Development of High-Throughput Quantitative Assays for Glucose Uptake in Cancer Cell Lines

Abstract Activating RAS mutations are among the most common pathogenetic events in a broad spectrum of hematologic malignancies and epithelial tumors. However, oncogenic RAS has thus far not proven to be a tractable target for therapeutic intervention. An alternative to direct targeting of known oncogenes is to perform “synthetic lethality” screens to identify genes that are selectively required for cell viability in the context of specific cancer-causing mutations. Using this approach, we have discovered a synthetic lethal interaction between mutant KRAS, the most frequently mutated oncogene in human cancer, and inactivation of the gene encoding the STK33 serine/threonine protein kinase. To identify genes that are essential for cell viability in the context of mutant KRAS, we performed high-throughput loss-of-function RNA interference (RNAi) screens in eight human cancer cell lines (mutant KRAS, n=4; wildtype KRAS, n=4), representing seven different tumor types (acute myeloid leukemia, multiple myeloma, colon cancer, breast cancer, ovarian cancer, prostate cancer, glioblastoma), as well as normal human fibroblasts and mammary epithelial cells. We screened each cell line with a subset of the short hairpin RNA (shRNA) library developed by the RNAi Consortium (http://www.broad.mit.edu/genome_bio/trc/rnai.html) that consists of 5,024 individual shRNA constructs targeting 1,011 human genes, including the majority of known and putative protein kinase and phosphatase genes and a selection of known cancer-related genes. In these cell lines, suppression of STK33 preferentially inhibited the viability and proliferation of cells that were dependent on mutant KRAS. The differential requirement for STK33 based on oncogenic KRAS dependency was confirmed in 16 additional cell lines using in vitro transformation assays and human tumor xenograft models. Biochemical analyses support the hypothesis that STK33 promotes cell growth and survival in a kinase activity-dependent manner by regulating the activity of S6K1 as well as BAD-induced apoptosis selectively in mutant KRAS-dependent cells. Notably, molecular genetic characterization of cancer cell lines and analysis of patient-derived genomic data sets indicate that STK33 is not frequently mutated or overexpressed in human tumors. These observations identify STK33 as a potential target for the treatment of mutant KRAS-driven cancers that may have a broad therapeutic index in normal versus malignant cells, and illustrate the potential of RNAi for discovering critical functional dependencies created by oncogenic mutations that cannot be identified using other genomic technologies.

Download Full-text

Selection of lung cancer-associated scFv antibodies from cultured cells

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e22137 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e22137-e22137

Author(s):

P. P. Massion ◽

T. V. Pedchenko ◽

D. V. Parekh ◽

R. Mernaugh

Keyword(s):

Lung Cancer ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

Cancer Cell Lines ◽

Normal Lung ◽

Antibody Library ◽

Lung Cancer Cell ◽

Cell Lysates

e22137 Background: Lung cancer is the most common cause of cancer-related deaths in the world. There is a critical need for new strategies of early lung cancer detection. The identification of tumor-associated antigens and corresponding antibodies is one approach to discovery of diagnostic biomarkers. We used a large phage-displayed recombinant antibody library and normal human lung epithelial and non-small cell lung cancer cell lines to select for and identify recombinant antibodies specific for proteins expressed, or over-expressed, in lung cancer. Methods: The antibody library was used to select for recombinant scFv antibodies reactive with proteins present, or aberrantly expressed, in non-small cell lung cancer cell lines (A549, H549, H157, H23) in comparison to normal lung cell lines (BEAS-2B, 16-HBE, KT). Soluble scFv antibodies were obtained through 2 rounds of phage antibody cross-absorption (on normal cell lines) and selection (on non-small lung cancer cell lines). Soluble scFv were assayed by a high-throughput fluorometric microvolume assay technology (FMAT) against normal and cancer lung cell line proteins. ScFv antibodies selected by FMAT were evaluated further with Western blot-based assays. Results: More than 100 scFv antibodies identified by FMAT bound preferentially to proteins in lung cancer. Of these, 46 scFv were assayed by a high throughput Western slot blot immunoassay against pooled normal lung and lung cancer cell lysates. Eight scFv were assayed in Western blot against individual lung normal and non-small lung cancer cell line lysates. Four of these demonstrated differential binding to normal and cancer cell lysates. Conclusions: In summary, we were able to detect cancer-associated antigens in lung cancer cell lines using a phage display antibody library. In combination with high-throughput fluorescent and Western blot assays, four unique scFv antibodies were selected that differentially bound to normal and lung cancer cell lysates. These scFv will be tested as candidate biomarkers of lung cancer in independent tissue and serum samples from patient with and without lung cancer to determine utility for use in lung cancer diagnosis. No significant financial relationships to disclose.

Download Full-text

Detection of Intracellular Granularity Induction in Prostate Cancer Cell Lines by Small Molecules Using the HyperCyt® High-Throughput Flow Cytometry System

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109335671 ◽

2009 ◽

Vol 14 (6) ◽

pp. 596-609 ◽

Cited By ~ 21

Author(s):

Mark K. Haynes ◽

J. Jacob Strouse ◽

Anna Waller ◽

Andrei Leitao ◽

Ramona F. Curpan ◽

...

Keyword(s):

Prostate Cancer ◽

Flow Cytometry ◽

Small Molecules ◽

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

Prostate Cancer Cell ◽

Cancer Cell Lines ◽

Phenotypic Change ◽

Prostate Cancer Cell Lines

Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt® high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of ~25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells. ( Journal of Biomolecular Screening. 2009:596-609)

Download Full-text

Novel three-dimensional cultures provide insights into thyroid cancer behavior

Endocrine Related Cancer ◽

10.1530/erc-19-0374 ◽

2020 ◽

Vol 27 (2) ◽

pp. 111-121 ◽

Cited By ~ 1

Author(s):

Mason A Lee ◽

Kensey N Bergdorf ◽

Courtney J Phifer ◽

Caroline Y Jones ◽

Sonia Y Byon ◽

...

Keyword(s):

Thyroid Cancer ◽

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

Drug Response ◽

3D Culture ◽

The United States ◽

Cancer Cell Lines ◽

Thyroid Cancer Cell ◽

Thyroid Cancer Cell Lines

Thyroid cancer has the fastest growing incidence of any cancer in the United States, as measured by the number of new cases per year. Despite advances in tissue culture techniques, a robust model for thyroid cancer spheroid culture is yet to be developed. Using eight established thyroid cancer cell lines, we created an efficient and cost-effective 3D culture system that can enhance our understanding of in vivo treatment response. We found that all eight cell lines readily form spheroids in culture with unique morphology, size, and cytoskeletal organization. In addition, we developed a high-throughput workflow that allows for drug screening of spheroids. Using this approach, we found that spheroids from K1 and TPC1 cells demonstrate significant differences in their sensitivities to dabrafenib treatment that closely model expected patient drug response. In addition, K1 spheroids have increased sensitivity to dabrafenib when compared to monolayer K1 cultures. Utilizing traditional 2D cultures of these cell lines, we evaluated the mechanisms of this drug response, showing dramatic and acute changes in their actin cytoskeleton as well as inhibition of migratory behavior in response to dabrafenib treatment. Our study is the first to describe the development of a robust spheroid system from established cultured thyroid cancer cell lines and adaptation to a high-throughput format. We show that combining 3D culture with traditional 2D methods provides a complementary and powerful approach to uncover drug sensitivity and mechanisms of inhibition in thyroid cancer.

Download Full-text