Proc. Nati. Acad. Sci. USA Vol. 78, No. 6, pp. 3901-3905, June 1981 Medical Sciences Role of basal lamina in neoplastic disorganization of tissue architecture (basement membrane/epithelial cell polarity/cell shape/immunofluorescence) DONALD E. INGBER*, JOSEPH A. MADRIt, AND JAMES D. JAMIESON* *Section of Cell Biology and tDepartment of Pathology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Communicated by Paul Greengard, March 13, 1981 ABSTRACT We have studied a transplantable carcinoma of ithelial form and orientation in embryogenesis (3) and is main- the rat pancreas [Reddy, J. K. & Rao, M. S. (1977) Science 198, tained throughout adult life. Thus, it is possible that neoplastic 78-80] that is composed ofcytologically differentiated acinar cells disorganization of epithelial architecture as well as malignant that have lost their epithelial orientation and do not form acini. invasion may result either from loss of maintenance of this ep- Light microscopy shows, however, consistent palisading, reorien- ithelial scaffolding or through the acquisition ofsome new trans- tation, and polarization of these cells in areas of contact with the formed cell product that compromises its structural integrity. vasculature. Electron microscopy reveals a normal basal lamina In order to investigate the role of BL in the maintenance of (BL) along the basal portions of repolarized tumor cells that is organized tissue structures as well as neoplastic disorganization, physically separate from the endothelial BL. We used indirect im- we studied rat exocrine munofluorescence to examine the distribution ofBL constituents, have a transplantable carcinoma ofthe laminin (Lm) and type IV collagen (type IV), within the different pancreas as a model system. This tumor is composed of cyto- microenvironments of this tumor. In normal pancreas, Lm and logically differentiated acinar cells that have lost their normal type IV are distributed linearly, outlining acini and blood vessels. epithelial organization. The tumor was discovered, with asso- In the tumor parenchyma, type IV is not detected, whereas Lm ciated metastatic foci, in nafenopin-treated rats in the laboratory appears in a punctate distribution outlining cells. Reorientation of Reddy and Rao (6) and was kindly provided to us for study. of tumor cells is observed only along linearly deposited Lm and As the BL is a complex of different collagenous and noncolla- type IV bordering vessels. These data indicate that this nonme- genous macromolecules, the distribution oftwo ubiquitous BL tastatic tumor has lost the ability to produce or maintain a com- constituents, the glycoprotein laminin (Lm) (7) and type IV col- plete BL within its disorganized parenchyma, while its cells retain lagen (type IV) (8), was studied in both the tumor and normal the capacity to produce and reorganize along linear BL when in pancreas, using indirect immunofluorescence. A preliminary contact with vascular adventitia. We suggest that failure to main- account of these studies has appeared in abstract form (9). tain a complete BL may be involved in the neoplastic disorgani- zation of normal tissue architecture as well as in the breakdown of boundaries during the development of invasive carcinomata. MATERIALS AND METHODS Experimental System. Weanling male F344 Sprague-Daw- ley rats (Harlan-Sprague-Dawley, Madison, WI) were inocu- Cells in a epithelium are highly organized and commonly ex- lated subcutaneously or intraperitoneally with a mechanically hibit polarized form as well as function. The cells always sit on prepared suspension of the pancreatic acinar carcinoma in is- top ofa continuous basement membranel or basal lamina (BL) otonic saline. All tumors used in this study were between the and are physically separated from the underlying connective 18th and 23rd passage, 1-4 cm in diameter, and displayed con- tissue. A normal adult epithelium is a dynamic structure (1), and sistent growth characteristics and morphology. In our labora- its orderly renewal (e.g., wound healing) requires the continued tory, the tumor grows as a nonmetastatic carcinoma. Normal presence of BL as an extracellular scaffolding or template that pancreas was obtained from either tumor-bearing animals or maintains the original architectural form and assures for accu- non-tumor-bearing animals; the distribution ofLm and type IV rate regeneration ofpreexisting structures (2). was identical in both. An epithelium may escape its normally tight growth con- Light Microscopy. Tissue specimens were fixed in 10% form- straints and produce a less ordered arrangement or piling up aldehyde in phosphate-buffered saline (PjNaCl) and embed- of atypical epithelial cells and so result in a disorganization of ded in paraffin, and sections were stained with hematoxylin and epithelial form termed dysplasia. As the epithelium becomes eosin. further disorganized, it reaches the arbitrary point at which it Electron Microscopy. Tumor tissue was fixed by perfusion is termed neoplastic. A histologic specimen ofthis tissue would through the left ventricle with 1% glutaraldehyde/3% (wt/vol) be designated as carcinoma in situ, a premalignant condition, formaldehyde in PJNaCl, treated with osmium tetroxide, as long as there is no morphologic evidence ofinvasion through stained en bloc with uranyl acetate, and embedded in Epon/ the underlying BL. Once physical penetration occurs, the neo- Araldite. Thin sections were stained with uranyl acetate and plasm becomes invasive and is malignant, because it is now free lead citrate (10) and were photographed on a Siemens 101 elec- to metastasize. tron microscope. Classically, the BL has been viewed as a host barrier through Fluorescence Microscopy. Tissue was processed according which a malignant tumor must gain the ability to invade. In fact, to the method of Beyer et al. (11) except that specimens were BL is normally a specialized product ofthe overlying epithelial cells (3-5), which also plays a central role as a stabilizer of ep- Abbreviations: BL, basal lamina; Lm, laminin; type IV, type IV colla- gen; PJNaCl, phosphate-buffered saline. The publication costs ofthis article were defrayed in part by page charge t "Basement membrane" is a light-microscopic term that at the level payment. This article must therefore be hereby marked "advertise- of electron microscopy refers to a basal lamina plus adjacent connec- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. tive tissue matrix. 3901 Downloaded by guest on October 1, 2021 3902 Medical Sciences: Ingber et al. Proc. Natl. Acad. Sci..USA 78 (1981) quenched after fixation with NaBH4 at 1 mg/ml in P/NaCl for center. Thus, examination of each acinus reveals a central area 40 min and placed either into'2 M sucrose/PJNaCl or in Tissue filled with zymogen granules surrounding a centroacinar lumen Tek embedding medium. Tissue in sucrose was handled ac- as well as a peripheral arrangement ofthe basally located nuclei cording to the method of Tokuyasu (12) and semithin sections along the outer margin of the acinus. (0.5-1.0 ,um) were cut at -90'C on a Sorvall MT-2B ultrami- On the other hand, the acinar cell tumor is characterized by crotome with a cryotomy attachment. Sections (2-4 ,um) of the a highly disorganized parenchyma with no evidence ofobvious tissue embedded in Tissue Tek were cut at -30'C on a Damon/ cell orientation (Fig. lb). However, consistent palisading and IEC cryostat. reorientation oftumor cells can be seen in areas ofdirect contact Methods for preparation of our antibodies to Lm and type with the vasculature. IV in rabbits, their characterization, and localization within BL Electron microscopy best displays this arrangement of cells have been published (13, 14). A fluorescein-conjugated sheep within the tumor. Epithelial cell repolarization can clearly be anti-rabbit immunoglobulin was used for detection of the pri- seen in tumor cells that line up along the abluminal side of a mary antibodies (13). Control experiments with preimmune tumor vessel because their nuclei are consistently located in serum in the first step or fluorescein-conjugated immunoglob- their basal portions while the zymogen granules fill the apical ulin alone were consistently negative. Localizations were done regions (Fig. 2a). Cells in the parenchyma of the tumor, how- on specimens from seven separate animals on seven different ever, are highly disorganized in that the apex ofone acinar tu- occasions and all findings were confirmed on cryostat sections mor cell is often abnormally juxtaposed with the basal or apical offrozen unfixed material. portion ofa neighbor. No centroacinar lumina or ductular struc- We used a Zeiss photomicroscope II equipped with phase- tures have been observed. While BL is not seen between the contrast optics and an epifluorescence illuminator containing cells within the tumor parenchyma (see also figure 4 in ref. 15), appropriate filters for fluorescein isothiocyanate. Images were a BL with characteristic morphology is closely apposed, to the recorded on Ektachrome 400 film. basal portions of the tumor cells in areas of cell repolarization adjacent to vascular adventitia (Fig. 2b). The tumor'BL and that RESULTS of the vessel are physically separated from each other by con- nective tissue matrix, and both basal laminae display occasional Light microscopy of normal rat pancreas displays the organi- discontinuities. zation of exocrine cells into acini (Fig. la). Each .acinar cell is Fig. 3a is a phase-contrast micrograph of a semithin section typically polarized, with its nucleus being located within the ofnormal rat pancreas that displays acini as well as neighboring basal portion ofthe cell while the zymogen granules fill the ap- capillaries filled with erythrocytes. The distribution of type IV ical region. These cells are consistently oriented within each within this same section is characterized by linear staining out- acinus with their bases at the periphery and apices towards the lining all acini and blood vessels (Fig. 3b). A phase-contrast view of the tumor (Fig. 3c) once again shows a disorganized *V e4.t *6'S FIG. 1. Light micrographs of normal pancreas (a) and acinar cell FnG.