Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment

Abstract.: The fatality of cancer predominantly results from the dissemination of primary tumor cells to distant sites and the subsequent formation of metastases. During tumor progression, some of the primary tumor cells as well as the tumor microenvironment undergo characteristic molecular changes, which are essential for the metastatic dissemination of tumor cells. In this review, we will discuss recent insights into pro-metastatic events occurring in tumor cells themselves and in the tumor stroma. Tumor cell-intrinsic alterations include the loss of cell polarity and alterations in cell-cell and cell-matrix adhesion as well as deregulated receptor kinase signaling, which together support detachment, migration and invasion of tumor cells. On the other hand, the tumor stroma, including endothelial cells, fibroblasts and cells of the immune system, is engaged in an active molecular crosstalk within the tumor microenvironment. Subsequent activation of blood vessel and lymph vessel angiogenesis together with inflammatory and immune-suppressive responses further promotes cancer cell migration and invasion, as well as initiation of the metastatic proces

VEGF family members : modulators of tumor angiogenesis and lymphangiogenesis

Members of the vascular endothelial growth factor (VEGF) family and their receptors (VEGFR) play an essential role in the development and maintenance of the blood and lymphatic vasculature. To date, five VEGFs have been identified in the mammalian genome, VEGF-A, -B, -C, -D, and placental growth factor (PlGF), which display distinct binding affinities for VEGFR-1, -2, and -3. In addition to their central function in physiological angiogenesis and lymphangiogenesis, VEGFs and VEGFRs are upregulated during carcinogenesis and are involved in the remodeling of the tumoral blood and lymphatic vasculature. By activating VEGFR-1 and –2, which are both expressed on blood endothelial cells, VEGF-A promotes the formation of new tumoral blood vessels and thereby accelerates tumor growth. In contrast, upregulation of VEGF-C, a ligand for lymphatic endothelial VEGFR-3 as well as for VEGFR-2, induces the formation of tumor-associated lymphatic vessels and thus promotes the passive metastatic dissemination of tumor cells to regional lymph nodes. Much less is known about the functional consequences of tumor-expressed VEGF-B and PlGF, two selective ligands for VEGFR-1, as well as VEGF-D, the second VEGFR-3- and -2-binding lymphangiogenic VEGF family member. Also, the biological effects of selective VEGFR-1, -2 or -3 signaling on tumor angiogenesis and tumor growth as well as tumor lymphangiogenesis and metastasis are incompletely studied. Only recently, the identification of VEGF-E, a selective ligand for VEGFR-2, as well as the generation of VEGF-C156S, a specific ligand for VEGFR-3, has enabled the study of the distinct roles of these receptors. To investigate the function of lymphangiogenic VEGF-D under physiological conditions, I analyzed transgenic mice, in which expression of VEGF-D is specifically targeted to β-cells of pancreatic islets of Langerhans (Rip1VEGF-D mice). In these mice, expression of VEGF-D induces the formation of large lymphatic lacunae surrounding most islets. A few of these lymphatic vessels may be dysfunctional, which causes intra-lymphatic accumulations of immune cells. Moreover, lymphatic lacunae often contain erythrocytes, which may result from blood-lymphatic vessel shunts found in the vicinity of some islets. However, the fact that erythrocytes are drained to regional lymph nodes demonstrates the draining capacity of the de novo formed lymphatic vessels. To address the impact of VEGF-D on tumorigenesis and metastasis, I crossed Rip1VEGF-D with Rip1Tag2 mice, a wellcharacterized transgenic model of poorly metastatic multistage β-cell carcinogenesis. Tumoral expression of VEGF-D in Rip1Tag2 mice promotes the growth of peri-tumoral lymphatic vessels that frequently contain leucocyte clusters and hemorrhages. Concomitantly, these double-transgenic mice exhibit a high incidence of regional lymph node and distant lung metastases. Since expression of VEGF-D does not significantly affect the invasiveness of tumors and all metastases are well differentiated, these data indicate that VEGF-D promotes lymphogenous metastasis by upregulating tumor-associated lymphangiogenesis. Interestingly, the presence of VEGF-D significantly represses tumor angiogenesis and tumor growth, yet the mechanisms of this inhibition are thus far uncharacterized. Notably, syngenic and allogenic subcutaneous transplantation of VEGF-D-producing Rip1Tag2 tumor cell lines results in the formation of tumors exhibiting a dense intra-tumoral lymphatic network but lacking peri-tumoral lymphatic vessels. In these transplanted tumors, no immune cell clusters or hemorrhages are formed in tumor-associated lymphatic vessels and tumor angiogenesis is unaffected by the expression of VEGF-D. These results demonstrate that the tumor microenvironment critically modulates VEGF-D-elicited effects. It has been recently shown that transgenic expression of VEGF-C during Rip1Tag2 tumorigenesis promotes metastasis to regional lymph nodes but not to the lungs by inducing peri-tumoral lymphangiogenesis. Tumor-associated lymphatic vessels of these mice neither contain immune cell accumulations nor hemorrhages, and tumor angiogenesis and tumor growth are not affected by the production of VEGF-C. Thus, by employing the Rip1Tag2 tumor model, I was able to identify not only similarities but also significant differences between VEGF-D and –C function. Since VEGF-C and –D can bind both VEGFR-3 and –2, it is not fully established whether selective activation of VEGFR-3 is sufficient to induce tumoral lymphangiogenesis and to promote lymphogenous metastasis. Therefore, I established transgenic mice expressing VEGF-C156S in the endocrine pancreas and crossed these mice with Rip1Tag2 animals. The analysis of single and double transgenic mice revealed that VEGF-C156S phenocopies VEGF-C in all investigated aspects. These results indicate that VEGFR-3 may be the predominant receptor mediating VEGF-C-elicited effects in Rip1Tag2 mice and that selective activation of VEGFR-3 is sufficient to promote tumor-associated lymphangiogenesis and metastasis. Hence, VEGFR-3 might represent a valuable target for future anti-metastatic strategies. To further understand the specific roles of VEGFR-1 and –2 signaling in physiological angiogenesis as well as in tumorigenesis, I established transgenic mouselines, which express the VEGFR-1-specific ligands VEGF-B167 and PlGF-1 as well as the selective VEGFR-2 ligand VEGF-ED1701 in β-cells of pancreatic islets (Rip1VEGF-B167, Rip1PlGF-1, and Rip1VEGF-ED1701 mice). These single transgenic mice were analyzed with regard to islet blood vessel morphology and density. In a second set of experiments, I crossed singletransgenic animals with Rip1Tag2 mice. These double-transgenic mice expressing either VEGF-B167, PlGF-1 or VEGF-ED1701 in tumor cells, were analyzed for changes in tumor angiogenesis, tumor growth, and tumor progression. The preliminary data provide evidence that β-cell-specific upregulation of VEGF-B167 does not critically affect physiological angiogenesis of single-transgenic mice but results in a significant increase in the tumor microvessel density of double-transgenic animals. However, tumor growth and tumor progression are not promoted by the stimulation of tumor angiogenesis. In contrast, overexpression of PlGF-1 in single-transgenic mice leads to a prominent dilation of blood capillaries, which may at least in part be caused by a significant reduction of stabilizing blood vessel-associated pericytes. Furthermore, tumoral expression of PlGF-1 significantly inhibits tumor angiogenesis and tumor growth, suggesting that this growth factor might be a natural inhibitor of pathological angiogenesis. Hence, although binding to the same receptor, VEGFB167 and PlGF-1 elicit opposing effects on the tumor blood vasculature. These results suggest that the two growth factors induce distinct signaling pathways via VEGFR-1, which might be considered when designing inhibitors of angiogenesis involving VEGFR-1. Importantly, the phenotype of VEGF-B167- and PlGF-1- expressing Rip1Tag2 mice is different from the recently described VEGF-A165 transgenic Rip1Tag2 mice, which exhibited accelerated tumor growth and early death. The analysis of VEGF-ED1701-expressing mice and effects induced by selective activation of VEGFR-2 signaling is currently underway

Clinical implications of increased lymph vessel density in the lymphatic metastasis of early-stage invasive cervical carcinoma: a clinical immunohistochemical method study

<p>Abstract</p> <p>Background</p> <p>Cervical cancer is the most common malignant gynecological cancer, and lymphatic metastasis can occur in the early stage of tumor growth. Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), a marker for lymphatic endothelium, provides powerful tools for studying tumor lymphangiogenesis. The purpose of this study is to investigate the clinical implications of lymphangiogenesis in the metastasis of early-stage invasive cervical carcinoma.</p> <p>Methods</p> <p>We used immunohistochemical (IHC) staining with the antibody against LYVE-1 to measure lymph vessel density in 41 cases of early-stage invasive cervical carcinoma and 12 cases of normal cervical samples. We then analyzed the correlation between lymph vessel density and clinicopathological features of the tumors.</p> <p>Results</p> <p>(1) The majority of peritumoral lymphatics were enlarged, dilated, and irregular. In contrast, intratumoral lymph vessels were small and collapsed. The peritumoral lymphatic vessel density (PLVD) was significantly higher than the intratumoral lymphatic vessel density (ILVD) (<it>P </it>< 0.01). (2) Both ILVD and PLVD were significantly higher than the LVD of the control cervixes (<it>P </it>< 0.01). (3) Both ILVD and PLVD were significantly associated with lymph node metastasis (ILVD, <it>P </it>< 0.05; PLVD, <it>P </it>< 0.01) and lymphatic vessel invasion (ILVD, <it>P </it>< 0.05; PLVD, <it>P </it>< 0.01). Both the ILVD and PLVD in patients with histological grade HG2 and HG3 were significantly higher than those with HG1 (<it>P </it>< 0.05).</p> <p>Conclusion</p> <p>Tumor lymphangiogenesis in early-stage invasive cervical carcinoma may play an important role in the process of lymphatic metastasis.</p

Computer simulation of glioma growth and morphology

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion

Suppressive Effects of Vascular Endothelial Growth Factor-B on Tumor Growth in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis

Peer reviewe

In vitro anti-angiogenic properties of LGD1069, a selective retinoid X-receptor agonist through down-regulating Runx2 expression on Human endothelial cells

<p>Abstract</p> <p>Background</p> <p>LGD1069 (Targretin^®) is a selective retinoid X receptor (RXR) ligand, which is used in patients for cutaneous T-cell lymphoma. Our published study reported that LGD1069 inhibited tumor-induced angiogenesis in non-small cell lung cancer. In present study, we found that LGD1069 suppressed the proliferation, adhesion, invasion and migration of endothelial cells directly, and affected the expression of vegf and some matrix genes.</p> <p>Methods</p> <p>Human umbilical vein endothelial cells (HUVECs) were used for <it>in vitro </it>study. MTT assay and Sulforhodamine B assay were used for cell viability assay; the tube formation assay was used to investigate the effect of LGD1069 on angiogenesis <it>in vitro</it>. <it>In vitro </it>adhesion, migration and invasion of HUVEC cells were analyzed by Matrigel adhesion, migration and invasion assay. Gene expressions were measured by RT-PCR and Western blot analysis.</p> <p>Results</p> <p>Our data showed here that LGD1069 inhibited the activation of TGF-β/Smad pathway significantly. Furthermore, it was demonstrated that expression of Runx2 was suppressed pronouncedly during incubation with LGD1069. Runx2 is a DNA-binding transcription factor which plays a master role in tumor-induced angiogenesis and cancer cells metastasis by interaction with the TGF-β/Smad pathway of transcriptional modulators.</p> <p>Conclusions</p> <p>Our results suggested that LGD1069 may impair angiogenic and metastatic potential induced by tumor cells through suppressing expression of Runx2 directly on human endothelial cells, which may point out new pathway through which LGD1069 display anti-angiogenic properties, and provide new molecular evidence to support LGD1069 as a potent anti-metastatic agent in cancer therapy.</p

I-BAR protein antagonism of endocytosis mediates directional sensing during guided cell migration

The I-BAR domain–containing protein, missing-in-metastasis, directs cell motility by opposing the endocytic activity of the endophilin–CD2AP–cortactin complex

Recombinant Human Endostatin Endostar Inhibits Tumor Growth and Metastasis in a Mouse Xenograft Model of Colon Cancer

To investigate the effects of recombinant human endostatin Endostar on metastasis and angiogenesis and lymphangiogenesis of colorectal cancer cells in a mouse xenograft model. Colon cancer cells SW620 were injected subcutaneously into the left hind flank of nude mice to establish mouse xenograft models. The mice were treated with normal saline or Endostar subcutaneously every other day. The growth and lymph node metastasis of tumor cells, angiogenesis and lymphangiogenesis in tumor tissue were detected. Apoptosis and cell cycle distribution were studied by flow cytometry. The expression of VEGF-A, -C, or -D in SW620 cells was determined by immunoblotting assays. Endostar inhibited tumor growth and the rate of lymph node metastasis (P < 0.01). The density of blood vessels in or around the tumor area was 12.27 ± 1.21 and 22.25 ± 2.69 per field in Endostar-treated mice and controls (P < 0.05), respectively. Endostar also decreased the density of lymphatic vessels in tumor tissues (7.84 ± 0.81 vs. 13.83 ± 1.08, P < 0.05). Endostar suppresses angiogenesis and lymphangiogenesis in the lymph nodes with metastases, simultaneously. The expression of VEGF-A, -C and -D in SW620 cells treated with Endostar was substantially lower than that of controls. Endostar inhibited growth and lymph node metastasis of colon cancer cells by inhibiting angiogenesis and lymphangiogenesis in a mouse xenograft model of colon cancer

Blocking TLR2 Activity Attenuates Pulmonary Metastases of Tumor

Background: Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis. Methods and Findings: In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8 + T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice. Conclusions and Significance: Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating