In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models

AbstractBackgroundOsteosarcoma is a common primary malignant bone cancer. Long noncoding RNA small nucleolar RNA host gene 15 (SNHG15) has been reported to play an oncogenic role in many cancers. Nevertheless, the role of SNHG15 in the doxorubicin (DXR) resistance of osteosarcoma cells has not been fully addressed.MethodsCell Counting Kit-8 assay was conducted to measure the half-maximal inhibitory concentration value of DXR in osteosarcoma cells. Western blotting was carried out to examine the levels of autophagy-related proteins and GDNF family receptor alpha-1 (GFRA1). Quantitative reverse transcription-polymerase chain reaction was performed to determine the levels of SNHG15, miR-381-3p, and GFRA1. The proliferation of osteosarcoma cells was measured by MTT assay. The binding sites between miR-381-3p and SNHG15 or GFRA1 were predicted by Starbase bioinformatics software, and the interaction was confirmed by dual-luciferase reporter assay. Murine xenograft model was established to validate the function of SNHG15 in vivo.ResultsAutophagy inhibitor 3-methyladenine sensitized DXR-resistant osteosarcoma cell lines to DXR. SNHG15 was upregulated in DXR-resistant osteosarcoma tissues and cell lines. SNHG15 knockdown inhibited the proliferation, DXR resistance, and autophagy of osteosarcoma cells. MiR-381-3p was a direct target of SNHG15, and GFRA1 bound to miR-381-3p in osteosarcoma cells. SNHG15 contributed to DXR resistance through the miR-381-3p/GFRA1 axis in vitro. SNHG15 depletion contributed to the inhibitory effect of DXR on osteosarcoma tumor growth through the miR-381-3p/GFRA1 axis in vivo.ConclusionsSNHG15 enhanced the DXR resistance of osteosarcoma cells through elevating the autophagy via targeting the miR-381-3p/GFRA1 axis. Restoration of miR-381-3p expression might be an underlying therapeutic strategy to overcome the DXR resistance of osteosarcoma.

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Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells

10.21203/rs.2.12812/v3 ◽

2019 ◽

Author(s):

Kazushige Yoshida ◽

Masanori Okamoto ◽

Jun Sasaki ◽

Chika Kuroda ◽

Haruka Ishida ◽

...

Keyword(s):

T Cells ◽

Cell Lines ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitors ◽

Checkpoint Inhibitors ◽

Tumour Volume ◽

Tumour Microenvironment ◽

Osteosarcoma Cell

Abstract Background: There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. Methods: In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFNγ for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry. Results: We discovered that IFNγ increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. Conclusions: Here we clarify for the first time an additional mechanism of anti-tumour effect—as exerted by anti-PD-1 antibody decreasing Treg— we anticipate that our findings will lead to the development of new methods for cancer treatment.

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Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells

10.21203/rs.2.12812/v1 ◽

2019 ◽

Author(s):

Kazushige Yoshida ◽

Masanori Okamoto ◽

Jun Sasaki ◽

Chika Kuroda ◽

Haruka Ishida ◽

...

Keyword(s):

T Cells ◽

Cell Lines ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitors ◽

Checkpoint Inhibitors ◽

Tumour Volume ◽

Osteosarcoma Cell ◽

The Difference

Abstract Background: There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. Methods: In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFNγ for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumor by Flowcytometry. Results: We discovered that IFNγ increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumor microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. Conclusions: Here we clarify for the first time an additional mechanism of anti-tumour effect—as exerted by anti-PD-1 antibody decreasing Treg— we anticipate that our findings will lead to the development of new methods for cancer treatment. keywords: PD-1, Treg, osteosarcoma, anti-PD-1 antibody.

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Characterization of five newly derived canine osteosarcoma cell lines

BMC Veterinary Research ◽

10.1186/s12917-019-2099-y ◽

2019 ◽

Vol 15 (1) ◽

Cited By ~ 1

Author(s):

Heather Wilson-Robles ◽

Kelli Franks ◽

Roy Pool ◽

Tasha Miller

Keyword(s):

Giant Cell ◽

Cell Lines ◽

Tumor Development ◽

Disease Process ◽

High Rate ◽

Osteosarcoma Cell ◽

Canine Osteosarcoma ◽

Cell Variant

Abstract Background Canine and human osteosarcomas (OS) are notably similar and have a high rate of metastasis. There is a poor understanding of the tumor development process, predisposing causes, and varying levels of aggression among different cell lines. By characterizing newly developed canine osteosarcoma cell lines, treatments for people and pets can be developed. Of the seven subtypes of OS, three are represented in this group: osteoblastic (the most common), fibroblastic, and giant cell variant. To our knowledge, there are no other giant cell variant canine OS cell lines in the published literature and only one canine fibroblastic osteosarcoma cell line. Understanding the differences between the histologic subtypes in dogs will help to guide comparative research. Results Alkaline phosphatase expression was ubiquitous in all cell lines tested and invasiveness was variable between the cell lines tested. Invasiveness and oxidative damage were not correlated with in vivo growth rates, where TOT grew the fastest and had the higher percentage of mice with metastatic lesions. TOL was determined to be the most chemo-resistant during cisplatin chemotherapy while TOM was the most chemo-sensitive. Conclusions Further comparisons and studies using these cell lines may identify a variety of characteristics valuable for understanding the disease process and developing treatments for osteosarcoma in both species. Some of this data was presented as a poster by KMF at the August 5th, 2017 National Veterinary Scholars Program in Bethesda, MA. Characterization of 5 newly generated canine osteosarcoma cell lines. Kelli Franks, Tasha Miller, Heather Wilson-Robles.

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Biological activity of gemcitabine against canine osteosarcoma cell lines in vitro

American Journal of Veterinary Research ◽

10.2460/ajvr.71.7.799 ◽

2010 ◽

Vol 71 (7) ◽

pp. 799-808 ◽

Cited By ~ 10

Author(s):

Melanie B. McMahon ◽

Misty D. Bear ◽

Samuel K. Kulp ◽

Michael L. Pennell ◽

Cheryl A. London

Keyword(s):

Biological Activity ◽

Cell Lines ◽

Osteosarcoma Cell ◽

Canine Osteosarcoma

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Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells

BMC Cancer ◽

10.1186/s12885-019-6499-y ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 5

Author(s):

Kazushige Yoshida ◽

Masanori Okamoto ◽

Jun Sasaki ◽

Chika Kuroda ◽

Haruka Ishida ◽

...

Keyword(s):

T Cells ◽

Cell Lines ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitors ◽

Checkpoint Inhibitors ◽

Tumour Volume ◽

Tumour Microenvironment ◽

Osteosarcoma Cell

Abstract Background There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. Methods In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFNγ for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry. Results We discovered that IFNγ increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. Conclusions Here we clarify for the first time an additional mechanism of anti-tumour effect—as exerted by anti-PD-1 antibody decreasing Treg— we anticipate that our findings will lead to the development of new methods for cancer treatment.

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ICG-001, an Inhibitor of the β-Catenin and cAMP Response Element-Binding Protein Dependent Gene Transcription, Decreases Proliferation but Enhances Migration of Osteosarcoma Cells

Pharmaceuticals ◽

10.3390/ph14050421 ◽

2021 ◽

Vol 14 (5) ◽

pp. 421

Author(s):

Geoffroy Danieau ◽

Sarah Morice ◽

Sarah Renault ◽

Régis Brion ◽

Kevin Biteau ◽

...

Keyword(s):

Cell Proliferation ◽

Mouse Model ◽

Cell Lines ◽

Gene Transcription ◽

Pediatric Population ◽

Wnt Signaling Pathway ◽

Osteosarcoma Cell ◽

Metastatic Dissemination

High-grade osteosarcomas are the most frequent malignant bone tumors in the pediatric population, with 150 patients diagnosed every year in France. Osteosarcomas are associated with low survival rates for high risk patients (metastatic and relapsed diseases). Knowing that the canonical Wnt signaling pathway (Wnt/β-catenin) plays a complex but a key role in primary and metastatic development of osteosarcoma, the aim of this work was to analyze the effects of ICG-001, a CBP/β-catenin inhibitor blocking the β-catenin dependent gene transcription, in three human osteosarcoma cell lines (KHOS, MG63 and 143B). The cell proliferation and migration were first evaluated in vitro after ICG-001 treatment. Secondly, a mouse model of osteosarcoma was used to establish the in vivo biological effect of ICG-001 on osteosarcoma growth and metastatic dissemination. In vitro, ICG-001 treatment strongly inhibits osteosarcoma cell proliferation through a cell cycle blockade in the G0/G1 phase, but surprisingly, increases cell migration of the three cell lines. Moreover, ICG-001 does not modulate tumor growth in the osteosarcoma mouse model but, rather significantly increases the metastatic dissemination to lungs. Taken together, these results highlight, despite an anti-proliferative effect, a deleterious pro-migratory role of ICG-001 in osteosarcoma.

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GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma

Journal of Hematology & Oncology ◽

10.1186/s13045-020-01012-y ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Jeong A. Park ◽

Nai-Kong V. Cheung

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitors ◽

Checkpoint Inhibitors ◽

Tumor Control ◽

Osteosarcoma Cell

Abstract Background The cure rate for metastatic osteosarcoma has not substantially improved over the past decades. Clinical trials of anti-HER2 trastuzumab or anti-GD2 dinutuximab for metastatic or refractory osteosarcoma were not successful, and neither was immune checkpoint inhibitors (ICIs). Methods We tested various target antigen expressions on osteosarcoma cell lines using flow cytometry and analyzed in vitro T cell engaging BsAb (T-BsAb)-dependent T cell-mediated cytotoxicity using 4-h 51Cr release assay. We tested in vivo anti-tumor activities of T-BsAb targeting GD2 or HER2 in established osteosarcoma cell line or patient-derived xenograft (PDX) mouse models carried out in BALB-Rag2−/−IL-2R-γc-KO (BRG) mice. We also generated ex vivo BsAb-armed T cells (EATs) and studied their tumor-suppressive effect against osteosarcoma xenografts. In order to improve the anti-tumor response, ICIs, anti-human PD-1 (pembrolizumab) or anti-human PD-L1 (atezolizumab) antibodies were tested their synergy with GD2- or HER2-BsAb against osteosarcoma. Results GD2 and HER2 were chosen from a panel of surface markers on osteosarcoma cell lines and PDXs. Anti-GD2 BsAb or anti-HER2 BsAb exerted potent anti-tumor effect against osteosarcoma tumors in vitro and in vivo. T cells armed with anti-GD2-BsAb (GD2-EATs) or anti-HER2-BsAb (HER2-EATs) showed significant anti-tumor activities as well. Anti-PD-L1 combination treatment enhanced BsAb-armed T cell function in vivo and improved tumor control and survival of the mice, when given sequentially and continuously. Conclusion Anti-GD2 and anti-HER2 BsAbs were effective in controlling osteosarcoma. These data support the clinical investigation of GD2 and HER2 targeted T-BsAb treatment in combination with immune checkpoint inhibitors, particularly anti-PD-L1, in patients with osteosarcoma to improve their treatment outcome.

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LncRNA RBM5-AS1 Promotes Osteosarcoma Cell Proliferation, Migration, and Invasion

BioMed Research International ◽

10.1155/2021/5271291 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Biyong Deng ◽

Runsang Pan ◽

Xin Ou ◽

Taizhe Wang ◽

Weiguo Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Western Blotting ◽

Migration And Invasion ◽

Osteosarcoma Cell ◽

Pediatric Age Group ◽

The Relationship

Purpose. Osteosarcoma (Os) is the most frequent malignant tumor of the bone in the pediatric age group, and accumulating evidences show that lncRNAs play a key role in the development of Os. Thus, we investigated the role of RBM5-AS1 and its molecular mechanism. Methods. The expression of RBM5-AS1 in Os tissues and cell lines was detected by real-time polymerase chain reaction (QPCR). The effect of RBM5-AS1 on the proliferation of Os cells was detected using CCK8 assays and flow cytometry. The effect of RBM5-AS1 on the migration and invasion of Os cells was detected by transwell assays. And we performed QPCR and western blotting assays to investigate the relationship between RBM5-AS1 and RBM5. Finally, western blotting assays were performed to explore the mechanism of RBM5. Results. LncRNA RBM5-AS1 was overexpressed in the Os tissues and cell lines. And lncRNA RBM5-AS1 promoted Os cell proliferation, migration, and invasion in vitro and tumor growth in vivo. LncRNA RBM5-AS1 targets RBM5 in Os cells. Conclusion. To sum up, the results showed that lncRNA RBM5-AS1 promotes cell proliferation, migration, and invasion in Os.

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Overexpression of the Ubiquitin Specific Proteases USP43, USP41, USP27x and USP6 in Osteosarcoma Cell Lines: Inhibition of Osteosarcoma Tumor Growth and Lung Metastasis Development by the USP Antagonist PR619

Cells ◽

10.3390/cells10092268 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2268

Author(s):

Mélanie Lavaud ◽

Mathilde Mullard ◽

Robel Tesfaye ◽

Jérôme Amiaud ◽

Mélanie Legrand ◽

...

Keyword(s):

Tumor Growth ◽

Cell Lines ◽

Lung Metastases ◽

Poor Response ◽

Response To Treatment ◽

Osteosarcoma Cell ◽

Sequencing Analysis ◽

Protein Ubiquitination

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan–Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

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