Figure 3 from <i>KRAS</i> Loss of Heterozygosity Promotes MAPK-Dependent Pancreatic Ductal Adenocarcinoma Initiation and Induces Therapeutic Sensitivity to MEK Inhibition
Authors: Fey Sigrid K. , Najumudeen Arafath K. , Watt Dale M. , Millett Laura M. , Ford Catriona A. , Gilroy Kathryn , Simpson Rosalin J. , McLay Kathy , Upstill-Goddard Rosanna , Chang David , Clark William , Nixon Colin , Birch Joanna L. , Barry Simon T. , Morton Jennifer P. , Campbell Andrew D. , Sansom Owen J. 
Published: January 2025

<p>Loss of WT KRAS induces increased MAPK signaling in KPC <i>Kras</i><sup>G12D/fl</sup>. <b>A,</b> Experimental schematic representing mice imaged once weekly by ultrasound from 6 weeks of age to clinical endpoint to follow tumor growth over time. <b>B,</b> Tumor volume relative to that of tumor at initial detection from KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> mice aged to clinical endpoint and measured once weekly by ultrasound imaging from 6 weeks of age. KPC <i>Kras</i><sup>G12D/fl</sup>, <i>n</i> = 8; KPC <i>Kras</i><sup>G12D/+</sup>, <i>n</i> = 10. FC, fold change. <b>C,</b> Kaplan–Meier survival curves for KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> mice aged to clinical endpoint. KPC <i>Kras</i><sup>G12D/fl</sup>, <i>n</i> = 29; KPC <i>Kras</i><sup>G12D/+</sup>, <i>n</i> = 23. *, <i>P</i> = 0.0396 log-rank (Mantel–Cox) test. Mice were censored when reaching clinical endpoint not associated with PDAC burden, such as primary tumors, which arose from other tissue, mostly mucocutaneous papillomas or lymphomas (typically thymic) but also gastric and lung tumors. MS, median survival. <b>D,</b> Incidence of metastasis (%) in KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> mice. KPC <i>Kras</i><sup>G12D/fl</sup>, <i>n</i> = 27; KPC <i>Kras</i><sup>G12D/+</sup>, <i>n</i> = 20. mets, metastasis. <b>E,</b> Migration speed of established cell lines derived from pancreatic cancers arising in KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> mice. <b>F,</b> Relative probe intensity in ddPCR from tumors arising in KPC <i>Kras</i><sup>G12D/+</sup> (<i>n</i> = 16) and KPC <i>Kras</i><sup>G12D/fl</sup> (<i>n</i> = 9) mice at clinical endpoint, comparing <i>Kras</i><sup>G12D</sup> to <i>Kras</i><sup>WT</sup> and <i>Kras</i><sup>G12D</sup> to <i>Gapdh</i> probe sets. Tumors from which cell lines analyzed in <b>I</b> were generated are highlighted in this analysis. <b>G,</b> Representative IHC images of CD3, CD4, and CD8a from PDAC arising in KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> mice taken at clinical endpoint. Representative of five mice per group. Scale bar, 200 μm. <b>H,</b> Bar graphs showing quantification of proportion of CD3-, CD4-, and CD8a-positive cells in PDAC tissue from KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> mice sampled at clinical endpoint (KPC <i>Kras</i><sup>G12D/+</sup>, <i>n</i> = 5; KPC <i>Kras</i><sup>G12D/fl</sup>, <i>n</i> = 5). Data are the mean ± SEM, represented in <b>G</b>. <b>I,</b> Immunoblotting of pMEK1/2, MEK1/2, pERK1/2, ERK1/2, and KrasG12D of KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> PDAC tissue lysates generated from mice at clinical endpoint. β-Actin was used as a loading control. Each lane represents PDAC tissue from an individual mouse of the indicated genotype. Bar graph shows quantification of pMEK1/2 levels, normalized to MEK1/2; pERK1/2 levels, normalized to ERK1/2; and KRAS-G12D levels, normalized to β-actin. Data represent the mean ± SEM. *, <i>P</i> = 0.0143; one-way Mann–Whitney <i>U</i> test. <b>J,</b> qRT-PCR analysis of <i>Etv4, Etv5, Dusp4, Dusp6, Spry1,</i> and <i>Spry2</i> in KPC <i>Kras</i><sup>G12D/+</sup> and KPC <i>Kras</i><sup>G12D/fl</sup> PDAC tissue. Transcript levels were normalized to <i>Gapdh</i> (KPC <i>Kras</i><sup>G12D/+</sup>, <i>n</i> = 5; KPC <i>Kras</i><sup>G12D/fl</sup>, <i>n</i> = 6). Data represent the mean ± SEM. <i>P</i> = 0.2 (<i>Etv4</i>); **, <i>P</i> = 0.0087 (<i>Etv5</i>); <i>P</i> = 0.4 (<i>Dusp4</i>); **, <i>P</i> = 0.0087 (<i>Dusp6</i>); *, <i>P</i> = 0.0152 (<i>Spry1</i>); and *, <i>P</i> = 0.0411 (<i>Spry2</i>); ns, nonsignificant; one-way Mann–Whitney <i>U</i> test. Rel., relative.</p>