Optimization of a Human Tumor-Derived 3D Extracellular Matrix with In Silico and In Vitro Validation of Synergistic Targeted Inhibitors in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), is characterized by high rates of recurrence, therapeutic resistance, and poor survival, underscoring the need for physiologically relevant preclinical models and novel therapeutic approaches. Myogel, a human tumor-derived extracellular matrix from uterine leiomyoma, provides a biologically relevant platform for 3D cancer modelling and more accurately recapitulates the human tumor microenvironment than conventional animal-derived systems. However, residual donor DNA and reliance on frozen storage may limit its standardization and broader applicability. In HNSCC and OSCC, BCL-XL and MCL-1 are co-dependent, key anti-apoptotic proteins whose compensatory and functionally redundant activity may drive resistance to single-agent inhibition, making dual targeting a relevant therapeutic strategy for effective apoptotic priming. In this thesis, Myogel was optimized by DNase I-mediated DNA depletion and lyophilization, and its functional performance was validated to establish a preclinical platform for 3D cancer modelling. The optimized matrix was then used to assess the anti-invasive effects of the BCL-XL inhibitor A-1331852 and the MCL-1 inhibitor MIK665, identified in an in-house high-throughput screen of patient-derived OSCC cell lines as the most synergistic combination, in HNSCC cell lines HSC-3 and UT-SCC-42A using spheroid invasion models. DNase I treatment (100 U/mL, 1 h) reduced residual DNA below the qPCR quantification limit, while automated capillary electrophoresis confirmed near-complete degradation of high-molecular-weight genomic DNA. Functional assays demonstrated that the optimized Myogel matrices retained biological performance comparable to untreated Myogel, as shown as shown by preserved HSC-3 cell viability on Myogel treated with different DNase I concentrations and maintained spheroid invasion capacity in both DNase-treated and DNase-treated lyophilized Myogel in HSC-3 and UT-SCC-42A spheroid models. In parallel, in silico ADMET profiling predicted favorable pharmacokinetic properties for both compounds, with no predicted hERG inhibition. In 3D spheroid invasion assays, combined A-1331852 and MIK665 treatment consistently produced the lowest mean invasion length across all Myogel conditions in both cell lines compared with single-agent treatments, although the differences did not reach statistical significance. These findings support DNase-treated Myogel, DNase-treated lyophilized Myogel as a DNA-depleted, functionally preserved human tumor-derived matrix for preclinical 3D cancer research and indicate that combined BCL-XL/MCL-1 inhibition warrants further mechanistic evaluation as a potential apoptosis-targeted therapeutic strategy in HNSCC models under optimized dosing conditions.