Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing Reveals the Heterogeneity in Differentiation Trajectory and Tumor Microenvironment Leading to More Aggressive Phenotypes of Papillary Thyroid Cancer in Children and Young Adult Patients

doi: 10.1002/advs.202417672

Figure Lengend Snippet: Subtyping of cancer‐associated fibroblasts and their heterogeneity between CAYA and ADULT. A) UMAP plots for the four different CAF subtypes in papillary thyroid tumors (top left), and each cell colored for group (top right), tissue origin (bottom left). UMAP representations with cells colored by the expression level of marker genes of PTC. The black lines represent the spatial density of the cells expressing the given gene higher than the mean level of expression (bottom). B) Bubble plot showing top six differentially expressed genes in each CAF subtypes and patient. The size of the dot indicates the fraction of cells expressing a particular marker, and the intensity of the color represents the level of mean expression. Cellular phenotypes and age group are indicated left side the dot map. C) Bar plot showing the gene count for the most significantly upregulated GO BP pathways in each subtype, calculated through GSVA and Empirical Bayes Statistics for differential expression. D) Multiplex immunofluorescence staining showed major CAF clusters existed in PTC tissues. A pseudocolored image depicting different markers identified by mxIHC (colored as indicated in the key) and the results from histocytometry‐based cell classification (anti‐LAMP5 for emCAF_LAMP5, anti‐CD36 for lpmCAF_CD36, anti‐SMMC for myoCAF_MYH11, anti‐FBLN1 for iCAF_CFD). E) The pseudotime trajectory analysis of thyrocytes and fibroblasts inferred by Monocle 2. Each color represents one cell subtype (left), each point corresponds to one single cell (right). F) Trace plots of the EMT score along pseudotime separated by branch point. G) Shaded line plot indicating the expression levels of the EMT score (left) and TGF‐β (right) along the pseudotime in CAYA and adult patients. H) UMAP reflection of EMT scored by Ucell. I) Rank plot of TF (transcription factor) activities in the four CAF types, scored by pySCENIC. J) The binomial distribution heatmap of transcription factors of age‐specific transcription factors in CAYA and adults.

Article Snippet: A sequential application of antibodies against ANGPTL4 (NBP2‐80039, NOVUS, USA), LAMP5 (NBP1‐84246, NOVUS, USA), CD31 (BD, USA), CD36 (18836‐1‐AP, Proteintech, China), FBLN1 (NBP1‐84725, NOVUS, USA), MYH11 (21404‐1‐AP, Proteintech, China), VEGFC (22601‐1‐AP, Proteintech, China), VEGFR3 (20712‐1‐AP, Proteintech, China), and Pan‐CK (BD, USA) was performed, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies and subsequent tyramide signal amplification.

Techniques: Expressing, Marker, Quantitative Proteomics, Multiplex Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing Reveals the Heterogeneity in Differentiation Trajectory and Tumor Microenvironment Leading to More Aggressive Phenotypes of Papillary Thyroid Cancer in Children and Young Adult Patients

doi: 10.1002/advs.202417672

Figure Lengend Snippet: Complex cell–cell communication networks in the PTC TME. A) Heatmap showing the strength of incoming and outgoing events in interactions between different clusters in the PTC. Histograms separately count the overall intensity of outgoing ( y ‐axis) and incoming ( x ‐axis) events for each cluster. B) Dot plots show gene expression levels of receptor–ligand pairs involved in interactions between different clusters in PTC. C) Interaction strength of emCAF, thyrocyte State 3, lymphatic EC, and vascular EC subclusters incoming and outgoing events between CAYA and adult PTC. D) Circle and chord diagram showing predicted cell–cell interactions of ANGPTL, TGF‐β, and VEGF signaling pathway between CAYA and adult PTC. The arrow width indicates the interaction strength levels. E) Bubble heatmap showing the mean interaction strength for selected ligand–receptor pairs between CAYA and adult in various cell–cell clusters. Dot size indicates P ‐value generated by permutation test, colored by interaction strength levels. F) Box plots showing the expression level of VEGFC, ANGPTL4, and FLT4 from PTCs in TCGA cohort between CAYA and adult. Violin plot showing expression of ANGPTL4 expression among different N and T stages. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; two‐sided t ‐test. Kaplan–Meier plots for overall survival of ANGPTL4‐high and ‐low in PTC patients. P ‐value was determined by Kaplan–Meier survival curves and log‐rank test. G) Representative micrographs from multiplexed IHC (mxIHC) labeled by ANGPTL4, LAMP5, CD31, and PANCK performed on serial sections. H) Representative images from multiplexed IHC (mxIHC) labeled by VEGGC and VEGFR3 performed on serial sections.

Article Snippet: A sequential application of antibodies against ANGPTL4 (NBP2‐80039, NOVUS, USA), LAMP5 (NBP1‐84246, NOVUS, USA), CD31 (BD, USA), CD36 (18836‐1‐AP, Proteintech, China), FBLN1 (NBP1‐84725, NOVUS, USA), MYH11 (21404‐1‐AP, Proteintech, China), VEGFC (22601‐1‐AP, Proteintech, China), VEGFR3 (20712‐1‐AP, Proteintech, China), and Pan‐CK (BD, USA) was performed, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies and subsequent tyramide signal amplification.

Techniques: Gene Expression, Generated, Expressing, Labeling

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing Reveals the Heterogeneity in Differentiation Trajectory and Tumor Microenvironment Leading to More Aggressive Phenotypes of Papillary Thyroid Cancer in Children and Young Adult Patients

doi: 10.1002/advs.202417672

Figure Lengend Snippet: Characteristics of FAP and serves as a target for PET imaging. A) Dot plot showing the correlation between FAP and LAMP5. The P value was generated by Pearson correlation. B) Violin plot of the relationship between the proportion of FAP and T stage (left) and N stage (right) in TCGA‐THCA database. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; two‐sided t ‐test. C) Overall survival curves (left) and Kaplan–Meier progress free survival curves (right) for FAP of the patients with PTC in TCGA‐THCA cohort for stratified by subclusters’ abundance using the optimal cut‐point for dichotomization. Statistical significance P value was assessed using log‐rank test. D) Related expression of FAP in CAYA and adult patient separated by tumor and para‐tumor tissues staining registration for FAP in the tumor and para‐tumor tissue from CAYA and adult patient (n=3). Overall survival curves (left) and Kaplan–Meier progress free survival curves (right) for FAP of the patients with PTC in TCGA‐THCA cohort for stratified by subclusters’ abundance using the optimal cut‐point for dichotomization. Statistical significance P ‐value was assessed using log‐rank test. F) Comparation of 18 F‐FDG‐PET and 68 Ga‐FAPI‐PET imaging.

Article Snippet: A sequential application of antibodies against ANGPTL4 (NBP2‐80039, NOVUS, USA), LAMP5 (NBP1‐84246, NOVUS, USA), CD31 (BD, USA), CD36 (18836‐1‐AP, Proteintech, China), FBLN1 (NBP1‐84725, NOVUS, USA), MYH11 (21404‐1‐AP, Proteintech, China), VEGFC (22601‐1‐AP, Proteintech, China), VEGFR3 (20712‐1‐AP, Proteintech, China), and Pan‐CK (BD, USA) was performed, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies and subsequent tyramide signal amplification.

Techniques: Imaging, Generated, Expressing, Staining