To date, anticancer therapies with evidenced efficacy in preclinical models fail during clinical trials. The shortage of robust drug screening platforms that accurately predict patient's response underlie these misleading results. To provide a reliable platform for tumor drug discovery, we herein propose a relevant humanized 3D osteosarcoma (OS) model exploring the potential of methacryloyl platelet lysates (PLMA)-based hydrogels to sustain spheroid growth and invasion. The architecture and synergistic cell-microenvironment interaction of an invading tumor was recapitulated encapsulating spheroids in PLMA hydrogels, alone or co-cultured with osteoblasts and mesenchymal stem cells. The stem cells alignment toward OS spheroid suggested that tumor cells chemotactically attracted the surrounding stromal cells, which supported tumor growth and invasion into the hydrogels. The exposure of established models to doxorubicin revealed an improved drug resistance of PLMA-based models, comparing with scaffold-free spheroids. The proposed OS models highlighted the feasibility of PLMA hydrogels to support tumor invasion and recapitulate tumor-stromal cell crosstalk, demonstrating the potential of this 3D platform for complex tumor modelling. STATEMENT OF SIGNIFICANCE: Cell invasion mechanisms involved in tumor progression have been recapitulated in the field of 3D in vitro modeling, leveraging the great advance in biomimetic materials. In line with the growing interest in human-derived biomaterials, the aim of this study is to explore for the first time the potential of methacryloyl platelet lysates (PLMA)-based hydrogels to develop a humanized 3D osteosarcoma model to assess tumor invasiveness and drug sensitivity. By co-culturing tumor spheroids with human osteoblasts and human mesenchymal stem cells, this study demonstrated the importance of the synergistic tumor cell-microenvironment interaction in tumor growth, invasion and drug resistance. The established 3D osteosarcoma model highlighted the feasibility of PLMA hydrogels as a relevant 3D platform for complex tumor modelling.

Three-dimensional (3D) in vitro tumor spheroids are becoming popular as pre-clinical platforms for testing the performance of existing drugs or for discovery of innovative anti-cancer therapeutics. This focus is correlated with in vitro 3D tumor models ability to mimic the multicellular compact structure and spatial architecture of human solid tumors. However, these microphysiological systems generally lack the pre-existence of tumor-ECM, a critical aspect that can affect the overall therapeutic performance and the decision of advancing candidate drugs to later stages of the pipeline. Aiming to face this drawback and mimic tumors-ECM, herein we rapidly fabricated in-air hyaluronan-methacrylate (HA-MA) and gelatin-methacrylate (GelMA) photocrosslinkable 3D spheroid microgels by using superhydrophobic surfaces. These platforms were used for establishing heterotypic 3D co-culture models of prostate cancer cells (PC-3) and human osteoblasts (hOB) to mimic prostate cancer-to-bone metastasis cellular heterogeneity and the tumor-ECM microenvironment. 3D microgel microtumors morphology, size and cell number were easily controlled via digital droplet generation on polystyrene superhydrophobic surfaces and under solvent-free conditions when compared to microfluidics or electrospray. Co-culture 3D microgels formed by 2.5%HA-MA-5%GelMA and 5%HA-MA-5%GelMA ratios showed the highest calcium deposition after 14 days of culture, evidencing osteoblasts viability and the establishment of functional mineralization in the 3D hydrogel matrix. Cisplatin cytotoxicity evaluation showed that 3D microgels are more resistant to platin chemotherapeutics than single or co-culture 3D multicellular spheroid counterparts. Overall, our findings indicate that solvent-free, in-air produced 3D microgel microenvironments are cost-effective and robust tumor mimicking platforms for in vitro high-throughput screening of therapeutics targeted to prostate-to-bone metastasis microenvironments. STATEMENT OF SIGNIFICANCE: The generation of robust microphysiological systems that recapitulate the complexity of the metastatic prostate-to-bone tumor microenvironment is crucial for pre-clinical evaluation of new therapeutics that can eradicate these secondary tumors. In this study, we employed superhydrophobic (SH) surfaces to rapidly fabricate photocrosslinkable hyaluronan-methacrylate/gelatin-methacrylate 3D spheroid microgels for prostate cancer cells and human osteoblasts co-culture models that simultaneously mimic the cellular and ECM tumor components. The use of SH platforms overcomes the issues of standard in-liquid microgel production technologies by providing a robust control over 3D microgels size/morphology and cell-cell co-encapsulation numbers, while avoiding the use of oil-based microgel droplets generation. Overall, SH surfaces allowed a solvent-free, cost-effective, reproducible and adaptable fabrication of heterotypic 3D spherical microgels for high throughput drug screening.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging diseases to treat with a very poor prognosis, exhibiting a 5-year survival rate of less than 10%. This dismal scenario arises from the late detection, rapid progression, and inefficacy of currently available treatments. Its aggressiveness and resistance are largely attributed to the dense fibrotic stroma and poor vascularization, hampering anti-cancer drugs delivery to malignant cells. In an attempt to accelerate the discovery and validation of new and more effective therapies, research has been focused on the development of increasingly biomimetic and predictive preclinical tumor models for the identification of new therapeutic targets and the screening of novel treatment approaches. Despite the efforts, most preclinical platforms still fail on resembling native tumor microenvironment (TME) of this neoplasia, especially the unique tumor-stroma organization and its complex biological interplay. A number of three-dimensional (3D) models have been fabricated aiming to recreate human TME namely, spheroids, hydrogel-based systems, cancer-on-a-chip, and 3D bioprinted constructs. Despite representing human tumors closer than the monolayer cultures, they generally still lack any prior representation of stroma components in a relevant manner. Therefore, this PhD thesis focuses on the bioengineering of advanced pancreatic cancer models, capable of more faithfully emulating the unique tumor-stroma interactions and architecture, where the core tumor mass is enveloped by a dense and fibrotic stroma compartment. A comprehensive overview of pancreatic TME and 3D in vitro tumor modeling is provided for contextualization of the current state-of-the-art of the field. Advances in cutting edge technologies such as 3D bioprinting and cryobioprinting to produce more complex human-scale 3D in vitro platforms are also highlighted. The bioengineering of tumor-stroma core-shell pancreatic cancer models by using scaffold-free and scaffold-based methods is focused with the aim to demonstrate the ability to produce tumor-stroma models, where the tumor core is encapsulated by a stroma compartment, and the crucial recapitulation of this hallmark on anti-cancer drugs response. Such core-shell structures were generated by using either ultra-low adhesion plates or superhydrophobic surfaces. The establishment of tumor-stroma models by using tissue-specific decellularized extracellular matrix (dECM) is herein also reported aiming to generate pancreas dECM-based miniaturized tumor-stroma platforms in a high-throughput manner and demonstrating the impact of PDAC stroma in chemoresistance. The screening of stroma-targeted therapies in combination with anti-cancer therapies was conducted and enabled the validation of the established tumor-stroma model potential for screening stroma ablation approaches to improve current treatments efficacy. The development of more complex and tissue-scale models for preclinical drug screening was performed through 3D bioprinting technology and the ability to produce and cryopreserve such platforms simultaneously during fabrication was carried out by employing 3D cryobioprinting technologies. 3D cryobioprinted and preserved tumor-stroma PDAC models showed potential to be used on-demand as off-the-shelf available platforms, emerging as an innovative alternative for preclinical drug-screening. Overall, the developed preclinical testing platforms level-up the current landscape of PDAC in vitro models being useful for unveiling innovative treatments for this disease and to potentially assist in bedside clinical decision making in the foreseeable future.

Necroptosis is an alternative pathway of regulated cell death, occurring independently of caspases, and mediated by receptor interacting serine/threonine-protein kinase-1 (RIP1), RIP3, and mixed lineage kinase domain-like (MLKL) protein. Necroptosis participates in the pathogenesis of several human conditions and activation may be beneficial for treating cancer. Despite this information, there are currently no pharmacological inhibitors or activators of necroptosis available with therapeutic properties. Colon cancer is the second most prevalent cancer worldwide and the third with the highest mortality rate. Cancer therapy has improved in recent years, but pro-apoptotic therapies still face cancer resistance. In this thesis, we started by screening three libraries of newly synthesized compounds for its ability to modulate necroptosis, using the murine fibrosarcoma L929 cell line as an in vitro model. We identified 7 compounds with the ability to inhibit TNF-α-induced necroptosis L929 cells by more than 70%. To further characterize compound activity, the half maximal effective concentration (EC50) for inhibiting necroptosis was determined. The compound with the lowest EC50 - MS-PSR90 - was further shown to reduce TNF-α-induced MLKL phosphorylation. Interestingly, one of the compounds - SAS9 - strongly induced necroptosis in both L929 cells and in human colorectal adenocarcinoma cells HT29; IC50 values were 16.96 and 49.73 μM, respectively. Interestingly, SAS9 concomitantly induced apoptosis in both cell lines, with increased caspase-3/7-like activity. In L929 cells, SAS9-induced necroptosis encompassed MLKL phosphorylation but not increased RIP1 expression. In conclusion, several novel modulators of necroptosis were identified in our screening, including a potential inducer of necroptosis. A better understanding of its mechanisms of action, as well as the investigation of pharmacokinetic properties, should help to determine their potential utility in cancer treatment.

Necroptosis is a caspase-independent form of regulated cell death executed via activation of receptor interacting protein 1 (RIP1) and RIP3, being negatively regulated by caspase-8. Neurodegenerative diseases are hereditary or sporadic conditions characterized by progressive cell death in selected areas of the nervous system, along with oxidative stress, mitochondrial dysfunction and neuroinflammation. Importantly, recent studies have demonstrated that necroptosis is involved in the pathogenesis of several disorders, including neurodegenerative diseases. Neuroinflammation, in turn, is a response to eliminate the original insult and damaged or dead cells. However, if tissue physiology is not restored, inflammation becomes a chronic condition, where microglia plays a major role. Studies have shown that defects in caspase-8 activation in microglia may promote inflammation through activation of necroptosis. In the present study, we used the murine BV2 microglia cell line as an in vitro model to screen a library of novel compounds, potential modulators of necroptosis, and evaluate if necroptosis modulation may contribute to attenuate microglia-mediated inflammation. Importantly, we identified one hit Oxa12 that inhibits this type of regulated cell death by preventing all necroptosis-associated events, including RIP1/RIP3 necrosome assembly, and MLKL phosphorylation. In addition, Oxa12 decreased TNF-α gene expression and secretion as well as JNK and p38 MAPK activation, while IκB phosphorylation was increased. In conclusion, we established a robust in vitro model of microglia necroptosis and identified a strong inhibitor of this regulated form of cell death that might be a promising candidate molecule for targeting RIP1/3-driven pathologies.

Osteosarcoma (OS) is the most common primary malignant tumor of bonethat mainly affects children and adolescents. The currently available therapiesare not effective and the search for new OS anticancer drugs is extremelyurgent. Understanding the mechanisms that underlie the tumor progression,invasion, and metastasis is an essential step toward effective cancertherapies. Tissue engineering has given a great contribution to thedevelopment of reliable and cost-effective platforms for drug screening andvalidation through 3D in vitro models that more faithfully mimic the in vivopathophysiology than conventional 2D models. The progress in the field offunctional and biomimetic biomaterials in the development of 3D tissuemodels has provided tools for a close recapitulation of the highly complex anddynamic tumor microenvironment. This review focuses on the most recentadvances in 3D in vitro osteosarcoma models, highlighting the crucial role ofthe extracellular matrix and stromal cells in tumor progression, how theycontribute to drug resistance and disease prevalence, and the future pathwaystoward an effective and personalized model for drug screening and validation.

Three-dimensional multicellular tumor models are receiving an ever-growing focus as preclinical drug-screening platforms due to their potential to recapitulate major physiological features of human tumors in vitro. In line with this momentum, the technologies for assembly of 3D microtumors are rapidly evolving towards a comprehensive inclusion of tumor microenvironment elements. Customized spherically structured platforms, including microparticles and microcapsules, provide a robust and scalable technology to imprint unique biomolecular tumor microenvironment hallmarks into 3D in vitro models. Herein, a comprehensive overview of novel advances on the integration of tumor-ECM components and biomechanical cues into 3D in vitro models assembled in spherical shaped platforms is provided. Future improvements regarding spatiotemporal/mechanical adaptability, and degradability, during microtumors in vitro 3D culture are also critically discussed considering the realistic potential of these platforms to mimic the dynamic tumor microenvironment. From a global perspective, the production of 3D multicellular spheroids with tumor ECM components included in spherical models will unlock their potential to be used in high-throughput screening of therapeutic compounds. It is envisioned, in a near future, that a combination of spherically structured 3D microtumor models with other advanced microfluidic technologies will properly recapitulate the flow dynamics of human tumors in vitro.

3D multicellular tumor spheroids (3D-MCTS) that closely mimic in vitro the complex lung tumor microenvironment (TME) are highly desirable for screening innovative anti-cancer therapeutics. Despite significant improvements in mimicking lung TME, few models have combined tumor-infiltrating mesenchymal stem cells from bone marrow (hBM-MSCs) with heterotypic 3D tumor spheroid models containing ECM mimetic components. Herein, we engineered hybrid 3D-MCTS that combine, for the first time, A549:fibroblasts:hBM-MSCs in heterotypic tri-culture, with bioinstructive hyaluronan microparticles that act as tumor-ECM mimetics and as cell-anchoring hotspots. The obtained results indicated that 3D microspheres provided proper support for cells to self-assemble into compact 3D microtissues and promoted an increase in CD44 expression, emulating the presence of native-ECM hyaluronan. 3D-MCTS size and sphere-like morphology was reproducible and tri-culture models presented the characteristic solid tumors necrotic core. Mesenchymal stem cells tracking demonstrated that hBM-MSCs migrate to different regions in 3D microtumors mass exhibiting dynamic interactions with cancer cells and stromal fibroblasts, alike in human tumors. Importantly, doxorubicin administration revealed hBM-MSCs effect on cytotoxic responses in 3D tri-culture models and in dual cultures of hBM-MSCs:A549 at 10:1 ratio. Such findings evidence the relevance of including hBM-MSCs in combination with cancer-stromal fibroblasts in 3D in vitro tumor models and the importance to test different cell-to-cell ratios to mimic tumor heterogeneity. In addition, bioinstructive hyaluronan-microparticles were also effective as cell-agglomerating scaffolds and showed potential to be used as an enabling technology for including different ECM components in 3D in vitro models in the future.

Prostate cancer is one of the most commonly diagnosed malignancy and a leading cause of death among men worldwide. Currently, despite many advances in medicine, the current treatments for this neoplasia are mostly ineffective. The development of advanced in vitro disease models that can recapitulate human prostate tumors may revert this scenario by accelerating the pre-clinical discovery of new therapies which can realistically impact patients’ life span. Up to now, regulatory agencies recommend that anti-tumor drug screening should be performed in two-dimensional (2D) cell cultures for gathering preliminary pre-clinical data. However, these models fail to mimic key characteristics of in vivo human tumors including their spatial distribution, cell-cell contacts and nutrients/oxygen gradients. Moreover, these 2D models utterly fail to replicate tumors extracellular matrix (ECM) and cellular heterogeneity. These intrinsic limitations cause a number of false positive/negative results and provide a poor correlation with clinical trials data. To overcome these issues, in vitro 3D tumor models were proposed as valuable alternatives. Such platforms are able to reproduce various aspects of human solid tumors microenvironment, including gene expression patterns, 3D cell-cell interactions, necrotic core formation and drug resistance phenotypes. The research work developed within the scope of this dissertation describes the production of a novel 3D prostate cancer in vitro tumor model that mimics prostate cancer bone metastasis cellular heterogeneity and ECM microenvironment. The model is comprised of human prostate cancer cells (PC-3) and human osteoblasts, encapsulated in spheroidal-shaped hydrogel microparticles. Such cell-laden spheroidal microcapsules were assembled on a quasi-superhydrophobic surface by unitary droplet dispensing through U.V. mediated photocrosslinking of methacrylated hyaluronic acid and methacrylated gelatin blends. The obtained results show that spheroidal microtumors were reproducible in terms of morphology, size and number of encapsulated cells. The selected HA-MA/GelMA formulations present the deposition of calcium after 14 days, when compared to the monocultures, thus evidencing the importance of osteoblasts inclusion. The evaluation of cisplatin cytotoxicity in heterotypic co-cultures showed that 2.5% HA-MA-5% GelMA microgels have higher drug resistance than 5% HA-MA-5% GelMA Overall, the findings indicate that quasi SH are suitable for rapid, and solvent-free, manufacture of 3D prostate tumor in vitro models that may serve as testing platforms for the discovery of new therapies for prostate cancer

In Alzheimer's disease (AD), amyloid β (Aβ)-triggered cleavage of TrkB-FL impairs brain-derived neurotrophic factor (BDNF) signaling, thereby compromising neuronal survival, differentiation, and synaptic transmission and plasticity. Using cerebrospinal fluid and postmortem human brain samples, we show that TrkB-FL cleavage occurs from the early stages of the disease and increases as a function of pathology severity. To explore the therapeutic potential of this disease mechanism, we designed small TAT-fused peptides and screened their ability to prevent TrkB-FL receptor cleavage. Among these, a TAT-TrkB peptide with a lysine-lysine linker prevented TrkB-FL cleavage both in vitro and in vivo and rescued synaptic deficits induced by oligomeric Aβ in hippocampal slices. Furthermore, this TAT-TrkB peptide improved the cognitive performance, ameliorated synaptic plasticity deficits and prevented Tau pathology progression in vivo in the 5XFAD mouse model of AD. No evidence of liver or kidney toxicity was found. We provide proof-of-concept evidence for the efficacy and safety of this therapeutic strategy and anticipate that this TAT-TrkB peptide has the potential to be a disease-modifying drug that can prevent and/or reverse cognitive deficits in patients with AD.