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1. Electron microscopy imaging and mechanical characterization of T47D multicellular tumor spheroids–Older spheroids reduce interstitial space and become stiffer

   https://doi.org/10.1371/journal.pone.0286291  

   Madhavan, Mathangi ; Jaiswal, Devina ; Karlberg, Sarah ; Duggan, Alexis ; Almarshad, Hassan A. ; Claffey, Kevin P. ; Hoshino, Kazunori ( May 2023 , PLOS ONE)

   Weihs, Daphne (Ed.)

   Multicellular cancer spheroids are an in vitro tissue model that mimics the three-dimensional microenvironment. As spheroids grow, they develop the gradients of oxygen, nutrients, and catabolites, affecting crucial tumor characteristics such as proliferation and treatment responses. The measurement of spheroid stiffness provides a quantitative measure to evaluate such structural changes over time. In this report, we measured the stiffness of size-matched day 5 and day 20 tumor spheroids using a custom-built microscale force sensor and conducted transmission electron microscopy (TEM) imaging to compare the internal structures. We found that older spheroids reduce interstitial spaces in the core region and became significantly stiffer. The measured elastic moduli were 260±100 and 680±150 Pa, for day 5 and day 20 spheroids, respectively. The day 20 spheroids showed an optically dark region in the center. Analyzing the high-resolution TEM images of spheroid middle sections across the diameter showed that the cells in the inner region of the day 20 spheroids are significantly larger and more closely packed than those in the outer regions. On the other hand, the day 5 spheroids did not show a significant difference between the inner and outer regions. The observed reduction of the interstitial space may be one factor that contributes to stiffer older spheroids. 

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2. Fluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids

   https://doi.org/10.1038/s41598-023-30403-7  

   Karrobi, Kavon ; Tank, Anup ; Fuzail, Mohammad Ahsan ; Kalidoss, Madhumathi ; Tilbury, Karissa ; Zaman, Muhammad ; Ferruzzi, Jacopo ; Roblyer, Darren ( March 2023 , Scientific Reports)

   Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylation (OXPHOS) while the spheroid core had changes consistent with a shift towards glycolysis. The MDA-MB-231 spheroids had a large shift consistent with increased OXPHOS with a more pronounced change at the higher collagen concentration. The MDA-MB-231 spheroids invaded into the collagen gel over time and cells that traveled the farthest had the largest changes consistent with a shift towards OXPHOS. Overall, these results suggest that the cells in contact with the extracellular matrix (ECM) and those that migrated the farthest had changes consistent with a metabolic shift towards OXPHOS. More generally, these results demonstrate the ability of multiphoton FLIM to characterize how spheroids metabolism and spatial metabolic gradients are modified by physical properties of the 3D ECM.

    

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3. Optical coherence tomography detects necrotic regions and volumetrically quantifies multicellular tumor spheroids

   Huang, Yongyang ; Wang, Shunqiang ; Guo, Qiongyu ; Kessel, Sarah ; Rubinoff, Ian ; Chan, Leo Li-Ying ; Li, Peter ; Liu, Yaling ; Qiu, Jean ; Zhou, Chao ( October 2017 , Cancer research)

   Three-dimensional (3D) tumor spheroid models have gained increased recognition as important tools in cancer research and anti-cancer drug development. However, currently available imaging approaches employed in high-throughput screening drug discovery platforms e.g. bright field, phase contrast, and fluorescence microscopies, are unable to resolve 3D structures deep inside (>50 μm) tumor spheroids. In this study, we established a label-free, non-invasive optical coherence tomography (OCT) imaging platform to characterize 3D morphological and physiological information of multicellular tumor spheroids (MCTS) growing from ~250 μm up to ~600 μm in height over 21 days. In particular, tumor spheroids of two cell lines glioblastoma (U-87 MG) and colorectal carcinoma (HCT 116) exhibited distinctive evolutions in their geometric shapes at late growth stages. Volumes of MCTS were accurately quantified using a voxel-based approach without presumptions of their geometries. In contrast, conventional diameter-based volume calculations assuming perfect spherical shape resulted in large quantification errors. Furthermore, we successfully detected necrotic regions within these tumor spheroids based on increased intrinsic optical attenuation, suggesting a promising alternative of label-free viability tests in tumor spheroids. Therefore, OCT can serve as a promising imaging modality to characterize morphological and physiological features of MCTS, showing great potential for high-throughput drug screening. 

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4. Probing Notch1-Dll4 signaling in regulating osteogenic differentiation of human mesenchymal stem cells using single cell nanobiosensor

   https://doi.org/10.1038/s41598-022-14437-x  

   Zhao, Yuwen ; Yang, Rui ; Bousraou, Zoe ; Richardson, Kiarra ; Wang, Shue ( June 2022 , Scientific Reports)

   Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

    

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5. The shape of (7) Iris as evidence of an ancient large impact?

   https://doi.org/10.1051/0004-6361/201834541  

   Hanuš, J. ; Marsset, M. ; Vernazza, P. ; Viikinkoski, M. ; Drouard, A. ; Brož, M. ; Carry, B. ; Fetick, R. ; Marchis, F. ; Jorda, L. ; et al ( April 2019 , Astronomy & Astrophysics)

   null (Ed.)

   Context. Asteroid (7) Iris is an ideal target for disk-resolved imaging owing to its brightness ( V ~ 7–8) and large angular size of 0.33′′ during its apparitions. Iris is believed to belong to the category of large unfragmented asteroids that avoided internal differentiation, implying that its current shape and topography may record the first few 100 Myr of the solar system’s collisional evolution. Aims. We recovered information about the shape and surface topography of Iris from disk-resolved VLT/SPHERE/ZIMPOL images acquired in the frame of our ESO large program. Methods. We used the All-Data Asteroid Modeling ( ADAM ) shape reconstruction algorithm to model the 3D shape of Iris, using optical disk-integrated data and disk-resolved images from SPHERE and earlier AO systems as inputs. We analyzed the SPHERE images and our model to infer the asteroid’s global shape and the morphology of its main craters. Results. We present the 3D shape, volume-equivalent diameter D eq = 214 ± 5 km, and bulk density ρ = 2.7 ± 0.3 g cm −3 of Iris. Its shape appears to be consistent with that of an oblate spheroid with a large equatorial excavation. We identified eight putative surface features 20–40 km in diameter detected at several epochs, which we interpret as impact craters, and several additional crater candidates. Craters on Iris have depth-to-diameter ratios that are similar to those of analogous 10 km craters on Vesta. Conclusions. The bulk density of Iris is consistent with that of its meteoritic analog based on spectroscopic observations, namely LL ordinary chondrites. Considering the absence of a collisional family related to Iris and the number of large craters on its surface, we suggest that its equatorial depression may be the remnant of an ancient (at least 3 Gyr) impact. Iris’s shape further opens the possibility that large planetesimals formed as almost perfect oblate spheroids. Finally, we attribute the difference in crater morphology between Iris and Vesta to their different surface gravities, and the absence of a substantial impact-induced regolith on Iris. 

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