Human Prostate DU145 Carcinoma Cells Implanted in Nude Mice Remove the Peritoneal Mesothelium to Invade and Grow as Carcinomas

Authors


Abstract

Implanted human, androgen-independent prostatic carcinoma cells (DU145) into athymic (NCr nu/nu) mice produce diverse tumors on the peritoneal surfaces of many organs. Light and ultrastructural observations show that the mesothelial covering these surfaces are typically microvilli-coated, squamous cells or secretory cuboidal cells. The peritoneal regions colonized by tumors lack mesothelial cells and are covered by actively replicating carcinoma cells that grow as poorly differentiated cell clusters made of cell aggregates to somewhat compact spheroids covered with pleiomorphic microvilli and containing an undifferentiated vascular supply. These xenografts clusters invade the diaphragm and develop into tumors with both a basal solid aspect and an upper region of cribriform morphology. Furthermore, each tumor contains two cell types: (1) a poorly differentiated clear cell type, which grows into intraperitoneal tumors and (2) a large, basophilic cell type, which invades the peritoneal stroma of organs, including of the diaphragm. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.

Prostate carcinoma is one of the most pervasive and pestilent malignancy encountered in the human males. The frequency and mortality rate of prostate cancer has increased over the past 40 years and is expected to rise steadily in subsequent years (Partin and Coffey, 1994; Turner et al., 1996). In the United States alone an estimated of 218,000 new cases and 32,000 deaths were attributed to prostate cancer in 2010 (Jemal et al., 2010). Males of African descent are almost twice as likely to develop prostate cancer as male Caucasians, while Asian males are half as likely to develop prostate cancer as Caucasian males (Jemal et al., 2010; Cancer Research UK, 2011; Chornokur et al., 2011).

Although prostate cancer in its early stages is responsive to standard treatments, patients with hormone-refractory prostate cancer have an overall median survival of 9–18 months, and no currently available treatment produces a survival advantage (Chen et al., 1998; Feldmann et al., 2000; Jamison et al., 2005). Deaths related to prostate cancer are invariably due to tumor invasion and metastasis to the lymph nodes, lungs, and bone marrow (Turner et al., 1996). Consequently, efforts to improve our understanding of the progression to the invasive and metastatic stages of prostate cancer should enhance the chances for developing meaningful therapeutic approaches.

An intraperitoneal xenograft model has often been employed to assess the invasiveness of human androgen independent prostate carcinoma cells (DU145) into the diaphragm (Turner et al., 1996, 1997; Fizazi and Navone, 2005). This model of invasion is used because of its reproducibility, it is quantifiable, and it correlates strongly with invasion from orthotopic tumor growth (Mamoune et al., 2004). While this model was originally employed to evaluate the metastatic mechanism of prostate tumor in nude mice (Kubota, 1994), the model has recently been employed to elucidate the molecular pathways involved in invasion (Turner et al., 1996, 1997; Chunthapong et al., 2004; Yue et al., 2010; Rybak et al., 2011) as well as the ability of drugs to modulate this invasion (Darnowski et al., 2004). However, in these studies, the accompanying imaging has been performed primarily at the level of light microscopy (LM). In this study, light, scanning, and electron microscopy techniques have been performed to characterize the colonization and invasion of the diaphragm and other peritoneal sites by DU145 cells. This investigation also demonstrates that two types of tumor morphology and invasive cell populations develop from the DU145 cell line originally implanted in nude mice. It is also illustrates that the original epithelium of the peritoneal surfaces is displaced and replaced by the DU145 cells of the growing tumors.

MATERIAL AND METHODS

Tumor Cells

The human androgen-independent prostate carcinoma cell line (DU145) was obtained from the American Type Culture Collection (Rockville, MD) and grown in McCoy's 5A medium (M5A, Gibco, Grand Island, NY) supllemented with 10% fetal bovine serum (FBS; Gibco; 2.2 g/L NaHCO3 and 50 μg/mL gentamicin sulfate (Fisher Scientific, Pittsburgh, PA). Cells were grown in a humidified atmosphere at 37°C in 5% CO2. When the tumor cells were used for experiments, they were harvested with trypsin-ethylenediamine tetraacetic acid (EDTA) (IRH Biosciences; Lenexa, KS), washed with PBS, pH 7.4 and resuspended in M5A. DU145 cells were counted with a hemocytometer using trypan blue (Gibco) exclusion to determine the number of viable cells.

Animals

Male athymic nude mice (NCr-nu/nu; 4 weeks old) were purchased from Taconic Farms (Germantown, NY) and maintained in microinsulator cages (within the AALAC accredited NEOUCOM Comparative Medicine Unit, Rootsown, OH) in a pathogen-free insulation facility. After a 1-week isolation period, 5 × 106 DU145 cells were injected intraperitoneally into the nude mice. Upon death of the mice (mean 60 days), autopsy was performed. All animal procedures were performed under protocols approved by the Northeastern Ohio Universities College of Medicine Animal Care and Use Committee.

Tumors

In all implanted mice, a visual examination revealed that several organs (kidney, diaphragm muscle, liver, and digestive tube) of the peritoneal cavity possessed minute to large tumors on their peritoneal mesothelium. Using a dissecting microscope, tumors were excised from their organ locations and washed in physiologic buffer to remove any blood. Some of the tumors were fixed in Carnoy's fixative for 3 hr, processed, and embedded in paraffin. Other specimens were embedded in paraffin, stained by hematoxylin and eosin (H&E) and were examined with LM.

Processing for Electron Microscopy

The remainder of the samples were fixed with 3.2% buffered glutaraldehyde solution (0.1 M Na cacodylate, pH 7.32) for 10 min at 4°C. After two rapid washes with sucrose cacodylate buffered solution (SC), the preparations were post-fixed for 30 min at room temperature in 1% aqueous osmium tetroxide-ruthenium tetroxide (1:1). Following fixation, the samples were washed four times with SC buffer and dehydrated through graded ethanol.

Scanning Electron Microscopy

Dehydrated samples were critical point dried in an E-3000 unit (Bio-Rad, Cambridge, MA). All samples were sputter-coated with a layer of gold-palladium to a thickness of 15–20 nm and observed with a Hitachi S-570 SEM equipped with tilting holder (Mountain View, CA) at 12–13 kV emission accelerating voltage at a distance of 12–15 mm from the specimens. Micrographs were obtained through a Polaroid attachment (Cambridge, MA.) using Polaroid type 55 film (Gilloteaux et al., 1998).

Transmission electron microscopy

Dehydrated samples were embedded in epoxy resin (Polysciences, PolyBed 812; Warrington PA) (Gilloteaux et al., 1999a,b). One-μm thick sections stained by toluidine blue (Aparicio and Marsden, 1969) of several pieces of each embedded specimen were examined with a Leitz and Olympus AX 70 Provis photomicroscope. Selected areas were chosen to obtain ultrathin sections. Ultrathin sections were collected on 50 and 100-mesh hexagonal copper grids (SPI, West Chester, PA), contrasted by uranyl acetate and lead citrate, before their examination in a Zeiss EM-10 Transmission electron microscopy (TEM), Carl Zeiss, Thornwood, NY. Digitized images were captured with an analySIS 2.1 software system© (Soft Imaging System GmbH, Lakewood CO and Münster, Germany) attachment to the TEM of the Pathology at the Pathology Department of the Children's Hospital Medical Center of Akron, Ohio.

RESULTS

Gross Appearance of the Tumors

All the mice implanted with DU145 cells possessed highly disseminated tumors on multiple parietal and visceral peritoneal surfaces. Many organs exhibited whitish-gray to pink tumors. Tumors also were found on the mesentery and the serosal surfaces of the tubular digestive tract (Fig. 1A), diaphragm (Figs. 1B, 2B), digestive glands, liver, pancreas, and gallbladder (Fig. 2A). Furthermore, small papillary tumors were found on the lower, dorsal surface of the abdominal cavity as well as the peritoneal or retroperitoneal covered surfaces, including the kidneys, urinary, and reproductive tracts. A few tumors also were discovered along the abdominal aorta wall, and attached to the capsule of lymph nodes. Most of the lymph nodes were abnormally swollen with some diameters as large as 2 mm, while others bore huge tumors attached to their capsules. Large tumors also were seen on the seminal vesicle wall. When these tumors reached a thickness of 1–3 mm, they often resembled sponges because of their white to red-speckled color, domed-shape, and their irregular tufts.

Figure 1.

AC: Three DU145 xenograft tumors in nu/nu mice attached via a short stalk to the small intestine wall (A) and with large bases to the diaphragm (B) and liver (C). In B, a dichotomous architecture is noted: the basal region of the tumor develops as carcinoma while the upper region is adenoid to adenocarcinoma. The two regions are separated by a narrow space. C: This pane depicts a solid carcinomatous mass with pleiomorphic cells (also shown with SEM in Fig. 3A). Scale bars in A and B equal 100 μm and scale bar in C is 25 μm.

Figure 2.

A–C: EM views of nu/nu mouse mesothelium covering the diaphragm. A: SEM view depicts an area of typically raised perikaryal morphology of the squamous mesothelial cells, their rich coat of entwined microvilli and an example of a large, reflective secreted body. B and C: TEM views of the mesothelial cells (Me) lining a continuous basement membrane composed of a basal lamina, its reticular matrix containing typically contrasted, discontinuous elastin bands made by a submesothelial layer of fibrocytes (F). Each mesothelial cell displays a large nucleus and active nucleolus, intricate microvilli, a narrow cytoplasm with small, round mitochondria, abundant reticulum, and secretory vesicles. The same secreted lamellar bodies that appear in A as snouts or reflective bodies are arrowed. L, peritoneal lumen. Scale bars in all micrographs equal 5 μm.

Histology

The majority of tumors covering the peritoneal surface of the diaphragm were easily detected as minute, superficial, flat to dome-shaped, or spheroid growths (Figs. 1A–C, 2E, 8D). These small, dome-shaped tumors are composed of compact cell aggregates with smooth to crypt covered surfaces and are found where the mesothelial cells are absent (Figs. 2E and 8D). However, some tumors are large and spherical to papillary in shape with pedunculate attachments to the diaphragms (Fig. 1A). These spherical tumors are composed of loosely associated DU145 cells, which sometimes give rise to fluid-filled, intercellular spaces or develop into poorly organized, endothelial-lined vessels (Fig. 8B).

Random cross sections of most tumors viewed with LM reveal closely clustered cells with tight basal attachments to the peritoneum and trabecular to crypt-covered surfaces (Fig. 1A–C). The basal regions of the tumors have always a solid, carcinomatous aspect in that they appear as compact clusters of cells, which are intimately attached with each other as well as the peritoneal connective tissue. Conversely, the upper portions of the tumors protrude into the peritoneal cavity and are composed of less intimately associated cells with crypt-like aspects. This adenoid architecture evolved from the compact basal region of the tumor and producing a cribriform epithelium out of the compact, pseudostratified aspect constituting the tumors formed by the clear, pale cells. Hematoxylin and eosin staining revealed that the tumors are composed of two populations. The majority of the tumors consist of cells that are lilac to pinkish-grey cells (pale, eosinophilic cells). These eosinophilic cells are especially prominent near the apices of the tumors while the second population of large basophilic cells predominates in the basal regions of those tumors (Fig. 1B). These basophilic DU145 cells (probably are putative tumor stem cells) are pleiomorphic, appear twice as large as the other cells and are disseminated along the basal zone of the tumor masses above the submesothelial areas. Subsequently, these basophilic cells develop into small nests, clusters, and then nodules, which invade the submesothelial region and then the muscle stroma (Gilloteaux et al., 2012).

Tumors associated with the gut mesentery or with the seminal vesicles appear as adenocarcinomatous with a rich blood supply captured from the serosae of the respective organs. These tumors connect to the organs through either a peduncular attachment (Fig. 1A) or a direct adherence of tumor cells to the adventitial layer of the organ.

As tumors on the diaphragm increase in size, larger, compact spheroids develop from dome-shaped aggregates on the basal region, while bush-like tumors with an appearance reminiscent of papillomatous adenomas form in the upper regions of the tumor. This dichotomous morphology appears to be directly proportional to the size of the tumors. This phenomenon is best illustrated in Fig. 1B where a tumor shows large, superficial dome region composed of an adenocarcinomatous mass separated by a space from the basal dome-shaped tumor mass from which it originated.

Following the attachment of the DU145 cells to the mesothelium, initial tumor growth is into the peritoneal cavity because tumor invasion is restricted by the mesothelial basement membrane and its submesothelial connective tissue. Hematoxylin and eosin staining of tumor sections revealed a population of large, basophilic DU145 cells disseminated along the basal zone of the tumor masses above the submesothelial areas, which may be responsible for invasion of the tumors (Fig. 1B). Subsequently, these basophilic cells develop into small nests, clusters and then nodules, which invade the submesothelial region and the muscle stroma (Gilloteaux et al., 2012, submitted for publication). In at least two of the mice, these tumor cells invaded the entire width of the diaphragm and eventually reached the pleural cavity. Other clumps of tumor cells, entered lymph vessels and were disseminated to other body sites.

The Typical, Intact Mesothelium Surfaces in the nu/nu Mice

Upon scanning electron microscopic (SEM) examination, the mesothelium of the diaphragm appears similar to that described in humans and most vertebrates. The peritoneal surfaces are covered by a single layer of mesothelial cells with a uniform morphologic population resembling paving stones that give these surfaces a typical cobblestone aspect (Figs. 2A, 3A–D, 4A,B).

Figure 3.

AE: Nude mouse peritoneal surfaces covered by growing DU 145 tumors (A, B, E) and typical mesothelial surfaces (C, D). In A: a spherical tumor growth over the liver; B: A serpentine tumor is seen on the rolling surface of the diaphragm. C, D: The mesothelial epithelium covering the liver with a uniform morphologic population of cells resembling paving stones giving these surfaces a typical cobblestone aspect. D: The mesothelial cells coating the liver possess long or short microvilli. E: A 1 μm-thick, toluidine blue stained section provides LM image of a tumor similar to that seen in B. Scale bars in A and D are 1 μm; scales bars in B–E equal 10 μm.

Figure 4.

A, B: SEM view of the mesothelial surface of the diaphragm muscle of a nu/nu mouse. A: A perpendicular view shows the polygonal profile of the cobblestone-like, epithelial cells, which are coated by microvilli. Some scattered, highly reflective secretory material has been expelled into the apical and intercellular, spaces. B: An enlarged view of the same mesothelium taken at a 15° tilt angle features a field of cell apices coated by irregular microvilli. The diverse stages of apices changes caused by secretory activity can be seen as well as a large reflective, folded, lamellar body in the process of being expelled is visible (see arrows in Fig. 2C). Scale in A equals 10 μm; scale in B is 1 μm.

The typical mesothelial lining the liver (Fig. 2A,C,D) and other peritoneal surfaces show homogeneous, flat to low cuboidal squamous cells coated by short to punctate microvilli (h < 0.5 μm). However, in the nude mice strain used in this investigation, the mesothelial cells that comprise the peritoneal serosal coating of the diaphragm (Figs. 2A–C, 3B,E, 4A,B, 8C,D) appear dome-shaped and are of greater height than those comprising the liver serosal layer (Fig. 3A,C,D). In contrast to the typical mesothelium covering the liver, tubular digestive tract and other organs, this murine mesothelium exhibits microvilli of diverse lengths and densities, which sometimes are entwined (Fig. 2A,B). In addition, apical secretory activity was captured during specimen processing. Apical excrescent bulges and expulsion of a highly reflective, vesicular material (d = 1.5–2 μm) can be seen. This extruded material is often retained in the intercellular, apical spaces of neighboring cells (Figs. 2A, 4A,B). This secretory activity appears to be widespread because of the frequency of apical depressions and operculate openings following secretion.

Viewed with the TEM the epithelial cells are seen to be 6 μm in height and 10–14 μm along their longest axis. These cells possess euchromatic nuclei with large, active nucleoli, and contain many small round mitochondria. Their apical surface is coated by an intricate coat of microvilli and microvilli-like extensions of variable height. The latter are quite intricate and are associated with an active secretory activity. This secretory activity entails the cytoplasmic production of abundant irregularly shaped vesicles, ranging from 200 to 1000 nm. These vesicles reach the surface, open to the extracellular space, and liberate their weakly electron dense content. These secretory vesicles have a size similar to those apical, highly reflective, vesicular bodies described by SEM in the previous paragraph (Fig. 2A–C).

The mesothelial cells are interconnected by typical junctional complexes and attach to their continuous basal lamina that is a portion of the basement membrane. The reticular layer contains a submesothelial layer contains a blood supply and active fibrocytes, which maintains an intricate network of collagen bundles and a discontinuous layer of elastic fibers. These elastic fibers appear as elongated electron densities ranging from 0.5 to 4 μm in length and about 0.5 μm in thickness and aligned parallel to the mesothelial surface plane (Figs. 2B–D, 8D). This submesothelial layer continues into the diaphragm muscle endomysial stroma where the skeletal myocytes are found.

The DU145 Tumor Surfaces

SEM images of the mesothelium of the diaphragm colonized with DU145 cells offer a stark morphological contrast to the typical mesothelium (Figs. 3A,B, 5A,B, 6A–F, 7A). Unlike the microvilli covered epithelial cells that give a cobblestone appearance to the normal epithelium (Fig. 4), tumor colonized mesothelium appears to be composed of fibroblasts, which give an apparent smooth surface to the mesothelium (Figs. 1A–C, 8E). At a higher magnification, the apparently smooth surfaces (Figs. 7B,C, 9A,B) are seen to contain troughs, cysts, and irregular, sometime papillary, spongy, or trabecules (Figs. 2E, 7B,C, 8A,C,D, 9C). All LM and SEM images of the surface morphology indicate that the tumor colonized diaphragms are not coated by mesothelial cells, but by the fibroblast-like colony of DU145 cells (Figs. 6A,B, 7A).

Figure 5.

A, B: SEM views depict DU145 tumors growing on the surface of the nu/nu mouse diaphragm. A: The overall view demonstrates the tortuous and pleiomorphic nature of the surface epithelial cells. Small crypt-like troughs with cell morphologies that resemble those of DU145 fibroblast (DU145-F) population. B: Enlarged view of the surface cells of the invading tumor exhibiting superficial, abraded areas, and lack distinct cell outlines. Tumor cells possess minute, aborted or no microvilli, while a remaining mesothelial cell (Me) (see Fig. 4B) is coated by microvilli and secretory activity. Notice that the DU145-F cells appear to be in the same plane as the mesothelial cell. Scale in A is 10 μm, in B equals 1 μm.

Figure 6.

A–E: SEM plate illustrating more of the pleiomorphic apical surface of a DU145 tumor growing on the surface of a mouse diaphragm. A: This low magnification view highlights the crypts on the tumor surface, which gives it a sponge-like appearance. B and C: Two panes demonstrate a tumor cell surface with cells with stellate shaped microvilli that are comparable to the epithelium-like population of the DU145 cells (DU145-E). D: A chain of smooth-surfaced cells making circular-shaped depressions or intracellular lumen-like structures. E and F: Tumor surface cells with conical, snout-shaped microvilli. Scale in A is 25 μm; scales in B, C, E, and F are 1 μm; the scale in D is 5 μm.

Figure 7.

A–D: SEM (A) and LM of 1 μm-thick, toluidine blue stained sections (B–D) of the surface of a DU145 carcinoma attached to the nu/nu mouse diaphragm. A: This view illustrates and suggests the dynamic activity of the growing surface cells and depicts a diverse surface apical coating. B–D: LM views complement the observations of pane A and verify that carcinoma cells line the peritoneal lumen (L). Examples of frequent replicating cells are denoted by arrows. Cancer cell damages or deaths create a cryptic or cribriform adenoid aspect. Capillaries (Cp) are noticed with either incomplete (C) or complete (D) endothelial linings. All the scale bars equal 10 μm.

Figure 8.

A–E: LM (A, C, and E) of 1 μm-thick, toluidine blue stained sections and TEM (B and D) views depict the colonization of the nu/nu mouse diaphragm mesothelium by the DU145 tumor cells. A: A cluster of pale-staining DU145 cells containing a basophilic cell (a putative DU145 cancer stem cell) proximal to the mesothelial surface. B: On the lower right of this pane, a DU-fibroblast-like (D) already attached to the submesothelium extends its processes and undermines the adjacent, darkly contrasted mesothelial cells (Me) from their attachment sites. Atop those cells, a mesothelial cell disconnected displays a lytic nucleus where the chromatin outlines its inner membrane and undergoes autoschizis cell death. C: This composite pane shows a clump of DU145 cells (D) colonizing the mesothelium lining. D: On the right side, a DU145 clump is bulldozing mesothelial cells from their attachment sites (arrowheads). A capillary (Cp) and submesothelial fibrocytes (F) adjacent to diaphragm myocytes are seen (also in Fig. 2E). E: This LM inset depicts a dome-shaped DU145 tumor on diaphragm. Scales in A–D is 10 μm; scale in E equals 25 μm.

Figure 9.

Schematic cartoon based on light and ultrastructural data depicting our interpretation of invasiveness and forced excision of the mesothelial cells (pale cells) by DU145 cells (dark cells) on the nu/nu mouse diaphragm and other peritoneal surfaces to form carcinomas.

As was the case for the LM images, SEM views demonstrate the surfaces of the mesothelia of tumors in the implanted mice differed in morphology from the normal mesenteric surfaces, and reinforced the concept that the basophilic stem cells and fibroblast-like population of DU145 cells are invading the diaphragm. On the liver mesothelial surface, tumor growth is usually seen as clumps of cells that organize into dome shapes and then become almost spherical in shape, while on other mesothelial surfaces, tumor growth can be quite irregular. In all tumors attached to the diaphragm (Figs. 1B, 2B–E, 5A,B, 6A–F, 7A), the metastatic surfaces appear in disarray with irregular, bulging, and spongy surfaces comprised of pleiomorphic cells with no secretory activity and few microvilli, if any. Many tumors also displayed a spherical bulging aspect on their surfaces (Figs. 1C, 3A, 9D) and exhibit an elongated morphology, swollen perikarya, and loss of surface specialization that strongly suggest they are dividing cells. However, these cells may represent either the fibroblast-like population of the DU145 cells or the epithelial population of the DU145 cells undergoing an epithelial to mesenchymal transition. All these SEM aspects are in contrast to the surfaces of the normal, quiescent Mesothelium, and typical human prostatic tumors. In Fig. 5B (left quadrant), the invasive DU145 cells have not covered a surface mesothelial cell appearing between the spreading arms of the growing tumor and showing the microvilli and surface secretory bulges. Another supplemental argument for the tumor cells to have replaced the mesothelium is that DU145 cells, as pleiomorphic, they show in Fig. 6B,C stellate-shaped microvilli, similar to those uniquely described as coating the epithelium of seminoma tumors (Tannenbaum, 1979).

Tumor Cells Invasion of the Peritoneal Surfaces

DU145 cells inoculated into the abdominal peritoneum may be found as isolated ascites or clumps of cells in the vicinity of the surface mesothelial cells. After establishing a stable focal, adhesion site, the DU145 cells with the epithelial phenotype assume a fibroblastic phenotype (or perhaps the DU145 cells with the fibroblastic phenotype themselves), insert their processes into spaces in the basal lamina underlying the mesothelial cells and efficiently scoop these mesothelial surface cells from their basement membrane. One can detect very subtle and delicate lamellipodia under the adjacent mesothelial cells where the detachment process is initiated (Fig. 8B,D). After removing the mesothelial cell, a space is created, which is now occupied by the tumor cell. Then the tumor cell replicates to form a tall mass of cancer cells. At the edge of the tumor mass, the mesothelial process continues to allow installation and attachment. Further growth on the same basal lamina components continues until the basement membrane and the subjacent, interrupted elastin-containing, submesothelial layer becomes a stromal layer for the growing carcinoma (Figs. 8A–D, 9). In all the ultrastructural micrographs illustrating this study (Figs. 1B, 7C, 10A,E) and in two other complementary studies (Gilloteaux et al., 2012, submitted for publication), the DU145 carcinoma cells are growing on the diaphragm or elsewhere on other peritoneal organs. One has not found a mesothelial cell between the DU145 carcinoma cells and their attachment on the submesothelium, that is, the connective tissue is freed from mesothelial covering. One can only interpret from these features and the observed carcinomas that the mesothelial covering cells have replaced by tumor cells, as suggested by an active excision of them by the DU145 cells as found by such complex picture noted in Fig. 8D.

The Blood Supply of the DU145 Tumors

Capillaries (or larger vessels) containing blood cells were readily visible in the lower portion of the tumors during cursory LM examination. However, these blood-containing vessels were much more difficult to detect in the upper regions of the tumor (Figs. 8B, 10A–D). Closer examination of these vessels at higher magnification in toluidine blue-stained, 1 μm-thick, plastic sections revealed the presence of endothelial cells lining some capillaries (Figs. 7B,D, 9C–E). However, in other blood supplies, especially those in the apical zones of the tumors, what one could call “blood spaces” were only lined by clear, pale tumor cells without any organized lining endothelium (Figs. 7C, 9B,E). The presence of lymph vessels could not be verified.

Figure 10.

A–E: LM of 1 μm-thick, toluidine blue, stained sections (A and B), and H& E-stained paraffin embedded sections (C–E) of the surface epithelium and blood supply to DU145 tumors growing on the diaphragm of a nu/nu mouse. A and B: These panes depict the apical region with pale and dark cells covering the tumors. Notice vascular spaces (arrows) containing circulating cells either without or partially delineated by endothelial linings. C: A capillary with endothelium can be found among tightly aggregated cells, near the surface of the growing tumor and resembles that showed in Fig. 7D (arrow). D and E: These panes illustrate a large, DU145 tumor attached to the pancreatic peritoneum with a blood supply, which has been branched into the tumor (arrows). E: This pane is an enlarged view of the mid section of the pane D to see blood spaces (primordial capillaries) that do not contain any endothelial lining (arrow). Notice the upper carcinoma region with round, scattered cribriform spaces among the tightly growing cancer cells. Scale bars in A–C and E equal 10 μm; scale bar in D is 50 μm.

DISCUSSION

The mesothelial surfaces in the nude mice are similar to those in humans (Leak and Rahil, 1978; Slater et al., 1989; Jonecko, 1990), other animals including mice (Carr et al., 1969; Watters and Buck, 1972; Schwarz, 1974; Baradi and Rao, 1976; Guggenheim et al., 1979; Hjelle et al., 1989; Gaudio et al., 1990; Ettarh and Carr, 1996; Bot et al., 2003; Michailova and Usunoff, 2006; Yung et al., 2006; Yung and Chan, 2007). The epithelial cells show secretory activity with the apical release of lamellar bodies analogous to those of alveolar or pneumocytes type II (Dobbie and Lloyd, 1989; Dobbie and Anderson, 1996a,b) as well as excrescences and expelled material similar to those observed in the gallbladder epithelium (Gilloteaux et al., 1993). Furthermore, the morphology of the epithelial cells of the mesothelium may vary in accordance with changes in their metabolic activity or following cell injury (Li and Jiang, 1993; Li et al., 1996; Michailova, 1997; Mutsaers, 2002; Yung et al., 2006; Yung and Chan, 2007).

All the mice implanted with DU145 cells possessed highly disseminated tumors on multiple parietal and visceral peritoneal surfaces. SEM and TEM of tumor bearing mice demonstrate the uniform morphology of the cobblestone-like, microvilli-coated epithelial cells of the mesothelium have been displaced by DU145 through epithelial damage (Groothuis et al., 1988; VanderWal et al., 1997), amoeboid characteristics of the tumor cells (Koga et al., 1980; Nakamura, 1994), cell surface modifications (Abercrombie and Ambrose, 1962; Abercrombie, 1967), lytic enzymes, or increased proliferation and movement mediated through paracrine activities of the adjacent tumor or stromal cells (Vo and Khan, 2011) such as hepatocyte growth factor (HGF) (Nishimura et al., 1998, 1999; Mamoune et al., 2004; Oosterhoff et al., 2005; Cai et al., 2008; Wang et al., 2010). A similar process of invasion was reported in the case of ovarian tumor cells in vitro (Iwanicki et al., 2011; Kenny et al., 2011). The presence of stellate-shaped microvilli is unique because they are seen only in some rare human seminomas (Tannenbaum, 1979).

LM of random hematoxylin and eosin stained cross sections of the majority of tumors reveal a mixed architecture (Fig. 1A–C). The obvious question is how did this morphological dichotomous tumor arise from a single tumor cell population?

The DU145 cell line is an androgen independent cell line derived from prostate carcinoma cells of a brain metastatic site of a 69-year-old, male, Caucasian patient (Stone et al., 1978). LM of the DU145 cell line shows pleiomorphic cells with at least two distinct phenotypes, elongated and polygonal (Peehl, 1994) and a small population of stem cells (Pfeiffer and Schalken, 2010). After serial passage of these cells through basement membrane-coated membranes, Chunthapong et al. (2004) were able to isolate a poorly invasive population with epithelial-like morphology (DU145-E) and a second, highly invasive population with elongated, fibroblastic-like morphology (DU145-F). These DU145-E cells possessed many filopodial extensions from the cell surface and many prominent infoldings or plicae and long, microvillar projections, while the DU145-F cells were more elongated, exhibited few if any cytoplasmic extensions, possessed filopodia extending from the leading edges of migrating cells and exhibited membranous folds and short microvilli on their cell surfaces.

E-cadherin is an important regulator of invasion in prostatic carcinomas (Morton et al., 1993; Lu, et al., 2003). DU145-E cells express a similar amount of E-cadherin as the parental DU145 cells, while the DU145-F cells express <0.1-fold of E-cadherin than the parental DU145 or DU145-E populations (Chunthapong et al., 2004). The levels of two E-cadherin-associated proteins, β-catenin and p120cas, also decreased, while the protein levels of cytokeratin 18 (an epithelial marker) and vimentin (a mesenchymal marker) remained constant in both populations and higher extracellular levels of pro-matrix metalloproteinase-2 (pro-MMP-2) were found in the DU 145-F population. This decreased expression of E-cadherin and increased vimentin expression was confirmed in DU145 cells forming subcutaneous tumors in SCID mice (Luo et al., 2006; Zhao et al., 2011) and suggest the DU145-F cells have undergone an epithelial-mesenchymal transition (EMT) to promote invasion and metastasis. Cancer cells may pass through EMTs to differing extents, with some cells retaining many epithelial traits while acquiring some mesenchymal ones and other cells shedding all vestiges of their epithelial origin and becoming fully mesenchymal (Kalluri and Weinberg, 2009).

As mentioned previously, the epithelial cells of the intact mesothelial surfaces (Figs. 2A–C, 3A–E, 4A,B, 8C,D) are displaced and replaced fibroblast-like tumor cells with very few microvilli (Fig. 5A,B). This process has been described in general by McCandless et al. (1997). The morphology of these cells closely resembled the DU145-F population of the parental DU145 cell line whose level of E-cadherin was decreased 10-fold while their extracellular levels of metalloproteinases were elevated. These fibroblast–like cells are believed to have undergone an EMT in an effort to enhance their migratory capacity, invasiveness, and to elevate their resistance to apoptosis (Kalluri and Weinberg, 2009). Paradoxically, these EMT-derived migratory cancer cells typically establish secondary colonies at distant sites that resemble the primary tumor from which they arose (Zeisberg et al., 2005; Coulson-Thomas et al., 2010). This observation suggests that the metastasizing cancer cells may shed their mesenchymal phenotype via a mesenchymal-epithelial transition (MET) during the course of secondary tumor formation (Zeisberg et al., 2005). If the fibroblast-like cells seen in our images maintain some of their epithelial and mesenchymal properties like the DU145-F population of the parental cell line, a MET is not too difficult to conceive.

Figure 8A shows a cluster of pale-staining DU145 cells surrounding a basophilic cell (a putative DU145 cancer stem cell) proximal to the mesothelial surface and suggests the interesting possibility that, in this model, successful metastasis may require the presence of both fibroblast-like cells (DU145-F) and one or more tumor stem cells. This could help explain why usually <100 tumors are formed from the 5 × 106 DU145 cells that are injected. If indeed, DU145 stem cells represent 0.01% of the population and the plating efficiency of these in forming holoclones in vitro is 35% (Pfeiffer and Schalken, 2010), one would expect a maximum of 175 colonies (5,000,000 × 0.0001 × 0.35 = 175). If it is assumed that the plating efficiency in vivo is only 1/3 as effective as in vitro, a maximum of 60 colonies (5,000,000 × 0.0001 × 0.12 = 60) is expected from an injection of 5 × 106 DU145 cells. Regardless of the plating efficiency, the basal regions of the resulting tumor surrounding the putative stem cell strongly resemble the holoclones produced in the DU145 experiments (Barrandon and Green, 1987). Subsequently, these basophilic cells develop into small nests, clusters, and then nodules, which invade the submesothelial region and then the muscle stroma. Conversely, the paraclones are highly irregular in shape and contain more flattened and scattered cells that resemble the morphology of the upper portions of the tumors that protrude into the peritoneal cavity and are composed of less intimately associated cells with crypts that announce a future adenocarcinomatous architecture.

The dichotomous morphology in the most advanced tumors results from a process akin to prostatic intraepithelial neoplasia (PIN) (McNeal, 1969; Bostwick et al., 1993; Helpap and Riede, 1995; Bostwick and Qian, 2004), studied morphologically by Kastendieck and Altenähr (1976) and recently reviewed by several authors (Montironi et al., 2000; Shin et al., 2000; Latour et al., 2008; Epstein, 2009; Dema et al., 2010; Shah et al., 2010). Prostatic lesions show isolated voids that appear as “sieve holes” within the tumors and evolve into a “cribriform acinar prostate carcinoma” through programmed cell death (Nagao et al., 2003; Shah et al., 2010). These areas can then evolve into adenoid or adenocarcinomatous aspect (Epstein, 2009; Dema et al., 2010) by increasing their number and enlargement of the voids (Figs. 1B, 7C,D, 10A,E) and have occurred (Fig. 10A,B,D) as the result of entotic deaths (Brouwer et al., 1984; Overholtzer et al., 2007). As noted, these cribriform structures are initially distributed in the upper regions of the DU145 carcinomas. They have been further described and discussed in other studies (Gilloteaux et al., 2012, submitted for publication).

Tumor growth and metastasis are dependent on the tumor's supply of blood vessels (Folkman, 1995). In this study, LM show blood-containing capillaries lined by endothelial cells (Figs. 7B,D, 9C–E) as well as blood-containing capillaries that appear to be lined by tumor cells without organized endothelium (Figs. 7C, 9B,E). SEM images (Figs. 6D, 7A) suggest these capillaries arise from a tumor cells that strongly resemble the DU145-F population. The chain-like structure of tumor cells that runs from the upper left to the lower right of Fig. 6D gives rise to a capillary-like structure in Fig. 7A. To explain these observations one needs to examine the process of tumor vascularization.

The architecture of tumor vasculature is different from normal vasculature because of its incomplete endothelial lining (Steinberg et al., 1990; McDonald and Foss, 2000). Tumor tissues progressively become hypoxic and necrotic due to rapid proliferation and insufficient blood supply and must constantly make new vasculature (Furuya et al., 2005). Angiogenesis is one method of developing vasculature with angiogenin expression being elevated in prostate cancer cells and their stem cells (Katona et al., 2005). Analysis of the tumor vascular bed of human prostate carcinomas by quantifying microvessel density count, proliferating capillary index, proliferating tumor versus endothelial cell index; and microvessel pericyte coverage index (MPI) indicates that prostate tumors are not very angiogenic and yet only one-third of the vasculature within these tumors is covered by pericytes (Eberhard et al., 2000). These observations suggest the majority of prostate tumor vasculature is being generated by a process other than angiogenesis.

Other mechanisms of tumor vascularization include vessel co-option, intussusception, recruitment of endothelial precursor cells (EPCs) and vasculogenic mimicry (VM) (Paulis et al., 2010). VM describes the ability of tumor cells to express multiple cellular phenotypes, gain endothelial characteristics, and form vascular-like networks (Maniotis et al., 1999; Folberg and Maniotis, 2004). In prostate tumor cells, VM involves cooperative interactions of distinct phenotypic subpopulations (Sharma et al., 2002) and higher expression of the basement membrane extracellular matrix components laminin5γ2, and metalloproteinase (MMP)-1, −2, −9, and −14 [membrane type (MT)1-MMP], which act cooperatively to form tubular networks of tumor cells, without endothelial cells or fibroblasts (Seftor et al., 2001; Kaminski et al., 2006). In this study, VM appears to be driven by the DU145-F population alone or in combination with the DU145 stem cell population (Jones et al., 2012). The DU145-F cell population fits the morphological profile and the genotypic profile in that it possesses both epithelial and mesenchymal markers, expresses EphA2, MT1-MMP and produces elevated extracellular levels of pro-MMP-2.

CONCLUSIONS

The results of this study suggest that DU145 cells implant into the mesothelium and form carcinomas. Subsequently, they can develop into adenoid or cribriform carcinomas (Gilloteaux et al., 2012, submitted for publication) before forming more aggressive and invasive adenocarcinomas, which are disseminated to other organs. Specifically, this more aggressive cancer cell phenotype would penetrate through the diaphragm muscle and its stroma via its stomata (Leak and Rahil, 1978; Abu-Hijleh et al., 1995) and metastasize into the pleural space as well as the visceral and parietal mesothelium (Meyer, 1966; Renshaw et al., 1996; Bubendorf et al., 2000; Adewuyi et al., 2011). While many of the ultrastructural observations shown in this study await confirmation with molecular markers, the model provides a unique opportunity to study tumor invasion and vascularization in vivo directly using molecular and immunohistochemical techniques in conjunction with morphological techniques or indirectly using remote imaging techniques.

Acknowledgements

The content of this study was part of an oral presentation made at the first International Symposium on “Innovative Anticancer Drugs and Strategies,” held in Newcastle upon Tyne, June 2010 sponsored by St Georges' University School of Medicine, Grenada W.I. and New York, IC-Med Tech, San Diego CA and Summa Research Foundation, Akron OH in memorial for our colleague H.S. Taper MD, PhD, recently deceased. The authors again recognize Summa Health System Research Foundation, Akron OH and the Hess-Roth Kaminsky and Maxon Urological Foundation of Erie, PA for their support in this investigation. Mr. S. Getch, Communication Specialist of Summa is recognized for his contribution and care in the setting and electronic assistance of the quality imagery illustrating this report.

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