1. Cancer Biology

Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging

  1. Takahisa Maruno
  2. Akihisa Fukuda  Is a corresponding author
  3. Norihiro Goto
  4. Motoyuki Tsuda
  5. Kozo Ikuta
  6. Yukiko Hiramatsu
  7. Satoshi Ogawa
  8. Yuki Nakanishi
  9. Yuichi Yamaga
  10. Takuto Yoshioka
  11. Kyoichi Takaori
  12. Shinji Uemoto
  13. Dieter Saur
  14. Tsutomu Chiba
  15. Hiroshi Seno  Is a corresponding author
  1. Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Japan
  2. Division of Hepatobiliary-Pancreatic Surgery and Transplantation, Department of Surgery, Kyoto University Graduate School of Medicine, Japan
  3. Department of Internal Medicine II, Klinikum rechts der Isar Technische Universität München, Germany
  4. Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Germany
  5. Kansai Electric Power Hospital, Fukushima-ku Osaka-shi, Japan
https://doi.org/10.7554/eLife.55117
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Cite this article
  1. Takahisa Maruno
  2. Akihisa Fukuda
  3. Norihiro Goto
  4. Motoyuki Tsuda
  5. Kozo Ikuta
  6. Yukiko Hiramatsu
  7. Satoshi Ogawa
  8. Yuki Nakanishi
  9. Yuichi Yamaga
  10. Takuto Yoshioka
  11. Kyoichi Takaori
  12. Shinji Uemoto
  13. Dieter Saur
  14. Tsutomu Chiba
  15. Hiroshi Seno
(2021)
Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging
eLife 10:e55117.
https://doi.org/10.7554/eLife.55117
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7 figures, 1 table and 4 additional files

Figures

Figure 1
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Dclk1+ cells presented in PanINs and pancreatic ductal adenocarcinomas (PDACs) also expressed cancer stem cell (CSC) markers.

(A–D) Histological analysis of PanINs developed in 3-month-old Pdx1-Flp; KrasFSF-G12D/+ (KF) mice. (A) Hematoxylin and Eosin (H and E) staining. (B) Alcian Blue staining. (C) Immunostaining for …

Figure 1—source data 1

This spreadsheet contains the source data for Figure 1D.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig1-data1-v2.docx
Figure 1—source data 2

This spreadsheet contains the source data for Figure 1G.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig1-data2-v2.docx
Figure 1—source data 3

This spreadsheet contains the source data for Figure 1H.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig1-data3-v2.docx
Figure 1—source data 4

This spreadsheet contains the source data for Figure 1I.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig1-data4-v2.docx
Figure 1—source data 5

This spreadsheet contains the source data for Figure 1J.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig1-data5-v2.docx
Figure 2
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Dclk1+ PanIN cells supply descendant PanIN cells.

(A) The scheme of Dclk1CreERT2-IRES-EGFP/+; Rosa26mTmG/+; Pdx1-Flp; KrasFSF-G12D/+ (DRKF) mouse constructs, flippase-mediated Kras activation, and CreERT2-driven reporter recombination. (B) The …

Figure 2—source data 1

This spreadsheet contains the source data for Figure 2G.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig2-data1-v2.docx
Figure 3 with 2 supplements
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Dclk1+ pancreatic ductal adenocarcinoma (PDAC) cells supply descendant PDAC cells in vivo.

(A) The scheme of Dclk1CreERT2-IRES-EGFP/+; Rosa26mTmG/+; Pdx1-Flp; KrasFSF-G12D/+; Trp53frt/frt (DRKPF) mouse constructs, flippase-mediated Kras activation and Trp53 deletion, and CreERT2-driven …

Figure 3—source data 1

This spreadsheet contains the source data for Figure 3I.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig3-data1-v2.docx
Figure 3—figure supplement 1
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Dclk1creERT2-IRES-EGFP mice had no leakiness of Cre expression.

(A) Immunofluorescence staining for Dclk1 (magenta), GFP (green), and Hoechst (blue) of Dclk1CreERT2-IRES-EGFP/+; Pdx1-Flp (DF) mice, Dclk1CreERT2-IRES-EGFP/+; Pdx1-Flp; KrasFSF-G12D/+ (DKF) mice …

Figure 3—figure supplement 1—source data 1

Analysis of the leak of Dclk1CreERT2.

The number of EGFP+ cells in Dclk1− cells of pancreatic epithelium in DRF, DRKF, and DRKPF mice with immunofluorescent staining.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig3-figsupp1-data1-v2.docx
Figure 3—figure supplement 2
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Dclk1+ cells supplied progeny in pancreatic ductal adenocarcinoma (PDAC) spheroids.

(A) Immunofluorescence staining for Dclk1 (magenta), GFP (green), and Hoechst (blue) of spheroids derived from PDAC of DRKPF mice. (B) Experimental strategy of Cre-mediated lineage tracing in …

Figure 3—figure supplement 2—source data 1

Lineage tracing of Dclk1+ cells in established spheroids from pancreatic ductal adenocarcinomas (PDACs) of DRKPF mice.

The number of EGFP+ cells before (day 0) and 3 days after 4-OHT administration (day 3).

https://cdn.elifesciences.org/articles/55117/elife-55117-fig3-figsupp2-data1-v2.docx
Figure 4
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Dclk1+ cells supplied progeny in liver tumors from splenic transplantation.

(A) Macroscopic image of liver tumors 2 months after splenic injection of pancreatic ductal adenocarcinoma (PDAC) spheroids derived from DRKPF mice. (B and C) Histological analysis of the metastatic …

Figure 4—source data 1

Lineage tracing of Dclk1+ cells in established mouse metastatic liver tumors.

Measurement of EGFP+ area in liver tumor area derived from spleen-injected pancreatic ductal adenocarcinomas (PDACs) before (day 0) and 14 days after tamoxifen injection. Image J was used for the measurement.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig4-data1-v2.docx
Figure 5
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Longitudinal imaging of PanINs and pancreatic ductal adenocarcinomas (PDACs) in live mice indicate Dclk1+ PDAC cells supply progeny PDAC cells.

(A) Photo of an abdominal imaging window (AIW) composed of a custom-made magnet ring and 12 mm cover glass. (B) Scheme of the microscopic observation of the pancreas through an AIW. CA: …

Figure 6
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Dclk1+pancreatic ductal adenocarcinoma (PDAC) cells have remarkable spheroid- and tumor-forming potential.

(A) Scheme of FACS and 3D culture of Dclk1+ and Dclk1− PDAC cells from DKPF mice. (B) FACS-sorting of PDAC cells on the basis of Epcam and Dclk1 expression in DKPF mice. (C and D) Representative …

Figure 6—source data 1

Growth of pancreatic ductal adenocarcinoma (PDAC) xenograft.

Measured value of increasing curve of subcutaneous tumor derived from Dclk1+ PDACs cells sorted by FACS.

https://cdn.elifesciences.org/articles/55117/elife-55117-fig6-data1-v2.docx
Figure 7
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Gene expression profile revealed Dclk1+pancreatic ductal adenocarcinoma (PDAC) cells possessed cancer stem cell potential.

(A) The heatmap of representative differentially expressed genes with a significant difference, p<0.01 (Student’s t-test) in gene expression. (B) Gene set enrichment analysis (GSEA) in Dclk1+ versus …

Tables

Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information
Gene Mus musculusDclk1NCBI Gene DatabaseNCBI Gene: 13175
Gene (Homo sapiens)DCLK1NCBI Gene
Database		NCBI Gene: 9201
Genetic reagent (Mus musculus)Dclk1CreERT2-IRES-EGFPGenerated in our laboratory Nakanishi et al., 2013N/A
Genetic reagent (Mus musculus)Pdx1-FlpSaur D. Schönhuber et al., 2014N/A
Genetic reagent (Mus musculus)KrasFSF-G12DSaur D. Schönhuber et al., 2014N/A
Genetic reagent (Mus musculus)Rosa26mTmGSaur D. Schönhuber et al., 2014N/A
Genetic reagent (Mus musculus)Trp53frtJackson Laboratory Schönhuber et al., 2014RRID:IMSR_JAX:017767
Genetic reagent (Mus musculus)CAnN.Cg-Foxn1nu/CrlCharles riverN/ABALB/c-nu
Genetic reagent (Mus musculus)NOD.CB17-Prkdcscid/JJackson LaboratoryRRID:IMSR_JAX:001303NOD scid
AntibodyRabbit polyclonal anti-Dcamkl1AbcamCat#: ab31704
RRID:AB_873537		1:200
AntibodyGoat polyclonal anti-Dcamkl1Santa Cruz BiotechnologyCat#: sc46312
RRID:AB_2090091		1:50
AntibodyRat monoclonal anti-Cd24AbcamCat#: ab64064
RRID:AB_2291132		1:100
AntibodyRat monoclonal anti-Cd44AbcamCat#: ab119863
RRID:AB_10898986		1:100
AntibodyRat monoclonal anti-Cd326Thermo Fisher ScientificCat#: 13-5791-82
RRID:AB_1659713		1:100
AntibodyRabbit polyclonal anti-Aldh1a1AbcamCat#: ab23375
RRID:AB_2224009		1:100
AntibodyRat monoclonal anti-Cd326 (EpCAM) eFluor450Thermo Fisher ScientificCat#: 48-5791-82
RRID:AB_10717090		1:50
AntibodyGoat polyclonal anti-GFPAbcamCat#: ab6673
RRID:AB_305643		1:100
AntibodyMouse monoclonal anti-CytokeratinDAKOCat#: IR05361-2J
RRID:AB_2868599		1:1
Sequence-based reagentDclk1-CreERT2 Forward1This paperPCR primersCGAGCTGGACGGCGACGTAAACG
Sequence-based reagentDclk1-CreERT2 Forward2This paperPCR primersGATGGACTCAAGAAGATCTCC
Sequence-based reagentDclk1-CreERT2 ReverseThis paperPCR primersAGTGACCCTTAGTGACCCTTAGT
Sequence-based reagentPdx1-Flp ForwardSaur D. Schönhuber et al., 2014PCR primersAGAGAGAAAATTGAAACAAGTGCAGGT
Sequence-based reagentPdx1-Flp ReverseSaur D. Schönhuber et al., 2014PCR primersCGTTGTAAGGGATGATGGTGAACT
Sequence-based reagentKras Common ForwardSaur D. Schönhuber et al., 2014PCR primersCACCAGCTTCGGCTTCCTATT
Sequence-based reagentKras WT ReverseSaur D. Schönhuber et al., 2014PCR primersAGCTAATGGCTCTCAAAGGAATGTA
Sequence-based reagentKras FSF MUT reverseSaur D. Schönhuber et al., 2014PCR primersGCGAAGAGTTTGTCCTCAACC
Sequence-based reagentp53-frt1Jackson LaboratoryPCR primersCAAGAGAACTGTGCCTAAGAG
Sequence-based reagentp53-frt2Jackson LaboratoryPCR primersCTTTCTAACAGCAAAGGCAAGC
Software, algorithmImage JNational Institutes of HealthRRID:SCR_003070https://imagej.net/
Software, algorithmInSight DeepSee LaserSpectra PhysicsRRID:SCR_012362http://www.scienceexchange.com/facilities/multiphoton-microscopy-core-rochester
Software, algorithmFlowJoFlowJo, LLCRRID:SCR_008520https://www.flowjo.com/

Additional files

Supplementary file 1

GO enrichment analysis up to 100 Go terms on DAVID, GO Biological process using 2171 genes significantly (p<0.01) highly expressed in Dclk1+ PDAC cells.

https://cdn.elifesciences.org/articles/55117/elife-55117-supp1-v2.docx
Supplementary file 2

Pathway analysis on DAVID, KEGG Pathway using 2171 genes significantly (p<0.01) highly expressed in Dclk1+ PDAC cells.

https://cdn.elifesciences.org/articles/55117/elife-55117-supp2-v2.docx
Supplementary file 3

GO analysis (GO Biological Process) and pathway analysis (KEGG Pathway) on genes that were significantly highly expressed (p<0.05) in the DCLK1 high expression group up to 50 Go terms or pathways.

https://cdn.elifesciences.org/articles/55117/elife-55117-supp3-v2.docx
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https://cdn.elifesciences.org/articles/55117/elife-55117-transrepform-v2.docx

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