Canine Osteosarcoma Cell Lines Research Articles

Proteomic Analyses Reveal the Role of Alpha-2-Macroglobulin in Canine Osteosarcoma Cell Migration.

Canine osteosarcoma (OSA) is an aggressive bone neoplasia with high metastatic potential. Metastasis is the main cause of death associated with OSA, and there is no current treatment available for metastatic disease. Proteomic analyses, including matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF/TOF MS), are widely used to select molecular targets and identify proteins that may play a key role in primary tumours and at various steps of the metastatic cascade. The main aim of this study was to identify proteins differently expressed in canine OSA cell lines with different malignancy phenotypes (OSCA-8 and OSCA-32) compared to canine osteoblasts (CnOb). The intermediate aim of the study was to compare canine OSA cell migration capacity and assess its correlation with the malignancy phenotypes of each cell line. Using MALDI-TOF/TOF MS analyses, we identified eight proteins that were significantly differentially expressed (p ≤ 0.05) in canine OSA cell lines compared to CnOb: cilia- and flagella-associated protein 298 (CFAP298), general transcription factor II-I (GTF2I), mirror-image polydactyly gene 1 protein (MIPOL1), alpha-2 macroglobulin (A2M), phosphoglycerate mutase 1 (PGAM1), ubiquitin (UB2L6), ectodysplasin-A receptor-associated adapter protein (EDARADD), and leucine-rich-repeat-containing protein 72 (LRRC72). Using the Simple Western technique, we confirmed high A2M expression in CnOb compared to OSCA-8 and OSCA-32 cell lines (with intermediate and low A2M expression, respectively). Then, we confirmed the role of A2M in cancer cell migration by demonstrating significantly inhibited OSA cell migration by treatment with A2M (both at 10 and 30 mM concentrations after 12 and 24 h) in a wound-healing assay. This study may be the first report indicating A2M's role in OSA cell metastasis; however, further in vitro and in vivo studies are needed to confirm its possible role as an anti-metastatic agent in this malignancy.

Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Potential Cytoprotective and Anti-Apoptotic Effect of Metamizole Alone and in Combination with Cytostatic Drugs Observed In Vitro in Canine (D-17) and Human (U-2 OS) Osteosarcoma Cell Lines.

Metamizole (dipyrone) is still a frequently used drug in human and veterinary medicine, especially for pain relief, sometimes also in osteosarcoma treatment. It has a very strong analgesic and antipyretic effect and weaker anti-inflammatory activity. Additionally, it has spasmolytic properties. It is used in many species, including dogs, cats, pigs, cattle, horses, and humans, in Europe, Canada, and South America. The aim of this study was to assess a potential synergism of metamizole as a non-steroidal anti-inflammatory drug with routinely used cytostatics (cisplatin, carboplatin, doxorubicin, and etoposide). In vitro studies were performed on a canine osteosarcoma D-17 cell line and a human U-2 OS cell line. We used the MTT method to assess cell viability, TUNEL staining to assess proapoptotic effects, and propidium iodide to analyse the cell cycle (potential arrest). The obtained results showed that metamizole at 50 μg/mL has potential cytoprotective and anti-apoptotic effects. Metamizole administered simultaneously with cytostatic drugs reduced their cytotoxic effect, which may suggest that such a combination of drugs used in the treatment of osteosarcoma may significantly reduce therapy effectiveness.

An evaluation of the combination effect of zoledronate and chemotherapeutic agents in canine osteosarcoma cells.

Osteosarcoma (OSA) is an aggressive form of bone cancer in both dogs and humans. The treatment options for metastatic (stage III) OSA are currently limited and the prognosis is poor. Zoledronate, a second generation amino-bisphosphonate, is commonly used for palliation of cancer induced bone pain. Zoledronate has also demonstrated anti-cancer properties and possibly enhances the cytotoxicity of doxorubicin in a canine histiocytosis cell line and human prostatic cancer cell line. The goal of this study was to evaluate the combination effect of zoledronate and various chemotherapeutic drugs in canine OSA cells. Canine OSA cell line (D17), cells from two canine primary OSAs, and MDCK, a canine kidney cell line, were used to evaluate the therapeutic potential of these drugs. Carboplatin, doxorubicin, vinorelbine, toceranib, and isophosphoramide mustard (active metabolite of ifosfamide) were used as chemotherapeutic agents. First, cells were treated with either zoledronate or chemotherapy drug alone for 72 hours. Cell viability was assessed using CellTiter Glo and IC5, IC10, IC20, and IC50 were calculated. Second, cells were treated with a combination of zoledronate and each chemotherapeutic agent at their IC5, IC10, IC20, and IC50 concentrations. After 72 hours, cell viability was assessed by CellTiter Glo. Zoledronate, carboplatin, doxorubicin, vinorelbine, and isophosphoramide mustard showed concentration dependent decrease in cell viability. Toceranib showed decreased cell viability only at higher concentrations. When zoledronate was used in combination with chemotherapy drugs, while it showed potential synergistic effects with toceranib, potential antagonistic effects with vinorelbine and isophosphoramide mustard were observed. However, the results differed by cell line and thus, further evaluation is warranted to understand the exact mechanism of action.

Investigating cell death responses associated with histotripsy ablation of canine osteosarcoma

Background Osteosarcoma (OS) is the most frequently occurring primary bone tumor in dogs and people and innovative treatment options are profoundly needed. Histotripsy is an emerging tumor ablation modality, and it is essential for the clinical translation of histotripsy to gain knowledge about the outcome of nonablated tumor cells that could remain postablation. The objective of this study was to characterize the cell death genetic signature and proliferation response of canine OS cells post a near complete histotripsy ablation (96% ± 1.5) and to evaluate genetic cell death signatures associated with histotripsy ablation and OS in vivo. Methods In the current study, we ablated three canine OS cell lines with a histotripsy dose that resulted in near complete ablation to allow for a viable tumor cell population for downstream analyses. To assess the in vivo cell death genetic signature, we characterized cell death genetic signature in histotripsy-ablated canine OS tumors collected 24-h postablation. Results Differential gene expression changes observed in the 4% viable D17 and D418 cells, and histotripsy-ablated OS tumor samples, but not in Abrams cells, were associated with immunogenic cell death (ICD). The 4% viable OS cells demonstrated significantly reduced proliferation, compared to control OS cells, in vitro. Conclusion Histotripsy ablation of OS cell lines leads to direct and potentially indirect cell death as evident by, reduced proliferation in remaining viable OS cells and cell death genetic signatures suggestive of ICD both in vitro and in vivo.

Establishment and Characterization of Cell Lines from Canine Metastatic Osteosarcoma.

Despite the advancements in treatments for other cancers, the outcomes for osteosarcoma (OSA) patients have not improved in the past forty years, especially in metastatic patients. Moreover, the major cause of death in OSA patients is due to metastatic lesions. In the current study, we report on the establishment of three cell lines derived from metastatic canine OSA patients and their transcriptome as compared to normal canine osteoblasts. All the OSA cell lines displayed significant upregulation of genes in the epithelial mesenchymal transition (EMT) pathway, and upregulation of key cytokines such as CXCL8, CXCL10 and IL6. The two most upregulated genes are MX1 and ISG15. Interestingly, ISG15 has recently been identified as a potential therapeutic target for OSA. In addition, there is notable downregulation of cell cycle control genes, including CDKN2A, CDKN2B and THBS1. At the protein level, p16INK4A, coded by CDKN2A, was undetectable in all the canine OSA cell lines, while expression of the tumor suppressor PTEN was variable, with one cell line showing complete absence and others showing low levels of expression. In addition, the cells express a variety of actionable genes, including KIT, ERBB2, VEGF and immune checkpoint genes. These findings, similar to those reported in human OSA, point to some genes that can be used for prognosis, targeted therapies and novel drug development for both canine and human OSA patients.

Evaluation of the Effect of Five Colombian Propolis Extracts on the Expression of Genes Associated with Cell Cycle and Inflammation in a Canine Osteosarcoma Cell Line

Evaluation of the Effect of Five Colombian Propolis Extracts on the Expression of Genes Associated with Cell Cycle and Inflammation in a Canine Osteosarcoma Cell Line

Utility of the second-generation curcumin analogue RL71 in canine histiocytic sarcoma.

Canine histiocytic sarcoma is an aggressive cancer, with a high rate of metastasis. Thus, novel therapeutic approaches are needed. Synthetic analogues of curcumin have elicited potent anti-cancer activity in multiple in vitro and in vivo models of human cancer. Furthermore, the compound 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidine-4-one (RL71) has recently exhibited potent cell cycle arrest and apoptotic induction in a canine osteosarcoma cell line. To determine its potency in canine histiocytic sarcoma cells, cell viability of DH82 and Nike cells was measured using the sulforhodamine B assay. Flow cytometry was then used to analyse both cell cycle distribution and apoptosis. Of the five curcumin analogues examined, RL71, had the highest potency with EC50 values of 0.66 ± 0.057 µM and 0.79 ± 0.13 µM in the DH82 and Nike cell lines, respectively. Furthermore, RL71 at the 1x EC50 concentration increased G2/M cell cycle arrest 2-fold, and at the 2x EC50 concentration increased the number of apoptotic cells 4-fold. These findings are consistent with previous work using RL71 in both canine and human cancer cell lines. Future research should be directed on time-dependent changes, and mechanistic investigation in greater detail to elucidate RL71 mechanisms of action.

The second-generation curcumin analogue RL71 elicits G2/M cell cycle arrest and apoptosis in canine osteosarcoma cells.

Canine osteosarcoma is an aggressive cancer, comprising 85% of canine bone neoplasms. Current treatment practices of surgery and chemotherapy increase 1-year survival by only 45%. The curcumin analogue RL71, has demonstrated potent in vitro and in vivo efficacy in several models of human breast cancer through increased apoptosis and cell cycle arrest. Thus, the present study aimed to investigate efficacy of curcumin analogues in two canine osteosarcoma cell lines. Osteosarcoma cell viability was assessed using the sulforhodamine B assay and mechanisms of action were determined by analysing the levels of cell cycle and apoptotic regulatory proteins via Western blotting. Further evidence was obtained using flow cytometry to detect cell cycle distribution and the number of apoptotic cells. RL71 was the most potent curcumin analogue with EC50 values of 0.64 ± 0.04 and 0.38 ± 0.009 μM (n = 3) in D-17 (commercial) and Gracie canine osteosarcoma cells, respectively. RL71 significantly increased the ratio of cleaved-caspase 3 to pro-caspase 3 and the level of apoptotic cells at the 2× and 5× EC50 concentration (p < 0.001, n = 3). Furthermore, at the same concentration, RL71 significantly increased the number of cells in the G2/M phase. In conclusion, RL71 has potent cytotoxic activity in canine osteosarcoma cells triggering G2/M arrest and apoptosis at concentrations achievable in vivo. Future research should further investigate molecular mechanisms for these changes in other canine osteosarcoma cell lines prior to in vivo investigation.

Canine osteosarcoma as a solid tumor model for evaluation of dual valent B7H3-CXCR2 CAR T cell therapy

Abstract To develop more effective solid tumor CAR T cell therapies, we utilized the spontaneous canine metastatic osteosarcoma (OS) animal model. Our studies evaluated the relative activity of single B7H3 and dual-valent B7H3-CXCR2 CAR constructs. Studies were also designed to determine if modifying the tumor microenvironment (TME) with oral immunotherapy improved CAR activity, based on recent studies of losartan and propranolol in dogs with OS and glioma. We evaluated B7H3 expression on canine OS cell lines using flow cytometry and OS biopsies using IHC. High levels of B7-H3 were observed on OS cell lines and OS biopsies, whereas normal canine tissues were negative or low. To assess the utility of the dual CAR we determined that canine OS cell lines secreted high IL8 spontaneously. To assess the functionality of canine B7-H3 CAR T cells, we generated canine CAR T cells from whole blood from dogs with cancer. We next determined that canine dual CAR T cells exerted specific killing and cytokine activity against B7H3+ canine OS cell lines. To further establish functionality, we evaluated the two CAR constructs in a mouse xenograft model of canine OS. We found that the dual CAR construct was significantly more active than the single CAR construct in slowing tumor growth. We found that losartan and propranolol blocked migration of mouse inflammatory monocytes to xenografted canine OS tumors. Future steps will be to evaluate the effectiveness of dual CAR T cells with TME modification in dogs with metastatic OS, to investigate the translational relevance in human patients. Results from these studies in dogs will likely give more clinically relevant results for evaluating new CAR T cell therapies and bridging results between mouse models and human clinical trials. The V Foundation, The Shipley Family Foundation, and NIH T32 training grant

Abstract 6233: Omacetaxine reduces c-MYC expression and demonstrates antitumor effects in osteosarcoma

Abstract Omacetaxine mepesuccinate (OMX), also known as homoharringtonine, is a protein translation inhibitor approved for use in chronic myeloid leukemia. Recent studies suggest OMX may be effective against other cancer types with high rates of protein translation, and may be successful against protein targets that are difficult to inhibit by reducing their translation rates. Using both canine and human osteosarcoma (OS) cell lines, we have investigated the efficacy of OMX against osteosarcoma and expression of c-MYC. OMX provides significant growth inhibition of OS cells with IC50 values in the low nanomolar range. OMX also reduces migration and invasion capabilities of OS cells. Strikingly, we observed reduction in levels of c-MYC protein in most canine and human OS cell lines after OMX exposure. This reduction was both dose and time dependent. We also investigated the efficacy of OMX against OS in an orthotopic mouse OS model. OMX is a promising candidate for treatment of OS in both dogs and humans, and potentially additional cancers dependent on c-MYC. Citation Format: Kristen B. Farrell, Douglas H. Thamm. Omacetaxine reduces c-MYC expression and demonstrates antitumor effects in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6233.

Abstract 2339: Exploration of multiple immunotherapy modalities in ostesarcoma

Abstract Osteosarcoma (OS) is the most common primary bone malignancy affecting long bones in children and the third most frequent in adults in the United States. It is highly invasive and metastatic. The survival rates over the last 20 years are unchanged. Therefore, it is essential to develop novel, targeted therapeutic approaches to treat OS. In pursuit of that goal, novel models are needed. Due to the high similarity in cancer genetics, the spontaneous occurrence of tumors, and the presence of an intact immune system, dogs are deemed an excellent cancer immunotherapy model. OS is also the most common malignant bone tumor (80%) in dogs. Monotherapies like oncolytic viruses and immune checkpoint inhibitors have been shown to be insufficient for effective cancer treatment. Therefore, we propose a combination of immune checkpoint inhibition and oncolytic virotherapy to target osteosarcoma tumor cells. In this combined therapy, the oncolytic virus will serve to both stimulate an inflammatory response and transmit the checkpoint inhibitor, which in turn will block PD-1-directed down-regulation of T cells, allowing the adaptive immune system to respond better to tumors. In order to evaluate this approach in the canine model, we have developed an oncolytic virus (CAV2-AU-M2) based on canine adenovirus type 2 and armed with an immune checkpoint inhibitor, an anti-canine PD1 single domain camelid antibody (sdAb). Four canine osteosarcoma cell lines, D17, D22, MCKOS, and CF11, were cultured in two-dimensional (2D) and three-dimensional (3D) cultures and were infected with CAV2-AU-M2 to analyze the infective and lytic effects of the virus. CAV2-AU-M2 infected and lysed the cells in 2D. The virus also produced anti-PD1 sdAb upon infecting the cells. Similarly, results were evident in 3D cultures. The virus infected the spheroids in the outer layers on Day 4 and slowly diffused towards the core in 7 days. The spheroids infected started showing some cell death on day 4, which progressed significantly by Day 7. This approach to oncolytic virotherapy makes the tumor more visible to the immune system and abrogates tumor-induced immune suppression. Citation Format: Aliaa A. Ismail, Daniel J. Patton, Theresa A. Higgins, Maninder Sandey, Bruce F. Smith, Payal Agarwal. Exploration of multiple immunotherapy modalities in ostesarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2339.

Isolation and Characterization of Novel Canine Osteosarcoma Cell Lines from Chemotherapy-Naïve Patients

The present study aimed to establish novel canine osteosarcoma cell lines (COS3600, COS3600B, COS4074) and characterize the recently described COS4288 cells. The established D-17 cell line served as a reference. Analyzed cell lines differed notably in their biological characteristics. Calculated doubling times were between 22 h for COS3600B and 426 h for COS4074 cells. COS3600B and COS4288 cells produced visible colonies after anchorage-independent growth in soft agar. COS4288 cells were identified as cells with the highest migratory capacity. All cells displayed the ability to invade through an artificial basement membrane matrix. Immunohistochemical analyses revealed the mesenchymal origin of all COS cell lines as well as positive staining for the osteosarcoma-relevant proteins alkaline phosphatase and karyopherin α2. Expression of p53 was confirmed in all tested cell lines. Gene expression analyses of selected genes linked to cellular immune checkpoints (CD270, CD274, CD276), kinase activity (MET, ERBB2), and metastatic potential (MMP-2, MMP-9) as well as selected long non-coding RNA (MALAT1) and microRNAs (miR-9, miR-34a, miR-93) are provided. All tested cell lines were able to grow as multicellular spheroids. In all spheroids except COS4288, calcium deposition was detected by von Kossa staining. We believe that these new cell lines serve as useful biological models for future studies.

Evaluation of the Apoptotic Effect of Sinapic Acid in D17 Canine Osteosarcoma Cell Line

Evaluation of the Apoptotic Effect of Sinapic Acid in D17 Canine Osteosarcoma Cell Line

Expression of Sex Hormone Receptors in Canine Osteosarcoma.

Simple SummaryOsteosarcoma is a bone tumor that arises from normal bone cells that become malignant. This is a disease that is relatively common in dogs and also affects humans. Dogs are a useful model for human osteosarcoma as they share many similarities including genetic changes, manner of spread and how they respond to treatment. In dogs, the tumors are frequently seen in large breeds and may be caused by environmental and genetic factors. Sex steroids have been shown to regulate bone metabolism directly and indirectly through receptors on bone. The aim of this study was to investigate the expression levels of sex hormone receptors in canine osteosarcoma tissue and cell lines. Our results demonstrate varying levels of receptors in tissue and a high expression of progesterone receptors in the canine osteosarcoma cell lines. Low oxygen levels further increased progesterone receptor expression. Lastly, estradiol decreased the expression of progesterone receptor in one of the cell lines, and progesterone decreased cell proliferation in the same cell line. Further investigation is recommended to determine whether or not sex steroid receptor levels and sensitivity to hormones are related.Sex steroids regulate bone metabolism directly and indirectly through receptors on bone. Estrogen receptors (ER-∝, ER-β), progesterone receptor (PR), and androgen receptor (AR), have been previously identified on human osteosarcoma (OSA) cells, and are considered to influence tumor growth, but their expression and role in canine OSA is unknown. The aim of this study was to characterize sex hormone receptor expression levels in naturally occurring OSA tissue and in three canine OSA cell lines. The expression of ER-α, ER-β, PR, and AR was investigated using RT-PCR. PR expression levels were also quantified in OSA cells cultured under hypoxic conditions or in the presence of estradiol. The effects of progesterone on cell proliferation were quantified. Results demonstrated varying expression levels of these receptors in five OSA subtypes. OSA cell lines demonstrated high gene expression levels of PR and low gene expression levels of ER-α and ER-β and no gene expression of AR. PR expression was increased in OSA cells cultured under hypoxic conditions in a HIF-∝ independent manner. Interestingly, one cell line expressed very high levels of PR, expression of which decreased in response to estradiol. In addition, progesterone decreased OSA cell proliferation in this particular cell line. Further investigation of the role of sex steroids, particularly PR and its ligands, in regulation of canine OSA is recommended.

Anticancer activity of an Artemisia annua L. hydroalcoholic extract on canine osteosarcoma cell lines

Since ancient times, Artemisia annua (A. annua) has been used as a medicinal plant in Traditional Chinese Medicine. In addition, recent studies have investigated the cytotoxic effects of A. annua extracts towards cancer cells. The leading aim of the present research is to evaluate the cytotoxic effects of an hydroalcoholic extract of A. annua on two canine osteosarcoma (OSA) cell lines, OSCA-8 and OSCA-40, focusing on the possible involvement of ferroptosis.The quantitative determination of artemisinin concentration in the extract, culture medium and OSA cells was carried out through the use of an instrumental analytical method based on liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometry (LC-DAD-MS/MS). OSCA-8 and OSCA-40 were exposed to different dilutions of the extract for the EC50 calculation then the uptake of artemisinin by the cells, the effects on the cell cycle, the intracellular iron level, the cellular morphology and the lipid oxidation state were evaluated. A concentration of artemisinin of 63.8 ± 3.4 μg/mL was detected in the extract. A dose-dependent cytotoxic effect was evidenced. In OSCA-40 alterations of the cell cycle and a significantly higher intracellular iron content were observed. In both cell lines the treatment with the extract was associated with lipid peroxidation and with the appearance of a “ballooning” phenotype suggesting the activation of ferroptosis. In conclusion the A. annua idroalcoholic extract utilized in this study showed anticancer activity on canine OSA cell lines that could be useful in treating drug resistant canine OSAs.

The genomic landscape of canine osteosarcoma cell lines reveals conserved structural complexity and pathway alterations.

The characterization of immortalized canine osteosarcoma (OS) cell lines used for research has historically been based on phenotypic features such as cellular morphology and expression of bone specific markers. With the increasing use of these cell lines to investigate novel therapeutic approaches prior to in vivo translation, a much more detailed understanding regarding the genomic landscape of these lines is required to ensure accurate interpretation of findings. Here we report the first whole genome characterization of eight canine OS cell lines, including single nucleotide variants, copy number variants and other structural variants. Many alterations previously characterized in primary canine OS tissue were observed in these cell lines, including TP53 mutations, MYC copy number gains, loss of CDKN2A, PTEN, DLG2, MAGI2, and RB1 and structural variants involving SETD2, DLG2 and DMD. These data provide a new framework for understanding how best to incorporate in vitro findings generated using these cell lines into the design of future clinical studies involving dogs with spontaneous OS.

Sorafenib and Doxorubicin Show Synergistic Effects in Human and Canine Osteosarcoma Cell Lines.

Osteosarcoma (OSA) is the most common bone tumor in both humans and dogs and has a nearly ten-fold higher incidence in dogs than humans. Despite advances in the treatment of other cancers, the overall survival rates for OSA have stagnated for the past four decades. Therefore, there is a great need to identify novel and effective treatments. We screened a series of tyrosine kinase inhibitors and selected sorafenib, a multi-kinase inhibitor, for further evaluation alone and in combination with cisplatin, carboplatin, and doxorubicin on canine and human OSA cell lines. Our data point to synergistic effects when sorafenib is combined with doxorubicin, but not when combined with cisplatin or carboplatin, in both human and canine OSA. Based on current findings, clinical trials using a combination of doxorubicin and sorafenib in proof-of-concept studies in dogs are warranted. These studies can be carried out relatively quickly in dogs where case load is high and, in turn, provide useful data for the initiation of clinical trials in humans.

Lipid droplets and perilipins in canine osteosarcoma. Investigations on tumor tissue, 2D and 3D cell culture models

Lipid droplets were identified as important players in biological processes of various tumor types. With emphasis on lipid droplet-coating proteins (perilipins, PLINs), this study intended to shed light on the presence and formation of lipid droplets in canine osteosarcoma. For this purpose, canine osteosarcoma tissue samples (n = 11) were analyzed via immunohistochemistry and electron microscopy for lipid droplets and lipid droplet-coating proteins (PLINs). Additionally, we used the canine osteosarcoma cell lines D-17 and COS4288 in 2D monolayer and 3D spheroid (cultivated for 7, 14, and 21 days) in vitro models, and further analyzed the samples by means of histochemistry, immunofluorescence, molecular biological techniques (RT-qPCR, Western Blot) and electron microscopical imaging. Lipid droplets, PLIN2, and PLIN3 were detected in osteosarcoma tissue samples as well as in 2D and 3D cultivated D-17 and COS4288 cells. In spheroids, specific distribution patterns of lipid droplets and perilipins were identified, taking into consideration cell line specific zonal apportionment. Upon external lipid supplementation (oleic acid), a rise of lipid droplet amount accompanied with an increase of PLIN2 expression was observed. Detailed electron microscopical analyzes revealed that lipid droplet sizes in tumor tissue were comparable to that of 3D spheroid models. Moreover, the biggest lipid droplets were found in the central zone of the spheroids at all sampling time-points, reaching their maximum size at 21 days. Thus, the 3D spheroids can be considered as a relevant in vitro model for further studies focusing on lipid droplets biology and function in osteosarcoma.

Expression of the hedgehog signalling pathway and the effect of inhibition at the level of smoothened in canine osteosarcoma cell lines.

Osteosarcoma (OSA) is the most common malignant bone cancer in dogs. Canine and human OSA share several features, including tumour environments, response to traditional treatment, and several molecular pathways. Hedgehog (Hh) signalling is known to contribute to tumorigenesis and progression of various cancers, including human OSA. This study aimed to identify the role of the Hh signalling pathway in canine OSA cell lines, including Abrams, D17, and Moresco, focusing on the signal transducer Smoothened (SMO). mRNA and protein levels of Hh pathway components, including SHH, IHH, SMO, and PTCH1, were aberrant in all examined OSA cell lines compared with canine osteoblast cells. The SMO inhibitor cyclopamine significantly decreased cell viability and colony-forming ability in the canine OSA cell lines in a dose-dependent manner. Moresco cells, which expressed the highest level of SMO protein, were the most sensitive to the anticancer effect of cyclopamine among the three canine OSA cell lines tested. Hh downstream target gene and protein expression in canine OSA cell lines were downregulated after cyclopamine treatment. In addition, cyclopamine significantly increased apoptotic cell death in Abrams and Moresco cells. The findings that Hh/SMO is activated in canine OSA cell lines and cyclopamine suppresses OSA cell survival via inhibition of SMO suggest that the Hh/SMO signalling pathway might be a novel therapeutic target for canine OSA.