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Cancer cells are continuously challenged by a limited oxygen, nutrients supply, and elevated protein synthesis. All these circumstances induce ER stress and Unfolded Protein Response (UPR). A huge amount of evidence demonstrates that the UPR is a crucial process for cancer cells to keep malignancy but also that, prolonged pharmacological induction of UPR, can be effective for promoting cancer cell death. Here we show that inhibition of the Hexosamine Biosynthetic Pathway (HBP) by means of a specific HBP inhibitor, FR054, causes a prolonged activation of the UPR that induces, in breast cancer (BC) cells, ROS accumulation and cell death. Conversely, such a ROS-dependent mechanism is somewhat inhibited in pancreatic cancer (PC) cells. However parallel inhibition of xCT/SLC7A11, involved in glutathione biosynthesis, by erastin (ERA), a recognized ferroptosis inducer, significantly enhanced the FR054 effect causing a noteworthy increase in cancer cell proliferation arrest and death.

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable clinical challenge due to its aggressiveness and resistance to treatment. Notably, PDAC exhibits a pronounced resistance to various therapeutic modalities, often attributed to a wide metabolic reprogramming involving also the Hexosamine Biosynthetic Pathway (HBP). Transcriptional and bioinformatics analyses, performed under inhibition of HBP in PDAC cell models, indicated that activation of ER stress and Unfolded Protein Response (UPR), due to HBP inhibition, activates an NRF2-dependent antioxidative response, characterized by activation of several genes involved in glutathione (GSH) metabolism. Indeed, inhibition of both HBP and the xCT protein, the cystine/glutamate antiporter involved in GSH synthesis, in PDAC cells significantly reduced cell proliferation, enhanced cell death, altered cellular redox status, heightened sensitivity to oxidative stress, increased dependence on glutamine metabolism, and induced ferroptosis. These findings underscore the potential of HBP inhibition to synergize with ERA, offering a promising therapeutic strategy for managing PDAC.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP). Methods: Transcriptional and bioinformatics analysis were performed to obtain information about the effect of the HBP inhibition in two cell models of PDAC. Cell count, western blot, HPLC and metabolomics analyses were used to determine the impact of the combined treatment between an HBP’s Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival. Results: Here we show that the combined treatment applied to different PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death. Conclusion: Our study discloses that HBP inhibition enhances, via UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.

Chitosan is a marine-origin polysaccharide obtained from the deacetylation of chitin, the main component of crustaceans’ exoskeleton, and the second most abundant in nature. Although this biopolymer has received limited attention for several decades right after its discovery, since the new millennium chitosan has emerged owing to its physicochemical, structural and biological properties, multifunctionalities and applications in several sectors. This review aims at providing an overview of chitosan properties, chemical functionalization, and the innovative biomaterials obtained thereof. Firstly, the chemical functionalization of chitosan backbone in the amino and hydroxyl groups will be addressed. Then, the review will focus on the bottom-up strategies to process a wide array of chitosan-based biomaterials. In particular, the preparation of chitosan-based hydrogels, organic–inorganic hybrids, layer-by-layer assemblies, (bio)inks and their use in the biomedical field will be covered aiming to elucidate and inspire the community to keep on exploring the unique features and properties imparted by chitosan to develop advanced biomedical devices. Given the wide body of literature that has appeared in past years, this review is far from being exhaustive. Selected works in the last 10 years will be considered.

Bioprinting is a challenging and dynamic field of tissue engineering that rapidly progresses in the latest 20 years. Firstly, the bioprinting idea has been strictly connected to the need of fabricating organ substitutes with minimal biological functionality for organ transplant’s purpose. In the latest years, thanks to the technological advances in biomaterial fields, bioprinting promoted the development of tissue constructs with biological functionality and architectural support. Current bioprinted models face the challenge to recapitulate the highly defined hierarchical organization of the cancer tissues. A gamechanger in bioprinting application is the possibility to recreate the tumor microenvironment in vitro, copycatting the multicellular organization of the cancer tissue, extracellular matrix properties, and tumor-metastatic architecture. In this chapter, an overview of 3D bioprinting technology applied to the most aggressive and complex cancer is proposed as a tool to fabricate viable constructs by depositing living cells with tunable biomaterials. The focus is on the platforms that are used to dissect the cell’s interactions in cancer and how these platforms are useful for the routine testing of drug compounds.

Early cancer detection is still a major clinical challenge. The development of innovative and noninvasive screening approaches for the detection of predictive biomarkers indicating the stage of the disease could save many lives. Traditional in vitro and in vivo models are not adequate to copycat the native tumor microenvironment and for the discovery of new biomarkers. Recent advances in microfluidics, biosensors, and 3D cell biology speed up the development of micro-physiological bioengineered systems that improve the discovery of new potential cancer biomarkers. This can accelerate the individualization of cancer treatments leading to precision medicine-oriented approaches that could improve patient prognosis. For this reason, it is necessary to develop point-of-care diagnostic tools that can be user-friendly, miniaturized, and easily translated into clinical practice. This chapter describes how far this new generation of cutting-edge technologies, such as microfluidics, label-free detection systems, and molecular diagnostics, are from being applied in the current clinical practice.

Since the formalization of the Central Dogma of molecular biology, the relevance of RNA in modulating the flow of information from DNA to proteins has been clear. More recently, the discovery of a vast set of non-coding transcripts involved in crucial aspects of cellular biology has renewed the enthusiasm of the RNA community. Moreover, the remarkable impact of RNA therapies in facing the COVID19 pandemics has bolstered interest in the translational opportunities provided by this incredible molecule. For all these reasons, the Italian Society of Biophysics and Molecular Biology (SIBBM) decided to dedicate its 17th yearly meeting, held in June 2022 in Rome, to the many fascinating aspects of RNA biology. More than thirty national and intemational speakers covered the properties, modes of action and applications of RNA, from its role in the control of development and cell differentiation to its involvement in disease. Here, we summarize the scientific content of the conference, highlighting the take-home message of each presentation, and we stress the directions the community is currently exploring to push forward our comprehension of the RNA World 3.0.

Adoptive cell therapy in solid tumors, such as melanoma, is impaired, but little is known about the role that the fibroblasts present in the tumor microenvironment could exert. However, the mechanism at play is not well understood, partly due to the lack of relevant pre-clinical models. Three-dimensional culture and microfluidic chips are used to recapitulate the dynamic interactions among different types of cells in the tumor microenvironment in controlled and physiological settings. In this brief report, we propose a reductionist melanoma-on-a-chip model for evaluating the essential role of fibroblasts in the antitumor activity of lymphocytes. To this end, 3D melanoma spheroids were monocultured and co-cultured with human dermal fibroblasts and the NK-92 cell migration towards the tumor compartment was tested in a commercially available microfluidic device. Utilizing confocal microscopy, we observed the different recruitment of NK-92 cells in the presence and absence of fibroblasts. Our results show that fibroblasts’ presence inhibits immune effector recruiting by exploiting a 3D pre-clinical tumor model.

Chemotherapeutic resistance is a major problem in effective cancer treatment. Cancer cells engage various cells or mechanisms to resist anti-cancer therapeutics, which results in metastasis and the recurrence of disease. Considering the cellular heterogeneity of cancer stroma, the involvement of stem cells is reported to affect the proliferation and metastasis of osteosarcoma. Hence, the duo (osteosarcoma: Saos 2 and human adipose-derived stem cells: ASCs) is co-cultured in present study to investigate the therapeutic response using a nonadherent, concave surface. Staining with a cell tracker allows real-time microscopic monitoring of the cell arrangement within the sphere. Cell–cell interaction is investigated by means of E-cadherin expression. Comparatively high expression of E-cadherin and compact organization is observed in heterotypic tumorspheres (Saos 2–ASCs) compared to homotypic ones (ASCs), limiting the infiltration of chemotherapeutic compound doxorubicin into the heterotypic tumorsphere, which in turn protects cells from the toxic effect of the chemotherapeutic. In addition, genes known to be associated with drug resistance, such as SOX2, OCT4, and CD44 are overexpressed in heterotypic tumorspheres post-chemotherapy, indicating that the duo collectively repels the effect of doxorubicin. The interaction between ASCs and Saos 2 in the present study points toward the growing oncological risk of using ASC-based regenerative therapy in cancer patients and warrants further investigation.

Cancer is a multifaceted pathology, where cellular and acellular players interact to drive cancer progression and, in the worst-case, metastasis. The current methods to investigate the heterogeneous nature of cancer are inadequate, since they rely on 2D cell cultures and animal models. The cell line-based drug efficacy and toxicity assays are not able to predict the tumor response to anti-cancer agents and it is already widely discussed how molecular pathway are not recapitulated in vitro so called flat biology. On the other side, animal models often fail to detect the side-effects of drugs, mimic the metastatic progression or the interaction between cancer and immune system, due to biologic difference in human and animals. Moreover, ethical and regulatory issues limit animal experimentation. Every year pharma/biotech companies lose resources in drug discovery and testing processes that are successful only in 5% of the cases. There is an urgent need to validate accurate and predictive platforms in order to enhance drug-testing process taking into account the physiopathology of the tumor microenvironment. Three dimensional in vitro tumor models could enhance drug manufactures in developing effective drugs for cancer diseases. The 3D in vitro cancer models can improve the predictability of toxicity and drug sensitivity in cancer. Despite the demonstrated advantages of 3D in vitro disease systems when compared to 2D culture and animal models, they still do not reach the standardization required for preclinical trials. This review highlights in vitro models that may be used as preclinical models, accelerating the drug development process towards more precise and personalized standard of care for cancer patients. We describe the state-of-the art of 3D in vitro culture systems, with a focus on how these different approaches could be coupled in order to achieve a compromise between standardization and reliability in recapitulating tumor microenvironment and drug response.

Mineralization is a natural process leading to the formation of mineralized tissue such as bone. The chief mineral component of bone is hydroxyapatite (HAp), which is deposited using an organic template like fibrillar Collagen I under physiological condition. Fibrous silk fibroin is structurally homologous to collagen and acts as nucleation site for HAp mineralization when immersed in simulated body fluid (SBF) or fetal bovine serum (FBS), therefore, considered as popular bone regeneration biomaterial. Hence, the mineralization behavior of silk fibroin self-assembled gellan gum enriched 3D hydrogels is investigated under conditions closer to physiological ones using SBF as well as FBS, and also in presence of cells (e.g. human adipose tissue-derived stem cells, ASCs). Incorporation of silk fibroin induces the mineralization in acellular spongy-like hydrogels in composition dependent manner, confirmed by SEM-EDS analysis. In contrast, ASCs mediated mineralization is found in all hydrogel compositions of 3 weeks post-culture under osteogenic conditions as demonstrated by gene expression profile and Alizarin Red S staining. This is perhaps due to the co-existence of fibroin and FBS together induce cell-mediated mineralization. The blending of fibroin offers cheap alternative strategy to improve or guide the repair of mineralized tissue using gellan gum-based biomaterials.

Inducing thermal gradients in two injected fluid systems results in the temporal formation of mixing conductive streams. If preserved through sol-gel transition, this mechanism can be used to drive and pattern non-living and living entities in mixed hydrogels. Interfaces are vital in nature, where gradients of non-living and living entities build distinct yet continuous integrated living tissues. However, the common tissue fabrication methodologies often result in dissimilar interfaces, lacking continuity through the interfaced engineered tissues. Thus, there is an urgent need for the fabrication of heterotypic but continuous engineered tissues with spatial control over biomimetic features. Here, we demonstrate the influence of gel injection temperature on the patterning of gradients of non-living and living entities. The experimental part was confirmed by numerical modelling, showing the formation of convective lines which spatially drive microscale microparticle and cells when different temperatures are applied in the sequential injection of two gels. Based on this finding, pure gellan gum (GG) and blended GG with methacrylated gelatin (GelMA) systems were used to program the formation of gradient features in hydrogels, such as microparticle and cells distribution patterns, polymeric bioactivity, degradation, controlled release, and stiffness. The correlation between gel injection temperature and gradients formation can be applied to tissue interface modelling, regeneration, drug release systems, and broader materials engineering fields.

The lack of robust and low-cost sorbent materials still represents a formidable technological barrier for long-term storage of (renewable) thermal energy and more generally for Adsorptive Heat Transformations—AHT. In this work, we introduce a novel approach for synthesizing cement-based composite sorbent materials. In fact, considering the number of available hygrosopic salts that can be accommodated into a cementitious matrix—whose morphological properties can be also fine-tuned—the new proposed in situ synthesis paves the way to the generation of an entire new class of possible sorbents for AHT. Here, solely focusing on magnesium sulfate in a class G cement matrix, we show preliminary morphological, mechanical and calorimetric characterization of sub-optimal material samples. Our analysis enables us to theoretically estimate one of the most important figures of merit for the considered applications, namely the energy density which was found to range within 0.088–0.2 GJ/m3 (for the best tested sample) under reasonable operating conditions for space heating applications and temperate climate. The above estimates are found to be lower than other composite materials in the literature. Nonetheless, although no special material optimization has been implemented, our samples already compare favourably with most of the known materials in terms of specific cost of stored energy. Finally, an interesting aspect is found in the ageing tests under water sorption-desorption cycling, where a negligible variation in the adsorption capability is demonstrated after over one-hundred cycles.

Osteosarcoma is one of the most common metastatic bone cancers, which results in significant morbidity and mortality. Unfolding of effectual therapeutic strategies against osteosarcoma is impeded because of the absence of adequate animal models, which can truly recapitulate disease biology of humans. Tissue engineering provides an opportunity to develop physiologically relevant, reproducible, and tunable in vitro platforms to investigate the interactions of osteosarcoma cells with its microenvironment. Adipose-derived stem cells (ASCs) are detected adjacent to osteosarcoma masses and are considered to have protumor effects. Hence, the present study focuses on investigating the role of reactive ASCs in formation of spheroids of osteosarcoma cells (Saos 2) within a three-dimensional (3D) niche, which is created using gellan gum (GG)-silk fibroin. By modifying the blending ratio of GG-silk, the optimum stiffness of the resultant hydrogels such as GG and GG75: S25 is obtained for cancer spheroid formation. This work indicates that the co-existence of cancer and stem cells can form a spheroid, the hallmark of cancer, only in particular microenvironment stiffness. The incorporation of fibrillar silk fibroin within the hydrophilic network of GG in GG75: S25 spongy-like hydrogels closely mimics the stiffness of commercially established cancer biomaterials (e.g., Matrigel, HyStem). The GG75: S25 hydrogel maintains the metabolically active construct for a longer time with elevated expression of osteopontin, osteocalcin, RUNX 2, and bone sialoprotein genes, the biomarkers of osteosarcoma, compared to GG. The GG75: S25 construct also exhibits intense alkaline phosphatase expression in immunohistochemistry compared to GG, indicating itspotentiality to serve as biomimetic niche to model osteosarcoma. Taken together, the GG-silk fibroin-blended spongy-like hydrogel is envisioned as an alternative low-cost platform for 3D cancer modeling.

The generation of invasive fluctuating protrusions is a distinctive feature of tumor dissemination. During the invasion, individual cancer cells modulate the morphodynamics of protrusions to optimize their migration efficiency. However, it remains unclear how protrusion fluctuations govern the invasion of more complex multi-cellular structures, such as tumors, and their correlation with the tumor metastatic potential. Herein, a reductionist approach based on 3D tumor cell micro-spheroids with different invasion capabilities is used as a model to decipher the role of tumor-associated fluctuating protrusions in cancer progression. To quantify fluctuations, a set of key biophysical parameters that precisely correlate with the invasive potential of tumors is defined. It is shown that different pharmacological drugs and cytokines are capable of modulating protrusion activity, significantly altering protrusion fluctuations, and tumor invasiveness. This correlation is used to define a novel quantitative invasion index encoding the key biophysical parameters of fluctuations and the relative levels of cell-cell/matrix interactions, which is capable of assessing the tumor's metastatic capability solely based on its magnitude. Overall, this study provides new insights into how protrusion fluctuations regulate tumor cell invasion, suggesting that they may be employed as a novel early indicator, or biophysical signature, of the metastatic potential of tumors.

Flat cell cultures or xenografts are inadequate tools to unravel cancer complex biology. 3D in vitro tumor models garnered interest since they recapitulate better dynamic mechanisms of cancer, but a gold standard model that faithfully mimics solid cancer is not available yet. 3D breast cancer model is fabricated using freeze-dried silk fibroin scaffolds. Breast cancer cell lines (MCF-7 and MDA-MB231) are seeded with normal mammary fibroblasts onto silk fibroin scaffold (1 and 2 mm thick). Cells proliferation is monitored by means of Alamar blue assay. 3D breast cancer models morphology is observed by confocal microscopy. Gene expression modulation concerning extracellular matrix markers is evaluated. Further, 3D bioengineered breast cancer models are treated with doxorubicin. Silk fibroin scaffolds allow the proliferation of cancer cells and fibroblasts. Cells growth is enhanced when cancer cells and fibroblasts are seeded together. Histological staining shows 3D cell organization. MMP-1, MMP-2, MMP-3, Col-1, and Fibronectin expression is upregulated in co-culture. After doxorubicin treatment, stronger reduction in cell activity is observed in 2 mm SF scaffold in comparison to 1 mm. The 3D in vitro breast cancer model obtained can easily be scaled-up and translated to the preclinical testing of novel chemotherapeutics.

Collagen is the main component of the extracellular matrix and it plays a key role in tumor progression. Commercial collagen solutions are derived from animals, such as rat-tail and bovine or porcine skin. Their cost is quite high and the product is stable only at low temperature, with the disadvantage of a short expiring date. Most importantly, lot-to-lot variability can occur and the reconstituted collagen gels differ significantly from native tissues in terms of both structure and stiffness. In this chapter, we describe a straightforward method to use native, collagen rich skin samples derived from by-products of the tanning industry. The protocol proposed preserves the microstructure of the ovine skin collagen network, offering structurally competent and more relevant model to investigate cell behavior in vitro. Other advantages of the proposed procedure consist in the cost-effectiveness of the process and an increased level of reproducibility. The decellularized ovine skin samples support the adhesion and growth of different cancer cell lines (pancreatic, breast and melanoma cells). The proposed decellularized skin scaffolds are meant as future low-cost competitors for conventional porous scaffold derived by biomaterials, since they offer a biomimetic environment for the cells.

The biomaterials-based three-dimensional (3D) cancer models enhance the investigation of the mechanisms underpinning the cancer progression and metastasis. In vitro 3D tumor models could substitute the use of two-dimensional cell culture and reduce the number of animals recruited for the preclinical trials of anticancer drug. Moreover, pharmaceutical companies are adopting 3D cancer models as platform for anticancer drug testing platforms. The spheroid is the gold standard for the drug screening 3D platform due to its easy handling and low cost. However, spheroids composed only with cancer cells, are not able to copy entirely the complexity of the tumor microenvironment (TME) due to the lack of expression of the extracellular matrix (ECM) proteins. On the other hand, tumor models based on microcarriers exhibit physiologically relevant cell-cell and cell-matrix interactions. In this perspective, biomaterials can be used as ECM substitute to mimic the cell-ECM interaction and structural complexity in order to reflect in vivo tumors. In this chapter, we focus on the different players of the TME such as cancer cells, fibroblasts, endothelial, and immune system cells. For each paragraph, the most interesting 3D tumor models (both spheroids- and microcarriers-based cancer models) are described in order to give an updated state-of-the-art overview of the field.

Decellularized matrices are steadily gaining popularity to study the biology of cells and tissues, as they represent a biomimetic environment in which cells can recapitulate certain behaviours that share similarities with those observed in vivo. Basically, biochemistry, microstructure and mechanics of the decellularized matrices are the most valuable properties that differentiate these culturing systems from conventional bidimensional models. Several procedures to decellularize tissues have been proposed so far, with the common aim to preserve the tissue chemical/physical properties of the original tissue. However, these processes are complex, time-consuming and expensive. In this work, we propose a cost-effective, easy-to-produce decellularized dermal matrix, derived from animal skin. The chemical/physical processes to obtain the matrices proved to not alter matrix structure and did not induce cytotoxicity issues. To test the validity of the decellularized matrices as a model to study the behaviour of tumour cells in vitro, we performed microstructural and mechanical investigations as well as cell proliferation assays. In particular, three different tumour cell lines were used, which proliferated and invaded the matrix with no additional treatments. Decellularized skin scaffold, presented in this work, could be a strong competitor for conventional 3D systems like synthetic porous scaffolds or hydrogels.

Many of the existing three-dimensional (3D) cancer models in vitro fail to represent the entire complex tumor microenvironment composed of cells and extra cellular matrix (ECM) and do not allow a reliable study of the tumoral features and progression. In this paper we reported a strategy to produce 3D in vitro microtissues of pancreatic ductal adenocarcinoma (PDAC) for studying the desmoplastic reaction activated by the stroma–cancer crosstalk. Human PDAC microtissues were obtained by co-culturing pancreatic cancer cells (PT45) and normal or cancer-associated fibroblasts within biodegradable microcarriers in a spinner flask bioreactor. Morphological and histological analyses highlighted that the presence of fibroblasts resulted in the deposition of a stromal matrix rich in collagen leading to the formation of tumor microtissues composed of a heterotypic cell population embedded in their own ECM. We analyzed the modulation of expression of ECM genes and proteins and found that when fibroblasts were co-cultured with PT45, they acquired a myofibroblast phenotype and expressed the desmoplastic reaction markers. This PDAC microtissue, closely recapitulating key PDAC microenvironment characteristics, provides a valuable tool to elucidate the complex stroma–cancer interrelationship and could be used in a future perspective as a testing platform for anticancer drugs in tissue-on-chip technology. Statement of Significance Tumor microenvironment is extremely complex and its organization is due to the interaction between different kind of cells and the extracellular matrix. Tissue engineering could give the answer to the increasing need of 3D culture model that better recapitulate the tumor features at cellular and extracellular level. We aimed in this work at developing a microtissue tumor model by mean of seeding together cancer cells and fibroblasts on gelatin microsphere in order to monitor the crosstalk between the two cell populations and the endogenous extracellular mat

Therapeutic approaches based on nanomedicine have garnered great attention in cancer research. In vitro biological models that better mimic in vivo conditions are crucial tools to more accurately predict their therapeutic efficacy in vivo. In this work, a new 3D breast cancer microtissue has been developed to recapitulate the complexity of the tumor microenvironment and to test its efficacy as screening platform for drug delivery systems. The proposed 3D cancer model presents human breast adenocarcinoma cells and cancer-associated fibroblasts embedded in their own ECM, thus showing several features of an in vivo tumor, such as overexpression of metallo-proteinases (MMPs). After demonstrating at molecular and protein level the MMP2 overexpression in such tumor microtissues, we used them to test a recently validated formulation of endogenous MMP2-responsive nanoparticles (NP). The presence of the MMP2-sensitive linker allows doxorubicin release from NP only upon specific enzymatic cleavage of the peptide. The same NP without the MMP-sensitive linker and healthy breast microtissues were also produced to demonstrate NP specificity and selectivity. Cell viability after NP treatment confirmed that controlled drug delivery is achieved only in 3D tumor microtissues suggesting that the validation of therapeutic strategies in such 3D tumor model could predict human response. Statement of Significance A major issue of modern cancer research is the development of accurate and predictive experimental models of human tumors consistent with tumor microenvironment and applicable as screening platforms for novel therapeutic strategies. In this work, we developed and validated a new 3D microtissue model of human breast tumor as a testing platform of anti-cancer drug delivery systems. To this aim, biodegradable nanoparticles responsive to physiological changes specifically occurring in tumor microenvironment were used. Our findings clearly demonstrate that the breast tumor microtissue

We fabricated three-dimensional microtissues with the aim to replicate in vitro the composition and the functionalities of the tumor microenvironment. By arranging either normal fibroblasts (NF) or cancer-activated fibroblasts (CAF) in two different three dimensional (3D) configurations, two kinds of micromodules were produced: spheroids and microtissues. Spheroids were obtained by means of the traditional cell aggregation technique resulting in a 3D model characterized by high cell density and low amount of extracellular proteins. The microtissues were obtained by culturing cells into porous gelatin microscaffolds. In this latter configuration, cells assembled an intricate network of collagen, fibronectin and hyaluronic acid. We investigated the biophysical properties of both 3D models in terms of cell growth, metabolic activity, texture and composition of the extracellular matrix (via histological analysis and multiphoton imaging) and cell mechanical properties (via Particle Tracking Microrheology). In the spheroid models such biophysical properties remained unchanged regardless to the cell type used. In contrast, normal-microtissues and cancer-activated-microtissues displayed marked differences. CAF-microtissues possessed higher proliferation rate, superior contraction capability, different micro-rheological properties and an extracellular matrix richer in collagen fibronectin and hyaluronic acid. At last, multiphoton investigation revealed differences in the collagen network architecture. Taken together, these results suggested that despite to cell spheroids, microtissues better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the stromal element of the tumor tissues. Statement of Significance This work concerns the engineering of tumor tissue in vitro. Tumor models serve as biological equivalent to study pathologic progression and to screen or validate the dr

The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure-activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1. H-indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however, the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15, apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.

In this work, a new model of breast cancer is proposed featuring both epithelial and stromal tissues arranged on a microfluidic chip. The main task of the work is the in vitro replication of the stromal activation during tumor epithelial invasion. The activation of tumor stroma and its morphological/compositional changes play a key role in tumor progression. Despite emerging evidences, to date the activation of tumor stroma in vitro has not been achieved yet. The tumor-on-chip proposed in this work is built in order to replicate the features of its native counterpart: multicellularity (tumor epithelial cell and stromal cell); 3D engineered stroma compartment composed of cell-assembled extracellular matrix (ECM); reliable 3D tumor architecture. During tumor epithelial invasion the stroma displayed an activation process at both cellular and ECM level. Similarly of what repeated in vivo, ECM remodeling is found in terms of hyaluronic acid and fibronectin overexpression in the stroma compartment. Furthermore, the cell-assembled ECM featuring the stromal tissue, allowed on-line monitoring of collagen remodeling during stroma activation process via real time multiphoton microscopy. Also, trafficking of macromolecules within the stromal compartment has been monitored in real time.