In vitro differentiation of rat seminiferous tubular segments from defined stages of the epithelial cycle morphologic and immunolocalization analysis

Rat seminiferous tubule segments have been cultured in chemically defined medium (F12/DMEM 1:1) without added hormones or growth factors. The segments (1-2 mm) were isolated from defined stages of the cycle of the seminiferous epithelium (VIII and XII) by transillumination-assisted microdissection. The precise stages were examined by phase contrast microscopy of live cells squashed carefully out from the adjacent segments between glass slides. The squash technique was also used for a primary screening of the cultured tubules. Pachytene primary spermatocytes from stages VIII to XII of the cycle were able to complete meiotic divisions in vitro. From stage XII, they differentiated up to step 5 spermatids, expressed their specific antigens, and developed characteristic movement patterns of the flagellum and of the chromatoid body. Preleptotene and zygotene spermatocytes from the same cell association differentiated synchronously, as judged by chromosome morphology, characteristic chromosome rotation in zygotene and early pachytene, and by development of specific antigen expression. The elongation phase of spermiogenesis did not proceed normally in vitro. The rate of differentiation was the same as observed earlier in vivo. Earlier studies with [3H]thymidine labeling and autoradiography only permitted follow-up of the development of preleptotene spermatocytes. With the present method, all stages of spermatogenesis can be traced in culture with great accuracy in experiments relating to local regulation of spermatogenesis.     

Flow cytometric DNA analysis in rectal carcinomas

The cellular DNA content was measured with flow cytometry from paraffin-embedded material in 329 patients and metastatic tumors of the liver from the rectum in 11 patients. The classification of the DNA ploidy pattern is as follows: A stem cell peak with a DNA index of 0.9-1.1 is defined as DNA diploid tumor and DNA aneuploid tumor is that with a DNA index greater than or equal to 1.1. There was a good correlation of DNA indices (r = 0.997) obtained from flesh and corresponding paraffin-embedded specimens. It is concluded that accurate determination of DNA index from paraffin-embedded materials is possible in the majority of cases. DNA ploidy of primary tumor cells correlated with clinicopathological findings such as lymphatic invasion, vascular invasion, lymph node metastasis and hepatic metastasis (p less than 0.01), but did not correlate with extramural carcinoma invasion. The cumulative survival rate (Kaplan-Meier) of curatively resected rectal carcinomas was worse in DNA aneuploid than in DNA diploid tumors (p less than 0.01). These observation showed that the determination of DNA ploidy in rectal carcinomas may prove to be of prognostic value.     

Flow cytometric DNA analysis in colorectal cancer

Flow cytometric DNA analysis for assessing malignant potential of colorectal carcinoma was investigated by paraffin-embedded materials. Preservation time of paraffin blocks and formalin fixation time of surgical specimens within 14 days do not influence the nuclear DNA content. There was seen a good correlation between the DNA contents of paraffin-embedded and fresh materials obtained from the same surgical specimens. Using deparaffinized tumor specimens, the nuclear DNA content was measured by flow cytometry in 144 patients with primary colorectal cancer, who had been treated from 1971 to 1985. Forty-four percent of colorectal cancer were diploid and 56% were aneuploid. There was no significant correlation between ploidy pattern and clinicopathological factors. However, the patients with aneuploid tumor had a significantly worse survival than those with diploid tumor (Generalized Wilcoxon test, p less than 0.001). The patients with aneuploid seemed to have an unfavorable survival than those with diploid in the same stage, and had a significantly worse survival in each group of negative nodes, P0 and H0. It is concluded, therefore, that the nuclear DNA content of colorectal cancer may be an important prognostic factor, being independent of pathological stage.     

Flow cytometric DNA analysis of thyroid carcinoma

Abnormal DNA content has been considered as an additional criterion for determining the biological behavior of a tumor. Flowcytometric DNA analysis was done on 121 patients with thyroid carcinoma encountered during the period between 1975 and 1987. Tumor tissues were sampled from paraffin-embedded blocks and the histology of thyroid carcinoma found to consist of 91 papillary, 23 follicular, 2 medullary, 1 squamous cell and 4 anaplastic carcinomas. The incidence of aneuploidy in thyroid carcinoma was 7.4 per cent (9 patients) while that of diploidy was 92.6 per cent (112 patients). The aneuploid specimens consisted of 6 papillary, 1 follicular, 1 medullary and 1 anaplastic carcinomas and, of 4 anaplastic carcinoma patients with subsequent death within 6 months, only 1 was aneuploid. As an indicator of proliferative potential, S-phase fraction (SPF) was also determined by flow cytometry, but this could not be used as an independent prognostic factor. The aneuploid patients showed a significantly decreased survival rate (p less than 0.01). Thus, although DNA measurement proved useful for predicting the survival of aneuploid patients, there is some discrepancy between DNA content and the biological behavior of the tumor.     

Flow cytometric DNA analysis of thyroid carcinoma

Abnormal DNA content has been considered as an additional criterion for determining the biological behavior of a tumor. Flowcytometric DNA analysis was done on 121 patients with thyroid carcinoma encountered during the period between 1975 and 1987. Tumor tissues were sampled from paraffin-embedded blocks and the histology of thyroid carcinoma found to consist of 91 papillary, 23 follicular, 2 medullary, 1 squamous cell and 4 anaplastic carcinomas. The incidence of aneuploidy in thyroid carcinoma was 7.4 per cent (9 patients) while that of diploidy was 92.6 per cent (112 patients). The aneuploid specimens consisted of 6 papillary, 1 follicular, 1 medullary and 1 anaplastic carcinomas and, of 4 anaplastic carcinoma patients with subsequent death within 6 months, only 1 was aneuploid. As an indicator of proliferative potential, S-phase fraction (SPF) was also determined by flow cytometry, but this could not be used as an independent prognostic factor. The aneuploid patients showed a significantly decreased survival rate (p<0.01). Thus, although DNA measurement proved useful for predicting the survival of aneuploid patients, there is some discrepancy between DNA content and the biological behavior of the tumor.     

Basal and FSH-stimulated steady state levels of SGP-2, alpha 2-macroglobulin, and testibumin in culture media of rat seminiferous tubules at defined stages of the epithelial cycle.

Production of several proteins by rat Sertoli cells is dependent on the stage of the cycle of the seminiferous epithelium. The authors have determined steady state levels and follicle-stimulating hormone responsiveness of three Sertoli cell products in culture media of rat seminiferous tubule segments at different stages of the epithelial cycle: SGP-2 (sulfated glycoprotein-2), alpha 2-macroglobulin, and testibumin. Basal SGP-2 levels were twofold higher in stages VII through VIII compared with stages XIII to I to VI (P less than 0.05). Highest basal alpha 2-macroglobulin levels were found in stages II through VIII; this was about 35% greater than in stages XIII through I of the cycle (P less than 0.05). Basal testibumin levels were twofold higher in stages II through VI compared with stages IX through XII of the cycle. Follicle-stimulating hormone had no effect on SGP-2, but by contrast it (50 mg/L) increased the level of alpha 2-macroglobulin significantly (P less than 0.05) in stages XIII through I. Follicle-stimulating hormone treatment (10 mg/L) elevated testibumin levels at each stage-pool by about 40% (P less than 0.05). The current results using staged tubular segments in vitro demonstrate cyclic basal steady-state levels of the three proteins along the seminiferous tubules and follicle-stimulating hormone regulation of alpha 2-macroglobulin and testibumin.     

Flow cytometric analysis of lymphoid cells in thymic epithelial neoplasms.

OBJECTIVE: There have been conflicts concerning the criteria for diagnosing malignant epithelial neoplasms of thymic origin. To differentiate thymic carcinomas from thymomas, the maturation stage of T-lineage lymphoid cells infiltrating thymomas and thymic carcinomas was examined by flow cytometry to associate it with the degree of tumor malignancy. METHODS: Multidimensional flow cytometric analysis was performed on the lymphoid cells extracted from 27 thymic epithelial neoplasms (14 encapsulated thymomas, ten invasive thymomas, and three thymic carcinomas) by using anti-CD3, -CD4, -CD8, -CD10, -CD20, -CD38, -CD45RA, and -CD45RO monoclonal antibodies. RESULTS: CD4 and CD8 were co-expressed on 76.8% of the lymphoid cells in encapsulated thymoma (N=14), 59.2% in invasive thymoma (N=10), and 6.7% in thymic cancer (N=3). The percentage of CD4- or CD8- single positive cells was 11.4% in encapsulated thymoma, 23.9% in invasive thymoma, and 77.7% in thymic cancer. The percentage of CD10-positive cells was 20.8% in encapsulated thymoma, 13.2% in invasive thymoma, and 6.0% in thymic cancer. The percentage of CD20-positive cell was 2.6% in encapsulated thymoma, 3.3% in invasive thymoma, and 31.6% in thymic cancer. There were significant statistical differences in the percentages of CD4/CD8 double positive cells, CD4- or CD8-single positive cells, CD10-positive cells and CD20-positive cells among the three groups. Two cases classified as invasive thymoma by pathohistological examination, however, showed the infiltration of mature lymphocytes like as thymic cancers. CONCLUSIONS: CD4+CD8+ or CD10+ T-lineage cells were the most reliable markers of the benignancy of thymic epithelial tumors. CD4- or CD8-single positive cells or CD20-positive cells were characteristic in thymic carcinoma. Flow cytometry on the maturity of lymphoid cells infiltrating thymic epithelial tumors was feasible for determining their degree of malignancy. Some invasive thymomas showed the intermediate characteristics with thymomatous epithelia and mature lymphoid cells.     

Flow cytometric analysis of DNA ploidy, cell cycle and cytomorphometry in sarcomatoid renal cell carcinoma.

In 5 patients with sarcomatoid renal cell carcinoma, the nuclear DNA content and size were determined by flow cytometry (FCM), and the prognostic value of DNA ploidy, the percentage of S-phase cells (SPF), and the ratio of modal nuclear size with clinicopathologic behavior was analyzed. Age and clinical stage have been shown to have a strong correlation with prognosis. Older patients with a high stage of cancer had poor outcome with a shorter survival time. In all 60% of the tumors were aneuploid. Tumor invasiveness was related to DNA ploidy. With increasing stage, the overall incidence of aneuploid rises. One alive patient had diploid DNA while 75% of the patients who died of sarcomatoid renal cell carcinomas had aneuploid DNA. Diploid sarcomatoid renal cell carcinomas show significantly lower SPF than aneuploid tumors. There was no significant association between the modal nuclear size and the invasiveness of tumors or survival time. This study suggests that FCM analysis of tumor DNA content and cell cycle could be regarded as an additional prognostic determinant of sarcomatoid renal cell carcinoma.     

Flow cytometric analysis of the DNA content of thymomas

In this study, the prognostic value of determining the nuclear DNA content of thymomas by flow cytometry was evaluated. Of a total 31 resected thymomas, 10 (32%) showed DNA aneuploidy, the presence of which was significantly correlated with an advanced clinical stage of disease. The patients with an aneuploid tumor had a poorer prognosis than those with a diploid tumor, demonstrating a survival rate of 50% at 7 postoperative years, which was considerably less favorable than that of the patients with a diploid tumor, being 100% in the same period (p<0.05). Moreover, patients with a high DNA index (DI), i.e., a DI1.5, tended to have a poorer prognosis than those with a low DI. These findings indicated that the DNA content can be an important prognostic index in patients with thymomas.     

Flow cytometric analysis of DNA aneuploidy in adrenal tumors]

Nuclear DNA content of paraffin-embedded tissue from 38 adrenal neoplasms and 9 histologically normal adrenal glands was analyzed using flow cytometry. Histological diagnosis of thirty-eight adrenal neoplasms were 3 adrenocortical carcinomas, 20 adrenocortical adenomas and 15 pheochromocytomas. In 33 cases (87%) of the 38 tumors the determination of DNA ploidy was possible. All 9 control specimens showed DNA diploid pattern in DNA histogram. In adrenocortical neoplasms the incidence of DNA aneuploidy was 0% (0 of 17) in adenomas and 100% (2 of 2) in carcinomas. All 17 adrenocortical adenomas which showed DNA diploid pattern are clinically benign. On the other hand, both 2 cases of adrenocortical carcinoma which showed DNA aneuploidy died within 1 year. These data suggest that DNA aneuploidy may be useful as a prognostic factor in adrenocortical neoplasm. With regard to pheochromocytoma, DNA aneuploidy was detected in 4 of 14 patients (29%). However, all 14 cases were clinically benign. In pheochromocytoma DNA aneuploidy was not found to be correlated with prognosis.     

Flow cytometric analysis of DNA content in children with neuroblastoma

Flow cytometric DNA content analyses were performed in a retrospective study of archival tumor specimens of 15 patients with diagnosed neuroblastoma treated at a single institution. Survival statistics were positively influenced by the presence of aneuploidy. Kaplan-Meier analysis indicated a significant survival advantage for patients with aneuploid tumors (P = .036). Survivors were significantly younger at the time of diagnosis than nonsurvivors, but age at diagnosis did not correlate with DNA content. We conclude that DNA content analysis may be of value in determining the prognosis for children with neuroblastoma.     

DNA-flow cytometry of defined stages of rat seminiferous epithelium: effects of 3 Gy of high-energy X-irradiation

Testes of adult Sprague-Dawley rats were irradiated locally by 3 Gy of 4 MeV X-rays produced by a linear accelerator. This type and dose of radiation gives an even distribution through the testis and selectively kills the proliferating spermatogonia. The seminiferous tubular cells were quantified by DNA flow cytometry at defined stages of the epithelial cycle at 7, 17, 22, 38, 52, and 80 days after irradiation. The flow cytometric technique was modified by using frozen instead of fresh samples. Freezing did not alter cell numbers when compared with fresh samples. At 7 days post-irradiation no significant changes were observed in any cell population by DNA flow cytometry, whereas histological analysis revealed a reduction in intermediate and type B spermatogonia. At 17 and 22 days post-irradiation, the number of cells at meiotic prophase (4C) was decreased, particularly in stages II-V of the cycle. In stages VII-VIII, cell numbers were 40 and 31%, and in stages IX-XIII, 24 and 43% of that in non-irradiated controls at 17 and 22 days, respectively. At 38 days after irradiation, both 4C and 1C (haploid) cells were decreased in number. The 4C cells were reduced to 24, 17, and 13% of that in non-irradiated controls in stages II-V, VII-VIII, and IX-XIII of the cycle, respectively. The corresponding numbers of 1C cells were 5, 17, and 4%. At 52 days after irradiation, 1C cells had declined to 38 and 19% of control values in stages II-V and IX-XIII, respectively. In stages II-V, 1C' cells (haploid cells with condensed nuclei) declined to 28% of controls at 52 days. The present data provide a quantitative basis for the use of X-ray-irradiated rat testes as a model system in experiments pursuing interactions between Sertoli cells and spermatogenic cells.     

Flow cytometric analysis of DNA ploidy in lymphomas of the thyroid

Four cases of primary non-Hodgkin's lymphoma (NHL) of the thyroid were studied using flow cytometric (FCM) DNA analysis of propidium iodide-stained nuclei retrieved from formalin-fixed, paraffin-embedded tissue. Two of the four cases were aneuploid and two were euploid. In the two euploid cases, both patients are alive and without evidence of recurrent disease after an average of 4 years follow-up. Of the two aneuploid cases, one patient is alive and free of recurrent disease after 1 year. In the other aneuploid case, the patient died of disseminated disease 8 months after presentation despite having a low-grade (follicular, predominantly small cleaved cell type) and low-stage (tumor confined to thyroid at presentation) lymphoma. These data suggest that the DNA ploidy of primary NHL of the thyroid can be determined using fixed, paraffin-embedded tissue. Our results also suggest that a large study to assess the prognostic value of this technique is warranted.     

Flow cytometric analysis of the human articular chondrocyte phenotype in vitro

OBJECTIVE: To develop flow cytometry for the study of human articular cartilage cell phenotype and to validate the method on chondrocytes cultured in different in-vitro systems. METHODS: Chondrocyte phenotype was modulated by culturing the cells under different in-vitro conditions: i.e. in monolayer and in suspension culture in gelled agarose. Monolayer cultured chondrocyte phenotype was assayed by immunohistochemical staining with monoclonal antibodies against chondrocyte-specific aggrecan, type II and I collagen. Flow cytometry was used to quantify the proportions of chondrocytes expressing these extracellular matrix molecules in both culture conditions. To exclude the effects of cell-harvesting methods on the presence of cell-bound ECM molecules, non-proteolytic isolation procedures were used to obtain the chondrocytes for flow cytometry. Subconfluent cells from monolayer cultures were detached with EDTA. Chondrocytes cultured in gelled agarose were obtained after the agarose was enzymatically digested with agarase. RESULTS: Immunohistochemical staining showed that monolayer-cultured chondrocytes, in the presence of serum, gradually lost the expression of chondrocyte-specific aggrecan and type II collagen, while type I collagen was increasingly expressed. Flow cytometry allowed monolayer cultured chondrocyte phenotype to be assessed reproducibly. Chondrocyte phenotype was characterized through the cell membrane-associated extracellular matrix antigens. EDTA, used to obtain single cells from monolayer cultures, did not affect the cell-associated matrix. Where the chondrocytes had been cultured in gelled agarose, flow cytometry allowed quantification of the percentages of chondrocytes maintaining or reexpressing their original phenotype. The agarase digestion procedure used to isolate the cells from the agarose gel did not affect the plasma membrane-associated extracellular matrix antigens. CONCLUSION: Flow cytometry allows quantification of cells expressing aggrecan, type II and I collagen in their cell-associated extracellular matrix. A continuously increasing number of specific monoclonal antibodies will broaden the range of applications offered by this method.     

Flow cytometric analysis of the nuclear DNA content of hepatocellular carcinoma

The nuclear DNA content of 77 resected specimens from 65 cases of hepatocellular carcinoma (HCC) was measured by means of flow cytometry. The DNA index (DI) was calculated and the correlation between the DNA ploidy pattern and clinicopathological findings was studied. In the cases of HCC with a diameter of less than 5 cm, the 3-year survival rate of the aneuploid cases was 44.5 per cent, which was significantly lower the 91.4 per cent of the diploid cases (p<0.001). Serum AFP levels were over 1000 ng/ml in 46.4 per cent of the aneuploid tumors and 18.5 per cent of the diploid tumors (p<0.05). The DI’s were investigated in several sites of the same tumor and no difference was seen among the different sites in 16 out of 17 tumors. From 8 recurrent cases out of 12 who underwent a second resection, seven did not show any significant differences in DI from their primary tumor. On the other hand, four cases of second primary tumors showed different DI’s to those of their first primary tumor. Intra-hepatic metastatic tumors exhibited the same DI’s as their primary tumors. Thus, the nuclear DNA ploidy pattern may serve as a stable and valuable marker in predicting the malignant potential and prognosis of HCC.     

Flow cytometric analysis of DNA content in testicular seminomas--correlation to mitotic count

The DNA ploidy of testicular seminomas was studied by flow cytometry using paraffin embedded samples. The mitotic count and DNA index (DI) for 27 seminomas were analyzed in 80 samples with a mean of 3.0 samples per case. Six anaplastic seminomas which were with 3 or more mitoses per a high power field were distinguished from 21 typical seminomas. DNA ploidy pattern was aneuploid in all seminomas except one case of anaplastic seminoma, and clonal heterogeneity in DNA content was found in 3 of 20 (15%) cases of which 2 or more samples were analyzed. Although the DI had no significant difference between those two groups of seminomas classified by mitotic count, the DI in anaplastic seminomas was ranged from 1.5 to 2.0 (median DI = 1.70), otherwise the DI in typical seminomas ranged from 1.5 to 3.5 (median DI = 1.89), particularly 9 cases in 21 (43%) typical seminomas distributed in hypertetraploid region. The median DI of stage I seminomas was 1.88 and that of stages II + III seminomas was 1.75, though there was also no significant correlation between DI and clinical stages. In general, it is postulated that the higher DI is paralleled to the more malignant nature of neoplasms, nevertheless this study suggested that the higher DI in seminomas is not always related to high malignant potentiality determined by histological type and clinical stage.     

Flow cytometric analysis of the nuclear DNA content of hepatocellular carcinoma

The nuclear DNA content of 77 resected specimens from 65 cases of hepatocellular carcinoma (HCC) was measured by means of flow cytometry. The DNA index (DI) was calculated and the correlation between the DNA ploidy pattern and clinicopathological findings was studied. In the cases of HCC with a diameter of less than 5 cm, the 3-year survival rate of the aneuploid cases was 44.5 per cent, which was significantly lower than the 91.4 per cent of the diploid cases (p less than 0.001). Serum AFP levels were over 1000 ng/ml in 46.4 per cent of the aneuploid tumors and 18.5 per cent of the diploid tumors (p less than 0.05). The DI's were investigated in several sites of the same tumor and no difference was seen among the different sites in 16 out of 17 tumors. From 8 recurrent cases out of 12 who underwent a second resection, seven did not show any significant differences in DI from their primary tumor. On the other hand, four cases of second primary tumors showed different DI's to those of their first primary tumor. Intra-hepatic metastatic tumors exhibited the same DI's as their primary tumors. Thus, the nuclear DNA ploidy pattern may serve as a stable and valuable marker in predicting the malignant potential and prognosis of HCC.     

Flow cytometric analysis of cellular DNA content in human astrocytomas and oligodendrogliomas

This report presents a flow-cytometric analysis of cellular DNA in biopsies and primary cell cultures of 21 human astrocytomas and 19 oligodendrogliomas. A distinct correlation between histological dedifferentiation and pathological DNA distribution was found. Classification was made according to increasing histological anaplasia, corresponding to a four-grade scale and proliferation index (PI). Four types of gliomas were defined according to characteristic DNA patterns and proliferative activities in comparison to their histological grading: 1. purely diploid DNA patterns with low 4C (premitotic) peaks and PI values up to 10 in well-differentiated gliomas; 2. increase of tetraploid cells and PI of 10-16 in tumors with histological grades II or II–III; 3. diploid-tetraploid DNA distribution with PI values up to 30–31 and malignancy grade III; 4. polyploid and aneuploid karyograms with excessive 4C increase, emerging in grade III and especially grade III–IV of these gliomas. Varying DNA distribution during tumor development could be observed in a malignant transformation of an oligodendroglioma I to a glioblastoma after a course of 3 1/2 years. A more detailed subdivision of these tumors according to their DNA content and proliferative activity was achieved. With the exception of occasional variation in karyograms, DNA distribution usually remained stable in primary tissue cultures (PTC).     

Flow cytometric DNA analysis of normal adrenal tissues and adrenal tumors

Flow cytometric DNA analysis (FCM) of adrenal tumors was studied to evaluate whether FCM will be a useful examination for differentiating between benign and malignant adrenal tumors. 10 specimens of surgically resected (for renal cell carcinoma confined within the middle or lower pole) normal adrenal glands and 20 specimens of surgically resected adrenal tumors were submitted for the study. Hyperplastic adrenal cortex as well as normal adrenal gland showed normal diploid pattern. On the other hand, some of the cortical adenomas showed tetraploid patterns, which has been known to be an index of malignancy in most of the flow cytometric intervention to other solid tumors. Conn adenomas were especially apt to show this tendency, in which as much as 86% showed tetraploid pattern. Proliferation Index (PI) (ratio of S + G2 +M cells for the whole population of analyzed cells) were as much as 9.45 +/- 6.97% in normal adrenal cells, whereas it was much higher in cortical adenomas (17.75 +/- 8.53%). As a matter of fact, PI of hyperplastic adrenal cortex was within the same range as that of the normal adrenal glands. In pheochromocytomas, aneuploid pattern, which has been believed to be a definite index of malignancy, was shown in 60% of the cases, tetraploid pattern in 20%, and normal diploid pattern in only 20%. As a matter of fact, a case of non functioning cortical adenocarcinoma and a case of malignant pheochromocytoma were judged to show typical aneuploid pattern. Thus, the application of the flow cytometric diagnosis for adrenal tumors was supposed to require some refinement in understanding the significance of aneuploidy or tetraploidy.