As an approach to isolating tumor cells from fine needle biopsy specimens, we investigated a dielectric cell preparation method using an in vivo xenographic tumor model. Cultured human MDA-MB-435 tumor cells were grown as solid tumors in nude mice and fine needle aspiration biopsies were conducted. Biopsied cells were suspended in sucrose medium and collected on slides patterned with microelectrode arrays (electrosmears) energized by electrical signals in the range 10 to 960 kHz. The unlabeled cells adhered to characteristic regions of the slides in accordance with their morphology as a result of dielectric forces. Tumor cells were trapped between 40 and 60 kHz and were separated according to whether they were mitotic, large and complex, or small. Damaged tumor cells were captured at between 60 and 120 kHz; granulocytes between 70 and 90 kHz; lymphocytes between 85 and 105 kHz; healthy erythrocytes between 140 and 180 kHz, and damaged erythrocytes above 180 kHz. Using intrinsic cell characteristics, the electrosmear presented cell subpopulations from fine needle aspiration biopsy specimens in a manner that is compatible with automated slide-based analysis systems. The approach has the potential to facilitate the analysis of the role of cell subpopulations in disease.

A rat hybridoma producing IgM monoclonal antibody (MAb) GP21:56 was generated with specificity for a high‐molecularweight, mucin‐like glycoprotein (gp580) present on highly metastatic 13762NF rat mammary adenocarcinoma cells. The hybridoma was made by fusing rat Y3 Ag1.2.3 myeloma cells with spleen cells from a rat immunized i. d. with purified gp580. The gp580 appeared to be of low immunogenicity in syngeneic F344 rats because a total of 27 fusions were required to produce one hybridoma with specificity for this glycoprotein. Immunoblotting of purified gp580 after electrophoresis in 1% agarose and antibody‐binding assays using purified gp580 linked to microtiter plates confirmed that MAb GP21:56 bound specifically to gp580. Other MAbs made against breast mucins were negative for gp580 reactivity. Enzymelinked immunoabsorbent assays (ELISA) and radiolabelled antibody binding assays demonstrated that MAb GP21:56 bound to 13762NF adenocarcinoma cell lines and clones in relation to their spontaneous metastatic potentials; significantly more MAb GP21:56 bound to highly metastatic MTLn3 cells than to low metastatic MTC cells, and MAb GP21:56 showed little reactivity towards the majority of other cell lines tested, whether of rodent or of human origin. Kinetic binding studies indicated that MAb GP21:56 does not have a high affinity for gp580 but, once bound, it shows high avidity for this sialogalactoprotein. Localization studies using frozen tissue sections of 13762NF tumors indicated that MAb GP21:56 reacts with tumor cells grown in vivo in an analogous manner to in vitro cultured cells. Using immunoperoxidase techniques, less than 50% of the highly metastatic MTLn3 tumor cells were stained, whereas approximately 20% of the intermediate metastatic MTF7 and MTLn2 cells and < 10% of low metastatic MTC and MTPa cells were stained with MAb GP21:56. The cell‐to‐cell reactivity was heterogeneous and mainly associated with the tumor‐cell surface and extracellular matrix.

With two-dimensional isoelectnic focusing sodium dode cyl sulfategel electrophoresis,32P-labeled cytosol proteins of Novikoff hepatoma and i8-hr regenerating rat liver were separatedinto 133 and 129 stained polypeptides,respec tively. Approximately i@3polypeptides of both tissues had essentially identical migration characteristics. Seven dense spots (molecularweight in thousands/pI)140/6.5,72/6.7, 64/7.2, 35/7.7, 29/7.3, 28/7.1 , and 27/7.2, that were found in the Novikoff hepatoma patterns were not present in the regenerating liver patterns. Six dense spots, 50/7.0, 50/7.4, 48/6.9, 43/8.2, 33/7.4, and 25/7.4, that were found in the regenerating livenwere not found in the Novikoff hepatoma. Three spots, 95/5.7, 4i/6.2, and 39/6.i , common to the Novikoff hepatoma and regenerating liver were not found in the normal liver. Autoradiogramsof 32P-labeled proteins revealed82 spots in Novikoff hepatomaand 88 spots in regenerating liven, 60 of which were common to both tissues; for Novikoff hepatoma, 5 g of cells were incubated with 300mCiof cannier-free 32p1, andfor regeneratingliver, 100 mCi cannier-free 32P@ were injected i.p. 15 hr after hepatectomyand 3 hr before sacrifice. Three 32P-labeled protein spots, 65/5.8, 25/5.5, and 25/6.0, were found only in the Novikoff hepatomaand 5 spots, 55/6.1, 45/7.0, 45/ 7.8, 42/5.9, and 24/5.5, were found in regenerating liver. Thesestudiesextend earlier reports of differencesin spot patterns of Novikoff hepatoma and normal liver cytosol using the additional parameterof 32P-Iabeling as a marker for phosphoproteins.

SUMMARY

The translational activities of cytoplasmic poly A(+) RNA of normal rat liver and Novikoff hepatoma cells in the wheat germ cell free system were found to be approximately 15–20 times greater than those of the corresponding nuclear poly A(+) RNA. The translationsl activities were 85 and 62 pmoles3H-leucine incorporated/µg cytoplasmic poly A(+) RNA for the liver and tumor respectively and 3–4 pmoles3H-leucine incorporated/µg nuclear poly A(+) RNA. Inasmuch as integrity of the ‘5′-cap’ of mRNA is essential for its translational activity, quantitative comparisons were made of its content in these RNA fractions. Of the total32P incorporated into the tumor cytoplasmic poly A(+) RNA, 0.41% was in the ‘5′-cap’; in nuclear poly A(+) RNA, the ‘5′-cap’ contained 0.11%. After periodate oxidation and labeling with KB3H4, m7 guanosine, the 5′-terminal nucleoside in both liver and Novikoff hepatoma nuclear poly A(+) RNA contained approximately 20% as much isotope as in the cytoplasmic poly A(+) RNA. These results suggest the lower translational activity of nuclear poly A(+) RNA is partly related to its lower content of the ‘5′-cap’. Molecular selection of poly A(+) RNA for transport out of the nucleus or further cytoplasmic processing may account for the higher percentage of the ‘5-cap’ and the greater translational activity of the cytoplasmic poly A(+) RNA. During these studies, it was also found that the m7 guanosine of the ‘5′-cap’ was not removed during translation of the mRNA in the wheat germ system; this result suggests that the ‘5′-cap’ may associate with allosteric binding sites of initiation factor(s).