Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids

Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed 1) a linear uptake that increased over time without saturation in tumor spheroids and 2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases.     

Glucose diffusivity in multicellular tumor spheroids

In order to understand the role of glucose limitations in controlling multicellular tumor spheroid growth, knowledge of the glucose diffusion coefficient is essential. The effective diffusivity of glucose in spheroids of rodent and human tumor cell lines has been determined by measuring the efflux of tritium labeled L-glucose from spheroids with time. When the rapid and irreversible binding of L-glucose in spheroids is properly taken into account, measurements of the efflux of this diffusion tracer from spheroids into label-free medium can be correlated to the diffusion equation in order to obtain the effective glucose diffusivity in spheroids. Such measurements have been made in EMT6/Ro mouse mammary tumor spheroids as well as in spheroids derived from human colon carcinoma cells (HT29, CO112, and WiDr) and from human squamous carcinoma cells (CaSki and A431). EMT6/Ro spheroids have a glucose diffusivity of 1.1 x 10(-6) cm2/s, while glucose diffusion coefficients in the human cell spheroids studied vary from 5.5 x 10(-7) cm2/s to 2.3 x 10(-7) cm2/s. These values are low enough to suggest that significant gradients in glucose concentration may exist in spheroids and tumors. It is thus believed that these glucose diffusivities, as well as their variation with cell line, may have important implications for the role played by glucose in the growth and cellular heterogeneity of spheroids and tumors.     

Repair of fractionated radiation in plateau phase cultures of human tumor cells and human multicellular tumor spheroids

We have measured potentially lethal damage repair (PLDR) after fractionated radiation, delivered at 24-h intervals in density-inhibited plateau phase cultures of four human tumor cell lines derived from tumors of differing radiocurability (two melanoma and two breast). The repair of potentially lethal damage conferred significant radioresistance on the human melanoma cells but not on the breast carcinoma cells. We examined the effects of fractionated radiation on human tumor cells adapted to become multicellular tumor spheroids (MTS). MTS derived from a human neuroblastoma were "cured" by a total fractionated radiation dose about 50% of that required for MTS derived from a human melanoma.     

Demonstration of an extracellular matrix in multicellular tumor spheroids

Multicellular spheroids of a human glioma cell line (U-118 MG) and a human thyroid cancer cell line ( HTh -7) were analyzed for the presence of extracellular matrix (ECM) using light microscopy, transmission electron microscopy, and indirect immunofluorescence staining for fibronectin, laminin, and collagen. These studies were supplemented by analyses of glycosaminoglycans using autoradiography or chemical methods after metabolic labeling with [35S]sulfate or [3H]glucosamine in conjunction with various extraction procedures. The results showed that both types of spheroids contained an ECM composed of fibronectin, laminin, collagen, and glycosaminoglycans. The organization of the ECM in the spheroids seemed to be similar to that of tumors in vivo. These findings help justify the use of the spheroid system as an in vitro model for the study of biological phenomena of human tumors in vivo. Furthermore, it is concluded that the formation of an ECM in vitro is not confined to normal cells but can be promoted in transformed cells using appropriate culture conditions.     

Oxygen tensions in two human tumor cell lines grown and irradiated as multicellular spheroids

Cells from two human cell lines were irradiated both as multicellular tumor spheroids (MTS) and in monolayer culture. Radiation response of MTS was quantified in terms of specific growth delay and proportion cured, and as clonogenic cell survival for monolayer cells. Radiation was applied either as a single or as a split dose with time intervals of 1, 2, and 4 h to determine the rate of sublethal damage repair. Using as endpoint the fraction of MTS cured at an iso-effect level, in MTS of NB-100 neuroblastoma cells repair of sublethal damage was complete within 1 h, whereas in MTS of HN-1 squamous cell carcinoma cells there was still some unrepaired damage left. At a larger dose for NB-100 MTS the repair curve showed a similar shape as for HN-1 spheroids. Using as endpoint specific growth delay, no difference in repair between the various time intervals was observed. In monolayer cells from both cell lines sublethal damage was not fully repaired in the time intervals used. Polarographic microelectrode measurements of oxygen tension inside MTS showed a marked difference in steepness of oxygen tension profiles between MTS from both cell lines. In HN-1 squamous cell carcinoma MTS with diameters up to 500 microns the central pO2 amounted to about 100 Torr, whereas in NB-100 neuroblastoma MTS with the same diameters central pO2-values lower than 30 Torr were observed. NB-100 MTS were irradiated with doses of 5 and 10 Gy gamma rays and subsequently the oxygen tension was measured 1 and 5 h after irradiation. A reoxygenation effect could not be observed, either after single dose or after split dose irradiation. If spheroids may be regarded as a suitable model for tumor responses in vivo, the results from these experiments indicate that reoxygenation is a process eluding polarographic measurements, or that no dramatic changes in oxygen tension are to be expected shortly after high single doses or early in a fractionation scheme.     

Repair of sublethal damage in two human tumor cell lines grown as multicellular spheroids

Multicellular tumor spheroids (MTS) provide a suitable in vitro model to study radiation sensitivity of tumor cells. Two cell lines of human origin, obtained from a neuroblastoma (NB-100) and a squamous cell carcinoma (HN-1), were exposed to graded doses (4-9 Gy) of radiation with 18 MV photons. Radiation was applied either as a single or as a split dose with an interval of 6 hr to determine the extent of sublethal damage repair. Treated spheroids regrew at approximately the same growth rate as control multicellular tumor spheroids, preceded by a static or regression phase. Radiation response was quantified in terms of regrowth delay, expressed as the time needed for treated spheroids to obtain an 8-fold increase of the initial volume at the time of irradiation. Data obtained from regrowth delay analysis were used to calculate the extent of sublethal damage repair, showing for the squamous cell carcinoma line a fractionally higher capacity to repair sublethal damage than the neuroblastoma line. Repair increased with larger dose fractions in both cell lines. Our results show that multicellular tumor spheroids from the two cell lines used in this study are best applicable at relatively high total radiation doses. This makes multicellular tumor spheroids a suitable model for the in vitro evaluation of clinical treatment rationales such as hyperfractionation.     

Bioactivity of well-defined green tea extracts in multicellular tumor spheroids

The effect of green tea extracts (GTE) of a reproducible, well-defined composition on cellular viability, proliferation, and antioxidant defense was investigated in multicellular spheroids derived from WiDr human colon adenocarcinoma cells. The maximum GTE concentration investigated, i.e. 100 micro g GTE/ml, was equivalent to the plasma concentration commonly measured in humans drinking 6-10 cups of green tea per day. This GTE concentration lead to a substantial retardation of spheroid volume growth with diameters reaching only half the size of untreated aggregates. Flow cytometric analysis and immunocytochemistry showed an enhanced accumulation of cells in G2/M and in the non-proliferating compartment, respectively. The emergence of central necrosis occurred at larger spheroid diameters compared to control conditions leading to a significant increase (p<0.05) in the thickness of the viable cell rim (mean +/- SD) from 240+/-49.9 micro m to 294+/-69.5 micro m. This was associated with an elevation of the intracellular GSH concentration and, thus, of cellular antioxidant defense, as shown by HPLC analysis. A considerable toxicity, however, was found at these GTE levels in single cells. Cells did not adhere to culture dishes nor did they aggregate to form spheroids when plated as a suspension with GTE already in the culture medium. The findings show that green tea constituents interfere with early phases of tumorigenesis at a cellular level, e.g., by reducing cell-substratum and cell-cell interaction, enhancing G2/M arrest, and retarding spheroid volume growth. The differences in GTE effects between single cells and cell spheroids underline the importance of inclusion of spheroids in pharmaco-/toxicological testing.     

Sublethal damage repair in two radioresistant human tumor cell lines irradiated as multicellular spheroids

Melanoma and lung adenocarcinoma may be amenable for radiotherapy if it were possible to increase the presently used total dose. In order to investigate this, spheroids from two cell lines of human origin, one obtained from a BRO melanoma and one from an NCI-H125 lung adenocarcinoma were exposed to graded doses (3-9 Gy) of radiation with 18-MV photons. Radiation was applied either as a single dose or as split doses with an interval of 6 h to determine the extent of sublethal damage repair. Radiation response was quantified in terms of spheroid cure and specific growth delay. Both cells lines have previously been shown to be less sensitive than a neuroblastoma and a squamous cell carcinoma cell line grown as spheroids. Data obtained from the growth delay analysis were used to calculate the extent of split-dose recovery. Repaired dose for BRO spheroids did not increase after 7 Gy, whereas in NCI-H125, the repaired dose showed a steady increase. Recovery ratios did not differ between the two cell lines, but were lower than reported for normal tissues. Both cell lines revealed a low repair capacity was expressed by the beta-value of the linear-quadratic (LQ) equation. However, repair capacity for sublethal damage as expressed by the dose repaired and the beta-value of the LQ equation was not different from values reported earlier by us for neuroblastoma and squamous cell carcinoma when grown as spheroids. This indicates that the low radiosensitivity for the cell lines used in this study is determined by the alpha-value of the LQ equation. Our results support the clinical finding that the application of increased total radiation doses in the treatment of melanoma and lung adenocarcinoma may be feasible if radiation is applied in multiple small fractions to ensure normal tissue sparing.     

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.     

Formation and growth of multicellular spheroids of human origin

Different types of human cells which normally grow as monolayers or suspension cultures were tested for their capacity to form and grow as spheroids. Sixteen out of the 27 tested tumour cell lines formed spheroids. Nearly all of these spheroids also grew. With only two exceptions the doubling times were longer when the tumour cells grew as spheroids than when they grew in conventional mass culture. Eleven out of 13 tested human non-tumour cells formed small spheroids but of these only the spheroids of lymphoid origin could grow. These lymphoid cells grew faster when aggregated to spheroids than when in single-cell suspension culture. None of the other non-tumour cells, which normally grew as monolayers, could grow as spheroids. The normally monolayer-cultured tumour cells formed symmetrical spheroids with smooth surfaces while the normally suspension-cultured cells formed irregular spheroids with rough surfaces. All large spheroids had a necrotic centre surrounded by a shell of viable cells. The thickness of the viable cell layer varied depending on cell type. The shape and organization of cells within the spheroids also varied largely. The results show that many types of human cells can be cultured as spheroids and that a wide spectrum of morphological appearances and growth rates can be obtained.     

Effects of anticancer agents on the shedding of cells from human multicellular tumor spheroids

We studied the effects of anticancer agents on the inhibition of cell shedding from the surface of multicellular tumor spheroids (MTS). MTS were produced from 2 human tumor cell lines; one melanoma and the other squamous cell lung cancer, by using liquid overlay culture technique. The cell shedding from the melanoma MTS was approximately 10-fold higher than the squamous cell carcinoma MTS. In the melanoma MTS, all 3 drugs studied - vincristine (VCR), doxorubicin (ADR) and cisplatin (DDP)-inhibited cell shedding and the degree of inhibition of cell shedding was drug concentration related. In the squamous cell carcinoma MTS, VCR was as active in inhibiting cell shedding as in the melanoma MTS, but ADR and DDP were less efficacious. When effects on cell shedding were compared with those on cell lethality, VCR produced inhibition of cell shedding at much lower concentrations than those producing cell kill effects. ADR and DDP produced cell lethality as effective as, or more effective than, inhibition of cell shedding. These data seem to parallel known effects of these agents on cell kill and inhibition of metastases. MTS may serve as an in vitro model for the study of cell shedding and metastasis.     

Pleiotropic effects of fenretinide in neuroblastoma cell lines and multicellular tumor spheroids

The efficacy and mechanism of action of fenretinide (4-HPR), a vitamin A analogue, was investigated in a panel of six neuroblastoma cell lines and multicellular tumor spheroids. The latter are three dimensional cell aggregates and as such, a model for micrometastases. In all cell lines, the production of reactive oxygen species (ROS) increased with 163-680% after 1 h of treatment with 4-HPR. In addition, a decrease of the mitochondrial membrane potential of 30-75% was observed after 4 h of incubation with 4-HPR. A 6-12-fold difference was observed between the IC50 values for cell proliferation and viability between the most sensitive (IMR32) and most resistant (NASS) cell line towards 4-HPR. Flow cytometric analysis showed an increased amount of apoptotic bodies and no cell-cycle arrest. The antioxidant Trolox completely inhibited the accumulation of 4HPR-induced ROS and prevented the 4HPR-associated cytotoxicity. In all neuroblastoma spheroids, 4-HPR induced a complete cytostasis at clinical relevant concentrations (3-10 microM). Immunohistochemical analysis of 4-HPR-treated spheroids showed a decreased staining for proliferation marker Ki-67 and an increased staining for cleaved-PARP, a marker of apoptosis. Our results suggest that 4-HPR might be a promising agent for the treatment of micrometastases and high-risk neuroblastoma.     

Radiosensitivity of quiescent and proliferating cells grown as multicellular tumor spheroids

The multicellular spheroid model partly mimics tumor microenvironments in vivo and has been reported in plenty of studies regarding radiosensitivity. However, clear isolation of quiescent and proliferating cells in live conditions has been quite difficult owing to technical limitations; therefore, comprehensive characterization could not be done thus far. In this study, we succeeded in separately isolating different cell types using a fluorescent ubiquitination‐based cell cycle indicator (Fucci) and determining their radiosensitivities. Unexpectedly, proliferating cells were more radioresistant than quiescent cells due to the contact effect when spheroids were disaggregated immediately after irradiation. However, the radiosensitivity of quiescent cells was not influenced by mild hypoxia (hypoxia‐inducible factor‐1α‐positive but pimonidazole‐negative), but their radioresistance became similar to that of proliferating cells due to potentially lethal damage repair when disaggregated 24 h after irradiation. The Fucci system further allowed long‐term observation of cell kinetics inside of the spheroid following irradiation using real‐time confocal fluorescence scanning. Repeated cycles of recruitment from the quiescent to the proliferating phase resulted in cell loss from the outside of the spheroid toward the inside, causing gradual shrinkage. Interestingly, the central region of the spheroid entered a dormant stage approximately 40 days after irradiation and survived for more than 2 months. Using the Fucci system, we were able to comprehensively characterize the radiosensitivity of spheroids for the first time, which highlights the importance of cell cycle kinetics after irradiation in determining the radiosensitivity under tumor microenvironments.     

Redox regulation of P-glycoprotein-mediated multidrug resistance in multicellular prostate tumor spheroids

Multicellular prostate tumor spheroids develop intrinsic P-glycoprotein (Pgp)-mediated multidrug resistance with the appearance of quiescent cell areas. We have investigated the effect of intracellular reactive oxygen species (ROS) on Pgp expression in large, quiescent and drug-resistant multicellular spheroids (diameter 250 +/- 50microm). Using the ROS-sensitive fluorescence dye 2;7;-dichlorodihydrofluorescein diacetate (H(2)DCFDA), we demonstrated that these tumor spheroids are characterized by reduced intracellular ROS compared with drug-sensitive small spheroids (diameter 60 +/- 20microm) consisting predominantly of proliferating cells. The prooxidants hydrogen peroxide, menadione and glyceraldehyde raised ROS in large tumor spheroids and significantly down-regulated Pgp within 24 hr. Comparable effects were achieved with the known Pgp-reversing agents sodium orthovanadate, quinidine and cyclosporin A but not with verapamil. Consequently, the retention and toxicity of the anthracycline doxorubicin was increased in tumor spheroids treated with prooxidants. Co-administration of prooxidants and the free radical scavenger ebselen did not alter Pgp levels, indicating that down-regulation of Pgp is mediated via ROS. Down-regulation of Pgp by H(2)O(2) was abolished when either forskolin, 8-Br-cAMP or IBMX, which raise intracellular cAMP levels, was co-administered, indicating that Pgp expression is regulated by protein kinase A (PKA). Furthermore, Pgp was down-regulated by the PKA inhibitors Rp-cAMPs and H89. Since prooxidants stimulated the growth of multicellular spheroids and down-regulated the cyclin-dependent kinase inhibitor p27(kip1), we conclude that ROS-mediated Pgp down-regulation may be paralleled by recruitment of drug-resistant quiescent cells in the depth of the tumor tissue for cell-cycle activity.     

Response of human neuroblastoma and melanoma multicellular tumor spheroids (MTS) to single dose irradiation

The growth characteristics of 6 human cell line derived multicellular tumor spheroids (MTS) were studied. Melanoma MTS (C32, HML-A, HML-B) were slow growing with baseline growth rates of 13.9 to 27.3 microns diameter/day. Neuroblastoma MTS (Lan-1, NB-100, NB-134) grew rapidly, with baseline growth rates of 32.1 to 40.3 microns diameter/day, that is, 1.2 to 2.9 times as fast as the melanomas. Delay constants were calculated for all six lines. The neuroblastomas were more sensitive to radiation than melanomas, as reflected in a greater value for the radiation-induced growth delay constant. One neuroblastoma line, Lan-1, was highly radioresponsive; that is, after a subcurative dose of radiation, the MTS diameter decreased beyond the original diameter, which was followed by recovery and regrowth. Irrespective of these initial changes in diameter, growth delay sensitivity (value of delay constant) was the same for Lan-1 and NB-100, an MTS line that did not show the responsive pattern.     

Role of intercellular communications in breast cancer multicellular tumor spheroids after chemotherapy

Tumor heterogeneity is an important feature that is especially involved in tumor aggressiveness. Multicellular tumor spheroids (MTS) may provide some benefits in different steps for investigation of the aggregation, organization, differentiation, and network formation of tumor cells in 3D space. This model offers a unique opportunity for improvements in the capability of a current strategy to detect the effect of an appropriate anticancer agent. The aim of this study was to investigate the cellular interactions and morphological changes following chemotherapy in a 3D breast cancer spheroid model. Distribution of the gap junction protein "connexin-43" and the tight junction protein "occludin" was investigated by immunohistochemistry. Cellular interactions were examined by using transmission and scanning electron microscopies as well as light microscopy with Giemsa staining after treating cells with doxorubicin, docetaxel, and doxorubicin/docetaxel combination. Statistical analyses showed significant changes and various alterations that were observed in all groups; however, the most prominent effect was detected in the doxorubicin/docetaxel combination group. Distinct composition as a vessel-like structure and a pseudoglandular pattern of control spheroids were detected in drug-administered groups. Immunohistochemical results were consistent with the ultrastructural changes. In conclusion, doxorubicin/docetaxel combination may be more effective than the single drug usage as shown in a 3D model. The MTS model has been found to be an appropriate and reliable method for the detection of the changes in the expression of cellular junction proteins as well as other cellular proteins occurring after chemotherapy. The MTS model can be used to validate the effects of various combinations or new chemotherapeutic agents as well as documentation of possible mechanisms of new drugs.     

Isolation of quiescent cells from multicellular tumor spheroids using centrifugal elutriation

A quiescent (nonproliferating) subpopulation was identified by flow cytometric analysis using two-step acridine orange staining in the EMT6/Rochester, N. Y. subline multicellular tumor spheroid, an in vitro culture system which provides a cellular microenvironment which mimics that of many of in vivo tumors. To isolate a viable quiescent cell subpopulation, centrifugal elutriation which allows for cell separation mainly on the basis of size was used. This technique provided single cells of relatively homogeneous cell volume which varied over a wide range (approximately 100 to 5000 cu microgram). Though the relatively small cell volume fractions were the most enriched (82%) in quiescent cells, such cells were also observed in significant numbers (congruent to 20%) even in the largest cell fractions. The cell clonogenicity of the various elutriation constant in fractions was also assessed and shown to be lowest (plating efficiency congruent to 20%) in the small spheroid cells but relatively constant in fractions containing intermediate and large cells (plating efficiency congruent to 50%). Continuous [3H]thymidine labeling indicated a slower rate of accumulation of labeled cells in the small spheroid cells, which may result from the transition of proliferating spheroid cells to the quiescent compartment during the course of labeling. These finding indicate the utility of centrifugal elutriation for quiescent cell characterization in in vitro tumor systems.     

Photodynamic therapy of transitional cell carcinoma multicellular tumor spheroids with hypericin

The objective of the present study was to evaluate the sensitivity of the bladder transitional cell carcinoma (TCC) to hypericin PDT in a 3-D system using multicellular tumor spheroids. The photodynamic response in RT-112 human bladder TCC spheroids was also compared to 2-D cultured monolayer cells. Following a 2-4 h incubation with 8-30 microM hypericin, spheroids or monolayer cells were irradiated at the light dose of 12 J/cm2, delivered at a fluence rate of 10-100 mW/cm2. The PDT effects were evaluated using a clonogenic assay. The results show that compared with the cells in a monolayer, cells in spheroids were dramatically less sensitive to hypericin PDT (<2000-fold). Studies of fluorescence microphotographs of centrally cut frozen sections of hypericin-exposed spheroids showed a gradient in hypericin concentration from the peripheral to the central region of the spheroid. Although it can be suggested that heterogeneity of drug uptake might be responsible for the observed resistance of spheroid to hypericin PDT, hypericin sensitized spheroids that were dissociated prior to light irradiation were as sensitive as the monolayer cells to hypericin PDT, suggesting that other factors such as oxygen depletion might be responsible for the resistance of spheroids to hypericin PDT.     

Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids

We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtk-expressing LM05e, LM3 and B16 spheroids were 16-, three- and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight- and five-fold more efficiently. Mixed populations of HSVtk- and their respective beta gal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk-expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the beta gal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis.