EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
52, 202-211 (19%))
Morphologic Characterization of Early Prostatic Carcinomas the ACI Rat: A Light and Electron Microscopic Study VIJAY
A. VARMA'AND
in
GARTH E. AUSTIN
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, and VA Medical Center, Decatur, Georgia 30033 Received March 23, 1989, and in revised form
October 25, 1989
The AC1 rat constitutes a unique model for human prostatic carcinogenesis. A high percentage of these animals spontaneously develop prostatic carcinomas in the ventral lobe as they age. The light microscopic appearance of these tumors is similar to the cribriform pattern of adenocarcinoma in human prostate. In order to further characterize this useful model, we carried out light and electron microscopy studies of the morphology of carcinomatous lesions developing in these animals. Sixteen rats ranging in age from 25 to 43 months were examined histologically, and ultrastructural studies were performed on eight of these cases. The neoplastic cells showed features of well-developed secretory epithelium including prominent Golgi apparatus, abundant rough endoplasmic reticulum, and numerous secretory vacuoles. Microvilli were numerous in some cells and focal apocrine secretory activity was present. Intraluminal crystals similar to those associated with human prostate carcinoma were observed in one of our cases. Prostate carcinomas developing in the AC1 rat share many of the ultrastructural features of human prostatic carcinoma. 8 19!30 Academic Press. Inc.
INTRODUCTION An important factor limiting progress in our understanding of human prostatic carcinogenesis has been the absence of an animal model with biological properties similar to those of the human disease. Specifically, an ideal animal model should exhibit spontaneous tumor development, increasing with age, and the tumors should be hormonally responsive and morphologically and biologically similar to their human counterparts. A possible solution to these requirements was provided by Shain et al. (1, 2) who described the spontaneous development of adenocarcinemas in the ventral lobes of the prostate in a high percentage of aging AC1 rats. While the light microscopic appearance of these tumors has been described by Shain and co-workers (l-3) and by Ward et al. (4), the electron microscopic (EM) appearance of these lesions remained uncharacterized. Because of the potential usefulness of this model and the importance of defining the extent of its resemblance to human prostatic carcinoma, we present a correlative study of the light and ultrastructural features of the early prostatic carcinomas developing in this strain of rats. MATERIALS AND METHODS ACI/rats (AXC/SH) were obtained as a kind gift from Dr. Sidney Shain of the Southwest Foundation for Research and Education (San Antonio, TX). All rats were the product of brother-sister matings in Dr. Shain’s colony, where they were fed Purina Laboratory Chow and maintained in a constant temperature environment with regulated 12-hr intervals of darkness and light (1, 3). At the age ’ To whom reprint requests should be addressed at the VA Medical Center. 202 OOW48OOBO $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of approximately 24 months the rats were transferred to the Animal Facility of the Research Service of the Atlanta VA Medical Center, where they received care as described above. Animals were sacrificed at 25 to 43 months of age by intraperitoneal injection of 3 ml of sodium pentabarbital(25 mg/ml). Tissues were obtained immediately after death and portions of the ventral lobes of the prostate gland were placed in neutral buffered formalin for light microscopic examination and in 4% cacodylatebuffered glutaraldehyde for electron microscopic examination. Tissue was processed for light microscopic examination by standard graded alcohol dehydration followed by embedding in paraffin, sectioning, and staining with hematoxylin and eosin. Tissue for ultrastructural examination was washed in sucrose cacodylate buffer, osmicated, dehydrated in graded ethanol solutions, and embedded in EMbed-812 (Electron Microscopy Sciences, Fort Washington, PA). One-micrometer sections stained with toluidine blue were examined for selection of blocks. Three hundred- to five hundred-angstrom sections of appropriate blocks were then stained with uranyl acetate and lead citrate and examined on an RCA EMU-4 electron microscope at SO-kV. RESULTS Light Microscopy
On gross examination of the dissected prostate glands, 14 of them appeared grossly unremarkable whereas the remaining 2 glands showed nodular enlargements of the right ventral lobes measuring 0.1 x 1.0 x 0.3 cm. However, light microscopic examination revealed intraacinar cribriform masses in the ventral prostatic lobes of all rats (Fig. 1). In most of the cases approximately 10 to 20%
FIG. 1. Photomicrograph of ventral lobe of AC1 rat prostate, showing focal proliferations of neoplastic epithelium within acini. Adjacent acini are lined by atrophic epithelium. Hemotoxylin and eosin. X91.
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of the surface area of the ventral lobe sections were involved, while the oldest rats showed involvement of up to 50% of the surface area. The cellular aggregates ranged from two to eight cells in thickness, forming complex gland-in-gland patterns. The lumina of these glands contained small amounts of homogeneous granular eosinophilic material, presumably representing epithelial secretory products. Examination of the two cases showing gross prostatic enlargement revealed ventral lobes comprising numerous enlarged and dilated neoplastic acini filled with acute inflammatory exudate and lined by epithelium exhibiting the proliferative changes described above. Some acini had been replaced by solid masses of neoplastic epithelial cells. Squamous metaplasia of the acinar cells was seen in other areas. The proliferative lesions consisted of a relatively monotonous population of slightly enlarged cells (Fig. 2). The cytoplasm was abundant and eosinophilic; some cells showed apical vacuolation. The nuclei were large with round to oval contours, finely dispersed chromatin, and prominent nucleoli. Few mitotic figures were identified. Scattered single cell necrosis was evident but there were no extensive areas of tissue necrosis. Despite the marked cellular proliferation, definite stromal invasion could not be identified; the basement membrane appeared intact and the basal cells were retained in all areas. Many of the uninvolved acini had an atrophic appearance and were lined by a single layer of flattened epithelial cells. Except for the two cases showing gross enlargement of the ventral lobes there was no associated inflammation and the stroma was free of significant pathologic change. The other lobes of the prostate glands as well as the seminal vesicles and the coagulation glands were free of any inflammatory or neoplastic changes.
FIG. 2. Higher power photomicrograph of neoplastic glands showing epithelial cells growing backto-back, forming a cribriform pattern. Occasional mitotic figures are evident (arrow). Hematoxylin and eosin. ~235.
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In one specimen, the neoplastic acini contained numerous intraluminal crystals (Fig. 3). These rectangular and rod-shaped crystals were deeply eosinophilic. The sides of the crystals appeared straight, smooth, and parallel while the ends were jagged, suggestive of fracture. They measured up to 8.0 pm in diameter and 72 pm in length and were refractile but not birefringent. Electron Microscopy
Four to twelve l-pm sections from each of these eight animals were first examined by light microscopy in order to select the neoplastic lesion for ultrastructural study. The proliferative lesions described above were readily identified in these sections. The neoplastic foci consisted of glands crowded with enlarged epithelial cells forming papillae and cribriform structures. In some areas bulbous protrusions of apical cytoplasm were evident, suggesting apocrine activity. The adjacent nonneoplastic glands were lined by low columnar epithelium. On ultrastructural examination the neoplastic cells appeared relatively monomorphic and retained the features of well-differentiated prostatic secretory epithelium. At low magnification crowded cells forming multiple lumina were observed (Fig. 4). The neoplastic epithelial cells were typically tall columnar in shape with well-developed intercellular junctional complexes (Fig. 5). Although the intercellular borders were straight, for the most part, complex interdigitations of the cytoplasm were noted in the basal portions of some epithelial cells. The luminal surface of the neoplastic epithelial cells was covered by variable numbers of microvilli. In some cells these were sparse and scattered, whereas in others they were numerous and crowded (Fig. 5). The terminal web, while discernable, was not prominent, even in cells showing numerous microvilli. Little or no glycocalyx could be visualized on the luminal aspect of the cell membranes. Numerous secretory vesicles were present within the apical portion of the
FIG. 3. Photomicrograph
of neoplastic prostatic a&i
showing rod-shaped crystals (H 19 E x364).
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FIG. 4. Electron micrograph showing epithelial cells forming lumina (L). Numerous secretory vesicles are evident in the apical portion of the cells (arrows) x4620.
cytoplasm of many of the cells (Fig. 4). These vesicles were membrane bound and filled with granular material. Apocrine secretory activity was prominent in other neoplastic cells and was characterized by large, bulbous protrusions of organellepoor granular cytoplasm extending into the lumina (Fig. 6). The Golgi apparatus was well developed and located between the nucleus and the luminal surface. Rough endoplasmic reticulum (RER) was extensive, coursing through most of the cytoplasm (Fig. 5). It contained finely granular material of an electron density lower than that of the remainder of the cytoplasm. In some cells the RER was distended with this material, forming large lakes. The cytoplasm also exhibited a variable number of lysosomes containing membrane fragments and electron-dense material. Large lipid bodies were prominent in other cells, occupying much of the cytoplasm. Mitochondria varied in number from cell to cell but were evenly distributed throughout the cytoplasm. They were normal in configuration with lamellar cristae. Nuclei were either basally or centrally located and were round to slightly oval in shape and occasionally grooved. The heterochromatin was marginated toward the nuclear membrane. Typically, the nuclei contained a single, eccentric nucleolus. An occasional nucleus contained a 700~nm reticular-granular inclusion. The basal lamina of the neoplastic acini measured up to 150 nm in thickness and was well preserved without disruptions (Fig. 7). Scattered basal cells could be seen between the epithelial cells and the basement membrane. These basal cells were smaller than the epithelial cells and were oval to polygonal in shape; they contained scanty, organelle-poor cytoplasm, but did not show features of myoepithelial cells, such as filament bundles with dense bodies or pinocytotic vesicles. The basal cells were attached to the epithelial cells by desmosomes.
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FIG. 5. Electron micrograph showing the luminal surface of neoplastic epithelial cells covered with numerous microvilli. Rough endoplasmic reticulum (R) and Golgi apparatus (G) are prominent. x3990.
Electron microscopy of the intraglandular crystals similar to those observed by light microscopy in the 43-month-old rat showed heavily but uniformly stained, rod-shaped structures (Fig. 8). These were embedded in granular material and did not possess a limiting membrane. The two ends of each crystal were jagged and occasional fine fracture lines were identified along the sides. No internal lattice structure was discernable. The benign acini within the glands were flattened, with decreased numbers of microvilli. The periglandular stroma was composed of well-differentiated smooth muscle cells, groups of which were separated by collagen fibers. Scattered fibroblasts were also identified within the stroma. DISCUSSION The present study provides, to our knowledge, the first detailed report on the ultrastructural appearance of spontaneous prostatic carcinoma arising in the rat and complements previous work on normal rodent prostate and human prostatic carcinoma. The ultrastructural appearance of the normal benign glands of the ventral lobes of rat prostate has already been well described (5-7). More recently, Bartsch and Rohr (8) compared the light and electron microscopic appearance of normal and hyperplastic prostatic tissue in the human, dog, and rat. Human prostatic carcinoma has also been thoroughly studied (9-l 1); however, to our knowledge, there have been no published reports on the ultrastructural appearance of prostatic carcinoma in the rat. Wakisaka et al. (12) described the electron microscopic appearance of stromal and myoepithelial cells in Dunning R-3327 rat prostatic
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FIG. 6. Low magnification electron micrograph showing tall columnar cells with apocrine snouts. x3675.
tumors, but ultrastructural features of adenocarcinoma cells in these tumors were neither described nor illustrated. Feuchter et al. (13) studied the in vitro ultrastructural appearance of a malignant prostatic epithelial cell line derived from the Copenhagen rat, but the ultrastructure of the tumors in vivo was not illustrated. There have been no previous descriptions of the ultrastructural appearance of the spontaneous prostatic carcinoma found in the AC1 rat. Our studies of early adenocarcinoma in the ventral prostatic lobes of the AXC/ SSh rat show that the neoplastic cells retain the essential ultrastructural features of normal epithelial cells of this gland. Like their benign counterparts, these malignant cells are columnar with well-differentiated secretory features. Particularly prominent in many cells are the abundant rough ER, distended with secretory material, and the well-developed Golgi apparatus. Some cells show prominent merocrine secretory activity as demonstrated by the numerous secretory vacuoles in the apical portion of the cytoplasm. The presence of apocrine snouts in other cells suggests retention of apocrine secretory activity as well. Our finding of both merocrine and apocrine secretory activity in neoplastic prostatic epithelium of the AC1 rat is reminiscent of similar observations which have been reported by Feuchter et al. (13) on benign prostatic epithelial cells of the Copenhagen rat. The finding of profuse, elongated microvilli in many of the neoplastic epithelial cells of the prostate was unexpected, since these organelles are generally associated with an absorptive and not a secretory function. However, previous studies by Feuchter et al. (13) and Guggenheim et al. (14) described the existence of such elongated microvilli in scattered epithelial cells of normal rat prostate. The significance of such structures in benign and malignant prostatic epithelium remains
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FIG. 7. Electron micrograph showing a distinct and continuous basal lamina (arrows) separating the epithelium from the stroma. x4760.
unclear. The nuclei of the neoplastic cells did not appear anaplastic. Although some nuclei had deep indentations or clefts, this was not a consistent feature. The distribution of heterochromatin and euchromatin and the appearance of the nucleoli were similar to what was described in nonneoplastic prostatic epithelium. Peculiar nonmembrane bound, fibrillary inclusions were identified in a few of the nuclei. These have been described in several benign and malignant cell types but their significance remains undetermined (15). These neoplastic lesions in the AC1 rat share many of the subcellular features of human prostatic carcinoma, including well-developed Golgi apparatus, secretory vacuoles, lysosomes, and lipid bodies. However, the abundant RER, distended with secretory material to form lakes, is not a feature of human prostatic epithelium, benign or malignant, although it is seen in the ventral lobe of the normal rat prostate. In adenocarcinoma of the human prostate the acini lack basal cells, and the basal lamina, when present, is disrupted (9). However, McNeal et al. (16), in a recent report on cribriform adenocarcinoma of human prostate, found intact basal laminae and basal cells in these lesions. Likewise, we found well-preserved basal laminae and basal cells in the cribriform neoplastic lesions in the AC1 rat prostate. The intraglandular lesions we observed in the rat model appear analogous to those described in human prostate (16). Although some authors speculate that the two lesions represent parts of a continuum in human prostate carcinoma (17), we did not observe any invasive carcinoma in our animal model. The preservation of basal cells is interesting, but we do not believe that this, in itself, indicates origin from secretory cells, as suggested by Heatfield ef al. in their description of human prostate carcinoma (18).
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FIG. 8. Electron micrograph x 12,600.
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showing rectangular crystals embedded within granular material.
The crystals that we observed in one of our specimens have not been previously described in rat prostate. The origin and chemical nature of these crystals are not clear. However, they bear a striking morphologic resemblance to the crystals described in association with human prostate carcinoma (19, 20). ACKNOWLEDGMENTS We thank Ms. Christine O’Connell and Ms. Peggy Miles for excellent technical assistance. This work was supported by a Biomedical Research Support Grant from Emory University (to G.E.A. and V.V.) and by a VA Merit Award (to G.E.A.). Also we thank Ms. Rosa J. Fowler for typing the manuscript.
REFERENCES 1. SHAIN, S. A., MCCULLOUGH, B., and SEGALOFF, A. (1975). Spontaneous adenocarcinoma of the ventral prostate of aged AXC rats. J. Natl. Cancer Inst. 55, 177-180. 2. SHAIN, S. A., MCCULLOUGH, B., NITCHUK, W. M., and BOESEL, R. W. (1977). Prostate carcinogenesis in the AXC rat. Oncology 34, 114-122. 3. SHAIN, S. A., MCCULLOUGH, B., and NITCHUK, W. M. (1979). Primary and transplantable adenocarcinomas of the AXC rat ventral prostate gland: Morphologic characterization and examination of C19-steroid metabolism by early-passage tumors. J. Natl. Cancer Inst. 62, 313-322. 4. WARD, J. M., REZNIK, G., STINSON, S. F., LATTUADA, C. P., LONGFELLOW, D. G., and CAMERON, T. P. (1980). Histogenesis and morphology of naturally occurring prostatic carcinoma in the ACYsegHapBR rat. Lab. Invest. 43, 517-522. 5. BRANDES, D. (1966). The fine structure and histochemistry of prostatic glands in relationship to sex hormones. Znt. Rev. Cytol. 20, 207-276. 6. BRANDES, D., and GROTH, D. P. (1961). The tine structure of the rat prostatic complex. Exp. Cell Res. 23, 159-175. 7. DIDIO, L. J. A. Correlative light and electron microscopy of the normal prostatic ventral lobe in rats. Amt. Anz. Bd 128, 17&190. 8. BARTSCH, G., and ROHR, H. P. (1980). Comparative light and electron microscopic study of the human, dog, and rat prostate. Ural. Znt. 35, 91-104.
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9. MAO, P., NAKAO, K., and ANGRIST, A. (1966). Human prostate carcinoma: An electron microscope study. Cancer Res. 26, 955-973. 10. TANNENBAUM, M., and TANNENBAUM, S. (1980). Ultrastructural pathology of human prostatic carcinoma. Pathology of human prostatic carcinoma. In B. F. Trump and R. Jones, Eds., “Diagnostic Electron Microscopy” Vo13, pp. 175-201. Wiley, New York. 11. FISHER, E. R., and SIERACKI, J. C. (1970). Ultrastructure of human normal and neoplastic prostate. Pathol. Annu. 5, l-25. 12. WAKISAKA, M., MIYAUCHI, T., HARA, S., MATSUZAKI, O., and SHIMAZAKI, J. (1986). Proliferation of Dunning R-3327 rat prostatic adenocarcinoma and of its sublines (CUA and CUB). Prostate 8, 381-391. 13. FEUCHTER, F. A., ROWLEY, D. R., and HEIDGER, P. M., JR. (1980). Isolation, in vitro cultivation, and electron microscopy of normal and malignant prostatic epithelial cells from the Copenhagen rat. Ural. Res. 8, 139-152. 14. GUGGENHEIM, R., BARTSCH, G., TANNENBAUM, M. and ROHR, H. P. (1979). Comparative scanning and transmission electron microscopy of prostatic gland in different species (mouse, rat, dog, man). Scanning Electron Microsc. 3, 721-728. 15. GHADIALLY, F. N. (1982). “Ultrastructural Pathology of the Cell and Matrix,” 2nd ed, pp. 118-121. Butterworths, London. 16. MCNEAL, J. E., REESE, J. H., REDWINE, E. A., FREIHA, F. S., and STAMEY, T. A. (1986). Cribriform adenocarcinoma of the prostate. Cancer 58, 1714-1719. 17. BOSTWICK, D. G., and BRAWER, M. K. (1987). Prostatic intra-epithelial neoplasia and early invasion in prostatic cancer. Cancer 59, 788-794. 18. HEATFIELD, B. M., SANEFUJI, H., and TRUMP, B. F. (1979). Studies on carcinogenesis of human prostate during long-term explant culture. Scanning Electron Microsc. 3, 645-656. 19. HOLMES, E. J. (1977). Crystalloids of prostatic carcinoma: Relationship to Bence-Jones crystals. Cancer 39, 2073-2080. 20. Ro, J. E., AYALA, A. G., ORDONEZ, N. G., CARTWEIGHT, J., and MACKAY, B. (1986). IntraIuminal crystalloids in prostatic adenocarcinoma. Immunohistochemical, electron microscopic and X-ray microanalytic studies. Cancer 57, 2397-2407.
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52, 202-211 (19%))
Morphologic Characterization of Early Prostatic Carcinomas the ACI Rat: A Light and Electron Microscopic Study VIJAY
A. VARMA'AND
in
GARTH E. AUSTIN
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, and VA Medical Center, Decatur, Georgia 30033 Received March 23, 1989, and in revised form
October 25, 1989
The AC1 rat constitutes a unique model for human prostatic carcinogenesis. A high percentage of these animals spontaneously develop prostatic carcinomas in the ventral lobe as they age. The light microscopic appearance of these tumors is similar to the cribriform pattern of adenocarcinoma in human prostate. In order to further characterize this useful model, we carried out light and electron microscopy studies of the morphology of carcinomatous lesions developing in these animals. Sixteen rats ranging in age from 25 to 43 months were examined histologically, and ultrastructural studies were performed on eight of these cases. The neoplastic cells showed features of well-developed secretory epithelium including prominent Golgi apparatus, abundant rough endoplasmic reticulum, and numerous secretory vacuoles. Microvilli were numerous in some cells and focal apocrine secretory activity was present. Intraluminal crystals similar to those associated with human prostate carcinoma were observed in one of our cases. Prostate carcinomas developing in the AC1 rat share many of the ultrastructural features of human prostatic carcinoma. 8 19!30 Academic Press. Inc.
INTRODUCTION An important factor limiting progress in our understanding of human prostatic carcinogenesis has been the absence of an animal model with biological properties similar to those of the human disease. Specifically, an ideal animal model should exhibit spontaneous tumor development, increasing with age, and the tumors should be hormonally responsive and morphologically and biologically similar to their human counterparts. A possible solution to these requirements was provided by Shain et al. (1, 2) who described the spontaneous development of adenocarcinemas in the ventral lobes of the prostate in a high percentage of aging AC1 rats. While the light microscopic appearance of these tumors has been described by Shain and co-workers (l-3) and by Ward et al. (4), the electron microscopic (EM) appearance of these lesions remained uncharacterized. Because of the potential usefulness of this model and the importance of defining the extent of its resemblance to human prostatic carcinoma, we present a correlative study of the light and ultrastructural features of the early prostatic carcinomas developing in this strain of rats. MATERIALS AND METHODS ACI/rats (AXC/SH) were obtained as a kind gift from Dr. Sidney Shain of the Southwest Foundation for Research and Education (San Antonio, TX). All rats were the product of brother-sister matings in Dr. Shain’s colony, where they were fed Purina Laboratory Chow and maintained in a constant temperature environment with regulated 12-hr intervals of darkness and light (1, 3). At the age ’ To whom reprint requests should be addressed at the VA Medical Center. 202 OOW48OOBO $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of approximately 24 months the rats were transferred to the Animal Facility of the Research Service of the Atlanta VA Medical Center, where they received care as described above. Animals were sacrificed at 25 to 43 months of age by intraperitoneal injection of 3 ml of sodium pentabarbital(25 mg/ml). Tissues were obtained immediately after death and portions of the ventral lobes of the prostate gland were placed in neutral buffered formalin for light microscopic examination and in 4% cacodylatebuffered glutaraldehyde for electron microscopic examination. Tissue was processed for light microscopic examination by standard graded alcohol dehydration followed by embedding in paraffin, sectioning, and staining with hematoxylin and eosin. Tissue for ultrastructural examination was washed in sucrose cacodylate buffer, osmicated, dehydrated in graded ethanol solutions, and embedded in EMbed-812 (Electron Microscopy Sciences, Fort Washington, PA). One-micrometer sections stained with toluidine blue were examined for selection of blocks. Three hundred- to five hundred-angstrom sections of appropriate blocks were then stained with uranyl acetate and lead citrate and examined on an RCA EMU-4 electron microscope at SO-kV. RESULTS Light Microscopy
On gross examination of the dissected prostate glands, 14 of them appeared grossly unremarkable whereas the remaining 2 glands showed nodular enlargements of the right ventral lobes measuring 0.1 x 1.0 x 0.3 cm. However, light microscopic examination revealed intraacinar cribriform masses in the ventral prostatic lobes of all rats (Fig. 1). In most of the cases approximately 10 to 20%
FIG. 1. Photomicrograph of ventral lobe of AC1 rat prostate, showing focal proliferations of neoplastic epithelium within acini. Adjacent acini are lined by atrophic epithelium. Hemotoxylin and eosin. X91.
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of the surface area of the ventral lobe sections were involved, while the oldest rats showed involvement of up to 50% of the surface area. The cellular aggregates ranged from two to eight cells in thickness, forming complex gland-in-gland patterns. The lumina of these glands contained small amounts of homogeneous granular eosinophilic material, presumably representing epithelial secretory products. Examination of the two cases showing gross prostatic enlargement revealed ventral lobes comprising numerous enlarged and dilated neoplastic acini filled with acute inflammatory exudate and lined by epithelium exhibiting the proliferative changes described above. Some acini had been replaced by solid masses of neoplastic epithelial cells. Squamous metaplasia of the acinar cells was seen in other areas. The proliferative lesions consisted of a relatively monotonous population of slightly enlarged cells (Fig. 2). The cytoplasm was abundant and eosinophilic; some cells showed apical vacuolation. The nuclei were large with round to oval contours, finely dispersed chromatin, and prominent nucleoli. Few mitotic figures were identified. Scattered single cell necrosis was evident but there were no extensive areas of tissue necrosis. Despite the marked cellular proliferation, definite stromal invasion could not be identified; the basement membrane appeared intact and the basal cells were retained in all areas. Many of the uninvolved acini had an atrophic appearance and were lined by a single layer of flattened epithelial cells. Except for the two cases showing gross enlargement of the ventral lobes there was no associated inflammation and the stroma was free of significant pathologic change. The other lobes of the prostate glands as well as the seminal vesicles and the coagulation glands were free of any inflammatory or neoplastic changes.
FIG. 2. Higher power photomicrograph of neoplastic glands showing epithelial cells growing backto-back, forming a cribriform pattern. Occasional mitotic figures are evident (arrow). Hematoxylin and eosin. ~235.
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In one specimen, the neoplastic acini contained numerous intraluminal crystals (Fig. 3). These rectangular and rod-shaped crystals were deeply eosinophilic. The sides of the crystals appeared straight, smooth, and parallel while the ends were jagged, suggestive of fracture. They measured up to 8.0 pm in diameter and 72 pm in length and were refractile but not birefringent. Electron Microscopy
Four to twelve l-pm sections from each of these eight animals were first examined by light microscopy in order to select the neoplastic lesion for ultrastructural study. The proliferative lesions described above were readily identified in these sections. The neoplastic foci consisted of glands crowded with enlarged epithelial cells forming papillae and cribriform structures. In some areas bulbous protrusions of apical cytoplasm were evident, suggesting apocrine activity. The adjacent nonneoplastic glands were lined by low columnar epithelium. On ultrastructural examination the neoplastic cells appeared relatively monomorphic and retained the features of well-differentiated prostatic secretory epithelium. At low magnification crowded cells forming multiple lumina were observed (Fig. 4). The neoplastic epithelial cells were typically tall columnar in shape with well-developed intercellular junctional complexes (Fig. 5). Although the intercellular borders were straight, for the most part, complex interdigitations of the cytoplasm were noted in the basal portions of some epithelial cells. The luminal surface of the neoplastic epithelial cells was covered by variable numbers of microvilli. In some cells these were sparse and scattered, whereas in others they were numerous and crowded (Fig. 5). The terminal web, while discernable, was not prominent, even in cells showing numerous microvilli. Little or no glycocalyx could be visualized on the luminal aspect of the cell membranes. Numerous secretory vesicles were present within the apical portion of the
FIG. 3. Photomicrograph
of neoplastic prostatic a&i
showing rod-shaped crystals (H 19 E x364).
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FIG. 4. Electron micrograph showing epithelial cells forming lumina (L). Numerous secretory vesicles are evident in the apical portion of the cells (arrows) x4620.
cytoplasm of many of the cells (Fig. 4). These vesicles were membrane bound and filled with granular material. Apocrine secretory activity was prominent in other neoplastic cells and was characterized by large, bulbous protrusions of organellepoor granular cytoplasm extending into the lumina (Fig. 6). The Golgi apparatus was well developed and located between the nucleus and the luminal surface. Rough endoplasmic reticulum (RER) was extensive, coursing through most of the cytoplasm (Fig. 5). It contained finely granular material of an electron density lower than that of the remainder of the cytoplasm. In some cells the RER was distended with this material, forming large lakes. The cytoplasm also exhibited a variable number of lysosomes containing membrane fragments and electron-dense material. Large lipid bodies were prominent in other cells, occupying much of the cytoplasm. Mitochondria varied in number from cell to cell but were evenly distributed throughout the cytoplasm. They were normal in configuration with lamellar cristae. Nuclei were either basally or centrally located and were round to slightly oval in shape and occasionally grooved. The heterochromatin was marginated toward the nuclear membrane. Typically, the nuclei contained a single, eccentric nucleolus. An occasional nucleus contained a 700~nm reticular-granular inclusion. The basal lamina of the neoplastic acini measured up to 150 nm in thickness and was well preserved without disruptions (Fig. 7). Scattered basal cells could be seen between the epithelial cells and the basement membrane. These basal cells were smaller than the epithelial cells and were oval to polygonal in shape; they contained scanty, organelle-poor cytoplasm, but did not show features of myoepithelial cells, such as filament bundles with dense bodies or pinocytotic vesicles. The basal cells were attached to the epithelial cells by desmosomes.
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FIG. 5. Electron micrograph showing the luminal surface of neoplastic epithelial cells covered with numerous microvilli. Rough endoplasmic reticulum (R) and Golgi apparatus (G) are prominent. x3990.
Electron microscopy of the intraglandular crystals similar to those observed by light microscopy in the 43-month-old rat showed heavily but uniformly stained, rod-shaped structures (Fig. 8). These were embedded in granular material and did not possess a limiting membrane. The two ends of each crystal were jagged and occasional fine fracture lines were identified along the sides. No internal lattice structure was discernable. The benign acini within the glands were flattened, with decreased numbers of microvilli. The periglandular stroma was composed of well-differentiated smooth muscle cells, groups of which were separated by collagen fibers. Scattered fibroblasts were also identified within the stroma. DISCUSSION The present study provides, to our knowledge, the first detailed report on the ultrastructural appearance of spontaneous prostatic carcinoma arising in the rat and complements previous work on normal rodent prostate and human prostatic carcinoma. The ultrastructural appearance of the normal benign glands of the ventral lobes of rat prostate has already been well described (5-7). More recently, Bartsch and Rohr (8) compared the light and electron microscopic appearance of normal and hyperplastic prostatic tissue in the human, dog, and rat. Human prostatic carcinoma has also been thoroughly studied (9-l 1); however, to our knowledge, there have been no published reports on the ultrastructural appearance of prostatic carcinoma in the rat. Wakisaka et al. (12) described the electron microscopic appearance of stromal and myoepithelial cells in Dunning R-3327 rat prostatic
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FIG. 6. Low magnification electron micrograph showing tall columnar cells with apocrine snouts. x3675.
tumors, but ultrastructural features of adenocarcinoma cells in these tumors were neither described nor illustrated. Feuchter et al. (13) studied the in vitro ultrastructural appearance of a malignant prostatic epithelial cell line derived from the Copenhagen rat, but the ultrastructure of the tumors in vivo was not illustrated. There have been no previous descriptions of the ultrastructural appearance of the spontaneous prostatic carcinoma found in the AC1 rat. Our studies of early adenocarcinoma in the ventral prostatic lobes of the AXC/ SSh rat show that the neoplastic cells retain the essential ultrastructural features of normal epithelial cells of this gland. Like their benign counterparts, these malignant cells are columnar with well-differentiated secretory features. Particularly prominent in many cells are the abundant rough ER, distended with secretory material, and the well-developed Golgi apparatus. Some cells show prominent merocrine secretory activity as demonstrated by the numerous secretory vacuoles in the apical portion of the cytoplasm. The presence of apocrine snouts in other cells suggests retention of apocrine secretory activity as well. Our finding of both merocrine and apocrine secretory activity in neoplastic prostatic epithelium of the AC1 rat is reminiscent of similar observations which have been reported by Feuchter et al. (13) on benign prostatic epithelial cells of the Copenhagen rat. The finding of profuse, elongated microvilli in many of the neoplastic epithelial cells of the prostate was unexpected, since these organelles are generally associated with an absorptive and not a secretory function. However, previous studies by Feuchter et al. (13) and Guggenheim et al. (14) described the existence of such elongated microvilli in scattered epithelial cells of normal rat prostate. The significance of such structures in benign and malignant prostatic epithelium remains
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FIG. 7. Electron micrograph showing a distinct and continuous basal lamina (arrows) separating the epithelium from the stroma. x4760.
unclear. The nuclei of the neoplastic cells did not appear anaplastic. Although some nuclei had deep indentations or clefts, this was not a consistent feature. The distribution of heterochromatin and euchromatin and the appearance of the nucleoli were similar to what was described in nonneoplastic prostatic epithelium. Peculiar nonmembrane bound, fibrillary inclusions were identified in a few of the nuclei. These have been described in several benign and malignant cell types but their significance remains undetermined (15). These neoplastic lesions in the AC1 rat share many of the subcellular features of human prostatic carcinoma, including well-developed Golgi apparatus, secretory vacuoles, lysosomes, and lipid bodies. However, the abundant RER, distended with secretory material to form lakes, is not a feature of human prostatic epithelium, benign or malignant, although it is seen in the ventral lobe of the normal rat prostate. In adenocarcinoma of the human prostate the acini lack basal cells, and the basal lamina, when present, is disrupted (9). However, McNeal et al. (16), in a recent report on cribriform adenocarcinoma of human prostate, found intact basal laminae and basal cells in these lesions. Likewise, we found well-preserved basal laminae and basal cells in the cribriform neoplastic lesions in the AC1 rat prostate. The intraglandular lesions we observed in the rat model appear analogous to those described in human prostate (16). Although some authors speculate that the two lesions represent parts of a continuum in human prostate carcinoma (17), we did not observe any invasive carcinoma in our animal model. The preservation of basal cells is interesting, but we do not believe that this, in itself, indicates origin from secretory cells, as suggested by Heatfield ef al. in their description of human prostate carcinoma (18).
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FIG. 8. Electron micrograph x 12,600.
VARMA
AND AUSTIN
showing rectangular crystals embedded within granular material.
The crystals that we observed in one of our specimens have not been previously described in rat prostate. The origin and chemical nature of these crystals are not clear. However, they bear a striking morphologic resemblance to the crystals described in association with human prostate carcinoma (19, 20). ACKNOWLEDGMENTS We thank Ms. Christine O’Connell and Ms. Peggy Miles for excellent technical assistance. This work was supported by a Biomedical Research Support Grant from Emory University (to G.E.A. and V.V.) and by a VA Merit Award (to G.E.A.). Also we thank Ms. Rosa J. Fowler for typing the manuscript.
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