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. Author manuscript; available in PMC: 2020 Feb 1.
Published in final edited form as: Cancer Lett. 2018 Nov 10;442:310–319. doi: 10.1016/j.canlet.2018.11.012

Fig. 4. SRC-3 silencing suppresses tumor growth in orthotopic PDAC mouse models.

Fig. 4.

(A) PANC-1-shCtrl and PANC-1-shSRC-3 cells with stable expression of luciferase were orthotopically injected into the pancreas of SCID mice. The tumor growth was monitored under IVIS live imaging system. Total flux (photons/second) was recorded. PANC-1-shSRC-3 group shows a dramatically suppressed tumor growth compared to the PANC-1-shCtrl group. (B) A live image of mouse tumors was taken right before sacrifice. (C) Tumors were harvested, photographed, and weighted. The statistic results of tumor weights are presented as mean±SEM, PANC-1-shCtrl group has a value of 1.09±0.18 g, whereas PANC-1-shSRC-3 group has a value of 0.22±0.11 g. Those values show a statistically significant difference by t-test, **, p<0.01. (D) A set of orthotopic PDAC mouse models were established by using Capan-2-shCtrl and Capan-2-shSRC-3 cells. After 8 weeks of tumor growth, tumors were harvested. The statistic values of tumor weights from Capan-2-shCtrl and Capan-2-shSRC-3 groups are 1.17±0.24 g and 0.23±0.7 g, respectively. A significant difference is revealed by t-test, **, p<0.01. (E) The SRC-3 and Ki-67 protein levels were measured by IHC. Mouse tumors established from PANC-1-shSRC-3 and Capan-2-shSRC-3 exhibits hardly detectable SRC-3 expression and less Ki-67 positive cells compared to the corresponding controls (scale bar: 50 μm). (F) The Ki-67-positive cells from multiple tumors (N>3) of each group were counted based on the photos taken under microscope. The numbers are 655.9±113.7 of PANC-1-shCtrl group, 161.0±3.4 of PANC-1-shSRC-3, 103.8±15.8 of Capan-2-shCtrl, and 36.0±4.9 of Capan-2-shSRC-3 (**, p<0.01, ***, p<0.001, by t-test). (G) Cell line PANC-1-Ind-shSRC-3 was constructed by introducing a doxycycline inducible anti-SRC-3 shRNA into PANC-1-Luciferase cells. The Western blotting results showed that SRC-3 expression levels in PANC-1-Ind-shSRC-3 cells were significantly knocked down when treated with doxycycline. (H) A set of orthotopic PDAC mouse models were made by inoculating PANC-1-Ind-shSRC-3 cells into SCID mouse pancreases. After 14 days of tumor growth, mice were randomly split into 2 groups (n =7). One group was feed with water containing doxycycline (200 ppm). The tumor size was monitored and recorded once a week by IVIS live imaging system. The tumor growth is remarkably delayed in the mouse group feed with doxycycline. (I) A live image of mouse tumors was pictured right before sacrifice to show the visible differences of tumor size between the groups feed with and without doxycycline. (J) The growth curves of PANC-1-Ind-shSRC-3 in the presence or absence of 1 μg/mL of doxycycline were measured by MTT assays. The original values of day 0 was set as 1. Doxycycline does not affect the growth of control cell line PANC-1-shCtrl, but significantly inhibits PANC-1-Ind-shSRC-3 cell growth. (K) An IHC staining was performed to demonstrate the SRC-3 protein levels and the amount of Ki-67 positive cells on above mouse tumors established with PANC-1-Ind-shSRC-3 cells. The tumor tissues from mice feed with doxycycline shows undetectable SRC-3 expression and less amount of Ki-67 positive cells. One representative photo from each group is presented (scale bar: 50 μm).