Fig 1.
Leader cells polarize and protrude along aligned fibers resulting in elongated tumor organoids.
(A) Representative immunofluorescence images of tumor organoids in aligned and random collagen fibers (confocal reflectance) (K14 = green, phalloidin = red, DAPI = blue, collagen = white). Scale bar = 200 μm. (B) Fiber angle frequency and alignment coefficient of collagen fiber orientation in aligned and random hydrogels. n = 39–40 per condition. (C) Representative rose plots of organoid protrusion angles in aligned or random collagen matrices. (D) Organoid morphology characterization: roundness (1 indicates perfectly round), and aspect ratio (higher value indicates more elongated shape) (E) Quantification of organoid protrusions for length and number of protrusions. n = 36 organoids from three different mice. All data shown as mean ± SEM. ns indicates p>0.05, ****p<0.0001, with an unpaired t-test.
Fig 2.
K14+ leader cells in tumor organoids generate increased traction forces and vinculin focal adhesions in 2D.
(A) Representative immunofluorescent image of tumor organoid on protein-patterned dot grid for 2D traction force measurement. (HexForce dots = red, K14 = green, Phalloidin = yellow, DAPI = blue) Scale bar = 500μm. White arrows highlight K14+ leader cells. (B) Analysis of cell traction forces exerted, cell area, and force/area ratio. n = 42 organoids from three mice. (C) Representative immunofluorescent image of K14+ leader and K14- follower cells for K14 (green) and vinculin (pink) with nuclei counterstain, DAPI (blue). White arrowheads highlight vinculin focal adhesions (pink). Scale bar = 15 μm. (D) Quantification for the number of focal adhesions and area in K14+ leader cells and K14- follower cells. n = 24 organoids from three mice. All data shown as mean ± SEM. ns indicates p>0.05, ****p<0.0001, with an unpaired T-test.
Fig 3.
Leader cells have increased integrin-α2 expression compared to follower cells.
Integrin gene expression panel from RNA extracted from (A) tumor organoids (n = 3 independent mouse models) and (B) BT549 (K14+ human breast cancer cell line) cells in aligned and random fibers. *p<0.05, with an unpaired T-test.
Fig 4.
Integrin-α2 colocalizes with K14 in leader cells.
(A) Representative immunofluorescent images of tumor organoids with integrin-α2 (red), K14 (green) and nuclei (blue). (B) Pearson’s R value from colocalization analysis of integrin-α2 and K1. n = 23 organoids from three mice. Data shown as mean ± SEM. (C) Representative immunofluorescent images of tumor organoids with activated integrin-β1 (9EG7, red), vinculin (pink), K14 (green) and nuclei (blue).