EMT transcriptional response are triggered in response to laser photoablation in 3D models of melanoma

Jun 30, 2022, 12:20 PM
10m
Room: S4 C

Room: S4 C

Speaker

Rodrigues, Daniel (3B's Research Group )

Description

Introduction: Lasers have been used for years in the field of cancer therapy. Nanosecond pulsed lasers, in particular, have been used in the generation of reactive oxygen species via plasma for the triggering of immunogenic cell death[1][2], for the delivery of biomolecules intracellularly via optoporation[3] and for tumor resection[4]. Notwithstanding this large range of uses, the full impact of nanosecond pulsed lasers on cellular mechanisms is not fully understood. Their effects on epithelial to mesenchymal transition (EMT), for instance, are unknown. While being a key mechanism in embryogenesis and wound healing, EMT has also been associated to tumor progression, invasion and resistance through the action of players like the transforming growth factor beta (TGF-β) family.
Here, the putative effects of nanosecond pulsed laser ablation on the expression of known EMT players in melanoma spheroids models are studied.
Methodology: Multicellular spheroids comprising the human melanoma cell line VMM-15 and human dermal fibroblasts (hDFbs) were produced while using monoculture spheroids of each cellular type as controls. Cellular aggregation was allowed to occur over 7 days, at which time partial ablation of the spheroids using a nanosecond 355nm laser was performed. Ablated spheroids were collected after allowed to recover for 3 hours or 3 days. Several key properties of the ablated spheroids where subject to evaluation. Morphology recovery was assessed using time-lapse microscopy. Gene and protein expression were analyzed by Real-Time PCR, Western Blot and immunohistochemistry.
Results: Ablated spheroids displayed variable angular openings of the wound surface, with greater values being verified for the multicellular spheroids. Laser ablation triggered an up-regulation of the gene expression of EMT mediator TGFβ1 across all conditions, which was also verified for its receptor TBFβ-R1. Canonical pathway transducer pSMAD2/3 presented a higher protein expression post-ablation. Additionally, increased gene expression was verified for PLOD2 which correlated with the increase of Col1 protein confirmed through western-blot. Further analysis of EMT players showed a decrease in the expression of epithelial marker E-Cadherin after ablation recovery, while an increase in the expression of mesenchymal marker N-Cadherin and ZEB1 was observed when compared to control. Gene expression of stem cell master regulators SOX2, OCT4 and NANOG was shown to be overexpressed in melanoma spheroids after laser ablation.
Conclusions: It was demonstrated that laser ablation triggers a phenotypical shift from an epithelial state into a mesenchymal state in melanoma cell spheroids mediated by the expression of TGF-β1. This was further reflected in the increased expression of key stem cell regulators linked to stemness. These results strongly suggest that nanosecond pulsed laser ablation is capable of promoting EMT in determined conditions.

[1] A. Lin, B. et al., Int. J. Mol. Sci. 2017, 18, 966.
[2] H. T. M. Nguyen et al., Nanoscale 2021, 13, 3644.
[3] P. Gupta et al., Analyst 2021, 146, 4756.
[4] J. Hornef et al., Sci. Rep. 2020, 10, 5122.

Acknowledgments: FCT/MCTES through the grants SFRH/BD/119756/2016 and IF/00347/2015 and EU Horizon 2020 research and innovation programme under the ERC grant CapBed (805411).

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