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.
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.
There is a high demand for non-invasive imaging techniques that allow observation of stem cells in their native
environment without significant input on cell metabolism, reproduction, and behavior. Easy accessible hair follicle
pluripotent stem cells in the bulge area and dermal papilla are potential sources for stem cell based therapy. It has been
shown that these cells are able to generate hair, non-follicle skin cells, nerves, vessels, smooth muscles etc. and may
participate in wound healing processes.
We report on the finding of nestin-GFP expressing stem cells in their native niche in the bulge of the hair follicle of
living mice by using high-resolution in-vivo multiphoton tomography. The 3D imaging with submicron resolution was
based on two-photon induced fluorescence and second harmonic generation (SHG) of collagen. Migrating stem cells
from the bulge to their microenvironment have been detected inside the skin during optical deep tissue sectioning.
We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.
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.
Fluorescent proteins have revolutionized the field of imaging. Our laboratory pioneered in vivo imaging with
fluorescent proteins. Fluorescent proteins have enabled imaging at the subcellular level in mice. We review here the
use of different vectors carrying fluorescent proteins to selectively label normal and tumor tissue in vivo. We show that
a GFP retrovirus and telomerase-driven GFP adenovirus can selectively label tumors in mice. We also show that a GFP
lentivirus can selectively label the liver in mice. The practical application of these results are discussed.
Tumor targeting Salmonella typhimurium has been developed. These bacteria were mutagenized and a strain auxotrophic for
leucine and arguine was selected. This strain was also engineered to express GFP. This train, termed A1, could target
prostate tumors in nude mouse models and inhibit their growth. A1 was passaged through a tumor and re-isolated and
termed A1-R. A1-R had greater antitumor efficacy and could cure breast, prostate, pancreatic, and lung tumors in nude
mouse models.
Although GFP and fluorescent proteins are used extensively for in vivo imaging, there are many misconceptions about
GFP imaging especially compared to luciferase. GFP is not toxic, indeed, transgenic animals with GFP expressed in
every cell (1) live as long as non-transgenic animals. Cancer cells with GFP are as aggressive and malignant as the cells
without GFP (2-4). Cell lines can be made very bright with fluorescent proteins with no toxicity. The in vivo signal
from fluorescent proteins is at least 1,000 times greater than luciferase (5). GFP is so bright that a single molecule of
GFP can be seen in a bacterium (6). GFP can be observed through the skin on deep organs (7). Skin autofluorescence
presents no problem for in vivo GFP imaging with proper filters (8). Fur can be rapidly clipped removing this
autofluorescence (9). GFP is readily quantified by the image area which correlates to tumor volume (10). There are
now numerous clones of GFP, RFP, YFP and proteins that change color (11) that can be used in vivo.
KEYWORDS: Tumors, Cancer, Green fluorescent protein, Fluorescent proteins, In vivo imaging, Whole body imaging, Proteins, Tissues, Tumor growth modeling, Microscopes
Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many
applications in small animals. Our laboratory pioneered in vivo imaging with fluorescent proteins (1)
including noninvasive whole-body imaging (2). Whole-body imaging with fluorescent proteins depends in
large part on the brightness of the protein. Brighter, red-shifted proteins can make whole-body imaging more
sensitive due to reduced absorption by tissues and less scatter. Non-invasive imaging with fluorescent
proteins has been shown to be able to quantitatively track tumor growth and metastasis, gene expression,
angiogenesis, and bacterial infection (3) even at subcellular resolution depending on the position of the cells
in the animal. Interference by skin autofluorescence is kept to a minimum with the use of proper filters. To
noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at
the cellular level in live animals in real time, we developed a new imageable three-color animal model. The
model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells
labeled with GFP in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Various in vivo
phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic
tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow as well as
drug response. This imageable technology should lead to many new insights of in vivo cancer cell biology.
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.
An important goal for in vivo imaging is to be able to non-invasively image single cells. The Olympus IV100 Laser
Scanning Microscope, with ultra-thin microscope objectives ("stick objectives"), was used for three-color whole-body
imaging of individual two-color cancer cells interacting with the GFP-expressing stromal cells. Cellular dynamics were
non-invasively imaged including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor
vasculature, and tumor blood flow. This imageable model should lead to a new paradigm of in vivo cancer cell biology
and to new visible real-time targets for cancer drug discovery.
We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.
KEYWORDS: Tumors, Green fluorescent protein, Fluorescent proteins, Visualization, In vivo imaging, Luminescence, Cancer, Proteins, Whole body imaging, Blood vessels
The revolution of in vivo cancer biology enabled by fluorescent proteins is described. The high extinction coefficients, quantum yields, and unique spectral properties of fluorescent proteins have been taken advantage of in order to visualize, in real time, the important aspects of cancer in living animals, including tumor cell trafficking, invasion, metastasis, and angiogenesis. Fluorescent proteins enable whole-body imaging of tumors on internal organs. These multicolored proteins have allowed the color-coding of cancer cells growing in vivo with distinction of different cell types, including host from tumor, with single-cell resolution.
In this work we studied in vivo the combined action of cyclophosphamide and the extract of mycelium of Pleurotus ostreatus on mice bearing melanoma B16-F0, expressing green fluorescent protein (GFP).
This model allows to recognize small-size tumors and metastases, unrecognizable by other methods.
It was found that combined administration of cyclophosphamide (300 mg/kg) and the extract of mycelium of Pleurotus ostreatus (100 mg/kg), administered for 10 days after cyclophosphamide injection, as well administration of cyclophosphamide alone, cause inhibition of tumor growth about 97%.
It was shown that administration of the extract of mycelium of Pleurotus ostreatus alone leads to inhibition of tumor growth of 61%.
It was found that in case of combined administration of cyclophosphamide and the extract of mycelium of Pleurotus ostreatus, leucopenia was less expressed than in case of administration of cyclophosphamide alone.
The new field of in vivo cell biology is being developed with multi-colored fluorescent proteins. With the use of fluorescent proteins, the behavior of individual cells can be visualized in the living animal. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of the tumor-stroma cell-cell interaction especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. Another example is the color-coding of cells with RFP or GFP such that both cell types and their interaction can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo with fluorescent proteins. Mice, in which the regulatory elements of the stem-cell marker nestin drive GFP expression, can be used to visualize hair follicle stem cells including their ability to form hair follicles as well as blood vessels. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Dual-color cells also enable the in vivo imaging of cell and nuclear deformation as well as trafficking in capillaries in living animals. Multiple-color labeling of cells will enable multiple events to be simultaneously visualized in vivo including cell-cell interaction, gene expression, ion fluxes, protein and organelle trafficking, chromosome dynamics and numerous other processes currently still studied in vitro.
Dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP) expressing transgenic mice has been shown to be a powerful technology to study tumor-host interaction. Host animals include mice which express the GFP transgene in essentially all cells as well as animals in which the regulatory elements of the stem cell marker nestin drive GFP. The general GFP-transgenic mouse is available in both the normal and athymic nude (nu/nu) background. These models show with great clarity the details of the tumor-stroma interaction especially tumor induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. GFP-expressing tumor vasculature could be visualized interacting with the RFP-expressing tumor cells transplanted to the nestin-driven GFP transgenic mice which expressed nestin-GFP in nascent blood vessels was shown as a marker of nascent tumor angiogenesis. Dual-color fluorescence imaging, which visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations (1).
KEYWORDS: Tumors, Green fluorescent protein, Luminescence, Blood vessels, Tumor growth modeling, In vivo imaging, Visualization, Lung, Cancer, Animal model studies
The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.
We report here a new approach to genetically engineering tumors to become fluorescence such that they can be imaged externally in freely-moving animals. We describe here external high-resolution real-time fluorescent optical imaging of metastatic tumors in live mice. Stable high-level green flourescent protein (GFP)-expressing human and rodent cell lines enable tumors and metastasis is formed from them to be externally imaged from freely-moving mice. Real-time tumor and metastatic growth were quantitated from whole-body real-time imaging in GFP-expressing melanoma and colon carcinoma models. This GFP optical imaging system is highly appropriate for high throughput in vivo drug screening.
Green fluorescent protein (GFP)-expressing cell-lines have been established by our laboratory that permit the visualization and imaging of primary tumors and micrometastases in live tissue and live animals. Hamster and human cancer cell-lines were transfected with vectors containing the humanized GFP cDNA. Stable high-level expression of GFP was maintained in subcutaneously and orthotopically growing tumors in nude or SCID mice. Subsequent micro-metastases were visualized by GFP fluorescence in live tissue of systematic organs down to the single-cell level. GFP-expressing lung and prostate cancer were visualized to metastasize widely throughout the skeleton when implanted orthotopically in nude mice. With these GFP-cell lines, we have developed models that closely mimic the clinic situation. We have now developed a mean to visualize the onset and progression of angiogenesis of growing and spreading tumors by injecting a fluorescent rhodamine dye to the GFP-tumor-bearing mice indicate that the onset and extent of tumor angiogenesis depends on the site and type of tumor growing in the animal. These models are ideal for studying the mechanisms of cancer metastasis and for discovery of angiogenesis and metastasis inhibitors.
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