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Image based detection and targeting of therapy resistance in pancreatic adenocarcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53, and SMAD42-4. To date, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavor. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression in both genetic models and patient derived xenografts. Specifically, we developed Msi reporter mice that allowed image based tracking of stem cell signals within cancers, revealing that Msi expression rises as PanIN progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbor the capacity to propagate adenocarcinoma, are enriched in circulating tumor cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in PanIN progression to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumors, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumor penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signaling as a central regulator of pancreatic cancer.

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Analysis of tumors from Msi  KPf/fC mice(a) Msi2 (green) and Keratin (red) immunofluorescent staining was performed on tissue sections from WT pancreas (Normal, n=3 samples), KRASG12D/+;Ptf1aCre/+ (PanIN, n=2 samples), and KRASG12D/+;p53f/f;Ptf1aCre/+ (PDAC, n=3 samples) mice with quantification of Msi2 fluorescence in Keratin positive cells. (b) Average weights of WT-KPf/fC (n=13) and Msi1−/−-KPf/fC tumors (n=9). See also Figure 2h-i. for tumor volume analysis (c) PAS and Alcian Blue stained sections of pancreata isolated from WT-KPf/fC represent areas used to identify the stages of PanINs (yellow boxes) and adenocarcinoma (red box). (d) Tumors from 11-13 week old WT-KPf/fC (n=6), Msi1−/−-KPf/fC (n=3), and Msi2−/−-KPf/fC (n=3) mice were stained and quantified for percent of Keratin+ tumor cells (red) expressing Ki67 (green); DAPI staining is shown in blue. (e) Average weights of WT-KPf/fC (n=5) and Msi2−/−-KPf/fC tumors (n=7). See also Figure 2h-I for tumor volume analysis. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test or One-way ANOVA. Source Data for all panels are available online.
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Figure 8: Analysis of tumors from Msi KPf/fC mice(a) Msi2 (green) and Keratin (red) immunofluorescent staining was performed on tissue sections from WT pancreas (Normal, n=3 samples), KRASG12D/+;Ptf1aCre/+ (PanIN, n=2 samples), and KRASG12D/+;p53f/f;Ptf1aCre/+ (PDAC, n=3 samples) mice with quantification of Msi2 fluorescence in Keratin positive cells. (b) Average weights of WT-KPf/fC (n=13) and Msi1−/−-KPf/fC tumors (n=9). See also Figure 2h-i. for tumor volume analysis (c) PAS and Alcian Blue stained sections of pancreata isolated from WT-KPf/fC represent areas used to identify the stages of PanINs (yellow boxes) and adenocarcinoma (red box). (d) Tumors from 11-13 week old WT-KPf/fC (n=6), Msi1−/−-KPf/fC (n=3), and Msi2−/−-KPf/fC (n=3) mice were stained and quantified for percent of Keratin+ tumor cells (red) expressing Ki67 (green); DAPI staining is shown in blue. (e) Average weights of WT-KPf/fC (n=5) and Msi2−/−-KPf/fC tumors (n=7). See also Figure 2h-I for tumor volume analysis. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test or One-way ANOVA. Source Data for all panels are available online.

Mentions: Because Msi expression rose during progression (Extended Data Fig. 1f-k, 4a), and marked therapy resistant cells, we tested if genetic or pharmacologic targeting of Msi could eradicate this ‘high risk’ population. Deletion of Msi1 led to a 5-fold reduction in tumor volume by MRI (Fig. 2a-b, Extended Data Fig. 4b, Supplementary Videos S2-S4). Histologically, adenocarcinoma areas comprised 67% of WT-KPf/fC but less than 10% of Msi1−/−KPf/fC pancreata; further while Msi1 loss allowed low grade PanINs to form, it largely blocked progression to adenocarcinoma (Fig. 2c-f, Extended Data Fig. 4 c, d). Finally, Msi1 deletion improved survival in orthotopic grafts: median survival for WT-KPf/fC graft recipients was 28.5 days, and for Msi1−/−-KPf/fC grafts was 70.5 days, representing a 2.5-fold increase in survival time and a 23-fold decrease in risk of death (Fig. 2g).


Image based detection and targeting of therapy resistance in pancreatic adenocarcinoma
Analysis of tumors from Msi  KPf/fC mice(a) Msi2 (green) and Keratin (red) immunofluorescent staining was performed on tissue sections from WT pancreas (Normal, n=3 samples), KRASG12D/+;Ptf1aCre/+ (PanIN, n=2 samples), and KRASG12D/+;p53f/f;Ptf1aCre/+ (PDAC, n=3 samples) mice with quantification of Msi2 fluorescence in Keratin positive cells. (b) Average weights of WT-KPf/fC (n=13) and Msi1−/−-KPf/fC tumors (n=9). See also Figure 2h-i. for tumor volume analysis (c) PAS and Alcian Blue stained sections of pancreata isolated from WT-KPf/fC represent areas used to identify the stages of PanINs (yellow boxes) and adenocarcinoma (red box). (d) Tumors from 11-13 week old WT-KPf/fC (n=6), Msi1−/−-KPf/fC (n=3), and Msi2−/−-KPf/fC (n=3) mice were stained and quantified for percent of Keratin+ tumor cells (red) expressing Ki67 (green); DAPI staining is shown in blue. (e) Average weights of WT-KPf/fC (n=5) and Msi2−/−-KPf/fC tumors (n=7). See also Figure 2h-I for tumor volume analysis. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test or One-way ANOVA. Source Data for all panels are available online.
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Figure 8: Analysis of tumors from Msi KPf/fC mice(a) Msi2 (green) and Keratin (red) immunofluorescent staining was performed on tissue sections from WT pancreas (Normal, n=3 samples), KRASG12D/+;Ptf1aCre/+ (PanIN, n=2 samples), and KRASG12D/+;p53f/f;Ptf1aCre/+ (PDAC, n=3 samples) mice with quantification of Msi2 fluorescence in Keratin positive cells. (b) Average weights of WT-KPf/fC (n=13) and Msi1−/−-KPf/fC tumors (n=9). See also Figure 2h-i. for tumor volume analysis (c) PAS and Alcian Blue stained sections of pancreata isolated from WT-KPf/fC represent areas used to identify the stages of PanINs (yellow boxes) and adenocarcinoma (red box). (d) Tumors from 11-13 week old WT-KPf/fC (n=6), Msi1−/−-KPf/fC (n=3), and Msi2−/−-KPf/fC (n=3) mice were stained and quantified for percent of Keratin+ tumor cells (red) expressing Ki67 (green); DAPI staining is shown in blue. (e) Average weights of WT-KPf/fC (n=5) and Msi2−/−-KPf/fC tumors (n=7). See also Figure 2h-I for tumor volume analysis. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test or One-way ANOVA. Source Data for all panels are available online.
Mentions: Because Msi expression rose during progression (Extended Data Fig. 1f-k, 4a), and marked therapy resistant cells, we tested if genetic or pharmacologic targeting of Msi could eradicate this ‘high risk’ population. Deletion of Msi1 led to a 5-fold reduction in tumor volume by MRI (Fig. 2a-b, Extended Data Fig. 4b, Supplementary Videos S2-S4). Histologically, adenocarcinoma areas comprised 67% of WT-KPf/fC but less than 10% of Msi1−/−KPf/fC pancreata; further while Msi1 loss allowed low grade PanINs to form, it largely blocked progression to adenocarcinoma (Fig. 2c-f, Extended Data Fig. 4 c, d). Finally, Msi1 deletion improved survival in orthotopic grafts: median survival for WT-KPf/fC graft recipients was 28.5 days, and for Msi1−/−-KPf/fC grafts was 70.5 days, representing a 2.5-fold increase in survival time and a 23-fold decrease in risk of death (Fig. 2g).

View Article: PubMed Central - PubMed

ABSTRACT

Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53, and SMAD42-4. To date, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavor. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression in both genetic models and patient derived xenografts. Specifically, we developed Msi reporter mice that allowed image based tracking of stem cell signals within cancers, revealing that Msi expression rises as PanIN progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbor the capacity to propagate adenocarcinoma, are enriched in circulating tumor cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in PanIN progression to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumors, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumor penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signaling as a central regulator of pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus