Periodic Reporting for period 1 - SkinModelOma (Multicompartment combinatory pre-clinical model for melanoma drug screening)
Reporting period: 2023-06-01 to 2025-05-31
Cutaneous melanoma is one of the most aggressive skin cancers, and its treatment development continues to face significant barriers due to limitations in current preclinical models. Traditional in vivo animal testing, while common, often lacks translational relevance due to the fundamental biological differences between human and animal skin. Furthermore, ethical and regulatory pressures aligned with the 3Rs principles (Replacement, Reduction, Refinement) are increasingly calling for alternative models that reduce animal use. In this context, there is an urgent need for innovative, human-relevant platforms that can bridge the gap between early-stage research and clinical application.
This project proposes the development of SkinModelOma, an ex vivo multicompartmental melanoma graft model designed to mimic human melanoma more accurately. It involves grafting a multicellular 3D spheroid—composed of melanoma cells, keratinocytes, fibroblasts, and monocytes—into human skin tissue. By integrating multiple human skin components and immune cells into a single, living structure, SkinModelOma aims to replicate the complex tumor microenvironment of melanoma more faithfully than any current model.
Through extensive histological and ultrastructural characterization, this model has already demonstrated features resembling in vivo melanoma lesions, such as extracellular matrix deposition, cell clustering, immune cell distribution, and proliferation markers. The successful development and grafting of these spheroids have enabled tissue integration and analysis of tumor-like behavior without the need for live animal models.
Critically, SkinModelOma accounts for biological diversity by incorporating age, gender, and skin type variability, factors that are rarely considered in preclinical testing but are essential for developing personalized therapies. From a strategic and political perspective, this project aligns with the European Union’s commitment to animal welfare, precision medicine, and gender-sensitive research. The development of a validated, human-derived melanoma model has the potential to significantly reduce animal testing, streamline the drug development process, and ultimately deliver more effective therapies to patients faster.
In terms of impact, the project is expected to: (i) Provide a reliable alternative to animal models for melanoma drug testing; (ii) Enhance the predictive accuracy of preclinical studies; (iii) Facilitate the screening of personalized therapies based on human skin diversity; (iv) Strengthen Europe’s leadership in 3D tissue engineering and ethical innovation in biomedicine. Moreover, by promoting an inclusive and human-centered approach to innovation, SkinModelOma stands to make a lasting contribution to both science and society.
This project proposes the development of SkinModelOma, an ex vivo multicompartmental melanoma graft model designed to mimic human melanoma more accurately. It involves grafting a multicellular 3D spheroid—composed of melanoma cells, keratinocytes, fibroblasts, and monocytes—into human skin tissue. By integrating multiple human skin components and immune cells into a single, living structure, SkinModelOma aims to replicate the complex tumor microenvironment of melanoma more faithfully than any current model.
Through extensive histological and ultrastructural characterization, this model has already demonstrated features resembling in vivo melanoma lesions, such as extracellular matrix deposition, cell clustering, immune cell distribution, and proliferation markers. The successful development and grafting of these spheroids have enabled tissue integration and analysis of tumor-like behavior without the need for live animal models.
Critically, SkinModelOma accounts for biological diversity by incorporating age, gender, and skin type variability, factors that are rarely considered in preclinical testing but are essential for developing personalized therapies. From a strategic and political perspective, this project aligns with the European Union’s commitment to animal welfare, precision medicine, and gender-sensitive research. The development of a validated, human-derived melanoma model has the potential to significantly reduce animal testing, streamline the drug development process, and ultimately deliver more effective therapies to patients faster.
In terms of impact, the project is expected to: (i) Provide a reliable alternative to animal models for melanoma drug testing; (ii) Enhance the predictive accuracy of preclinical studies; (iii) Facilitate the screening of personalized therapies based on human skin diversity; (iv) Strengthen Europe’s leadership in 3D tissue engineering and ethical innovation in biomedicine. Moreover, by promoting an inclusive and human-centered approach to innovation, SkinModelOma stands to make a lasting contribution to both science and society.
The project was structured into three main technical phases: (1) development of the multicellular 3D spheroid, (2) grafting and integration of the spheroid into human skin tissue, and (3) validation of the ex vivo melanoma model (SkinModelOma) as a platform for therapeutic screening.
1. Development and Characterization of Multicellular Spheroids
- Monoculture Spheroid (A375 melanoma cells): Successfully established spheroids with an optimal size (~400 µm), compact structure, high sphericity (>0.95) and increasing metabolic activity over time.
- Co-culture Optimization: Melanoma + Keratinocytes: Identified 2:1 as the optimal cell ratio, maintaining structural integrity and allowing future therapeutic access.
- Triple and Quadruple Co-culture (A375 + HaCaT + HDF ± Monocytes): Achieved structurally stable spheroids with high sphericity and biologically relevant architecture, including cellular clustering and enhanced extracellular matrix (ECM) production.
Structural Characterization: (i) Histological and immunohistochemical analyses (H&E, Masson's Trichrome, Ki-67, S-100) revealed proliferative zones, collagen/keratin deposition specially for triple and quadruple conditions, and melanoma-related protein (S100) high expressed also by the triple and quadruple conditions; (ii) Electron microscopy and advanced 3D imaging confirmed ultrastructural features, including intense lipid droplets secretion, tight cellular junctions, and spatial organization of cell populations; (iii) Fibroblasts showed morphological changes suggestive of differentiation toward cancer-associated fibroblasts (CAFs), and monocytes localized peripherally, indicating possible immune compartmentalization.
2. Spheroid Grafting into Ex Vivo Human Skin
Initial Grafting Attempts:
- Topical deposition and surgical incision methods failed to integrate spheroids into skin due to the barrier function of the stratum corneum and poor healing kinetics, respectively.
- Injection Method Standardization: Optimized injection parameters including volume, depth, and needle type.
Histological analysis post-injection confirmed successful deposition of spheroids within the dermis and observed skin responses such as fibrosis, tumor spread, spongiosis, and hyperkeratosis over time.
3. Nanomedicine Screening and Model Validation
- Nanoparticle Selection: Evaluated cytotoxicity and uptake efficiency via resazurin assay and flow cytometry, selecting candidates with high cellular uptake and low toxicity.
- Tissue Biocompatibility and Drug Delivery: Applied nanoparticles to the grafted skin model and tracked tissue viability.
Immunofluorescence analysis confirmed nanoparticle penetration and their distribution within the tissue.
Topical application versus intradermal injection: topically the drug release is intense into the epidermis with a minimal drug content surpassing the dermis, on the other hand intradermal injection helps the drug to be released in the dermis with some drug being detected into the receptor (basolateral) compartment.
- Efficacy Evaluation: Final phase involved assessing the therapeutic impact of selected nanomedicines on tumor growth and progression in the ex vivo graft model;
Notable changes included reduction in proliferation markers, structural disintegration of tumor spheroids, and tissue remodeling, demonstrating the model's applicability for therapeutic evaluation.
1. Development and Characterization of Multicellular Spheroids
- Monoculture Spheroid (A375 melanoma cells): Successfully established spheroids with an optimal size (~400 µm), compact structure, high sphericity (>0.95) and increasing metabolic activity over time.
- Co-culture Optimization: Melanoma + Keratinocytes: Identified 2:1 as the optimal cell ratio, maintaining structural integrity and allowing future therapeutic access.
- Triple and Quadruple Co-culture (A375 + HaCaT + HDF ± Monocytes): Achieved structurally stable spheroids with high sphericity and biologically relevant architecture, including cellular clustering and enhanced extracellular matrix (ECM) production.
Structural Characterization: (i) Histological and immunohistochemical analyses (H&E, Masson's Trichrome, Ki-67, S-100) revealed proliferative zones, collagen/keratin deposition specially for triple and quadruple conditions, and melanoma-related protein (S100) high expressed also by the triple and quadruple conditions; (ii) Electron microscopy and advanced 3D imaging confirmed ultrastructural features, including intense lipid droplets secretion, tight cellular junctions, and spatial organization of cell populations; (iii) Fibroblasts showed morphological changes suggestive of differentiation toward cancer-associated fibroblasts (CAFs), and monocytes localized peripherally, indicating possible immune compartmentalization.
2. Spheroid Grafting into Ex Vivo Human Skin
Initial Grafting Attempts:
- Topical deposition and surgical incision methods failed to integrate spheroids into skin due to the barrier function of the stratum corneum and poor healing kinetics, respectively.
- Injection Method Standardization: Optimized injection parameters including volume, depth, and needle type.
Histological analysis post-injection confirmed successful deposition of spheroids within the dermis and observed skin responses such as fibrosis, tumor spread, spongiosis, and hyperkeratosis over time.
3. Nanomedicine Screening and Model Validation
- Nanoparticle Selection: Evaluated cytotoxicity and uptake efficiency via resazurin assay and flow cytometry, selecting candidates with high cellular uptake and low toxicity.
- Tissue Biocompatibility and Drug Delivery: Applied nanoparticles to the grafted skin model and tracked tissue viability.
Immunofluorescence analysis confirmed nanoparticle penetration and their distribution within the tissue.
Topical application versus intradermal injection: topically the drug release is intense into the epidermis with a minimal drug content surpassing the dermis, on the other hand intradermal injection helps the drug to be released in the dermis with some drug being detected into the receptor (basolateral) compartment.
- Efficacy Evaluation: Final phase involved assessing the therapeutic impact of selected nanomedicines on tumor growth and progression in the ex vivo graft model;
Notable changes included reduction in proliferation markers, structural disintegration of tumor spheroids, and tissue remodeling, demonstrating the model's applicability for therapeutic evaluation.
The SkinModelOMA project presents a groundbreaking advance in melanoma research by developing the first immune-competent, patient-relevant ex vivo 3D melanoma model that faithfully simulates the tumor microenvironment (TME) in human skin. Through an integrated series of scientific work packages and milestones, the project successfully established a multicellular, multicompartmental graft model that combines tumor biology, skin tissue engineering, and nanomedicine screening in a single, modular platform.
SkinModelOMA introduces several results that go beyond the state of the art:
- First ex vivo immune-competent melanoma skin model, incorporating patient-derived components and immune cells;
- Allows Rapid, high-throughput spheroid generation, completed within seven days, compatible with clinical workflows;
- Extensive biological validation, using multimodal imaging, immunohistochemistry, and ultrastructural analysis;
- Personalization potential, through modular design adaptable to different skin types, ages, and genders;
- Ethically aligned, offering a robust alternative to animal testing and supporting the EU’s 3Rs principles.
The model’s fidelity to human biology, combined with its adaptability and scalability, makes it a powerful platform for functional precision oncology, drug development, and translational research. It also opens the door to future exploration of angiogenesis, immunotherapy responses, tumor resistance mechanisms, and personalized medicine approaches.
By delivering a clinically relevant, ethically sound, and scientifically advanced tool, SkinModelOMA contributes significantly to the future of cancer research and therapeutic innovation.
SkinModelOMA introduces several results that go beyond the state of the art:
- First ex vivo immune-competent melanoma skin model, incorporating patient-derived components and immune cells;
- Allows Rapid, high-throughput spheroid generation, completed within seven days, compatible with clinical workflows;
- Extensive biological validation, using multimodal imaging, immunohistochemistry, and ultrastructural analysis;
- Personalization potential, through modular design adaptable to different skin types, ages, and genders;
- Ethically aligned, offering a robust alternative to animal testing and supporting the EU’s 3Rs principles.
The model’s fidelity to human biology, combined with its adaptability and scalability, makes it a powerful platform for functional precision oncology, drug development, and translational research. It also opens the door to future exploration of angiogenesis, immunotherapy responses, tumor resistance mechanisms, and personalized medicine approaches.
By delivering a clinically relevant, ethically sound, and scientifically advanced tool, SkinModelOMA contributes significantly to the future of cancer research and therapeutic innovation.