Title: DIAGNOSTIC BIOMARKERS FOR COLORECTAL CANCER
1DIAGNOSTIC BIOMARKERS FOR COLORECTAL CANCER
- Identifying Protein based Serum Plasma
Biomarkers using a Proteomics-based Approach
Ms. Aisha Q. Butt Prof. Martin Clynes Dr. Paul
Dowling National Institute for Cellular
Biotechnology (NICB)
2Objectives
- Colorectal Cancer Facts Diagnostic Limitation
- Hypothesis Aims
- Research Strategy
- Biomarker Discovery
- Cell Culture
- Nano-HPLC Mass Spectrometry
- Bioinformatics
- Biomarker Verification
- ELISA
- Immunohistochemistry
- Conclusion Future Work
-
3Colorectal Cancer Facts Figures
- 2nd most common cause of death in Republic of
Ireland. - Approximately, 2000 new CRC cases diagnosed each
year. - Ireland Highest bowel cancer incidence, lower
survival rate and higher mortality rates in
Western Europe both in men and women.
- 1 Million new cases 1/2 million deaths p/yr
- 3rd most common malignancy in world.
- Primary Cancer mortality cause in USA, Europe
Korea
- Prediction By 2020, new CRC cases projected to ?
by 79 in men 56 in women.
4- CRC Diagnosing Limitations
- CRC highest cure rates if detected early.
- Colonoscopy
- Gold standard for diagnosis of colonic neoplasia
- But its not a perfect test
- Lengthy waiting lists for colonoscopy services in
Ireland - Lack of sensitivity low compliance Most of
current diagnostics detect cancer after it has
already spread to other parts of the body
- Devise screening programme based on
- Panel of Protein Biomarkers
- Used in combination with Colonoscopy
- To offer most accurate test for early diagnosis
of CRC -
- Novel high value biomarker diagnostics test kit
based on blood or serum could - Prescreen high risk groups
- Eliminate waiting lists
- Permit rapid access to suspects
5Research Strategy
- Cancer Secretome comprising all proteins
released by tumour cell Attracting much
attention recently. - Our Approach Collection of Conditioned Media
from 4 CRC cell lines HCT116, HT-29, SW480 WiDr
using Cell Culturing Techniques. - Proteins identified from conditioned media using
Mass Spectrometry evaluated using specific
criteria to identify most likely candidate for
further investigations. - Criteria Being detectable in all cell lines
- Likelihood that proteins are shed/secreted by
tumour cells - Proteins primary localisation molecular
function within the cell - Literature search
- Bioinformatics allowed data sets to be cross
compared from the shortlisted data, few
proteins were verified using - ELISA kit
- Immunohistochemistry
6Biomarker Discovery
- Cell Culture
- Four CRC cell lines cultured in respective media-
HCT116, HT-29, SW480 WiDr - Assessment of Cell Viability performed on derived
Conditioned Media - Protein Concentration
- Proteins in CMs centrifuged down in CENTRICONS
(Sartorius Stedim Biotech) - Concentrated proteins by 40 fold (4mls to 100µl)
- Protein Precipitation Clean Up
- Using Ready PrepTM 2-D Clean-Up Kit protein
samples subjected to precipitation and cleaning
using buffers and reagents yielding 30µl sample
for each cell line - In Solution Digestion / Double Digestion
- Double Digestion carried out using LysC Trypsin
for denaturation digestion of proteins into
peptides. - Sample Clean Up
- Using PepClean TM C-18 Resin Spin Columns,
tryptic peptide mixtures were further cleaned up
for removal of any solvents and buffers. - Sample speed vacuumed down and resuspended in
Mass Spec. compatible buffer.
7LC-MS Bioinformatics
- Ettan MDLC system (GE Healthcare, Piscataway, NJ)
- applied for desalting and separation of LysC
and tryptic peptide mixtures. - Two experiments set up
- 10µl tryptic peptide sample loaded onto LC column
with 1hour gradient time - 20µl tryptic peptide sample loaded onto LC column
with 3hour gradient time
- MDLC-MS results analysed using Bioworks Browser
software suite TM (Thermo Fisher Scientific, USA) - Proteins in samples searched against Swiss Prot
human protein database (Dec, 2009 65,533
entries 20,339 human proteins). - The stringent protein identification criteria
based on multiple filters - Distinct peptides
- Delta CN (0.040)
- Xcorr vs. Charge state (1.50, 2.20, 2.50, and
3.00) - Peptide Probability (0.05)
- Number of distinct peptides (2)
8Results Discussion
- Conditioned Media Cell Viability
- Mean Average 89.875 Viable Cells 10.125 Dead
Cells - Cell counts performed using haemocytometer and
trypan blue dye on CM derived from 4 CRC cell
lines using NICB-SOP-003-01.
9Mass Spectrometry Sample Gradient Time
- An approximate gt2.5 fold increase in the total
proteins identified on MS using 1hr vs. 3hr
sample gradient time. - Stringent Multiple filters applied Distinct
Peptides, Delta CN (0.040), Xcorr vs. Charge
state (1.50, 2.20, 2.50, and 3.00), Peptide
Probability (0.05) and Number of distinct
peptides (2). - Data Cross compared using Bioinformatics Excel
tools - 92 common proteins identified within 4 CRC cell
lines.
Cell Lines Quantity-Gradient Total Proteins Filtered Proteins
HCT 116 10ul - 1Hour 2076 148
20ul - 3Hour 8120 603
HT 29 10ul - 1Hour 1851 75
20ul - 3Hour 3889 185
SW-480 10ul - 1Hour 1953 119
20ul - 3Hour 3949 255
WiDr 10ul - 1Hour 2086 200
20ul - 3Hour 5162 384
10Cellular Localisation Biological Process
- 92 Common proteins further analysed classified
using Human Protein Reference Database on basis
of - Cellular Localisation
- Biological Processes
- Motifs
- 51 Proteins localization is based within
cytoplasm and 23 proteins are associated with
cell growth maintenance.
- 92 Common proteins further analysed classified
using Human Protein Reference Database on basis
of - Cellular Localisation
- Biological Processes
- Motifs
- 51 Proteins localization is based within
cytoplasm and 23 proteins are associated with
cell growth maintenance.
11Protein Motifs
- Proteins secreted/shed by the tumour cells (and
so will be detectable in the circulatory system)
an important criterion to identify the most
likely candidate biomarkers for further
investigations. - Proteins having signal peptide on their motifs
are secretory proteins, whereas all other
proteins are shed by the tumour cells.
12Biomarker Verification
- Stringent classification of 92 common proteins on
the basis of - Proteins Cellular Localisation
- Biological Process
- Proteins Motifs
- Allowed to choose 2 proteins
- Protein X
- Protein Y
- Why these 2 Proteins?
- Presence of signal peptide motif on Protein X
- Relevance and association of Protein Y with other
cancer types (Literature Search)
13ELISA Test Serum Plasma Samples
- Protein X levels in serum plasma samples
measured using double-antibody sandwich ELISA
system (Bender MedSystems, Austria) - Serum samples 16 Advanced stage 8 Healthy
controls - Plasma samples 9 Early stage 8 Healthy
controls
(Early Stage )
14Receiver Operating Characteristics ROC Curve
- ROC curve analysis of Protein X for
discriminating CRC patients from healthy controls
for advanced stage serum (AUC - 0.825) and early
stage plasma samples (AUC - 0.639).
Protein X Diagnostic efficacy in Serum samples
Protein X Diagnostic efficacy in Plasma samples
15Immunohistochemistry CRC Tissue Sections
- CRC tissue sections compared to normal colon
tissues via Protein Y expression using
Immunohistochemical staining. - Protein Y positive expression observed in a
moderately differentiated adenocarcinoma
(intermediate differential grade) - Protein Y positive staining in the cytoplasm -
Granular in nature - A lot less nuclear staining in areas of tumour
compared to normal tissue
16Conclusion
- This pilot study has shown that cancer secretome
from the tumour cells presents a promising
reservoir of biomarkers with soluble-secreted
proteins and shed membrane proteins. - And, the use of these secreted proteins to go
back on clinical serum or plasma samples to
distinguish patients with or without CRC is a
promising diagnostic approach. - Also, both Protein X and Protein Y could serve as
potential biomarkers if used in combination with
few other biomarkers for the early diagnosis of
colorectal cancer.
17Further Work...
- Further work and efforts are required to fully
validate the biomarkers detected in this study
with - Large sized clinical samples
- Multiple medical centre samples
- ELISA Test for Protein Y protein on serum
plasma samples - These biomarkers detected can be used in
combination with a panel of other protein
biomarkers and colonoscopy to improve the overall
accuracy and speed for detecting CRC at an early
stage and prioritise high-risk individuals.
18Summary
- Colorectal Cancer Diagnostic Limitations
- Aim Protein based Biomarkers for early detection
of CRC - Biomarker Discovery Research Strategy
- Cell Culture
- Nano-HPLC Mass Spectrometry
- Bioinformatics
- Biomarker Verification
- ELISA
- Immunohistochemistry
- Conclusion Future Work
-
19Thank You !