Laser ablation triggers EMT associated transcriptional response in melanoma tumor spheroids

Nanosecond pulsed lasers have become widely adopted in the field of cancer therapy, namely to generate reactive oxygen species via plasma for the triggering of immunogenic cell death^[1][2], to deliver biomolecules intracellularly via optoporation^[3] or for tumor ablation^[4]. Despite this range of applications, the full impact of nanosecond pulsed lasers on cellular mechanisms is not fully understood.   

Epithelial to mesenchymal transition(EMT) is a key process in embryogenesis and wound healing but its role in cancer is tied to progression, invasion and resistance through the action of players like the transforming growth factor beta(TGF-β) family.

Here we proceeded to evaluate the effects of nanosecond pulsed laser ablation in the expression of known EMT players in tumor spheroids. For this effect, spheroids comprising the human melanoma cell line VMM-15, human dermal fibroblasts(hDFbs) or a mixture of both where cultured for 7 days, at which time partial ablation of the spheroids using a nanosecond 355nm laser was performed. Ablated spheroids were collected either 3 hours or 3 days later. Morphology recovery was assessed using time-lapse microscopy. Gene and protein expression were analyzed by Real-Time PCR, Western Blot and immunohistochemistry.

Ablated spheroids displayed variable angular openings of the wound surface, with greater angular openings being verified for spheroids comprising both tumor cells and hDFbs. Wound closure was faster for spheroids containing fibroblasts, with Picrosirius red staining showing higher collagen deposition where laser ablation had been performed. Analyzing the gene expression of the EMT mediator TGFβ1 confirmed that laser ablation triggered an up-regulation of this gene across all conditions. In addition, increased gene expression for TGFβ1R, COL1α1, PLOD2, COL3α1 and FN-EDA was also observed. When looking into the expression of key proteins such as smooth muscle actin and phosphorylated SMAD 2/3, a tendency for higher expression upon laser ablation was confirmed.

Overall, the aforementioned results show that laser ablation triggers the upregulation of known EMT players in melanoma cell spheroids. Additionally, we have shown that the ablation recovery time is highly dependent on the presence of a fibroblast component within the spheroids which can be an effect of varying biomechanical cues due to differential extracellular matrix deposition.