Silvia Sartoris

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.

IFN-γ-mediated upmodulation of MHC class I expression activates tumor-specific immune response in a mouse model of prostate cancer

De novo expression of B7-1 impaired tumorigenicity of TRAMP-C2 mouse prostate adenocarcinoma (TRAMP-C2/B7), but it did not elicit a protective response against TRAMP-C2 parental tumor, unless after in vitro treatment with IFN-gamma. TRAMP-C2 cells secrete TGF-beta and show low MHC-I expression. Treatment with IFN-gamma increased MHC-I expression by induction of some APM components and antagonizing the immunosuppressant activity of TGF-beta. Thus, immunization with TRAMP-C2/B7 conferred protection against TRAMP-C2-derived tumors in function of the IFN-gamma-mediated fine-tuned modulation of either APM expression or TGF-beta signaling. To explore possible clinical translation, we delivered IFN-gamma to TRAMP-C2 tumor site by means of genetically engineered MSCs secreting IFN-gamma.

The emerging immunological role of post-translational modifications by reactive nitrogen species in cancer microenvironment.

Under many inflammatory contexts, such as tumor progression, systemic and peripheral immune response is tailored by reactive nitrogen species (RNS)-dependent post-translational modifications, suggesting a biological function for these chemical alterations. RNS modify both soluble factors and receptors essential to induce and maintain a tumor-specific immune response, creating a “chemical barrier” that impairs effector T cell infiltration and functionality in tumor microenvironment and supports the escape phase of cancer. RNS generation during tumor growth mainly depends on nitric oxide production by both tumor cells and tumor-infiltrating myeloid cells that constitutively activate essential metabolic pathways of l-arginine catabolism. This review provides an overview of the potential immunological and biological role of RNS-induced modifications and addresses new approaches targeting RNS either in search of novel biomarkers or to improve anti-cancer treatment.

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.

MHC: orchestrating the immune response

The major histocompatibility complex (MHC) is one of the most interesting and intriguing genetic systems, and continues to amaze geneticists, biochemists and particularly, immunologists and pathologists. The latest achievements on the structure and function of MHC genes and their products were discussed at a recent workshop.*

Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis

Expression of HLA and CD1b molecules was investigated in the THP‐1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2 – 3 days, cell surface expression of HLA class II and CD1b was drastically down‐modulated, whereas HLA class I expression was up‐modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down‐modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down‐modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN‐γ, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP‐1 cells to process and present mycobacterial antigens to HLA‐DR‐restricted antigen‐specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen‐presenting molecules.

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.

Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

Transcriptional regulation of MHC class II genes

SummaryMHC class II molecules play a fundamental role in the homeostasis of the immune response, functioning as receptors for antigenic peptides to be presented to regulatory T cells. Both quantitative and qualitative alterations in the expression of these molecules on the cell surface dramatically affect the onset of the immune response, and may be the basis of a wide variety of disease states, such as autoimmunity, immunodeficiencies, and cancer. Most regulation of MHC class II molecule expression is under the control of transcription mechanisms which are both cell type and development specific. In the last few years classical genetics together with molecular biology have greatly contributed to the widening of our knowledge on the regulatory mechanisms operating in the control of class II gene expression. This review deals with the lates developments in this fundamental area of immunology.

Efficacy Assessment of Interferon-Alpha–Engineered Mesenchymal Stromal Cells in a Mouse Plasmacytoma Model

Bone marrow mesenchymal stromal cells (BM-MSCs) may survive and proliferate in the presence of cycling neoplastic cells. Exogenously administered MSCs are actively incorporated in the tumor as stromal fibroblasts, thus competing with the local mesenchymal cell precursors. For this reason, MSCs have been suggested as a suitable carrier for gene therapy strategies, as they can be genetically engineered with genes encoding for biologically active molecules that can inhibit tumor cell proliferation and enhance the antitumor immune response. We used BM-MSCs engineered with the murine interferon-alpha (IFN-α) gene (BM-MSCs/IFN-α) to assess in a mouse plasmacytoma model the efficacy of this approach toward neoplastic plasma cells. We found that IFN-α can be efficiently produced and delivered inside the tumor microenvironment. Subcutaneous multiple administration of BM-MSCs/IFN-α significantly hampered the tumor growth in vivo and prolonged the overall survival of mice. The antitumor effect was associated with enhanced apoptosis of tumor cells, reduction in microvessel density, and ischemic necrosis. By contrast, intravenous administration of BM-MSCs/IFN-α did not significantly modify the survival of mice, mainly as a consequence of an excessive entrapment of injected cells in the pulmonary vessels. In conclusion, BM-MSCs/IFN-α are effective in inhibiting neoplastic plasma cell growth; however, systemic administration of engineered MSCs needs to be improved to make this approach potentially suitable for the treatment of multiple myeloma.