No abstract

Three-dimensional (3D) tissue cultures are replacing conventional two-dimensional (2D) cultures for applications in cancer drug development. However, direct comparisons of in vitro 3D models relative to in vivo models derived from the same cell lines have not been reported because of the lack of sensitive optical probes that can extract high-content information from deep inside living tissue. Here we report the use of biodynamic imaging (BDI) to measure response to platinum in 3D living tissue. BDI combines low-coherence digital holography with intracellular Doppler spectroscopy to study tumor drug response. Human ovarian cancer cell lines were grown either in vitro as 3D multicellular monoculture spheroids or as xenografts in nude mice. Fragments of xenografts grown in vivo in nude mice from a platinum-sensitive human ovarian cell line showed rapid and dramatic signatures of induced cell death when exposed to platinum ex vivo, while the corresponding 3D multicellular spheroids grown in vitro showed negligible response. The differences in drug response between in vivo and in vitro growth have important implications for predicting chemotherapeutic response using tumor biopsies from patients or patient-derived xenografts.

Mammalian target of rapamycin (mTOR) is a protein that regulates cell growth in response to altered nutrient and growth factor availability. Our objective was to assess activated mTOR and its intracellular intermediates p70, and 4EBP1 in placental and invasive trophoblast cells in a hypoxia‐induced model of intrauterine growth restriction (IUGR) in rats. Rats were treated with hypoxia (9%) for 4 days. Placental and fetal weights, as well as conceptus numbers were recorded at the time of necropsy. Immunohistochemistry was used to determine the level of trophoblast invasion and apoptosis. Western blots were used to determine the activation of mTOR, p70, and 4EBP1 in the placenta and the uterine mesometrial compartment. We observed (1) decreased placental (21%) and fetal (24%) weights (P < 0.05); (2) decreased trophoblast invasion; (3) significantly increased active 4EBP1 (28%; P < 0.05) in invasive trophoblast cells yet no changes in the activation of mTOR and p70 proteins; and (4) a significant decrease in the activation of mTOR (48%; P < 0.05) with no differences in p70 or 4EBP1 activation in the placenta. We conclude that the development of IUGR is correlated with decreased activation of the mTOR protein in the placenta and increased 4EBP1 activity in the invading trophoblast. These results provide important insight into the physiological relevance of these pathways. Furthermore, modification of these and other related targets during gestation may alleviate IUGR severity.

Abstract. Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a highcontent 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications.

Digital holography provides improved capabilities for imaging through dense tissue. Using a short-coherence source, the digital hologram recorded from backscattered light performs laser ranging that maintains fidelity of information acquired from depths much greater than possible by traditional imaging techniques. Biodynamic Imaging (BDI) is a developing technology for live-tissue imaging of up to a millimeter in depth that uses the hologram intensity fluctuations as label-free image contrast and can study tissue behavior in native microenvironments. In this paper BDI is used investigate the change in adhesion-dependent tissue response in 3D cultures. The results show that increasing density of cellular adhesions slows motion inside tissue and alters the response to cytoskeletal drugs. A clear signature of membrane fluctuations was observed in mid frequencies (0.1 – 1 Hz) that was enhanced by the application of cytochalasin-D that degrades the actin cortex inside the cell membrane. This enhancement feature is only observed in tissues that have formed adhesions, because cell pellets initially do not show this signature, but develop this signature only after incubation enables adhesions to form.