Unravelling Lung Cancer Heterogeneity and Associated Therapeutic Responses using in vivo and ex vivo Model Systems

Lung cancer is the major cause of cancer-related deaths worldwide (Ferlay et al., 2015; Siegel et al., 2018). Tobacco smoking is the primary risk factor, contributing to nearly 85% of the lung cancer deaths (Furrukh, 2013). Lung cancer is histologically and genetically a heterogeneous disease (Gridelli et al., 2015). Comprehensive genomic profiling has led to the classification of lung cancer into distinct molecular subtypes, and the development of targeted therapies against specific genetic alterations (Bronte et al., 2010). However, as of yet, no clinically approved therapies available for the treatment of lung cancer patients carrying common driver genes, namely mutations in oncogenic KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog), and the tumor suppressor STK11 (Serine/Threonine Kinase 11, also known as LKB1). This thesis investigates the role of niche-specific lung progenitor cells in defining NSCLC histotype spectra and immune microenvironment heterogeneity, using a murine model driven by oncogenic Kras and loss of the tumor suppressor Lkb1. In addition, using Kras-driven NSCLC mouse models, this thesis also demonstrates histotype-associated spatial oncogenic signaling heterogeneity and its importance in defining therapeutic responses. Furthermore, it establishes Kras-driven NSCLC tumor tissue slice cultures and shows their utility as ex vivo models in predicting spatial responses to drug combinations. Finally, by performing drug screening on murine NSCLC primary cell cultures coupled with in vivo acute drug response analysis, this thesis identifies tumor subtype-selective therapeutic sensitivity and resistance mechanisms, as well as effective drug combinations.Keuhkosyöpä on syöpäperäisten kuolemien suurin syy maailmanlaajuisesti (Ferlay et al., 2015; Siegel et ai., 2018). Tupakan tupakointi on ensisijainen riskitekijä, joka myötävaikuttaa lähes 85 prosenttiin keuhkosyöpäkuolemista (Furrukh, 2013). Keuhkosyöpä on histologisesti ja geneettisesti heterogeeninen sairaus (Gridelli ym., 2015). Kattava genominen profilointi on johtanut keuhkosyövän luokitteluun erillisiin molekyylialatyyppeihin ja kohdennettujen terapioiden kehittämiseen tiettyjä geneettisiä muutoksia vastaan ​​(Bronte et al., 2010). Kuitenkin vieläkään kliinisesti hyväksyttyjä terapioita ei ole saatavilla keuhkosyöpäpotilaille, joilla on tavallisia kuljettajageenejä, nimittäin onkogeenisen KRAS: n (Kirsten Rat Sarcoma Viral Oncogene Homolog) ja kasvaimen suppressori STK11 (Serine / Threonine Kinase 11, myös tunnetaan nimellä LKB1). Tämä opinnäytetyö tutkii niche-spesifisten keuhkojen progenitorisolujen roolia määriteltäessä NSCLC-histotyyppisiä spektrejä ja immuuni-mikroympäristön heterogeenisyyttä käyttämällä hiirimallia, jota onkologinen Kras ja peräsuolen vaimennin Lkb1. Lisäksi tässä tutkimuksessa käytetään Kras-ohjattujen NSCLC GEM -mallien avulla loogisesti tyypillistä spatiaalisen onkogeenisen signaloinnin heterogeenisyyttä ja sen merkitystä terapeuttisten vasteiden määrittämisessä. Lisäksi se luo Kras-ohjatut NSCLC-tuumorikudosviipaleet ja osoittaa niiden käyttökelpoisuuden ex vivo -malleina spatiaalisten vasteiden ennustamiseksi lääkekombinaatioille. Lopuksi, tekemällä huumeiden seulonta hiiren NSCLC-primaarisoluviljelmillä, joihin on kytketty in vivo akuutti lääkeanalyysianalyysi, tässä työssä tunnistetaan kasvain-alatyyppiselektiivinen terapeuttinen herkkyys ja vastustusmekanismit sekä tehokkaat lääkeyhdistelmät

Utility of Neutrophil Lymphocyte Ratio (NLR) and Platelet Lymphocyte Ratio (PLR) as A Predictor of Mortality in COVİD-19

Coronavirus-19 pandemic has stricken our world since December 2019; the disease, first reported in China, is now a pandemic. More than 400 million people have been affected, and 5 million people have succumbed to the disease. Hence, it is the need of the era to find readily available laboratory parameters to assess the mortality chances in these patients. Our study aims to determine the utility of NLR and PLR ratios as a predictor of severity and clinical outcome of COVID-19 patients.100 patients admitted to a tertiary care hospital in Karnataka, India, during the months April to July 2020 were studied. Only patients with a positive RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) report for COVID-19 were included. Demographic data, comorbidities, and mortality status were collected from electronic hospital records. Lab parameters including- Total Count (TC), Absolute count of neutrophils and lymphocytes, platelet count were taken. NLR and PLR were derived from available lab parameters. Patients were categorized into varying severity depending on their SpO2 levels at admission. Neutrophil count (P=0.001) and NLR (P=0.002) were associated with an increased risk of mortality and disease severity. An increase in PLR ratio (P=0.05) shows a mild association with mortality but not with disease severity (P=0.096). In contrast, comorbidities, increasing age, and gender did not show any statistical significance for mortality. The presence of statistical significance concerning NLR and PLR should be utilized as an aid by clinicians to assess disease severity and chances of mortality. As new variants of the disease are uprising and a single therapeutic measure is not available currently for the treatment of COVID-19, clinicians should be well informed about how to monitor the disease in a cost-effective and easily accessible way to reduce the disease mortality and morbidity

Spa-RQ: an Image Analysis Tool to Visualise and Quantify Spatial Phenotypes Applied to Non-Small Cell Lung Cancer

To facilitate analysis of spatial tissue phenotypes, we created an open-source tool package named 'Spa-RQ' for 'Spatial tissue analysis: image Registration & Quantification'. Spa-RQ contains software for image registration (Spa-R) and quantitative analysis of DAB staining overlap (Spa-Q). It provides an easy-to-implement workflow for serial sectioning and staining as an alternative to multiplexed techniques. To demonstrate Spa-RQ's applicability, we analysed the spatial aspects of oncogenic KRAS-related signalling activities in non-small cell lung cancer (NSCLC). Using Spa-R in conjunction with ImageJ/Fiji, we first performed annotation-guided tumour-by-tumour phenotyping using multiple signalling markers. This analysis showed histopathology-selective activation of PI3K/AKT and MAPK signalling in Kras mutant murine tumours, as well as high p38MAPK stress signalling in p53 null murine NSCLC. Subsequently, Spa-RQ was applied to measure the co-activation of MAPK, AKT, and their mutual effector mTOR pathway in individual tumours. Both murine and clinical NSCLC samples could be stratified into 'MAPK/mTOR', 'AKT/mTOR', and 'Null' signature subclasses, suggesting mutually exclusive MAPK and AKT signalling activities. Spa-RQ thus provides a robust and easy to use tool that can be employed to identify spatially-distributed tissue phenotypes

Development of an adenosquamous carcinoma histopathology - selective lung metastasis model

Peer reviewe

Spa-RQ : an Image Analysis Tool to Visualise and Quantify Spatial Phenotypes Applied to Non-Small Cell Lung Cancer

To facilitate analysis of spatial tissue phenotypes, we created an open-source tool package named 'Spa-RQ' for 'Spatial tissue analysis: image Registration & Quantification'. Spa-RQ contains software for image registration (Spa-R) and quantitative analysis of DAB staining overlap (Spa-Q). It provides an easy-to-implement workflow for serial sectioning and staining as an alternative to multiplexed techniques. To demonstrate Spa-RQ's applicability, we analysed the spatial aspects of oncogenic KRAS-related signalling activities in non-small cell lung cancer (NSCLC). Using Spa-R in conjunction with ImageJ/Fiji, we first performed annotation-guided tumour-by-tumour phenotyping using multiple signalling markers. This analysis showed histopathology-selective activation of PI3K/AKT and MAPK signalling in Kras mutant murine tumours, as well as high p38MAPK stress signalling in p53 null murine NSCLC. Subsequently, Spa-RQ was applied to measure the co-activation of MAPK, AKT, and their mutual effector mTOR pathway in individual tumours. Both murine and clinical NSCLC samples could be stratified into 'MAPK/mTOR', 'AKT/mTOR', and 'Null' signature subclasses, suggesting mutually exclusive MAPK and AKT signalling activities. Spa-RQ thus provides a robust and easy to use tool that can be employed to identify spatially-distributed tissue phenotypes.Peer reviewe

Receptor Tyrosine Kinase Signaling Networks Define Sensitivity to ERBB Inhibition and Stratify Kras Mutant Lung Cancers

Most non-small cell lung cancers (NSCLC) contain nontargetable mutations, including KRAS, TP53, or STK11/LKB1 alterations. By coupling ex viva drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from Kras(G12D/+);Lkb1(fl/fl) (KL) tumors or AC cultures from Kras(G12D/+);p53(fl/fl) (KP) tumors. Although p53-null cells readily propagated as conventional cultures, Lkb1-null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTK): activation of ERBBs in KL cultures, or FGFR in AC niltures. Furthermore, pan-ERBB inhibition reduced the clonogenidty of KL cultures, which was exacerbated by combinatorial MEK inhibition, whereas combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as a valid diagnostic approach to identify tumors sensitive to ERBB network inhibition.Peer reviewe

Capturing complex tumour biology in vitro: Histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means

Capturing complex tumour biology in vitro : histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1 alpha. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.Peer reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival