A microbial beacon for cancer detection Primary Metastasis - PowerPoint PPT Presentation

A microbial beacon for cancer detection

Primary Metastasis cancer 1 WHO Cancer Fact Sheet N o 297

Primary Metastasis cancer Late treatment Bad prognosis 1 WHO Cancer Fact Sheet N o 297

Primary Metastasis cancer Late treatment General Bad and early prognosis 1 WHO Cancer Fact Sheet N o 297

Circulating tumor cells Primary Metastasis in blood cancer Late treatment General Bad and early prognosis 1 WHO Cancer Fact Sheet N o 297

Circulating tumor cells Primary Metastasis in blood cancer Late treatment Better Early General Bad prognosis treatment and early prognosis 1 WHO Cancer Fact Sheet N o 297

Circulating tumor cells Primary Early detection Metastasis in blood cancer of circulating tumor cells (CTCs) Late treatment Better Early General Bad prognosis treatment and early prognosis 1

Early and Universal Diagnosis not yet Reliable • Inconsistent results by existing tests • Not used in clinical practice  Need to find more stable markers 2

Early and universal diagnosis not yet reliable MicroBeacon • Inconsistent results by existing detects two general tests cancer markers • Not used in clinical practice  Need to find more stable markers 2

Marker 1: Elevated Lactate Production Rate by Cancer Cancer cell Non-cancer cell L-Lactate MicroBeacon 3

Marker 1: Elevated Lactate Production Rate by Cancer Signal 1 Cancer cell Non-cancer cell L-Lactate MicroBeacon 3

Marker 1: Elevated Lactate Production Rate by Cancer Signal 1 Cancer cell Non-cancer cell Challenge: Only three-fold [1] L-Lactate difference in production rate! MicroBeacon [1] Anal. Chem. 82.12 (2010): 5082 3

Marker 2: Cancer C ell’s sTRAIL Susceptibility Cancer cell Death Receptor Non-cancer cell sTRAIL Phosphatidylserine MicroBeacon 4 4

Marker 2: Cancer C ell’s sTRAIL Susceptibility Cancer cell Death Receptor Non-cancer cell sTRAIL Phosphatidylserine MicroBeacon 4 4

Marker 2: Cancer C ell’s sTRAIL Susceptibility Apoptotic cell Cancer cell Death Receptor Non-cancer cell sTRAIL Phosphatidylserine MicroBeacon 4 4

Marker 2: Cancer C ell’s sTRAIL Susceptibility Signal 2 Cancer cell Death Receptor Non-cancer cell sTRAIL Phosphatidylserine MicroBeacon 4

Two-step Sequential Filtering Density Lactate GFP sensor sensor Lactate Apoptosis sTRAIL Phosphatidyl- serine 5

Single-cell Analysis Setup for Detecting Scarce CTCs MicroBeacon Blood Microfluidic chip 6

Single-cell Analysis Setup for Detecting Scarce CTCs MicroBeacon Blood Microfluidic chip 6

Single-cell Analysis Setup for Detecting Scarce CTCs MicroBeacon Blood Microfluidic chip 6

Single-cell Analysis Setup for Detecting Scarce CTCs MicroBeacon Blood Microfluidic chip 6

Single-cell Analysis Setup for Detecting Scarce CTCs MicroBeacon Blood Microfluidic chip Cancer 6

Marker 1 Lactate Production Rate

Single Cell Model • System of 10 ODEs and 40 parameters • Mechanistic assumptions based on reaction rates • Characterization of lactate sensor • Preliminary characterization of density sensor 7

Model Predicts: Simple System Will Not Work Threshold 8

Incoherent Feed-forward Loop (IFFL) [1] Threshold [1] Molec. cell 36.5 (2009): 894-899. 9

Implementation of the Lactate Sensor X Y Z 10

Two Promoters Needed for Incoherent Feed-forward Loop Single input promoter controlled by LldR, responsive to lactate Hybrid promoter controlled by LldR and LacI 11

Two Promoters Needed for Incoherent Feed-forward Loop Single input promoter controlled by LldR, responsive to lactate Hybrid promoter controlled by LldR and LacI 11

Single Input Promoter P lld O1 gfp O2 12

Single Input Promoter P lld O1 gfp O2 12

Single Input Promoter: Repression and Activation LldR lactate P lld O1 gfp O2 13

Single Input Promoter: Repression and Activation Activation LldR Repression lactate P lld O1 gfp O2 13

Design of Promoter Library 14

Design of Promoter Library Different: O2 O1 J23117 • Core promoter 14

Design of Promoter Library Different: O2 O1 J23117 • Core promoter spacer J23117 O1 O2 Architecture • 14

Design of Promoter Library Different: O2 O1 J23117 • Core promoter spacer J23117 O1 O2 Architecture • Spacing • 14

BBa_K1847009 BBa_K1847007 BBa_K1847008 Different architecture BBa_K1847004 BBa_K1847002 BBa_K1847003 BBa_K1847005 BBa_K1847006 Strength of core promotor 15

BBa_K1847009 BBa_K1847007 BBa_K1847008 Different architecture BBa_K1847004 BBa_K1847002 BBa_K1847003 BBa_K1847005 BBa_K1847006 Strength of core promotor 15

Increased ON/OFF Ratio LldR lactate P lld O1 gfp O2 16

Model Predicts: Lactate Import by LldP is Important Intracellular 17

Experiments Confirm: LldP is Essential for Sensing LldP P const lldP LldR lactate P lld O1 gfp O2 18

Detection System Works in Mammalian - E. coli Co-culture Jurkat, E. coli Jurkat, E. coli P lldR ::gfp, lldR P lldR ::gfp, lldR, lldP 19

Two Promoters Needed for Incoherent Feed-forward Loop Single input promoter controlled by LldR, responsive to lactate Hybrid promoter controlled by LldR and LacI 20

Hybrid Promoter Fluo. 10 4 (Au. norm.) 21

Marker 2 sTRAIL Sensitivity

How Can One Detect PS Exposure on the Target Cell? Death receptor Cancer cell Phosphatidylserine Apoptotic cancer cell Annexin V- Alexa 488 [1] JBC 272.42: 26159 22

Annexin V Displayed on Bacterial Outer Membrane AD: AutoDisplay [1] An-V: Annexin V [1] J. Bact. 179,3 794 23

How to Detect E. coli Bound to a Cancer Cell Density 24

How to Detect E. coli Bound to a Cancer Cell Density Quorum sensing 24

Genetic Design: Quorum Sensing 25

Genetic Design: Combined Circuit 26

Why is LuxR the Output of the Lactate Sensor? 27

Two Additional Models Compartment model • Multi-compartment ODE system with instant diffusion within a compartment • System design and worst-case testing 3D spatio-temporal model • Whole system with biological properties System of 27 ODEs and PDEs and 42 • parameters in multiple domains • Validation in more realistic environment 28

An AND Gate is Insufficient AND gate Sequential filtering 29

Expected Behavior of MicroBeacon 30

Compartment Model: Incoherent Feed-forward Loop is Necessary Without With Feed-forward Feed-forward Measurement Measurement 31

3D Spatio-temporal Model 32

3D Spatio-temporal Model: MicroBeacon is Reliable 33

Achievements Designed, characterized, and submitted over 17 new biobricks Demonstrated that MicroBeacon detects lactate produced by cancer cells Developed and used 3 different models to understand, evaluate, and optimize our design Collaborated with 3 other iGEM teams Outreach: Interviews, teaching in school, newspapers 34

Attributions Lab, poster, safety Lab, logo design Lab, human practices, poster Modeling, poster, Modeling, logo design, Lab, organization, organization, chip wiki development sponsoring 35

Thank you • Instructors : Sven Panke, Jörg Stelling, Kobi Benenson, Prof. Savas Tay • Advisors : Daniel Gerngross, Sabine Österle, Lukas Widmer, Margaux Dastor, Christian Jordi, Janina Linnik • Other Support : Erica Montani, Verena Jäggin, Michal Stanczak, Markus Jeschek, Matthias Mehlig, Myriam Moisan, Tino Frank, Johannes Thoma, Gaspar Morgado, Michael Junkin, Bartolomeo Angelici, Max Endele, Martin Etzrodt 36

Sponsors 37