Original Article/PancreasTumor–stromal cross-talk modulating the therapeutic response in pancreatic cancer
Introduction
Pancreatic cancer is one of the most lethal types of cancer [1]. Although substantial improvements in therapy have been made over the last three decades, the current 5-year survival rate is only 7% [2]. At the time of diagnosis only 15% of the patients are eligible for curative surgical resection [3]. Late diagnosis, rapid tumor progression, early metastasis, resistance to radiation and poor response to chemotherapeutic reagents are the main reasons for the poor clinical outcome of patients with pancreatic cancer.
The tumor's microenvironment is very important for chemotherapy. One of the major contributors towards the tumor's chemoresistance is the stromal component which represents up to 90% of the tumor volume [4], [5]. Understanding the complex interaction between tumor and stromal cells has been very challenging and many different experimental, pathological and clinical approaches have been made.
Tumor stroma is very heterogeneous and characterized by dense desmoplastic reactions, involving various extracellular matrix (ECM) proteins, cancer associated fibroblasts (CAFs), endothelial cells, immune cells and neural networks. The key players producing ECM proteins, such as collagen type I and fibronectin have been identified to be CAFs [6]. These cells interact with tumor cells in a paracrine way [7] and by direct cell–cell contact [8]. It has been previously shown that CAFs inhibit apoptosis of pancreatic cancer cells and interact via various mitogenic substances, stimulate tumor growth, invasion and resist chemotherapy [9]. Therefore, new therapeutic approaches are being developed specifically targeting the stroma.
Although stroma-targeted therapies are at the center of attention by now, recent findings of in vivoand in vitro models have questioned the purely beneficial synergy of stroma and tumor cells [4]. Froeling et al. showed that non-malignant stromal cells have a protective character and reduce the total number of tumor cells in a three dimensional co-culture model [10]. Moreover, a targeted therapeutic approach reducing the stroma volume by inhibiting the Sonic hedgehog (Shh) pathway resulted in an increased proliferation, more aggressive tumor and a reduced survival in mice [11]. In a different mouse model, the depletion of α-smooth muscle actin (α-SMA) active myofibroblasts led to a smaller tumor size, but with a higher insensitivity to gemcitabine and decreased overall survival [12].
Additionally, results of phase I and II studies of different stromal targeted therapies remained inconclusive [13]. Whilst the reduction of stroma increased the overall survival in a phase I hyaluronidase study [14], the overall survival was decreased significantly in a phase II study using the Shh inhibitor Saridegib so that the phase II study had to be suspended [15]. Moreover, a clinical study including 66 patients who underwent surgical resection, proved a strong and significant positive correlation between high stromal density and longer disease-free and overall survival [16]. Based on these results, it can be assumed that CAFs retain some protective elements and targeting the stromal component of the tumor has to be considered cautiously.
One central way of tumor–stromal cross-talk is provided by paracrine interactions. Mediators such as platelet derived growth factor (PDGF), TNF-α, TNF-β and pro-inflammatory cytokines such as IL-1 and IL-6 have been identified to play a vital role in the tumor–stromal interaction [17], [18], [19]. All of them influenced tumor and stroma proliferation, invasion and migration. Although the influence of various mediators has been studied extensively, more attention should be drawn to the effects of these mediators when CAFs and stromal cells interact and are simultaneously exposed to chemotherapeutic agents.
Many research groups have studied the effect of gemcitabine and other drugs on pancreatic cell lines in the absence of CAFs [20], [21], [22]. The interaction of cancer cells and CAFs is not clear. Preceding experiments examined the therapeutic response of cancer cells, when exposed to CAF pre-conditioned medium [23], [24], [25], [26]. In another case the effect of ECM protein pre-coated plates on cancer cells was investigated [27]. The actual response of standard chemotherapeutic agents on monocultures of CAFs has not yet been studied.
Moreover, when handling CAFs, most studies used CAFs isolated from rats [28], [29], mice [5] or immortalized human CAFs [24], [30], [31], [32]. Fewer groups have been using primary derived native CAFs as their cell growth is slow and replicative potential is limited [28], [33], [34]. It has been reported that immortalization of CAFs induced significant genetic instability with a preferential selection of malignant subclones [35]. Therefore, functional tests investigating the chemotherapeutic effects on CAFs and on co-cultures of CAFs with cancer cells should be conducted with native human CAFs.
This study proposed a novel method by which the efficacy of chemotherapeutic substances can be studied in the context of tumor–stromal interactions. We used primary derived native CAFs from six different patients and analyzed the way in which tumor and stromal cells are affected when exposed to the following three different therapeutic agents: gemcitabine, nab-paclitaxel and the γ-secretase-inhibitor (GSI) DAPT. The latter provided an example for a small molecule targeted therapy inhibiting the Notch pathway. Lastly, the correlation between chemoresistance and IL-6 concentrations were also investigated.
Section snippets
Cell lines
AsPC and Panc-1 were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin and cotrimoxazol at a concentration of 110 µg/mL. Cell lines were incubated in a humidified atmosphere at 37 °C with 5% CO2 and passaged by trypsin/EDTA detachment, centrifugation and resuspension in growth medium.
Reagents
RPMI 1640, FBS superior and trypsin/EDTA solution were purchased from Biochrom (Berlin, Germany) and penicillin/streptomycin from Life Technologies
Characterization of CAFs
Primary patient derived stromal cells were immunohistochemically analyzed with respect to the three markers typically expressed by CAFs: α-SMA, GFAP and vimentin [36], [37]. Staining was positive in all cases, indicating that all stromal cells isolated were CAFs (Fig. 2B-D). Since all markers stained positive for 100% of CAFs isolated, contamination with other cell types (i.e. immune and tumor cells) could be excluded.
Visualizing the morphology of co-cultures by crystal violet staining
Monocultures of CAFs were observed to form a homogenous two dimensional layer
Discussion
The microenvironment of pancreatic cancer is highly complex and it is important to understand the interactions between cancer cells, CAFs, endothelial, immune and endocrine cells [38]. A particular interest has been drawn to the stromal component of the tumor. Since this makes up the majority of the tumor mass in many cases [4], stromal is believed to play a pivotal role in the tumor's chemoresistance [5].
So far paracrine effects between CAFs and cancer cells have been studied extensively with
Acknowledgments
We thank Anja Schirmeier for the close collaboration by isolating primary cultures of cancer associated fibroblasts.
Contributors
BM and SRB proposed the study. NCCM and VHE performed the research. NCCM and SRB wrote the first draft. NCCM, VHE, RSA, SRB and BM collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. NCCM is the guarantor.
Funding
The study was supported by a grant from Charité – Universitaetsmedizin Berlin in house funding.
Ethical approval
This study was approved by the Ethics Committee of Charité – Universitaetsmedizin Berlin.
Competing interest
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
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