Uptake of IgG in osteosarcoma correlates inversely with interstitial fluid pressure, but not with interstitial constituents.

The uptake of therapeutic macromolecules in solid tumours is assumed to be hindered by the heterogeneous vascular network, the high interstitial fluid pressure, and the extracellular matrix. To study the impact of these factors, we measured the uptake of fluorochrome-labelled IgG using confocal laser scanning microscopy, interstitial fluid pressure by the 'wick-in-needle' technique, vascular structure by stereological analysis, and the content of the extracellular matrix constituents collagen, sulfated glycosaminoglycans and hyaluronan by colourimetric assays. The impact of the microenvironment on these factors was studied using osteosarcomas implanted either subcutaneously or orthotopically around the femur in athymic mice. The uptake of IgG was found to correlate inversely with the interstitial fluid pressure and the tumour volume in orthotopic, but not subcutaneous tumours. No correlation was found between IgG uptake and the level of any of the extracellular matrix constituents. The content of both collagen and glycosaminoglycans depended on the site of tumour growth. The orthotopic tumours had a higher vascular density than the subcutaneous tumours, as the vascular surface and length were 2-3-fold higher. The data indicate that the interstitial fluid pressure is a dominant factor in controlling the uptake of macromolecules in solid tumours; and the site of tumour growth is important for the uptake of macromolecules in small tumours, extracellular matrix content and vascularization.     

The failure of human leukocyte interferon to influence the growth of human glioma cell populations: in vitro and in vivo studies

Five high-grade (3 grade III and 2 grade IV) astrocytoma tumour cell populations were treated with a preparation of Human Leukocyte Interferon either in monolayer cell culture or as multicellular spheroids in vitro or as xenografts growing in immune-deprived mice in vivo. A moderate and transient sensitivity was seen in one grade III tumour when tested in both of the in vitro assays, but no inhibition of growth was seen in vivo. Two tumours which were apparently resistant to Interferon treatment responded to orthodox chemotherapy. When used in conjunction with BCNU, Interferon was not effective in prolonging delay in tumour growth. It is concluded that Interferon is unlikely to be an effective agent in the treatment of malignant brain tumours.     

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.     

Evaluation of the cytotoxicity of the indoloquinone eo9 in a human colon adenocarcinoma model

The development of anti-cancer drugs that are active in regions of low oxygen tension within solid tumours is an important goal for the chemotherapist. Equally as important is the utilization of appropriate model systems for their selection. This study describes morphological characteristics of multicellular spheroids derived from the human carcinoma cell line, DLD-1 and the evaluation of an investigational bioreductive alkylating agent (EO9) in monolayers, spheroids and xenografts. Histological examination of the cell line in vitro revealed typical features of glandular epithelium with microvilli on free surfaces and cell junction formation. Spheroids had acina formation, extensive necrosis and hypoxia at the time of treatment suggesting the spheroid model to be more representative of solid tumour geometry than more conventional in vitro test systems. EO9 is active against this cell line grown as a monolayer (IC50=0.76 mug ml-1) but is inactive against spheroids or established solid tumours in vivo. The suitability of this system for evaluating bioreductive drugs is discussed.     

131I-meta-iodobenzylguanidine therapy in neuroblastoma spheroids of different sizes.

Mathematical models have predicted that targeted radiotherapy of neuroblastoma with metaiodobenzylguanidine (mIBG) is less likely to cure small rather than large micrometastases if 131I is the conjugated radionuclide. This study uses multicellular tumour spheroids as an in vitro model to test the hypothesis that smaller tumours of sub-millimetre dimensions are relatively resistant to 131I-mIBG. Spheroids of the human neuroblastoma cell line SK-N-BE(2c), either 250 microns or 400 microns diameter, were incubated with 131I-mIBG at concentrations of up to 6.0 MBq ml-1. Using both regrowth delay and spheroid 'cure' as endpoints, the greater vulnerability of larger spheroids was confirmed. From this in vitro result we conclude that when used in vivo 131I-mIBG may spare smaller micrometastases. Therefore, either a radionuclide such as 211At which emits a shorter path length radiation should be conjugated to mIBG, or targeted radiotherapy should be combined with a treatment such as total body irradiation, the efficacy of which is not reduced in smaller tumours.     

An experimental trial of cyclic nucleotides on multicellular spheroids derived from human brain tumours

The effects of cyclic nucleotides, dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate (db-cAMP and db-cGMP), on the growth rate of multicellular tumour spheroids were evaluated by comparing the growth delay and colony forming efficiency in vitro. Multicellular tumour spheroids were derived directly from human brain tumours. To compare the chemotherapeutic effect of cyclic nucleotides, CCNU was used as a known effective cytotoxic drug on malignant gliomas.Significant growth delay was obtained by db-cAMP (p<0.001) while CCNU was tumouricidal rather then producing a delay in growth.of the tumpur spheroids. Db-cGMP found not to be effective in decreasing the growth rate of the tumour spheroids in vitro (p>0.2).The role of cyclic nucleotides in brain tumours is discussed on a review basis. address for offprints     

Inhibition of P-glycoprotein function by XR9576 in a solid tumour model can restore anticancer drug efficacy

Resistance to cancer chemotherapy involves both altered drug activity at the designated target and modified intra-tumour pharmacokinetic properties (e.g. uptake, metabolism). The membrane transporter P-glycoprotein (P-gp) plays a major role in pharmacokinetic resistance by preventing sufficient intracellular accumulation of several anticancer agents. Whilst inhibiting P-gp has great potential to restore chemotherapeutic effectiveness in blood-borne cancers, the situation in solid tumours is less clear. Therefore, the degree of resistance tumours pose to the cytotoxicity of vinblastine and doxorubicin was characterised using the multicellular tumour spheroid model. Tumour spheroids were generated from either drug-sensitive MCF7(WT) breast cancer cells or a resistant P-gp-expressing variant (NCI/ADR(Res)). Drug-induced cytotoxicity in tumour spheroids was measured using an outgrowth assay and compared with that observed in monolayer cultures. As anticipated, the 3-D organisation of MCF7(WT) in tumour spheroids was associated with a reduction in the potency of doxorubicin and vinblastine-i.e. the inherent multicellular resistance phenomenon. In contrast, tumour spheroids from NCI/ADR(Res) cells did not display multicellular resistance. However their constitutive expression of P-gp reduced the potency of both anticancer drugs. Moreover, the highly potent P-gp inhibitor, the anthranilic acid derivative, XR9576, was able to restore the cytotoxic efficacy of both drugs in tumour spheroids comprising NCI/ADR(Res) cells. The results suggest that inhibition of P-gp in solid tumours is achievable and that generation of potent inhibitors will provide a significant benefit towards restoration of chemotherapy in solid tissues.     

Characterization of cell lines originating from an invasive transitional cell carcinoma

A reporter gene (lacZ) was introduced into a human transitional cell cancer cell line (Hu1703He) by means of liposomal transfection. The lacZ-transfected cell line induced subcutaneous tumours in nude rats and cells from one rat tumour were then established as a monolayer culture. The two lacZ-transfected cell lines both stained positive for CK7 and negative for CK14 and additionally formed spheroids in three-dimensional cultures. Insignificant genomic changes occurred in the tumour cells after incubation in nude rats, while the lacZ transfection caused alterations that probably correspond to increased invasiveness and tumourigenicity in vitro and in vivo. Most important is the observation that lacZ transfection of this human TCC cell line does not reduce its invasion potential in vitro or in vivo. The lacZ reporter gene may thus be exploited to facilitate the identification and quantification of migrating tumour cells and subsequently for studies of invasion in in vitro coculture systems. The observation that the spheroidal growth is reduced after transfection of the cell line, in contrast to increased invasion and cellular growth in monolayer, is an observation indicating that a three-dimensional arrangement mimicking the in vivo conditions offers important regulating factors to cellular growth.     

Diversity of cell-mediated adhesions in breast cancer spheroids

Due to their three dimensional (3D) architecture, multicellular tumor spheroids mimic avascular tumor areas comprising the establishment of diffusion gradients, reduced proliferation rates and increased drug resistance. We have shown recently that the spontaneous formation of spheroids is restricted to a limited number of cell lines whereas the majority grow only as aggregates of cells with loose cell-cell contacts when cultured in 3D. However, by the addition of reconstituted basement membrane (rBM, Matrigel), aggregates can be transformed into spheroids with diffusion barriers and development of quiescent therapy-resistant cells. In this report, we investigated adhesion molecules responsible for rBM-driven versus spontaneous spheroid formation in a diverse population of eight breast tumor cell lines relevant for in vitro and in vivo antitumor drug testing. Inhibition of spheroid formation was monitored in the presence of adhesion molecule functional blocking antibodies and after siRNA-mediated down-regulation of E- and N-cadherin and integrin beta1 adhesion receptors. We identified that E-cadherin mediates the spontaneous formation of spheroids in MCF7, BT-474, T-47D and MDA-MB-361 cells, whereas N-cadherin is responsible for tight packing of MDA-MB-435S cells. In contrast, the matrix protein-induced transformation of 3D aggregates into spheroids in MDA-MB-231 and SK-BR-3 cells is mediated primarily by the collagen I/integrin beta1 interaction with no cadherin involvement. A combination of both, homophilic E-cadherin and integrin beta1/collagen I interaction establishes spheroids in MDA-MB-468 cells. These findings indicate that an evolutionary diverse and complex pattern of interacting cell surface proteins exists in breast cancer cells that determines the 3D growth characteristic in vitro, thereby influencing small molecule or antibody permeation in preclinical in vitro and in vivo tumor models.     

Macromolecule uptake in human melanoma xenografts. relationships to blood supply, vascular density, microvessel permeability and extracellular volume fraction

The uptake of albumin-Evans blue in human melanoma xenografts was studied and related to blood supply, vascular density, microvessel permeability and extracellular volume fraction in an attempt to identify transport barriers limiting the delivery of macromolecular therapeutic agents to tumours. Three melanoma lines (A-07, R-18, U-25) were included in the study. Tissue concentrations of albumin-Evans blue were determined by spectrophotometry. The [86Rb] uptake method was used to measure tumour blood supply. Vascular density was determined by stereological analysis of histological sections. Microvessel permeability was measured by using the indicator diffusion method. Contrast-enhanced magnetic resonance imaging was used to measure tumour extracellular volume fraction. The fractional volume of the extracellular space governed the uptake of albumin-Evans blue in the tumours. The uptake of albumin-Evans blue in the extracellular space was primarily limited by transport in the vasculature and not by transport across the microvascular wall or the transport through the interstitium. Our study thus suggests that novel strategies for improving the delivery of macromolecular therapeutic agents to tumours should focus on enhancing the tumour blood supply, increasing the half-life of the therapeutic agent in the blood plasma and/or enhancing the volume of the extracellular space available to macromolecules rather than on increasing the permeability of the microvascular wall or improving diffusion conditions in the tumour interstitium.     

CB 1954 revisited

We have assessed the antitumour activity of the nitrophenylaziridine CB 1954 in vitro and in vivo. For EMT6 mouse mammary tumour multicellular spheroids under hypoxic conditions in vitro, a 6-h exposure to 40 micrograms/ml reduced the surviving fraction to as low as 10(-3) and the growth delay was 5.4 days. Oxic cells were twofold less sensitive. Phenyl AIC protected oxic and hypoxic cells equally. Under oxic conditions minimal cell killing was seen with HT29 cells, either in multicellular spheroids or in monolayer; a 6-h exposure to 40 micrograms/ml gave a spheroid growth delay of 1.5-1.7 days. No growth delay was seen with single maximum tolerated doses of CB 1954 against HT29 grown as a xenograft in immunosuppressed mice. Only minimal growth delays of 1-2 days were seen with similar doses against the EMT6 tumour and the RIF-1 and KHT sarcomas in mice. Little activity was seen with maximum tolerated doses given once a day for 5 days against EMT6 and RIF-1. No chemosensitization was measurable with CCNU, cyclophosphamide or melphalan in the KHT tumour.     

Anti-tumoural activity of peripheral blood mononuclear cells against melanoma cells: discrepant in-vitro and in-vivo effects

As tumour cells use multiple mechanisms to escape from chemotherapeutic drugs, the anti-tumoural activity of naive mouse peripheral blood mononuclear cells was examined in this study, using a mouse melanoma cell subline resistant to doxorubicin (B16R). Multicellular spheroids are known to be the most adapted in-vitro model to mimic solid tumours in vivo and are used to investigate many aspects of tumour biology. For in-vitro studies, murine peripheral blood mononuclear cells recovered by Ficoll gradient centrifugation after caudal puncture were co-cultured with multicellular tumour spheroids of B16R cells. Morphological investigations show that peripheral blood mononuclear cells were gathered and focused around the spheroids after 14 h of co-culture and contacts were established within 32 h. Between 38 and 62 h of co-culture, the size of the spheroids decreased significantly. The peripheral blood mononuclear cells exerted cytolytic effects that correlated with the induction of cell death in spheroids of B16R melanoma cells. Immunological investigations to localize and identify peripheral blood mononuclear cells that exerted anti-tumoural effects have shown that spheroids were deeply infiltrated by monocytes/macrophages at a stage in which a significant cytolytic activity and a strong cell death rate were observed. For in-vivo studies, intratumoural injections of syngeneic naive peripheral blood mononuclear cells were administered. A weak potential in-vivo anti-tumoural effect of these cells was observed (inhibition of B16R melanoma growth by 20-25%) but the median survival time of mice treated with peripheral blood mononuclear cells did not increase compared with untreated control mice. Thus, despite anti-tumoural activities of peripheral blood mononuclear cells against the poorly immunogenic and highly metastatic chemoresistant B16 melanoma cells in vitro, a potential anti-melanoma effect in vivo, if present, did not increase the life span of B16R melanoma-bearing mice.     

Induction of differentiation in neoplastic cells

There is now clear evidence that cells cultured from human and animal tumours can be induced to differentiate in vitro by recognised hormones, regulatory peptides, polar solvents and cytotoxic drugs. Examples can be found from several different types of tumour with the bulk of the data deriving from neuroblastoma and myeloid leukaemia. There is no clear correlation of inducer with cell type, other than some specific peptides like MSH, and agents such as dimethyl sulphoxide and dexamethasone have wide ranging activity. Steroid hormone action may require interaction between different cell types, and the inability of tumours to differentiate in situ may implicate reduced cell-cell interaction, possibly due to degradation of extracellular matrix, or to alteration of the stromal phenotype by tumour-derived factors such as peptides or prostaglandins. When differentiation has been demonstrated, it has been possible, in some cases, to correlate increased differentiation with reduced malignancy by in vitro characterisation or tumorigenicity. Conditions which induce differentiation in rat mammary carcinoma and mouse myeloma also reduce tumour growth in vivo. Clinical trials have not provided any conclusive evidence for a therapeutic benefit so far, but relatively few trials have been carried out. There is clearly a need for further investigation both in vitro and in vivo to select optimal conditions and combinations of agents for clinical evaluation.     

Multicellular spheroids grown directly from human tumour material

Human tumour cells from surgical material were grown as multicellular spheroids. In 16 out of 20 cultures spheroids with a diameter of more than 250 microns could be observed. In 6 out of 20 cultures more than 30 spheroids with a diameter of at least 300 microns were obtained, i.e. 30% of the cultures fulfilled the criterion for a possible chemosensitivity test on primary cell spheroids. A study of stained sections from the spheroids and the respective tumours, showed that the morphology of the spheroid was very similar that of the tumour from which the cells were derived. Samples from 9 malignant melanomas, 4 bladder carcinomas, 2 renal-cell carcinomas, 2 ovarian carcinomas, 1 lymphoma, 1 colon carcinoma, and 1 schwannoma were tested for spheroid growth. Spheroids were obtained from at least one representative of all these tumour types. However, investigations involving larger numbers of tumours are needed to find out which tumour types are most suitable for further biological characterization of primary cell spheroids and tests for therapy response.     

Deriving cell survival curves from the overall responses of irradiated tumours: analysis of published data for tumour spheroids

Curves of growth delay (GD) or 'cure' after graded doses of radiation have been analysed for 16 lines of human and animal tumours grown as multicellular spheroids in vitro. Dose-survival curves were derived for those cellular units from which spheroids regrow after unsuccessful irradiation (spheroid-regenerating cellular units, SRU). For 10 sets of data from 6 spheroid lines, the Do's and extrapolation numbers of the SRU derived by GD could be compared with the response of the clonogenic cells of the spheroids. For Do, a good correlation (r = 0.910) was found between the two; this was true also for Do derived from curves of spheroid 'cure' (7 sets of data from 6 spheroid lines) and clonogenic cells (r = 0.986). Using GD, the correlation of extrapolation numbers was less good (r = 0.682), the values for SRU commonly being higher than those for clonogenic cells. This may reflect features of the growth curves of spheroids after the lower range of doses of radiation. For human and animal tumour spheroids of 250 microns or less, derived Do ranged from 0.5 to 2.5 Gy. For spheroids of 350 microns or more, derived Do for animal tumour lines ranged from 3.4 to 4.2 Gy, for human lines from 1.5 to 2.1 Gy.     

The radiobiology of human neuroblastoma

The radiation response of a human neuroblastoma xenograft HX138 has been studied in vitro and in vivo using single cells in suspension, multicellular spheroids, and xenografts in immune-suppressed mice. End-points used were growth delay and clonogenic cell survival. Growth delay experiments with spheroids and xenografts showed a high degree of radioresponsiveness. Cell survival curves obtained from all systems were characterised by the lack of a shoulder. An increase in Do of the cell survival curve was seen after irradiation of intact spheroids and xenografts, perhaps due to the presence of a contact effect. Cellular capacity for split-dose recovery in vitro was modest. Delayed assay experiments using spheroids and xenografts showed some potentially lethal damage (PLD) repair in vitro but not in vivo. The results show this human tumour line to be intrinsically highly radiosensitive, with a limited repair capacity.     

Extracellular matrix components in breast carcinomas

A malignant process interferes with the normal 'programme' of extracellular matrix biosynthesis and can modify extensively the structure and composition of the matrix. This effect appears to be attributable to several processes such as direct production of some selected matrix macromolecules by malignant cells or indirectly by the production of factors by malignant cells interfering with the regulation of normal matrix production. Other possibilities may also exist, such as the direct action of an environmental carcinogen on otherwise normal mesenchymal cells. The result is a more or less profound modification of tissue structure and composition with possible feedback effects on the malignant process. Some examples will be discussed such as elastin production by some tumours as well as the biosynthesis of some other selected matrix macromolecules as tenascin and osteopontin by breast tumours. Although the detailed mechanisms of these specific matrix productions is not yet completely elucidated, the rapidly increasing knowledge on the regulation of specific matrix production process and deranged matrix production might represent a new area of crosstalk between cancer research and matrix biology.     

Rat mammary adenocarcinoma heat-stress proteins in vivo

Rat 13762NF mammary adenocarcinoma cells (clone MTC) were heated to 42 degrees C either in vivo as a subcutaneous tumour in the rat mammary fat pad or in vitro as attached cells. Labelling in vivo or in vitro detected very similar heat-stress proteins (hsp) at 160, 112, 90, 70 and 56 kDa. Syngeneic rat endothelial and macrophage cells synthesized several cellular proteins in vitro differently than did the tumour cells in vitro, but both types of normal cells were similar to tumour cells in the hsp synthesized. Although the quantitative aspects of induction and repression of hsp may depend on cell type and microenvironment, the major tumour hsp being studied for function in vitro were qualitatively similar to those produced and labelled in vivo in response to a similar heat dose. Hsp were similar in both normal cells and tumour cells from the same host. These observations support the concept that hsp function in fundamental processes in the different microenvironmental and metabolic conditions found in vivo and in vitro. In addition, these observations suggest that prediction of tumour thermal response by measuring hsp levels may be influenced by host cell components.