Single-cell analysis demonstrates how nutrient deprivation creates apoptotic and quiescent cell populations in tumor cylindroids

Understanding how quiescent and apoptotic populations form in tumors is necessary because these cell types can considerably diminish therapeutic efficacy. Most cancer therapeutics are ineffective against quiescent cells because they target rapidly proliferating cells. Distinguishing apoptosis is important because apoptotic cells are committed to death and do not require treatment. Regrowth of quiescent cell can lead to tumor re-occurrence and metastasis, which are the leading causes of cancer mortality. We hypothesized that cylindroid cultures and acridine orange staining could be used to determine how nutrient diffusion creates apoptotic and quiescent regions in tumors. To test this hypothesis we developed a microscopy technique to measure cellular DNA and RNA content in single cells using thin cylindroids and acridine orange staining. Cell classification was compared to flow cytometry of cells grown in defined monolayer cultures. The presence of apoptosis was confirmed by morphological nuclear analysis. The effect of diffusion was determined by varying incubation time, cylindroid size, and exposing cylindroids to nutrient-deficient media. Four overlapping regions were identified as a function of cylindroid radius: an outer viable/quiescent region; a second quiescent/apoptotic region; a third late-stage apoptotic region; and an inner dead region. In monolayer cultures the absence of glutamine and growth factors induced apoptosis and hypoxia induced quiescence. Treating with nutrient-deficient media suggested that cells became quiescent near the periphery because of glucose and oxygen limitations, and became apoptotic and died further from the edge because of glutamine and growth factor limitations. These results show that cellular microenvironments can be identified in cylindroids using simple acridine orange staining and that single cell fluorescence can be measured in three-dimensional culture. The developed techniques will be useful for developing cancer therapies and determining how cell death and apoptosis are induced in three-dimensional tumor tissue.     

Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro

Multi-drug resistance greatly limits the efficacy of conventional blood-born chemotherapeutics, which have limited ability to penetrate tumor tissue and are ineffective at killing quiescent cells far from tumor vasculature. Nonpathogenic, motile bacteria can overcome both of theses limitations. We hypothesize that the accumulation of S. typhimurium in tumors is controlled by two mechanisms: (1) chemotaxis towards compounds produced by quiescent cancer cells and (2) preferential growth within tumor tissue. We tested this hypothesis by quantifying the relative contributions of these mechanisms using the tumor cylindroid model, which mimics the microenvironments of in vivo tumors. Time-lapse fluorescence microscopy was used to measure the accumulation of GFP-labeled S. typhimurium into cylindroids of different size. Cylindroids larger than 500 microm in diameter contain quiescent cells, whereas cylindroids smaller than 500 microm do not. Spatio-temporal profiles of bacterial concentration were fit to a mathematical model to calculate two parameters that describe bacterial interaction with tumors: intratumoral bacterial growth, M, and intratumoral bacterial chemoattraction, K. It was observed that S. typhimurium is attracted to cylindroids and accumulate at long time points in the central region of large cylindroids. Both intratumoral bacterial growth and chemotaxis were significantly greater in large cylindroids, suggesting that quiescent cells secrete bacterial chemoattractants and the presence of necrotic and quiescent cells enable S. typhimurium to replicate in tumor tissue. In this study, several mechanisms of S. typhimurium accumulation in solid tumors have been quantified, which we believe is an important step in the development of bacterial-based therapeutics to target tumor quiescence.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Formation of cylindrical multicellular aggregate (cylindroid) and expression of liver specific functions of primary rat hepatocytes

In our studies of the development of a hybrid artificial liver, we investigated the formation of cylindrical multicellular aggregate (cylindroid) of primary rat hepatocytes on a pressed sheet of polyurethane foam (pressed–PUF) as a culture substratum. Hepatocytes formed cylindroids by attaching to a pressed–PUF surface, peeling off from the surface and aggregating. The diameter and length of most cylindroids were approximately 200–500 μm and 500 μm–2 mm, respectively. The activities of liver specific functions (albumin secretion and ammonia metabolism) of hepatocyte cylindroids were equivalent to or higher than those of hepatocyte spheroids. These results suggest that hepatocyte cylindroids can maintain highly differentiated functions longer than hepatocyte spheroids, and that a PUF/cylindroid culture may be effective to develop of a hybrid artificial liver. This revised version was published online in August 2006 with corrections to the Cover Date.     

Autowaves in a model of invasive tumor growth

A mathematical model for invasive tumor growth is proposed, which takes into account cell division, death, and motility. The model includes local cell density and the distribution of nutrient (oxygen) concentration. Cancer cells die in the absence of nutrients; therefore, the distribution of oxygen in tissue substantially affects both the tumor proliferation rate and its structure. The model adequately describes the experimentally measured rate of tumor proliferation. The existence of autowave solutions is demonstrated, and their properties are investigated. The results are compared with the properties of the Kolmogorov-Petrovskii-Piskunov and Fisher equations. It is shown that the nutrient distribution influences the selection of speed and the convergence of the initial conditions to the automodel solution.     

The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of mammalian cells in perfusion reactors

A general methodology is proposed to estimate the actual specific growth and death rate of mammalian cells in continuous perfusion reactors from the monitoring of the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the culture medium. The procedure is illustrated on a perfusion culture of human tumor kidney cells growing on microcarriers and producing prourokinase (PUK). The intracellular LDH content of living attached cells is checked to be constant during the culture. However, cells detached from the microcarriers, and counted dead because of the uptake of trypan blue, have only released part of their intracellular LDH. In the culture medium, LDH is relatively stable as the loss of activity does not exceed 5% per day. The time variation of the LDH concentration in the medium is used to calculate the total amount of lysed and actually produced cells in the reactors, hence, the actual specific rates of cell growth and death. It is thus found that the stationary phase observed after 400 h of perfusion culture is the result of equal growth and death rates, with a daily renewal of living cells on the microcarriers near 10%. Moreover, for the cell line tested, the production of PUK is associated with cellular growth.     

Gender dimorphism of tumor growth: role of gonadal hormones in differential regulation of apoptosis of a murine T cell lymphoma

The present investigation was undertaken to study if a gender-dependent differential induction of tumor cell apoptosis is responsible for the manifestation of gender dimorphism observed in the growth of a transplantable murine T cell lymphoma, designated as Dalton’s lymphoma (DL). Tumor cell samples obtained from male tumor-bearing mice showed a higher number of cells with apoptotic morphology compared to that observed in female tumor-bearing mice. In this report we demonstrate that male hormone androgen and female hormone estrogen can differentially modulate tumor cell proliferation and apoptosis through alteration in the expression pattern of cell death regulating genes: p53 and CAD. DL cells were shown to express mRNA for androgen and estrogen receptors. Further these gonadal hormones also induced tumor cells to produce tumor growth regulating proteins: VEGF, TGF-β, IL-2, IL-2R, SOCS, Hsp-70 and IFN-γ which in turn either through autocrine action on tumor cells or via TAM-derived NO were observed to regulate tumor cell apoptosis leading to gender dimorphism of tumor growth. This study also discusses the possible mechanism involved. The study has clinical significance as these results will helps in understanding the mechanism of gender dimorphism with respect to the progression of T-cells tumors.     

Increased rate of tumor cell death caused by polyamine synthesis inhibitors

This investigation was designed to determine whether cell death plays a role in the antiproliferative action exerted by polyamine synthesis inhibitors. To estimate the rate of tumor cell death, we measured the loss of 125I from mice harboring Ehrlich ascites tumor cells in which DNA was labeled with 5-125I-iodo-2'-deoxyuridine. DL-alpha-difluoromethylornithine (0.85 mumoles/g body weight/6 h), and enzyme-activated irreversible inhibitor of ornithine decarboxylase, and methylglyoxal-bis(guanylhydrazone) (45 nmoles/g body weight/6 h), an inhibitor of S-adenosylmethionine decarboxylase, were both found to increase the rate of 125I excretion. Our data suggest that these polyamine synthesis inhibitors provoke an increase in the rate of tumor cell death beyond that normally occurring during growth, methylglyoxal-bis(guanylhydrazone) being considerably more potent than DL-alpha-difluoromethylornithine. These in vivo data were corroborated by a study where the host-mediated responses did not have to be considered. Thus, Ehrlich ascites tumor cells were adapted for suspension growth in culture and treated with methylglyoxal-bis(guanylhydrazone) or DL-alpha-difluoromethylornithine. The growth kinetics and the colony forming efficiency of the drug-treated cells clearly show that polyamine synthesis inhibitors not only slow the growth rate but also cause an increase in tumor cell death.     

Induction of entosis by epithelial cadherin expression

Cell engulfment typically targets dead or dying cells for clearance from metazoan tissues. However, recent evidence demonstrates that live cells can also be targeted and that engulfment can cause cell death. Entosis is one mechanism proposed to mediate the engulfment and killing of live tumor cells by their neighbors, an activity often referred to as cell cannibalism. Here we report that the expression of exogenous epithelial cadherin proteins (E- or P-cadherin) in human breast tumor cells lacking endogenous expression of epithelial cadherins induces entosis and inhibits transformed growth. Entosis induced by cadherin expression is associated with the polarized distribution of Rho and Rho-kinase (ROCK) activity within entotic cells, which is dependent on p190A RhoGAP activity. ROCK inhibition or downregulation of p190A RhoGAP expression reduces entosis and increases the transformed growth of epithelial cadherin-expressing tumor cells. These data define new cell systems for the study of entosis, and identify entosis as a mechanism of cell cannibalism that is induced by the establishment of epithelial adhesion and inhibits transformed growth.     

Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release

The death of Medicago sativa L. cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death. Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated. A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated. The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation. Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process. Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture. When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days. Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size Copyright 1999 John Wiley & Sons, Inc.     

Mathematical description and analysis of cell cycle kinetics and the application to Ehrlich ascites tumor

The linear and nonlinear aspects of the dynamics of the cell cycle kinetics of cell populations are studied. The dynamics are represented by difference equations. The characteristics of cell population systems are analyzed by applying the model to Ehrlich ascites tumor. The model applied for the simulations of the growth of Ehrlich ascites tumor cells incorporates processes of cell division, cell death, transition of cells to resting states and clearance of dead cells. Comparison of the results obtained with the model and the experimental data suggests that the duration of the mean generation time of the proliferating EAT cells increases with aging of the tumor. An attempt is made to relate the prolongation of cell mean generation time with processes of cell death and dead cell clearance. Studying the transition of cells to the resting states, it becomes apparent that in fact transition of proliferating cells to the resting states occurs somewhere close to the end of the cell cycle and with a rate that varies with the age of the tumor. Time course behavior of the cell age, cell size, and cell DNA distribution with aging of the tumor are obtained. Variations in average size and average DNA contents are determined.     

Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells.

Tumor vascularization is the rate-limiting step for the progression of cancer. Differential steps of tumor-induced angiogenesis were studied by a novel in vitro confrontation culture of avascular multicellular prostate tumor spheroids and embryoid bodies grown from pluripotent embryonic stem (ES) cells. Vascularization in embryoid bodies started on day 5 of cell culture and was paralleled by down-regulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). In parallel, a dissipation of gradients in the pericellular oxygen pressure was observed as measured by O(2)-sensitive microelectrodes. After 24--48 h of confrontation culture, cells positive for platelet endothelial cell adhesion molecule (PECAM-1) became visible in the contact region between the embryoid body and the tumor spheroid and sprouted within the confrontation cultures during subsequent days. Tumor-induced angiogenesis resulted in growth stimulation of tumor spheroids, disappearance of central necrosis and a reduction of the pericellular oxygen pressure. Furthermore, tumor vascularization resulted in elevated levels of HIF-1 alpha, VEGF, heat shock protein 27 (HSP27), and P-glycoprotein. Tumor-induced angiogenesis may augment the oxygen consumption in tumors resulting in an increased expression of hypoxia-related, proangiogenic genes as well as of HSP27 and P-glycoprotein, which are involved in a multidrug resistance phenotype.     

Reduced growth of Ehrlich ascites tumor cells in creatine depleted mice fed β-guanidinopropionic acid

The effect of implantation of Ehrlich ascites tumor (EAT) cells of creatine distribution was investigated. It was also studied how depletion of creatine by feeding creatine-analogue β-guanidinopropionic acid (β-GPA) affects the growth of EAT cells in mice. Enhanced mobilization of creatine from host tissues to EAT cells against a greater concentration gradient was observed. The creatine (but not creatinine) level in blood plasma was lowered to 22% of the normal value by β-GPA feeding alone and assimilation of ¹⁴C-creatine into EAT cells was inhibited. The growth of EAT cells was significantly reduced and the duration of survival of mice after implantation of EAT cells was extended when the creatine concentration was decreased. A decrease in daily food consumption and the degree of muscle atrophy after implantation of EAT cells was less in β-GPA than control groups. In the creatine-depleted mice, the rate of increase in total EAT cell number and the volume of abdominal ascites were approximately half of the control values, and more dead EAT cells were observed. These results suggest that supplementation of β-GPA inhibits creatine transfer to EAT cells and reduces the growth of cancer cells.     

Low-grade cribriform ductal carcinoma in situ of the breast. Fine needle aspiration cytology in three cases.

This paper presents the cytologic features of fine needle aspiration biopsy specimens from three cases of ductal carcinoma in situ characterized by small and uniform tumor cells growing in a predominantly cribriform pattern without comedo necrosis (low-grade cribriform ductal carcinoma in situ). On cytology, most of the tumor cells were clustered in three-dimensional ductal structures. Occasionally in the clusters the tumor cells were seen bordering central lumina, quite similar to the architecture in histology. A few single tumor cells and no myoepithelium were seen. The background was clear or slightly hemorrhagic, without necrosis. The tumor cells were uniform and had a cylindroid shape, with round or oval nuclei. Morphometrically the mean largest nuclear diameter was 1.5-1.6 times that of a red blood cell. The chromatin was finely granular, with a minute nucleolus and slight condensation along the nuclear membrane. In cut sections all three tumors showed strong immunoreactivity for neuron-specific enolase. Unless the cribriform growth pattern is recognized in the smear, the cytologic diagnosis of this entity is difficult.     

Stable hybridoma cultivation in a pilot-scale acoustic perfusion system: long-term process performance and effect of recirculation rate

Perfusion systems have the possibility to be operated continuously for several months. It is important that the performance of the cell retention device does not limit the operation time of a perfusion process used in the production of active pharmaceutical ingredients. Therefore, the aim of this study was to investigate the reliability and long-term stability of an acoustic perfusion process using the 200 L/d BioSep. As the BioSep is an external device, it is possible that dependent on the recirculation rate nutrient gradients occur in the external loop, which could affect the cell metabolism. Therefore, the effect of possible nutrient gradients on cell metabolism, viability and productivity was studied by varying the recirculation rate. In this study, it is shown that a perfusion process using a pilot-scale acoustic cell-retention device (200 L/d) is reliable and simple to operate, resulting in a stable 75-day cultivation of a hybridoma cell line producing a monoclonal antibody. The recirculation rate had a significant effect on the oxygen concentration in the external loop, with oxygen being depleted within the cell-retention device at recirculation rates below 6 m3/m(reactor)3.d (=600 L/d). The oxygen depletion at low circulation rates correlated with a slightly increased lactate production rate. For all other parameters no effect of the recirculation rate was observed, including cell death measured through the release of lactate dehydrogenase and specific productivity. A maximum specific productivity of 12 pg/cell.d was reached.     

CIRCADIAN RHYTHM IN GROWTH AND DEATH OF ANABAENA FLOS-AQUAE (CYANOBACTERIA)

Circadian periodicity in cell division and death was investigated in the cyanobacterium Anabaena flos-aquae (Lyngb.) Bréb in a phosphorus (P)-limited, N₂-fixing chemostat culture. When entrained under 12:12 h LD cycles, not only cell division but also cell death showed a clear circadian rhythm in this filamentous cyanobacterium. The rhythm persisted under continuous light and was temperature compensated. Circadian rhythm was clearly observed in the steady-state cell number and instantaneous growth rate, μ(t), which reached a maximum at about 2 h before sunset and a minimum at about 2 h before sunrise. The number of dead cells and the instantaneous death rate γ(t) also showed a circadian periodicity; the peak of γ(t) occurred approximately 8 h before that of μ(t). Therefore, cell growth and death in A. flos-aquae appear to be under the control of circadian clocks, and thus it seems that their death is programmed cell death.