CDK2 Depletion Impairs Tumour Growth in the 3D Luminal Breast Cancer Model via CRISPR-CAS9 Gene Editing

Abstract

Objective: Cyclin-Dependent Kinase 2 (CDK2) is a key regulator of the G1/S transition and genomic stability. While recent stud- ies have linked CDK2 activity to therapy resistance in hormone receptor-positive breast cancer, its functional role under tumour- relevant conditions remains unclear. This study aims to elucidate the context-specific requirement of CDK2 in luminal breast cancer models. Material and Methods: CDK2 was knocked out in T47D cells using CRISPR-Cas9. Phenotypic effects were evaluated in both 2D mono- layer and 3D spheroid cultures. Quantitative PCR validated gene knockout. Colony formation and GFP-based competition assays assessed 2D proliferation, while 3D spheroid size was quantified using ImageJ. Results: CDK2 depletion resulted in a 40% reduction in 2D colony formation and a 50% decrease in spheroid size. The fitness disad- vantage was more pronounced in 3D cultures, suggesting increased dependency on CDK2 in complex microenvironments. Conclusion: CDK2 plays a critical role in sustaining tumour growth and structural organization, particularly in 3D environments that mimic the tumour microenvironment. These findings highlight CDK2 as a potential therapeutic target in luminal breast cancer, especially in the context of anti-oestrogen resistance.