Bioengineering strategies for cancer therapy and modelling

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisCancer is a global pandemic with a high incidence among the world population and effective treatments are for the most part elusive. The tumor microenvironment is a highly complex and heterotypic mixture of cells that interact to regulate central control mechanisms, driving immunosuppression and promoting both survival and invasion of cancer cells into surrounding tissues. It has been this complexity that has made finding effective therapeutics such a demanding task and therefore cancer still remains a burden worldwide in health as well as in economic terms. While the progression in the field of cancer research has been clear over the years, there are still several challenges that need to be addressed. Herein, two different sides to this disease are explored: treatment and in vitro models. Adoptive T cell therapy has shown impressive results, however not without its limitations. The use of the T cell mitogen IL-2 within culture systems is known to lead to early exhaustion of T cell subsets while high density of co-stimulating molecules has been linked to undesired immune responses. As an alternative, a nanoparticle system based on the natural polymer gellan-gum was proposed, with tailorable surface functionalization with co-stimulatory molecules. High levels of T cell expansion were observed over the studied period, with secreted IL-2 levels overcoming those of commercial alternatives. With this system, increased expression of cytotoxic molecules Granzyme B and Perforin were also detected in vitro. On the other spectrum, 3D cancer models have sustained a great number of developments observed by an increase in similarity towards native tissues; however, a requirement for even more complex architectures capable of better mimicking cellular interactions is still present. Therefore, an assembloid-based approach was proposed to develop a 3D in vitro melanoma model to further study cellular interactions. These heterotypic tumor assembloids presented a complex architecture capable of sustaining endothelial cell function as well as a high expression of stemness-related markers. These models were subjected to functionality assays where they showed a capacity for “cooperative invasion” which was coincident with an observed increased production of MMP-2. To further unravel the role of stromal cells in the invasive potential of cancer cells a 3D chemotaxis chamber was developed to study cellular interactions observed in the tumor microenvironment, where stem cells and fibroblasts showed to have a crucial role. Ultimately, this thesis allowed to explore biomedical engineering approaches to further contribute to the knowledge in the field opening new doors to be explored in the future.O cancro é uma pandemia global com uma elevada incidência e cujo desenvolvimento de tratamentos eficazes continua a ser difícil. O microambiente tumoral é uma mistura altamente complexa e heterotípica de células que interagem para regular mecanismos centrais que provocam imunossupressão promovendo a sobrevivência e invasão de células tumorais para os tecidos circundantes. É esta complexidade que tem tornado desafiante encontrar terapias eficazes, tornando esta doença um fardo a nível global em termos de saúde e economia. Enquanto a progressão na área da investigação oncológica tem sido clara ao longo dos anos, existem ainda vários desafios que precisam de serem encarados para permitir futuros desenvolvimentos. Aqui, foram exploradas duas vertentes diferentes desta doença: o tratamento e os modelos in vitro. A terapia celular adotiva tem demonstrado resultados impressionantes, no entanto não sem as suas limitações. O uso do mitogénio IL-2 nestes sistemas de cultura é conhecido por levar rapidamente à exaustão das células T, enquanto o uso de moléculas co-estimulatórias em elevadas densidades está associado a respostas imunes não desejadas. Como alternativa, foi proposto um sistema de nanopartículas baseado no polímero natural goma gelana e funcionalizado com moléculas co estimulatórias. Foram observados elevados níveis de expansão de células T e quantidade de IL-2 secretada superior à de alternativas comerciais. Foi ainda verificado in vitro um aumento de expressão das moléculas citotóxicas Grazima B e Perforina. No outro espectro, têm sido desenvolvidos modelos tumorais 3D com uma cada vez maior similaridade para tecidos nativos; no entanto, a necessidade de arquiteturas ainda mais complexas capazes de melhor representar interações celulares persiste. Assim, foi proposta uma abordagem baseada em “assemblóides” para obter modelos 3D in vitro de melanoma para estudar interações celulares. Estes “assemblóides” tumorais heterotípicos apresentaram uma arquitetura complexa capaz de suportar a função de células endoteliais, bem como a elevada expressão de marcadores de pluripotência. Estes modelos foram sujeitos a ensaios de funcionalidade onde mostraram a capacidade de “invasão cooperativa” que foi coincidente com uma produção aumentada de MMP-2. Para tornar mais claro o papel das células estaminais no potencial invasivo de células tumorais, uma câmara 3D de quimiotaxia foi desenvolvida para estudar as interações celulares observadas no microambiente tumoral onde as células estaminais e fibroblastos mostraram ter um papel determinante. Em última análise, esta tese permitiu explorar abordagens da engenharia biomédica de forma a contribuir para o conhecimento da área e abrir novas portas a serem exploradas no futuro

Pharmacodynamic therapeutic drug monitoring for cancer: challenges, advances, and future opportunities

In the modern era of cancer treatment, with targeted agents superseding more traditional cytotoxic chemotherapeutics, it is becoming increasingly important to use stratified medicine approaches to ensure that patients receive the most appropriate drugs and treatment schedules. In this context, there is significant potential for the use of pharmacodynamic biomarkers to provide pharmacological information, which could be used in a therapeutic drug monitoring setting. This review focuses on discussing some of the challenges faced to date in translating preclinical pharmacodynamic biomarker approaches to a clinical setting. Recent advances in important areas including circulating biomarkers and pharmacokinetic/pharmacodynamic modeling approaches are discussed, and selected examples of anticancer drugs where there is existing evidence to potentially advance pharmacodynamic therapeutic drug monitoring approaches to deliver more effective treatment are discussed. Although we may not yet be in a position to systematically implement therapeutic drug monitoring approaches based on pharmacodynamic information in a cancer patient setting, such approaches are likely to become more commonplace in the coming years. Based on ever-increasing levels of pharmacodynamic information being generated on newer anticancer drugs, facilitated by increasingly advanced and accessible experimental approaches available to researchers to collect these data, we can now look forward optimistically to significant advances being made in this area

Quantitative Mechanistic Modeling in Support of Pharmacological Therapeutics Development in Immuno-Oncology

Following the approval, in recent years, of the first immune checkpoint inhibitor, there has been an explosion in the development of immuno-modulating pharmacological modalities for the treatment of various cancers. From the discovery phase to late-stage clinical testing and regulatory approval, challenges in the development of immuno-oncology (IO) drugs are multi-fold and complex. In the preclinical setting, the multiplicity of potential drug targets around immune checkpoints, the growing list of immuno-modulatory molecular and cellular forces in the tumor microenvironment—with additional opportunities for IO drug targets, the emergence of exploratory biomarkers, and the unleashed potential of modality combinations all have necessitated the development of quantitative, mechanistically-oriented systems models which incorporate key biology and patho-physiology aspects of immuno-oncology and the pharmacokinetics of IO-modulating agents. In the clinical setting, the qualification of surrogate biomarkers predictive of IO treatment efficacy or outcome, and the corresponding optimization of IO trial design have become major challenges. This mini-review focuses on the evolution and state-of-the-art of quantitative systems models describing the tumor vs. immune system interplay, and their merging with quantitative pharmacology models of IO-modulating agents, as companion tools to support the addressing of these challenges

Anti-tumor immunity induced by a novel nanoparticulate vaccine : a mechanistic approach

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016Biodegradable polymeric nanoparticles (NP) are promising tools for tumor eradication. Different nanovaccines based on the aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) have been developed and optimized to investigate how different methods for antigen association to nanoparticulate carriers affect antigen uptake by antigen presenting cells (APC) and the generation and nature of antigen-specific immune responses. The different experimental parameters have been tested in order to predict the effect of the nature of protein association (adsorbed vs. entrapped) and polymers/surfactant concentrations, on NP average mean diameter, polydispersity index, surface charge, encapsulation efficiency, protein integrity and surfactant residual amount. This development procedure allowed for the rational identification of particle composition and experimental conditions that led to the antigenassociated nanocarrier presenting the ideal product specifications previously identified for optimal immune cell modulation, having in consideration literature evidences and our previous data. We used PLGA and pegylated (PEG)-PLGA as a matrix polymer. When preparing NP with double-emulsion solvent-evaporation technique, we used α-lactalbumin (LALBA) as an antigen, glycol chitosan (CS) to increase the viscosity of the internal aqueous phase (IP) and polyvinyl alcohol (PVA) or Pluronic F127 (PF127) to stabilization the interphase of double emulsion. Various concentrations of surfactants has been used (2, 4, 5, 8, 10, 12 % v/v) to best meet the criteria of the most optimal nanoparticulate delivery system. We aimed for NP below 200 nm, polydispersity index (PDI) under 0.2 and ζ potential close to neutrality. The formulations with 10 % v/v PVA solution in IP and 0.3 % v/v PVA or PF127 in external aqueous phase (EP) presented stable and repetitive NP formulations with desired physiochemical properties. The highest encapsulation efficacy (EE) values were obtained for the formulation prepared with PVA (EntrapLALBA(PVA)), reaching 80 % of the integrated protein, while NP with PF127 (EntrapLALBA(PF127)) presented around 70 % EE. As regards the protein adsorbed, NP formulations prepared with PVA (AdsLALBA(PVA)) or PF127 (AdsLALBA(PF127), reached the values of 40 % and 50 %, respectively. Dynamic light scattering (DLS) and laser doppler velocimetry (LDV) were used to determine average particle size, polydispersity (PDI) and ζ potential, respectively. EntrapLALBA(PVA) presented the average size of 171 nm with PDI around 0.15 and ζ potential 0.51 mV. EntrapLALBA(PF127) had 185 nm with PDI 0.17 and ζ potential 0.37 mV. On the other side AdsLALBA(PVA) and AdsLALBA(PF127) presented slightly biggersize 188 nm and 195 nm, respectively, with more negative ζ potential, -1,89 and -1,42 mV, respectively. NP size, PDI and geometry were also confirmed by atomic force microscopy (AFM). Protein integrity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fourier transform infrared spectroscopy (FTIR) was further used to study the interaction of protein adsorption onto surfaces of polymeric NP. The presence of bands at the wavelengths 1662 cm-1 and 1562 cm-1, that are specific for primary amides and amines, were detected in formulations AdsLALBA(PVA) and AdsLALBA(PF127), where LALBA was adsorbed onto the surface of the NP. While in NP EntrapLALBA(PVA) and EntrapLALBA(PF127) this bands were absent indicating the protein incorporation into the polymeric matrix. Differential scanning calorimetry (DSC) showed that the formulation process did not alter the structure of the polymers used in the preparation of NP. The ability of the optimal formulations for systemic delivery and activation of DC was further evaluated in vitro and in vivo on bone marrow derived dendritic cells (BMDC). Similar activation and maturation profile of APC were obtained after the internalization of NP with antigen entrapped (EntrapNP) or adsorbed onto NP surface (AdsNP) evaluated at 3, 6, 16 and 24 h (ImageStreamTM and flow cytometry). The DC-NP interaction increased with the incubation time, presenting internalization values between 50-60% and 30-40%, in vitro and in vivo, respectively. Interestingly, MHCII was upregulated after the immunization with AdsNP and MHCI after the vaccination with EntrapNP, suggesting more efficient crosspresentation. To evaluate the activation of antigen-specific immune response, we replaced LALBA for ovalbumin (OVA). It is a highly immunogenic model antigen for which, in immunology, we have well-established animal models that can help to study the effect of the antigen on immune system without any unspecific immune response. Phenotype, frequency and efficacy of immune cell stimulation induced by NP loaded with OVA (EntrapNP or AdsNP) and with/without TLR ligands (CpG and Monophosphoryl Lipid A (MPLA)) were characterized and their specificity clarified by the engrafted OT-I (CD8+) and OT-II (CD4+) T cells. EntrapNP with OVA and adjuvants presented the strongest antigen-specific cytotoxic immune response, following NP with AdsOVA-Ajds. Moreover, long-lasting memory of cytotoxic T lymphocytes (CTL), was evaluated 8 weeks after a single immunization in response to the different ways of antigen delivery, PBS, OVA in solution, OVA & Adjs in solution and NP AdsOVA, AdsOVA-Adjs, EntrapOVA, or EntrapOVA-Adjs. Immunizations where we used antigen alone did not induce efficient CTL reactivation. Overall, Entrap OVA-Adj NP presented the most robust immune response in inducing the memory, followed by AdsOVAAdjs NP and OVA & Adjs in solution. To explore whether the immunization with formulated NP results in efficient crosspriming, an in vivo killing assay in steady-state conditions was performed. 24 h prior to immunization, animals were engrafted with CD8+ (OT-I) cells. 5 days p.i., mice were injected with an equal mix of targeted and untargeted CD45.1 splenocytes. Targeted cells were pulsed with OVA peptide, SIINFEKL, and labeled with higher dose of CFSE (CFSEhi) while untargeted control cells were labeled with a lower dose of CFSE (CSFElow). The in vivo clearance of both type of grafted cells was evaluated 16 h later in LN and spleens by flow cytometry. All NPs, with or without adjuvants, generated the most efficient in vivo killing activity towards the SIINFEKL-pulsed cells, as shown by the disappearance of the CFSEhi peak of cells. Indicating the successful induction of cross-priming and activation of antigenspecific CTL that could potentially lead to a broad and effective immune response pivotal in case of tumor rejections. To test the efficacy of the therapeutic vaccine and to have a good read-out of the antigen-specific T cells, we used B16.MO5 melanoma-bearing mice using different vaccination schedules, single and three time immunization with OVA and adjuvants- loaded NP. The groups immunized with PBS and Empty NP served as a control and presented similar tumor growth. All treated groups showed a significant reduction in tumor growth. EntrapOVA-Adjs NP (3x immunization) presented the slowest tumor growth (more than 12x smaller final tumor volume), followed by the group immunized with AdsOVA-Adjs NP (3x immunization) (more than 9x smaller final tumor volume) and single vaccination with EntrapOVA-Adjs NP (more than 8x smaller final tumor volume). The highest amount of tumor infiltrating lymphocytes (TILs) at the tumor site was detected for EntrapOVA-Adjs NP (3x immunization), suggesting the best remission and survival prognosis of the treated group. Two inflammatory cytokines (TNF-α and IFN-γ) produced by TILs were quantified in tumor microenvironment. 4-time high levels of TNF-α upon 3-time immunizations were detected in tumor microenvironment which kept the tumor growth under control. On the other side in the spleens, repeated vaccination prevented upregulation of both inflammatory markers, TNF-α and IFN-γ, indicating absence of systemic inflammation. Overall, the present work shows that the design of safe but efficacious nanoparticulate cancer vaccines requires a deep understanding of NP biological effect on immune cells, both under steady-state and cancerous environment. A detailed characterization of the imune cell-related pathways modulated by the optimal antigen-loaded NP and their correlation with the nature and anti-tumor efficacy of the induced immune response was performed in vivo in healthy and pathological conditions, showing that the nature of antigen delivery is crucial for a specific T cell activation and targeted cytotoxic effect

Combinatorial Low-Binding Affinity Polymersomes for Targeting Dendritic Cells: Towards Cancer Vaccine Development

Conventional DNA cancer vaccines fail to adequately stimulate the adaptive immune system and to establish proper immunisation. This is reflected in clinical practice, where only a handful of them have been approved by the FDA. Within this project the use of the pH-sensitive polymer Poly (2-(methacryloyloxyethyl phosphorylcholine)-poly(2-(diisopropylamino-ethyl methacrylate) (PMPC-PDPA) has been investigated for DNA antigen encapsulation and intracellular delivery. By implementing a dendritic cellular model (DC2.4), it was demonstrated the expression and surface presentation of the antigen model (SIINFEKL). Furthermore, exploratory experiments highlighted the inflammatory power of the immunomodulator cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) in in vitro settings, with potential implications for in vivo cancer vaccines. Moreover, current strategies of design for active targeting nanoparticles (NPs) are suboptimal and characterised by off-target binding and side effects. In this work, it was demonstrated a paradigm shift in the design of active targeting nanoparticles based on the concepts of the ‘range selectivity’ theory. Specific Ligands for the phenotypic targeting of dendritic cells (DCs) were selected (PMPC, mUNO and PEP4) and conjugated to the respective polymer, such as PMPC-PDPA or polyethylene glycol-poly (lactic acid) (PEG- PLA). Multivalent and multiplexing POs were prepared and tested in vitro, proving experimentally the validity of computational hypotheses. Multivalent and combinatorial POs were also intradermally injected into animal models to further corroborate in vitro experimental evidence. It was envisioned that the implementation of empirical observation combined with in silico simulation would help to define the optimal range of the number of ligands on a vesicle for the phenotypic targeting of DCs, ultimately improving the intracellular co-delivery of antigen and adjuvant for the development of a cancer vaccine

The Shaping of Cancer by the Tumour Microenvironment and Its Relevance for Cancer Therapy

In this book, we present a compilation of original research articles as well as review articles that are focused on improving our understanding of the molecular and cellular mechanisms by which cancer cells adapt to their microenvironment. These include the interplay between cancer cells and the surrounding microenvironmental cells (e.g., macrophages, tumor-infiltrating lymphocytes and myeloid cells) and microenvironmental environments (e.g., oxidative stress, pH, hypoxia) and the implications of this dynamic interaction to tumor radioresistance, chemoresistance, invasion and metastasis. Finally, the importance and relevance of these findings are translated to cancer therapy

Molecular expression and immunotherapeutic potential of the novel tumour associated antigen T21

The application of immunotherapeutic approaches to target tumour cells by harnessing the body’s most important defence the immune system, could provide opportunities to oppose cancer. A prerequisite of tumour immunotherapy lies in the identification of antigens that are distinctively expressed in cancer and can elicit immune responses in the tumour-bearing host. Testis clone 21 (T21) was first identified as a promising prostate-associated tumour antigen identified using modified SEREX expression cloning showing restricted protein expression to normal testis and prostate, breast, kidney, ovary, melanoma, colon and stomach cancer among others. More recently, T21 was found to share significant sequence similarity with a centrosomal protein called CEP290. This study proposed to address the current understanding of T21 through analysis of gene sequence similarities observed with CEP290 and begin to extrapolate possible functional attributes of T21 and the role it may play in cancer progression. Furthermore this investigation aimed to evaluate T21 as a prospective prognostic indicator of prostate cancer and in addition, investigate T21 as a potential target for immunotherapy. In silico sequence analysis revealed that T21 contained a 93bp region, a consequence of an alternative splicing event resulting in the retention of a CEP290 intronic region making its translation to protein unique. This finding was supported by experimental observations using PCR and northern blotting. Using this T21 “unique region” sequence, T21 mRNA and protein was shown to be expressed in some normal tissues including prostate, stomach and spinal cord when compared to testis therefore T21 cannot be considered a cancer testis antigen as previously suggested

GENOME WIDE DISCOVERY OF DISEASE MODIFIERS

Disease modifiers are genes that when activated can alter the expression of a phenotype associated with a disease. This can be done directly through affecting the expression of another gene that is causing the disease, or indirectly by affecting other factors that contribute to the phenotype’s variability. Identification of disease modifiers is of great interest from both treatment and genetic counseling perspectives. We set here to develop computational approaches to identify and study disease modifiers. We focus on two research avenues for studying disease modifiers: (1) One aimed at identifying and investigating modifiers of cancer, a complex disease influenced by multiple genetic and environmental factors, and (2) the other focuses on the identification of disease modifiers for monogenetic disorders which involve a single disease causing gene. Towards the first aim of studying cancer modifiers we take four complimentary approaches. (a) First, we developed a computational approach to identify metabolic drivers of cancer that when applied to colorectal cancer, successfully identified FUT9 as a gene that strongly modifies tumors aggressiveness. (b) Second, to study metabolic pathway-level modifications in cancer, we developed an algorithm that summarizes cancer modifications to generate pathway compositions that best capture cancer associated alterations, which, as we show, enhances cancer classification and survival prediction. (c) Third, to identify modifiers of cancer immunotherapy treatment, we developed a new computational approach that robustly predicts the response to immune checkpoint blockage therapy. (d) Fourth, to identify modifiers of cancer radiotherapy treatment we built a robust predictor of rectal cancer patients’ response to chemo-radiation-therapy (CRT), identifying a signature of genes that may serve a potential targets for modifying patients’ response to CRT. Towards the second aim of studying genetic modifiers of Mendelian diseases, we developed a computational approach for identifying a specific expression pattern associated with genes that are modifying disease severity. We show that we can successfully prioritize genes that are modifying disease severity in cystic fibrosis and spinal muscular atrophy, where we have identified a new modifier and validated it experimentally. As will become evident from reading my dissertation, my work has naturally focused on developing a variety of computational approaches to analyze research questions that were of interest to me. Obviously, my work has greatly benefited and has been significantly enriched by close collaboration with many experimental labs that have kindly embarked on testing the predictions made, and to whom I am indebted. In sum, we developed methods to identify and study disease modifiers for both cancer and Mendelian diseases. The applications of these methods generates a few promising leads for advancing the treatment for these diseases and improving clinical decision-making

The role of STEAP2 in aggressive prostate cancer traits and androgen responses

The prognosis of localised prostate cancer is generally promising, as many tumours remain dormant and therefore do not require immediate intervention. In contrast, once metastasised, the prognosis for aggressive prostate cancer is often poor, highlighting the need for novel, effective treatment approaches. The expression of the six transmembrane epithelial antigen of the prostate2 (STEAP2) cell surface protein is increased in aggressive prostate cancer compared to normal prostate tissue. In vitro studies have shown STEAP2 to aid in prostate cancer progression, and as such this molecule shows promise as a potential novel therapeutic target in the treatment of advanced disease. The aim of this thesis was to develop a comprehensive understanding of the mechanistic role of STEAP2 in promoting aggressive prostate cancer traits and evaluate if its knock-out has the capacity to reduce the invasive potential of prostate cancer cells in vitro. As prostate cancer is a largely androgen dependent disease, this thesis also aimed to evaluate the effects of STEAP2 inhibition on the expression of the androgen receptor and androgen-regulated genes. This study developed and optimised a protocol for generating a set of 3D prostate cancer spheroids to provide more representative models of the in vivo prostate cancer environment. In this thesis, one commercial anti-STEAP2 polyclonal antibody and a panel of anti-STEAP2 monoclonal antibodies were selected for proof-of-concept studies where their effects on reducing prostate cancer cell viability were assessed. Receptor internalisation of STEAP2 was evaluated upon anti-STEAP2 monoclonal antibody binding to determine its suitability for use with antibody-drug conjugate technology. STEAP2 expression was knocked out using CRISPR/Cas9 genome engineering technology in two prostate cancer cell lines to evaluate its impact on cell proliferation, migration and invasion. Furthermore, gene expression profiling was conducted to explore interactions between STEAP2, the androgen receptor and a panel of androgen-regulated genes (PSA, FKBP5, GPRC6A and TMPRSS2) following: 1) anti-STEAP2 antibody treatment, 2) STEAP2-knockout and 3) the growth of prostate cancer cells in androgen-depleted conditions. The data presented in this thesis demonstrate that inhibition of STEAP2 by both the polyclonal anti-STEAP2 antibody and lead anti-STEAP2 monoclonal antibody significantly reduced prostate cancer cell viability. STEAP2 receptor internalisation was triggered following treatment of prostate cancer cells with the anti-STEAP2 monoclonal antibody, demonstrating its potential utility with antibody-drug conjugate technology in the future. STEAP2 knockout prostate cancer cells exhibited decreased cell proliferation, migration and invasion in comparison to wild-type cells. These promising findings highlight the therapeutic value of STEAP2-knockout in inhibiting invasive tumour cell traits. Gene expression data from both STEAP2-knockout cells and androgen-depleted cells suggest that STEAP2 may be involved in crosstalk between the androgen receptor and androgen-regulated genes. STEAP2 could therefore provide a novel target in conjunction with current conventional androgen deprivation therapy. In conclusion, the in vitro findings presented herein suggest STEAP2 as a viable target for the development of more tailored and personalised therapeutic agents to improve the clinical management of men with aggressive prostate cancer

Strategies for targeting the tumour microenvironment using high fidelity 3D culture systems

The heterogenous tumour microenvironment (TME) has been well established in the progression of cancer and patient outcome. In particular, the success of anticancer therapy is influenced by various constituents and intercommunicating networks within the TME. Drug development research often focuses on targeting cancer cells alone without considering all the cellular and non-cellular components of the TME, which plays a crucial role in chemoresistance and disease relapse. Thus, there is a necessity to understand the individual features of the TME in order to deliver a more personalised therapy that improves the rate of successful outcomes while reducing the recurrence of the disease. However, the mechanisms of acquired drug resistance and pharmacokinetics within the tumour still remains elusive. Currently, there is a lack of high-throughput methods suitable to study the functional effects of anticancer drugs on tumour samples where the interplay between cancer cells and the TME remains intact. This thesis embodies the work in the development of the ALTEN (Alginate-based Tissue Engineering) platform, a biomimetic hydrogel system for rapid functional testing of anticancer drugs in explanted tumours. The alginate hydrogels provides a 3D scaffold that resembles the native extracellular matrix (ECM) that preserve the original characteristics of the tumour to permit detailed assessment of the TME and the complex molecular interactions between cell species. Here, we use high-resolution single-cell RNA-seq technologies to analyse the molecular effects of anticancer treatments within the tumour to study the mechanisms of acquired drug resistance. The combination of ALTEN and scRNA-seq technology enable high-throughput and high-resolution screening of tumour explants exposed to cytotoxic agents and immunomodulators. The impact of drug therapies within an intact TME could be evaluated to 1) determine treatment efficacy and 2) characterise drug resistant cancer populations. In summary, the ALTEN platform provides an innovative avenue allowing engineering of patient tumour explants to investigate the efficacy of therapies in a high-throughput manner and provide preclinical information on optimising treatments. The integration of both the high-fidelity drug testing capacity of ALTEN and the high-resolution molecular phenotyping scRNA-seq aims to assist oncologists in providing personalised therapies to improve patient outcome