Emergent Properties of Tumor Microenvironment in a Real-life Model of Multicell Tumor Spheroids

Multicellular tumor spheroids are an important {\it in vitro} model of the pre-vascular phase of solid tumors, for sizes well below the diagnostic limit: therefore a biophysical model of spheroids has the ability to shed light on the internal workings and organization of tumors at a critical phase of their development. To this end, we have developed a computer program that integrates the behavior of individual cells and their interactions with other cells and the surrounding environment. It is based on a quantitative description of metabolism, growth, proliferation and death of single tumor cells, and on equations that model biochemical and mechanical cell-cell and cell-environment interactions. The program reproduces existing experimental data on spheroids, and yields unique views of their microenvironment. Simulations show complex internal flows and motions of nutrients, metabolites and cells, that are otherwise unobservable with current experimental techniques, and give novel clues on tumor development and strong hints for future therapies.Comment: 20 pages, 10 figures. Accepted for publication in PLOS One. The published version contains links to a supplementary text and three video file

384 hanging drop arrays give excellent Z ‐factors and allow versatile formation of co‐culture spheroids

We previously reported the development of a simple, user‐friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti‐cancer drug sensitivity testing. The 384 hanging drop array plate allows for high‐throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell‐based high‐throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence‐ and colorimetric‐based assays through Z ‐factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. Biotechnol. Bioeng. 2012; 109:1293–1304. © 2011 Wiley Periodicals, Inc. This paper characterizes the robustness of the high‐throughput 384 hanging drop array spheroid formation and culture plate in terms of assay performance. The versatility of the platform was further demonstrated through 3D patterning of multiple cell types into concentric layers and as Janus spheroids. The system is envisioned to deliver valuable insights into 3D cellular behavior as well as more accurate readouts from 3D cell‐based high‐throughput screening and testing.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90601/1/24399_ftp.pd

Can macroalgae provide promising anti-tumoral compounds? A closer look at Cystoseira tamariscifolia as a source for antioxidant and anti-hepatocarcinoma compounds

Marine organisms are a prolific source of drug leads in a variety of therapeutic areas. In the last few years, biomedical, pharmaceutical and nutraceutical industries have shown growing interest in novel compounds from marine organisms, including macroalgae. Cystoseira is a genus of Phaeophyceae (Fucales) macroalgae known to contain bioactive compounds. Organic extracts (hexane, diethyl ether, ethyl acetate and methanol extracts) from three Cystoseira species (C. humilis, C. tamariscifolia and C. usneoides) were evaluated for their total phenolic content, radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, and antiproliferative activity against a human hepatocarcinoma cell line (HepG2 cells). C. tamariscifolia had the highest TPC and RSA. The hexane extract of C. tamariscifolia (CTH) had the highest cytotoxic activity (IC50 = 2.31 mu g/mL), and was further tested in four human tumor (cervical adenocarcinoma HeLa; gastric adenocarcinoma AGS; colorectal adenocarcinoma HCT-15; neuroblastoma SH-SY5Y), and two non-tumor (murine bone marrow stroma S17 and human umbilical vein endothelial HUVEC) cell lines in order to determine its selectivity. CTH strongly reduced viability of all tumor cell lines, especially of HepG2 cells. Cytotoxicity was particularly selective for the latter cells with a selectivity index = 12.6 as compared to non-tumor cells. Incubation with CTH led to a 2-fold decrease of HepG2 cell proliferation as shown by the bromodeoxyuridine (BrdU) incorporation assay. CTH-treated HepG2 cells presented also pro-apoptotic features, such as increased Annexin Wpropidium iodide (PI) binding and dose-dependent morphological alterations in DAPI-stained cells. Moreover, it had a noticeable disaggregating effect on 3D multicellular tumor spheroids. Deme boxy cystoketal chromane, a derivative of the meroditerpenoid cystoketal, was identified as the active compound in CTH and was shown to display selective in vitro cYtotoxicitY towards HepG2 cells

Three-dimensional constructs using hyaluronan cell carrier as a tool for the study of cancer stem cells

Background: Cancer research focuses increasingly on cancer stem cell study as those cells are thought to be the root of chemo and radioresistance of the most aggressive cancer types. Nevertheless, two-dimensional (2D) cell culture and even three-dimensional (3D) spheroid models, with their limited ability to reflect cell-extracellular matrix interactions, are not ideal for the study of cancer stem cells (CSCs). In this study, we establish a 3D in vitro cancer model using a synthetic and natural scaffold with tunable features and show that U87 cells cultured in this system acquire a stem-cell like phenotype. Methods: U87 astrocytoma cells were grown on polycaprolactone (PCL)-2D flat substrates (2D) and PCL-3D scaffolds (3D) eventually containing hyaluronic acid (3D-HA). Cell viability, growth patterns, morphology, and cell surface marker expression (CD44, RHAMM and CD133) were studied to assess the effect of 3D culture and presence of HA. Results: 3D scaffold, but most prominently presence of HA induced changes in cell morphology and marker expression; 3D-HA cultures showed features of aggregates; moreover, markedly increased expression of Nestin, CD44, RHAMM, and CD133 in 3D-HA scaffolds were found. Conclusions: the behavior of U87 in our 3D-HA model is more similar to tumor growth in vivo and a stem-like phenotype is promoted. Thus, the 3D-HA scaffold could provide a useful model for CSCs study and anti-cancer therapeutics research in vitro and may have preclinical application for the screening of drug candidates. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1249-1257, 2015.Contract grant sponsors: National R&D&i Plan (2008-2011; CIBER-BBN), Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and Instituto de Salud Carlos III (European Regional Development Fund)Martínez Ramos, C.; Lebourg, M. (2015). Three-dimensional constructs using hyaluronan cell carrier as a tool for the study of cancer stem cells. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 103(6):1249-1257. https://doi.org/10.1002/jbm.b.33304S12491257103

Predicting protein–ligand binding affinity and correcting crystal structures with quantum mechanical calculations: lactate dehydrogenase A

Accurately computing the geometry and energy of host–guest and protein–ligand interactions requires a physically accurate description of the forces in action. Quantum mechanics can provide this accuracy but the calculations can require a prohibitive quantity of computational resources. The size of the calculations can be reduced by including only the atoms of the receptor that are in close proximity to the ligand. We show that when combined with log P values for the ligand (which can be computed easily) this approach can significantly improve the agreement between computed and measured binding energies. When the approach is applied to lactate dehydrogenase A, it can make quantitative predictions about conformational, tautomeric and protonation state preferences as well as stereoselectivity and even identifies potential errors in structures deposited in the Protein Data Bank for this enzyme. By broadening the evidence base for these structures from only the diffraction data, more chemically realistic structures can be proposed

Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers

Objective: Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer. Experimental design: Lactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA. Results: Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells. Conclusions: Loss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia

Study of the chemotactic response of multicellular spheroids in a microfluidic device

YesWe report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it.This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBERBBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Progress in melanoma modeling in vitro

Melanoma is one of the most studied neoplasia, although laboratory techniques used for investigating this tumor are not fully reliable. Animal models may not predict the human response due to differences in skin physiology and immunity. In addition, international guidelines recommend to develop processes that contribute to the reduction, refinement and replacement of animals for experiments (3Rs). Adherent cell culture has been widely used for the study of melanoma to obtain important information regarding melanoma biology. Nonetheless, these cells grow in adhesion on the culture substrate which differs considerably from the situation in vivo. Melanoma grows in a 3D spatial conformation where cells are subjected to a heterogeneous exposure to oxygen and nutrient. In addition, cell-cell and cell-matrix interaction play a crucial role in the pathobiology of the tumor as well as in the response to therapeutic agents. To better study melanoma new techniques, including spherical models, tumorospheres, and melanoma skin equivalents have been developed. These 3D models allow to study tumors in a microenvironment that is more close to the in vivo situation, and are less expensive and time consuming than animal studies. This review will also describe the new technologies applied to skin reconstructs such as organ-on-a-chip that allows skin perfusion through microfluidic platforms. 3D in vitro models, based on the new technologies, are becoming more sophisticated, representing at a great extent the in vivo situation, the "perfect" model that will allow less involvement of animals up to their complete replacement, is still far from being achieved. This article is protected by copyright. All rights reserved

Lactate::fueling the fire starter

It is becoming increasingly appreciated that intermediates of metabolic pathways, besides their anabolic and catabolic functions, can act as signaling molecules and influence the outcome of immune responses. Although lactate was previously considered as a waste product of glucose metabolism, accumulating evidence has highlighted its pivotal role in regulating diverse biological processes, including immune cell polarization, differentiation and effector functions. In addition, lactate is a key player in modulating tumor immune surveillance. Hence, targeting lactate-induced signaling pathways is a promising tool to reduce inflammation, to prevent autoimmunity and to restore anti-tumor immune response. This article is characterized under: Biological Mechanisms > Metabolism