Julia Wulfkuhle

Laser-capture microdissection

Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h.

PROTEOMIC APPLICATIONS FOR THE EARLY DETECTION OF CANCER

The ability of physicians to effectively treat and cure cancer is directly dependent on their ability to detect cancers at their earliest stages. Proteomic analyses of early-stage cancers have provided new insights into the changes that occur in the early phases of tumorigenesis and represent a new resource of candidate biomarkers for early-stage disease. Studies that profile proteomic patterns in body fluids also present new opportunities for the development of novel, highly sensitive diagnostic tools for the early detection of cancer.

Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance

Side-population (SP) cells within cancers and cell lines are rare cell populations known to enrich cancer stem-like cells. In this study, we characterized SP cells from the human breast cancer cell line MCF7 as a model for cancer stem-like cells. Compared with non-SP cells, MCF7 SP cells had higher colony-formation ability in vitro and greater tumorigenicity in vivo, suggesting that MCF7 SP cells enrich cancer stem-like cells. cDNA microarray analysis of the SP cells indicated higher expression of ATP-binding cassette transporters and genes involved in quiescence, which were confirmed by quantitative RT-PCR and flow cytometry cell cycle analysis. To identify signal pathways important for cancer stem-like cells, we analyzed cDNA microarray data and identified nine pathways that were altered in the SP cells. To analyze the protein signaling networks, we used reverse-phase signaling pathway protein microarray technology and identified three signaling proteins that are significantly different between MCF7 SP and non-SP cells. Notably, signaling of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR), signal transduction and activator of transcription (STAT3), and phosphatase and tensin homolog (PTEN) was confirmed to be critical for MCF7 SP cell survival and proliferation by pathway specific inhibitors, selected gene knockdown, and in vivo tumorigenicity assay. The STAT3 pathway was found to be positively regulated by mTOR signaling, whereas PTEN served as a negative regulator of both STAT3 and mTOR signaling. This study suggests the existence of prosurvival signaling pathways critical for cancer stem-like cell maintenance, which could be selectively targeted for inhibiting cancer stem-like cells for improved treatment.

Protein microarrays: Meeting analytical challenges for clinical applications

Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.

Use of Reverse Phase Protein Microarrays and Reference Standard Development for Molecular Network Analysis of Metastatic Ovarian Carcinoma

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient’s metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

New approaches to proteomic analysis of breast cancer

Proteomic based approaches are beginning to be utilized to study the natural history and treatment of breast cancer. A variety of proteomics approaches are under study, and are summarized herein. Two‐dimensional gel electrophoresis (2D‐PAGE) is still the foundation of most proteomics studies. We present an analysis of 2D‐PAGE studies reported to date in breast cancer, including those examining normal/tumor differences and selected populations of breast cells. Newer technologies such as laser capture microdissection and highly sensitive mass spectrometry methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Novel technologies still in developmental phases will enable identification of validated targets in small biopsy specimens, including high density protein arrays, antibody arrays and lysate arrays. Surface‐enhanced laser desorption/ionization time‐of‐flight (SELDI‐TOF) analysis enables the high throughput characterization of lysates from very few tumor cells and may be best suited for clinical biomarker studies. We present SELDI‐TOF data herein to show the accuracy of the method in a small cohort of breast tumors, as well as its potential discriminatory capability. Such technologies are expected to supplement our armamentarium of mRNA‐based assays, and provide critical information on protein levels and post‐translational modifications.

Multiplexed cell signaling analysis of human breast cancer applications for personalized therapy.

Phosphoprotein driven cellular signaling events represent most of the new molecular targets for cancer treatment. Application of reverse-phase protein microarray technology for the study of ongoing signaling activity within breast tumor specimens holds great potential for elucidating and profiling signaling activity in real-time for patient-tailored therapy. Analysis of laser capture microdissection primary human breast tumors and metastatic lesions reveals pathway specific profiles and a new way to classify cancer based on functional signaling portraits. Moreover, the data demonstrate the requirement of laser capture microdissection for analysis and reveal the metastasis-specific changes that occur within a new microenvironment. Analysis of biopsy material from clinical trials for targeted therapeutics demonstrates the feasibility and utility of comprehensive signal pathway activation profiling for molecular analysis.

Proteomic approaches to the diagnosis, treatment, and monitoring of cancer.

The field of proteomics holds promise for the discovery of new biomarkers for the early detection and diagnosis of disease, molecular targets for therapy and markers for therapeutic efficacy and toxicity. A variety of proteomics approaches may be used to address these goals. Two-dimensional gel electrophoresis (2D-PAGE) is the cornerstone of many discovery-based proteomics studies. Technologies such as laser capture microdissection (LCM) and highly sensitive MS methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Newer technologies such as reverse phase protein arrays will enable the identification and profiling of target pathways in small biopsy specimens. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis enables the high throughput characterization of lysates from very few tumor cells or body fluids and may be best suited for diagnosis and monitoring of disease. Such technologies are expected to supplement our arsenal of mRNA-based assays, and we believe that in the future, entire cellular networks and not just a single deregulated protein will be the target of therapeutics and that we will soon be able to monitor the status of these pathways in diseased cells before, during and after therapy.

Early detection: Proteomic applications for the early detection of cancer

The ability of physicians to effectively treat and cure cancer is directly dependent on their ability to detect cancers at their earliest stages. Proteomic analyses of early-stage cancers have provided new insights into the changes that occur in the early phases of tumorigenesis and represent a new resource of candidate biomarkers for early-stage disease. Studies that profile proteomic patterns in body fluids also present new opportunities for the development of novel, highly sensitive diagnostic tools for the early detection of cancer.

The needle in the haystack: Application of breast fine‐needle aspirate samples to quantitative protein microarray technology

There is an unmet clinical need for economic, minimally invasive procedures that use a limited number of cells for the molecular profiling of tumors in individual patients. Reverse‐phase protein microarray (RPPM) technology has been applied successfully to the quantitative analysis of breast, ovarian, prostate, and colorectal cancers using frozen surgical specimens.