Extracellular matrix composition and interstitial pH modulate NHE1-mediated melanoma cell motility.

The activity of the Na+/H+ exchanger NHE1 is required for human melanoma cell adhesion and migration. The goal of the present study was to suppress mouse melanoma (B16V) cell invasion in vivo by inhibiting NHE1. Intravital observations in mobilized left liver lobes of laparotomized male Sprague-Dawley rats disclosed that five minutes after intra-arterial administration of the B16V cell suspension, cells adhered to the endothelia of liver sinusoidal capillaries and started to migrate into the surrounding liver tissue. In the presence of the NHE1-specific inhibitor cariporide, migration/invasion was reduced by about 50% while adhesion was not lowered. Time-lapse video microscopy and adhesion/invasion assays revealed that in vitro, blockade of NHE1 by cariporide i) significantly decreased the migratory speed of the cells and ii) completely inhibited the invasive behavior of both an artificial, basement membrane-like and a dermis-like matrix. Cells were more motile on the basement membrane and more invasive on the dermis-like matrix. Small-animal PET (positron-emission tomography) analyses of B16V metastasis in female C57BL/6 mice showed that, although NHE1 inhibition hardly affected the percentage of animals developing metastases or relapses, metastases seem to get directed to the lungs in cariporide-treated animals while animals feeding on the standard diet show metastases spread all over the body. We conclude that i) B16V cells prefer to invade a dermis-like rather than a basement membrane-like matrix; ii) the extracellular matrix (ECM) composition strongly impacts on NHE1-dependent in vitro cell motility and invasion; and iii) the lungs are metastasis­prone and impair the efficiency of cariporide due to their ECM composition and the pulmonary interstitial (extravascular) pH.

CXCR4 regulates the early extravasation of metastatic tumor cells in vivo.

Recent studies have demonstrated that the chemokine receptor CXCR4 plays a crucial role in organ-specific metastasis formation. Although a variety of studies showed the expression of chemokine receptors, in particular, CXCR4, by gastrointestinal tumors, the precise mechanisms of chemokine receptor-mediated homing of cancer cells to specific sites of metastasis remained elusive. Here, we used liver metastatic human HEP-G2 hepatoma and HT-29LMM colon cancer cells expressing functional CXCR4 to dissect the metastatic cascade by intravital fluorescence microscopy. Immunohistochemistry revealed that the CXCR4 ligand CXCL12 is expressed by endothelial cells and likely Kupffer cells lining the liver sinusoids. Tumor cell adhesion and extravasation in vivo was quantitatively analyzed using intravital fluorescence microscopy. Treatment of cells with an anti-CXCR4 antibody did not affect cell adhesion but significantly impaired tumor cell extravasation (HEP-G2; isotype control: 22.3% +/- 4.3% vs anti-CXCR4: 6.0% +/- 5.0%, P < .001). In addition, pretreatment of tumor cells with the ligand CXCL12 enhanced the activation of the small GTPases Rho, Rac, and cdc42 as well as tumor cell extravasation without affecting tumor cell adhesion within liver sinusoids. Taken together, the findings of the present study provide first in vivo insights into the early events of chemokine ligand/receptor-mediated liver metastasis formation of tumor cells and define tumor cell extravasation rather than tumor cell arrest as the rate-limiting event.