Teaching self-destructing Salmonella new tricks to fight cancer

Teaching self-destructing Salmonella new tricks to fight cancer

Wei Kong and Roy Curtiss IIIArizona State University, Tempe,

Arizona, USA

Cancer is one of the leading causes of death in the world

An estimated 289,000 Americans are expected to die from lung and other three most common cancers (colon, breast and pancreatic) in 2014

http://www.lung.org

Tumor hypoxia- a major hazard of cancer therapy

http://www.mc.pref.osaka.jp

Development of novel therapies targeting hypoxic cancer cells

Bacteria have been investigated as anticancer agents:

•Salmonella •Escherichia •Clostridium•Bifidobacterium•Caulobacter•Listeria•Proteus•Streptococcus, etc.

Bacteria

Live, attenuated or genetically-engineered

Vector

Immunotherapeutic agents

Toxin

Spore

Motile S. Typhimurium exists as facultative anaerobes, allowing them to survive in both oxygenated and hypoxic conditions.

S. Typhimurium strains have been shown to preferentially accumulate in tumours.

Importantly, S. Typhimurium was shown not only to colonize large established tumors but also exhibit the property to invade and affect metastases.

Salmonella as anti-cancer therapy

Self-destructing Salmonella - Programmed regulated lysis system

For antigen delivery:

For DNA vaccine delivery:

Programmed regulated lysis system for antigen delivery

In vitro with arabinose

Plasmid

araC PBADmurA asd P22PR

Ptrc Ag

In vivo without arabinose

Chromosome

araC PBAD murA araC PBADC2 araC PBAD lacI

SCV

nucleus

Host cell

RASV multiplyand antigen synthesis

Endosome escapingRelease Ag by lysis

The

gut e

pith

eliu

m

RASV

DAP-less and Muramic acid-less Death in Host Strain with Regulated Lysis System Vector

11283

11283(pYA3681)

LB Agar LB Agar + 0.2% Arabinose

+ 50g/ml DAP

LB Agar + 0.2% Arabinose

LB Agar

Recombinant host-vector strain displaying arabinose-dependent growth and regulated cell lysis in the absence of arabinose

The genotype of 11283: asdA::araC PBAD c2 PmurA::araC PBAD murA (wza-wcaM) relA::araC PBAD lacI TTpmiPrfc::TT araC PBAD rfcpagP::Ptrc lpxE

pYA3681 araC PBAD regulated murA+ asdA+

Self-destructing Salmonella for DNA vaccine delivery- Vector

The synthesis of EGFP from pYA4545 harboring EGFP gene in INT-407 and Vero cell line

5’-ggggactttcc ggggactttcc tccccacgcg ggggactttcc gccacgggc ggggactttcc ggggactttcc

pYA4685 (pYA4545-EGFP)

INT-407

Vero

DTS (II)pYA4272

(pYA3650-EGFP)

Genotype of the ideal RASV strain: asdA::TT araC PBAD c2 PmurA::TT araC PBAD murArelA (wza-wcaM) pmi pagP::Ptrc lpxE fljBfliCendA sifAPhilA::Ptrc lacO hilAtlpA sseL

Self-destructing Salmonella for DNA vaccine delivery- Strain

Importin family members

RAN

Transcription factors

RASV harboring DNA vaccine vector pYA4545

∆tlpA∆sseL

PhilA :: Ptrc lacOhilA (Hyper-invasive)

Apoptosis/pyroptosis

∆sifA

Salmonella cell lysis

∆asdA∆PmurA

pYA4545

Host Cell

SCV

nucleus

NLS

Programmed Salmonella regulated lysis system delivering antigens or DNA vaccine induced mucosal and systemic antibody and T-cell

responses protective immunity against various diseases

Teaching self-destructing Salmonella new tricks to fight cancer

Exhibit diminished toxicity of lipid A to lessen inflammatory responses.

Rapid release of vacuolar Salmonella from the endosome to increase the efficacy of delivery and expression of a DNA vaccine.

Allow in vivo maximized Salmonella localization in tumors.

11371 (Ptar::Ptrc lacOtar)3761 Chemo attractants: 100 μM Aspartate

3.0 cm 4.3 cm

A. Bacterium swimming test on soft agar plate

113713761

Swim

min

g D

ista

nce

(cm

)

B. Graph of swimming test result

*The data of graph were collected from 4 independent experiments, ρ < 0.05

Modulate Salmonella swimming behavior for enhanced tumor targeting and tumor homing

• ΔPtar::PtrcΔlacO tar• ΔPtsr::Ptrc ΔlacOtsr• Δtrg

Genetically modification of chemoreceptor genes*

The

ratio

of S

alm

onel

la c

olon

izati

on in

tum

or v

s. s

plee

n

3761

3761+ htrA

3761 + purA

Reduced normal tissue fitness and enhance tumor fitness by genetically modifying RASV strains

Deletion of purA gene:•eliminate the production of adenosine monophosphate (AMP)

Deletion of htrA gene: •Eliminate a bifunctional stress protein HtrA that is required by many bacterial pathogens to successfully cause infection

tumor specific fitness of indicated strains was determined in BALB/c CT-26 subcutaneous tumor bearing mice post IT injection

Day 0 Day 4 Day 8

BSG

Χ11517(pYA4545)

χ11517(pYA4545 + FasL)

11517

PmurA::TT araC PBAD murA asdA::TT araC PBAD c2 (araC PBAD)::P22 PR araBAD (wza-wcaM)pmi relA::araC PBAD lacI TT pagP::Plpp lpxE endAPhilA::PtrclacOhilA Ptar::Ptrc lacO tar Ptsr::Ptrc lacO tsr trg

Anti-tumor efficacy assay of anti-cancer Salmonella strains by intra-tumoral injection using whole body bioluminescence imaging

breast cancer subcutaneous mouse model

National Institutes of Health (NIH/NIAID)

U. S. Department of Agriculture (USDA)

Acknowledgements

Project of programmed regulated lysis system:

Dr. Roy Curtiss IIIDr. Shamaila AshrafDr. Keith AmeissDr. Xiangmin ZhangDr. Maria Dolores Juarez-Rodriguez Project of RASV anti-cancer therapeutic vaccine:

Dr. Roy Curtiss IIIMr. Matthew BrovoldMr. Jeffrey TullyMr. Lee BensonMs. Chelsea Warren

National Institutes of Health (NIH/NCI)