Supplementary Figure 1 from Preparing the “Soil”: The Primary Tumor Induces Vasculature Reorganization in the Sentinel Lymph Node before the Arrival of Metastatic Cancer Cells
We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.
B45 The c-Met tyrosine kinase receptor and its ligand, the hepatocyte growth factor (HGF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF-Met pathway is frequently activated in a variety of cancers, and uncontrolled c-Met activation correlates with highly invasive tumors and poor prognosis. Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the c-Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as an Fc-fused protein in a mammalian expression system and was designated Cgen-241A. The biological activity of Cgen241A was assessed in various cell-based assays, reflecting different outcomes of Met activation. Cgen241A significantly inhibited HGF-mediated c-Met phosphorylation as well as cell proliferation and survival, indicating an anti-mitogenic activity. In addition, Cgen241A showed a profound inhibitory effect on cell scattering, invasion and urokinase upregulation, indicating anti-tumorigenic and anti-metastatic activities. These inhibitory effects of Cgen-241A were shown in multiple human and non-human cell types, representing different modes of ligand-dependent and ligand-independent c-Met activation. In agreement, binding studies revealed that Cgen241A binds both HGF and c-Met receptor in its membranal form, pointing to a dual mechanism of action. Preliminary results show that Cgen-241A inhibits xenograft tumor growth in vivo, suggesting a therapeutic potential. Taken together, our results indicate that Cgen241A is a potent antagonist of the HGF-Met pathway. Its dual mechanism of action may confer a therapeutic advantage, due to its ability to inhibit the diverse modes of c-Met activation existing in human malignancies.
A monoclonal antibody, H23, that specifically recognizes a breast‐tumor‐associated antigen, was used to isolate a cDNA insert that codes for the antigenic epitope. Nucleotide sequencing of this cDNA, as well as a longer 850‐bp cDNA insert, shows that they are composed of 60‐bp (G + C)‐rich tandem repeating units. The coding strand was determined and codes for a proline‐rich 20‐amino‐acid repeat motif. A comparison of the highly conserved repeat unit with the deduced flanking amino acid sequences demonstrates conservation of specific subregions of the repeat consensus within the flanking amino acids. Hybridization of the 60‐bp cDNA probe with RNAs extracted from a variety of primary and metastatic human tumors yields relatively high levels of hybrid with the breast carcinomas, as compared to lower hybrid levels with RNAs from other epithelial tumors. RNA extracted from breast tissue adjacent to the tumor or from benign breast tumors, demonstrates low or undetectable levels of hybridization. Probing Southern blots with the 60‐bp repeat shows that the tumor antigen is highly polymorphic and contains a variable number of tandem repeats (VNTRs). The VNTR nature of the gene was confirmed by probing Southern blots with unique genomic sequences that are physically linked to an isolated gene fragment that also contains the tandem repeat array. Mouse cells transfected with this gene fragement produce tumor antigen that is readily detected by H23 monoclonal antibodies. The allelic forms seen in 10 different primary human tumors demonstrate 100% concordance with the various mRNA species expressed. These studies are extended to the protein forms detected by immunoblot analyses that show both a correlation of the expressed tumor antigen species with the allelic forms as well as significantly increased expression in breast cancer tissue. The above studies unequivocally establish the over‐expression of a VNTR gene coding for an epithelial tumor antigen in human breast cancer tissue.
Metastasizing tumor cells migrate through the surrounding tissue and extracellular matrix toward the blood vessels, in order to colonize distant organs. They typically move in a dense environment, filled with other cells. In this work we study cooperative effects between neighboring cells of different types, migrating in a maze-like environment with directional cue. Using a computerized model, we measure the percentage of cells that arrive to the defined target, for different mesenchymal/amoeboid ratios. Wall degradation of mesenchymal cells, as well as motility of both types of cells, are coupled to metabolic energy-like resource level. We find that indirect cooperation emerges in mid-level energy, as mesenchymal cells create paths that are used by amoeboids. Therefore, we expect to see a small population of mesenchymals kept in a mostly-amoeboid population. We also study different forms of direct interaction between the cells, and show that energy-dependent interaction strength is optimal for the migration of both mesenchymals and amoeboids. The obtained characteristics of cellular cluster size are in agreement with experimental results. We therefore predict that hybrid states, e.g. epithelial-mesenchymal, should be utilized as a stress-response mechanism.
The present study describes experiments of the effect of Candida albicans metabolite(s) (arcsf) on cellular actin, and constitutes a continuation of our previous investigations regarding the effect of the fungus on actin. Western blot analysis of HEp2 cells exposed to arcsf or subfraction of arcsf (<100 kDa) revealed that the amount of soluble actin was decreased, and total actin increased, as compared to untreated cells. Transmission electron microscopy observations of HEp2 cells interacted with C. albicans or arcsf, and gold-labeled specifically for actin, revealed more intensive labeling, with labeled particles clustered in groups. Confocal laser scanning microscopy analysis has shown, as well, that exposure of HEp2 cells to the <100-kDa subfraction resulted in actin rearrangement, as did the unfractionated arcsf. Our results point to: (1) C. albicans metabolite(s) affects cellular actin by increasing the transition of soluble actin to the insoluble form; (2) the effect is associated with the subfraction of arcsf which is smaller than <100 kDa.
