Preclinical evaluation of a novel CEA‐targeting near‐infrared fluorescent tracer delineating colorectal and pancreatic tumors

Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA‐targeted near‐infrared fluorescent (NIRF) tracer, based on a disulfide‐stabilized single‐chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell‐based plate assays and orthotopic colorectal (HT‐29, well differentiated) and pancreatic (BXPC‐3, poorly differentiated) xenogeneic human–mouse models. NIRF signals were visualized using the clinically compatible FLARE? imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor‐to‐background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent‐guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.     

Necrosis avid near infrared fluorescent cyanines for imaging cell death and their use to monitor therapeutic efficacy in mouse tumor models

Quantification of tumor necrosis in cancer patients is of diagnostic value as the amount of necrosis is correlated with disease prognosis and it could also be used to predict early efficacy of anti-cancer treatments. In the present study, we identified two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW (800CW), which possess strong necrosis avidity. In vitro studies showed that both dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane integrity. Affinity for cytoplasmic proteins was confirmed using quantitative structure activity relations modeling. In vivo results, using NIRF and optoacoustic imaging, confirmed the necrosis avid properties of HQ5 and 800CW in a mouse 4T1 breast cancer tumor model of spontaneous necrosis. Finally, in a mouse EL4 lymphoma tumor model, already 24 h post chemotherapy, a significant increase in 800CW fluorescence intensity was observed in treated compared to untreated tumors. In conclusion, we show, for the first time, that the NIRF carboxylated cyanines HQ5 and 800CW possess strong necrosis avid properties in vitro and in vivo. When translated to the clinic, these dyes may be used for diagnostic or prognostic purposes and for monitoring in vivo tumor response early after the start of treatment.     

Preclinical evaluation of near‐infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence‐guided surgery

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence‐guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near‐infrared (NIR) fluorescently labeled as a new tool for fluorescence‐guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux‐Alexa‐647) and the co‐injected control human IgG Alexa Fluor 750 conjugate (hIgG‐Alexa‐750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC‐1 and MIA PaCa‐2 PDAC tumors. Cetux‐Alexa‐647, but not the control hIgG‐Alexa‐750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC‐1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux‐Alexa‐647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux‐Alexa‐750). Fluorescence‐guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux‐800CW), enabled a real‐time delineation of AsPC‐1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux‐800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence‐guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.     

Time‐domain in vivo near infrared fluorescence imaging for evaluation of matriptase as a potential target for the development of novel,inhibitor‐based tumor therapies

Proteolytic enzymes expressed on the surface of tumor cells, and thus easily accessible to external interventions, represent useful targets for anticancer and antimetastatic therapies. In our study, we thoroughly evaluated matriptase, a trypsin‐like transmembrane serine protease, as potential target for novel inhibitor‐based tumor therapies. We applied time‐domain near infrared fluorescence (NIRF) imaging to characterize expression and activity of matriptase in vivo in an orthotopic AsPC‐1 pancreatic tumor model in nude mice. We show strong and tumor‐specific binding of intravenously injected Cy5.5 labeled antimatriptase antibody (MT‐Ab*Cy5.5) only to primary AsPC‐1 tumors and their metastases over time within living mice, taking into account fluorescence intensities and fluorescence lifetimes of the applied probes. Specific binding of MT‐Ab*Cy5.5 to tumor sites was confirmed by ex vivo NIRF imaging of tumor tissue, NIRF microscopy and by coregistration of the in vivo acquired NIRF intensity maps to anatomical structures visualized by flat‐panel volume computed tomography (fpVCT) in living mice. Moreover, using an activatable synthetic substrate S*DY‐681 we could clearly demonstrate that matriptase is proteolytically active in vitro as well as in vivo in tumor‐bearing mice, and that application of synthetic active‐site inhibitors having high affinity and selectivity toward matriptase can efficiently inhibit its proteolytic activity for at least 24 hr. We thus successfully applied NIRF imaging in combination with fpVCT to characterize matriptase as a promising molecular target for inhibitor‐based cancer therapies.     

Vasohibin‐2 plays an essential role in metastasis of pancreatic ductal adenocarcinoma

Vasohibin‐2 (VASH2) is expressed in various cancers and promotes their progression. We recently reported that pancreatic cancer patients with higher VASH2 expression show poorer prognosis. Herein, we sought to characterize the role of VASH2 in pancreatic cancer. We used LSL‐Kras^G12D; LSL‐Trp53^R172H; Pdx‐1‐Cre (KPC) mice, a mouse model of pancreatic ductal adenocarcinoma (PDAC), and cells isolated from them (KPC cells). Knockdown of Vash2 from PDAC cells did not affect their proliferation, but decreased their migration. When Vash2‐knockdown PDAC cells were orthotopically inoculated, liver metastasis and peritoneal dissemination were reduced, and the survival period was significantly prolonged. When KPC mice were crossed with Vash2‐deficient mice, metastasis was significantly decreased in Vash2‐deficient KPC mice. VASH2 was recently identified to have tubulin carboxypeptidase activity. VASH2 knockdown decreased, whereas VASH2 overexpression increased tubulin detyrosination of PDAC cells, and tubulin carboxypeptidase (TCP) inhibitor parthenolide inhibited VASH2‐induced cell migration. We next clarified its role in the tumor microenvironment. Tumor angiogenesis was significantly abrogated in vivo when VASH2 was knocked down or deleted. We further examined genes downregulated by Vash2 knockdown in KPC cells, and found chemokines and cytokines that were responsible for the recruitment of myeloid derived suppressor cells (MDSC). Indeed, MDSC were accumulated in PDAC of KPC mice, and they were significantly decreased in Vash2‐deficient KPC mice. These findings suggest that VASH2 plays an essential role in the metastasis of PDAC with multiple effects on both cancer cells and the tumor microenvironment, including tubulin detyrosination, tumor angiogenesis and evasion of tumor immunity.     

Intraoperative fluorescence delineation of head and neck cancer with a fluorescent Anti‐epidermal growth factor receptor nanobody

Intraoperative near‐infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real‐time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)‐targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti‐EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12‐800CW and R2‐800CW). Orthotopic tongue tumors were induced in nude mice using the OSC‐19‐luc2‐cGFP cell line. Tumor‐bearing mice were injected with 25 µg 7D12‐800CW, R2–800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12‐800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12‐800CW. A significantly higher tumor‐to‐background ratio (TBR) was observed in mice injected with 7D12–800CW compared to mice injected with R2‐800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR‐specific nanobody. After injection of 75 µg 7D12‐800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR‐targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.     

Clostridium perfringens enterotoxin C‐terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy‐resistant ovarian cancer

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin‐3‐ and ‐4‐overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy‐naïve and chemotherapy‐resistant human ovarian cancer xenografts or patient‐derived xenografts (PDXs) were treated with the carboxyl‐terminal binding domain of the Clostridium perfringens enterotoxin (c‐CPE) conjugated to FITC (FITC‐c‐CPE) or the near‐infrared (NIR) fluorescent tag IRDye CW800 (CW800‐c‐CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC‐c‐CPE and the CW800‐c‐CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800‐c‐CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor‐to‐background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c‐CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c‐CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real‐time detection of micrometastatic ovarian disease overexpressing the Claudin‐3 and ‐4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.     

Dual wavelength tumor targeting for detection of hypopharyngeal cancer using near-infrared optical imaging in an animal model

Optical imaging is a promising technique to visualize cancer tissue during surgery. In this study, we explored the use of combinations of near-infrared (NIR) fluorescence agents that emit fluorescence signal at different wavelengths and each target specific tumor characteristics. Two combinations of agents (ProSense680 combined with 2DG CW800 and MMPSense680 combined with EGF CW800) were used to detect hypopharyngeal cancer in an animal model. ProSense680 and MMPSense680 detect increased activity of cathepsins and matrix metalloproteinases, respectively. These enzymes are mainly found in the invasive tumor border due to degradation of the extracellular matrix. 2DG CW800 detects tumor cells with high glucose metabolism and EGF CW800 is internalized by the epidermal growth factor receptor of tumor cells. Whole-body imaging revealed clear demarcation of tumor tissue using all four agents. The tumor-to-background ratio (standard deviation, p-value) was 3.69 (0.72, p < 0.001) for ProSense680; 4.26 (1.33, p < 0.001) for MMPSense680; 5.81 (3.59, p = 0.02) for 2DG CW800 and 4.84 (1.56, p < 0.001) for EGF CW800. Fluorescence signal corresponded with histopathology and immunohistochemistry, demonstrating signal of ProSense680 and MMPSense680 in the invasive tumor border, and signal of 2DG CW800 and EGF CW800 in the tumor tissue. In conclusion, we demonstrated the feasibility of dual wavelength tumor detection using different targeting strategies simultaneously in an animal model. Combined targeting at different wavelengths allowed simultaneous imaging of different tumor characteristics. NIR fluorescence optical imaging has the potential to be translated into the clinic in order to improve the complete removal of tumors by real-time image-guided surgery.     

Nuclear expression of Y‐box binding protein‐1 is associated with poor prognosis in patients with pancreatic cancer and its knockdown inhibits tumor growth and metastasis in mice tumor models

The objective of this study was to examine the implication of Y‐box‐binding protein‐1 (YB‐1) for the aggressive phenotypes, prognosis and therapeutic target in pancreatic ductal adenocarcinoma (PDAC). YB‐1 expression in PDAC, pancreatic intraepithelial neoplasia (PanIN) and normal pancreas specimens was evaluated by immunohistochemistry, and its correlation with clinicopathological features was assessed in patients with PDAC. The effects of YB‐1 on proliferation, invasion and expressions of cell cycle‐related proteins and matrix metalloproteinases (MMPs) were analyzed by WST‐8, cell cycle and Matrigel invasion assays, Western blotting and quantitative RT‐PCR in PDAC cells transfected with YB‐1‐siRNAs. To verify the significance of YB‐1 for tumor progression in vivo, the growth and metastasis were monitored after intrasplenic implantation of ex vivo YB‐1 siRNA‐transfected PDAC cells, and YB‐1‐targeting antisense oligonucleotides were intravenously administered in nude mice harboring subcutaneous tumor. The intensity of YB‐1 expression and positivity of nuclear YB‐1 expression were higher in PDAC than PanIN and normal pancreatic tissues. Nuclear YB‐1 expression was significantly associated with dedifferentiation, lymphatic/venous invasion and unfavorable prognosis. YB‐1 knockdown inhibited cell proliferation via cell cycle arrest by S‐phase kinase‐associated protein 2 downregulation and consequent p27 accumulation, and decreased the invasion due to downregulated membranous‐type 2 MMP expression in PDAC cells. Tumor growth and liver metastasis formation were significantly suppressed in nude mice after implantation of YB‐1‐silenced PDAC cells, and the YB‐1 targeting antisense oligonucleotide significantly inhibited the growth of subcutaneous tumors. In conclusion, YB‐1 may be involved in aggressive natures of PDAC and a promising therapeutic target.     

Decreased TIP30 expression predicts poor prognosis in pancreatic cancer patients

Pancreatic ductal adenocarcinoma (PDAC) is known for its aggressive growth, and is characterized by early tissue invasion and metastasis with poor prognosis. Identifying prognostic markers and delineating the underlying mechanisms that promote progression of PDAC are important for the treatment of pancreatic cancer. TIP30, a newly identified tumor suppressor, appears to be involved in multiple processes during tumor development and metastasis. Here, we investigated the expression of TIP30 in PDAC and its prognostic value in PDAC patients. We examined the expression of TIP30 by immunohistochemistry in tissue microarrays containing 106 surgically resected PDAC. Kaplan–Meier analysis and Cox proportional hazards regression modeling analysis showed that TIP30 expression independently predicted better survival in pancreatectomy patients (p < 0.01). Moreover, decreased TIP30 expression was associated with lymph node metastasis (p < 0.05) and loss of E‐cadherin expression (r = 0.329, p < 0.01). Suppression of TIP30 resulted in upregulation of Snail and subsequent downregulation of E‐cadherin in SW1990 cells containing high‐level of endogenous TIP30. However, in the PANC‐1 cells containing low level of endogenous TIP30, suppressing TIP30 caused upregulation of Slug instead of Snail, followed by upregulation of MMP9 rather than E‐cadherin. Taken together, our work reveals that decreased TIP30 expression is able to enhance invasion and metastasis of pancreatic cancer cells through upregulation of the Snail family members and may serve as an independent predictor for poor outcomes in PDAC patients.     

Imaging the distribution of an antibody-drug conjugate constituent targeting mesothelin with 89Zr and IRDye 800CW in mice bearing human pancreatic tumor xenografts

Mesothelin is a tumor differentiation antigen expressed by epithelial tumors, including pancreatic cancer. Currently, mesothelin is being targeted with an antibody-drug conjugate (ADC) consisting of a mesothelin-specific antibody coupled to a highly potent chemotherapeutic drug. Considering the toxicity of the ADC and reduced accessibility of pancreatic tumors, non-invasive imaging could provide necessary information. We therefore developed a zirconium-89 (⁸⁹Zr) labeled anti-mesothelin antibody (⁸⁹Zr-AMA) to study its biodistribution in human pancreatic tumor bearing mice. Biodistribution and dose-finding of ⁸⁹Zr-AMA were studied 144 h after tracer injection in mice with subcutaneously xenografted HPAC. MicroPET imaging was performed 24, 72 and 144 h after tracer injection in mice bearing HPAC or Capan-2. Tumor uptake and organ distribution of ⁸⁹Zr-AMA were compared with nonspecific ¹¹¹In-IgG. Biodistribution analyses revealed a dose-dependent ⁸⁹Zr-AMA tumor uptake. Tumor uptake of ⁸⁹Zr-AMA was higher than ¹¹¹In-IgG using the lowest tracer dose. MicroPET showed increased tumor uptake over 6 days, whereas activity in blood pool and other tissues decreased. Immunohistochemistry showed that mesothelin was expressed by the HPAC and CAPAN-2 tumors and fluorescence microscopy revealed that AMA-800CW was present in tumor cell cytoplasm. ⁸⁹Zr-AMA tumor uptake is antigen-specific in mesothelin-expressing tumors. ⁸⁹Zr-AMA PET provides non-invasive, real-time information about AMA distribution and tumor targeting.     

Mass profiling of serum to distinguish mice with pancreatic cancer induced by a transgenic Kras mutation

Mass spectrometry (MS) has the unique ability to profile, in an easily accessible body tissue (peripheral blood/serum,) the sizes and relative amounts of a wide variety of biomolecules in a single platform setting. Using electrospray ionization (ESI)‐MS, we distinguished individual serum from wild‐type control mice from serum of mice containing an oncogenic Kras mutation, which leads to development of pancreatic ductal adenocarcinoma (PDAC) similar to that observed in humans. Identification of differences in significant ESI‐MS sera mass peaks between Kras‐activated mice and control mice was performed using t tests and a “nested leave one out” cross‐validation procedure. Peak distributions in serum of control mice from mice with Kras‐mutant‐dependent PDAC were distinguished from those of pancreatic intraepithelial neoplasia (PanIN) lesions (p = 0.00024). In addition, Kras mutant mice with PDAC were distinguished from Kras mutant mice with PanIN alone (p = 0.0057). Test specificity, a measure of the false positives, was greater for the control vs. Kras mutated mice, and the test sensitivity, a measure of false negatives, was greater for the PDAC vs. PanIN containing mice. Receiver‐operating characteristic (ROC) curve discriminatory values were 0.85 for both comparisons. These studies indicate ESI‐MS serum mass profiling can detect physiological changes associated with pancreatic cancer initiation and development in a GEM (genetic engineered mouse) model that mimics pancreatic cancer development in humans. Such technology has the potential to aid in early detection of pancreatic cancer and in developing therapeutic drug interventions.     

Fatty pancreas: A possible risk factor for pancreatic cancer in animals and humans

Obesity, type 2 diabetes mellitus (T2DM) and aging are associated with pancreatic cancer risk, but the mechanisms of pancreatic cancer development caused by these factors are not clearly understood. Syrian golden hamsters are susceptible to N‐nitrosobis(2‐oxopropyl)amine (BOP)‐induced pancreatic carcinogenesis. Aging, BOP treatment and/or a high‐fat diet cause severe and scattered fatty infiltration (FI) of the pancreas with abnormal adipokine production and promote pancreatic ductal adenocarcinoma (PDAC) development. The KK‐A^y mouse, a T2DM model, also develops severe and scattered FI of the pancreas. Treatment with BOP induced significantly higher cell proliferation in the pancreatic ducts of KK‐A^y mice, but not in those of ICR and C57BL/6J mice, both of which are characterized by an absence of scattered FI. Thus, we hypothesized that severely scattered FI may be involved in the susceptibility to PDAC development. Indeed, severe pancreatic FI, or fatty pancreas, is observed in humans and is associated with age, body mass index (BMI) and DM, which are risk factors for pancreatic cancer. We analyzed the degree of FI in the non‐cancerous parts of PDAC and non‐PDAC patients who had undergone pancreatoduodenectomy by histopathology and demonstrated that the degree of pancreatic FI in PDAC cases is significantly higher than that in non‐PDAC controls. Moreover, the association with PDAC is positive, even after adjusting for BMI and the prevalence of DM. Accumulating evidence suggests that pancreatic FI is involved in PDAC development in animals and humans, and further investigations to clarify the genetic and environmental factors that cause pancreatic FI are warranted.     

Diagnostic potential of hypermethylation of the cysteine dioxygenase 1 gene (CDO1) promoter DNA in pancreatic cancer

DNA markers for pancreatic ductal adenocarcinoma (PDAC) are urgently needed for detection of minimally invasive disease. The epigenetic relevance of the cysteine dioxygenase 1 gene (CDO1) has been never investigated in PDAC. Three studies, including cellular experiments, tissue validation, and pilot testing for pancreatic cytology, were carried out. Promoter DNA methylation value (MV) of CDO1 was quantified by quantitative methylation‐specific PCR. CDO1 expression was consistent with its promoter DNA methylation in 7 PDAC cell lines. In 160 retrospectively collected primary PDAC tumor tissues, MV was significantly higher compared to the corresponding noncancerous pancreas (area under the receiver operating characteristic curve [AUC] = 0.97, P < .0001), and CDO1 hypermethylation was highly specific to PDAC tumor tissues. CDO1 hypermethylation group (MV over 19) was significantly associated with diverse prognostic factors in PDAC. Surprisingly, it was significantly higher in prospectively collected PDAC cytology samples (n = 37), including both pancreatic juice (n = 12) and endoscopic ultrasound‐fine needle aspiration (EUS‐FNA) cytology (n = 25) compared to pancreatic benign diseases (AUC = 0.96, P < .0001). Detection of PDAC was confirmed by DNA testing in 35 of 37 patients (95% sensitivity); thus, it was more sensitive than cytology (33%) or EUS‐FNA cytology (88%). Promoter DNA methylation of CDO1 is extremely specific for PDAC tumors, and accumulates with PDAC tumor progression. It could be a definitive diagnostic marker of PDAC in pancreatic juice or EUS‐FNA cytology.     

Highly sensitive detection of early-stage pancreatic cancer by multimodal near-infrared molecular imaging in living mice

Pancreatic cancer is a serious disease with poor patient outcome, often as a consequence of late diagnosis in advanced stages. This is in large part due to the lack of diagnostic tools for early detection. To address this deficiency, we have investigated novel molecular near-infrared fluorescent (NIRF) in vivo imaging techniques in clinically relevant mouse models of pancreatic cancer. Genome wide gene expression profiling was used to identify cathepsin cystein proteases and matrix metalloproteinases (MMP) as targets for NIRF imaging. Appropriate protease activatable probes were evaluated for detection of early-stage pancreatic cancer in mice with orthotopically implanted pancreatic cancer cell lines. Mice with pancreatitis served as controls. Whole body in vivo NIRF imaging using activatable cathepsin sensitive probes specifically detected pancreatic tumors as small as 1-2 mm diameter. Imaging of MMP activity demonstrated high specificity for MMP positive tumors. Intravital flexible confocal fluorescence lasermicroscopy of protease activity enabled specific detection of pancreatic tumors at the cellular level. Importantly, topical application of NIRF-probes markedly reduced background without altering signal intensity. Taken together, macroscopic and confocal lasermicroscopic molecular in vivo imaging of protease activity is highly sensitive, specific and allows discrimination between normal pancreatic tissue, inflammation and pancreatic cancer. Translation of this approach to the clinic could significantly improve endoscopic and laparoscopic detection of early-stage pancreatic cancer.     

Predictive imaging of chemotherapeutic response in a transgenic mouse model of pancreatic cancer

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual‐modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1‐specific contrast agent (MN‐EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran‐coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1‐specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2‐weighted MRI and optical imaging before and 24 hr after injection of the MN‐EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT‐PCR. We observed that the average delta‐T2 in the gemcitabine‐treated group was significantly reduced compared to the control group indicating lower accumulation of MN‐EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine‐treated group compared to the control, which was confirmed by qRT‐PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN‐EPPT‐enhanced in vivo MR and optical imaging. These results suggest that the uMUC1‐targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response.     

Expression of classical human leukocyte antigen class I antigens,HLA‐E and HLA‐G,is adversely prognostic in pancreatic cancer patients

The expression of classical human leukocyte antigen class I antigens (HLA‐I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA‐I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA‐I and non–classical HLA‐I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA‐I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune‐related genes to characterize PDAC tumor microenvironments. Lower expression of HLA‐I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA‐E and HLA‐G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA‐I antigens was significantly correlated with shorter survival. Higher HLA‐I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD‐L1 and PD‐L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA‐I in three PDAC cell lines. It also upregulated surface expression of HLA‐E, HLA‐G and immune checkpoint molecules, including PD‐L1 and PD‐L2. These results suggest that the higher expression of HLA‐I, HLA‐E and HLA‐G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA‐I expression on PDAC cells.     

Suppression of pancreatic cancer growth and metastasis by HMP19 identified through genome‐wide shRNA screen

Therapeutic effectiveness against metastatic or even locally advanced pancreatic ductal adenocarcinoma (PDAC) is dismal, with 5‐year survival less than 5%. Even in patients who undergo potentially curative resection, most patients' tumors recur in the liver. Improving therapies targeting or preventing liver metastases is crucial for improving prognosis. To identify genes suppressing metastasis, a genome‐wide shRNA screen was done using the human non‐metastatic PDAC cell line, S2‐028. After identification of candidates, functional validation was done using intrasplenic and orthotopic injections in athymic mice. HMP19 strongly inhibited metastasis but also partially attenuated tumor growth in the pancreas. Knockdown of HMP19 increased localization of activated ERK1/2 in the nucleus, corresponding to facilitated cell proliferation, decreased p27^Kip1 and increased cyclin E1. Over‐expression of HMP19 exerted the opposite effects. Using a tissue microarray of 84 human PDAC, patients with low expression of HMP19 showed significantly higher incidence of liver metastasis (p = 0.0175) and worse prognosis (p = 0.018) after surgery. HMP19, a new metastasis/tumor suppressor in PDAC, appears to alter signaling that leads to cell proliferation and appears to offer prognostic value in human PDAC.     

Targeting of the P2X7 receptor in pancreatic cancer and stellate cells

The ATP‐gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu‐1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu‐1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7^‐/‐ animals. PancTu‐1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120‐treated mice showed reduced bioluminescence compared to saline‐treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120‐treated tumours.