Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

Extracellular matrix and mouse mammary cell function: Comparison of substrata in culture

Summary Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveoluslike structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominately in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.     

In vitro culture of cryopreserved bovine mammary cells on collagen gels: synthesis and secretion of casein and lactoferrin

The preparation, cryopreservation, and culture on type I collagen gels of lactating bovine mammary cells with prolonged milk protein synthesis and secretion in vitro is described. Cryopreserved cells prepared as acinar fragments from either lactating or developing mammary glands attached to the collagen substratum within 24-48 hr after plating in serum and hormone supplemented medium. During continued culture in hormone-supplemented (insulin, cortisol, and prolactin) serum-free medium outgrowth of cells from the attached acinar fragments was observed beginning on day 2, with continued outgrowth to near confluence by day 6. Two morphologically distinct cell types were evident; initial outgrowth was by large polygonal cells that were subsequently overlain by spindle-shaped cells. Cells from both lactating and developing mammary glands sustained substantial milk protein secretion for at least 14 days in culture. Alpha S1-casein synthesis and secretion in cultures of lactating mammary cells was dependent on a critical minimum cell population density, below which alpha S1-casein was not secreted. In contrast, lactoferrin (LF) secretion into the medium increased linearly with the increase in cell population density. Cells cryopreserved up to 16 months secreted LF at levels comparable to fresh cultures of the same cells.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Morphology and lactose synthesis in tissue culture of mammary alveoli isolated from lactating mice

Summary Mammary epithelial cells from lactating mice synthesize and secrete lactose in culture and retain many features of their in vivo morphology if mammary glands are only partially dissociated to alveoli, rather than completely dissociated to single cells. After 5 d in culture lactose synthesis by alveoli cultured on floating collagen gels is 10 to 20 times higher than in cultures of single cells on floating collagen gels. Moreover, mammary alveoli in culture retain sensitivity to lactogenic hormones; the synthesis of lactose by alveoli depends on the continued presence of insulin and either hydrocortisone or prolactin. In addition, within alveoli the original juxtaposition of constituent epithelial cells is retained, and cells are cuboidal and have many microvilli and fat droplets. In contrast, alveoli on attached gels flatten and lose their secretory morphology. These results indicate that the shape of the cells, presence of lactogenic hormones, and maintenance of epithelial:epithelial cell contacts are required for maintenance of mammary epithelial cell differentiation in culture. This research was supported by Grants CA-16392 and AG-02909 from the National Institutes of Health and Institutional Grant IN 119 from the American Cancer Society.     

Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce gamma-casein, a member of the casein gene family, only if they are maintained on floating collagen gels (Emerman, J.T., and D.R. Pitelka, 1977, In Vitro, 13:316-328). In this paper we show: (a) Cells on floating collagen gels secrete not only gamma-casein but also alpha 1-, alpha 2-, and beta-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross- linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. alpha-Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.     

Rapid and reversible regulation of collagen XII expression by changes in tensile stress

We studied the expression of the fibril-associated collagen XII by fibroblasts cultured on attached (stretched) or floating (relaxed) collagen I gels. Accumulation of collagen XII in the medium as determined by semiquantitative immunoblotting was 8-16 times higher under stretched compared to relaxed conditions. Northern blot experiments showed that tensile stress controls collagen XII expression at the mRNA level. Tenascin-C mRNA levels were also influenced, whereas relative amounts of fibronectin and matrix metalloproteinase-2 mRNA were barely affected. The response to a change in tensile stress is rapid, since de novo biosynthesis of collagen XII was fully down-regulated 12 h after relaxation of a stretched culture. To demonstrate that the effect is also reversible, we mounted collagen gels with attached cells to movable polyethylene plugs. The cultures were relaxed or stretched at intervals of 24 and 48 h, and media samples were analyzed every 24 h. By ELISA, the amount of collagen XII secreted into the medium was found to increase or decrease in accordance with the tensile stress applied. This is evidence that the mechanical stimulus per se, rather than an indirect secondary effect, was responsible for the observed changes in collagen XII production.     

Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and α-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and alpha-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the cent of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures. These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins.     

Synthesis of novel calcium-dependent proteins associated with mammary epithelial cell migration and differentiation

A class of proteins from mouse mammary epithelial cells has been isolated which, like the calcium-binding protein calmodulin (CaM), binds to phenothiazine in a calcium-dependent manner. These proteins do not bind to phenothiazine through binding to CaM; we infer that they are calcium-binding proteins, and that they may be related to the similarly isolated 'calcimedins' of Moore, P D & Dedman, J, J biol chem 257 (1982) 9663 [8]. In primary cultures of mouse mammary cells on collagen gels, synthesis of certain of these proteins is associated with the spreading of cells to form monolayers; failure of cells to spread and differentiate, through omission of serum from culture medium, results in the inhibition of calcium-binding protein synthesis, with the exception of CaM and a 15 kD species. The CaM/15 kD pair are prominent during all phases of culture, and are secreted during the secretory differentiation phase of culture (floating gels). We propose that these calcium-binding proteins play a specific role in the motility of mammary epithelial cells and that they may also be involved in mammary secretory differentiation.     

Lactating goat mammary gland cells in culture.

1. Isolated mammary gland cells were cultured embedded in collagen gels or as monolayers on floating collagen gels. Under these conditions the cells were able to grow for at least 6 weeks during five passages. Growth was sustained in M199/F12 (1:1) supplemented with insulin, hydrocortisone, epidermal growth factor, tri-iodothyronine, estradiol and bovine serum albumin. 2. The cells secreted lactose into the medium in significant amounts throughout the culture period. 3. Prolactin had a slightly stimulatory effect as had fetal bovine serum on growth and protein synthesis, but none of these factors were obligatory in this respect. Insulin-like growth factor I (Somatomedin C) could replace high concentrations of insulin whereas bovine growth hormone had no detectable effect. 4. Depending on the hormone content of the medium and the age of the culture, different labelling patterns of the arachidonic acid-containing phospholipids were observed. The effect of prolactin on phosphatidyl inositol and arachidonic acid metabolism was studied.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.     

The effects of a collagenous extracellular matrix on fibroblast membrane organization : An ESR spin label study

Human skin fibroblasts, both in suspension and cultured within a three-dimensional collagen matrix have been examined by electron spin resonance ESR using the probe 5-doxyl stearic acid. The order of the plasma membrane was found to be strongly influenced by the collagen matrix, being greater for cells within the collagen gel than in suspension. The collagen cultures used in this study were either left attached to the walls of the plastic culture dish (‘attached’ gels) or dislodged and allowed to float freely in the culture medium (‘floating’ gels). Membrane order increased with time in attached gels, reaching a steady value after 2–3 h. A further increase in order was observed when floating gels were prepared 24 h later. Cell morphology within the collagen gel culture was observed to vary considerably, with time and mode of culture. Increased order, over that observed in suspension, was also found for cells attached to other substrata. The data indicate that the increase in membrane order observed in cells embedded within a three-dimensional collagen gel matrix compared with cells in suspension does not correlate with a particular cell morphology in the gel, but rather appears to result from the establishment of adhesive interactions with the surrounding collagen fibres.     

Xanthine oxidase,an indicator of secretory differentiation in mammary cells

Elevated levels of xanthine oxidase were found in (1) lactating mouse mammary glands, compared with virgin and midpregnant glands; and (2) primary mouse mammary cells cultured on floating collagen gels, compared with non-secretory cells on attached gels. In primary culture, increase in xanthine oxidase activity above a basal level coincided with secretory activity as measured by casein production; intracellular levels of casein and xanthine oxidase showed a high degree of correspondence. It is suggested that xanthine oxidase levels can be used as an indicator of in vivo and in vitro secretory differentiation in mammary epithelial cells.     

Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture

Epithelial cells dissociated from mammary glands of midpregnant mice and cultured with lactogenic hormones on plastic or collagen gel substrates have been shown to vary in their extent of differentiation, as identified by the presence of secretory organelles and accumulation and secretion of casein. Morphological and biochemical differentiation was obtained on floating collagen gels. At least four unique factors provided by the floating collagen gel substrates are not found on plastic substrates: access of nutrients to basolateral cell surfaces, close proximity of cells to the medium surface and gas phase, interaction of epithelial cells with stromal elements, and substrate flexibility permitting cell shape change. In this study, we have attempted to assess the relative contributions of these factors in the ultrastructural differentiation of mammary cells in culture. None of these factors alone is responsible for the differentiation achieved when all are present. The novel aspect of this research is the identification of the cells' apparent requirements for basolateral access to nutrients and for freedom to assume a preferred shape in order to achieve differentiation.     

Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.