Molecular determinants of ovarian cancer plasticity.

During development, the formation and remodeling of primary vascular networks occurs by vasculogenesis and angiogenesis. Recently, the term "vasculogenic mimicry" has been used by our laboratory and collaborators to reflect the embryonic-like ability of aggressive, but not nonaggressive, melanoma tumor cells to form a pattern of matrix-rich networks (containing channels) surrounding spheroids of tumor cells in three-dimensional culture, concomitant with their expression of vascular cell markers. Ovarian cancer is usually diagnosed as advanced stage disease in most patients when widespread metastases have already been established within the peritoneal cavity. In this study, we explored whether invasive ovarian carcinoma cells could engage in molecular vasculogenic mimicry reflected by their plasticity, compared with their normal cell counterparts. The data revealed that the invasive ovarian cancer cells, but not normal ovarian surface epithelial cells, formed patterned networks containing solid and hollow matrix channels when grown in three-dimensional cultures containing Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. Immunohistochemical analysis showed that matrix metalloproteinases (MMP)-1, -2, and -9, and MT1-MMP were discretely localized to these networks, and the formation of the networks was inhibited by treatment with MMP inhibitors. Furthermore, the RNase protection assay revealed the expression of multiple vascular cell-associated markers by the invasive ovarian cancer cells. In patient tumor sections from high-stage, high-grade ovarian cancers, 7 to 10% of channels containing red blood cells were lined by tumor cells. By comparison, all vascular areas in benign tumors and low-stage cancers were endothelial lined. These results may offer new insights and molecular markers for consideration in ovarian cancer diagnosis and treatment strategies based on molecular vascular mimicry by aggressive tumor cells.

A novel immunological model for the study of prostate cancer.

The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.

Fine structural studies of induced tumors arising within the prostatic complex of Lobund-Wistar rats.

BACKGROUND: Morris Pollard, Phyllis Luckert, and colleagues have reported the occurrence of spontaneously arising tumors of the prostatic complex in aged Lobund-Wistar (L-W) rats, and have also shown that the genesis of such tumors may be accelerated by means of intravenous administration of methylnitrosourea, followed by androgen supplementation. METHODS: Light and electron microscopic investigations of the tumors arising under this regime were conducted, with the objective of documenting morphological changes attending the transformation process; 10 tumor samples were used for the electron microscopic studies. RESULTS: All tumors studied were adenocarcinomas arising within the prostatic complex of induced animals. These tumors varied in size, degree of differentiation, and invasiveness. Foci of prostatic intraepithelial neoplasia were noted in light microscopic studies as well. Consistent fine structural features exhibited by cells of the induced adenocarcinomas included a large nuclear to cytoplasmic ratio; large irregular nuclei with heavily marginated chromatin; conspicuous nucleoli; abundant ribosomes and polysomes and a paucity of rough endoplasmic reticulum; and numerous cytoplasmic vesicles and lipid inclusions. Numerous, short microvilli extended from the cell surface into a copious surrounding extracellular matrix. CONCLUSIONS: Thus, these tumors shared many of the fine structural features characteristic of the Dunning (rat) and human prostatic adenocarcinomas.

Gross and microscopic pathology of induced prostatic complex tumors arising in Lobund-Wistar rats.

The necessity for additional animal models for prostate cancer has recently been stressed. The Pollard model of chemically induced prostate cancer has received attention in this regard although the histiogenetic origin of these tumors has come under question. We independently studied this model for the development of tumors in the prostate region. The tumors, all of which were adenocarcinomas, first became grossly evident 5 months after induction and ultimately occurred in 71% of the animals. Seventy-three % of the tumors involved only the seminal vesicle, 22% involved other portions of the prostatic complex as well as the seminal vesicle, and 5% were located in the coagulating gland (anterior prostate). Although the majority of tumors arose in or involved the seminal vesicle, this may still be a useful model for the study of human prostate cancer because the tumors are adenocarcinomas, occur in the large majority of animals, are hormonally induced, and have the propensity to metastasize.

Peptidergic nerves in the ureter.

Vasoactive intestinal peptide (VIP) and substance P were demonstrated in the pig ureter by immunohistochemical techniques. Nerves containing these materials were related mainly to the smooth muscle layer in the normal and obstructed ureter. In isolated ureteral segments, VIP caused relaxation at doses exceeding 0.18 micrograms/ml, with no significant difference seen in the effect on normal and obstructed ureter. Vasoactive intestinal polypeptide may play a role in the regulation of ureteral smooth muscle tone.

Cell wall deficient bacteria as a cause of idiopathic hematuria.

Idiopathic hematuria in the absence of bacteriuria is a medical challenge. Routine cultures of catheterized bladder and endoscopically obtained ureteral urine specimens from a 22-year-old woman with a 6-week history of hematuria showed no growth after 24 to 48 hours of incubation. However, bacterial variants were grown on enriched media. Colonies were typical cell wall deficient/defective bacteria. Phase and electron microscopy of cystoscopic urine specimens obtained by retrograde ureteral catheterization as well as phase microscopy of the cultures revealed the classic morphology of these organisms. When the variant cultures were subcultured the organisms reverted to their related walled forms, that is Streptococcus agalactiae and Staphylococcus haemolyticus. Because the colonies of these organisms showed various patterns of biochemical reactivity, each phenotype was tested against 15 antimicrobials. Collectively, all biotypes had a common susceptibility to only nitrofurantoin. The patient was treated with nitrofurantoin for 6 weeks. Four days after initiation of therapy she had complete remission of hematuria. During the next 3 years she remained well and free of hematuria.

Stereological study of Leydig cell density in the guinea pig testis.

Stereological analysis of Leydig cell and macrophage volume density along the long axis of the guinea pig testis was assessed quantitatively by histometric point counting. Morphological identification of Leydig cells was accomplished by staining for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas macrophages were identified by vital staining (trypan blue). The average percentage of Leydig cell density constitutes about 10.17 +/- 0.23% at the cranial level (level 1) and about 8.8 +/- 0.21% at the caudal level (level 4). Leydig cell density through four testicular levels showed no significant difference among levels 1, 2, and 3, whereas level 4 was significantly different. The percentage of macrophage density, on the other hand, was approximately 0.4% +/- SEM per cross-sectional profile. It may thus be concluded that the macrophage density within the guinea pig testis does not bias histometric studies of the Leydig cell population to any significant degree, but that regional differences in Leydig cell volume density could influence validity of sampling if tissue procurement is from different testicular levels in successive experiments.

Characterization of the heterogeneity of R3327 rat prostatic tumors derived from single-cell clones.

Prostatic adenocarcinoma is characterized by cellular diversity, which is well demonstrated in the Dunning R3327 rat prostatic adenocarcinoma. This heterogeneity may arise from epigenetic influences, ie, cellular adaptation or selection, and/or from genetic changes. To investigate the question of genetic instability, four tissue culture cell lines were derived from single cells isolated from the uncloned late (UCL) passage of the Dunning R3327H prostate cell culture. Each of these clonally derived tissue cultures was injected into castrated and intact young adult male rats for tumor production. Uncloned early (UCE) and UCL passage tissue cultures were also propagated as solid tumors. Tumors and the cultures from which they were derived were examined for evidence of phenotypic and genetic changes using morphological and cytometric methods. Transmission and scanning electron microscopy revealed only slight differences among the cell cultures. A single population of diploid cells was demonstrated in each of the cell cultures by propidium iodide staining and subsequent flow cytometric measurement of DNA content/nucleus. Tumors of unicellular as well as multicellular origin exhibited extreme heterogeneity of histological features, both among animals as well as within a single tumor. Tumors were surveyed and tissue types were characterized and cataloged. Clone 3 was generally better differentiated than the others; tumors from castrated animals were better differentiated than those from intact animals. Flow cytometry revealed multiple hyperdiploid cell populations that were variable from one sample to another. We concluded that changes in genotype as well as phenotype occurred in the tumors derived from single cells. Some of these changes may have occurred in the cells while still in culture.

Androgen receptor binding characteristics in the cytosol of the rat dorsolateral prostate gland and the Dunning R-3327 prostatic adenocarcinoma.

The incidence of prostatic cancer is highly correlated with advanced age, and it has been suggested that changes in androgen binding may be important in age-associated alterations in growth regulatory mechanisms of prostatic epithelial cells. In this study the effects of age on androgen binding characteristics in the dorsolateral prostate glands of young and aged Copenhagen rats were determined and the binding properties in the Dunning R3327/130 subline of rat prostatic adenocarcinoma were characterized. Tritium-labeled and nonlabeled methyltrienolone analogs (R1881) were used to study the binding properties of 5 alpha-dihydrotestosterone receptor in the cytosol of tumors and prostate glands. Binding of R1881 was low but specific for the androgen receptor as shown by competition studies in which nonlabeled R1881, 5 alpha-dihydrotestosterone, and testosterone competed successfully with 3H-R1881 for binding sites, but 17 beta-estradiol and low levels of progesterone did not. In Copenhagen dorsolateral prostate, Scatchard analysis suggested a single class of binding sites. In young animals (three to five months) the average binding capacity was 10.36 fmol/mg cytosol protein with a dissociation constant (Kd) of 2.28 nmol/L. The dorsolateral prostate of aged rats (11-16 months) showed no significant difference in specific binding characteristics as compared to the younger age group. Specific binding of 3H-R1881 in R3327/130 tumor was saturable with a single class of high-affinity binding sites having an average binding capacity of 64.77 fmol/mg cytosol protein and a Kd of 2.76 nmol/L. These data show that the tumor had approximately 6.5 times the number of binding sites as did the normal Copenhagen rat dorsolateral prostate gland. However, no age-related changes were detected through 11-16 months of age in the androgen binding characteristics of normal rat dorsolateral prostate gland that could be correlated with the higher concentration of androgen binding sites in the R3327/130 tumor subline.

Isolation, in vitro cultivation, and electron microscopy of normal and malignant prostatic epithelial cells from the Copenhagen rat.

Procedures are described for the isolation and cultivation of normal rat prostatic epithelial cells. The techniques, which involve collagenase digestion and Ficoll purification of the epithelial population, are efficient, inexpensive, and produce pure monolayers. Included is a scanning and transmission electron microscopic study comparing cells isolated in vitro to rat prostatic epithelial cells in situ. Further ultrastructural comparisons are made to a malignant cell line, the Dunning R3327H Copenhagen rat prostatic adenocarcinoma.

Effects of estrogen upon the fine structure of epithelium and stroma in the rat ventral prostate gland.

We studied the effects of estradiol upon the fine structure of the rat ventral prostate, with special emphasis on the interstitial tissue. Intact and castrated rats were injected with estradiol or testosterone in combination, or with peanut oil (control). Estrogen exerted an antiandrogenic effect on the epithelium of intact animals resulting in lower epithelial height and reduction in the number of cell organelles and secretory bodies; however, the prostate gland did not atrophy to the same degree as that in castrated controls. In castrates, estradiol had no discernible effect upon the glandular epithelium. Although testosterone alone was able to restore glandular morphology to normal in these animals, estrogen in combination prevented regeneration of rough endoplasmic reticulum and elicited the formation of large lipid-like inclusions and the accumulation of secretory bodies in the apical zone of many cells. Intact animals treated with these hormones in combination exhibited many of the same effects except that a more normal configuration of rough endoplasmic reticulum was present. In castrates, the prostatic stroma became thickened, with a large increase in fibrous material between and surrounding each acinus, although smooth muscle cells retained their normal cytology. With estradiol treatment, singly or in combination with testosterone, smooth muscle cells increased in size and number and organelles decreased in number, cytoplasm became more electron dense, and nuclei became more heterochromatic. Surface vesicles were profuse in smooth muscle cells in animals treated with estradiol alone; larger phagocytic-like vacuoles were characteristic of the glands of animals treated with estrogen and testosterone in combination. In these latter treatment groups, testosterone prevented the overall hypertrophy and hyperplasia of the interstitial tissue; to a degree, endogenous testosterone levels in intact animals treated with estradiol alone prevented these effects as well.

Spermatic granuloma of vas deferens after vasectomy in rhesus monkeys and men: light and electron microscopic study.

Spermatic granuloma of the vas deferens is a common complication of vasectomy which has received scant morphologic study. This study investigated the light and electron microscopic structure of such granulomas detected in rhesus monkeys (Macaca mulatta) and man after various modes of vasectomy and postoperative periods. Unilateral experimental vasectomy in monkeys was performed by either silk ligation or clasp occlusion; in 4 of 13 ligated animals and 5 of 5 clasp vasectomized animals granulomas developed at the site of fasectomy. In man, portions of the vas deferens were excised adjacent to the site of vasectomy preparatory to vasovasostomy. Of 5 patients studied, unilateral spermatic granulomas developed in 3. Such granulomas in both monkey and man were characterized by (1) masses of sperm surrounded by epithelioid cells and connective tissue, and (2) multiple epithelial-lined channels which often contained sperm and spermiophages. In both species, fine structural characteristics of the epithelium lining such channels closely resembled those of the principal cells of the normal vas. Spermiophagic cells included macrophages, epithelioid cells, and, in the monkey only, neutrophils. Lymphocytic invasion was a common feature of the human granulomas but was found only occasionally in the monkey granulomas. As a greater number of granulomas are studied in humans and monkeys, it is hoped that the processes underlying granuloma formation and the role of such granulomas in the development of complications after vasectomy will be clarified.

Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment.

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM)electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.

Smooth muscle within ovarian decidual nodules: a link to leiomyomatosis peritonealis disseminata?

Evidence is presented for the coexistence of smooth muscle and decidual cells in nodules on and within the ovarian tunica albuginea at term. Routine histologic techniques and electron microscopy have been employed in characterizing the morphology of the nodules. Recent literature concerning the frequency of ovarian decidualization during pregnancy is discussed with respect to the possible relationship of such decidualization to the histogenesis of leiomyomatosis peritonealis disseminata (LPD). The hypothesis that LPD may represent "disseminated fibrosing decidua" is discussed in light of finding collagen fibrils, secretory decidual cells, and smooth muscle cells in these nodules. It is concluded that the present case does not represent "fibrosing decidua." The authors agree with others who have proposed that the smooth muscle in ovarian decidua and LPD result from proliferation of stem cells which may reside in the subperitoneal stroma in association with ectopic endometrial stroma and which may respond to the hormones of pregnancy.

Decidual cells in the human ovary at term. I. Incidence, gross anatomy and ultrastructural features of merocrine secretion.

Decidual tissue occurring within the human ovarian cortex was examined by light and electron microscopy. Of 21 ovarian specimens obtained at term (36-42 weeks of gestation), decidual cells were confirmed in each. Decidual cells were found within the tunica albuginea as single cells, in nodules, in polyps or in confluent sheets. Decidual cells exhibited several characteristics of cells engaged in secretory activity: abundant rough and smooth endoplasmic reticulum, numerous profiles of the Golgi complex and a large, euchromatic nucleus devoid of heterochromatin and displaying a prominent fibrous lamina. Peduncular protrusions at the periphery of the cell contained numerous dense bodies 0.4-0.9 micron in diameter. These dense bodies were bounded by a single membrane and contained granular subunits 30-60 nm in diameter. These granular subunits were observed in the process of apparent exocytosis, as well as free in the extracellular space. Secretory bodies and their granular content also were observed both in the region of the Golgi complex and partially extruded into peduncular processes. By far the greatest number of secretory bodies occurred within peduncular processes where they may be stored prior to release. Migration of a secretory body into a peduncular process and exocytosis from such a process appears to be an unusual mode of meocrine secretion, perhaps unique to decidual cells.

Vasectomy in rhesus monkeys. III. Light microscopic studies of testicular morphology.

Rhesus monkeys were randomly assigned to undergo various surgical procedures. The animals were followed from one to sixty-six weeks postvasectomy, at which time they were sacrificed and their tissues prepared for light and electron microscopy. Vasectomy in the rhesus monkey, as in certain other species, appears to be a procedure not attended with widespread testicular atrophy or histologic evidence of impaired spermatogenic potential utilizing the procedures and postoperative periods studied. Why certain animals exhibited focal degenerative changes is unclear; perhaps a certain population, yet to be defined, is more sensitive to such procedures, resulting in testicular alterations. It is important that such a population and such changes be defined to predict more accurately the possibility of successful vasovasostomy and reestablishment of fertility.