Tesis sobre el tema "Lung Stem Cell"

Small cell lung cancer (SCLC) is a highly aggressive malignancy with extreme mortality and morbidity. Although initially chemo- and radio-sensitive, almost inevitable recurrence and resistance occurs. SCLC patients often present with metastases, making surgery not feasible. Current therapies, rationally designed on underlying pathogenesis, produce in vitro results, however, these have failed to translate into satisfactory clinical outcomes. Recently, research into cancer stem cells (CSCs) has gained momentum and form an attractive target for novel therapies. Based on this concept, CSCs are the cause of neoplastic tissue development that are inherently resistant to chemotherapy, explaining why conventional therapies can shrink the tumour but are unable to eliminate the tumour completely, leading to eventual recurrence. Here I demonstrate that SCLC H345 and H69 cell lines contain a subset of cells expressing CD133, a known CSC marker. CD133+ SCLC sub-population maintained their stem cell-like phenotype over a prolonged period of culture, differentiated in appropriate conditions and expressed the embryonic stem cell marker Oct-4 indicating their stem-like phenotype. Additionally, these cells displayed augmented clonogenic efficacy, were chemoresistant and tumorigenic in vivo, distinct from the CD133- cells. Thus, the SCLC CD133 expressing cells fulfil most criteria of CSClike definition. The molecular mechanisms associated with CD133+ SCLC chemoresistance and growth is unknown. Up-regulated Akt activity, a known promoter of resistance with survival advantage, was observed in CD133+ SCLC cells. Likewise, these cells demonstrated elevated expression of Bcl-2, an anti-apoptotic protein compared to their negative counterpart explaining CD133+ cell chemoresistance phenotype. Additionally, CD133+ cells revealed greater expression of neuropeptide receptors, gastrin releasing peptide (GRP) and V1A receptors compared to the CD133- cells. Addition of exogenous GRP and arginine vasopressin (AVP) to CD133+ SCLC cells promoted their clonogenic growth in semi-solid medium, illustrating for the first time neuropeptide dependent growth of these cells. A novel peptide (peptide-1) was designed based on the known structure of the substance P analogues that have shown benefit in animal models and in early clinical trials. This compound inhibited the growth of SCLC cells in in vitro with improved potency and stability compared to previous analogues and reduced tumorigenicity in vivo. Interestingly, peptide-1 was more effective in CD133+ cells due to increased expression of neuropeptide receptors on these cells. In conclusion, my results show that SCLC cells retain a sub-population of cells that demonstrate CSC-like phenotype. Preferential activation of Akt and Bcl-2 survival pathways and enhanced expression of neuropeptide receptors contribute to CD133+ SCLC chemoresistance and growth. Therefore, it can be proposed that CD133+ cells are the possible cause of SCLC development, treatment resistance and disease recurrence. Despite being chemoresistant, CD133+ cells demonstrated sensitivity to peptide-1. The identification of such new analogue that demonstrates efficacy towards resistant CD133+ SCLC cells is a very exciting step forward in the identification of a potential new therapy for resistant disease.

Stem cell therapies and tissue engineering strategies are required for the clinical treatment of respiratory diseases. Previous studies have established protocols for the differentiation of airway epithelium from stem cells but have involved costly and laborious culture methods. The aim of this thesis was to achieve efficient and reproducible maintenance and differentiation of embryonic stem cells to airway epithelium, in 2D and 3D culture, by developing appropriate bioprocessing technology. Firstly, the 2D differentiation process of human and murine ES cells into pulmonary epithelial cells was addressed. The main finding in was that the proportion of type II pneumocytes, the major epithelial component of the gas-exchange area of lung, differentiated with this method was higher than that obtained in previous sudies, 33% of resultant cell expressed the specific marker surfactant protein C (SPC) compared with up to 10%. Secondly, the maintenance and differentiation was carried out in 3D. A protocol was devised that maintained undifferentiated human ES cells in culture for more than 200 days encapsulated in alginate without any feeder layer or growth factors. For ES cell differentiation in 3D, a method was devised to provide a relatively cheap and simple means of culture and use medium conditioned by a human pneumocyte tumour cell line (A549). The differentiation of human and murine ES cells into pulmonary epithelial cells, particularly type II pneumocytes, was found to be upregulated by culture in this conditioned medium, with or without embryoid body formation. The third step was to test whether this differentiation protocol was amenable to scale-up and automation in a bioreactor using cell encapsulation. It was possible to show that encapsulated murine ES cells cultured in static, co-culture or rotating wall bioreactor (HARV) systems, differentiate into endoderm and, predominantly, type I and II pneumocytes. Flow cytometry revealed that the mean yield of differentiated type II pneumocytes was around 50% at day 10 of cultivation. The final stage of the work was to design and produce a perfusion system airlift bioreactor to mimic the pulmonary microenvironment in order to achieve large scale production of biologically functional tissue. The results of these studies thus provide new protocols for the maintenance of ES cells and their differentiation towards pulmonary phenotypes that are relatively simple and cheap and can be applied in bioreactor systems that provide for the kind of scale up of differentiated cell production needed for future clinical applications.

Stem Cell Factor (SCF) is widely recognised as a crucial growth factor for mast cells and has been shown to mediate the development, differentiation and proliferation of mast cells from precursors. These effects of SCF are thought to be mediated by the c-KIT receptor. Whether SCF directly activates mast cells is more contentious and most of the data suggests that SCF can ‘prime’ mast cells for enhanced responses to IgE-dependent activation. The principal aim of the present work was to determine whether SCF activates human lung mast cells. Our studies showed that SCF was an effective direct activator of human lung mast cells. Although not as effective as anti-IgE at inducing histamine release, nonetheless, in about a third of all mast cell preparations, SCF induced substantial levels of release. Even more strikingly, SCF was as effective as anti-IgE at inducing prostaglandin D2 (PGD2) generation from mast cells. By contrast, SCF was relatively ineffective at inducing cytokine generation from human lung mast cells, a feature shared by other stimuli such as anti-IgE. The effects of SCF on histamine and PGD2 release were blocked by the c-KIT inhibitors imatinib, dasatinib and nilotinib suggesting that SCF acts through the c-KIT receptor to activate mast cells. Further studies were performed to evaluate the mechanism by which SCF and anti-IgE drive PGD2 generation from mast cells. In particular, the role of the enzyme cyclooxygenase (COX) on PGD2 generation was evaluated. Studies utilising COX-1 and COX-2 selective inhibitors demonstrated that COX-1 was the isoform that drives PGD2 generation from mast cells. This finding was further supported by Western blotting studies showing that COX-1 is the principal isoform expressed by mast cells. In total, these findings indicate that SCF can directly activate human lung mast cells through the c-KIT receptor to generate substantial amounts of PGD2 and variable amounts of histamine. The data also show that COX-1 is the principal isoform involved in PGD2 generation from human lung mast cells. Overall these studies indicate that SCF is a far more effective activator of human lung mast cells than hitherto appreciated.

Human embryonic stem cells (hESCs) show significant therapeutic potential in treating degenerative disorders. This is in part because of their ability to produce a limitless supply of starting cells and their potential to differentiate into more than 200 different cell types. The aim of the current research was to generate a robust stage wise protocol for the differentiation of hESCs to respiratory epithelial cells. The epithelial cells could then be used either for transplantation studies or, as an in vitro model for drug toxicity testing. In order to achieve this goal, we must identify the key steps in lung development and apply these to the differentiation protocol. In this study, we maintained Shef3 hESCs in their undifferentiated pluripotent state to expand the cells prior to the differentiated towards the definitive endoderm (DE) lineage. I used a two-stage protocol based on culture with a novel glycogen synthase kinase-3 (GSK-3) inhibitor (termed 1m), along with Activin-A. We confirmed the status of the cells by a combination of immunostaining and PCR. We showed loss of the pluripotency markers (Sox2 and Oct3/4) and gain of DE markers (Sox17, FoxA2 and CXCR4). After the induction of DE from hESCs, we then treated the cells with transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) pathway inhibitors (SB431542 and Noggin respectively). This combinatorial treatment resulted in the differentiation into the anterior foregut endoderm (AFE) lineage based on expression of Pax9 and FoxA2 plus the up-regulation of Sox2. Further differentiation of AFE derivatives into more mature epithelial cells, termed lung progenitor cells (LPCs), was achieved following the treatment of AFE cells with a cocktail of trophic factors (BMP4, EGF, bFGF, FGF10, KGF and Wnt3a) yielded a population of NKX2.1-positive and FoxA2-positive cells that potentially corresponded to the lung lineage. Finally, prolonged treatment with FGF10 and FGF2 on LPC derived hESCs induced proximal (CC10, MUC5AC) and distal (SPB, SPC) airway epithelial cells. In addition, we also utilised the ectopic expression of an adenovirus expressing NKX2.1 to promote lung maturation. In conclusion, we have generated a protocol for the differentiation of hESCs into mature lung-like cells. The generation of these cells in vitro could potentially lead to a better in vitro model for toxicity testing and the development of novel therapies for promoting regeneration of lungs in patients with severe lung disorders.

Tumor heterogeneity has long been observed and recognized as a challenge to cancer therapy. The cancer stem cell (CSC) model is one of the hypotheses proposed to explain such a phenomenon. A specific cancer stem cell marker has not been determined for non-small cell lung cancers (NSCLC), preventing the definitive evaluation of whether the biology of NSCLC is governed by the CSC model. This study aimed to analyze the expression of candidate CSC markers and using the identified putative marker, to isolate CSC and determine the applicability of the CSC model in NSCLC. The expression or activities of four putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) were measured by flow cytometry in eight NSCLC cell lines before and after chemotherapy for 24 hours. Markers with increased expression after treatment were considered potential CSC markers and used for isolating tumor cell subpopulations from the untreated cell lines by fluorescence-activated cell sorting (FACS). Confirmatory analyses using widely acceptable methodology were performed to test for CSC properties in the marker+ and marker- subpopulations. Isolated subpopulations were further characterized by functional and phenotypic studies. Flow cytometry showed amongst the 4 markers, only ALDH1 expression was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly more and larger cell spheres in non-adherent, serum-free conditions than ALDH1- cells. Likewise, higher in vitro tumorigenic ability was observed in ALDH1+ subset using colony formation assay. Furthermore, a higher resistance to cytotoxic drugs was observed in ALDH1+ compared to ALDH1- cells. In vivo studies also showed ALDH1+ cells showed higher tumorigenicity than ALDH1- cells; as few as 2,500 ALDH1+ cells formed tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH- cells with even ten times the number of cells. Also, expression analysis revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. In view of previous studies reporting CD44 as a CSC marker in lung cancer, double marker selection of putative CSC was performed to compare ALDH1+CD44+ and ALDH1-CD44+ subpopulations. Results of the spheroid body formation assay and cisplatin treatment experiments revealed the ALDH1+CD44+ subpopulation possessed higher self-renewal ability and chemo-resistance. Cell migration and invasion assays showed differences between the ALDH1+CD44+ and ALDH1- CD44+ subpopulations. The significance of these observations require further investigation. In conclusion, the result showed that ALDH1 could be a marker for NSCLC stem cells as evidenced by enhanced self-renewal and differentiation abilities in ALDH1+ subpopulation. Furthermore, this study observed the presence of at least two potential stem cell subpopulations in NSCLC cells with differential selfrenewal, chemotherapy resistance and cell mobility properties. Further investigations are required to validate these observations and to investigate the underlying mechanisms. Better understanding of these issues would help to solve the challenges brought by tumor heterogeneity in lung cancer therapy.
published_or_final_version
Pathology
Master
Master of Philosophy

Recent advances in understanding lung biology have shown evidence for the existence of resident lung stem cells. Independent studies in identifying and characterizing these somatic lung stem cells have shown the potential role of these cells in lung repair and regeneration. Understanding the functional characteristics of these tissue resident stem/progenitor cells has gained much importance with increasing evidence of cancer stem cells, cells in a tumor tissue with stem cell characteristics. Lung cancer is most commonly characterized by loss of p53 function which results in uncontrolled cell divisions. Incidence of p53 point mutations is highest in lung cancer, with a high percentage of missense mutations as a result of tobacco smoking. Certain point mutations in p53 gene results in its oncogenic gain of functions (GOF), with enhanced tumorigenic characteristics beyond the loss of p53 function. However, there are no available data on characterization of lung stem cells carrying GOF mutations and correlating them with those of normal stem cells, in this study, for the first time we show that percentage of Sca-1 expressing subpopulation is significantly higher in the lungs of mice carrying p53 GOF mutations than those in lungs isolated from p53+/+ wild type mice. Further, we successfully established lung cells differentially expressing two cell surface markers, Sca-1 and PDGFR-α, with results demonstrating existence of different subpopulations of cells in the lung. Results from our project demonstrate the importance of p53 GOF mutations as correlated with specific lung cell subpopulations.

Development of resistance to radiation and chemotherapy turns the treatment of solid cancers into a therapeutic challenge. One of the most exciting breakthroughs being explored in cancer research today is the concept of cancer stem cells (CSCs). CSCs are a minority of cells within a tumor that are the source of tumor cell renewal and thereby determine the behavior of tumors, including proliferation, spreading and response to therapy. CSCs are highly resistant to conventional treatment and are therefore emerging as the preferred target of drug therapies in order to obtain eradication of tumors. In this study, we examined the activation of the DNA damage response pathway in CSCs derived from non-small cell lung cancer (NSCLC-CSCs) and their differentiated counterparts after treatment with chemotherapeutic agents commonly used in clinic for lung cancer treatment. Our data show that NSCLC-CSCs possess a highly active DNA damage pathway compared to differentiated progeny and preferentially activate the checkpoint kinase Chk1 in response to DNA damage caused by chemotherapy. This indicates that Chk1 is most likely the main player of drug resistance in NSCLC-CSCs and its targeting might yield significant therapeutic gains. We demonstrate that chemical Chk1 inhibitors dramatically induce NSCLC-CSC death in vitro in combination with DNA damaging drugs. Cell death is induced through a premature activation of the cell cycle regulatory proteins Cdc2 and Cyclin B1, which in turn forces cells with damaged DNA to enter aberrant mitosis, a mechanism known as mitotic catastrophe. Moreover, our results indicate that final cell death occurs through apoptosis. Combination therapy studies have been successfully carried out also in vivo. Chk1 inhibition enhanced the antitumoral effect of conventional chemotherapy in mice xenograft tumor models by increasing tumor latency, potently abrogating tumor growth and reducing tumor mass. We also found a significant reduction of NSCLC-CSCs in xenograft-derived cells, confirming that combination treatment actually targets and reduces the NSCLC-CSCs compartment in vivo. The importance of DNA repair as a resistance mechanism in cancer is a clinically relevant topic and we believe that the combination of selective Chk1 inhibitors with anti-cancer drugs could represent a new therapeutic approach for targeting NSCLC-CSCs and thereby for effective treatment of lung tumors.

Current diagnostic tools do not allow prognostic evaluation of patients with early stage lung cancer or selection of patients that might benefit from adjuvant chemotherapy. Therefore, the identification of novel prognostic markers in early-stage lung cancer is paramount. In this scenario, the transcription factor HOXB7, belonging to the homeobox family, has been shown to correlate with poor prognosis in different types of cancer and recently also in stage I lung adenocarcinoma. To better understand the prognostic implication of alterations in HOXB7 expression in lung cancer, we performed a bioinformatics analysis of multiple lung cancer expression datasets in order to identify gene sets representing cancer-relevant biological functions enriched in high-HOXB7 expressing tumors. We found several gene sets enriched in high-HOXB7 expressing tumors representing molecular mechanisms involved in epithelial to mesenchymal transition, in cancer progression, and, interestingly, in stemness and cellular reprogramming. Based on these results, we hypothesized that HOXB7 may have a role in the expansion of the stem cell compartment in cancer, a mechanism that has been shown to be a hallmark of enhanced tumorigenicity and of increased metastatic potential. Analysis of the stem-related surface marker CD90 revealed that overexpression of HOXB7 in lung cells increases the CD90high sub population. CD90high, but not CD90low cells, are able to form spheroids, which is an hallmark of stemness. Indeed, the sphere forming efficiency of normal lung BEAS-2B cells was 22% and 1.64% in CD90high and CD90low populations, respectively. In addition, we found that silencing of LIN28B counteracts the expansion of the CD90high population. LIN28B was recently described as an oncogene that regulates the cancer stem cell compartment. We found that LIN28B is under the direct transcriptional control of HOXB7. Therefore, we propose a novel molecular mechanism driven by HOXB7 and can increase stem-like properties in lung cells. We further demonstrated that the HOXB7-LIN28B axis plays an important role in reprogramming of adult cells into induced pluripotent stem cells (iPS). Indeed, HOXB7 may enhance the reprogramming efficiency achieved by the three genes OCT4, KLF4, SOX2 in both mouse embryonic fibroblast and human epithelial BEAS-2B cells by substituting MYC in the transcription factor cocktail of reprogramming factors used by Yamanaka. Of note, LIN28B silencing strongly decreases the number of reprogrammed colonies in high-HOXB7 expressing cells. These findings suggest that HOXB7, through transcriptional induction of the LIN28B gene, activates a program relevant for stem/iPS cell biology and for tumor progression, possibly opening a new line of research for the development of more effective therapies for metastatic lung cancer patients.

An emerging concept in cancer initiation and development is that cells with characteristics of the stem cell phenotype drive tumour growth and progression. Accumulating evidence reveals that solid tumours such as brain and breast cancer contain primitive cancer stem cells that have high repopulation capacity. Recent studies have demonstrated that adult stem cells can be identified by a side population (SP) phenotype. The work in this thesis was the first in investigating the existence of cancer stem cells in human lung cancer. This study used flow cytometry and the Hoechst 33342 dye efflux assay to isolate and characterise SP cells from various human lung cancer cell lines. In addition, the existence of SP cells in lung cancer tissues obtained from surgical resection was also investigated. Results indicated that all six human lung cancer cell lines contained an SP that could be reliably detected under the experimental conditions used. Evidence was found for asymmetric division by the SP to generate a population resembling the original unsorted population. SP cells and non-SP cells were characterised at the molecular level to compare mRNA expression between the two populations. SP cells from each cell line displayed elevated expression of ATP-Binding Cassette (ABC) transporters associated with multi-drug resistance. In particular, expression of ABCG2 (BRCP1) defines the SP phenotype. Human telomerase reverse transcriptase (hTERT) expression was higher in the SP cells, suggesting this fraction may represent a reservoir with high proliferative potential for generating cancer cells. mRNA levels of MCM7, a member of the mini-chromosome maintenance (MCM) family of proteins, a critical component of the DNA replication complex, was lower in SP cells suggesting that a majority of the cells from the SP fraction were in G₀ of the cell cycle. Levels of mRNA expression of BMI-1, a pathway associated with stem cell self-renewal, were higher in the SP cells compared to the non-SP cells in four of the six cell lines. The Notch-1 pathway was another self-renewal pathway that had increased mRNA expression in five of six cell lines. Functional characterisation of the SP and non-SP were investigated in both in vitro and in vivo. The Matrigel [Trademark] invasion assay demonstrated SP cells as having higher potential for invasiveness than non-SP cells, suggesting there exist a population of stem-like cells within a lung tumour that is involved in the initiation of invasion and metastasis. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that SP cells were more tumourigenic compared to non-SP cells. Using a cell viability assay, SP cells were determined to exhibit higher resistance to drugs used to treat lung cancer, some of which are substrates for ABC transporters. Staining patterns of other putative stem cell-related surface markers such as CD24, CD34, CD44 and nestin were also examined and compared between the SP and non-SP. Using the Hoechst efflux assay, SP cells were also isolated from patient tissues, which contained a low but persistent percentage (0.025 - 1.08) of SP cells. Taken together, these studies suggested that SP is an enriched source of tumour re-initiating cells with stem cell properties and may be an important target for effective therapy and a useful tool to investigate the tumourigenic process.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

[ES] El cáncer de pulmón es el tipo de cáncer más frecuentemente diagnosticado y la principal causa de muerte debida a cáncer en el mundo, con sólo un 15% de pacientes con una supervivencia mayor a 5 años tras el diagnóstico. La resección quirúrgica es el tratamiento estándar para los pacientes en estadios tempranos con un buen ECOG, pero el 75% de los pacientes son diagnosticados en estadios avanzados, cuando la intervención quirúrgica no es posible y entre un 35% y un 50% de los pacientes operados recaen tras una cirugía aparentemente exitosa. En los últimos años, se han logrado importantes avances en el desarrollo de la inmunoterapia y de tratamientos contra mutaciones conductoras, pero muchos pacientes todavía desarrollan resistencia, progresan y mueren. Esta resistencia terapéutica ha sido asociada a las células madre tumorales (CMTs), una población tumoral con propiedades de célula madre capaz de sobrevivir a las terapias convencionales y regenerar el tumor incluso cuando son indetectables. En esta tesis doctoral, se establecieron cultivos primarios de pacientes de cáncer de pulmón no microcítico (CPNM) resecados, usando ensayos de formación de tumoresferas para el enriquecimiento en CMTs y condiciones de adherencia para los controles. Las tumoresferas derivadas de pacientes mostraron capacidad de autorenovación y crecimiento exponencial ilimitado, alta resistencia a agentes quimioterápicos, capacidad de invasión y diferenciación in vitro y un elevado potencial tumorigénico in vivo. Usando PCR cuantitativa, se analizaron los perfiles de expresión de los cultivos y se determinó que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 e ITGA6 eran los genes más diferencialmente expresados entre tumoresferas y células adherentes. Los análisis de inmunoblot e inmunofluorescencia confirmaron que las proteínas codificadas por estos genes se encuentran aumentadas en tumoresferas de los pacientes con adenocarcinoma y mostraron patrones de expresión y localización diferencial entre éstas y los controles en adherencia. El valor pronóstico de los genes significativamente sobreexpresados en tumoresferas fue evaluado in silico en una cohorte de 661 pacientes con CPNM procedente del TCGA. De todos ellos, CDKN1A, SNAI1 y ITGA6 mostraron estar relacionados con el pronóstico de los pacientes de acuerdo a un análisis de regresión de Cox y fueron seleccionados para construir una firma de expresión génica, denominada firma de CMTs. Los análisis de supervivencia por Kaplan-Meier mostraron que los pacientes con valores elevados de la firma tienen una supervivencia global (SG) menor para la cohorte completa de CPNM [37,7 vs. 60,40 meses, p = 0,001] y para la subcohorte de adenocarcinoma (ADC) [36,6 vs. 53,5 meses, p = 0,003], pero no para la de los epidermoides. Además, el análisis multivariante mostró que la firma de CMTs es un marcador pronóstico independiente para la SG de los pacientes en la cohorte completa [hazard ratio (HR): 1,498; intervalo de confianza (IC) 95%, 1,167-1,922; p = 0,001] y la subcohorte de ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Esta firma fue también analizada en un grupo independiente de 245 pacientes procedentes del Consorci Hospital General Universitari de València, confirmando su valor pronóstico en los pacientes con ADC [42,90 vs. no alcanzado (NA) meses, p = 0,020]. En resumen, nuestros hallazgos aportan información pronóstica relevante para los pacientes con ADC de pulmón y establecen las bases para el desarrollo de nuevos tratamientos.
[CAT] El càncer de pulmó és el tipus de càncer més diagnosticat i la principal causa de mort deguda a càncer en el món, amb només un 15% de pacients amb una supervivència major a 5 anys després del diagnòstic. La resecció quirúrgica és el tractament estàndard per als pacients en estadis primaris amb un bon ECOG, però el 75% dels pacients són diagnosticats en estadis avançats, quan la intervenció quirúrgica no és possible i entre un 35% i un 50% dels pacients operats recauen després d'una cirurgia aparentment satisfactòria. En els últims anys, s'han aconseguit importants avanços en el desenvolupament de la immunoteràpia i de tractaments contra mutacions conductores, però molts pacients encara desenvolupen resistència, progressen i moren. Aquesta resistència a les teràpies ha estat relacionada amb les cèl·lules mare tumorals (CMTs), una població tumoral amb propietats de cèl·lula mare capaç de sobreviure a les teràpies convencionals i regenerar el tumor fins i tot quan són indetectables. En aquesta tesi doctoral, es van establir cultius primaris de pacients de càncer de pulmó no microcític (CPNM) ressecats, usant assajos de formació de tumoresferes per a l'enriquiment en CMTs i condicions d'adherència per als controls. Les tumoresferes derivades de pacients van mostrar capacitat d'autorenovació, creixement exponencial il·limitat, alta resistència a agents quimioteràpics, capacitat d'invasió i diferenciació in vitro i un elevat potencial tumorigènic in vivo. Usant PCR quantitativa, es van analitzar els perfils d'expressió dels cultius i es va determinar que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 i ITGA6 eren els gens més diferencialment expressats entre tumoresferes i cèl·lules adherents. Les anàlisis de immunoblot i immunofluorescència van confirmar que les proteïnes codificades per aquests gens es troben augmentades en tumoresferes dels pacients amb adenocarcinoma i van mostrar patrons d'expressió i localització diferencial entre aquestes i els controls en adherència. El valor pronòstic dels gens significativament sobreexpressats en tumoresferes va ser avaluat in silico en una cohort de 661 pacients amb CPNM procedent del TCGA. De tots ells, CDKN1A, SNAI1 i ITGA6 van mostrar estar relacionats amb el pronòstic dels pacients d'acord a una anàlisi de regressió de Cox i van ser seleccionats per a construir una signatura d'expressió gènica, denominada signatura de CMTs. Les anàlisis de supervivència per Kaplan-Meier van mostrar que els pacients amb valors elevats de la signatura tenen una supervivència global (SG) menor per a la cohort completa de CPNM [37,7 vs. 60,40 mesos, p = 0,001] i per a la subcohort d'adenocarcinoma (ADC) [36,6 vs. 53,5 mesos, p = 0,003], però no per a la dels escamosos. A més, l'anàlisi multivariant va mostrar que la signatura de CMTs és un marcador pronòstic independent per a la SG dels pacients en la cohort completa [hazard ratio, (HR): 1,498; interval de confiança (IC) 95%, 1,167-1,922; p = 0,001] i la subcohort d'ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Aquesta signatura va ser també analitzada en un grup independent de 245 pacients procedents del Consorci Hospital General Universitari de València, confirmant el seu valor pronòstic en els pacients amb ADC [42,90 vs. no arribat (NA) mesos, p = 0,020]. En resum, els nostres resultats aporten informació pronòstica rellevant per als pacients amb ADC de pulmó i estableixen les bases per al desenvolupament de nous tractaments.
[EN] Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-related death worldwide, with approximately 15% of patients surviving 5 years after diagnosis. Curative surgery is the standard of care for early-stage patients with a good performance status, but 75% are diagnosed at advances stages, when surgery is not possible, and 35-50% of the resected patients relapse after an apparently successful surgical treatment. Significant advances in the development of therapies against driver mutations and immune-based treatments for these patients have been achieved in recent years, but many patients still develop treatment resistance, progress, and die. The high resistance against these therapies has been associated to cancer stem-like cells (CSCs), a population with stem properties which is able to survive after conventional treatments and regenerate tumor even when are undetectable. In this thesis, primary cultures from early-stage non-small cell lung cancer (NSCLC) patients were established, using sphere-forming assays for CSCs enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using RTqPCR, gene expression profiles were analyzed, and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected as the best contributors to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in silico in a cohort of 661 NSCLC patients from TCGA. Based on a Cox regression analysis, CDKN1A, SNAI1 and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSCs score. Kaplan-Meier survival analysis showed that patients with high CSCs score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months, p = 0.001] and in the adenocarcinoma (ADC) subcohort [36.6 vs. 53.5 months, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both, the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167-1.922; p = 0.001], and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. This score was also analyzed in an independent group of 245 patients from Consorci Hospital General Universitari de València, confirming its prognostic value in the ADC subtype [42.90 vs. not reached (NR) months, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung ADC patients and the basis for developing novel therapies.
Herreros Pomares, A. (2020). Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137036
TESIS

ABSTRACT Introduzione. Riparare il tessuto danneggiato per evitare complicanze postoperatorie e risparmiare il tessuto sano per garantire una buona funzionalità respiratoria sono due degli obiettivi del chirurgo toracico. Negli anni sono stati introdotti nella pratica clinica numerosi prodotti sintetici che riescono a simulare le caratteristiche meccaniche e fisiologiche del tessuto danneggiato, ma a fronte degli elevati costi, non sempre garantiscono risultati stabili. Il trapianto autologo di cellule staminali adipose (ASC) viene proposto come nuova strategia per promuovere la riparazione dei tessuti dopo l'intervento chirurgico. Progetto di Ricerca. Il lipocentrifugato con le ASC è stato sottoposto ad una valutazione preclinica mediante saggi in vitro e in vivo per testare il potenziale riparativo sul tessuto polmonare e per chiarire il supposto effetto tumorigenico su eventuali residui neoplastici. Lo studio clinico è stato eseguito su 20 interventi di metastasectomia polmonare eseguita mediante laser di ultima generazione (2010nm wavelength). I principali end-point dello studio erano: 1) la fattibilità dell’applicazione di cellule staminali nella metastasectomia polmonare mediante laser Thulium (2010nm), 2) incidenza delle perdite aeree postoperatorie prolungate (>7 giorni) 3) fattibilità del prelievo di cellule staminali a livello della toracotomia sulla base del conteggio delle cellule staminali 4) impatto di questo tipo di procedura sulla funzionalità polmonare. Risultati Clinici. Non abbiamo avuto alcun decesso perioperatorio. Dopo rimozione di lesioni centroparenchimali, 4 pazienti hanno sviluppato perdite aeree prolungate. In un paziente il drenaggio pleurico è stato rimosso in POD 13 e 3 pazienti sono stati dimessi con il tubo toracico collegato alla valvola di Heimlich. Non sono stati necessari re interventi per revisione dell’aerostasi. Un paziente ha sviluppato un ematoma nel sito di prelievo del tessuto adiposo. 4 casi di febbre sono stati risolti con terapia antibiotica. Le cellule staminali mesenchimali sono state rilevate in concentrazione> 1% in 13 casi. Età, sesso chemioterapia preoperatoria e indice di massa corporea non hanno influenzato il numero di cellule staminali. Nessuna significativa riduzione in termini di funzione polmonare è stata rilevata dopo l'intervento chirurgico. In nessun caso sono stati utilizzati materiali sintetici. Conclusioni. Il nostro studio mostra che il tessuto grasso raccolto a livello dello strato sottocutaneo della toracotomia è una fonte di cellule staminali. I vantaggi portati da questa tecnica combinata possono essere misurati in termini di funzione respiratoria. Con un follow-up di 16 mesi non è stata osservata alcuna recidiva a livello del sito di applicazione del centrifugato adiposo.
ABSTRACT Introduction. Repair damaged tissue to prevent postoperative complications and save healthy tissue to ensure good respiratory function are two of the objectives of the thoracic surgeon. Over the years have been introduced in clinical practice numerous synthetic products that are able to simulate the mechanical and physiological characteristics of the damaged tissue, but in the face of high costs, do not always guarantee stable results. The autologous adipose stem cells (ASC) is proposed as a new strategy to promote the repair of tissues after surgery. Research Project. The lipocentrifugated with the ASC has been subjected to a pre-clinical evaluation using in vitro and in vivo assays to test the reparative potential on lung tissue and to clarify the supposed tumorigenic effect of any residual neoplastic. The clinical study was performed on 20 pulmonary metastasectomies performed using last generation laser (wavelength 2010nm). The main endpoints of the study were: 1) the feasibility of pulmonary metastasectomy with Thulium laser (2010nm), 2) incidence of postoperative prolonged air leak (> 7 days) 3) feasibility of the harvesting of stem cells at the level of thoracotomy on the basis the cell count stem 4) impact of this type of procedure on respiratory function. Clinical Results. We had no perioperative death. After removal of lesions located deep in the parenchyma, 4 patients developed prolonged air leaks. In one patient the pleural drain was removed in POD 13 and 3 patients were discharged with a chest tube connected to the valve Heimlich. Redo surgery to improve the aerostasis was not required. One patient developed a hematoma at the site of collection of adipose tissue and 4 cases of fever have been resolved with antibiotic therapy. The mesenchymal stem cells were detected in concentrations> 1% in 13 cases. Age, sex, preoperative chemotherapy and body mass index did not affect the number of stem cells. No significant reduction in terms of lung function was measured after surgery. No synthetic materials were used. Conclusions. Our study shows that the fat tissue collected at the level of the subcutaneous layer of the thoracotomy is a useful source of stem cells. The advantages brought by this technique can be measured in terms of respiratory function. With a median follow-up of 16 months no recurrence was observed at the site of application of centrifuged fat graft.

Neste trabalho de tese aplicamos a espectroscopia de Ressonância Paramagnética Eletrônica (RPE) junto com a chamada técnica do grupo repórter, aliada a simulação espectral por meio do programa NLSL, para investigar a interação da nicotina dependente com membranas. Também empregamos Ressonância Ferromangnética (RFM), que é uma técnica similar em muitos aspectos ao RPE, para investigamos células-tronco marcadas com nanopartículas superparamagnéticas de óxido de ferro (NSOF), previamente preparada no laboratório de nanotecnologia do Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). Os resultados relativos à primeira parte da tese, mostraram que a nicotina quando em meio ácido interage com a região da cabeça polar dos fosfolipídios estudados e tem ação penetrante em meio básico. A interação da nicotina com o DPPC resulta em uma desestabilização da fase gel do fosfolipídio. Este fato pode estar relacionado com a ocorrência de doenças respiratórias em tabagistas devido ao fato de que o DPPC é o maior constituinte do complexo surfatante pulmonar. Na segunda parte, fomos capazes de marcar células-tronco com nanopartículas superparmangéticas e também quantificá-las pelo cálculo da área, dupla integral do espectro de RFM das NOSF. Finalmente, fizemos uma caracterização das nanopartículas por meio de experimentos de RFM dependente da variação de temperatura, que confirmaram o comportamento superparamagnético das NOSF.
In this work, we made use of the Electron Paramagnetic Resonance (EPR) spectroscopy along with the so-called reporter group technique and extensive spectral simulations by means of the software NLSL to investigate the interaction of nicotine with model membranes. Besides that we also employed Ferromagnetic Resonance (FMR), which is a technique similar in many aspects to EPR, to investigate stem cells labeled with superparamagnetic nanoparticles of iron oxide (NSOF) previously made at the Nanotechnology Laboratory of the Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). The results concerning the first part of our work showed that nicotine in acidic pH value interacts with the polar headgroup region of the phospholipids under investigation, whereas it penetrates the model membrane in alkaline pH value. Also, the interaction of nicotine with DPPC resulted in destabilization of the phospholipid gel phase. This fact could be related to some of the effects of nicotine in respiratory diseases since DPPC is the major constituent of the pulmonary surfactant complex. In the second part of our work, we were able to successfully label stem cells with the superparamagnetic nanoparticles and also quantify that labeling by calculating the area under the double integrated NSOF FMR spectra. We also performed a characterization of the nanoparticles by means of temperature-variation FMR experiments, which showed that the iron oxide nanoparticled indeed had a superparamagnetic behavior.

Les progéniteurs bronchioloalvéolaires situés aux jonctions bronchioloalvéolaires peuvent être impliquésdans l'embryogenèse ou la régénération. Ces cellules non décrites chez les gros animaux peuventparticiper au développement de nouvelles thérapies contre les maladies pulmonaires aiguës ouchroniques. Dans cette étude, nous avons établi la présence de progéniteurs bronchioloalvéolaires dansles poumons des ovins nouveaux nés. Ces cellules ont été identifiées par leur co-expression desprotéines CCSP, SP-C et CD34. Une population mineure de cellules CD34pos/SP-Cpos/CCSPpos (0,33% ±0,31) était présente ex vivo. Le tri magnétique des cellules CD34pos a permis l’isolement des progéniteursSP-Cpos/CCSPpos (> 80%). Ces cellules étaient maintenues et amplifiées in vitro en interfaceliquide/liquide. De même, ces progéniteurs étaient capables de se différencier soit en cellules de Clarasoit en AEC II dans différents milieux de cultures synthétiques et diverses matrices extracellulaires. Enoutre, les progéniteurs bronchioloalvéolaires obtenus ex vivo et in vitro exprimaient les gènes NANOG,Oct4 et BMI1 spécifiques aux cellules souches. Nous rapportons ainsi, pour la première fois dans ungrand animal, l’existence de cellules progénitrices bronchioloalvéolaires à fort potentiel de doubledifférenciation et d’expression de certains gènes de cellules souches
Bronchioloalveolar stem cells located at the bronchiolar/alveolar junction may be involved inembryogenesis or regeneration. These cells have not yet been described in large animals, and they mayenable the development of new therapeutics to treat acute or chronic lung disease. In this study, weaimed to establish the presence of bronchioloalveolar stem cells in ovine lungs and to characterize theirstemness properties. Lung cells were studied using immunohistochemistry on frozen sections of the lung,and immunocytochemistry and flow cytometry were conducted on derived cells. The stem cells wereidentified by co-expression of CCSP, SP-C and the CD34 hematopoietic stem-cell marker. A minorpopulation of CD34pos/SP-Cpos/CCSPpos cells (0.33% ± 0.31) was present ex vivo in cell suspensions fromdissociated lungs. Using CD34 magnetic positive cell sorting, undifferentiated SP-Cpos/CCSPpos cellswere purified (>80%) and maintained in culture. Using synthetic media and various extracellular matrixes,SP-Cpos/CCSPpos cells differentiated into either Clara cells or alveolar epithelial type-II cells. Furthermore,bronchioloalveolar stem cells obtained ex vivo and in vitro expressed the stem cell-specific genesNANOG, OCT4 and BMI1. We report for the first time in a large animal the existence ofbronchioloalveolar stem cells with dual differentiation potential and the expression of stem cell-specificgenes

[ES] El cáncer de pulmón es el tumor más frecuente y también el que presenta mayor mortalidad en términos absolutos, suponiendo hasta más del 18% de los fallecimientos por cáncer en el mundo al año. El cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los tumores de pulmón. Tanto las alteraciones genéticas como la heterogeneidad y el microambiente tumoral influyen en la agresividad de los tumores de CPNM. Un factor que contribuye a la heterogeneidad tumoral es la presencia de células madre tumorales (CSC, Cancer Stem Cells). La metástasis y recurrencia tumoral después del tratamiento han sido atribuidas al crecimiento y supervivencia de esta subpoblación celular. La falta de marcadores específicos de CSC de pulmón representa una dificultad para identificarlas, y los marcadores de superficie conocidos hasta el momento no son válidos para separar poblaciones de CSC, por lo que se hace necesaria la generación de nuevos ensayos experimentales para aislar CSC de manera más robusta. La identificación de CSC podría ser la base para el diseño de nuevas estrategias terapéuticas personalizadas, basadas en la selección de una combinación más racional de fármacos, que tendría como objetivo eliminar la población de CSC en los tumores de CPNM. Los objetivos de esta tesis doctoral han sido el aislamiento y caracterización de CSC derivadas de tumores de pacientes con CPNM, y de líneas de cultivo establecidas; la caracterización de proteínas involucradas en la transición epitelial mesenquimal (EMT) en CPNM que pueden ser relevantes para la adquisición y el mantenimiento de características de las células madre y para la progresión de la enfermedad; y el desarrollo de modelos experimentales de ratón a partir de xenoinjertos derivados de tumores de pacientes con CPNM (PDX) para la identificación de nuevos biomarcadores y para estudiar el fenotipo y evolución de los tumores de pacientes. En este trabajo se han optimizado los protocolos para la obtención de tumoresferas (ESF) con propiedades de células madre a partir de tejido tumoral de pacientes con CPNM y de líneas células de CPNM establecidas. Se ha confirmado el fenotipo característico de célula madre en las ESF mediante el análisis de la expresión de marcadores de superficie de CSC como CD326, CD166, CD44 y CD133, su capacidad de autorrenovación y diferenciación, así como la capacidad de iniciar tumores en ratones inmunodeprimidos. Se ha observado además que la señalización mediada por TGF-ß1 podría estar contribuyendo a un aumento de la población CSC, y que habría una relación entre el proceso de promoción de la EMT inducida por TGF-ß1 y las propiedades de CSC. El análisis de genes involucrados en la EMT y CSC en las ESF mostró que la expresión de CDKN1A, NOTCH3, CD44, NANOG, SNAI1 e ITGA6 es característica de las ESF, y por tanto la expresión combinada de estos genes podría identificar la subpoblación de CSC. Al correlacionar la expresión de estos genes con la supervivencia de los pacientes, se obtuvo una firma con valor pronóstico en ADC basada en la expresión de los genes CDKN1A, SNAI1 e ITGA6. Se ha estudiado además el papel de proteínas involucradas en la EMT cuya expresión podría promover la migración e invasión celular en CPNM. De esta manera se ha identificado que el factor de transcripción JunB y el factor de traducción eIF5A2 están involucrados en la iniciación de la EMT mediada por TGF-ß1. Finalmente, se han establecido 9 modelos de xenoinjertos derivados de tumores de pacientes con CPNM (PDX). La correlación del éxito de implantación del tumor en ratón con variables clínico-patológicas de los pacientes mostró que los tumores primarios que generaron PDX derivaban de pacientes con peor pronóstico. También se observó que la elevada expresión de Vimentina, Ezrina y Ki67 en tumores sugiere una mayor agresividad y, por tanto, estudiar su expresión podría utilizarse en
[CA] El càncer de pulmó és el tumor més frequent i també el que presenta major mortalitat en termes absoluts, suposant fins més del 18% de les defuncions per càncer en el món a l'any. El càncer de pulmó no microcític (CPNM) representa quasi el 85% de tots els tumors de pulmó. Tant les alteracions genètiques com l'heterogeneïtat i el microambient tumoral influeixen en l'agressivitat dels tumors de CPNM. Un factor que contribueix a l'heterogeneïtat tumoral és la presència de cèl·lules mare tumorals (CSC, Cancer Stem Cells). La metàstasi i recurrència tumoral després del tractament han sigut atribuïdes al creixement i supervivència d'aquesta subpoblació cel·lular. La falta de marcadors específics de CSC de pulmó representa una dificultat per a identificar-les, i els marcadors de superfície coneguts fins al moment no són vàlids per a separar poblacions de CSC, per la qual cosa es fa necessària la generació de nous assajos experimentals per a aïllar CSC de manera més robusta. La identificació de CSC podria ser la base per al disseny de noves estratègies terapèutiques personalitzades, basades en la selecció d'una combinació més racional de fàrmacs, que tindria com a objectiu eliminar la població de CSC en els tumors de CPNM. Els objectius d'aquesta tesi doctoral han sigut l'aïllament i caracterització de CSC derivades de tumors de pacients amb CPNM, i de línies de cultiu establides de CPNM; la caracterització de proteïnes involucrades en la transició epitelial mesenquimal (EMT) en CPNM que poden ser rellevants per a l'adquisició i el manteniment de característiques de les cèl·lules mare i per a la progressió de la malaltia; i el desenvolupament de models experimentals de ratolí a partir de xenotrasplantaments derivats de tumors de pacients amb CPNM (PDX) per a la identificació de nous biomarcadors i per a estudiar el fenotip i evolució dels tumors de pacients. En aquest treball s'han optimitzat els protocols per a l'obtenció de tumoresferes (ESF) amb propietats de cèl·lules mare a partir de teixit tumoral de pacients amb CPNM i de línies cel·lulars de CPNM establides. S'ha confirmat el fenotip característic de cèl·lula mare en les ESF mitjançant l'anàlisi de l'expressió de marcadors de superfície de CSC com CD326, CD166, CD44 i CD133, la seua capacitat d'autorenovació i diferenciació, així com la capacitat d'iniciar tumors en ratolins immunodeprimits. S'ha observat a més que la senyalització mediada per TGF-ß1 podria estar contribuint a un augment de la població CSC, i que hi hauria una relació entre el procés de promoció de l'EMT induïda per TGF-ß1 i les propietats de CSC. L'anàlisi de gens involucrats en l'EMT i CSC en les ESF va mostrar que l'expressió de CDKN1A, NOTCH3, CD44, NANOG, SNAI1 i ITGA6 és característica de les ESF, i per tant l'expressió combinada d'aquests gens podria identificar la subpoblació de CSC. En correlacionar l'expressió d'aquests gens amb la supervivència dels pacients, es va obtindre una signatura amb valor pronòstic en ADC basada en l'expressió dels gens CDKN1A, SNAI1 i ITGA6. S'ha estudiat a més el paper de proteïnes involucrades en l'EMT que la seua expressió podria promoure la migració i invasió cel·lular en CPNM. D'aquesta manera s'ha identificat que el factor de transcripció JunB i el factor de traducció eIF5A2 estan involucrats en la iniciació de l'EMT mediada per TGF-ß1. Finalment, s'han establit 9 models de xenotrasplantaments derivats de tumors de pacients amb CPNM (PDX). La correlació de l'èxit d'implantació del tumor en ratolí amb variables clinicopatològiques dels pacients va mostrar que els tumors primaris que van generar PDX derivaven de pacients amb pitjor pronòstic. També es va observar que l'elevada expressió de Vimentina, Ezrina i Ki67 en tumors suggereix una major agressivitat i, per tant, l'avaluació de la seua expressió podria utilitzar-se en combinació com a marcador de pr
[EN] Lung cancer is the most frequent and the most mortal tumor in absolute terms, accounting for up to more than 18% of cancer deaths worldwide per year. Non-small cell lung cancer (NSCLC) accounts for almost 85% of all lung tumors. Apart from genetic alterations, both tumor heterogeneity and tumor microenvironment influence the aggressiveness of NSCLC tumors. A factor that contributes to tumor heterogeneity is the presence of cancer stem cells (CSC). Tumor metastasis and recurrence after treatment have been attributed to the growth and survival of this cell subpopulation. The lack of specific markers for lung CSC represents a drawback for their identification, and the surface markers known to date are not valid for separating CSC subpopulations, so it is necessary to generate new experimental assays to isolate CSC in a more robust way. The identification of CSC could be the basis for the design of new personalized therapeutic strategies, based on the selection of a more rational combination of drugs, which would aim to eliminate the CSC population in NSCLC tumors. The objectives of this doctoral thesis have been the isolation and characterization of CSC derived from tumors of patients with NSCLC, and from established NSCLC cell lines; the characterization of proteins involved in the epithelial-mesenchymal transition (EMT) in NSCLC that may be relevant for the acquisition and maintenance of stem cell characteristics and for disease progression; and the development of experimental mouse models from patient-derived xenografts (PDX) of NSCLC for the identification of new biomarkers and to study the phenotype and evolution of patient tumors. In this work, the protocols for obtaining tumorspheres (SPH) with stem cell properties from tumor tissue of NSCLC patients and from established NSCLC cell lines have been optimized. The characteristic stem cell phenotype in SPH has been confirmed by analyzing the expression of CSC surface markers such as CD326, CD166, CD44 and CD133, their capacity for self-renewal and differentiation, as well as the ability to initiate tumors in immunosuppressed mice. It has also been observed that TGF-ß1-mediated signaling could be contributing to an increase in CSC population, and that there would be a relationship between the process of promoting TGF-ß1-induced EMT and CSC features. The analysis of genes involved in EMT and CSC in the SPH showed that the expression of CDKN1A, NOTCH3, CD44, NANOG, SNAI1 and ITGA6 is characteristic of the SPH, and therefore the combined expression of these genes could identify the CSC subpopulation in NSCLC tumors. By correlating the expression of these genes with the survival of the patients, a signature with prognostic value in ADC was obtained based on the expression of CDKN1A, SNAI1 and ITGA6. The role of proteins involved in EMT whose expression could promote cell migration and invasion in NSCLC has also been studied. Thus, it has been identified that the JunB transcription factor and the eIF5A2 translation factor are involved in the initiation of EMT mediated by TGF-ß1. Finally, 9 PDX models from tumors of NSCLC patients have been established. The correlation of implantation success of the tumor in mice with the clinical-pathological variables of the patients showed that primary tumors that generated PDX were derived from patients with bad prognosis. It was also observed that higher expression of Vimentin, Ezrin and Ki67 in tumors suggests higher aggressiveness and, therefore, the study of their expression could be used in combination as a prognostic marker to evaluate the progression of the disease.
Pardo Sánchez, JM. (2021). Aproximaciones moleculares para la identificación de nuevos biomarcadores y dianas terapéuticas del cáncer de pulmón no microcítico: células madre tumorales, transición epitelial-mesenquimal y modelos de xenoinjertos derivados de pacientes [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172736
TESIS

Il carcinoma polmonare non a piccole cellule (NSCLC) rappresenta circa l 80% di tutti i tumori al polmone ed è il cancro più comune e più mortale al mondo. Il trattamento convenzionale per il NSCLC in stadio avanzato è stato basato per molto tempo sull uso della chemioterapia, ma con basso impatto sulla sopravvivenza . Una migliore comprensione dei meccanismi molecolari coinvolti nel processo di tumorigenesi e una maggiore capacità nell identificazione di specifiche alterazioni genetiche come bersagli terapeutici, hanno portato ad un significativo avanzamento verso lo sviluppo di terapie più efficaci. Il recettore del fattore di crescita dell epidermide (EGFR) è spesso over-espresso nel NSCLC ed è considerato un promettente bersaglio terapeutico per il trattamento di questo tumore. La presenza di mutazioni nel gene EGFR sono un importante predittore di risposta agli inibitori dell EGFR. Sebbene gli inibitori dell EGFR di prima generazione hanno mostrato incoraggianti risposte cliniche nei tumori al polmone, quasi tutti i pazienti sviluppano resistenza al trattamento nel corso del tempo. La resistenza ai trattamenti potrebbe dipendere anche dalla presenza delle cellule staminali tumorali (CSCs), una sottopopolazione di cellule intrinsecamente resistenti. Così, lo studio delle cellule staminali tumorali del polmone, potrebbe essere uno strumento efficace per l identificazione e validazione di bersagli terapeutici innovativi contribuendo all'introduzione di importanti miglioramenti nell ambito dell oncologia clinica. Pertanto, la terapia mirata verso l EGFR continua ad evolvere in seguito alla scoperta della sensibilità agli inibitori tirosin-chinasici da parte di pazienti caratterizzati da mutazioni attivanti del gene EGFR. Tuttavia, circa il 10-20% dei pazienti privi della mutazione dell EGFR, beneficiano anch essi del trattamento con gli inibitori TKIs, suggerendo che potrebbero esistere altri determinanti di risposta al trattamento, indipendenti dalla mutazione del recettore. Questo progetto, quindi, è stato focalizzato sull analisi della via di segnale dell EGFR e sullo studio della sensibilità delle cellule staminali tumorali di polmone e di modelli murini da esse derivati, agli inibitori dell EGFR, al fine di identificare possibili biomarcatori predittivi di risposta agli TKIs, in cellule prive della mutazione dell EGFR. Questo studio ha portato all identificazione della fosforilazione dell EGFR al residuo tirosina 1068, ma non 1173, come potenziale marcatore di risposta all Erlotinib nelle cellule staminali tumorali di polmone e negli xenografts da esse derivati. Inoltre, anche linee cellulari commerciali di polmone sensibili all Erlotinib, esprimevano pEGFR-tyr-1068 indipendentemente dalla mutazione dell EGFR, così, l espressione di pEGFR-tyr1068 nelle cellule staminali tumorali di polmone è risultata essere associata ad una risposta positiva al trattamento con l Erlotinib. La valutazione, mediante immunoistochimica, dello stato di fosforilazione dell EGFR in pazienti con mutazione e senza mutazione del recettore, ha portato a correlare solo pEGFR-tyr1068 e non pEGFR-tyr1173, con la mutazione dell EGFR. In base a questi dati, quindi, è possibile ipotizzare che l identificazione del livello di fosforilazione dell EGFR al residuo tirosina 1068 nei tumori dei pazienti, permetterebbe di individuare tumori con e senza mutazione dell EGFR ma caratterizzati da attivazione del recettore, in grado probabilmente di rispondere in modo positivo al trattamento con l Erlotinib. Questi studi potrebbero avere importanti implicazioni terapeutiche per il trattamento dei tumori al polmone e potrebbero permettere ai pazienti con NSCLC di essere selezionati per terapie più efficaci e meno tossiche.

Mestrado em Bioquímica Clínica
A resistência tumoral é o maior problema relacionado com a eficácia do tratamento de cancro de pulmão, tipo de cancro com a maior taxa de mortalidade a nível mundial. Atualmente, acredita-se que uma subpopulação de células tumorais, as células estaminais cancerígenas (CECs) que possuem capacidade de autorrenovação e capacidade de sustentar o crescimento tumoral, seja parcialmente responsável pela resistência tumoral face à terapia. De facto, CECs pulmonares isoladas de tumores de pacientes com cancro de pulmão revelaram-se particularmente químio-resistentes. Embora os mecanismos subjacentes à resistência não serem completamente compreendidos, a sobre-expressão de bombas de efluxo, de proteínas anti-apoptóticas e alta eficiência na reparação do ADN parecem fazer parte das propriedades das CECs responsáveis pela resistência aos agentes químio-terapêuticos. É pretendido, neste projeto, isolar e caracterizar populações de CECs pulmonares e, tendo em conta os seus mecanismos de resistência, identificar possíveis alvos terapêuticos de forma a sensibilizá-las aos fármacos atualmente utilizados clinicamente. Linhas celulares pulmonares cancerígenas (NCI-H460, A549) foram incubadas com os fármacos cisplatina ou doxorrubicina durante três semanas, seguindo-se um período de recuperação, para isolar uma possível população de CECs. Durante este período, a morfologia celular foi acompanhada e registada. Para medir o efeito de fármacos foram feitos ensaios de crescimento/viabilidade celular (ensaio à base de resazurina e ensaio de sulforodamina B) tanto nas células parentais como nas selecionadas. Realizou-se, ainda, qRT-PCR e Western blot para averiguar a existência de possíveis mecanismos de resistência nas células selecionadas. Utilizou-se citometria de fluxo e qRT-PCR para procurar marcadores de estaminalidade (como, ABCG2 e Sox2) e o ensaio de formação de colónias para verificar o enriquecimento de CECs após uma exposição prolongada aos fármacos. A exposição aos fármacos levou a uma alteração temporária da morfologia celular, onde as células apareceram com uma estrutura do tipo mesenquimal. A exposição à cisplatina conduziu a um aumento na capacidade das células NCI-H460 resistirem tanto ao agente de seleção como à gencitabina e à doxorrubicina, contudo o mesmo não se verificou em relação ao 5-FU. Após o tratamento com cisplatina, registou-se um aumento das proteínas anti-apoptóticas, Bcl-XL e XIAP, e da glicoproteína-P, em comparação com as células NCI-H460 parentais. Houve um ligeiro aumento na percentagem de células a expressar ABCG2 e, com menor intensidade, CD133. Relativamente à expressão génica do Bmi-1 e do Sox2, não foi registado nenhum aumento de expressão após o contacto com cisplatina. As células resistentes não demonstraram mais capacidade para formar colónias que as células parentais. Possivelmente, o aumento da resistência das células após o tratamento com cisplatina deve-se ao aumento de expressão das proteínas Bcl-XL, XIAP e glicoproteína-P. Como trabalho futuro ir-se-á silenciar estas proteínas através de iRNAs, numa tentativa de sensibilizar as células resistentes e validar, assim, estas moléculas como possíveis alvos terapêuticos para ultrapassar a resistência das células pulmonares cancerígenas à quimioterapia.
Tumour drug resistance is a major issue in the management of lung cancer, the worldwide leading cause of cancer-related deaths. It is currently believed that a small sub-population of tumour cells, the cancer stem cells (CSCs) that possess self-renewal capacity and are able to sustain tumour growth, are partially responsible for tumour drug resistance. Indeed lung CSCs isolated from patients’ tumours have been shown to be particularly chemoresistant. Although the mechanisms underlying this resistance are not fully understood, over-expression of efflux pumps, over-expression of anti-apoptotic proteins and efficient DNA repair seem to be involved in resistance of CSCs to chemotherapeutic agents. In this project we aim to isolate and characterize putative lung CSC populations taking into account the chemoresistance mechanisms of these cells and to identify potential therapeutic targets to render them more sensitive to the chemotherapeutic drugs used in the clinic. Lung cancer cells (NCI-H460, A549) were incubated with the drugs cisplatin or doxorubicin, for three weeks followed by a drug-free recovery period, in order to isolate a putative CSC enriched population. Cell morphology was monitored and recorded throughout the experiment. Drug-selected and parental cells were incubated with chemotherapeutic agents and multiwell based cell growth/viability assays (resazurin-based and SRB assays) were performed. Western Blot and qRT-PCR were performed to identify possible chemoresistance mechanisms present in the putative CSC enriched populations. Flow cytometry analysis and qRT-PCR for stem cell markers (e.g. ABCG2 and Sox2) and colony-forming assay were performed in order to assess enrichment of putative CSCs upon prolonged drug exposure. Drug treatment led to a transient alteration in cell morphology in both cell lines, whereby cells acquired a more mesenchymal-like structure. In NCI-H460 cells, cisplatin exposure led to increased resistance towards the selecting drug but also to doxorubicin and gemcitabine, although not for 5-FU. Increased expression of the apoptosis-related proteins Bcl-XL and XIAP and of the drug efflux pump P-glycoprotein was verified in the cisplatin-selected population, when compared to the parental NCI-H460 cell line. There was an apparent increase in the percentage of cells expressing the putative stem cell marker ABCG2, and to a much lesser extent CD133, upon drug treatment. Bmi-1 and Sox2 gene expression do not appeared to be up-regulated in selected cells and colony-forming assay did not show any differences between NCI-H460 parental and resistant cells. The verified increased drug resistance after cisplatin treatment is possibly due to overexpression of Bcl-XL, XIAP and P-glycoprotein. We will now perform RNAi approaches to inhibit the combined expression of these proteins, in an attempt to chemosensitize resistant cells and to validate these molecules as therapeutic targets for overcoming chemoresistance in lung cancer cells.

The cancer stem cell hypothesis implicates that tumor cell population is heterogeneous with relatively well-differentiated cells and poorly-differentiated cells. Only the small population of tumourigenic poorly-differentiated CSCs can escape the normal limits of self-renewal and has the ability to proliferate and maintain the malignant growth of the tumor. One characteristic of stem cell is that the ability to exclude DNA dyes, such as Hoechst 33342 via the over-expression of ATP-binding cassette transporters (ABC transporters) on the cell membrane. It makes the detecting of the stem cell possible. After the Hoechst 33342 staining, stem cells extrude this dye and show a typical profile of low fluorescence in Hoechst red versus Hoechst blue bivariate dot plots. These low Hoechst 33342 stained cells are named as side population (SP) cells. This characteristic has enabled purification and characterization of CSCs when carried out alone or in combination with stem cell surface markers. The CSC hypothesis could have a fundamental influence on cancer therapy. CSCs have shown significant substantial resistance to conventional chemotherapy in contrast to the differentiated cancer cells. It is essential to design a complete therapy strategy first to reduce or minimize proliferating cell mass and then to differentiate or eliminate CSCs, so that the relapses of metastatic cancers could be prevented. This work aimed at investigating if Hedgehog pathway inhibitor GDC-0449 is effective in the lung cancer cell lines HCC (adeno-carcinoma) and H1339 (small cell lung carcinoma) and also the cisplatin resistant lung cancer cells, and if possible effects of GDC-0449 are mediated via SPs. Furthermore, the effect of GDC-0449 on the calcium homeostasis was also investigated. GDC-0449 showed dose-dependent inhibitory effects on cell growth in both HCC and H1339 cells. The combination of GDC-0449 and cisplatin gave an additional inhibitory effect. GDC- 0449 could also inhibit the cell growth in cisplatin resistant HCC and H1339 cells. SP cells as cancer stem-cell-like cells could be found in HCC and H1339 cells. Only the SP cells showed the repopulation ability but not the non-SP cells. GDC-0449 could inhibit the SP cell fraction in both HCC and H1339 cells. So the inhibitory effect of GDC-0449 on cell growth may be mediated via SP. GDC-0449 affected the expression of the Hh pathway components in both HCC and H1339 cells. In HCC cells, GDC-0449 inhibited the activity of the Hh pathway and the down- regulation of Shh, Patched and Gli-1 could be shown. In H1339 cells, GDC-0449 could also inhibit the pathway activity and decrease the expression of Gli-1 in an autocrine pattern due to the over-expression of Shh. The inhibition of Hh pathway increased the expression of Bmi-1 to compensate the loss of Hh pathway function. The Hh pathway activity could be detected only in SP cells from HCC and H1339 cells. The application of GDC-0449 on HCC and H1339 naïve and cisplatin resistant cells increased [Ca2+]c by decreasing [Ca2+]ER. GDC-0449 induced Ca2+ release from ER into cytoplasm in HCC and H1339 naïve and cisplatin resistant cells. The Ca2+ overload could lead to apoptosis, which was related to the cell growth inhibitory effect of GDC-0449 in lung cancer cells. The expression of SERCA and IP3R was not detectably influenced by GDC-0449. The effect of GDC-0449 on lung cancer cell Ca2+ -regulating machinery was not via the alternation of the expression of ER Ca2+ regulating channels.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by persistent airway obstruction that is only partially reversible. It is the fourth leading cause of death and is predicted to be the third by 2030. The progression of the disease involves chronic inflammation, oxidative stress, excess protease activity, increased lung cell apoptosis and accelerated lung aging, but the exact pathogenesis is still unclear. The major cause of COPD is cigarette smoking(CS). Although COPD is associated with increasing social and economical burden, there have been few advances in pharmacological therapy of COPD. Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells which can be isolated from a broad range of sources including bone marrow (BM) and adipose tissue. Administration of BM-derivedMSCs (BM-MSC) or adipose tissue-derived MSCs was reported to attenuate CS-induced emphysema in murine models. Induced pluripotent stem cell-derived MSC (IPSC-MSC) are MSCs differentiated from induced pluripotent stem cells(IPSCs), which are pluripotent cells generated by somatic cell reprogramming in vitro. IPSC-MSCs have several advantages over BM-MSC, including more abundant sources and high capacity of doubling without loss of differentiation potency. A general exploration and comparison on the effects of human IPSC-MSC and BM-MSC treatments were carried out in a 56-day CS-exposed rat model. Compared to BM-MSC, IPSC-MSC showed a higher capacity to reside in lung tissue. The two treatments shared similar efficacy to attenuate CS-induced lung cell apoptosis, to restore CS-induced reduction of lungIL-10and to alleviate CS-induced elevation of systemic TGF-β1. In addition, IPSC-MSC was found to cause reduction in CS-induced elevation of systemic oxidative stress and reversal of CS-induced reduction of lung adiponectin. Furthermore, in order to understand the possible paracrine mechanism involved, human airway epithelial cells were treated with IPSC-MSC or BM-MSC-conditioned medium in a cell culture system in the presence of cigarette smoke medium (CSM). Potentiation rather than attenuation of CSM-induced release of pro-inflammatory cytokine IL-8, MCP-1 and IL-6 was observed with IPSC-MSC or BM-MSC conditioned medium. It is currently unknown whether cultured IPSC-MSCs or BM-MSCs will release pro-inflammatory mediators into the conditioned medium or not. In order to study CS-induced oxidative stress and inflammation in a short time frame, anacute (5-day) CS-exposed rat model was established in juvenile and adult groups. An age-dependent alteration of CS-induced oxidative and inflammatory responses was demonstrated in this model. In summary, our in vivo rat model provides a platform for elucidating the effects of stem cell treatment in CS-induced oxidative stress and inflammation, leading to lung damage. Our findings suggest that treatment of IPSC-MSC or BM-MSC might be able to slow down CS-induced disease progression, possibly through anti-oxidant, anti-inflammatory and anti-apoptotic properties. However, caution should be taken as our in vitro data revealed that conditioned medium from MSCs may provoke pro-inflammatory responses. Further studies on the regulation of the activity of MSCs in vivo will be needed before developing IPSC-MSC into cell therapies for COPD to halt the progression over time.
published_or_final_version
Medicine
Master
Master of Philosophy

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
During early lung and thyroid development two populations of Nkx2-1-expressing progenitors in the foregut endoderm give rise to the entire lung and thyroid epithelium. To date, little is known about the genetic programs of these progenitors since they are few in number, present only fleetingly in the embryo, and have not been studied in pure preparations. We demonstrate here the purification, and directed differentiation in culture of primordial lung and thyroid progenitors derived from mouse embryonic stem (ES) cells. We find that sequential stage-specific and time-dependent inhibition of Transforming Growth Factor β and Bone Morphogenetic Protein signaling, followed by combinatorial stimulation of BMP and FGF signaling is necessary for the efficient specification of these cells from definitive endodermal precursors in vitro. Employing a novel Nkx2-1GFP knock-in reporter ES cell line, these progenitors can be isolated, expanded in culture, and display a global transcriptome that overlaps that of an in vivo developing lung epithelium. Moreover, these progenitors can be induced to sequentially express a broad repertoire of lineage markers indicative of the full diversity of lung and thyroid epithelial differentiation. Thus, we have derived the first population of progenitors that recapitulates the developmental milestones of lung/thyroid development.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2020
Cataloged from student-submitted PDF of thesis. Vita.
Includes bibliographical references.
Lung adenocarcinoma (LADC) remains the most common and lethal cancer type worldwide. Although recent breakthroughs using a new class of immune-modulatory therapeutics have improved patient survival in the clinic, the majority still invariably succumb to this disease, highlighting the importance of improving treatment strategies. A wide variety of models have been developed to study LADC. Cell line- and transplant-based models offer rapid and flexible platforms for discovering and testing novel therapeutics using patient-derived specimens. On the other hand, genetically engineered mouse models (GEMMs) recapitulate key aspects of human LADC including initiation from normal pulmonary epithelial cells and progression into a malignant state. The development of organoid technology has revolutionized the way we model cancer and a vast number of other biological phenomena.
Organoids are cultured miniature organs derived from normal adult stem cells that display self-renewal, differentiation capacity and remarkable genetic stability. These features have facilitated the creation of next generation cancer models that combine the best features of their predecessors. The alveolar type 2 (AT2) cell represents the most prominent cell-of-origin of LADC. These cells serve as stem cells in the adult lung to support tissue turnover during homeostasis and regeneration after injury. They accomplish this by self-renewing and differentiating into alveolar type 1 (AT1) cells. Using organoid technology, we have developed an improved system to cultivate alveolar organoids from normal murine lungs. We demonstrated that these organoids are positive for AT2 and AT1 markers and completely lack expression of basal and club cell makers. Critically, we observed long-term proliferative potential in these organoids.
Using this improved culture system, we generated organoid models of LADC, representing three distinct molecular subclasses of this disease. We found that Kras-, Braf-, and Alk-mutant organoids with Trp53 deficiency displayed mitogen independent growth in vitro. Most strikingly, Krasmutant Trp53-inactivated organoids orthotopically transplanted into immunocompetent recipient mice formed tumors that displayed histopathological characteristics of human LADC. Taken together, the work presented here demonstrates the power of organoid technology for building clinically relevant and experimentally flexible cancer models.
by Santiago Naranjo.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology

Multiple research problems related to the lung involve a need to take into account the spatiotemporal dynamics of the underlying component cells. Two such problems involve better understanding the nature of the allergic inflammatory response to explore what might cause chronic inflammatory diseases such as asthma, and determining the rules underlying stem cells used to engraft decellularized lung scaffolds in the hopes of growing new lungs for transplantation. For both problems, we model the systems computationally using agent-based modeling, a tool that enables us to capture these spatiotemporal dynamics by modeling any biological system as a collection of agents (cells) interacting with each other and within their environment. This allows to test the most important pieces of biological systems together rather than in isolation, and thus rapidly derive biological insights from resulting complex behavior that could not have been predicted beforehand, which we can then use to guide wet lab experimentation. For the allergic response, we hypothesized that stimulation of the allergic response with antigen results in a response with formal similarity to a muscle twitch or an action potential, with an inflammatory phase followed by a resolution phase that returns the system to baseline. We prepared an agent-based model (ABM) of the allergic inflammatory response and determined that antigen stimulation indeed results in a twitch-like response. To determine what might cause chronic inflammatory diseases where the twitch presumably cannot resolve back to baseline, we then tested multiple potential defects to the model. We observed that while most of these potential changes lessen the magnitude of the response but do not affect its overall behavior, extending the lifespan of activated pro-inflammatory cells such as neutrophils and eosinophil results in a prolonged inflammatory response that does not resolve to baseline. Finally, we performed a series of experiments involving continual antigen stimulation in mice, determining that there is evidence in the cytokine, cellular and physiologic (mechanical) response consistent with our hypothesis of a finite twitch and an associated refractory period. For stem cells, we made a 3-D ABM of a decellularized scaffold section seeded with a generic stem cell type. We then programmed in different sets of rules that could conceivably underlie the cell's behavior, and observed the change in engraftment patterns in the scaffold over selected timepoints. We compared the change in those patterns against the change in experimental scaffold images seeded with C10 epithelial cells and mesenchymal stem cells, two cell types whose behaviors are not well understood, in order to determine which rulesets more closely match each cell type. Our model indicates that C10s are more likely to survive on regions of higher substrate while MSCs are more likely to proliferate on regions of higher substrate.

Bioengineering
Ph.D.
Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases and the third leading cause of death in the US, estimated to increase in magnitude in the future. Current treatment approaches are palliative in nature and restricted to controlling symptoms and reducing the risk of complications. Lung transplantation is an option for certain patients, but this option is limited by the shortage of donor organs and the possibility of rejection and the need for life-long immune-suppression. Therefore, current studies focus on cell based therapies for lung repair and regeneration. In addressing the issue of cell sourcing for such approaches, I tested the hypothesis that the efficiency of directed pulmonary differentiation of mouse embryonic stem cells (mESC) can be enhanced by employing certain micro-environmental cues, found in the developing lung. Such micro-environmental cues will provide appropriate physicochemical signals at the right time during the embryonic development and thus modulate fate decisions of progenitor cells during tissue assembly and maturation. In this study, I explored the effects of matrix stiffness on cell fate decisions in mESC, first into definitive endoderm and then into lung alveolar epithelial cells. I engineered bio-activated polyacrylamide (PA) gels with varying elastic moduli, mimicking those of physiologic tissues, and covalently modified the surfaces with fibronectin to provide optimal stem cell adhesion. My studies demonstrated, for the first time, a biphasic stiffness-dependent enhancement of endodermal differentiation of mESCs, with an optimum at ~ 20 kPa. This effect was qualitatively similar in three different mESC lines. By contrast, increasing matrix stiffness favored mESC differentiation towards a mesodermal phenotype. The enhanced endodermal differentiation of mESCs was abolished in the presence of a specific inhibitor of ROCK, suggesting that this process is mediated through cytoskeletal signaling. The subsequent differentiation of mESC-derived endodermal cells towards pulmonary epithelial cells was no longer dependent on the stiffness of the matrix. In this dissertation I demonstrate for the first time the feasibility of utilizing developmental and physiological / physicochemical cues, such as matrix stiffness, to selectively modulate and enhance mESC differentiation towards endodermal and pulmonary lineages. The impact of the results will be relevant for optimizing cell-based lung therapies and for effectively engineering lung and other endoderm-derived organs.
Temple University--Theses

Abstract Bone marrow mesenchymal stromal cells (MSCs) and mononuclear cells (BM-MNCs) have shown great therapeutic potential in various clinical settings. Although intravascular transplantation of the cells constitutes the optimal delivery route, massive pulmonary entrapment, with the threat of embolization, remains a major obstacle for using this type of therapy. Because pulmonary entrapment is at least partially mediated by adhesion molecules, cell surface modification could enhance pulmonary passage. We used a porcine model of allogeneic MSC and autologous BM-MNC transplantation and radionuclide labelling to track the cells. The role of the transplantation route on lung entrapment, biodistribution, safety and BM-MNC targeting to the injured brain was studied. Effects of pronase detachment on the lung passage of MSCs were studied in porcine and murine models; a rat model of acute limb injury was used to further evaluate tissue targeting. Treatment with pronase to detach cell surface molecules and the effect on stem cell potential was assessed in vitro. Intra-arterial administration of MSCs diminishes their lung deposition; intravascular transplantation did not cause pulmonary embolisms. Intra-arterially transplanted BM-MNCs did not reach the brain in significant numbers. Transient proteolytic modification of MSCs with pronase decreased lung accumulation and tissue targeting without affecting their therapeutic characteristics. Intra-arterial transplantation increases lung passage of MSCs. Although thromboembolic events were not observed, further studies are warranted to ensure the safety of this route of MSC delivery. Pronase detachment is a promising method to enhance the potential of systemic MSC therapies
Tiivistelmä Luutytimen mesenkymaaliset kantasolut (MSC) ja mononukleaariset solut (BM-MNC) ovat osoittautuneet tehokkaiksi useissa kliinisissä käyttöaiheissa. Solujen systeeminen annostelu verenkiertoon olisi käytännön kannalta paras soluterapian toteutukseen, mutta solujen merkittävä taipumus jäädä keuhkoihin loukkuun ja veritulppariski muodostavat haasteen. Keuhkohakeutumisen tiedetään ainakin osin johtuvan solujen pintamolekyyleistä ja näiden muokkaaminen voisi parantaa solujen keuhkoläpäisevyyttä. Tutkimuksessa käytettiin koe-eläimenä sikaa, jolle istutettiin systeemisesti allogeenisia mesenkymaalisia kantasoluja tai autologisia luuytimen mononukleaarisia soluja; solujen kudoshakeutumisen seuranta toteutettiin isotooppileimauksella- ja kuvannuksella. Tutkimuksessa arvioitiin annostelureitin vaikutusta keuhkoläpäisevyyteen, solujen kudojakautumista, toimenpiteen turvallisuutta sekä mononukleaarisolujen hakeutumista vaurioituneeseen aivokudokseen. Pronaasikäsittelyn vaikutusta mesenkymaalisten kantasolujen keuhkoläpäisevyyteen arvioitiin sika- ja hiirimallissa; rotan raajavauriomallia käytettiin lisäksi pronaasin kudoshakeutumisvaikutusten arvioimiseen. Pronaasikäsittelyn vaikutuksia solujen pintarakenteisiin ja toiminnallisuuteen arvioitiin in vitro- kokeissa. Mesenkyymalisten kantasolujen annostelu valtimonsisäisesti paransi solujen keuhkoläpäisevyyttä; tutkimuksissa käytetyt solut eivät aiheuttaneet keuhkoveritulppia. Valtimonsisäisesti annostellut mononukleaarisolut eivät hakeutuneet vaurioituneeseen aivokudokseen sikamallissa. Pronaasikäsittely muovasi solujen pintaproteiineja palautuvasti ja tämä lisäsi huomattavasti mesenkymaalisten kantasolujen keuhkoläpäisevyyttä ja kudoshakeutumista vaikuttamatta solujen toiminnallisuuteen. Mesenkymaalisten kantasolujen annostelu valtimonsisäisesti voi parantaa solujen keuhkoläpäisevyyttä. Tutkimuksessa ei todettu keuhkoveritulppaa tai muita tromboembolisia tapahtumia, mutta lisätutkimuksia tarvitaan MSC- terapian turvallisuuden takaamiseksi. Pronaasikäsittelyn tulokset mesenkymaalisten kantasolujen systeemisen annostelun parantamisessa olivat lupaavia

Chronic obstructive pulmonary disease (COPD) including emphysema is a devastating condition, increasing in prevalence in the US and worldwide. There remains no cure for COPD, rather only symptomatic treatments. Due to unique challenges of the lung, translation of therapies for acute lung injury to target chronic lung diseases like COPD has not been successful. We have been investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. During the course of this work we have developed and characterized a lug derived ECM hydrogel that exhibits “injectability,” allowing cells or dugs to be delivered in a liquid and encapsulated at body temperature. The hydrogel self assembles in <5 minutes and achieves mechanical stiffness similar to other soft tissue ECM hydrogels. The hydrogel can support 3D cell growth and encapsulated cell viability. Encapsulated hMSCs can also still be activated by simulated inflammatory environments. Naïve mouse macrophages exposed to the fully formed gel were not significantly induced to express markers for pro or anti-inflammatory polarized phenotypes, but increased expression for several secreted inflammatory mediators was observed. We also investigated a novel approach for preparing and solubilizing the isolated ECM proteins, using digestion time as a variable for controlling hydrogel density (interconnectivity), mechanical stiffness, component protein size distribution, and cell behavior on fully formed gels. The potential future impact for the presented research includes optimization for future animal studies, expansion to additional applications, and the development of new derivative materials.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation caused by a combination of airways disease (bronchiolitis) and parenchymal destruction (emphysema), whose relative proportion varies from patient to patient. An abnormal immune response to tobacco smoke is one of the main factors, both in the lungs and circulating blood. Both the relation between the pulmonary and systemic inflammation and the regenerative capacity of the lung are also unclear. OBJECTIVES: Divided into 3 specific objectives: 1. To explore and contrast the molecular pathogenesis of emphysema and bronchiolitis in COPD. 2. To characterize and contrast the cellular pulmonary and systemic immune response in COPD patients and healthy controls smokers and non-smokers. 3. To identify, characterize and compare the immunomodulatory capacity of a putative resident stem cell population in the lung. METHODS: For the first objective differential expression and gene co-expression in bronchiolitis and emphysema were analyzed by lung transcriptomics. For the second, flow cytometry from lung tissue and blood was performed to evaluate the cellular immune response. For the third objective, a new cell culture methodology was developed. RESULTS: Specific results for each objective 1. Emphysema signature is different from bronchiolitis with an up-regulated expression of ontologies related with B-cell homing and activation. 2. The lung of mild COPD patients has increase macrophages and decrease T lymphocytes associated to both COPD and smoking status, that is not co-related with systemic immune response. 3. A putative lung resident stem cell population was identify with transcriptional signature of mesenchymal origin and immunomodulatory properties. CONCLUSIONS: The lung immune response is heterogeneous and associated with both the lung regenerative capacity and the clinical heterogeneity/complexity of COPD.
INTRODUCCIÓN: La enfermedad pulmonar obstructiva crónica (EPOC) se caracteriza por una limitación crónica del flujo aéreo causada por una combinación de enfermedad de las vías aéreas (bronquiolitis) y destrucción del parénquima (enfisema). La contribución relativa de cada patología varia entre pacientes. Un respuesta anormal y exagerada frente al humo del tabaco es uno de los principales factores de riesgo. La relación entre la respuesta inmune pulmonar y sistémica y la capacidad regenerativa del pulmón no se encuentran determinadas. OBJETIVOS: Hay 3 objetivos específicos: 1. Comparar las diferencias moleculares entre la patogénesis del enfisema y la bronquiolitis en la EPOC. 2. Caracterizar y comparar la respuesta inmune celular a nivel pulmonar y en sangre periférica en pacientes con EPOC y controles sanos fumadores y no fumadores. 3. Identificar, caracterizar y comparar la actividad inmunomoduladora de una población de células madre residentes en pulmón. MÉTODOS: Para el primer objetivo la expresión diferencial y la co-expresión de los genes se realizó mediante análisis del transcriptoma pulmonar. Para el segundo, se utilizó citometría de flujo para la determinación celular de las poblaciones de la respuesta inmune en pulmón y sangre. Para el tercer objetivo, se ha desarrollado una nueva metodología de cultivo celular. RESULTADOS: Resultados específicos por objetivos 1. La caracterización molecular es diferentes entre la bronquiolitis y el enfisema con un incremento en la expresión de genes relacionados con la activación y movilidad de los linfocitos B. 2. El pulmón de los pacientes con EPOC leve contiene un incremento de macrófagos y una reducción de linfocitos T asociada tanto a la EPOC como al consumo de tabaco. 3. Se ha identificado una población de células madre residentes en pulmón con una transcriptómica de origen mesenquimal y propiedades inmunomoduladoras. CONCLUSIONES: la respuesta inmune pulmonar es heterogénea y está asociada tanto con la capacidad regenerativa como con la complexidad de la EPOC.

The number of deaths around the globe due to lung diseases is over 9.5 million each year, including the paediatric population, and it is expected to increase. Currently the only treatment for many end-stage debilitating lung diseases is orthotopic lung transplantation. Nonetheless, there are several problems associated with this procedure, including shortage of donor organs, the risk of allograft rejection, organ failure and the lifelong burdening regimen of immunosuppressive drugs that the patients must take. One potential solution is the transplantation of a bioengineered lung made from patient’s own cells. With this approach, damaged or immunologically incompatible lungs of allogeneic or xenogeneic origin can be decellularised to generate scaffolds that retain the macro/micro architecture and topological specificity, including a perfusable vascular bed. These acellular scaffolds could be then seeded with patient specific cells of epithelial and endothelial origin and transplanted back to the patient. In this project, human amniotic fluid stem cells (hAFSC) were reprogrammed into induced pluripotent stem cells (iPSC) using clinically relevant episomes, and cell lines were thoroughly characterised. The potential of the iPSC to be directly differentiated into endodermal and mesodermal lineages such as the alveolar epithelium and vasculature respectively was then assessed. In parallel, a novel automatic decellularisation/recellularisation bioreactor was designed in which the aforementioned cells were seeded and cultured, under dynamic conditions. The combination of the acellular constructs, the lung epithelial cells from iPSC as well as vascular cells all put together represents a proof of concept towards the translation of tissue engineered lungs to the clinics.

Allogenic stem cell transplantation represents an important therapeutic option for the treatment of a number of haematological diseases. Particularly in the context of onco-haematological diseases this treatment has been shown to improve outcome thanks to the graft versus tumour (GVT) effect. Although recent improvements in transplantation procedures, still some limitations to the applicability of these treatment persist. First, not all patients may have available an HLA (human leukocyte antigens) identical donor and secondly, conditioning regimens before transplantation may be too toxic for elderly patients or for patients with other co-morbidities. To overcome this limitations in last few years many researchers have been exploiting new approaches. The introduction of not fully matched transplantations (mismatched or haploidentical) and reduced intensity regiments have partially overcome the limitations. In the onco-haematological context the most common complications of allogenic stem cell transplantation are relapse, opportunistic infections and activation of transplanted immune system against the normal tissues of the host (graft versus host disease – GVHD). All of these represent alterations of the normal functions of the immune system and demonstrate the importance of monitoring immunity in allo-transplanted patients. Studies in these field are mostly limited to the evaluation of the first year after transplantation and data from longer follow up are limited. In this study we monitored patients undergoing haematopoietic stem cell transplantation from an alternative donor after reduced intensity regimen over one year after transplantation (up to 4-5 years after transplantation). Particularly we evaluated 6 patients (age at transplantation 34; range 15-49) undergoing haploidentical stem cell transplantataion associated with T cell depletion in vitro (immunomagnetic selection of CD34 stem cells) and in vivo (anti CD52 antibody – Alemtuzumab) followed by infusion of CD8 depleted donor lymphocytes and 18 patients (age at transplantation 40; range 22-60) undergoing transplantation from a match unrelated donor (MUD) followed by in vivo T cell depletion (anti thymocyte globulins – ATG). All the patients have been analysed at the times of clinical remission and not under pharmacological treatment. In order to compare data obtained by the single cohorts to a normal situation we also evaluated 10 healthy donors (age 37; range 24-55). The evaluation of the immune recovery has been carried out through 4 different techniques: - analysis of chimerism through amplification of 9 different short tandem repeats (STR); - evaluation of the lymphoid sub population B, T and NK (and their maturation stages) through flow cytometry immunophenotype; - analysis of the thymic productivity trough quantification of the episomal DNA sjTREC (signal joint T-cell receptor excision circle); - evaluation of the receptorial complexity of the T and B cell compartment trough analysis of the CDR3 (complementary determinating region 3) of the β chain of the T cell receptor and of the heavy chain of the immunoglobulins. Our results show no significant differences between the two groups of patients analysed in the long term immune reconstitution, neither respectively the counts of the lymphoid sub population nor regarding the complexity values for the B and T cell receptors comparing the data to the ones from healthy subjects. We show a reduction in the thymic productivity that persist over 3 years post transplantation although there are no difference comparing the two cohorts of patients. Even though the counts of the main populations normalize between 1 and 2 years after transplantation the analysis of the maturation of the B and T cell compartments highlights the persistence of alterations in the normal maturation process in all the follow up points. Particularly both patients undergoing haploidentical or MUD transplantation present an increase in the B naïve subpopulation and a decrease in the B memory subsets demonstrating an alteration in the normal B cell development probably due to an alteration in the normal function of the germinal center. Concerning the T cell compartment it has been seen a decrease in the production of naïve T cells, that reflects the low thymic production, and an increase in the effector and terminal memory compartment. These might be a mechanism involved in the control of the oncological disease . In conclusion, patients that do not relapse and do not experience other clinical problems are able to recover immunity in the long term although thymic production remains low. No significant difference are found between the two types of transplantation highlighting that haploidentical transplantation is a good alternative to HLA identical transplantation implying less problems for donor recruitment. Analysis of the maturation steps of the B and T cell compartment demonstrated the persistence of alterations long term after transplantation indicating that this evaluation should be further studied in order to elucidate the mechanism at the basis of the immune recovery. Moreover this could be a good marker for monitoring the clinical course of the patients.

O câncer de pulmão está entre os tipos de câncer com maiores índices de mortalidade no mundo. Na década de 90, houve a descoberta de populações de células tumorais com características de células-tronco e implicou na hipótese das células-tronco cancerosas (CTCs). Se a hipótese das CTCs for correta, a recorrência/recidiva dos cânceres é gerada por uma porcentagem de células (CTCs) que prolifera pouco e é resistente a quimioterapia convencional. Desta forma, a busca por novos alvos terapêuticos que tenham como alvo estas CTCs tem enorme implicação no desenvolvimento de novos fármacos ou modalidades terapêuticas contra o câncer. O guaraná (Paullinia cupana Mart var. sorbilis) tem demonstrado efeitos promissores sobre o câncer, em especial efeitos quimiopreventivos e antineoplásicos. Portanto, objetivando-se neste projeto avaliar a presença de CTCs a partir de células tumorais de pulmão de camundongo, onde se possa conseguir rapidamente uma alta porcentagem deste tipo celular para posteriores estudos, além de avaliar os efeitos do extrato etanólico do guaraná e de suas frações butanólica e aquosa, sobre as células tumorais e em especial verificar os efeitos sobre a população rica em CTCs, onde foi possível observar a presença de possíveis CTCs, a partir de cultivo de células tumorais de pulmão de camundongo, como também que o guaraná apresentou um efeito sobre estas possíveis CTCs devido a diminuição da expressão dos genes ABCG2 e ALDH1a1.
Lung cancer is among the cancers with the highest mortality rates in the world. In the 90\'s, there was the discovery of tumor cell populations with stem cell characteristics implied in the case of cancer stem cells (CSCs). If the hypothesis is correct CSCs of the recurrence / relapse of cancers is generated by a percentage of cells (CSCs) that growth low and are more resistant to conventional chemotherapy. Thus, the search for new therapeutic targets that target these CSCs has huge implications in developing new drugs or therapeutic approaches against cancer. Guarana (Paullinia cupana Mart var. Sorbilis) has shown promising effects on cancer, especially chemopreventive and anticancer effects. Therefore, the objective in this project is to evaluate the presence of CSCs from tumor cells of mouse lung, where it can quickly get a high percentage of this cell type for further studies and to evaluate the effects of ethanol extract of guarana and its butanol and aqueous fractions on tumor cells and in particular to verify the effect on the population rich in CSCs, where it was possible to observe the possible presence of CSCs from cultivation of tumor cells from mouse lung, as well as guarana presented an effect on these CSCs possible to decreased expression of ABCG2 and ALDH1a1 genes.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Tumor hypoxia is common and can contribute to drug resistance. To take advantage of hypoxia, bioreductive drugs that are activated to cytotoxic metabolites in a hypoxic tumor environment have been developed. The cytotoxicity of one such drug, AQ4N, was assessed under normoxic and hypoxic conditions across a panel of tumor cell lines. AQ4N showed significantly increased cytotoxicity under hypoxia in rat 9L gliosarcoma and H460 human non-small cell lung carcinoma cell cultures, but not in cultures often other human cancer cell lines. Thus, the bioreductive activation of AQ4N is not widespread in cancer cell lines. Protein levels of the quinone reductase DT-diaphorase were poorly correlated with AQ4N chemosensitivity across the cell line panel, and AQ4N chemosensitivity was unaffected by DT-diaphorase inhibitors, indicating little contribution of DT-diaphorase to AQ4N cytotoxicity. The vasodilator hydralazine decreased tumor perfusion and increased tumor hypoxia in 9L tumor xenografts, and to a lesser extent in H460 xenografts, but did not increase AQ4N-dependent anti-tumor activity. Combining of AQ4N with the anti-angiogenic drug axitinib did not augment AQ4N anti-tumor activity beyond that of axitinib alone, despite the increased hypoxic environment. Thus, AQ4N activation in vivo requires tumor hypoxia that is more extensive or prolonged than can readily be achieved by vasodilation or anti-angiogenic drug treatment. Cancer stem-like cells have been proposed to be critical for tumor growth, initiation, and drug resistance. Stem-like cells from six human tumor cell lines were isolated at frequencies ranging from 12-69% based on their characteristic holoclone morphology. All but one tumor cell line yielded holoclones with the capacity for self-renewal. Tumor xenografts grown from H460 holoclones showed significant increases in microvessel density and tumor blood perfusion compared to parental H460 tumors. Microarray analysis identified genes commonly dysregulated in holoclone-derived H460 tumors, including a network of genes associated with angiogenesis. These and other genes may serve as therapeutic targets to eliminate cancer stem-like cells or inhibit angiogenesis in H460 tumors. Together, these studies advance efforts to improve bioreductive drug activity through anti-angiogenesis and elucidate the role of stem-like cells in tumor angiogenesis.

Mathematical models apply to a multitude physiological processes and are used to make predictions and analyze outcomes of these processes. Specifically, in the medical field, a mathematical model uses a set of initial conditions that represents a physiological state as input and a set of parameter values are used to describe the interaction between variables being modeled. These models are used to analyze possible outcomes, and assist physicians in choosing the most appropriate treatment options for a particular situation. We aim to use mathematical modeling to analyze the dynamics of processes involved in the inflammatory process. First, we create a model of hematopoiesis, the processes of creating new blood cells. We analyze stem cell collection regimens and statistically sample parameter space in order to create a model accounts for the dynamics of multiple patients. Next, we modify an existing model of the wound healing response by introducing a variable for two inflammatory cell types. We analyze the timing of the inflammatory response and introduce the presence of systemic estrogen in the model, as there is evidence that the presence of estrogen leads to a more efficient wound healing response. Last, we mathematically model the gas exchange process in the lungs and body in order to lay the foundation for a model of the inflammatory response in the lung under conditions of mechanical ventilation. We introduce normal and ventilation breathing waveforms and a third state of hemoglobin in a closed loop partial differential equations model. We account for gas exchange in the lung and body compartments in addition to introducing a third discretized well-mixing compartment between the two. We use ordinary and partial differential equations to model these systems over one or more independent variables, as well as classical analysis techniques and computational methods to analyze systems. Statistical sampling is also used to investigate parameter values in order for the mathematical models developed to account for patient-to-patient variability. This alters the traditional mathematical model, which yields a single set of parameter values that represent one instance of the physiology, into a mathematical model that accounts for many different instances of physiology.}

O enfisema pulmonar define-se como a destruição das paredes alveolares e consequente dispneia progressiva. Este trabalho objetivou a adequação de um modelo de transplante celular in vivo de BMMC em camundongos com enfisema pulmonar. O enfisema foi induzido por instilação nasal de elastase (4 UI por animal). O diâmetro alveolar médio para os grupos não tratados e tratados com elastase apresentou diferença estatisticamente significativa, e mudanças no padrão de expressão de metaloproteinases envolvidas no processo inflamatório foram detectadas, indicando que a instilação de uma dose de elastase promove lesão semelhante ao enfisema pulmonar. Infundiu-se 0,4ml de BMMC (7x106 céls./ml) nestes animais. No grupo tratado com células, detectou-se mudanças morfométricas e no padrão de expressão de metaloproteinases, indicando melhora na evolução da lesão pulmonar 21 dias após a infusão. Foram ainda avaliadas duas e três doses do pool de BMMC, porém os resultados das análises mostraram que não há diferenças entre estre grupos e os grupos controle.
Pulmonary emphysema is defined as the destruction of the alveolar walls and consequent progressive dyspnea. This study aimed the adequacy of a model of BMMC transplantation in vivo in mice with pulmonary emphysema. Emphysema was induced by nasal instillation of elastase (4 IU per animal). The mean linear intercept for the groups untreated and treated with elastase showed a statistically significant difference, and changes in the pattern of expression of metalloproteinases involved in inflammation were detected, indicating that the instillation of a dose of elastase promotes lung damage similar to emphysema. 0.4 ml of BMMC (7x106 céls. / ml) was infused in these animals. In the group treated with cells there were detected and morphometric changes in the pattern of expression of metalloproteinases, indicating an improvement in the evolution of lung injury 21 days after infusion. Were also evaluated two and three doses of the pool BMMC, but the results of the analysis showed no differences between experimental and the control groups.

Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.

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Currently the Chronic Obstructive Pulmonary Disease (COPD) has a high prevalence and a high economic and social cost. In this context, several experimental models have been proposed, aiming at the discovery of new therapeutic approaches. Accordingly, the use of mesenchymal stem cells (MSC) is an innovative and accessible treatment of pulmonary acute and chronic disease, as they have important therapeutic potentials (immunoregulation, anti-fibrogenic, inducing proliferation of tissue progenitor cells, anti-apoptotic and pro-angiogenic and chemoattraction). Therapy with low levellaser (LLL) is a relatively new effective therapy, with very low cost and no side effects. In this project we aim to study some parameters in animals with COPD undergoing therapies with LLL (15 days before the experiment) and MSC obtained from human fallopian tube (administered 2 times: 15 days and 7 days before the experiment). The protocol used for the induction of COPD consists in submitting C57BL/6 mice for 75 days (2 times / day) to inhaled cigarette smoke. On day 76ththe animals were sacrified and structural and functional parameters of lungs were evaluated. Our results indicate that the treatment with LLL and MSC greatly reduces lung inflammation (as demonstrated through BAL cell counting and histomorphometric analysis in lung parenchyma), BAL pro-inflammatory cytokines (IL-1β, IL-6, IL-10, TNF-α and KC) and the lung expression of NF-B, NFAT and IL-10. Furthermore, these therapies also reduced airway mucus secretion and collagen deposition.
Atualmente a Doença Pulmonar Obstrutiva Crônica (DPOC) apresenta alta prevalência e um elevado custo econômico e social. Neste contexto, vários modelos experimentais têm sido propostos, objetivando o descobrimento de novas opções terapêuticas. Neste sentido, o uso de células-tronco mesenquimais (CTM) constitui uma estratégia inovadora e acessível para tratamento de doenças pulmonares de caráter agudo e crônico. Essas células-troncos possuem um importante potencial terapêutico (imunorregulação, anti-fibrogênica, indutora da proliferação de células progenitoras teciduais, anti-apoptótica, pró-angiogênica e de quimioatração). A terapia com Laser de Baixa Potência (LBP) é uma terapia relativamente nova e eficaz, de baixíssimo custo, sem efeitos colaterais e de possível utilização no tratamento das doenças crônicas pulmonares. No presente estudo visamos estudar alguns parâmetros em animais com DPOC submetidos às terapias com laser de diodo (660nm), 30 mW, 60 s por ponto (3 pontos por aplicação) por 15 dias antes do experimento e com CTMs obtidas da tuba uterina humana (104 células administradas i.p. ou i.n. 2 vezes: 15 dias e 7 dias antes do experimento).O protocolo utilizado para a indução da DPOC consistiu em nebulizar camundongos fêmeas C57BL/6 com fumaça de cigarro por 75 dias (2 vezes/dia). No dia 76, os animais foram sacrificados e avaliados os parâmetros funcionais e estruturais pulmonares.Nossos resultados indicam que os tratamentos com CTM e LBP reduziram a inflamação pulmonar (demonstrado pela contagem total e diferencial de células do lavadobroncoalveolar (LBA) e análise histomorfométrica do parênquima pulmonar), os níveis de citocinas quimiocinas (IL-1β, IL-6, IL-10, IFN-, TNF-α e, KC) e a expressão de NF-B, NFAT e IL-10 no pulmão. Além disso, essas terapias também reduziram a secreção de muco e deposição de colágeno nas vias aéreas.

La pollution atmosphérique est responsable d’environ 7 millions de décès prématurés par an au niveau mondial. Les particules fines (PM2.5) en particulier, jouent un rôle majeur dans le développement de pathologies respiratoires chroniques telles que la bronchopneumopathie obstructive chronique et l’asthme. Plusieurs études ont récemment mis en évidence que ces maladies pouvaient prendre racine dès l’enfance, au cours du développement pulmonaire. Il existe cependant peu de modèles précliniques permettant d’étudier ces étapes précoces et critiques en santé respiratoire. Les cellules souches pluripotentes induites humaines (hiPSC) sont capables de se différencier vers tous les types cellulaires de l’organisme. Elles offrent ainsi une formidable opportunité de modéliser in vitro le développement pulmonaire et plus particulièrement celui de l’épithélium bronchique, dans le cadre de ces pathologies respiratoires chroniques.Grâce à un protocole de différenciation de hiPSC vers un épithélium bronchique fonctionnel, nous pouvons mimer les étapes clés de la morphogenèse bronchique in vitro en reproduisant les stades d’endoderme définitif, d’intestin primitif antérieur, de progéniteurs bronchiques jusqu’au stade d’épithélium bronchique composé de cellules multiciliées, à mucus, basales, club et neuroendocrines. En exposant à différents stades du protocole cet épithélium à des particules fines de pollution atmosphérique, nous avons pu mettre en évidence une cytotoxicité provoquée par de fortes doses de PM2.5, une induction de mécanismes de stress oxydant et d’inflammation, ainsi qu’un impact sur le processus de différenciation : diminution des marqueurs d’intestin primitif antérieur et de progéniteurs bronchiques aux étapes précoces ; modifications de la proportion des marqueurs de cellules terminales aux étapes tardives.Les résultats de ce travail ouvrent de nouvelles perspectives dans l’étude de l’impact de la pollution atmosphérique sur le développement pulmonaire. En enrichissant ce modèle d’autres compartiments cellulaires du poumon, en développant de nouveaux systèmes d’exposition et en variant le fond génétique des hiPSC, les propriétés d’auto-renouvellement virtuellement illimité de ces cellules souches pourraient permettre une évaluation à grande échelle de l’impact de nombreux facteurs environnementaux, au cours du développement
ABSTRACTAir pollution is one of the largest environmental cause of disease and every year, 7 millions of premature deaths are attributable to air pollution worldwide. Ambient fine particulate matter plays a major role in the development of chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD) or asthma. Recently, several studies have highlighted pediatric roots in the trajectories of these diseases. However, only few preclinical models are available to study these early developmental stages yet critical in respiratory health. Human induced pluripotent stem cells (hiPSC) are able to differentiate into all cell types of human body. Thus, they offer a great opportunity to recapitulate lung development in vitro, especially bronchial airway development.Thanks to our differentiation protocol of hiPSC into functional airway epithelium, we can mimic in vitro key bronchial development steps: definitive endoderm, anterior foregut endoderm, lung progenitors towards functional epithelial cells such as ciliated cells, goblet cells, basal cells, club cells and neuroendocrine cells. PM2.5 exposure performed at different steps of the protocol showed high doses related cytotoxicity, oxidative stress and inflammatory responses. An effect on differentiation process was also observed with a decrease of anterior foregut endoderm and lung progenitors markers along with a modification in differentiated cells proportion.These results open new possibilities to study air pollution impact on lung development. Thanks to hiPSC self-renewal property, high scale exposure studies of environmental factors impact on lung development could emerge by adding new cell types to this model, developing new exposure systems and modifying genetic background of hiPSC