Imaging tumor angiogenesis with fluorescent proteins

We have developed three unique mouse models to image angiogenesis with fluorescent proteins, which are described in this review. First, we have adapted the surgical orthotopic implantation (SOI) model to image angiogenesis of human tumors labeled with green fluorescent protein (GFP) transplanted in nude mice. The nonluminous induced capillaries are clearly visible by contrast against the very bright tumor fluorescence examined either intravitally or by whole-body imaging in real time. Intravital images of an SOI model of human pancreatic tumors expressing GFP visualized angiogenic capillaries at both primary and metastatic sites. Whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period in an SOI model of human breast cancer expressing GFP. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold and enabling vessels to be externally visualized in GFP-expressing tumors growing on internal organs. The second model utilizes dual-color fluorescence imaging, effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice that express GFP in all cells. This dual-color model visualizes with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis. The GFP-expressing tumor vasculature, both nascent and mature, are readily distinguished interacting with the RFP-expressing tumor cells. Using a spectral imaging system based on liquid crystal tunable filters, we were able to separate individual spectral species on a pixel-by-pixel basis. Such techniques non-invasively visualized the presence of host GFP-expressing vessels within an RFP-labeled orthotopic human breast tumor by real-time whole-body imaging. The third model involves a transgenic mouse in which the regulatory elements of the stem cell marker nestin drive GFP. The nestin-GFP mouse expresses GFP in areas of the brain, hair follicle stem cells, and in a network of blood vessels in the skin interconnecting hair follicles. RFP-expressing tumors transplanted to nestin-GFP mice enable specific visualization of nascent vessels in skin-growing tumors such as melanoma. Thus, fluorescent proteins expressed in vivo offer very high resolution and sensitivity for real-time imaging of angiogenesis.     

A highly metastatic Lewis lung carcinoma orthotopic green fluorescent protein model

The Lewis lung carcinoma has been widely used for many important studies. However, the subcutaneous transplant or orthotopic cell-suspension injection models have not allowed the expression of its full metastatic potential. A powerful new highly metastatic model of the widely-used Lewis lung carcinoma is reported here using surgical orthotopic implantation (SOI) of tumor fragments and enhanced green fluorescent protein (GFP) transduction of the tumor cells. To achieve this goal, we first developed in vitro a stable high-expression GFP transductant of the Lewis lung carcinoma with the pLEIN retroviral expression vector containing the enhanced Aequorea victoria GFP gene. Stable high-level expression of GFP was found maintained in vivo in subcutaneously-growing Lewis lung tumors. The in vivo GFP-expressing tumors were harvested and implanted as tissue fragments by SOI in the right lung of additional nude mice. This model resulted in rapid orthotopic growth and extensive metastasis visualized by GFP-expression. 100% of the animals had metastases on the ipsilateral diaphragmatic surface, contralateral diaphragmatic surface, contralateral lung parenchima, and in mediastinal lymph nodes. Heart metastases were visualized in 40%, and brain metastases were visualized in 30% of the SOI animals. Mice developed signs of respiratory distress between 10–15 days post-tumor implantation and were sacrificed. The use of GFP-transduced Lewis lung carcinoma transplanted by SOI reveals for the first time the high malignancy of this tumor and provides an important useful model for metastasis, angiogenesis and therapeutic studies.     

Therapeutic potential of mesenchymal stem cells producing interferon-alpha in a mouse melanoma lung metastasis model

Adult stem cells represent a potential source for cell-based therapy of cancer. The present study evaluated the potential of bone marrow-derived mesenchymal stem cells (MSC), genetically modified to express interferon (IFN)-alpha, for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A recombinant adeno-associated virus (rAAV) 6 vector encoding IFN-alpha was used to transduce mouse bone marrow-derived MSC ex vivo. Expression and bioactivity of the transgenic protein from rAAV-transduced MSC were confirmed prior to in vivo studies. A lung metastasis model of melanoma was developed by i.v. injection of B16F10 cells into 8-week-old C57BL/6 mice. Ten days later, MSC transduced with rAAV-IFN-alpha or green fluorescent protein were intravenously injected. One cohort of mice was sacrificed to determine the effects of the therapy at an earlier time point, and another cohort was observed for long-term survival. Results indicated that systemic administration of MSC producing IFN-alpha reduced the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC-IFN-alpha-treated animals indicated an increase in apoptosis and a decrease in proliferation and blood vasculature. These data demonstrate the potential of adult MSC constitutively producing IFN-alpha to reduce the growth of lung metastasis in melanoma.     

Optically imageable metastatic model of human breast cancer

We report an optically imageable orthotopic metastatic nude mouse model of the human breast cancer MDA-MB-435 expressing green fluorescent protein (GFP). We demonstrate fluorescent imaging of primary and metastatic growth in live tissue and in intact animals. Fragments of tumor tissue expressing GFP were sutured into the pocket in the right second mammary gland. Tumor tissue was strongly fluorescent, enabling whole-body imaging of tumor growth by week 5. Neovascularization of the primary tumor was also visualized by whole-body imaging by contrast of the vessels to the fluorescent tumor. At autopsy, the MDA-MB-435-GFP was found to have metastasized to various organs, including the lung in 55% of the animals, the lymph nodes in 15% of the animals including axillary nodes, and the liver in 10% of the animals. These metastases could be visualized in fresh tissue by fluorescent imaging. Detailed fluorescence analysis visualized extensive metastasis in the thoracic cavity and the lymphatic system. Large metastatic nodules in the lung involved most of the pulmonary parenchyma in all lobes. Lymph node metastasis was found mainly in the axillary area. In the liver, fluorescent macroscopic metastatic nodules were found under the capsule. The metastatic pattern in the model thus reflected clinical metastatic breast cancer and provides a powerful model for drug discovery for this disease. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metastasis-suppressed C8161 melanoma cells arrest in lung but fail to proliferate

The incidence of melanoma continues to increase at a rapid rate. As for most cancers, it is melanoma metastases, rather than the primary malignancy, that is the principal cause of death. We previously showed that the introduction of a normal copy of chromosome 6 into the metastatic human melanoma cell line C8161 suppresses metastasis at a step subsequent to tumor cells entering the bloodstream. To better define the step(s) in metastasis blocked by the addition of chromosome 6 we engineered cells that constitutively express green fluorescent protein (GFP). When these tagged, chromosome 6 hybrid cells were injected intravenously into athymic mice, grossly detectable metastases did not form. However, fluorescence microscopy revealed micro-metastases (single cells or clusters of <10 cells) in the lungs, suggesting that these cells lodged in the lungs but failed to proliferate. Cells isolated from lung up to 60 days post-injection grew in culture and/or formed tumors when injected into the skin, indicating that they were still viable, but dormant. This result implies that the gene(s) on chromosome 6 interfere specifically with growth regulatory response in the lung, but not in the skin. Thus, the gene(s) responsible for metastasis suppression represents a new class of metastasis inhibitors acting at the final stages of the metastatic cascade - that is, affecting the ability of the cells to survive and proliferate at a specific secondary site. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alteration of angiogenic patterns on B16BL6 melanoma development promoted in Matrigel

Because the progression and metastasis of solid tumors depend on their local microcirculation, we sought to characterize tumor angiogenesis three dimensionally in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into the subcutis in the skin on the back of syngeneic C57BL/6 mice. We found that B16 with Matrigel grew significantly faster than B16 alone and had altered tumor angiogenesis. Tumor vessels apparently grew vigorously in the opposite direction of the tumor without invading the tumor mass until at least day 10 of injection. In addition, vascular branching resulted not only from sprouting as was seen in B16 without Matrigel but also from vascular splitting, either because of compression from outside the vessels or from septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other tumor cells, including Lewis lung carcinoma and ASH-1 hybridoma cell lines, both of which were tested under the same conditions. The reduction in various angiogenic factors in Matrigel did not affect the angiogenic patterns and tumor growth. We hypothesize that tumor vessels may vigorously alter their angiogenic patterns in response to the local microenvironment.     

Recombinant human prothrombin kringle-2 inhibits B16F10 melanoma metastasis through inhibition of neovascularization and reduction of matrix metalloproteinase expression

Angiogenesis, a multi-step process which involves endothelial cell proliferation, adhesion, migration, and basement membrane (BM) degradation, is essential for tumor metastasis. Here we show that recombinant human prothrombin kringle-2 (rk-2) inhibited bovine capillary endothelial cell migration with an IC₅₀ (concentration for half maximal inhibition) of 38 nM and inhibited adhesion to extracellular matrix (ECM) proteins. Because tumor metastasis requires angiogenesis, we examined whether rk-2 could inhibit metastases induced by injection of B16F10 melanoma cells into mice. The results revealed that the metastatic tumors in mouse lung were markedly decreased in a dose-dependent manner and acute lung injury induced by B16F10 melanoma metastasis was diminished by systemic rk-2 treatment. In immunohistochemical analysis, rk-2 reduced expression of vascular endothelial growth factor, which is a potent angiogenic activator and neovascularization in the mouse lung. Also, rk-2 diminished the expression of matrix metalloproteinase-2 and -9 in the mouse lung which induces tumor metastasis and angiogenesis. These data suggest that inhibition of B16F10 melanoma metastasis by rk-2 was caused by inhibition of neovascularization and reduction of matrix metalloproteinase expression.     

纳米金结合恩度抑制小鼠黑色素瘤肺转移

目的:观察纳米金结合恩度对小鼠黑色素瘤肺转移的影响并探讨其机制。方法:C57BL/6小鼠24只,构建小鼠黑色素瘤B16-F10细胞自发性肺转移模型,随机分为恩度组、纳米金组、纳米金结合恩度组和模型组。成瘤后各组小鼠均经尾静脉注射相应药物0.1 mL(每天1次)。9 d后麻醉小鼠并切取原位肿瘤,继续饲养2周后处死小鼠取肺组织观察转移情况。HE染色观察原位肿瘤坏死情况,免疫染色检测肿瘤CD31、碳酸酐酶IX(CA-IX)、vimentin和闭锁小带蛋白1(ZO-1)的表达。结果:与模型组相比,药物处理组小鼠的肺转移灶均明显减少,其中纳米金结合恩度组的肺转移抑制率最高,肿瘤坏死明显减少,肿瘤中CD31、CA-IX和vimentin的表达量显著降低,ZO-1的表达量显著升高。结论:与单用恩度或纳米金相比,纳米金结合恩度可明显抑制小鼠黑色素瘤肺转移,其机制可能为使肿瘤新生血管减少及血管正常化,改善肿瘤缺氧,从而抑制肿瘤上皮-间充质转化,使肿瘤细胞间紧密连接增加而侵袭性减低。     

GFP-transfected tumor cells are useful in examining early metastasis <Emphasis Type="Italic">in vivo</Emphasis>, but immune reaction precludes long-term tumor development studies in immunocompetent mice

To develop effective therapeutic strategies aimed at treating tumor metastasis, critical steps in this process must be better understood. For this purpose we have established a new model to visualize and quantify early metastasis. Murine CT-26 colon adenocarcinoma cells were stably transfected with green fluorescent protein (GFP). Tumor cells were intraportally delivered to the liver of Balb/c mice and subsequently tracked by intravital fluorescence microscopy. Coinjection of fluorescent beads and in vivo propidium iodide staining allowed examination of initial tumor cell arrest, extravasation, viability and proliferation. Results showed that GFP-transfection compared to conventional labeling procedures (Calcein, cytoplasmic microspheres) did not alter early metastatic properties. However, the long-term development of liver metastases expressing GFP was markedly reduced compared to wild type CT-26 tumor cells. An increase in the size and the number of liver metastases in T- and B-cell-deficient SCID mice suggested an immune response to the GFP transfected cells responsible for the reduced metastatic growth in wild-type mice. Based on our findings, this model can be used to examine the early steps of metastasis in vivo. However, in immunocompetent mice, the use of GFP-labeled tumor cells should be limited to tracking cell arrest and extravasation, whereas evaluations of long-term metastatic growth should be performed in immunodeficient mice. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Fluorescent Orthotopic Mouse Model for Reliable Measurement and Genetic Modulation of Human Neuroblastoma Metastasis

Neuroblastoma is the most common extra-cranial solid tumor of infancy and childhood, and majority of patients die from the metastatic disease. Orthotopic xenograft mouse models are valuable tools for improving our understanding and control of neuroblastoma metastasis, because they readily represent genetic diversity and allow spontaneous metastasis. Intra-adrenal injection is commonly used for establishing the orthotopic animal models since human neuroblastoma frequently originates in the adrenal gland. However, it is unclear whether the metastatic potential of neuroblastoma can be reliably determined in adrenally-injected mice because their gland size is so small. In this study, we developed and characterized a fluorescent orthotopic xenograft animal model of NB69-derived human neuroblastoma. By comparing animals receiving adrenal injection and adrenal overlay, with the latter mimicking injection spillover, we found that the metastatic potential of neuroblastoma can be reliably determined in animal lungs. Furthermore, the lung metastasis can be genetically modulated in these animals. The results also show that the expression of Renillagreen fluorescent protein (GFP) was exceptionally stable in NB69 cells, allowing rapid and sensitive detection of lung metastases at the macroscopic level. Additional features of our model include 100 tumor take, a 1-week tumor latency, resemblance to tumor behaviors in neuroblastoma patients, and the ability to monitor the expression of a gene of interest with GFP. This animal model of human neuroblastoma will be useful for studying genes involved in the metastatic process and for evaluating anti-metastasis agents in pre-clinical trials. This revised version was published online in July 2006 with corrections to the Cover Date.     

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model

The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.     

Metastatic patterns of lung cancer visualized live and in process by green fluorescence protein expression

We demonstrate here the visualization of human lung cancer metastasis live and in process in nude mice by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell line Anip 973 stably transfected with the humanized GFP-S65T cDNA was selected for very bright green fluorescence. GFP-transfected lung cancer cells were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, the resulting tumor had reached over 1 cm in diameter and had very bright GFP fluorescence. Fragments of subcutaneous tumor were implanted onto the visceral pleura of the left lung of nude mice by surgical orthotopic implantation (SOI) of histologically-intact tissue via transverse thoracotomy. The ipsilateral resulting tumor was highly fluorescent due to GFP expression. GFP expression allowed the visualization of the advancing margin of the ipsilateral tumor into the fresh normal lung tissue. Lymphogenous and direct-seeding metastases in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity and contralateral lung in the SOI-treated mice were brightly visualized by GFP expression in fresh tissue. GFP-transfected and untransfected tumor had similar metastatic characteristics suggesting that GFP expression had no effect on metastasis itself. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process.     

The matrix metalloproteinase inhibitor batimastat inhibits angiogenesis in liver metastases of B16F1 melanoma cells

Matrix metalloproteinases (MMPs) have been shown to contribute functionally to tumor metastasis. MMP inhibitors are thus being assessed for clinical utility as anti-metastatic therapeutics. Batimastat (BB-94) is a synthetic MMP inhibitor that has been shown to inhibit tumor growth and metastasis in mice. Here we assessed the ability of batimastat to inhibit liver metastases of murine B16F1 cells, after injection of cells in mice via mesenteric vein to target the liver. We then determined which of the sequential steps in metastasis were affected by batimastat, in order to identify its mechanism of action in vivo. Intravital videomicroscopy was used to assess the effect on extravasation, and a cell accounting procedure was used to determine the effect on initial survival of cells. Stereological quantification of functional blood vessels was used to determine the effect on tumor vascularity, thereby avoiding problems associated with immunohistochemical detection of liver sinusoidal endothelial cells. We found that batimastat (50 mg/kg i.p. 5 h prior to and after cell injection, daily thereafter) resulted in a 23% reduction in mean diameter of liver metastases (equivalent to a 54% reduction in tumor volume), while not reducing the number of metastases. Extravasation of cells from the liver circulation was not affected: at 8, 24 and 48 h after injection of cells, the same proportion of cells had extravasated from treated vs. control mice. Batimastat also did not inhibit early survival of cells. However, batimastat-treated mice had a significantly reduced percentage vascular volume within liver metastases, indicating inhibition of angiogenesis. This study demonstrates in vivo that the mechanism by which batimastat limits growth of B16F1 metastases in liver is not by affecting extravasation, but by inhibiting angiogenesis within metastases. This finding suggests that MMP inhibitors may be appropriate for use in patients with metastatic cells that have already extravasated in secondary sites.     

A novel noninvasive model of endometriosis for monitoring the efficacy of antiangiogenic therapy

Endometriosis, the presence of ectopic endometrial tissue, is a common disease associated with high morbidity and socioeconomic problems. Angiogenesis, the formation of new blood vessels, plays an important role in the formation and growth of endometriotic lesions. We have created a novel, noninvasive model to monitor the growth of these lesions and the associated angiogenesis in vivo. First, we generated luciferase-expressing transgenic mice by inserting the human ubiquitin C promoter coupled to the firefly luciferase reporter. Injection of luciferin in these mice causes full-body bioluminescence, which can be detected using a low-light CCD camera. Endometrial tissue from these transgenic mice was surgically implanted into nonluminescent recipients. Bioluminescence of lesions was noninvasively imaged after intravenous or intraperitoneal injection of luciferin. Transabdominal luminescence compared well with the location of the transgenic endometriotic lesions, and lesion size correlated with the intensity of luminescence. Systemic treatment with the angiogenesis inhibitors caplostatin and endostatin peptide mP-1 delayed and suppressed the onset and intensity of the luminescent signal. Caplostatin suppressed the growth of endometriotic lesions by 59% compared with controls. This novel, noninvasive model of endometriosis provides a means to study early angiogenesis in vivo and to monitor endometriotic growth and the efficacy of systemic antiangiogenic therapy.     

Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.     

In vivo imaging with fluorescent proteins: the new cell biology

We propose a new cell biology where the behavior of 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 tumor-stroma interactions and 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 can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo. Mice in which the regulatory elements of the stem-cell marker nestin drive GFP enable nascent vasculature to be visualized interacting with transplanted RFP-expressing cancer cells. Nestin-driven GFP expression can also be used to visualize hair follicle stem cells. 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. Multiple-color labeling of cells will enable multiple events to be simultaneously visualized in vivo including gene expression, ion fluxes, protein and organelle trafficking, chromosome dynamics and numerous other processes currently still only studied in vitro.     

Analysis of the tumor vasculature and metastatic behavior of xenografts of human melanoma cell lines transfected with vascular permeability factor.

Vascular permeability factor (VPF) is an important mediator of vascular development in tumors. Some human melanoma cell lines have a low VPF expression level in culture, but this level is upregulated when growing as a tumor in nude mice. Other melanoma lines have a constitutively high VPF expression. To compare the biological behavior of tumors with these two expression patterns, a human melanoma cell line with an inducible VPF expression was transfected with VPF expression constructs. In this way, several lines were obtained that constitutively produce either the soluble VPF121 or the matrix-associated VPF189 variant at levels of 4 to 30 times the VPF level in mature tumors derived from the parental line. The recombinant VPF RNA, which lacks most of the 5' noncoding sequences present in the endogenous VPF mRNA, was much more efficiently translated than the endogenous messenger. Upon injection in nude mice, all VPF-transfected lines developed tumors with aberrations in vascularization and in distribution of matrix components. In these tumors the blood vessels were hyperpermeable for an i.v. injected protein tracer. Transfection did not influence the in vitro growth rate of the cell lines, but the tumors from the VPF-transfected lines had higher growth rates in vivo than tumors from the parental line or the vector-transfected line. Although the incidence of lung metastasis was similar in all lines, the number of metastases per affected lung was significantly increased in mice carrying VPF-transfectant tumors. We conclude that the pattern and the level of VPF expression in a tumor are important determinants of the architecture and functionality of the vascular bed, but that overexpression of VPF does not necessarily lead to an increase of microvascular density or metastatic spread. The role of VPF in melanoma progression is obviously complex and may be difficult to derive in its generality from a single experimental model.     

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.