Integrating Cytomics with Proteomics
J. Paul RobinsonProfessor of Immunopharmacology
Professor of Biomedical Engineering
Harvard UniversityNovember, 2004
Cytome - Cytomics• Cytomes can be defined as cellular systems and subsystems
and functional components of the body.
• Cytomics is the study of the heterogeneity of cytomes or more precisely the study of molecular single cell phenotypes resulting from genotype and exposure in combination with exhaustive bioinformatics knowledge extraction.
• The word Cytomics was first used in 2001 by:Davies E, Stankovic B, Azama K, Shibata K, Abe S.“Novel components of the plant cytoskeleton: A beginning to plant "cytomics"
Plant Science, Invited Review, Plant Science (160)2 (2001) pp. 185-196.
Cytomics links technology to biology – it relates measurement and detection to structure and function. It Integrates flow and image cytometry with proteomics.
Environment – Cytome (cell) – GenomeA web of interactions
Instead of concentrating on molecular targets within the relatively infinite network of highly redundant molecular pathways of cells, one could focus on the end result, represented by molecular phenotypes of cells as a consequence of both genotype
and environment.
Environment From Gene to ProteinGenome - Proteome
CellCytome
Exploring the Human CytomeTechnology
• Advanced microscopy techniques:– LM, EM, Confocal and laser scanning microscopy, spectral
imaging, FRET, SEM, TEM, digital microscopy, …
• High Content Screening:– High speed and large volume screening, …
• Flow Cytometry– Fast imaging in flow, …
• Biomolecular analysis techniques:– Single-cell polymerase chain reaction (PCR), labeling of
biomolecules by quantum dots, protein identification, etc
• Bioinformatics:– Data exploration, statistics and data management, …
Predictive Medicine by Cytomics(= therapy-dependent individualized disease
course prediction)
• Diseases represent molecular changes in cellular systems of organisms (cytomes)
• Cytomics: study of molecular cell phenotypes in combination with exhaustive bioinformatic knowledge extraction
• Cell phenotypes result from genotype and exposure
• Goal: individualized predictions >95% correct
Systems Integration• Analytical Cytology
– Flow cytometry– Single cell analysis systems– Tissue analysis– High Content Analysis (screening)
• Image Analysis – Single cells– Tissues and sections– Cell culture systems
• Proteomics
Study approaches• Study 1: Cell growth in 3D matrix
• Study 2: 3D matrix isolation and characterization
• Study 3: Standard cell line characterization
(Study 4: toxins of phenotypically separated Listeriamonocytogenes)
Cellular Heterogeneity• Identification and characterization of cellular systems is
advanced• We can separate and very highly define cellular systems
by with multivariate analysis
High-resolution cytology segmentation
ConventionalRGB Image
Spectrallysegmented Image
Wavelength (nm)
CharacteristicSpectra
High spectral resolution increases utility of spectrally responsive indicator dyesSlide from Dr. Richard Levenson, CRi, Inc.,35B Cabot Rd.,Woburn, MA 01801, www.cri-inc.com
Count
log green log red
dead
live
reduce temperatureless than 15°C
liquid
solid hydrogel
Flow Cytometry
Confocal Microscopy
Analysis of complex Microbial Systems
Extracellular Matrix (ECM)
Tissue Engineering ApplicationsScaffold to support tissue growth Structure and function are closely relatedNeed in vitro models to study such as
MatrigelVitrogen
Small Intestinal Submucosa (SIS)Basal Lamina of the Avian OvarianFollicle
Study 1: Cell growth in 3D matrix
With Professor Sophie LelièvreProfessor of Veterinary Medicine
Purdue University
Three-dimensional models of breast epithelial cell culturesrecapitulate tissue differentiation and tumor formation
1 10
normal
tumor
days
acini
Invasive tumornodule
In the presence of extracellular matrix enriched in laminin, non-neoplastic breast epithelial cells (HMT-3522 S1) form phenotypically normal tissue-like glandular structures (acini) while malignant breast epithelial cells (HMT-3522 T4-2) develop into tumor-like nodules. Cells are plated as single cells at day 1 and full differentiation and formation of large tumors is seen at day 10 of culture. A) In 3D culture, S1 cells arrest growth and differentiate after a few days of proliferation. B) In 3D culture, S1-derived T4 –2 cells continue to proliferate to form tumor-like nodules.
A
B
Cross-section indicatesthe presence of a central lumen
S1 phenotype
T4-2 phenotype
days6 8 10
30 microns
As tumors develop they enter in contact with cells from nonmalignant tissue
Contact co-culture: T4-2 cells grow toward S1 glandular structures and surround these structures. T4-2 cells were added to pre-formed S1 acini and cultured for 10 days. T4-2 cells were stained with DiI(red) prior to being plated, and S1 cells are stably transfected with green fluorescent protein(GFP). Images show co-cultures of S1 and T4-2 cells at days 6, 8, and 10. At day 6:Tumor cells (red) proliferate when in contact with S1 structures (green, arrow). Day 8:T4-2 cells expand over several S1 structures (5 glandular structures can be seen in green). Day 10: A single tumor (red) is in development; at least four S1 glandular structures (green) have been engulfed.
S1 phenotype(normal)
T4-2 phenotype(tumor)
Flow Sorting followed by protein profiling
• Sort dsRed stained tumor cells (T4-2_ from GFP-non-malignant cells (S1) population after 3 weeks of co-culture
• Analyze the changes in protein expression profile compared to the control (separately cultured) T4-2 and S1 cell populations
Phenotypically S1 Normal Malignant T4-2 PhenotypeDifferences
HMT-3522 epithelial cells
Study 2Professor Eli Asem
School of Veterinary Medicine, Purdue University
Isolation and Characterization of Extracellular matrix
Basement Membrane• The maintenance of normal function of vertebrate cells
depends on the integrity of the extracellularmicroenvironment of the tissue/organ.
• Basement membranes (basal laminae) are specialized ECM sheets that participate in numerous physiological processes and play key roles in regulating proliferation and differentiation of cells.
• Only some of the proteins of basement membranes have been identified due to the complex protein composition and the unavailability of pure preparations.
• Recently a pure preparation was identified and shown to be biologically active
Study 3
• A “standardized” cell culture environment
• Establishing a “normal” profile for HepG2 cell line
• Interest from pharmaceutical companies for some relatively fast classification systems
A Study of HepG2 cells
• HepG2 - cell line that is a human, primary liver cancer • Human hepatocellular liver carcinoma cell line, Epithelial
cells.• An established human hepatocarcinoma cell line with
epithelial morphology. HepG2 cells are used routinely for a variety of biochemical and cell biological studies. HepG2 is the most commonly used cell line for examining the regulation of hepatic protein synthesis by cytokines .
# cells sorted3 x 106
# cells sorted1 x 106
Cells were sorted on a Beckman-Coulter Altra cell sorter prior to PF2D
Cell/system complexity can be reduced using tools such as flow cytometry
Figures from Roederer, et al
Concluding Thoughts
Beckman-Coulter automated PF2D protein separation system
Cell Sorter
• Developing an understanding of complex biological systems should be approached by first considering the heterogeneity of the system• Using a cytomics approach reduces the complexity at an earlier stage• Tools such as the PF2D offer a significant opportunity for cell biologists to approach proteomic solutions
Acknowledgements• Contributors – Collaborators
Dan Hirleman (ME)Yinlong Sun (CS)Kinam Park (IP)
• PostdocsGerald J. Gregori (Microbiology)Valery Patsekin (Photonics)Tytus Bernas (Proteomics)Sang Youp Lee (Fluidics)Bartek Rajwa (Biophysics)
• Graduate StudentsWamiq Ahmed (Computational)Murugesan Venkatapathi (M.Eng)Bulent Bayraktar (Computational)Silas Leavesley (BioEng)Jia Liu (Cell Biology)Connie Paul (Pharmaceutical)
• StaffJennie SturgisKathy RaghebCheryl HoldmanGretchen LawlerSteve Kelley
Departments & CentersPurdue University Cytometry LabsBasic Medical ScienceBiomedical Engineering Electrical & Computing EngineeringMechanical EngineeringBindley Bioscience CenterDiscovery ParkPurdue Cancer Center
Funding:NIH, NSF, USDA, Purdue UniversityCorporate: Beckman-Coulter, Point-Source,Parker-Hannifin, Bio-Rad, Polysciences
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