The two major theories of cancer metastasis, the seed and soil hypothesis and the mechanical trapping theory, view tumor cell adhesion to blood vessel endothelia and cancer cell aggregation as corresponding key components of the metastatic process. Here, we demonstrate in vitro, ex vivo, and in vivo that metastatic breast and prostate carcinoma cells form multicellular homotypic aggregates at the sites of their primary attachment to the endothelium. Our results suggest that metastatic cell heterotypic adhesion to the microvascular endothelium and homotypic aggregation represent two coordinated subsequent steps of the metastatic cascade mediated largely by similar molecular mechanisms, specifically by interactions of tumor-associated Thomsen-Friedenreich glycoantigen with the ␤-galactoside-binding protein, galectin-3. In addition to inhibiting neoplastic cell adhesion to the endothelium and homotypic aggregation, disrupting this line of intercellular communication using synthetic ThomsenFriedenreich antigen masking and Thomsen-Friedenreich antigen mimicking compounds greatly affects cancer cell clonogenic survival and growth as well. Thus, ␤-galactoside-mediated intravascular heterotypic and homotypic tumor cell adhesive interactions at the sites of a primary attachment to the microvascular endothelium could play an important role during early stages of hematogenous cancer metastasis.

In this report, we challenge a common perception that tumor embolism is a size-limited event of mechanical arrest, occurring in the first capillary bed encountered by blood-borne metastatic cells. We tested the hypothesis that mechanical entrapment alone, in the absence of tumor cell adhesion to blood vessel walls, is not sufficient for metastatic cell arrest in target organ microvasculature. The in vivo metastatic deposit formation assay was used to assess the number and location of fluorescently labeled tumor cells lodged in selected organs and tissues following intravenous inoculation. We report that a significant fraction of breast and prostate cancer cells escapes arrest in a lung capillary bed and lodges successfully in other organs and tissues. Monoclonal antibodies and carbohydrate-based compounds (anti-Thomsen-Friedenreich antigen antibody, anti-galectin-3 antibody, modified citrus pectin, and lactulosyl-l-leucine), targeting specifically beta-galactoside-mediated tumor-endothelial cell adhesive interactions, inhibited by >90% the in vivo formation of breast and prostate carcinoma metastatic deposits in mouse lung and bones. Our results indicate that metastatic cell arrest in target organ microvessels is not a consequence of mechanical trapping, but is supported predominantly by intercellular adhesive interactions mediated by cancer-associated Thomsen-Friedenreich glycoantigen and beta-galactoside-binding lectin galectin-3. Efficient blocking of beta-galactoside-mediated adhesion precludes malignant cell lodging in target organs.

Thomsen-Friedenreich antigen (TF-Ag) is expressed in many carcinomas, including those of the breast, colon, bladder, and prostate. TF-Ag is important in adhesion and metastasis and as a potential immunotherapy target. We hypothesized that passive transfer of JAA-F11, an anti-TF-Ag monoclonal antibody, may create a survival advantage for patients with TF-Ag-expressing tumors by cytotoxicity, blocking of tumor cell adhesion, and inhibition of metastasis. This was tested using in vitro models of tumor cell growth; cytotoxicity assays; in vitro, ex vivo, and in vivo models of cancer metastasis; and, finally, in vivo effects in mice with metastatic breast cancer. Unlike some anti-TF-Ag antibodies, JAA-F11 did not enhance breast carcinoma cell growth. JAA-F11 did not induce the killing of 4T1 tumor cells through complement-dependent cytotoxicity or apoptotic mechanisms. However, JAA-F11 blocked the stages of metastasis that involve the adhesion of human breast carcinoma cells to human endothelial cells (human umbilical vein endothelial cells and human bone marrow endothelial cells 60) in in vitro static adhesion models, in a perfused ex vivo model, and in murine lung vasculature in an in vivo metastatic deposit formation assay. JAA-F11 significantly extended the median survival time of animals bearing metastatic 4T1 breast tumors and caused a > 50% inhibition of lung metastasis.

Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-L-leucine (Lac-L-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-L-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-L-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-L-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-L-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting β-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.

Angiosarcoma (ASA) in humans and hemangiosarcoma (HSA) in dogs are deadly neoplastic diseases characterized by an aggressive growth of malignant cells with endothelial phenotype, widespread metastasis, and poor response to chemotherapy. Galectin-3 (Gal-3), a beta-galactoside-binding lectin implicated in tumor progression and metastasis, endothelial cell biology and angiogenesis, and regulation of apoptosis and neoplastic cell response to cytotoxic drugs, has not been studied before in tumors arising from malignant endothelia. Here, we tested the hypothesis that Gal-3 could be widely expressed in human ASA and canine HSA and could play an important role in malignant endothelial cell biology. Immunohistochemical analysis demonstrated that 100% of the human ASA (10 of 10) and canine HSA (17 of 17) samples analyzed expressed Gal-3. Two carbohydrate-based Gal-3 inhibitors, modified citrus pectin (MCP) and lactulosyl-l-leucine (LL), caused a dose-dependent reduction of SVR murine ASA cell clonogenic survival through the inhibition of Gal-3 antiapoptotic function. Furthermore, both MCP and LL sensitized SVR cells to the cytotoxic drug doxorubicin to a degree sufficient to reduce the in vitro IC(50) of doxorubicin by 10.7-fold and 3.6-fold, respectively. These results highlight the important role of Gal-3 in the biology of ASA and identify Gal-3 as a potential therapeutic target in tumors arising from malignant endothelial cells.

Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2 x 10(3) microm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2 x 10(3) to 0 microm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human.

It is well established that after metastatic cancer cells escape the primary tumour and enter the circulation, their interactions with microvascular endothelium of a target organ constitute an essential rate-limiting step in haematogenous cancer metastasis. However, the physiological and biochemical processes supporting neoplastic cell arrest and retention in the microcirculation are still poorly understood. In this study, we present experimental evidence that microvascular endothelium of metastasis-prone tissues undergoes activation in response to desialylated cancer-associated carbohydrate structures such as Thomsen-Friedenreich (TF) antigen (Galβ1-3GalNAc) expressed on circulating glycoproteins and neoplastic cells. The metastasis-associated endothelium activation, manifested by marked increase in endothelial cell surface galectin-3 expression, causes gradual decrease in cancer cell velocities (from 72 × 10 2 ± 33 × 10 2 µm s −1 to 7.6 × 10 2 ± 1.9 × 10 2 µm s −1 , mean ± S.D.) accompanied by a corresponding increase in the percentage of rolling cells (from 3.3% ± 1.2% to 24.3% ± 3.6%, mean ± S.D.), and results in human breast and prostate carcinoma cell arrest and retention in the microvasculature. This process, which could be of high importance in haematogenous cancer metastasis, was inhibited efficiently by an anti-TF antigen function-blocking antibody. Carbohydrate-mediated endothelial activation could be a process of physiological significance as it probably occurs in the interactions between a variety of circulating constituents and the vessel wall.

At present, there is no efficient curative therapy for cancer patients with advanced metastatic disease. Targeting of antiapoptotic molecules acting on the mitochondrial apoptosis pathway could potentially augment antimetastatic effect of cytotoxic drugs. Similarly to Bcl-2 family members, beta-galactoside-binding lectin galectin-3 protects cancer cells from apoptosis induced by cytotoxic drugs through the mitochondrial pathway. In this study, we tested the hypothesis that inhibiting galectin-3 antiapoptotic function using a synthetic low-molecular weight carbohydrate-based compound lactulosyl-L-leucine (Lac-L-Leu) will augment apoptosis induced in human cancer cells by paclitaxel and increase its efficacy against established metastases. Treatment with synthetic glycoamine Lac-L-Leu alone reduced the number of established MDA-MB-435Lung2 pulmonary metastases 5.5-fold (P = .032) but did not significantly affect the incidence of metastasis. Treatment with paclitaxel alone (10 mg/kg three times with 3-day intervals) had no significant effect on the incidence or on the number of MDA-MB-435Lung2 metastases. Treatment with Lac-L-Leu/paclitaxel combination decreased both the number (P = .02) and the incidence (P = .001) of pulmonary metastases, causing a five-fold increase in the number of metastasis-free animals from 14% in the control group to 70% in the combination therapy group. The median number of lung metastases dropped to 0 in the combination therapy group compared with 11 in the control (P = .02). Synergistic inhibition of clonogenic survival and induction of apoptosis in metastatic cells by Lac-L-Leu/paclitaxel combination was functionally linked with an increase in mitochondrial damage and was sufficient for the antimetastatic activity that caused a reversal and eradication of advanced metastatic disease in 56% of experimental animals.