Fig 1: LncRNA FOXD2‐AS1 was highly expressed in glioma tissues and cells as well as GSCs. A, LncRNA FOXD2‐AS1 expression predicted in GSE104267 (p = 1.482E‐07). The box on the left represents expression of lncRNA FOXD2‐AS1 in normal tissues and the box on the right represents expression in cancer tissues. B, A box plot displaying lncRNA FOXD2‐AS1 expression in glioma samples (left) and normal samples (right) from TCGA database using GEPIA. C, Survival curve of patients with glioma regarding lncRNA FOXD2‐AS1 expression from TCGA database using GEPIA (HR = 1.8, p = 0.032). D, LncRNA FOXD2‐AS1 expression in glioma tissues and normal tissues detected by RT‐qPCR (n = 26). E, A Kaplan–Meier one‐way survival analysis according to lncRNA FOXD2‐AS1 expression, followed by log‐rank test (n = 26). F, FOXD2‐AS1 expression in glioma cell lines determined using RT‐qPCR. G, Expression of lncRNA FOXD2‐AS1 and GSC markers (OCT4, SOX2, Nanog, Nestin and CD133) determined using RT‐qPCR. H, Expression of Nestin and GFAP in GSCs determined by immunofluorescence. I, CD133+ GSCs selected using flow cytometry. J, LncRNA FOXD2‐AS1 expression in GSCs determined using RT‐qPCR. * p < 0.05. Measurement data were expressed as mean ± standard deviation. Paired t‐test was used to analyse comparison within group. Unpaired t‐test was adopted to analyse differences between two groups. Differences among multiple groups were analysed by one‐way ANOVA, followed by a Tukey multiple comparisons post‐test. Comparisons among multiple groups at different time points were performed with two‐way ANOVA. Cell experiments were conducted three times independently.
Fig 2: Silencing lncRNA FOXD2‐AS1 suppressed stemness and proliferation but induced apoptosis and differentiation of U251 GSCs. U251 GSCs were transfected with lncRNA FOXD2‐AS1 shRNAs and sh‐NC. A, Silencing efficiency of lncRNA FOXD2‐AS1 shRNA in U251 GSCs determined using RT‐qPCR. B, The number of main spheres every 1000 GSCs and sphere at the second passage every 100 GSCs. C, Expression of GSC markers (OCT4, SOX2, Nanog, Nestin and CD133) determined using RT‐qPCR. D, CD133+ cells in U251 GSCs measured by flow cytometry. E, U251 GSC proliferation detected using colony formation in soft agar. F, U251 GSC apoptosis determined using flow cytometry. G‐H, GFAP and CD133 expression in GSCs determined by immunofluorescence staining (400 ×). * p < 0.05. Measurement data were expressed as mean ± standard deviation and analysed by unpaired t‐test. Cell experiments were conducted in triplicate.
Fig 3: LncRNA FOXD2‐AS1 elevation induced GSC stemness and inhibited GSC differentiation via activation of the NOTCH signalling pathway via TAF‐1 recruitment. GSCs were transfected with oe‐NC, oe‐lncRNA FOXD2‐AS1 or oe‐lncRNA FOXD2‐AS1 + sh‐TAF‐1. A, Western blot analysis of expression of NOTCH pathway related genes (JAG1, PS1 and HES1). B, The number of main spheres every 1000 GSCs and sphere at the second passage every 100 GSCs. C, OCT4, SOX2, Nanog, Nestin and CD133 expression detected using RT‐qPCR. D, CD133+ cells in U251 GSCs assessed by flow cytometry. E, U251 GSC proliferation measured by colony formation in soft agar. F, U251 GSC apoptosis evaluated by flow cytometry. G, GFAP and CD133 expression in GSCs assessed by immunofluorescence staining. * p < 0.05. Measurement data were expressed as mean ± standard deviation and analysed by one‐way ANOVA, followed by a Tukey multiple comparisons post‐test. Cell experiments were repeated in triplicate.
Fig 4: Automatic recognition of tumor-infiltrating CD8+ T cells, CD133+ CSCs, and CK19+ tumor budding in PDAC based on artificial intelligence. Images in Panels A, D, E, F, and G were all obtained from the same slide of the same PDAC patient and used as an example to illustrate the analytic process. (A) Representative mIF images of PDAC tissues. CK19+ tumor cells, CD133+ CSCs, CD8+ T cells, and nuclei are annotated in red, green, yellow, and blue, respectively. Scale bar, 100 μm. (B) Representative IHC images of PDAC tissues for CK19, CD133, and CD8. Scale bar, 100 μm. (C) Brief introduction of whole landscape quantitative analysis system of Tissue Gnostics (Zeiss). (D) Schematic illustration for cellular segmentation, CK19+ cell recognition, and CK19+ cell counting based on an automatic system. The DAPI+ and CK19+ cells are presented as dot plots. The red rectangle frame (formed by dash lines) in the upper dot plots represents DAPI-positive cell subsets based on the threshold of DAPI intensity and the red rectangle frame (formed by dash lines) in the lower dot plots represents CK19-positive cells subsets based on the threshold of CK19 intensity. Scale bar, 100 μm. (E) Representative IHC and mIF images for CK19+ tumor budding detection. The red arrows indicate CK19+ tumor budding determined by CK19 IHC staining and the white arrows indicate CK19+ tumor budding determined by CK19 mIF staining. Scale bar, 100 μm. (F) Recognition of CK19+ tumor budding based on automatic system. The region formed by green dash lines indicate CK19+ tumor cells and the region formed by red dash lines indicate tumor budding. Scale bar, 100 μm. (G, H) Recognition of PDAC with different histologic grades based on machine learning. The CK19+ PDAC was recognized and marked with a white mask by artificial intelligence. Consistency between results of IHC evaluation by experienced pathologists and number of mIF assessments by machine learning was compared and a Spearman correlation test was performed (H). The number next to the blue points in Figure 2H mean patient sample counts with the indicated IHC grade. (I–L) Consistency between results of the IHC evaluation by experienced pathologists and the number of mIF assessments by automatic counting was compared and the Spearman correlation test was performed. CK19+ tumor cells (I), CD133+ CSCs (J), CD8+ T cells (K), and tumor budding (L).
Fig 5: Correlation of tumor-infiltrating CD8+ T cells, CD133+ CSCs, and tumor budding, and clinical outcome. (A) Representative mIF images for co-staining of CK19, CD8, and CD133 on the same slide. CD8 in yellow, CK19 in red, CD133 in green, and nuclei in blue for multi-color IF staining. Scale bar, 100 μm. (B) mIF staining for low and high CD8+ T cells in a retrospective primary training cohort (left). Scale bar, 100 μm. CD8 in yellow and nuclei in blue for multi-color IF staining. Overall survival and relapse-free survival of patients grouped by different CD8+ T cell infiltrating status (right). (C) mIF staining for CD133+ CSCs in a retrospective primary training cohort (left). CD133 in green and nuclei in blue for multi-color IF staining. Scale bar, 100 μm. Overall survival and relapse-free survival of patients grouped by different CD133+ CSC infiltrating status (right). (D) mIF staining for CK19+ tumor budding in a retrospective primary training cohort (left). CK19 in red and nuclei in blue for multi-color IF staining. Scale bar, 100 μm. Overall survival and relapse-free survival of patients grouped by different CK19+ tumor budding infiltrating status (right). (E) mIF staining for CD8+ T cells in a retrospective validation cohort (left). CD8 in yellow and nuclei in blue for multi-color IF staining. Scale bar, 100 μm. Overall survival and relapse-free survival of patients grouped by different CD8+ T cell infiltrating status (right). (F) mIF staining for CD133+ CSCs in a retrospective validation cohort (left). CD133 in green and nuclei in blue for multi-color IF staining. Scale bar, 100 μm. Overall survival and relapse-free survival of patients grouped by different CD133+ CSC infiltrating status (right). (G) mIF staining for CK19+ tumor budding in a retrospective validation cohort (left). CK19 in red and nuclei in blue for multi-color IF staining. Scale bar, 100 μm. Overall survival and relapse-free survival of patients grouped by different CK19+ tumor budding infiltrating status (right). (H) Flow cytometry detection for CD8+ T cells of fresh specimens from another prospective validation cohort. Representative dot plots are shown (left). Gated at CD45+ cells. Overall survival and relapse-free survival of patients grouped by different CD8+ T cell infiltrating status (right). (I) Flow cytometry detection for CD133+ CSCs of fresh specimens from another prospective validation cohort. Representative histograms are shown (left). Gated at EpCAM+ epithelial cells. Overall survival and relapse-free survival of patients grouped by different CD133+ CSC infiltrating status (right). (J) IHC staining for CK19+ tumor budding in another prospective validation cohort. Representative IHC image of tumor budding are shown (left). Scale bar, 100 μm. Overall survival and relapse-free survival of patients grouped by different CK19+ tumor budding infiltrating status (right).
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