Head and neck squamous cell carcinoma (HNSCC) undergoes stepwise progression from normal tissues to precancerous leukoplakia, primary HNSCC, and metastasized tumors. To delineate the heterogeneity of tumor cells and their interactions during the progression of HNSCC, we employ single-cell RNA-seq profiling for normal to metastasized tumors. We can identify the carcinoma in situ cells in leukoplakia lesions that are not detected by pathological examination. In addition, we identify the cell type subsets of the Galectin 7B (LGALS7B)-expressing malignant cells and CXCL8-expressing fibroblasts, demonstrating that their abundance in tumor tissue is associated with unfavorable prognostic outcomes. We also demonstrate the interdependent ligand-receptor interaction of COL1A1 and CD44 between fibroblasts and malignant cells, facilitating HNSCC progression. Furthermore, we report that the regulatory T cells in leukoplakia and HNSCC tissues express LAIR2, providing a favorable environment for tumor growth. Taken together, our results update the pathobiological insights into cell-cell interactions during the stepwise progression of HNSCCs.

Recapitulating the in vivo microenvironments of damaged tissues through modulation of the physicochemical properties of scaffolds can boost endogenous regenerative capacity. A series of critical events in tissue healing including immune-responses, angiogenesis, and stem cell homing and differentiation orchestrate to relay the regeneration process. Herein, we report hierarchically structured ('microchanneled') 3D printed scaffolds (named 'μCh'), in contrast to conventional 3D printed scaffolds, induce such cellular responses in a unique way that contributes to accelerated tissue repair and remodeling. The μCh reduced the extracellular trap formation of anchored neutrophils at the very beginning (24 h) of implantation while increasing the number of live cells. Among the macrophages covered the surface of μCh over 7 days a major population polarized toward alternativelly activated phase (M2) which contrasted with control scaffolds where classically activated phase (M1) being dominant. The mesenchymal stem cells (MSCs) recruited to the μCh were significantly more than those to the control, and the event was correlated with the increased level of stem cell homing cytokine, stromal derived factor 1 (SDF1) sequestered to the μCh. Furthermore, the neo-blood vessel formation was more pronounced in the μCh, which was in line with the piling up of angiogenic factor, vascular endothelial growth factor (VEGF) in the μCh. Further assays on the protein sequestration to the μCh revealed that a set of chemokines involved in early pro-inflammatory responses were less found whereas representative adhesive proteins engaged in the cell-matrix interactions were significantly more captured. Ultimately, the fibrous capsule formation on the μCh was reduced with respect to the control, when assessed for up to 21 days, indicating less severe foreign body reaction. The tissue healing and regenerative capacity of the μCh was then confirmed in a critically sized bone model, where those series of events observed are essential to relay bone regeneration. The results over 6 weeks showed that the μCh significantly enhanced the early bone matrix deposition and accelerated bone regeneration. While more in-depth studies remain as to elucidate the underlying mechanisms for each biological event, the molecular, cellular and tissue reactions to the μCh were coherently favorable for the regeneration process of tissues, supporting the engineered scaffolds as potential therapeutic 3D platforms.