Roberto Chignola

Forecasting the growth of multicell tumour spheroids: implications for the dynamic growth of solid tumours.

The growth dynamics of multicell tumour spheroids (MTS) were analysed by means of mathematical techniques derived from signal processing theory. Volume vs. time trajectories of individual spheroids were fitted with the Gompertz growth equation and the residuals (i.e. experimental volume determinations minus calculated values by fitting) were analysed by fast fourier transform and power spectrum. Residuals were not randomly distributed around calculated growth trajectories demonstrating that the Gompertz model partially approximates the growth kinetics of three‐dimensional tumour cell aggregates. Power spectra decreased with increasing frequency following a 1/fδ power‐law. Our findings suggest the existence of a source of ‘internal’ variability driving the time‐evolution of MTS growth. Based on these observations, a new stochastic Gompertzian‐like mathematical model was developed which allowed us to forecast the growth of MTS. In this model, white noise is additively superimposed to the trend described by the Gompertz growth equation and integrated to mimic the observed intrinsic variability of MTS growth. A correlation was found between the intensity of the added noise and the particular upper limit of volume size reached by each spheroid within two MTS populations obtained with two different cell lines. The dynamic forces generating the growth variability of three‐dimensional tumour cell aggregates also determine the fate of spheroid growth with a strong predictive significance. These findings suggest a new approach to measure tumour growth potential.

Effects of wheat germ agglutinin on human gastrointestinal epithelium: Insights from an experimental model of immune/epithelial cell interaction

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only approximately 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.

Oscillating growth patterns of multicellular tumour spheroids

Abstract. The growth kinetics of 9L (rat glioblastoma cell line) and U118 (human glioblastoma cell line) multicellular tumour spheroids (MTS) have been investigated by non‐linear least square fitting of individual growth curves with the Gompertz growth equation and power spectrum analysis of residuals. Residuals were not randomly distributed around calculated growth trajectories. At least one main frequency was found for all analysed MTS growth curves, demonstrating the existence of time‐dependent periodic fluctuations of MTS volume dimensions. Similar periodic oscillations of MTS volume dimensions were also observed for MTS generated using cloned 9L cells. However, we found significant differences in the growth kinetics of MTS obtained with polyclonal cells. Our findings demonstrate that the growth patterns of three‐dimensional tumour cell cultures are more complex than has been previously predicted using traditional continuous growth models.

Emergent properties of tumor microenvironment in a real-life model of multicell tumor spheroids.

Multicellular tumor spheroids are an important 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.

Temperature-dependent decay of wheat germ agglutinin activity and its implications for food processing and analysis

Abstract A recently-described immunoenzymatic (ELISA) method for the quantitative determination of biologically-active wheat germ agglutinin (WGA) in unknown samples has been applied to measure the concentration of active WGA in raw and cooked wheat-derived foodstuffs. The method exploits the binding specificity of WGA to ovalbumin as a first step followed by identification of bound lectin with polyclonal antibodies. Purified WGA was used to obtain calibration curves. Detectable amounts of WGA were found in raw foodstuffs and wheat flours, whilst variable amounts of agglutinin were found in wholemeal pasta probably as a consequence of thermal inactivation during food processing. The thermal gradient gel electrophoresis (TGGE) technique was therefore applied to analyse the thermal stability of WGA. The biological activity of WGA decreased as a function of heating temperatures and time of exposure to thermal treatment in an S-shaped fashion with an inflection point around 65 °C. As a consequence, WGA might represent a biochemical “indicator” allowing one to determine the thermal treatment undergone by wheat-derived foods during processing.

Analysis of CIITA encoding AIR-1 gene promoters in insulin-dependent diabetes mellitus and rheumatoid arthritis patients from the Northeast of Italy: Absence of sequence variability

Qualitative and/or quantitative alterations in the expression of the MHC class II molecules affect the onset and maintenance of the immune response and may be the basis of a wide variety of disease states, such as autoimmunity and immunodeficiency.CIITA is a major physiological regulator of the expression of MHC class II genes. The availability of CIITA ap- pears generally essential for MHC class II gene expression, and hence its own transcriptional regulatory mechanisms result of fundamental importance for a correct homeostasis of the immune response. Therefore, it is possible to hypothesize that variability at the CIITA-encoding locus, AIR-1, could constitute an additional source of susceptible traits to autoimmune diseases. Mutations at AIR-1/CIITA promoters could modulate expression of CIITA. Variations in CIITA expression could influence the qualitative and quantitative expression of MHC class II molecules at cell surface. We have analyzed sequence variation at AIR-1/CIITA promoters by PCR-SSCP in 23 IDDM and 30 RA patients compared to a sample of 19 unaffected normal controls and 16 unaffected IDDM family members, for a total of 88 Caucasian subjects from the Northeast of Italy. No sequence difference was found at the four AIR-1/CIITA promoters between autoimmune patients and normal controls. Moreover, the promoters resulted invariant within the entire group of 88 subjects analyzed, comprising patients and controls. This finding suggests a possible selective advantage in maintaining CIITA upstream regulatory sequences invariant.

Self-potentiation of ligand-toxin conjugates containing ricin A chain fused with viral structures.

A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell-free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10-20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90-240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins.

Estimating the growth kinetics of experimental tumors from as few as two determinations of tumor size: implications for clinical oncology

Clinical information on tumor growth is often limited to a few determinations of the size of the tumor burden taken at variable time. As a consequence, fitting of growth equations to clinical data is hampered by the small number of available data. On the other hand, characterising the tumor growth kinetics in terms of clinically relevant parameters, such as the doubling time of the tumors, is increasingly required to optimize and personalise treatments. A computational method is presented which can estimate the growth kinetics of tumors from as few as two determinations of its size taken at two successive time points, provided the size at which tumor growth saturates is known. The method is studied by using experimental data obtained in vitro with multicell tumor spheroids and in vivo with tumors grown in mice, and its outputs are compared to those obtained by fitting of experimental data with the Gompertz growth equation. Under certain assumptions and limitations the method provides comparable estimates of the doubling time of tumors with respect to the classical nonlinear fitting approach. The method is then tested against simulated tumor growth trajectories spanning the range of tumor sizes observed in the clinics. The simulations show that a relative classification of tumors on the basis of their growth kinetics can be obtained even if the size at which tumor growth saturates is not known. This result opens the possibility to classify patients bearing fast or slow growing tumors and, hence, to adapt therapeutic regimens under a more rationale basis.

Ab initio phenomenological simulation of the growth of large tumor cell populations

In a previous paper we have introduced a phenomenological model of cell metabolism and of the cell cycle to simulate the behavior of large tumor cell populations (Chignola and Milotti 2005 Phys. Biol. 2 8). Here we describe a refined and extended version of the model that includes some of the complex interactions between cells and their surrounding environment. The present version takes into consideration several additional energy-consuming biochemical pathways such as protein and DNA synthesis, the tuning of extracellular pH and of the cell membrane potential. The control of the cell cycle, which was previously modeled by means of ad hoc thresholds, has been directly addressed here by considering checkpoints from proteins that act as targets for phosphorylation on multiple sites. As simulated cells grow, they can now modify the chemical composition of the surrounding environment which in turn acts as a feedback mechanism to tune cell metabolism and hence cell proliferation: in this way we obtain growth curves that match quite well those observed in vitro with human leukemia cell lines. The model is strongly constrained and returns results that can be directly compared with actual experiments, because it uses parameter values in narrow ranges estimated from experimental data, and in perspective we hope to utilize it to develop in silico studies of the growth of very large tumor cell populations (10(6) cells or more) and to support experimental research. In particular, the program is used here to make predictions on the behavior of cells grown in a glucose-poor medium: these predictions are confirmed by experimental observation.

Active soybean lectin in foods: quantitative determination by ELISA using immobilised asialofetuin

A recently described immunoenzymatic method for the quantitative determination of biologically active lectins in unknown samples has been adapted to measure the concentration of active soybean lectin (SBA) in foodstuffs. The method was developed by using purified SBA to build up reference standard curves and to determine the specificity and the sensitivity of the assay. Detectable amounts of soybean lectin were found in soy-based edible products such as hamburger, milk and sprouts and they have been compared to those determined in standard hemagglutination tests. Particular attention has been given to SBA quantitation in soy sprouts as a function of germination time. SBA was not degraded during germination although the hamagglutination activity of sprout extracts rapidly decreased. SBA expression was modulated during the first 10 days of germination, a time period spanning that used to produce soy-sprouts for alimentary purposes.