Flux Balance Analysis of ccRCC cells

(SKRC-7 and VHL-7 cell lines)

 Clear cell Renal Cell Carcinoma (ccRCC)
• RCC account for 80% all kidney cancer, 70% ccRCC

• Most aggressive RCC

• Highest rates of metastasis

• High mortality

• Hallmark: mutation in Von Hippel Lindau gene (VHL)

Image: http://emedicine.medscape.com/article/1612043-overview
 VHL gene

image: http://www.usmleforum.com/files/forum/2014
Flux Balance Analysis
• Analyze flux through metabolic network

• Constraint-based approach
Mathematical representation of GSMM

• Represent metabolic reaction in

Stoichiometric (S) matrix

S = Stoichiometric matrix
v = flux vector
𝑑𝑥
= concentration change over time
𝑑𝑡
 Flux Balance Analysis

(Orth JD, Nature 2010)

Expression data of 2 cell lines

SKRC-7
ccRCC cancer cell with mutation in VHL

VHL-7
rescued cell, isogenic pair of SKRC-7
 Metabolic model used

INIT algorithm : Model used:

• 82 different human cell types iRenalCancer1410
• 16 different human cancers

http://www.metabolicatlas.org/
Model set up

Cell medium
(metabolite constraint)

Gene expression integration Fetal Bovine Serum

constraint
(SKRC-7 and VHL-7)
???

INIT renal cancer

model
 Continuous gene expression values as constraints

E-Flux

(Colijn et al, PLOS Computational Biology 2009)

Gene expression integration
Culture media constraint
L-Arginine Hydrochloride 241.86 L-Leucine 50
L-Asparagine Monohydrate 56.81 L-lysine Monohydrochloride 40
L-Aspartic Acid 20 L-Methionine 15
D-Biotin 0.2 Niacinamide (Nicotinamide) 1
Choline Chloride 3 D-Calcium Pantothenate 0.25
L-Cysteine Monohydrochloride Monohydrate 65.19 L-Phenylalanine 15
Folic Acid 1 L-Proline 20
D-Glucose anhydrous 2000 Pyridoxine Monohydrochloride 1
L-Glutamic Acid 20 Riboflavin 0.2
L-Glutamine 300 L-Serine 30
Glycine 10 Thiamine Monohydrochloride 1
Glutathione Reduced 1 L-Threonine 20
L-Histidine Monohydrochloride Monohydrate 20.27 L-Tryptophane 5
Myo-inositol 35 L-Tyrosine Disodium Salt, Dihydrate 28.83
L-Isoleucine 50 L-Valine 20

Normalization:
Mij = normalized value for metabolite i in reaction j
ci c = vector of metabolite concentration
𝑀𝑖𝑗 = ∗ 𝑘 ∗ 𝑔𝑒𝑛𝑒𝑗 genej = gene expression for reaction j
|c| k = constant ; k = 1000
Fetal Bovine Serum (FBS) constraint
iRenalCancer Human Serum Normalization:
1410 Database

𝑘 ∗ 0.1
𝐹𝑖𝑗 = ∗ 𝑔𝑒𝑛𝑒𝑗
string 𝑛
Exchange matching Human serum
metabolites metabolites
(with all synonyms) Fij = normalized value for metabolite i in
reaction j

genej = gene expression for reaction j

Manual search
n = number of metabolites

k = constant ; k = 1000
190 FBS
metabolites
Analysis Gene
exp

media serum
INIT
model

Model 1 Model 2 Model 3

(median) (maximum) (Boolean)

1. Predict metabolite essentiality in SKRC7

2. Reaction(s) contributing to higher biomass production in SKRC7
Metabolite essentiality prediction in SKRC-7
1. Decrease FBS concentration to 10 %

2. Set constraint of one metabolite (from culture media

list) in SKRC-7 to VHL-7 constraint

3. Optimize biomass production

Metabolite essentiality prediction in SKRC-7
In silico mechanism of cycteine dependency in SKRC-7
Higher growth in SKRC-7
in silico prediction of higher biomass production in SKRC-7
Models comparison
Conclusion
Integration of gene expression and environtment data to metabolic model can be used to
reconstruct a predictive cell-line specific model

in silico mechanism which cannot described without full picture of metabolic network

Recommendations
Experimental validation

Comparison with previous algorithms