Dominance of a tumor subpopulation line in mixed heterogeneous mouse mammary tumors

When mixtures of cell lines 168 and 4T07, both derived from the same mouse mammary tumor, were injected into syngeneic mice, the resulting tumors, analyzed over a large size range by colony-forming assays in selective media, consisted primarily of line 4T07, even when the ratio injected was 100:1 or greater in favor of line 168. This result indicated a suppression of growth of line 168, since the volume-doubling time of line 168 tumors in the absence of line 4T07 was one-half that of line 4T07 tumors. That growth suppression was not due to inhibition of line 168 by immunity induced to line 4T07 was shown in two ways: (a) line 168 tumors grew almost as well in mice preimmunized with line 4T07 as in controls, whereas line 4T07 tumor growth was strongly inhibited in preimmunized mice; and (b) the final composition (favoring line 4T07) in mixed tumors was similar in tumors grown in mice immunosuppressed by irradiation to that in nonirradiated controls. The strong suppression of line 168 did not occur when the two cell lines were injected simultaneously at different s.c. sites, nor did it occur when line 168 cells were injected in mixtures with lethally irradiated line 4T07 cells. Line 4T07 cells also suppressed the growth of line 168 cells in monolayer cultures. It was not likely that suppression was due to competition for growth factors, since the effect required cell contact. Suppression probably was not mediated through junctional communication, since these cells do not engage in metabolic cooperation. We suggest that a growth-inhibitory factor produced by line 4T07 mediates the suppression of 168 cells.     

Factors affecting growth and drug sensitivity of mouse mammary tumor lines in collagen gel cultures

A series of mouse mammary tumor subpopulation lines were compared for growth properties and sensitivity to chemotherapeutic drugs when grown as boluses in a collagen gel matrix versus in monolayer culture. Although the cell lines exhibited characteristic rates of bolus expansion in collagen, this growth was not paralleled by an exponential increase in cell number with time. Cell boluses contained a higher proportion of cells in G0-G1 phases of the cell cycle than did the same cell lines in monolayer cultures. Histological examination revealed areas of necrosis in boluses. Thus cells growing in collagen cultures resembled cells growing as solid tumors and cells from other three-dimensional culture systems. The growth of cell boluses in collagen gel cultures was reduced nonexponentially by melphalan, methotrexate, and 5-fluorouracil in contrast to the exponential decrease in growth measured in cloning assays. The lowest concentration to which cells first responded to drug was in general similar for collagen gel assays and for cloning assays. The rank order of sensitivity of different cell lines in the two assays was identical for methotrexate (four cell lines), similar for melphalan (four of five lines), but quite different for 5-fluorouracil. In contrast to cloning assays cell boluses continued to grow, albeit at a reduced rate, in the presence of high drug concentrations. This was not due to either diminished drug availability in collagen gel or drug penetration into the bolus.     

Clonal variation in the sensitivity of a murine mammary carcinoma to melphalan

The sensitivity to melphalan of clones derived from individual lung colonies produced by i.v. injection of cells of the MT murine mammary carcinoma (caMT) and its melphalan-resistant sub-line (MTME16) has been examined. A degree of clonal heterogeneity was observed which was greater than could be explained by experimental variation. The distribution of melphalan sensitivities in both wild-type caMT and MTME16 raises questions as to the validity of a two-compartment model of drug-resistance development in tumours. A more complex model, possibly involving a continuous spectrum of drug sensitivity, is required. Differences in the sensitivity of the clonal lines of wild-type caMT in various passages were observed and this would appear to be due to phenotypic instability in these lines. This suggests that to use survival data from clones which have been passaged many times for predicting the response of the parent tumour may be misleading.     

Morphology of MXT mouse mammary tumors. Correlation with growth characteristics and hormone sensitivity

The transplantable MXT mouse mammary tumor has been a useful tool for studying endocrine mechanisms underlying mammary tumor growth. It is our experience, however, that this model is unstable during serial transplantation. This paper analyses this variability from the viewpoints of histology and estrogen receptor content and indicates that these parameters should always be checked before planning experimental work. It is advised that a more homogeneous material is needed and that this goal should be achieved by clonal selection before transplantation.     

Immunological studies on mouse mammary tumors. II. Characterization of tumor-specific antibodies against mouse mammary tumors

After injection of isografted mammary tumor MM2 sensitized with rabbit antiserum, resistance was induced in C3H/He mice after repeated challenges with MM2. The serum taken from these mice was found to be cytotoxic against MM2 cells. The serum also inhibited the outgrowth of transplant of primary tissue culture cells of spontaneous mammary tumor of C3H/He mice. A series of transplanted mammary tumors recently converted into ascitic form in our laboratory (MM3, MM4-1, MM4-2, MM4-3, MM6, MM8, MM9, MM11, MM12 and MM13), and Ehrlich ascites tumor were found to be susceptible to this serum, as tested by trypan blue uptake in vitro. Outgrowth of these tumors was also inhibited when tumor premixed with this serum was injected. No cytotoxic effect was observed against normal mouse mammary gland cells of a C3H/He mouse. Sera obtained from hyperimmunized syngeneic C3H/He mice were able to fix complement with MM2 tumors. They were partially inactivated by heating at 56° C and by treatment with 2-mercaptoethanol. After gel filtration through Sephadex G-200, complement fixing and cytotoxic activity were found in both 19S and 7S fractions. The 7S fractions, after DEAE cellulose column chromatography, gave a precipititin line at the IgG position in immunoelectrophoresis. From the above evidence, it is concluded that the target cells of the cytotoxic factors of this serum are primary cultured cells or isografted cells of mammary tumors of C3H/He mice. The cytotoxic factors present in the serum are considered to be antibodies against isografts of mammary tumors in C3H/He mice.     

Localization of tissue copper in mouse mammary tumors

The purpose of this study was to determine the copper deposition and localization during the evolution of two murine mammary adenocarcinomas. In the normal tissue, the copper was located within the cytoplasm, whereas it was intra- and perinuclear in the tumors. The more angiogenic and metastatic tumor showed the higher percentage of copper-positive cells. In the tumor, copper deposits correlated well with its angiogenic and metastatic ability, but additional factors would be required for the process to be induced.     

Quantification of mouse mammary tumor virus structural proteins in hormone-induced mammary tumors of low mammary tumor mouse strains

The expression of the mouse mammary tumor virus (MMTV) in hormone-induced mammary tumors was investigated by means of a radioimmunoassay for two major MMTV proteins, gp52 and p27. MMTV proteins were isolated on lectin affinity- and ion-exchange chromatography columns. The purified viral proteins were electrophoretically homogeneous and retained immunoreactivity after labelling with 125iodine. Standard competition assays showed that group-specific antigenic determinants were reacting. Mammary tumors were induced in three strains of mice with a low natural incidence of mammary tumors, C57BL, O20 and C3Hf, by a combined hormone treatment, consisting of hypophyseal isografts and administration of progesterone and estrone. Mammary tumors and mammary glands of hormone-treated animals were extracted and used for competition radioimmunoassays. In general, the tumorigenic hormone treatment resulted in enhanced amounts of MMTV proteins in the mammary glands, compared to the amounts found in lactating mammary glands of untreated animals. The levels of MMTV proteins in the mammary tumors were lower than in the mammary glands.     

Heterogeneity of expression and induction of mouse mammary tumor virus antigens in mouse mammary tumors

Seven spontaneous BALB/cfC3H mouse mammary tumors were heterogeneous in expression of murine mammary tumor virus-associated cell surface antigens. To determine the basis of this heterogeneity, cells from spontaneous tumors and from five subpopulations isolated from a single spontaneous tumor were examined for expression of viral antigens under both conventional conditions and conditions known to induce synthesis of murine mammary tumor virus antigens (5-iodo-2'-deoxyuridine and dexamethasone). Induction with 5-iodo-2'-deoxyuridine resulted in further manifestation of the antigenic heterogeneity of spontaneous tumors. The five subpopulations from a single tumor differed in the amount of viral antigens present in untreated and in induced cultures. Coculturing showed that viral antigen expression was independent in each subpopulation within a heterogeneous mixture and was not influenced by the presence of other subpopulations with different potentials for viral antigen synthesis. The expression of murine mammary tumor virus structural antigens, a protein with a molecular weight of 28,000 and a glycoprotein with a molecular weight of 52,000, differed within the heterogeneous subpopulations, and was noncoordinate. The data suggest that the antigenic heterogeneity in spontaneous tumors reflects the existence of cells within them that differ in both expression of viral antigens and in their response to inducers of viral antigen synthesis.     

Factors that affect metastasis in virus-induced mouse mammary tumors

Two hundred and forty-one mammary tumor-bearing breeding female mice of the BALB/cf C3H and BALB/ cfRIII strains, carrying milk-transmitted murine mammary tumor virus (MuMTV) of C3H and RIII origin, respectively, were studied to evaluate the factors that affect tumor metastasis. Only lung metastases were considered and the following factors taken in account: MuMTV inducing variant (C3H, RIII), number of deliveries, tumor histotype , number of tumors per mouse, clinical duration of tumors, tumor size, and tumor growth rate. Only the number of deliveries, the tumor size and the number of tumors per mouse were found to significantly influence the rate of metastasis. The tumor growth rate affects concurrently with tumor size the metastatic process. In fact, the larger the tumor the higher the tumor growth rate. This direct relationship is significant (P less than 0.01) and, in case of lung metastases at autopsy, there was a prevalence of large tumors (greater than 2 cm) and high growth rate (greater than 0.3 mm/day). The discordance of these data with those concerning mammary tumor metastasis in virgin females of the same two strains, the enhancing effect of pregnancies on metastatic spread of tumors, and the significance of results for the understanding of the general mechanisms of tumor metastasis are discussed.     

Translocations involving chromosome 1 in hormone-independent GR mouse mammary tumors

Large marker chromosomes were found in 2 hormone-independent (HI) GR mouse mammary tumors, i.e. a tumor (TSl 75) that was already HI at the first transplantation, and a tumor (TSl 66) that became HI at the 19th passage. These marker chromosomes appear to be translocations to distal and proximal ends of chromosome 1, respectively. The translocation in tumor TSl 66 may be t(15:1).     

Combined endocrine therapy and chemotherapy of mouse mammary tumors

Hormone-responsive mammary tumors of GR mice were treated with tamoxifen, cyclophosphamide, or with both drugs combined. Tamoxifen alone or cyclophosphamide alone caused inhibition of tumor growth, but more growth inhibition was obtained with the combined therapy.     

Influence of pituitary grafts or prolactin administrations on the hormone sensitivity of ovarian hormone-independent mouse mammary MXT tumors

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.