Growth kinetics of a rat mammary tumour transplanted into immune-suppressed mice

The growth kinetics of a transplanted rat mammary tumour and its xenografts in immune-suppressed mice were studied using the technique of labelled mitoses. Growth of the tumour in rats was regular and uniform but when transplanted into mice the growth of the implants was irregular and generally slower. The second passage tumours in mice showed rapid and regular growth with a volume doubling time approximately half that for the tumours in the rat. Values for the G1 phase duration, intermitotic time and growth fraction were greater for the tumours in the mouse although the data for the first passage in mice were difficult to interpret. The kinetic changes between the rat and mouse hosts primarily reflect a large and variable amount of cell loss from the first passage xenografts in the mouse, with adaptation by the second passage towards reduced cell loss.     

GROWTH KINETICS OF A RAT MAMMARY TUMOUR TRANSPLANTED INTO IMMUNE-SUPPRESSED MICE

The growth kinetics of a transplanted rat mammary tumour and its xenografts in immune-suppressed mice were studied using the technique of labelled mitoses. Growth of the tumour in rats was regular and uniform but when transplanted into mice the growth of the implants was irregular and generally slower. The second passage tumours in mice showed rapid and regular growth with a volume doubling time approximately half that for the tumours in the rat. Values for the G₁ phase duration, intermitotic time and growth fraction were greater for the tumours in the mouse although the data for the first passage in mice were difficult to interpret. The kinetic changes between the rat and mouse hosts primarily reflect a large and variable amount of cell loss from the first passage xenografts in the mouse, with adaptation by the second passage towards reduced cell loss.     

CYTOKINETICS OF TUMOUR AND ENDOTHELIAL CELLS AND VASCULARIZATION OF LUNG METASTASES IN C3H/HE MICE

A model of lung metastases was developed using intravenous injection of tumour cell aggregates of spontaneous C3H/He mammary tumours in syngeneic mice. the growth rate of lung tumours decreased with increasing tumour volume, with mean host survival of 46 days. the cytokinetics of individual tumours ranging between 0.004 and 4.2 mm³ in volume were studied. the labelling index (LI) ranged between 12 and 17%, the DNA synthesis time (T_s) being 9–10 hr. the growth fraction (GF) ranged between 26 and 38%. the cell cycle time (T_c) was found to be 18–19 hr. the LI and the GF decreased with increasing tumour volume doubling time (T_d). No correlation was found between the tumour volume and T_c. the LI of endothelial cells within these tumours, ranging between 0.004 and 4.2 mm³ in volume was 14–15% and endothelial cell proliferation was not affected by tumour growth. Vascular parameters were also determined for these tumours as a function of tumour volume. Vascular volume increased with increasing tumour size while the percentage of capillary vessels decreased. the cellular volume to capillary volume ratio increased with increasing tumour volume. Necrosis was observed in 0.27 mm³ tumours and increased with increasing tumour size. The results from these studies suggested that the age-dependent decrease in proliferative activity of tumour cells growing in the lung is related to change in effective vascularity.     

CYTOKINETIC ANALYSIS OF THE JB-1 ASCITES TUMOUR AT DIFFERENT STAGES OF GROWTH

The cell kinetics of the murine JB-1 ascites tumour have been investigated on days 4, 7 and 10 after transplantation of 2·5 × 10⁶ cells. The experimental data, growth curve, percentage of labelled mitoses curves, continuous labelling curves and cytophotometric determination of single-cell DNA content have been analysed by means of a mathematical model for the cell kinetics. The important result was the existence of 8% non-cycling cells with G₂ DNA content in the 10-day tumour, while only 0·2 and 0% were observed in the 7- and 4-day tumours, respectively. The doubling times determined from the growth curve were 22·8, 70 and 240 hr, respectively, in the 4-, 7- and 10-day tumours. Growth fractions of 76, 67 and 44% were calculated for the same tumour ages. The mean cell cycle time increased from 14 to 44 hr from day 4 to 7 due to a proportional increase in the mean transit time of all phases in the cell cycle. In the 10-day tumour, the mean cell cycle changed to 41 hr and T _G1 decreased to 0·5 hr. The cell production rate was 4·3%/hr in the 4-day tumour, 1·2%/hr in the 7-day tumour and 1·0%/hr in the 10-day tumour. The cell loss rates in the same tumours were 1·3, 0·2 and 0·7%/hr, respectively. The analysis made it probable that the mode of cell loss was an age-specific elimination of non-cycling cells with postmitotic DNA content.     

Cell kinetics and radiation biology

The cell cycle, the growth fraction and cell loss influence the response of cells to radiation in many ways. The variation in radiosensitivity around the cell cycle, and the extent of radiation-induced delay in cell cycle progression have both been clearly demonstrated in vitro. This translates into a variable time of expression of radiation injury in different normal tissues, ranging from a few days in intestine to weeks, months or even years in slowly proliferating tissues like lung, kidney, bladder and spinal cord. The radiosensitivity of tumours, to single doses, is dominated by hypoxic cells which arise from the imbalance between tumour cell production and the proliferation and branching of the blood vessels needed to bring oxygen and other nutrients to each cell. The response to fractionated radiation schedules is also influenced by the cell kinetic parameters of the cells comprising each tissue or tumour. This is described in terms of repair, redistribution, reoxygenation and repopulation. Slowly cycling cells show much more curved underlying cell survival curves, leading to more dramatic changes with fractionation, dose rate or l.e.t. Rapidly cycling cells redistribute around the cell cycle when the cells in sensitive phases have been killed, and experience less mitotic delay than slowly proliferating cells. Reoxygenation seems more effective in tumours with rapidly cycling cells and high natural cell loss rates. Compensatory repopulation within a treatment schedule may spare skin and mucosa but does not spare slowly proliferating tissues. Furthermore, tumour cell proliferation during fractionated radiotherapy may be an important factor limiting the overall success of treatment.     

Changes In the Proliferation Characteristics of A Solid Transplantable Tumour of the Mouse With Time After Transplantation

The aim of the present investigation was to study changes in cell proliferation kinetics with time after transplantation in a solid transplantable mammary carcinoma of the C3H mouse. From growth curves and tumour doubling times, three different experimental times were chosen; the exponential phase of growth on the 7th day post-transplantation, during the phase of retardation on the 21st day after transplantation and in the stationary phase of growth on the 30th day after transplantation. It was found that the cell loss factor contributed primarily to the retardation of volume growth. the lowering of the growth fraction played a minor part in the delay of growth while the duration of cell cycle was not prolonged before the plateau phase of growth on the 30th day after transplantation was reached.     

Age-related changes in the cell proliferation of ATPC+ mouse ascites tumour

The growth of ATPC+, an ascites tumour derived from a spontaneous mammary carcinoma in BALB/c+ mice, was studied at different ages. It was observed that the number of cells increases rapidly during the first 5 days after implantation. Thereafter, the cell number increases more slowly, reaching a plateau after 8 days. This slowing-down is not due to a reduction in the growth fraction but to a lengthening of the cell cycle. Between 5 and 8 days the duration of all phases increases, including the S phase, which increases from 5.2 h in 5-day tumours to 8.2 h in 8-day tumours. In 12-day tumours both the cell cycle and S phase are only slightly longer than in 8-day tumours whereas the growth fraction is reduced. The slowing-down of cell growth can be attributed to growth fraction reduction rather than cell loss, which is maximal in the 5-day tumour. At this age the time course of the percent labelled cells and of the number of grains/nucleus suggests reutilization of [3H]-thymidine. Incorporation of [3H]-thymidine/cell decreases sharply in 12-day tumours due to a reduced availability of thymidine, which is degraded to thymine in the in vivo ascitic fluid faster than in 8-day tumours. This indicates an age-related change in the ascitic fluid composition.     

The Growth of the Emt6 Tumour In the Lungs of Balb C Mice Following Intravenous Inoculation of Tumour Cells From Culture

The growth of the EMT6 tumour in the lungs of Balb C mice has been studied following intravenous inoculation of different numbers of tumour cells taken from culture. At various times after injection of cells into mice, cell suspensions have been prepared from pairs of lungs and the number of in vitro colony forming cells assayed by plating into petri dishes. Following intravenous injection of 10⁵ cells, the time required for doubling of the number of clonogenic tumour cells appearing in the cell suspension is around 17 hr until such time that the total tumour cell population per set of lungs reaches 10⁸ cells (at 10–12 days). This doubling time has to be corrected for changes in ability to extract cells from the lungs into the cell suspension at various times and also for possible changes in plating efficiency in vitro. When these correction factors are applied, the most likely value for the doubling time of clonogenic tumour cells in the lungs is in the range 20–24 hr. This is a similar figure to that previously deduced for the EMT6 flank tumour during its microscopic period of growth. After reaching a total size of 10⁸ tumour cells, the time for doubling of the number of clonogenic tumour cells in the lung increases. During the later stages of tumour growth a good correlation is seen between total lung tumour weight and the number of clonogenic cells present. For the final 3–4 days of the initial period of rapid tumour growth, it is possible to carry out a haemocytometer count of tumour cells in the lung suspension and hence surviving fraction experiments may be carried out after various forms of treatment. In this way the response to treatment of microscopic tumour foci may be determined.     

Comparison of pulmonary endothelial cell and fibroblast proliferation using time-lapse cinematographic analysis

Time-lapse cinematography was used to study and compare the proliferation and migration activity of pulmonary endothelial cells and fibroblasts, two cell types with very different structural and functional properties. Endothelial cells were found to have a mere rapid growth rate than fibroblasts. Contributing to the shorter population doubling time of the endothelial cells were lower interdivision times and a tendency for these cells to remain in division cycle with successive generations of growth. Striking differences between endothelial cells and fibroblasts were seen in migration behaviour. Endothelial cells had lower migration rates and tended to remain within a restricted growth area, whereas fibroblasts migrated freely throughout the growth area.     

Cell kinetics of a rat thyroid transplantable tumour

Changes in morphology and cell kinetics are described in a rat thyroid transplantable tumour (TTT) during the first few transplant generations. The growth of TTT in animals was possible only with an increased circulation level of the thyroid stimulating hormone (TSH). With serial transplantation subcutaneously in isologous animals, the morphology of TTT changed dramatically from that of a follicular tumour in the 3rd passage to become, by the 9th generation, a poorly differentiated tumour with a trabecular arrangement of cells. This change in tumour morphology was accompanied by an increase in the number of proliferating cells--mitotic index (MI), [3H]thymidine labelling index (LI), growth fraction (GF)--and cell loss factor (O) as well as a decrease in the cell cycle time (Tc) and potential population doubling time (TPD). TTT belongs to the class of tumours with a low proliferative activity and might be used in a variety of cell kinetic, radiobiological and chemotherapy studies.     

Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.     

Amine dependence of proliferative activity in two transplantable lines of mouse colonic carcinoma

Serotonin, histamine and their antagonists have previously been shown to influence both the cell proliferation rate and the volumetric growth rate of colonic tumours. Of these earlier studies, those on cell proliferation could not distinguish between direct effects on tumour cells and indirect effects on the host, whereas those on the volumetric growth rate of tumours, whilst suggesting an outcome related to the individual properties of the tumour rather than the host, could not distinguish between influences on cell gain, cell loss or stromal changes. In an attempt to distinguish between these possibilities the current experiments on the mitotic rate in two lines of transplantable mouse colonic carcinoma were undertaken. One line of tumour proved to be sensitive to inhibition by a histamine H2 receptor antagonist and a dopamine D2 antagonist but resistant to serotonin antagonists; the inhibition by histamine antagonists was surmountable by co-administration of histamine. The other line proved to be highly sensitive to the inhibitory effects of serotonin antagonist and less so to antagonists of the other two amines and in this case the effect of serotonin antagonists was surmountable by serotonin. These results suggest that the variations between different colonic tumours in the response to amine antagonists is due to differences in the extent of inhibition of cell proliferation rather than differences in cell loss or stromal effects. Thus it appears likely that amine antagonists are able to directly interfere with the proliferation of some colonic tumour cells.     

PV1 down‐regulation via shRNA inhibits the growth of pancreatic adenocarcinoma xenografts

PV1 is an endothelial‐specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour‐bearing mice by single‐dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down‐regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC‐1 and BxPC‐3). The effect observed is because of down‐regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down‐regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.     

Optimal conditions of fixation for immunohistochemical staining of proliferating cell nuclear antigen in tumour cells and its cell cycle related immunohistochemical expression

Abstract. Comparison of the results of immunohistochemical expression, such as proliferating cell nuclear antigen (PCNA) in archival material of tumours, with the clinical course is extremely valuable in determining the biological malignant potential of newly detected tumours. To obtain stable and reproducible results of immunohistochemical expression of the PCNA of tumours, we studied the optimal conditions of fNation, processing and staining of samples using animal-implanted MBT-2 cells derived from chemical-induced mouse bladder carcinoma and PC10, a monoclonal antibody for PCNA. The intensity of staining and PCNA positive rates were stable and reproducible when resected specimens from the tumours were covered with gauze wetted with physiological saline at room temperature before fixation for less than 12 h, fixation in formaldehyde was less than 48 h, and paraffin-embedded sections were dried for less than 1 h. The most clear staining of PCNA positive nuclei was observed when 10% neutral buffered formaldehyde was used as a fixative. The PCNA positive rates obtained under these conditions was compared with the bromodeoxyuridine (BrdUrd) labelling indices. Although the average PCNA positive rate was significantly higher than the BrdUrd labelling index (P?0.01), a significant correlation between PCNA positive rates and BrdUrd labelling indices was observed. In order to study the cell cycle related expression of PCNA, Ehrlich ascites tumour cells were separated by centrifugal elutriation. PCNA positive nuclei were observed in all phases of the cell cycle including G,. Occurrence of PCNA positive G₁ cells was expected at a half-life of the PCNA-protein of 20 h and a tumour cell doubling time of about 24h. Thus, the percentage of PCNA positive nuclei in a conventionally paraffin-embedded specimen of a tumour reflects both the growth fraction and the doubling time of the tumour and it may be a useful parameter of the biological malignant potential of tumours.     

Cell proliferation of carcinoma in Bilharzial bladder: an autoradiographic study

The rate of cell production in thirty-five cases of carcinoma in Bilharzial bladder was evaluated from the labelling index after in vitro incubation with [3H]TdR. Squamous cell carcinoma was the most frequent histological type in this series and had a median LI of 8.0% which corresponds to a potential doubling time of 5.9 days. In squamous cell tumours the LI increased with the histological grade. Transitional cell tumours had a somewhat greater LI. In all histological types the LI was significantly greater in the deep infiltrating parts of the tumour than in the superficial parts. The discrepancy between the estimated potential doubling time and the growth rate normally attributed to such tumours suggests the existence of an extensive cell loss factor. Areas of focal or diffuse mucosal hyperplasia were associated with increased LI.     

CELL PROLIFERATION OF CARCINOMA IN BILHARZIAL BLADDER: AN AUTORADIOGRAPHIC STUDY

The rate of cell production in thirty-five cases of carcinoma in Bilharzial bladder was evaluated from the labelling index after in vitro incubation with [³H]TdR. Squamous cell carcinoma was the most frequent histological type in this series and had a median LI of 8.0% which corresponds to a potential doubling time of 5.9 days. In squamous cell tumours the LI increased with the histological grade. Transitional cell tumours had a somewhat greater LI. In all histological types the LI was significantly greater in the deep infiltrating parts of the tumour than in the superficial parts. The discrepancy between the estimated potential doubling time and the growth rate normally attributed to such tumours suggests the existence of an extensive cell loss factor. Areas of focal or diffuse mucosal hyperplasia were associated with increased LI.     

Growth and vascular structure of human melanoma xenografts

The growth and the vascular structure of five human melanomas grown in athymic nude mice were studied. Four growth parameters (tumour volume doubling time, fraction of cells in S-phase, growth fraction, cell-loss factor) were analysed against each of four vascular parameters (length of vessels with diameters in the range 5-15 micron, total vessel length, total vessel surface, total vessel volume--all per unit of histologically intact tumour volume). Statistically significant linear correlations between the parameters were found for any of the combinations. However, there was a consistent trend in the data: the tumour volume doubling time and the cell-loss factor tended to decrease while the fraction of cells in S-phase and the growth fraction tended to increase with increasing vascular density, whichever vascular parameter was considered. This finding indicates that the vascular density is among the factors which are decisive for the growth rate of tumours. However, the present work does not exclude the possibility that intrinsic properties of the tumour cells may also be important.     

Growth and Vascular Structure of Human Melanoma Xenografts

Abstract The growth and the vascular structure of five human melanomas grown in athymic nude mice were studied. Four growth parameters (tumour volume doubling time, fraction of cells in S-phase, growth fraction, cell-loss factor) were analysed against each of four vascular parameters (length of vessels with diameters in the range 5–15 μm, total vessel length, total vessel surface, total vessel volume-all per unit of histologically intact tumour volume). Statistically significant linear correlations between the parameters were found for any of the combinations. However, there was a consistent trend in the data: the tumour volume doubling time and the cell-loss factor tended to decrease while the fraction of cells in S-phase and the growth fraction tended to increase with increasing vascular density, whichever vascular parameter was considered. This finding indicates that the vascular density is among the factors which are decisive for the growth rate of tumours. However, the present work does not exclude the possibility that intrinsic properties of the tumour cells may also be important.     

Analysis of cell flow and cell loss following X-irradiation using sequential investigation of the total number of cells in the various parts of the cell cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. The generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G2 blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. The experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.