Gastroenterology

Gastroenterology

Volume 137, Issue 3, September 2009, Pages 1102-1113
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
Combined Targeted Treatment to Eliminate Tumorigenic Cancer Stem Cells in Human Pancreatic Cancer

https://doi.org/10.1053/j.gastro.2009.05.053Get rights and content

Background & Aims

Pancreatic cancers contain exclusively tumorigenic cancer stem cells (CSCs), which are highly resistant to chemotherapy, resulting in a relative increase in CSC numbers during gemcitabine treatment. Signaling through sonic hedgehog and mammalian target of rapamycin (mTOR), respectively, may be essential for CSC self-renewal and could represent putative targets for novel treatment modalities.

Methods

We used in vitro and in vivo models of pancreatic cancer to examine the effects of sonic hedgehog inhibition (cyclopamine/CUR199691) and mTOR blockade (rapamycin) on the tumorigenic CSC population.

Results

Surprisingly, neither cyclopamine nor rapamycin alone or as supplements to chemotherapy were capable of effectively diminishing the CSC pool. Only the combined inhibition of both pathways together with chemotherapy reduced the number of CSCs to virtually undetectable levels in vitro and in vivo. Most importantly, in vivo administration of this triple combination in mice with established patient-derived pancreatic tumors was reasonably tolerated and translated into significantly prolonged long-term survival.

Conclusions

The combined blockade of sonic hedgehog and mTOR signaling together with standard chemotherapy is capable of eliminating pancreatic CSCs. Further preclinical investigation of this promising approach may lead to the development of a novel therapeutic strategy to improve the devastating prognosis of patients with pancreatic cancer.

Section snippets

Human Pancreatic Cancer Cell Line

The highly metastatic human pancreatic cancer cell line L3.6pl was maintained as previously described.5 CSC spheres were cultured in Dulbecco's modified Eagle medium/F12 supplemented with B27 (Gibco, Karlsruhe, Germany) and FGF-2 (PeproTech EC, London, England).

Primary Human Pancreatic Cancer Cells

Human pancreatic tumors were obtained with written informed consent from all patients. Tissue fragments were minced and enzymatically digested with collagenase (Stem Cell Technologies, Vancouver, British Columbia, Canada) for 90 minutes

In Vitro Evaluation of Anti-CSC Agents

First we screened for potentially effective substances for targeted elimination of CD133+ pancreatic CSCs by means of flow cytometry following 48 hours of therapy.5 Consistent with previous reports, we observed a marked enrichment of CD133+ cells following gemcitabine therapy (Figure 1A).5 Because a gemcitabine concentration of 100 ng/mL was the lowest concentration capable of achieving a strong relative enrichment for CD133+ cells through depletion of CD133 cells and similar dosages have been

Discussion

Considering the still devastating prognosis of patients with pancreatic cancer,1 the development of novel therapeutic strategies is a prerequisite to eventually achieve better outcomes. Previous studies including our own were the first to show that pancreatic cancers contain a rare population of undifferentiated cells that are characterized by expression of CD133 or CD44/CD24,5, 14 are exclusively tumorigenic, and are highly resistant to chemotherapy.5, 9 Treatment of pancreatic cancer with the

Acknowledgments

The authors thank Ana Castillo, Sonia Alcalá, Aurora Garcia, Martin Luckner, Virginia Granda, and Karin Nispel for their excellent technical assistance. This report includes data generated during the doctoral thesis of M.-T.M. at the Medical School of the Ludwig-Maximilian-University Munich (manuscript in preparation).

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    Conflicts of interest The authors disclose the following: Ludwig-Maximilian-University has filed a patent application for the use of the described treatment modality for epithelial cancer. The authors (M.-T.M., P.C.H., S.H., and C.H.) are inventors of this patent and may receive royalties from licensees. The remaining authors disclose no conflicts.

    Funding Supported by the Dr Mildred Scheel Foundation (108168 to C.H.), Friedrich-Baur Foundation, ERC Advanced Investigator Grant (Pa-CSC 233460), and Ludwig-Maximilian University (research grant to M.-T.M.).

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