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Image-based detection and targeting of therapy resistance in pancreatic adenocarcinoma

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 resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53 and SMAD4 (refs 2, 3, 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.

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Figure 1: Msi reporter+ pancreatic cancer cells are enriched for tumour-initiating capacity.
Figure 2: Loss of Msi1 or Msi2 impairs tumour initiation and progression in a genetic mouse model of pancreatic cancer.
Figure 3: Msi controls expression of key oncogenic and epigenetic signals.
Figure 4: Targeting MSI inhibits pancreatic cancer growth in patient-derived xenografts.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray and RNA-seq data have been deposited in the Gene Expression Omnibus under accession numbers GSE73312 and GSE75797.

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Acknowledgements

We are grateful to I. Verma, M. Karin, and D. Cheresh for advice and comments on the manuscript, A. Luo and T. Wang for technical support, G. Yeo for advice on Msi targeting, K. Jenne for advice on MRI imaging, N. Patel and P. Mischel for reagents and experimental advice, and E. O’Conner and K. Marquez for cell sorting. R.F. is a recipient of a California Institute for Regenerative Medicine interdisciplinary stem cell training program fellowship and received support from T32 HL086344 and T32 CA009523, C.K. received support from T32 GM007752, N.K.L. received support from T32 GM007752 and a National Research Service Award F31 CA206416, J.L.K. received support from National Institutes of Health (NIH)-F32CA136124 and an Advanced Postdoctoral Fellowship from the Juvenile Diabetes Research Foundation, and B.Z. received support from T32 GM007184-33 (Duke University). F.P. is a recipient of a California Institute for Regenerative Medicine interdisciplinary stem cell training program fellowship and the University of California San Diego Clinical and Translational Research Institute KL2 Award. T.I. is the recipient of a California Institute for Regenerative Medicine interdisciplinary stem cell training program fellowship, J.B. is supported by a postdoctoral fellowship from National Cancer Center, and T.R. was supported in part by a Leukemia and Lymphoma Society Scholar Award. P.M.G. and M.A.H. are supported by a Specialized Program of Research Excellence (SPORE) in Pancreatic Cancer, CA127297, a TMEN Tumor Microenvironment Network U54, a National Cancer Institute Cancer Center Support Grant P30 CA36727, and an Early Detection Research Network (EDRN) U01 CA111294, M.Sa. is supported by NIH DK078803 and NIH CA194839, J.K.S. is supported by NIH K08CA168999, R.S. is supported by the Clinical and Translational Research Institute (CTRI) grant UL1TR001442, and A.M.L. is supported by donations from Ride the Point. This work was also supported by CA155620 to A.M.L., DK63031, HL097767, DP1 CA174422, and R35 CA197699 to T.R., and CA186043 to A.M.L. and T.R.

Author information

Authors and Affiliations

Authors

Contributions

R.F. designed and performed all experiments related to Msi expression and deletion, whole genome and target analysis, and ASO delivery in pancreatic cancer; N.K.L. designed and performed all live imaging of Msi reporter pancreatic tumours, and provided functional analysis of cancer stem cells, circulating tumour cells, and therapy resistance; R.F., N.K.L., and M.K. helped write the paper; D.V.J. performed histological analysis, and provided mouse and xenograft models; F.P., T.I., J.B., C.K., and B.Z. provided experimental data and advice; R.S. performed all bioinformatics analysis; M.Y., S.S., and H.O. provided Msi1−/− mice and CLIP-seq analysis; M.V. and D.P. performed pathology/in situ hybridization analysis; M.Sc. performed MRI analysis; J.K. and M. Sa. provided experimental advice, tumour samples, and mouse models; J.S., A.M.L., M.V., P.A.G., and M.A.H. provided patient samples; Y.K. and R.M. designed, synthesized, and screened MSI ASOs, and provided advice on ASO-related experiments. A.M.L. and T.R. conceived the project, planned and guided the research, and wrote the paper.

Corresponding authors

Correspondence to Andrew M. Lowy or Tannishtha Reya.

Ethics declarations

Competing interests

T.I. and T.R. are named inventors on a patent application number 61/178,370 titled ‘Diagnostic and treatment for chronic and acute myeloid leukemia’. Y.K. and A.R.M. are employees and shareholders of Ionis Pharmaceuticals.

Extended data figures and tables

Extended Data Figure 1 The Musashi genes MSI1 and MSI2 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 cells; yellow arrow indicates a MSI1+ 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 cells while yellow dotted regions indicate MSI2+ 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 cells; yellow arrow indicates a MSI1+ 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+ 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 cells; yellow arrows indicate MSI1+ 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+ cells. d, Quantification of MSI1 and MSI2 expression in four patients comparing primary pancreatic adenocarcinoma to the patient-matched liver metastasis; four images analysed per patient. e, Quantification of the frequency of MSI1+ and MSI2+ cells in four patients comparing primary pancreatic adenocarcinoma to the patient-matched liver metastasis; four images analysed 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 ± s.e.m. Total magnification ×200 (ac). Source data for all panels are available online.

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Extended Data Figure 2 Validation of Msi1 and Msi2 reporter mice.

a, FACS analysis of Msi2 reporter expression in haematopoietic 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+ haematopoietic 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 (possibly mesenchymal population). f, Immunofluorescence analysis of Msi1 and Msi2 expression overlap in isolated EpCAM+ KPf/fC cells (n = 3, 1,000 total cells analysed from 3 independent experiments). Data are represented as mean ± s.e.m. 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 tumour; VE-cadherin (magenta), Hoescht (blue), Msi reporter (green). See also Fig. 1c, d. Source data for all panels are available online.

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Extended Data Figure 3 Analysis of stem cell traits in Msi1 and Msi2 reporter+ KPf/fC populations.

a, ALDH expression in reporter+ tumour 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+ tumour cells (n = 3 each; 90 total cells analysed from 3 REM1-KPf/fC and 150 total cells analysed from 3 REM2-KPf/fC). (c) Average Msi expression in ALDH+ cells from REM1-KPf/fC and REM2-KPf/fC tumours (n = 3 independent experiments for each genotype). d, e, Representative images of spheres formed from (d) Msi1 and (e) Msi2 reporter+ and reporter tumour cells. See also Fig. 1g, h. f, g, In vivo tumour growth of Msi2 reporter+ or Msi reporter KPf/fC cells at (f) 500 or (g) 1,000 cells (n = 16). See also Fig. 1i. (h) Survival of mice orthotopically transplanted with 10,000 Msi2 reporter+ and reporter KPf/fC tumour cells (n = 6). See also Fig. 1j. Log-rank (Mantel–Cox) survival analysis (P < 0.05). i, j, Reporter frequency in REM2-KPf/fC mice treated with vehicle or 200 mg per kg (body weight) gemcitabine (n = 3 each). See also Fig. 1m, n for high-dose (500 mg per kg (body weight)) gemcitabine. Data are represented as mean ± s.e.m. ***P < 0.001 by Student’s t-test or one-way ANOVA. k, Msi2 reporter KPf/fC cells do not turn on Msi2 expression after 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 h. Representative series of images from 10 μM gemcitabine treatment. Reporter cells (red); GFP reporter+ cells (green); tracking of Msi2 reporter cells (white arrows); tracking of Msi2 reporter+ cells (yellow arrows) (n = 3 independent experiments). Source data for all panels are available online.

Source data

Extended Data Figure 4 Analysis of tumours 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/+ (pancreatic ductal adenocarcinoma, n = 3 samples) mice with quantification of Msi2 fluorescence in keratin+ cells. b, Average weights of WT-KPf/fC (n = 13) and Msi1−/−-KPf/fC tumours (n = 9). See also Fig. 2a, b for tumour 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, Tumours from 11- to 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 percentage of Keratin+ tumour cells (red) expressing Ki67 (green); DAPI staining is shown in blue. e, Average weights of WT-KPf/fC (n = 5) and Msi2−/−-KPf/fC tumours (n = 7). See also Fig. 2h, i for tumour volume analysis. Data are represented as mean ± s.e.m. *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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Extended Data Figure 5 Selection for escaper Msi-expressing cells in Msi1, Msi2 single and double knockout KPf/fC mice.

ac, 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). eg, Immunohistochemical staining for (e) IgG control, (f, red) Msi1, and (g, red) Msi2 in a 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 tumours (n = 35). Source data for all panels are available online.

Source data

Extended Data Figure 6 Genome-wide analysis of Msi controlled programs in pancreatic cancer.

a, Genome-wide expression analysis of dissociated pancreatic tumours. Microarray analysis was performed on RNA from three 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 PaCa-2 cells. c, Gene changes specific to MSI1-knockdown (turquoise) or MSI2-knockdown (purple) in MIA PaCa-2 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 PaCa-2 cells. Within scatterplots, lighter colour corresponds to a probability > 0.5 and the darker colour corresponds to a probability > 0.75. Source data for all panels are available online.

Extended Data Figure 7 Molecular targets of Msi signalling.

a, b, Quantitative 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). e, Analysis of direct Msi targets: Msi consensus binding sites in 3′ UTR of BRD4, HMGA2, and cMET transcripts. f, g, Phospho-cMet staining in WT-KPf/fC and (f) Msi1−/−-KPf/fC, (g) Msi2−/−-KPf/fC mice; keratin (magenta), phospho-cMet (green), DAPI (blue). See Fig. 3b–c for quantified data. h, Colony formation of MIA PaCa-2 cells infected with empty vector or cMET overexpression vector (three independent experiments) shows no impact of overexpressed cMet on control MIA PaCa-2 (control for cMet-mediated rescue of MSI knockdown in Fig. 3f). Data are represented as mean ± s.e.m. ***P < 0.001, ****P < 0.0001 by Student’s t-test. Source data for all panels are available online.

Source data

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 tumour cells before and after transplantation. See also Fig. 4a, b for patients 1 and 2. d, e, MSI1 expression after free uptake of (d) control ASO or (e) MSI1-ASO2 in human pancreatic cancer line (n = 3 per condition). See also Fig. 4c for impact of MSI1-ASO1. fj, ASO delivery in vivo. f, Target knockdown efficacy of lead-optimized ASO in KPf/fC stem cells. Malat1 expression in EpCAM+/ALDH+ and EpCAM+/ALDH cells after systemic delivery of control ASO or lead-optimized Malat1-ASO in autochthonous KPf/fC model (n = 3 independent experiments). See also Fig. 4h for target knockdown in unfractionated EpCAM+ cells. g, h, Analysis of potential toxicity of MSI-ASO: g, cage weight of mice receiving daily treatment of MSI1 ASO-1 (50 mg per kg (body weight)) or vehicle by intraperitoneal injection; four mice per cage; cage weight was measured every 3 days; h, average body weight of mice after 3 weeks of daily treatment with MSI1 ASO-1 (50 mg per kg (body weight)) or vehicle by intraperitoneal injection (n = 4 mice/cohort). In vivo delivery of MSI1 ASOs (50 mg per kg (body weight)) had no deleterious impact on body weight and maintained plasma chemistry markers (AST, ALT, BUN, T.Bil) within 3× upper limit of normal. i, j, Representative images of in situ hybridization for Malat1 (purple) in pancreatic tumours isolated from KPf/fC mice treated by daily intraperitoneal injection with (i) control ASO (50 mg per kg (body weight)) or (j) Malat1-ASO (50 mg per kg (body weight)) for 14 days. Source data for all panels are available online.

Source data

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 ten 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 (black dotted outlines) and acinar cells (blue squares) in caerulein-treated 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 with normal pancreas (n = 14). Data are represented as mean ± s.e.m. ****P < 0.0001 by Student’s t-test. Source data for all panels are available online.

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Supplementary information

Live imaging of tumor bearing REM2-KPf/fC mouse

4D view of Msi2 reporter+ pancreatic cancer cells within tumor mass. VE-cadherin (magenta), Hoechst (blue) and Msi2 reporter (green). (MOV 13841 kb)

MRI of wild type mouse

3-D projection of MRI image from control mouse with normal pancreas. (MPG 12540 kb)

MRI of WT-KPf/fC mouse

3-D projection of MRI image from WT-KPf/fC mouse (11 weeks) (MPG 20443 kb)

MRI of Msi1-/--KPf/fC mouse

3-D projection of MRI image from Msi1-/--KPf/fC mouse (11 weeks) (MPG 11032 kb)

MRI of WT-KPf/fC mouse

3-D projection of MRI image from WT-KPf/fC mouse, (13 weeks) (MPG 14210 kb)

MRI of Msi2-/--KPf/fC mouse

3-D projection of MRI image from Msi2-/--KPf/fC mouse, (13 weeks) (MPG 12749 kb)

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Fox, R., Lytle, N., Jaquish, D. et al. Image-based detection and targeting of therapy resistance in pancreatic adenocarcinoma. Nature 534, 407–411 (2016). https://doi.org/10.1038/nature17988

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