Neoplastic cell spreading in rodent transplantable tumor models. I. Ehrlich tumor

Ehrlich ascites tumor cells were ectopically transplanted in femoral muscles of tumor-free Swiss and BALB/c mice with the same modality used for i.p. serial transplantations of the ascitic form. A solid tumor developed (100% takes as i.p. grafts) locally invading surrounding tissues and leading to death within 30-40 days (12-14 days in ascitic form). These animals were killed when showing signs of debilitation by tumor growth (1 mo.). The recipients' own thoracic and abdominal organs (lung, liver, spleen, and kidney plus peritoneal fluid) as well as the solid tumor were removed to obtain imprints and smears fixed and stained for cytology (May Grünwald Giemsa). Tumor-free mice were used as a control and i.p. transplanted mice were sacrificed on day 8. Disseminated tumor cells were seen in recipient organ imprints and peritoneal fluid smears scattered among local normal cells. Host defense cells with prevalence of neutrophils were observed infiltrating the solid tumor or adjacent to disseminated tumor cells. According to previous findings, organ imprints of i.p. transplanted mice showed disseminated tumor cells and host defense cells. Surprisingly, in liver imprints of ectopically transplanted BALB/c mice, numerous megakaryocytes were detected. This tumor and host organ imprint assay offers the possibility to monitor in vivo the phenomenon of metastatic tumor spread.     

Intraperitoneal transplantation of Yoshida ascites hepatoma cells after fine needle aspiration from tumor bearing rats organs

The authors tested the ability of scattered tumor cells to re-form a tumor in vivo. Disseminated tumor cells are morphologically visible stained with May-Grünwald Giemsa in the lung, liver, kidney, and spleen of Yoshida ascites tumor bearing rats. Free tumor cells can easily be fine needle aspirated from those organs and injected in syngeneic Wistar rats. All the host rats show ascites tumor take after intraperitoneal transplantation of each aspirated sample. This biological model might be useful to study in vivo a wide range of properties of neoplastic and non-neoplastic host cells.     

Morphological characterization of lymphoid cells conjugates with scattered tumor target cells in organ imprints of Yoshida ascites bearing rats

The authors analysed morphologically the ability of host effector cells to bind disseminated tumor target cells (pre-lysis) in vivo. Scattered tumor cells are morphologically visible stained with May Grünwald-Giemsa in the lung, liver, kidney, and spleen imprints of Yoshida ascites tumor bearing rats. Lymphoid cells can be seen binding disseminated tumor target cells in organs but not in the peritoneal cavity. This model may provide a useful technique for morphological studies on the in vivo conjugation capacity of host effector cells against tumor target cells.     

Morphological analysis of circulating tumour cells in patients undergoing surgery for non‐small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method

V. Hofman, E. Long, M. Ilie, C. Bonnetaud, J. M. Vignaud, J. F. Fléjou, S. Lantuejoul, E. Piaton, N. Mourad, C. Butori, E. Selva, C. H. Marquette, M. Poudenx, S. Sibon, S. Kelhef, N. Vénissac, J. P. Jais, J. Mouroux, T. J. Molina, P. Vielh and P. Hofman
Morphological analysis of circulating tumour cells in patients undergoing surgery for non‐small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method Background and objective: Recurrence rates after surgery for non‐small cell lung cancer (NSCLC) range from 25 to 50% and 5‐year survival is only 60–70%. Because no biomarkers are predictive of recurrence or the onset of metastasis, pathological TNM (pTNM) staging is currently the best prognostic factor. Consequently, the preoperative detection of circulating tumour cells (CTCs) might be useful in tailoring therapy. The aim of this study was to characterize morphologically any circulating non‐haematological cells (CNHCs) in patients undergoing surgery for NSCLC using the isolation by size of epithelial tumour cell (ISET) method. Methods: Of 299 blood samples tested, 250 were from patients with resectable NSCLC and 59 from healthy controls. The presence of CNHCs was assessed blindly and independently by 10 cytopathologists on May‐Grünwald–Giemsa stained filters and the cells classified into three groups: (i) malignant cells, (ii) uncertain malignant cells, and (iii) benign cells. We assessed interobserver agreement using Kappa (κ) analysis as the measure of agreement. Results: A total of 123 out of 250 (49%) patients showed CNHCs corresponding to malignant, uncertain malignant and benign cells, in 102/250 (41%), 15/250 (6%) and 6/250 (2%) cases, respectively. No CNHCs were detected in the blood of healthy subjects. Interobserver diagnostic variability was absent for CNHCs, low for malignant cells and limited for uncertain malignant and benign cells. Conclusion: Identification of CTCs in resectable NSCLC patients, using ISET technology and according to cytopathological criteria of malignancy, appears to be a new and promising field of cytopathology with potential relevance to lung oncology.     

Fine needle aspiration cytology of infantile haemangioendothelioma of the liver: a report of two cases

E. Sigamani, V. K. Iyer and S. Agarwala Fine needle aspiration cytology of infantile haemangioendothelioma of the liver: a report of two cases Background: Fine needle aspiration cytology (FNAC) of infantile haemangioendothelioma of the liver (IHL) has not previously been described because routine use of FNAC is contraindicated due to the risk of bleeding. Methods and materials: Two patients presented with progressively increasing right upper quadrant abdominal mass. The index case was a girl aged two and a half years with a large single mass in the right lobe of the liver. The second was a 3‐month‐old girl in whom ultrasonography revealed multiple hypoechoic lesions in the liver. Ultrasound‐guided fine needle aspiration had been performed on both patients. May‐Grünwald‐Giemsa stained smears from these two patients were reviewed and correlated with histopathology. Results: Both aspirates showed predominantly normal hepatocytes and bile ductules amongst which tumour cells were admixed. The latter were oval to spindle‐shaped with scant cytoplasm and wavy, kinked and indented nuclear outlines. The non‐epithelial character of the tumour cells was apparent and helped to rule out hepatoblastoma. One case showed extramedullary haemopoiesis. The diagnosis of IHL was established on subsequent excision in the first case and a wedge biopsy in the second case. CD34 and factor VIII R antigen were positive in the tumour cells. Conclusion: Radiological diagnosis of IHL is possible in a majority of cases, but sometimes features may overlap with hepatoblastoma and fine needle aspiration may be performed inadvertently. Characteristic kinked nuclei and intermixed normal liver tissue might suggest IHL in the differential diagnosis of a spindle cell vasoformative tumour.     

In vivo 5-flourouracil-induced apoptosis on murine thymocytes: involvement of FAS,Bax and Caspase3

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. It was reported as the major mechanism of anticancer drug-induced cells death. Unfortunately, many of these drugs are non-specific and cause severe side effects. The effects of 5-fluorouracil (5-FU) on the apoptotic events in normal murine thymus were evaluated using an in vivo model. A single dose of 5-FU (150 mg/kg ip) was injected to CF-1 mice. A multiparametric analysis of thymic weight, cellularity, viability, architectural organization, apoptosis, DNA fragmentation, and the expression of several apoptotic proteins was evaluated in 10 days time-course study post-5-FU dosing. Total organ weights, thymocyte counts, and cell viabilities diminished drastically from the second day. The thymus architecture assessed through electron scanning microscopy revealed deep alterations and the lost of cell-cell contact between the first and the third days. DNA fragmentation and apoptotic indexes (May Grünwald Giemsa staining, double fluorescent dyes, and TdT-mediated dUTP nick-end labeling assay) revealed that cell death was maximal on the second day (three times over control). Furthermore, the pro-apoptotic proteins FAS and Bax were strongly up-regulated during the first 2 days. The aforementioned morphological and biochemical changes were also accompanied within the same period by caspase 3 activation. This study revealed that in vivo apoptosis in normal thymus after 5-FU administration is related to FAS, Bax, and Caspase 3 co-expressions under the current experimental conditions, these findings, therefore, contribute to a new insight into the molecular mechanisms involved during 5-FU administration upon the thymus and the possible events committed in the lymphophenia associated with chemotherapy.     

Thyroid tuberculosis – role of PCR in diagnosis of a rare entity

N. Gupta, K. Sharma, A. Barwad, M. Sharma, A. Rajwanshi, P. Dutta and A. Sharma
Thyroid tuberculosis – role of PCR in diagnosis of a rare entity Background: Tuberculosis is a rare cause of granulomatous thyroiditis, whose diagnosis may be difficult with routine cytopathology and staining for acid‐fast bacilli (AFB). Study design: Amongst 7962 cases of various thyroid lesions subjected to fine needle aspiration cytology (FNAC) over a period of 12 years, 34 cases (0.43%) were found to have cytological features of granulomatous inflammation with or without necrosis, which could be due to tuberculosis, granulomatous thyroiditis or other causes of granulomatous inflammation such as sarcoidosis or fungal infections. DNA was extracted from the material available on May‐Grünwald–Giemsa‐stained smears from the archival material. PCR for Mycobacterium tuberculosis was performed for insertion sequence IS6110. Results: The age of the patients ranged from 32 to 58 years (median 48 years); 24 were female and 10 male. FNAC from thyroid swellings showed epithelioid granulomas with giant cells and/or necrosis. Although acid‐fast bacilli were only seen in smears in two cases, 19/34 (55.9%) showed the presence of 123 bp DNA band under ultraviolet transillumination. Five control cases were negative. Conclusion: Our study of archival cytological material illustrates the importance of PCR as a potentially useful tool for the detection of M. tuberculosis DNA from FNAC of thyroid lesions, which could provide an alternative for rapid diagnosis of thyroid tuberculosis in AFB‐negative cases.     

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions

OBJECTIVE: To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. STUDY DESIGN: Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. RESULTS: Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. CONCLUSION: Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.     

The role of micronucleus scoring in fine needle aspirates of ductal carcinoma of the breast

S. Samanta, P. Dey and R. Nijhawan The role of micronucleus scoring in fine needle aspirates of ductal carcinoma of the breast Aims and objectives: Micronucleus (MN) scoring was carried out in benign (fibroadenoma) and malignant (infiltrating ductal carcinoma) breast lesions to evaluate the role of MN as a biomarker in breast carcinomas. We also compared MN scores among different cytological grades of breast carcinoma. Materials and methods: A total of 31 archival cases of fibroadenoma (FA) and 40 cases of infiltrating ductal carcinoma (IDC) were selected. The best May‐Grünwald–Giemsa (MGG) stained fine needle aspiration cytology (FNAC) smear of each case was selected. The MN scoring was carried out independently by two observers on 1000 epithelial cells in oil immersion magnification (100× objective). The MN scores in FA and IDC were compared. The IDC cases were graded and the MN scores in different cytological grades of IDC were compared. Results: The mean MN scores (± standard deviation) in FA and IDC were 0.6 (± 1.1) and 13.6 (± 12.8), respectively, which were significantly different (P < 0.0001). There were seven grade 1, 13 grade 2, and 20 grade 3 IDCs. The mean MN scores (± standard deviation) of grade 1, 2 and 3 IDC were 4.3 (± 2.3), 11.95 (± 9.2) and 21.1 (± 16.7), respectively. An analysis of variance (anova ) test showed a significant difference in MN score between all the grades of IDC (P < 0.05). However, there was no significant difference between fibroadenoma and grade 1 IDC. The Pearson’s correlation coefficient showed positive correlations between MN scoring in the different grades of IDC. Conclusions: MN scoring on routinely stained smears of IDCs was significantly higher than in fibroadenoma and was relatively easy, reliable and reproducible. As MN scoring of grade 1 IDC was similar to fibroadenoma, a larger study should be conducted to compare grade 1 IDC with other benign breast lesions.     

Breakdown of the endothelial barrier function in tumor cell transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.     

Light microscopical detection of leukocyte cell surface antigens with a one-nanometer gold probe.

The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated. Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter. Cytocentrifuge preparations were made from the cell suspensions. Silver enhancement was performed on all preparations. Then they were counterstained with May-Grünwald Giemsa and examined in light microscopy. The immunostaining appeared as fine dark granules on the surface membrane of the cells. Labeling conditions were determined which gave a dense specific immunostaining and a low background. High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples. The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles. Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy. We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens.     

Fine needle aspiration cytology in HIV‐related lymphadenopathy: experience at a single centre in north India

P. K. Sarma, A. K. Chowhan, V. Agrawal and V. Agarwal
Fine needle aspiration cytology in HIV‐related lymphadenopathy: experience at a single centre in north India Objective: Fine needle aspiration (FNA) is emerging as a rapid and minimally invasive tool in evaluating lymphadenopathy associated with human immunodeficiency virus (HIV). We evaluated the role of FNA in differentiating various causes of lymphadenopathy in patients with HIV and correlated the cytological diagnosis with CD4 counts. Methods: Seventy‐nine HIV‐positive patients (median age 35 years, 68 male) underwent ultrasound‐guided (n = 16) and unguided (n = 63) FNA from 1999 to 2006. Smears were stained with May–Grünwald–Giemsa, haematoxylin & eosin and Papanicolaou stains. Ziehl–Neelsen (ZN) staining for acid‐fast bacilli (AFB) was performed in all cases. Staining for fungus was performed whenever required. Results: The aspirates were adequate in 75 cases (95%). Non‐specific reactive hyperplasia was the most common FNA diagnosis (39, 52%) followed by granulomatous necrotizing lymphadenitis (15, 20%), necrotizing lymphadenitis (13, 17.3%) and granulomatous lymphadenitis (4, 5.2%). Fungal infection and non‐Hodgkin lymphoma (NHL) were seen in two patients each. ZN staining was positive for AFB in 25 (33.3%) cases. One of these was morphologically interpreted as reactive hyperplasia, 12 as necrotizing lymphadenitis and 12 as granulomatous necrotizing lymphadenitis. Both patients with NHL had CD4 counts below 100/dl. Necrotizing lymphadenitis and granulomatous lymphadenitis were significantly associated with CD4 counts below and above 200/dl, respectively (P = 0.0002). Conclusions: FNA is an important tool for assessing the cause of lymphadenopathy in HIV patients. Necrotizing inflammation is more often seen in patients with low CD4 counts. AFB are commonly found in necrotic aspirates with or without granulomas. However, a stain for AFB should be performed in all aspirates from HIV‐related lymphadenopathy including reactive hyperplasia.     

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.     

Infection of bovine oviduct cell cultures with Chlamydophila abortus

Bovine infertility is a major cause of loss in the livestock industry. In the present study bovine oviduct cell cultures were infected with a Chlamydophila abortus strain. A direct evaluation of infection was performed by means of May Grünwald-Giemsa and immunocytochemistry for chlamydial LPS, which revealed inclusion bodies and vacuolisation. SEM and TEM analysis of infected cells showed various degrees of cell damage and conglutination of microvilli. This finding suggests that cattle infertility may result from an alteration of oviduct environment caused by multiplication of C. abortus. This microorganism, among other infectious agents, could be considered a potential causative agent of bovine infertility.     

Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion

Tumors develop through multiple stages, implicating multiple effectors, but the tools to assess how candidate genes contribute to stepwise tumor progression have been limited. We have developed a novel system in which progression of phenotypes in a mouse model of pancreatic islet cell tumorigenesis can be used to measure the effects of genes introduced by cell-type-specific infection with retroviral vectors. In this system, bitransgenic mice, in which the rat insulin promoter (RIP) drives expression of both the SV40 T antigen (RIP-Tag) and the receptor for subgroup A avian leukosis virus (RIP-tva), are infected with avian viral vectors carrying cDNAs encoding candidate progression factors. Like RIP-Tag mice, RIP-Tag; RIP-tva bitransgenic mice develop isolated carcinomas by ∼14 wk of age, after progression through well-defined stages that are similar to aspects of human tumor progression, including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. When avian retroviral vectors carrying a green fluorescent protein marker were introduced into RIP-Tag; RIP-tva mice by intra-cardiac injection at the hyperplastic or early dysplastic stage of tumorigenesis, approximately 20% of the TVA-positive cells were infected and expressed green fluorescent proteins as measured by flow cytometry. Similar infection with vectors carrying cDNA encoding either of two progression factors, a dominant-negative version of cadherin 1 (dnE-cad) or Bcl-xL, accelerated the formation of islet tumors with invasive properties and pancreatic lymph node metastasis. To begin studying the mechanism by which Bcl-xL, an anti-apoptotic protein, promotes invasion and metastasis, RIP-Tag; RIP-tva pancreatic islet tumor cells were infected in vitro with RCASBP-Bcl-xL. Although no changes were observed in rates of proliferation or apoptosis, Bcl-xL altered cell morphology, remodeled the actin cytoskeleton, and down-regulated cadherin 1; it also induced cell migration and invasion, as evaluated using two-chamber transwell assays. In addition, myosin Va was identified as a novel Bcl-xL-interacting protein that might mediate the effects of Bcl-xL on tumor cell migration and invasion.     

Combined effects of doxorubicin and STI571 on growth, differentiation and apoptosis of CML cell line K562

STI571 (imatinib mesylate; Gleevec) is an inhibitor that targets the tyrosine kinase activity of Bcr-Abl present in chronic myelogenous leukemia (CML) cells. Some preclinical studies have demonstrated that the combination of STI571 with chemotherapeutic drugs results in enhanced toxicity in Bcr-Abl-positive leukemias. We investigated the potential benefit of using STI571 to down-regulate Bcr-Abl activity for the enhancement of doxorubicin anti-proliferative action in K562 cell line derived from blast crisis of CML. At low concentrations of both drugs (40 nM doxorubicin combined with STI571 in the range of 100-150 nM), the antiproliferative effects were mainly due to cellular differentiation as assessed by benzidine staining for hemoglobin synthesis level and real-time PCR for gamma-globin expression. Higher concentrations of STI571 used in combinations with doxorubicin caused mainly apoptosis as shown by DNA degradation and nuclear fragmentation visualized by fluorescence microscopy after DAPI staining, changes in cell morphology observed after Giemza-May Grünwald staining and cellular membrane organization estimated by flow cytometry after Annexin V staining. As compared with either drug alone, cotreatment with STI571 and DOX induced stronger cellular responses. A low concentration of STI571 in combination with a low concentration of DOX might be tested as an alternative approach to increasing the efficacy of chemotherapy against CML.     

Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial-specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic-free Ca(2+) [Ca(2+)](c). In the present study we show that the mitochondria-to-nucleus stress signaling induces invasive phenotypes in otherwise non-invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor-specific markers cathepsin L and transforming growth factor beta (TGFbeta) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4- to 6-fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca(2+)-dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with approximately 70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.     

New insights into germ cell tumor formation.

Recent years have witnessed a number of new findings with significant implications for our understanding of the development of germ cell tumors. This communication reviews some of these recent insights with an emphasis on mechanisms that may convert a germ cell into a tumor cell. Three aspects are discussed in this review: (1) the early origin of germ cell tumors from primordial germ cells through an aberrant mitosis-to-meiosis switch; (2) errors during meiosis, which promote tumorigenic transformation of germ cells; and (3) the role of small RNAs such as oncomirs (miRNAs) and oncopirs (piRNAs) in germ cell tumor formation. Since much has been learned using a variety of organismal models, data obtained in experiments with mice, nematodes, fruit flies, and human data will be considered. Only exemplary references are included.