Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.     

Serum level of apoptosis inhibitor of macrophage in dogs with histiocytic sarcoma and its association with the disease

Histiocytic sarcoma (HS) is a rare neoplasm of macrophages or dendritic cells with a poor prognosis in dogs. As the apoptosis inhibitor of macrophage (AIM) is characteristically expressed in canine macrophages, we hypothesised that AIM is involved in the development or progression of HS in dogs. In this study, AIM expression in the tumour region and serum AIM levels in dogs with HS was assessed. Additionally, the effects of AIM overexpression on HS cell viability were investigated using a HS cell line that was selected from five validated HS cell lines. Immunohistochemistry showed that AIM expression was observed in the cytoplasm of the HS cells. CD36, a candidate AIM receptor, was also observed on the cell membrane of HS cells. When the serum AIM level was detected in 36 dogs with HS and 10 healthy dogs via western blot analysis, the AIM levels in the HS dogs were significantly higher than those in the controls. AIM mRNA expression in the 5 HS cell lines varied but was higher than that in the other tumour-derived lines. Among the five HS cell lines, DH82 originally had lower AIM and the highest CD36 expression. When AIM was overexpressed in DH82, therein cell growth speed and invasion, apoptosis inhibition and phagocytic activity were strongly upregulated. These data suggest that elevated intra-tumour expression of AIM could induce the progression of HS cells in dogs. Moreover, elevated serum AIM levels in dogs with HS could serve as a biomarker of HS.     

Properties of nine continuous B-cell lines established from enzootic bovine leukosis tumors.

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.     

Establishment of cell line and in vivo mouse model of canine Langerhans cell histiocytosis

A cell line named FB‐LCH01, derived from a dog diagnosed with Langerhans cell histiocytosis (LCH), was established and characterized. FB‐LCH01 had C‐shaped nucleoli, characterized by modal chromosome aberrations. The original tumour cells as well as established FB‐LCH01 cells were immunopositive for human leukocyte antigen‐DR, Iba‐1 and E‐cadherin, and immunonegative for CD163 and CD204, suggesting Langerhans cell origin. Furthermore, the characteristics of FB‐LCH01 were compared with those of two canine histiocytic sarcoma cell lines (PWC‐HS01 and FCR‐HS02) established previously. Expression of E‐cadherin was detected only in FB‐LCH01, but not in PWC‐HS01 and FCR‐HS02. All (n = 9) the severe combined immunodeficiency mice inoculated with the FB‐LCH01 cells developed subcutaneous tumour masses after 3 weeks. Eight of nine mice also developed metastatic lesions in the lymph nodes (8/8; 100%), lung (5/8; 62.5%), stomach (5/8; 62.5%), heart (4/8; 50%), pancreas (4/8; 50%), kidney (3/8; 37.5%), skin (3/8; 37.5%) and bone marrow (1/8; 12.5%). Tumour cells were pleomorphic and round‐ to polygonal‐shaped with prominent anisocytosis and anisokaryosis. The xenotransplanted tumour cells maintained the immunohistochemical features of the original tumour with persistent E‐cadherin expression at injection site and some visceral organs. In conclusion, the established cell line as well as the mice xenotransplant model in this study reflect the nature of canine LCH and may serve as promising models for investigating the patho‐tumorigenesis and therapy of the disease.     

Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Previously, the kinase inhibitor dasatinib was shown to have potent growth inhibitory activity against HS cells in vitro, possibly via targeting the EPHA2 receptor. Here, the in vivo effect of dasatinib in HS cells was investigated using a xenograft mouse model. Moreover, the expression status of EPHA2 was examined in six HS cell lines, ranging from insensitive to highly sensitive to dasatinib. In the HS xenograft mouse model, dasatinib significantly suppressed tumor growth, as illustrated by a decrease in mitotic and Ki67 indices and an increase in apoptotic index in tumor tissues. On Western blot analysis, EPHA2 was only weakly detected in all HS cell lines, regardless of sensitivity to dasatinib. Dasatinib likely results in the inhibition of xenograft tumor growth via a mechanism other than targeting EPHA2. The findings of this study suggest that dasatinib is a targeted therapy drug worthy of further exploration for the treatment of canine HS.     

B-congenic chickens differ in macrophage inflammatory responses

The influence of the chicken major histocompatibility (B) complex (MHC) on monocyte and macrophage recruitment and activation was examined using fully developed 15I5-B congenic White Leghorn lines (ten backcross generations). The phagocytic activity of Sephadex-elicited peritoneal macrophages for sheep red blood cells (SRBCs) was highest in lines 15.7-B2 and 15.P-B13 and lowest in 15.15I-B5 and 15.N-B21. The same pattern of phagocytic activity was obtained when LPS (E. coli) was used as the in vivo elicitor-activator of peritoneal macrophages. Lines with B2 and B13 haplotypes had elevated percentages of phagocytic macrophages and a higher internalization activity per cell than did B5 and B21 congenic chickens. Differential peritoneal macrophage function between congenic lines was further supported by quantitation of superoxide anion release. B2 and B13 haplotypes were associated with high activity in contrast with B5, which was low, and 15I5 (B15) and B21 which were intermediate for superoxide anion release by macrophages. In vitro activation of blood monocytes with LPS resulted in similar line differences for SRBC phagocytic activity as were observed with in vivo Sephadex and LPS activation. In contrast, chemotaxis of blood mononuclear leukocytes to f-met-leu-phe produced a reciprocal response pattern among the haplotypes. Cells from lines with haplotypes B5 and B21 were superior to those of B2, B13, and B15 congenic lines in their directed migration towards this chemoattractant. All functional differences occurred despite similarities among lines in the cellular profiles of both elicited peritoneal exudate cells and isolated blood mononuclear cells.     

Canine oral primary melanoma cells exhibit shift to mesenchymal phenotype and phagocytic behaviour

Canine oral malignant melanoma (COMM) is a potentially lethal cancer disease. We established primary cell lines from mostly amelanotic primary COMM and metastases and assessed lesions and derived cells for Melan A, PNL2 and CD146 expression. Then, migration and invasion of CD146‐enriched vs ‐depleted COMM cells were analysed. Epithelial‐to‐mesenchymal transition (EMT) was addressed by Vimentin‐staining and MMP2/MMP9 zymography. Phagocytic behaviour was analysed by histopathological examination and phagocytosis assay. While Melan A‐ and PNL2‐staining yielded inconsistent data, 100% of COMM sections and primary cells showed CD146 expression, suggesting that this protein may serve as a prognostic marker. An overall correlation between CD146‐expression and migration/invasion was not observed. All primary cell lines consistently expressed Vimentin and secreted biologically active MMP2, indicating that they had undergone EMT. Importantly, COMM sections exhibited cell‐in‐cell structures, and all primary cell lines exhibited phagocytic activity, supporting the concept that cell cannibalism may have a role in COMM progression.     

Canine histiocytic sarcoma cell lines with SHP2 p.Glu76Gln or p.Glu76Ala mutations are sensitive to allosteric SHP2 inhibitor SHP099

Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain‐containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto‐inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open‐active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099‐sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS.     

Suppression of canine myeloid cells by soluble factors from cultured canine tumor cells

BackgroundCancer profoundly affects immunity and causes immunosuppression that contributes to tumor escape, metastases and resistance to therapy. The mechanisms by which cancer cells influence immune cells are not fully known but both innate and adaptive immune cells can be altered by cancer. Myeloid cells are innate immune cells that comprise the mononuclear phagocytic system (MPS) and include monocytes, macrophages, dendritic cells (DCs) and their progenitors. Myeloid cells play important roles in both the promotion and regulation of immune responses. Dysregulated myeloid cells are increasingly being recognized as contributing to cancer-related immunosuppression. This study investigated whether soluble factors produced by canine tumor cells inhibited canine myeloid cell function.MethodsThese studies investigated the utility of using the canine DH82 cell line for assessment of canine myeloid responses to tumor-derived soluble factors (TDSFs). Phenotypic comparisons to canine bone marrow-derived DCs (BM-DCs) and bone marrow-derived macrophages (BM-MΦs) were performed and expression of myeloid cell markers CD11b, CD11c, CD80, and major histocompatibility complex (MHC) class II were evaluated by flow cytometry. Phenotypic and functional changes of DC populations were then determined following exposure to tumor-conditioned media (TCM) from canine osteosarcoma, melanoma and mammary carcinoma cell lines.ResultsWe found that the canine BM-DCs and the DH82 cell line shared similar CD11b, CD11c and MHC II expression and morphologic characteristics that were distinct from canine BM-MΦs. Myeloid cells exposed to TDSFs showed decreased expression of MHC class II and CD80, had reduced phagocytic activity and suppressed the proliferation of responder immune cells.ConclusionThese results show that soluble factors secreted from canine tumor cells suppress the activation and function of canine myeloid cells. Our results suggest that, similar to humans, dysregulated myeloid cells may contribute to immunosuppression in dogs with cancer.     

Malignant histiocytosis in cattle

Malignant histiocytosis was diagnosed in 4 cows. In all cases the tumor tissues were composed of cytologically atypical histiocytes with evidence of erythrophagocytosis. The tumor in case 1 appeared highly anaplastic with marked nuclear pleomorphism, and had areas of spindle cell differentiation, but had no relation to malignant fibrous histiocytoma. The neoplastic tissue in case 2, characterized by cohesive growth of tumor cells, was distinguishable from anaplastic carcinoma cells by cytokeratin immunostaining. There were many hemosiderin-laden neoplastic cells suggestive of high phagocytic activity in a lymph node of case 3. The neoplastic cells in case 4, frequently multinucleated, were less atypical than in the other cases. All cases expressed histiocyte-associated markers (lysozyme and HAM56), and were negative for cytokeratin, S100, and T- and B-cell lineage-specific markers (CD3 and CD79a). The most frequent HAM56 immunoreactivity was detected in case 4, and the giant, multinucleated forms, reminiscent of epithelioid cell differentiation. seemed not to indicate cytological pleomorphism as a result of neoplastic transformation.     

Production and characterization of monoclonal antibodies against chicken lymphocyte surface antigens

A panel of monoclonal antibodies (mAbs) with specificity for chicken lymphocyte surface antigens was established and characterized based on their reactivities against chicken lymphoid cells and tumor cell lines on flow cytometry. Three mAbs (7-3G-2, 7-2E-8, and JB-2) reacted preferentially with thymocytes, however, none of them reacted with Marek's disease derived T lymphoblastoid cell lines. Four mAbs (6-27A-1, 4-5C-5, Lc-4, and Lc-6) reacted with spleen cells and peripheral blood leukocytes as well as thymocytes. All seven mAbs reacted with chicken embryonic thymocytes from day 12 of embryonic life onward. All mAbs showed no reactivity against bursal lymphocytes.     

Oncolysis of canine tumor cells by myxoma virus lacking the serp2 gene

Objective-To determine the oncolytic efficacy of an attenuated form of myxoma virus lacking the serp2 gene in canine tumor cells. Sample-Primary cells were isolated from tumors that were surgically removed from dogs and from connective tissue obtained from the cadaver of a dog. Cells of various established cell lines from tumors and nontumorous tissues were obtained. Procedures-Experiments were performed with cells in monolayer culture. Cell cultures were inoculated with wild-type myxoma viruses or myxoma viruses lacking the serp2 gene, and measures of cytopathic effects, viral growth kinetics, and cell death and apoptosis were determined. Results-Myxoma viruses replicated in cells of many of the primary and established canine tumor cell lines. Canine tumor cells in which expression of activated protein kinase B was upregulated were more permissive to myxoma virus infection than were cells in which expression of activated protein kinase B was not upregulated. Myxoma viruses lacking the serp2 gene caused more cytopathic effects in canine tumor cells because of apoptosis than did wild-type myxoma viruses. Conclusions and Clinical Relevance-Results of the present study indicated myxoma viruses lacking the serp2 gene may be useful for treatment of cancer in dogs. Impact for Human Medicine-Results of the present study may be useful for development of novel oncolytic treatments for tumors in humans.     

Successful isolation,cultivation and partial characterization of naturally occurring ovine squamous cell carcinomata

Seventeen epithelial cell lines have been successfully established from naturally occurring ovine squamous cell carcinomata. Culture establishment was most successful when tumor tissue was directly explanted rather than treated enzymatically. Success in establishing cultures also appeared to be related to the site on the body from which the tumor biopsy was taken, with tumors derived from the nose being most readily cultured. Several of the cell lines were successfully transplanted to nude mice where the growth patterns observed in the original host, i.e. expansive or invasive, were maintained. All cell lines assumed one of two distinct morphological types; however, no association could be seen between morphology and pattern of in vivo growth.     

Establishment and characterisation of ovine blood monocyte-derived cell lines

Studies of the important functions in host defense assured by macrophages, both as functional elements and as potential targets for intracellular pathogens, are often inhibited by the lack of a source of large numbers of uniform, well-characterised cells. To address this lack for ovine studies, we have established cell lines from spontaneously-proliferating adherent mononuclear cells from sheep blood. Eight such lines which have been continuously cultured for over 400 passages have phagocytic activities and cytochemical characteristics indicating that they retain the nature of mononuclear phagocytes. They display typical functional membrane proteins such as CD14, Fc receptors and MHC class II. Such cells can facilitate in vitro studies of pathogen-monocyte interactions and can furnish copious amounts of cells for transfer experiments.     

Establishment and characterization of a chicken mononuclear cell line

A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.     

Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages

Histiocytic disorders of dogs include histiocytoma, localized histiocytic sarcoma (HS), disseminated HS (malignant histocytosis), and the reactive histiocytoses: cutaneous and systemic. A common element to these diseases is proliferation of dendritic cells (DC) of either Langerhans cell (epithelial DC) or interstitial DC lineage. In this report, 17 dogs with hemophagocytic HS are described. Breeds affected included Bernese Mountain Dog (6), Golden Retriever (4), Rottweiler (3), Labrador Retriever (2), a mixed-breed dog, and a Schnauzer, which were from 2.5 to 13 years old. The dogs presented with Coombs negative responsive anemia in 16/17 dogs (94%), thrombocytopenia in 15/17 dogs (88%), hypoalbuminemia in 16/17 dogs (94%), and hypocholesterolemia in 11/16 dogs (69%). All dogs died or were euthanized. The clinical course ranged from 2 to 32 weeks (mean 7.1 weeks). Diffuse splenomegaly with ill-defined masses was consistently present. Microscopic lesions were prevalent in spleen, liver, lung, and bone marrow. Metastasis occurred by insidious intravascular invasion with minimal mass formation. Histiocytes were markedly erythrophagocytic and accompanied by foci of extramedullary hemopoiesis. Cytologically, the histiocytes varied from well differentiated to atypical, with atypia more prevalent in spleen than bone marrow. These tumors arose from splenic red pulp and bone marrow macrophages, which expressed major histocompatibility complex class II and the beta2 integrin, CD11d. They had low and/or inconsistent expression of CD1 and CD11c, which are dominantly expressed by canine nonhemophagocytic HS of DC origin. Canine histiocytic proliferative diseases now encompass proliferation of all members of the myeloid histiocytic lineage: Langerhans cells, interstitial DC, and macrophages.