Evaluation of (<i>fli:GFP</i>) <i>Casper</i> Zebrafish Embryos as a Model for Human Conjunctival Melanoma

Pontes, Kelly Cristine de Sousa; Groenewoud, Arwin; Cao, Jinfeng; Ataíde, Lívia Maria Silva; Snaar‐Jagalska, Ewa; Jager, Martine J.

doi:10.1167/iovs.17-22023

Cited by 15 publications

(15 citation statements)

References 42 publications

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“…New treatments for CM are being actively looked for, for example by knocking down the over-expressed gene EZH2 in CM cell lines [10], using e.g., zebrafish xenograft models for faster and more predictive drug testing [11], and by CM-specific gene discovery for targeted therapy [12]. However, cross-talk between different signalling pathways makes specific gene targeting approaches quite challenging [13,14].…”

Section: Introductionmentioning

confidence: 99%

“…We hence developed a new orthotopic model for CM by RO injection of CM cells, mimicking primary tumour spread. We also investigated a previously-developed ectopic model, generated by intravenous cell injection: circulating cancer cells usually form tumour lesions in the tail of the embryo [11,42]. Using three different treatment modalities of TLD1433 in two different tumour models, we established a testing platform in which the anti-tumour efficacy of this PS can be observed.…”

Section: Introductionmentioning

confidence: 99%

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TLD1433 Photosensitizer Inhibits Conjunctival Melanoma Cells in Zebrafish Ectopic and Orthotopic Tumour Models

Chen

Ramu

Aydar

et al. 2020

Cancers

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The ruthenium-based photosensitizer (PS) TLD1433 has completed a phase I clinical trial for photodynamic therapy (PDT) treatment of bladder cancer. Here, we investigated a possible repurposing of this drug for treatment of conjunctival melanoma (CM). CM is a rare but often deadly ocular cancer. The efficacy of TLD1433 was tested on several cell lines from CM (CRMM1, CRMM2 and CM2005), uveal melanoma (OMM1, OMM2.5, MEL270), epidermoid carcinoma (A431) and cutaneous melanoma (A375). Using 15 min green light irradiation (21 mW/cm2, 19 J.cm−2, 520 nm), the highest phototherapeutic index (PI) was reached in CM cells, with cell death occurring via apoptosis and necrosis. The therapeutic potential of TLD1433 was hence further validated in zebrafish ectopic and newly-developed orthotopic CM models. Fluorescent CRMM1 and CRMM2 cells were injected into the circulation of zebrafish (ectopic model) or behind the eye (orthotopic model) and 24 h later, the engrafted embryos were treated with the maximally-tolerated dose of TLD1433. The drug was administrated in three ways, either by (i) incubating the fish in drug-containing water (WA), or (ii) injecting the drug intravenously into the fish (IV), or (iii) injecting the drug retro-orbitally (RO) into the fish. Optimally, four consecutive PDT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW/cm2, 114 J.cm−2, 520 nm). This PDT protocol was not toxic to the fish. In the ectopic tumour model, both systemic administration by IV injection and RO injection of TLD1433 significantly inhibited growth of engrafted CRMM1 and CRMM2 cells. However, in the orthotopic model, tumour growth was only attenuated by localized RO injection of TLD1433. These data unequivocally prove that the zebrafish provides a fast vertebrate cancer model that can be used to test the administration regimen, host toxicity and anti-cancer efficacy of PDT drugs against CM. Based on our results, we suggest repurposing of TLD1433 for treatment of incurable CM and further testing in alternative pre-clinical models.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

TLD1433 Photosensitizer Inhibits Conjunctival Melanoma Cells in Zebrafish Ectopic and Orthotopic Tumour Models

Chen

Ramu

Aydar

et al. 2020

Cancers

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“…As to melanoma, zebrafish is a powerful model in studying melanocyte biology, identifying novel disease genes, and discovering new therapeutics that regulate melanocyte and melanoma development (Lally et al, 2007;Ceol et al, 2008;Van Rooijen et al, 2017). In this study, microinjection of melanoma cells to the yolk sack of 2 dpf embryos, a well-established tumor cell xenograft zebrafish model (Van Der Ent et al, 2014;Saltari et al, 2016;Pontes et al, 2017), was used to determine the in vivo anti-melanoma activity of A B C FIGURE 3 | Shikonin inhibited melanoma cell migration and invasion. (A) A single scratch was created in the confluent monolayer of A375 or A2058 cells.…”

Section: Discussionmentioning

confidence: 97%

Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

Cao

Liu

Lai³

et al. 2020

Front. Pharmacol.

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Background: Malignant melanoma is an extremely aggressive and metastatic cancer, and highly resistant to conventional therapies. Signal transducer and activator of transcription 3 (STAT3) signaling promotes melanoma development and progression, which has been validated as an effective target in melanoma treatment. Natural naphthoquinone shikonin is reported to exert anti-melanoma effects. However, the underlying mechanisms have not been fully elucidated. Purpose: This study aims to evaluate the anti-melanoma activities of shikonin and explore the involvement of STAT3 signaling in these effects. Methods: Zebrafish tumor model was established to evaluate the anti-human melanoma effects of shikonin in vivo. MTT assay and colony formation assay were employed to investigate the anti-proliferative effects of shikonin on human melanoma A375 and A2058 cells. Flow cytometry was used to analyze cell cycle distribution and apoptosis induction. Wound healing assay and Transwell chamber assay were conducted to examine the cell migratory and invasive abilities. Immunofluorescence assay was used to observe F-actin, Tubulin, and STAT3 localization. Western blotting was used to determine the expression levels of proteins associated with apoptosis and key proteins in the STAT3 signaling pathway. Immunoblotting was performed in DSS cross-linked cells to determine the homo-dimerization of STAT3. Gelatin zymography was employed to evaluate the enzymatic activity of MMP-2 and MMP-9. Transient transfection was used to overexpress STAT3 in cell models. Results: Shikonin suppressed melanoma growth in cultured cells and in zebrafish xenograft models. Shikonin induced melanoma cells apoptosis, inhibited cell migration and invasion. Mechanistic study indicated that shikonin inhibited the phosphorylation and homo-dimerization of STAT3, thus reduced its nuclear localization. Further study showed that shikonin decreased the levels of STAT3-targeted genes Mcl-1, Bcl-2, MMP-2,

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“…Virus‐containing medium was replaced with fresh medium containing blasticidin‐S (2 μg/ml) to select transduced cells. Transduction of the cells with the tdTomato‐expressing virus did not alter the growth pattern of parental cells .…”

Section: Methodsmentioning

confidence: 93%

“…The (fli:GFP) Casper zebrafish embryos were maintained according to standard protocols (http://zfin.org, in the public domain) , in compliance with Dutch animal welfare regulations. The research followed the statement on the use of animals in research as published by the Association for Research in Vision and Ophthalmology and has been described previously . To test drug toxicity, non‐injected 3 days post‐fertilization (dpf) (fli:GFP) Casper zebrafish embryos were put into a 24‐well plate; each well contained 1 ml of egg water and different concentrations of GSK503.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of EZH2 in conjunctival melanoma offers a new therapeutic target

Cao

Pontes

Heijkants

et al. 2018

The Journal of Pathology

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Malignant melanoma of the conjunctiva (CM) is an uncommon but potentially deadly disorder. Many malignancies show an increased activity of the epigenetic modifier enhancer of zeste homolog 2 (EZH2). We studied whether EZH2 is expressed in CM, and whether it may be a target for therapy in this malignancy. Immunohistochemical analysis showed that EZH2 protein expression was absent in normal conjunctival melanocytes and primary acquired melanosis, while EZH2 was highly expressed in 13 (50%) of 26 primary CM and seven (88%) of eight lymph node metastases. Increased expression was positively associated with tumour thickness (p =0.03). Next, we targeted EZH2 with specific inhibitors (GSK503 and UNC1999) or depleted EZH2 by stable shRNA knockdown in three primary CM cell lines. Both pharmacological and genetic inactivation of EZH2 inhibited cell growth and colony formation and influenced EZH2‐mediated gene transcription and cell cycle profile in vitro. The tumour suppressor gene p21/CDKN1A was especially upregulated in CM cells after EZH2 knockdown in CM cells. Additionally, the potency of GSK503 against CM cells was monitored in zebrafish xenografts. GSK503 profoundly attenuated tumour growth in CM xenografts at a well‐tolerated concentration. Our results indicate that elevated levels of EZH2 are relevant to CM tumourigenesis and progression, and that EZH2 may become a potential therapeutic target for patients with CM. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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Evaluation of (fli:GFP) Casper Zebrafish Embryos as a Model for Human Conjunctival Melanoma

Cited by 15 publications

References 42 publications

TLD1433 Photosensitizer Inhibits Conjunctival Melanoma Cells in Zebrafish Ectopic and Orthotopic Tumour Models

TLD1433 Photosensitizer Inhibits Conjunctival Melanoma Cells in Zebrafish Ectopic and Orthotopic Tumour Models

Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin

Overexpression of EZH2 in conjunctival melanoma offers a new therapeutic target

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