Fig 2: Elastase I-driven oncogenic KRASG12V expression induces SDC-like tumors in the SMGs.(A) The diagram of the Ela-CreERT and LGL-KRasG12V transgenes2630. Expression of the TAM-inducible Cre recombinase (CreERT) is driven by elastase I promoter/enhancer. KRASG12V expression is blocked by a loxP-GFP-Stop-loxP cassette (LGL). A mouse BAC of 222-kb carrying the intact elastase I gene was used to provide the endogenous native context of elastase I gene26. CreERT removes STOP cassette (LGL) through recombination, allowing the KRASG12V expression. (B) Elastase I-driven recombination in mTmG and Rosa26R reporter mice ascertains ductal original. Green fluorescence was exclusively detected in the ductal cells of mTmG;Ela-CreERT reporter mice after vehicle (panel a) or TAM-feeding (panel b). Scale bar: 50 µm (panel a). ß-galactosidase activity was observed in the SMG ductal cells of Rosa26R;Ela-CreERT mice after TAM-feeding (panel d) compared to control mice (panel c). Scale bar: 200 µm. (C) Characterization of SMGs of LGL-KRasG12V;Ela-CreERT mice on day-24 post TAM-feeding. Gross anatomy revealed large ventrolateral cervical masses in SMGs of LGL-KRasG12V;Ela-CreERT mice on day-24 (panel b). H&E and immunohistochemical (IHC) staining showed the microscopic abnormalities in the SMGs of TAM-fed LGL-KRasG12V;Ela-CreERT mice on day-24 (panels d, f, h). Scale bars: 800 µm (in panels c and d); 100 µm (panels e–h). (D) Wet weight of SMG from LGL-KRasG12V;Ela-CreERT mice at 15-days post TAM-feeding (n = 13) or not (n = 7). ***p < 0.001. (E) Overall survival of mice after KRASG12V induction is drastically reduced. Percent survival of Ela-CreERT versus LGL-KRasG12V;Ela-CreERT mice after TAM administration. Median survival of LGL-KRasG12V;Ela-CreERT mice was 28-days. n = 13 per group; p < 0.0001 by Log-rank (Mantel-Cox) test. (F) KRASG12V is activated in the SMG tumors of LGL-KRasG12V;Ela-CreERT mice following TAM-gavage. Transgenic mice of indicated genotypes were gavaged with TAM. SMG samples were harvested on day-24 and whole tissue lysates (1 mg) from each sample were pulled down with Raf-1 RBD agarose beads. Pulldown reactions were resolved by SDS-PAGE (12%) and Western blotting was performed with an anti-KRAS antibody to detect active (GTP-bound) KRASG12V.

Fig 3: Cell proliferation starting from day-9 following oncogenic KRAS activation.(A) Nuclear Ki-67-positive cells increased mainly at the ductal cells starting from day-9 post KRASG12V induction. Scale bars: 250 µm; inset: 200X; A: acini; D: duct; black arrow: nuclear Ki-67-positive ductal cell; red arrow: nuclear Ki-67-positive fibroblast; red arrowhead: blood vessel. The relative fold change in the nuclear Ki-67-positive staining with the level in the SMGs of LGL-KRasG12V;Ela-CreERT mice prior to Tam-feeding (Ctrl) set as 1. Results are shown as mean ± S.D. enumerated from 6 randomly selected non-overlapping fields (10X) by Image-Pro Premier 9.0; *p < 0.05, **p < 0.01. (B) Elastase I accumulation in the SMG ductal cells of LGL-KRasG12V;Ela-CreERT mice decreased during the course of tumorigenesis (left panels). Panel d (enlarged from boxed region in panel b): elastase I staining was lost in a GCT (shown by dotted line, panel d) near a dyad structure of a striated duct (blue arrow) and a blood vessel (red arrow); residual elastase I staining was visualized in some GCTs (label with G). Scale bar: 100 µm (panels a–c:); 100 µm (panel d, enlarged from b). The relative fold change in the elastase I-positive staining (right panel). Quantitative analyses of elastase I-positive staining with level at control SMGs set as 1. Results are shown as mean ± S.D. enumerated from 5 randomly selected non-overlapping fields (10X) by Image-Pro Premier 9.0; **p < 0.01. (C) Loss of AQP5 staining during KRASG12V-induced tumorigenesis. Immunochemical staining with an anti-AQP5 antibody in the SMGs of LGL-KRasG12V;Ela-CreERT mice at indicated time points following TAM-feeding. Scale bar: 50 µm. (D) Gradual loss of a-smooth muscle actin (aSMA) expression during early tumorigenesis. The earliest loss of aSMA occurred on day-6 in GCTs near a dyad structure of a striated duct (blue arrow) and a blood vessel (red arrow). Scale bar: 50 µm; G: GCT; T: tumor cell masses.