Thore Nederman

Penetration of substances into tumor tissue—A methodological study on cellular spheroids

SummaryThe penetration of [3H]thymidine, [3H]d-leucine, [125I]albumin, and the drugs [3H]5-fluorouracil and [3H]vinblastine into human glioma spheroids (in vitro tumor models) was studied by a method based on rapid freezing, freeze drying, vapor fixation, wax embedding, dry sectioning, and contact autoradiography. No significant disturbances in the distribution of water soluble substances were observed. Thymidine andd-leucine penetrated the whole spheroids relatively fast, whereas albumin showed reduced penetration. the concentration of albumin was highest at the periphery of the spheroids, but only smaller amounts were detected in the deeper regions. A significant difference between the penetration patterns of the drugs studied was also observed. Fluorouracil penetrated rather freely, but the penetration of vinblastine was limited.

Penetration and binding of vinblastine and 5-fluorouracil in cellular spheroids.

SummaryThe kinetics of the penetration and binding of the two commonly used antitumour drugs vinblastine and 5-fluorouracil in nonvascularized tumour tissue were studied. Multicellular human tumour spheroids (glioma U-118 MG and thyroid cancer HTh-7) were used as a model system. Radiolabelled drugs were used in all studies. To avoid disturbances in the distribution of unbound drugs a dry histological technique was used in combination with contact autoradiography. In addition, quantitative measurements of the accumulation and binding of the drugs were made. The results showed that vinblastine penetrated the spheroids less efficiently than 5-fluorouracil. Vinblastine required about 2 h to be isotropically distributed within the studied spheroids, while only a few minutes were required for 5-fluorouracil. Vinblastine seemed to be accumulated in the peripheral parts of the spheroids within 15 min. High concentrations of 5-fluorouracil, isotropically distributed in the spheroids, were observed after 2 h of incubation. Significant amounts (about half) of the accumulated drugs resisted gentle washing for 3x20 s plus 15 min in fresh medium. The limited penetration of vinblastine correlated well with a previously observed high resistance of spheroids to treatments of short duration with this drug.

Tumour spheroid technology in cancer therapy research

GENERAL GROWTH PATTERN Spheroids have an outer layer of proliferating cells and an inside layer of mainly quiescent cells. In the central areas of spheroids with a diameter greater than 400-700 pm, there are often massive necrotic areas. This growth pattern is similar to that ofsolid tumour nodules containing proliferating cells close to the capillaries, quiescent cells next to these and necrotic areas at larger distances. Spheroid cells can be further divided into subpopulations as, for example, quiescent cells close to the necrotic centre which are irreversibly growth inhibited and quiescent cells closer to the surface that have the capacity to enter the cell cycle. The pattern is general although there are large cell-type dependent variations in growth rate, cell morphology and the thickness of the viable cell layers [l-7]. The spheroids contain an extensive extracellular matrix composed of, for example, fibronectin, laminin and collagen in a proteoglycan gel. There are differences between different types of spheroids also in this aspect and the differences correlate with differences in growth [8-l 11.

An in vitro bioassay for quantitation of human interferons by measurements of antiproliferative activity on a continuous human lymphoma cell line.

Interferons have, in addition to their antiviral effects, been shown to possess several non-antiviral activities. In this study, an in vitro bioassay for interferon alpha (IFN-alpha) preparations based on their antiproliferative effect in cultured Daudi cells has been developed. Briefly, about 10(5) cells per ml treated with different concentrations of IFN were incubated under standard culture conditions for 3 days. Two different end points, i.e. incorporation of [3H]thymidine and final cell density, were used and responses were evaluated according to established pharmacopoeial principles for quantification of biomolecules. Both methods gave similar results. However, measurement of final cell density yielded the most precise results. The proposed assay, with an effective assay range of 1-10 IU/ml (approximately 0.2-2 x 10(-12)M, had a high sensitivity and precision as well as a good reproducibility. Compared with antiviral assays, it is less resource demanding. In conclusion, the in vitro bioassay described is well suited for potency determinations of IFN-alpha and probably also IFN-beta preparations.

Penetration of substances into tumor tissue: A methodological study with microelectrodes and cellular spheroids

SummaryA new method was tested for studies of penetration of substances into tumorlike tissue. The penetration of the ions K+, Cl−, and Ca2+ through several layers of tumor cells was demonstrated by using double barrelled, ion sensitive microelectrodes with extra thin tip diameters. Spheroids consisting of human glioma, U-118 MG, and human thyroid cancer, HTh-7, cells were used as models of tumor tissue. A microelectrode was inserted into the center of a spheroid. Thereafter, the concentration of the test substance was increased in the surrounding medium. The change in concentration inside the spheroid was recorded and the penetration pattern evaluated. All three types of tested ions penetrated easily through the spheroids. The K+ ions penetrated most efficiently, and the Ca2+ ions showed the slowest penetration. The Ca2+ ions penetrated somewhat more slowly in the U-118 MG spheroids (which had rather small extracellular spaces) than in the HTh-7 spheroids (which had larger extracellular spaces). Ion sensitive electrodes, which are easily available, were used in this study only to demonstrate the principle. We hope that the method described can be used for penetration studies of various substances. For example, all substances that can be detected by enzyme microelectrodes could be studied. The main advantage of the method is that the complete penetration pattern can be studied as a function of time in individual spheroids. Previously described methods require histological procedures for each analyzed penetration time.

Penetration of substances into tumour tissue

SummaryIn order to achieve a better understanding of factors involved in drug penetration into poorly vascularized tumour tissue, the penetration of some model substaces was studied in vitro. Multicellular human tumour spheroids were used as model system. The test substances were [3H]thymidine and [14C]glucose, both of which are capable of passing easily through cell membranes, and [3H]thymidine-5′-triphosphate, [3H]sucrose and [3H]inulin, all of which are unable to pass directly through cell membranes. The penetration of these substances was studied using a dry histological and autoradiographical method preserving the distribution of water-soluble substances. The two thymidine compounds penetrated very efficiently into the spheroids, and their penetration patterns were rather similar. The saccharides differed somewhat in their penetration properties. Glucose had the fastest penetration and inulin the slowest. After 15 min, however, inulin was also found isotropically distributed within the spheroids. Thus, extracellular penetration seemed to be a possible way for a substance to reach the central parts of a spheroid. The differences between the saccharides could be due to some extent to differences in molecular weight and solubility.

The quantitation of human growth hormone by a radioreceptor assay using an established human cell line

Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The assay should fulfil established pharmacopoeial requirements for quantitation of hormones. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 X 10(7) cells ml-1 were incubated with 5-20 X 10(-12) M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations.

Binding of Epidermal Growth Factor (EGF) to a Cultured Human Glioma Cell Line

We have studied binding of 125I-EGF to the human malignant glioma cell line U-343 MG aCl2:6, which is planned to be used as a model system in studies of toxic effects of EGF conjugates. Special care has been taken to fulfil the requirements for a correct Scatchard analysis of binding parameters. Binding as a function of time, temperature and pH was investigated as well as dissociation and internalization of bound EGF. The stability of EGF during incubation was also determined. After binding to the receptor, EGF is rapidly internalized and degraded at physiological temperature. We found that binding experiments should be performed at 4 degrees C, since at this temperature practically no internalization took place, whereas dissociation occurred. From displacement experiments using increasing concentrations of unlabelled EGF competing with 125I-EGF for binding, binding parameters were calculated using a computerized, nonlinear, least-squares regression analysis of binding data. We found that EGF bound to a class of high affinity receptors with an apparent dissociation constant KD of about 4 x 10(-10) M. The mean number of receptors was 25,000 per cell. In experiments where receptors were saturated with 125I-EGF an additional class of low affinity receptors was detected. This had an apparent KD of 1 x 10(-8) M with a mean receptor number per cell of 780,000. We also noticed enhanced dilution-induced dissociation of bound 125I-EGF in the presence of excess unlabelled EGF, suggesting negative cooperativity.

Radioreceptor assay for formulations of salmon calcitonin

Abstract Receptors on cultured human lymphocytes (IM-9) have been shown to bind salmon calcitonin (sCT) specifically. The aim of the present study was to develop an in vitro assay for sCT based on this binding. The assay should fulfil pharmacopoeial requirements for quantitation of biologicals and should be validated against an established in vivo assay of CT. The binding of 125I-sCT was studied as a function of time and concentration of unlabelled sCT and other peptides. Also dissociation of bound sCT and degradation of sCT in the incubation medium were studied. From these studies, conditions for a radioreceptor assay for sCT were chosen. Concentrations from 7 × 10−11 to 2 sx 10−9 M or from 1 to 30 mIU ml−1 caused a linear reduction of binding of 125I-sCT. This concentration interval was chosen for a three-dose assay. The results obtained with the radioreceptor assay correlated with results from the established in vivo bioassay but were more precise. Since the in vitro method is also less resource demanding than the in vivo bioassay, it is suggested that the former is suitable as a screening method for potency determinations of sCT preparations.

In-vitro bioassays for cholecystokinin.

Two in‐vitro methods for the bioassay of cholecystokinin (CCK) are described. They should be useful for determinations of potencies of pharmaceutical formulations of CCK. Both are based on the hormones ability to stimulate exocrine pancreas. The stimulatory effect on 45Ca efflux from preloaded suspended acinar cells or on release of amylase from pancreatic acini from the guinea‐pig has been recorded. The assay based on stimulation of 45Ca outflux is more specific and precise than the method using measurements of amylase secretion. Therefore, the calcium assay was further validated against the conventional guinea‐pig gall‐bladder contraction assay. Both methods gave similar potency readings but the results from the in‐vitro assay were more precise.