Characterization of tumor-infiltrating lymphocytes from murine mammary adenocarcinomas.

The aim of this investigation was to assess the in vitro functional and phenotypic characteristics of lymphocytes isolated from C3H mouse mammary adenocarcinomas. A protocol was developed for the expansion of TILs in long-term culture. The homing pattern of TILs prepared and grown in this manner was studied. Cells that had been in culture for up to 96 days accumulated at higher levels in mammary tumors than in corresponding normal mammary tissue 24 hr after adoptive transfer. The ability of cultured TILs to lyse YAC-1 cells was determined. Peak activity was demonstrated by lymphocytes that had been in culture for three days. By two weeks in culture the level of cytotoxicity returned to that of fresh TILs, and after 45 days it was negligible. T cells were the major constituents in all preparations. The relative frequency of CD8+ cells remained fairly constant over time in culture, but that of CD4+ cells declined. At all time points the CD4:CD8 ratio for TILs was less than 1. The percentage of ASGM1+ bright cells among fresh TILs was low. It increased dramatically within 3 days, remained high for about 7 weeks, and then declined rapidly to pre-culture levels. An unusual large cell characterized by the presence of an intensely PAS positive peripheral region was observed.

Preferential homing of tumor-infiltrating lymphocytes in tumor-bearing mice.

In view of the current interest in the use of lymphoid cells in adoptive immunotherapy of patients with advanced cancer, we have studied the homing patterns of various lymphoid effector cells in mammary-tumor-bearing mice. Single-cell suspensions of total splenocytes, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells were prepared from the spleens of C3H/OuJ mice. Tumor-infiltrating lymphocytes (TIL) were isolated from mammary adenocarcinomas excised from retired breeder females of the same substrain. Effector cells were labeled with indium-111 and injected via a tail vein into female C3H/OuJ mice bearing one or more mammary tumors. Twenty-four hours after administration, total splenocytes, NK cells, and LAK cells distributed themselves evenly between normal mammary tissue and mammary adenocarcinomas. Only TIL had a higher concentration in tumors than in corresponding normal mammary tissue. The ability of the different lymphocyte preparations to lyse YAC-1 cells was determined by means of a 4-h 51Cr-release cytotoxicity assay. Cells harvested from LAK cell cultures and further enriched by centrifugation through a discontinuous Percoll gradient and interleukin-2 (IL-2)-stimulated TIL demonstrated the highest levels of cytotoxicity, while total splenocytes and fresh TIL were characterized by the lowest levels. Since IL-2-stimulated TIL were highly cytotoxic and exhibited better tumor localization than both NK cells and LAK cells in this system, they may be the lymphoid effectors of choice for adoptive immunotherapy of advanced cancer.

Migratory patterns of different indium-111 labeled leukocyte populations (chiefly lymphocytes) from control and thymectomized rats.

The blood clearance and 24-hr organ distribution of five lymphocyte-rich suspensions harvested from different source organs were compared in the Wistar rat, after labeling with [111In]tropolone and intravenous injection. Peripheral blood lymphocytes (chiefly T cells) had the highest levels in the blood and lymph nodes, a relatively higher level in intestine and a lower level in the spleen than other suspensions. Thymocytes cleared promptly from the blood, accumulated markedly in the liver, moderately in the spleen, but very poorly in other organs, including the thymus. Splenic cells (rich in B cells) sequestered predominantly in the spleen and liver. Splenic cells nonadherent to nylon wool (T-cell enriched) had a relatively high uptake in lymph nodes and marrow. Splenic cells from thymectomized rats (NK cell enriched) localized predominantly in the liver and spleen, and poorly in lymph nodes. We conclude that the source organ for harvesting rat lymphocytes is an important determinant in their localization, as well as other factors such as the techniques of isolation, the toxicity and stability of the radioactive label and the cell radiation dose from internalized radioactivity.

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells.

We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h 51Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.

Decreased natural cytotoxicity in mice with high incidence of mammary adenocarcinoma.

In an attempt to gather evidence relevant to the question of whether natural killer (NK) cells play a role in resisting the development of primary tumors, we compared natural cell-mediated cytotoxicity in two substrains of C3H mice. Animals of the C3H/OuJ substrain are at high risk for the formation of mammary adenocarcinomas, while C3Heb/FeJ mice have a low incidence of such tumors. Natural cytotoxicity of splenic mononuclear cells was lower in the high-risk substrain, suggesting that a lesion in NK cell activity may be involved in murine mammary tumorigenesis. This difference was observed in animals between 5 and 37 weeks of age. There was no significant difference in the number of splenic large granular lymphocytes between the substrains. A significant difference in the ability of splenic lymphocytes from the two substrains to bind to the target cells was noted. Since the binding capacity of lymphocytes was greater in mice with reduced NK cell activity, the lesion in cytotoxicity may exist at a postbinding step in the lytic sequence. It is felt that the C3H mouse may provide a useful model for studying the role of NK cells in controlling primary tumors.

The influence of epidermal growth factor on surface morphology of fetal rat hepatocytes in primary culture.

In an attempt to understand the hormonal regulation of somatomedin secretion in the fetus, we have confirmed that epidermal growth factor (EGF) stimulates fetal rat hepatocytes in primary culture to secrete somatomedin in a time and a dose-dependent fashion. Transmission electron microscopy (TEM) revealed that the cultured cells had ultrastructural features consistent with those of fetal hepatocytes. Scanning electron microscopy (SEM) showed that cells grown in either Medium 199 or EGF supplemented Medium 199 formed cellular aggregates within 6 h. The surface features of cells in control and experimental cultures were indistinguishable up until 24 h after exposure to EGF. At this point in time, morphological differences between treatment groups were first apparent with SEM. In the presence of EGF, cellular aggregates were thicker, cells were more rounded in contour, and the number of microvilli and cytoplasmic excrescences (blebs) was greater than in control cultures. These differences were further accentuated at 48 h after exposure to the growth factor. Since the appearance of microvilli and blebs coincides with increasing production of somatomedin, they may represent morphological evidence of secretory activity.

Fine structure analysis and surface characteristics of mouse mammary gland adenocarcinomas.

Mammary adenocarcinomas from C3H/HeJ mice carrying the mammary tumor virus were studied by means of light, transmission, and scanning electron microscopy. The histological appearance of the tumors was variable; however, 60% were classified as type B adenocarcinomas, while 20% were type A and 20% were composed of equal regions characteristic of both types. Four populations of tumor cells were observed with the TEM. The most abundant of these, primitive glandular cells, were characterized by large, regular, euchromatic nuclei and cytoplasm containing numerous free ribosomes, little rough endoplasmic reticulum, few mitochondria, small Golgi complex and a variable number of type A virus particles. Specialized glandular cells contained highly pleomorphic nuclei, many lysosomes, lipid droplets, multivesicular bodies, profiles of rough endoplasmic reticulum and granules resembling secretory proteins. Myoepithelial cells and dark glandular cells with abundant organelles, large Golgi complexes, dense cytoplasmic matrix and very heterochromatic nuclei were observed infrequently. The SEM revealed tumor cells to be variable in size, shape and surface characteristics. Most cells were rough in texture, displaying irregular ridges, small blebs and a few short microvilli. The contours of some cells were smooth, and a few cells had short, irregular microvilli on limited regions of their surfaces. Cells lining ducts within the tumor had microvilli on their apical surface, but the number, size, shape and distribution of microvilli varied considerably. Cells lining ducts from non-tumor-bearing animals displayed less variation in size, shape and surface morphology.

Auxin: its role in genetic tumor induction.

Seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown on nutrient medium supplemented with indoleacetic acid (IAA) and scored at regular intervals for the incidence of tumor formation. IAA at 2 x 10(-5)m caused a significant reduction in the rate of tumor formation. Plants were also grown on nutrient medium under two different intensities of illumination, and the endogenous level of IAA was determined in 23-day-old seedlings. Those grown under 2000 ft-c of illumination had a higher incidence of tumors and a significantly lower level of endogenous IAA than those grown under 500 ft-c of illumination. A correlation in time between decline in the endogenous level of IAA and onset of tumor formation was demonstrated in greenhouse-grown plants.

Endogenous levels of auxin and tumorigenesis in a Nicotiana amphiploid.

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown aseptically on nutrient medium in a controlled environment chamber. At regular intervals after the seeds had been sown, the incidence of tumor formation was scored and plants were harvested. Indole-3-acetic acid was extracted, purified, and assayed spectrofluorometrically. A close correlation in time between onset of tumor formation and decline in endogenous level of indole-3-acetic acid was demonstrated.