Objectives Melanoma skin cancer is an aggressive tumour, whose incidence has been steadily increasing over the last 50 years, now representing 3% of total tumours. Despite the new immunomodulating therapies, the prognosis of metastatic melanoma continues to be very unfavorable, so that it is necessary to develop further therapeutic options. The overall goal of this project is to establish new advanced immune-competent 3D human melanoma models, as useful and more predicting tools for preclinical studies. In particular, we focused on a 3D skin equivalent melanoma model, where melanoma cells are co-seeded with primary keratinocytes, which in turn form a fully stratified epithelium on a fibroblasts-enriched dermal compartment. Methods We established two different 3D human melanoma models: 1. A commercially available model, containing A375 melanoma cells (Mattek, USA), was used to test the anti-proliferative effect of innovative drug-loaded nanoparticles (HNE-NCs). This model is able to recapitulate the main melanoma progression stages occurring in vivo: the radial, the vertical, and the metastatic growth phases. We performed two diverse treatments, one in the medium, mimicking the parenteral administration, and the other onto the epidermal surface, mimicking the topical treatment. IHC analysis was performed in order to examine proliferation index (Ki67) and tumor invasivity (S100). 2. A home-made model, where pools of normal human Keratinocytes (KCs), derived from neonatal foreskins, were stratified onto a collagen-based dermis enriched with primary pooled human fibroblasts (HDFs). We assessed this novel 3D melanoma model not only with commercially available A375 cells, but also with two primary melanoma cells that we have previously isolated from patients derived melanoma skin tumours. In vitro 3D tissues were morphologically characterized by H&E staining; IHC analysis (Ki67 and S100) was also performed. Results We proved the anti-proliferative effect of our innovative drug-loaded nanoparticles (HNE-NCs) versus the growth of the commercially available melanoma model, as demonstrated by morphological (H&E) and IHC analysis (Ki67 and S100 markers). Interestingly, we also obtained encouraging results with the topic administration. We observed that the different tissue morphology and proliferation/invasion markers expression of the home-made 3D models, correlate with the diverse malignant features of patients derived primary melanoma cells. Conclusions Our engineered 3D human skin melanoma models can be suitable for cancer progression studies, targets and drugs discovery and development. The use of patients derived cells will allow achieving personalized cancer treatments. Moreover, the insertion of immune cells of the patients also inside the models could pave the way towards the study of immune-modulating drugs as well.
3D advanced melanoma models
DIANZANI, Chiara;CAVALLI, Roberta;CASTELLANO, ISABELLA;ANNARATONE, LAURA;RIBERO, Simone;RUSSO, ROSALIA;SENETTA, REBECCA;DAGA, MARTINA;BARRERA, Giuseppina;PIZZIMENTI, Stefania
2016-01-01
Abstract
Objectives Melanoma skin cancer is an aggressive tumour, whose incidence has been steadily increasing over the last 50 years, now representing 3% of total tumours. Despite the new immunomodulating therapies, the prognosis of metastatic melanoma continues to be very unfavorable, so that it is necessary to develop further therapeutic options. The overall goal of this project is to establish new advanced immune-competent 3D human melanoma models, as useful and more predicting tools for preclinical studies. In particular, we focused on a 3D skin equivalent melanoma model, where melanoma cells are co-seeded with primary keratinocytes, which in turn form a fully stratified epithelium on a fibroblasts-enriched dermal compartment. Methods We established two different 3D human melanoma models: 1. A commercially available model, containing A375 melanoma cells (Mattek, USA), was used to test the anti-proliferative effect of innovative drug-loaded nanoparticles (HNE-NCs). This model is able to recapitulate the main melanoma progression stages occurring in vivo: the radial, the vertical, and the metastatic growth phases. We performed two diverse treatments, one in the medium, mimicking the parenteral administration, and the other onto the epidermal surface, mimicking the topical treatment. IHC analysis was performed in order to examine proliferation index (Ki67) and tumor invasivity (S100). 2. A home-made model, where pools of normal human Keratinocytes (KCs), derived from neonatal foreskins, were stratified onto a collagen-based dermis enriched with primary pooled human fibroblasts (HDFs). We assessed this novel 3D melanoma model not only with commercially available A375 cells, but also with two primary melanoma cells that we have previously isolated from patients derived melanoma skin tumours. In vitro 3D tissues were morphologically characterized by H&E staining; IHC analysis (Ki67 and S100) was also performed. Results We proved the anti-proliferative effect of our innovative drug-loaded nanoparticles (HNE-NCs) versus the growth of the commercially available melanoma model, as demonstrated by morphological (H&E) and IHC analysis (Ki67 and S100 markers). Interestingly, we also obtained encouraging results with the topic administration. We observed that the different tissue morphology and proliferation/invasion markers expression of the home-made 3D models, correlate with the diverse malignant features of patients derived primary melanoma cells. Conclusions Our engineered 3D human skin melanoma models can be suitable for cancer progression studies, targets and drugs discovery and development. The use of patients derived cells will allow achieving personalized cancer treatments. Moreover, the insertion of immune cells of the patients also inside the models could pave the way towards the study of immune-modulating drugs as well.
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