We have developed a technology for analysis and sorting of live cells according to secreted molecules. An artificial affinity matrix, specific for the secreted product of interest, is created on the cell surface, and the cells are allowed to secrete for a defined time period. The secreted molecules bind to the affinity matrix on the secreting cell and are subsequently labeled with specific fluorescent or magnetic staining reagents for cytometric analysis and cell sorting. Crossfeeding of the secreted products to other cells is prevented by decreasing the permeability of the incubation medium. This approach will have a wide range of applications in biotechnology and biomedical research. Here, we describe analysis and sorting of hybridoma cells, according to secreted antibodies, and of activated T lymphocytes, according to secreted cytokines.Proteins and other cellular products can be expressed either intracellularly, on the cell surface, or by secretion into the extracellular space. Despite its biological significance, only a few methods are presently available for the analysis of secreted products on the single cell level. This situation is mainly because secreted cellular products are hard to assign to the secreting cell, especially in a quantitative way. In the currently available protocols, the plaque assay (1), the ELISPOT assay (2), and the microdroplet technology (3), secreting cells are fixed in or on support matrices, which trap the secreted products for analysis. These approaches impose severe limitations because the secreted molecules still dissociate from the cell, and trapping matrix and cells must form a unit for further analysis and sorting of the cells. Here we describe another concept for cytometry and sorting of live cells based on secreted products. Basically, the secreted product is retained on the cell surface of the secreting cell, making it accessible to the powerful technologies for detection of surface markers. As shown schematically in Fig. 1, an affinity matrix for the secreted product is generated by attaching a specific antibody to the cell surface. Subsequently, the cells are allowed to secrete their products under defined conditions into a medium of low permeability for the secreted product. After removal of the cells from the incubation medium, they are stained for the secreted product, which is now bound to the cell-surface affinity matrix, with specific fluorochrome-, hapten-, or particle-labeled "detection" antibodies or other staining reagents. Preparation of Medium of Low Permeability. Sixty grams of gelatin (from bovine skin, 75 bloom, Sigma) was dissolved in 100 ml of warm PBS and dialyzed extensively, first against PBS and then against RPMI 1640 medium (GIBCO). For use, this stock solution was diluted (with RPMI medium/5% fetal calf serum) to a final concentration of 25% or 40% gelatin. MATERIAL AND METHODSEmbedding of Cells in Gelatin. A cell suspension of 106_107 cells in 100 ml of PBS/BSA was mixed with 1 ml of gelatinous medium (25 or 40% gelatin) prewarmed to 3...
Two lines of evidence are reported which suggest that the highly metastatic variant ESb of the T-cell lymphoma Eb is derived from spontaneous fusion with a host macrophage. Firstly, ESb cells are shown to express the macrophage differentiation antigen Mac-1 which was not found on Eb cells or on any other tumor cells tested except the macrophage tumor line Pu5. Secondly, the progression from low to high metastatic capacity could be reproduced in vitro following hybridization of thioguanine-resistant Eb cells (EbTGR) with syngeneic bone-marrow-derived macrophages. Two HAT medium-selected hybrid tumor lines (Eb-F1 and Eb-F2) could be established. They were found to express cell surface markers of both parental lines: T lymphoid differentiation antigens from T-lymphoma and macrophage antigens (Mac-1, class II MHC antigens) from the normal cell fusion partner. The antigens were identified on the hybrids and subclones thereof by means of monoclonal antibodies and 3 different detection assays: cytofluorography, complement-dependent cytotoxicity and immunoprecipitation followed by gel electrophoresis. Animals inoculated s.c. with the parental line EbTGR developed local tumors but not metastases and survived for more than 40 days. In contrast, animals inoculated similarly with Eb-F1 or Eb-F2 cells quickly developed metastases in visceral organs and died as early as 10-14 days following inoculation. In many but not all respects, the in vitro-derived T-lymphoma-macrophage hybrids resembled the spontaneous in vivo-derived variant ESb. These findings, together with the presence of Mac-1 antigen on ESb cells, suggest (1) that ESb variant cells may be derived from spontaneous fusion with a host cell, most likely a macrophage and (2) that somatic cell fusion may be an important mechanism of genetic rearrangements leading to metastatic variants. The new highly metastatic tumor lines which were developed under well-defined in vitro conditions, and their subclones, may become very useful tools for studying the contribution of specific genetic traits and of membrane-related structures to various steps of the metastatic process.
Although tumor heterogeneity (1) and progression (2) have important biological implications and therapeutic consequences, little is yet known about basic mechanisms of tumor diversification and variant generation. Somatic hybridization between tumor cells and normal host cells has been reported (3) to occur in vivo and could potentially contribute to the diversification and generation of highly malignant variants. To test this possibility we attempted the in vitro fusion of tumor cells with defined normal host cells such as macrophages, which are often present within growing tumors. We herein report on two highly metastatic hybrid lines independently obtained after in vitro hybridization of a nonmetastatic murine lymphoma line (Eb) (4, 5) with syngeneic bone marrow-derived macrophages. Interestingly, such high metastatic hybridomas were found to express a tumor antigen similar to that expressed by spontaneous in vivo derived high metastatic variants (ESb) (6-8) of the same tumor. Materials and MethodsThe two cell types to be fused were carefully selected. The lymphoma line was made thioguanine resistant (EbTGR), and thus HAT (hypoxanthine, aminopterin, thymidine)-sensitive, without mutagenesis, by growing it in gradually increasing concentrations of thioguanine up to 4 #g/ml, followed by cloning. G-banding caryotype analysis revealed TGR that the clones were unstable and heterogeneous while the noncloned Eb line was stable, homogeneous, and identical in all eight marker chromosomes with the parental line Eb (8). As fusion partner for the Eb "rcR line we used in vitro differentiated, bone marrow-derived macrophages precuitured for 15 d in L ceil-conditioned medium. As described elsewhere (9), these cultures consisted predominantly of macrophages that were highly spread out and had phagocytic activity. Tumor macrophage fusions with polyethylene glycol (PEG) (Merck AG, Darmstadt, FRG) (1,000 tool wt; 45% in RPMI, pH 7.5, or in 0.15 M Hepes) were performed with a modified procedure (10). Fusion cultures a-GR ro ha es and PEG consisted of Eb , mac p g , , while control cultures had either Eb T°R cells and PEG, macrophages and PEG, or Eb vGa cells and macrophages without PEG. This work was supported by grant SFB 136 (Deutsche Forschungsgemeinschaft) and by a visitors grant from the DKFZ Heidelberg. J. ExP. MED.
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