Basal Lamina and Tissue Recognition in Malignant Mammary Tumors1

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[CANCER RESEARCH 40. 1600-1611. May 1980] 0008-5472/80/004-OOOOS02.00 Basal Lamina and Tissue Recognition in Malignant Mammary Tumors1

Dorothy R. Pitelka, Susan T. Hamamoto, and Barbara N. Taggart

Department of Zoology and Cancer Research Laboratory. University of California. Berkeley, California 94720

ABSTRACT surfaces (4, 9, 10, 34). In normal adult epithelium, however, it forms a continuous boundary between the entire epithelial The basal lamina is important in normal epithelial morpho tissue and other cell types, and it is respected as a barrier by genesis and provides a barrier between normal adult epithelium both the epithelial cells and all others except macrophages and and connective tissue that is not penetrated except by cells of migrating leukocytes, which can penetrate it without evident hematogenous origin. Invasion and metastasis by malignant damage. epithelium therefore are thought to require alterations in basal It has long been recognized that either breaching or absence lamina structure or disposition. We have investigated this ques of tumor basal lamina may be a necessary factor in certain tion in primary mammary tumors and in their spontaneous phases of epithelial malignancy, presumptively when carcino pulmonary mÃ©tastasesin mice. The commonest structural ab mas invade adjacent tissues, and certainly when cells are normality of the tumor basal lamina in both sites is hypertrophy released from the parent tumor into the lymph or blood stream. in the form of folding, multilayering, or irregular thickening, Basal lamina of the blood vessel is also involved in the latter without observable failure of the barrier function. Interruptions case, and endothelial basal lamina must again be breached in the continuity of the basal lamina are extremely rare and do when a blood-borne tumor embolus invades extravascular not appear to be significant sites of emigration of malignant space in a secondary site. Finally, if a primary or metastatic cells. Mammary tumor epithelium thus maintains an effective carcinoma invades normal epithelium, the basal lamina bound basal lamina barrier while invading nonepithelial tissues of ary of the invaded tissue must also be violated. Explanations of either body wall or lung. Even intraarteriolar metastic foci these various events could include the failure of motile tumor secrete basal lamina between themselves and the arteriolar cells to respect intact basal lamina, degradation of basal lamina endothelium, showing that the tumor cells recognize the latter by appropriate extracellular enzymes, or abnormalities in the as nonepithelial. Extravascular mÃ©tastasesaggressively invade quantity or quality of the epithelial basal lamina secreted by the pulmonary alveolar air spaces. Here the cells of the two tumor cells. Evidence supporting each of these mechanisms is epithelia share the same basal lamina and collaborate to form reported in the literature, to be discussed later, but proof of luminal tight junctions, showing that the tumor recognizes the their involvement in spontaneous invasion and metastasis by pulmonary tissue as epithelial but not as nonmammary. Some autochthonous tumors is lacking. evidence suggests that a merger of the two different epithelia We have examined primary mammary tumors and their spon may involve local fusion of the tumor basal lamina with that of taneous pulmonary mÃ©tastases in mice overtly infected with the lung, followed by degradation of the lamina in the fused the MTV3 to determine the ultrastructural disposition of mam zone only; similar fusion of basal lamina between adjacent mary epithelial basal lamina in both sites and of pulmonary nests within a mammary tumor is also suggested. We conclude epithelial and endothelial basal lamina in invaded lungs. We will that retention of the basal lamina reinforces the tendency of present evidence that, whereas morphological discontinuity of tumor epithelium to grow as cords of cells in contact, while the tumor basal lamina is rare in these tissues and probably qualitative changes in the lamina contribute to the mode in not a major factor in invasion, other abnormal properties of the which the tumor invades epithelial tissues. basal lamina in both primary and metastatic tumors may influ ence their growth pattern and invasive behavior. Our work INTRODUCTION confirms and extends the observations of Brooks (6), who The epithelial basal lamina2 is a thin layer of material, mainly described the ultrastructure of metastatic mammary tumors in the mouse lung, including the invasion of pulmonary epithelium. peptides and carbohydrates (27), secreted by epithelial tissues Part of the work to be presented here has been published in at their interface with the connective tissue matrix. Similar abstract form (39). We discuss in another paper (42) the laminae are formed by mesothelium and endothelium, and an histological organization and the distribution of epithelial cell individual basal lamina surrounds each muscle cell, adipose junctions in these primary and metastatic adenocarcinomas. cell, or Schwann cell. The basal lamina is important in epithelial- The basal lamina in the normal mammary gland, to be reviewed mesenchymal morphogenetic interactions (2, 24), in regener below for comparative purposes, has been described else ation of damaged tissue (30, 48, 51) and, at least in some where (41). tissues, in filtration of macromolecules (14). Transitory inter ruptions in its continuity apparently contribute to morphoge netic changes in several fetal or neonatal epithelial tissues by MATERIALS AND METHODS allowing direct contact of epithelial and mesenchymal cell The tissues examined and methods used for this study are 1 Supported by NIH Grant CA05388. described in detail in a companion paper (42). All tissues were ' Also called basement membrane or basement lamina Because the histolo- from female Crgl mice infected with MTV. Spontaneous pul glst's basement membrane includes the basal lamina plus an adjoining cell-free monary mÃ©tastases were identified and removed at autopsy zone containing collagen fibers and other materials, the common and more restrictive term 'basal lamina' is used here. Received September 24, 1979; accepted February 11. 1980. ;l The abbreviation used is: MTV, mammary tumor virus.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. Basal Lamina in Malignant Mammary Tumors from 69 mammary tumor-bearing females of the C3H, C3Hf, still confined to the epithelial compartment, demonstrating that BALB/cfC3H, BALB/cNIV, A, GR, and Rill strains. Primary the physical integrity of the basal lamina bounding the cyst was tumor samples were taken from some of the animals with not destroyed during cytolysis. mÃ©tastases and from similar females without mÃ©tastasesde The most frequent structural deviation of the basal lamina in tected at autopsy. All mÃ©tastases were confirmed by light both primary and metastatic tumors is hypertrophy. It takes the microscope examination of 1- to 2-jum sections of samples form of extensive folding (Fig. 4), multiple layering (Fig. 5), or fixed in glutaraldehyde-paraformaldehyde, postfixed in osmium irregularly increased thickness (Fig. 10). Any of these variants tetroxide, and embedded in Epon. Twenty-two of the samples can be found at the interfaces between mammary epithelium were further studied by electron microscopy of thin sections and stroma within an established tumor, between an invasive stained with uranyl acetate and lead citrate. In addition, large primary tumor and connective tissue or muscle of the body pieces from 6 lungs were embedded in paraffin and serially wall, and between metastatic mammary epithelium and tissues sectioned for histological study. Forty-four primary tumors were of the lung. The presence or absence of peripheral cells examined in thick Epon section, and 20 of these were examined identifiable as myoepithelial has no observable influence on in thin section. the morphology of the tumor basal lamina. A striking form of To see whether transplanted tumors in relatively early pas hypertrophy seen by Brooks (6) in pulmonary mÃ©tastasesis the sages displayed altered morphological or malignant properties, development of whorls of evenly spaced layers of basal lamina we also examined various generations of 3 C3H transplantable displaying a distinct periodic structure. We found numerous tumor lines, MT3, MT4, and MT6, now in their 13th, 18th, and such whorls in one primary tumor but have not encountered 8th passages, respectively. them elsewhere. Samples of both primary and metastatic tumors for this study Actual interruptions in the continuity of the basal lamina, were generally taken from firm, nonnecrotic areas near the leaving epithelial cell surfaces exposed to interstitial matrix or periphery, including for each tumor some samples selected to cells, are extremely rare in the primary tumors and spontaneous include the interface between tumor and surrounding tissues. mÃ©tastasesthat we have examined. Most of those identified display no other abnormality than the localized absence of the RESULTS lamina. In some instances, small processes either from the exposed epithelial surface or from nearby fibroblasts extend Normal Mammary Gland. A continuous basal lamina sepa through the gap (see Fig. 14), but we have never found per rates the mammary epithelium from the cells and fibers of the suasive evidence of migration of epithelial cells into the con stroma. It appears in cross-section as a finely filamentous nective tissue. Since both stromal and epithelial cells may be dense layer, about 30 nm in average thickness, and a less so undifferentiated as to lack diagnostic ultrastructural fea dense layer of similar depth between it and the basal plasma- tures when seen in section, the occasional egress of single lemma of the mammary cells (Fig. 1). In major ducts, most of epithelial cells could escape our scrutiny; frequent or large- the cells abutting the basal lamina are myoepithelial; in small scale infiltration of surrounding tissue through breaks in the ducts and alveoli, the myoepithelial layer is discontinuous, and basal lamina, however, almost certainly could not. luminal epithelial cells also rest on the basal lamina. Myoepi More pronounced than structural deviations of the basal thelial cells frequently, and luminal epithelial cells occasionally, lamina are abnormalities in its disposition, especially at sites of are attached to the basal lamina and external connective tissue confrontation of mammary and pulmonary tissues. The sites elements by hemidesmosomes (Fig. 1, inset). During lactation, that appear most significant to the invasive behavior of the when the gland is packed with secretory alveoli, the distance mammary tumor are its interfaces with pulmonary arteriolar between external surfaces of the basal laminae of adjacent endothelium and with pulmonary alveolar epithelium. alveoli, or of alveoli and adjacent capillaries, may be as little as Intravascular Metastatic Foci. Since this study was con 50 nm (Fig. 1); separation of the 2 surfaces appears always to cerned with spontaneous metastasis, all metastatic tumors be maintained, however. During postlactational regression of examined necessarily had been established long enough to secretory lobules, the alveolar basal lamina persists as a pro reach a size detectable at autopsy, with the exception of a fusely folded layer around surviving terminal ducts (40). Thick small focus found in a random lung sample from the host of a halos of pleated material are still evident 1 month after weaning. transplanted tumor (Fig. 6). Within the lung tissues excised Hyperplastic alveolar nodules, the most common preneo- with macroscopic mÃ©tastases, however, small foci often were plastic lesion in these mice, resemble normal mammary tissue present at some distance from any larger tumor. In virtually all in the structure and arrangement of basal lamina. such cases examined in either serial histological or Epon thick Primary and Metastatic Tumors. Since primary and estab sections, the smallest foci observed were within the arteriole lished metastatic tumors do not differ appreciably in histological accompanying a small bronchiole (Figs. 6 and 8). Intraarteriolar and ultrastructural organization (42), the 2 will be considered tumor nests were also present in many large, established together in this section. Tumor epithelium is separated from mÃ©tastases(Fig. 7). Small arterioles often were packed with connective tissue by a basal lamina that often is thin and healthy-looking tumor cells, distending the vessel wall (Fig. 6). discrete (Fig. 2), like that of normal or hyperplastic mammary Rupture of the muscularis and elastic layers of the wall had tissues. occurred in many of these vessels, allowing the tumor to Advanced necrosis often brings about disruption of the basal expand, by spilling or proliferation or both, out into the pul lamina along with adjacent tissues. Occasionally, however, monary connective tissue (Figs. 6 to 8). More or less extensive localized cell lysis leaves patches of naked but intact lamina. proliferation of malignant cells through the arterial system took Such an area from a small cystic structure is illustrated in Fig. place in some lungs (Fig. 9). In an extreme case, serial histo 3. MTV particles and cell debris in the lumen of the cyst are logical sections revealed tumor growth throughout most of the

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. D. R. Pitelka et al. arteries of an entire lung lobe, with little extravascular invasion. internal lumina and MTV particles, abutting on a pulmonary A tumor embolus growing within a vessel must confront the alveolar air space otherwise lined by intact types I and II free apical surface of the vascular endothelium, a surface that pneumonocytes. The basal lamina of the tumor in all such normal epithelium never sees. The endothelium in some of cases is continuous with that of the normal pulmonary epithe these areas is destroyed, but as frequently it persists (Figs. 10 lium. and 11 ) except at the site of rupture of the vessel wall. When A possible example of an early stage in air space invasion is it does, tumor epithelium and vascular endothelium are invari illustrated in Fig. 17. A blunt cytoplasmic process from one of ably, in our experience, separated by a basal lamina, often the cells at the periphery of a large tumor mass extends toward hypertrophied (Fig. 10). Where the endothelium has disap a distorted (presumably by tumor expansion) pulmonary alveo peared, a single or hypertrophied basal lamina usually is pres lus. At this level, the basal lamina enclosing the tumor is ent between the tumor and the elastic layer of the vessel wall. deflected to follow the contour of the pulmonary alveolus. In Where the tumor extends through the ruptured wall, its extra- the gateway (Fig. 17, arrows) between the 2 tissues, the basal vascular portion as a rule is separated from the lung connective surface of a type I pneumonocyte, which invariably rests on a tissue by basal lamina, like other tumor nests within the lung. basal lamina in normal circumstances, is associated only with In 2 of the arteriolar foci that we examined, the tumor basal a fragmentary remnant of one (Fig. 17, inset). The picture lamina was locally interrupted just at the site of wall rupture, suggests that local back-to-back fusion of tumor and pulmo where the endothelium was absent and the mammary tissue nary basal laminae had occurred, followed by dissolution or faced the elastic and collagenous fibers of the wall. rearrangement within the fusion zone to create an open path The smallest tumor focus that we found (Fig. 8), taken from between the 2 epithelial compartments and a single, continuous a lung with multiple mÃ©tastases, differed from all others in basal lamina enclosing them both. If this is correct, breaching displaying frequent discontinuities of its basal lamina, both of the fused basal lamina need not involve any break in conti within and outside of the arteriole. Only a few vestiges of nuity of the barrier between either of the epithelia and the arteriolar endothelium were present, and the space between pulmonary connective tissue or any migration of tumor cells tumor epithelium and the internal elastic layer of the wall was through nonepithelial tissue, and our inability to find evidence occupied by collagen fibers. Basal lamina was present only of such events reflects the real situation. intermittently at the tumor-collagen interface and was often The appearance of degenerative changes, including the deg poorly defined (Figs. 12 and 13). Occasional contact between radation of surfactant granules, in pulmonary epithelial cells a tumor cell surface and a smooth muscle cell process extend close to or in contact with tumor cells has already been de ing between elastic fibers was evident. The basal lamina of the scribed by Brooks (6). We assume that most such cells even extravascular part of the tumor (Fig. 8) was similarly incom tually die, since identifiable pneumonocytes within tumor plete, and contact between tumor and lung cells was frequent masses are found mainly near the periphery. (Fig. 14). Nowhere, however, did we identify more than a small Fusion of Mammary Basal Lamina within a Tumor. In pseudopod extending into the stromal space from the naked addition to the continuous basal lamina boundary between tumor surface. lr>this and other intravascular tumors examined, mammary epithelium and adjacent nonepithelial tissues, round true epithelial lumina and tight junctions were present, and or irregular spaces lined by a single or multiple basal lamina other junctions often linked cells not fronting on a lumen; in are common within masses of tumor epithelial cells (Figs. 18 other words, the internal organization of the small foci resem and 19). Inevitably, some such configurations are explainable bled that of established tumors. as sections cut tangential to an irregular periphery, depressions Basal Lamina Fusion and Lung Invasion. A phenomenon in the lamina-coated surface then appearing as isolated already reported by Brooks (6) in metastatic tumors is the spaces. However, the isolated pockets are too frequent and invasion of pulmonary alveolar air spaces by mammary cells, too often distributed at all depths within an epithelial mass to the cells of the 2 epithelia then sharing the same basal lamina. admit such an explanation for all cases. Cells associated with This relationship can be found somewhere in nearly every large the pockets sometimes resemble myoepithelial cells in their metastatic tumor in our material; pulmonary and mammary cells abundant filament content. The smallest pockets typically are face the same lumen and collaborate to form tight junctions irregular in shape, following the contours of the enclosing cell (Figs. 15 and 16). Since the normal pulmonary epithelium and surfaces. Larger ones occasionally become spherical as the invading mammary epithelium are both provided with their though engorged, and they may be filled with lamellar or own basal laminae, contact between the 2 cell types should unorganized basal lamina-like material. require breaching of both laminae. To search for relevant That such pockets may originate from incomplete fusion of evidence, we routinely traced the basal lamina at tumor-lung the basal lamina of adjacent tumor nests is suggested by tissue interfaces while viewing sections in the electron microscope. configurations such as that illustrated in Fig. 20, a section from Any area that was particularly interesting, or too complex to a small, well-differentiated tumor that arose in the outgrowth of follow on the viewing screen, was recorded as a montage of a transplanted hyperplastic alveolar nodule. The periphery of micrographs; the interdigitations of confronting tissues then this neoplastic alveolar structure is in intermittent back-to-back could be followed accurately over considerable distances and contact with that of other, similar structures on at least 3 sides. the identities of both tissues could be firmly established. The interstitial space is divided into a series of segments, lined An exhaustive search of sections and montages has yielded by multiple basal lamina and separated by bridging epithelial no good evidence of migration of tumor cells through either or myoepithelial cells. As noted earlier, peripheral fusion of tumor or pulmonary basal lamina or through intervening inter closely packed alveoli or ductules does not occur in normal stitial space. Instead, a frequent occurrence near the periphery mammary epithelium. The apparently isolated pockets in Fig. of metastatic tumors is a mammary tumor mass, containing 20 may in fact have been continuous with larger interstitial

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. Basal Lamina in Malignant Mammary Tumors spaces; however, expansion of the areas of fusion would result in mammary but not most other adult basal laminae (11)]. in sequestration of the pockets. Although there appears to be considerable similarity among all Transplanted Tumors. Tumors of lines MT3 and MT6 resem basal laminae, those of different epithelia differ significantly in ble the less differentiated primary tumors in histological and composition (27). ultrastructural organization (42). They also retain a single or Whatever the composition of hypertrophied basal lamina, its hypertrophied basal lamina, whether invading the s.c. connec frequency in a variety of carcinomas suggests that the normal tive tissue, the mammary fat pad, or the body wall. A small habit of basal lamina production at every epithelial-stromal pulmonary metastasis found in a fourth-generation MT3 host interface is often exaggerated rather than lost by malignant did not differ from mÃ©tastasesof primary tumors (Fig. 6). epithelial cells. The hypertrophied lamina appears to maintain The anaplastic tumor line MT4, in which we have not seen its barrier function, inasmuch as transit of cells through it has typical tumor lumina (42), does not form identifiable basal not been reported. Thus, as long as it persists, tumor growth lamina. Both within the tumor and between it and endothelial and invasion must take place by extension or budding of or stromal cells, intercellular spaces contain either a homoge cohesive cell aggregates rather than by single-cell infiltration. neous material of high density or unorganized, flocculent ma Basal Lamina Discontinuities. Ultrastructural studies of hu terial unlike hypertrophied basal lamina. Interdigitation of tumor man breast tumors have revealed occasional interruptions in cells and host fibroblasts occurs sporadically at the tumor the basal lamina in benign lesions and more frequent and periphery, but invasiveness is, if anything, less pronounced extensive discontinuities or complete absence of basal lamina than in the other transplant lines or primary tumors. in tumors of increasing grades of malignancy (1,7,16, 20, 21, 22, 37, 45, 54). Basal lamina interruptions have also been DISCUSSION pictured in chemically induced mammary tumors of rats (17) and mice (46) and in other malignant or benign epithelial tumors We have presented evidence that both primary mammary of humans and experimental animals (e.g., Refs. 5, 18, 25, and tumors and their spontaneous pulmonary mÃ©tastasesin MTV- 26). infected mice typically maintain an uninterrupted basal lamina Most authors cited consider that these basal lamina lesions barrier between epithelium and surrounding nonepithelial tis provide pathways for active emigration of malignant cells, sue, even while invading the body wall or the lung. Deviations which then can disperse through the surrounding tissue. Such in morphology or disposition of the basal lamina were frequent, a mechanism could account for the histological pattern of however. These included: hypertrophy of basal lamina or mor diffuse infiltrative invasion familiar to pathologists. Several ul- phologically similar material at the interface between tumor trastructural studies of infiltrating mammary carcinomas (1, 7, epithelium and connective tissue; production of single or hy 20) have demonstrated epithelial cells in the stroma, alone or pertrophied basal lamina at a novel site, the interface between in single file, without any basal lamina, whereas similar isolated tumor epithelium and the luminal surface of arteriolar endothe- epithelial cells with an enclosing lamina have not been reported. lium; continuity of the basal laminae of tumor epithelium and Direct evidence for involvement of epithelial cell locomotion in normal pulmonary alveolar epithelium; and intraepithelial pock invasion or metastasis is scant, however (52). It is possible that ets lined by basal lamina. Discontinuities of the lamina occurred protracted or extensive absence of the basal lamina removes but, except in a single small metastatic focus, they were rare certain constraints on growth patterns, permitting cells in some and small. In view of the role of the basal lamina in morpho tissue environments to grow out as naked rows or sheets (seen genesis and in maintaining the exclusiveness of epithelial tis in cross-section as singles or rows, respectively). However, sues, it is to be expected that each of these abnormal proper such freedom is not requisite for epithelial cancer; the highly ties may influence the course of malignant epithelial growth. malignant mammary carcinoma of MTV-infected mice can in Hypertrophy. Most ultrastructural studies of primary breast vade surrounding tissues aggressively while continuing to pro and other epithelial cancers have reported some degree of duce and maintain a continuous basal lamina. Even in our folding, multilayering, or thickening of the basal lamina (1, 12, exceptional metastatic focus with multiple discontinuities in the 16, 21, 22, 25, 29, 35), especially in early stages. In some lamina, egress of epithelial cells was not observed. cases, this tendency becomes extreme, with large interstitial We do not know whether discontinuity of the basal lamina in or intratumor spaces filled with diffuse or organized basal the cases cited above results from defects in the ability of the lamina-like material (e.g., Ref. 6). More moderate thickening, tumor to secrete it or from the activity of lytic substances in the duplication, or folding of the basal lamina of various epithelia surrounding tissues. Production of collagenase by various tu or endothelia is commonly associated with aging, atrophy, and mors has been reported, and a transplantable fibrosarcoma certain diseases, especially diabetes (19, 38, 43). The folded has recently been shown to produce an enzyme specific for lamina of mammary tumors is remarkably similar to that of type IV collagen (31 ). However, we have seen that basal lamina regressed normal secretory tissues in the mouse (40). Marti may survive local cytolysis. nez-Hernandez eia/. (33) concluded that alveolar basal lamina One malignant event that undeniably depends on basal lam was being degraded after weaning, because electron micro ina lesions is the release of potential metastatic emboli from a scope immunolabeling of the structure was diffuse even where parent carcinoma into blood or lymph. Cells are released more micrographs showed a folded dense layer; the same technique or less liberally by most cancers, whether they metastasize or yielded discrete, intense labeling of normal basal lamina before not (23, 44). We have found no solid evidence, in our own regression. This suggests the possibility that the folded or study or in the literature, of the nature of this event. The hypertrophied lamina may be deficient in some antigenic con explanation that appears most promising is enzymatic destruc stituents. Normal basal lamina contains type IV collagen and tion or mechanical tearing of the basal lamina in a zone of other glycoproteins (27, 28) and glycosaminoglycans [present advancing necrosis (53). Patchy cytolysis, or a fortuitous dis-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. D. R. Pitelka et al. tribution of tumor cells not linked by junctions, could then free Our evidence leads us to postulate that spontaneously me- viable single cells or, more probably, clusters of linked cells tastasizing mouse mammary carcinoma acquires access to the into fluid that ultimately would drain out of the tumor (blood if pulmonary epithelium when the basal lamina at the advancing walls of vessels are locally affected by necrosis, or lymph). edge of the tumor meets, locally fuses with, and becomes Gullino (23) has demonstrated significant convective currents continuous with the lamina of the pulmonary alveolar epithe of interstitial fluid in transplanted rat mammary tumors. lium. This appears to be a selective fusion, since we have not Intravascular MÃ©tastases.In published studies of the lodg found any case of fusion with the equally accessible basal ment and growth of blood-borne tumor emboli in laboratory laminae of pulmonary capillaries, pleural mesothelium, or vas animals, either i.v. injection of cell suspensions or spontaneous cular smooth muscle cells. There is also reason to think that metastasis by transplanted tumors have been used in order to the basal laminae of neighboring cords of tumor cells may fuse. identify and time early events. It is reported that clumped cells It is not uncommon in mice for the basal laminae of normal are more successful than single cells in establishing metastatic pulmonary alveolar epithelium and closely adjacent capillary foci (15, 47) and that capillaries and arterioles are the usual endothelium to fuse, back to back, and a single, thick basal sites of arrest (3, 36). Most investigators consider that invasion lamina separates endothelium and epithelium in the glomerulus. of extravascular space occurs next, when the tumor cells either In these cases, the fused lamina remains intact as a distinct destroy endothelium and its basal lamina or actively migrate boundary between the 2 tissues, respected by both. Back-to- and penetrate both layers (8, 32). Baserga and Saffioti (3), back fusion followed by dissolution of the fused area (as using a transplanted bladder carcinoma, and Nakamura et al. suggested by our observations) and actual merging of 2 differ (36), with injected ascites hepatoma, also observed intravas- ent epithelia, now enclosed in a single continuous lamina [as cular proliferation of tumor cells, leading to swelling of the demonstrated by both Brooks (6) and ourselves], is a different vessel and rupture of its wall. We found evidence of this phenomenon. process in enough affected lungs to suggest that it is a common Almost all of the epithelia of the body, and therefore their phenomenon in the initiation of spontaneous mÃ©tastases of basal laminae, are ultimately continuous with one another, but mouse mammary cancer. However, the same phenomenon internal contact between the epithelia of different organs is might occur also as a late event if retrograde growth of tumor effectively prevented by connective tissue sheaths. Close pack through the the arterial system leads to the distention and ing of identical or related epithelial units within an organ (e.g., rupture of other arterioles. We plan experiments with primary pancreas, kidney, gastric mucosa, mammary gland), however, mammary tumor cells injected into the tail vein to identify the often brings their basal surfaces close enough together so that critical early steps in lung colonization. occasional contact of basal laminae must be feasible. Back-to- Previous investigators examining intravascular tumor foci back fusion and merger of units under these circumstances have not reported basal lamina formation at this site. The could play havoc with tissue organization and activity, and the secretion of basal lamina by mammary tumor cells within the barrier function of the basal lamina presumably has evolved pulmonary arteriole (regardless of whether it is an early event) partly as a protection against just such an occurrence. Availa shows that the malignant epithelium recognizes the apical ble information on basal lamina composition is not yet adequate surface of the arteriolar endothelium as foreign, in the absence to explain the physicochemical basis of its barrier role. of either connective tissue matrix or an endothelial basal lamina Our observations and the published data on selective inva at this surface. The presence of the tumor lamina evidently sion of epithelial tissues imply that malignant epithelium rec precludes contact between the 2 cell types and prohibits any ognizes normal epithelium generically but fails to make specific tendency that might otherwise exist for the tumor cells to distinctions among different types; it indiscriminately forms penetrate the intact endothelium. Conceivably, it may foster epithelial junctions with nonhomologous cells and anneals their proliferation of cells in the tumor. basal laminae with its own. Cells of the normal epithelium are Basal Lamina and Tissue Recognition. The preferential displaced and finally destroyed. An important result of this infiltration of pulmonary epithelium by metastatic mammary sequence of events may be that the proliferating tumor gains tumor cells, first observed by Brooks (6), is not an isolated ready access to the vascular network of the resident epithelium. phenomenon. Dingemans (13) found that cells from a trans Furthermore, epithelial lumina provide unencumbered space planted mouse mammary carcinoma, after injection into mes- for tumor growth. It appears likely that qualitative abnormalities enteric veins, invaded the liver via the sinusoids and grew in the basal lamina secreted by the malignant epithelium ac among the normal liver cells, forming tight junctions with them count for an important aspect of invasive behavior if its com at canalicular lumina. We have observed the same relationship position is sufficiently altered to allow merging with laminae of when mammary tumor cells injected i.p. invade the liver." Tickle other epithelia while remaining incompatible with those of me- et al. (49, 50) found that cells of 3 mammalian carcinomas, sothelial, endothelial, or other cell types. including human breast tumors, did not invade the mesen- We conclude: (a) that focal or general loss of the basal chyme when implanted in the surface of developing chick wing lamina of mammary carcinomas is not a necessary prelude to bud but positioned themselves in the chick ectoderm, often on invasive growth, nor is tumor cell emigration an inevitable its basal lamina, and formed desmosomes with ectodermal consequence when it occurs; (b) that normal or hypertrophied cells. Sarcoma, neuroblastoma, trophoblast, and cultured fi- basal lamina, together with the cohesiveness of cells linked by broblastic cell lines moved into the mesenchyme or, in some specialized junctions, limits the pattern of tumor growth but not cases, both layers. the growth itself; and (c) that basal lamina may be important in the initial recognition of resident epithelium by metastatic mam mary cells and the preferential infiltration of epithelial compart ' Unpublished observations. ments that follows.

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ACKNOWLEDGMENTS carcinoma of the thyroid. Cancer (Phila.), 35. 1280-1292. 1975. 26. Kaye. G. I.. Fenoglio, C. M., Pascal, R. R., and Lane, N. Comparative electron microscopic features of normal, hyperplastic, and adenomatous The authors are grateful for the technical assistance of John R. Underhill and human colonie epithelium. Variations in cellular structure relative to the Joan G. Duafala. process of epithelial differentiation. Gastroenterology. 64: 926-945, 1973. 27. Kefalides, N. A. Current status of chemistry and structure of basement membrane. In: N. A. Kefalides (ed.), Biology and Chemistry of Basement REFERENCES Membranes, pp. 215-228. New York: Academic Press. Inc., 1978 28. Kidwell, W. R., and Wicha, M. S. Basement membrane collagen synthesis 1. Ahmed, A. Atlas of the Ultrastructure of Human Breast Diseases. Edinburgh: and its recognition by rat mammary ducts and alveoli in culture. Proc Am Churchill Livingstone Ltd. 1978. Assoc. Cancer. Res., 20. 147, 1979. 2. Banerjee, S. 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Cancer Res., 20. 235, 1979. 5. Bockman, D. E., Black. O., Jr., Mills, L. R.. Mainz, D. L., and Webster, P. D. 32. Ludatscher, R. M., Luse. S. A., and Suntzeff, V. An electron microscopic Fine structure of pancreatic adenocarcinoma induced in rats by 7,12-di- study of pulmonary tumor emboli from transplantable Morris Hepatoma methylbenz[a]anthracene. J. Nati. Cancer Inst.. 57. 931-936. 1976. 5123. Cancer Res., 27. 1939-1952. 1967. 6. Brooks, R. E. Mouse mammary tumor mÃ©tastases in lung: an electron 33. Martinez-Hernandez, A.. Fink, L. M., and Pierce. G. B. Removal of basement microscopic study. Cancer Res., 30. 2156-2165, 1970. membrane in the involuting breast. Lab. Invest.. 34: 455-462. 1976. 7. Carter, D., Yardley. J. H.. and Shelley, W. M. Lobular carcinoma of the 34. Mathan, M., Hermos, J. A., and Trier, J. S. Structural features of the breast. 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Electronmicroscopic studies on extravasation of tumor mammaires primaires d'embryons de souris mÃ¢leset femelles au stade de cells and early foci of hematogenous mÃ©tastases Gann Monogr., 20. 57- la differentiation sexuelle. C. R. Acad. Sci. [D] (Paris), 280 1821-1824, 72, 1977. 1975. 37. Ozzello, L. Ultrastructure of the human mammary gland. Pathol. Annu.. 6. 10. Cutler, L. S. Intercellular contacts at the epithelial-mesenchymal interface of 1-59, 1971. the developing rat submandibular gland in vitro. J. Embryol. Exp. Morphol.. 38. Pierce, G. B. Epithelial basement membrane: origin, development, and role 39. 71-77, 1977. in disease. In: E. A. Balazs (ed.). Chemistry and Molecular Biology of the 11. David, G., and Bernfield, M. R. Collagen reduces glycosaminoglycan deg Intercellular Matrix, Vol. 1, pp. 471-506. New York: Academic Press, Inc., radation by cultured mammary epithelial cells: possible mechanism for basal 1970. lamina formation. Proc. Nati. Acad. Sei. U. S. A., 76: 786-790, 1979. 39. Pitelka, D. R. Cell recognition by malignant mammary epithelium. J. Cell 12. Dees, J. H., Reuber, M. D., and Trump, B. F. Adenocarcinoma of the kidney. Biol.. 70. 66a, 1976. I. Ultrastructure of renal adenocarcinomas induced in rats by W-(4'-fluoro-4- 40. Pitelka. D. R., and Hamamoto. S. T. Form and function in the mammary biphenyDacetamide. J. Nati. Cancer Inst.. 57 779-794. 1976. gland: the interpretation of ultrastructure. J. Dairy Sci., 60. 643-654. 1977. 13. Dingemans, K. P. Invasion of liver tissue by blood-borne mammary carci 41. Pitelka, D. R.. Hamamoto. S. T.. Duafala. J G.. and Nemanic. M. K Cell noma cells. J. Nati. Cancer Inst., 53. 1813-1824, 1974. contacts in the mouse mammary gland. I Normal gland in postnatal devel 14. Farquhar, M. G. Structure and function in glomerular capillaries: role of the opment and the secretory cycle. J. Cell Biol., 56. 797-818, 1973. basement membrane in glomerular filtration. In: N. A. Kefalides (ed.), Biology 42. 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Cancer (Phila.), 37. 828-840. 1976. to cancer in MCA-induced mammary tumorigenesis. Lab. Invest., 33. 33- 46. Tarin. D. Fine structure of murine mammary tumors: the relationship between 42, 1975. epithelium and connective tissue in neoplasms induced by various agents. 18. Frei. J. V. The fine structure of the basement membrane in epidermal tumors. Br. J. Cancer, 23. 417-426. 1969 J. Cell Biol.. 15: 335-342, 1962. 47. Thompson. S. C. The colony forming efficiency of single cells and cell 19. Glassock, R. J. Changes of glomerular basement membrane in disease. In: aggregates from a spontaneous mouse mammary tumour using the lung N. A. Kefalides (ed.), Biology and Chemistry of Basement Membranes, pp. colony assay. Br. J. Cancer, 30. 332-336, 1974. 421-441. New York: Academic Press. Inc.. 1978. 48. Thorning, D.. and Vracko, R. Renal glomerular basal lamina scaffold. Em 20. Goldenberg. V E., Goldenberg. N. S., and Sommers, S. C. Comparative bryologie development, anatomy, and role in cellular reconstruction of rat ultrastructure of atypical ductal hyperplasia, intraductal carcinoma, and glomeruli injured by freezing and thawing. Lab. Invest., 37. 105-119, 1977. infiltrating ductal carcinoma of the breast. Cancer (Phila.), 24: 1152-1169. 49. Tickle, C.. Crawley, A., and Goodman, M. Cell movement and the mechanism 1969. of invasiveness: a survey of the behaviour of some normal and malignant 21. Gould, V. E., Miller, J., and Jao, W. Ultrastucture of medullary, intraductal. cells implanted into the developing chick wing bud J. Cell Sci.. 37 292- tubular, and adenocystic breast carcinomas: comparative patterns of myo- 322, 1978. 9 epithelial differentiation and basal lamina deposition. Am. J. Pathol., 78: 50. Tickle, C., Crawley, A., and Goodman, M. Mechanisms of invasiveness of 401-416, 1975. epithelial tumours: ultrastructure of the interactions of carcinoma cells with 22. Gould, V. E.. and Snyder, R. W. 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MAY 1980 1605

Fig. 1. The peripheries of 2 alveoli in normal lactating C3H mouse mammary gland lie very close together; between the arrows, their basal laminae (BL) are separated by only 50 nm. Elsewhere the narrow interstitial space contains collagen fibrils cut at various angles. Small myoepithelial cell processes (My) are shown in both alveoli, x 30,000. Inset, a typical hemidesmosome of a normal ductal myoepithelial cell. The dense plaque on the cytoplasmic side of the cell membrane, the thin dense layer just external to the membrane, and the increased density of the basal lamina in this area are characteristic, x 180,000. Fig. 2. Section from a pulmonary metastasis in a C3Hf mouse, showing the periphery of a tumor nest at the bottom, with its thin basal lamina. At the top is the cavity of a large blood sinus, lined by intact endothelium (En) with its own basal lamina. Between the 2 laminae are parts of several pulmonary connective tissue cells, x 12.000. Fig. 3. Section from a primary tumor in a C3Hf mouse. Parts of the edge of one tumor nest, with its distinct basal lamina, are just visible at the bottom. Separated from this by interstitial space with collagen fibrils is the basal lamina of a small cyst, enclosing remnants of cytolyzed cytoplasm and a number of spherical MTV virions with dense nucleoids. x 24,000. Fig. 4. Folded basal lamina at the periphery of a C3H tumor nodule. In the adjacent interstitium are dense cross-sections of collagen fibrils and an abundance of flocculent material resembling the basal lamina in density, x 30,000. Fig. 5. Multilayered basal lamina at the surface of a tumor from a different C3H female. Cross-sectioned dense collagen fibrils are present between layers, x 12.000. Figs. 6 to 9 and 11. Photomicrographs. Epon thick sections. Azure ll-methylene blue. Fig. 6. Mammary tumor focus from the lung of a C3H female bearing a 32-day-old s.c. transplant of MT3 tumor in its fourth generation. The distinct, parallel dark lines are the elastic layers of an arteriole accompanying a small bronchiole (not shown). The intravascular tumor has spread into the lung tissue through the ruptured vessel wall, x 220. Fig. 7. Part of an intraarteriolar tumor with extravascular growth through the ruptured wall. Central necrosis within the arteriole is evident. There were numerous tumor masses in the nearby lung tissue, x 140. Fig. 8. Small tumor focus in the lung of a BALB/cNIV female; no other mammary tumor was observed in this small tissue block. Lung cells around the tumor are compacted. The ruptured elastic layers of the arteriole (right) are retracted and much folded. The larger extravascular nodule (left) is well circumscribed. The edge of a bronchiole is visible at the upper right, x 560. Fig. 9. Mammary tumor, with several large lumina, in an arteriole in a C3H lung; a bronchiole is adjacent. This tumor may have grown through the arteriolar system from a large metastasis a short distance away, x 140. Fig. 10. The edge of a C3H mammary tumor within a pulmonary arteriole is separated by an irregular, thickened layer of basal lamina-like material from the persisting arteriolar endothelium. Two endothelial cells interdigitale and are attached together by an extensive junction (J). Plasmalemmal vesicles are numerous along the basal surface of the endothelium but absent from the apical surface, facing the tumor. Elastic (El) and collagen fibers separate endothelium from 2 smooth muscle cells. Basal laminae of the endothelium and muscle cells are absent or fragmentary, x 30,000. Fig. 11. Enlarged view of part of Fig. 6. The arteriolar endothelium is visible as a layer of flattened cells with flattened, dense nuclei immediately within the inner elastic layer of the wall; smooth muscle cell nuclei are distinguishable between the 2 elastic layers. The extravascular extension of the tumor lies within the pulmonary connective tissue and is distinctly demarcated from it. x 560. Fig. 12. Part of the small intraarteriolar tumor focus shown in Fig. 8. The edge of the tumor cell at the top is in contact with cross-sectioned collagen fibrils. Only an ill-defined basal lamina (left) and questionable fragments elsewhere (arrows) are present; cross-sectioned collagen fibrils fill the space between the tumor and the elastic layer of the vessel below. Small processes from the smooth muscle cell at the bottom interdigitale with fibers of the elastic layer, x 32,000. Fig. 13. Opposite side of the same intravascular focus. The basal lamina of the tumor cells again is indistinct or absent. At the arrow, a short, blunt projection from a tumor cell meets a slender process from a smooth muscle cell, x 12,000. Fig. 14. Part of the edge of the extravascular part of the same tumor focus. On the right and left, the tumor cells are bordered by diffuse basal lamina. In between, a tumor cell surface parallels that of a cell of the lung for some distance at the left, and a pseudopod from the adjacent cell extends for about 1.5 /im between lung cells. The apical surface of the labeled tumor cell (T) faced a well-formed lumen (out of this field), with microvilli, tight junctions, and MTV particles, x 24,000. Fig. 15. Pulmonary metastasis in a C3H mouse. At the top, the endothelium of a capillary runs parallel to the very thin cytoplasm of a type I pulmonary epithelial cell (/); the narrow space between the 2 contains the basal laminae, not distinct at this low magnification. Below the air space, a type II pulmonary epithelial cell (//) (left) contains laminated bodies identifiable as surfactant granules. The remaining cells are part of a large mammary tumor mass (T). Lung and tumor epithelial cells are joined at the luminal border by typical junctional complexes, visible here in tangential section as convoluted dense bands (J). x 19,000. Fig. 16. Section similar to that in Fig. 15, showing intimate contact of a pulmonary type II epithelial cell at the top with cells of a tumor (T). Some of the tumor cells facing this air space were producing MTV virions (arrow), x 9,000. Fig. 17. Continuity of the basal laminae (small arrows) of a large tumor mass (J~)and a distorted pulmonary alveolus (Ai is shown here. The alveolar air space contains numerous normal or condensed tubular myelin bodies characteristic of discharged surfactant material. The lumen in this section is bounded on the lower side by the cytoplasm of one or more type I epithelial cells (/). That on the right rests on the continuous basal lamina, but the type I cell closest to the tumor cell, between the large arrows, is associated with only a short fragment (inset, arrows). Fig. 17. x 12,000; inset, x 30,000. Fig. 18 Section of a C3Hf primary tumor, showing several small, irregular cavities (arrows) lined by basal lamina and usually filled with additional basal lamina-like material, x 12,000. Fig. 19. Part of a somewhat larger cavity from the same C3Hf tumor. The basal lamina is distinct, and material within the cavity is fibrillar. Two of the adjoining cells appear to be myoepithelial (My), x 15,000. Fig. 20. Section of a small tumor within a C3H nodule transplant. Cells of the central alveolar structure contain secretory material (S) and clusters of intracytoplasmic MTV particles (V). Series of pockets of interstitial space, lined by multiple layers of basal lamina and sometimes containing fibroblastic cell processes, interrupt the continuity of this alveolus with others seen here on 3 sides, x 5,000.

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Cancer Res 1980;40:1600-1611.

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