Collectively, the results of this study point to the promising role of verteporfin-based PDT to eradicate stubborn populations that are non-responsive to gemcitabine and perhaps other standard chemotherapy agents for treatment of pancreatic cancer. Our results indicate that PDT with verteporfin overcomes multiple mechanisms of gemcitabine resistance and is effective against the troublesome pancreatic cancer cell populations that are non-responsive to gemcitabine. It has been previously shown that gemcitabine resistant pancreatic cancer cells are also more invasive and migratory, thus further underscoring the importance of our finding that verteporfin PDT can target this potentially lethal population (48
). Gemcitabine resistance in pancreatic cancer cells has previously been linked with Bcl-XL, a mitochondrial anti-apoptotic factor (6
). Our results () show that verteporfin PDT targets this very factor, thus optimally leveraging the inherent preferential localization of this photosensitizer to the mitochondria to attack a key site for undermining this gemcitabine resistance mechanism exploited by pancreatic cancer cells. Interestingly, comparison of PDT treatment in naïve cells () and as part of a regimen in cells that have been subject to high dose gemcitabine () indicates that PDT efficacy is agnostic to prior exposure to gemcitabine treatment. Furthermore the contrasting sites and mechanisms of each modality may have interesting implications that warrant further investigation in more complex preclinical models that would allow for a broader application.
The results of this study in five pancreatic cancer cell lines point to the broad conclusion that these cells are generally responsive to PDT with significant populations that are non-responsive to gemcitabine. However, evaluation of response in this diverse and well-characterized panel of cell lines exhibiting a range of disease histopathology allows for focused correlation between treatment response and tumor grade, stage, and specific immunocytochemical features (37
). In general, our results are consistent with an inverse correlation between the grade and histologic differentiation and sensitivity to therapeutics. Of the cell lines tested, the two adenocarcinoma lines derived from tumors with poor histologic differentiation (AsPC-1 and PANC-1) (39
), both very clearly exhibit the least sensitivity to PDT and gemcitabine treatment. AsPC-1 and PANC-1 are also derived from the highest grade tumors (G2 and G3 respectively, WHO grading system) (38
). Capan-2, which are of moderate histologic differentiation, also exhibit less sensitivity to gemcitabine than either Capan-1 (well differentiated) or BxPC-3 (adenosquamous) but share approximately the same verteporfin PDT dose response relationship with these cell lines. Of the five cell lines used in this study there is also a correlation between the cell lines which have been reported by Sipos et al to have the strongest expression of vimentin (38
), AsPC-1 and PANC-1, and the least sensitivity to therapeutics. Additionally, Capan-2, AsPC-1 and PANC-1, the cell lines with poor gemcitabine response, and with the exception of Capan-2, poor response to verteporfin PDT, have all been shown to overexpress cytokeratin (CK) 8, CK18, and CK19, in contrast to Capan-1 and BxPC-3 which stain positively for these markers in less than 10% of cells. While it is too early to make any firm conclusions from these in vitro studies these findings point to the utility of further investigation of these targets in more elaborate in vivo studies to identify populations that will benefit most from verteporfin PDT and gemcitabine treatments.
This study also brings to light provocative observations on the critical role of the tumor microenvironment in drug response that are relevant to the present discussion as to the role of the rigid fibrotic stroma that surrounds pancreatic tumors serving potentially, as both a barrier to drug penetration and a signaling partner. While recent studies have made the case that enhancing gemcitabine delivery through the hypovascular stroma improves treatment response (14
), it has also been asserted that the role of drug penetration is overstated as pancreatic cancer cells are innately non-responsive to therapeutics, even when barriers to delivery are not present (11
). In this study we show that PANC-1 cells, a primary tumor derived cell line (which would exist in this characteristic stroma-rich environment) actually become more resistant to gemcitabine treatment when grown in contact with a bed of laminin-rich basement membrane. In these experiments examining the role of stromal signaling in a culture geometry where drug penetration is explicitly not a factor, we demonstrate that indeed the mere presence of extracellular matrix changes treatment sensitivity. It is important to note that in contrast to previous studies in which we and others have utilized overlay growth on GFR Matrigel for three-dimensional cell culture, the brief period of overlay growth gel surface in the present study allows for adhesion to the matrix and early events in the change of phenotype in the matrix rich environment without formation of large multicellular 3D nodules, which require extended periods in culture. Our results (), using the laminin-rich basement membrane Matrigel, are consistent with a previous report from Miyamoto et al that the presence of laminin in cultures of PANC-1 and other pancreatic cell lines reduced cytotoxicity of 5-FU (15
). This observation could be explained by increased treatment resistance through interaction of cancer cells with the stroma via cell-matrix receptors such as integrins (49
). It is further interesting to note that while gemcitabine treatment is clearly less effective in Matrigel adherent cells, verteporfin PDT efficacy is only modestly decreased indicating that the mechanism of cytotoxicity of verteporfin PDT, by direct damage to the mitochondria, may bypass the mechanisms through which matrix interactions lead to an apparent decrease in gemcitabine sensitivity observed here. Runnels et al have shown that BPD-PDT decreases the function of α5
integrin, thus potentially disrupting this resistance pathway (45
). This observation suggests the utility of further investigation using more fully developed methods for three-dimensional culture of pancreatic cancer cell lines in extracellular matrix and quantitative methods for analysis of growth and treatment response in 3D systems, building on recent studies from our own lab in other 3D and in vivo
disease models (31
These early studies, combined with ongoing clinical work point to the promising role of PDT in the therapeutic armamentarium for pancreatic cancer. The demonstrated capability of verteporfin-based PDT to bypass gemcitabine resistance could be especially important given the widespread lack of response to this front line therapeutic. Building on the central findings of this study, further investigation into PDT and gemcitabine combination regimens is warranted to identify the optimal dose and scheduling of each modality in more sophisticated pre-clinical models and also to investigate different routes of delivery. For example, nanoconstructs that allow delivery of multiple therapeutics (50
) could be used for simultaneous delivery of BPD, gemcitabine and other agents.