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FEBS Open Bio May 2020The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In...
The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer-associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2-derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha-smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR-27a and let-7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR-27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR-27a in canine fibroblasts and involved in their activation. Importantly, miR-27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin-6 (IL-6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR-27a may play an important role in the tumour-microenvironment crosstalk.
Topics: Animals; Cancer-Associated Fibroblasts; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Coculture Techniques; Dog Diseases; Dogs; Endothelial Cells; Exosomes; Fibroblasts; Mast Cells; Mastocytoma, Skin; MicroRNAs; Signal Transduction; Tumor Microenvironment
PubMed: 32133790
DOI: 10.1002/2211-5463.12831 -
European Journal of Immunology Jun 2021The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for...
The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for therapy. However, its specific inhibition with antibodies is complicated by the fact that its two subunits are present in other cytokines: p40 in IL-23 and p35 in IL-35. This has led to erroneous conclusions like the alleged implication of IL-12 in experimental autoimmune encephalomyelitis (EAE). Here, we report the development of a mouse anti-mouse IL-12 vaccine and the production of monoclonal antibodies (mAbs) that do not react with p40 or p35 (in IL-35) but specifically recognize and functionally inhibit the IL-12 heterodimer. Using one of these mAbs, MM12A1.6, that strongly inhibited IFN-γ production and LPS-induced septic shock after viral infection, we demonstrate the critical role played by IL-12 in the rejection of male skin graft by female C57BL/6 syngeneic recipients and in the clearance of an immunogenic mastocytoma tumor variant by DBA/2 mice, but not in a parent to F1 immune aggression model nor in MOG-induced EAE, which was clearly prevented by anti-p40 mAb C17.8. Given this selective inhibition of IL-12, these mAbs provide new options for reassessing IL-12 function in vivo.
Topics: Animals; Antibodies, Monoclonal; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Graft Rejection; Humans; Hybridomas; Interleukin-12; Mastocytoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Multiple Sclerosis; Neoplasms, Experimental; Nidovirales; Nidovirales Infections; Protein Subunits; Sepsis; Skin Transplantation
PubMed: 33788263
DOI: 10.1002/eji.202048936 -
Scientific Reports Nov 2020Cutaneous mast cell tumours (MCTs) are common skin neoplasms in dogs. MicroRNAs (miRNAs) are post-transcriptional regulators involved in several cellular processes, and...
Cutaneous mast cell tumours (MCTs) are common skin neoplasms in dogs. MicroRNAs (miRNAs) are post-transcriptional regulators involved in several cellular processes, and they can function as tumour promoters or suppressors. However, the role of miRNAs in canine MCTs has not yet been elucidated. Thus, the current study aimed to characterize miRNA profiles and to assess their value as biomarkers for MCTs. miRNA expression profiles were assessed in formalin-fixed, paraffin-embedded samples by next-generation sequencing. Ten samples were MCT tissues, and 7 were healthy adjacent tissues. Nine dysregulated miRNAs (DE-miRNAs) were then validated using RT-qPCR in a larger group of MCT samples, allowing the calculation of ROC curves and performance of multiple factor analysis (MFA). Pathway enrichment analysis was performed to investigate miRNA biological functions. The results showed that the expression of 63 miRNAs (18 up- and 45 downregulated) was significantly affected in MCTs. Five DE-miRNAs, namely, miR-21-5p, miR-92a-3p, miR-338, miR-379 and miR-885, were validated by RT-qPCR. The diagnostic accuracy of a panel of 3 DE-miRNAs-miR-21, miR-379 and miR-885-exhibited increased efficiency in discriminating animals with MCTs (AUC = 0.9854) and animals with lymph node metastasis (AUC = 0.8923). Multiple factor analysis revealed clusters based on nodal metastasis. Gene Ontology and KEGG analyses confirmed that the DE-miRNAs were involved in cell proliferation, survival and metastasis pathways. In conclusion, the present study demonstrated that the miRNA expression profile is changed in the MCT microenvironment, suggesting the involvement of the altered miRNAs in the epigenetic regulation of MCTs and identifying miR-21, miR-379 and miR-885 as promising biomarkers.
Topics: Animals; Biomarkers, Tumor; Dog Diseases; Dogs; Factor Analysis, Statistical; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Lymphatic Metastasis; Male; Mastocytoma; MicroRNAs; Oligonucleotide Array Sequence Analysis; Skin Neoplasms; Tumor Microenvironment
PubMed: 33144602
DOI: 10.1038/s41598-020-75877-x -
BMC Veterinary Research Oct 2020The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of...
BACKGROUND
The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior.
METHODS/RESULTS
WWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs.
CONCLUSIONS
These data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.
Topics: Animals; Cell Line, Tumor; Dogs; Gene Expression Regulation, Neoplastic; Mast Cells; Mastocytoma; Skin Neoplasms; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase
PubMed: 33129329
DOI: 10.1186/s12917-020-02638-3 -
Journal of Cutaneous Pathology Nov 2021We present a case of an adult male with a solitary mast cell tumor of the skin with unusual nuclear pleomorphism and mitotic activity. The tumor was excised, recurred...
We present a case of an adult male with a solitary mast cell tumor of the skin with unusual nuclear pleomorphism and mitotic activity. The tumor was excised, recurred within 2 years, was reexcised after 4 years and did not recur >6 years after diagnosis. The tumor showed progressive cytonuclear atypia and a high mitotic and proliferation rate by Ki67-staining from the onset. No KIT mutations were identified in the tumor and bone marrow. Serum tryptase levels and a bone marrow aspirate and trephine biopsy were normal. Although the histomorphology of the skin tumor was consistent with mast cell sarcoma, the clinical behavior without systemic progression argued against this diagnosis. The tumor was finally considered as atypical mastocytoma, borderline to mast cell sarcoma. Currently, the patient is in close follow-up and still in complete remission.
Topics: Adult; Diagnosis, Differential; Humans; Male; Mast-Cell Sarcoma; Mastocytoma, Skin
PubMed: 34152029
DOI: 10.1111/cup.14088 -
BMC Veterinary Research Oct 2021While lymphadenectomy of metastatic lymph nodes (LNs) has been associated with improved outcome, the clinical utility of prophylactic lymphadenectomy in dogs with stage...
A retrospective study on prophylactic regional lymphadenectomy versus nodal observation only in the management of dogs with stage I, completely resected, low-grade cutaneous mast cell tumors.
BACKGROUND
While lymphadenectomy of metastatic lymph nodes (LNs) has been associated with improved outcome, the clinical utility of prophylactic lymphadenectomy in dogs with stage I cutaneous mast cell tumors (cMCTs) remains a controversial topic. To assess the therapeutic role of lymphadenectomy of uninvolved regional LNs, the long-term outcome of cMCT-bearing dogs with cytologically negative and surgically unresected regional LNs (observation only, OO) was compared with that of dogs with surgically resected and histologically negative regional LNs (prophylactic regional lymphadenectomy, PRL).
RESULTS
A retrospective analysis of 64 dogs with a low-grade, completely resected stage I cMCT was performed: 35 (54.7%) dogs were subjected to OO and 29 (45.3%) underwent PRL. Dogs were monitored for a median of 813 and 763 days in the OO group and PRL group, respectively. The number of dogs undergoing MCT progression was significantly higher in the OO group (P = 0.028) and curve comparison revealed a tendency to a better time to progression in the PRL group (P = 0.058). No significant difference in survival time (P = 0.294) was observed between dogs in the OO and PRL groups.
CONCLUSIONS
Our results showed that lack of immediate lymphadenectomy was associated with a higher risk for tumor progression. This preliminary judgement, reinforced by the findings that lymphadenectomy was well tolerated in all cases, and that histopathology provides the definitive assessment of the nodal pathological status, may suggest that prophylactic lymphadenectomy is indicated in the management of stage I MCTs. Larger prospective studies are warranted for generating clinical evidence of this latter hypothesis.
Topics: Animals; Case-Control Studies; Dog Diseases; Dogs; Lymph Node Excision; Lymph Nodes; Lymphatic Metastasis; Mastocytoma; Retrospective Studies; Skin Neoplasms
PubMed: 34649575
DOI: 10.1186/s12917-021-03043-0 -
Journal of Veterinary Diagnostic... Sep 2019DNA amplification by PCR detects exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from...
DNA amplification by PCR detects exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from formalin-fixed, paraffin-embedded (FFPE) canine MCTs, blocking PCR amplification and, consequently, preventing mutation detection. We used a commercial kit to extract DNA from FFPE canine MCTs. Two independent PCR assays, each with one primer set, were used to amplify target genes ( and ) directly after FFPE DNA extraction. PCR amplification failed with at least one primer set in 153 of 280 samples (54.6%, 95% CI: 48.8-60.5%). One or 2 DNA washing steps were required to remove PCR inhibitors in 130 of 280 (46.4%) and 23 of 280 (8.2%) of these cases, respectively. DNA concentration and quality (A/A and A/A) either pre- or post-washing were not associated with ability of the samples to be amplified by PCR using both and primer sets. Low-grade and subcutaneous MCTs were less likely to amplify directly after DNA extraction and without any washing steps compared to high-grade MCTs using gene primers.
Topics: Animals; Circulating Tumor DNA; Dog Diseases; Dogs; Formaldehyde; Mast Cells; Mastocytoma; Mutation; Nucleic Acid Amplification Techniques; Paraffin Embedding; Polymerase Chain Reaction; Proto-Oncogene Proteins c-kit; Skin Neoplasms
PubMed: 31378162
DOI: 10.1177/1040638719867743 -
Veterinary and Comparative Oncology Sep 2020Lymph node (LN) metastasis is a negative prognostic factor in dogs with cutaneous mast cell tumours (cMCTs). While elective lymphadenectomy of metastatic LNs improves...
Lymph node (LN) metastasis is a negative prognostic factor in dogs with cutaneous mast cell tumours (cMCTs). While elective lymphadenectomy of metastatic LNs improves outcome, the benefit of adjuvant medical therapy in dogs with early metastatic (HN2) LNs is debated. The aim of this retrospective multicentre study was to evaluate the therapeutic benefit of adjuvant medical therapy following surgical removal of the primary low-grade cMCT (Patnaik grade 1-2 and Kiupel low-grade) and lymphadenectomy of HN2 LNs by analysing survival rates and patterns of recurrence. Seventy-three dogs were included: 42 received adjuvant medical treatment (chemotherapy and/or kinase inhibitors), and 31 did not. The median follow-up time for medically treated dogs was 619 days: two experienced local recurrence, three nodal relapse and four distant relapse. For dogs undergoing surgery only, the median follow-up time was 545 days. None of them experienced local recurrence, nodal, or distant relapse. Time to progression was significantly shorter in dogs receiving adjuvant medical treatment (P = .021). A similar tendency was observed for overall survival (P = .056). The current study shows that dogs with low-grade cMCTs, that undergo surgical excision of the primary tumour and elective lymphadenectomy of the HN2 regional LN harbour a good prognosis. The use of adjuvant medical treatment in these dogs does not seem to provide any benefit in terms of progression and survival.
Topics: Animals; Chemotherapy, Adjuvant; Dog Diseases; Dogs; Female; Italy; Lymphatic Metastasis; Male; Mastocytosis; Neoplasm Recurrence, Local; Retrospective Studies; Skin Neoplasms; Survival
PubMed: 31930651
DOI: 10.1111/vco.12566