Tumor Spheroid Culture Research Articles

Surface Acoustic Waves (SAW)-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures.

Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose–response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell culture models, which may have potential applications in both longitudinal 3D cell cultures in cancer biology and in regenerative medicine.

Abstract 4672: Development of 2D - cell strain and 3D - tumor spheroid models for Precision Medicine

Abstract Background Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patient's malignancy. Available cell line models are greatly limited in recapitulating the genetic profile of the tumor and tumor microenvironment of patients and therefore limit preclinical evaluation of new targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to use of two dimensional (2D) monolayer cultures as the initial screening method that frequently produces inaccurate results and does not predict chemoresistance. The ability of three dimensional (3D) cultured tumor cells derived directly from patients to recapitulate tumor-like microenvironment suggests that engineered tumor models may provide more accurate and reproducible information for drug testing. At the Institute for Precision Medicine, we grow patient-derived 2D cell strains and engineer 3D tumor spheroids for patient`s tumor specific drug testing. Design Fresh tissue samples from 30 different solid tumors were digested in Type II or IV Collagenase (Gibco) and then re-suspended in growth factor reduced Matrigel (BD), plated on plastic, and overlaid with cell type specific cell growth media tsCM, which consists of either serum free Advanced DMEM/F12 (Gibco) with multiple growth factors optimized to propagate benign primary tumor cells, or FBS enriched DMEM/F12 media. Cultures were maintained at 37°C in 5% CO2. Once established, early cell strain culture passage and tumor spheroids size < 500 μm were cryopreserved. Specific drug testing was performed following 3 models: 2D monolayer culture, 2D monolayer culture converted in a 3D system using the hanging drop system from Biomatrix 3D®, and engineered 3D tumor spheroids. Results Our success rate in generating 2D- cell strains and 3D tumor spheroids is about 30%-40%. In the initial 30 samples, we were able to establish 11 continuous 2D and 3D tumor spheroid cultures derived from distinct primary and metastatic cancers including prostate, kidney, bladder and endometrium, as well as sarcomas and a neuroendocrine tumor. Tumor content in biopsy represents a major factor for successful growth. Histology and cytology examination demonstrates that 3D tumor spheroids recapitulate harvested tumors. Next generation sequencing shows the presence of same genomic alterations compared with original tumor without generating additional mutations. Conclusion We have developed individual patient-derived 2D tumor cell strains and 3D engineered tumor spheroids as well as models for patient`s tumor specific drug testing. This effort has been implemented for Precision Care and Clinical Trials at our Institution. Citation Format: Chantal Pauli, Jonathan Pauwels, Myriam Kossai, Nikolai Steklov, Andrea Sboner, Rema Rao, Kenneth Hennrick, Brian Robinson, Juan Miguel Mosquera, Himisha Beltran, Mark A. Rubin. Development of 2D - cell strain and 3D - tumor spheroid models for Precision Medicine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4672. doi:10.1158/1538-7445.AM2015-4672

Alginate core-shell beads for simplified three-dimensional tumor spheroid culture and drug screening.

We demonstrate that when using cell-laden core-shell hydrogel beads to support the generation of tumor spheroids, the shell structure reduces the out-of-bead and monolayer cell proliferation that occurs during long-term culture of tumor cells within core-only alginate beads. We fabricate core-shell beads in a two-step process using simple, one-layer microfluidic devices. Tumor cells encapsulated within the bead core will proliferate to form multicellular aggregates which can serve as three-dimensional (3-D) models of tumors in drug screening. Encapsulation in an alginate shell increased the time that cells could be maintained in three-dimensional culture for MCF-7 breast cancer cells prior to out-of-bead proliferation, permitting formation of spheroids over a period of 14 days without the need move the cell-laden beads to clean culture flasks to separate beads from underlying monolayers. Tamoxifen and docetaxel dose response shows decreased toxicity for multicellular aggregates in three-dimensional core-shell bead culture compared to monolayer. Using simple core-only beads gives mixed monolayer and 3-D culture during drug screening, and alters the treatment result compared to the 3-D core-shell or the 2-D monolayer groups, as measured by standard proliferation assay. By preventing the out-of-bead proliferation and subsequent monolayer formation that is observed with core-only beads, the core-shell structure can obviate the requirement to transfer the beads to a new culture flask during drug screening, an important consideration for cell-based drug screening and drugs which have high multicellular resistance index.

Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment.

Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the "single file" pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12.

A novel three-dimensional microfluidic platform for on chip multicellular tumor spheroid formation and culture

The formation of three-dimensional (3D) multicellular cell spheroids such as microspheres and embryoid bodies has recently gained much attention as a useful cell culture technique, but few studies have investigated the suitability of glass for spheroids formation and culture. In this work, we present a novel three-dimensional microfluidic device made of poly(dimethylsiloxane) (PDMS) and glass for the easy and rapid synthesis and culture of tumor spheroid. The cell culture unit is composed of an array of microwells on the bottom of a glass plate, bigger microwells and elastomeric microchannels on the top of a PDMS plate. Cell suspension can be easily introduced into the cell culture unit and exchange with the external liquid environment by the microfluidic channels. A single tumor spheroid can be formed and cultured in each glass cell culture chamber, the surface of which was modified with poly(vinyl alcohol) to render it to be resistant to cell adhesion. As the cell culture medium could be replaced, spheroids of the human breast cancer (MCF-7) cells were cultured on the chip for 3 days, reaching the diameters of about 150 μm. Furthermore, the MCF-7 cells were successfully cultured on the chip in 2D and 3D culture modes. Results have shown that glass is well suitable for multicellular tumor spheroids culture. The established platform provides a convenient and rapid method for tumor spheroid culture, which is also adaptable for anticancer drug screening and fundamental biomedical research in cell biology.

Low-Cost Media Formulation for Culture of Brain Tumor Spheroids (Neurospheres)

Recent studies have found that the biological features of primary tumors are faithfully recapitulated when a patient's tumor is processed and then maintained as a 3-D spheroid in specialized cell culture media. However, a major drawback for maintenance and routine passage of primary tumors as spheroids has been the high cost of custom-formulated media compared to regular serum-supplemented media. Here we report the formulation of a cost-effective, serum-free medium in which high-grade primary brain tumor (glioblastoma) explants can be established and maintained as spheroids. Based on DMEM, this formulation requires only supplementation with several amino acids, vitamins, synthetic EGF, and bFGF, with most of the cost being associated with the growth factors. A simple addition of BSA (fraction V) obviated the need for numerous other components (or human serum) commonly used in the specialized commercial media formulations.

PDMS well platform for culturing millimeter‐size tumor spheroids

Multicellular tumor spheroids are widely used as in vitro models for testing of anticancer drugs. The advantage of this approach is that it can predict the outcome of a drug treatment on human cancer cells in their natural three-dimensional environment without putting actual patients at risk. Several methods were utilized in the past to grow submillimeter-size tumor spheroids. However, these small models are not very useful for preclinical studies of tumor ablation where the goal is the complete destruction of tumors that can reach several centimeters in diameter in the human body. Here, we propose a PDMS well method for large tumor spheroid culture. Our experiments with HepG2 hepatic cancer cells show that three-dimensional aggregates of tumor cells with a volume as large as 44 mm(3) can be grown in cylindrical PDMS wells after the initial culture of tumor cells by the hanging drop method. This is a 350 times more than the maximum volume of tumor spheroids formed inside hanging drops (0.125 mm(3)).

The Effect of Quercetin and Hyperthermia on spheroid model of DU145 Prostate Carcinoma Cell line

The purpose of this study is to investigate the effects of Quercetin as an inhibitor of heat shock proteins and hyperthermia on the induced DNA damages and colony formation ability of DU145 tumor spheroid culture. DU145 cells were cultured as spheroids. On day11, spheroids with mean diameter 100 μm were treated with different concentration of Quercetin for 24 hours and then exposed to hyperthermia at 43 o C for 1 hour. After heat exposure, the colony forming ability and the induced DNA damages were examined using colonogenic and alkaline comet assay methods, respectively. Our results showed that DMSO diluent in combination with hyperthermia had no significant effect on the number of colonies and the level of DNA damages as compared to control (p>0.05). Furthermore, number of colonies decreased and DNA damages increased by increasing Quercetin concentration in combined treatment of DU145 cells with Quercetin and hyperthermia in spheroid cultures. Quercetin as an inhibitor of heat shock protein 70 production in cells exposed to hyperthermia can increase DNA damages and decrease colony numbers of the prostate cancer cells in a dose-dependent manner and there is a correlation between the increase of DNA damages and decrease of colony numbers.

Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines In Vitro: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere®

While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of in vivo anti-tumor activity. In this work, a series of complementary assays was established for evaluating the in vitro efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the in vivo tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional in vitro and in vivo models for high-throughput assessment of therapeutic candidates.

Phenotypic Switch in Blood: Effects of Pro-Inflammatory Cytokines on Breast Cancer Cell Aggregation and Adhesion

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLex expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of ‘triple-negative’ breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments.

Cell behavior observation and gene expression analysis of melanoma associated with stromal fibroblasts in a three‐dimensional magnetic cell culture array

A three-dimensional (3D) multicellular tumor spheroid culture array has been fabricated using a magnetic force-based cell patterning method, analyzing the effect of stromal fibroblast on the invasive capacity of melanoma. Formation of spheroids was observed when array-like multicellular patterns of melanoma were developed using a pin-holder device made of magnetic soft iron and an external magnet, which enables the assembly of the magnetically labeled cells on the collagen gel-coated surface as array-like cell patterns. The interaction of fibroblast on the invasion of melanoma was investigated using three types of cell interaction models: (i) fibroblasts were magnetically labeled and patterned together in array with melanoma spheroids (direct-interaction model), (ii) fibroblasts coexisting in the upper collagen gel (indirect-interaction model) of melanoma spheroids, and (iii) fibroblast-sheets coexisting under melanoma spheroids (fibroblast-sheet model). The fibroblast-sheet model has largely increased the invasive capacity of melanoma, and the promotion of adhesion, migration, and invasion were also observed. In the fibroblast-sheet model, the expression of IL-8 and MMP-2 increased by 24-fold and 2-fold, respectively, in real time RT-PCR compared to the absence of fibroblasts. The results presented in this study demonstrate the importance of fibroblast interaction to invasive capacity of melanoma in the 3D in vitro bioengineered tumor microenvironment.

Development of Multicellular Tumor Spheroid (MCTS) Culture from Breast Cancer Cell and a High Throughput Screening Method Using the MTT Assay

In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures’ response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.

Development of a three-dimensional microfluidic system for long-term tumor spheroid culture

Abstract In this paper, we present a low-cost three-dimensional microfluidic system made of poly(dimethylsiloxane) for tumor spheroid formation and cultivation. Tumor spheroids reaching diameters of up to 300 μm were cultured in perfusion microchambers providing extremely low shear stress. Spheroids were cultured for 15 days and high viability of the cells was observed within this time. The presented microsystem is a prospective solution for development of microfluidic platforms for anticancer drug screening.

Abstract 4483: Establishing patient-derived colorectal cancer stem cell models with a PIK3CA mutation for the development of inhibitory drugs as targeted therapies

Abstract Emerging evidences suggest that cancer stem cells (CSC) may be critically responsible for tumor initiation, progression, metastasis, and drug resistance. It becomes important to ask whether anti-cancer agents are able to target the tumor-initiating subpopulation in relevant CSC models. In this study, we first established xenograft tumors in NOD/SCID mice from a colorectal cancer patient specimen and demonstrated that CD133/EpCAM-expressing CSC population was highly tumorigenic. We then sought to propagate the CSC population under a serum-free condition. In culture, the tumor cells formed non-adherent, 3-dimensional spheroids, a fraction of which retained expression of the CSC markers. When exposed to a serum-containing medium, tumor spheroid cells differentiated into epithelial-like adherent cells with an increase in cell proliferation rate. In comparison with the differentiated progeny, tumor spheroid cells exhibited resistance to the standard-of-care agent oxaliplatin and, in limiting dilution assays in mice, displayed substantially higher tumorigenic potential. In contrast to the tumors originated from the differentiated cells, tumor spheroid cell-derived tumors recapitulated not only the CSC frequency marked by CD133/EpCAM expression, but also the histological characters of the original tumor. Similarly, only were the fragments of spheroid cell-derived xenograft tumors capable of regenerating highly proliferative tumors in secondary transplantation. Thus, the tumor spheroid culture is indeed enriched of drug resistant, self-renewing, and tumor-initiating CSC populations. Mutation profiling of frequently mutated oncogenes using Sequenom OncoCarta™ panel identified a mutation in the kinase domain of PIK3CA (H1047R) in the cultured CSCs. This mutation has been reported present in a large number of colon cancer patients and likely functions as an oncogene (Samuels et al., Science 304:554; 2004). We further demonstrated that a dual mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitor (PF-04691502) exhibited a more potent effect on inhibition of in vitro proliferation of the mutated CSCs compared to the chemotoxic agent oxaliplatin. Collectively, our findings suggest that CSC models provide a novel avenue to drug sensitivity and efficacy studies. The well-characterized CSC model systems may assist in the development of more effective therapy against the subpopulation of tumors driven by the CSCs bearing specific mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4483.

Pioglitazone modulates tumor cell metabolism and proliferation in multicellular tumor spheroids

The anti-diabetic thiazolidinedione compound pioglitazone, a peroxisome proliferator-activated receptor-gamma agonist, and selective cyclooxygenase-2 inhibitors are clinically used in patients with advanced malignancies. Several previously published in vivo and in vitro studies showed growth inhibitory effects on different cancer cell lines. However, the underlying mechanisms are fairly unclear. Here, we analyzed the effects of pioglitazone in combination with other drugs in a three-dimensional multicellular tumor spheroid culture system (MCTS) generated from the two prostate carcinoma cell lines PC3 and LNCaP. As expected, pioglitazone also inhibited tumor cell proliferation in the MCTS system. Further studies revealed that pioglitazone lowered the pH of the culture medium, decreased oxygen consumption and increased lactate secretion in both tumor cell lines. Other glitazones, troglitazone and ciglitazone, had similar effects. The combination of pioglitazone with 2-deoxyglucose, a potent inhibitor of glycolysis, had an additive effect on the inhibition of cell proliferation and led to MCTS disintegration. Our data propose a new mechanism of growth inhibition by pioglitazone through modulation of the tumor cell metabolism.

Docetaxel suppresses invasiveness of head and neck cancer cells in vitro

The combination of docetaxel, cisplatin, and fluorouracil significantly enhances the survival of head and neck cancer patients compared to cisplatin and fluorouracil. We hypothesized that docetaxel may affect invasiveness of the head and neck cancer cells in addition to its tumor-killing effect. Two different head and neck cancer cell lines (HEp-2 and Ca9-22) were treated with docetaxel at IC(10) and IC(50) concentrations. Cell migration and invasive growth was evaluated by wound healing assay and three-dimensional (3D) culture of multicellular tumor spheroids, respectively. Expression levels of possible downstream effectors for cell migration/invasiveness were measured by immunoblotting in conditions with or without docetaxel. Docetaxel, but not cisplatin, suppressed filopodia formation compared with no treatment (control) condition. Consistent with this, docetaxel suppressed two-dimensional (2D) cell migration and 3D cell invasion compared with control or cisplatin. Only docetaxel treated cells exhibited thick tubulin bundle and had lower activity of Cdc42, a member of the Rho family of small GTPases. In conclusion, Docetaxel treatment suppressed migration and invasiveness of head and neck cancer cells in vitro, which is likely to be mediated by regulating Cdc42 activity.

Three-dimensional modeling of transport of nutrients for multicellular tumor spheroid culture in a microchannel

The growth dynamics of avascular tumors in a microchannel bioreactor is investigated. A three-dimensional flow and nutrient transport model, incorporating the multicellular tumor spheroid (MTS) growth model, has been developed to study the influence of nutrients (oxygen and glucose) supply and distribution on the MTS growth. Numerical simulations based on the EMT6/Ro tumor cells show that the continuous-flow perfusion is more efficient to deliver nutrients to the MTS than the diffusion-only static culture. It is further demonstrated that as long as there is bulk flow, the growth of a single tumor spheroid at the early stage is insensitive to the flow velocity and the channel size. For multiple tumor spheroids in the same microchannel, however, increasing the perfusion velocity can improve the nutrient environment for the disadvantageous downstream tumor spheroid. The flow shear stress exerting on the MTSs in the current microchannel bioreactor is estimated to be far below the critical value to affect the MTS growth, which means that there is still much room for increasing perfusion velocity to satisfy the higher nutrient requirement by the growing tumor spheroids.

Efficient infection and persistence of a herpesvirus saimiri-based gene delivery vector into human tumor xenografts and multicellular spheroid cultures

Herpesvirus saimiri (HVS) has the ability to infect a variety of human cell lines and establish a persistent infection by virtue of episomal maintenance. Moreover, the viral episome provides sustained expression of a heterologous transgene. HVS-based vectors can also persist for a long term in tumor xenografts generated from HVS-infected human carcinoma cell lines. The viral episome remains latent within the xenograft allowing long-term transgene expression. These properties, in addition to its ability to incorporate large amounts of heterologous DNA, make HVS an attractive potential gene delivery vector. Here we report on the further evaluation of such HVS-based vectors. We demonstrate for the first time that HVS can efficiently infect solid tumor xenografts derived from a variety of human carcinoma cells via direct intratumoral injections. Furthermore, HVS can efficiently infect spheroid cultures, a three-dimensional cell culture system that closely resembles a tumor. Upon infection of both the tumor xenografts and spheroid cultures, HVS-based vectors can establish a persistent episomal infection within the tumor xenograft allowing expression of a heterologous transgene. These results suggest that HVS-based vectors may be suitable for cancer gene therapy applications.

Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells.

Tumor vascularization is the rate-limiting step for the progression of cancer. Differential steps of tumor-induced angiogenesis were studied by a novel in vitro confrontation culture of avascular multicellular prostate tumor spheroids and embryoid bodies grown from pluripotent embryonic stem (ES) cells. Vascularization in embryoid bodies started on day 5 of cell culture and was paralleled by down-regulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). In parallel, a dissipation of gradients in the pericellular oxygen pressure was observed as measured by O(2)-sensitive microelectrodes. After 24--48 h of confrontation culture, cells positive for platelet endothelial cell adhesion molecule (PECAM-1) became visible in the contact region between the embryoid body and the tumor spheroid and sprouted within the confrontation cultures during subsequent days. Tumor-induced angiogenesis resulted in growth stimulation of tumor spheroids, disappearance of central necrosis and a reduction of the pericellular oxygen pressure. Furthermore, tumor vascularization resulted in elevated levels of HIF-1 alpha, VEGF, heat shock protein 27 (HSP27), and P-glycoprotein. Tumor-induced angiogenesis may augment the oxygen consumption in tumors resulting in an increased expression of hypoxia-related, proangiogenic genes as well as of HSP27 and P-glycoprotein, which are involved in a multidrug resistance phenotype.

Cytoreductive effects of anti-transferrin receptor immunotoxin in a multicellular tumor spheroid model.

We have evaluated the sensitivity to immunotoxins (IT) of monolayer and of 200-250 microns multicellular tumor spheroid (MTS) cultures obtained with human breast (MCF7) and glioblastoma (U118) tumor cells and with rat glioblastoma (9L) cells. Monolayer MCF7 and U118 cells were highly sensitive to antitransferrin receptor (anti-TfnR) ricin A chain (RTA)-IT (Tfn-RTA and MAb OKT9-RTA) treatment in the presence of the intracellular RTA-IT enhancing agent human serum albumin-monensin (HSA-Mo) conjugate. A 790- to 2000-fold higher concentration of anti-TfnR IT was instead required to reduce by 50% the volume of individually treated MCF7 spheroids, as evaluated by applying the Gompertz growth model. Monolayer 9L cells showed 230- to 5700-fold lower sensitivity to Tfn-RTA IT than MCF7 and U118 monolayers, yet 9L spheroid cells were almost as sensitive to anti-TfnR IT as monolayer 9L cultures. Binding studies performed with [125I]-Tfn and FITC-labelled anti-TfnR MAb revealed that 9L monolayers and MTS expressed 4.1-fold and 8.8-fold lower amounts of TfnR than MCF7 monolayers and MTS, respectively. However, Tfn bound to TfnR sites of 9L and of MCF7 cells with comparable affinity. Experiments carried out with the diphtheria toxin mutant CRM107 linked to Tfn confirmed the pattern observed with RTA-IT. Monolayers and spheroids showed no considerable differences in sensitivity to ricin toxin. Collectively, these results indicated that the efficacy of IT against 3-D tumors is heavily influenced by the number of target Ag expressed by the tumor cells, as well as by the affinity of IT/toxin-cell interaction.