Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 7, 2018; 24(41): 4663-4678
Published online Nov 7, 2018. doi: 10.3748/wjg.v24.i41.4663
Typing of pancreatic cancer-associated fibroblasts identifies different subpopulations
Michael Friberg Bruun Nielsen, Michael Bau Mortensen, Sönke Detlefsen
Michael Friberg Bruun Nielsen, Sönke Detlefsen, Department of Pathology, Odense University Hospital, Department of Clinical Research, University of Southern Denmark, Odense Pancreas Center (OPAC), Odense C 5000, Denmark
Michael Bau Mortensen, Department of Surgery, HPB Section, Odense University Hospital, Department of Clinical Research, University of Southern Denmark, Odense Pancreas Center (OPAC), Odense C 5000, Denmark
Author contributions: Mortensen MB and Detlefsen S initiated the study; Detlefsen S conceptualized the study; Nielsen MFB and Detlefsen S designed and coordinated the experiments; Nielsen MFB and Detlefsen S performed a majority of the experiments; Nielsen MFB and Detlefsen S evaluated the results; Nielsen MFB, Mortensen MB and Detlefsen S discussed and interpreted the data; Nielsen MFB and Detlefsen S wrote the manuscript; Nielsen MFB, Mortensen MB and Detlefsen S critically revised the manuscript.
Supported by Aase-and-Ejnar Danielsen’s Foundation, No. 10-001452; Brødrene Hartmann’s Foundation, No. A28308; the Foundation of 17.12.1981, No. 19024005; Karen S. Jensens Grant, No. 27-A1433; University of Southern Denmark Faculty Scholarship; Odense University Hospital Free Research Fund, No. 29-A1500, 22-A1133 and 49-A2379; Odense University Hospital Ph.D. stipend, No. 1032; and Odense Pancreas Center (OPAC).
Institutional review board statement: The study was approved by the Ethical Committee of the Region of Southern Denmark (project ID: S-20140168 and project ID: S-20150130).
Conflict-of-interest statement: The authors declare no conflict of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Sönke Detlefsen, MD, PhD, Associate Professor, Department of Pathology, Odense University Hospital, University of Southern Denmark, J.B. Winsløws Vej 15, Odense C 5000, Denmark.
Telephone: +45-65414806 Fax: +45-65912943
Received: July 23, 2018
Peer-review started: July 23, 2018
First decision: August 25, 2018
Revised: October 12, 2018
Accepted: October 21, 2018
Article in press: October 21, 2018
Published online: November 7, 2018
Research background

The prognosis of pancreatic cancer (PC) patients remains extremely poor, and unlike other major forms of cancer, there has been no significant improvement in survival rates in recent years. This poor prognosis is mainly due to late-stage diagnosis and limited response to treatment. Hence, continued research into this devastating disease is urgently needed.

PC is characterized by abundant desmoplasia in the stroma surrounding the cancer cells. The desmoplastic stroma consists predominantly of the extracellular matrix (ECM) produced by cancer-associated fibroblasts (CAFs). The exact role of the desmoplastic stroma in PC progression remains unclear. Some studies have indicated that high stromal activity had a negative prognostic impact in resected PC patients, whereas stromal depletion of the entire fibroblast (FB) population promoted tumour growth in genetically engineered mouse models. We hypothesize that these conflicting studies could be explained by CAF heterogeneity in the desmoplastic stroma of PC, with some CAFs promoting and other CAFs hampering tumour growth.

Research motivation

CAFs are the main effector cells in the desmoplastic reaction in PC. However, it is currently unclear whether CAFs are promoters or inhibitors of tumour growth. Extensive effort has been made to design therapies that target the stromal compartments, including CAFs, in PC, but to date, these efforts have had limited success. This limited success is highlighted by a continually high mortality rate among PC patients. Identification of a panel of markers that could distinguish CAF subtypes would allow researchers to perform subsequent studies to determine the prognostic significance and precise functional properties of these subtypes in PC. Further, it could be speculated that future targeted therapies should be designed to specifically modulate the activity of certain CAF subtypes in PC.

Research objectives

The present study aimed to determine whether it is possible to identify markers that can distinguish different immune phenotypic subpopulations of CAFs in PC. After examining 45 CAF and ECM markers, we found that CD10, CD271, cytoglobin, DOG1, miR-21, nestin, and tenascin C are significantly differentially expressed in the juxtatumoural stroma versus the peripheral stroma in PC. Furthermore, a panel of the markers cytoglobin, CD271, and miR-21 allows the distinction of juxtatumoural and peripheral CAFs (j-CAFs and p-CAFs) in PC. Future studies should examine whether j-CAFs and p-CAFs hold prognostic value and/or have different functional properties in PC.

Research methods

The present study was predominantly based on immunohistochemistry (IHC), immunofluorescence (IF), double-IF (d-IF), histochemistry, quantitative reverse transcription PCR (qPCR) and in situ hybridization (ISH). We defined four different stromal compartments in surgical specimens of PC: the juxtatumoural, peripheral, lobular and septal stroma. Tissue microarrays were produced that contained all of the pre-defined compartments. Using a semi-quantitative 4-tiered scoring system, we evaluated the expression of 37 FB markers and 8 ECM markers to evaluate the compartment-specific expression of each individual marker.

Research results

In this study, we found that CD10, CD271, cytoglobin, DOG1, miR-21, nestin, and tenascin C exhibited significant differences in expression profiles between the juxtatumoural and peripheral compartments of the PC stroma. CD10, cytoglobin, DOG1, miR-21, and nestin were all expressed at significantly higher levels in j-CAFs than in p-CAFs. Similarly, tenascin C was more abundantly expressed in juxtatumoural ECM than in peripheral ECM. CD271 was the only of the examined markers to be expressed at higher levels in p-CAFs than in j-CAFs. A combination of the markers cytoglobin, CD271, and miR-21 can be used to identify the different immune phenotypic subpopulations of CAFs in PC.

Research conclusions

In the present study, by using d-IF for multiple combinations of markers as well as conventional IHC, IF, and ISH, we were able to identify different immune phenotypic subpopulations of CAFs in the PC stroma. Using a panel of immunohistochemical biomarkers, we could distinguish two immunophenotypically different populations of CAFs: Juxtatumoural CAFs (j-CAFs), which were in very close vicinity to the cancer cells, and peripheral CAFs (p-CAFs), which were located > 100 μm away from the cancer cells. Interestingly, some of the markers that we identified to be predominantly expressed in j-CAFs (CD10, miR-21) have previously been demonstrated to have negative prognostic value in PC, whereas CD271, a marker that we found to be expressed mainly in p-CAFs, has been shown to hold positive prognostic value in PC. These findings may indicate that j-CAFs may be involved in an “unholy alliance” with the cancer cells, whereas p-CAFs may promote reduction of tumour growth. Hence, CAF heterogeneity in PC may explain some of the previously published, seemingly conflicting data regarding the role of CAFs in PC progression. The findings from this study could indicate that the composition of CAF subtypes in the desmoplastic stroma of PC could affect the outcomes of individual patients. Furthermore, in the future, the stromal CAF composition may possibly be used as a marker to evaluate whether a specific PC patient might benefit from stroma-modulating therapies.

Research perspectives

This study indicates that it is too simplistic to view the CAF population in PC as a homogeneous cell population. Instead, at least two immune phenotypic subpopulations of CAFs can be characterized by different biomarker profiles. Future studies should determine whether the different CAF subpopulations in PC hold prognostic value or have different functional properties. Furthermore, CAF heterogeneity could provide an opportunity for the development of therapies aiming at the modulation of only one CAF subpopulation instead of targeting the entire CAF population in PC.