Replacing in vivo skin cancer studies by tumor organoids

Preclinical cancer research has relied on animal models and on the analysis of immortalized two-dimensional (2D) cancer cell lines in vitro for decades. Although animal models are time- consuming and costly, and cancer cell lines may not recapitulate the pathophysiological features of the original tumor, these two approaches have tremendously contributed to our understanding of cancer development and progression, and have led to a multitude of different cancer therapies. In recent years novel 3D cancer organoid cultures have been developed, which combine the experimental feasibility and tractability of cancer cell lines with the complex cellular heterogeneity of in vivo model systems. These patient-derived cancer organoids (PDOs) are excellent in vitro models that precisely recapitulate the pathophysiological nature of tumorigenesis. Cancer organoids have been established from various types of cancers, however not from human cutaneous squamous cell carcinoma (cSCC). Many of these PDOs were generated by submerged culture, which typically solely involves epithelial cells. We will employ airliquid interface (ALI) culture methodology to generate PDOs that include epithelial cells alongside integrated stroma and immune cells, thereby allowing to assess the contribution of the tumor stroma to cancer growth and treatment response. Specifically, we aim at establishing cancer organoids from human cSCC biopsies comprising both cancer and stroma cells (i) to replace in vivo skin cancer studies in animals, (ii) to develop an in vitro drug screening pipeline for novel targets as well as for the development of personalized cancer treatments, and (iii) to dissect the interactions of distinct cancer- associated fibroblast (CAF) subsets with tumor and immune cells, since single-cell RNA sequencing (scRNA-Seq) predicts distinct functions for three CAF subsets in cSCC according to their expression profiles. Intriguingly, one CAF subset might affect immune surveillance and the response to immunotherapy, which we aim at dissecting in PDOs. Maintaining the cellular heterogeneity of the original tumor provides a unique opportunity to dissect complex cancer cell-stroma interactions, and to advance cancer research and precision medicine.