1 Department of Biochemistry, the University of Hong Kong, Pokfulam, Hong Kong

1 Department of Biochemistry, the University of Hong Kong, Pokfulam, Hong Kong

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain

Bin Yu1#, Mei Yang1,2#, Lei Shi1, Yandan Yao2, Qinqin Jiang1, Xuefei Li3, Lei-Han Tang3, Bo-Jian Zheng4, Kwok-Yung Yuen4, David K. Smith5, Erwei Song2*, Jian-Dong Huang1*

1 Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong.

2 Department of Breast Surgery, SunYat-Sen Memorial Hospital, SunYat-Sen University, Guangzhou, China

3 Department of Physics, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China

4 Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong.

5 Labyrinth Bioinformatics and Education, Kennedy Town, Hong Kong

# Equal contribution;

* Correspondence and requests for materials should be addressed to J.D.H. () or E.S. ()

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S1

Construction of alternate asd gene expression cassettes The PW construct uses the sense PpepT promoter that contains the constitutive promoter P2 and the FNR regulated promoter P1. In the EW construct a sense FNR regulated promoter PansB and antisense promoter PSodA were used.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S2

Loading control of Fig. 1b. Proteins were loaded into SDS-PAGE gel and then stained by Coomassie blue for the indication of total protein amount.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S3

Quantitative determination of bacteria infection rate of Fig. 2a. Bacteria invasion assay was performed as described in results and methods section. After infection, breast cancer cells were collected, stained using an anti-Salmonella antibody (red) and phalloidin to indicate cancer cells (green) and observed by confocal microscopy. Bacteria invaded breast cancer cells were quantified under microscope. ***, P<0.001.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S4

The distribution of Salmonella in tumor sections. Nude mice with an MBA-MB-231 tumor of approximately 500mm3 received temporal vein injections of YB1, SL7207. Mice were euthanized at the indicated time points and Salmonella bacteria accumulation in tumor was demonstrated in tissue sections by immuno-staining. Brown color indicates bacteria and blue indicates cancer cell nuclear.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S5

Salmonella distribution in liver sections. Nude mice with an MBA-MB-231 tumor of approximately 500mm3 received temporal vein injections of YB1, SL7207. Mice were euthanized at the indicated time points and liver sections were made. Upper panel: Immuno-staining showed the bacteria distribution in liver. Brown color indicates bacteria and blue indicates cancer cell nuclear. Lower panel: H&E staining showing the liver destroyed by SL7207.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S6

YB1 colonization of the hypoxic region YB1 and PBS treated tumor bearing mice were i.p. injected with hypoxyprobe-1 before being sacrificed. Tumor samples were removed, prepared and visualized with anti-salmonella or anti-hydroxyprobe-1 antibodies as noted in Materials and Methods. The transverse sections show an overview of the hypoxic area and YB1 distribution in the tumor. PBS treated tumor-bearing mice were used as a control.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S7

Cytotoxicity of 5-FU on breast cancer cells in vitro.Breast cancer cell MDA-MB-231 cells were incubated with differentconcentrations of 5-FU (1, 10, 20, 50, 100, 200, 500, 1000g/ml). . Rates ofgrowth inhibition were determined by MTT assay at day 1, day 2 and day 3.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Fig. S8

Body weight comparison after bacterial treatment. MBA-MB-231 tumor bearing nude mice with approximately 500mm3in tumor size received injections of VNP20009, YB1, or PBS (n=5, mean±sd). Body weight was measured every 2 days. **, YB1 group vs. PBS group, P<0.01;***, P<0.001; ##, VNP20009 group vs. PBS group, P<0.01; ###, P<0.001.

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Table S1

Bacteria and plasmids in this experiment

Relevant genotype or characteristics / Ref. or source
Strain
S. typhimurium
SL7207 / hisG46 DEL407 [aroA::Tn10 {Tcs}]; wild type in this study / 1
VNP20009 / YS72; ∆purI, ∆msbB / ATCC
YB1 / SL7207; CmR; ∆asd::cm-PpepT-asd-sodA / This study
YB1-myc / SL7207; CmR; ∆asd::cm-PpepT-asd-myc-sodA / This study
YB-asd / SL7207; CmR; ∆asd / This study
YB-pw / SL7207; CmR; ∆asd::cm-PpepT-asd / This study
YB-myc-pw / SL7207; CmR; ∆asd::cm-PpepT-asd-myc / This study
YB-ew / SL7207; CmR; ∆asd::cm-PansB-asd-sodA / This study
YB-myc-ew / SL7207; CmR; ∆asd::cm-PansB-asd-myc-sodA / This study
Plasmid
pBluescript II SK / ApR; cloning vector / Stratagene
ploxp-cm-loxp / ApR, CmR ; pBSK derivative containing loxp-cm-loxp fragment / 2, 3
pSim6 / ApR; Lambda-red recombinase plasmid / 4
p705Cre-Km / KmR; cre-recombinase expressing plasmid / 2
pYB1 / ApR; CmR; pBSK derivative with cm-PpepT-asd-sodA fusion / This study
pYB1-myc / ApR; CmR; pBSK derivative with cm-PpepT-asd-myc-sodA fusion / This study
pYB-pw / ApR; CmR; pBSK derivative with cm-PpepT-asd fusion / This study
pYB-myc-pw / ApR; CmR; pBSK derivative with cm-PpepT-asd-myc fusion / This study
pYB-ew / ApR; CmR; pBSK derivative with cm-PansB-asd-sodA fusion / This study
pYB-myc-ew / ApR; CmR; pBSK derivative with cm-PansB-asd-myc-sodA fusion / This study

Explicit hypoxia targeting with tumor suppression by creating an “obligate” anaerobic Salmonella Typhimurium strain---- by Yu et al.

Supplementary Table S2

Primers used in this work

Primers / Sequence (5’-3’) / Purpose
pepT-F / ATTTGCGGCCGCGTAAACGCAACGGATGGCTGACCGC / pepT promoter
pepT-R / CCCAAGCTTCTTTTCGTGACAACATTATTAATAAG
asd-C-F / CCCAAGCTTTGGAGCGAAACCGATGAAAAATGTTGGTTTTATCGGCTGGC / asd gene with or without myc-tag
asd-C-R / CCGCTCGAGCTACGCCAACTGGCGCAGCATTCGA
asd-myc-C-R / CCGCTCGAGCTACAGATCTTCTTCGCTAATCAGTTTCTGTTCTTCCGCCAACTGGCGCAGCATTCGA
ansB-F / ATTTGCGGCCGCTTTTTTGACCTGCCTCAAACTTTGTAGATCTCCAAAATATATTCACGTTGTAAATTG / ansB promoter
ansB-R / CCCAAGCTTCGCTACGCATTATCCCTTAGCTCTGTATGGGAAATTTGACGTTAAACAATTTACAACGTGAATA
sodA-F / GACGAAAAGTACGGCATTGATAATCATTTTCAATATCATTTAATTAACTATAATGAACCAAC / sodA promoter
sodA-R / TCGAGTTGGTTCATTATAGTTAATTAAATGATATTGAAAATGATTATCAATGCCGTACTTTTCGTCTGCA
cm-F / ATTTGCGGCCGCCCGATCATATTCAATAACCCT / chloramphenicol resistance gene
cm-R / ATTTGCGGCCGCGACTAGTGAACCTCTTCGAGGG
asd-F / GTATGGTGAAGGATGCGCCACAGGATACTGGCGCGCATACACAGCACATCTCTTTGCAGGAAAAAACCGATCATATTCAATAACCCT / knock-out asd gene from SL7207 chromosome
asd-R / ATGGCGGCGCTGACGCGCCTTATCCGGCCTACAGAACCACACGCAGGCCCGATAAGCGCTGCAATAGCCGACTAGTGAACCTCTTCGAGGG
YB1-F / GCTGGCGGCGGCAGTGCGCATCATTCAGGGTTCCGCGACCGTGGCGTGTTAGGGTTTTCCCAGTCACGACGTT / knock-in oxygen response cassette to SL7207 chromosome
YB1-R / TGCAATTAGCGCATTAATCACGTCTCTATCGATACGACTGGACATGGTTTGAGCGGATAACAATTTCACACAGG
YB1-test-F / GATTCTGGTCGCTTGTCTGG / Verification of insertion
YB1-test-R / ACATTCCAGTTTGCCGACTT

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