Advanced Cutaneous Squamous Cell Carcinoma: Drug Response Profiling, Development of Three-Dimensional Cell Culture Models, and Factors Governing Response to Immunotherapy

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and is a disease with globally rising incidence. Approximately 3–5% of patients with cutaneous squamous cell carcinoma develop advanced disease, accounting for roughly 1% of all cancer deaths in Australia. Advanced cutaneous squamous cell carcinoma comprises locally advanced cSCC (lacSCC) and metastatic cSCC (mcSCC), and includes large primary tumors, tumors with regional nodal metastasis, as well as distant metastatic disease. Due to the mortality and morbidity associated with advanced cSCC, proper risk stratification and early intervention are paramount. Additionally, novel treatment approaches are required for patients unresponsive to immune checkpoint inhibitors (ICI) or with innately resistant de novo primary skin cancers (DPSCs). A detailed overview of the current state of cSCC research and the context of this work is provided in Chapter I.

Epithelial-mesenchymal transition (EMT) is a driver of metastasis in many carcinomas, though its role as a potential biomarker for metastasis and therapeutic target remains unclear in cSCC. In Chapter II, a systematic overview of the clinical and experimental evidence for the role of EMT in cSCC is provided, with critical appraisal of type and quality of the methodology used. This information was then used as rationale for potential drug targets against advanced cSCC. All primary literature encompassing clinical and cell-based or xenograft experimental studies reporting on the role of EMT markers or related signaling pathways in the progression of cSCC were considered. From the clinical studies, a timeline illustrating the alteration of EMT markers and related signaling was evident based on clinical progression of the disease. The experimental studies reveal connections of EMT with a multitude of factors such as genetic disorders, cancerassociated fibroblasts (CAFs), and matrix remodeling via matrix metalloproteinases and urokinase plasminogen activator. Additionally, EMT was found to be closely tied to environmental factors as well as to stemness in cSCC via NFκB and β-catenin. It was concluded that EGFR, TGF-βR, PI3K/AKT and NFκB signaling are the four canonical signaling pillars that induce EMT in cSCC and could be valuable therapeutic targets.

In Chapter III, the use of small molecule PI3K inhibitors (PI3Ki) in combination with CDK inhibitors (CDKi) as well as the impact of PI3Ki on EMT markers was investigated against in-house cell line models of mcSCC. The use of this drug combination is based on the frequent dysregulation of cell cycle and PI3K signaling in lacSCC and mcSCC and the role of PI3K in governing EMT (Chapter II). A combination drug approach might overcome the underwhelming clinical performance of inhibitors targeting PI3K/AKT/mTOR or cell cycle signaling pathways. Cell viability, migration, cell signaling, cell cycle distribution, and apoptosis were assessed using two-dimensional (2D) and three-dimensional (3D, spheroid) culture models of our mcSCC cell lines, UW-CSCC1 and UW-CSCC2. Distinct advantages of 3D cell culture models include recapitulation oxygen, nutrient and drug diffusion which result in higher physiological relevance compared to 2D cell culture models. Some synergistic cytotoxic behavior of the combination was observed against 2D cell culture models of UW‑CSCC2, but not UW‑CSCC1; a relationship inverted in spheroid models. Synergistic combinations of CDKi and PI3Ki caused increased cell cycle arrest in G1 and G2, reduced motility, and efficiently induced apoptotic cell death. The differential responses correlated with driver gene mutation profiles of the cell lines. Overall, we provide sufficient evidence to support continued investigation of CDKi and PI3Ki drug combinations, particularly for tumors with mutations in cell cycle control genes.

In Chapter III, the differential drug responses in 3D spheroid compared to 2D models highlighted a need for more physiologically relevant cell culture models. Additionally, the literature review conducted in Chapter II yielded cancer-associated fibroblasts (CAFs) as another factor contributing to EMT in cSCC. Multi-cellular tumor spheroids (MCTS) have found widespread use in pre-clinical research. However, their complex 3D structure makes immunofluorescent staining and imaging challenging. Moreover, MCTS have not been established for la/mcSCC models. Thus, a methodology for reproducibly establishing such complex co-culture models for subsequent characterization was required. Chapter IV comprises a protocol that was developed for whole spheroid staining and automated imaging using laser-scanning confocal microscopy including detailed steps for cell culture, seeding of spheroids, transfer of MCTS for adhesion to multi-well chamber slides and fixation. Immunofluorescent staining based on optimized reagent concentrations and incubation times, and confocal imaging facilitated by glycerol-based optical clearing are then described. This methodology was then employed to investigate the potential effect CAF subtype has on cSCC EMT and invasion. For this purpose, MCTS containing mcSCC cell lines (which differed in EMT phenotype) and either dermal or lymph node-derived fibroblasts were utilized. The imaging technology developed and described above was used to showcase the unique spatial architecture of these MCTS, investigate the expression of EMT and CAF markers, and investigate the invasion of MCTS embedded in a collagen matrix. This showed for the first time that mcSCC/CAF-derived MCTS self-organize to form unique spatial architectures for each cell line combination according to the EMT phenotype of the cancer cell line used.

While the development of novel therapies against cSCC (Chapter III) and the expansion of in vivo models (Chapter IV) are important, gaining a better understanding of established therapies such as immunotherapy is warranted to ensure proper patient selection for optimal therapeutic outcome. Immunotherapy has demonstrated significant clinical benefit in advanced cSCC. However, there are limited data for patients treated outside of clinical trials which is particularly relevant in advanced cSCC most often seen in elderly patients with significant comorbidities. In Chapter V, we describe our experience with immunotherapy comprising immune checkpoint inhibitors (ICIs) Pembrolizumab and Cemiplimab in a cohort of patients with advanced cSCC in Australia. Among the 51 patients treated with immunotherapy, there was an objective response rate of 53% and disease control rate of 67%. The most significant predictor of response for this cohort was sex, with male patients more likely to have better responses compared to female patients as well as improved progression-free survival and overall survival. Differential expression analysis of immune-related genes demonstrated an impaired CD8 T-cell response in female patients proposing a potential mechanism for the worse patient outcome observed in female patients. Curiously, while receiving ICIs as part of the study, four patients developed new (up to 3) primary cSCC tumors (de novo primary skin cancers (DPSCs)) unrelated to the initial lesion. The DPSCs were analyzed for their expression of immune-related genes compared to a cohort of non-metastatic primary cSCC with similar clinicopathological characteristics. These investigations found evidence for significant changes in the immune-related transcriptome DPSCs when compared to treatment naïve primary cSCC that have never metastasized. Consequently, we propose further investigations on incidence and biological drivers of skin-associated DSPCs.

In conclusion, the work presented in this thesis consolidates previous research on EMT in cSCC (Chapter II), investigates PI3Ki as modulators of EMT in our patient-derived cell line models and presents a novel drug combination (of PI3Ki and CDKi) for further development (Chapter III). Additionally, a novel methodology for the characterization of MCTS was developed, utilized to show how CAFs impact EMT status of cSCC cells, and to resolve the unique spatial architecture of MCTS (Chapter IV). Lastly, the outcome of female patients with advanced cSCC treated with ICI is worse potentially due to a perturbed T-cell response (Chapter V). Overall, the findings presented as part of this work highlight the complexity of the disease and rationalize the need for further investigation to gain a better understanding of disease progression, improve existing treatment options, and develop effective novel treatments.