Most cancer biologists still rely on conventional two-dimensional (2D) monolayer culture techniques to test in vitro anti-tumor drugs prior to in vivo testing. However, the vast majority of promising preclinical drugs have no or weak efficacy in real patients with tumors, thereby delaying the discovery of successful therapeutics. This is because 2D culture lacks cell–cell contacts and natural tumor microenvironment, important in tumor signaling and drug response, thereby resulting in a reduced malignant phenotype compared to the real tumor. In this sense, three-dimensional (3D) cultures of cancer cells that better recapitulate in vivo cell environments emerged as scientifically accurate and low cost cancer models for preclinical screening and testing of new drug candidates before moving to expensive and time-consuming animal models. Here, we provide a comprehensive overview of 3D tumor systems and highlight the strategies for spheroid construction and evaluation tools of targeted therapies, focusing on their applicability in cancer research. Examples of the applicability of 3D culture for the evaluation of the therapeutic efficacy of nanomedicines are discussed.

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments.

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.

We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element–induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants.We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.

Exposure to hypoxia and subsequent development of pulmonary hypertension is associated with an impairment of the nitric oxide (NO) mediated response to endothelium-dependent vasodilators. Inhaled NO may reach resistive pulmonary vessels through an abluminal route. The aim of this study was to investigate if continuous inhalation of NO would attenuate the development of pulmonary hypertension in rats exposed to chronic hypoxia.In conscious rats previously exposed to 10% 02 for 3 wk, short-term inhalation of NO caused a dose-dependent decrease in pulmonary artery pressure (PAP) from 44±1 to 32±1 mmHg at 40 ppm with no changes in systemic arterial pressure, cardiac output, or heart rate. In normoxic rats, acute NO inhalation did not cause changes in PAP. In rats simultaneously exposed to 10% 02 and 10 ppm NO during 2 wk, right ventricular hypertrophy was less severe (P < 0.01), and the degree of muscularization of pulmonary vessels at both alveolar duct and alveolar wall levels was lower (P < 0.01) than in rats exposed to hypoxia alone. Tolerance to the pulmonary vasodilator effect of NO did not develop after prolonged inhalation. Brief discontinuation of NO after 2 wk of hypoxia plus NO caused a rapid increase in PAP. These data demonstrate that prolonged inhalation of low concentrations of NO induces sustained pulmonary vasodilation and reduces pulmonary vascular remodeling in response to chronic hypoxia. (J. Clin. Invest. 1994. 94:578-584.)