Fig 2: Epithelial-mesenchymal transition increases tumor cell adherence to collagen through α1β1 integrin.(A) Cell adherence was quantified 1 hour after the indicated KP cells were seeded on wells coated with collagen 1 (Col1), collagen IV (Col4), fibronectin, BSA, or nothing, and OD595 values are shown in the heatmap. E, epithelial; M, mesenchymal. n = 4. (B) Cell spreading assay on Col1. Cells were plated in Col1-coated plates for 1 hour, and percentages of spread cells were quantified. Scale bar: 50 μm. (C) Quantitative reverse transcription PCR analysis of the mRNA levels of ZEB1 and Col1 receptors in KP cell lines (Pearson correlation). (D) Western blot analysis of Itga1 and Itgb1 in KP cell lines classified as epithelial or mesenchymal. β-Actin was used as a loading control. (E) Heatmap illustration of gene expression levels in lung adenocarcinoma and squamous cell carcinoma data sets (n = 1016 tumors) in The Cancer Genome Atlas. An epithelial-mesenchymal transition score was correlated with the levels of collagen receptors using the Pearson coefficient (r value). (F) Cell adherence was quantified 1 hour or 2 hours after the indicated cells were seeded on uncoated wells or on wells coated with Col1. n = 4. **P < 0.01. (G) Cell spreading assay on Col1. Percentages of spread cells were quantified. Scale bar: 100 μm. ***P < 0.001. (H) Quantitative reverse transcription PCR analysis of Col1 receptors. (I) Western blot analysis of Itga1 in 393P_Vec and 393P_ZEB1 cells. Data are shown as the mean ± SEM from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. *P < 0.01; ***P < 0.001. Two-tailed Student’s t test.

Fig 3: Itgb1knockdown reduces β1-integrin protein levels. a. CaMKII-AAVs containing Cre were infused bilaterally into PL of Itgb1-flox mice. (left) Images from Allen Brain Atlas (from left: + 2.71 mm, + 2.22 mm, and + 1.98 mm relative to bregma) depicting the PL (purple). (right) Overlays of viral vector spread (red) encompassing the PL from mice in this report on images from the Mouse Brain Library (Rosen et al., 2000). All cohorts exhibited consistent viral vector localization. (top right) Representative blots. b. Viral-mediated Itgb1 silencing significantly reduced β1-integrin protein levels in PFC tissue punches from both adolescents and adults. Incomplete protein loss was expected, given that samples contained transduced and unaffected cells, and glial integrins were spared. There were no age-based differences in β1-integrin levels. c. β1-integrin protein levels correlated with phosphorylation (activation) of the neuronal substrate p190RhoGAP. Bars represent means, and symbols represent individual mice. *p < 0.05 relative to 1.0; “n.s.” refers to no significant difference between ages; “adol.” refers to “adolescent.

Fig 4: (A) Western blot showing the expression of ITGB1, ITGA3 and PDCD6IP in exosomes and whole cell lysates of LNCaP and PC3 cells. (B) Effect of blocking antibodies of ITGA3 and ITGB1, which were added combined with LNCaP-medium (CM-L) or PC3-medium (CM-P) on the migration and invasion of prEC. As negative controls, isotype control was added. Statisticial differences between controls and between antibody and CM-L or CM-P are indicated in the graph (*p<0.05) (C) Exosomal uptake of LNCaP and PC3 exosomes in prEC cells as measured by FACS analysis. Values represent means of 3 independent experiments. (D) Effect of exosomes from LNCaP and CP3 cells on prEC invasion, with/without blocking antibodies against ITGA3 and ITGB1. As antibody-controls, isotype control was added. Statisticial differences are indicated in the graph (*p<0.05).

Fig 5: Expression of mesenchymal and hematopoietic stem cell markers in hypo- and normo-VICs. (a) Quantifications of mRNA levels for THY1, CD34, Oct4, CD29, ENG, VCAM1, CD44, MCAM, and ALCAM (n = 3). Expression levels were normalized to those of GAPDH. Data are presented as means ± SE. (b). Validation of CD29, VCAM1, and CD34 upregulation in hypo-VICs by western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s.: Not significant.