Intercellular interactions between mast cells and stromal fibroblasts obtained from canine cutaneous mast cell tumours.

Mast cell tumours (MCTs) are the most frequent malignant skin neoplasm in dogs. Due to the difficulty in purifying large numbers of canine neoplastic mast cells, relatively little is known about their properties. A reproducible in vitro model is needed to increase the understanding about the phenotype and functional properties of neoplastic mast cells. In the present study, we describe the establishment of primary cocultures of neoplastic mast cells from canine cutaneous MCTs and cancer-associated fibroblasts. We confirmed the inability of canine neoplastic mast cells to remain viable for long periods in vitro without the addition of growth factors or in vivo passages in mice. Using a transwell system, we observed that mast cell viability was significantly higher when there is cell-to-cell contact in comparison to non-physical contact conditions and that mast cell viability was significantly higher in high-grade than in low-grade derived primary cultures. Moreover, the use of conditioned medium from co-cultured cells led to a significantly higher tumoral mast cell viability when in monoculture. Signalling mechanisms involved in these interactions might be attractive therapeutic targets to block canine MCT progression and deserve more in-depth investigations.

Proteomic Analysis Identifies FNDC1, A1BG, and Antigen Processing Proteins Associated with Tumor Heterogeneity and Malignancy in a Canine Model of Breast Cancer.

New insights into the underlying biological processes of breast cancer are needed for the development of improved markers and treatments. The complex nature of mammary cancer in dogs makes it a great model to study cancer biology since they present a high degree of tumor heterogeneity. In search of disease-state biomarkers candidates, we applied proteomic mass spectrometry imaging in order to simultaneously detect histopathological and molecular alterations whilst preserving morphological integrity, comparing peptide expression between intratumor populations in distinct levels of differentiation. Peptides assigned to FNDC1, A1BG, and double-matching keratins 18 and 19 presented a higher intensity in poorly differentiated regions. In contrast, we observed a lower intensity of peptides matching calnexin, PDIA3, and HSPA5 in poorly differentiated cells, which enriched for protein folding in the endoplasmic reticulum and antigen processing, assembly, and loading of class I MHC. Over-representation of collagen metabolism, coagulation cascade, extracellular matrix components, cadherin-binding and cell adhesion pathways also distinguished cell populations. Finally, an independent validation showed FNDC1, A1BG, PDIA3, HSPA5, and calnexin as significant prognostic markers for human breast cancer patients. Thus, through a spatially correlated characterization of spontaneous carcinomas, we described key proteins which can be further validated as potential prognostic biomarkers.

Galectin-3 immunolabelling correlates with BCL2 expression in canine cutaneous mast cell tumours.

Mast cell tumour (MCT) is the most frequent skin neoplasm in dogs. These tumours are characterised by variable behaviour and clinical presentation that make prognosis an important and challenging task in the veterinary practice. Galectin-3 (Gal-3) is known to influence several biological processes that are important in the cancer context and has been described as a prognostic marker for several human cancers. The aim of the present work was to characterise Gal-3 immunolabelling in canine cutaneous MCTs and to investigate its value as a prognostic marker for the disease. Thirty-four random cases of canine cutaneous MCT that were surgically treated with wide margins were included in this study. Gal-3 expression was evaluated using immunohistochemistry and the results were compared with the expression of apoptosis-related proteins, Ki67 index, histopathological grades, mortality due to the disease and post-surgical survival. The majority of the MCTs (65.8%) were positive for Gal-3. Gal-3 immunolabelling was variable among the samples (2.7%-86.8% of the neoplastic cells). The protein was located in the cytoplasm or in the cytoplasm and the nucleus. Gal-3 positivity was correlated with BCL2 expression (P < 0.001; r = 0.604), but not with Ki67 and BAX. No significant differences were detected between histological grades or in the survival analysis. Gal-3 expression correlates with BCL2 expression in MCTs. Although an efficient marker for several human neoplasms, the results presented herein suggest that Gal-3 immunolabelling is not an independent prognostic indicator for this disease.

Liquid biopsy based on small extracellular vesicles predicts chemotherapy response of canine multicentric lymphomas.

Lymphoma is the most common type of canine hematological malignancy where the multicentric (cMCL) form accounts for 75% of all cases. The standard treatment is the CHOP chemotherapy protocols that include cyclophosphamide, doxorubicin, vincristine and prednisone, where the majority of dogs achieve complete/partial response; however, it is very important to predict non-responsive cases to improve treatment and to develop new targeted therapies. Here we evaluate a liquid biopsy approach based on serum Small Extracellular Vesicles enriched for exosomes (SEVs) to predict cMCL chemotherapy response. Nineteen dogs at the end of the 19-week chemotherapy protocol (8 Complete Response and 11 Progressive Disease) were evaluated for serum SEVs size, concentration and screened for 95 oncomirs. PD patients had higher SEVs concentration at the diagnosis than CR patients (P = 0.034). The ROC curve was significant for SEVs concentration to predict the response to CHOP (AUC = 0.8011, P = 0.0287). A potential molecular signature based on oncomirs from SEVs (caf-miR-205, caf-miR-222, caf-mir-20a and caf-miR-93) is proposed. To the best of our knowledge, this is the first study demonstrating the potential of a liquid biopsy based on SEVs and their miRNAs content to predict the outcome of chemotherapy for canine multicentric lymphomas.

Identification of two molecular subtypes in canine mast cell tumours through gene expression profiling.

Mast cell tumours (MCTs) are common neoplasms in dogs and are usually regarded as potentially malignant. Several studies have attempted to identify biomarkers to better predict biological behaviours for this tumour. The aim of this study was to identify pathways connected to clinical and histopathological malignancies, shorter survival times, and poor prognoses associated with MCTs. We performed genome-wide gene expression analyses on tissues obtained from 15 dogs with single MCTs, and identified two distinct tumour subtypes-high-risk and low-risk-associated with differences in histological grades, survival times, Ki67 indices, and occurrence of death due the disease. Comparative analyses of RNA sequence profiles revealed 71 genes that were differentially expressed between high- and low-risk MCTs. In addition to these analyses, we also examined gene co-expression networks to explore the biological functions of the identified genes. The network construction revealed 63 gene modules, of which 4 were significantly associated with the more aggressive tumour group. Two of the gene modules positively correlated with high-risk MCTs were also associated with cell proliferation and extracellular matrix-related terms. At the top of the extracellular matrix module category, genes with functions directly related to those of cancer-associated fibroblasts (CAFs) were identified. Immunohistochemical analyses also revealed a greater number of CAFs in high-risk MCTs. This study provides a method for the molecular characterisation of canine MCTs into two distinct subtypes. Our data indicate that proliferation pathways are significantly involved in malignant tumour behaviours, which are known to be relevant for the induction and maintenance of MCTs. Finally, animals presenting high-risk MCTs overexpress genes associated with the extracellular matrix that can be robustly linked to CAF functions. We suggest that CAFs in the MCT stroma contribute to cancer progression.

Intratumoral collagen index predicts mortality and survival in canine cutaneous mast cell tumours.

BACKGROUND: Mast cell tumours (MCTs) constitute almost 25% of cutaneous neoplasms in dogs. Their biological behaviour is predicted using histopathological grading which is based on several subjective criteria that are vulnerable to intra- and interobserver variability. To improve the prediction of the biological behaviour, several complementary markers have been studied. The integrity of the extracellular matrix (ECM) may play a protective role against tumoral progression, and favour cellular proliferation, angiogenesis, invasion and metastases when altered. HYPOTHESIS/OBJECTIVES: To evaluate the quantification of collagen and elastic fibres as prognostic markers for MCTs. ANIMALS: Thirty-eight random cases of canine cutaneous MCT surgically treated with wide margins were included. METHODS AND MATERIALS: Intratumoral collagen and elastic fibres were identified and quantified on histological sections stained with Masson's trichrome, Picrosirius red and Verhoeff; the results were compared with histopathological grades, mortality due to the disease and postsurgical survival. RESULTS: Morphometric analysis revealed a significant relationship between histopathological grade and intratumoral collagen index (CoI). In addition, the CoI was considered an independent indicator for mortality and postsurgical survival. CONCLUSIONS AND CLINICAL IMPORTANCE: These results support the importance of the CoI in the grading and prognosis of MCTs, suggesting that preservation and/or synthesis of collagen have the potential to become targets for MCT therapeutics.

Nanog Expression and Proliferation Indices in Canine Cutaneous Mast Cell Tumors.

Mast cell tumors are one of the most frequent skin tumors in dogs. Treatment decisions often depend on a wide range of clinical information and the main criteria for prognostic formulation are histological grade, mitotic count, Ki67 index, and KIT immunostaining pattern. NANOG is a pluripotency factor expressed by normal and cancer stem cells, which is a prognostic marker and a potential therapeutic target for several human tumors. In the present study, mast cell tumor samples from 41 dogs were evaluated for NANOG and Ki67 by immunohistochemistry. All samples were positive for NANOG but its expression was not correlated with Ki67 index and no significant differences were found with respect to histopathological grades, disease-related mortality, or survival. Our results suggest that, although related to pluripotency, NANOG expression does not correlate with proliferative activity, and is not a reliable prognostic factor for canine cutaneous mast cell tumors.

Liver transcriptomic networks reveal main biological processes associated with feed efficiency in beef cattle.

BACKGROUND: The selection of beef cattle for feed efficiency (FE) traits is very important not only for productive and economic efficiency but also for reduced environmental impact of livestock. Considering that FE is multifactorial and expensive to measure, the aim of this study was to identify biological functions and regulatory genes associated with this phenotype. RESULTS: Eight genes were differentially expressed between high and low feed efficient animals (HFE and LFE, respectively). Co-expression analyses identified 34 gene modules of which 4 were strongly associated with FE traits. They were mainly enriched for inflammatory response or inflammation-related terms. We also identified 463 differentially co-expressed genes which were functionally enriched for immune response and lipid metabolism. A total of 8 key regulators of gene expression profiles affecting FE were found. The LFE animals had higher feed intake and increased subcutaneous and visceral fat deposition. In addition, LFE animals showed higher levels of serum cholesterol and liver injury biomarker GGT. Histopathology of the liver showed higher percentage of periportal inflammation with mononuclear infiltrate. CONCLUSION: Liver transcriptomic network analysis coupled with other results demonstrated that LFE animals present altered lipid metabolism and increased hepatic periportal lesions associated with an inflammatory response composed mainly by mononuclear cells. We are now focusing to identify the causes of increased liver lesions in LFE animals.

Constitutive androstane receptor ligands modulate the anti-tumor efficacy of paclitaxel in non-small cell lung cancer cells.

BACKGROUND: Lung tumors are the leading cause of cancer deaths worldwide and paclitaxel has proven to be useful for patients with lung cancer, however, acquired resistance is a major problem. To overcome this problem, one promising option is the use of Constitutive Androstane Receptor (CAR) ligands in combination with chemotherapeutics against cancer cells. Therefore, we wish to elucidate the effects of CAR ligands on the antineoplastic efficacy of paclitaxel in lung cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Our results from cell viability assays exposing CAR agonist or inverse-agonist to mouse and human lung cancer cells modulated the antineoplastic effect of paclitaxel. The CAR agonists increased the effect of Paclitaxel in 6 of 7 lung cancer cell lines, whereas the inverse-agonist had no effect on paclitaxel cytotoxicity. Interestingly, the mCAR agonist TCPOBOP enhanced the expression of two tumor suppressor genes, namely WT1 and MGMT, which were additively enhanced in cells treated with CAR agonist in combination with paclitaxel. Also, in silico analysis showed that both paclitaxel and CAR agonist TCPOBOP docked into the mCAR structure but not the inverse agonist androstenol. Paclitaxel per se increases the expression of CAR in cancer cells. At last, we analyzed the expression of CAR in two public independent studies from The Cancer Genome Atlas (TCGA) of Non Small Cell Lung Cancer (NSCLC). CAR is expressed in variable levels in NSCLC samples and no association with overall survival was noted. CONCLUSIONS/SIGNIFICANCE: Taken together, our results demonstrated that CAR agonists modulate the antineoplastic efficacy of paclitaxel in mouse and human cancer cell lines. This effect was probably related by the enhanced expression of two tumor suppressor genes, viz. WT1 and MGMT. Most of NSCLC cases present CAR gene expression turning it possible to speculate the use of CAR modulation by ligands along with Paclitaxel in NSCLC therapy.