Cellular interactions in the tumor microenvironment: the role of secretome

Over the past years, it has become evident that cancer initiation and progression depends on several components of the tumor microenvironment, including inflammatory and immune cells, fibroblasts, endothelial cells, adipocytes, and extracellular matrix. These components of the tumor microenvironment and the neoplastic cells interact with each other providing pro and antitumor signals. The tumor-stroma communication occurs directly between cells or via a variety of molecules secreted, such as growth factors, cytokines, chemokines and microRNAs. This secretome, which derives not only from tumor cells but also from cancer-associated stromal cells, is an important source of key regulators of the tumorigenic process. Their screening and characterization could provide useful biomarkers to improve cancer diagnosis, prognosis, and monitoring of treatment responses.Agência financiadora Fundação de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) FAPESP 10/51168-0 12/06048-2 13/03839-1 National Council for Scientific and Technological Development (CNPq) CNPq 306216/2010-8 Fundacao para a Ciencia e a Tecnologia (FCT) UID/BIM/04773/2013 CBMR 1334info:eu-repo/semantics/publishedVersio

Renormalized and Entropy Solutions of Tumor Growth Model with Nonlinear Acid Production

This paper establishes the existence of renormalized and entropy solutions for a system of nonlinear reaction-diffusion equations which describes the tumor growth along with acidification and interaction. Under the assumptions of L1 data and no growth conditions with zero Dirichlet boundary conditions, we prove the existence of renormalized and entropy solutions for the considered mathematical model

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

Glioma-associated mesenchymal stem cells have profound effects on brain tumors

GBMs have proven to be a major pathology with vast infiltration potential and extreme chemo- and radio-resistance leading to devastating outcomes. The GBMs, however, are not only detrimental on the host alone but also they interact with the microenvironment to manifest some of their pathological hallmarks. Within the tumor microenvironment, MSCs have gained a strong attention in the recent years. The role of MSCs within the brain tumor niche has been partially explained including pro- and anti-tumorigenic effects. It was, however, a controversial issue as to what makes MSCs pro- or anti-tumorigenic. Moreover, the contribution of MSCs in the brain tumor histopathology was not yet fully uncovered. Here I investigated the role of MSCs in the brain tumor pathology and the signaling mechanisms. It was shown earlier in our lab that MSCs act as anti-tumorigenic in the presence of serum and anti-tumorigenic in serum-free conditions. Which condition is more relevant to the pathological situation was an open question. When I co-inoculated MSCs with GBMs, they homed the tumor satellites, where they are probably not in contact with blood-borne factors. Therefore, I concluded that serum-free conditions are more relevant to in vivo pathological situation. MSCs promoted viability of many primary GSC lines under serum-free conditions. The induction of survival and proliferation was mediated by the increase in the levels of QKIs in GBMs upon exposure to MSC-derived soluble factors. In turn, QKIs increase the levels of EGFR causing an overresponse to growth factors. Moreover, increased QKI-levels in GBMs mediate the chemoresistance against TMZ. In addition to soluble factors, MSCs signal via exosomes. Those exosomes carry EFNA3 mRNA from MSCs to GBMs. EFNA3 expression in turn induces cellular migration. The inhibition of EFNA3 transfer via genetically engineered antibodies against exosome docking sites on the recipient GBM cells or against EFNA3 (if it is translated and integrated on the surface) as well as EGFR-blockade might prove useful for future therapeutic approaches against GBMs. There are no well-established mouse models to study the interaction between MSCs and GBMs at the tumor satellite. Therefore, I sought to establish such a model by expressing HSV-TK in GBMs and inducing cell death via GCV administration either by osmotic pumps or via systemic injection and established a model where one can study tumor satellites. Additionally, I established a model to study the mRNA transfer from MSCs to GBMs. For this, I expressed Cre-recombinase in MSCs, and flipped- and floxed-GFP in GBMs. The Cre-recombinase mRNA is packed into exosomes and delivered to GBMs, labelling GBMs with GFP for the rest of their lives. This enables us to demonstrate the transfer of mRNA as well as to track individual GBM cells, contacted by the MSCs, in terms of their migratory behaviors in vivo. All in all, I uncovered a previously unknown action of MSCs in GBM pathology defining two targetable systems (EGFR and EFNA3) and established two models to study in vivo interaction of MSCs with GBMs

Tackling Metastasis with Gold Nanoparticles: Inhibition of Intercellular Communication Mediated by Cancer Cells-Derived Exosomes

Metastases are accountable for at least 66.7% of cancer deaths, implying a terminal illness for the great majority of patients diagnosed with metastatic cancer. The last and rate-limiting step in invasion-metastasis cascade is colonization. Colonization is the process by which disseminated tumour cells penetrate distant tissues and adapt to the foreign “soil” in order to prosper. To hasten colonization, cancer cells often secrete soluble factors and extracellular vesicles such as exosomes through systemic circulation, which ultimately lead to creation of tumour permissive microenvironments at secondary organs, pre-metastatic niches. Exosomal integrins α6β1 and α6β4 secreted by MDA-MB-231 cells, a human breast cancer cell line, were found to specifically fuse with lung-resident fibroblasts and epithelial cells. The cargo release within recipient cells activates Src and upregulates pro-inflammatory S100 genes, which generates lung pre-metastatic niches and promotes lung tropic metastasis. In this case, cell-cell communication via exosomes between primary tumour cells and microenvironment of distant organs is a key mediator of metastasis progression. Taken into account the recent bet on antisense oligonucleotides therapeutics and the advent of nanotechnology, a gold-nanoconjugate with gene silencing activity was produced. This was achieved by functionalizing gold nanoparticles with an antisense oligonucleotide designed to silence ITGA6, the gene that encodes the α6 subunit, present in integrins α6β1 and α6β4. The silencing efficacy was evaluated using MDA-MB-231 cells at the RNA and protein levels, and the existence of phenotypical changes between untreated and treated cells was assessed. The nanoconjugate showed silencing activity. Reduced cellular levels of integrins α6β1 and α6β4 may lead to diminished packaging of these receptors into exosomes and impairment of integrin-mediated lung tropic metastasis. This nanoscale approach has potential to be employed to prevent lung metastasis, but further studies are required.Pelo menos 66.7% das mortes por cancro são atribuídas à presença de metástases, implicando uma doença terminal para a grande maioria dos pacientes diagnosticados com cancro metastático. O passo limitante da cascata de invasão-metastização é o último passo, a colonização. A colonização é o processo pelo qual células tumorais disseminadas penetram tecidos distantes e se adaptam a “solo” desconhecido de modo a prosperar. Para acelerar o processo de colonização, as células cancerígenas libertam frequentemente fatores solúveis e vesículas extracelulares como os exossomas através da circulação sistémica, o que conduz à criação de microambientes permissivos ao desenvolvimento tumoral noutros órgãos, os nichos pré-metastáticos. Descobriu-se que as integrinas exossomais α6β1 e α6β4, libertadas por células do cancro da mama, se ligam especificamente a fibroblastos e células epiteliais residentes nos pulmões. A libertação do conteúdo exossomal nas células recetoras ativa a proteína Src e aumenta a expressão dos genes pro-inflamatórios S100. Esta interação leva à formação de nichos pré-metastáticos no pulmão e promove a metastização com tropismo para o pulmão. Neste caso, a comunicação célula-célula via exossomas entre o tumor primário e microambientes de órgãos distantes é o mediador chave da progressão metastática. Tendo em conta a recente aposta nas terapêuticas de oligonucleótidos antisense e o advento da nanotecnologia, foi produzido um nanoconjugado de ouro com atividade de silenciamento génico. Isto foi alcançado através da funcionalização de nanopartículas de ouro com um oligonucleótido antisense desenhado para silenciar ITGA6, o gene que codifica para a subunidade α6 das integrinas α6β1 e α6β4. A eficácia do silenciamento foi avaliada ao nível do RNA e da proteína usando células MDA-MB-231, e foi verificada também a existência de alterações fenotípicas entre células tratadas e não tratadas. O nanoconjugado demonstrou atividade de silenciamento génico. A redução dos níveis de integrinas α6β1 e α6β4 pode levar à diminuição do empacotamento destes recetores nos exossomas e travar a metastização com tropismo para o pulmão mediada por integrinas. Esta estratégia à nanoescala tem potencial para ser utilizada para prevenir a metastização nos pulmões, mas são necessários mais estudos

Increasing pH in cancer: enabling a new therapeutic paradigm using novel carbonate nanoparticles

Enormous progress has been made to treat cancer, and yet the mortality rate of cancer remains unacceptably high. High clinical resistance to molecularly targeted therapeutics has pushed interest again towards inhibiting universal biochemical hallmarks of cancer. Recent evidence suggests that malignant tumors acidify the local extracellular environment to activate proteases for degrading the tumor matrix, which facilitates metastasis, and explains why more aggressive tumors are more acidic. Current therapies have only focused on using the low pH for enhancing drug release in tumors, thereby still relying on the traditional paradigm of intracellular inhibition of pathways, a method that continues to have mixed results. In this dissertation we explore the development of a novel platform that can be made to monitor and modify the critical tumor extracellular environment. The platform enables a shift in the paradigm of current cancer therapy from a predominantly intracellular approach to an extracellular synergistic method of targeting cancer. In the process, we demonstrate the synthesis of a novel type of CaCO3 nanoparticle, its stabilization, the increase of pH in vivo, a mathematical justification and simulation, and the subsequent inhibition of tumor growth and metastasis. By neutralizing the tumor extracellular microenvironment, the platform aims to prevent tumor progression and metastasis as well as minimize the traditional intracellular based pathways of resistance. Because of the broad universal aspect of low extracellular pH in cancer, particularly in the more malignant tumors, we expect this platform will also have wide applicability, with particular potency on the most aggressive of cancers

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

Tumor Cell Migration and Interaction with ECM and Stroma in 3D Tissue Models

Tumors are often described as ”wounds that do not heal”. Tumor progression and woundhealing both feature sustained proliferative signaling, evasion from immune destruction, cell death resistance, inflammation, angiogenesis, extracellular matrix(ECM) remodeling, and activated cell migration. Unlike normal wound healing which often ends up with restored tissue homeostasis, pathological ECM remodeling is frequently implicated in fibrotic diseases and solid tumors. In addition, the dysregulated ECM signatures are directly associated with poor prognosis and also immunotherapy failure in certain types of cancers. In this dissertation, we used 3D in vitro cocultures to understand how tumor cells co-op with stromal/immune components, how ECM remodeling is hijacked, and how ECM architecture impacts tumor progression. We first investigated the impact of fibroblasts. Fibroblasts are the most abundant cell types in the tumor stroma. The density of cancer-associated fibroblasts(CAFs) has been shown directly correlated with poor prognosis in some types of solid tumors. To uncover potential mechanisms behind the quantitative relationship between CAFs and tumor dissemination, we developed our coculture model by varying the density ratios between normal human lung fibroblasts and breast cancer cells(MDA-MB-231s). We found that fibroblasts increase tumor cell motility and facilitate the transition from confined to diffusive tumor cell motions, indicative of an uncaging effect. Furthermore, the ECM is globally and locally remodeled substantially with the presence of fibroblasts. Moreover, these fibroblast-mediated phenomena are in part dependent on matrix metalloproteinases. We then investigated the impact of macrophages. In this study, we developed a 3D collagen co-culture system to mimic the melanoma microenvironment and investigate how interactions between melanoma cancer cells, fibroblasts, and macrophages shape the early stages of macrophage immune activity. In this in vitro model, we captured the macrophage immunosuppressive transition. Macrophages in the model displayed increased motility and acquired a phenotype that was similar to tumor-associated macrophages(TAMs) from melanoma tumors. This model may provide a platform for further studies on TAMs targeted immune therapy in melanoma. In the end, we investigated the impact of ECM architecture in tumor progression. Reconstruction of a biomimetic scaffold is critical in 3D in vitro models. Here we introduce a new type of thick collagen bundles that highly mimic in vivo ECM structure. We fabricated this type of thickened collagen bundles by introducing mechanical agitation during the transient gelation process. Thickened collagen patches are interconnected with a loose collagen network, highly resembling collagen architecture in human skin scars. This type of thickened collagen bundles promotes tumor cell dissemination. The effect is significantly augmented in the presence of fibroblasts. The application of this type of collagen triggers different morphology and migration behaviors of tumor cells and highlights the importance of mesoscale architectures. Overall, this dissertation investigated the roles of stromal and non-stromal components in tumor progression through 3D in vitro models. The coculture models established in this dissertation may be further extended to test novel therapeutics targeted at CAFs, TAMs or ECM architecture

Identifying and targeting vulnerabilities in cancers that metastasize to the bone and bone marrow

Evolving technologies enable scientist to ask and seek answers to advanced questions, above and beyond. During the past decade single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of biology, in particular within cancer research. Contributing with its high-resolution has unraveled complicated biological circuits, along with a profound understanding of heterogeneity, cellular and molecular processes, including gene expression profiles and transcriptional states. Especially in the quest of understanding cancer metastasis, aggressive disease phenotype and treatment resistance. In Paper I we utilize scRNA-seq on tissue material from untreated human primary clear cell renal cell carcinoma (ccRCC) patients. We dissect the tumor microenvironment (TME) and discover a metastatic gene signature that is upregulated in bone metastatic ccRCC patients that could serve as a predictive tool. We describe an immunosuppressive microenvironment with tumor associated macrophages (TAMs) with a M2 phenotype overexpressing TREM2, further associated with low survival outcome. Computational interactive receptor-ligand analysis reveals CD70 (on tumor) and CD27 (on cytotoxic T cells and regulatory T cells) as a potential pharmaceutical target. In Paper II, a continuation of Paper I, we aimed to understand genetic alterations involved in metastatic disease from the primary site, namely bone metastatic ccRCC, including comparison of a normal bone marrow to malignant. Scrutinizing the complex TME we detect, as well here, an immunosuppressive microenvironment with transcriptionally different TAMs, exhausted T cell phenotype and a tumor associated mesenchymal stem/stromal cell (TA-MSC) population that upregulates bone remodeling genes. Concluding that the relevance of TA-MSC subset causes excessive bone resorption via RANK/RANKL/OPG signaling pathway. Finally, in Paper III, we evaluate brequinar, a DHODH inhibitor, as a therapeutic agent in high-risk neuroblastoma (NB). We find DHODH as an independent risk factor in high-risk NB demonstrating prolonged survival in preclinical models when inhibited. In combination with temozolomide, already in clinical use for these children, a synergistic effect is achieved in vivo, proposing a promising approach to validate clinically. Taken together, the presented thesis has provided comprehensive insights in the progression of ccRCC in a primary and a metastatic setting, including the identification of potential vulnerable targets. Additionally, a potential combination treatment in vitro and in vivo is presented as a promising treatment option in high-risk NB by inhibiting DHODH together with standard care of treatment agent temozolomide