Cytometry in the brain: studying differentiation to diagnostic applications in brain disease and regeneration therapy.

During brain development, a population of uniform embryonic cells migrates and differentiates into a large number of neural phenotypes - origin of the enormous complexity of the adult nervous system. Processes of cell proliferation, differentiation and programmed death of no longer required cells, do not occur only during embryogenesis, but are also maintained during adulthood and are affected in neurodegenerative and neuropsychiatric disease states. As neurogenesis is an endogenous response to brain injury, visible as proliferation (of to this moment silent stem or progenitor cells), its further stimulation can present a treatment strategy in addition to stem cell transfer for cell regeneration therapy. Concise techniques for studying such events in vitro and in vivo permit understanding of underlying mechanisms. Detection of subtle physiological alterations in brain cell proliferation and neurogenesis can be explored, that occur during environmental stimulation, exercise and ageing. Here, we have collected achievements in the field of basic research on applications of cytometry, including automated imaging for quantification of morphological or fluorescence-based parameters in cell cultures, towards imaging of three-dimensional brain architecture together with DNA content and proliferation data. Multi-parameter and more recently in vivo flow cytometry procedures, have been developed for quantification of phenotypic diversity and cell processes that occur during brain development as well as in adulthood, with importance for therapeutic approaches.

Cytomics - importance of multimodal analysis of cell function and proliferation in oncology.

Cancer is a highly complex and heterogeneous disease involving a succession of genetic changes (frequently caused or accompanied by exogenous trauma), and resulting in a molecular phenotype that in turn results in a malignant specification. The development of malignancy has been described as a multistep process involving self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and finally tissue invasion and metastasis. The quantitative analysis of networking molecules within the cells might be applied to understand native-state tissue signalling biology, complex drug actions and dysfunctional signalling in transformed cells, that is, in cancer cells. High-content and high-throughput single-cell analysis can lead to systems biology and cytomics. The application of cytomics in cancer research and diagnostics is very broad, ranging from the better understanding of the tumour cell biology to the identification of residual tumour cells after treatment, to drug discovery. The ultimate goal is to pinpoint in detail these processes on the molecular, cellular and tissue level. A comprehensive knowledge of these will require tissue analysis, which is multiplex and functional; thus, vast amounts of data are being collected from current genomic and proteomic platforms for integration and interpretation as well as for new varieties of updated cytomics technology. This overview will briefly highlight the most important aspects of this continuously developing field.

Cardiac troponin I release after transcatheter atrial septal defect closure depends on occluder size but not on patient's age.

OBJECTIVE: To examine whether transcatheter closure of secundum atrial septal defect (ASD) with the Amplatzer septal occluder leads to more myocardial injury in children than in adults. DESIGN: In a prospective study with children and adults cardiac troponin I (cTnI) serum concentrations were determined by immunoassay (AxSYM, Abbott Laboratories) before, during, and up to 20 months after surgical or transcatheter ASD closure. PATIENTS: Four groups of patients were studied: transcatheter ASD closure (group 1: 22 children, age range 3.26-14.7 years; group 2: 22 adults, 18.0-67.3 years), surgical ASD closure (group 3: 18 children, 3.12-13.5 years), and diagnostic catheterisation (group 4: 12 children, 2.68-15.0 years). RESULTS: cTnI concentrations were significantly increased after occluder implantation with higher serum concentrations in children than in adults (immediately after implantation: group 1, 3.2 (4.4) microg/l; group 2, 1.1 (4.2) microg/l; four hours after implantation: group 1, 4.8 (5.0) microg/l; group 2, 1.7 (2.3) microg/l; both p < 0.01, group 1 v group 2; one day after implantation: group 1, 3.0 (5.7) microg/l; group 2, 2.2 (5.2) microg/l) but were less than 20% of those after surgical ASD closure (group 3; p < 0.001) where the highest cTnI concentration was found (37.1 (26.3) microg/l). Diagnostic catheterisation (group 4) was not associated with detectable cTnI increase. From the cTnI concentrations the total amount of cTnI released after ASD closure was estimated for each patient. This was dependent on the size of the occluder (p < 0.05) but not on the patient's age or procedural duration. CONCLUSION: In regard to interventional ASD closure our data do not provide evidence that the child's myocardium is more vulnerable. Transcatheter ASD closure induces minor myocardial lesion, the extent of which depends on the size of the Amplatzer septal occluder but is irrespective of the patient's age.

Flow cytometric DNA analysis of benign hyperfunctioning parathyroid glands: significant difference in the S phase fraction and proliferative index between adenomas and hyperplasias.

AIMS: Flow cytometric DNA analysis was performed to measure the DNA content of benign parathyroid tumours in patients with primary hyperparathyroidism. METHODS: DNA analysis of paraffin-embedded parathyroid samples was performed on 51 parathyroid glands from 29 patients after parathyroidectomy. Histopathology showed parathyroid adenoma in 25 cases and hyperplasia in four patients. DNA ploidy status, DNA index (DI), percentage of cells in S phase and proliferative index (PI) were determined. RESULTS: Normal cells from normal glands were all diploid. DNA cytometry showed 12 aneuploid and 13 diploid adenomas. There were 12 diploid and four aneuploid hyperplastic glands. Incidence of aneuploid DNA histograms did not show a statistically significant difference between adenomas and hyperplasias (P=0.216). Mean S phase fraction was 3.45% in adenomas and 1.53% in hyperplasias (P= 0.015). Mean PI was 6.48% in adenomas and 2.78% in hyperplastic parathyroid glands. This difference was statistically significant (P=0.006). Diploid cases had a mean PI of 4.78% and aneuploid glands a mean PI of 7.7% (P=0.08). Aneuploid DNA content did not reveal statistically significant correlation with age, gender, pre-operative Ca, alkaline phosphatase, i-PTH levels, and tumour size. The mean S phase fraction and PI were 2.25% and 4.78% in diploid glands, and 4.5% and 7.7% in aneuploid cases. CONCLUSION: Aneuploid DNA content may be present in benign parathyroid diseases, but not in normal parathyroid glands. Aneuploid DNA histograms and higher PI occur more often in adenomas compared with hyperplasias, but the nuclear DNA analysis is unable to make a distinction between adenomas and hyperplasias.

Prognostic value of DNA ploidy status in patients with rectal cancer.

BACKGROUND: A retrospective study was performed to measure the prognostic value of DNA ploidy status and proliferative index (PI) for survival in patients with rectal cancer. PATIENTS AND METHODS: Fifty-two patients underwent curative surgery for rectal carcinoma. Ten tumors were in Stage I, 25 cancers were in Stage II, and 17 of them were in Stage III. Using flow cytometry the nuclear DNA content of the tumor cells was measured. RESULTS: There were 25 DNA diploid and 27 DNA aneuploid carcinomas. Aneuploid DNA content did not show higher occurrence in advanced tumors. The mean survival was 59 months in the case of DNA diploid carcinoma, while it was 47 months in the case of DNA aneuploid cancer. The mean PI of the DNA diploid cancers was 8%. The PI of DNA aneuploid tumors was 22%. High PI (PI > 10%) was observed in 32 carcinomas while low PI (PI < 10%) occurred in 20 cases. Patients with aneuploid DNA content and high PI had significantly worse survival compared to patients with diploid DNA content while low PI. Locoregional and distant metastases occurred more frequently in patients with aneuploid tumor. By univariate analysis, tumor size, lymph node involvement, DNA ploidy status and PI all correlated with prognosis. However, multivariate analysis showed that TNM stage and PI were the only significant prognostic factors for survival. CONCLUSION: The survival and disease-free survival of patients with diploid DNA content was better compared to aneuploid cases. The results suggest that DNA ploidy status is important in determining the biological behaviour of rectal carcinomas, although the multivariate analysis did not prove its significant influence. The PI were independent negative prognostic factors for survival.

Preoperative prediction of postoperative edema and effusion in pediatric cardiac surgery by altered antigen expression patterns on granulocytes and monocytes.

Postoperative edema and effusion (POEE) following cardiopulmonary bypass (CPB) surgery in children retards recovery and may aggravate postpericardiotomy (PPS), capillary leak syndrome (CLS), or multiorgan failure (MOF). Compared with complication-free children, POEE affected children have different preoperative serum levels of circulating cytokines and adhesion molecules. These levels may be used preoperatively to assess POEE, but their determination is time consuming, costly, and a substantial blood volume is required. Altered serum levels of cytokines and adhesion molecules also may be reflected in altered antigen expression on circulating blood leukocytes. The predictive potential of flow cytometric (FCM) leukocyte immunophenotyping was explored as a sensitive and fast method that required small blood samples. Blood samples taken 24 h preoperatively from 49 patients (3-18 years old) were stained with monoclonal antibodies for adhesion molecules (ICAM-1, LFA-1, Mac-1) or constitutive/activation markers (CD4, CD14, CD16, CD25, CD54, CD69, HLA-DR) and measured on a microbead calibrated FCM. Neutrophils, monocytes, and eosinophils from POEE patients express higher preoperative levels of LFA-1, monocytes, HLA-DR, and other activation markers (all P < 0.03). Over 89% of the patients were classified correctly by using two discriminant analysis methods (sensitivity, >76%; specificity, >86%; positive prediction, >80%; negative prediction, >83%). Granulocytes and monocytes of postoperative POEE patients exhibit significant preoperative immune activation, suggesting an increased risk for patients with atopic/allergic predisposition. Surgical trauma and CPB cause additional immune activation, leading to POEE by a summative response. Most patients at risk for POEE can be identified preoperatively by using data pattern analysis on FCM-derived parameters.

TGF beta 1 induces caspase-dependent but death-receptor independent apoptosis in lymphoid cells.

Transforming growth factor beta 1 (TGF beta 1) is an antiproliferative and proapoptotic cytokine for normal B-cells, however many B-cell lymphomas have lost their response to TGF beta 1. The aim of this study was to identify the sequence of events in apoptosis induced by TGF beta 1 in an EBV negative, human B-cell lymphoma line (HT58). The proportion of apoptotic cells increased gradually (up to 72 hr) at an optimal dose range of 0.5-1.0 ng/ml. The induced cell death required the action of downstream caspases. Caspase activation was accompanied by an increase in the permeability of mitochondrial membranes, but there was no change in the expression of certain members of Bcl-2 family (Bcl-2, Bax, Bcl-XL). Similarly, none of the death receptors or ligands were involved in apoptosis induction. Further study will include the participation of TGF beta 1 target genes in the pore formation of mitochondrial membranes and/or the elimination of a putative survival signal.

Role of the basement membrane in tumor cell dormancy and cytotoxic resistance.

OBJECTIVES AND METHODS: Tumor dormancy and resistance to cytotoxic agents are key limiting events in the treatment of malignant diseases. To determine whether both are influenced by the extracellular milieu in which tumors reside, HT1080 human fibrosarcoma, MCF-7 breast carcinoma and OSCORT osteosarcoma cell proliferation, viability, apoptosis and cytoreductive-treatment-induced death were investigated in the presence or absence of extracellular matrix (ECM). RESULTS: ECM-adherent, but not plastic-adherent HT1080 cells formed a multicellular network accompanied by reduced proliferation and lowered DNA synthetic capacity. The number of cells in S-phase was dramatically reduced. Viable cells entered a state of dormancy reminiscent of that observed in the step of metastasis after extravasation, i.e. prior to the initiation of progressive growth. Such ECM-induced dormancy could be reversed by plating cells on plastic, but only after a 48-hour lag period. No difference was indicated in clonogenicity of HT1080 cells originated from plastic or ECM gel. However, the cells released from ECM gel showed significantly reduced migration ability. The resistance of anchored cells against cytotoxic damage was increased by ECM gel. Examination of cytoreductive treatment revealed that ECM adherence at the time of injury is partially protective, a property which was also moderately apparent when injured cells were transferred to the basement membrane. CONCLUSIONS: Taken together, these results suggest that the ECM plays a key role in tumor dormancy and cytotoxic resistance, both explorable at the molecular level using our in vitro model system.

Formaldehyde promotes and inhibits the proliferation of cultured tumour and endothelial cells.

Formaldehyde was applied in various doses (0.1-10.0 mM) to HT-29 human colon carcinoma and HUV-EC-C human endothelial cell cultures. Cell number, apoptotic and mitotic index as well as proportion of cells in S-phase was investigated by morphological methods and flow cytometry. Ten mM of formaldehyde caused high degree of cell damage and practically eradicated the cell cultures. One mM of formaldehyde enhanced apoptosis and reduced mitosis in both types of cell cultures, in a moderate manner. The low dose (0.1 mM) enhanced cell proliferation and decreased apoptotic activity of the cultured cells, the tumour cells appeared to be more sensitive. The possible role of this dose-dependent effect of formaldehyde in various pathological conditions, such as carcinogenesis and atherogenesis is discussed with emphasis on the eventual interaction between formaldehyde and hydrogen peroxide.

Induction of apoptosis in MDR1 expressing cells by daunorubicin with combinations of suboptimal concentrations of P-glycoprotein modulators.

The application of most agents with the capacity to reverse multidrug resistance (MDR) via modulation of the multidrug transporter P-glycoprotein (Pgp) was shown to be associated with toxic side-effects. For this reason, we have investigated the effect of combinations of suboptimal concentrations of Pgp blockers on the induction of apoptosis and growth arrest in daunorubicin (D) treated, MDR1 gene transfected cells. We used verapamil, PSC833 and Cremophor EL as Pgp modulators, which affect the function of Pgp by different mechanisms. Treatment of NIH3T3/MDR1 cells with combinations of suboptimal concentrations of Pgp modulators in the presence of D caused apoptosis and G(2) arrest to the same extent as optimal concentrations of singly used blockers. We conclude that combinations of suboptimal concentrations of Pgp modulators may cause effective sensitization of resistant tumor cells, and at the same time, may avoid the frequently observed toxic effects experienced in clinical trials with a single modifier applied at the optimal dose.

Anti-apoptotic and apoptotic action of (-)-deprenyl and its metabolites.

The mode of cytoprotective action of the monoamine oxydase B inhibitor (-)-deprenyl was studied using A-2058 human melanoma cells in culture. Serum deprivation caused apoptosis of the cultured cells, which could be decreased by administration of 10(-9) - 10(-13)M (-)-deprenyl. The known metabolites of (-)-deprenyl, (-)-desmethyl-deprenyl, (-)- and (+)-methylamphetamine failed to exert the same effect. The anti-apoptotic activity of (-)-deprenyl was prevented by the simultaneous application of the microsomal drug-metabolizing enzyme inhibitor SKF-525A. These results show that (-)-deprenyl needs metabolic conversion in order to be anti-apoptotic, but the effective metabolite is still unknown. On the other hand, higher dose (10(-13)M) of (-)-deprenyl, (-)-desmethyl-deprenyl, (-)- and (+)-methylamphetamine induced apoptosis in the non-serum-deprived A-2058 cell culture. SKF-525A did not prevent the apoptosis-inducing effect of (-)-deprenyl, which means that no metabolic changes are needed for this activity. High dose (10(-3)M) of (-)-deprenyl induced very high Caspase 3 activity in non-serum-deprived A-2058 cell culture, low doses (10(-9) - 10(-3) M) of (-)-deprenyl maintained Caspase 3 activity on control level in case of serum-deprivation.

Low degree of activation of circulating neutrophils determined by flow cytometry during cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Enhanced expression of adhesion molecules LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) following cardiac surgery with cardiopulmonary bypass (CPB) is held responsible for postoperative complications. Surface expression of these molecules, intracellular pH (pH(i)), and oxidative burst capacity was analyzed to test for neutrophil activation during pediatric cardiac surgery. METHODS: Blood samples were drawn from 36 patients (age: 3--16 years) 24 h preoperatively, after onset of anesthesia, after connection to CPB (CPB1, before and after passing CPB, n = 15), at reperfusion (CPB2), and up to 7 days postoperatively. Cells adhering to CPB filters were isolated (n = 11). Antigen expression, pH(i), and oxidative burst capacity on neutrophils was analyzed by flow cytometry. RESULTS: During surgery, oxidative burst capacity was at low level with a mild increase only 1 day after surgery. pH(i) was decreased throughout the surgery. Surgery induced more than 36% decrease of LFA-1 and Mac-1 expression (P < 0.03). Up to postoperative day 7, no increase of antigen expression above baseline was found. Neutrophils isolated from filters of the CPB had increased LFA-1 and Mac-1 expression (all P < 0.05). Integrin expression on neutrophils passing the CPB at CPB1 was decreased (P < 0.05). CONCLUSION: Reduced adhesion molecule expression on neutrophils may be due to selective filtration of highly adhesive cells. This, in combination with low-level oxidative burst capacity, induced by immunosuppressive cytokines (e.g., interleukin-10), reduced the neutrophil activity. Our data indicate that increased activity of circulating neutrophils cannot exclusively be held responsible for postoperative complications after surgery with CPB.

Constitutive intracellular expression and activation-induced cell surface up-regulation of CD44v3 in human T lymphocytes.

The cell adhesion molecule CD44 exists in multiple isoforms generated by alternative RNA splicing. Increased expression of CD44 isoforms containing exon v6 and v9 has been reported to be associated with the activated state of T lymphocytes. Using monoclonal antibodies against variant exon products we studied the expression of another variant exon, v3 on resting and in vitro activated human peripheral blood T cells. We found that CD44v3, in parallel with CD44v6, is up-regulated at the surface of normal T cells stimulated by anti-CD3 antibody or by the phorbol ester PMA, as well as on PMA-stimulated T cell leukemia lines CCRF-CEM and MOLT-4. Beside the cell surface, we demonstrated CD44v3 intracellularly in both resting and activated T cells by flow cytometry and immunomorphology. Reverse transcription-PCR and Western blot analyses confirmed the constitutive expression of CD44v3 in these cells. The increase in the cell surface expression of CD44v3 on stimulated T lymphocytes was inhibited by cycloheximide and brefeldin A, indicating the requirement of de novo protein synthesis and endoplasmic reticulum Golgi transport. Our studies establish CD44v3 as an additional activation marker for human T cells, with a yet unidentified function.

Flow cytometric evidence of apoptosis in human pancreatic cancer xenografts treated with Sandostatin (octreotide).

BACKGROUND: The antiproliferative effect of octreotide (Sandostatin) is partly attributed to induction of apoptosis in the given tumors. In this work, apoptosis was assessed in human pancreatic carcinoma xenografts after a 4-week high-dose Sandostatin treatment. MATERIALS AND METHODS: Subcutaneously growing human pancreatic cancer xenografts (PZX-5) in immunosuppressed mice were treated with 500 micrograms/kgb.w. Sandostatin twice a day i.p. for 4 weeks. Apoptosis was evaluated by means of conventional histology, Apoptag-immunohistochemistry and flow cytometry. RESULTS: The Sandostatin-treatment resulted in a decreased tumor volume in 9 out of 16 animals. Immunohistochemical detection of apoptosis by Apoptag revealed a 75-fold increase of the positively stained tumorous nuclei (210.9 +/- 53.9 per square mm) versus nontreated tumors (2.8 +/- 0.5 per square mm). The sub-G1 fraction was 3.61 +/- 0.4% in untreated samples while it doubled after treatment (p < 0.001). CONCLUSION: A 4-week octreotide (Sandostatin) treatment induced significantly increased apoptosis in human pancreatic carcinoma xenografts evidenced by morphological studies and Apoptag-immunohistochemistry, and these results were clearly reinforced by flow cytometry.

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes.

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.

Human pancreatic leiomyosarcoma (PZX-7) growing as a serially transplantable xenograft in immunosuppressed mice.

BACKGROUND: Several human leiomyosarcoma xenografts have been established, but pancreatic smooth muscle sarcomas have never been serially transplanted and investigated. METHOD: Immunosuppression of CBA/CA mice was achieved by thymectomy, whole-body irradiation, and bone marrow reconstruction. Tumor fragments were subcutaneously implanted from a Grade III pancreatic leiomyosarcoma and serially passaged for more than 24 mo. The xenografted tumors were characterized by morphological, morphometrical, biochemical, and flow cytometric methods. RESULTS: The tumor has retained its characteristic morphology and no further differentiation occurred. The mitotic counts and the amount of the connective tissue all remained constant. The calculated volume doubling time was 11.3 d. Immunohistochemically, the tumor proved to be p53-negative, but the strong expression of the bcl-2 remained as a constant feature throughout successive transplantations. The DNA index and the proliferation indices did not change significantly with the time (mean DI: 1.65, range: 1.561-1.70; mean PI: 17.9%, range: 15.3-20.7%). Lactose dehydrogenase (LDH) isoenzyme electrophoresis evidenced a retained human pattern of the tumor even after 32 mo of transplantations. CONCLUSION: The first human pancreatic leiomyosarcoma xenograft (PZX-7) growing in immuno-suppressed mice is described and characterized.

A specifically radiolabeled somatostatin analog with strong antitumor activity induces apoptosis and accumulates in the cytosol and the nucleus of HT29 human colon carcinoma cells.

The new heptapeptide somatostatin analog TT-232 decreases proliferation of HT-29 human colon carcinoma cells in vitro by reducing mitotic and increasing apoptotic activity. We have synthesized and characterized a specifically tritium labeled 3H-Tyr3-TT-232 (30 Ci/mmol) to investigate the effect and the fate of this antitumor peptide on human colon tumor cells. 3H-labeled TT-232 could be detected on the cell surface, on cytoplasmic membranes and also in the nucleus of HT-29 cells, 1-6 h after the administration of 0.5 and 50 microg/mL [3H]TT-232. Binding and internalization of TT-232 to human colon tumor cells at a relatively high dose provide further evidence for the existence of low-affinity somatostatin receptors in such cells, which might mediate the apoptosis-inducing effect. Our data suggest the possible use of TT-232 in the treatment of human colon tumors.

Establishment and long-term xenografting of human pancreatic carcinomas in immunosuppressed mice: changes and stability in morphology, DNA ploidy and proliferation activity.

The prognosis of pancreatic carcinoma is grave, therefore the experimental model systems remain major tools for testing new treatment modalities. We have developed human pancreatic cancer lines growing in immunosuppressed mice and characterized them by morphological and flow-cytometric studies. Immunosuppression has been achieved in young (4-week-old) CBA/CA mice by thymectomy, whole-body irradiation and bone marrow reconstruction. Twelve surgically removed human pancreatic carcinomas were implanted subcutaneously and serially transplantable xenografts have been established. Altogether 129 samples derived from 59 generations have been analyzed. Out of 12 carcinomas, 6 developed continuously growing and transplantable xenografts (PZX-2, PZX-5, PZX-11, PZX-15, PZX-16, PZX-20; take rate: 50%). They were successfully maintained for 9-16 passages, for 18-25 months. New subpopulations developed during transplantations in 3 tumor lines and these morphological changes have been reflected by the appearance of an aneuploid peak in flow cytometry. In 1 tumor line, however, DNA aneuploidy was observable despite the unaltered histology. The PZX-20 line retained its original morphology and aneuploid pattern during 9 consecutive passages and over 18 months. The results indicate that the artificially immunosuppressed CBA/CA mice are suitable hosts for accepting and maintaining human pancreatic carcinomas. During successive xenograftings the regular morphological characterization must be supplemented by flow cytometry, because new tumorous clones may develop despite the unchanged histological picture.

The site-dependent growth characteristics of a human xenotransplanted basaloid squamous cell carcinoma.

PURPOSE: To improve our understanding of the aggressive behaviour of basaloid squamous cell carcinoma (BSCC) certain biological features related to malignancy were compared in the basaloid and in the squamous cell population of this tumour. METHODS: Growth rate, cell population kinetics parameters, ploidy and collagenase activity were measured in BSCC xeno-transplanted subcutaneously or into oral submucosa. RESULTS: The basaloid component of BSCC showed a growth advantage in the subcutaneous location and formed a mainly aneuploid population (69.3%) without any sign of invasiveness. However the transplantation of this tumour into the oral submucosa resulted in the reappearance of the squamous carcinoma cell population containing diploid and aneuploid cells in equal proportion. The diploid cells in the tumour growing in the subcutis were in G1 phase, whereas 30% of the diploid and aneuploid cells growing in the oral submucosa were in the growing (S+G2) phases of the cell cycle. The mixed tumour cell population in the oral submucosa produced 92-kDa collagenase IV, indicating a potential to infiltrate surrounding tissues. CONCLUSIONS: The biological behaviour of a human oral carcinoma (BSCC) in a xenograft model depends on the site of the transplantation. The aggressive malignancy of BSCC may be associated with the capacity of the basaloid cell population to generate squamous cells that are able to produce the 92-kDa type of collagenases in an appropriate microenvironment.

Relationship between the survival and the clinicopathological parameters of the patients with tumors in the pancreatic head region.

Retrospective study was performed to assess the possible prognostic factors for survival in patients after radical surgery with carcinoma of the pancreatic head region. Twenty-nine patients underwent pancreaticoduodenectomy for cancers of the pancreatic head (n = 22) and the papilla of Vater (n = 7). Using flow cytometry, authors measured the nuclear DNA content of tumor cells. DNA ploidy status was evaluated from paraffin-embedded tumor tissues. Fourteen DNA diploid and eight DNA-aneuploid pancreatic carcinomas occurred. Six DNA diploid and one DNA-aneuploid tumors were diagnosed in the group of papilla of Vater. Mean survival of patients with the carcinoma of pancreatic head was 9.3 months. Survival of the patients with the cancer of papilla of Vater was 20.5 months. The mean survival was 10 months in case of DNA-diploid pancreatic carcinoma, and it was 8 months in case of DNA-aneuploid cancer. The survival of the patients with DNA* diploid Vater papilla tumor was 17 months, and it was 40 months with the DNA-aneuploid cancer. The mean proliferative index (PI) of DNA-diploid pancreatic cancers was 9.7%, whereas that of the DNA-aneuploid cases was 13.3%. The mean PI of DNA-diploid tumors of papilla of Vater was 7.5% and that of the DNA aneuploid cases was 28%. There was no significant correlation between the PI and the survival. DNA-ploidy status and PI had no significant effect on the survival in patients with carcinoma of the pancreatic head region.