Blocking of spleen cell activity against target mammary tumor cells by viral antigens.

Spleen cells from BALB/c females exposed to or neonatally infected with mammary tumor virus (MTV) are cytotoxic to MTV-induced mammary tumor cells in microcytotoxocity assay. This activity can be partially or completely blocked by pretreatment of spleen cells with MTV purified from milk. Murine leukemia virus (MuLV) has no effect. T cell responses of virgin and multiparous BALB/cfC3H females are effectively blocked. Non-T cell responses of multiparous BALB/cfC3H females or of virgin BALB/c females are blocked by some but not all of the MTV antigen preparations. MuLV, but not MTV, can block activity of spleen cells from MuLV-sensitized donors against target MuLV-producing tumor cells.     

Growth effect of lithium on mouse mammary epithelial cells in serum-free collagen gel culture

The effect of lithium on the growth of mammary epithelial cells from adult virgin and midpregnant BALB/c or BALB/cfC3H mice was tested in a serum-free collagen gel culture system. The serum-free medium consisted of a 1:1 mixture of Ham's F12 and Dulbecco's Modified Eagle's medium supplemented with insulin, transferrin, cholera toxin, epidermal growth factor (EGF), and bovine serum albumin fraction V (BSA V). A multifold increase in cell number occurred during 10–12 days of culture in this medium. In dose-response studies in which the concentration of each component of this serum-free medium was varied in turn, the addition of LiCL (10 mM) enhanced growth at most concentrations of each factor. However, LiCL could not enhance growth in the absence of insulin or BSA V, but could replace EGF. The optimal concentration of LiCl was 5–10 mM; higher concentrations (20–80 mM) were toxic. KCl (1–10 mM) when added to the serum-free medium slightly stimulated growth; the addition of NaCl to the medium had little effect on growth. LiCl did not enhance the growth of cells from spontaneous mammary tumors of BALB/cfC3H mice.     

Relationship Between Organization of Mammary Tumors and the Ability of Tumor Cells to Replicate Mammary Tumor Virus and to Recognize Growth-Inhibitory Contact Signals In Vitro

Mammary tumor virus (MTV) replication was confined primarily to cells organized as acini in intact mouse mammary glands. Primary mammary tumors maintained a high degree of acinar organization and cells therein continued to replicate MTV vegetatively. Nonacinar mammary cells, derived by serial transplantation of acinar tumor cells, no longer actively replicated MTV. This suggests that phenotypic differences exist among mammary epithelial cells in their ability to support virus replication, that a fundamental relationship exists between the organization of epithelium for secretion and active virus replication, and that this relationship is not altered as a primary consequence of neoplastic transformation. Mammary epithelial cells from pregnant, non-tumor-bearing, MTV-infected BALB/cfC3H mice or from acinar mammary tumors from a number of mouse strains were grown in primary monolayer cultures. Such cell cultures under the influence of insulin and cortisol exhibited the ability to organize into discrete three-dimensional structures called "domes." MTV replication in such cultures took place primarily in cells within the organized domes. Cells cultured from nonacinar tumors did not exhibit any propensity to organize into domes, nor did they replicate MTV in primary culture. This suggests that the cell organizational requirement for MTV replication observed in vivo is conserved in primary culture. Dome formation is not an effect of virus replication, as cells from uninfected BALB/c animals organized into domes in culture without concomitant MTV replication. Growth-regulating signals, exerted between contiguous cells in cultures of non-MTV-infected mammary epithelium, were not modified by the occurrence of active virus replication nor as a direct consequence of neoplastic transformation. Cells derived from nontumor BALB/cfC3H glands and from spontaneous tumors exhibited cell growth kinetics, saturation densities, and deoxyribonucleic acid synthesis kinetics nearly identical to those of noninfected normal mammary epithelium in primary culture. Cell to cell growth regulatory signals were modified in cultures of nonalveolar tumor cells wherein evidence of overgrowth is documented.     

In vitro studies of splenocyte cytotoxicity against syngeneic mammary tumors of mice

Summary Spleen cells from BALB/c mice immunized with D7T4S (MTV-negative) or D14 (MTV-positive) mammary tumors exhibited marked cytotoxic activity for the corresponding tumor cells in a terminal ⁵¹ -Cr-labeling cytotoxicity assay. A pronounced, seemingly nonspecific cytotoxic effect was displayed by splenocytes derived from normal BALB/c and BALB/cfC3H mice subjected to various surgical procedures 10–14 days before testing. Possible mechanisms underlying this phenomenon are discussed.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Antibody in the sera of tumor-bearing mice that mediates spleen cell cytotoxicity toward the autologous tumor.

Pretreatment of MTV-induced BALB/cfC3H mammary tumor cells with autologous serum results in increased spleen cell cytotoxic activity and the recruitment of previously inactive spleen cells to cytotoxic activity against the target cells. These recruiting antibodies are tumor-specific for individual tumors; pretreatment with such serum of target cells of an MTV-induced mammary tumor obtained from a different BALB/cfC3H female results in blocking of spleen cell activity. The autologous recruiting factors are active at dilutions of 1000 or more of whole serum are found in the 19S fraction after gel filtration.     

Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one-year-old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.     

Identification of the mammary tumor virus envelope glycoprotein (gp52) on mouse mammary epithelial cell surface.

Lactoperoxidase radioiodination of mammary epithelial cells cultured in monolayers followed by SDS-PAGE analysis revealed only a few distinct peaks. One of these, identified as major envelope glycoptrotein (gp 52) of MTV, is present on the surface of mammary epithelial cells (both tumor and normal) from chronically infected BALB/cfC3H mice but not on the surface of normal mammary epithelial cells from virus-free $\text{sol}\text{BALB}\text{c}$ mice. Its presence on the cell surface is influenced by both hormones and cell density, the same factors which greatly control the production and release of intact MTV virions into culture media. This suggests a correlation between abundance of radioiodinatable gp 52 on the cell surface and MTV found in culture media.     

Cytogenesis of murine mammary tumors in BALB/cfC3H and BALB/cfRIII strains

Biological and morphological differences in the mammary tumors of BALB/cfC3H and BALB/cfRIII mice are due to differences in the causative viruses. The C3H and RIII variants of the murine mammary tumor virus (MuMTV) might give origin to different mammary tumors by transforming different types of cell, i.e. epithelial or myoepithelial cells. The nature (epithelial or myoepithelial) of the neoplastic cells has been investigated by demonstrating their plasma membrane ATPase activities. We found that in normal murine mammary gland both epithelium and myoepithelium have Mg++ dependent ATPase activity, while the myoepithelium shows in addition an Na+K+ dependent ATPase activity. It is suggested that the results obtained exclude the participation of myoepithelium to the neoplastic growth and we ascribe the differences in mammary tumors of the two strains of mice to differences in the mechanisms of action of the virus variants.     

Direct detection of exogenous mouse mammary tumor virus sequences in lymphoid cells of BALB/cfC3H female mice.

The presence of exogenous mouse mammary tumor virus (MMTV) (C3H) DNA sequences in lymphoid tissue (spleen, bone marrow, and thymus) and nonlymphoid tissue (liver and kidney) of BALB/cfC3H female mice was directly assessed by DNA hybridization methods. Lymphoid tissues were found positive for integrated MMTV(C3H) sequences in females as young as 4 weeks. In most samples, the level of splenic MMTV(C3H) infection was low (2 to 5%). Infection remained throughout the life of the animal. The percentage of spleen samples found positive for exogenous viral infection was significantly higher in females bearing mammary tumors, whether virgin or multiparous. Liver and kidney DNAs were negative for exogenous MMTV sequences, suggesting tissue type selectivity in MMTV infection.     

Possible role of genetic cell variants in the viral induction of mouse mammary tumors

Out of three attempts to induce neoplasia in normal C57Bl mammary epithelial cells with the mouse mammary tumor virus (MuMTV) only one presented signs of tumorigenicity. Immunofluorescence showed that virus synthesis took place in all three sublines but tumorigenicity as detected by cell aggregation viability (CAV) and transplantation into syngeneic mice failed to occur in two of them. By comparison, cells from a BALB/c spontaneous mammary tumor that do not express MuMTV were 100% tumorigenic, whereas cells from a BALB/cfC3H tumor with a 95% virus-producing cell population had a normal CAV and were tumorigenic only in 60% of the test animals. This lack of correlation suggested that many of the virus-producing cell were not neoplastic and that neoplasia might occur under virus stimulation only if a restricted population of genetic cell variants existed. Accelerated tissue culture passages of virus-free C57Bl and BALB/c normal mammary cells resulted in their spontaneous neoplasia at Passages 23 and 50, respectively; when duplicated cells cryopreserved in early passages were revived and cultivated in the same manner, neoplasia occurred at Passages 27 and 58. The similarity of the passage numbers appears to confirm the existence of genetic cell variants among the normal cell population.     

Tumor antigens: Biological activity of components of soluble antigens of MTV-induced mammary tumors

Summary Antigens (AMMT) of MTV-induced mammary tumors of BALB/cfC3H CRGL mice were solubilized by treatment of homogenates of the tumor with 1 M perchloric acid. The soluble antigens exhibited biological activity by their ability to induce DNA synthesis in spleen cells of mice bearing syngeneic transplants of the tumor. AMMT-induced DNA synthesis, however, was abrogated by serum from tumor-bearing mice. AMMT neutralized complement-dependent rabbit anti-ML(r) antibody activity and intracytoplasmic fixed cell immunofluorescent activity of the same antibodies. One component of AMMT has an electrophoretic mobility pattern identical to that of ML(r) antigen. Thus, AMMT may share antigenicity with MTV-specific antigens.     

Possible role of genetic cell variants in the viral induction of mouse mammary tumors

Summary Out of three attempts to induce neoplasia in normal C57B1 mammary epithelial cells with the mouse mammary tumor virus (MuMTV) only one presented signs of tumorigenicity. Immunofluorescence showed that virus synthesis took place in all three sublines but tumorigenicity as detected by cell aggregation viability (CAV) and transplantation into syngeneic mice failed to occur in two of them. By comparison, cells from a BALB/c spontaneous mammary tumor that do not express MuMTV were 100% tumorigenic, whereas cells from a BALB/cfC3H tumor with a 95% virus-producing cell population had a normal CAV and were tumorigenic only in 60% of the test animals. This lack of correlation suggested that many of the virus-producing cells were not neoplastic and that neoplasia might occur under virus stimulation only if a restricted population of genetic cell variants existed. Accelerated tissue culture passages of virus-free C57B1 and BALB/c normal mammary cells resulted in their spontaneous neoplasia at Passages 23 and 50 respectively; when duplicated cells cryopreserved in early passages were revived and cultivated in the same manner, neoplasia occurred at Passages 27 and 58. The similarity of the passage numbers appears to confirm the existence of genetic cell variants among the normal cell population. This investigation was supported by U.S. Public Health Service Grant R01-CA-08515 from the National Cancer Institute.     

Comparative expression of endogenous retrotransposons in adult mouse mammary gland during normal development and differentiation

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and post-lactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.     

The role of progesterone in pregnancy-induced aggression in mice

A series of six experiments was performed in order to explore the potential involvement of progesterone (P) in pregnancy-induced aggression (PIA) displayed by Rockland-Swiss mice toward adult male intruders. In Experiment 1, circulating levels of P and aggression were low on gestation Days 6 and 10 while both the behavior and the steroid reached peak levels by gestation Day 14. By gestation Day 18 (the day prior to parturition), serum P was at its lowest level yet aggressive behavior was still intense. Also, individual differences in the display of fighting behavior by pregnant females were not related to circulating P. Experiments 2 and 3 showed that supplemental P treatment to early pregnant female mice did not advance the onset of aggression. Experiment 4 showed that P treatment promoted the onset and elevated the incidence of aggression in virgin mice, but only in those females with intact ovaries. Experiment 5 showed that the aggressive behavior of P-stimulated virgin females was qualitatively and quantitatively different from that exhibited by pregnant mice in that the former exhibited fewer attacks and lunges than the latter. Finally, Experiment 6 showed that the removal of P from aggressive, P-stimulated virgins dramatically attenuated levels of the behavior. This contrasts sharply with the continued fighting behavior observed in late pregnant P-deficient mice. Thus, although P augments aggression in female mice it apparently is not a sufficient stimulus for producing pregnancy-like aggressive behavior.     

Neoplastic transformation of mouse mammary epithelial cells by deregulated myc expression.

A spontaneously immortalized, nontumorigenic mouse mammary epithelial cell line (MMEC) was transfected with an activated myc construct by electroporation. Constitutive expression of myc in MMEC resulted in anchorage independence in soft agar and tumorigenicity in nude mice. The myc-expressing MMEC showed higher saturation density, faster growth rate, and partial abrogation of serum-derived growth factor(s) requirement compared with parent MMEC. Epidermal growth factor or transforming growth factor alpha stimulated the anchorage-independent growth, but not the anchorage-dependent growth, of MMEC-myc cells. Type 1 transforming growth factor beta, on the other hand, inhibited both the anchorage-independent and anchorage-dependent growth of MMEC-myc cells. These results demonstrate that deregulated expression of myc results in neoplastic transformation iin mammary epithelial cells. Accompanying the transformation is altered sensitivity to polypeptide growth factors.     

Cellular stimuli for rejection of sarcoma I tumor allografts by BALB/c recipient mice

We have evaluated the role of passenger leukocytes in Sarcoma I (SaI) tumor allograft rejection by BALB/c recipient mice. SaI tumor cells grown in tissue culture expressed low levels of class I MHC antigens as determined by flow cytometry. Ascites-derived tumor cells contained additional cell populations of A/J host origin which expressed high levels of class I and class II alloantigens. Tissue culture-derived SaI grafts grew progressively or were rejected in delayed fashion by some BALB/c hosts. In contrast, ascites-derived tumor allografts were rejected rapidly by 100% of recipient mice. Passenger cells appear to be responsible for these striking differences in immunogenicity because the addition of allogeneic A/J splenocytes to the tissue culture form of SaI caused rapid rejection. When tissue culture- and ascites-derived tumors simultaneously were grafted on opposite shoulders of recipient mice, both tumors were rejected. These data indicate that passenger cells provide the primary stimulus for SaI tumor allograft rejection by BALB/c mice. The mechanism of SaI enhancement by anti-Ia antibodies may be analogous to the prolonged survival of endocrine grafts after depletion of Ia+ passenger leukocytes.     

A 3D in situ cell counter reveals that breast tumor cell (MDA‐MB‐231) proliferation rate is reduced by the collagen matrix density

Many cell types require the biophysical and biochemical cues within the 3D extracellular matrix (ECM) to exhibit their true physiologically relevant behavior. As a result, cell culture platforms have been evolving from traditional 2D petridish plates into 3D biomatrices, and there is a need for developing analytic tools to characterize 3D cell culture. The existing cell counting method, using a hemocytometer or coulter counter, requires that cells are suspended in fluids prior to counting. This poses a challenge for 3D cell culture as cells are embedded in a 3D biomatrix. We use a facile 3D cell counting method that overcomes this limitation and allows for in situ cell counting in a 3D cell culture using equipment that is commonly available in a biology lab. Using a breast tumor cell line, MDA‐MB‐231, as a model system, we demonstrated that MDA‐MB‐231 cells (1) grow slower within a 3D collagen matrix than on a 2D substrate for an extended growth time (a week) with a comparable, initial cell‐to‐cell distance, (2) their cell growth rate decreases with the increase of collagen concentration, showing a linear growth rate rather than an exponential growth rate. Further work using flow cytometry showed that the observed growth rate reduction was consistent with the retardation of the transition to S (synthesis) phase in the cell cycle. This work demonstrates the validity of the 3D cell counting method and the importance of cell–ECM interactions in cell proliferation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:990–996, 2015     

Expression of keratins and other cytoskeletal proteins in mouse mammary epithelium during the normal developmental cycle and primary culture

Mammary epithelium is composed of ductal, alveolar, and myoepithelial cells, and undergoes dramatic responses in growth, differentiation, and function to hormonal stimuli during the four stages of the mammary developmental cycle represented in virgin, pregnant, lactating, and involuting animals. To determine if progression of the epithelium through the cycle is accompanied by changes in cytoskeletal composition, particularly the keratins, the polypeptides in cytoskeletal extracts from BALB/c mouse mammary tissues were analyzed by one- and two-dimensional gel electrophoresis combined with immunoblots using polyclonal and monoclonal antikeratin antibodies. The major polypeptides in cytoskeletal fractions enriched in intermediate filaments included seven acidic and three basic components ranging in molecular weight from 40,000 to 90,000. Two major polypeptides of Mr 50,000 and 40,000, along with two minor components of Mr 57,000 and 55,000 were identified as keratins. The polypeptide profiles of mammary glands from virgin, pregnant, lactating, and involuting mice were very similar, indicating a remarkable stability of cytoskeletal composition during hormonal shifts and periods of minimal or maximal cell growth and differentiated function. The data also suggest that ductal and alveolar cells express the same set of cytoskeletal polypeptides, including keratins. Mammary cells grown in primary culture exhibited a loss or reduction in most of the basic polypeptides, a large increase in an acidic Mr 55,000 keratin, and the appearance of a prominent acidic polypeptide of Mr 46,000. The latter results demonstrate that keratin expression in mouse mammary epithelial cells is subject to regulation by certain environmental factors.