Identification of cancer stem cells derived from a canine lung adenocarcinoma cell line

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

Isolation of Canine Mammary Cells With Stem Cell Properties and Tumour-Initiating Potential

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.     

Expression of Bcl-2 in feline lymphoma cell lines

BACKGROUND: The Bcl-2 gene is a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli, including chemotherapy and gamma irradiation. OBJECTIVE: The purpose of this study was to determine feline Bcl-2 expression level in feline lymphoma cells using an immunoblot assay with anti-human and anti-canine Bcl-2 monoclonal antibodies. METHODS: About 708 base pairs containing the coding sequence of the feline Bcl-2 gene were transformed into Escherichia coli. The recombinant Bcl-2 was used as a positive control for an immunoblot assay using mouse monoclonal antibodies against human and canine Bcl-2. An immunoblot assay using the monoclonal antibodies was carried out to determine the level of feline Bcl-2 expression in lymphoma and lymphocytic leukemia cell lines. RESULTS: The recombinant feline Bcl-2 protein produced in E. coli had a molecular weight of about 26 kDa and was detected by immunoblot assay by using anti-human Bcl-2 mouse monoclonal antibody. Feline Bcl-2 expression was high in lymphoma cell lines (FL-74-UDC-1 and FT-1) and low in the cell line from peripheral blood mononuclear cells from a healthy cat (FeTJ-1) but not low in freshly isolated peripheral blood mononuclear cells from a healthy cat. The anti-human Bcl-2 mouse monoclonal antibody was found to cross-react with feline Bcl-2. CONCLUSIONS: These results confirm the expression of Bcl-2 in T-cell lymphoma cell lines and indicate that it is suitable to detect feline Bcl-2 using an immunoblot assay. Pending further evaluation, Bcl-2 expression might be useful in the differential diagnosis of feline tumors.     

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye‐based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage‐independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct‐4 and CD133 gene than those of adherent‐cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent‐cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent‐cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.     

The Utility of Flow Cytometry for the Diagnosis of Cranial Mediastinal Malignancy in Canine Patients

Introduction:  The most common neoplasms located in the anterior mediastinum in the canine are thymoma and lymphoma. Distinguishing between the two is a diagnostic challenge. Treatment and prognosis for these diseases differs significantly. Thymomas contain a population of normally developing T cells. The majority of these T cells exhibit an immature phenotype, characterized by co‐expression of CD4 and CD8. This phenotype is rarely seen on neoplastic lymphocytes. The purpose of this study was to determine if analysis by flow cytometry could discriminate thymoma from lymphoma based on these cell surface markers.
Methods:  Fine needle aspirates were obtained from ten canine patients with mediastinal masses. Cells were analyzed by flow cytometry using a panel of T and B cell markers.
Results:  Six cases with 10% or greater CD4 + CD8+ cells were diagnosed with thymoma and were confirmed by histopathology. Four cases had fewer than 5% CD4 + CD8+ cells, having lymphocytes expressing CD4 only (3 cases) or CD21, a B cell marker(1 case). These were confirmed as lymphoma by cytology and/or a clonality assay. The sensitivity and specificity of this assay when used in the diagnostic work‐up for suspected thymoma was 100%.
Conclusion:  Flow cytometry may provide important, complementary information in the diagnostic work‐up of the canine patient with a mediastinal mass.     

Parathyroid hormone-related protein (PTHrP) produced by dog lymphoma cells

Parathyroid hormone-related protein (PTHrP) was investigated in a canine lymphoma case with hypercalcemia by means of immunoradiomentric assay (IRMA) and molecular analysis. The plasma calcium level of the patient dog was 13.7 mg/dl. The PTHrP concentration examined by IRMA was 6.1 pmol/L in the plasma sample from the dog, but it was undetectable (< 1.1 pmol/L) in plasma samples from 4 lymphoma cases without hypercalcemia or 5 normal dogs. The PTHrP concentration examined in the culture supernatant of the lymphoma cells from this case was 1.3 pmol/L, whereas those of the lymphoma cells from a lymphoma case without hypercalcemia was undetectable. PTHrP mRNA was clearly detected not only in the lymphoma cells from this dog with hypercalcemia but also in lymphoma cells from 4 lymphoma cases without hypercalcemia and 2 canine lymphoma cell lines.     

Phenotypic characterization of canine lymphoma, using monoclonal antibodies and a microlymphocytotoxicity assay

Cells acquired from lymph node biopsy specimens obtained from 58 dogs scheduled to undergo chemotherapy for lymphoma were immunophenotyped, using a microlymphocytotoxicity (MLCT) assay comprising a panel of well-characterized monoclonal antibodies (MAB) specific for canine cell surface antigens. Cells from 54 of the dogs concurrently were tested cytofluorometrically, using surface immunoglobulin (SIg) as a marker for B cells and the MAB DT2 specific for peripheral blood T cells. The MLCT results indicated frequent coexpression of antigens identified by DT2 antibody and, to a lesser extent, by 1A1 antibody on SIg-positive cells, suggesting that these antigens may be associated with other types of less-differentiated lymphoid cells, in addition to being associated with mature T cells. Class-II major histocompatibility antigens, as recognized by MAB H81.98.71, HB10a, and H40.315.7, were detected on most SIg-positive cells, but generally were lacking on SIg-negative, DT2-negative cells. The MAB Wig4, reactive with canine monocytes, recognized relatively few cells (11 of 58). Response to chemotherapy was not correlated with reactivity to MAB DLy6 specific for resting lymphocytes or to MAB W3G10 specific for a polymorphic antigen associated with the canine major histocompatibility complex. The MLCT assay appears to be efficient, rapid, and inexpensive for immunophenotyping cells from lymphoma biopsy specimens.     

Development of the polymerase chain reaction assay based on the canine genome database for detection of monoclonality in B cell lymphoma

From the canine genome database and its bioinformatic analysis, we identified conserved sequences within the vast majority of 61 variable segments and 1 joining segment of the immunoglobulin heavy chain (IgH) gene, and designed optimal primers for polymerase chain reaction (PCR) amplification directed at these conserved sequences to evaluate the monoclonality of IgH in canine B cell lymphoma. Using the primers, a PCR-based assay was performed on fine-needle aspiration samples of normal, hyperplasia, and malignant lymph nodes and lymphoma cell lines. All fine-needle aspiration samples of five B cell lymphoma cases and the B cell lymphoma line GL-1 exhibited clonal amplification, whereas no amplification was observed in the samples from normal and hyperplasia lymph nodes, cases of T cell lymphoma, and the T cell lymphoma line CL-1. The primers we designed clearly distinguished malignant B lymphocytes from normal, reactive, and malignant T lymphocytes, indicating a potential utility of the primers for PCR-based routine clinical examination for canine B cell lymphoma.     

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

Expression of O6-methylguanine-DNA methyltransferase causes lomustine resistance in canine lymphoma cells

The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.     

Substance P: a neurogenic mediator of acute cellular inflammation in the dog?

Substance P (SP) is a neuropeptide that has recently been implicated in the pathogenesis of neurogenic inflammation. SP has been shown to activate polymorphonuclear leukocytes (PMN) as well as other inflammatory cells. The present study investigated the direct stimulatory and priming effects of SP on canine PMN aggregation and migration. Direct stimulation of cell migration by SP was present at an unphysiologically high concentration of the mediator. However, when micromolar concentrations of SP were added to PMN prior to stimulation with sub-optimal concentrations of leukotriene B4 (LTB4), the cells exhibited enhanced aggregation and migration, i.e. priming, when stimulated with the latter. Since SP has been reported to act via the formyl-Met-Leu-Phe (fMLP) chemotaxin receptor, this mediator was also studied and found not to possess any effects similar to SP. Thus, the results indicate that SP acts as a primer of canine PMN functions in vitro via a receptor different from that for fMLP. Before ascribing SP a mediator role in canine neurogenic inflammation, in vivo studies determining the concentrations of, and responses to SP in inflamed tissue should be performed.     

A canine lymphocyte surface antigen detectable by a monoclonal antibody (DT200).

A murine monoclonal antibody (DT200) was raised against a 210,000-dalton (210 K) lymphocyte surface protein (a member of the lymphocyte antigen known as T200) which was purified from a canine lymphoid tumor by preparative slab gel electrophoresis. In immunoblotting studies of electrophoretically separated plasma membranes from five cases of canine lymphoma, the antibody detected two antigenically intact peptides at 95 and 110 K which, based on previous polyclonal anti-210 K antiserum immunoblotting and peptide mapping studies, may represent the protease-resistant fragment of the canine T200 molecule. Since DT200 retains its reactivity in formalin-fixed, paraffin-embedded tissue sections, 13 dogs with malignant lymphoma and a panel of normal lymphoid and nonlymphoid tissues were studied using an indirect immunoperoxidase technique. The antigen was localized predominantly to the surface membrane of lymphoid cells. DT200 reacted strongly with all five histological subtypes of lymphoma tested while moderate reactivity was detected in normal B and T cell areas of lymph node, spleen and tonsil. Thymocytes and selected hemopoietic precursors were weakly reactive with DT200 while plasma cells, mature granulocytes, red cells and megakaryocytes were unstained. It was concluded that DT200 is a useful reagent for the diagnosis of malignant lymphoma particularly in extranodal sites and may prove valuable in the investigation of the structure and function of T200 in the dog.     

Identification and characterisation of side population cells in the canine pituitary gland

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression.     

Exclusion of cytoplasmic fragments in flow cytometric analysis of lymph node samples from dogs with lymphoma using membrane-permeable violet laser-excitable DNA-binding fluorescent dye (DyeCycle Violet)

Background: Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes. Objective: The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present. Methods: Single‐cell suspensions of neoplastic cells were prepared from biopsy samples and fine‐needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser‐excitable (405 nm) membrane‐permeable DNA‐binding fluorescent dye (DyeCycle Violet [DCV]), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7‐amino‐actinomycin D (7‐AAD), an argon‐excitable (488 nm) membrane‐impermeable DNA‐binding fluorescent dye. Multiparameter flow cytometry was used for analysis based on selective uptake and laser‐activated fluorescence of these dyes. Results: Cytoplasmic fragments, which were DCV‐negative and CD45‐positive, and dead cells, which were positive for 7‐AAD, were efficiently separated from neoplastic cells. Conclusion: Staining with DCV is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma.     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.