Three-dimensional spatial configuration of tumour cells confers resistance to chemotherapy independent of drug delivery.

Anticancer drug discovery has been hampered by the lack of reliable preclinical models, which routinely use cells grown in two-dimensional (2D) culture systems. However, many of the characteristics of cells in 2D culture do not translate into the findings in animal xenografts. Three-dimensional (3D) growth may be responsible for some of these changes, and models using cells grown in 3D may form a more representative step in tumouricidal validation prior to animal implantation and human testing. For the 3D model, we cultured 143.98.2, SaOS2 or U2OS osteosarcoma cells seeded in porous Bombyx mori silk sponges. We conducted real-time PCR on cells grown in 2D culture and 3D scaffolds for the proliferation markers cyclin B1 and E2F1 and the actin regulator RhoA, and found a significant decrease in expression levels for the 3D tumour models (p = 0.02, < 0.001 and 0.008 for cyclin B1, E2F1 and RhoA for 143.98.2; p = 0.02, 0.002 and 0.02 for cyclin B1, E2F1 and RhoA for U2OS, respectively). In contrast, p21 was upregulated when SaOS2 and U2OS were cultured in the 3D scaffolds (p < 0.001) and there was no increase in DNA quantity during the culture period. We correspondingly observed G1 arrest when cell cycle analysis was conducted. Cytotoxicity results for cells treated with serial dilutions of doxorubicin and cisplatin showed that cells in 3D scaffolds were less sensitive to drug treatment than in 2D culture, and the difference was more pronounced for cell cycle specific agents. Copyright © 2013 John Wiley & Sons, Ltd.

The dominant role of IL-8 as an angiogenic driver in a three-dimensional physiological tumor construct for drug testing.

The induction of angiogenesis and the promotion of tumor growth and invasiveness are processes critical to metastasis, and are dependent on reciprocal interactions between tumor cells and their microenvironment. The formation of a clinically relevant tumor requires support from the surrounding stroma, and it is hypothesized that three-dimensional (3D) tumor coculture models offer a microenvironment that more closely resembles the physiological tumor microenvironment. In this study, we investigated the effects of tissue-engineered 3D architecture and tumor-stroma interaction on the angiogenic factor secretion profiles of U2OS osteosarcoma cells by coculturing the tumor cells with immortalized fibroblasts or human umbilical vein endothelial cells (HUVECs). We also carried out Transwell migration assays for U2OS cells grown in monoculture or fibroblast coculture systems to study the physiological effect of upregulated angiogenic factors on endothelial cell migration. Anti-IL-8 and anti-vascular endothelial growth factor (VEGF)-A therapies were tested out on these models to investigate the role of 3D culture and the coculture of tumor cells with immortalized fibroblasts on the efficacy of antiangiogenic treatments. The coculture of U2OS cells with immortalized fibroblasts led to the upregulation of IL-8 and VEGF-A, especially in 3D culture. Conversely, coculture with endothelial cells resulted in the downregulation of VEGF-A for cells seeded in 3D scaffolds. The migration of HUVECs through the Transwell polycarbonate inserts increased for the 3D and immortalized fibroblast coculture models, and the targeted inhibition of IL-8 greatly reduced HUVEC migration despite the presence of VEGF-A. A similar effect was not observed when anti-VEGF-A neutralizing antibody was used instead, suggesting that IL-8 plays a more critical role in endothelial cell migration than VEGF-A, with significant implications on the clinical utility of antiangiogenic therapy targeting VEGF-A.

Interstitial fluid pressure as an alternate regulator of angiogenesis independent of hypoxia driven HIF-1α in solid tumors.

We previously showed that interstitial fluid pressure (IFP) may be an alternate regulator of angiogenesis in solid tumors. Given the accepted link between hypoxia-induced factor and angiogenesis this study investigated the effect of IFP on hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) in human osteosarcoma xenografts in SCID mice and in different hypoxic environments. Tumors were grown either at heterotopic (flank) or orthotopic (medullary canal of the proximal tibia) sites in the host animal. Microfluidic probes determined pH, O(2)-saturation, IFP, and peripheral blood flow perfusion continuously. We assessed tumor growth in the orthotopic site (n = 15) by softex radiographs weekly, 3D microCT, histological evaluation, and for molecular responses. An increased cytoplasmic immunohistostaining of cells for HIF-1α (p = 0.03) and VEGF-A (p = 0.004) on the outer periphery was noted compared to the tumor center, with VEGFR2 uniformly stained throughout. This paralleled a raised state of interstitial hypertension (p = 0.007) in the tumor center relative to the peripheral surface but was inconsistent with a state of hypoxia (p = 0.03) in the tumor center. In vitro culture of human osteosarcoma cell lines (HOS, U2OS) and a human osteoblast control at 0- and 20-mmHg of hydrostatic pressure revealed suppression of HIF-1α (p = 0.02) and VEGF-A (p = 0.02) gene expression when IFP was raised, while the effect on VEGFR1 was equivocal. This study proposes an alternative regulatory angiogenic pathway via the influence of IFP on cancer cell function. The identification of a mechanistic cellular link to the physical parameter becomes an important tool to evaluate cancer cell growth within solid tumors.

Three-dimensional porous silk tumor constructs in the approximation of in vivo osteosarcoma physiology.

The lack of good preclinical models has hampered anticancer drug discovery. Standard preclinical protocols require the growth of cells in high throughput two-dimensional (2D) culture systems. However, such in vitro drug testing methods yield drug efficacy results that differ greatly from animal models. Conversely, it is much more difficult and expensive to use animal models for large-scale molecular biology research. It is conceivable that three-dimensional (3D) growth may be responsible for some of these changes. Porous silk sponges were fabricated through freeze drying and seeded with 143.98.2 osteosarcoma cells. Molecular profiles were obtained by carrying out real-time polymerase chain reaction for angiogenic growth factors and proliferation markers for osteosarcoma cells grown under 2D, 3D, and SCID mouse xenograft conditions. The angiogenic factor expression profiles for cells grown in 2D differed greatly from the 3D silk scaffold model (P < 0.05 for bFGF, HIF-1α, IL-8, and VEGF-A), whereas 3D tumor model profiles were found to be able to approximate that for the in vivo tumor better with no statistically different expression of HIF-1α and VEGF-A between the two. Immunohistochemistry staining for HIF-1α, VEGF-A, and VEGF receptor on osteosarcoma cells grown on the scaffolds validated the results obtained with the gene expression profiles. The results suggest that 3D tumor models could be used to bridge the gap between in vitro and in vivo tumor studies, and aid in the study of mechanisms activated during tumorigenesis for the development of novel targeted chemotherapy.