinoma. All PCR-positive tumours were also positive for p14. Multiple nucleotide alignment has shown similarity to MMTV ranging from 98% to 100%. All the 102 examined FMCs were submitted to immunohistochemistry for molecular phenotyping. Of the nine MMTV-like positive FMCs, six were basal-like and three luminal-like. Our results demonstrate MMTV-like sequences and protein in FMCs of different geographic areas. Molecular phenotyping could contribute to understand the possible role of MMTV-like virus in FMC tumor biology.

PIP4Ks impact on PI3K, FOXP3, and UHRF1 signaling and modulate human regulatory T cell proliferation and immunosuppressive activity.

Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2 They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linked with susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.

An Immunohistochemical Study of the PTEN/AKT Pathway Involvement in Canine and Feline Mammary Tumors.

Phosphatase and tensin homolog deleted on chromosome10 (PTEN), phospho-v-Akt murine thymoma viral oncogene homolog (AKT), and the Rapamycin-Insensitive Companion of mTOR (Rictor) expression was investigated by immunohistochemistry in 10 canine mammary adenomas (CMAs), 40 canine mammary carcinomas (CMCs), and 30 feline mammary carcinomas (FMCs). All the CMAs, 25 of 40 CMCs (63%) and 7 of 30 FMCs (23%), were PTEN-positive. In dogs, no CMAs and 15 of 25 CMCs (37%) expressed phospho-AKT (p-AKT), while 24 of 30 FMCs (82%) were p-AKT-positive. One of 10 CMAs (10%), 24 of 40 CMCs (60%) and 20 of 30 FMCs (67%) were Rictor-positive. In the dog, PTEN expression correlated with less aggressive tumors, absence of lymphatic invasion, and longer survival. P-AKT expression correlated with more aggressive subtype, lymphatic invasion, and poorer survival and Rictor expression with lymphatic invasion. In cats, PTEN correlated with less aggressive carcinomas, absence of lymphatic invasion, and better survival. P-AKT and Rictor expression correlated with poorer survival. PTEN expression was inversely correlated with p-AKT and Rictor in both species, while p-AKT positively correlated with Rictor expression. A strong PTEN/AKT pathway involvement in behavior worsening of CMT and FMTs is demonstrated, providing a rationale for further studies of this pathway in veterinary oncology.

M1 and M2 tumour-associated macrophages subsets in canine malignant mammary tumours: An immunohistochemical study.

Among the innate and adaptative immune cells recruited to the tumour site, tumour associated macrophages (TAMs) are particularly abundant and by simplified classification can be classified into (M1) and (M2) TAMs. In the present study, we quantified by immunohistochemistry ionized calcium binding adaptor molecule 1 (Iba1)-positive total and CD204-positive M2-polarized TAMs in 60 canine malignant mammary tumours (CMMTs) to analyze the relationship between M1 or M2 response and the histopathologic features of examined CMMTs, the dogs' body condition score (BCS) and the progression of the neoplastic disease. The mean number of total and CD204+ TAMS were significantly higher in solid and in grade III than in grades I and II carcinomas. Moreover, the mean number of CD204-positive TAMs was significantly higher in CMMTs with lymphatic invasion and necrosis rather than CMMTs without. The presence of higher number of CD204-positive M2-polarized TAMs was associated with a worst outcome of the neoplastic disease: bitches bearing CMMTs with a prevalent M2-polarized TAM response had a median cancer-specific survival time of 449 days, while in animals with a M1-polarized TAM response the median cancer-specific survival time was 1209 days. The results of our study confirm that in CMMTs the presence of a M2-polarized TAMs response might affect the tumour development and behaviour. Finally, it strongly suggests the potential of CD204 expression as a prognostic factor.

Canine Mammary Carcinoma With Vacuolated Cytoplasm: Glycogen-Rich Carcinoma, a Histological Type Distinct From Lipid-Rich Carcinoma.

Lipid-rich carcinoma is a rare histotype of canine mammary tumors with cytoplasmic vacuolation. In humans, glycogen-rich carcinoma, secretory carcinoma, and myoepithelial neoplasms are included in the differential diagnosis for lipid-rich carcinoma. The aim of the study was to investigate the existence of histotypes other than lipid-rich in canine mammary carcinomas with vacuolated cytoplasm using a diagnostic algorithm based on histopathology, histochemistry, immunohistochemistry, and ultrastructure and to evaluate the molecular phenotype of these neoplasms. Ten mammary carcinomas were collected, histologically reviewed, and subjected to histochemistry (PAS, PAS with diastase, Alcian blue, Sudan III [1 case], and Congo red [1 case]); immunohistochemistry for CK19, CK5/6, CK14, p63, calponin, vimentin, ER, PR, and HER2; and transmission electron microscopy (TEM). Cytokeratin immunolabeling demonstrated the epithelial origin of all tumors. Sudan III and TEM confirmed the diagnosis of lipid-rich carcinoma in 8 tumors (one amyloid-producing). One tumor was reclassified as a glycogen-rich carcinoma based on PAS reactivity that was diastase-labile, and a second tumor was reclassified as a carcinoma-and-malignant myoepithelioma based on the differentiation markers. Lipid-rich carcinomas were basal-like (5/8), null-type (2/8), and luminal A phenotype (1/8). The glycogen-rich carcinoma was basal-like, while the carcinoma-and-malignant myoepithelioma was luminal A. Vacuolated morphology of neoplastic cells in canine mammary carcinoma can indicate either a neoplasm of luminal epithelial origin with cytoplasmic lipid or glycogen, or vacuolated neoplastic suprabasal myoepithelial cells. Glycogen-rich carcinoma is a novel histological type that should be considered in the differential diagnosis for canine mammary carcinomas with vacuolated cytoplasm.

Cytologic grading of canine and feline spindle-cell sarcomas of soft tissues and its correlation with histologic grading.

In humans, soft tissue spindle cell sarcomas (STSCS) grading is considered a useful parameter in determining prognosis and therapy, and it is recognized as an important prognostic factor in canine STSCS. The purpose of this study was to assess the utility and the accuracy of a cytologic grading system on fine needle aspiration cytology (FNAC) smears of canine and feline cutaneous and subcutaneous STSCS .Thirty-three cases of cytologically diagnosed STSCS were included. The smears and their tumour sections were cytologically and histologically graded, according to established methods in human oncology.Canine STSCS showed a cyto/histologic concordance in 12/20 cases (60%). Concordance was observed in 4/8 (50%) of grade 1, in 8/12 (67%) of grade 2, and in 0 cases of grade 3. Feline STSCS showed concordance in 11/13 cases (85%). Concordance was observed in 5/6 (83%) of grade 1, in 4/4 (100%) of grade 2, and in 2/3 (66.6%) of grade 3 cases. The overall concordance in the entire canine and feline population was 70%. The gradewise concordance was 65% in grade 1, 75% in grade 2, and 66% in grade 3 cases. The overall concordance is similar to that reported in humans. Although a wider population is required to strengthen our findings, these results suggest that cytologic grading of STSCSs may be a useful tool for therapeutic and prognostic evaluations in dogs and cats.