Influence of BTK on EMT, CSC enrichment and immune system/tumor micromilieu in HNSCC

Background Constitutive active kinases are key players in carcinogenesis and the inhibition of these kinases are widely used for molecular tumor therapy. Recently, we identified Bruton’s Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC). To further investigate the function of BTK for tumorgenesis we tested 3 HNSCC cell lines in vitro for their capability to metastasize, further differentiate and modulate immune system via influencing tumor micromilieu. M&M: 2 different 3D culture systems were used: Cells as colonies in Matrigel and as floating spheres on ultra low attachment surface plates (ULAs). Cells were cultured for 72 hours after treatment with BTK-inhibitor (AVL-292) or control. Markers associated with epithelial-mesenchymal-transition (EMT), immune checkpoint molecules (BTLA, CTLA4, PD-1) and Cancer-Stem-Cells (CSC) were analyzed via Western blotting and flow cytometry. Cytokines (IFNg, TNFα, IL6) were determined using ELISA assays. ERK and NF-kB activation were analyzed.

Results ALDH1A1+ and CD44+/CD24- cells were defined as CSC. ALDH1A1+ cells decreased after BTK inhibition. CD44+/CD24- cells showed altered mean fluorescent intensities (MFI) after culturing on ULAs or in Matrigel. Different trends in the expression of EMT markers (vimentin, E-cadherin, N-cadherin, snail, slug, twist) were observed after BTK inhibition. We found a significant reduction of cytokine release by tumor cells. In contrast the expression of immune checkpoint molecules was not altered. We found a significant reduction of (un)phosphorylated NF-kB levels after BTK inhibition. ERK pathway remained unaffected.

Conclusion BTK plays an important role in HNSCC in terms of EMT and CSC enrichment, affects NF-kB signaling pathway and cytokine milieu (microenvironment) in HNSCC cells.

Publikationsverlauf

Artikel online veröffentlicht:
12. Mai 2023