| dc.description.abstracteng | Pancreatic cancer has the poorest survival rate of the most common types of cancers with a 5 year survival rate of only 3-5% after diagnosis. Since there has been no significant improvement in the clinical outcome for several decades, there is an urgent need for novel concepts in the understanding of the pathophysiology, diagnosis and treatment of PDAC.
Therefore, the aim of the study was to characterize the in vivo tumor progression and metastatic behavior of PDAC orthotopic mouse models, developing PDAC tumors with different grades of differentiation and to assess novel imaging strategies as well as a new combination therapy in some of these models.
To this end, PDAC xenografts were extensively characterized after orthotopic transplantation of five different human PDAC cell lines with known grade of differentiation (AsPC-1, MIA PaCa-2 and PANC-1 as poorly differentiated, BxPC-3 as moderately differentiated and Capan-1 as well differentiated) in the head of the pancreas of nude mice. Tumors that developed in each mouse model showed distinct tumor take rates and different time of tumor development, independently from the grade of differentiation. All of them presented with invasion into the stomach and duodenum, resembling the classic features of PDAC progression in humans. The differentiated tumors were composed of tumor cells organized in duct-like clusters (Capan-1), as seen in humans, and in round clusters (BxPC-3), whereas the poorly differentiated tumors (AsPC-1, MIA PaCa-2 and PANC-1) showed a typical unorganized morphology. Moreover, by analyzing the presence of PSCs and collagen deposition, in each PDAC mouse model different degrees of a desmoplastic reaction, another hallmark of human PDAC, were observed. Similar to human PDAC, high amounts of collagen fibers around the duct-like cell clusters were only found in Capan-1 tumors.
Then, two EGFR expressing PDAC models, AsPC-1 and MIA PaCa-2, were applied for testing the suitability of near-infrared fluorescent (NIRF) labeled Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), as a new tool for fluorescence-guided surgery (FGS). Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux-Alexa-647) and the co-injected control human IgG Alexa 750 conjugate (hIgG-Alexa-750) (25 µg each) were studied over 48 h by NIRF imaging (Optix MX2 and IVIS) in mice bearing orthotopic AsPC-1 and MIA PaCa-2 tumors. Results obtained here showed that Cetux-Alexa-647 but not the control hIgG-Alexa-750 fluorescence was specifically detected in vivo and ex vivo in the primary pancreatic tumors and metastases as small as 1 mm, with maximum intensities at 24 h. LT analysis and NIRF microscopy of tumor sections confirmed the binding specificity of Cetux-Alexa-647 to PDAC cells, whereas a multimodal approach applying 3D NIRF imaging and CT of AsPC-1 mice receiving both, the Cetux-Alexa-647 and iodine containing CT contrast agent, confirmed that the Cetux-Alexa-647 signal localized at the primary pancreatic tumor in correlation with the anatomical structure of the mice. Moreover, during fluorescence-guided dissection, performed 24 h after injection of the probe using the Quest Spectrum clinical device for FGS, Cetuximab conjugated to IRDye-800CW (Cetux-800CW) enabled fast and straightforward real time delineation of AsPC-1 tumor margins and also of small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part, was stained with Cetux-800CW. Tumor cells invading into the stomach and duodenum were also detected. Moreover, ex vivo scans of organs with detected metastases during fluorescence-guided dissection, also revealed high signals only at the metastatic sites and not in the healthy tissue. Taken together, NIRF labeled Cetuximab might be a promising tool for the visualization of tumor margins and metastatic sites during FGS, allowing a precise surgical resection and consequently aiding the decrease of disease recurrence.
Finally, a combination of Erlotinib, a tyrosine kinase inhibitor (TKi) of EGFR, and Cariporide, a specific inhibitor of NHE1, was tested in vivo in the highly metastatic AsPC-1 mouse model to determine their effect on tumor growth, invasion and metastatic spread. 10 days after cell transplantation AsPC-1 mice were divided in 4 groups and treated for 20 days with Erlotinib (50 mg/kg) or Cariporide (3 mg/kg) only or with the combination of both. The control group received saline solution and Captisol 6%. Primary tumor and tumor spread in different peritoneal organs (liver, spleen, kidney, diaphragm, mesentery and site of surgical incision) were analyzed. Quantification of the metastatic spread was difficult to obtain, due to the low number of metastatic findings in each organ and to the high variation of the data within each treated group. However, Erlotinib/Cariporide-double treated mice presented with less metastatic spread to different organs such as liver, kidney and mesentery, and tumor infiltration of the site of surgical incision. Moreover, Erlotinib/Cariporide-double therapy showed a slight but no significant inhibitory effect on tumor growth as compared to controls, and decreased the grade of tumor infiltration in the stomach. Individual therapies with either Erlotinib and Cariporide did not show any therapeutic response, neither to tumor growth nor to tumor invasion in the stomach, but the metastatic spread to the kidney (Erlotinib treated group) and to the diaphragm (Cariporide-treated group) was slightly reduced. Taken together, this study provides a preclinical proof-of-concept for a novel anti-metastatic activity of Erlotinib/Cariporide-double therapy based on the inhibition of both, EGFR, whose overexpression usually correlates with an advanced cancer disease, and NHE1, a protein interconnected with the EGFR downstream signaling and implicated in the pH regulation of cell invasion.
Overall, these studies confirm that PDAC orthotopic bearing mice are useful tools for testing novel imaging and therapeutic strategies, as well as for studying the pathophysiology of the disease, such as expression of tumor markers and stroma interaction. | de |