Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging
- Kyoto University Graduate School of Medicine, Japan
- Klinikum rechts der Isar Technische Universität München, Germany
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Although rigorous efforts identified the presence of 'cancer stem cells (CSCs)' in PDAC and molecular markers for them, stem cell dynamics in vivo have not been clearly demonstrated. Here we focused on Doublecortin-like kinase 1 (Dclk1), known as a CSC marker of PDAC. Using genetic lineage tracing with a dual-recombinase system and live imaging, we showed that Dclk1+ tumor cells continuously provided progeny cells within pancreatic intraepithelial neoplasia, primary and metastatic PDAC and PDAC-derived spheroids in vivo and in vitro. Furthermore, genes associated with CSC and epithelial mesenchymal transition were enriched in mouse Dclk1+ and human DCLK1-high PDAC cells. Thus, we provided direct functional evidence for the stem cell activity of Dclk1+ cells in vivo, revealing the essential roles of Dclk1+ cells in expansion of pancreatic neoplasia in all progressive stages.
Data availability
Microarray data have been deposited in GEO under accession codes GSE139167.
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Gene expression profiles of Dclk1+ and Dclk1- PDAC cellsNCBI Gene Expression Omnibus, GSE139167.
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Expression data from Mayo Clinic Pancreatic Tumor and Normal samplesNCBI Gene Expression Omnibus, GSE16515.
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Integrative Survival-Based Molecular Profiling of Human Pancreatic CancerNCBI Gene Expression Omnibus, GSE32676.
Article and author information
Author details
Funding
Grants-in-Aid KAKENHI (26293173)
- Hiroshi Seno
Naito Foundation (N/A)
- Hiroshi Seno
Princess Takamatsu Cancer Research Fund (13-24514)
- Tsutomu Chiba
Princess Takamatsu Cancer Research Fund (17-24924)
- Hiroshi Seno
Takeda Science Foundation (201749741)
- Hiroshi Seno
Uehara Memorial Foundation (201720143)
- Hiroshi Seno
Mochida Foundation (201356)
- Tsutomu Chiba
Mochida Foundation (2017bvAg)
- Hiroshi Seno
Mitsubishi Foudation (281119)
- Hiroshi Seno
Mitsubishi Foudation (201910037)
- Hiroshi Seno
European Research Council (648521)
- Dieter Saur
Grants-in-Aid KAKENHI (15H06334)
- Takahisa Maruno
Deutsche Forschungsgemeinschaft (1374/4-2)
- Dieter Saur
Grants-in-Aid KAKENHI (16K09394)
- Akihisa Fukuda
Grants-in-Aid KAKENHI (16K15427)
- Hiroshi Seno
Grants-in-Aid KAKENHI (17H04157)
- Hiroshi Seno
Grants-in-Aid KAKENHI (19H03639)
- Akihisa Fukuda
apan Agency for Medical Research and Development (19cm0106142h0002)
- Hiroshi Seno
apan Agency for Medical Research and Development (19cm6010022h0002)
- Akihisa Fukuda
Kobayashi Foundation for Cancer Research (N/A)
- Hiroshi Seno
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were approved by the animal research committee of the Kyoto University and performed in accordance with Japanese government regulations. All surgery was performed under Isoflurane anesthesia, and every effort was made to minimize suffering.
Human subjects: Surgically resected specimens of pancreatic cancer tissues were obtained from patients who had been admitted to Kyoto University Hospital. Written informed consent was obtained from all patients and study protocol (#G1200-1) was approved by Ethics Committee of Kyoto University Hospital.
Copyright
© 2021, Maruno et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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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
Further reading
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TAK1 is a serine/threonine protein kinase that is a key regulator in a wide variety of cellular processes. However, the functions and mechanisms involved in cancer metastasis are still not well understood. Here, we found that TAK1 knockdown promoted esophageal squamous cancer carcinoma (ESCC) migration and invasion, whereas TAK1 overexpression resulted in the opposite outcome. These in vitro findings were recapitulated in vivo in a xenograft metastatic mouse model. Mechanistically, co-immunoprecipitation and mass spectrometry demonstrated that TAK1 interacted with phospholipase C epsilon 1 (PLCE1) and phosphorylated PLCE1 at serine 1060 (S1060). Functional studies revealed that phosphorylation at S1060 in PLCE1 resulted in decreased enzyme activity, leading to the repression of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. As a result, the degradation products of PIP2 including diacylglycerol (DAG) and inositol IP3 were reduced, which thereby suppressed signal transduction in the axis of PKC/GSK-3β/β-Catenin. Consequently, expression of cancer metastasis-related genes was impeded by TAK1. Overall, our data indicate that TAK1 plays a negative role in ESCC metastasis, which depends on the TAK1-induced phosphorylation of PLCE1 at S1060.
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Cell crowding is a common microenvironmental factor influencing various disease processes, but its role in promoting cell invasiveness remains unclear. This study investigates the biomechanical changes induced by cell crowding, focusing on pro-invasive cell volume reduction in ductal carcinoma in situ (DCIS). Crowding specifically enhanced invasiveness in high-grade DCIS cells through significant volume reduction compared to hyperplasia-mimicking or normal cells. Mass spectrometry revealed that crowding selectively relocated ion channels, including TRPV4, to the plasma membrane in high-grade DCIS cells. TRPV4 inhibition triggered by crowding decreased intracellular calcium levels, reduced cell volume, and increased invasion and motility. During this process, TRPV4 membrane relocation primed the channel for later activation, compensating for calcium loss. Analyses of patient-derived breast cancer tissues confirmed that plasma membrane-associated TRPV4 is specific to high-grade DCIS and indicates the presence of a pro-invasive cell volume reduction mechanotransduction pathway. Hyperosmotic conditions and pharmacologic TRPV4 inhibition mimicked crowding-induced effects, while TRPV4 activation reversed them. Silencing TRPV4 diminished mechanotransduction in high-grade DCIS cells, reducing calcium depletion, volume reduction, and motility. This study uncovers a novel pro-invasive mechanotransduction pathway driven by cell crowding and identifies TRPV4 as a potential biomarker for predicting invasion risk in DCIS patients.
