Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures

Gebhard, Christiane; Gabriel, Cordula; Walter, Ingrid

doi:10.1111/ahe.12190

Cited by 35 publications

(24 citation statements)

References 56 publications

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“…Among these, Col I is considered the most promising osteospecific marker in the literature concerning human OSA (31), and was therefore a logical choice to assess in canine OSA. Both canine OSA cell lines were indeed highly positive with antibodies to Col I, consistent with the fundamental characteristic of collagen production by this tumor histotype (41,42). Osteocalcin was detected in most but not all tumor cell lines, which is in agreement with findings from immunohistochemical assays of primary tumor tissue (40,42), and studies of human OSA (31).…”

Section: Discussionsupporting

confidence: 83%

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

et al. 2019

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Osteosarcoma (OSA) is a malignant tumor of middle-aged dogs and adolescent humans. The clinical outcome of OSA has not improved over more than three decades, and dogs typically succumb to metastatic disease within 6 months despite tumor resection through limb amputation and adjuvant chemotherapy. Therefore, undetectable tumor cells with potential to form metastases are present at diagnosis. An assay to identify canine immortalized and primary OSA cells through flow cytometric detection of intracellular collagen 1 (Col I) and osteocalcin was optimized, and applied to blood samples from tumorbearing dogs for detection of circulating tumor cells (CTCs). Spiking variable number of OSA cells into normal dog blood recovered 50-60% of Col I positive cells with high forward and variable side light scatter. An algorithm to exclude nonviable, doublet, and autofluorescent cells was applied to sequential blood samples from three dogs obtained prior to and after limb amputation, and at approximately, triweekly intervals over 121, 142, and 183 days of chemotherapy, respectively. Dogs had >100 CTC/10 6 leukocytes prior to amputation, variably frequent CTC during chemotherapy, and an increase up to 4,000 CTC/10 6 leukocytes within 4 weeks before overt metastases or death. Sorted CTCs were morphologically similar to direct tumor aspirates and positive for Col I. Although preliminary, findings suggest that CTCs are frequent in canine OSA, more numerous than carcinoma CTC in humans, and that an increase in CTC frequency may herald clinical deterioration. This assay may enable enumeration and isolation of OSA CTC for prognostic and functional studies, respectively. OSTEOSARCOMA (OSA) is a primary bone cancer that is common in middle-aged to older, large breed dogs, and less common in humans, where children and young adults are most often affected. Therapy consisting of limb amputation or limbsparing tumor resection followed by chemotherapy (typically platinum or doxorubicin-based) is the gold standard, but the clinical outcome of OSA in dogs and humans remains poor (1-3). Dogs with OSA of the appendicular skeleton treated with amputation and chemotherapy had median survival times (STs) of 235-540 days (4), while in humans 5-year survival has been stagnant at about 60% (5). Although the median ST is relatively homogeneous, the range of ST in dogs with OSA treated identically varies from 2 to >30 months, illustrating that there is extensive heterogeneity in tumor biology and/or host response (6-8). Metastatic disease is the usual cause of spontaneous death or euthanasia, but fewer than 10% of dogs have radiographic evidence of metastasis at the time of diagnosis (2).Computed tomography also lacks sensitivity for identifying metastases, and histologic grading schemes for canine OSA poorly predict biological behavior (9-11).

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Section: Discussionsupporting

confidence: 83%

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

et al. 2019

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“…These data confirm that 3D culture is the most useful tool to assess the ability of tumour cells to perform VM, providing a more similar physiological environment, enhancing spheroid formation . This is the first evidence for the establishment of a scaffold‐free spheroid system in canine OSA with the ability to mimic the architecture of the in vivo tumour . In non‐adherent in vitro conditions, it has been already showed that D17 cells assume stem cell characteristics which are absent in monolayer culture, also increasing collagen I and osteocalcin expression with respect to D17 monolayer .…”

Section: Discussionsupporting

confidence: 74%

“…Significant differences between treated and untreated control cells are shown by asterisks (*P < .05, **P < .01) evidence for the establishment of a scaffold-free spheroid system in canine OSA with the ability to mimic the architecture of the in vivo tumour. 47 In non-adherent in vitro conditions, it has been already showed that D17 cells assume stem cell characteristics which are absent in monolayer culture, also increasing collagen I and osteocalcin expression with respect to D17 monolayer. 31 Because tumour cell spheroids are generally less sensitive to drugs when compared to cells treated in monolayer culture, investigations on the molecular mechanisms of 17-AAG activity in canine OSA 3D cultures could offer new opportunities for the development of therapeutic strategies based on the synergy between VM-blocking and anti-angiogenic properties of Hsp90 inhibition, considering that its anti-tumour activity on OSA cells has been previously established.…”

Section: Discussionmentioning

confidence: 99%

Establishment of three‐dimensional canine osteosarcoma cell lines showing vasculogenic mimicry and evaluation of biological properties after treatment with 17‐AAG

Massimini

Maria

Malatesta

et al. 2019

Vet Comparative Oncology

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Vasculogenic mimicry (VM) is an alternative type of blood perfusion characterized by formation of non‐endothelial cell‐lined microcirculatory channels and is responsible for aggressive tumour biology and increased tumour‐related mortality. VM‐correlated genes are associated with vascular endothelial grown factor receptor 1 (VEGFR1), and hypoxia‐related (hypoxia inducible factor 1 α—HIF1α) signalling pathways, whose molecules are client proteins of Hsp90 (heat shock protein 90) and are potential therapeutic targets. This pilot study was aimed to investigate vasculogenic mimicry in a three‐dimensional (3D) cell culture system of two aggressive canine osteosarcoma (OSA) cell lines (D22 and D17), and to evaluate the response of these cells to 17‐AAG (17‐N‐allylamino‐17‐demethoxygeldanamycin) treatment in relation to tubular‐like structure formation in vitro. Only D17 cell line formed hollow matrix channels in long‐term 3D cultures and assumed endothelial morphology, with cells expressing both Hsp90 and VEGFR1, but lacking expression of endothelial marker CD31. 17‐AAG treatment inhibited migration of D17 OSA cells, also decreasing VM markers in vitro and inducing a reduction of HIF1α transcript and protein in this cell line. Taken together, these preliminary data indicate that the biological effects of 17‐AAG on D17 3D culture and on HIF1α regulation can provide interesting information to translate these findings from the basic research to clinical approach for the treatment of canine OSA as a model in comparative oncology.

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“…Contrary to the method used by Gebhard et al, who harvested MCS using a plastic pipette of 2 mm tip diameter and centrifuged them to cell pellets (13), in the present investigation MCS were carefully harvested using a pasteur pipette and were not centrifuged. The use of a plastic pipette and centrifugation disrupted BHY MCS structure in the present investigation.…”

Section: Discussionmentioning

confidence: 80%

Generation of highly differentiated BHY oral squamous cell carcinoma multicellular spheroids

et al. 2017

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Abstract. Three-dimensional (3D) multicellular spheroids (MCS) are considered suitable models in cancer research and anticancer drug development. Although studying the complex tumour characteristics from all different degrees of malignancy is vital, MCS generation has only been described in a few moderately-and poorly differentiated oral squamous cell carcinoma (OSCC) cell lines. No previous study has demonstrated the MCS formation in a highly differentiated OSCC cell line. For the first time, the present study aimed to generate MCS from the highly differentiated OSCC cell line BHY. BHY spheroids were grown in three independent experiments in 96-well plates through the use of the liquid overlay technique. Although BHY cells are grow slowly and are difficult to culture, they formed compact MCS within 24 h. After 3 days of incubation, no further increase in spheroid size was observed. MCS were harvested, paraffin-embedded and 2 µm tissue sections were prepared for further analysis. The diameter and volume of each spheroid were determined. BHY MCS diameter ranged between 46.76 and 233.26 µm, with a volume range from 5.35x10 4 -6.65x10 6 µm³. In conclusion, using the liquid overlay technique, the highly differentiated OSCC cell line BHY forms different sized spheroids, which may be used for further investigations.

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Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures

Cited by 35 publications

References 56 publications

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

Establishment of three‐dimensional canine osteosarcoma cell lines showing vasculogenic mimicry and evaluation of biological properties after treatment with 17‐AAG

Generation of highly differentiated BHY oral squamous cell carcinoma multicellular spheroids

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