Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models

Abstract

Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients’ care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.

Review of the Tumor Microenvironment in Basal and Squamous Cell Carcinoma

“…Thus, about 75% of all deaths due to skin cancer are caused by SCC [33]. The development of SCC is gradual, stemming from an uncontrolled proliferation of epidermal keratinocytes and progression through dysplasia and actinic keratosis as the tumor cells accumulate genetic mutations in genes such as TP53, CDKN21, NOTCH, and RAS [33,34]. During tumorigenesis, the cancer cells enable growth, angiogenesis, and metastasis through autocrine and paracrine signaling [35].…”

3D modeling of normal skin and cutaneous squamous cell carcinoma. A comparative study in 2D cultures, spheroids, and 3D bioprinted systems

“…Moreover, in the absence of fibroblasts, HaCaT skin keratinocytes tend to form a thin layer of epithelium and subsequently lose their proliferative capacity, as demonstrated in a study by Stark et al [20]. Therefore, the approach presented here contributes significantly to the development of in vitro 3D models for studying normal skin development, including the formation of the epidermis and the dermal layer, and regeneration, and investigating tumor cell organization and drug response [14,21,22].…”

“…However, many existing 3D tumor models lack diversity in cell types, limiting their ability to represent the complex tumor microenvironment accurately. Currently available models of cSCC include 2D and 3D tissue cultures, syngeneic and transgenic mouse models, as well as xenotransplants derived from cell lines and patient-derived xenografts [14]. To date, only one 3D tricellular cSCC model has been described, posing an important limitation owing to the lack of comparisons to other models [15,16].…”

Ex vivo therapeutic screening of metastatic cSCC: A review of methodological considerations for clinical implementation

“…lymph nodes) or distant metastatic tissue. These models have been reviewed extensively elsewhere 15 . Briefly, established cell lines derived from primary cSCC have been cultured as monolayers, spheroids, and as part of more complex organotypic (skin equivalent) cultures comprised of tumour cells seeded atop fibroblast‐contracted collagen I matrices.…”

“…Direct functional testing of therapeutics on patient‐derived disease models may circumvent these limitations. Existing patient‐derived models of metastatic cSCC have facilitated the identification of candidate therapeutics, 13–15 but these models lack both precision medicine capability and key features that govern therapeutic sensitivity. Short‐term ex vivo culture could overcome these limitations to permit superior recapitulation of in vivo tumours with patient‐specific detail as has been achieved for other solid tumours including melanoma and oral squamous cell carcinoma 16,17 .…”