<p>Cancer patients undergoing chemotherapy have an elevated risk for thrombosis. Although thrombosis is a common complication in cancer patients, the mechanisms of chemotherapy-induced thrombosis remain unclear. We investigated the procoagulant effects of lung cancer chemotherapy agents (carboplatin, paclitaxel, cisplatin, and gemcitabine) on endothelial cells, A549 cells, and monocytes. We also investigated the <em>in </em>vivo procoagulant effects of the aforementioned chemotherapeutic agents as well as the anti-angiogenic agent bevacizumab. Tissue factor (TF) activity, TF antigen and phosphatidylserine (PS) levels were measured on chemotherapy-treated human umbilical vein endothelial cells (HUVEC), A549 cells, and monocytes. Treatment of HUVECs, A549 cells, and monocytes with lung cancer single agent and combination chemotherapy resulted in significant increases in TF activity. However, only cisplatin- and gemcitabine- treated monocytes were found to have increased TF antigen levels. PS exposure was increased only on HUVEC and monocytes treated with cisplatin/gemcitabine combination therapy. Interestingly, addition of paclitaxel to carboplatin resulted in reduced levels of PS exposure on monocytes. This study is the first to explore the procoagulant effects of lung cancer chemotherapy agents on monocyte and A549 cell TF activity levels, as well as to investigate the mechanisms by which lung cancer agents may promote TF decryption on these cell lines<strong>.</strong> Our <em>in vivo</em> results demonstrated that treatment of healthy mice with bevacizumab, paclitaxel and carboplatin moderately increased plasma TAT levels in healthy mice. These studies reveal potential mechanisms by which lung cancer chemotherapy may increase the risk of thrombosis. These studies reveal potential mechanisms by which lung cancer chemotherapy agents induce a hypercoagulable state.</p> / Master of Science (MSc)

<p>Brain metastases are most common in adults suffering from lung cancer, predicting uniformly poor patient outcome and short survival time. Despite their frequency and severity, very little is known about the tumorigenesis of brain metastases. Previously developed primary brain tumour-initiating cell (BTIC) models were used to determine the presence of a stem-like population in brain metastases from the lung. Use of clinical samples and the NCI-H1915 cell line allowed for the development of useful strategies for study of brain metastasis.</p> <p>The sphere formation capacity and expression of known BTIC markers in brain metastases was suggestive of a self-renewing population. Differentiation studies demonstrated that neither clinical samples nor NCI-H1915 cells had neural lineage potential. Intracranial xenotransplant of clinical samples and NCI-H1915 cells into NOD-SCID mice led to formation of multiple focal masses throughout the ventricles; the tumours were also serially transplantable, further implicating a TIC population. Of known BTIC markers, only CD15 expression levels and patterns were similar enough in clinical samples and NCI-H1915 cells to warrant prospective sorting experiments in the cell line. Use of CD15 failed to identify a CSC or TIC population in NCI-H1915 cells.</p> <p>These findings suggest that a TIC population is present in brain metastases; however, this remains to be identified. It is recommended that due to the limitations of cell surface markers, the study of brain metastasis should use a selective gene expression approach, in order to target genes and pathways essential to metastasis. It was shown that NCI-H1915 cells could be useful for such an approach, studying the effects on proliferation, sphere formation, and tumour formation capacity of brain metastases from the lung. Further study using this model could ultimately lead to the disruption of pathways essential to the metastatic process, transforming a uniformly fatal disease into a more localized and treatable one.</p> / Master of Science (MSc)

<p><strong><em>Purpose</em></strong></p> <p>To examine the potential of the anti-diabetic agent Metformin (MET) to enhance responses of NSCLC to ionizing radiation (IR).</p> <p><strong><em>Experimental Design</em></strong></p> <p>Human NSCLC A549, H1299 and SK-MES cells were treated with IR, MET or the mTOR inhibitor rapamycin and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays. A549 and H1299 cells were grafted into flanks of immunosuppressed mice and treated with MET and/or IR. Tumours were analyzed by immunoblotting and immunohistochemistry.</p> <p><strong><em>Results</em></strong></p> <p>MET(2.5uM-5mM) caused dose-dependent inhibition of proliferation (10-70%)in all lines, inibited clonogenic survival and sensitized cells to IR. In A549 cellsMET caused inhibition of proliferation comparable to rapamycin, stimulated expression and activation of the ATM and AMPK-p53-p21^cip1and inhibited the Akt-mTOR-4-EBP1 pathway.MET caused G1 arrest of cell cycle, enhanced apoptosis and induced sustained DNA repair foci of gH2AX. MET and IR alone inhibited xenograft growth and combined treatment enhanced that further. IR and MET induced sustained enhancement of expression and activity of ATM-AMPK-p53-p21^cip1and inhibitionof Akt-mTOR-4-EBP1 pathways in tumours also. MET reduced expression of angiogenesis and enhanced expression of apoptosis markers in both control and radiated tumours.</p> <p><strong><em>Conclusions</em></strong></p> <p>Clinically achievable(uM) doses ofMET inhibit human NSCLC cell and tumour growth and sensitize them to IR.This is accompanied by desirable modulation of molecular signals, inhibition of angiogenesis and induction of apoptosis. Our results suggest that MET could be a clinically useful adjunct to radiotherapy in NSCLC and support clinical investigation of MET in combination with radiotherapy.</p> / Master of Science (MSc)

<p> It has been suggested that combination treatment of high dose rate (HDR) intraluminal brachytherapy and PDT (Photodynamic therapy) in non-small cell lung cancer (NSCLC) may improve the efficacy of treatment, reduce the toxicity and improve quality of life for patients. To provide a cellular basis for this approach we have examined the in vitro sensitivity of normal lung fibroblasts (MRC5) and four NSCLC cell lines (SKMES-1, A549, NCIH460 and NCIH23) following, UVC treatment, HDR radiation, HDR radiation with Photofrin alone, PDT and combined HDR radiation and PDT. Cell sensitivity was measured using clonogenic survival. HDR radiation was cobalt-60 gamma rays (1.5-1.9 Gy/min). For PDT treatment, cells were exposed to 2.5 J.lg/ml Photofrin for 18-24 h followed by light exposure (20mW/cm2). D37 values calculated from the survival curves indicated a 2-fold difference in sensitivity to UVC, 6-fold difference in HDR radiation sensitivity and an 8-fold difference in PDT sensitivity. All cell lines showed a similar Photofrin uptake per cell when measured by flow cytometry using 488nm excitation and 620-675 nm emission wavelengths. Photofrin alone at concentrations up to 10 J.lg/ml had no significant effect on the survival of the NSCLC cell lines, whereas 10 J.lg/ml ofPhotofrin alone reduced survival significantly in MRC5 cells. A radiosensitizing effect of Photofrin was detected in MRC5 and NCIH460 cells, but not in A549, SKMES-1 and NCI-H23 cells. For combined treatment cells were exposed to Photofrin and then either exposed to light and 15-30 minutes later exposed to HDR radiation or exposed to HDR radiation and 15-30 minutes later exposed to light. Results showed that although light followed by gamma rays resulted in a somewhat greater tumor cell kill compared to gamma rays followed by light this difference was not significant for any of the cell lines tested. However, this difference was significant when data for all NSCLC cell lines were pooled. The combined treatment with high dose rate HDR radiation and PDT was not significantly different from an additive effect of the individual treatment modalities for in vitro survival of 4 NSCLC cells. In contrast the combined treatment was less than additive for the MRCS cells suggesting that the combined treatment would have the potential advantage of doing less damage to the normal lung cells and suggests that equivalent tumour cell kill in vivo may be possible at reduced systemic effects to patients. In preliminary experiments we have started to examine the effects of Photofrin-mediated PDT on the extra cellular signal-activated protein kinase (ERK) signaling pathway in NSCLC cells. The use of multiple NSCLC cell lines allows for the possible identification of cell line specific changes involved in resistance to PDT and HDR radiation and this will be explored in future work. </p> / Thesis / Master of Science (MSc)

No description available.

Cancer is one of the main causes of death in the US. Lung cancer remains the highest killing form of cancer. Lung screening rates are low amongst the general population and even lower in minority populations. It is not well known what the barriers are for lung cancer screening. In order to investigate barriers, I created questions to add onto an existing questionnaire survey for an ongoing lung cancer screening research project. These questions focused on social determinants of health and the survey was administered to patients who were non-adherent to lung cancer screening. Patients reported cost concerns for screening, potential medical care costs, and ability to attend medical appointments as barriers to lung cancer screening. Both non-white and female patients reported more difficulties attending appointments than their white and male counterparts. Patient physician relationship and perceived racial discrimination were not barriers to lung cancer screening in the patient population surveyed. Further research needs to investigate specific details on these barriers to create interventions to increase lung cancer screening rates. / Urban Bioethics

Introduction Neutropenia is a life-threatening and dose-limiting toxicity of palliative lung cancer chemotherapy. Whilst some neutropenias are inevitable, evidence suggests that patients with a previous neutropenic event are 50% more likely to have a further neutropenic event. The aim of this research is to evaluate the variables associated with the risk of secondary neutropenic events and the role of chemotherapy dose reductions. Methods A retrospective analysis was carried out on 361 biochemical neutropenic events in palliative lung cancer patients across 5 sites in South Yorkshire and Bassetlaw. Predictors for a secondary neutropenic event were investigated in univariate and multivariate logistic regression analysis. The predictive model was validated through discrimination statistics, described by Receiver Operating Characteristic Area Under Curve (ROC-AUC). Results The incident rate for secondary neutropenic events was 32.7%. Patients with a successful intervention received a higher mean Relative Dose Intensity (RDI) of 75.65% compared to 65.05%, across the 2 chemotherapy cycles. The univariate analysis found that the biochemical type of neutropenia (depth and length of suppression) (p=0.003), dose reduction of drug 1 (p=0.042), average dose reduction (p=0.019), and cumulative dose reduction (p=0.018) were significant at reducing the risk of secondary neutropenia. Granulocyte-Colony Stimulating Factor did not offer a protective effect. The final logistic regression model evaluated 357 events and included all variables due to significant interrelationship. The model had a ROC-AUC of 0.76 (0.71-0.81) (p= 0.0021), explaining 27% of the variance. Conclusion Appropriate dose reductions play a vital role in preventing secondary neutropenic events and delivering optimal RDIs. The results of this study can aid in identifying high-risk patients.

[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. / [CA] 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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137036

No / Improved understanding of the involvement of matrix metalloproteinases (MMPs), including membrane-type MMPs (MT-MMPs), in human tumours has potential diagnostic, prognostic and therapeutic implications. We assessed the relationship between MT-MMP expression and clinicopathological parameters in human non-small cell lung cancer (NSCLC) and histologically normal lung tissue by quantitative Real Time PCR (qRT-PCR). All MT-MMPs (MMPs 14-17, 24 and 25) were detected by qRT-PCR with significantly higher MMP-14, -15 and -17 expression observed in tumour relative to normal lung specimens. MMP-16 was undetectable in normal lung but expressed in 8% tumours. MMP-15 demonstrated significant overexpression in adenocarcinomas relative to squamous cell carcinomas and normal lung tissue. MMP-14 mRNA expression strongly correlated to MMP-14 proteolytic activity in preclinical tumour models, indicating that qRT-PCR may predict MMP-14 activity levels in NSCLC. These data suggest that MMP-14, -15 and -17 may be good markers of disease, or therapeutic targets for treatment of human NSCLC.

Yes / Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzyme activity which affects the inflammatory response. Inflammation is associated with increasing cancer incidence. Pre-clinical and clinical studies have shown that NSAID treatment could cause an anti-tumor effect in cancers. In the present study, blood was taken from healthy individuals (n = 17) and patients with respiratory diseases or lung cancer (n = 36). White blood cells (WBC) were treated with either a micro-suspension, i.e., bulk (B) or nano-suspension (N) of aspirin (ASP) or ibuprofen (IBU) up to 500 μg/ml in the comet assay and up to 125 μg/ml in the micronucleus assay. In this study results were compared against untreated lymphocytes and their corresponding treated groups. The results showed, that NSAIDs in their nano form significantly reduced the DNA damage in WBCs from lung cancer patients in bulk and nano compared to untreated lymphocytes. Also, there was a decrease in the level of DNA damage in the comet assay after treating WBCs from healthy individuals, asthma and COPD groups with aspirin N (ASP N) but not with IBU N. In addition, the number of micronuclei decreased after treatment with NSAIDs in their nano form (ASP N and IBU N) in the healthy as well as in the lung cancer group. However, this was not the case for micronucleus frequency in asthma and COPD patients. These data show that lymphocytes from different groups respond differently to treatment with ASP and IBU as measured by comet assay and micronucleus assay, and that the size of the suspended particles of the drugs affects responses. / The present study was part funded by United Kingdom India Education Research Initiative (UKERI) SA 07-067.