Cell kinetic studies on the JB-1 ascites tumour of the mouse

Abstract. The FLM method, modified by double labelling with [³H]- and [¹⁴C]-thymidine, has been applied to the 4-day old JB-1 ascites tumour of the mouse. It results in well separated waves of purely [³H]- and purely [¹⁴C]-labelled mitoses, which show a remarkable asymmetry with long tails to the right. The following values for the mean transit times of the cells have been derived from this FLM curve, for a tumour age of 4–6 days: T_C= 32.5 hr, T_S= 16.7 hr, T_G1= 3.7 hr, T_G1= 11.0 hr and T_M= 1.1 hr. A further evaluation of the FLM curve, however, is difficult, due to the non-stationary growth of the tumour. A number of other experimental findings (growth curve, decrease of the labelling and mitotic index with increasing tumour age, two single-labelled FLM curves starting 4 and 6 days after tumour inoculation) indicate that the cell cycle time increases during the experimental period of the double-labelled FLM curve (about 2 days). A lengthening of the cycle time should result in an increasing enlargement of the areas under the waves of the modified FLM curve. However, such an increase in area has not been found; the areas are constant. All the results of the present cell kinetic studies would be consistent if it were postulated that the cell cycle time lengthens with increasing tumour age up to about 4 days after inoculation, then remains relatively constant at between 4 and 6 days and thereafter increases again. Short-term double labelling experiments suggest that this is actually the case. Under the assumption of nearly constant phase durations during the 5th and 6th day of tumour growth further conclusions can be drawn from the modified FLM curve. In particular, it follows that the transit times of the cells through successive cycle phases are uncorrelated and the variances of the transit times through a cycle phase are proportional to the duration of this phase.     

The Effect of X-Irradiation On Cell Loss In Five Solid Murine Tumours, As Determined By the 125Iudr Method

The rate of cell loss in irradiated RIF-1, EMT6, KHJJ, B16 and KHT tumours was studied using the ¹²⁵IUdR loss technique. Administration of ¹²⁵IUdR preceded localized tumour irradiation by 2 days. Loss of tumour radioactivity was measured for 6–8 days after irradiation. the blood flow to some tumours was occluded during, and for 30 min following, injection of the label to measure the amount of radioactivity entering the tumour as a result of reutilization of label from the gut epithelia and influx of labelled host cells. Irradiation did not significantly alter the amount of radioactivity entering these clamped tumours during the 8–10 days after injection of ¹²⁵IUdR. This permitted comparison of irradiated and control groups based on the loss of radioactivity from the non-occluded tumours. Irradiation of RIF-1, EMT6, KHJJ or B16 tumours with doses of 600, 1400, 2400 or 4400 rads produced no significant increase in the rate of loss of tumour radioactivity. This suggested that, in the population of labelled cells, cell lysis following irradiation proceeded slowly. In contrast, KHT tumours showed a significant increase in loss rate following each radiation dose, although the increase was dose-independent. In all tumour systems, the constant rate of cell loss after radiation appeared to coincide with published reports of tumour growth responses after irradiation. the present data suggest that the manner of expression of radiation-induced cell killing results from the cellular proliferative status, i.e. whether a cell is cycling or non-cycling.     

State-vector models of the cell cycle 1. Parametrization and fits to labeled mitoses data

In this paper we study the Hahn model of the cell cycle from the point of view that a cell population's age distribution is more relevant to labeled mitoses data than is the distribution of its transit times.Closed-form relationships are derived between the transition probabilities of the Hahn model and the transit time of the mean of a cohort of labeled cells (with the variance of their transit time through mitosis). Constraints result which define the acceptable values for the number of ages in the state vector and the length of the time step (rarely does the dimension of the state vector equal the number of time steps in the generation time).A generalization to distinct probabilities for G₁, S and G₂M is presented, and the automatic fitting of fraction-labeled mitoses (FLM) data is described. The doubling time of the population is used to define the daughter factor, via the largest eigenvalue of the state transition matrix. The performance of the generalized Hahn model is compared to that of other commonly used fitting methods using two sets of FLM data from the literature. The synthesis of continuous labeling curves is discussed as an independent check of the parametrization. Based on the stable age distribution resulting from fits to experimental FLM data, it is shown that a nonlinear relationship exists between biological age and time.     

The effects of ionizing radiation on cell cycle progression in ataxia telangiectasia

Although ataxia telangiectasia (AT) cells are more sensitive than normal cells to killing by ionizing radiation, their DNA synthesis is more resistant to inhibition by radiation. It was thought that this anomaly in DNA synthesis was likely to perturb cell cycle progression. Flow cytometry and the fraction of labelled mitoses (FLM) were used to investigate effects of irradiation in normal and AT cell lines. The FLM indicated that radiation apparently induced a longer G2 delay in normal cells than in AT cells. However, flow cytometry showed that radiation induced much larger and more prolonged increases in the proportion of G2 cells in AT than in normals. AT populations also showed much larger postirradiation decreases in viable cell numbers. These data suggest that a large proportion of the radiosensitive AT cells are not reversibly blocked in G2 but die there, and never proceed through mitosis. The less radiosensitive normal cells are delayed in G2 and then proceed through mitosis. We suggest that the apparently shorter radiation-induced mitotic delay seen in AT cells by FLM is not real but is an artifact arising from perturbation of steady state conditions by selective elimination of a particular cohort of AT cells. Accumulation of AT cells in G2 is compatible with radiosensitivity of these cells and may arise from a defect in DNA repair or an anomaly in DNA replication.     

Modelling the Cell Cycle and Cell Movement in Multicellular Tumour Spheroids

This paper analyses a recent mathematical model of avascular tumour spheroid growth which accounts for both cell cycle dynamics and chemotactic driven cell movement. The model considers cells to exist in one of two compartments: proliferating and quiescent, as well as accounting for necrosis and apoptosis. One particular focus of this paper is the behaviour created when proliferating and quiescent cells have different chemotactic responses to an extracellular nutrient supply. Two very different steady-state behaviours are identified corresponding to those cases where proliferating cells move either more quickly or more slowly than quiescent cells in response to a gradient in the extracellular nutrient supply. The case where proliferating cells move more rapidly leads to the commonly accepted spheroid structure of a thin layer of proliferating cells surrounding an inner quiescent core. In the case where proliferating cells move more slowly than quiescent cells the model predicts an interesting structure of a thin layer of quiescent cells surrounding an inner core of proliferating and quiescent cells. The sensitivity of this tumour structure to the cell cycle model parameters is also discussed. In particular variations in the steady-state size of the tumour and the types of transient behaviour are explored. The model reveals interesting transient behaviour with sharply delineated regions of proliferating and quiescent cells.     

Culture of low passage colorectal cancer cells and demonstration of variation in selected tumour marker expression

There is increasing evidence that a tumour comprises of heterogeneous population of cells. Thus, studying homogenous cell lines in vitro may yield results that are not reflective of the true situation in a tumour and studying low passage cell lines maintained in a heterogeneous population before they transform away from the original state may provide a more complete picture of colorectal cancer. A method was developed to isolate and establish low passage colorectal cancer cell lines from tumour biopsies. The media contents, combination of antimicrobials and specimen collection and transport conditions employed, successfully eliminated microbial contamination which is frequently present in samples obtained from the gastrointestinal tract. A variety of growth forms indicating a heterogeneous mixture of cells was seen in the initial cultures. Using fluorescence immunocytochemistry, primary tumour cultures were shown to variably express selected tumour markers, carcinoembryonic antigen and C2 antigen. These low passage cell lines growing in a heterogeneous environment would more closely reflect the characteristics of the cells of the original tumour.     

Induction of VX2 carcinoma in rabbit liver: comparison of two inoculation methods

Direct injection of VX2 cell suspension into the liver is simple and widely used. Implantation of a fragment of VX2 tumour into the liver using a surgical technique has also been developed in the last decade. In this study, we compared these two methods in order to find a better modality for establishing VX2 liver mass. Forty rabbits, each weighing 2.8-3.2 kg, were divided into two groups, 20 rabbits in each. In Group 1, a tumour cell suspension containing 1 x 10(6) cells in a volume of 0.1 ml, was injected slowly into the liver parenchyma using a 27-gauge needle during laparotomy. In Group 2, a 1 mm(3) fragment of VX2 carcinoma was inoculated into the sub-capsule of the left anterior lobe of the liver. In Group 1, three rabbits showed no tumour growth and 10 rabbits showed evidence of leakage and tumour seeding outside of the liver. In Group 2, all but one rabbit showed tumour growth and none showed evidence of tumour seeding. The leakage rates were 50% and 0% for Group 1 and Group 2, respectively. Overall, the success inoculation rate was 35% for Group 1 and 95% for Group 2. In conclusion, to create the VX2 liver tumour model in rabbits, direct implantation of VX2 tumour fragment into the liver achieved better results than injecting cell suspension of VX2 tumour into the liver.     

Discrepancies in ploidy determination due to specimen sampling errors

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.     

THE FINE STRUCTURE OF THE CELLS IN MOUSE SARCOMA 37 ASCITIC FLUIDS

The tumour cells and the reaction cells in Sarcoma 37 ascitic fluids have been studied in thin sections with the electron microscope. The reaction cells were either leucocytes or much larger acidophilic peritoneal cells of the same dimensions as the tumour cells; the peritoneal cells formed as much as 20 per cent of the large cell population. The fine structure of the cells is described and some new observations recorded. It has been found that the cell membrane of eosinophil granulocytes has a laminated composition and the characteristic granules of these cells a double limiting membrane. The pores in the double nuclear membrane of the peritoneal cells have been observed to have a fine line running across them. In the tumour cells, a rounded granular body with a central dense area has been found in the region of the centrosome; these cells were also seen to contain rows of parallel smooth surfaced cisternae lying 150 mµ apart similar to those hitherto only observed in spermatids. There was a feltwork of fine filaments in the cytoplasm of the centrosome region of the tumour cells. The cytoplasmic fine structure underlying the basophilia of the tumour cells and the acidophilia of the peritoneal cells is compared and discussed.     

Monitoring of lung tumour cell growth in artificial membranes

Morbidity of many tumour types is associated with invasion of tumour cells through the basement membrane and subsequent metastasis to vital organs. Tumour invasion is frequently detected late on as many patients present with advanced disease. The method of detecting invasion is through conventional histological staining techniques, which are time consuming and require processing of the sample. This can affect interpretation of the results. In this study, a new imaging technique, optical coherence tomography (OCT), was used to monitor lung tumour cell growth in two artificial membranes composed of either collagen type I or Matrigel. In parallel, standard histological section analysis was performed to validate the accuracy of the monitoring by OCT. Cross-sectional images from OCT revealed that lung tumour cells infiltrated only when low cell seeding density (5 x 10(5)) and low collagen concentration (1.5 mg/ml) were combined. The cells could be easily differentiated from the artificial membranes and appeared as either a brighter layer on the top of the membrane or brighter foci embedded within the darker membrane. These cell-membrane morphologies matched remarkably to the standard histological section images. Our results suggest that OCT has a great potential to become a useful tool for fast and robust imaging of cell growth in vivo and as a potential assessment of cell invasion.     

Density-dependent quiescence in glioma invasion: instability in a simple reaction-diffusion model for the migration/proliferation dichotomy

Gliomas are very aggressive brain tumours, in which tumour cells gain the ability to penetrate the surrounding normal tissue. The invasion mechanisms of this type of tumour remain to be elucidated. Our work is motivated by the migration/proliferation dichotomy (go-or-grow) hypothesis, i.e. the antagonistic migratory and proliferating cellular behaviours in a cell population, which may play a central role in these tumours. In this paper, we formulate a simple go-or-grow model to investigate the dynamics of a population of glioma cells for which the switch from a migratory to a proliferating phenotype (and vice versa) depends on the local cell density. The model consists of two reaction-diffusion equations describing cell migration, proliferation and a phenotypic switch. We use a combination of numerical and analytical techniques to characterize the development of spatio-temporal instabilities and travelling wave solutions generated by our model. We demonstrate that the density-dependent go-or-grow mechanism can produce complex dynamics similar to those associated with tumour heterogeneity and invasion.     

DNA tumour viruses promote tumour cell invasion and metastasis by deregulating the normal processes of cell adhesion and motility

Approximately 15-20% of global cancer incidence is causally linked to viral infection, yet the low incidence of cancers in healthy infected individuals suggests that malignant conversion of virus-infected cells occurs after a long period as a result of additional genetic modifications. There are four families of viruses that are now documented to be involved in the development of human cancers which include members of the polyomavirus, hepadnavirus, papillomavirus and herpesvirus families. Although a number of these viruses are implicated in the aetiology of lymphomas or leukaemias, the vast majority are associated with malignancies of epithelial cells. In epithelial tissues, several classes of proteins are involved in maintaining tissue architecture, including those that promote cell-cell adhesion, and others, which mediate cell-matrix interactions. Proteins representative of all classes are frequently altered in malignant tumour cells that possess invasive and metastatic properties. Malignant tumour cells acquire mechanisms to degrade basement membranes and invade the underlying tissue. Many viruses encode proteins which engage signalling pathways that affect one or more of these mechanisms. It is believed that activation of these processes by chronic viral infection can, under certain circumstances, promote tumour cell invasion and metastasis. This review will take a brief look at the current knowledge of viral-induced alterations in cell motility and invasiveness in the context of tumour invasion and metastasis.     

Do neuroendocrine cells in human prostate cancer express androgen receptor?

The presence of androgen receptors (AR) in neuroendocrine cells was investigated in benign tissue of 10 prostatectomy specimens, in 12 prostatic adenocarcinomas with focal neuroendocrine differentiation and in 1 case of a pure neuroendocrine small cell carcinoma of the prostate. Neuroendocrine cells were defined by their reactivity with an antibody to chromogranin A. Monoclonal antibody F39.4 directed against the amino-terminal domain of the AR molecule was used to detect AR. AR and chromogranin A were simultaneously visualized with a double immunofluorescence technique. The results indicate that chromogranin positive cells in both benign and malignant prostatic tissue lack detectable expression of AR. No effect of endocrine therapy was noted. These results are in agreement with the hypothesis that prostatic neuroendocrine tumour cells represent an androgen insensitive cell population, which incidentally may expand to replace the androgen-sensitive tumour cell population during androgen ablation therapy.     

Cell kinetics of hypoxic cells in a murine tumour in vivo: flow cytometric determination of the radiation-induced blockage of cell cycle progression

Cells from the small cell population of viable cells in the large necrotic centre of murine M8013 tumours were investigated with respect to their cell kinetics. Flow cytometry (FCM) of this part of subcutaneously transplanted tumours revealed the presence of tumour cells with G1, S and G2 + M phase DNA-contents. These severely hypoxic cells could have stopped cell cycle progression due to the nutritional deprivation, irrespective of their position within the cell cycle. Labelling methods, used to disclose the cell kinetics of this cell population, are hampered by the absence of a transport system in these large necrotic areas. Therefore, FCM was used to monitor radiation-induced changes in the cell cycle distribution. From this investigation it was concluded that hypoxic cells in the necrotic centre of the M8013 tumour progress through the cell cycle. As well as a cell population with a cell cycle time (Tc) of approximately 84 hr, a subpopulation with a Tc of approximately 21 hr occurred.     

Proton magnetic relaxation time of normal and tumor cells

A proton NMR analysis of an in vitro culture of cells in heavy water has been made. The relaxation times of L-strain cells 929, He-La, transformed and normal embryonic human cells, C3H mice and isolated Yoshida sarcoma tumour cells, as well as of Yoshida sarcoma tumour tissue were determined. It turned out that spin-lattice (T1) and spin-spin (T2) relaxation times are characteristic of every cell and fairly different from those of corresponding tissues, which may be used for NMR identification of cells (NMR cytology). Furthermore, it has incontestably been proved that there is an ordered water fraction of cells, which is very slowly exchanged with surrounding heavy water.     

Cell population dynamics during the differentiative phase of tissue development

The dynamics of a cell population whose numbers are growing exponentially have been described well by a mathematical model based on the theory of age-dependent branching processes. Such a model, however, does not cover the period following exponential growth when cell differentiation curtails population size. This paper offers an extension to the branching process model to remedy this deficiency. The extended model is ideal for describing embryonic growth; its use is illustrated with data from embryonic retina. The model offers a better computational framework for the interpretation of a variety of data (growth curves of cell numbers, DNA histograms, thymidine labelling indices, FLM curves, BUdR-labelled mitoses curves) because age-distributions can be calculated at any stage of development, not just during exponential growth. Proportions of cells in the various phases of the cell cycle can be computed as growth slows. Such calculations show the gradual transition from a population dominated by cells which are young with respect to cell cycle age to one dominated by those which are old, and the effects such biases have on the proportions of cells in each phase.     

Transit times through the cycle phases of jejunal crypt cells of the mouse. Analysis in terms of the mean values and the variances.

Mean transit times as well as variances of the transit times through the individual phases of the cell cycle have been determined for the crypt epithelial cells of the jejunum of the mouse. To achieve this the fraction of labelled mitoses (FLM) technique has been modified by double labelling with [3H] and [14C]thymidine. Mice were given a first injection of [3H]thymidine, and 2 hr later a second injection of [14C]thymidine. This produces a narrow subpopulation of purely 3H-labelled cells at the beginning of G2-phase and a corresponding subpopulation of purely 14C-labelled cells at the beginning of the S-phase. When these two subpopulations progress through the cell cycle, one obtains FLM waves of purely 3H- and purely 14C-labelled mitoses. These waves have considerably better resolution than the conventional FLM-curves. From the temporal positions of the observed maxima the mean transit times of the cells through the individual phases of the cycle can be determined. Moreover one obtains from the width of the individual waves the variances of the transit times through the individual phases. It has been found, that the variances of the transit times through successive phases are additive. This indicates that the transit times of cells through successive phases are independently distributed. This statistical independence is an implicit assumption in most of the models applied to the analysis of FLM curves, however there had previously been no experimental support of this assumption. A further result is, that the variance of the transit time through any phase of the cycle is proportional to the mean transit time. This implies that the progress of the crypt epithelial cells is subject to an equal degree of randomness in the various phases of the cycle.     

CELL PROLIFERATION IN EMT6 TUMOURS TREATED WITH SINGLE DOSES OF X-RAYS OR HYDROXYUREA II. COMPUTER SIMULATIONS

A computer simulation technique was used to analyse data on the proliferation of clonogenic cells in EMT6 tumours treated with 5 mg/mouse of hydroxyurea (HU) or 3·0 Gy (300 rads) X-rays. This simulation technique is able to determine the respective roles of selective killing, blocks in cell progression and recruitment of the treated population. When the technique was applied to tumours treated with HU, it was possible to prove that both a G₁/S block and recruitment occurred. These phenomena could not have been demonstrated quantitatively, or even qualitatively, without the use of the simulation. After irradiation, blocks in cell progression and differences in the proliferative patterns of the surviving clonogenic cells and the total tumour cell population were found.     

Cell kinetics of hypoxic cells in a murine tumour in vivo: flow cytometric determination of the radiation-induced blockage of cell cycle progression

Abstract. Cells from the small cell population of viable cells in the large necrotic centre of murine M8013 tumours were investigated with respect to their cell kinetics. Flow cytometry (FCM) of this part of subcutaneously transplanted tumours revealed the presence of tumour cells with G1, S and G2 + M phase DNA-contents. These severely hypoxic cells could have stopped cell cycle progression due to the nutritional deprivation, irrespective of their position within the cell cycle.
Labelling methods, used to disclose the cell kinetics of this cell population, are hampered by the absence of a transport system in these large necrotic areas. Therefore, FCM was used to monitor radiation-induced changes in the cell cycle distribution. From this investigation it was concluded that hypoxic cells in the necrotic centre of the M8013 tumour progress through the cell cycle. As well as a cell population with a cell cycle time (T_c) of approximately 84 hr, a subpopulation with a T_c of approximately 21 hr occurred.     

T and B cells in B-chronic lymphocytic leukaemia: Faust, Mephistopheles and the pact with the Devil

A large number of human malignancies are associated with decreased numbers of circulating T cells. B-CLL, in this regard, represents an anomaly since there is not only high numbers of circulating B cells, characteristic of the malignancy, but also a massive expansion of both CD4 and CD8 T cells. These T cells for the most part may probably not represent a leukaemia-specific TCR-dependent expansion. On the contrary, these T cells, especially the CD4 subset, might support a "microenvironment" sustaining the growth of the leukaemic B cell clone. Conversely, the leukaemic B cells may produce membrane-bound as well as soluble factors that stimulate the proliferation of these T cells in an antigen independent manner. In addition to these T cells lacking anti-leukaemic reactivity, there exist spontaneously occurring leukaemia-specific T cells recognizing several leukaemia-associated antigens, e.g. the tumour derived idiotype, survivin and telomerase. Both CD4 and CD8 leukaemia-specific T cells have been identified using proliferation and gamma-IFN assays. These reactive T cells can lyse autologous tumour cells in an MHC class I and II restricted manner. Spontaneously occurring leukaemia-specific T cells are more frequently noted at an indolent stage rather than in progressive disease. Preliminary results from vaccination trials using whole tumour cell preparations as vaccine have demonstrated that vaccination may induce a leukaemia-specific T cell response, which might be associated with clinical benefits. Extended clinical trials are required to establish the therapeutic effects of vaccination in B-CLL. Studies in our laboratory as well as those of others indicate that whole tumour cell antigen in the form of apoptotic bodies or RNA loaded on to dendritic cells may be a suitable vaccine candidate. Patients with low stage disease may maximally benefit from this form of therapy.