Effects of vitamin D and retinoids on the differentiation and growth in vitro of canine osteosarcoma and its clonal cell lines

Although canine osteosarcoma is one of the most malignant, aggressive and lethal neoplasms originating from undifferentiated bone cells, it may retain some capacity for normal differentiation. The purpose of this study was to ascertain if the residual capacity for differentiation could be used to suppress its malignant properties. We tested the efficacy of vitamin D and retinoids in inducing differentiation and inhibiting growth of the POS canine osteosarcoma and four of its clonal cell lines, POS 14A (fibroblast type), POS 53B (chondroblast type), POS 53C (undifferentiated type) and POS 53D (osteoblastic type). Treatment with 10(-10)to 10(-8)M concentrations of calcitriol, OCT, cholecalciferol, all-trans retinoic acid (ATRA) and 9-cis retinoic acid for 48-120 hours changed the morphology of POS, POS 53B, POS 53C and POS 53D cells to cells that were elongated and spindle-shaped. Increased number of cytoplasmic organelles and pronounced nuclear activities were induced by concentrations of 10(-8)M and 10(-7)M for 120 hours. All drugs at concentrations of 10(-10)to 10(-8)M for 72 hours inhibited POS growth dose-dependently. OCT significantly reduced the cell number in all cell lines when used at concentrations between 10(-9)and 10(-8)M for 72 hours and exerted significant anti-proliferative effects for eight days culture. This study demonstrated that changed morphology and inhibition of growth was induced by treatment of the cells with these vitamins, that the loss of control of differentiation in the neoplasia was not irreversible and that these drugs may be useful in the clinic.     

Induction of functional differentiation and growth inhibition in vitro of canine osteosarcoma by 22-oxacalcitriol, calcitriol and all-trans retinoic acid

The effects of 22-oxacalcitriol (OCT), calcitriol and all-trans retinoic acid (ATRA) on the induction of functional differentiation and growth inhibition of the canine osteosarcoma cell line POS were investigated in vitro via bone differentiation markers and proliferation assays, respectively. The intracellular alkaline phosphatase (ALP) activity and the gamma-carboxyglutamic acid osteocalcin (GLA-OC) and procollagen type I C peptide (PIP) production were used as markers of differentiation. Treatment with 10(-8) M concentrations of all drugs for 72 h significantly inhibited growth (P < 0.0001) and increased ALP activity and GLA-OC and PIP production in POS. OCT, calcitriol and ATRA significantly increased the: ALP activity from 1.58 +/- 0.14 mumol/min/mg protein (mean +/- SD; control) to 2.50 +/- 0.09 (P < 0.0001), 2.30 +/- 0.14 (P < 0.0001) and 2.00 +/- 0.14 (P = 0.0008), respectively; GLA-OC production from 0.71 +/- 0.01 ng/ml (control) to 2.87 +/- 0.01 (P < 0.0001), 2.87 +/- 0.11 (P < 0.0001) and 1.36 +/- 0.06 (P < 0.0001), respectively; and PIP production from 433.91 +/- 23.29 ng/ml (control) to 536.54 +/- 15.46 (P = 0.0002), 497.06 +/- 1.99 (P = 0.0028) and 481.66 +/- 0.01 (P = 0.0104), respectively. This study demonstrated that treatment with these drugs induced a phenotypic maturation of POS cells into cells that exhibit the properties of functionally mature bone cells with parallel growth inhibition. The effects of these drugs on functional differentiation may be useful to complement the progression of a normal osteogenic differentiation process in the sarcoma.     

Effect of all-trans and 9-cis retinoic acid on growth and metastasis of xenotransplanted canine osteosarcoma cells in athymic mice

OBJECTIVE: To determine effects of all-trans and 9-cis retinoic acid (RA) on tumor growth and metastatic ability of canine osteosarcoma cells transplanted into athymic (nude) mice. ANIMALS: Forty-five 5-week-old female BALB/c nude mice. PROCEDURE: 1 X 10(7) POS osteosarcoma cells were transplanted subcutaneously into the intrascapular region of mice. All-trans RA (3 or 30 microg/kg of body weight in 0.1 ml of sesame oil), 9-cis RA (3 or 30 mg/kg in 0.1 ml of sesame oil), or sesame oil (0.1 ml; control treatment) were administered intragastrically 5 d/wk for 4 weeks beginning 3 days after transplantation (n = 4 mice/group) or after formation of a palpable tumor (5 mice/group). Tumor weight was estimated weekly by measuring tumor length and width, and retinoid toxic effects were evaluated daily. Two weeks after the final treatment, mice were euthanatized, and number of mice with pulmonary metastases was determined. RESULTS: Adverse treatment effects were not detected. Tumor weight was less in mice treated with either dose of 9-cis RA than in control mice, although this difference was not significant. Treatment with 30 mg of 9-cis RA/kg initiated after tumor formation significantly reduced the incidence of pulmonary metastasis, compared with the control group. CONCLUSIONS AND CLINICAL RELEVANCE: 9-cis RA decreased the incidence of pulmonary metastasis in nude mice transplanted with canine osteosarcoma cells and may be a potential adjunct therapy for treatment of osteosarcoma in dogs.     

Inhibitory effects of 22-oxa-calcitriol and all- trans retinoic acid on the growth of a canine osteosarcoma derived cell-line in vivo and its pulmonary metastasis in vivo

Pulmonary metastasis is a major cause of death and a major obstacle to the successful treatment of canine osteosarcoma. However, the residual capacity of the neoplasia for differentiation and its susceptibility to undergo apoptosis may be used to suppress its growth and metastatic properties. The highly metastasizing POS (HMPOS) canine osteosarcoma cell line which preferentially metastasize to the lungs was used to test the possible efficacy of 22-oxa-calcitriol (OCT) and all- trans retinoic acid (ATRA) to inhibit growth and pulmonary metastasis of the subcutaneously grown osteosarcoma in nude mice. Treatments in vitro, morphologically elongated and increased alkaline phosphatase activity and staining of cells. Tumour growth in vivo was inhibited significantly and the combination treatment of OCT and ATRA (OCT + ATRA) exerted a synergistic and stronger suppression at concentration of 1.0 microg kg(-1)body weight when given subcutaneously three times a week for 5 weeks. The subcutaneous tumours of the control mice consisted of osteoblast-like cells and isolated chondroblast-like cells, but formed several areas of osteoid and increased amount of collagen tissue in all treated mice. Pinpoint macrometastatic nodules developed only in all control mice. Micrometastatic nodule developed only in two of six mice treated with ATRA. However, nodule size and number, and lung wet weight were all reduced significantly. Metastasis were not seen in the mice treated with OCT or OCT + ATRA. This study demonstrated that inhibition of growth and pulmonary metastasis was induced by subcutaneous treatment with these drugs and suggest that both its differentiating and apoptotic inducing activities may be responsible for the antitumour effects. These drugs may be useful in the clinic as an adjunct for the treatment of canine osteosarcoma.     

Differentiation induction of canine osteosarcoma cell lines by retinoids

The effect of two retinoids, all- trans and 9- cis retinoic acid, on the differentiation of three canine osteosarcoma cells (OOS, HOS, and POS) was examined using markers specifically expressed by phenotypic osteoblasts. Both retinoids induced morphologic differentiation in all the canine osteosarcoma cells. Retinoids enhanced cell flattening and spreading, as well as reduction in cell overlapping. Alkaline phosphatase (ALP) activity and ALP staining was enhanced in OOS, and HOS cells, but decreased in POS cells. These results may suggest that OOS and HOS cells have immature osteoblastic properties and POS cells have mature osteoblastic properties. Retinoids decreased osteocalcin production in all the osteosarcoma cells. They induced an increase in production of type I collagen in HOS and POS cells, but a decrease in OOS cells. These results indicate that retinoids induce differentiation of canine osteosarcoma cells, resulting in an altered expression of their malignant phenotype.     

Retinoid receptors and the induction of apoptosis in canine osteosarcoma cells

Retinoids, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA), induced morphological changes and apoptosis-like cell death characterized by cell shrinkage, chromatin condensation and nuclear disintegration in three canine osteosarcoma cells, OOS, HOS and POS, at a concentration of 10(-5) M. Both retinoid receptors, RARs and RXRs, were identified in these cells. 9-cis-RA bound to both the RXRs and the RARs, whereas ATRA bound to only the RARs in these cells. Those results indicate that the induction of apoptosis in canine osteosarcoma cells may be mediated by the specific control of RARs and RXRs.     

1,25-Dihydroxyvitamin D3, recombinant human transforming growth factor-beta 1, and recombinant human bone morphogenetic protein-2 induce in vitro differentiation of canine osteosarcoma cells.

Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), recombinant human transforming growth factor (rhTGF)-beta 1 and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1,25(OH)2D3 increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-beta 1 increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1,25(OH)2D3 or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1,25(OH)2D3 seemed to be more effective than rhTGF-beta 1. These agents are potential inducers of apoptosis.     

鸡胚原始生殖细胞定向诱导分化成骨细胞的研究

探讨体外培养的鸡胚胎原始生殖细胞(EPGCs)的多向分化潜能。分别从发育至19期和28期的鸡胚胎性腺中获取EPGCs,体外培养传代。通过地塞米松、β-甘油磷酸钠和维生素C诱导EPGCs向成骨细胞分化,分别进行碱性磷酸酶改良钙钴法染色鉴定、钙结节Von Kossa's染色鉴定和免疫组化鉴定。结果表明:EPGCs被诱导21 d后,定向分化成成骨细胞,诱导率达到80%,碱性磷酸酶改良钙钴法染色、钙结节Von Kossa's染色和免疫组化鉴定均呈阳性,阳性率为75%-85%。由此可得出在诱导条件下,EPGCs体外可定向诱导分化为成骨细胞。     

9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression

Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-α (TNF-α) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-α production in various cell lines. The aim of the present study was to determine whether oocyte TNF-α concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-α production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-α (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-α (RA+TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA downregulated (P < 0.01) both basal and TNF-α-induced TNF-α mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA+TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 ± 6.9 vs. 23.6 ± 8.0%) and total cell number (138.4 ± 19.2 vs. 120.2 ± 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 ± 2.0 vs. 5.6 ± 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-α (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 ± 11.1 vs. 15.6 ± 9.0%), increased total cell number (124.6 ± 36.5 vs. 106.9 ± 31.1), and reduced apoptosis (5.8 ± 2.0 vs. 8.1 ± 3.1%) in blastocysts exposed to TNF-α (TNF group). Caspase 3 (0.8-fold in RA+TNF vs. 2.2-fold in TNF) and TNF-α (0.3-fold in RA+TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-α-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-α mRNA expression.     

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.     

Conversion of Goat Fibroblasts into Lineage‐Specific Cells Using a Direct Reprogramming Strategy

Direct reprogramming is an efficient strategy to convert one cell type to another. In this study, due to the failure of maintaining the undifferentiated state of goat embryotic stem‐ and induced pluripotent stem‐like cells in vitro, we explored an alternative way to directly convert goat fibroblasts to lineage‐specific cells. The ‘Yamanaka factors’ was ectopically expressed in fibroblasts for a short term to situate cells in a metastable state. By culturing with lineage‐specific media for 1–2 weeks, the cardiomyocyte‐like cells and neurocyte‐like cells were generated and confirmed by the quantitative RT‐PCR and immunocytochemical staining. The metastable‐state cells could also be converted into oocyte‐like cells (OLCs) after culturing in media with retinoic acid (RA) and bovine follicular fluid (bFF) for 2–3 weeks. The generated OLCs were surrounded by cumulus granulosa cell‐like cells and formed a structure resembling goat cumulus‐oocyte complex from ovaries. This primary follicular structure could be developed further in oocyte mature medium and expressed germ cell‐specific markers. In addition, we found that the induction efficiency was higher and OLC cell size was bigger in bFF than in RA treatment. Altogether, the direct reprogramming of goat fibroblasts into lineage‐specific cells can facilitate stem cell research in domestic animals.     

视黄酸诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

体外胚胎干细胞（embryonic stem cells,ESCs）向生殖细胞分化可用于治疗不育症,同时也为揭示种系世代的分子机制提供最佳模型。试验旨在探讨视黄酸（retinoic acid,RA）诱导鸡胚胎干细胞（chicken embryonic stem cells,cESCs）向雄性生殖细胞（male germ cells,MGCs）分化的作用效果。利用胰蛋白酶消化法从新鲜种蛋X期鸡胚中分离胚胎干细胞,以鸡胚成纤维（chicken embryo fibroblast,CEF）细胞为滋养层,进行体外培养,利用形态法、碱性磷酸酶（alkaline phosphatase,AKP）染色和胚胎阶段特异性表面抗原（embryo specific surface antigen 1,SSEA-1）检测对获得的胚胎干细胞进行鉴定。结果表明,获得典型的呈巢状或岛状的cESCs克隆,细胞AKP染色呈蓝紫色,表明其具有较高的内源性AKP活性;SSEA-1鉴定结果呈阳性,显示cESCs克隆具有多能性。采用10^-5 mol/L RA诱导鸡胚胎干细胞向雄性生殖细胞分化,镜下观察细胞形态变化,分别于诱导第0、2、4、6、8、10天提取细胞总RNA,反转录成cDNA,用于实时荧光定量PCR检测生殖细胞标志基因的表达。结果表明,在此诱导过程中,作为胚胎干细胞标志基因Nanog、Sox2表达量持续显著下降,而生殖细胞特异性基因Dazl、Stra8、c-kit、integrin α6表达量呈持续上升趋势;免疫细胞化学检测可观察到特异基因相关蛋白的阳性克隆。本研究成功分离出cESCs,可体外培养并保持未分化状态及多能性。10^-5 mol/L RA能够促进cESCs向雄性生殖细胞方向分化,可以引起生殖细胞相应基因的表达,为进一步研究雄性生殖细胞的形成和调控机制提供参考。     

Osteosarcoma tissues and cell lines from patients with differing serum alkaline phosphatase concentrations display minimal differences in gene expression patterns

Serum alkaline phosphatase (ALP) concentration is a prognostic factor for osteosarcoma in multiple studies, although its biological significance remains incompletely understood. To determine whether gene expression patterns differed in osteosarcoma from patients with differing serum ALP concentrations, microarray analysis was performed on 18 primary osteosarcoma samples and six osteosarcoma cell lines from dogs with normal and increased serum ALP concentration. No differences in gene expression patterns were noted between tumours or cell lines with differing serum ALP concentration using a gene‐specific two‐sample t‐test. Using a more sensitive empirical Bayes procedure, defective in cullin neddylation 1 domain containing 1 (DCUN1D1) was increased in both the tissue and cell lines of the normal ALP group. Using quantitative PCR (qPCR), differences in DCUN1D1 expression between the two groups failed to reach significance. The homogeneity of gene expression patterns of osteosarcoma associated differing serum ALP concentrations are consistent with previous studies suggesting serum ALP concentration is not associated with intrinsic differences of osteosarcoma cells.     

Effect of retinoids on growth inhibition of two canine melanoma cell lines

Two new canine melanoma cell lines (CMM1 and CMM2) were established from the patients with oral malignant melanomas. Histopathological type of both CMM1 and CMM2 was a mixed cell type consisted of spindle-shaped cells, polygonal cells, and oval cells. Doubling time of CMMI and CMM2 were 18.4 +/- 1.96 hr and 21.0 +/- 0.73 hr, respectively. The effect of two kinds of retinoids (all-trans retinoic acid and 9-cis retinoic acid) on the proliferation of these cells were examined by morphological changes, proliferation assay and apoptosis assay. However, the retinoids did not suppress growth rate of these cells. This result suggests that retinoids used in this study did not induce differentiation, apoptosis, and growth inhibition of the canine melanoma cell lines.     

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

不同代次大鼠骨髓间充质干细胞和脂肪间充质干细胞成骨分化比较

本研究旨在观察不同代次骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ADSCs)体外培养的生长特点和体外诱导成骨能力。通过密度梯度离心和贴壁培养法分离培养大鼠骨髓间充质干细胞和脂肪间充质干细胞,用含地塞米松、抗坏血酸、β-甘油磷酸钠的培养液定向诱导传代细胞向成骨细胞分化,并利用茜素红染色、碱性磷酸酶染色及PCR方法检测成骨细胞。结果表明骨髓及脂肪间充质干细胞呈成纤维细胞样生长,增殖能力强,生长迅速。第5、10、15、20代BMSCs及ADSCs经诱导培养后茜素红染色呈阳性并且出现"矿化"、碱性磷酸酶活性强,随着细胞代次的递增,诱导后细胞碱性磷酸酶活性呈递减趋势;诱导后的两类细胞传代后细胞仍能继续分化,并形成正常的"矿化"结节,且碱性磷酸酶染色均弱于初次诱导。结果提示,BMSCs及ADSCs易于分离培养及体外扩增,诱导条件下成骨能力强且成骨细胞传代培养仍具有成骨能力,适合作为再生医学骨组织工程的种子细胞。     

Use of alkaline phosphatase staining to differentiate canine osteosarcoma from other vimentin-positive tumors

Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone neoplasia. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, and plasma cell myeloma. The purpose of this study is to determine the sensitivity and specificity of alkaline phosphatase (ALP) staining to differentiate OSA from other tumors that express vimentin by immunocytochemistry or immunohistochemistry. ALP is a hydrolytic enzyme present in multiple tissues including liver, kidney, intestine, placenta, and bone. Hypothetically, neoplasms actively producing bone should be specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 8-10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase substrate. A positive reaction stains the membrane of the cells gray to black. Samples were counterstained with a Romanowsky's stain to determine whether the sample was of representative cellularity. A total of 61 vimentin-positive neoplasms have been evaluated and confirmed histopathologically. Tumors that expressed vimentin and were positive for ALP included 33 OSAs, one multi-lobular tumor of bone, one amelanotic melanoma, and one chondrosarcoma. Tumors that expressed vimentin and were negative for ALP included chondrosarcomas (three of four), multiple fibrosarcomas, and multiple synovial cell sarcomas. The sensitivity is 100%, and the specificity is 89%. In conclusion, ALP appears to be a highly sensitive and fairly specific marker in the diagnosis of OSA.     

Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioural differences in vitro

Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma‐bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration.