Tumor DDR1 deficiency reduces liver metastasis by colon carcinoma and impairs stromal reaction.

Tumor DDR1 acts as a key factor during the desmoplastic response surrounding hepatic colorectal metastasis. Hepatic sinusoidal cell-derived soluble factors stimulate tumor DDR1 activation. DDR1 modulates matrix remodeling to promote metastasis in the liver through the interaction with hepatic stromal cells, specifically liver sinusoidal endothelial cells and hepatic stellate cells.

Decreased expression of the ß2 integrin on tumor cells is associated with a reduction in liver metastasis of colorectal cancer in mice.

BACKGROUND: Lymphocyte Function-Associated Antigen-1 (LFA-1; CD18/CD11a) is one of the main adhesion molecules used by immune cells to infiltrate the liver under inflammatory conditions. Recently, the expression of this integrin has also been reported on several solid tumors, including colorectal cancer. However, its functional role in the metastatic progression to the liver remains unknown. Using in vitro assays and an experimental orthotopic in vivo model of liver metastasis, we aimed to elucidate the role of tumor LFA-1 in the metastatic progression by means of the partial depletion of the ß2 subunit of LFA-1, required for integrin activation, firm adhesion and signaling. METHODS: To do so, we evaluated the effects of ß2 reduction on the murine colon carcinoma C26 cell line on their pro-metastatic features in vitro and their metastatic potential in vivo in a mouse model of colon carcinoma metastasis to the liver. RESULTS: The reduction in ß2 integrin expression correlated with a slower proliferation, and a reduced adhesion and migration of C26 cells in an in vitro setting. Additionally, tumor cells with a reduced in ß2 integrin expression were unable to activate the liver sinusoidal endothelial cells (LSECs). This resulted in a recovery of the cytotoxic potential of liver lymphocytes which is compromised by LSECs activated by C26 cells. This was related to the abrogation of RNA expression of inflammatory and angiogenic cytokines by C26 cells after their activation with sICAM-1, the main ligand of ß2αL. Furthermore, in vivo tumor cell retention and metastasis were profoundly reduced, along with a decrease in the recruitment and infiltration of myeloid derived suppressor cells (MDSCs) and lymphocytes to the liver. CONCLUSION: Taken together, our findings uncovered the modulatory role for the tumor ß2 subunit of the LFA-1 integrin in the metastatic progression of colorectal cancer to the liver by impairing activation of liver endothelium and thus, the local immune response in the liver. Besides, this integrin also showed to be critical in vivo for tumor cell retention, cytokine release, leukocyte recruitment and metastasis development. These data support a therapeutical potential of the integrin LFA-1 as a target for the treatment of colorectal liver metastasis.

Discoidin domain receptor 2 deficiency predisposes hepatic tissue to colon carcinoma metastasis.

BACKGROUND: The transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a major mechanism for stroma development in hepatic metastasis, but their regulatory pathways remain unclear. Transdifferentiated HSCs from fibrotic liver express high levels of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2), but it is unclear if DDR2 plays a direct profibrogenic role in the tumour microenvironment. AIM: To assess the impact of DDR2 on the prometastatic role of HSC-derived myofibroblasts. METHODS: Hepatic metastases were induced in DDR2(-/-) and DDR2(+/+) mice by intrasplenic injection of MCA38 colon carcinoma cells, and their growth and features were characterised. Stromagenic, angiogenic and cancer cell proliferation responses were quantified in metastases by immunohistochemistry. The adhesion-, migration- and proliferation-stimulating activities of supernatants from primary cultured DDR2(-/-) and DDR2(+/+) HSCs, incubated in MCA38 cell-conditioned medium, were evaluated in primary cultured liver sinusoidal endothelium cells (LSECs) and MCA38 cells. Gene expression signatures from freshly isolated DDR2(-/-) and DDR2(+/+) HSCs were compared and DDR2-regulated genes were studied by RT-PCR under basal conditions and after stimulation with MCA38 tumour-conditioned media. RESULTS: Metastases were increased three fold in DDR2(-/-) livers, and contained a higher density of α-smooth muscle actin-expressing myofibroblasts, CD31-expressing microvessels and Ki67-expressing MCA38 cells than metastases in DDR2(+/+) livers. Media conditioned by MCA38-activated DDR2(-/-) HSCs significantly increased adhesion, migration and proliferation of LSECs and MCA38 cells, compared with DDR2(+/+) HSCs. DDR2 deficiency in HSCs led to decreased gene expression of interferon γ-inducing factor interleukin (IL)-18 and insulin-like growth factor-I; and increased gene expression of prometastatic factors IL-10, transforming growth factor (TGF)ß and vascular endothelial growth factor (VEGF), bone morphogenetic protein-7 and syndecan-1. MC38 tumour-conditioned media further exacerbated expression changes in DDR2-dependent IL-10, TGFß and VEGF genes. CONCLUSION: DDR2 deficiency fosters the myofibroblast transdifferentiation of tumour-activated HSCs, generating a prometastatic microenvironment in the liver via HSC-derived factors. These findings underscore the role of stromal cells in conditioning the hepatic microenvironment for metastases through altered receptor-stroma interactions.

Loss of discoidin domain receptor 2 promotes hepatic fibrosis after chronic carbon tetrachloride through altered paracrine interactions between hepatic stellate cells and liver-associated macrophages.

Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl(4)) to DDR2(+/+) and DDR2(-/-) mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after chronic CCl(4) administration, DDR2(-/-) livers had increased collagen deposition, gelatinolytic activity, and HSC density. Increased basal gene expression of osteopontin, transforming growth factor-ß1, monocyte chemoattractant protein-1, and IL-10 and reduced basal gene expression of matrix metalloproteinase-2, matrix metalloproteinase-13, and collagen type I in quiescent DDR2(-/-) HSCs were amplified further after chronic CCl(4). In concordance, DDR2(-/-) HSCs isolated from chronically injured livers had enhanced in vitro migration and proliferation, but less extracellular matrix degradative activity. Macrophages from chronic CCl(4)-treated DDR2(-/-) livers showed stronger chemoattractive activity toward DDR2(-/-) HSCs than DDR2(+/+) macrophages, increased extracellular matrix degradation, and higher cytokine mRNA expression. In conclusion, loss of DDR2 promotes chronic liver fibrosis after CCl(4) injury. The fibrogenic sinusoidal milieu generated in chronic DDR2(-/-) livers recruits more HSCs to injured regions, which enhances fibrosis. Together, these findings suggest that DDR2 normally orchestrates gene programs and paracrine interactions between HSCs and macrophages that together attenuate chronic hepatic fibrosis.

A Synthetic Analog of Resveratrol Inhibits the Proangiogenic Response of Liver Sinusoidal Cells during Hepatic Metastasis.

We utilized Fas21, a resveratrol analog, to modulate the function of hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) during the angiogenic phase of murine liver metastasis by B16 melanoma and 51b colorectal carcinoma. Preangiogenic micrometastases were treated with Fas21 (1 mg/kg/day) or vehicle during the development of intra-angiogenic tracts. Mice treated with Fas21 showed reduced liver tumor foci in both liver metastasis models. Micrometastases were classified immunohistochemically, as well as according to their position coordinates and connection to local microvasculature. The volume of liver occupied by sinusoidal-type foci, containing infiltrating angiogenic capillaries, decreased by ~50% in Fas21-treated mice compared to vehicle-treated ones in both tumor metastasis models. The volume of portal foci, containing peripheral neoangiogenesis within a discontinuous layer of myofibroblasts, was similar in all experimental groups in both tumor metastasis models, but displayed enhanced necrotic central areas devoid of angiogenesis following Fas21 treatment. As a result, sinusoidal tumors from mice treated with Fas21 showed a 50% reduction in desmin(+)/asma(+) HSCs and CD31(+) vessel density, and a 45% reduction in intrametastatic VEGF mRNA compared with sinusoidal tumors from vehicle-treated mice. Necrotic portal metastases increased 2-4-fold in treated mice. In vitro, Fas21 reduced VEGF secretion by HSCs and 51b cells dose-dependently. Additionally, HSCs migration in response to tumor soluble factors was dose-dependently diminished by Fas21, as was LSEC migration in response to HSCs and tumor soluble factors. Resveratrol analog Fas21 inhibits the proangiogenic response of HSCs and LSECs during the development of murine liver metastasis.

Resveratrol prevents inflammation-dependent hepatic melanoma metastasis by inhibiting the secretion and effects of interleukin-18.

BACKGROUND: Implantation and growth of metastatic cancer cells at distant organs is promoted by inflammation-dependent mechanisms. A hepatic melanoma metastasis model where a majority of metastases are generated via interleukin-18-dependent mechanisms was used to test whether anti-inflammatory properties of resveratrol can interfere with mechanisms of metastasis. METHODS: Two experimental treatment schedules were used: 1) Mice received one daily oral dose of 1 mg/kg resveratrol after cancer cell injection and the metastasis number and volume were determined on day 12. 2) Mice received one daily oral dose of 1 mg/kg resveratrol along the 5 days prior to the injection of cancer cells and both interleukin-18 (IL-18) concentration in the hepatic blood and microvascular retention of luciferase-transfected B16M cells were determined on the 18th hour. In vitro, primary cultured hepatic sinusoidal endothelial cells were treated with B16M-conditioned medium to mimic their in vivo activation by tumor-derived factors and the effect of resveratrol on IL-18 secretion, on vascular cell adhesion molecule-1 (VCAM-1) expression and on tumor cell adhesion were studied. The effect of resveratrol on melanoma cell activation by IL-18 was also studied. RESULTS: Resveratrol remarkably inhibited hepatic retention and metastatic growth of melanoma cells by 50% and 75%, respectively. The mechanism involved IL-18 blockade at three levels: First, resveratrol prevented IL-18 augmentation in the blood of melanoma cell-infiltrated livers. Second, resveratrol inhibited IL-18-dependent expression of VCAM-1 by tumor-activated hepatic sinusoidal endothelium, preventing melanoma cell adhesion to the microvasculature. Third, resveratrol inhibited adhesion- and proliferation-stimulating effects of IL-18 on metastatic melanoma cells through hydrogen peroxide-dependent nuclear factor-kappaB translocation blockade on these cells. CONCLUSIONS: These results demonstrate multiple sites for therapeutic intervention using resveratrol within the prometastatic microenvironment generated by tumor-induced hepatic IL-18, and suggest a remarkable effect of resveratrol in the prevention of inflammation-dependent melanoma metastasis in the liver.

Inhibition of COX-2 Impairs Colon Cancer Liver Metastasis through Reduced Stromal Cell Reaction.

Liver colonization is initiated through the interplay between tumor cells and adhesion molecules present in liver sinusoidal endothelial cells (LSECs). This crosstalk stimulates tumor COX-2 upregulation and PGE2 secretion. To elucidate the role of the LSEC intercellular adhesion molecule-1 (ICAM-1) in the prometastatic response exerted by tumor and stromal COX-2, we utilized celecoxib (CLX) as a COX-2 inhibitory agent. We analyzed the in vitro proliferative and secretory responses of murine C26 colorectal cancer (CRC) cells to soluble ICAM-1 (sICAM-1), cultured alone or with LSECs, and their effect on LSEC and hepatic stellate cell (HSC) migration and in vivo liver metastasis. CLX reduced sICAM-1-stimulated COX-2 activation and PGE2 secretion in C26 cells cultured alone or cocultured with LSECs. Moreover, CLX abrogated sICAM-1-induced C26 cell proliferation and C26 secretion of promigratory factors for LSECs and HSCs. Interestingly, CLX reduced the protumoral response of HSC, reducing their migratory potential when stimulated with C26 secretomes and impairing their secretion of chemotactic factors for LSECs and C26 cells and proliferative factors for C26 cells. In vivo, CLX abrogated the prometastatic ability of sICAM-1-activated C26 cells while reducing liver metastasis. COX-2 inhibition blocked the creation of a favorable tumor microenvironment (TME) by hindering the intratumoral recruitment of activated HSCs and macrophages in addition to the accumulation of fibrillar collagen. These results point to COX-2 being a key modulator of processes initiated by host ICAM-1 during tumor cell/LSEC/HSC crosstalk, leading to the creation of a prometastatic TME in the liver.

Inhibition of DDR1 reduces invasive features of human A375 melanoma, HT29 colon carcinoma and SK-HEP hepatoma cells.

DDR1 is a receptor tyrosine kinases for collagen and an adverse prognostic factor in primary and metastatic tumors.Despite this, DDR1 signaling and its functional consequences in tumor development remain unclear. RT-PCR and Western blot show that A375, colon carcinoma HT29 and liver carcinoma SK-HEP human cell lines express functional DDR1 that phosphorylates in response to collagen type I. Chemical inhibition of DDR1 phosphorylation or DDR1 mRNA silencing reduced AKT and ERK phosphorylation, expression of ICAM1 and VCAM1, Ki67 and secretion of MMP9. DDR1 silenced cells showed reduced adhesion to collagen type I, MMP-dependent invasion, and chemotactic and proliferative responses to collagen type I. Our work indicates an essential role for DDR1 signaling in key prometastatic features of collagen type I in human carcinoma cells.

Silencing of sinusoidal DDR1 reduces murine liver metastasis by colon carcinoma.

Liver metastasis depends on the collagenous microenvironment generated by hepatic sinusoidal cells (SCs). DDR1 is an atypical collagen receptor linked to tumor progression, but whether SCs express DDR1 and its implication in liver metastasis remain unknown. Freshly isolated hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs), that conform the SCs, expressed functional DDR1. HSCs expressed the largest amounts. C26 colon carcinoma secretomes increased DDR1 phosphorylation in HSCs and KCs by collagen I. Inhibition of kinase activity by DDR1-IN-1 or mRNA silencing of DDR1 reduced HSCs secretion of MMP2/9 and chemoattractant and proliferative factors for LSECs and C26 cells. DDR1-IN-1 did not modify MMP2/9 in KCs or LSECs secretomes, but decreased the enhancement of C26 migration and proliferation induced by their secretomes. Gene array showed that DDR1 silencing downregulated HSCs genes for collagens, MMPs, interleukins and chemokines. Silencing of DDR1 before tumor inoculation reduced hepatic C26 metastasis in mice. Silenced livers bore less tumor foci than controls. Metastatic foci in DDR1 silenced mice were smaller and contained an altered stroma with fewer SCs, proliferating cells, collagen and MMPs than foci in control mice. In conclusion, hepatic DDR1 promotes C26 liver metastasis and favors the pro-metastatic response of SCs to the tumor.

Innate Lymphoid Cells in the Malignant Melanoma Microenvironment.

The role of innate lymphoid cells (ILCs) in cancer progression has been uncovered in recent years. ILCs are classified as Type 1, Type 2, and Type 3 ILCs, which are characterized by the transcription factors necessary for their development and the cytokines and chemokines they produce. ILCs are a highly heterogeneous cell population, showing both anti- and protumoral properties and capable of adapting their phenotypes and functions depending on the signals they receive from their surrounding environment. ILCs are considered the innate counterparts of the adaptive immune cells during physiological and pathological processes, including cancer, and as such, ILC subsets reflect different types of T cells. In cancer, each ILC subset plays a crucial role, not only in innate immunity but also as regulators of the tumor microenvironment. ILCs' interplay with other immune and stromal cells in the metastatic microenvironment further dictates and influences this dichotomy, further strengthening the seed-and-soil theory and supporting the formation of more suitable and organ-specific metastatic environments. Here, we review the present knowledge on the different ILC subsets, focusing on their interplay with components of the tumor environment during the development of primary melanoma as well as on metastatic progression to organs, such as the liver or lung.

Liver sinusoidal endothelial cell ICAM-1 mediated tumor/endothelial crosstalk drives the development of liver metastasis by initiating inflammatory and angiogenic responses.

The prometastatic stroma generated through tumor cells/host cells interaction is critical for metastatic growth. To elucidate the role of ICAM-1 on the crosstalk between tumor and primary liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), implicated in tumor adhesion and angiogenesis, we performed in vitro cocultures and an in vivo model of liver metastasis of colorectal cancer (CRC). ICAM-1 blockade in the LSECs decreased the adhesion and transmigration of tumor cells through an LSEC in vitro and vivo. Cocultures of C26 cells and LSECs contained higher amounts of IL-1ß, IL-6, PGE-2, TNF-α and ICAM-1 than monocultures. C26 cells incubated with sICAM-1 secreted higher amounts of PGE-2, IL-6, VEGF, and MMPs, while enhanced the migration of LSECs and HSCs. HSCs cultures activated by media from C26 cells pretreated with sICAM-1 contained the largest amounts of VEGF and MMPs. C26 cell activation with sICAM-1 enhanced their metastasizing potential in vivo, while tumor LFA-1 blockade reduced tumor burden and LSECs and HSC-derived myofibroblasts recruitment. In vivo ICAM-1 silencing produced similar results. These findings uncover LSEC ICAM-1 as a mediator of the CRC metastatic cascade in the liver and identifies it as target for the inhibition of liver colonization and metastatic progression.

Ocoxin Oral Solution Exerts an Antitumoral Effect in Pancreatic Cancer and Reduces the Stromal-Mediated Chemoresistance.

OBJECTIVES: Pancreatic carcinoma is one of the most aggressive cancers overcoming chemoresistance. Thus, novel compounds to complement the current antitumor agents are in need. Ocoxin oral solution (OOS) has proven antioxidant, anti-inflammatory, and antistromagenic properties. The aim of this study was to analyze the effect of OOS in an experimental pancreatic cancer model and its implication in stroma-related chemoresistance to paclitaxel and gemcitabine. METHODS: Murine pancreatic carcinoma 266-6 cells were treated with OOS to analyze cell cycle and to perform a mRNA comparative microarray study. Then the viability was assessed in combination with paclitaxel and/or gemcitabine. Chemoresistance induced by the medium taken from fibroblast cultures was also investigated on 6 human pancreatic carcinoma cell lines. Furthermore, an experimental model of pancreatic cancer was carried out to study the effect of OOS in vivo. RESULTS: Ocoxin oral solution enhances the cytotoxic effect of paclitaxel and gemcitabine, while it ameliorates the chemoresistance induced by fibroblast-derived soluble factors in human pancreatic cancer cells. The OOS also promotes the regulation of the expression of genes that are altered in pancreatic carcinoma and slows down 266-6 cell pancreatic tumor development in vivo. CONCLUSIONS: Ocoxin oral solution could be a potential complement to the chemotherapeutic drugs for pancreatic adenocarcinoma.

Ocoxin oral solution® as a complement to irinotecan chemotherapy in the metastatic progression of colorectal cancer to the liver.

Colorectal cancer (CRC) is an aggressive disease in which patients usually die due to its metastatic progression to the liver. Up to date, irinotecan is one of the most used chemotherapeutic agents to treat CRC metastasis with demonstrated efficacy. However, the severity of the side effects constitute the main limitation to its use in the treatment. Consequently, new complementary therapies are being developed to avoid these adverse effects while maintaining the efficacy of the antitumoral drugs. Ocoxin oral solution (OOS®) is a nutritional mixture containing biologically active compounds with demonstrated antitumoral and immunomodulatory effects. Thus, we aimed to analyze the effect of OOS® as a suitable complement to irinotecan therapy in the treatment of CRC metastasis to the liver. First, the effect of OOS®, irinotecan and the combination of both on the viability of C26 cells was tested in vitro and in vivo. Second, the expression of caspase-3, Ki67 and the macrophage infiltration by F4/80 marker was quantified in liver tissue sections by immunohistochemistry. Finally, mRNA microarray study was carried out on tumor cells isolated from tumor-bearing livers collected from mice subjected to the above treatments. Our results show that OOS® administered as a complementary therapy to irinotecan reduced tumor cell viability in vitro. Moreover, irinotecan administered either alone or in combination with 100 µl OOS® from the 7th day after tumor cell inoculation decreased the metastatic growth in the liver. Besides, several genes with binding and catalytic activities showed to be deregulated by RNA microarray analysis. In conclusion, OOS®, when administered as a complement to irinotecan, reduced the metastatic development of colorectal cancer to the liver. Additionally, the overall health state of the animals improved. These results point out OOS® as a potential supplement to the anti-tumoral treatments used in clinical settings in patients suffering from disseminated colorectal cancer.

Ocoxin® oral solution slows down tumor growth in an experimental model of colorectal cancer metastasis to the liver in Balb/c mice.

Liver metastatic disease is the main cause of death in colorectal cancer (CRC) patients. During metastatic spread of the disease an imbalance in the oxidative stress and inflammation plays a crucial role in tumor progression. In order to improve the efficacy of current therapies, new complementary therapeutic approaches are being analyzed including biologically active compounds with low side effects. The anti-inflammatory and anti-oxidant properties of Ocoxin® oral solution (OOS) prompt us to analyze its effect on the metastatic development of CRC to the liver. First, in vitro effect of OOS in tumor cell viability and migration was analyzed. Second, in vivo effect of different dosage patterns and concentrations in the development of hepatic metastasis was analyzed by intra-splenic inoculation of C26 colon carcinoma cells in Balb/c mice. Third, the expression of alpha smooth muscle actin, caspase-3 and Ki-67 expression was quantified by immunohistochemistry, then gene expression levels of inflammatory factors were measured by quantitative RT-PCR. According to our results, OOS reduced tumor cell viability and migration in vitro. Moreover, in vivo daily administration of OOS from the 7th day after tumor cell inoculation decreased the total area and size of metastatic foci in the liver. Furthermore, cell proliferation and fibroblast recruitment was decreased in tumor foci while a higher number of apoptotic cells were observed. Finally, RNA levels for the inflammatory mediators COX-2, IFNγ, IL1ß, IL6 and TNFα were reduced in total liver. In conclusion, OOS reduced the metastatic development of colorectal cancer to the liver by increasing apoptosis, and decreasing tumor cell proliferation and fibroblast recruitment in the tumor foci, as well as the expression of inflammatory mediators in total liver. These results point out OOS as a potential supplement to be applied as complementary therapy for the treatment of liver metastasis from colorectal cancer.

Role of discoidin domain receptor 2 in wound healing.

Until recently, collagen interactions with cells had been ascribed to integrins. The identification of the Discoidin Domain Receptor (DDR) family as collagen receptors represents a new paradigm in the regulation of collagen-cell interactions. How DDR signaling is biochemically linked to specific cell regulatory functions remains largely unknown. Moreover, the characteristic slow and substained phosphorylation of DDRs and the elevated expression of DDR2 in the myofibroblasts of healing wounds suggest a role for DDR2 in physiological and pathological wound healing. In fact, DDR2 signaling regulates cell proliferation and extracellular matrix synthesis, which are key aspects of fibroblast contribution to tissue healing. In this review we summarize evidence in favor of this concept.

Downregulation of discoidin domain receptor 2 in A375 human melanoma cells reduces its experimental liver metastasis ability.

Discoidin domain receptors (DDR1 and DDR2) are tyrosine kinase receptors for fibrillar collagen implicated in postnatal development, tissue repair, and primary and metastatic cancer progression. While DDR1 has been described in tumor cells, DDR2 has been localized in the tumor stroma, but its presence in the tumor cells remains unknown. The aim of this study was to elucidate the role of DDR2 signaling in tumor cells during hepatic metastasis progression. DDR2 expression and phosphorylation in cultured human A375 melanoma cells was documented by Western blot analysis. A375 cells were stably transfected with a small interfering RNA (siRNA) against DDR2 and two clones were selected: A375R2-70 and A375R2-40, with 70 and 40% of the DDR2 protein expression respectively, compared to mock-transfected cells (A375R2-100). Development of experimental liver metastasis by intrasplenic inoculation of A375R2-70 and A37R2-40 clones was reduced by 60 and 75%, respectively, measured as tumor volume, compared to livers injected with A375R2-100 cells. Accordingly, A375R2-70 and A37R2-40 clones showed reduced in vitro gelatinase activity and JNK phosphorylation, compared to mock transfected cells, with maximal inhibition in A375R2-40. Additionally, A375 melanoma, SK-HEP hepatoma and HT-29 colon carcinoma human cell lines transiently transfected with siRNA against DDR2 also showed reduced proliferation and migration rates compared to mock-transfected ones. In conclusion, DDR2 promotes A375 melanoma metastasis to the liver and the underlying mechanism implicates regulation of metalloproteinase release, cell growth and chemotactic invasion of the host tissue.

Impaired dermal wound healing in discoidin domain receptor 2-deficient mice associated with defective extracellular matrix remodeling.

BACKGROUND: The wounding response relies on tightly regulated crosstalk between recruited fibroblasts and the collagenous extracellular matrix (ECM). Discoidin domain receptor 2 (DDR2) is a tyrosine kinase receptor for fibrillar collagen expressed during pathologic scarring, for example wound healing, arthritis and cancer. We have previously shown that DDR2 phosphorylation drives key wounding responses in skin fibroblasts including proliferation, chemotactic migration and secretion of both metalloproteinases and fibrillar collagen. In this study we compared healing of cutaneous wounds in DDR2+/+ and DDR2-/- mice and analyzed specific fibroblast responses. RESULTS: Cutaneous wound healing was significantly delayed in DDR2-/- mice compared with DDR2+/+ animals. Reduced α-smooth muscle actin (αSMA) expression and matrix metalloproteinase 2 (MMP2) activity in the DDR2-/- wound extracts indicated defective recruitment of skin fibroblasts. DDR2-/- wounds showed decreased tensile strength during healing, which correlated with a significant reduction in collagen content and defective collagen crosslinking. Non-wounded skin in DDR2-/- mice expressed less mRNA of the crosslinking enzymes lysyl oxidase (LOX), lysyl hydroxylase1 (LH1) and matricellular 'secreted protein, acidic and rich in cysteine' (SPARC; also known as osteonectin). Skin fibroblasts isolated from DDR2-/- mice displayed altered mRNA expression of a cluster of collagens, proteoglycans, integrins and MMPs that have been previously correlated with DDR2 expression, and reduced LOX, LH1 and SPARC mRNA levels and proteins. Stable reconstitution of wild-type DDR2 by retroviral infection restored LOX, LH1 and SPARC mRNA and protein levels in DDR2-/- fibroblasts. Contraction of collagen gels was reduced in DDR2-/- fibroblasts, accompanied by significantly reduced phosphorylated SrcY418. Inhibition of either LOX activity by ß-aminoproprionitrile or MMP activity by N-[(2R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001) reduced collagen gel contraction by skin fibroblasts after DDR2 induction with soluble collagen type I. CONCLUSIONS: DDR2 contributes to skin fibroblast responses during tissue injury. Defective synthesis of collagen type I, crosslinking molecules and MMP2 predispose DDR2-/- mice to defective dermal wounding.

Endostatin inhibits murine colon carcinoma sinusoidal-type metastases by preferential targeting of hepatic sinusoidal endothelium.

An angiogenic response originating from peritumoral sinusoids and portal tracts that leads to the formation of metastases with sinusoidal- and portal-type angiogenic patterns, respectively, occurs during the course of liver colonization by murine 51b colon carcinoma (51b-CC) cells. We found a 5-fold increase in endogenous endostatin levels from hepatic blood over baseline (25 +/- 6 ng/mL) when micrometastatic foci had a detectable size and a 14-fold increase when macrometastases were developed. Despite this endogenous endostatin production, subcutaneous administration of recombinant human endostatin (rh-E; 50 mg/kg) decreased metastasis number by 60% when dosed from days 1 to 20 after 51b-CC cell injection, by 40% when given from days 10 to 20, and by 30% when administered as a single dose 30 minutes before 51b-CC cell injection compared with controls. In addition, administration of rh-E from days 10 to 20 decreased overall metastasis volume by 90% compared with controls. rh-E increased the number of necrotic sinusoidal-type metastases by 7-fold and decreased their intrametastatic CD31(+)-microvessel density by 80% without affecting portal-type metastases. Flow cytometry showed rh-E binding to mouse liver sinusoidal cells but not to CD45(+) cells (leukocytes and Kupffer cells) or 51b-CC cells. Furthermore, rh-E induced sinusoidal endothelium cell apoptosis. In conclusion, despite the direct correlation between metastasis development and endogenous endostatin generation in the liver, administration of rh-E inhibited micrometastasis generation and macrometastasis growth very efficiently. The antiangiogenic mechanism was selective for sinusoidal-type metastases, in which the neovasculature originating from sinusoidal endothelium cells was targeted by rh-E.

Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis.

Myofibroblasts infiltrate malignant liver tumors, although their pathogenic implications are unclear. Immunohistochemical detection of alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP), and CD31 and CD34 expression was used to analyze the contribution of myofibroblasts to angiogenesis in hepatic metastasis produced by intrasplenically-injected B16 melanoma (B16M). Because activated hepatic stellate cells (HSCs) are oxygen-sensing myofibroblasts producing vascular endothelial growth factor (VEGF), the effect of B16M and human A375 melanoma supernatants on VEGF production by immortalized rat HSC line T6 and primary cultured human HSCs also was studied under an hypoxic atmosphere mimicking a tumor microenvironment. Myofibroblast infiltration preceded endothelium recruitment in avascular micrometastasis and generated specific stroma for sinusoidal-type and portal-type angiogeneses. Thereafter, myofibroblasts and endothelial cells colocalized within both angiogenic patterns and their numerical densities correlated with metastasis development. Myofibroblasts often were GFAP-positive, suggesting an HSC origin. Melanoma supernatants stimulated VEGF messenger RNA and protein synthesis by HSCs. These effects were potentiated by hypoxia. VEGF up-regulation was accompanied by increased expression of cyclooxygenase type 2 (COX-2) and PGE2 synthesis. HSC production of VEGF decreased under COX-2 inhibition, whereas it was increased by exogenous PGE2. The high VEGF expression in HSCs induced by melanoma factors and hypoxia resulted in mitogenic, antiapoptotic, and motogenic stimulation of both murine hepatic sinusoidal endothelium and human umbilical vein endothelium. In conclusion, temporal and positional relationships evolve between myofibroblast and endothelium recruitment during metastasis development. Mechanistically, hypoxic induction of VEGF in tumor-activated HSCs may create a proangiogenic microenvironment, facilitating endothelial cell recruitment and survival during hepatic metastasis transition from an avascular to a vascular stage.