The establishment of primary cell culture from canine mammary gland tumor

2021 ◽  
pp. 1-9
Author(s):  
Sena Ardicli ◽  
Hale Samli ◽  
Gülnaz Mecitoglu ◽  
Buse Vatansever ◽  
Ayse Meric Mutlu
Keyword(s):  
Mammary Gland ◽  
Tumor Cells ◽  
Cell Lines ◽  
Morphological Characteristics ◽  
Passage Time ◽  
Surgical Removal ◽  
Freezing And Thawing ◽  
Logarithmic Growth Phase ◽  
Benign Mixed Tumor ◽  
Canine Mammary Gland

BACKGROUND: In dogs, an insufficient variety of cell lines commercially available or difficulties in obtaining the existing cell lines developed from various studies results in a limited number of cytotoxicity and related molecular studies integrated with clinical practice. Hence, the doses of many drugs or supportive treatments used in canine tumor cases are adjusted based on studies in humans. OBJECTIVE: A cell line was established from a benign mixed tumor of the canine mammary gland. METHODS: Following surgical removal of the tumor, mechanical dissociation, and PBS washing, a culture process of the tumor cells was performed, including the passaging, freezing, and thawing stages. After several passages, the morphological characteristics of the cells at the logarithmic growth phase were observed under a phase-contrast microscope. RESULTS: The microscopy of the cells cultured on plastic dishes revealed monolayer colonies. The average passage time, which was 5–6 days in the first three passages, decreased to 2–3 days after the third passage. Microscopic examination of tumor cells revealed an adherent, stellated, and spindle-shaped structure. CONCLUSIONS: No difference was observed in the viability and morphology of the cells thawed even after the long period of freezing (∼18 months). The different canine cell lines can provide promising molecular applications that can be adapted into practical clinics in veterinary science.

scholarly journals LncRNA-42060 Regulates Tamoxifen Sensitivity and Tumor Development via Regulating the miR-204-5p/SOX4 Axis in Canine Mammary Gland Tumor Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Enshuang Xu ◽  
Mengxin Hu ◽  
Reidong Ge ◽  
Danning Tong ◽  
Yuying Fan ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mammary Gland ◽  
Tumor Cells ◽  
Luciferase Reporter ◽  
Drug Resistant ◽  
Mammary Gland Tumor ◽  
Luciferase Reporter Gene ◽  
Gland Tumor ◽  
Canine Mammary Gland ◽  
Resistant Cells

Tamoxifen is the drug of choice for endocrine therapy of breast cancer. Its clinical use is limited by the development of drug resistance. There is increasing evidence that long non-coding RNAs (lncRNAs) are associated with tumor drug resistance. Therefore, we established two TAM-resistant cell lines, CHMpTAM and CHMmTAM. The different expression levels of lncRNA and miRNA in CHMmTAM and CHMm were screened by RNA sequencing, and the lncRNA-miRNA interactions were analyzed. LncRNA ENSCAFG42060 (lnc-42060) was found to be significantly upregulated in drug-resistant cells and tumor tissues. Further functional validation revealed that the knockdown of lnc-42060 inhibited proliferation, migration, clone formation, restoration of TAM sensitivity, and reduction of stem cell formation in drug-resistant cells, whereas overexpression of lnc-4206 showed opposite results. Bioinformatics and dual-luciferase reporter gene assays confirmed that lnc-42060 could act as a sponge for miR-204-5p, further regulating SOX4 expression activity and thus influencing tumor cell progression. In conclusion, we screened lncRNAs and miRNAs associated with TAM resistance in canine mammary gland tumor cells for the first time. lnc-42060 served as a novel marker that may be used as an important biomarker for future diagnosis and treatment.

scholarly journals Adhesional Function of Canine Mammary Gland Tumor Cells Expressing Sialyl Lewis X

2009 ◽  
Vol 71 (9) ◽  
pp. 1225-1228 ◽  
Author(s):  
Takayuki NAKAGAWA ◽  
Yoshifumi ENDO ◽  
Manabu WATANABE ◽  
Manabu MOCHIZUKI ◽  
Ryohei NISHIMURA ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Tumor Cells ◽  
Sialyl Lewis X ◽  
Mammary Gland Tumor ◽  
Gland Tumor ◽  
Lewis X ◽  
Sialyl Lewis ◽  
Canine Mammary Gland

scholarly journals STUDY OF EFFECTS OF CIRCADIAN RHYTHMS ON SOLID TUMOR CELLS PROLIFERATION AND CHEMORESISTANCE

2020 ◽  
Vol 66 (5) ◽  
pp. 563-571
Author(s):  
Anna Danilova ◽  
N. Avdonkina ◽  
Ye. Gubareva ◽  
I. Baldueva ◽  
Anton Zozulya ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Solid Tumor ◽  
Lung Tumor ◽  
Morphological Characteristics ◽  
Biological Properties ◽  
Physiological Processes

Circadian clock is a complex mechanism regulating many different physiological processes. Preclinical, epidemiological and clinical studies demonstrate association between circadian rhythms disruption and tumor initiation. Study of modulation of solid tumor cells biological properties through enhancement of clock mechanisms could attribute to the development of more effective chemo- and hormone therapy approaches. Aim: Evaluate the effects of ovarian and lung tumor cells synchronization with dexamethasone in vitro on cells sensitivity to cisplatin. Materials and methods: Metastatic ovarian cancer (n=3) and lung cancer (n=3) cell lines were obtained from patients tumors. Tumor cell cultivation was performed in accordance with the protocol. Artificial synchronization was performed with dexamethasone 200 nM introduction to the cell cultures. Doses of cisplatin used were 1.5 and 3.0 mg/ml. xCELLigence Real-Time Cell Analysis and Cell-IQ was used to measure proliferation and chemoresistance of tumor cells. Results: Each cell-line had individual morphological characteristics and proliferation parameters. Preliminary incubation with dexamethasone (2 h) had a stimulating effect on proliferation of all tumor cell lines (Slope min -4.3(0.3)хЕ ‘х10-3 - max 36.8(0.6)хЫх10'3, min 2.2(0.2)хЕ1х10'3- max 50.4(0.8)хЕ1х10'3), and increased their sensitivity to cisplatin (min -43(2.6)хЕ1х10-3 - max 57.5(0.6)хЕ1х10-3 и min -217,3(2,2) -1,9(0,1)хч-1х10-3 - max -1,9(0,1)хч'1х10'3, respectively. Conclusion: These results should be the platform for future studies of the interaction of clock mechanisms, cell cycle regulation and viability of tumor cells.

scholarly journals In vitro antiproliferation activity of Typhonium flagelliforme leaves ethanol extract and its combination with canine interferons on several tumor-derived cell lines

2020 ◽  
Vol 13 (5) ◽  
pp. 931-939
Author(s):  
Bambang Pontjo Priosoeryanto ◽  
Riski Rostantinata ◽  
Eva Harlina ◽  
Waras Nurcholis ◽  
Rachmi Ridho ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Synergistic Effect ◽  
Cell Lines ◽  
Ethanol Extract ◽  
Phytochemical Screening ◽  
Mixed Tumor ◽  
In Ovo ◽  
Benign Mixed Tumor ◽  
Canine Mammary Gland

Background and Aim: Tumor disorder is one of the degenerative diseases that affected human and animals and recently is tend to increase significantly. The treatment of tumor diseases can be performed through surgical, chemotherapy, radiotherapy, biological substances, and herbs medicine. Typhonium flagelliforme leaves extract known to have an antiproliferation activity, while interferons (IFNs) one of the cytokines that first used as an antiviral agent was also known to have antitumor activity. Nowadays, the treatment of tumors using a traditional way, including the use of herbal substances, becomes popular. Some limitations of the antitumor activity due to resistant development of the cell to some substances were one of the problems on why the treatment of cancer was unsuccessful. This study aimed to elaborate the synergistic effect on the antiproliferation and anti-angiogenesis activities of the combinations between T. flagelliforme leaves ethanol extract and canine natural (natural canine IFN [nCaIFN]) and recombinant (recombinant canine IFN [rCaIFN]) IFNs on tumor-derived cell lines to find the new potential antitumor substances. Materials and Methods: The extraction of T. flagelliforme leaves was performed using the maceration method and followed by phytochemical screening assays. According to the result of LC50 by the brine shrimp lethality test, the dose used for T. flagelliforme extract was 120 ppm while the dose of IFNs was 102 U/ml. The tumor-derived cell lines (canine squamous cell carcinoma [CSCC], canine mammary gland benign mixed tumor/MCM-IPB-B3, and feline squamous cell carcinoma [FSCC]) and normal rabbit endothelial cells were cultured and maintained on Dulbecco's Modified Eagle's Medium DMEM/Ham-F12 medium supplemented with 10% fetal calf serum, antibiotic, and antifungal. The antiproliferation activity was assayed by calculated the total cell number after treated with the tested substances. The antiangiogenesis assay was performed using in vitro method on rabbit normal endothelial cells and in ovo using chicken chorioallantoic membrane (CAM). Results: The phytochemical screening test of the T. flagelliforme leaves ethanol extract indicated that the compound consisted of flavonoid, steroid, and tannin. The antiproliferation activity was increased in the combination of substances compared to the single exposure of each substance on all tested tumor-derived cell lines. There was no significantly different on the antiproliferation activity between a combination of T. flagelliforme with nCaIFN or rCaIFN in every single tested cell lines, but the comparison of this activity among the three tumor-derived cell lines seem that the antiproliferation activity is more effective on CSCC cell lines compared to the canine mammary gland benign mixed tumor and FSCC cell lines. A similar pattern of synergistic effect was also detected on the anti-angiogenesis activity in vitro using rabbit endothelial cells as well as in ovo assays. The most effective of the in vitro and in ovo anti-angiogenesis activity was observed on the combination substances between T. flagelliforme extract and rCaIFN compared to other treatments. Conclusion: There was a synergistic effect on the antiproliferation and antiangiogenesis activities of the combination between T. flagelliforme and canine IFNs (natural and recombinant) and this result could be developed as another alternative on the cancer treatments.

scholarly journals 5-Azacytidine treatment induces demethylation of DAPK1 and MGMT genes and inhibits growth in canine mammary gland tumor cells

2018 ◽  
Vol Volume 11 ◽  
pp. 2805-2813 ◽  
Author(s):  
Xiaoli Ren ◽  
Huatao Li ◽  
Xianyi Song ◽  
Yuhong Wu ◽  
Yun Liu
Keyword(s):  
Mammary Gland ◽  
Tumor Cells ◽  
Mammary Gland Tumor ◽  
Gland Tumor ◽  
Canine Mammary Gland

scholarly journals Proteomics Approach of Rapamycin Anti-Tumoral Effect on Primary and Metastatic Canine Mammary Tumor Cells In Vitro

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1213
Author(s):  
Patrícia F. Lainetti ◽  
Antonio F. Leis-Filho ◽  
Priscila E. Kobayashi ◽  
Laíza S. de Camargo ◽  
Renee Laufer-Amorim ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Statistical Analysis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Phosphoglycerate Mutase ◽  
Preclinical Model ◽  
Metabolic Processes ◽  
Control Versus

Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due to drug antitumor potential, the aim of this study was to evaluate its effect on a preclinical model of primary mammary gland tumors and their metastases from female dogs. Four cell lines from our cell bank, two from primary canine mammary tumors (UNESP-CM1, UNESP-CM60) and two metastases (UNESP-MM1, and UNESP-MM4) were cultured in vitro and investigated for rapamycin IC50. Then, cell lines were treated with rapamycin IC50 dose and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN and 4EBP1 gene expression and global proteomics by mass spectrometry. MTT assay demonstrated rapamycin IC50 dose for all different tumor cells between 2 and 10 μM. RT-qPCR from cultured cells, control versus treated group and primary tumor cells versus metastatic tumor cells, did not shown statistical differences. In proteomics were found 273 proteins in all groups, and after data normalization 49 and 92 proteins were used for statistical analysis for comparisons between control versus rapamycin treatment groups, and metastasis versus primary tumor versus metastasis rapamycin versus primary tumor rapamycin, respectively. Considering the two statistical analysis, four proteins, phosphoglycerate mutase, malate dehydrogenase, l-lactate dehydrogenase and nucleolin were found in decreased abundance in the rapamycin group and they are related with cellular metabolic processes and enhanced tumor malignant behavior. Two proteins, dihydrolipoamide dehydrogenase and superoxide dismutase, also related with metabolic processes, were found in higher abundance in rapamycin group and are associated with apoptosis. The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor and metastatic tumors cells in vitro, however, concentrations needed to reach the IC50 were higher when compared to other studies.

scholarly journals Neuroendocrine Carcinomas of the Canine Mammary Gland: Histopathological and Immunohistochemical Characteristics

2021 ◽  
Vol 7 ◽  
Author(s):  
Karen Yumi Ribeiro Nakagaki ◽  
Maíra Meira Nunes ◽  
Ana Paula Vargas Garcia ◽  
Marina De Brot ◽  
Geovanni Dantas Cassali
Keyword(s):  
Mammary Gland ◽  
Mitotic Index ◽  
Histological Grade ◽  
Morphological Characteristics ◽  
Neuroendocrine Differentiation ◽  
Neuroendocrine Neoplasms ◽  
Typical Carcinoid ◽  
Grade Ii ◽  
Canine Mammary Gland ◽  
Neuroendocrine Carcinomas

Invasive mammary carcinomas with neuroendocrine differentiation are rare in women and were reported only once in female dogs. For the present study, ten cases of solid mammary carcinoma positive for chromogramin A in immunohistochemistry were selected. Histopathological characteristics of these tumors were described and immunohistochemical evaluation was performed with chromogranin A, synaptophysin, CD56, NSE, PGP 9.5, pancitokeratin, Ki67, estrogen receptor (ER), and progesterone receptor (PR). The average animal age was 13.2 years old and the average tumor size was 4.8 cm. In total, 70% of the neoplasms were classified as grade III and 30% as grade II by the Nottingham histological grade system. High mitotic index was observed with a mean of 27.5 mitoses in 10 high magnification fields. Only one case showed typical carcinoid tumor characteristics. In addition, vascular invasion was shown in 3 tumors. All carcinomas were positive for chromogran A, while only two cases were reactive to synaptophysin. For PGP 9.2, NSE and CD56, we observed positivity of 100, 90, and 70%, respectively, in the samples, being that no tumor was positive for all the neuroendocrine markers. All neoplasms showed ER and PR in at least 10% of neoplastic cells, while Ki67 varied from 29 to 95%, with mean mitotic index of 67%. Four of the ten animals died within 1 year of the tumor diagnosis. Neuroendocrine neoplasms occur in the canine mammary gland and are propably underdiagnosed. This is due to their non-specific morphological characteristics and the low use of neuroendocrine immunohistochemistric markers the diagnostic routine. More studies are necessary to determine the prognosis of this new histological type.

scholarly journals Establishment and Characterization of Canine Mammary Gland Carcinoma Cell Lines with Vasculogenic Mimicry Ability in vitro and in vivo

2019 ◽  
Author(s):  
Patricia de Faria Lainetti ◽  
Andressa Brandi ◽  
Antonio Fernando Leis Filho ◽  
Maria Carolina Mangini Prado ◽  
Priscila Emiko Kobayashi ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Cell Lines ◽  
Vasculogenic Mimicry ◽  
Preclinical Model ◽  
Mammary Gland Carcinoma ◽  
Gland Carcinoma ◽  
Canine Mammary Gland

Mammary neoplasms affect a population of uncastrated and elderly female dogs and most of these neoplasms are malignant. In order to study this disease cell culture presents itself as a promising preclinical model, creating the opportunity to deposit cell lines at a cell bank, allowing a great repetition of the assays and making the validation of the results more reliable. Including, in vitro experiments for vasculogenic mimicry (VM) evaluation. VM is related to cancer cells capable of generate vascular-like structures without endothelial cells, mimicking the vasculogenic process. The aim of this study was to establish and characterize ten cell lines from canine mammary gland tumour according to immunophenotype and tumorigenicity, and with its ability to form vasculogenic mimicry-like structures in vitro. Fifteen samples from canine mammary gland carcinoma were collected and cultured in vitro and ten cell lines were established and characterized. Cells were evaluated for morphology, phenotype, vascular mimicry and tumorigenicity. All cell lines presented spindle shape morphology and expressed concomitant pan-cytokeratin and CK8/18. Four cell lines had vasculogenic mimicry ability and two cell showed in vivo tumorigenic potential. Cell characterization of those lines will help to create a database for more knowledge of mammary gland carcinomas in dogs, including studies of tumor behavior and new therapeutic targets.

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