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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.

No MeSH data available.


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Validation of Msi1 and Msi2 reporter mice(a) FACs analysis of Msi2 reporter expression in hematopoietic stem cells, progenitors and lineage-positive differentiated cells. (b) Representative image of Msi1 expression in FACs sorted YFP+ neuronal cells; YFP (green), Msi1 (red), and DAPI (blue). (c) Representative image of Msi2 expression in FACs sorted GFP+ hematopoietic cells; GFP (green), Msi1 (red), and DAPI (blue). (d-e) Msi-expression in keratin+ cells. (d) Msi1-YFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM1-KPf/fC mice; (e) Msi2-GFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM2-KPf/fC mice. DAPI staining is shown in blue. Rare cells (<5%) were found to be keratin-negative (possibly mesenchymal population). (f) Immunofluorescence analysis of Msi1 and Msi2 expression overlap in isolated EpCAM+ KPf/fC cells (n=3, 1000 total cells analyzed from 3 independent experiments). Data are represented as mean ± SEM. (g-h) Survival of Msi reporter-KPf/fC and WT-KPf/fC mice. Survival curves of (g) Msi1YFP/+-KPf/fC (REM1-KPf/fC, n=21) or WT-KPf/fC (n=18) mice, and (h) Msi2GFP/+-KPf/fC (REM2-KPf/fC, n=65) or WT-KPf/fC (n=54) mice. (i) Live image of Msi2 reporter cells in REM2-KPf/fC tumor; VE-cadherin (magenta), Hoescht (blue), Msi reporter (green). See also Figure 1c-d. Source Data for all panels are available online.
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Figure 6: Validation of Msi1 and Msi2 reporter mice(a) FACs analysis of Msi2 reporter expression in hematopoietic stem cells, progenitors and lineage-positive differentiated cells. (b) Representative image of Msi1 expression in FACs sorted YFP+ neuronal cells; YFP (green), Msi1 (red), and DAPI (blue). (c) Representative image of Msi2 expression in FACs sorted GFP+ hematopoietic cells; GFP (green), Msi1 (red), and DAPI (blue). (d-e) Msi-expression in keratin+ cells. (d) Msi1-YFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM1-KPf/fC mice; (e) Msi2-GFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM2-KPf/fC mice. DAPI staining is shown in blue. Rare cells (<5%) were found to be keratin-negative (possibly mesenchymal population). (f) Immunofluorescence analysis of Msi1 and Msi2 expression overlap in isolated EpCAM+ KPf/fC cells (n=3, 1000 total cells analyzed from 3 independent experiments). Data are represented as mean ± SEM. (g-h) Survival of Msi reporter-KPf/fC and WT-KPf/fC mice. Survival curves of (g) Msi1YFP/+-KPf/fC (REM1-KPf/fC, n=21) or WT-KPf/fC (n=18) mice, and (h) Msi2GFP/+-KPf/fC (REM2-KPf/fC, n=65) or WT-KPf/fC (n=54) mice. (i) Live image of Msi2 reporter cells in REM2-KPf/fC tumor; VE-cadherin (magenta), Hoescht (blue), Msi reporter (green). See also Figure 1c-d. Source Data for all panels are available online.

Mentions: To address these questions, we first analyzed MSI expression in human pancreatic cancers. MSI1 and MSI2 were expressed in all primary tumor samples analyzed, with expression increasing during progression (Extended Data Fig. 1). To track the function of Msi-expressing cells, we developed Msi knock-in reporters (Reporter for Musashi, REM) in which fluorescent signals reflected endogenous Msi expression (Fig. 1a-b; Extended Data Fig. 2a-c). To define if Msi-expressing cells contribute to pancreatic cancer, we crossed REM mice to the KrasLSL-G12D/+;p53f/f; Ptf1aCRE/+ model13-15 (Extended Data Fig. 2d-h). In vivo imaging of living tumors revealed clear Msi1 and Msi2 reporter activity within remarkable spatially restricted domains frequently surrounded by blood vessels (Fig. 1c-d; Extended Data Fig. 2i, Supplementary Video S1). Cells with high levels of Msi reporter expression were rare, and detected in 1.18% and 9.7% of REM1 and REM2 cancers (Fig. 1e-f). Because cancer stem cells can be similarly rare16,17, we tested if Msi-expressing cells have preferential capacity for tumor propagation18. Consistent with this possibility, Msi+ cells expressed ALDH19, and were dramatically more tumorigenic in vitro and in vivo (Fig. 1g-i; Extended Data Fig. 3a-g). Most importantly, Msi2+ cells were highly lethal: while 100% of mice orthotopically transplanted with Msi2+ cells developed invasive tumors and died, none of the mice receiving Msi2− cells showed signs of disease (Fig. 1j, Extended Data Fig. 3h). Given the suggestion that certain markers may not consistently enrich for tumor propagating ability20, our findings indicate that Msi-expression can identify cancer stem cells at least in some contexts, and that Msi2 + cells preferentially drive pancreatic cancer growth, invasion and lethality.


Image based detection and targeting of therapy resistance in pancreatic adenocarcinoma
Validation of Msi1 and Msi2 reporter mice(a) FACs analysis of Msi2 reporter expression in hematopoietic stem cells, progenitors and lineage-positive differentiated cells. (b) Representative image of Msi1 expression in FACs sorted YFP+ neuronal cells; YFP (green), Msi1 (red), and DAPI (blue). (c) Representative image of Msi2 expression in FACs sorted GFP+ hematopoietic cells; GFP (green), Msi1 (red), and DAPI (blue). (d-e) Msi-expression in keratin+ cells. (d) Msi1-YFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM1-KPf/fC mice; (e) Msi2-GFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM2-KPf/fC mice. DAPI staining is shown in blue. Rare cells (<5%) were found to be keratin-negative (possibly mesenchymal population). (f) Immunofluorescence analysis of Msi1 and Msi2 expression overlap in isolated EpCAM+ KPf/fC cells (n=3, 1000 total cells analyzed from 3 independent experiments). Data are represented as mean ± SEM. (g-h) Survival of Msi reporter-KPf/fC and WT-KPf/fC mice. Survival curves of (g) Msi1YFP/+-KPf/fC (REM1-KPf/fC, n=21) or WT-KPf/fC (n=18) mice, and (h) Msi2GFP/+-KPf/fC (REM2-KPf/fC, n=65) or WT-KPf/fC (n=54) mice. (i) Live image of Msi2 reporter cells in REM2-KPf/fC tumor; VE-cadherin (magenta), Hoescht (blue), Msi reporter (green). See also Figure 1c-d. Source Data for all panels are available online.
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Figure 6: Validation of Msi1 and Msi2 reporter mice(a) FACs analysis of Msi2 reporter expression in hematopoietic stem cells, progenitors and lineage-positive differentiated cells. (b) Representative image of Msi1 expression in FACs sorted YFP+ neuronal cells; YFP (green), Msi1 (red), and DAPI (blue). (c) Representative image of Msi2 expression in FACs sorted GFP+ hematopoietic cells; GFP (green), Msi1 (red), and DAPI (blue). (d-e) Msi-expression in keratin+ cells. (d) Msi1-YFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM1-KPf/fC mice; (e) Msi2-GFP reporter (green, white arrows) and keratin (red) staining was performed on tissue sections of REM2-KPf/fC mice. DAPI staining is shown in blue. Rare cells (<5%) were found to be keratin-negative (possibly mesenchymal population). (f) Immunofluorescence analysis of Msi1 and Msi2 expression overlap in isolated EpCAM+ KPf/fC cells (n=3, 1000 total cells analyzed from 3 independent experiments). Data are represented as mean ± SEM. (g-h) Survival of Msi reporter-KPf/fC and WT-KPf/fC mice. Survival curves of (g) Msi1YFP/+-KPf/fC (REM1-KPf/fC, n=21) or WT-KPf/fC (n=18) mice, and (h) Msi2GFP/+-KPf/fC (REM2-KPf/fC, n=65) or WT-KPf/fC (n=54) mice. (i) Live image of Msi2 reporter cells in REM2-KPf/fC tumor; VE-cadherin (magenta), Hoescht (blue), Msi reporter (green). See also Figure 1c-d. Source Data for all panels are available online.
Mentions: To address these questions, we first analyzed MSI expression in human pancreatic cancers. MSI1 and MSI2 were expressed in all primary tumor samples analyzed, with expression increasing during progression (Extended Data Fig. 1). To track the function of Msi-expressing cells, we developed Msi knock-in reporters (Reporter for Musashi, REM) in which fluorescent signals reflected endogenous Msi expression (Fig. 1a-b; Extended Data Fig. 2a-c). To define if Msi-expressing cells contribute to pancreatic cancer, we crossed REM mice to the KrasLSL-G12D/+;p53f/f; Ptf1aCRE/+ model13-15 (Extended Data Fig. 2d-h). In vivo imaging of living tumors revealed clear Msi1 and Msi2 reporter activity within remarkable spatially restricted domains frequently surrounded by blood vessels (Fig. 1c-d; Extended Data Fig. 2i, Supplementary Video S1). Cells with high levels of Msi reporter expression were rare, and detected in 1.18% and 9.7% of REM1 and REM2 cancers (Fig. 1e-f). Because cancer stem cells can be similarly rare16,17, we tested if Msi-expressing cells have preferential capacity for tumor propagation18. Consistent with this possibility, Msi+ cells expressed ALDH19, and were dramatically more tumorigenic in vitro and in vivo (Fig. 1g-i; Extended Data Fig. 3a-g). Most importantly, Msi2+ cells were highly lethal: while 100% of mice orthotopically transplanted with Msi2+ cells developed invasive tumors and died, none of the mice receiving Msi2− cells showed signs of disease (Fig. 1j, Extended Data Fig. 3h). Given the suggestion that certain markers may not consistently enrich for tumor propagating ability20, our findings indicate that Msi-expression can identify cancer stem cells at least in some contexts, and that Msi2 + cells preferentially drive pancreatic cancer growth, invasion and lethality.

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