Fig. 5: Lin28b-positive CAFs promote growth of pancreatic cancer.
Before the experiment started, CAFs were cultured with 15376T-CM for 6 days to induce Lin28b expression. CM for CAFs culture was replaced daily (a, b, e–h, k–p). a 15376T were co-cultured with or without 15376CAFs in transwell chambers and treated with complete media (25 mM glucose) or glucose-limited medium (2 mM glucose) for 2 days. The cells were counted to calculate the cell proliferation. b Tumors were co-cultured with 15376CAFs or Lin28b-KO 15376CAFs in transwell chambers for 2 days. The cells were counted to calculate the cell proliferation. c, d Lin28b-WT was stable expressed in 14837CAFs (c) and 15376CAFs (d). Tumors were co-cultured with 14837CAFs (c), Lin28b-WT-expressing 14837CAFs (c), 15376CAFs (d) or Lin28b-WT-expressing 15376CAFs (d) in transwell chambers for 2 days. The cells were counted to calculate the cell proliferation. e–g 15376T was orthotopically co-injected with 15376CAFs or Lin28b-KO 15376CAFs into C57BL/6J mice (n = 6 mice). After 1 week, the pancreas was weighed (e) and analyzed by Ki-67 and CK19 IHC staining (f). Scale bar: 30 μM. The proportion of ki67-positive (g) cell was shown (n = 10 views per group). Data are shown as mean ± s.d. h, i 14837T was orthotopically co-injected with or without 14837CAFs (h), Lin28b-WT-expressing 14837CAFs (h), 15376CAFs (i) or Lin28b-WT-expressing 15376CAFs (i) into C57BL/6J mice (n = 6 mice). After 1 week, the pancreas was weighed. j Tumors were co-cultured with 15376CAFs or Fzd4-KO 15376CAFs in transwell chambers for 2 days. The cells were counted to calculate the cell proliferation. k Tumors were co-cultured with Fzd4+ CAFs or Fzd4- CAFs in transwell chambers for 2 days. The cells were counted to calculate the cell proliferation. l–n 15376T was orthotopically co-injected with 15376CAFs or Fzd4-KO 15376CAFs into C57BL/6J mice (n = 6 mice). After 1 week, the pancreas was weighed (l) and analyzed by Ki-67 and CK19 staining (m). Scale bar: 30 μM. The proportion of ki67-positive (n) cell was shown (n = 10 views per group). o 15376T cells were orthotopically injected into WT or FSP-Cre;Lin28bfl/fl mice (n = 6 mice). After 2 weeks, the tumors were analyzed by Lin28b and α-SMA immunofluorescence staining. Representative images are shown. Scale bar: 30 μM. p–r 15376T or Lin28b-KO 15376T were orthotopically injected into WT or FSP-Cre;Lin28bfl/fl mice (n = 6 mice). After 2 weeks, the pancreas was weighed (p) and analyzed by Ki-67 and CK19 immunofluorescence staining (q). Scale bar: 30 μM. The proportion of ki67-positive (r) cell was shown (n = 10 views per group). Four biologically independent experiments were performed (a–d, j, k). Data are shown as mean ± s.d. P-value were determined by one-way ANOVA with Tukey’s multiple comparison test (a–d, h–k, p, r) or two-tailed unpaired Student’s t-tests (e, g, l, n).
