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. 2018 Sep 14;7(1):73–91. doi: 10.1016/j.jcmgh.2018.09.003

Figure 3.

Figure 3

Morphological analysis of patient-derived 3D organoids. Biopsies and 3D organoids representing (A) normal mucosa and (B–E) tumors from indicated patients were morphologically analyzed by H&E staining, IHC for p53, Ki-67, and cleaved LC3. (A) Normal mucosa gave rise to non-neoplastic 3D organoids with a clear concentric stratification or stratified squamous cell differentiation gradient, approximately reproducing all layers of a stratified squamous epithelium with basaloid and spinous cells in the periphery and stratum corneum-like squames in the center. Tumor biopsies gave rise to both (B) neoplastic and (C) non-neoplastic 3D structures. (B) Tumor-derived neoplastic 3D structures (SCC 3D organoids) feature lack clear differentiation pattern although the eosinophilic cytoplasm of most cells point to a squamous differentiation, and thus, compatible with poorly differentiated squamous cell carcinoma. There is mild to moderate nuclear atypia noted. (C) Tumor-derived non-neoplastic 3D organoids contain cells with very little or no nuclear atypia (patient 12). The majority of non-neoplastic structures feature focal incomplete keratinization without clear stratification (patient 5). The minority of non-neoplastic structures display stratification (ie, a concentric complete keratinization [patient 4]), a rare cystic form with squamous differentiation with very little or no terminally differentiated squamous (horny layer-like) cells. Scale bar = 100 μm for tissue sections; (A, C) 20 μm for 3D organoids. Scale bar = 100 μm for tissue sections (patient 4); 20 μm for 3D organoids (patient 4); and (B) 50 μm for 3D organoids (patients 10 and 16). (D) Ki67 labeling index was determined in tumor-derived neoplastic (SCC compatible) and non-neoplastic 3D structures (10 per each group) from multiple patients as biological replicates (3 independent patients for neoplastic 3D organoids and 7 independent patients for non-neoplastic 3D organoids). Data were presented as mean ± SD. Student’s t test was used to examine statistical significance. *P < .05, vs non-neoplastic organoids. (E) Cleaved LC3 was stained in tumor biopsies and corresponding SCC 3D organoids. Scale bar = 100 μm for tissue sections; and 50 μm for 3D organoids.