Influence of fetal bovine serum and hormones on primary vs. long-term cultures of human breast cancers

Summary We investigated the influence of fetal bovine serum, complemented or otherwise with estradiol or insulin or both, on the proliferation of mammary cancer cells from long-term and primary cultures. The long-term culture corresponded to mouse MXT and MCF-7 cell lines whereas the primary culture corresponded to primitive breast cancers squashed onto histologic slides and maintained in cultures for between 12 and 48 h. Cell proliferation was evaluated by means of digital cell image analysis of Feulgen-stained nuclei. Our results show that the addition of estradiol and insulin slightly but nevertheless significantly increases the proportion of cells still living at Hour 48 of culture. Fetal bovine serum, necessary for the growth of MXT and MCF-7 mammary cells, was highly cytotoxic with respect to the primary cultures of the 20 breast cancers under study. We are now conducting new experiments using chemically defined media to study the influence of new antineoplastic compounds on primary cultures of breast cancers.     

Virulence of chick embryo fibroblast-passaged flury HEP rabies virus and its revertants in mice

Chick embryo fibroblast-passaged Flury high egg passage (HEP) rabies virus failed to kill nude mice or cyclophosphamide-treated mice when inoculated intracerebrally. The virus regained neurovirulence for adult mice after three passages in mouse neuroblastoma C1300 cells (NA cells). However, even after 20 passages in NA cells, the virulence could not be increased to the level shown by the virus passaged several times in suckling mice. Some physiological and biological properties of the virus showing and not showing mouse virulence after five serial passages and after one single passage in NA cells, respectively, were compared.     

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human lung cancers serially transplanted in nude mice.

Gamma-glutamyl transpeptidase (y-GT) activity and distribution were investigated in different types of human lung cancers (three epidermoid carcinomas, one large cell carcinoma) which were maintained by serial transplantation in nude mice. All transplanted tumour fragments were positive for the enzyme. In the epidermoid carcinomas, y-GT levels were related to the degree of tumour differentiation. Enzyme activity in tumour fragments was always higher than that found in normal adult human lung tissue, and was, in general, maintained throughout the transplantation series.     

Effects of lossy image compression on quantitative image analysis of cell nuclei

OBJECTIVE: To investigate whether statistically significant changes occur in quantitative image analysis of cell nuclei when lossy image compression techniques are used. STUDY DESIGN: Thirty-five stoichiometric, Feulgen-stained samples of rat hepatocytes, human thyroid and ovarian cancer cell nuclei were used. Image analysis was performed by a computerized system that used AutoCyte LINK V1.1.1.56 software (Burlington, North Carolina, U.S.A.) for image acquisition and Zeiss Vision KS 400 V3.0 software (Oberkochen, Germany) for quantitative image analysis. After lossy JPEG compression of acquired images at different quality levels, some densitometric features were selected and measurements performed. RESULTS: We observed that nearly all the standard densitometric features showed statistically significant changes when images were compressed with the lossy JPEG algorithm. However, most invariant densitometric moment features remained free of statistically significant changes. CONCLUSION: The standard densitometric measurements that we used do not tolerate lossy compression. However, analyses using invariant densitometric features may be performed on images compressed with lossy JPEG, resulting in simpler, less expensive systems demanding less network bandwidth.     

裸鼠体内高转移人肺癌模型的筛选

将人肺巨细胞癌ＰＬＡ－８０１Ｄ裸鼠皮下移植瘤作为瘤源,建立了ＰＬＡ－８０１Ｄ－ＡＳ裸鼠腹水鼠模型,其转移率及转移程度明显增高;以ＰＬＡ－８０１Ｄ－ＡＳ腹水瘤模型的肺转移灶细胞进行裸鼠皮下连续传代,建立了肺转移率及程度高于ＰＬＡ－８０１Ｄ的ＰＬＡ－８０１ＤＬ皮下移植瘤株。本文从肿瘤的异质性以及肿瘤与宿主相互作用的角度讨论了其肺转移提高的原因。     

Comparison of fine needle aspirates of breast cancers to imprint smears by means of digital cell image analysis

Morphonuclear assessments were performed using the SAMBA 2005 cell image processor on cell nuclei in fine needle aspirates and corresponding imprint smears from 17 not-otherwise-specified (NOS) breast carcinomas to study the influence of cell sampling on the morphonuclear measurements. Fourteen parameters related to densitometric (nuclear DNA content), morphometric (nuclear area) and textural (chromatin organization and distribution) characteristics were computed for each nucleus. The results demonstrated that such morphonuclear features evolved significantly and positively with respect to conventional histopathologic grading. The method of cell sampling significantly influenced the results, but without altering the general conclusions regarding evolution of the morphonuclear features.     

Carcinogen metabolism in human skin grafted onto athymic nude mice: a model system for the study of human skin carcinogenesis

Human skin grafted onto athymic nude mice maintains its major histological features and may provide a useful system with which to assess the carcinogen interaction with human skin. Significant differences were observed in basal levels of cytochrome P-450 and cytochrome P-448-dependent monooxygenase activities between human grafted and nude mouse epidermis. Topical application of crude coal tar (CCT) to human skin transplanted onto nude mice resulted in 3.9 & 3.5; 3.2 & 2.9 and 1.1 & 1.2 fold increases in mouse and human epidermal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin deethylase (ERD) and ethoxycoumarin deethylase (ECD) activities, respectively. CCT applied topically to mouse skin resulted in 27.8 & 6.4; 12.8 & 3.3 and 1.7 & 2.6 fold increases in mouse and human epidermal AHH, ERD and ECD activities, respectively. Topical application of coal tar either onto human transplanted skin or to mouse skin also resulted in substantial induction of hepatic and pulmonary AHH and ERD activities. These studies indicate that human skin grafted onto nude mice preserves its metabolic capacity and offers a useful model system with which to assess the effects of polycyclic aromatic hydrocarbons and CCT on cutaneous xenobiotic metabolism in the human population.     

Morphometry and cytometry correlated to quantitative autoradiography

Studies performed in various cell systems and designed to establish correlations between morphometric and functional parameters of individual cells are reviewed. Functional parameters were evaluated by utilizing quantitative 14C-autoradiography and measuring DNA, RNA and protein synthesis. Morphologic parameters were derived from the scanning of Feulgen-stained nuclei and the calculation of features related to shape, optical density and texture. A series of correlations between parameters of these two groups of features was established. Results such as the possibility of allocating cells to the G1, S and G2 phases by textural and densitometric features alone, without making use of the total DNA content and nuclear size, point to the power of this approach. The data, however, are not yet comprehensive enough to allow the interpretation of morphologic parameters in terms of cellular function on a large scale. It is emphasized that the measurement of the RNA synthesis rate will further promote the functional understanding of structural details and may help in making this approach useful for diagnostic purposes.     

Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue

The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.     

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human lung cancers serially transplanted in nude mice

Summary Gamma-glutamyl transpeptidase (y-GT) activity and distribution were investigated in different types of human lung cancers (three epidermoid carcinomas, one large cell carcinoma) which were maintained by serial transplantation in nude mice. All transplanted tumour fragments were positive for the enzyme. In the epidermoid carcinomas, y-GT levels were related to the degree of tumour differentiation. Enzyme activity in tumour fragments was always higher than that found in normal adult human lung tissue, and was, in general, maintained throughout the transplantation series.This work was supported by a grant from A.S.F.C., Switzerland, and the Jubiläumsspende 1970 der Zürcher KantonalbankN.F. was supported during part of the work by a Royal Society European Science Exchange Programme Fellowship     

Digital imaging analysis of normal, hyperplastic and malignant endometrial cells in endometrial brushing samples

Sixty cytologic specimens obtained by endometrial brushing (using the Gynecyte device) were quantitated by digital imaging techniques. These samples included 25 from normal endometria, 6 from persistent proliferative endometria, 14 from cystic or adenomatous hyperplasias and 15 from carcinomas. The morphometric parameters surveyed included mean cell area, nuclear area, perimeter and long and short axes. The amount of hematoxylin dye in the nuclei was expressed by mean transmittance (mean of gray levels) and chromatin index (standard deviation of gray levels). The frequency distributions of cells derived from normal tissue and persistent proliferative endometrium were quite similar. The quantitative parameters of cystic and adenomatous hyperplasia, although intermediate between those of normal endometrium and carcinoma, were closer to the former than to the latter. Using stepwise discriminant analysis of the morphometric parameters, 83% of the specimens were correctly classified into the categories of normal/persistent proliferative, hyperplasia and carcinoma. The accuracy was improved to 88% when densitometric parameters were added. This study demonstrates the potential application of digital imaging techniques to the distinction and classification of normal, hyperplastic and malignant endometrial cells.     

Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats

The progressive morphological changes in the liver during neoplastic transformation have been studied by histological, cytophotometric and morphometric methods in male Wistar rats treated with two carcinogens: N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1). Cytophotometric and morphometric analysis of hepatocyte nuclei using Feulgen-stained tissue sections were performed in morphologically normal hepatic parenchyma and in early preneoplastic foci composed of altered hepatocytes. Foci of clear cells, mixed cells and large basophilic cells possessed a ploidy distribution similar to the surrounding non-transformed parenchyma, while the small hyperbasophilic cell foci were predominantly diploid. These findings confirm that the foci composed of PAS-negative, small hyperbasophilic cells with an unique diploid content may represent one of the earliest stages in the neoplastic transformation.     

肺癌转移瘤动物模型的建立

目的通过尾静脉注射,建立一种符合临床特征的肺腺癌转移瘤动物模型,为下一步的肺腺癌转移机制的研究提供可靠的实验造模方法。方法取对数生长期的A549细胞,11只SPF级、4~6周龄BALB/c裸鼠,分别以1×106个细胞/只注射入裸鼠尾静脉。接种后每天观察小鼠状态。分别于接种肿瘤细胞后第4、5、6、7周随机处死2只,余3只小鼠处于濒死状态时处死。解剖小鼠,观察肺部有无转移、转移结节的数目及全身其他器官的转移情况,并做病理取材,HE染色观察。结果注射过程中小鼠均存活。未处死的3只分别于第11、13、14周出现恶液质。第4周肺部未见转移结节;第5周出现镜下肺部转移结节;第6周肉眼可见肺部转移结节;第7周转移结节数增多;第11周出现纵隔淋巴结转移。第11、13、14周出现肺部结构大量破坏,弥漫性的肿瘤细胞浸润,出现淋巴结浸润,病理证实为腺癌。结论通过尾静脉注射A549细胞可以成功建立人肺腺癌转移瘤模型。     

Nuclear morphometric features of epithelial cells lining keratocysts

OBJECTIVE: To investigate the nuclear morphometric features of epithelial cells lining keratocysts and some other odontogenic cysts. STUDY DESIGN: All cases were selected from the archives of the Department of Pathology, Gülhane Military Medical Academy, as follows: 20 keratocysts and 10 dentigerous and 10 radicular cysts. Nuclear morphometric variables were measured on hematoxylin and eosin-stained histologic slides. Basal and intermediate cells of the epithelium were evaluated separately. Nuclei of the cells were outlined interactively and measured using a specially written macro program. Area, feret ratio (ratio of the longest nuclear axis to the shortest one) and circularity (F circle) of the nuclei were calculated. Additionally, nuclear densitometric analysis was performed on the keratocyst cases. RESULTS: The number of cells in the basal layer (cell density) was higher in keratocysts than in other cysts. The mean nuclear area of basal cells was smaller than of intermediate cells in both keratocysts and other cysts (P < .001). The feret ratio values revealed that basal cell nuclei of keratocysts were more ovoid as compared to those of other cysts (P < .001). Nuclear densitometric findings showed that the DNA indices of all keratocyst cases were close to 1.0, and the cells were considered diploid. CONCLUSION: Increased cell density, a more ovoid nuclear shape and more variation in the size of basal layer cell nuclei in keratocysts were helpful in differentiating these lesions from other odontogenic cysts.     

Deletion of self-reactive T cells in nude mice grafted with neonatal allogeneic thymus

The cellular basis of tolerance induction has been investigated in BALB/c(H-2d, thy 1.2, M1s1b2a) nude mice grafted with thymus of neonatal AKR/J mice(H-2k,Thy1.1,M1s1a2b). The spleen cells from nude mice grafted with AKR/J thymus showed a significantly decreased level of primary cytotoxic T cell response when stimulated with AKR/J cells, although these cells lysed well target cells of a third party C57BL/6 when stimulated with C57BL/6 cells. Consistent with CTL responses, T cells bearing V beta 6, that is important for recognizing M1s1a-encoded products of the thymic phenotype, were virtually abolished in the spleen and lymph node cells of nude mice 8 wk after grafting with AKR/J thymus. However, a substantial number of V beta 6-bearing T cells were detected in the peripheral organs of nude mice 23 wk after grafting with AKR/J thymus and in those of nude mice grafted with AKR/J fetal thymus depleted of macrophages/dendritic cells by incubating with 2'-deoxyguanosine in vitro before grafting. On the other hand, T cells bearing V beta 3, which are selectively related to M1s2a-encoded products of the host phenotype, were expressed neither on the peripheral T cells of nude mice grafted with AKR/J thymus at any stage after grafting nor on those of nude mice grafted with 2'-deoxyguanosine-treated AKR/J thymus. These data suggested that both V beta 6 and V beta 3 T cells were eliminated in the thymus of nude mice grafted with AKR/J thymus, presumably on the basis of interaction with both of graft-derived persisting and host-derived hemopoietic cells in the thymus and that thymic epithelium appears to have little capacity to eliminate T cells reactive to minor lymphocyte stimulating-encoded products.     

Relationship between computerized morphonuclear image analysis and histopathologic grading of breast cancer

A pilot study analyzed the relationship between several morphonuclear parameters and the Bloom-Richardson score for 37 invasive, not-otherwise-specified (NOS) ductal breast carcinomas. The SAM-BA 200 cell image processor and its software were used to measure the nuclear features on Feulgen-stained imprint smears. Two parameters representing the numbers of large and dense chromatin clots and two parameters describing the heterogeneity of the chromatin among nuclei in a specimen evolved in a continuous manner parallel with the Bloom-Richardson score from stages NOS-4 to NOS-8. The systematic measurement of these four parameters on a large series of breast cancers may be able to define an objective and reproducible "scale" of differentiation that could be a helpful tool for pathologists and clinicians.     

Sensitivity of Seescan TV image analysis in discriminating between normal, dysplastic and malignant oral smears

OBJECTIVE: Smears from premalignant and malignant lesions may contain large proportions of normal cells together with atypical cells; that could reduce the sensitivity of cytologic diagnosis. The present study assessed the performance of the Seescan TV image analysis system (TVIAS) in distinguishing between normal, premalignant and malignant oral smears. The sensitivity of Seescan TVIAS was tested using both white and monochromatic light. STUDY DESIGN: Nuclear area (NA) and corrected integrated optical density (IOD) of 50 Feulgen-stained nuclei were measured in smears collected from normal oral mucosa (n = 6), lesions displaying epithelial dysplasia (n = 5) and invasive squamous cell carcinoma (n = 5) using a Seescan TVIAS with both white and monochromatic light. RESULTS: There was a significant increase (P < .001) in mean IOD for nuclei in smears from dysplastic lesions and carcinomas as compared with normal smears. For smears from carcinomas, the mean NA was significantly elevated as compared with dysplastic (P < .001) and normal smears (P < .01). Mean NA for dysplastic smears was significantly reduced as compared with normal smears. While all smears from premalignant and malignant lesions contained mostly normal nuclei, a significant proportion of abnormal nuclei was identified in each smear. CONCLUSION: Although oral smears contain large amounts of normal cells, the Seescan TVIAS could successfully identify dysplastic and malignant cells on the basis of both IOD and NA values with or without the use of a monochromatic filter.     

The use of the digital cell image analysis of Feulgen-stained nuclei to detect apoptosis

Cell death is an essential event in the functioning of multicellular organisms. It plays a role opposite to that of mitosis in the regulation of cell populations. In the present work, we describe an original methodology which permits the easy detection and count of apoptotic cells in a given tissue. This methodology is based on the digital cell image analysis of Feulgenstained nuclei, which also permits the calculation of the proliferation index, i.e. the precentage of cells in the S phase of the cell cycle. This percentage of cells in the S phase is strongly related to the mitotic index. Our methodology, which involves the multivariate analysis of 14 morphonuclear parameters computed by means of the digitized cell image analysis of Feulgen-stained nuclei, was applied here to a well-known biological apoptosis model, namely glucocorticoid-treated rat thymocytes. The parameters that permitted the detection of apoptotic cells were the integrated optical density, a parameter that describes the nuclear DNA content, and the run length percentage and long run length parameters which are related to the pattern of chromatin condensation. This determination can be carried out on a relatively small number of cells.