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PubMed2003

A theoretical analysis of the changes in basic multicellular unit activity at menopause.

Bone loss at menopause is an important contributor to the development of osteoporosis in women. Although alterations in bone remodeling are the implied process through which bone is lost at menopause, how menopause influences basic multicellular units (BMUs), the teams of cells that perform bone remodeling, is not completely clear. In this analysis we utilize a computer simulation of BMU activity to evaluate the changes that occur at menopause. Transient and maintained changes in both the rate of bone turnover (expressed as the BMU birthrate or origination frequency) and the focal bone balance (differences between the amount of bone formed and resorbed at each remodeling site) are considered. The magnitude of the change in BMU activity is determined parametrically through comparison to lumbar spine bone mineral density data present in the literature. We find that a change in bone turnover that is maintained after menopause, a transient change in focal bone balance at menopause, or a combination of the two is consistent with bone loss patterns seen clinically. Understanding the changes in BMU activity that occur at menopause could lead to improved strategies to treat and prevent postmenopausal osteoporosis.

Bone Density↗
PubMed1999

The relationship between basic multicellular unit activation and origination in cancellous bone.

Activation frequency is often used as a measure of basic multicellular unit (BMU) activity in cancellous bone. However, activation frequency expresses the rate of BMU appearance in a histologic slide and not the rate of origination, which is a more physiologic indicator of remodeling activity and is necessary for the development of BMU-level bone remodeling simulations. Using identical assumptions to those for calculating the activation frequency, it is shown that the origination frequency in cancellous bone is equal to the activation frequency divided by the total distance traveled by the BMU and its width.

Bone Development↗
PubMed2005

Extravascular transport of drugs in tumor tissue: effect of lipophilicity on diffusion of tirapazamine analogues in multicellular layer cultures.

The extravascular diffusion of antitumor agents is a key determinant of their therapeutic activity, but the relationships between physicochemical properties of drugs and their extravascular transport are poorly understood. It is well-known that drug lipophilicity plays an important role in transport across biological membranes, but the net effect of lipophilicity on transport through multiple layers of tumor cells is less clear. This study examines the influence of lipophilicity (measured as the octanol-water partition coefficient P) on the extravascular transport properties of the hypoxic cytotoxin tirapazamine (TPZ, 1) and a series of 13 neutral analogues, using multicellular layers (MCLs) of HT29 human colon carcinoma cells as an in vitro model for the extravascular compartment of tumors. Flux of drugs across MCLs was determined using diffusion chambers, with the concentration-time profile on both sides of the MCL measured by HPLC. Diffusion coefficients in the MCLs (D(MCL)) were inversely proportional to M(r)(0.5) (M(r), relative molecular weight), although this was a minor contributor to differences between compounds over the narrow M(r) range investigated. Differences in lipophilicity had a larger effect, with a sigmoidal dependence of D(MCL) on log P. Correcting for M(r) differences, lipophilic compounds (log P > 1.5) had ca. 15-fold higher D(MCL) than hydrophilic compounds (log P < -1). Using a pharmacokinetic/pharmacodynamic (PK/PD) model in which diffusion in the extravascular compartment of tumors is considered explicitly, we demonstrated that hypoxic cell kill is very sensitive to changes in extravascular diffusion coefficient of TPZ analogues within this range. This study shows that simple monosubstitution of TPZ can alter log P enough to markedly improve extravascular transport and activity against target cells, especially if rates of metabolic activation are also optimized.

Antineoplastic Agents↗
PubMed1998

Induction of apoptosis in the centre of multicellular tumour spheroids of colorectal adenocarcinomas--involvement of CD95 pathway and differentiation.

Multicellular tumour spheroids (MCTS) are three-dimensional cell culture systems which are widely used in cancer research. They are characterized by an outer zone of proliferating cells, an inner region of differentiating quiescent cells and an area of so-called necrotic cell death in their centre. The exact cause of this cell death, a controversy for many years, was the aim of the present study. Our data show that cell death in the centre of MCTS of three colorectal adenocarcinoma cell lines (HRT-18, HT-29 and CX-2) was induced by apoptosis. Apoptotic cells were initially distributed at random but accumulated very quickly in the quiescent and central area at day 4-5, suggesting a time- rather than size-dependent synchronization of apoptosis parallel to the formation of the proliferation gradient in MCTS. To study mechanisms inducing apoptosis, the Fas-pathway was investigated. A cell-cell contact-dependent expression of CD95 was found in all MCTS. FasL was not detected in monolayer cultures, but was expressed in spheroids of HRT-18 and CX-2. We found that TNF alpha and TGF beta 1 activated the CD95 pathway in all three cell lines. Since both TNF-alpha and TGF-beta are known to be inducible by hypoxia in a variety of cell types, we suggest that these hypoxia-induced factors sensitize the CD95 pathway in the quiescent area of MCTS. Furthermore, a loss of the heat shock proteins 27, 32, 60, 73 and 90 was observed in the quiescent area of spheroids. This suggests that tumour cell differentiation in the inner region of MCTS may be an additional factor inducing apoptosis.

Journal Article↗
PubMed2002

Antimicrobial peptides of multicellular organisms.

Multicellular organisms live, by and large, harmoniously with microbes. The cornea of the eye of an animal is almost always free of signs of infection. The insect flourishes without lymphocytes or antibodies. A plant seed germinates successfully in the midst of soil microbes. How is this accomplished? Both animals and plants possess potent, broad-spectrum antimicrobial peptides, which they use to fend off a wide range of microbes, including bacteria, fungi, viruses and protozoa. What sorts of molecules are they? How are they employed by animals in their defence? As our need for new antibiotics becomes more pressing, could we design anti-infective drugs based on the design principles these molecules teach us?

Amino Acid Sequence↗
PubMed1980

Multicellular tumour spheroids: a model for combined in vivo/in vitro assay of tumour immunity.

Multicellular tumour spheroids (MTS) from 4 mouse tumours (Line 1 lung carcinoma; a fibrosarcoma, FSA; a mammary carcinoma, MCa-11; and SV40-transformed fibroblasts, SV-A31) WEre injected into the abdominal cavity of normal, immunized or tumour-bearing syngeneic mice, recovered after 4-48 h, and their growth measured in vitro for 7-16 days. Both normal and immunized mice inhibited MTS growth, but there was no correlation between the two types of inhibition, suggesting that different immunological processes were involved. For example, the greatest inhibition by normal mice was seen for the weakly immunogenic MCa-11, and the highly immunogenic tumour, SV-A31, was only moderately inhibited. However, the summed inhibition of MTS growth in normal and sensitized hosts corresponded to the behaviour of tumours as s.c. transplants; i.e., was inversely related to the malignancy of the same tumours. The inhibition of MTS by mice bearing identical early tumours (FSA or MCa-11) was comparable to that in immunized mice. Histological sections of SV-A31 MTS in normal or immunized hosts revealed the infiltration of MTS by various types of host cells, mostly polymorphonuclears, macrophages and lymphocytes.

Animals↗
PubMed1981

Response of EMT6 multicellular tumour spheroids to hyperthermia and cytotoxic drugs.

The response of multicellular tumour spheroids of the EMT6 cell line to combinations of hyperthermia and Bleomycin (BLM) or Adriamycin (ADM) has been investigated. Using this model system, we have demonstrated enhanced BLM cytotoxicity at 43 degrees C and also heat-induced drug tolerance to BLM at 43 degrees C. ADM cytotoxicity was not significantly increased after 43 degrees C x 1 h but after 6 h at 42 degrees C greatly enhanced cell-killing was evident. These results are discussed in relation to our previous data for EMT6 cells growing either as monolayer cultures in vitro or as solid tumours in mice.

Animals↗
PubMed1982

In vitro cytotoxic drug sensitivity testing of human tumour xenografts grown as multicellular tumour spheroids.

Tumour cells from 7 patients with ovarian carcinoma and from 22 different human tumour xenografts representing a wide range of histological sub-types have been examined for multicellular spheroid forming ability. Spheroid formation was limited to cells derived from xenografts. Of the 22 lines tested, 5 formed spheroids capable of growth in isolation. There was no clear relationship between histological type and spheroid-forming ability. The plating efficiency of tumour cells obtained from spheroids was always greater than for the cells obtained from the dissociated tumour of origin and was in some cases as much as 6-fold greater. Spheroid growth was nearly exponential for 4 cell lines. Volume growth delay was used to investigate the activity of melphalan, adriamycin, the Vinca alkaloids, CCNU and cisplatin. Differences between lines in drug response broadly reflected patient and in vivo xenograft response.

Animals↗
PubMed1983

Enhancement of thermal killing by polyamines V. The response of EMT6 multicellular tumour spheroids versus monolayer cells.

Polyamines, especially spermine, are very effective in enhancing thermal killing of mammalian cells cultured as a monolayer. The response of EMT6 multicellular tumour spheroids to heat in the presence of spermine was studied using cell survival and growth delay as endpoints. Compared to cells in a monolayer, spheroids were found to be highly resistant to combined heat and spermine. In spite of this, considerable enhancement of thermal killing by spermine was observed when the combined treatment was prolonged for a few hours. These results, together with data obtained using labelled spermine, suggest that difficulties in penetration of spermine into the inner cells of the spheroids contribute to the resistance of the latter. A method of circumventing this difficulty is discussed.

Animals↗
PubMed1984

The radiation response of V79 and human tumour multicellular spheroids--cell survival and growth delay studies.

Chinese hamster cells (V79 379A) cells from a human small cell carcinoma of the lung (ME/MAR) and two xenografted human melanomas (HX117 and HX118) have been grown as multicellular spheroids in vitro. The radiation response of these four cell types has been compared when grown as spheroids (200 or 400 micron in diameter) and as single cells from disaggregated spheroids. The radiation sensitivity of the three human lines irradiated as single cells in air, is similar. In comparison, the V79 cells are more radioresistant. Only the V79 and HX118 cells show a spheroid size dependent radiation response. The radiation response of spheroids has been assayed using both cell survival and growth delay. V79, ME/MAR and HX117 cells demonstrate a good correlation between the two endpoints whereas with HX118 there appears to be greater cell kill for a given level of growth delay. This may be because HX118 is efficient in the repair of potentially lethal damage (PLD). The results support the view that extrinsic factors such as three dimensional contact, hypoxia and repair of PLD can be important and together with the intrinsic cell radiosensitivity will determine the radiation response of tumours.

Animals↗
PubMed1986

Growth and radiosensitivity of malignant melanoma multicellular spheroids initiated directly from surgical specimens of tumours in man.

The growth and radiosensitivity of multicellular spheroids initiated directly from disaggregated surgical specimens of four human malignant melanomas were studied. The spheroids were grown in liquid-overlay culture for up to 6 passages. Cell survival following irradiation was measured by using the Courtenay soft agar colony assay. The four melanomas formed spherical, densely packed spheroids. The volumetric growth rate as well as the plating efficiency in soft agar usually increased with increasing passage number. The radiosensitivity differed significantly among the melanomas. The survival curves for single cells from disaggregated spheroids in the first passage were always similar to those for single cells isolated directly from the surgical specimens. Two of the melanomas showed a significant contact effect as spheroids whereas the other two did not. The spheroids of two of the melanomas showed lower D0 in the third and the sixth passage than in the first passage, whereas the spheroids of the other two melanomas showed similar survival curves in the first and the third passage. There was no clear relationship between the changes in radiosensitivity and the changes in growth rate or plating efficiency. It is concluded that spheroids in the first passage, but not spheroids in later passages, may have the potential to identify differences in clinical radioresponsiveness among tumours.

Cell Division↗
PubMed1987

Use of a tritiated thymidine suicide technique in the study of the cytotoxic drug response of cells located at different depths within multicellular spheroids.

A technique using 'tritiated thymidine suicide' has been established as a means of studying the response to cytotoxic drugs of cells at different depths within multicellular tumour spheroids. Because of the characteristic spatial arrangement of cycling cells (mostly in the outer regions) and non-cycling cells (mostly at the inner regions) of spheroids, cells surviving after long term (24 h) exposure of spheroids to high doses of 3HTdR will be those located furthest from the surface. By comparing the drug response of cells from 3HTdR pre-treated and untreated spheroids, the individual response of total cells, cells near to the surface and cells lying deeper within the viable rim of spheroids can therefore be deduced. In this study, large spheroids of about 800 micron in diameter of a mouse mammary cell line, EMT6/Ca/VJAC, and of a human small cell lung cancer cell line, POC, have been used. Using clonogenic assay, the response of these two cell types to adriamycin (ADM), nitrogen mustard (HN2), CCNU and vincristine (VCR) (POC only) were measured. The preliminary part of this study has confirmed that the cells killed are those which incorporate 3HTdR during the DNA synthesis period; the cells killed are mainly located in the outer regions of spheroids i.e. surviving cells are mostly located in the inner part of the viable rim and 3HTdR pretreatment does not sensitise surviving cells to subsequent cytotoxic drug treatment. Results from large EMT6 spheroids agree with our previous findings (obtained using a selective disaggregation method) that cells in the outer regions of spheroids are more sensitive to ADM and HN2 than cells in the inner regions whilst the opposite is true for CCNU. For POC spheroids, cells in the outer region of spheroids are more sensitive to ADM and VCR than cells in the inner region whilst a reverse trend is seen for the response to CCNU. The response to HN2 is similar at all depths. Amongst the factors governing the response of cells in spheroids to cytotoxic drugs, the responses to ADM and VCR are thought to be largely dictated by cell cycle distribution and limited drug penetrability, whilst for HN2 the response may be determined by the factor of cell cycle distribution. For CCNU, we believe that the cellular response is largely dependent upon microenvironmental factors prevailing within spheroids.

Animals↗
PubMed1989

Growth and radiation sensitivity of the MLS human ovarian carcinoma cell line grown as multicellular spheroids and xenografted tumours.

The growth characteristics and the radiation sensitivity of multicellular spheroids of the MLS human ovarian carcinoma cell line grown in spinner culture in atmospheres of 5% CO2 in air or 5% CO2, 5% O2 and 90% N2 were studied and compared to that of MLS xenografted tumours. The spheroids grew exponentially with a volume-doubling time of approximately 24 h up to a diameter of approximately 580 microns and then the growth rate tapered off, more for spheroids grown at the low than at the high oxygen tension. Thirty days after initiation, the spheroid diameters were approximately 1,500 microns at the low and 2,100 microns at the high oxygen tension. The tumour volume-doubling times were approximately 8 days (V less than 200 mm3) and 17 days (V = 1,000-4,000 mm3). The histological appearance of the spheroids and the tumours was remarkably similar; both developed large central necrosis and both were composed of epithelial cells and showed pseudoglandular structures with lumen. The spheroids were slightly less differentiated than the tumours. The intrinsic, cellular radiation sensitivity was independent of whether the cells were grown in vitro as spheroids or in vivo as tumours, as revealed by irradiating single cells from dissociated spheroids and tumours under aerobic conditions and intact spheroids and tumours under hypoxic conditions. Studies of 1,600 microns spheroids grown in 5% CO2 in air showed that the intrinsic radiation sensitivity of the chronically hypoxic cells was the same as that of acutely hypoxic cells. The fraction of radiobiologically hypoxic cells under these conditions was approximately 15% and similar to those of 9% (V = 200 mm3) and 28% (V = 2,000 mm3) found for the tumours. Spheroids with diameter of 1,200 microns did not show survival curves parallel to those for acutely hypoxic cells, i.e. they did not contain a measurable fraction of clonogenic cells at complete radiobiological hypoxia. The final portion of their survival curves represented partially hypoxic cells; the OERs were 1.6 and 1.3 for spheroids grown at the high and the low oxygen tension, respectively. The considerable similarity between the spheroids and the tumours suggests that MLS spheroids constitute a valuable in vitro model for studies of human tumour radiation biology and related physiological processes. MLS spheroids may be particularly useful in studies of therapeutic consequences of partial radiobiological hypoxia since complete hypoxia and different levels of partial hypoxia can be studied separately by varying spheroid size and the oxygen tension in the culture medium.

Cell Line↗
PubMed1992

Use of a high frequency ultrasound microscope to image the action of 2-nitroimidazoles in multicellular spheroids.

A system was designed to allow imaging of control and drug treated multicellular spheroids with a high frequency backscatter ultrasound microscope. It allowed imaging of individual spheroids under good growth conditions. Since little data were available on cellular toxicity of ultrasound at these high frequencies (80 MHz), studies were undertaken to evaluate effects on cell survival, using a colony forming assay. No toxicity was observed on cell monolayers subjected to pulsed ultrasound at the intensities used for imaging experiments. Spheroids were also subjected to pulsed ultrasound and no growth delay was observed when exposed spheroids were compared with mock-exposed spheroids. Imaging studies were performed and pictures of untreated spheroids were obtained in which the necrotic and viable regions are clearly distinguishable. When the hypoxic cell cytotoxin 1-methyl-2-nitroimidazole (INO2) was added to the spheroid, dramatic changes were observed in the backscatter signal. The interior viable cells of the spheroid were selectively affected. Changes in the backscatter signal were also observed when the reduction product 1-methyl-2-nitrosoimidazole (INO) was added to spheroids. With INO however, the changes were located at the periphery of the spheroid, presumably due to the high reactivity of INO which limits diffusion of the drug into the spheroid. The present work demonstrates the potential usefulness of ultrasound backscatter microscopy in following the action of selected drugs in this in vitro tumour model.

Animals↗
PubMed1992

Enhancement of transforming growth factor-alpha synthesis in multicellular tumour spheroids of A431 squamous carcinoma cells.

Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha (TGF-alpha) was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.

Blotting, Northern↗
PubMed1995

The novel fluorinated 2-nitroimidazole hypoxia probe SR-4554: reductive metabolism and semiquantitative localisation in human ovarian cancer multicellular spheroids as measured by electron energy loss spectroscopic analysis.

The novel fluorinated 2-nitroimidazole SR-4554 is undergoing preclinical development as a magnetic resonance spectroscopy and imaging probe for hypoxic tumour cells. We have used electron energy loss spectroscopic analysis (EELS) to show selective reduction and differential subcellular localisation of SR-4554 in human ovarian multicellular spheroids. SR-4554 was demonstrated to be metabolised by these A2780 cells under hypoxic but not under normal aerobic cell culture conditions. The EELS technique illustrated that the relative amount of drug within the cytoplasm of cells from both the inner region (150-160 microns from edge) and outer edge of the spheroid did not differ significantly after an initial 3 h incubation with drug. In contrast, an 8-fold differential between the amount of drug retained in the cytoplasm (primarily ribosomes and endoplasmic reticulum) of cells from the inner vs outer regions of the spheroids was observed following a subsequent 2 h 'chase' culture in drug-free medium. Within cells from the hypoxic region of the spheroid, SR-4554 was mainly associated with the endoplasmic reticulum, nucleus and the cytoplasmic side of intracellular vesicles and also to a lesser extent with the nuclear periphery. Interestingly, the drug was only weakly associated with the mitochondria and plasma membrane of the cells. The characteristics of cellular and subcellular distribution of SR-4554 are consistent with the hypothesis that 2-nitroimidazole compounds undergo hypoxia-mediated enzymatic reduction to reactive species. These reactive species are selectively retained in the cells in which they are metabolised through covalent association with subcellular components. These findings provide additional support for the clinical development of the drug as a non-invasive probe for tumour hypoxia and at the same time illustrate the utility of the EELS technique for examining the heterogeneity of drug distribution both between and within cells.

Aerobiosis↗
PubMed1995

Heterogeneous response of individual multicellular tumour spheroids to immunotoxins and ricin toxin.

The cytoreductive effects of anti-transferrin receptor (anti-TfnR) immunotoxins (ITs) and of ricin toxin against tumour micromasses have been evaluated in a multicellular tumour spheroid (MTS) model. More than 600 (656) MTSs obtained with human breast carcinoma (MCF7) or rat glioblastoma (9L) cell lines were treated individually with ITs or toxin and the effects induced by the treatment were measured for each MTS as volume variation vs time by applying the Gompertz growth model. Two dose-dependent patterns of MTS growth were observed in MTSs of both cell lines in response to IT or toxin treatment: (1) complete inhibition of MTS growth ('sterilisation'); and (2) partial/complete inhibition ('heterogeneous response'). Within the range of IT or toxin concentrations resulting in partial inhibition of MTS growth, the sensitivity of treated MTSs was extremely heterogeneous (the cytoreductive effects varying between 0.1 and 4 logs of cells killed for a given IT or toxin concentration). Analysis of the post-treatment regrowth kinetics indicated that treated non-sterilised and control MTSs reached the same final limiting volumes. However, the doubling time estimated for the surviving cells of treated MCF7 and 9L MTSs ranged between 15 and 50 h, indicating that each MTS had individual growing potential. In conclusion, our results indicate that at substerilising IT concentrations individual heterogenicity of MTSs may greatly influence the cytoreductive potential of ITs. An implication of our study is that the efficacy of an IT treatment in eradicating disseminated micrometastases may not be predictable a priori. The MTS model that we describe in this paper may help in dissecting out factors limiting the effect of ITs in three-dimensional tumours.

Animals↗
PubMed1996

Daunorubicin and doxorubicin but not BCNU have deleterious effects on organotypic multicellular spheroids of gliomas.

In the present study organotypic multicellular spheroids (OMS) were used to study the effects of chemotherapeutic agents on malignant gliomas. Compared with the frequently used cell line models, OMS have several advantages with respect to the preservation of the cellular heterogeneity and the structure of the original tumour. OMS prepared from seven glioma specimens were treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), daunorubicin or doxorubicin. After exposure to these drugs, the histology and cell proliferation of the OMS were analysed by immunohistochemistry and image analysis. Furthermore, the expression of P-glycoprotein (P-gp) and multidrug resistance-related protein (MRP), which both can contribute to resistance to daunorubicin and doxorubicin, were immunohistochemically investigated. We found that OMS from gliomas are sensitive for daunorubicin and doxorubicin but not for BCNU in terms of tissue destruction and decrease in cell proliferation. In addition, all gliomas were P-gp and MRP negative, which is in accordance with the sensitivity for daunorubicin and doxorubicin. Considering the potential use of several new alternative drug delivery methods, such as intratumoural implantation of drug-impregnated polymers or liposomal encapsulation of cytostatic drugs, daunorubicin and doxorubicin might be effective in the treatment of malignant gliomas.

ATP Binding Cassette Transporter, Subfamily B, Mem↗