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BackgroundPancreatic 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).MethodsTranscriptional 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.ResultsHere 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.ConclusionOur 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.

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 fibroblastsâ presence inhibits immune effector recruiting by exploiting a 3D pre-clinical tumor model., This research was funded by FCT through project BREAST-IT (PTDC/BTM-ORG/28168/2017) under the Compete2020 program. This work was partially supported by IET A. F. Harvey Engineering Research Award 2018 (ENG The Cancers 2022) and "Associazione Italiana Ricerca sul Cancro (AIRC)" IG-2017 No. 20259 (D.S.). D.C. acknowledges the financial support from FCT (CEECIND/00352/2017 and PTDC/BTM-ORG/28070/2017). The support of EU Framework Programme for Research and Innovation H2020 on FoReCaST (grant agreement No. 668983) is significantly acknowledged.

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.

Breast cancer is still the leading cause of women's death due to relapse and metastasis. In vitro tumor models are considered reliable tools for drug screening and understanding cancer-driving mechanisms due to the possibility of mimicking tumor heterogeneity. Herein, a 3D breast cancer model (3D-BCM) is developed based on enzymatically-crosslinked silk fibroin (eSF) hydrogels. Human MCF7 breast cancer cells are encapsulated into eSF hydrogels, with and without human mammary fibroblasts. The spontaneously occurring conformational change from random coil to β-sheet is correlated with increased eSF hydrogels’ stiffness over time. Moreover, mechanical properties analysis confirms that the cells can modify the stiffness of the hydrogels, mimicking the microenvironment stiffening occurring in vivo. Fibroblasts support cancer cells growth and assembly in the eSF hydrogels up to 14 days of culture. Co-cultured 3D-BCM exhibits an upregulated expression of genes related to extracellular matrix remodeling and fibroblast activation. The 3D-BCM is subjected to doxorubicin and paclitaxel treatments, showing differential drug response. Overall, these results suggest that the co-culture of breast cancer cells and fibroblasts in eSF hydrogels allow the development of a mimetic in vitro platform to study cancer progression. This opens up new research avenues to investigate novel molecular targets for anti-cancer therapy.

Simple Summary: Gliomas can be difficult to discern clinically and radiologically from other brain lesions (either neoplastic or non-neoplastic) since their clinical manifestations as well as preoperative imaging features often overlap and appear misleading. Radiomics could be extremely helpful for non-invasive glioma differential diagnosis (DDx). However, implementation in clinical practice is still distant and concerns have been raised regarding the methodological quality of radiomic studies. In this context, we aimed to summarize the current status and quality of radiomic studies concerning glioma DDx in a systematic review. In total, 42 studies were selected and examined in our work. Our study revealed that, despite promising and encouraging results, current studies on radiomics for glioma DDx still lack the quality required to allow its introduction into clinical practice. This work could provide new insights and help to reach a consensus on the use of the radiomic approach for glioma DDx. Abstract: Radiomics is a promising tool that may increase the value of imaging in differential diagnosis (DDx) of glioma. However, implementation in clinical practice is still distant and concerns have been raised regarding the methodological quality of radiomic studies. Therefore, we aimed to systematically review the current status of radiomic studies concerning glioma DDx, also using the radiomics quality score (RQS) to assess the quality of the methodology used in each study. A systematic literature search was performed to identify original articles focused on the use of radiomics for glioma DDx from 2015. Methodological quality was assessed using the RQS tool. Spearman’s correlation (ρ) analysis was performed to explore whether RQS was correlated with journal metrics and the characteristics of the studies. Finally, 42 articles were selected for the systematic qualitative analysis. Selected articles were grouped and summarized in terms of those on DDx between glioma and primary central nervous system lymphoma, those aiming at differentiating glioma from brain metastases, and those based on DDx of glioma and other brain diseases. Median RQS was 8.71 out 36, with a mean RQS of all studies of 24.21%. Our study revealed that, despite promising and encouraging results, current studies on radiomics for glioma DDx still lack the quality required to allow its introduction into clinical practice. This work could provide new insights and help to reach a consensus on the use of the radiomic approach for glioma DDx.

Glioblastoma multiforme (GBM) typically exhibits substantial intratumoral heterogeneity at both microscopic and radiological resolution scales. Diffusion Weighted Imaging (DWI) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) are two functional MRI techniques that are commonly employed in clinic for the assessment of GBM tumor characteristics. This work presents initial results aiming at determining if radiomics features extracted from preoperative ADC maps and post-contrast T1 (T1C) images are associated with pathomic features arising from H&E digitized pathology images. 48 patients from the public available CPTAC-GBM database, for which both radiology and pathology images were available, were involved in the study. 91 radiomics features were extracted from ADC maps and post-contrast T1 images using PyRadiomics. 65 pathomic features were extracted from cell detection measurements from H&E images. Moreover, 91 features were extracted from cell density maps of H&E images at four different resolutions. Radiopathomic associations were evaluated by means of Spearman’s correlation (ρ) and factor analysis. p values were adjusted for multiple correlations by using a false discovery rate adjustment. Significant cross-scale associations were identified between pathomics and ADC, both considering features (n = 186, 0.45 < ρ < 0.74 in absolute value) and factors (n = 5, 0.48 < ρ < 0.54 in absolute value). Significant but fewer ρ values were found concerning the association between pathomics and radiomics features (n = 53, 0.5 < ρ < 0.65 in absolute value) and factors (n = 2, ρ = 0.63 and ρ = 0.53 in absolute value). The results of this study suggest that cross-scale associations may exist between digital pathology and ADC and T1C imaging. This can be useful not only to improve the knowledge concerning GBM intratumoral heterogeneity, but also to strengthen the role of radiomics approach and its validation in clinical practice as “virtual biopsy”, introducing new insights for omics integration toward a personalized medicine approach.

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 international 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.

uncorrected proof version, 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., Portuguese Foundation for Science and Technology (FCT) under the program CEEC Individual 2017 (CEECIND/00352/2017). D.C., V.B., A.C.L., C.M.A., and S.C.K. also thank the support from the FCT under the scope of the projects 2MATCH (PTDC/BTM-ORG/28070/2017) and BREAST-IT (PTDC/ BTM-ORG/28168/2017) funded by the Programa Operacional Regional do Norte supported by European Regional Development Funds (ERDF). The authors also thank the financial support from the European Union Framework Program for Research and Innovation Horizon 2020 on FoReCaST project under (Grant Number: 668983), and the Institution of Engineering and Technology (IET) for the funding provided under the ENG ThE CANCER project (Grant Number: IET Harvey Engineering Research Award 2018). J.M.O. is grateful for the FCT distinctions (IF/00423/2012 and IF/01285/2015)

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., This work is supported by the European Union Framework Programme for Research and Innovation Horizon 2020 (nº 668983 — FoReCaST; FROnTHERA—NORTE-01-0145-FEDER-000023), Investigator FCT program (IF/01214/2014—V.M.), FCT2015 (IF/01285/2015—J.M.O.) and PTDC/BTMORG/28168/2017 (V.B. and S.C.K.).

Simple Summary An accurate survival analysis is crucial for disease management in glioblastoma (GBM) patients. Due to the ability of the diffusion MRI techniques of providing a quantitative assessment of GBM tumours, an ever-growing number of studies aimed at investigating the role of diffusion MRI metrics in survival prediction of GBM patients. Since the role of diffusion MRI in prediction and evaluation of survival outcomes has not been fully addressed and results are often controversial or unsatisfactory, we performed this systematic review in order to collect, summarize and evaluate all studies evaluating the role of diffusion MRI metrics in predicting survival in GBM patients. We found that quantitative diffusion MRI metrics provide useful information for predicting survival outcomes in GBM patients, mainly in combination with other clinical and multimodality imaging parameters. Abstract Despite advances in surgical and medical treatment of glioblastoma (GBM), the medium survival is about 15 months and varies significantly, with occasional longer survivors and individuals whose tumours show a significant response to therapy with respect to others. Diffusion MRI can provide a quantitative assessment of the intratumoral heterogeneity of GBM infiltration, which is of clinical significance for targeted surgery and therapy, and aimed at improving GBM patient survival. So, the aim of this systematic review is to assess the role of diffusion MRI metrics in predicting survival of patients with GBM. According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, a systematic literature search was performed to identify original articles since 2010 that evaluated the association of diffusion MRI metrics with overall survival (OS) and progression-free survival (PFS). The quality of the included studies was evaluated using the QUIPS tool. A total of 52 articles were selected. The most examined metrics were associated with the standard Diffusion Weighted Imaging (DWI) (34 studies) and Diffusion Tensor Imaging (DTI) models (17 studies). Our findings showed that quantitative diffusion MRI metrics provide useful information for predicting survival outcomes in GBM patients, mainly in combination with other clinical and multimodality imaging parameters.

The lack of validation and standardization represents the main drawback for a clear role of whole-body diffusion weighted imaging (WB-DWI) for prediction and assessment of treatment response in Hodgkin’s lymphoma (HL). We explored the reliability of an automatic approach based on the WB-DWI technique for prediction and assessment of response to treatment in patients with HL. The study included 20 HL patients, who had whole-body positron emission tomography (PET)/ magnetic resonance Imaging (MRI) performed before, during and after chemotherapy. Using the syngo.via MR Total Tumor Load tool, we automatically extracted values of diffusion volume (DV) and its associated histogram features by WB-DWI images, and evaluated their utility in predicting and assessing interim and end-of-treatment (EOT) response. The Mann–Whitney test followed by receiver operator characteristic (ROC) analysis was performed between features and their inter-time point percentage differences for patients having a complete or partial treatment response, revealing that several WB-DWI associated features allowed for prediction of interim response and both prediction and assessment of EOT response. Our proposed method offers huge advantages in terms of saving time and work, enabling clinicians to draw conclusions relating to HL treatment response in a fully automatic way, and encloses, also, all DWI advantages compared to PET/ computed tomography (CT).

Glioblastoma (GBM) is the most lethal primary brain tumor of the central nervous system in adults. Despite advances in surgical and medical neuro-oncology, the median survival is about 15 months. For this reason, initial diagnosis, prognosis, and targeted therapy of GBM represent very attractive areas of study. Aptamers are short three-dimensional structures of single-stranded nucleic acids (RNA or DNA), identified by an in vitro process, named systematic evolution of ligands by exponential enrichment (SELEX), starting from a partially random oligonucleotide library. They bind to a molecular target with high affinity and specificity and can be easily modified to optimize binding affinity and selectivity. Thanks to their properties (low immunogenicity and toxicity, long stability, and low production variability), a large number of aptamers have been selected against GBM biomarkers and provide specific imaging agents and therapeutics to improve the diagnosis and treatment of GBM. However, the use of aptamers in GBM diagnosis and treatment still represents an underdeveloped topic, mainly due to limited literature in the research world. On these bases, we performed a systematic review aimed at summarizing current knowledge on the new promising DNA and RNA aptamer-based molecules for GBM diagnosis and treatment. Thirty-eight studies from 2000 were included and investigated. Seventeen involved the use of aptamers for GBM diagnosis and 21 for GBM therapy. Our findings showed that a number of DNA and RNA aptamers are promising diagnostic and therapeutic tools for GBM management.

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.

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 3Â 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., This research was supported by the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement n° 668983 — FoReCaST and project FROnTHERA (NORTE-01-0145-FEDER-000023).

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 standardmodel that faithfullymimics solid cancer is not available yet. 3D breast cancermodel 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 2mm 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 2mm SF scaffold in comparison to 1mm. The 3D in vitro breast cancer model obtained can easily be scaled-up and translated to the preclinical testing of novel chemotherapeutics., This work was supported by EU-Horizon 2020 grant FoReCaST—Forefront Research in 3D Disease Cancer Modelsasin vitroScreening Technologies (H2020-WIDESPREAD-2014-668983). The authors also acknowledge the FRONTHERA project (Frontiers of technology for theranostics of cancer,metabolic and neurodegenerative diseases) (NORTE-01-0145-FEDER-0000232) and Fundação Ciência e tecnologia (FCT grantagreement: PTDC/BTM-ORG/28168/2017 to VB and SK).

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.

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.

"Available online 28 October 2020", 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 authors are grateful for the Portuguese Foundation for Science and Technology (FCT) distinctions attributed to R. F. Canadas (SFRH/BD/92565/2013), D. Caballero (CEECIND/00352/2017), and to J. M. Oliveira (IF/00423/2012, IF/01285/2015). R. F. Canadas and J. M. Oliveira are also thankful to FCT, Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa Operacional Competitividade e Internacionalização (POCI) for funding the B-Liver Project (PTDC/EMD-EMD/29139/2017). D. Caballero and S.C. Kundu also acknowledge the FCT for the financial support under the scope of the 2MATCH Project (02/SAICT/2017 – n° 028070). V. Brancato and S.C. Kundu acknowledge FCT project BREAST-IT (grant agreement: PTDC/BTM-ORG/28168/2017). The authors are also thankful to FCT for supporting the project Hierarchitech (M-ERA-NET/0001/2014). Pedro Patrício and Nuno A. M. Araújo acknowledge financial support from the FCT under Contracts no. PTDC/FIS-MAC/28146/2017 (LISBOA-01-0145-FEDER-028146) and UID/FIS/00618/2019. The authors acknowledge that this material and collaboration is based in part upon work supported by FLAD (2016/CON15/CAN6). This work was in part supported by European Research Council Grant agreement ERC-2012-ADG 20120216-321266 for project ComplexiTE.

Prova tipográfica, 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., European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement nº 668983 — FoReCaST (Forefront Research in 3D Disease Cancer Models as in vitro Screening Technologies) and FRONTHERA project (Frontiers of technology for theranostics of cancer, metabolic and neurodegenerative diseases; grant agreement: NORTE-01-0145-FEDER-0000232). JMO is thankful to FCT - Fundação para a Ciência e a Tecnologia, Portugal (IF/00423/2012 and IF/01285/2015) for financial support. VB and SCK also acknowledge FCT project BREAST-IT (grant agreement: PTDC/BTM-ORG/28168/2017)