Fig 1.
Decreased membranous expression of p120ctn in DBPDE-induced premalignant and cancer lesions.
Staining for p120ctn was performed on oral mucosal tissue harvested from C57Bl/6 mice treated with DBPDE (n = 15) or DMSO (n = 10) as control mice. Representative samples are shown for: (A) Normal oral tissue from control mice, (B) premalignant tissue with decreased p120ctn expression, (C) invasive tumor with decreased p120ctn expression. (D) Quantification of p120ctn mean fluorescent intensity. Scale Bar = 20uM.
Table 1.
p120ctn protein expression changes in DBPDE mice (n = 15) versus controls (DMSO) (n = 10).
Fig 2.
Ki67 increases in DBPDE treated mice.
(A) Ki67 staining of normal tissues demonstrates a low proliferative index in the basal layers of the epithelium. (B) Dysplastic lesions have an increase in proliferation that is expanded beyond just the basal layer. (C) Regions of invasive cancer have wide-spread increases in proliferation. Scale bar = 50uM. n = 15. (D) Quantification of the percentage of Ki67 positive cells measured by counting 40x images (n = 3).
Fig 3.
Increased NFkB and immune cell presence in DBPDE-induced mice.
(A) Normal oral epithelium in DBPDE treated mice are negative or weakly positive for total p65 NFkB protein levels as measured by IHC staining. (B) A representative image of a dysplastic epithelium from DBPDE-treated mice that has increased NFkB staining. (C) NFkB staining is increased in invasive cancers of DBPDE-treated mice. Ly6G was used as a marker for MDSCs, primarily of the neutrophil precursor lineage. (D) Normal oral epithelia do not have a significant presence of Ly6G positive cells. (E) DBPDE-induced dysplasia and (F) invasive cancers do have an increased number of Ly6G-positive cells suggesting a role for MDSCs in tumor development in DBPDE treated mice. Scale bar = 50uM. n = 15. (G) Quantification of Ly6G recruitment measured by counting Ly6G cells/20x image (n = 3).
Fig 4.
Increased vascular density surrounding DBPDE-induced lesions as well as in p120ctn-null lesions.
CD31 staining reveals that there is an increase of vascular development (neovascularization) in the oral tissues of DBPDE-treated mice. (A) CD31 staining of vasculature reveals a regular, single layer of vasculature below the oral cavity epithelium. (B) CD31 increases occur very early and can be seen adjacent to in hyperplastic/mildly dysplastic epithelium. (C) In invasive cancer regions, the vasculature can be seen throughout the tumor. n = 15 for DBPDE mice and n = 10 for controls. (D) Quantification of CD31 staining in DBPDE treated mice as measured by fold difference in CD31 stained area (n = 3). (E) Normal epithelia have a regular vascular pattern underlying the basal layer as measured by CD31. (F) As dysplasia progresses, the blood vessels become very prominent and extend to the tips of the stromal stalks that form. (G) In invasive cancer lesions, the vasculature is fully integrated with the tumor cells. Scale bar = 50uM. n = 5 for p120ctn knockout mice and controls. (H) Quantification of CD31 staining in p120ctn knockout mice as measured by fold difference in CD31 stained area (n = 3).
Fig 5.
p120ctn expression increases in BRB treated mice.
IF for p120ctn was performed on oral mucosal tissue samples harvested from C57Bl/6 mice treated with DBPDE with BRB (n = 10) and without BRB (n = 7) administration. Mice treated with DBPDE alone, panels (A) and (B), have a relatively low expression level of p120ctn while DBPDE + BRB treated mice have relatively increased levels of p120ctn, panels (C) and (D). Panels (A) and (C) represent papillomas while panels (B) and (D) represent invasive cancer. Scale bar = 20uM. (E) Quantification of the mean intensity of p120ctn IF from representative images of papillomas and invasive cancers from all DBPDE treated mice, with and without BRB. (F) Western blot analysis for p120ctn protein levels in DBPDE treated NOK-hTERT cells. b-actin serves as a loading control (G) Quantification of the p120ctn band intensities from western blot analysis, normalized to b-actin (n = 3). (H) Western blot analysis for p120ctn protein levels in DBPDE and/or BRBE treated NOK-hTERT cells. b-actin serves as a loading control (I) Quantification of the p120ctn band intensities from western blot analysis, normalized to b-actin (n = 3).
Fig 6.
High EGFR expression in DBPDE-treated mice.
(A) EGFR expression in normal epithelia is highest in the basal layer and decreases as cells become more differentiated and superficial. (B) EGFR expression is high in hyperplastic and dysplastic epithelium. (C) EGFR expression is high in invasive cancers. n = 15. *p<0.05.
Fig 7.
Schematic of the effects DBPDE treatment has on the tumor and its microenvironment.
DBPDE treatment induced signaling changes in cells of premalignant and invasive cancer lesions resulting in increased NFkB and EGFR expression, increased proliferation, and reduced p120ctn expression. In the microenvironment, increased Ly6G cell (neutrophil lineage) recruitment and neovascularization was detected. These changes, except for EGFR expression, are common between DBPDE treatment and p120ctn decreased expression.