Patient-derived Lung Cancer Cell Research Articles

Abstract A104: Development of pathophysiologically relevant in vitro tumor models: Cytotoxicity of anticancer drugs in two dimensional and three dimensional cell line cultures versus primary colorectal tumor cells

Abstract Chemotherapy is an effective modality for cancer therapy. However, treatment of solid tumors is hampered by several factors, such as development of acquired resistance, nonspecific drug effects, etc. Moreover, high attrition rate of anticancer drugs is an additional clinical hurdle often encountered in cancer patients. One of the potential ways to overcome these issues is to use in vitro tumor models that reliably capture in vivo tumor conditions for effective drug selection in the preclinical stages. Although two-dimensional (2D) cultures of cancer cells are widely used for drug screening and target validation; they are less predictive in vitro tumor models than three-dimensional (3D) cultures and primary cultures of patient-derived tumor cells. Cytotoxic drugs, nanoparticles, and antitumor antibodies display altered activity in 2D, 3D, and primary cancer cell cultures, potentially due to cellular and molecular differences in these culture types. Recently, the use of multicellular 3D spheroid cultures of cancer cells have gained considerable attention among researchers as more predictive in vitro tumor models to study the effects of tumor microenvironment on cancer cell response to drugs. In addition to 3D cultures, patient-derived tumor cells are also becoming increasingly popular in drug research for their relative closeness to tumors. It is expected that drugs characterized using these pathophysiologically relevant in vitro tumor models will improve their translation to higher disease models, and significantly reduce the cost associated with failure of drugs in the clinical trials. In this study, we compare the cytotoxic effects of 12 standard anticancer drugs in 2D and 3D cultures of colorectal carcinoma (CRC) cells to >500 primary CRC cultures. Furthermore, using primary cultures of patient-derived ovarian, breast, and lung cancer cells, we present the potential use of primary tumor cells as in vitro predictive model for chemotherapy. Our data indicates that compared to 2D cultures, 3D cultures of CRC cells better reflect the drug effects seen in primary CRC cultures. Our cytotoxicity data from primary cultures also show how the drug sensitivity profiles differ across histologically different solid tumors types. Acknowledgements This work was supported by grants from the Czech Ministry of Education, Youth and Sports (CZ.1.07/2.3.00/30.0041 to V.D.; LO1304 to M.H.), the Czech Ministry of Health (NT14282 to M.H.), and the Technological Agency of the Czech Republic (TE01020028 to M.H.). Citation Format: Viswanath Das, Marta Zbozinkova, Martin Holoubek, Petr Dzubak, Marian Hajduch. Development of pathophysiologically relevant in vitro tumor models: Cytotoxicity of anticancer drugs in two dimensional and three dimensional cell line cultures versus primary colorectal tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A104.

Abstract 5102: Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway

Abstract Kras controls many cellular processes such as cell migration and survival. Mutation of Kras to a constitutively active form is a common alteration in lung cancer. Being constantly active results in a hyper activation of downstream signaling cascades, which leads to enhanced tumorigenicity and high metastatic potential. How constitutive Kras activation translates into these different functions, however, remains unclear. Using patient-derived lung cancer cell lines expressing oncogenic KrasV12 we discovered that Kras expression, not transcription, gets up-regulated under long-term culture in 1% O2. We confirmed the in vivo relevance of this finding, staining mouse lung tumors for Kras and Hif1α, as an indicator for hypoxic areas, and found heterogeneous Kras levels throughout the tumors with hot-spots that overlaid with Hif1α expression. Up-regulation of KrasV12 also correlated with a significant increase in growth of tumor-like aggregates in soft-agar assays and a morphological change from adherent single-cells into sphere-like aggregates in cell culture. This morphology was reversible upon knock-down of Kras. Separation of single clones confirmed the relation between adherent and sphere-like morphologies and the level of KrasV12 expression. Interestingly, the expression level of Kras correlated with clonal migration behavior. Low Kras expressers were highly motile whereas high Kras expressers remained stationary in aggregates. In agreement, exogenous expression of KrasV12 in motile, low Kras expressers converted their morphology and migration behavior to sphere-like, stationary aggregates. Using Western blot analysis we found that these changes are due to differences in Erk activation down-stream of Kras. In high Kras expressers Erk is highly phosphorylated which leads to higher Myosin II activity and thus to a contractile, sphere-like morphology with low mobility. Low Kras levels result in less Erk activation and therefore lower Myosin II activity which leads to an adherent, motile phenotype. Using a specific Erk inhibitor, we could convert high Kras expressers into adherent, motile low Kras clones. At the same time we found an adhesion-dependent feedback mechanism which regulates cellular Kras levels. Protein analysis of cells cultured in low adhesion dishes formed spheres and dramatically increased their Kras content. Most interestingly, this increase was reversible by stimulation of adhesion. Additionally, blocking FAK activation in low Kras expressers resulted in up-regulation of Kras and Erk phosphorylation which went along with a switch to sphere-like morphology. Based on these findings we propose a novel mechanism for controlling cancer cell migration by a hypoxia-sensitive, adhesion-dependent Kras switch which makes the cell very adaptable to different environments during the whole process of metastasis. Citation Format: Anette C. Schafer, Meghan Discoll, Ashwathi Mohan, Andrew Ludlow, Wesley Burford, Jerry W. Shay, Gaudenz Danuser. Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5102. doi:10.1158/1538-7445.AM2015-5102

Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

Abstract A25: Systematic analysis of gene-drug interactions in isogenic cells reveals novel unexpected dependencies in lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, killing more people than colon, breast and prostate cancer together. Although the majority of genes that cause lung cancer have been identified, the translation of this knowledge into effective treatments has proven challenging. Thus, an interrogation of functional gene-drug interactions and identification of cancer vulnerabilities for approved or experimental drugs can help bridge the gap between cancer genomics and effective treatments. We constructed a genetically tractable and flexible lung cancer model consisting of a panel of isogenic cell lines that captures parts of the molecular heterogeneity and complexity of lung adenocarcinoma. This panel was screened against a collection of drugs, mainly kinase inhibitors and lung cancer chemotherapeutics. The screen revealed several known or expected gene-drug interactions, thus providing the proof of principle for our approach. For instance, RAS mutant cells were resistant to p38 inhibitors, presumably as RAS is able to activate p38 MAPK. Also the resistance of RAS mutant cells to VEGFR inhibitors can be assigned to the fact that RAS is a downstream effector of VEGFR. On the other hand, we observed that DNA-PK(cs)-deficient cells display hypersensitivity to the DNA-damaging agents etoposide and doxorubicin. Interestingly, we also found strong sensitivity of ATM loss-of-function cells to CHK1/2 inhibitor. As CHK activates DNA-PK(cs) and this substrate was shown to be synthetically lethal with ATM, this may provide plausible explanation for the mechanism. Importantly, we have identified several unexpected vulnerabilities to multiple protein kinase inhibitors that are currently in clinical trials. These sensitivities were consequently reproduced in a panel of patient derived lung cancer cell lines and the physiological relevance was demonstrated. Currently, we are investigating the mechanisms responsible for the exquisite dependency observed. Overall, these data may provide a direct and testable rational to apply these kinase inhibitors in stratified lung cancer patient cohorts with the relevant genetic makeup. Citation Format: Michal Smida, Ferran Fece de la Cruz, Iris Uras, Sebastian MB Nijman. Systematic analysis of gene-drug interactions in isogenic cells reveals novel unexpected dependencies in lung cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A25.

Identification of CCDC6-RET Fusion in the Human Lung Adenocarcinoma Cell Line, LC-2/ad

Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non-small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion-positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.

Flow Cytometric Technique for Quantitating Cytotoxic Response to Photodynamic Therapy

A simple flow cytometric technique for rapid measurement of multilog cytotoxic responses to photosensitization of cellular systems is described. This technique is particularly useful for cell lines with a low colony-forming efficiency, for which a nonclonogenic assay is required. The assay separates cell-sized objects from cellular debris by gating on forward scatter versus side scatter, identifies viable cells by positive calcein AM and negative ethidium homodimer-1 staining and measures cell concentration relative to an internal standard of polystyrene beads. Large numbers of cells can be analyzed rapidly. Two patient-derived small cell lung cancer cell lines, NCI-H209 and SV-E, were used to test the technique. Photordiation survival curves of the response of these cell lines to 5-aminolevulinic acid-induced protoprophyrin IX photosensitization correlated with the extent of photosensitizer accumulation. There was good agreement between the results obtained using the tritiated thymidine incorporation assay and the flow cytometric cytotoxicity assay. The technique can be used to measure cytotoxic responses to photosensitization of cell lines regardless of their plating efficiencies.