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. 2016 Jun 16;534(7607):407-411.
doi: 10.1038/nature17988. Epub 2016 Jun 6.

Image-based Detection and Targeting of Therapy Resistance in Pancreatic Adenocarcinoma

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Free PMC article

Image-based Detection and Targeting of Therapy Resistance in Pancreatic Adenocarcinoma

Raymond G Fox et al. Nature. .
Free PMC article

Abstract

Pancreatic intraepithelial neoplasia is a pre-malignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53 and SMAD4 (refs 2-4). So far, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavour. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression both in genetic models and in 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 pancreatic intraepithelial neoplasia progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbour the capacity to propagate adenocarcinoma, are enriched in circulating tumour 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 the progression of pancreatic intraepithelial neoplasia to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumours, 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 tumour 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 signalling as a central regulator of pancreatic cancer.

Figures

Extended Data Figure 1
Extended Data Figure 1. The Musashi genes MSI1 and MSI1 are expressed in human pancreatic adenocarcinoma
(a, top row) Representative images of a primary patient pancreatic adenocarcinoma sample stained with anti-keratin (green), DAPI (blue), and anti-MSI1 (red) antibodies. White arrows indicate MSI1 negative cells; yellow arrow indicates a MSI1 positive cell (a, bottom row) Representative images of a primary patient pancreatic adenocarcinoma sample stained with anti-keratin (green), DAPI (blue), and anti-MSI2 (red) antibodies. White dotted regions indicate MSI2 negative cells while yellow dotted regions indicate MSI2 positive cells. (b, top row) Representative images of a primary patient pancreatic adenocarcinoma sample stained with anti-keratin (green), DAPI (blue), and anti-MSI1 (red) antibodies. White arrows indicate MSI1 negative cells; yellow arrow indicates a MSI1 positive cell. (b, bottom row) Representative images of a primary patient pancreatic adenocarcinoma sample stained with anti-keratin (green), DAPI (blue), and anti-MSI2 (red) antibodies. Yellow dotted region indicates MSI2 positive cells. (c, top row) Representative images of a matched liver metastasis from a patient with pancreatic adenocarcinoma stained with anti-keratin (green), DAPI (blue), and anti-MSI1 (red) antibodies. White arrows indicate MSI1 negative cells; yellow arrows indicate MSI1 positive cells. (c, bottom row) Representative images of a matched liver metastasis from a patient with pancreatic adenocarcinoma stained with anti-keratin (green), DAPI (blue), and anti-MSI2 (red) antibodies. Yellow dotted region indicates MSI2 positive cells. (d) Quantification of MSI1 and MSI2 expression in four patients comparing primary pancreatic adenocarcinoma to the patient matched liver metastasis; 4 images analyzed per patient. (e) Quantification of the frequency of MSI1 and MSI2 positive cells in four patients comparing primary pancreatic adenocarcinoma to the patient matched liver metastasis; 4 images analyzed per patient. (f) MSI1 and (g) MSI2 expression in normal pancreas (n=1), PanIN (n=9), and pancreatic adenocarcinoma samples (n=9). (h) Quantification of MSI2 expression from a human tissue array comparing Grade 1 (well-differentiated, n=9), Grade 2 (moderately differentiated, n=12), and Grade 3 (poorly differentiated, n=16) adenocarcinoma relative to normal pancreas (n=14) and normal adjacent pancreas (n=16). (i) MSI1 and (j) MSI2 expression in well-differentiated, moderately differentiated, and poorly differentiated human pancreatic cancer cell lines (n=3 independent experiments). (k) Colony formation of well-differentiated, moderately differentiated, and poorly differentiated human pancreatic cancer cell lines (n=3 independent experiments). Data are represented as mean ± SEM. Total Magnification 200x A-B. Source Data for all panels are available online.
Extended Data Figure 2
Extended Data Figure 2. 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.
Extended Data Figure 3
Extended Data Figure 3. Analysis of stem cell traits in Msi1 and Msi2 reporter+ KPf/fC populations
(a) ALDH expression in reporter positive tumor cells sorted from REM1-KPf/fC (top row) and REM2-KPf/fC (bottom row) mice; ALDH1 (red), DAPI (blue) and GFP or YFP (green). (b) Average ALDH expression in bulk or Msi1 and Msi2 reporter positive tumor cells (n=3 each; 90 total cells analyzed from 3 REM1-KPf/fC and 150 total cells analyzed from 3 REM2-KPf/fC). (c) Average Msi expression in ALDH+ cells from REM1-KPf/fC and REM2-KPf/fC tumors (n=3 independent experiments for each genotype). (d-e) Representative images of spheres formed from (d) Msi1 and (e) Msi2 reporter+ and reporter- tumor cells. See also Figure 1g-h. (f-g) In vivo tumor growth of Msi2 reporter+ or Msi reporter- KPf/fC cells at (f) 500 or (g) 1000 cells (n=16). See also Figure 1i. (h) Survival of mice orthotopically transplanted with 10,000 Msi2 reporter+ and reporter- KPf/fC tumor cells (n=6). See also Figure 1j. Log-rank (Mantel-Cox) survival analysis (p<0.05). (i-j) Reporter frequency in REM2-KPf/fC mice treated with vehicle or 200mg/kg Gemcitabine (n=3 each). See also Figure 1m-n for high dose (500mg/kg) Gemcitabine. Data are represented as mean ± SEM. *** P < 0.001 by Student's t-test or One-way ANOVA. (k) Msi2 reporter-negative KPf/fC cells do not turn on Msi2 expression following in vitro gemcitabine treatment, suggesting that Msi-reporter+ cells are differentially resistant to Gemcitabine. Low passage Msi2 reporter KPf/fC cells loaded with DiI were live-imaged continuously for up to 48 hours. Representative series of images from 10μM gemcitabine treatment. Reporter-negative cells (red); GFP reporter-positive cells (green); tracking of Msi2 reporter-negative cells (white arrows); tracking of Msi2 reporter-positive cells (yellow arrows) (n=3 independent experiments). Source Data for all panels are available online.
Extended Data Figure 4
Extended Data Figure 4. Analysis of tumors from Msi null 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.
Extended Data Figure 5
Extended Data Figure 5. Selection for escaper Msi expressing cells in Msi1, Ms2 single and double knockout KPf/fC mice
(a-c) Immunohistochemical staining for (a) IgG control (n=4), or (b-c, red) Msi2 in 13 week old WT-KPf/fC (n=4) and Msi2−/−KPf/fC (n=4) mice. (d) Immunohistochemical staining for Msi2 (red) in 22 week old Msi2−/−KPf/fC mouse (n=1). (e-g) Immunohistochemical staining for (e) IgG control, (f, red) Msi1 and (g, red) Msi2 in 15-week-old Msi1f/fMsi2−/− double knockout KPf/fC mouse (n=1). (h) Survival curves of Msi1f/fMsi2−/−-KPf/fC (n=6) or WT-KPf/fC tumors (n=35). Source Data for all panels are available online.
Extended Data Figure 6
Extended Data Figure 6. Genome wide analysis of Msi controlled programs in pancreatic cancer
(a) Genome wide expression analysis of dissociated pancreatic tumors. Microarray analysis was performed on RNA from 3 pairs of WT-KPf/fC and Msi1−/−-KPf/fC matched littermates. Heat map shows differential expression of selected mRNAs identified as part of a stem cell associated gene signature. (b) Concordantly (upper right and lower left quadrants) and discordantly (upper left and lower right quadrants) regulated genes (red) in MSI1-knockdown and MSI2-knockdown MIA PaCa2 cells. (c) Gene changes specific to MSI1-knockdown (turquoise) or MSI2-knockdown (purple) in MIA PaCa2 cells. (d) Heat maps indicating concordant, MSI1 specific, and MSI2 specific genes. (e) Venn diagram displaying the intersection of probe sets that are differentially regulated in MSI1-knockdown, MSI2-knockdown and double knockdown of MSI1 and MSI2 in MIA PaCa2 cells. Within scatterplots, lighter color corresponds to a probability >0.5 and the darker color corresponds to a probability >0.75. Source Data for all panels are available online.
Extended Data Figure 7
Extended Data Figure 7. Molecular targets of Msi signaling
(a-b) Real-time PCR analysis of (a) Msi1 and (b) Msi2 expression in MIA PaCa-2 human pancreatic cancer cells relative to normal pancreas (n=3 independent experiments). (c-d) Analysis of shRNA knockdown efficiency in GFP+ sorted MIA PaCA-2 cells infected with GFP tagged lentiviral shRNA against scrambled control sequences, (c) MSI1 or (d) MSI2 (n=3 independent experiments). Analysis of direct Msi targets (e) Msi consensus binding sites in 3'UTR of Brd4, Hmga2 and c-Met transcripts. (f-g) Phospho-c-Met staining in WT-KPf/fC and (f) Msi1−/−-KPf/fC, (g) Msi2−/−-KPf/fC mice; Keratin (magenta), phospho-c-Met (green), DAPI (blue). See Figure 3b-c for quantified data. (h) Colony formation of MIA PaCa-2 cells infected with empty vector or c-MET overexpression vector (3 independent experiments) shows no impact of overexpressed c-Met on control MIA PaCa-2 (control for c-Met mediated rescue of MSI knockdown in Figure 3f). Data are represented as mean ± SEM. *** P < 0.001, **** P < 0.0001 by Student's t-test. Source Data for all panels are available online.
Extended Data Figure 8
Extended Data Figure 8. Analysis of impaired pancreatic cancer growth with shMSI and MSI1-ASOs
(a) Schematic for inhibiting MSI in primary patient-derived xenografts. (b-c) Frequency of GFP+ patient tumor cells before and after transplantation. See also Figure 4a-b for Patient#1, #2. ASO delivery in vivo (d, e) MSI1 expression following free uptake of (d) control ASO or (e) MSI1-ASO2 in human pancreatic cancer line (n=3 per condition). See also Figure 4c for impact of MSI1-ASO1. Target knockdown efficacy of lead optimized ASO in KPf/fC stem cells (f) Malat1 expression in EpCAM+/ALDH+ and EpCAM+/ALDH- cells following systemic delivery of lead-optimized control ASO or Malat1-ASO in autocthonous KPf/fC model (n=3 independent experiments) See also Figure 4h for target knockdown in unfractionated Epcam+ cells. Analysis of potential toxicity of MSI-ASO (g) Cage weight of mice receiving daily treatment of MSI1 ASO-1 (50mg/kg) or vehicle by IP injection; 4 mice per cage; cage weight was measured every 3 days. (h) Average body weight of mice following 3 weeks of daily treatment with MSI1 ASO-1 (50mg/kg) or vehicle by IP injection (n=4 mice/cohort). In vivo delivery of MSI1 ASOs (50mg/kg) had no deleterious impact on body weight and maintained plasma chemistry markers (AST, ALT, BUN, T.Bil) within 3x ULN (upper limit of normal). (i-j) Representative images of in situ hybridization for Malat1 (purple) in pancreatic tumors isolated from KPf/fC mice treated by daily IP injection with (i) control ASO (50mg/kg) or (j) Malat1-ASO (50mg/kg) for 14 days. Source Data for all panels are available online.
Extended Data Figure 9
Extended Data Figure 9. Elevated expression of Msi in pancreatitis
Msi2 expression in a caerulein-induced mouse model of pancreatitis, and in human pancreatitis. (a) Msi2 staining and (b) quantification of 10 images per group in pancreas from PBS-treated (a, top panels, n=1) and caerulein-treated mice (a, bottom panels, n=1). (c) Msi2 immunohistochemical staining in islets (purple circles) and acinar cells (blue squares) in caerulein or PBS treated mice (n=1 for each group). (d) Immunofluorescent staining of Msi2 (green) in DBA+ ductal cells (red) treated with PBS (left panels) or caerulein (right panels) (n=1 for each group); DAPI is shown in blue. (e) MSI2 expression in human tissue arrays from patients presenting with mild chronic inflammation (n=4) and chronic pancreatitis (n=6) compared to normal pancreas (n=14). Data are represented as mean ± SEM. **** P < 0.0001 by Student's t-test. Source Data for all panels are available online.
Figure 1
Figure 1. Msi reporter positive pancreatic cancer cells are enriched for tumor initiating capacity
(a-b) Design of Msi reporter constructs (REM1, Msi1eYFP/+; REM2, Msi2eGFP/+). (c-d) Live images of Msi reporter cells in (c) REM1-KPf/fC and (d) REM2-KPf/fC tumors; VE-cadherin (magenta), Hoechst (blue), Msi reporter (green). (e-f) Msi1 and Msi2 reporter expression in dissociated tumors (n=6). (g-h) Sphere-forming ability of Msi-reporter+ and reporter- cells (g, n=8; h, n=6). (i) In vivo growth of Msi2 reporter+ tumor cells (n=8). (j) Survival of mice orthotopically transplanted with Msi2 reporter+ and reporter- KPf/fC tumor cells (n=6). Log-rank (Mantel-Cox) survival analysis (p<0.05). (k) Reporter frequency in primary tumors (n=3), and CTCs from ascites (n=3) or peripheral blood (n=4). (l) Average frequency of tumor-spheres from Msi2 reporter+ and reporter- CTCs (n=3 technical replicates). (m-n) Reporter frequency in REM2-KPf/fC mice treated with vehicle or 500mg/kg Gemcitabine (n=6). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by Student's t-test or One-way ANOVA. Source Data for all panels are available online.
Figure 2
Figure 2. Loss of Msi1 or Msi2 impairs tumor initiation and progression in a genetic mouse model of pancreatic cancer
(a) Coronal and sagittal MRI images of normal, WT-KPf/fC and Msi1−/−-KPf/fC mice with 3-dimensional volume rendering of tumor mass (red). (b) Average volumes of isolated WT-KPf/fC (n=13) and Msi1−/−-KPf/fC tumors (n=9). (c-d) Histology and (e-f) quantification of PanIN and/or adenocarcinoma areas in WT-KPf/fC and Msi1−/−-KPf/fC tumors. (g) Survival of mice orthotopically grafted with Msi1−/−-KPf/fC or WTKPf/fC tumors (n=16). Analysis of Msi2−/−-KPf/fC tumors (h) by MRI and (i) after isolation, WT-KPf/fC (n=5), Msi2−/−-KPf/fC (n=7). (j-m) Histology of WT-KPf/fC and Msi2−/−-KPf/fC pancreatic tumors (40x magnification); (k) Adenocarcinoma, liver invasion (green arrows), (l) adenocarcinoma (yellow arrows), (m) PanINs (blue arrows). (n-o) quantification of PanIN and/or adenocarcinoma areas in WT-KPf/fC and Msi2−/−-KPf/fC tumors (n=6). (p) Survival of autochthonous Msi2−/−-KPf/fC (n=19) or WT-KPf/fC (n=32) mice. Log-rank (Mantel-Cox) survival analysis (p<0.0001). Data represented as mean ± SEM. ** P < 0.01, *** P < 0.001 by Student's t-test. Source Data for all panels are available online.
Figure 3
Figure 3. Msi controls expression of key oncogenic and epigenetic signals
(a) Msi RIP-PCR for indicated transcripts. (b-c) Frequency of phospho-c-Met+ cells in WT-KPf/fC, Msi1−/−-KPf/fC, and Msi2−/−-KPf/fC mice, (b, n=8; c, n=6). (d) Schematic of c-MET exons and 3'UTR. CLIP tags (red triangles) indicate MSI1 binding in 3'UTR. (e) c-MET 3'UTR luciferase reporter activity in the presence or absence of MSI1 or MSI2 (n=3 independent experiments). (f) Colony formation of MSI1 or MSI2 knockdown cells with or without c-MET (n=4 independent experiments). (g-h) FACs analysis of tumors from Gemcitabine-treated REM2-KPf/fC mice, in the presence or absence of Crizotinib and iBet762; Vehicle (n=7), Gemcitabine (n=3), Gemcitabine+iBet762 (n=3), Gemcitabine+Crizotinib (n=3). Data represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t-test or One-way ANOVA. ns, not significant. Source Data for all panels are available online.
Figure 4
Figure 4. Targeting MSI inhibits pancreatic cancer growth in patient-derived xenografts
(a-b) Frequency of GFP+ tumor cells before and after transplantation. (c) MSI1 expression following MSI1-ASO free uptake in human pancreatic cancer line (n=3 independent experiments/dose). (d) Colony formation of control or MSI1-ASO treated human pancreatic cancer line (n=3 independent experiments). (e) In vivo growth of human cell line-derived tumors in control or MSI1-ASO treated mice (n=10). (f) Relative tumor volume and (g) rate of growth of KPf/fC-derived tumors in control or MSI1-ASO treated mice (n=8). (h) Malat1 expression in autocthonous KPf/fC tumors following systemic delivery of lead-optimized control or Malat1-ASO (n=6). Data represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by One-way ANOVA. ns, not significant. Source Data for all panels are available online.

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