Enzymes, either acid phosphatase or horscradish pcroxidase, were conjugated to antibodics with bifunctional reagents. The conjugates, cnzymatically and immunologically active, werc employed in the immunohistochcmical localization of tissue antigens utilizing the reaction product of the enzymatic reaction as the marker. Tissues reacted with acid phosphataselabcled antibodies directed against basemcnt membrane werc stained for the enzyme with Gomori's method, and those reacted with peroxidase-labcled antibody were stained with Karnovsky's method. The reaction products of the enzymes localized in the basement membrane. Unlike thc preparations of the fluorescent antibody technique, enzyme-labelcd antibody prcparations were permanent, could be observed with an ordinary microscope, and could be examined with the electron microscope. In the latter, specific localization of antibody occurred in the basement membrane and in the endoplasmic reticulum of cells known to synthesize basemcnt mcmbrane antigens. The method is sensitive bccause of the amplifying effect of the enzymatic activity. The ultrastructural preservation and localization were better with acid phosphatase-labeled antibody than with peroxidase-labclcd antibody, but acid phosphatase conjugated antibody was unstable and difficult to prepare. Peroxidaseantibody conjugatcs were stable and could bc stored for several months at 4°C, or indcfiniely in a frozen state.

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Monolayer cultures of a mouse teratocarcinoma were established in vitro. These cultures contained embryonal carcinoma, the malignant stem cell, and its differentiated progeny: parietal yolk sac, neuroepithelial, and mesenchymal cells. Tissues such as squamous epithelium, cartilage, striated muscle, neuroepithelium, and glands were produced from embryonal carcinoma that was maintained under conditions of long term culture. Frequent subcultivation with pancreatin allowed the establishment of cell lines of embryonal carcinoma which have been maintained for more than 18 months in vitro and continue to produce differentiated cells under specific culture conditions. Chromosomally these lines of embryonal carcinoma have a stem line of 39 chromosomes. Two lines of parietal yolk sac cells have been established which produce basement membrane, are not tumorigenic, and chromosomally are hypotetraploid. This system may yield information concerning neoplastic differentiation and its possible use in therapy for cancer.

The malignant growth potential of embryonal carcinoma cells may be controlled by environmental factors. For example, embryonal carcinoma cells placed into normal blastocysts may not exhibit the continued growth expected of malignant cells but rather may lose all aspects of the malignant phenotype and become apparently normal embryonic cells. Loss of the malignant phenotype of embryonal carcinoma cells occurs early in these I jected blastocysts and has been used as the basis of assays to study the mechanisms of regulation of embryonal carcinoma by the blastocyst. In this regard, P19, an embryonal carcinoma that makes midgestation chimeras, was regulated by blastocele fluid plus contact with trophectoderm but not by blastocele fluid plus contact with inner cell mass (ICM). In contrast, ECa 247, which makes trophectoderm, was regulated by exposure to blastocele fluid plus contact with trophectoderm or ICM. During the course of these experiments, dead embryonal carcinoma and ICM cells were observed, and blastocele fluid was then shown to kill ECa 247 and normal ICM cells of early blastocysts with trophectodermal potential. P19 cells and ICM cells with potential to make the embryo were not killed by blastocele fluid. Programmed cell death occurs in the ICM of the blastocyst during the transition from early (when ICM has the potential to make trophectoderm) to late (when the ICM lacks the potential to make trophectoderm). It is postulated that this programmed cell death is designed to eliminate redundant 1CM cells with trophectodermal potential, and its mechanism of action is mediated by epigenetic factors in blastocele fluid.zP T E _G

This report focuses on the regulation of murine melanoma by the embryonic skin. A surgical technique was developed to allow inijection of B16 melanoma cells into the embryo in utero. A significant decrease in incidence of tumors was noted, which correlated with the time of arrival of normally migrating premelanocytes into the skin. Media were conditioned from skin explanted at the time premelanocytes arrive in it; these media inhibited the growth of melanoma cells in vitro. Under optimal conditions the growth of melanoma cells ceased; the cells had altered morphology and failed to proliferate when placed in fresh growth media.The discovery that cancer cells could differentiate into benign cells and tissues (1, 2) and that the process could be modulated (3) led to the concept that direction of differentiation of malignant to benign cells might serve as an alternative to cytotoxic therapy for cancer (4). In 1974 Brinster produced a chimeric mouse by injecting embryonal carcinoma cells into a blastocyst and putting the injected blastocyst into the uterus of a mouse made pseudopregnant (5). Thus, the blastocyst could regulate embryonal carcinoma cells and their progeny so they behaved as apparently normal embryonic cells. Regulation of tumor (6) and colony (7) formation of embryonal carcinoma by the blastocyst was tissue specific in that it was dependent upon close correspondence of the embryonic field (8) and the malignant cells. Further, contact of the cancer cells with trophectoderm in the presence of blastocele fluid was required (9). The problems occasioned by the paucity of trophectodermal cells made further study of the mechanism of embryonic regulation of embryonal carcinoma extremely difficult.Accordingly, other embryonic fields were examined to see if they could regulate tumor or colony formation of their closely related cancers. The neural-stage mouse embryo regulated neuroblastoma cells (10) MATERIALS AND METHODSIn the surgical approach to mouse embryos in utero, C57BL/6 female mice (The Jackson Laboratory) pregnant for 10 or 14 days (day 1 is the day of observation of the mating plug) were anesthetized with 0.4 ml of Avertin injected intraperitoneally. Avertin is composed of 2.5 g of 2,2,2,-tribromoethanol in 5 ml of 2-methyl-2-butanol and 200 ml of distilled water.A midline abdominal incision was made, a uterine horn was exposed, and the skin and viscera were packed out of the operative field with saline-soaked gauze. The animal was placed under a dissecting microscope using the lowest power objectives and x 10 eyepieces. An assistant observed the operative field through a large magnifying glass. A pursestring suture (6-0 silk) was placed in the uterine muscle, and the muscle inside this suture was divided with fine forceps to expose the fetal membranes. When the yolk sac was reached and before herniation of the embryo could occur, tension was placed on the purse-string suture, and a dam was firmly pressed down over the incision and held in place by the assistant. The dam was a piece of wire b...