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.

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Objective. To determine the long-term clinical and immunologic outcomes in a well-characterized cohort of 47 patients with mixed connective tissue disease (MCTD), including reactivity with U small nuclear RNP (snRNP) polypeptides.Methods. Patients were followed up over a period of 3-29 years with immunogenetic and systematic clinical and serologic analysis. Sera were analyzed for reactivity with snRNP polypeptides U1-70 kd, A, C, B/B , and D, for anti-U1 RNA, and for anticardiolipin antibodies (aCL).Results. The typical core clinical features of MCTD tended to develop over time; features of inflammation as well as Raynaud's phenomenon and esophageal hypomotility diminished, while pulmonary hypertension, pulmonary dysfunction, and central nervous system disease persisted, following treatment. A favorable outcome was observed in 62% of patients; 38% had continued active disease or had died, with death associated with pulmonary hypertension and aCL. All patients had autoantibodies to the U1-70 kd polypeptide of snRNP, and most were positive for anti-U1 RNA. An orderly progression of intramolecular spreading of autoantibody reactivity against snRNP polypeptides was observed, as was the novel finding of "epitope contraction" followed by disappearance of anti-snRNP autoantibodies during prolonged remission.Conclusion. These patients demonstrated the typical immunogenetic, clinical, and serologic findings of MCTD, and the condition rarely evolved into systemic lupus erythematosus or systemic sclerosis. The majority of patients had favorable outcomes, with pulmonary hypertension being the most frequent disease-associated cause of death. Intramolecular spreading of autoantibody reactivity against snRNP polypeptides was observed, followed by "epitope contraction" and ultimate disappearance of anti-snRNP autoantibodies during prolonged disease remission.

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Purpose: The cellular targeting and tumor imaging properties of a novel ErbB-2-avid peptide, discovered from bacteriophage display, were evaluated in human breast carcinoma cells and in breast carcinoma^xenografted mice. Experimental Design: The affinity of the ErbB-2 targeting peptide KCCYSL and its alanine substituted counterparts for the extracellular domain (ECD) of purified recombinant ErbB-2 (ErbB-2-ECD) was assessed by fluorescence titration. Binding of the KCCYSL peptide to breast and prostate carcinoma cells was analyzed by confocal microscopy. A DOTA(GSG)-KCCYSL peptide conjugate was radiolabeled with 111 In, and stability, target binding, and internalization were analyzed in vitro. In vivo biodistribution and single-photon emission computed tomography imaging studies were done with the radiolabeled peptide in MDA-MB-435 human breast tumorb earing severe combined immunodeficient mice. Results: KCCYSL peptide exhibited high affinity (295 F 56 nmol/L) to ErbB-2-ECD. Substitution of alanine for lysine, tryptophan, and cysteine reduced the peptide affinity f 1-to 2.4-fold, whereas replacing leucine completely abolished binding. Both biotin- KCCYSL and 111 In-DOTA(GSG)-KCCYSL were capable of binding ErbB-2^expressing human breast carcinoma cells in vitro. Approximately 11% of the total bound radioactivity was internalized in the carcinoma cells. Competitive binding studies indicated that the radiolabeled peptide exhibited an IC 50 value of 42.5 F 2.76 nmol/L for the breast carcinoma cells.

There is an increasing medical need to detect and spatially localize early and aggressive forms of prostate cancer. Affinity ligands derived from bacteriophage (phage) library screens can be developed to molecularly target prostate cancer with fluorochromes for optical imaging. Toward this goal, we used in vivo phage display and a newly described micropanning assay to select for phage that extravasate and bind human PC-3 prostate carcinoma xenografts in severe combined immune deficiency mice. One resulting phage clone (G1) displaying the peptide sequence IAGLATPGWSHWLAL was fluorescently labeled with the near-infrared fluorophore AlexaFluor 680 and was evaluated both in vitro and in vivo for its ability to bind and target PC-3 prostate carcinomas. The fluorescently labeled phage clone (G1) had a tumor-to-muscle ratio of approximately 30 in experiments. In addition, prostate tumors (PC-3) were readily detectable by optical-imaging methods. These results show proof of principle that disease-specific library-derived fluorescent probes can be rapidly developed for use in the early detection of cancers by optical means.

Ro, or Sjogren syndrome type A (SS-A), antigen is the most prevalent of the human systemic autoimmune specificities and exists as an inabundant ribonucleoprotein complex (RNP) composed of a 60,649-Da protein, as defined here by cDNA cloning, and the human Y RNAs. The recombinant 60-kDa Ro protein and human Y1 RNA were reconstituted in vitro, and the binding was enhanced by divalent cations. A region of the Ro amino acid sequence revealed a resemblance to the RNP consensus motif found in most RNA-binding proteins. In addition, Ro contained a potential "zinc-binding finger" motif that was distinct from the RNP consensus region and that may participate in the interaction with human Y RNAs or with other proteins. The recombinant Ro fusion protein also proved useful for the detection of autoantibodies in the sera of patients with autoimmune disorders. Possible functions of the Ro RNPs and their relationship to RNA polymerase III transcription are discussed.