MyCTC chip: microfluidic-based drug screen with patient-derived tumour cells from liquid biopsies.

Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns, often leading to ineffective treatment. Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies, yet when disease progression accelerates, tissue biopsies are not performed on a regular basis. It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities, providing valuable information about previously underappreciated treatment opportunities. Here, we present MyCTC chip, a novel microfluidic device enabling the isolation, culture and drug susceptibility testing of cancer cells derived from liquid biopsies. Cancer cell capture is achieved through a label-free, antigen-agnostic enrichment method, and it is followed by cultivation in dedicated conditions, allowing on-chip expansion of captured cells. Upon growth, cancer cells are then transferred to drug screen chambers located within the same device, where multiple compounds can be tested simultaneously. We demonstrate MyCTC chip performance by means of spike-in experiments with patient-derived breast circulating tumour cells, enabling >95% capture rates, as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian, tubal and endometrial cancer, where sensitivity to specific chemotherapeutic agents was identified. Together, we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities.

The metastatic spread of breast cancer accelerates during sleep.

The metastatic spread of cancer is achieved by the haematogenous dissemination of circulating tumour cells (CTCs). Generally, however, the temporal dynamics that dictate the generation of metastasis-competent CTCs are largely uncharacterized, and it is often assumed that CTCs are constantly shed from growing tumours or are shed as a consequence of mechanical insults1. Here we observe a striking and unexpected pattern of CTC generation dynamics in both patients with breast cancer and mouse models, highlighting that most spontaneous CTC intravasation events occur during sleep. Further, we demonstrate that rest-phase CTCs are highly prone to metastasize, whereas CTCs generated during the active phase are devoid of metastatic ability. Mechanistically, single-cell RNA sequencing analysis of CTCs reveals a marked upregulation of mitotic genes exclusively during the rest phase in both patients and mouse models, enabling metastasis proficiency. Systemically, we find that key circadian rhythm hormones such as melatonin, testosterone and glucocorticoids dictate CTC generation dynamics, and as a consequence, that insulin directly promotes tumour cell proliferation in vivo, yet in a time-dependent manner. Thus, the spontaneous generation of CTCs with a high proclivity to metastasize does not occur continuously, but it is concentrated within the rest phase of the affected individual, providing a new rationale for time-controlled interrogation and treatment of metastasis-prone cancers.

Transcription factors AP-2α and AP-2ß regulate distinct segments of the distal nephron in the mammalian kidney.

Transcription factors AP-2α and AP-2ß have been suggested to regulate the differentiation of nephron precursor populations towards distal nephron segments. Here, we show that in the adult mammalian kidney AP-2α is found in medullary collecting ducts, whereas AP-2ß is found in distal nephron segments except for medullary collecting ducts. Inactivation of AP-2α in nephron progenitor cells does not affect mammalian nephrogenesis, whereas its inactivation in collecting ducts leads to defects in medullary collecting ducts in the adult. Heterozygosity for AP-2ß in nephron progenitor cells leads to progressive distal convoluted tubule abnormalities and ß-catenin/mTOR hyperactivation that is associated with renal fibrosis and cysts. Complete loss of AP-2ß in nephron progenitor cells caused an absence of distal convoluted tubules, renal cysts, and fibrosis with ß-catenin/mTOR hyperactivation, and early postnatal death. Thus, AP-2α and AP-2ß have non-redundant distinct spatiotemporal functions in separate segments of the distal nephron in the mammalian kidney.

An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression.

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.

Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors.

Macrophages undergoing M1- versus M2-type polarization differ significantly in their cell metabolism and cellular functions. Here, global quantitative time-course proteomics and phosphoproteomics paired with transcriptomics provide a comprehensive characterization of temporal changes in cell metabolism, cellular functions, and signaling pathways that occur during the induction phase of M1- versus M2-type polarization. Significant differences in, especially, metabolic pathways are observed, including changes in glucose metabolism, glycosaminoglycan metabolism, and retinoic acid signaling. Kinase-enrichment analysis shows activation patterns of specific kinases that are distinct in M1- versus M2-type polarization. M2-type polarization inhibitor drug screens identify drugs that selectively block M2- but not M1-type polarization, including mitogen-activated protein kinase kinase (MEK) and histone deacetylase (HDAC) inhibitors. These datasets provide a comprehensive resource to identify specific signaling and metabolic pathways that are critical for macrophage polarization. In a proof-of-principle approach, we use these datasets to show that MEK signaling is required for M2-type polarization by promoting peroxisome proliferator-activated receptor-γ (PPARγ)-induced retinoic acid signaling.

Distinct effects of complement and of NLRP3- and non-NLRP3 inflammasomes for choroidal neovascularization.

NLRP3 inflammasome activation and complement-mediated inflammation have been implicated in promoting choroidal neovascularization (CNV) in age-related macular degeneration (AMD), but central questions regarding their contributions to AMD pathogenesis remain unanswered. Key open questions are (1) whether NLRP3 inflammasome activation mainly in retinal pigment epithelium (RPE) or rather in non-RPE cells promotes CNV, (2) whether inflammasome activation in CNV occurs via NLRP3 or also through NLRP3-independent mechanisms, and (3) whether complement activation induces inflammasome activation in CNV. Here we show in a neovascular AMD mouse model that NLRP3 inflammasome activation in non-RPE cells but not in RPE cells promotes CNV. We demonstrate that both NLRP3-dependent and NLRP3-independent inflammasome activation mechanisms induce CNV. Finally, we find that complement and inflammasomes promote CNV through independent mechanisms. Our findings uncover an unexpected role of non-NLRP3 inflammasomes for CNV and suggest that combination therapies targeting inflammasomes and complement may offer synergistic benefits to inhibit CNV.

Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells.

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.

Targeting Platelet-Derived Growth Factor Receptor ß(+) Scaffold Formation Inhibits Choroidal Neovascularization.

Neovascular age-related macular degeneration is among the most common causes of irreversible blindness and manifests with choroidal neovascularization (CNV). Anti-vascular endothelial growth factor-A therapies are only partially effective and their chronic administration may impair functions of the choriocapillaris and retina. Thus, novel therapeutic targets are needed urgently. We have observed in a laser-induced model of CNV that a platelet-derived growth factor receptor ß positive (PDGFRß(+)) scaffold is formed before infiltration of neovessels into this scaffold to form CNV lesions, and that this scaffold limits the extent of neovascularization. Based on these observations we hypothesized that ablation of proliferating PDGFRß(+) cells to prevent the formation of this scaffold might inhibit CNV growth and present a novel therapeutic approach for neovascular age-related macular degeneration. To test this hypothesis we targeted proliferating PDGFRß(+) cells through independent distinct approaches after laser injury: i) by using an inducible genetic model to inhibit specifically proliferating PDGFRß(+) cells, ii) by treating mice with a neutralizing anti-PDGFRß antibody, iii) by administering an anti-PDGF-AB/BB aptamer, and iv) by using small chemical inhibitor approaches. The results show that therapeutic targeting of proliferating PDGFRß(+) cells potently inhibits the formation of the pericyte-like scaffold, with concomitant attenuation of CNV. Moreover, we show that early inhibition of PDGFRß(+) cell proliferation before neovessel formation is sufficient to inhibit scaffold formation and neovascularization.

Loss of stromal JUNB does not affect tumor growth and angiogenesis.

The transcription factor AP-1 subunit JUNB has been shown to play a pivotal role in angiogenesis. It positively controls angiogenesis by regulating Vegfa as well as the transcriptional regulator Cbfb and its target Mmp13. In line with these findings, it has been demonstrated that tumor cell-derived JUNB promotes tumor growth and angiogenesis. In contrast to JUNB's function in tumor cells, the role of host-derived stromal JUNB has not been elucidated so far. Here, we show that ablation of Junb in stromal cells including endothelial cells (ECs), vascular smooth muscle cells (SMCs) and fibroblasts does not affect tumor growth in two different syngeneic mouse models, the B16-F1 melanoma and the Lewis lung carcinoma model. In-depth analyses of the tumors revealed that tumor angiogenesis remains unaffected as assessed by measurements of the microvascular density and relative blood volume in the tumor. Furthermore, we could show that the maturation status of the tumor vasculature, analyzed by the SMC marker expression, α-smooth muscle actin and Desmin, as well as the attachment of pericytes to the endothelium, is not changed upon ablation of Junb. Taken together, these results indicate that the pro-angiogenic functions of stromal JUNB are well compensated with regard to tumor angiogenesis and tumor growth.

Repression of malignant tumor progression upon pharmacologic IGF1R blockade in a mouse model of insulinoma.

NVP-AEW541, a specific ATP-competitive inhibitor of the insulin-like growth factor-1 receptor (IGF1R) tyrosine kinase, has been reported to interfere with tumor growth in various tumor transplantation models. We have assessed the efficacy of NVP-AEW541 in repressing tumor growth and tumor progression in the Rip1Tag2 transgenic mouse model of pancreatic ß-cell carcinogenesis. In addition, we have tested NVP-AEW541 in Rip1Tag2;RipIGF1R double-transgenic mice which show accelerated tumor growth and increased tumor malignancy compared with Rip1Tag2 single-transgenic mice. Previously, we have shown that high levels of IGF-2, a high-affinity ligand for IGF1R, are required for Rip1Tag2 tumor cell survival and tumor growth. Unexpectedly, treatment of Rip1Tag2 mice with NVP-AEW541 in prevention and intervention trials neither did affect tumor growth nor tumor cell proliferation and apoptosis. Yet, it significantly repressed progression to tumor malignancy, that is, the rate of the transition from differentiated adenoma to invasive carcinoma. Treatment of Rip1Tag2;RipIGF1R double-transgenic mice resulted in moderately reduced tumor volumes and increased rates of tumor cell apoptosis. Sustained expression of IGF-2 and of the IGF-2-binding form of insulin receptor (IR-A) in tumor cells suggests a compensatory role of IR-A upon IGF1R blockade. The results indicate that inhibition of IGF1R alone is not sufficient to efficiently block insulinoma growth and imply an overlapping role of IGF1R and insulin receptor in executing mitogenic and survival stimuli elicited by IGF-2. The reduction of tumor invasion upon IGF1R blockade on the other hand indicates a critical function of IGF1R signaling for the acquisition of a malignant phenotype.

Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis.

BACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing ß-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.

Pericytes regulate the blood-brain barrier.

The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.

A bioluminescent mouse model of pancreatic {beta}-cell carcinogenesis.

The Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis has been instrumental in identifying several hallmarks of cancer, including tumor cell evasion from apoptosis, tumor angiogenesis and tumor invasion. Moreover, Rip1Tag2 mice have been helpful in the development and testing of innovative cancer therapies and tumor imaging protocols. However, based on tumor localization in the mouse, primary tumor growth and metastatic dissemination cannot be easily monitored in a longitudinal axis by non-invasive and low-technology approaches. Here, we report the generation of a new transgenic mouse line as a versatile tool to study beta-cell carcinogenesis. Transgenic expression of a bicistronic messenger RNA encoding simian virus large T antigen and firefly luciferase in pancreatic beta-cells recapitulates insulinoma development in a reproducible multistage process. In the mouse line called RipTag-IRES-Luciferase line (RTL) 1, the beta-cell-specific expression of luciferase allows the non-invasive monitoring of primary tumor growth over time in vivo and the detection and quantification of disseminated tumor cells and micrometastases in distant organs ex vivo. When crossed to mouse lines in which the expression of cancer 'modifier' genes has been manipulated, tumor initiation and tumor progression are similarly affected as previously reported for Rip1Tag2 mice, indicating a robust tumor progression pathway shared between the two different transgenic mouse lines. Together, the data indicate that the RTL1 mouse line will be of great value to study anti-tumoral therapeutic approaches as well as to define the functional roles of cancer- and metastasis-related genes when crossed to appropriate transgenic or gene-targeted mouse lines.

Differential effects of the vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 on tumor angiogenesis and tumor lymphangiogenesis.

Halting tumor growth by interfering with tumor-induced angiogenesis is an attractive therapeutic approach. Such treatments include humanized antibodies blocking the activity of vascular endothelial growth factor (VEGF)-A (bevacizumab), soluble VEGF receptor (VEGFR) constructs (VEGF-Trap), or small-molecule inhibitors of VEGFR signaling, including PTK787/ZK222584 (PTK/ZK), sorafenib, and sunitinib. PTK/ZK has been shown previously to specifically block VEGF-induced phosphorylation of VEGFR-1, -2 and -3 and thereby to inhibit endothelial cell proliferation, differentiation, and tumor angiogenesis. We have investigated the effect of PTK/ZK on tumor angiogenesis and tumor lymphangiogenesis using the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis. In Rip1Tag2 mice, tumor angiogenesis is predominantly mediated by VEGF-A, and as expected, PTK/ZK efficiently impaired tumor blood vessel angiogenesis and tumor growth. Double-transgenic Rip1Tag2;Rip1VEGF-C and Rip1Tag2;Rip1VEGF-D mice not only exhibit VEGF-A-dependent blood vessel angiogenesis but also tumor lymphangiogenesis induced by the transgenic expression of VEGF-C or -D. In these mouse models, PTK/ZK also repressed tumor blood vessel angiogenesis and tumor growth yet failed to affect tumor lymphangiogenesis and lymphogenic metastasis. Adenoviral delivery of soluble VEGFR-3 also did not prevent tumor lymphangiogenesis in these mice. In contrast, spontaneous tumor lymphangiogenesis, as observed by the stochastic expression of VEGF-C and -D in tumors of neural cell adhesion molecule-deficient Rip1Tag2 mice, was repressed by PTK/ZK and soluble VEGFR-3. The results indicate that the time of onset and the levels of VEGF-C/D expression may be critical variables in efficiently repressing tumor lymphangiogenesis and that pathways other than VEGFR signaling may be involved in tumor lymphangiogenesis.

NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin.

Loss of expression of the cell-cell adhesion molecule E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT) in development and in the progression from epithelial tumours to invasive and metastatic cancers. Here, we demonstrate that the loss of E-cadherin function upregulates expression of the neuronal cell adhesion molecule (NCAM). Subsequently, a subset of NCAM translocates from fibroblast growth factor receptor (FGFR) complexes outside lipid rafts into lipid rafts where it stimulates the non-receptor tyrosine kinase p59(Fyn) leading to the phosphorylation and activation of focal adhesion kinase and the assembly of integrin-mediated focal adhesions. Ablation of NCAM expression during EMT inhibits focal adhesion assembly, cell spreading and EMT. Conversely, forced expression of NCAM induces epithelial cell delamination and migration, and high NCAM expression correlates with tumour invasion. These results establish a mechanistic link between the loss of E-cadherin expression, NCAM function, focal adhesion assembly and cell migration and invasion.

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis.

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

Increased tumor cell dissemination and cellular senescence in the absence of beta1-integrin function.

Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization, as well as transduction of signals into cells, to promote various aspects of cellular behavior, such as proliferation or survival. Integrins participate in many aspects of tumor biology. Here, we have employed the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis to investigate the role of beta(1)-integrin in tumor progression. Specific ablation of beta(1)-integrin function in pancreatic beta cells resulted in a defect in sorting between insulin-expressing beta cells and glucagon-expressing alpha cells in islets of Langerhans. Ablation of beta(1)-integrin in beta tumor cells of Rip1Tag2 mice led to the dissemination of tumor cell emboli into lymphatic blood vessels in the absence of ongoing lymphangiogenesis. Yet, disseminating beta(1)-integrin-deficient beta tumor cells did not elicit metastasis. Rather, primary tumor growth was significantly impaired by reduced tumor cell proliferation and the acquisition of cellular senescence by beta(1)-integrin-deficient beta tumor cells. The results indicate a critical role of beta(1)-integrin function in mediating metastatic dissemination and preventing tumor cell senescence.

Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis.

In many human carcinomas, expression of the lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) correlates with up-regulated lymphangiogenesis and regional lymph node metastasis. Here, we have used the Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis to investigate the functional role of VEGF-D in the induction of lymphangiogenesis and tumor progression. Expression of VEGF-D in beta cells of single-transgenic Rip1VEGF-D mice resulted in the formation of peri-insular lymphatic lacunae, often containing leukocyte accumulations and blood hemorrhages. When these mice were crossed to Rip1Tag2 mice, VEGF-D-expressing tumors also exhibited peritumoral lymphangiogenesis with lymphocyte accumulations and hemorrhages, and they frequently developed lymph node and lung metastases. Notably, tumor outgrowth and blood microvessel density were significantly reduced in VEGF-D-expressing tumors. Our results demonstrate that VEGF-D induces lymphangiogenesis, promotes metastasis to lymph nodes and lungs, and yet represses hemangiogenesis and tumor outgrowth. Because a comparable transgenic expression of vascular endothelial growth factor-C (VEGF-C) in Rip1Tag2 has been shown previously to provoke lymphangiogenesis and lymph node metastasis in the absence of any distant metastasis, leukocyte infiltration, or angiogenesis-suppressing effects, these results reveal further functional differences between VEGF-D and VEGF-C.

Moderate antiangiogenic activity by local, transgenic expression of endostatin in Rip1Tag2 transgenic mice.

Many previous reports have demonstrated that systemic administration of endostatin (ES), a proteolytic cleavage product of collagen type XVIII and an endogenous angiogenesis inhibitor, represses tumor angiogenesis in different preclinical tumor models with varying efficacy. For example, systemic delivery of recombinant ES to rat insulin promoter 1 (Rip1)T-antigen 2 (Tag2)-transgenic mice, a mouse model of pancreatic beta-cell carcinogenesis, has repressed tumor angiogenesis efficiently and with it, tumor growth. Here, we report that the transgenic expression of ES in Rip1ES-transgenic mice only interferes moderately with tumor growth in Rip1Tag2;Rip1ES double-transgenic mice. Tumor incidence is not reduced by the local expression of ES, and tumor outgrowth and progression to tumor malignancy are only retarded slightly. A significant effect of local ES expression on tumor angiogenesis is only apparent during the early stages of tumor development, where less angiogenic hyperplastic lesions are observed. Although efficiently produced and secreted by transgenic beta cells, locally expressed ES appears to be sequestered in the microenvironment, and its systemic levels are not increased. The results indicate that the antiangiogenic functions of ES critically depend on the mode of delivery and the site of expression: although its systemic application represses tumor angiogenesis and tumor growth efficiently, locally expressed ES appears to be less effective, and hence, additional mechanisms of solubilization or activation of latent ES seem to be required. These results have important implications about the modes of delivery used in antiangiogenic, therapeutic strategies, which are based on the antiangiogenic activities of ES.

Loss of neural cell adhesion molecule induces tumor metastasis by up-regulating lymphangiogenesis.

Reduced expression of neural cell adhesion molecule (NCAM) has been implicated in the progression to tumor malignancy in cancer patients. Previously, we have shown that the loss of NCAM function causes the formation of lymph node metastasis in a transgenic mouse model of pancreatic beta cell carcinogenesis (Rip1Tag2). Here we show that tumors of NCAM-deficient Rip1Tag2 transgenic mice exhibit up-regulated expression of the lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and -D (17% in wild-type versus 60% in NCAM-deficient Rip1Tag2 mice) and, with it, increased lymphangiogenesis (0% in wild-type versus 19% in NCAM-deficient Rip1Tag2 mice). Repression of VEGF-C and -D function by adenoviral expression of a soluble form of their cognate receptor, VEGF receptor-3, results in reduced tumor lymphangiogenesis (56% versus 28% in control versus treated mice) and lymph node metastasis (36% versus 8% in control versus treated mice). The results indicate that the loss of NCAM function causes lymph node metastasis via VEGF-C- and VEGF-D-mediated lymphangiogenesis. These results also establish Rip1Tag2;NCAM-deficient mice as a unique model for stochastic, endogenous tumor lymphangiogenesis and lymph node metastasis in immunocompetent mice.