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PubMedDate not supplied

Autologous confrontation of brain tumor derived spheroids with human dermal spheroids.

An in vitro model is presented to test the invasiveness of human brain tumour derived cells. Spheroids of brain tumours are confronted with autologous spheroids of human dermal fibroblasts. Aggregates of seven tested brain tumours, respectively 5 benign and 2 malignant ones are confronted for 7 days in vitro. The aggregates of the malignant tumours do invade into the dermal spheroids and progressive replacement of the dermal aggregate is observed. The aggregates of the benign tumours do not invade, a clearcut border between the tumour compartment and the dermal one is seen. This model will be applied for clinical testing of the invasiveness of selected tumours and will offer a good model to study mechanisms of invasion.

Brain Neoplasms↗
PubMed1978

The multicellular spheroid as a model tumor allograft. II. Characterization of spheroid-infiltrating cytotoxic cells.

Alloimmune lymphoid cells infiltrating multicellular spheroids of EMT6 mammary sarcoma cells (a solid tumor allograft model) have been characterized according to their morphological and functional properties. Both lymphocytes and macrophages were found within spheroids at the time of peak tumor cell damage. Cytotoxic cells specific for allograft antigens were also present. Using a short-term 51-Cr release assay, the cells responsible for cytotoxicity were characterized as a nonadherent, nonphagocytic T cell population. Velocity sedimentation cell separation further demonstrated that these cytotoxic cells had the physical properties of small lymphocytes. Some evidence for selective spheroid infiltration by specifically alloimmune cells was also obtained. The possible relationship of this cellular infiltrate to graft damage is discussed.

Animals↗
PubMed1974

Bacteriophages of Rhodopseudomonas spheroides: isolation and characterization of a Rhodopseudomonas spheroides bacteriophage.

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.

Adsorption↗
PubMed1985

The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs--an in vitro study using EMT6 multicellular spheroids.

Multicellular spheroids of the EMT6/Ca/VJAC mouse mammary tumour cell line have been used in an investigation of the effect of tumour geometry on the response of tumour cells to 3 cytotoxic drugs, adriamycin (ADM), nitrogen mustard (HN2) and CCNU. In addition to the inherent cellular drug response, factors related to spheroid structure, namely cell-cycle distribution, intercellular contact, drug penetration and microenvironment (pH, oxygen, glucose, etc.) are believed to influence the response of cells within spheroids to cytotoxic drugs. Selective enzymatic dissociation (with bacterial neutral protease) has been used to separate the cells within large (approximately 800 micron in diameter) spheroids into 4 distinct subpopulations. The cells within the subpopulations have been characterized by their DNA content, RNA content, tritiated thymidine labelling index, cell size and clonogenic capacity. It was found that cells at the surface of spheroids are relatively larger and more proliferative than cells towards the centre while their clonogenic capacity is similar. Studies on the responses of EMT6/Ca/VJAC log and plateau-phase monolayer cells have been carried out in parallel and have shown that cycling cells are more sensitive to ADM and HN2 than are non-cycling cells but somewhat less sensitive for the response to CCNU. Since the response patterns of cells from different regions of spheroids to HN2, treated either before disaggregation (intact spheroid) or after disaggregation (isolated spheroid cells), are similar and the surviving fraction increases from the surface towards the centre of the spheroid, cell cycle distribution is thought to be the only factor involved in the cytotoxicity of HN2 towards cells within the spheroids. Although the patterns of response to ADM of cells within intact spheroids and isolated spheroid cells are similar to those for HN2, the initial slope of the curve for intact spheroids is much steeper than that of the isolated spheroid cells. Therefore, in addition to the factor of cell-cycle distribution, drug penetration also appears to be involved in the action of ADM on spheroids, while the factors of intercellular contact and microenvironment appear to be relatively less important. The reverse pattern was found for the response of cells within different regions of spheroids to CCNU, treated as intact spheroids or as isolated spheroid cells (i.e., greater killing of inner compared with outer cells).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Resistance of V79 multicell spheroids to mitoxantrone: drug uptake and cytotoxicity.

We have previously shown that V79 multicell spheroids are resistant to the anthracenedione mitoxantrone (1). In this paper we demonstrate that this resistance is not solely due to restricted drug penetration into the spheroid, but also to an altered intrinsic resistance of the cells when grown as a three-dimensional structure. We have studied the uptake and toxicity of mitoxantrone in V79-OCF4 monolayers, 100 micron spheroids, 650 micron spheroids, and outer and inner cells of 650 micron spheroids. The LD90 for cells exposed as monolayers to mitoxantrone for two hours was 0.016 microgram/ml, 0.055 microgram/ml for 100 micron spheroids, 1.5 micrograms/ml for outer spheroid cells and 6.2 micrograms/ml for inner spheroid cells. Uptake of [14C]mitoxantrone was linear for all populations with no plateau up to the highest doses used. The uptake of drug required to kill 90% of the cells in a population (UP90) of monolayers was 3.7 ng/10(6) cells, 10.7 ng/10(6) cells for 100 micron spheroids, 169 ng/10(6) cells for outer spheroid cells, and 146 ng/10(6) cells for inner spheroid cells. The relative resistance of spheroids compared to monolayers, based on drug concentration in the medium, was 3.4 for small spheroids, 92 for outer cells, and 390 for inner cells. When cell survival was normalized to drug uptake, the relative resistance of spheroids to monolayers was 2.9 for small spheroids, 46 for outer cells and 40 for inner cells of large spheroids. The data demonstrate that the resistance of multicell spheroids to mitoxantrone is not solely due to restricted drug penetration into the cell aggregate but is also due to a spheroid-induced altered intrinsic resistance of the V79 cells.

Cell Survival↗
PubMedDate not supplied

Autologous spheroid culture: a screening tool for human brain tumour invasion.

Spheroids are three-dimensional cell aggregates expressing histotypic organisation in vitro comparable to tissue continuity in vivo. They can be prepared from normal tissue and from tumour fragments. In the experiments presented here, dermal human spheroids and brain tumour spheroids are prepared from the same patient. The dermal tissue originates from the border of the incision wound made to effect a stereotactic brain tumour biopsy. The tumour originates from a fragment of the collected stereotactic biopsy. The dermal fragment and the brain biopsy are explanted in vitro to form confluent monolayers. At confluency, the dermal cells are transferred into small Erlenmeyer flasks and rotated at 37 degrees C for 1-2 days and rotation mediated spheroids are formed. Small flaps of the tumour monolayer are placed on a semisolid non-adhesive substrate, reorganise and form agar overlay spheroids. After spheroid formation, a dermal spheroid is confronted with a brain tumour derived spheroid. The confronting pair, after adhering to each other, present an invasion model in vitro. The dermal spheroid functions as the autologous host for the brain tumour spheroid. Putative invasive cells present in the reaggregated brain spheroid will invade the dermal spheroid and destroy it. If no invasive cells are present in the tumour derived spheroid no morphologic changes will be seen in the dermal spheroid; 24 tested brain biopsy spheroids demonstrated a clear correlation between malignancy in situ and invasiveness in vitro. So it can be concluded that the autologous confrontation of brain tumour derived spheroids with dermal spheroids derived from the patient has a predictive value concerning malignant evolution and mimics the situation of the tumour in situ.

Brain Neoplasms↗
PubMed1986

Growth and characterization of multicellular tumor spheroids of human bladder carcinoma origin.

We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGH-U1 cells placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and and consistently than cells derived from monolayer culture. Spheroid diameter increased at a rapid rate of approximately 100 microns/d in multiwell dishes, and necrosis occurred only in spheroids of diameter greater than 1 mm. Spheroids placed in spinner culture at a higher concentration (approximately 1.5 spheroids/ml) grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture was maintained at approximately 190 microns over a wide range of spheroid diameters (400 to 1000 microns). Sequential trypsinization of spheroids, which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids. This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human bladder biopsies to form spheroids.

Anthraquinones↗
PubMedDate not supplied

Multicellular tumor spheroids in radiotherapy research (review).

Culturing of human tumor cells as multicellular spheroids can be a tool to study radiation responses. The degree of structural and functional differentiation in the primary tumor may be retained in spheroids rather than in conventional monolayer cultures. In the liquid overlay culture technique spheroids can be individually assessed for their responses to treatment, whereas in spinner flasks, large quantities of similarly sized spheroids can be produced. Studying the response of spheroids to irradiation can be performed on single cells obtained after disaggregation of these spheroids, or on intact spheroids, using cure and growth delay as endpoints. Clonogenic cell survival is especially difficult to perform on spheroids of human tumor cells. Modern calculation methods, however, may offer promising correlates between growth curves and single cell survival. Spheroids of human tumor cell lines show tumor type dependent radiation responses, offering an approach for comparison of radiosensitivity of tumor cell lines of different histologic origin. Contact effect, as a modifying factor of radiation response in spheroids, has especially been studied in murine cell lines. The use however, of human tumor cell lines, may offer new insight in this phenomenon. Radiobiologic hypoxia has been observed in spheroids of both murine and human origin. Reoxygenation after irradiation has also been described by radiobiologic parameters. So far, no physiologic reoxygenation processes after radiation treatment have been identified. In view of the clinical relevance of oxygen to radiation responses and treatment outcome, reoxygenation processes should be further elucidated in spheroids of human origin. Repair of potentially lethal damage in spheroids has been reported for only one murine cell line. In an indirect manner it has also been studied in spheroids of human origin. Sublethal damage repair has been studied rather extensively in murine cell line spheroids. However, only recently it has been reported in human tumor spheroids in relation to the clinical curability of the tumors of origin. Use of human tumor cell lines to study radiation responses of spheroids is necessary to determine tumor type dependent differences in several radiation related phenomena, such as reoxygenation, contact effect, and repair processes.

Animals↗
PubMed1986

Oxygenation and differentiation in multicellular spheroids of human colon carcinoma.

Oxygenation and development of necrosis were evaluated in multicellular spheroids of poorly differentiated (HT29) and moderately well-differentiated (Co112) human adenocarcinoma of the colon. Spheroids were grown in vitro under well-controlled oxygen and nutrient conditions in spinner flasks up to sizes of 2800-micron diameter after 5 wk of culture. Morphological studies showed that the Co112 spheroids contained pseudoglandular structures with lumen, very similar to the characteristics of the original tumor specimen from the patient and to the cells when grown as xenograft tumors in nude mice. Microelectrodes were used to measure the oxygen tension (PO2) profile within individual spheroids at different stages of growth. Histological sections through the centers of spheroids were measured to determine the thickness of the viable rim of cells surrounding spheroid necrotic centers in order to estimate the size of the severely hypoxic zone of cells by comparison with the PO2 profiles of the same spheroids. The data demonstrate significant differences between these two human colon tumor spheroid systems. Both spheroid types exhibited steep PO2 gradients at relatively small sizes of less than 600-micron diameter, but for any given size in this range, the more differentiated Co112 spheroids were more hypoxic. Although severe hypoxia (PO2, less than 10 mm of Hg) was present in both spheroid types at larger sizes, there was a significant difference in the central PO2 values which were between 5 and 10 mm of Hg in large Co112 spheroids but remained at or close to 0 mm of Hg in large HT29 poorly differentiated human colon tumor spheroids. The presence of pseudoglandular structures and lumen in the Co112 spheroids was associated with changes in the shape of PO2 profiles. Such profiles have not previously been seen in other poorly differentiated human or rodent tumor spheroids. Furthermore, the PO2 profiles of both of these human tumor spheroid types were often continuously curving with a very shallow gradient in the inner edge of the viable rim of cells surrounding the necrotic center. Regulation of oxygen consumption and/or diffusion in these inner regions of human spheroids could produce these continuously curving PO2 gradients.

Adenocarcinoma↗
PubMed1992

Radiosensitivity, repair capacity, and stem cell fraction in human soft tissue tumors: an in vitro study using multicellular spheroids and the colony assay.

Radiation doses necessary to control 50% of spheroids (SCD50) were determined for five human soft tissue tumor lines after single dose and fractionated irradiation. Spheroids with 1000-1500 cells were used throughout. A similar number of cells per spheroid resulted in different sized spheroids for the respective cell lines. The parameters alpha, beta, and the number of regenerating cellular units per spheroid (SRU) were estimated from the spheroid control data using a direct fit according to the linear quadratic model assuming Poisson statistics. The number of spheroid regenerating cellular units was also determined from the growth delay at doses required for 10% spheroid control. In addition, alpha, beta, and the fraction of clonogenic cells of the five cell lines were obtained from a soft agar colony forming assay. The most precise parameter for radiation sensitivity was the SCD50, with a coefficient of variation smaller than 5%. SCD50 values ranged from 5.9 to 11.0 Gy for the five soft tissue tumor lines. Two of the five cell lines showed significantly higher alpha values and lower calculated survival fractions after 2 Gy (SF2) in the soft agar clonogenic assay than in the spheroid control assay. This points to a resistance-enhancing effect in the spheroid system. Whereas the fractions of SRU from the number of cells per spheroid, estimated from the spheroid control and growth delay assays, agreed well, no significant correlation existed between the fraction of SRU and the fraction of clonogenic cells in the soft agar colony forming assay. The alpha/beta ratios as a descriptive measure of the fractionation sensitivity of the tumor cell spheroids in the spheroid control assay corresponded well with those derived from the dose-cell survival data using a soft agar colony forming assay. Two of the five cell lines showed high fractionation sensitivities with alpha/beta values smaller than 5 Gy while those of the remaining three ranged from 7.8 to 10.8 Gy. Spheroids are structurally more similar to in vivo tumors than monolayer cultures. From the observed lack of correlation in the radiosensitivity parameters alpha and SF2 as well as in the fraction of SRU or clonogenic cells obtained from the spheroid control assay or the colony forming assay, one would expect even greater differences between results from colony forming assays and the radiosensitivity of in vivo tumors, at least for human soft tissue sarcomas.

Cell Aggregation↗
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↗
PubMed1999

Growth and characterization of LNCaP prostate cancer cell spheroids.

Cells from the prostate tumor cell line LNCaP have been grown as spheroids. The growth kinetics of the spheroids have been characterized by fitting a Gompertz equation to spheroid growth curves. The proliferation state of cells within spheroids of different diameters was assessed by bromodeoxyuridine staining. Scanning and electron transmission microscopy were performed to determine the ultrastructure of the spheroids. Prostate-specific antigen (PSA) secretion was monitored throughout spheroid growth. Consistent with Gompertzian kinetics, the volume of LNCaP spheroids initially increased exponentially and then reached a plateau. The doubling time during the exponential phase was 29 +/- 4 h. A core of nonproliferating cells was seen in spheroids with a diameter of 400 microm; at a diameter of 600 microm, a necrotic core had formed. In smaller, 200-microm diameter spheroids, a core of nonproliferating cells was not seen, but proliferating cells were concentrated at the spheroid periphery. Electron microscopy showed that the spheroids were enveloped by an extracellular matrix and that cell adhesion within the spheroids was due in part to desmosomes. PSA secretion by the spheroids could be modeled as originating from a spherical shell whose thickness was independent of overall spheroid diameter. The shell thickness obtained by fitting an appropriate equation to the data was consistent with that determined from the bromodeoxyuridine studies. LNCaP cells exhibit several important features of prostate cancer cells; in vivo, they are androgen responsive, and they express prostatic acid phosphatase, PSA, and prostate-specific membrane antigen. LNCaP spheroids provide a simple but relevant model for the study of drug delivery and response in prostate cancer.

Bromodeoxyuridine↗
PubMed2006

Increased cell compaction can augment the resistance of HT-29 human colon adenocarcinoma spheroids to ionizing radiation.

One of the most important questions in tumor biology is the understanding of the mechanisms responsible for the resistance of cancer cells to radiotherapy. In the present study, the possible role played by cell-cell interactions in determining the response of tumor cells to ionizing radiation was investigated. HT-29 colon adenocarcinoma spheroids were irradiated with a dose of 15 Gy in two different stages of growth characterized by diverse degrees of compaction: loosely organized spheroids (early spheroids) and compacted spheroids (late spheroids). Morphological analyses demonstrated that late spheroids were less damaged than early spheroids. Moreover, analyses of the cell cycle and cell death showed that ionizing radiation induced necrosis in early spheroids and apoptosis in late ones. From these results it can be concluded that late, compacted spheroids are more resistant to ionizing radiation than early, loose spheroids. In order to understand the mechanisms regulating this compaction-induced resistance of late spheroids, E-cadherin/beta-catenin complex expression and distribution were analyzed. In late spheroids, E-cadherin/beta-catenin complexes were shown to be tethered to the cytoskeleton, and since this organization has been demonstrated to strengthen cell-cell adhesion in other systems, it can be postulated that the same is true in HT-29 spheroids. In conclusion, it can be hypothesized that compaction of HT-29 spheroids is mediated by the reorganization of E-cadherin/beta-catenin complexes on the plasma membrane and that this compaction may be responsible for the increase in resistance of HT-29 spheroids to ionizing radiation.

Adenocarcinoma↗
PubMed1997

Inverse correlation of cell proliferation and expression of progesterone receptors in tumor spheroids and monolayer cultures of human meningiomas.

OBJECTIVE: The progesterone receptor (PgR) can be detected in 60 to 70% of meningiomas using immunohistochemistry] in situ. Whereas in monolayer tissue cultures the PgR is only rarely expressed, we were able recently to demonstrate the preservation of the PgR in fragment spheroid cultures of meningiomas. The aim of the present study was to evaluate the stability of PgR expression in meningioma spheroids in vitro and the correlation of PgR expression and cell proliferation in spheroids and whether meningioma cells reaggregated to spheroids from monolayer cultures to reexpress the PgR again. METHODS: Tumor fragment spheroids (Weeks 1-6) and cell monolayers (Passages 1 and 3) of 15 PgR-positive meningiomas were investigated by immunohistochemistry for the expression of PgRs and their proliferative activity, as demonstrated by positivity for the proliferation-related antigen Ki-67. To study PgR reexpression in reaggregated spheroids, Northern blots were performed. In addition, a reverse transcriptase-polymerase chain reaction technique was established and evaluated in combination with immunohistochemistry. Growth of meningioma spheroids was quantified in the presence of progesterone and the specific antagonist onapristone. RESULTS: The PgR remained stable in spheroids for 6 weeks in 9 of 13 cases that were able to be evaluated. All tumor fragment spheroids exhibited a proliferation index of 5 to 40% Ki-67-positive cells. Monolayer cell cultures, on the other hand, failed to express PgRs but revealed higher proliferation indices (40-90%) to a significant extent. The detection of PgR messenger ribonucleic acid in reaggregated spheroids by means of reverse transcriptase-polymerase chain reaction correlated to the nuclear expression of PgR in immunohistochemistry. Neither progesterone nor its antagonist onapristone altered spheroid growth in vitro. CONCLUSION: The expression of the PgR in meningiomas is preserved in spheroid cultures with low proliferation indices for at least 6 weeks, whereas monolayer cell cultures with a high proliferative activity lack PgR expression. The inverse pattern of Ki-67-positive cells in the outer regions of the spheroids and PgR-expressing tumor cells in the spheroid centers leads us to the conclusion that proliferating meningioma tumor cells do not express PgRs. This might also explain why tumor cell growth in vitro was neither affected by progesterone nor by onapristone. Monolayer cell cultures can be reaggregated to spheroids, the consequence being a reexpression of PgRs and, therefore, a down-regulation of proliferation.

Antineoplastic Agents↗