A leishmaniose tegumentar americana (LTA) é uma doença infecciosa, crônica, não contagiosa, causada por diferentes espécies de protozoários do gênero Leishmania. A interação entre a espécie de Leishmania e os mecanismos da resposta imune do hospedeiro resulta em um amplo espectro de manifestações clínicas, histopatológicas e imunopatológicas na infecção humana. No Brasil, esta endemia representa um sério problema de saúde pública, sendo o estado do Maranhão responsável por 13% da casuística nacional. O objetivo deste estudo foi caracterizar o perfil imunopatológico de lesões cutâneas de 22 pacientes com LTA, causada por Leishmania (Viannia) sp, oriundos do estado do Maranhão. A reação de imunoistoquímica em biópsias de pele foi realizada empregando os anticorpos monoclonais anti-humano CD68, CD1a, fator XIIIa, CD4, CD8, IL-10 e IFN-g e os anticorpos policlonais anti-Lisozima, Foxp3 e TGF-b. A análise histológica das lesões demonstrou a presença de intenso infiltrado inflamatório na derme composto de macrófagos, linfócitos e plasmócitos com formação de granuloma na maioria dos casos. Formas sugestivas de Leishmania foram observadas em 90,9% dos casos examinados. A análise imunoistoquímica mostrou densidade superior de macrófagos ativados (4824,0 células/mm²) quando comparados aos não ativados 3458,9 células/mm²). Foi detectada densidade de células de Langerhans (CD1a+) de 310,4 células/mm² na epiderme e células dendríticas dérmicas (fator XIIIa+) de 633,6 células/mm² na derme. Foi verificada ainda, predominância de linfócitos T CD8+ (2374,1células/mm²) em relação a T CD4+ (1267,6 células/mm²). A densidade de células Treg Foxp3+ foi de 301,3 células/mm². A análise de correlação mostrou correlação positiva entre células TGF-b+ e T CD8+ e fator XIIIa+; e uma correlação negativa entre a densidade de células IFN-g+ e TGF-b+. As lesões de pacientes infectados com L. (Viannia) sp, apresentaram maior densidade de células Treg Foxp3+, e IL-10+ e IFN-g+ quando comparadas a de pacientes infectados por Leishmania (Viannia) braziliensis. A densidade de células TGF-b+ mostrou-se semelhante nos dois grupos de pacientes. A avaliação dos marcadores de acordo com o tempo de evolução da doença, não mostrou diferenças entre as densidades de células imunomarcadas. Esses dados indicam que embora exista um perfil imunopatológico associado ao controle da infecção, a presença de uma resposta regulatória no sítio da lesão pode estar relacionada à persistência parasitária. / American tegumentary leishmaniasis (ATL) is a chronic, non contagious, infectious disease caused by different protozoan parasite species of the genus Leishmania. The interaction between Leishmania species and host response results in a wide spectrum of clinical, histopathological and immunopathological presentations in humans infection. In Brazil, this endemic disease is a serious public health problem and Maranhão State is responsible for 13% of Brazilian cases. The aim of this study was to characterize the immunopathology of lesions from 22 biopsies of patients with ATL, caused by Leishmania (Viannia) sp, from Maranhão State. Immunohistochemistry was carried out by incubating skin biopsies with monoclonal mouse anti- human CD68, CD1a, XIIIa factor, CD4, CD8, IL-10 and IFN-g and polyclonal rabbit\'s anti- human lysozyme, Foxp3 and TGF-b. Analysis of skin biopsies demonstrated a dense inflammatory infiltrate in the dermis, characterized by the presence of mononuclear cells, mainly lymphocytes, macrophages, plasma cells and granulomas in most samples. Suggestive forms of amastigotes were detected in 90.9% samples observed. Immunohistochemistry showed high levels of activated macrophages (4824.0 cell/mm²) when compared to inactive macrophages (3458.9 cells/mm²). In the epidermis, the densities of Langerhans Cells (CD1a+) were 310.4 cells/mm², while in the dermis the densities of dermal dendritic cells (factor XIIIa+) were 633,6 cells/mm². High density of CD8 T+ lymphocytes (2374.1 cell/mm²) has been found compared to CD4+ T lymphocytes (1267.6 cell/mm²). The cellular density of Treg (Foxp3+) was 301 cells/mm². Among cell markers there were positive correlations between TGF-b-producing cells and CD8+ T lymphocytes and between TGF-b-producing cells and dendritic dermal cells, as well as a negative correlation between TGF-b-producing cells and IFN-g-producing cells. Patients infected with L. (Viannia) sp. presented increased densities of Treg cells and IL-10 and IFN-g- producing cells compared to Leishmania (Viannia) braziliensis infected patients. The density TGF-b+ producing cells was similar in both groups of patients. Evaluation of Immunological markers according time of infection showed no differences in the densities of immunostain cell. Results suggest that the profile of immunopathological response is associated to the control of infection, although the presence of a regulatory response at the site of infection can be responsible to the persistence of parasite in skin.

Realizou-se estudo comparando o uso da reação em cadeia da polimerase à evolução clínica e painel de exames tradicionais para diagnóstico em pacientes com suspeita clínica de tuberculose cutânea e micobacteriose atípica. Observou-se sensibilidade da reação em cadeia da polimerase de 88%, especificidade de 83%, valor preditivo positivo de 82%, valor preditivo negativo de 88% e acurácia de 85% com concordância pelo teste de McNemar (p= 0,655). Os exames do painel de maior acurácia, após a reação em cadeia da polimerase, foram o teste tuberculínico com acurácia de 79% e a presença de dermatite crônica granulomatosa com reação em cadeia da polimerase positiva com acurácia também de 79%, ambos com concordância pelo teste de McNemar (p= 0,179 e p= 0,655, respectivamente) / A study was performed comparing the polymerase chain reaction and the traditional panel of exams for the diagnosis in patients with a clinical suspicion of cutaneous tuberculosis and atypical mycobacteria infection to the clinical evaluation. It was observed that the sensitivity of the PCR was 88%, the specificity was 83%, the positive predictive value was 82%, the negative predictive value was 88% and the accuracy was 85% in agreement with the McNemar test (p=0.655). The panel exams of second highest accuracy, were the tuberculin test with an accuracy of 79% and the chronic granulomatous dermatitis with positive PCR, also with an accuracy of 79%, both in agreement with the McNemar test (p=0.179 and p=0.655, respectively)

A Leishmaniose Tegumentar Americana (LTA) representa um sério problema de saúde pública. No Brasil muitas espécies são reconhecidas como patogênicas para o homem, portanto o diagnóstico diferencial é necessário para compreender o perfil epimemiológico da LTA em áreas endêmicas. Com o objetivo de identificar espécies de Leishmania utilizando ferramentas moleculares, cinquenta e três biópsias de pele de pacientes com LTA, fixadas em formalina e incluídas em parafina dos Estados do Pará (N=33) e Maranhão (20) foram submetidos a diferentes protocolos da Reação em Cadeia da Polimerase (PCR) para a identificação dos agentes causadores. Biopsias foram desparafinizadas e o DNA foi extraído usando o protocolo de fenol-clorofórmio, quantificado e submetido a reações de PCR, com base na sequência de nucleotídeos codificadora do RNA que compõe a subunidade menor do ribossomo (SSUrDNA) e da enzima Glicose 6-Fosfato Desidrogenase (G6PD). O alvo G6PD foi utilizado tanto em reações de PCR convencional (cPCR), como de PCR quantitativo (qPCR). As reações de cPCR e Nested PCR SSUrDNA apresentaram resultado positivo para o gênero Leishmania em 40 (83,3%) das amostras submetidas. Vinte e sete desses produtos de PCR foram sequenciados, sendo 17 identificados como L.(L.) amazonensis e 10 como L.(Viannia) sp. A cPCR G6PD identificou 9 amostras como L.(V.) braziliensis , sendo 7 do Maranhão (36%) e 2 do Pará (6%). O DNA de L.(Viannia) sp. foi quantificado em quatro amostras do Maranhão através da reação de qPCR G6PD, mesmo esse alvo sendo de cópia única. Esses resultados indicam que sequências especificas de Leishmania sp. presentes em múltiplas cópias devem ser escolhidas para a aplicação de cPCR em DNA provindo de amostras parafinadas,uma vez que é freqüente casos de LTA com baixo parasitismo e consequentemente, pequenas concentrações de DNA. E ainda a cPCR SSUrDNA pode ser um bom alvo para estudos diagnósticos e epidemiológicos.A qPCR G6PD permitiu a detecção, identificação e quantificação de L. (Viannia) sp. em uma única etapa de amplificação em quatro amostras que presentaram resultados positivos somente na Nested ou Semi-Nested PCR, demonstrando uma maior sensibilidade oferecida pela q PCR / American Cutaneous Leishmaniasis (ACL) presents a serious problem of public healthy. In Brazil many species are recognized as pathogenic to humans, therefore differential diagnostic is necessary to understand the epidemiological profile of ACL in endemic areas. Fifty-three paraffinembedded skin biopsies of ACL patients from Pará (N=33) and Maranhão (20) States, were submitted to different protocols of polymerase chain reaction (PCR) for identification of their causative agents. Biopsies were deparaffinized and DNA were extracted using phenol-chloroform, quantified and submitted to PCR reaction, using small subunit coding sequence (SSUrDNA) and enzyme glucose-6-phosphate dehydrogenase (G6PD). The target of G6PD was used both in conventional PCR reactions (cPCR) and quantitative PCR (qPCR). The reactions of cPCR and Nested PCR SSUrDNA showed positive result for the genus Leishmania in 40 (83.3%) of the samples. Twenty-seven positive samples were submitted to sequencing and 10 were identified as L. (Viannia) sp. and 17 as L. (L.) amazonensis. The G6PD PCR identified 9 samples as L. (V.) braziliensis, 2 from Pará (6%) and 7 from Maranhão (35%).Four samples were quantified in G6PD qPCR, even this is a single copy. These results indicate that specific sequences from Leishmania sp. present in multiple copies should be chosen in relation to those from unique copies in paraffin-embedded tissues, once is frequent cases of ACL with low parasitism, consequently small DNA concentrations and that SSUrDNA can be a good target to diagnostic and epidemiologic studies of ACL. The qPCR allowed the detection and identification of L. (Viannia) sp. in a single round of amplification in four samples that when showed positive results only in the Nested or Semi Nested cPCR suggesting a higher sensitivity offered by qPCR

Orientador: Silvia Cristina Mangini Bocchi / Resumo: Introdução. Verifica-se eficácia no uso da laserterapia transcutânea (Intravenous Laser Irradiation of Blood – ILIB) na saúde dos indivíduos, entretanto, existem escassas evidências científicas para tratamento dos efeitos colaterais de quimioterápicos citotóxicos. Objetivo. Avaliar a eficácia dos protocolos ILIB 30’ e 60’ com comprimento de onda de 660nm sobre os efeitos colaterais no trato gastrointestinal (náuseas, vômitos, diarreias e constipação) e no tecido hematopoiético (plaquetopenias, neutropenias e alterações da hemoglobina), em pacientes oncológicos recebendo tratamento quimioterápico endovenoso. Método. Ensaio clínico, randomizado, unicego e aprovado por Comitê de Ética em Pesquisa (CAE 82323318.90000.5411, Parecer 2.512.164) e pelo Registro Brasileiro de Ensaios Clínicos (RBR – 7y8rtz). Estudo realizado em serviço ambulatorial de quimioterapia de hospital público do estado de São Paulo, Brasil. A amostra constituiu de 55 pacientes ³ 18 anos, com tumores sólidos, a partir do segundo ciclo de tratamento quimioterápico com fármacos endovenosos citotóxicos para o trato gastrointestinal e tecido hematopoiético, assim alocados nos grupos de seguimentos: 21 no controle, 21 no ILIB 30’ e 13 no ILIB 60’. A aplicação deu-se por via transcutânea sobre a artéria radial, utilizando-se aparelho de laser de baixa intensidade com comprimento de onda de 660nm. Considerou-se eficácia a manutenção ou aumento dos parâmetros mínimos hematológicos (hemoglobina, plaquetas e neutrófilos... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Le mélanome est le cancer de la peau le plus dangereux : il est capable de métastaser rapidement vers les ganglions et les viscères, et est réfractaire aux chimio/radiothérapies. De nouvelles thérapies ciblant la kinase BRAFV600 retrouvée dans 60% des mélanomes ont montré des effets spectaculaires en termes de survie globale et sans progression de la maladie. L’efficacité de ces thérapies est compromise par l’apparition fréquente de résistances. Les cellules cancéreuses sont ancrées au sein d’un microenvironnement avec lequel elles interagissent. Elles profitent de facteurs solubles du stroma et de l’adhésion à la matrice extracellulaire (MEC) pour survivre face aux thérapies. La protéine matricellulaire SPARC orchestre les interactions entre les cellules saines ou cancéreuses et leur microenvironnement. Absente dans les mélanocytes, son expression est initiée et augmentée dans les mélanomes, corrélée à la progression tumorale et à un mauvais pronostic clinique. Lorsqu’elle est sécrétée par les cellules de mélanome, elle active la kinase AKT, déstabilise le suppresseur de tumeur p53 et favorise la prolifération/survie tumorale. Nous avons montré que le module SPARC/AKT est un nouveau déterminant de la résistance innée ou acquise des mélanomes mutés BRAFV600 aux anti-BRAF, et mis en évidence l’intérêt du ciblage de SPARC en combinaison avec des anti-BRAF ou -MEK pour optimiser/restaurer la sensibilité des mélanomes à ces inhibiteurs. Nous avons aussi montré que les fibroblastes ganglionnaires ont des caractéristiques de fibroblastes activés, et confèrent une résistance aux anti-BRAF aux cellules de mélanomes en générant une MEC permissive à laquelle elles adhérent. / Melanoma is the most dangerous form of skin cancer due to its high metastatic potential and its resistance to both classical chemo- and radiotherapies. New targeted therapies directed against the V600E oncogenic form of BRAF found in about 60% of patients have demonstrated spectacular efficacy both in terms of progression free and overall survival. However most patients invariably relapse after a few months due to resistance mechanisms. Cancer cells are anchored and interact constantly with their microenvironment. Both soluble factors and the extracellular matrix produced by stromal cells have been shown to contribute to cancer cell resistance to therapies. SPARC is a matricellular protein that orchestrates interactions between normal and/or cancer cells and their microenvironment. While it is absent in melanocytes, SPARC expression increases in melanoma and is correlated with both tumoral progression and a bad prognosis. When SPARC is secreted by melanoma cells, it activates the AKT kinase, destabilizes the p53 tumor suppressor and promotes proliferation and survival. Here we identify the couple SPARC/AKT as a new actor contributing to both innate and acquired resistance to BRAFV600E inhibitors. In addition, we demonstrate that targeting SPARC in melanoma cells increases their sensitivity to both BRAF and MEK inhibitors. Finally we show that lymph node fibroblasts share features of activated fibroblasts and confer resistance to BRAF inhibitors to melanoma cells through the production of a permissive extracellular matrix.

During dexterous manipulation with the fingertips, forces are applied to objects' surfaces. To achieve grasp stability, these forces must be appropriate given the properties of the objects and the skin of the fingertips, and the nature of the task. It has been demonstrated that tactile sensors in the fingertips provide crucial information about both object properties and mechanical events critical for the control of fingertip forces, while in certain tasks vision may also contribute to predictions of required fingertip actions. This thesis focuses on two specific aspects of the sensory control of manipulation: (i) how individual fingers are controlled for grasp stability when people restrain objects subjected to unpredictable forces tangential to the grasped surfaces, and (ii) how tactile sensors in the fingertips encode direction of fingertip forces and shape of surfaces contacted by the fingertips. When restraining objects with two fingers, subjects adjust the fingertip forces to the local friction at each digit-object interface for grasp stability. This is accomplished primarily by partitioning the tangential force between the digits in a way that reflects the local friction whereas the normal forces at the involved digits are scaled by the average friction and the total load. The neural control mechanisms in this task rely on tactile information pertaining to both the friction at each digit-object interface and the development of tangential load. Moreover, these mechanisms controlled the force application at individual digits while at the same time integrating sensory inputs from all digits involved in the task. Microneurographical recordings in awake humans shows that most SA-I, SA-II and FA-I sensors in the distal phalanx are excited when forces similar to those observed during actual manipulation are applied to the fingertip. Moreover, the direction of the fingertip force influences the impulse rates in most afferents and their responses are broadly tuned to a preferred direction. The preferred direction varies among the afferents and, accordingly, ensembles of afferents can encode the direction of fingertip forces. The local curvature of the object in contact with the fingertip also influenced the impulse rates in most afferents, providing a curvature contrast signals within the afferent populations. Marked interactions were observed in the afferents' responses to object curvature and force direction. Similar findings were obtained for the onset latency in individual afferents. Accordingly, for ensembles of afferents, the order by which individual afferents initially discharge to fingertip events effectively represents parameters of fingertip stimulation. This neural code probably represents the fastest possible code for transmission of parameters of fingertip stimuli to the CNS.

The RAS-MAPK syndromes are a group of clinically and genetically related disorders, characterized by cardiac defects, facial dysmorphism, cutaneous abnormalities and neurocognitive impairment. The pathogenesis is dysregulation of the RAS-MAPK pathway, and several genes within the pathway are involved. The present thesis aimed at identifying genetic causes in three of the RAS-MAPK syndromes - Noonan syndrome (NS), cardio-facio-cutaneous syndrome (CFC) and Neurofibromatosis-Noonan syndrome (NFNS) - and at correlating genotype with phenotype. A mutation analysis of six genes associated with the RAS-MAPK syndromes in NS and CFC patients revealed mutations in 10/31 patients. The results suggested more complex genetic overlap and genetic heterogeneity among these syndromes than previously believed. Subsequently, gene dosage imbalances of seven RAS-MAPK-syndrome-related genes were investigated in mutation-negative patients. A multiplex ligation-dependent probe amplification strategy was developed that excluded copy number changes of these genes as a common mechanism in NS. Genetic causes of clinical variability in NS were investigated where an atypical and severe NS patient was described. In addition, multiple café-au-lait (CAL) spots affected the patient and four otherwise healthy family members. Molecular analysis of four candidate genes revealed a previously described de novo PTPN11 mutation and an inherited NF1 variant in the patient. Neither of them explained the CAL spots trait, which consequently represented a distinct entity. The results suggested that the atypical and severe phenotype in the patient could be a consequence of an additive effect. Finally, a family displaying NFNS was investigated clinically and molecularly revealing a novel mutation in the GAP-domain of NF1. Furthermore, the results suggested that other RAS-MAPK-syndrome-related genes are not involved in NFNS. A proposal of prioritizing the GAP-domain of NF1 in NFNS was presented. Conclusively, these studies contribute to further understanding of the RAS-MAPK syndromes and facilitate the diagnostic process and future prognosis prediction.

Non-melanoma skin cancer (NMSC), comprised of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is the most common cancer in Caucasians. Ultraviolet radiation (UVR) exposure is the most important environmental risk factor for both BCC and SCC development. However, the precise relationship between UVR and the risk of NMSC is complex, and the relationship may differ by skin cancer type. It has been hypothesized that intermittent patterns and childhood sunlight exposure are important for BCC while continuous (chronic) and lifelong (i.e. childhood and adulthood) sunlight exposure is important for SCC. Epidemiologic studies have demonstrated that cutaneous human papillomavirus (HPV) infection may also be a risk factor for developing NMSC. However, the pathway by which cutaneous HPV is associated with NMSC remains unclear. It is hypothesized that UVR exposure may interact synergistically with cutaneous HPV in NMSC development. The goal of the research study was to evaluate the relationship between levels of sunlight exposure and BCC and SCC and to investigate differences in sunlight-associated BCC and SCC risk by genus-specific cutaneous HPV serostatus. To address these goals, we conducted a clinic based case-control study of histologically confirmed BCC and SCC cases recruited from a university dermatology clinic and controls with no history of cancer and screened negative for current skin cancer. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI) for the associations between measures of sunlight exposure and BCC and SCC. Multiplicative interactions were tested by placing an interaction term for the product of genus-specific HPV seroreactivity and sunlight related factors in the logistic regression models. Measures of both intermittent and continuous patterns of sunlight exposure were associated with both types of skin cancer (i.e. BCC and SCC). Specifically, history of blistering sunburn (a marker of intermittent sunlight exposure) and occupational sunlight exposure (i.e. having a job in the sun for at least 3 months for >10 years) were both associated with BCC and SCC. The major differences in patterns of sunlight exposure between BCC and SCC were observed for sunlight exposure in one's thirties. Additionally, sunlight exposure in one's twenties was associated with SCC, regardless of pattern of exposure; similar associations were not observed for BCC. Measures of timing of sunlight exposure consistently demonstrated that childhood/adolescent sunlight exposure was more important for SCC than BCC. These included number of moles on the forearms and entire body (measure of increased childhood sunlight exposure), and younger age at first and tanning bed use. Younger age at first blistering sunburn was statistically significantly associated with both BCC and SCC. NMSC cases were more likely to be seropositive for cutaneous HPV antibodies compared to controls. Compared to tanning, having a propensity to sun burn (p=0.006), or poor tanning ability (p=0.003) were significantly associated with a higher seroprevalence to genus beta HPV types within SCC cases. Statistically significant interactions were observed between poor tanning ability and genus-specific seropositivity with NMSC. Specifically, the associations between poor tanning ability and BCC (p interaction=0.02) and SCC (p interaction=0.01) were significantly stronger among individuals that were seropositive for antibodies to genus alpha HPV types. Similarly, the association between poor tanning ability and SCC was stronger among those seropositive for genus beta HPV types (p interaction=0.001). No additional significant interactions were observed for BCC or SCC between cutaneous sensitivity, history of blistering sunburn, or cumulative sunlight exposure and genus-specific seroreactivity. In conclusion, associations with patterns of sunlight exposure appeared to be similar between BCC and SCC cases. With the exception of age at first blistering sunburn, factors measuring timing of sunlight exposure demonstrated stronger and statistically significant relationships with SCC. Additionally, of the sunlight related factors measured, only the associations between poor tanning ability and BCC and SCC were significantly modified by HPV seropositivity to types in genera alpha or beta.

Chytridiomycosis is an emerging cutaneous fungal disease that contributes to recent global declines and extinction of amphibian species, caused by infection of the skin with a fungus known as Batrachochytrium dendrobatidis (Bd). Many species of frogs secrete antimicrobial peptides onto their skin that are capable of killing Bd. This thesis is an investigation of the effect of corticosteroids on cutaneous innate immunity in frogs, in the context of infection with Bd. The general hypothesis was that injections of glucocorticoids would impair the cutaneous synthesis of these antimicrobial peptides, thereby increasing susceptibility to Bd infection. The objective of the first experiment was to measure and compare gene expression levels of cutaneous AMP’s in frogs treated with glucocorticoids with sham-treated controls. Wild-caught Lithobates pipiens were acclimatized and administered either the corticosteroid methylprednisolone or saline every 48 hours. Norepinephrine-elicited cutaneous secretions were collected prior to the first injection of corticosteroid or saline, and then every 8 days for 40 days. Gene expression of the AMP’s brevinin and ranatuerin in the cutaneous secretions was quantified relative to the reference genes EF1-α and RPL8 using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Corticosteroid treatment was associated with a significant (P<0.027) increase in brevinin gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin expression followed a similar but nonsignificant trend. The second experiment was a pilot study intended to establish a Bd challenge protocol in L. pipiens. Frogs were immersed in water containing 0, 104, 105 or 106 zoospores of Bd strain JEL 423. Cutaneous swabs were collected prior to challenge and tested for Bd by qPCR; unexpectedly, some tested positive, indicating pre-challenge infection. The analysis was complicated by an identified cross-reactivity of the assay with other fungi. The findings of the first experiment refuted the hypothesis, and suggested that corticosteroids promote rather than impair AMP gene expression in the skin of L. pipiens, under these experimental conditions. Further, the second study demonstrated that none of the frogs showed clinical abnormalities or died, despite exposure to Bd zoospores and despite molecular and histologic evidence of cutaneous Bd infection in some frogs. / NSERC Discovery Grant

LKB1 is the second most commonly altered tumour suppressor gene in lung adenocarcinoma, the most prevalent form of lung cancer. LKB1 is a "master kinase" that has been shown to phosphorylate up to 13 downstream targets. We hypothesised that LKB1 loss is associated with an increased dependency on alternative, targetable pathways. The overall aims of this project were to better understand the role of LKB1 loss in lung cancer and to identify novel approaches to selectively target LKB1 mutated cells. We generated isogenic cells with or without LKB1 and used these to study the effect of LKB1 on cell proliferation. Importantly, we used a range of models including 2D culture, 3D spheroids and, sub-cutaneous and orthotopic xenograft models. To understand the role of LKB1 loss in lung cancer, the effect of LKB1 on mRNA expression was analysed using whole genome RNA Sequencing. To identify novel approaches to selectively target LKB1 mutated cells, we used biological screening methods and also investigated the effect of several metabolic inhibitors. We found that loss of LKB1 expression had no effect on cell proliferation in 2D culture, but was associated with increased growth in 3D spheroids, sub-cutaneous and orthotopic xenografts, as well as greater metastasis in a lung orthotopic model. Gene ontology analysis of the transcriptome identified that genes associated with cAMP signalling and cytoskeletal organisation were differentially expressed between LKB1 deficient and proficient cells. We confirmed that cAMP signalling was increased in LKB1 deficient cells, though there was no difference in sensitivity between LKB1 deficient and proficient cells to cAMP signalling modulators. The bioactive small molecule screen showed that LKB1 deficient cells underwent apoptosis more slowly and therefore, were less sensitive to many compounds, compared with LKB1 proficient cells. Screening in 3D spheroids was a novel approach that we used to identify microtubule inhibitors as potentially selective compounds acting in LKB1 deficient cells. Our RNASeq data suggests that there was a metabolic shift from oxidative phosphorylation to aerobic glycolysis in LKB1 deficient cells, although this did not affect sensitivity to complex I inhibitors. Importantly, LKB1 deficient cells were more sensitive to glucose and glutamine deprivation which suggests that targeting these metabolic pathways may hold the greatest promise to selectively inhibit proliferation in LKB1 mutated cells.