Design of new strategies for breast cancer diagnosis

Dissertação de mestrado em BioengenhariaCancer represents a public health problem worldwide due to the high incidence, prevalence and mortality in the human population, mainly in developed countries. Adoption of a cancerassociated lifestyle and population aging are considered as the main reasons for the increase in the burden of cancer. Early diagnosis is important since treatment is most effective. The difficulty of diagnosing cancer patients at an early stage of the disease may lead to lower rates of successful treatment, so new diagnostic tools are required. Currently, diagnoses of many types of cancers include only non-invasive examinations and biopsies. Cancer diagnosis can be very sensitive if based on molecular features, but the lack of molecular tools makes the cancer understanding at this level a difficult task. Elucidating the molecular features of specific tumors can increase our knowledge about the mechanisms underlying disease development and progression. In this work, cell-based systematic evolution of ligands by exponential enrichment (Cell- SELEX) was used as an approach to evolve aptamers capable of specifically discriminate between human breast carcinoma MDA-MB-435 cells and mouse embryonic fibroblast 3T3 cells, used as non-target cells. The selected aptamers were characterized taking into account their primary sequences and homologies between them; the conserved domains of secondary structures were also analyzed. Two different sequences, selected against a different breast cancer cell lines, and presenting near 100% homology were used as a comparison when the sequences that target MDA-MB-435 were used. Then, the carbodiimide methodology was utilized for the conjugation of amine labeled aptamer with silica particles. For the characterization of this ligation, the colorimetric method was used. Binding assays were performed using FAM (Fluorescein Amidite) aptamer and aptamerfunctionalized silica particles. For the aptamer 1A selected for breast cancer cell line MDA-MB-435 results were promising. Hybridization seems to occur with the breast cancer cells visible under microscope or flow cytometry. For the negative control cell line no fluorescence was detected. Unlike, the aptamer 2A did not exhibit fluorescence when incubated with target cells. This could indicate that aptamer 1A have a good affinity and selectivity to discriminate markers in the cell surface meaning a successful Cell-SELEX for the cell lines in study.O cancro representa um problema de saúde pública em todo o Mundo devido à sua alta incidência, prevalência e mortalidade na população, principalmente em países desenvolvidos. A adoção de um estilo de vida de risco e o envelhecimento são consideradas as principais razões para o aumento da incidência do cancro. Um diagnóstico precoce é muito importante para um tratamento mais eficaz. A dificuldade de diagnóstico de um paciente com cancro numa fase inicial da doença pode levar a menores taxas de sucesso do tratamento, por isso novas ferramentas de diagnóstico são necessários. Atualmente, o diagnóstico dos vários tipos de cancro incluem exames não invasivos e biópsias. O diagnóstico do cancro pode ser muito sensível se baseado em características moleculares, mas a falta de ferramentas moleculares faz com que a compreensão do cancro a este nível seja uma tarefa difícil. A elucidação das características moleculares de tumores específicos pode aumentar o nosso conhecimento acerca de mecanismos subjacentes ao desenvolvimento e progressão da doença. Neste trabalho foi utilizado o Cell-SELEX para selecionar aptamers capazes de especificamente discriminar entre células do carcinoma da mama humano MDA-MB-435 e fibroblastos embrionários de rato 3T3 como células não alvo. Os aptamers selecionados foram caracterizados tendo em conta as suas sequências primárias e as homologias entre elas; também as regiões conservadas das estruturas secundárias foram analisadas. Duas sequências diferentes, selecionadas para uma linha de cancro de mama diferente e apresentando uma homologia de quase 100% foram usadas como comparação onde as sequências com o alvo MDA-MB-435 forem usadas. Depois, a metodologia de carbodiimide foi utilizada para a conjugação do aptamer marcado com partículas de sílica. Para a caracterização desta ligação, um método colorimétrico foi utilizado. Os ensaios de ligação foram feitos utilizando o aptamer marcado com FAM e as partículas de sílica funcionalizadas com o aptamer. Para o aptamer 1A selecionado para a linha celular MDA-MB-435 os resultados foram promissores. Parece ocorrer hibridização com as células de cancro da mama visíveis quer por microscopia quer por citometria. Para a linha celular de controlo nenhuma fluorescência foi detetada. Ao contrário, o aptamer 2A não apresentou nenhuma fluorescência quando incubado com as células alvo. Isto pode indicar que o aptamer 1A tem uma boa afinidade e seletividade para descriminar marcadores na superfície das células o que significa um Cell-SELEX bem-sucedido para as linhas celulares em estudo

Glucose metabolism reprogramming in bladder cancer: hexokinase 2 (HK2) as prognostic biomarker and target for bladder cancer therapy

Proliferating cancer cells are able to reprogram their energy metabolism, favouring glycolysis even in the presence of oxygen and fully functioning mitochondria. Research is needed to validate the glycolysis-related proteins as prognostic/predictive biomarkers in urothelial bladder carcinoma (UBC), a malignancy tagged by high recurrence rates and poor response to chemotherapy. Here, we assessed GLUT1, HK2, PFKL, PKM2, phospho-PDH, and LDHA immunoexpression in 76 UBC samples, differentiating among urothelial, fibroblast, and endothelial cells and among normoxic versus hypoxic areas. We additionally studied the functional effects of the HK2 inhibitor 2-deoxy-D-glucose (2DG) in “in vitro” and “in vivo” preclinical UBC models. We showed that the expression of the glycolysis-related proteins is associated with UBC aggressiveness and poor prognosis. HK2 remained as an independent prognostic factor for disease-free and overall survival. 2DG decreased the UBC cell’s viability, proliferation, migration, and invasion; the inhibition of cell cycle progression and apoptosis occurrence was also verified. A significant reduction in tumour growth and blood vessel formation upon 2DG treatment was observed in the chick chorioallantoic membrane assay. 2DG potentiated the cisplatin-induced inhibition of cell viability in a cisplatin-resistant subline. This study highlights HK2 as a prognostic biomarker for UBC patients and demonstrates the potential benefits of using 2DG as a glycolysis inhibitor. Future studies should focus on integrating 2DG into chemotherapy design, as an attempt to overcome cisplatin resistance.The work presented herein was performed at the Life and Health Sciences Research Institute (ICVS), University of Minho. Financial support was provided by the Scientific Microscopy Platform of ICVS, member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122), by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020, and by the projects NORTE 01-0145-FEDER-000039 and NORTE-01-0145-FEDER-000055, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.A. was supported by FCT (fellowship ref. SFRH/BPD/116784/2016). C.G. was supported by Programme NORTE 2020 [CTTI-117/21-ICVS(1)] and FCT (contract ref. 2021.02600.CEECIND). D.F. was supported by “Liga Portuguesa contra o Cancro—Núcleo Regional do Norte” (fellowship ref. LPCC-NRN).info:eu-repo/semantics/publishedVersio

Novel biorecognition elements against pathogens in the design of state-of-the-art diagnostics

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020.info:eu-repo/semantics/publishedVersio

Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with Kd values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis. © 2021, The Author(s).Tis study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Joaquim Barbosa (JB) and Diana A. Sousa (DAS) acknowledge FCT for the Grants SFRH/BD/51109/2010 and PD/BD/139083/2018, respectively.info:eu-repo/semantics/publishedVersio

Electrochemical aptasensor for the detection of the key virulence factor YadA of Yersinia enterocolitica

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and of LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Exosomes modified with anti-MEK1 siRNA lead to an effective silencing of triple negative breast cancer cells

Available online 28 September 2023Triple negative breast cancer (TNBC) is a highly heterogenous disease not sensitive to endocrine or HER2 therapy and standardized treatment regimens are still missing. Therefore, development of novel TNBC treatment approaches is of utmost relevance. Herein, the potential of MAPK/ERK downregulation by RNAi-based therapeutics in a panel of mesenchymal stem-like TNBC cell lines was uncovered. Our data revealed that suppression of one of the central nodes of this signaling pathway, MEK1, affects proliferation, migration, and invasion of TNBC cells, that may be explained by the reversion of the epithelial-mesenchymal transition phenotype, which is facilitated by the MMP-2/MMP-9 downregulation. Moreover, an exosome-based system was successfully generated for the siRNA loading (iExoMEK1). Our data suggested absence of modification of the physical properties and general integrity of the iExoMEK1 comparatively to the unmodified counterparts. Such exosome-mediated downregulation of MEK1 led to a tumor regression accompanied by a decrease of angiogenesis using the chick chorioallantoic-membrane model. Our results highlight the potential of the targeting of MAPK/ERK cascade as a promising therapeutic approach against TNBC.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2020 unit. CAM and immunohistochemistry experiments have been funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122); by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/ 2020 and UIDP/50026/2020 and by the project NORTE-01-0145- FEDER-000039, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Débora Ferreira is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Débora Ferreira also acknowledges “Liga Portuguesa contra o cancro - Núcleo Regional do Norte (LPCC-NRN)” for her fellowship. The authors thank Diana Vilas Boas (CEB/University of Minho) for confocal microscopy technical support. Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral program in Applied and Environmental Microbiology (DP_AEM). Julieta Afonso acknowledges the Postdoctoral fellowship SFRH/BPD/116784/2016 funded by FCT. The work performed in the Kalluri Laboratory was supported by funds from UT MD Anderson Cancer Center and the Sid Richardson Foundation.info:eu-repo/semantics/publishedVersio

Sistema de libertação decorado com péptidos para tratamento de cancro de mama triplo negativo

Doctoral program in BioengineeringO cancro de mama é um grande problema de saúde a nível mundial, particularmente o cancro de mama triplo negativo (CMTN) que é responsável por aproximadamente 10-20% de todos os casos de cancro de mama. Apesar das novas opções de tratamento disponíveis, a quimioterapia continua a ser a base de tratamento do cancro e como tal, a procura por novos ligandos e com elevada especificidade, como por exemplo os aptâmeros e péptidos, é crucial para o desenvolvimento de novas terapias dirigidas ao alvo. A combinação de cell-SELEX com o sequenciamento de nova geração e análise bioinformática levou à identificação de sequências de aptâmeros (Apt1 and Apt2) com grande afinidade e especificidade para a linha celular metastática CMTN MDA-MB-231. A possível translação para a prática clínica ficou demonstrada para o Apt2. Adicionalmente foram usadas bibliotecas de phage display para selecionar péptidos com afinidade para linhas celulares CMTN. Dois novos péptidos (4T1pep1 e 4T1pep2) exibiram ligação específica à linha celular CMTN 4T1, que é um modelo preciso para cancro de mama em estágio IV. As análises bioinformáticas sugeriram que ambos os péptidos reconhecem Mucin-16 humana, proteína que está implicada no cancro de mama. A identificação de dois novos péptidos (231Pep1 e 231Pep2) visando especificamente a linha celular MDA-MB-231 é também aqui descrita. As abordagens de bioinformática mostraram que o 231Pep1 e 231Pep2 reconhecem especificamente os biomarcadores associados com cancro de mama TIMP-1 e PAI1, respetivamente. No que diz respeito à libertação controlada de drogas, os exossomas apresentam várias propriedades vantajosas. Nesse sentido, desenvolveu-se com sucesso uma plataforma baseada no uso de exossomas para incorporação de moléculas de RNAi para o silenciamento da via MAPK/ERK em células CMTN. O silenciamento mediado por este sistema levou a uma regressão do tumor e a uma diminuição da angiogénese in vivo. Alguns dados preliminares sobre o papel da via PI3K/AKT/mTOR em células de cancro de mama mostraram resultados diferentes, dependendo do estado da mutação PIK3CA. Além disso, a combinação simultânea de moléculas de RNAi direcionadas para as vias acima descritas levou a um aumento da apoptose em células MDA-MB-231 em comparação com o efeito isolado. Em suma, os resultados reunidos nesta tese contribuíram para o objetivo de desenvolver novas abordagens terapêuticas para CMTN através da identificação de novos ligandos específicos para posterior incorporação em exossomas, bem como avaliação da supressão das vias de sinalização complexas MAPK/ERK e PI3K/AKT/mTOR.Breast cancer is a major health problem worldwide. In particular, triple negative breast cancer (TNBC) accounts for approximately 10-20% of all breast cancers and, despite the novel treatment options available, cytotoxic chemotherapy remains the mainstay of treatment. Therefore, finding novel and specific targeting ligands, including aptamers and peptides, is crucial for the development of new targeted therapies. The combination of cell-SELEX with high throughput sequencing and bioinformatic analysis led to the identification of ssDNA aptamer sequences (Apt1 and Apt2) with high affinity and specificity towards the metastatic TNBC cell line MDA-MB-231. The potential translation application to a clinical setting was proven for Apt2. In addition, phage-based libraries were also used to select peptides with affinity for TNBC cell lines. Two novel peptides (4T1pep1 and 4T1pep2) were found to exhibit specific binding to the 4T1 cell line, an accurate xenograft model for stage IV of human breast cancer. Bioinformatic analysis suggested that both peptides target the human Mucin-16, which is implicated in breast cancer. The identification of two novel peptides (231Pep1 and 231Pep2) specifically targeting MDA-MB-231 cell line was also herein reported. Bioinformatic approaches showed that 231Pep1 and 231Pep2 specifically target the breast cancer associated biomarkers TIMP-1 and PAI1, respectively. Regarding drug delivery, exosomes present several distinct advantageous properties. Hence, a successful exosome-based platform for the loading of RNAi-based therapeutics was developed for the downregulation of MAPK/ERK cascade in a panel of TNBC cell lines. This exosome-mediated downregulation led to a tumor regression and a decrease of angiogenesis in vivo. Some preliminary data on the role of PI3K/AKT/mTOR in breast cancer cells showed different outcomes depending on the PIK3CA mutation status. Moreover, the simultaneous combination of RNAi-therapeutics targeting the abovementioned pathways led to an enhanced apoptosis induction in MDA-MB-231 cells compared with the effect of single inhibitors. Overall, the results gathered in this thesis contributed to the goal of developing novel therapeutic approaches for TNBC through the identification of novel TNBC-specific ligands for posterior incorporation into exosomes, as well as the suppression assessment of the complex signaling cascades MAPK/ERK and PI3K/AKT/mTOR.I would like to acknowledge my fellowship (UMINHO/BD/21/2016) supported by the doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, as well as the strategic funding of UIDB/04469/2020 unit

New advances in exosome-based targeted drug delivery systems

Online ahead of printIn recent years, various drug nano-delivery platforms have emerged to enhance drug effectiveness in cancer treatment. However, their successful translation to clinics have been hampered by unwanted side effects, as well as associated toxicity. Therefore, there is an imperative need for drug delivery vehicles capable of surpassing cellular barriers and also efficiently transfer therapeutic payloads to tumor cells. Exosomes, a class of small extracellular vesicles naturally released from all cells, have been exploited as a favorable delivery vehicle due to their natural role in intracellular communication and biocompatibility. In this review, information on exosome biogenesis, contents, forms of isolation and their natural functions is discussed, further complemented with the various successful methodologies for therapeutic payloads encapsulation, including distinct loading approaches. In addition, grafting of molecules to improve pharmacokinetics, tumor homing-ligands, as well as stimuli-responsive elements to enhance cell specificity are also debated. In the end, the current status of clinical-grade exosome-based therapies is outlined.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and projects POCI-01-0145-FEDER-016390 (CancelStem), CENTRO-01-0145-FEDER-000012-HealthyAging2020, and CIBB (FCT reference UIDB/04539/2020). Débora Ferreira (DF) is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Lactoferrin perturbs intracellular trafficking, disrupts cholesterol-rich lipid rafts and inhibits glycolysis of highly metastatic cancer cells harbouring plasmalemmal V-ATPase

Available online 16 September 2022The milk-derived bovine lactoferrin (bLf) is an iron-binding glycoprotein with remarkable selective anticancer activity towards highly metastatic cancer cells displaying the proton pump V-ATPase at the plasma membrane. As studies aiming to dissect the bLf mechanisms of action are critical to improve its efficacy and boost its targeted clinical use, herein we sought to further uncover the molecular basis of bLf anticancer activity. We showed that bLf co-localizes with V-ATPase and cholesterol-rich lipid rafts at the plasma membrane of highly metastatic cancer cells. Our data also revealed that bLf perturbs cellular trafficking, induces intracellular accumulation of cholesterol and lipid rafts disruption, downregulates PI3K and AKT or p-AKT and inhibits glycolysis of cancer cells harbouring V-ATPase at the plasma membrane lipid rafts. Altogether, our results can lay the foundation for future bLf-based targeted anticancer strategies as they unravel a novel cascade of molecular events that explains and further reinforces bLf selectivity for cancer cells displaying plasmalemmal V-ATPase.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and “Contrato-Programa” UIDB/04050/2020; and by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral pro- gramme in Applied and Environmental Microbiology (DP_AEM). Joana P. Guedes acknowledges the PhD fellowship SFRH/BD/132070/2017 funded by FCT. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norteinfo:eu-repo/semantics/publishedVersio