Modelling the internalization of labelled cells in tumour spheroids

In this paper a mathematical model is developed to describe the migration of labelled particles within a multicell spheroid. In the model, spatial variations in cell proliferation and death create an internal velocity field which leads to redistribution of the labelled and unlabelled cells. By applying a range of numerical and analytical techniques to the model equations, it is possible to show that, whilst the speed with which the labelled cells migrate through the tumour is independent of the type of cells that are labelled, their limiting distribution depends crucially on whether inert polystyrene microspheres or live tumour cells are labelled. These predictions are shown to be in good qualitative agreement with independent experimental results.     

The migration of cells in multicell tumor spheroids

A mathematical model is proposed to explain the observed internalization of microspheres and 3H-thymidine labelled cells in steady-state multicellular spheroids. The model uses the conventional ideas of nutrient diffusion and consumption by the cells. In addition, a very simple model of the progress of the cells through the cell cycle is considered. Cells are divided into two classes, those proliferating (being in G1, S, G2 or M phases) and those that are quiescent (being in G0). Furthermore, the two categories are presumed to have different chemotactic responses to the nutrient gradient. The model accounts for the spatial and temporal variations in the cell categories together with mitosis, conversion between categories and cell death. Numerical solutions demonstrate that the model predicts the behavior similar to existing models but has some novel effects. It allows for spheroids to approach a steady-state size in a non-monotonic manner, it predicts self-sorting of the cell classes to produce a thin layer of rapidly proliferating cells near the outer surface and significant numbers of cells within the spheroid stalled in a proliferating state. The model predicts that overall tumor growth is not only determined by proliferation rates but also by the ability of cells to convert readily between the classes. Moreover, the steady-state structure of the spheroid indicates that if the outer layers are removed then the tumor grows quickly by recruiting cells stalled in a proliferating state. Questions are raised about the chemotactic response of cells in differing phases and to the dependency of cell cycle rates to nutrient levels.     

Design of polypeptide-functionalized polystyrene microspheres

In this contribution, the principle of spontaneous surface segregation has been applied for the preparation of polypeptide-functionalized polystyrene microspheres. For that purpose, an amphiphilic diblock copolymer was introduced in the mixture styrene/divinylbenzene and polymerized using AIBN as initiator. During the polymerization, cross-linked particles were obtained in which the diblock copolymer was encapsulated. The amphiphilic diblock copolymers used throughout this study contain a hydrophilic polypeptide segment, either poly(L-lysine) or poly(L-glutamic acid) and a hydrophobic polystyrene block. After 4 h of polymerization, rather monodisperse particles with sizes of approximately 3-4 microm were obtained. Upon annealing in hot water, the hydrophilic polypeptides migrate to the interface, hence, either positively charged or neutral particles were obtained when poly(L-lysine) is revealed at the surface and exposed to acidic or basic pH, respectively. On the opposite, negatively charged particles were achieved in basic pH water by using poly(L-glutamic acid) as additive. The surface chemical composition was modified by changing the environment of the particles. Thus, exposure in toluene provoked a surface rearrangement, and due to its affinity, the polystyrene block reorients toward the interface.     

Visualizing the effect of tumor microenvironments on radiation-induced cell kinetics in multicellular spheroids consisting of HeLa cells

In this study, we visualized the effect of tumor microenvironments on radiation-induced tumor cell kinetics. For this purpose, we utilized a multicellular spheroid model, with a diameter of ∼500 μm, consisting of HeLa cells expressing the fluorescent ubiquitination-based cell-cycle indicator (Fucci). In live spheroids, a confocal laser scanning microscope allowed us to clearly monitor cell kinetics at depths of up to 60 μm. Surprisingly, a remarkable prolongation of G2 arrest was observed in the outer region of the spheroid relative to monolayer-cultured cells. Scale, an aqueous reagent that renders tissues optically transparent, allowed visualization deeper inside spheroids. About 16 h after irradiation, a red fluorescent cell fraction, presumably a quiescent G0 cell fraction, became distinct from the outer fraction consisting of proliferating cells, most of which exhibited green fluorescence indicative of G2 arrest. Thereafter, the red cell fraction began to emit green fluorescence and remained in prolonged G2 arrest. Thus, for the first time, we visualized the prolongation of radiation-induced G2 arrest in spheroids and the differences in cell kinetics between the outer and inner fractions.     

Penetration of anti-melanoma immunotoxin into multicellular tumor spheroids and cell kill effects

Summary In order to gain a better understanding of the interaction between immunotoxins and tumor cells at the level of three-dimensional tumor mass, we evaluated the cell kill effects of monoclonal antimelanoma-antibody/ricin-A-chain immunotoxin (ITN) on melanoma cells in multicellular tumor spheroids (MTS) as well as the penetration of ITN into MTS. For Minor melanoma cells in monolayer the ITN exerted cytotoxic effects after as little as 1 h of exposure. Increasing exposure time resulted in progressive increases in cytotoxic activity. In contrast, the cell kill effects of ITN were markedly delayed and reduced when Minor cells were in MTS. The ITN cytotoxic effects on the melanoma MTS were more than 100 fold less than those in monolayer. Patterns of ITN-induced cytotoxicities for Minor and for another melanoma cell line, DND-1A, were comparable. The native ricin A was more active against PC-10 squamous lung cancer cells than Minor cells, whereas the ITN was more cytotoxic against Minor cells than PC-10 cells, thus exhibiting selectivity. An autoradiographic study revealed time-dependent penetration of radiolabeled ITN from the surface of Minor MTS into the core. Incubation for 1 h resulted in the penetration of ITN into only the two or three outer layers of the Minor MTS, and low grain counts. Prolonged exposure resulted in inhomogeneous penetration of ITN into almost the entire melanoma MTS. Penetration of ITN into PC-10 MTS was extremely poor. The reduced cytotoxicity of ITN on melanoma cells in MTS as compared to cells grown in monolayer appears to correlate with its inhomogeneous distribution in the MTS. The delayed cytotoxicity of ITN is also consistent with its slow penetration into the core of the MTS.     

A homogeneous fluorescence-based method to measure antibody internalization in tumor cells

We have developed a simple fluorescence-based method to monitor antibody internalization. Panitumumab was dual-labeled with the fluorophore IRDye 800CW and quencher IRDye QC-1 to yield the biomolecular probe Pan800QC. The fluorescence of IRDye 800CW is quenched by IRDye QC-1 on the same intact antibody. After incubation with epidermal growth factor receptor (EGFR)-expressing cells, internalization of Pan800QC was detected by an increase in fluorescence signal due to enzymatic digestion of the antibody and separation of IRDye 800CW and IRDye QC-1. By optimizing reaction conditions, a signal-to-background ratio of 8.5 was obtained. This homogeneous assay can be applied in the characterization and screening of internalizing antibodies.     

Morphological and functional characteristics of cells infiltrating and destroying tumor multicellular spheroids in vivo.

EMT6 mammary sarcoma cells were grown in vitro as multicellular spheroids to model for the heterogeneity of microenvironments and structural changes which develop in many tumors, including micrometastases. Spheroids of 700-900 micron diameter were implanted into and recovered at different times from the peritoneal cavities of sensitized or nonsensitized allogeneic and syngeneic mice. The colony forming efficiency of spheroid tumor cells recovered at 24 and 48 h from sensitized allogeneic mice was markedly decreased as compared with those from nonsensitized allogeneic or syngeneic animals. These recovered spheroids were extensively infiltrated by both lymphocytes and macrophages, which ultrastructurally had very close membrane associations with tumor cells. Host cells recovered from spheroids exhibited cytotoxic activity in an in vitro 51Cr release assay. Thus, multicellular spheroids in vivo provide a unique experimental model to study the functional capacity of host cells within a spheroical tumor. Although lacking the stroma and the vasculature of in vivo solid tumors, this model does have many similarities to in vivo tumors and is thus suitable for studying the tumor cell-host cell interactions within the tumor microenvironment. In addition, the system offers the potential for quantitative study of the effects of treatment modalities on tumor cell-host cell interactions.     

3D in vitro co-culture models based on normal cells and tumor spheroids formed by cyclic RGD-peptide induced cell self-assembly

Objectives

To design novel 3D in vitro co-culture models based on the RGD-peptide-induced cell self-assembly technique.

Results

Multicellular spheroids from M-3 murine melanoma cells and L-929 murine fibroblasts were obtained directly from monolayer culture by addition of culture medium containing cyclic RGD-peptide. To reach reproducible architecture of co-culture spheroids, two novel 3D in vitro models with well pronounced core–shell structure from tumor spheroids and single mouse fibroblasts were developed based on this approach. The first was a combination of a RGD-peptide platform with the liquid overlay technique with further co-cultivation for 1–2 days. The second allowed co-culture spheroids to generate within polyelectrolyte microcapsules by cultivation for 2 weeks. M-3 cells (a core) and L-929 fibroblasts (a shell) were easily distinguished by confocal microscopy due to cell staining with DiO and DiI dyes, respectively.

Conclusions

The 3D co-culture spheroids are proposed as a tool in tumor biology to study cell–cell interactions as well as for testing novel anticancer drugs and drug delivery vehicles.

     

Interaction of polystyrene microspheres with liver cells: roles of membrane receptors and serum proteins

Our previous studies have demonstrated that serum would play an important role in the hepatic disposition of polystyrene microspheres (MS) and that complement C3 should be involved as the serum opsonin. In this study, we tried to identify the entity of other serum opsonins and dysopsonin for the hepatic uptake of MSs with particle sizes of 50 nm (MS-50) and 500 nm (MS-500) by isolated liver perfusion studies using a recirculation procedure in rats. Pretreatment of the liver by trypsin significantly suppressed the serum-dependent hepatic uptake of both MSs, suggesting that some protein components on the cell surface should be necessary for the serum-dependent phagocytosis of MSs. Pretreatment of the serum by the anti-fibronectin antibody resulted in a significant reduction in the hepatic disposition of MS-500 (49% of control), suggesting that fibronectin should also work as the opsonin for the hepatic uptake of MS-500. The hepatic disposition of both MSs in the presence of serum was inhibited by the addition of N-acetylgalactosamine into the perfusate, suggesting the possible involvement of lectin in the serum-dependent hepatic uptake of MSs. Furthermore, a more intensive hepatic disposition of MSs was observed in the presence of plasma compared with that in the presence of serum in the perfusate, suggesting the possible involvement of blood coagulation factors, such as fibrinogen, as the opsonin in the hepatic disposition of MSs.     

Modulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by cell cycle inhibitors

The effects of cell cycle inhibition on the expression of the multidrug resistance transporter P-glycoprotein (P-gp) as well as of the cyclin-dependent kinase (CDK) inhibitors p27(Kip1) and p21(WAF-1) were investigated in DU-145 prostate tumor spheroids. With increasing spheroid size the number of cells in the G0/G1 phase augmented, whereas the number of cells in the G2/M phase and the S phase of the cell cycle declined. The number of G0/G1 cells was elevated after incubation with either mimosine, staurosporine or serum-free medium. Mitomycin C and roscovitine increased the number of S phase cells. Roscovitine additionally increased cells in the G2/M phase. Incubation in serum-free medium upregulated p21(WAF-1), p27(Kip1) and P-gp. Mimosine treatment resulted in upregulation of p27(Kip1) and P-gp, whereas p21(WAF-1) remained unchanged. Upon roscovitine treatment p27(Kip1) and p21(WAF-1) were downregulated, whereas P-gp was unaltered. Mitomycin C treatment resulted in downregulation of p27(Kip1) and p21(WAF-1); no significant change in P-gp levels was observed. Staurosporine induced upregulation of p21(WAF-1) whereas p27(Kip1) remained unaltered. P-gp was downregulated upon staurosporine treatment, which was owing to an elevation of intracellular reactive oxygen species by this compound. It is concluded that upregulation of P-gp in G0/G1 phase cells requires coexpression of the CDK inhibitor p27(Kip1) but not the CDK inhibitor p21(WAF-1).     

Migration capacity of tumor cells treated with immune sera

The action of antitumour sera on the adhesive properties of the L-cells and the cells of Ehrlich's ascitic carcinoma. It was shown that after the treatment with immune sera the tumour cells lost their capacity to form rosettes with sheep red blood cells, to adhere to plastics and glass; when administered into the mouse organism these cells were worse retained in the lungs, spleen and the liver. The data obtained indicated that the antibodies could play a significant role in the tumour cell metastasis.     

Semiautomatic growth analysis of multicellular tumor spheroids

Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to analyze the size of complete MCTS populations in culture containers with transparent, flat bottoms. MCTS sediments are digitally scanned and spheroid volumes are calculated by computerized image analysis. The equipment includes regular office hardware (personal computer, flatbed scanner) and software (Adobe Photoshop, Microsoft Excel, ImageJ). The accuracy and precision of the method were tested using industrial precision steel beads with known diameter. In summary, in comparison with other methods, this approach provides benefits in terms of semiautomation, noninvasiveness, and low costs.     

Use of multicellular tumor spheroids to dissect endothelial cell-tumor cell interactions: a role for T-cadherin in tumor angiogenesis

This study addresses establishment of an "in vitro" melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.     

Regulation of the mouse mammary tumor virus receptor by phosphorylation and internalization in mammary epithelial cells

The mouse mammary tumor virus enters mammary epithelial cells via a plasma membrane protein that binds to a viral envelope glycoprotein, gp52. In intact cells, this gp52 receptor can be phosphorylated by activators of protein kinase A and protein kinase C (PKC), but this modification does not occur in response to epidermal growth factor, whose receptor is a tyrosine kinase, or to gp52. Phosphorylation of the gp52 receptor rapidly leads to internalization and gradual loss of binding activity. Both the phosphorylation and the intrnalization induced by PKC are abolished by prior downregulation of this kinase. Although the physiological function of the gp52 receptor is unknown, its binding to gp52 can stimulate several biological activities, including amino acid accumulation. Receptor processingimpairs this gp52-induced amino acid uptake, as well as viral infection, by depleting the binding protein at the cell surface. In contrast, PKC augments insulin-induced amino acid transport, and PKC downregulation abolishes the action of insulin, suggesting that insulin and gp52 utlize partially separate pathways leading to amino acid transport. These data further suggest that PKC may be involved in this insulin-stimulated activity. © 1994 Wiley-Liss, Inc.     

The appearance and internalization of transferrin receptors at the margins of spreading human tumor cells

Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin receptor, the distribution of transferrin receptors on the surfaces of human epidermoid carcinoma A431 cells has been mapped at high resolution. On prefixed cells and cells incubated at 5 degrees C, the receptors are predominantly within and around clathrin-coated microdomains near the free cell margin. By preincubating the cells with saturating concentrations of free antibody at 5 degrees C and warming them to 37 degrees C in the presence of the gold complexes, the appearance of new receptors in the membrane has been followed. The majority first appear near the free cell margin and then move centripetally. At first, they are monodisperse, but as they move toward the site of internalization they form loose aggregates. Within the immediate vicinity of the clathrin-coated microdomains the migrating receptors form closely packed, ordered aggregates. These observations indicate recycling transferrin receptors move to their site of internalization without cross-linking.     

Migration studies and histology of injectable microspheres of different sizes in mice

Injectable dermal filler materials consist of either fluids, biological fragments, or suspensions of particles or microspheres. Particles and microspheres are said to "migrate," but migration can occur only when they are injected into blood vessels. To evaluate biocompatibility and transport, five nonresorbable polymethylmethacrylate microspheres of various sizes, suspended in different carriers, as well as resorbable polylactic acid and dextran microspheres were injected subcutaneously into mice. The five implantation sites were the right cheek, right axilla, right groin, urethra, and the right quadriceps muscle of the thigh. These sites were excised along with the local lymph nodes, lungs, liver, and spleen at 1, 3, 6, and 9 months after injection. Polymethylmethacrylate microspheres of 4 microm and 8 microm were phagocytosed but not transported to lymph nodes or distant organs. Larger microspheres of 20, 40, and 100 microm were encapsulated by connective tissue, macrophages, and giant cells. Polylactic acid microspheres caused a mild inflammatory response and had disappeared at 6 months. Dextran microspheres caused a pronounced foreign-body reaction and were phagocytosed at 9 months. The extremely large carbon-coated spheres of 200 to 500 microm in diameter "migrated" up to 1 cm from the implantation site. With the exception of an erroneous intravenous injection, no migration or transportation of any of the injected microspheres to lymph nodes or filter organs was seen. Obviously, the collagen glue released no microspheres. After subdermal injection, the collagen carrier substance kept the microspheres apart as a scaffold for tissue ingrowth, whereas all other carrier substances, such as gelatin, hyaluronic acid, or alginate, separated soon after injection, thereby causing agglomeration of the microspheres.