BackgroundThere is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed.ResultsTo address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease.ConclusionsWe have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.

A dosage formula has been derived from a retrospective analysis of carboplatin pharmacokinetics in 18 patients with pretreatment glomerular filtration rates (GFR) in the range of 33 to 136 mL/min. Carboplatin plasma clearance was linearly related to GFR (r = 0.85, P less than .00001) and rearrangements of the equation describing the correlation gave the dosage formula dose (mg) = target area under the free carboplatin plasma concentration versus time curve (AUC) x (1.2 x GFR + 20). In a prospective clinical and pharmacokinetic study the formula was used to determine the dose required to treat 31 patients (GFR range, 33 to 135 mL/min) with 40 courses of carboplatin. The target AUC was escalated from 3 to 8 mg carboplatin/mL/min. Over this AUC range the formula accurately predicted the observed AUC (observed/predicted ratio 1.24 +/- 0.11, r = 0.886) and using these additional data, the formula was refined. Dose (mg) = target AUC x (GFR + 25) is now the recommended formula. AUC values of 4 to 6 and 6 to 8 mg/mL. min gave rise to manageable hematological toxicity in previously treated and untreated patients, respectively, and hence target AUC values of 5 and 7 mg/mL min are recommended for single-agent carboplatin in these patient groups. Pharmacokinetic modeling demonstrated that the formula was reasonably accurate regardless of whether a one- or two-compartment model most accurately described carboplatin pharmacokinetics, assuming that body size did not influence nonrenal clearance. The validity of this assumption was demonstrated in 13 patients where no correlation between surface area and nonrenal clearance was found (r = .31, P = .30). Therefore, the formula provides a simple and consistent method of determining carboplatin dose in adults. Since the measure of carboplatin exposure in the formula is AUC, and not toxicity, it will not be influenced by previous or concurrent myelosuppressive therapy or supportive measures. The formula is therefore applicable to combination and high-dose studies as well as conventional single-agent therapy, although the target AUC for carboplatin will need to be redefined for combination chemotherapy.

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K d = 1.7 nmol/L) and proliferation of human tumor cells with GI 50 values of approximately 2 to 40 nmol/L, inducing G 1 -G 2 arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI 50 . This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1A, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro , WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials. [Cancer Res 2008;68(8):2850-60]