The ability of the surgeon to accurately visualize tumor margins and identify metastases is necessary for accurate staging and the success of any cancer operation. Fluorescence imaging, because of its high sensitivity, low cost, portability, and real-time capabilities has great potential to improve surgical outcomes. In our laboratory, we have developed a variety of tumor specific antibodies and nanobodies conjugated to near infrared dyes to label GI cancers including pancreatic, colon, and gastric cancers in mouse models of cancer. While most of the studies are pre-clinical in nature, several antibodies and small peptides are now in human clinical trials for fluorescence guided surgery (FGS). In addition to labelling tumors for accurate and complete resection, it is also vital to preserve nearby anatomical structures, such as nerves, to decrease the morbidity of surgery and reduce complications. Preclinical studies that have led to the current technology for fluorescence imaging that is now available in the operating room will be reviewed as well as current clinical trials for FGS.
Pancreatic cancer and colon cancer metastasis are recalcitrant cancers that are often difficult to detect. Mucin 5AC (MUC5A) and Mucin 4 (MUC4) have been found to be overexpressed in pancreatic and colon cancers, respectively, while having minimal expression in normal tissue. Using Mucin antibodies conjugated to a fluorescent dye, we demonstrate their specific labeling of human derived pancreatic and colon cancers in both subcutaneous and orthotopic mouse models. Tumor-specific fluorescent antibodies are clinically promising tools for improving both oncologic resection and patient survival.
Accelerating innovation in the space of fluorescence imaging for surgical applications has increased interest in safely and expediently advancing these technologies to clinic through Food and Drug Administration- (FDA-) compliant trials. Conventional metrics for early phase trials include drug safety, tolerability, dosing, and pharmacokinetics. Most procedural imaging technologies rely on administration of an exogenous fluorophore and concurrent use of an imaging system; both of which must receive FDA approval to proceed to clinic. Because fluorophores are classified as medical imaging agents, criteria for establishing dose are different, and arguably more complicated, than therapeutic drugs. Since no therapeutic effect is desired, medical imaging agents are ideally administered at the lowest dose that achieves adequate target differentiation. Because procedural imaging modalities are intended to enhance and/or ease proceduralists’ identification or assessment of tissues, beneficial effects of these technologies may manifest in the form of qualitative endpoints such as: 1) confidence; 2) decision-making; and 3) satisfaction with the specified procedure. Due to the rapid expansion of medical imaging technologies, we believe that our field requires standardized criteria to evaluate existing and emerging technologies objectively so that both quantitative and qualitative aspects of their use may be measured and useful comparisons to assess their relative value may occur. Here, we present a 15-item consensus-based survey instrument to assess the utility of novel imaging technologies from the proceduralist’s standpoint.
Tumor-specific targeting with fluorescent probes can enhance contrast for identification cancer during surgical resection and visualize otherwise invisible tumor margins. Fluorescent probes derived from nanobodies, the smallest naturally occurring antigen binding molecules, can be used for in-vivo labeling with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe clearly localized to the pancreatic cancer tumors and had a tumor-to-background ratio of 2 or greater by 1 hour. Tumor-specific fluorescent nanobodies are clinically promising molecules for same-day labeling and imaging of tumors.
Introduction The 5 year survival rate of pancreatic cancer is <10%. Most patients have metastatic disease at time of diagnosis, often to the liver. Innovative imaging modalities, i.e. fluorescence guided surgery (FGS), may better appreciate metastatic disease and guide treatment. Mucin 4 (MUC4), a glycoprotein, is found in 89% of pancreatic cancers and absent in normal pancreatic tissue making it a candidate for tumor targeting in FGS. In the present study, a fluorescently-labeled MUC4 antibody preferentially targets patient pancreatic cancer in a mouse model. Methods and Materials A MUC4 antibody was conjugated to the infrared dye IRDye800CW (LICOR, Lincoln, NE) to synthesize MUC4-IR800. A high MUC4 expressing patient-derived hepatic metastatic pancreatic tumor (Panc Met) was divided into 1mm3 tumor fragments and implanted under the skin of the nude mouse. After the tumors grew ~5mm3, two mice received 50 μg and two mice received 75 μg of MUC4-IR800 via tail vein injection. Daily in-vivo imaging was performed with the Pearl Trilogy Imager (LICOR, Lincoln, NE) for 3 days. Tumor to background ratios (TBR) were calculated using skin as background. Results MUC4-IR800 selectively imaged the Panc Met tumors (see figure below). TBRs for all time points and doses were <2. The 75 μg arm had higher TBRs at 24 and 72 hours. At 48 hours, the TBRs were the same. Conclusion This present study demonstrated the successful targeting of a patient hepatic metastatic pancreatic cancer mouse model with MUC4-IR800. This has potential to improve metastatic pancreatic cancer detection. Future studies will be conducted with orthotopic models.
Background: Colon-cancer liver metastases is the frequent cause of death due to difficulties in visualizing margins of the metastases resulting in incomplete resection. To perform safer and more reliable liver surgery, indocyanine green (ICG) labeling has been used to visualize liver tumors and liver segment, but it is difficult to distinguish between a liver metastasis and its adjacent liver segment with traditional use of ICG alone. We have previously developed a method to label a liver metastasis with a tumor-specific fluorescent conjugated antibody and the adjacent liver segment with ICG in order to perform image guided metastasectomy. Methods: Nude mice were surgically orthotopically implanted with a human coloncancer cell-line or colon-cancer liver metastases derived from patients. After liver tumor growth, mice received near-infrared conjugated anti-CEA or anti-CEACAM antibody to label the liver metastases. ICG was intravenously injected after ligation of the left or left lateral Glissonean pedicle resulting in specific labeling of the segment adjacent to the tumor with preserved blood-flow in the liver. Imaging was performed with the FLARE Imaging Systems. Results: The liver metastasis was brightly labeled with near infrared fluorescence with selective tumor targeting by the fluorescent anti-CEA or anti-CEACAM antibody, which was imaged on the 700 nm channel. The adjacent liver segment with preserved bloodflow in the liver had a bright fluorescence ICG 800 nm signal, while the left or left lateral segment had no fluorescence signal. Overlay of the images showed clear color-coded differentiation between the tumor and the liver segment, enabling image guided metastasectomy. Conclusions: Color coded imaging of the liver metastasis and adjacent liver segment in the present review can be used in the future for improved liver metastasectomy in the clinic.
Real-time intraoperative image-guided cancer surgery promises to improve oncologic outcomes. Tumor-specific antibodies conjugated with near-infrared (NIR) fluorophores have demonstrated the potential to enhance visualization of solid tumor margins and metastatic disease; however, multiple challenges remain, including improvement in probe development for clinical utility. We have developed an NIR-IR800 dye on a PEGylated linker (sidewinder) conjugated to the humanized anti-carcinoembryonic antigen (CEA) antibody (M5A) with extended in vivo serum and tumor persistence. The anti-CEA M5A-sidewinder has a high dye-to-antibody ratio (average of 7 per antibody) that allows, in an orthotopic implanted human pancreatic cancer mouse model increased tumor fluorescence, higher tumor-to-background ratio and extends the surgical scheduling window compared to current antibody dye conjugates. These preclinical results demonstrate the potential of this probe for fluorescence-guided surgery of CEA-positive gastrointestinal cancers.
In proof-of-concept studies, the anti-CEA M5A-IR800 conjugate demonstrated rapid and effective near infrared (NIR) imaging of human colon cancer and pancreatic cancer primary and metastatic lesions in mouse models. A limitation observed from these studies is the antibody-dye conjugate’s rapid clearance from the blood due to the increased hydrophobicity of the IR800 dye. This is a bottleneck for clinical applications, requiring high doses to be administered and a short surgical time window for intraoperative imaging. As a result, we developed a new prototype anti-CEA-swPEG-IR800 conjugate, that incorporates a PEGylated sidearm linker to shield or mask the IR800 dye’s hydrophobicity, a novel approach to extend the blood circulation half-life and in doing so increase tumor sensitivity as well as lower normal hepatic uptake. Results of the anti-CEA-swPEG-IR800 in an orthotopic human pancreatic cancer mouse model demonstrated exceptional optical imaging at lower doses, a much longer in vivo half-life enabling increased tumor fluorescence and higher tumor to background ratios. We propose that our novel anti-CEA-swPEG-IR800 is capable of enhanced optical imaging than currently available agents and will become the next generation optical imaging agent for safe and effective intraoperative image-guided surgery in CEA expressing GI cancers.
Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.
Negative surgical margins are critical to prevent recurrence in cancer surgery. This is because with current technology in many cases negative margins are impossible due the inability of the surgeon to detect the margin. Our laboratory has developed fluorophore-labeled monoclonal antibodies to aid in cancer visualization in orthotopic nude mouse models of human gastrointestinal (GI) cancer in order to achieve negative margins in fluorescence-guided surgery (FGS). The technologies described herein have the potential to change the paradigm of surgical oncology to engender significantly improved outcomes.
Labeling of metastatic tumors can aid in their staging and resection of cancer. Near infrared (NIR) dyes have been used in the clinic for tumor labeling. However, there can be a nonspecific uptake of dye by the liver, lungs, and lymph nodes, which hinders detection of metastasis. In order to overcome these problems, we have used two NIR dyes (DyLight 650 and 750) conjugated to a chimeric anti-carcinoembryonic antigen antibody to evaluate how polyethylene glycol linkage (PEGylation) can improve specific tumor labeling in a nude mouse model of human pancreatic cancer. The conjugated PEGylated and non-PEGylated DyLight 650 and 750 dyes were injected intravenously into non-tumor-bearing nude mice. Serum samples were collected at various time points in order to determine serum concentrations and elimination kinetics. Conjugated PEGylated dyes had significantly higher serum dye concentrations than non-PEGylated dyes (p=0.005 for the 650 dyes and p<0.001 for the 750 dyes). Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with conjugated PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (p<0.001 for the 650 dyes; p=0.01 for 750 dyes). PEGylation of the NIR dyes also decreased their accumulation in lymph nodes, liver, and lung. These results demonstrate enhanced selective tumor labeling by PEGylation of dyes conjugated to a tumor-specific antibody, suggesting their future clinical use in fluorescence-guided surgery.
The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.
Early detection is important for many solid cancers but the images provided by ultrasound, magnetic resonance imaging (MRI), and computed tomography applied alone or together, are often not sufficient for decisive early screening/diagnosis. We demonstrate that MRI augmented with fluorescence intensity (FI) substantially improves detection. Early stage murine pancreatic tumors that could not be identified by blinded, skilled observers using MRI alone, were easily identified with MRI along with FI images acquired with photomultiplier tube detection and offset laser scanning. Moreover, we show that fluorescence lifetime (FLT) imaging enables positive identification of the labeling fluorophore and discriminates it from surrounding tissue autofluorescence. Our data suggest combined-modality imaging with MRI, FI, and FLT can be used to screen and diagnose early tumors.
We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 µm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 µm. This compares favorably with the 100- to 300 µm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40-µm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.
Although side effects of cancer chemotherapy are well known, "opposite effects" of chemotherapy which enhance the
malignancy of the treated cancer are not well understood. We have observed a number of steps of malignancy that are
enhanced by chemotherapy pre-treatment of mice before transplantation of human tumor cells. The induction of
intravascular proliferation, extravasation, and colony formation by cancer cells, critical steps of metastasis was
enhanced by pretreatment of host mice with the commonly-used chemotherapy drug cyclophosphamide.
Cyclophosphamide appears to interfere with a host process that inhibits intravascular proliferation, extravasation, and
extravascular colony formation by at least some tumor cells. Cyclophosphamide does not directly affect the cancer cells
since cyclophosphamide has been cleared by the time the cancer cells were injected. Without cyclophosphamide
pretreatment, human colon cancer cells died quickly after injection in the portal vein of nude mice. Extensive
clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours. The number of apoptotic cells
rapidly increased within the portal vein within 12 hours of injection. However, when the host mice were pretreated with
cyclophosphamide, the cancer cells survived and formed colonies in the liver after portal vein injection. These results
suggest that a cyclophosphamide-sensitive host cellular system attacked the cancer cells. This review describes an
important unexpected "opposite effects" of chemotherapy that enhances critical steps in malignancy rather than
inhibiting them, suggesting that certain current approaches to cancer chemotherapy should be modified.
KEYWORDS: Cancer, Lymphatic system, Skin, Imaging systems, Tumors, Real time imaging, Green fluorescent protein, Blood vessels, Skin cancer, In vivo imaging
With the use of fluorescent cells labeled with green fluorescent protein (GFP) in the nucleus and red
fluorescent protein (RFP) in the cytoplasm and a highly sensitive small animal imaging system with both
macro-optics and micro-optics, we have developed subcellular real-time imaging of cancer cell trafficking in
live mice. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice.
The mice were imaged with an Olympus OV100 small animal imaging system with a sensitive CCD camera
and four objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We
observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by
various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time
dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei
following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during
extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. We have also
developed real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with GFP
and/or RFP were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked
through lymphatic vessels where they were imaged via a skin flap in real-time at the cellular level until they
entered the axillary lymph node. The bright dual-color fluorescence of the cancer cells and the real-time
microscopic imaging capability of the Olympus OV100 enabled imaging the trafficking cancer cells in both
blood vessels and lymphatics. With the dual-color cancer cells and the highly sensitive imaging system
described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time.
Here we describe our cumulative experience with the development and preclinical application of several highly
fluorescent, clinically-relevant, metastatic orthotopic mouse models of pancreatic cancer. These models utilize the
human pancreatic cancer cell lines which have been genetically engineered to selectively express high levels of the
bioluminescent green fluorescent (GFP) or red fluorescent protein (RFP). Fluorescent tumors are established
subcutaneously in nude mice, and tumor fragments are then surgically transplanted onto the pancreas. Locoregional
tumor growth and distant metastasis of these orthotopic implants occurs spontaneously and rapidly throughout the
abdomen in a manner consistent with clinical human disease. Highly specific, high-resolution, real-time visualization of
tumor growth and metastasis may be achieved in vivo without the need for contrast agents, invasive techniques, or
expensive imaging equipment. We have shown a high correlation between florescent optical imaging and magnetic
resonance imaging in these models. Alternatively, transplantation of RFP-expressing tumor fragments onto the pancreas
of GFP-expressing transgenic mice may be used to facilitate visualization of tumor-host interaction between the
pancreatic tumor fragments and host-derived stroma and vasculature. Such in vivo models have enabled us to serially
visualize and acquire images of the progression of pancreatic cancer in the live animal, and to demonstrate the real-time
antitumor and antimetastatic effects of several novel therapeutic strategies on pancreatic malignancy. These fluorescent
models are therefore powerful and reliable tools with which to investigate human pancreatic cancer and therapeutic
strategies directed against it.
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