Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.

Human cells have twenty-three pairs of chromosomes. In cancer, however, genes can be amplified in chromosomes or in circular extrachromosomal DNA (ecDNA), although the frequency and functional importance of ecDNA are not understood. We performed whole-genome sequencing, structural modelling and cytogenetic analyses of 17 different cancer types, including analysis of the structure and function of chromosomes during metaphase of 2,572 dividing cells, and developed a software package called ECdetect to conduct unbiased, integrated ecDNA detection and analysis. Here we show that ecDNA was found in nearly half of human cancers; its frequency varied by tumour type, but it was almost never found in normal cells. Driver oncogenes were amplified most commonly in ecDNA, thereby increasing transcript level. Mathematical modelling predicted that ecDNA amplification would increase oncogene copy number and intratumoural heterogeneity more effectively than chromosomal amplification. We validated these predictions by quantitative analyses of cancer samples. The results presented here suggest that ecDNA contributes to accelerated evolution in cancer.

Ratiometric activatable cell-penetrating peptides label pancreatic cancer, enabling fluorescence-guided surgery, which reduces metastases and recurrence in orthotopic mouse models.

BACKGROUND: The aim of this study was to evaluate the efficacy of using matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)-cleavable ratiometric activatable cell-penetrating peptides (RACPPs) conjugated to Cy5 and Cy7 fluorophores to accurately label pancreatic cancer for fluorescence-guided surgery (FGS) in an orthotopic mouse model. METHODS: Orthotopic mouse models were established using MiaPaCa-2-GFP human pancreatic cancer cells. Two weeks after implantation, tumor-bearing mice were randomized to conventional white light reflectance (WLR) surgery or FGS. FGS was performed at far-red and infrared wavelengths with a customized fluorescence-dissecting microscope 2 h after injection of MMP-2 and MMP-9-cleavable RACPPs. Green fluorescence imaging of the GFP-labeled cancer cells was used to assess the effectiveness of surgical resection and monitor recurrence. At 8 weeks, mice were sacrificed to evaluate tumor burden and metastases. RESULTS: Mice in the WLR group had larger primary tumors than mice in the FGS group at termination [1.72 g ± standard error (SE) 0.58 vs. 0.25 g ± SE 0.14; respectively, p = 0.026). Mean disease-free survival was significantly lengthened from 5.33 weeks in the WLR group to 7.38 weeks in the FGS group (p = 0.02). Recurrence rates were lower in the FGS group than in the WLR group (38 vs. 73 %; p = 0.049). This translated into lower local and distant recurrence rates for FGS compared to WLR (31 vs. 67 for local recurrence, respectively, and 25 vs. 60 % for distant recurrence, respectively). Metastatic tumor burden was significantly greater in the WLR group than in the FGS group (96.92 mm(2) ± SE 52.03 vs. 2.20 mm(2) ± SE 1.43; respectively, χ (2) = 5.455; p = 0.02). CONCLUSIONS: RACPPs can accurately and effectively label pancreatic cancer for effective FGS, resulting in better postresection outcomes than for WLR surgery.

Fluorescence-guided surgery with a fluorophore-conjugated antibody to carcinoembryonic antigen (CEA), that highlights the tumor, improves surgical resection and increases survival in orthotopic mouse models of human pancreatic cancer.

BACKGROUND: We have developed a method of distinguishing normal tissue from pancreatic cancer in vivo using fluorophore-conjugated antibody to carcinoembryonic antigen (CEA). The objective of this study was to evaluate whether fluorescence-guided surgery (FGS) with a fluorophore-conjugated antibody to CEA, to highlight the tumor, can improve surgical resection and increase disease-free survival (DFS) and overall survival (OS) in orthotopic mouse models of human pancreatic cancer. METHODS: We established nude-mouse models of human pancreatic cancer with surgical orthotopic implantation of the human BxPC-3 pancreatic cancer. Orthotopic tumors were allowed to develop for 2 weeks. Mice then underwent bright-light surgery (BLS) or FGS 24 h after intravenous injection of anti-CEA-Alexa Fluor 488. Completeness of resection was assessed from postoperative imaging. Mice were followed postoperatively until premorbid to determine DFS and OS. RESULTS: Complete resection was achieved in 92 % of mice in the FGS group compared to 45.5 % in the BLS group (p = 0.001). FGS resulted in a smaller postoperative tumor burden (p = 0.01). Cure rates with FGS compared to BLS improved from 4.5 to 40 %, respectively (p = 0.01), and 1-year postoperative survival rates increased from 0 % with BLS to 28 % with FGS (p = 0.01). Median DFS increased from 5 weeks with BLS to 11 weeks with FGS (p = 0.0003). Median OS increased from 13.5 weeks with BLS to 22 weeks with FGS (p = 0.001). CONCLUSIONS: FGS resulted in greater cure rates and longer DFS and OS using a fluorophore-conjugated anti-CEA antibody. FGS has potential to improve the surgical treatment of pancreatic cancer.

Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. METHODS: Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. RESULTS: The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. CONCLUSION: Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model.

In vivo fluorescence imaging of gastrointestinal stromal tumors using fluorophore-conjugated anti-KIT antibody.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are frequently characterized by KIT overexpression. Tumor-free margins and complete cytoreduction of disease are mainstays of treatment. We hypothesized that fluorescently labeled anti-KIT antibodies can label GIST in vivo. METHODS: KIT K641E(+/-) transgenic mice that spontaneously develop cecal GISTs were used in this study, with C57BL/6 mice serving as controls. Alexa 488 fluorophore-conjugated anti-KIT antibodies were delivered via the tail vein 24 h prior to fluorescence imaging. Following fluorescence laparoscopy, mice were sacrificed. The gastrointestinal tracts were grossly examined for tumors followed by fluorescence imaging. Tumors were harvested for histologic confirmation. RESULTS: KIT K641E(+/-) mice and C57BL/6 control mice received anti-KIT antibody or isotope control antibody. Fluorescence laparoscopy had a high tumor signal-to-background noise ratio. Upon blinded review of intravital fluorescence and bright light images, there were 2 false-positive and 0 false-negative results. The accuracy was 92 %. The sensitivity, specificity, positive and negative predictive values were 100, 87, 85, and 100 %, respectively, for the combined modalities. CONCLUSIONS: In this study, we present a method for in vivo fluorescence labeling of GIST in a murine model. Several translatable applications include: laparoscopic staging; visualization of peritoneal metastases; assessment of margin status; endoscopic differentiation of GISTs from other benign submucosal tumors; and longitudinal surveillance of disease response. This novel approach has clear clinical applications that warrant further research and development.

In vivo serial selection of human pancreatic cancer cells in orthotopic mouse models produces high metastatic variants irrespective of Kras status.

INTRODUCTION: Kras mutations have been thought to play an important role in pancreatic cancer progression. In this study, we evaluated how serially passaging primary pancreatic tumors with and without Kras mutations, in nude mice, can generate more aggressive variants of human pancreatic cancer. MATERIALS & METHODS: Orthotopic mouse models of human pancreatic cancer were established by injecting 1 × 10(6) cells of the Kras wildtype BxPC-3 cell line, expressing red fluorescent protein or the Kras mutant Panc-1 cell line expressing green fluorescent protein, into the pancreas. Pancreatic tumors were harvested from premorbid mice to establish cell lines. One million passaged cells were then orthotopically injected into another set of mice. Serial passaging continued until decreasing lifespan of the implanted mice stabilized, which occurred by six passages. Mice harboring serially-passaged cell lines were followed with weekly imaging. RESULTS: Serially passaging generated more aggressive variants of both human pancreatic cancer cell lines, one of which was Kras wild-type (BXPC-3) and the other Kras mutant, Panc-1, which displayed faster tumor growth and shortened survival time. Overall survival decreased from 18 wk in mice with the parental cell line (passage 0) tumor to ∼6 wk in mice by passage 6. Average time to metastasis was shortened from 14 wk to ∼3 wk or less. At termination, mice with the passaged tumor demonstrated a greater extent of distant metastasis. CONCLUSIONS: Serial passaging of tumor creates more aggressive variants of human pancreatic cancer cell lines regardless of Kras mutation. The aggressive variants can be used to study the molecular basis of highly malignant pancreatic cancer and to screen for effective agents against this disease.

Fluorescence-guided surgery of human colon cancer increases complete resection resulting in cures in an orthotopic nude mouse model.

BACKGROUND: We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes in fluorescent orthotopic nude mouse models of human colon cancer. METHODS: We established fluorescent orthotopic mouse models of human colon cancer expressing a fluorescent protein. Tumors were resected under bright light surgery (BLS) or FGS. Pre- and post-operative images with the OV-100 Small Animal Imaging System (Olympus Corp, Tokyo Japan) were obtained to assess the extent of surgical resection. RESULTS: All mice with primary tumor that had undergone FGS had complete resection compared with 58% of mice in the BLS group (P = 0.001). FGS resulted in decreased recurrence compared with BLS (33% versus 62%, P = 0.049) and lengthened disease-free median survival from 9 to >36 wk. The median overall survival increased from 16 wk in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS (P = 0.049). CONCLUSIONS: Surgical outcomes in orthotopic nude mouse models of human colon cancer were significantly improved with FGS. The present study can be translated to the clinic by various effective methods of fluorescently labeling tumors.

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression [corrected].

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.

Tumor-specific fluorescence antibody imaging enables accurate staging laparoscopy in an orthotopic model of pancreatic cancer.

BACKGROUND/AIMS: Laparoscopy is important in staging pancreatic cancer, but false negatives remain problematic. Making tumors fluorescent has the potential to improve the accuracy of staging laparoscopy. METHODOLOGY: Orthotopic and carcinomatosis models of pancreatic cancer were established with BxPC-3 human pancreatic cancer cells in nude mice. Alexa488-antiCEA conjugates were injected via tail vein 24 hours prior to laparoscopy. Mice were examined under bright field laparoscopic (BL) and fluorescence laparoscopic (FL) modes. Outcomes measured included time to identification of primary tumor for the orthotopic model and number of metastases identified within 2 minutes for the carcinomatosis model. RESULTS: FL enabled more rapid and accurate identification and localization of primary tumors and metastases than BL. Using BL took statistically significantly longer time than FL (p<0.0001, fold change and 95% CI for BL vs. FL: 8.12 (4.54,14.52)). More metastatic lesions were detected and localized under FL compared to BL and with greater accuracy, with sensitivities of 96% vs. 40%, respectively, when compared to control. FL was sensitive enough to detect metastatic lesions <1mm. CONCLUSIONS: The use of fluorescence laparoscopy with tumors labeled with fluorophore-conjugated anti-CEA antibody permits rapid detection and accurate localization of primary and metastatic pancreatic cancer in an orthotopic model. The results of the present report demonstrate the future clinical potential of fluorescence laparoscopy.

Knockdown of the ß(1) integrin subunit reduces primary tumor growth and inhibits pancreatic cancer metastasis.

To address the role of ß(1) integrins in pancreatic cancer progression, we stably knocked down ß(1) integrin subunit expression in human FG-RFP pancreatic cancer cells using lentiviral-based RNA interference. We then examined the effects of ß(1) integrin subunit knockdown on pancreatic cancer cell adhesion, migration and proliferation on tumor microenvironment-specific extracellular matrix proteins in vitro and on tumor progression in vivo using a clinically relevant fluorescent orthotopic mouse model of pancreatic cancer. Knockdown of the ß(1) integrin subunit inhibited cell adhesion, migration and proliferation on types I and IV collagen, fibronectin and laminin in vitro. In vivo, knockdown of the ß(1) integrin subunit reduced primary tumor growth by 50% and completely inhibited spontaneously occurring metastasis. These observations indicate a critical role for the ß(1) integrin subunit in pancreatic cancer progression and metastasis in particular. Our results suggest the ß(1) integrin subunit as a therapeutic target for the treatment of pancreatic cancer, especially in the adjuvant setting to prevent metastasis of this highly aggressive cancer.

Fluorescence laparoscopy imaging of pancreatic tumor progression in an orthotopic mouse model.

BACKGROUND: The use of fluorescent proteins to label tumors is revolutionizing cancer research, enabling imaging of both primary and metastatic lesions, which is important for diagnosis, staging, and therapy. This report describes the use of fluorescence laparoscopy to image green fluorescent protein (GFP)-expressing tumors in an orthotopic mouse model of human pancreatic cancer. METHODS: The orthotopic mouse model of human pancreatic cancer was established by injecting GFP-expressing MiaPaCa-2 human pancreatic cancer cells into the pancreas of 6-week-old female athymic mice. On postoperative day 14, diagnostic laparoscopy using both white and fluorescent light was performed. A standard laparoscopic system was modified by placing a 480-nm short-pass excitation filter between the light cable and the laparoscope in addition to using a 2-mm-thick emission filter. A camera was used that allowed variable exposure time and gain setting. For mouse laparoscopy, a 3-mm 0° laparoscope was used. The mouse's abdomen was gently insufflated to 2 mm Hg via a 22-gauge angiocatheter. After laparoscopy, the animals were sacrificed, and the tumors were collected and processed for histologic review. The experiments were performed in triplicate. RESULTS: Fluorescence laparoscopy enabled rapid imaging of the brightly fluorescent tumor in the pancreatic body. Use of the proper filters enabled simultaneous visualization of the tumor and the surrounding structures with minimal autofluorescence. Fluorescence laparoscopy thus allowed exact localization of the tumor, eliminating the need to switch back and forth between white and fluorescence lighting, under which the background usually is so darkened that it is difficult to maintain spatial orientation. CONCLUSION: The use of fluorescence laparoscopy permits the facile, real-time imaging and localization of tumors labeled with fluorescent proteins. The results described in this report should have important clinical potential.

Half-antibody functionalized lipid-polymer hybrid nanoparticles for targeted drug delivery to carcinoembryonic antigen presenting pancreatic cancer cells.

Current chemotherapy regimens against pancreatic cancer are met with little success as poor tumor vascularization significantly limits the delivery of oncological drugs. High-dose targeted drug delivery, through which a drug delivery vehicle releases a large payload upon tumor localization, is thus a promising alternative strategy against this lethal disease. Herein, we synthesize anti-carcinoembryonic antigen (CEA) half-antibody conjugated lipid-polymer hybrid nanoparticles and characterize their ligand conjugation yields, physicochemical properties, and targeting ability against pancreatic cancer cells. Under the same drug loading, the half-antibody targeted nanoparticles show enhanced cancer killing effect compared to the corresponding nontargeted nanoparticles.

Cryopreservation of Viable Human Tissues: Renewable Resource for Viable Tissue, Cell Lines, and Organoid Development.

The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor™ media, and frozen using a slow freezing method (-1°C/min in -80°C) for 24 hours and then stored in liquid nitrogen. After 15-18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.

Development of the transgenic cyan fluorescent protein (CFP)-expressing nude mouse for "Technicolor" cancer imaging.

A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non-transgenic nude mice with the transgenic CK/ECFP mouse in which the beta-actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA-1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color-coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color-coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment.

Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer.

BACKGROUND: Pancreatic cancer is a devastating disease characterized by dismal 5-year survival rates and limited treatment options. In an effort to provide useful models for preclinical evaluation of new experimental therapeutics, we and others have developed orthotopic mouse models of pancreatic cancer. The utility of these models for pre-clinical testing is dependent upon quantitative, noninvasive methods for monitoring in vivo tumor progression in real time. Toward this goal, we performed whole-body fluorescence imaging and ultrasound imaging to evaluate and to compare these noninvasive imaging modalities for assessing tumor burden and tumor progression in an orthotopic mouse model of pancreatic cancer. METHODS: The human pancreatic cancer cell line XPA-1, engineered for stable, high-level expression of red fluorescent protein (RFP), was implanted into the pancreas of nude mice using orthotopic implantation. The tumors were allowed to grow over a period of one to several weeks during which time the mice were imaged using both fluorescence imaging and ultrasound imaging to measure tumor burden and to monitor tumor growth. RESULTS: Whole-body fluorescence imaging and ultrasound imaging both allowed for the visualization and measurement of orthotopic pancreatic tumor implants in vivo. The imaging sessions were well-tolerated by the mice and yielded data which correlated well in the quantitative assessment of tumor burden. Whole-body fluorescence and two-dimensional ultrasound imaging showed a strong correlation for measurement of tumor size over a range of tumor sizes (R(2) = 0.6627, P = 0.003 for an exposure time of 67 msec and R(2) = 0.6553, P = 0.003 for an exposure time of 120 msec). CONCLUSION: Our findings suggest a complementary role for fluorescence imaging and ultrasound imaging in assessing tumor burden and tumor progression in orthotopic mouse models of human cancer.

Fluorescent LYVE-1 antibody to image dynamically lymphatic trafficking of cancer cells in vivo.

BACKGROUND: The lymphatic system is a major route for cancer cell dissemination, and a potential target for antitumor therapy. Despite ongoing interest in this area of research, the real-time behavior of cancer cells trafficking in the lymphatic system is poorly understood due to lack of appropriate tools to image this process. MATERIALS AND METHODS: We have used monoclonal-antibody and fluorescence technology to color-code lymphatic vessels and the cancer cells inside them in a living animal. Monoclonal anti-mouse LYVE-1 antibody was conjugated to a green fluorophore and delivered to the lymphatic system of a nude mouse, allowing imaging of mouse lymphatics. Tumor cells engineered to express red fluorescent protein were then imaged traveling within the labeled lymphatics in real time. RESULTS: AlexaFluor-labeled monoclonal anti-mouse LYVE-1 created a durable signal with clear delineation of lymphatic architecture. The duration of fluorescent signal after conjugated LYVE-1 delivery was far superior to that of fluorescein isothiocyanate-dextran or control fluorophore-conjugated IgG. Tumor cells engineered to express red fluorescent protein delivered to the inguinal lymph node enabled real-time tracking of tumor cell movement within the green fluorescent-labeled lymphatic vessels. CONCLUSIONS: This technology offers a powerful tool for the in vivo study of real-time trafficking of tumor cells within lymphatic vessels, for the deposition of the tumor cells in lymph nodes, as well as for screening of potential antitumor lymphatic therapies.

The cyan fluorescent protein (CFP) transgenic mouse as a model for imaging pancreatic exocrine cells.

CONTEXT: The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. OBJECTIVE: To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. DESIGN: Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA). INTERVENTION: All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. MAIN OUTCOME MEASURE: Determination of fluorescence intensity of different organs. RESULTS: Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. CONCLUSIONS: The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.

Real-Time Temperature Mapping in Ultra-Low Freezers as a Standard Quality Assessment.

Adequate preservation of biospecimens has been proven to be critical to obtain reliable and reproducible results in genomics, transcriptomics, proteomics, and many other assays. Most biological assays can be performed on specimens preserved in -80°C ultra-low freezers, but their quality can be influenced by temperature variability within storage units. Thus, regulatory standards such as those from the College of American Pathologists (CAP), the federal Clinical Laboratory Improvement Amendments, and standards from the Food and Drug Administration require temperature mapping, a standard quality assessment for accreditation when using ultra-low freezers for long-term biospecimen storage. The current mapping methods, providing annual/periodic data, may not be adequate indicators of temperature stability within the different zones of the freezers. In addition, they frequently require manual handling of biospecimens periodically, as they require freezers to be emptied or rearranged temporarily for the installation of temperature probes, risking the integrity of biospecimen quality. In this article, we describe a novel monitoring methodology based on real-time temperature reading of multiple zones by permanently installing thermocouples. An online cloud-based application records temperature variations within 1 minute intervals, and its 24/7 alert system triggers text alarm messages to a predefined set of users when temperature values increase above preset defaults. This provides an opportunity to take remedial action and to obtain a better-quality assessment. Our results indicate that real-time temperature monitoring at multiple zones of a freezer with a 1 minute resolution is a stable and sustainable methodology and, most importantly, lowers the risk of compromising the quality of the biospecimen. The design and use of the real-time monitoring system for ultra-low freezers is one of the acceptable methods by CAP to ensure the stability of biospecimen quality during long-term storage.

Tumor growth inhibition by mSTEAP peptide nanovaccine inducing augmented CD8+ T cell immune responses.

Poly(lactic-co-glycolic) acid (PLGA) has been successfully used in drug delivery and biomaterial applications, but very little attention has been directed towards the potential in vivo effects of peptide-loaded PLGA nanoparticles (NPs), specifically the potency of intravenous (IV) STEAP peptide-loaded PLGA-NP (nanovaccine) dosing and whether STEAP-specific CD8+ T cells directly play a key role in tumor inhibition. To address these concerns, syngeneic prostate cancer mouse models were established and treated with either mSTEAP peptide emulsified in incomplete Freund's adjuvant (IFA) via subcutaneous (SC) injection or mSTEAP peptide nanovaccine containing the same amount of peptide via IV or SC injection. Meanwhile, mice were treated with either CD8b mAb followed by nanovaccine treatment, free mSTEAP peptide, or empty PLGA-NPs. Immune responses in these mice were examined using cytotoxicity assays at 14 days after treatment. Tumor size and survival in various treatment groups were measured and monitored. The results demonstrated that mSTEAP peptide nanovaccine resulted in tumor inhibition by eliciting a significantly stronger CD8+ T cell immune response when compared with the controls. Moreover, the survival periods of mice treated with mSTEAP nanovaccine were significantly longer than those of mice treated with mSTEAP peptide emulsified in IFA or the treatment controls. Additionally, it was observed that the peptide nanovaccine was mainly distributed in the mouse liver and lungs after IV injection. These findings suggest that the peptide nanovaccine is a promising immunotherapeutic approach and offers a new opportunity for prostate cancer therapies.

Common bile duct injection as a novel method for establishing red fluorescent protein (RFP)-expressing human pancreatic cancer in nude mice.

CONTEXT: In our previous pancreatic cancer mouse models, we have used surgical orthotopic implantation of human pancreatic tumors to establish clinically relevant fluorescent mouse models of pancreatic cancer. OBJECTIVE: Since exocrine pancreatic cancer is thought to arise from the cells lining the ducts of the pancreas, we hypothesized that direct injection of tumor cells into the common bile duct would also result in pancreatic tumor formation and metastasis. INTERVENTION: In this study we injected a suspension of the low passage human pancreatic cancer cell line xPA-1 transfected with red fluorescent protein into the common bile duct of nude mice. MAIN OUTCOME MEASURE: Pancreatic tumor growth and metastasis formation was monitored by intravital and whole body fluorescent imaging. Single fluorescent pancreatic cancer cells were imaged in the pancreatic duct shortly after injection using the Olympus OV100 Whole Mouse Imaging System. RESULTS: Five days after tumor cell injection in the common bile duct, tumor colonies could be imaged forming within the pancreatic duct. Metastases in the liver were imaged 14 days post common bile duct injection. By day 28, massive tumors were imaged encompassing the entire pancreas. By day 42, RFP-expressing metastases were imaged in the omentum and liver. CONCLUSION: Common bile duct injection is a novel technique for the development of fluorescent mouse models of metastatic pancreatic cancer.