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

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite ((Formula presented.)) into the less bioavailable elemental selenium (Se0) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of (Formula presented.) exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with (Formula presented.). The results show substantial biochemical changes in SeITE01 cells when exposed to (Formula presented.). The initial uptake of (Formula presented.) by SeITE01 cells (3h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against (Formula presented.) are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of (Formula presented.) to Se0. In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from (Formula presented.) toxicity while it is converting into SeNPs

po 126 survival probability of human breast carcinoma cells to radiation treatment role of cell fusion and of a syncytin1 homologous protein

Introduction The success of radiotherapy depends on the ability to inhibit tumour growth, and relapse after therapy is determined by cells that retain their clonogenic potential. The radiation sensitivity of isolated tumour cell clones in vitro is routinely determined with clonogenic assays. In solid tumours, however, clonogenic cells are not isolated and we carried out experiments to measure the influences of cell-cell contact on their proliferative potential. To this end we developed a new experimental approach to measure the effects of radiation on tumour cell populations. The observations can be understood with the help of a novel stochastic model with a well-defined biological basis. Material and methods T47D cells (human breast carcinoma) were grown at various concentrations in F(flat)-bottom and V-bottom wells of 96-well culture plates. The spheroid outgrowth method was also used to obtain densely-packed tissue cell cultures. A Gammacell40 irradiator equipped with a 137Cs source was used to treat cell cultures. Cell fusion was assessed by confocal microscopy. Syncytin 1 expression was assessed by RT-PCR and by flow cytometry using an anti-HERV antibody (clone ab7115, Abcam). Results and discussions The probability of cell survival after 8 Gy radiation treatment increased ~4.7 times when the cells were grown in V-bottom wells as compared to cells grown in F-bottom wells (p(survival)=0.0113 and 0.0024, respectively). Microscopic inspections of tissue-like cultures showed that after treatment cell populations were mostly composed of giant cells with multiple nuclei. Cytoplasmic bridges joining different cells were clearly visible. Giant cells and cytoplasmic bridges disappeared at later times (>600 hours) when the cells displayed normal morphology and started to proliferate again. Sequence analysis of cloned RT-PCR products showed that cells expressed a Syncytin 1 homologous protein (Sp). Flow cytometry assays confirmed cytoplasmic expression of Sp and revealed that Sp translocated to the cell surface of irradiated cells committed to death. The fraction of cells surviving 8 Gy treatment was significantly reduced in cultures treated with anti-Sp antibodies. Conclusion Our experimental findings indicate that recovery of breast tumours from radiation is very likely to involve complex pathways that act at the cell population level and that include events of cell fusion mediated by a protein homologous to Syncytin 1

Planar AFM macro-probes to study the biomechanical properties of large cells and 3D cell spheroids

The ability to measure mechanical response of cells under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Living cells have been mechanically investigated by several approaches. Among them, atomic force microscopy (AFM) is widely used thanks to its high versatility and sensitivity. In the case of large cells or 3D multicellular aggregates, standard AFM probes may not be appropriate to investigate the mechanical properties of the whole biological system. Owing to their size, standard AFM probes can compress only a single somatic cell or part of it. To fill this gap, we have designed and fabricated planar AFM macro-probes compatible with commercial AFM instruments. The probes are constituted of a large flat compression plate, connected to the chip by two flexible arms, whose mechanical characteristics are tuned for specific biological applications. As proof of concept, we have used the macro-probes to measure the viscoelasticity of large spherical biological systems, which have a diameter above 100 \u3bcm: human oocytes and 3D cell spheroids. Compression experiments are combined with visual inspection, using a side-view configuration imaging, which allows us to monitor the sample morphology during the compression and to correlate it with the viscoelastic parameters. Our measurements provide a quantitative estimate of the relaxation times of such biological systems, which are discussed in relation to data present in literature. The broad applicability of the AFM macro-probes can be relevant to study the biomechanical features in any biological process involving large soft materials. Statement of Significance: The understanding of the role of physical factors in defining cell and tissue functions requires to develop new methods or improve the existing ones to accurately measure the biomechanical properties. AFM is a sensitive and versatile tool to measure the mechanical features from single proteins to single cells. When cells or cell aggregates exceed few tens of microns, AFM suffers from limitations. On these biological systems the control of the contact area and the application of a precise uniform compression becomes crucial. A modification of the standard cantilevers fabrication allowed us obtaining AFM macro-probes, having large planar contact area and spring constant suitable for biological investigations. They were demonstrated valuable to characterize the mechanical properties of large hierarchical biological systems

A New Computational Tool for the Phenomenological Analysis of Multipassage Tumor Growth Curves

Multipassage experiments are performed by subcutaneous implantation in lab animals (usually mice) of a small number of cells from selected human lines. Tumor cells are then passaged from one mouse to another by harvesting them from a growing tumor and implanting them into other healthy animals. This procedure may be extremely useful to investigate the various mechanisms involved in the long term evolution of tumoral growth. It has been observed by several researchers that, contrary to what happens in in vitro experiments, there is a significant growth acceleration at each new passage. This result is explained by a new method of analysis, based on the Phenomenological Universalities approach. It is found that, by means of a simple rescaling of time, it is possible to collapse all the growth curves, corresponding to the successive passages, into a single curve, belonging to the Universality Class U2. Possible applications are proposed and the need of further experimental evidence is discussed

Cancer recurrence times from a branching process model

As cancer advances, cells often spread from the primary tumor to other parts of the body and form metastases. This is the main cause of cancer related mortality. Here we investigate a conceptually simple model of metastasis formation where metastatic lesions are initiated at a rate which depends on the size of the primary tumor. The evolution of each metastasis is described as an independent branching process. We assume that the primary tumor is resected at a given size and study the earliest time at which any metastasis reaches a minimal detectable size. The parameters of our model are estimated independently for breast, colorectal, headneck, lung and prostate cancers. We use these estimates to compare predictions from our model with values reported in clinical literature. For some cancer types, we find a remarkably wide range of resection sizes such that metastases are very likely to be present, but none of them are detectable. Our model predicts that only very early resections can prevent recurrence, and that small delays in the time of surgery can significantly increase the recurrence probability.Comment: 26 pages, 9 figures, 4 table

Anti-cancer drug validation: the contribution of tissue engineered models

Abstract Drug toxicity frequently goes concealed until clinical trials stage, which is the most challenging, dangerous and expensive stage of drug development. Both the cultures of cancer cells in traditional 2D assays and animal studies have limitations that cannot ever be unraveled by improvements in drug-testing protocols. A new generation of bioengineered tumors is now emerging in response to these limitations, with potential to transform drug screening by providing predictive models of tumors within their tissue context, for studies of drug safety and efficacy. Considering the NCI60, a panel of 60 cancer cell lines representative of 9 different cancer types: leukemia, lung, colorectal, central nervous system (CNS), melanoma, ovarian, renal, prostate and breast, we propose to review current Bstate of art^ on the 9 cancer types specifically addressing the 3D tissue models that have been developed and used in drug discovery processes as an alternative to complement their studyThis article is a result of the project FROnTHERA (NORTE-01-0145-FEDER-000023), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This article was also supported by the EU Framework Programme for Research and Innovation HORIZON 2020 (H2020) under grant agreement n° 668983 — FoReCaST. FCT distinction attributed to Joaquim M. Oliveira (IF/00423/2012) and Vitor M. Correlo (IF/01214/2014) under the Investigator FCT program is also greatly acknowledged.info:eu-repo/semantics/publishedVersio

Fuori dalla pura politica. Laboratori globali della soggettivit\ue0

The essay discusses some important contributions to the critical debates surround- ing the issues of political subjectivity and subjectivation (A. Badiou, J. Rancie\u300re, Ch. Mouffe, E. Laclau, E\u301. Balibar). According to the authors the main limit of these con- tributions lies in the idea of a \uabpure politics\ubb (S. Z\u30ciz\u30cek), constructed upon the model of the modern State. This is clear both in theories of political subjectivity focused on the absolute present of an event and in theories that stress the importance of political and institutional articulation. Against the background of an analysis of current political developments in Latin America, the authors outline a different theory of political sub- jectivation, attempting to take stock of the crisis and transformations both of the State and of contemporary capitalism