Patient-derived osteosarcoma cells are resistant to methotrexate

Cavalcanti, Amanda dos Santos; Meohas, Walter; Ribeiro, Gabriele de Oliveira; Lopes, Armando; Gholamin, Sharareh; Razavi, Seyed Mostafa; Kasai-Brunswick, Taís Hanae; Avan, Amir; Guimarães, João Antônio Matheus; Duarte, Maria Eugênia Leite; Kahn, Suzana Assad

doi:10.1371/journal.pone.0184891

Cited by 11 publications

(12 citation statements)

References 35 publications

(45 reference statements)

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“…High level of in-vitro acquired resistance was obtained with MTX (5/6 cell lines), while only one of the six lines developed resistance to DOXO. This is consistent with observation in other models derived from patient samples which showed resistance to MTX while still sensitive to DOXO and CISP [14]. Continuous in-vitro exposure to the drug induced large CNA changes translated at transcriptomic GE mRNA level, in a drug and cell line dependent manner [21].…”

Section: Discussionsupporting

confidence: 89%

“…Similarly, MMP3 decrease in MTX-resistant cells suggested less invasive potential [34,35]. In our in-vitro resistant models, no modification of cancer stem cell markers was observed as it has been seen in PDX models [14]. We also observed in the DOXO-resistant line compared to its parental counterpart an enrichment of the HLA cluster genes on cytoband 6p21.3.…”

Section: Discussionsupporting

confidence: 70%

“…Decreased mRNA expression of RFC and increased mRNA expression of DHFR, although not significant, might have participated in the slight MTX resistance observed in the DOXO-resistant line (Figure 1B). One cancer stem cell marker SOX2 was up-regulated in HOS-R/DOXO compared to its parental line (log2 FoldChange 1.89, adjusted p value 6,45×10 -04 ), while none of the cancer stem cell markers (SOX2, OCT4, SSEA4, NANOG and ABCG2) [14] were modified in MTX-resistant lines.…”

Section: Resultsmentioning

confidence: 99%

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In-Vitro and In-Vivo Establishment and Characterization of Bioluminescent Orthotopic Chemotherapy-Resistant Human Osteosarcoma Models in NSG Mice

Costa

Marchais

Gomez‐Brouchet

et al. 2019

Cancers

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Osteosarcoma, the most common bone malignancy with a peak incidence at adolescence, had no survival improvement since decades. Persistent problems are chemo-resistance and metastatic spread. We developed in-vitro osteosarcoma models resistant to chemotherapy and in-vivo bioluminescent orthotopic cell-derived-xenografts (CDX). Continuous increasing drug concentration cultures in-vitro resulted in five methotrexate (MTX)-resistant and one doxorubicin (DOXO)-resistant cell lines. Resistance persisted after drug removal except for MG-63. Different resistance mechanisms were identified, affecting drug transport and action mechanisms specific to methotrexate (RFC/SCL19A1 decrease, DHFR up-regulation) for MTX-resistant lines, or a multi-drug phenomenon (PgP up-regulation) for HOS-R/DOXO. Differential analysis of copy number abnormalities (aCGH) and gene expression (RNAseq) revealed changes of several chromosomic regions translated at transcriptomic level depending on drug and cell line, as well as different pathways implicated in invasive and metastatic potential (e.g., Fas, Metalloproteinases) and immunity (enrichment in HLA cluster genes in 6p21.3) in HOS-R/DOXO. Resistant-CDX models (HOS-R/MTX, HOS-R/DOXO and Saos-2-B-R/MTX) injected intratibially into NSG mice behaved as their parental counterpart at primary tumor site; however, they exhibited a slower growth rate and lower metastatic spread, although they retained resistance and CGH main characteristics without drug pressure. These models represent valuable tools to explore resistance mechanisms and new therapies in osteosarcoma.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

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In-Vitro and In-Vivo Establishment and Characterization of Bioluminescent Orthotopic Chemotherapy-Resistant Human Osteosarcoma Models in NSG Mice

Costa

Marchais

Gomez‐Brouchet

et al. 2019

Cancers

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“…Osteosarcoma is one of the most common cancers in adolescence and young adults and often leads to lethal pulmonary metastasis [ 24 , 25 , 26 ]. The current standard treatment for osteosarcoma is a combination of radical surgery and chemotherapy such as doxorubicin and methotrexate [ 26 , 27 , 28 ]; however, the efficacy is still limited, indicating that developing a novel therapeutic strategy is an emergent issue [ 29 , 30 ]. Recently, our group reported that dual immune checkpoint blockade of anti-PD-L1 (P1) and anti-CTLA-4 (C4) antibodies combined with single delivery of 10 Gy X-ray irradiation enhanced the systemic antitumor response both at local and distant sites in murine osteosarcoma [ 31 ].…”

Section: Introductionmentioning

confidence: 99%

Radiation Dose Escalation Is Crucial in Anti-CTLA-4 Antibody Therapy to Enhance Local and Distant Antitumor Effect in Murine Osteosarcoma

Takenaka

Takahashi

Tamari

et al. 2020

Cancers

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We previously reported that a combination of 10 Gy of X-ray irradiation and dual immune checkpoint blockade with anti-CTLA-4 (C4) and anti-PD-L1 antibodies produced a significant shrinkage of irradiated and unirradiated tumors (abscopal effect) and prolonged overall survival. However, the optimal radiation delivery regimen combined with single immune checkpoint blockade of C4 for inducing a maximum systemic antitumor response still remains unclear, particularly for patients with osteosarcoma. We used syngeneic C3H mice that were subcutaneously injected with LM8 osteosarcoma cells into both legs. C4 was administered three times, and one side of the tumor was irradiated by X-ray beams. The optimal radiation dose required to induce the abscopal effect was explored with a focus on the induction of the type-I interferon pathway. Radiation delivered in a single fraction of 10 Gy, 4.5 Gy × 3 fractions (fx), and 2 Gy × 8 fx with C4 failed to produce significant inhibition of unirradiated tumor growth compared with monotherapy with C4. Dose escalation to 16 Gy in a single fraction, or the equivalent hypofractionated dose of 8 Gy × 3 fx, which significantly increased secretion of IFN-β in vitro, produced a dramatic regression of both irradiated and unirradiated tumors and prolonged overall survival in combination with C4. Furthermore, irradiation at 16 Gy in both a single fraction and 8 Gy × 3 fx diminished regulatory T cells in the unirradiated tumor microenvironment. These results suggest that total dose escalation of radiation is crucial in C4 therapy to enhance the antitumor response in both local and distant tumors and prolonged overall survival regardless of fractionation for osteosarcoma.

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“…. Results of one latest study shows that primary OS cells are resistant to methotrexate (MTX) treatment and MTX does not alter Sox-2 and OCT-4 expression, which are used to identify OSC from OS [12], suggesting OSC is resistant to MTX. Studies have been performed to explore the underlying mechanism of OSC resistant to chemotherapy.…”

mentioning

confidence: 99%

Untargeted LC–MS/MS analysis reveals metabolomics feature of osteosarcoma stem cell response to methotrexate

et al. 2020

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Background: Cancer stem cell (CSC) is identified in osteosarcoma (OS) and considered resistant to chemotherapeutic agents. However, the mechanism of osteosarcoma stem cell (OSC) resistant to chemotherapy remains debatable and vague, and the metabolomics feature of OSC is not clarified. Materials and methods: OSC was isolated by using sphere forming assay and identified. Untargeted LC-MS/MS analysis was performed to reveal the metabolomics feature of OSC and underlying mechanisms of OSC resistant to methotrexate (MTX). Results: OSC was efficiently isolated and identified from human OS 143B and MG63 cell lines with enhanced chemoresistance to MTX. The untargeted LC-MS analysis revealed that OSC showed differential metabolites and perturbed signaling pathways, mainly involved in metabolisms of fatty acid, amino acid, carbohydrate metabolism and nucleic acid. After treated with MTX, metabolomics feature of OSC was mainly involved metabolisms of amino acid, fatty acid, energy and nucleic acid. Moreover, compared with their parental OS cells response to MTX, the differential metabolites and perturbed signaling pathways were mainly involved in metabolism of amino acid, fatty acid and nucleic acid. What's more, Rap1 signaling pathway and Ras signaling pathway were involved in OS cells and their SCs response to MTX. Conclusion: Sphere-forming assay was able to efficiently isolate OSC from human OS cell lines and the untargeted LC-MS/MS analysis was suggested a sufficient methodology to investigate metabolomics features of OS cells and OSCs. Moreover, the metabolomics features of OSCs response to MTX might reveal a further understanding of chemotherapeutic resistance in OS.

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Patient-derived osteosarcoma cells are resistant to methotrexate

Cited by 11 publications

References 35 publications

In-Vitro and In-Vivo Establishment and Characterization of Bioluminescent Orthotopic Chemotherapy-Resistant Human Osteosarcoma Models in NSG Mice

In-Vitro and In-Vivo Establishment and Characterization of Bioluminescent Orthotopic Chemotherapy-Resistant Human Osteosarcoma Models in NSG Mice

Radiation Dose Escalation Is Crucial in Anti-CTLA-4 Antibody Therapy to Enhance Local and Distant Antitumor Effect in Murine Osteosarcoma

Untargeted LC–MS/MS analysis reveals metabolomics feature of osteosarcoma stem cell response to methotrexate

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