Acta Pharmaceutica Sinica B 2023;13(3):1014e1027

Chinese Pharmaceutical Association

Institute of Materia Medica, Chinese Academy of Medical Sciences

Acta Pharmaceutica Sinica B

w w w. e l s ev i e r. c o m / l o c a t e / a p s b
w w w. s c i e n c e d i r e c t . c o m

REVIEW

Recent advances in bacterial therapeutics based

on sense and response
Zhuo Fenga,y, Yuchen Wanga,y, Haiheng Xua, Yunfei Guoa, Wen Xiaa,
Chenxuan Zhaoa, Xiaozhi Zhaoc,*, Jinhui Wua,b,*

a
State Key Laboratory of Pharmaceutical Biotechnology, Chemistry and Biomedicine Innovation Center, Medical
School of Nanjing University, Nanjing 210093, China
b
Jiangsu Key Laboratory for Nano Technology, Nanjing University, Nanjing 210093, China
c
Department of Andrology, Drum Tower Hospital, Medical School of Nanjing University, Nanjing 210093, China

Received 7 May 2022; received in revised form 26 June 2022; accepted 18 August 2022

KEY WORDS Abstract Intelligent drug delivery is a promising strategy for cancer therapies. In recent years, with the
rapid development of synthetic biology, some properties of bacteria, such as gene operability, excellent
Cancer therapy;
tumor colonization ability, and host-independent structure, make them ideal intelligent drug carriers and
Bacterial drug delivery
system;
have attracted extensive attention. By implanting condition-responsive elements or gene circuits into bac-
Synthetic biology; teria, they can synthesize or release drugs by sensing stimuli. Therefore, compared with traditional drug
Responsive elements; delivery, the usage of bacteria for drug loading has better targeting ability and controllability, and can
Gene circuits; cope with the complex delivery environment of the body to achieve the intelligent delivery of drugs. This
Intelligential delivery review mainly introduces the development of bacterial-based drug delivery carriers, including mecha-
nisms of bacterial targeting to tumor colonization, gene deletions or mutations, environment-
responsive elements, and gene circuits. Meanwhile, we summarize the challenges and prospects faced
by bacteria in clinical research, and hope to provide ideas for clinical translation.

ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical
Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

*Corresponding authors. Tel.: þ025 83592629.

E-mail addresses: zhaoxz@nju.edu.cn (Xiaozhi Zhao), wuj@nju.edu.cn (Jinhui Wu).
y
These authors made equal contributions to this work.
Peer review under responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.

https://doi.org/10.1016/j.apsb.2022.09.015
2211-3835 ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting
by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Bacterial therapeutics in cancer
1. Introduction
Cancer has become the second leading cause of human death1.
The development of anti-tumor drugs is urgent. In the develop-
ment history of anti-tumor drugs, conventional small molecules
were first used in tumor treatment. However, small molecules can
not only kill tumor cells but also indiscriminately attack normal
tissues, resulting in intolerable toxic reactions in patients2.
Therefore, antitumor effects that can lead to the elimination of
tumor cells without causing normal tissue damage have long been
pursued. Nanocarriers based on proteins, micelles, and liposomes3
have emerged aiming to reduce the damage to normal tissue.
Based on the characteristics of tumor and tumor microenviron-
ment (TME), nanocarriers can be modified with specific ligands to
improve the efficiency of targeting. Nonetheless, the efficiency
and accuracy of targeted delivery of nanocarriers are still unsat-
isfactory. In particular, most of the nanoparticles circulating in the
blood are usually cleared by the mononuclear phagocyte system
in vivo, which forms a strict biological barrier for the transport of
nanomedicines4. In addition, since the anti-tumor effects of most
nanoparticles are mediated by small-molecule drugs, drug resis-
tance to tumors is still inevitable5. Unlike conventional ap-
proaches to targeting cancer cells, immunotherapy is highly
specific, such as chimeric antigen receptor (CAR) T-cell therapy
and checkpoint inhibitors, because it exploits the specific recog-
nition of tumor antigens by the host immune system. However,
due to the suppressive microenvironment and low immune cell
infiltration, immunotherapy is not effective in most solid
tumors6e9. In recent studies, bacterial delivery systems combined
with chemotherapy and immunotherapy have provided an inter-
esting idea for tumor treatment and have attracted widespread
attention.
Bacteria are the oldest, the most widely distributed, and most
abundant life forms on earth, and are also closely related to humans.
Bacteria colonize various human physiological organs and participate
in the regulation of organ function after human beings are born10e12. At
the end of the nineteenth century, Dr. William B. Coley observed tumor
regression in acute Streptococcal infection of inoperable sarcoma,
which led to the study of bacterial application in cancer therapy13.
Bacteria can be used as an effective tool for cancer therapy. However,
the application of bacteria was limited because of their toxicity. With
the rapid development of synthetic biology, many strains have been
engineered to control their safety and once again entered the stage of
tumor treatment, such as Escherichia coli14e16, Salmonella
typhimurium17e19, and Listeria monocytogenes20e22. Based on the
colonization in tumor tissue, engineered bacteria can perceive the
external conditions, and responsively synthesize or release drugs to
improve the targeting delivery of drugs, reduce the damage to normal
tissues, and provide a unique idea for tumor-targeted therapies. Starting
from the mechanism of bacterial colonization targeting the tumor, this
review introduces the recent advances of the engineered bacteria based
on synthetic biology to function as a precise drug delivery carrier to
synthesize and secrete drugs in tumor tissues.
2. The mechanisms of bacterial colonization in tumor
The phenomenon that bacteria can target tumors for colonization
is important for bacteria used as drug delivery vehicles in tumor
therapy. Most of the bacteria used in tumor treatment possess
flagella, such as E. coli, S. typhimurium, Clostridium novyi-
NT23,24, and L. monocytogenes25. The bacterial flagellum plays an
essential role in bacterial colonization targeting tumors, mainly
1015
performing bacterial chemotaxis, motility, and invasion functions,
which are important for bacterial colonization in tumors26e29. In
this section, we focus on the motility of flagella and discuss the
mechanism of bacterial colonization in the tumor.
2.1. The structure of flagella
The flagella are generally composed of three adjacent sub-
structures: the basal body, hook, and helical filament, whose
length is around 20 mm30,31. The flagellar motor located in basal
body is essential for bacterial motility, and consists of five pro-
teins: MotA, MotB, FliG, FliM, and FliN respectively (Fig. 1).
Among these, MotA and MotB, as the stator, drive flagellar
rotation by conducting the passage of protons. FliG, FliM, and
FliN act as the rotor to regulate the direction of flagellar move-
ment32. MotB is physically linked to the peptidoglycan layer
through a peptidoglycan-binding domain and therefore fixes the
stator in place. In addition, MotA combines directly with the
carboxyl (C)-terminus of FliG and forms a ring of FliG molecules,
C-ring. Existing data suggest that MotA pushes the FliG so that
the flagellar motor starts rotating when protons go through the
MotA and MotB channels, which results in flagellum rotation33,34.
Research showed that the speed of bacterial flagellum can reach
18,000 rpm with protons driving35e37.
2.2. Motility
Motility is an important factor affecting the ability of bacterial
colonization in tumors. Rotation of the flagella allows rapid
movement of bacteria to evade clearance by the immune sys-
tem36e38. In addition, bacterial motility endows them with the
ability to overcome the permeation limitations of cancer chemo-
therapeutics, allowing them to distribute evenly inside the tumors.
Studies have shown that the motility of S. typhimurium and E. coli
is positively correlated with their ability to colonize tumors. By
comparing the colonization ability of Salmonella and E. coli with
different motile speeds in tumor tissue, the results showed that the
fastest moving Salmonella showed good colonization ability, at
least 80 times that of E. coli. And in Salmonella-based tumor
therapeutic, knocking out the bacteria flagellum leads to a
decrease in the therapeutic efficacy39,40.
Based on the outstanding motility of Salmonella, in situ cancer
vaccine development is available. VNP2000941 is an attenuated
Salmonella strain for cancer treatment with superb motility and
colonization targeting the tumor. The accumulation in the tumor
was 1000 times higher than in the normal tissue42. Importantly, it
has been well documented that motility is significant for the
efficient distribution and accumulation of VNP20009 in tumor43.
The antigen-presenting cells (APCs) in the TME are usually in a
dysfunctional state6,44, while a large number of functioning APCs
are present around the tumor45. Based on these theoretical foun-
dations, our research group46 constructed a motile bacterium that
can absorb antigens. Since most tumor antigens are electronega-
tive, a layer of antigen-adsorbing cationic polymer nanoparticles
is coated on VNP20009 to absorb tumor antigens released by
radiotherapy (Fig. 2). Then, using the bacterial motility,
VNP20009 can transport the antigens to peritumoral functioning
APCs, thereby causing a strong adaptive immune response and
forming an immune memory. When CT26 tumors are re-
challenged, tumor growth was significantly inhibited. Overall,
our work exploits the motility of bacteria to increase the radio-
immunotherapy effect.
1016
Figure 1 The structure of flagellum. Bacteria rotate clockwise
(CW) or counterclockwise (CCW) by adjusting different structural
patterns. When MtoA is located outside the C ring, the flagellum is in
a clockwise rotation. When MtoA is located inside the C ring, the
flagellum rotates counterclockwise.
2.3. Chemotaxis
Bacterial chemotaxis is one of the crucial mechanisms of its
colonization in the tumor. Motility is rarely random47. A motile
bacteria can sense stimuli and alter the direction of motility to
improve its chances of migrating to a better residence35. Tumor
vasculature appears irregular, disorganized, and discontinuous48.
This unique pathological alteration at the tissue level limits the
supply of oxygen and nutrients but may provide an opportunity for
bacterial colonization. In addition, hypoxia is an important factor
in immune cell dysfunction49. Compared with the normal tissues,
the clearance of bacteria is therefore slower at the tumor site,
which provides favorable conditions for bacterial colonization. In
contrast, hypoxia is an important niche for the proliferation of
facultative anaerobes. Hypoxia enables tumor cells and immune
cells to obtain energy mainly through glycolysis and glucose up-
take at tumor sites is much higher than in normal tissues50.
Intracellular bacteria such as Salmonella and Listeria can exploit
the intermediates of glycolysis as an important carbon source for
their proliferation, while non-intracellular bacteria can use glucose
for proliferation51. Altogether, TME provides a good haven for
bacteria due to its features such as hypoxia, accumulation of
glucose and amino acids, and dysfunction of immune cells.
Zhuo Feng et al.
2.4. Invasiveness
The invasiveness refers to the capacity of bacteria to actively enter
host cells. The invasion of Salmonella also depends on flagella,
which can be regarded as bacterial tentacles that can sense the
surface of host cells and initiate the bacterial invasion of host
cells40,52. Type III secretion system (T3SS) apparatus is also an
indispensable structure for Salmonella invasion. Effectors
including SopB, SopE, SopE2, SipA, SipC, and SopA, encoded by
Salmonella pathogenicity island 1 (SPI-1) and secreted by T3SS,
can rearrange the actin cytoskeleton of host cells to drive bacterial
invasion. SopE is essential for Salmonella to invade host cells due
to its regulation of Rho-GTPases and regulate GDP/GTP nucle-
otide exchange, which resulted in depolymerizing microtubules
and eventually endocytosing Salmonella53e56. While the invasion
of Salmonella was abolished by knocking down the bacterial
flagellum and sopE genes, respectively40,53.
Based on the mechanism of invasion, Salmonella could be
exploited for intracellular drug delivery40,57, induction of tumor-
specific antigen expression58, and vaccine development59e61.
Avogadri et al.58 took advantage of Salmonella to invade mela-
noma cells that can present antigenic determinants of bacterial
origin and inducted Salmonella-specific CD4þ and CD8þT cell
production. To improve the therapeutic performance of S. typhi-
murium, tumor-bearing mice need to be vaccinated against Sal-
monella to stimulate the host immune response and generate
immune memory before intratumoral Salmonella injection. A
considerable anti-tumor effect was exhibited after intratumoral
injection of Salmonella. The mechanism of bacterial invasion can
also be used to develop tumor vaccines, Carleton et al.62 con-
structed an expression vector encoded fusion protein SopE-OVA
to introduce into Salmonella, which can deliver specific antigen
OVA to the DCs (dendritic cells) cytosol via T3SS and resulted in
successful induction of OVA specific CD8þ production.
3. Gene deletion or mutation
In bacterial-based cancer therapy, the primary problem is to
overcome the toxicity of bacteria. Gene deletion or mutation is the
best means of addressing it. Most of the attenuated strains now
used in cancer clinical research are based on the deletion and
mutation of virulence genes in the wild type, which reduces the
expression of virulence factors and also avoids biological
contamination. The genotype of VNP20009 is msbB‒ and purI‒.
The deletion of msbB leads to loss of myristoylation of lipid A and
reduces the production of TNF-a by 10,000 times41,63. Moreover,
Deletion of purI impairs adenine synthesis and renders bacteria
unable to survive without exogenous purine supplementation.
Compared with other strains, VNP20009 did not happen any side
effects in higher doses of intravenous injection64. In conclusion,
the safety profile of Salmonella has been significantly improved.
Gene deletion or mutation to enhance bacteria’s original
properties can also be used to treat tumors. Various types of cells
in TME need to synthesize a large number of nutrients for pro-
liferation, such as proteins, lipids, and nucleic acids. Therefore,
the metabolism of amino acids at the tumor site is extremely
vigorous, resulting in the accumulation of a large number of
biological ammonia65e67. L-Arginine is essential for T cell pro-
liferation, and increasing the concentration at the tumor site can
prolong T cell survival and proliferation and enhance the anti-
Bacterial therapeutics in cancer 1017

Figure 2 (A) Using bacterial motility, VNP20009 can transport antigens to functional peritumoral APCs and increase the radio-
immunotherapy. (B) Representative photographs of 32 days after corresponding treatment in CT26 tumor-bearing mice. (C, D) Average tu-
mors growth curves of primary and secondary CT26 tumors. The radiotherapy (RT) combined with polyamidoamine dendrimer (NPþ, positively
charged nanoparticles), dead NPþ-coated VNP20009 (dBþ), toll-like receptor agonists (CpG) and NPþ-coated VNP20009 (Bþ), respectively in
(B), (C) and (D). Reprinted with the permission from Ref. 46. Copyright ª 2022 Springer Nature.

tumor effect68. Since E. coli can convert ammonia to L-arginine
with a low efficiency69,70, Canale et al.71 upgraded the original
gene circuit to improve the production of L-arginine by E. coli.
They first deleted the gene encoding the arginine repressor argi-
nine repressor (ArgR), and then integrated the gene ArgAfbr,
which encodes a feedback-resistant mutated version of the argi-
nine synthase enzyme ArgA, into the bacterial genome. In this
way, engineered bacteria produced arginine levels at least 150
times higher than non-engineered bacteria. When the engineered
bacteria were injected intratumorally and combined with PD-L1
antibody, the number of CD4þ and CD8þ T cells in the tumor
tissue is increased and immunological memory is formed, which
inhibits tumor growth and metastasis. In addition, this method was
used to reduce intestinal ammonia absorption, improving hyper-
ammonemia and survival in mice72. In conclusion, attenuated
bacteria based on gene deletion or mutation have been widely
used in tumor treatment (Table 114,19,23,27,41,63,73e83).
4. The condition-responsive element
With the methods of synthetic biology, researchers can implant
condition-responsive gene elements into bacteria that endow them
with the capacity to sense external conditions. Bacteria start to
synthesize or release drugs via responsive gene elements after
sensing the conditions, which achieve specificity and controlla-
bility of drug delivery. According to the different response con-
ditions, the condition-responsive bacteria used in tumor therapy
can be divided into two categories, namely TME-responsive
bacteria and exogenous inducer-responsive bacteria (Fig. 3).
1018
4.1. The TME-responsive element
TME is an important reason for the difficulty of tumor immuno-
therapy. However, the characteristics of TME are more specific
such as hypoxic niche, glucose concentration, and acidic micro-
environment. Researchers make use of these characteristics as the
stimulation conditions for condition-responsive genetic elements
that allow bacteria to initiate drug synthesis or release after
reaching the TME, which can be reduced the off-target phenom-
ena in drug delivery and improved the accuracy of drug delivery.
4.1.1. The hypoxia-response element
The hypoxic niche is one of the obvious characteristics dis-
tinguishing tumor tissues from normal tissues and can become a
potential target for cancer therapy. The concentration of oxygen in
tissues is determined by both the rate of diffusion and the rate of
consumption of oxygen, and most tissue oxygen concentrations
are around 2%e9% which is much lower than 2% in solid tumors
because of the abnormal tissue density and metabolism of tumor
cells49,50,84. Conversely, researchers can design hypoxia-
responsive gene elements to control drug delivery and bacterial
toxicity in bacterial-based cancer therapy, thereby achieving the
goal of reducing side effects and accurate therapeutic14,17,76,85.
Facultative anaerobes such as S. typhimurium and E. coli can
be good carriers for cancer therapy due to intratumor hypoxia86.
Several studies have reported on the construction of hypoxia-
response bacterial vectors.
It has been demonstrated that aspartate b-semialdehyde de-
hydrogenase, encoded by the asd gene, engages in the biosynthetic
pathway for the synthesis of diaminopimelic acid (DAP), which is
an essential component of the peptidoglycan of the cell wall of
Gram-negative bacteria87. In the asd gene deletion SL7207, an
attenuated Salmonella strain for tumor targeting treatment, Yu
et al.76 reconstructed asd gene under the control of the hypoxia-
responsive promoter fnrS, while the transcription of antisense
asd was under the control of an aerobic promoter PsodA. In this
way, any leakage of asd expression is prevented under aerobic
conditions. If asd is not transcribed and DAP is not supplied in the
environment, SL7207 will not survive in normal tissues, reducing
the damage to the body. Bacteria can achieve in situ drug delivery
by perceiving hypoxic conditions. Ryan et al.85 placed the gene
encoding a cytotoxic protein Hemolysin E (HlyE) under the
regulation of hypoxic inducible promoter fnrS and introduced into
Salmonella (Fig. 3A). When the engineered bacteria enter the
TME, under the stimulation of hypoxic conditions, they begin to
secrete HlyE In 4T1tumour-bearing mice, the engineered Salmo-
nella results in a significant increase in tumor necrosis, and the
tumor growth is reduced by w50% compared with the control
group.
4.1.2. The acidic-responsive element
The unique metabolic pattern of tumor cells is closely related to
the low pH of the TME. Glycolysis increases lactate accumulation
and maintains the pH of the TME at 5.8e6.6, showing weakly
acidic, which is regarded as an important factor of immune
dysfunction88,89. Nonetheless, lower pH still confers the speci-
ficity to TME, and this property can therefore be exploited to
develop tumor-specific drug delivery systems. Flentie et al.90
designed a promoterless transposon reporter containing bacterial
luciferase. By co-culturing Salmonella containing transposon
insertion mutations with melanoma or colon cancer cells, the
bacterial gene expression library was analyzed, and the pH-
Zhuo Feng et al.
responsive STM1787 gene was screened. Then they constructed
the plasmid expressing Shiga toxin under the regulation of
STM1787 promoter to make engineered bacteria release toxin
proteins after reaching the TME. The engineered Salmonella
presented a significant and specific antitumor effect in vitro and
in vivo. Additionally, Qing et al.16developed a pH-responsive
therapeutic platform in attenuated E. coli. They placed the gene
of cytolysin A (ClyA) under the control of the adiA promoter that
is sensitive to acid responsiveness which resulted in releasing
specifically therapeutic protein after sensing the acidic condition
(Fig. 3B). In CT26 tumor-bearing mice, this strategy greatly
inhibited tumor progression and prevent tumor metastasis.
4.1.3. The glucose-responsive element
Even under aerobic conditions, tumor cells and most activated
immune cells at tumor sites mainly supply energy through
glycolysis, a phenomenon known as the Warburg effect68,91e94.
Glycolysis results in rapid depletion of glucose at the tumor site,
so the targeting of drug delivery can also be improved by engi-
neered bacteria that can sense glucose concentration95. Therefore,
Panteli et al.96 invented a drug delivery carrier based on E. coli
responsive to the concentration of glucose. They constructed a
vector plasmid expressing a fusion protein Trz1 which combines
the periplasmic domain of chemotactic receptor Trg and the
cytoplasmic domain of osmoporin sensor EnvZ, to endow bacteria
to sense glucose concentration, and then induce expression GFP
through the osmoporin promoter POmpC (Fig. 3C). They propose
a way to initiate drug delivery by sensing the changes of glucose
concentration in TME.
In conclusion, researchers can take advantage of the specificity
of TME to control the drug delivery, which shows the potential for
TME-responsive bacteria in tumor-specific therapeutic and has
good application prospects.
4.2. The exogenous inducer-responsive element
Different from the TME-responsive element, the exogenous
inducer-responsive element performs better initiative and
controllability and achieves precise drug delivery by artificially
creating response conditions. Here we divide exogenous inducers
into chemical inducers and physical inducers to introduce their
application in tumor therapy.
4.2.1. Exogenous chemical inducer-responsive element
In a sequence of symbolic research published in 1961, Jacob and
Monod proposed a profound theory of gene regulation: the operon
model97e99. The operons, a small sequence of DNA (26bp), are
the most important form of gene expression in prokaryotes, which
are binding sites for regulatory proteins on DNA molecules to
regulate the initiation of transcription on adjacent promoters. The
operons determine whether the RNA polymerase can make contact
with the promoter on the DNA sequence, thereby moving along
the DNA molecule to initiate RNA transcription. Gene regulatory
elements composed of operons are involved in the expression of
most prokaryotic genes. In bacterial-based cancer therapeutic,
most of the expression elements introduced into bacteria are
induced and expressed by exogenous inducers, which can strictly
control the expression and delivery of drugs. Common exogenous
inducers that used in drug delivery include L-arabinose85,100e107,
Isopropyl-beta-D-thiogalactopyranoside108 (IPTG), tetracy-
cline109,110, and salicylic acid (ASA)111e113.
Bacterial therapeutics in cancer 1019

Table 1 Attenuated bacterial strains used for cancer therapy (modified from Ref. 73).
Bacteria Strain Mutated/deleted Phenotype description Ref.
Salmonella A1-R Dleu/Darg Impair leucine and arginine synthesis 27
typhimurium VNP20009 DmsbB/DpurI The ability to induce TNF-a production reduced by 10,000 times; 41,63
Impair adenine synthesis
SHJ2037 DrelA/DspoT Unable to produce ppGpp (a global regulator involved in bacterial 19,41
adaptation to extreme environments); reduction in bacterial
invasion
SL3261 aro- Defective in aromatic amino-acid biosynthesis 74
MvP728 DpurD/DhtrA Defective in purine biosynthesis and heat-shock protein production 75
in response to stress stimuli
YB1; ST8 Dasd Defective in DAP (in diaminopimelic acid) synthesis, results in 76
bacterial lysis bacterial lysis in the absence of exogenous DAP
SB824 DsptP Increased bacterial inflammatory response 77
Listeria DP-L4027 DLLO (hly) Defective phagolysosome release 78
monocytogenes DP-L4029 DactA Defective surface-bound ActA polypeptide, constitutive LLO 79
activity at physiologic pH
DP-L4364 DlplA Defective in bacterial invasion of nonphagocytic cells, limited the 80
capacity to bacterial proliferation in vivo
CRS207 DactA/DinlB Impaired the capacity about cell-to-cell spread and invasion of
nonphagocytic cells
DP-L4405 DinlA Defective InlA-mediated invasion 81
Escherichia coli SYNB1891 DthyA/DdapA Impaired thymidylate and DAP (diaminopimelic acid) synthesis 14
SYNB1020 DthyA Impair thymidylate synthesis 71,72
MG1655 rph-1 Impair pyrimidine utilization 82,83
ECN1917 ArgRe/ArgAfbr Increase the expression of L-arginine 71
Clostridium novyi Remove the lethal toxin gene 23

Nguyen et al.101 constructed a pBAD vector plasmid expressing
ClyA and introduced it into Salmonella (Fig. 3D). After adminis-
tering 60 mg/day of arabinose through the tail vein for four
consecutive days, significant anti-tumor effects were observed in
CT26 tumor-bearing mice, which were positively correlated with the
concentration of arabinose. In another study, Danino108 et al.
invented a visualization of E. coli for tumor diagnosis. They Inte-
grated the LuxCDABE gene cassette into an IPTG-inducible
expression vector and fluorescent gene expression was induced by
intravenous administration of IPTG. In the mouse colorectal cancer
metastasis model, the bacterial fluorescent signal was detected in
about 88% of the metastases, as small as 1 mm in diameter. In a recent
study, Cheng114 et al. constructed IPTG-induced plasmids expressing
fusion proteins ClyA-SpC and Cly-SnC catchers into E. coli. After
IPTG induction and ultracentrifugation, outer membrane vesicles
(OMVs) are obtained and their surface contained ClyA-SpC and Cly-
SnC catchers, while SpT- or SnT-labeled antigens can be coupled to
the surface of OMVs by forming an amide bond between the catcher
and the tag. Engineered OMVs loaded with different tumor antigens
reduce pulmonary melanoma metastasis and inhibit subcutaneous
colorectal cancer growth.
Compared with L-arabinose and IPTG, tetracycline is an ideal
chemical inducer, which can achieve the induction effect at a low
dose, and its metabolic cycle is sufficient to achieve the thera-
peutic effect of the drug. The tetracycline-responsive expression
element contains two promoters, tetA, and tetR, respectively115.
Inspired by this, Jiang et al.109 regulate the expression of ClyA
and bioluminescent reporter genes via two promoters, respec-
tively. In the mouse CT26 tumor-bearing model, intravenous
administration of tetracycline induced Salmonella at the tumor site
to deliver ClyA, and the phenomenon of Salmonella tumor colo-
nization was confirmed by fluorescence signal.
Although tetracycline has a good induction efficiency, it can
inevitably cause body damage at high doses, and it is not suitable
for clinical research. ASA-responsive regulatory factors NahR116
play a role in controlling the naphthalene degradation pathway
in Pseudomonas putida. NahR and target promoter Psal or Pnah
can be applied to control heterologous gene expression. Leventhal
et al.14 utilized an ASA-inducible vector to achieve the expression
of cyclic di-AMP (CDA) synthetases. Like tetracycline, ASA also
has a long metabolic cycle, which is sufficient to induce the
expression of target proteins. Besides, ASA shows a better safety
profile due to the absence of high-dose toxicity.
4.2.2. Exogenous physical inducer-responsive element
In addition to the above chemical inducers, bacterial drug carriers
responsive to radiotherapy117e119 and ultrasound120 have also
been developed. Unlike chemical inducers, physical inducers do
not need to care about the metabolism of inducers in vivo.
Based on radiotherapy-responsive bacterial drug delivery ve-
hicles, the RecA promoter plays a vital role, which endows bac-
teria to perceive the DNA damage caused by radiotherapy, and
then start the expression of the target protein. Ganai et al.117
engineered VNP20009 to secrete TNF-related apoptosis-
inducing ligand (TRAIL) under the control of the radiation-
inducible RecA promoter (Fig. 3E). Under the induction of g ra-
diation as low as 2Gy, the expression of TRAIL can be induced to
kill tumor cells. Experimental results show that the progress of
breast tumors is significantly delayed, and the death rate has been
reduced by 76%.
Compared with radiotherapy, ultrasound can achieve the pur-
pose of inducing drug expression more simply and effectively and
has almost no damage to the body. The ultrasound-induced drug
delivery mainly depends on heat generation, and the expression of
the purpose genes is regulated by controlling the temperature. Abedi
et al.120 put the GFP gene under the control of the pL/pR thermal
induction promoters (Fig. 3F), whose function is regulated by the
TcI42 repressor. Elevated temperature induced by ultrasound can
1020 Zhuo Feng et al.

Figure 3 The condition-responsive element. It consists of two parts, TME-responsive and exogenous inducer-responsive element. (A) The
expression of cytotoxic protein Hemolysin E (HlyE) under the regulation of hypoxic inducible promoter fnrS. (B) The gene of cytolysin A (ClyA)
under the control of the adiA promoter. (C) The expression of GFP under the regulation of the osmoporin promoter POmpC. (D) The expression of
ClyA after IPTG induction. (E) TNF-related apoptosis-inducing ligand (TRAIL) under the control of the radiation-inducible RecA promoter. (F)
The expression of GFP under the control of the pL/pR thermal induction promoters.

relieve the repression with TcI42 and achieve target protein
expression. This strategy proposes a novel ultrasound-responsive
bacterial drug delivery approach. Interestingly, the temperature-
responsive element is also used to prevent environmental pollu-
tion caused by the excretion of microbial drugs in the body121,122.
5. Gene circuit
Gene circuits are based on condition-responsive elements. By
connecting multiple genetic elements in series and parallel from
multiple response elements or in combination with other synthetic
biology methods, it is possible to cope with a more complex
physiological environment of the body, achieve precise drug de-
livery or reduce side effects. Therefore, engineered bacteria based
on gene circuits can realize intelligent drug delivery. Here, ac-
cording to the different types of gene circuits, we mainly introduce
self-lysis, hypoxia-responsive, exogenous chemical inducer-
responsive, and ultrasound-responsive gene circuits (Fig. 4).
5.1. Self-lysis
The bacterial self-lysis provides a new idea and method for drug
delivery. The conditions-responsive element satisfies the purpose
of specific drug delivery to a certain extent, but the delivery effect
of some drug molecules is insufficient. These molecules tend to
have the characteristics such as large molecular weight, difficulty
to secrete, and poor performance to cross cell membranes. Based
on the phenomenon123 that phage invasion of bacteria will cause
bacterial lysis and death, the researchers constructed a self-lysis
gene circuit by combining phage lysis genes with condition-
responsive elements. The bacteria possessed the self-lysis gene
circuit and can be lysed to release the drug, which on the one hand
improves the delivery efficiency of this part of the drug, and on the
other hand, limits the potential danger caused by bacterial
proliferation.
5.1.1. Quorum-sensing lysis
The phenomenon of quorum sensing was first discovered in a
marine luminescent bacterium Vibrio Fischer, and it is a
physiological behavior that regulates gene expression according to
the density of bacteria. The researchers constructed gene circuits
based on this phenomenon to confer quorum-sensing capabilities
to a variety of other bacteria. The autoinducer is N-acyl-homo-
serine lactone (AHL) in quorum-sensing gene circuits. The
regulation of gene expression mediated by AHL includes the
transcriptional regulator LuxR and the AHL synthase LuxI. AHL
is catalyzed by LuxI, and combines with LuxR to form a complex,
which binds to the promoter sequence of the target gene to initiate
the expression of the target protein. When the bacterial density is
low, LuxI only has a small amount of constitutive expression, and
cannot form enough AHL and LuxReAHL complexes; but when
the bacteria grow to a certain density, a large amount of AHL and
LuxReAHL complexes are produced in the bacterial population.
It promotes the expression of downstream genes in the form of
positive feedback124e126.
Taking advantage of the aforementioned quorum-sensing phe-
nomenon, Din et al.127 constructed a quorum-sensing self-lysis gene
circuit in Salmonella to achieve drug delivery (Fig. 4A). The circuit
consists of a common Luxl promoter that drives the expression of
LuxR and phage lysis gene 4X174E, which can promote drug
release in tumor tissues. Based on the above-mentioned mechanism,
AHL can spread to neighboring bacteria and provide an intercellular
synchronization mechanism. In this gene circuit, LuxI promoter
was used to regulate the expression of HlyE and GFP. In the MC26
subcutaneous tumor model, engineered Salmonella inhibited tumor
growth and prolonged survival in mice.
Quorum-sensing lysis gene circuit can also be applied to
deliver larger nanobodies. Chowdhury et al.128 constructed the
expression vector of LuxI promoter to regulate 4X174E and LuxI
based on the principle of quorum-sensing. When the bacterial
density reaches a threshold, self-lysis delivery of anti-CD47
nanobodies to block the “don’t eat me”129 signal can be
released. The phagocytosis of bone-marrow-derived macrophages
was increased by 60% in vitro, and in the A20 tumor-bearing
mouse model, increased activation of tumor-infiltrating T cells
stimulated rapid tumor regression, prevented metastasis, and
resulted in long-term survival. In addition, other researchers took
advantage of the quorum-sensing lysis gene circuit to deliver anti-
Bacterial therapeutics in cancer
PD-L1/PD-1 and anti-CTLA4 nanobodies in tumor therapy have
also been reported130.
5.1.2. The condition-responsive lysis
In addition to the self-lysis gene circuit based on quorum sensing,
the condition-responsive element can also be combined with the
self-lysis gene circuit to construct a condition-responsive self-lysis
circuit, which can realize precise drug delivery after sensing the
conditional stimuli. Raman et al.40 constructed an intracellular
self-lysis gene circuit by combining two elements that respond to
different conditions in VNP20009. They first integrated the
constitutive two-chain active caspase-3 (CT-Casp3) gene into the
pBAD plasmid and induced expression by exogenous arabinose;
meanwhile, the lysis gene 4X1174 was placed in the sseJ pro-
moter, a type of promoter that regulates the expression of Sal-
monella enterica pathogenicity island 2 (SPI-2) gene after
Salmonella invades the interior of the cell56,131,132. Compared
with the control, the results showed that the engineered Salmo-
nella could significantly inhibit the growth of liver cancer, prevent
the occurrence of lung metastasis and prolong survival in 4T-1
tumor-bearing mice. Altogether, intracellular delivery of protein
drugs can also be achieved by this strategy.
5.2. Hypoxia-responsive gene circuit
An ideal bacterial drug carrier should have dependable safety and
definite targeting. The combination of gene response elements and
gene deletion or mutation can achieve targeted drug delivery while
improving the safety of drug delivery. Leventhal et al.14 developed
an intracellular drug delivery method that mainly targets immune
cells at the tumor site. By engineering E. coli 1917, the CDA
synthase dacA gene from Listeria is placed under the control of
the hypoxia-responsive promoter fnrS, inserted into the bacterial
genome of the dapA/ and thyA/ double mutants, and finally
removed all antibiotic resistance genes, and finally removed all
antibiotic resistance genes (Fig. 4B). Due to the lack of inva-
siveness of E. coli, engineered bacteria can only be phagocytosed
by immune cells such as macrophages and DCs after entering the
tumor site, to achieve the purpose of targeting immune cells to
deliver CDA. Meanwhile, the engineered bacteria can stably
synthesize CDA under the induction of hypoxia in the tumor tis-
sue, and mutations of dapA/ and thyA/ make them unable to
survive in vivo. In a variety of mouse tumor models, the engi-
neered E. coli obtained obvious anti-tumor immunity.
5.3. Exogenous inducer-response gene circuits
5.3.1. Exogenous chemical inducer-responsive gene circuits
Although bacterial therapy has undergone extensive clinical and
preclinical research, balancing the interaction between bacteria
and the immune system is extremely important to improve
safety133. Capsular polysaccharide (CAP), tightly regulated by the
kfiC gene134, protects bacteria from various immune factors and
promotes bacterial survival and colonization in tumor
tissues135e137. After knocking out the kfiC gene (OkfiC ) in E.
coli, Harimoto et al.15 introduced a gene vector for IPTG-induced
kfiC expression to control the thickness of the CAP (Fig. 4C).
When the IPTG concentration increases, the thickness of the CAP
increases to allow bacteria to escape the attack of the immune
system; but as the IPTG concentration gradually decreases, the
expression of the CAP is inhibited, resulting in the gradual thin-
ning of the CAP, which is rapidly cleared by the host and then
1021
improve security. In addition, they also constructed a Theta toxin
(TT) expression vector under the LuxI promoter that is responsive
to AHL. In CT26 tumor-bearing mice, the engineered E. coli
significantly markedly inhibited tumor progression through the
dual induction of IPTG and AHL. In another research, Royo
et al.111 constructed an ASA-induced high-expression gene circuit.
When ASA or salicylate exists, on the one hand, NahR can pro-
mote the expression of XylS2 protein controlled by the Psal pro-
moter, and on the other hand, it can promote the XylS2 regulatory
protein to activate the Pm promoter to achieve a higher level of
target protein expression. Compared with the tetracycline-induced
expression vector, the expression efficiency of the target protein is
increased by about 20e120 times, the prodrug transformation at
the tumor site is significantly improved, and the tumor growth is
significantly inhibited.
5.3.2. The ultrasound-responsive gene circuits
The ultrasound-responsive element mentioned in Section 4.2.2 can
induce the expression of target genes simply, directly, and non-
invasively. However, the ultrasonic induction process is short-
lived, so by designing a reasonable ultrasound-responsive gene
circuit, a long-term drug release can be maintained after a short-
term induction. Abedi et al.120 constructed a gene circuit by
upgrading the ultrasound-responsive element, which achieved a
short-term ultrasound induction leading to a lasting drug release
effect. They first constructed a serine integrase Bxb1, a gene that
can recognize specific DNA sequences namely attP and attB sites,
which can be placed on either side of any DNA sequence and
mediate its inversion, resulting in a stable switch function138,139,
under the control of the pL/pR phage lambda thermally inducible
promoters. The attP and attB sites were then placed on either side
of the P7 promoter, a mesomorphic promoter that strongly drives
the expression of the gene payload without undue stress on the
cell140, to regulate the expression of immune checkpoint inhibitor
nanobodies (Fig. 4D). Thus, continuous expression of non-invasive
inducing drugs can be achieved through this gene circuit. In the
A20 tumor-bearing mouse model, the ultrasound-responsive gene
circuit can achieve long-term release of anti-CTLA4 nanobodies
after ultrasound induction, subsequently increasing T cell infiltra-
tion and inhibiting tumor growth beyond 50%.
6. Clinical challenges and prospects
Twenty years ago, the attenuated Salmonella strain VNP20009
was tested in phase I clinical trials64. Intravenous injection of
different doses of VNP20009 did not cause tumor regression in 24
patients with metastatic melanoma and one patient with renal cells
carcinoma (RCC), but the experiment verified the ability of the
bacteria to target tumors, and at a certain dose, intravenous in-
jection is safe feasible18,42. Since then, clinical research based on
bacterial cancer therapeutic has been carried out. However,
considering the past clinical translation, the application of bacteria
to the clinical treatment of tumors still faces many difficulties
(Table 22,18,20e22,64,141e145).
6.1. Safety
Although there have been no deaths in clinical trials, side effects
caused by bacteria were unavoidable, such as nausea, vomiting
and persistent bacteremia observed at the individual maximum
tolerated dose64. The security concerns of bacterial therapy go far
1022
Figure 4 The self-lysis, hypoxia-responsive, exogenous chemical inducer-responsive, and ultrasound-responsive gene circuits.
beyond that. First, in the process of genetically modifying certain
bacteria, the researchers also conferred antibiotic resistance to the
bacteria while constructing the plasmid expression vector, which
may cause the horizontal transfer of bacterial resistance. There-
fore, in the production process of engineered bacteria, eliminating
bacterial resistance to antibiotics is essential. Second, plasmids are
genetic elements independent of bacterial genomes and may be
lost when bacteria are under environmental stress. To avoid this
phenomenon, expression elements can be integrated into the
genome. Traditional pharmaceutical production requires strict
exclusion of microbial contamination. During the production
process, contaminating microorganisms can be specifically
removed by heating or filtration. However, these methods cannot
be used for the production of bacterial therapeutics. Strict pro-
duction specifications and reasonable contamination prevention
therefore need to be explored.
Eliminating the virulence of bacteria thoroughly is not advis-
able in pursuit of safety, because the virulence of bacteria is a
powerful weapon for bacteria to fight against the immune clear-
ance of the body. Once thoroughly eliminated, the bacteria will be
quickly removed and the effect of drug delivery will not be ach-
ieved43,134. Therefore, it is extremely important to balance the
virulence and safety of bacteria. Harimoto’s research in Section
5.3.1 provides valuable ideas for the clinical application of bac-
teria. In the future, we can control the virulence of bacteria on
demand via gene circuits, which can effectively balance the safety
and efficacy of bacterial therapy.
Exploiting bacterial byproducts is also a potential strategy to
improve safety, such as minicells and OMVs. Minicells are low
activity, non-nucleated, non-dividing cells, generally less than
500 nmol/L in diameter. It arises primarily as a result of mutations
in genes associated with normal bacterial division146. The bacte-
rial Min system consists of three genes: minC, minD, and minE.
Mutations in the minC or minD genes cause cells to divide un-
equally. Similar to their parent bacterial cells, Minicells also
Zhuo Feng et al.
contain peptidoglycan, ribosomes, proteins, RNA, and plasmids,
but lack chromosomal DNA. Minicells cannot grow or divide, but
still maintain other cellular activities, including ATP synthesis,
mRNA translation, transcription, and plasmid DNA translation.
Therefore, minicells can not only deliver general chemothera-
peutic drugs but also can be used to deliver gene expression
vectors. However, Minicells lack targeting, and in order to target
the drug delivery, the surface of minicells is generally modified
with bispecific antibodies147e149. Compared with Minicells,
OMVs have a smaller particle size, ranging in size from 30 to
250 nm, and are natural non-replicating particles secreted by
Gram-negative bacteria. OMVs are limited in drug delivery due to
their high immunogenicity but can be used as adjuvants for tumor
vaccine development114,150.
6.2. The ability of bacteria to colonize tumors
Intravenous or intratumoral administration is commonly used in
clinical bacteria therapeutics. In phase I clinical trial of
VNP20009, colonization of the tumor site was observed in only 2
of 25 patients who received the bacteria intravenously64. In many
studies, it has been shown that the colonization effect of the
intratumoral injection is better than that of intravenous injec-
tion71,151. For example, in another phase I clinical trial of
VNP20009, bacterial colonization of the tumor site was observed
in two of three patients by intratumoral injection18. Therefore, an
appropriate mode of administration should be selected. In addi-
tion, to increase bacterial colonization at tumor sites, reducing
bacterial clearance is another considerable strategy. Harimoto
et al.15 used synthetic biology methods to control the expression of
bacterial CAP, enhance the ability of bacteria to resist immune
clearance, and improve their colonization at tumor sites. The
number of engineered bacteria was three times that of unengi-
neered bacteria by intratumoral injection. Hence, it can also
Bacterial therapeutics in cancer 1023

reduce the clearance of engineered bacteria at the tumor site to
improve the drug delivery efficiency.
6.3. Anti-tumor effect
The antitumor response elicited by the application of engineered
bacteria alone is limited64,152. Moreover, in terms of tumor size,
human tumor tissues are much larger in volume than that of the
mouse, and the number of bacteria required to elicit a potent
antitumor response is very essential. Collectively, combination
therapy is a necessary measure. Whether the amount of loaded
drug can elicit a potent antitumor effect is a challenge for bacterial
therapeutic. It is also necessary to develop more effective anti-
cancer drugs. The gene operability of bacteria can make up for the
lack of quantity, and with this feature, we can realize the
continuous delivery of drugs. TAPET-CD, a Salmonella
VNP20009 strain expressing cytosine deaminase, can target tu-
mors and convert 5-FC into 5-FU. In a clinical study, intratumoral
injected TAPET-CD resulted in three times the concentration of 5-
FU at the tumor site compared to plasma18. This study demon-
strated that bacteria colonizing human tumors can express a large
number of functional enzymes. In addition, Bacillus Calm-
etteeGuérin (BCG) is a live attenuated strain of Mycobacterium
tuberculosis variant bovis and has been approved by the FDA to
treat high-risk, non-muscle invasive bladder cancer153. Finally,
although bacteria have a good ability to colonize tumors, not all
tumor sites are suitable for bacterial colonization. A study has
reported154 that among the seven common tumors in humans
including breast cancer, lung cancer, pancreatic cancer, colorectal
cancer, cervical cancer, osteosarcoma, and glioma, only breast
cancer and lung cancer have the largest number and variety of
bacterial colonization. This report limits the scope of application
of bacteria in cancer treatment to a certain extent but has a guiding
significance for the selection of bacteria in the study of tumor
therapy.
7. Conclusions
By means of synthetic biology, researchers can make bacteria into
vehicles that can sense conditional stimuli and synthesize or release
drugs in response. And drug delivery system based on engineered
bacteria can effectively deal with the complex delivery environ-
ment of the body, and improve the efficiency and accuracy of drug
delivery. But engineered bacteria-based drug delivery still faces
many challenges, such as stable gene circuits, efficient colonization
in the tumor, and robust efficacy. Clinical experimental data show
that engineered bacteria alone are not enough for tumor therapy. To
achieve a better treatment effect, the combination of other treat-
ment methods may achieve better results. In the future, it is
believed that with the discovery of high-efficiency anti-tumor drugs
and the steady development of synthetic biology, engineered bac-
teria can perform drug delivery as well as intelligent robots.

Table 2 Clinical trials with engineered tumor-targeting bacteria (modified from Ref. 2).
Bacteria strain Cancer type Dosage and Administration Date Ref.
VNP20009 Phase I: 24 patients with i.v. 1.5  l05‒3  l08 CFU/m2 2001 64
melanoma and 1 RCC
VNP20009 Phase I: 4 patients with i.v. 3  l08 CFU/m2 2002 141
superficial solid tumors
VNP20009 (expressing Phase I: 3 patients with head and i.t.3  l06, l  l07 or 3  l07 CFU/m2 2003 18
cytosine deaminase) neck SCC or oesophageal
adenocarcinoma
S. typhimurium (expressing Phase I: 22 patients with liver PO. l  l05‒l  l010 CFU/m2 2010
IL-2) cancer
VNP20009 (expressing L Phase I:50 patients in treating i.v. unknow 2021
-methioninase) advanced solid tumors
C. novyi-NT Phase I: 2 patients with colorectal i.v. 1  106 spores/kg (1 dose) 2006
cancer
C. novyi-NT Phase I: 5 patients with solid i.v. 1  105e1  107 spores/kg 2010
tumor malignancies
C. novyi-NT Phase I: 24 patients with solid i.t. 1  106e3  106 spores/kg 2013 142
tumor malignancies
C. novyi-NT Phase Ib: 24 patients with solid i.t. 3  104 spores/kg 2018
tumor malignancies
Listeria (expressing Phase II: 93 patients with i.v. 1  109 CFU/dosage 2011 21,22
mesothelin) metastatic pancreatic cancer
Listeria (expressing Phase II: 303 patients with 2nd- i.v. 1  109 CFU/dosage 2013 22,143,144
mesothelin) line, 3rd-line and greater
metastatic pancreatic cancer
Listeria (expressing Phase I: 60 patients with solid i.v. 1  109 CFU/dosage 2014 20,145
mesothelin) tumor malignancies
Listeria Phase I/II: patients with non- i.v. 1  108, 5  108 CFU/dosage 2019
small cell lung cancer
E. coil (expressing CDA) Phase I: patients with metastatic i.t. 3  105e1  106 CFU/dosage 2019
solid neoplasm
CFU, colony-forming units; i.v., intravenous; i.t., intratumoural; PO., per os (oral administration).
1024
Acknowledgments
This paper was supported by National Natural Science Foundation
of China (Nos. 32171372 and 31872755); Jiangsu Outstanding
Youth Funding (BK20190007, China) and Logistics research
projects (BWS20J017, China).
Author contributions
The manuscript of this review was composed by Zhuo Feng,
Yuchen Wang, Haiheng Xu, Yunfei Guo, Wen Xia, Chenxuan
Zhao under the guidance and advice of Profs. Xiaozhi Zhao and
Jinhui Wu.
Conflicts of interest
The authors declare no conflicts of interest.
References
1. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022.
CA Cancer J Clin 2022;72:7e33.
2. Zhou S, Gravekamp C, Bermudes D, Liu K. Tumour-targeting bac-
teria engineered to fight cancer. Nat Rev Cancer 2018;18:727e43.
3. Quader S, Kataoka K. Nanomaterial-enabled cancer therapy. Mol
Ther 2017;25:1501e13.
4. Blanco E, Shen H, Ferrari M. Principles of nanoparticle design for
overcoming biological barriers to drug delivery. Nat Biotechnol
2015;33:941e51.
5. Pan H, Zheng M, Ma A, Liu L, Cai L. Cell/bacteria-based bioactive
materials for cancer immune modulation and precision therapy. Adv
Mater 2021;33. e2100241.
6. Jhunjhunwala S, Hammer C, Delamarre L. Antigen presentation in
cancer: insights into tumor immunogenicity and immune evasion.
Nat Rev Cancer 2021;21:298e312.
7. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V,
Havel JJ, et al. Cancer immunology. Mutational landscape de-
termines sensitivity to PD-1 blockade in non-small cell lung cancer.
Science 2015;348:124e8.
8. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK,
et al. Mismatch repair deficiency predicts response of solid tumors to
PD-1 blockade. Science 2017;357:409e13.
9. Mizukoshi E, Kaneko S. Immune cell therapy for hepatocellular
carcinoma. J Hematol Oncol 2019;12:52.
10. Cubillos-Ruiz A, Guo T, Sokolovska A, Miller PF, Collins JJ, Lu TK,
et al. Engineering living therapeutics with synthetic biology. Nat Rev
Drug Discov 2021;20:941e60.
11. Tamburini S, Shen N, Wu HC, Clemente JC. The microbiome in early
life: implications for health outcomes. Nat Med 2016;22:713e22.
12. Stewart CJ, Ajami NJ, O’Brien JL, Hutchinson DS, Smith DP,
Wong MC, et al. Temporal development of the gut
microbiome in early childhood from the TEDDY study. Nature
2018;562:583e8.
13. Coley WB. The treatment of malignant tumors by repeated in-
oculations of erysipelas. With a report of ten original cases. 1893.
Clin Orthop Relat Res 1991;262:3e11.
14. Leventhal DS, Sokolovska A, Li N, Plescia C, Kolodziej SA,
Gallant CW, et al. Immunotherapy with engineered bacteria by tar-
geting the STING pathway for anti-tumor immunity. Nat Commun
2020;11:2739.
15. Harimoto T, Hahn J, Chen YY, Im J, Zhang J, Hou N, et al. A
programmable encapsulation system improves delivery of therapeutic
bacteria in mice. Nat Biotechnol 2022;40:1259e69.
16. Qin W, Xu W, Wang L, Ren D, Cheng Y, Song W, et al. Bacteria-
elicited specific thrombosis utilizing acid-induced cytolysin A
Zhuo Feng et al.
expression to enable potent tumor therapy. Adv Sci (Weinh) 2022;9:
e2105086.
17. Friedlos F, Lehouritis P, Ogilvie L, Hedley D, Davies L, Bermudes D,
et al. Attenuated Salmonella targets prodrug activating enzyme
carboxypeptidase G2 to mouse melanoma and human breast and
colon carcinomas for effective suicide gene therapy. Clin Cancer Res
2008;14:4259e66.
18. Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C,
et al. Pilot trial of genetically modified, attenuated Salmonella
expressing the E. coli cytosine deaminase gene in refractory cancer
patients. Cancer Gene Ther 2003;10:737e44.
19. Zheng JH, Nguyen VH, Jiang SN, Park SH, Tan W, Hong SH, et al.
Two-step enhanced cancer immunotherapy with engineered Salmo-
nella typhimurium secreting heterologous flagellin. Sci Transl Med
2017:9.
20. Le DT, Dubenksy Jr TW, Brockstedt DG. Clinical development of
Listeria monocytogenes-based immunotherapies. Semin Oncol 2012;
39:311e22.
21. Brockstedt DG, Giedlin MA, Leong ML, Bahjat KS, Gao Y,
Luckett W, et al. Listeria-based cancer vaccines that segregate
immunogenicity from toxicity. Proc Natl Acad Sci U S A 2004;101:
13832e7.
22. Le DT, Wang-Gillam A, Picozzi V, Greten TF, Crocenzi T, Springett G,
et al. Safety and survival with GVAX pancreas prime and Listeria
monocytogenes-expressing mesothelin (CRS-207) boost vaccines for
metastatic pancreatic cancer. J Clin Oncol 2015;33:1325e33.
23. Dang LH, Bettegowda C, Huso DL, Kinzler KW, Vogelstein B.
Combination bacteriolytic therapy for the treatment of experimental
tumors. Proc Natl Acad Sci U S A 2001;98:15155e60.
24. Staedtke V, Roberts NJ, Bai RY, Zhou S. Clostridium novyi-NT in
cancer therapy. Genes Dis 2016;3:144e52.
25. Radoshevich L, Cossart P. Listeria monocytogenes: towards a com-
plete picture of its physiology and pathogenesis. Nat Rev Microbiol
2018;16:32e46.
26. Min JJ, Kim HJ, Park JH, Moon S, Jeong JH, Hong YJ, et al.
Noninvasive real-time imaging of tumors and metastases using
tumor-targeting light-emitting Escherichia coli. Mol Imaging Biol
2008;10:54e61.
27. Zhao M, Yang M, Li XM, Jiang P, Baranov E, Li S, et al. Tumor-targeting
bacterial therapy with amino acid auxotrophs of GFP-expressing Sal-
monella typhimurium. Proc Natl Acad Sci U S A 2005;102:755e60.
28. St Jean AT, Zhang M, Forbes NS. Bacterial therapies: completing the
cancer treatment toolbox. Curr Opin Biotechnol 2008;19:511e7.
29. Kasinskas RW, Forbes NS. Salmonella typhimurium lacking ribose
chemoreceptors localize in tumor quiescence and induce apoptosis.
Cancer Res 2007;67:3201e9.
30. Hu H, Santiveri M, Wadhwa N, Berg HC, Erhardt M, Taylor NMI.
Structural basis of torque generation in the bi-directional bacterial
flagellar motor. Trends Biochem Sci 2022;47:160e72.
31. Evans LD, Hughes C, Fraser GM. Building a flagellum outside the
bacterial cell. Trends Microbiol 2014;22:566e72.
32. Macnab RM. How bacteria assemble flagella. Annu Rev Microbiol
2003:57.
33. Kojima S, Blair DF. Conformational change in the stator of the
bacterial flagellar motor. Biochemistry 2001;40:13041e50.
34. Wadhwa N, Berg HC. Bacterial motility: machinery and mecha-
nisms. Nat Rev Microbiol 2022;20:161e73.
35. Jarrell KF, McBride MJ. The surprisingly diverse ways that pro-
karyotes move. Nat Rev Microbiol 2008;6:466e76.
36. Minamino T, Imada K. The bacterial flagellar motor and its structural
diversity. Trends Microbiol 2015;23:267e74.
37. Nord AL, Sowa Y, Steel BC, Lo CJ, Berry RM. Speed of the bacterial
flagellar motor near zero load depends on the number of stator units.
Proc Natl Acad Sci U S A 2017;114:11603e8.
38. Berg HC. The rotary motor of bacterial flagella. Annu Rev Biochem
2003;72:19e54.
39. Chen J, Qiao Y, Chen G, Chang C, Dong H, Tang B, et al. Salmonella
flagella confer anti-tumor immunological effect via activating
Bacterial therapeutics in cancer
Flagellin/TLR5 signalling within tumor microenvironment. Acta
Pharm Sin B 2021;11:3165e77.
40. Raman V, Van Dessel N, Hall CL, Wetherby VE, Whitney SA,
Kolewe EL, et al. Intracellular delivery of protein drugs with an
autonomously lysing bacterial system reduces tumor growth and
metastases. Nat Commun 2021;12:6116.
41. Clairmont C, Lee KC, Pike J, Ittensohn M, Low KB, Pawelek J, et al.
Biodistribution and genetic stability of the novel antitumor agent
VNP20009, a genetically modified strain of Salmonella typhimurium.
J Infect Dis 2000;181:1996e2002.
42. Luo X, Li Z, Lin S, Le T, Ittensohn M, Bermudes D, et al. Antitumor
effect of VNP20009, an attenuated Salmonella, in murine tumor
models. Oncol Res 2001;12:501e8.
43. Toley BJ, Forbes NS. Motility is critical for effective distribution and
accumulation of bacteria in tumor tissue. Integr Biol 2012;4:165e76.
44. Maier B, Leader AM, Chen ST, Tung N, Chang C, LeBerichel J,
et al. A conserved dendritic-cell regulatory program limits anti-
tumour immunity. Nature 2020;580:257e62.
45. Bell D, Chomarat P, Broyles D, Netto G, Harb GM, Lebecque S,
et al. In breast carcinoma tissue, immature dendritic cells reside
within the tumor, whereas mature dendritic cells are located in per-
itumoral areas. J Exp Med 1999;190:1417e26.
46. Wang W, Xu H, Ye Q, Tao F, Wheeldon I, Yuan A, et al. Systemic
immune responses to irradiated tumours via the transport of antigens
to the tumour periphery by injected flagellate bacteria. Nat Biomed
Eng 2022;6:44e53.
47. Szurmant H, Ordal GW. Diversity in chemotaxis mechanisms among
the bacteria and archaea. Microbiol Mol Biol Rev 2004;68:301e19.
48. Tozer GM, Kanthou C, Baguley BC. Disrupting tumour blood ves-
sels. Nat Rev Cancer 2005;5:423e35.
49. Kaymak I, Williams KS, Cantor JR, Jones RG. Immunometabolic
interplay in the tumor microenvironment. Cancer Cell 2021;39:28e37.
50. Bertout JA, Patel SA, Simon MC. The impact of O2 availability on
human cancer. Nat Rev Cancer 2008;8:967e75.
51. Jiang L, Wang P, Song X, Zhang H, Ma S, Wang J, et al. Salmonella
typhimurium reprograms macrophage metabolism via T3SS effector
SopE2 to promote intracellular replication and virulence. Nat Com-
mun 2021;12:879.
52. Misselwitz B, Barrett N, Kreibich S, Vonaesch P, Andritschke D,
Rout S, et al. Near surface swimming of Salmonella typhimurium
explains target-site selection and cooperative invasion. PLoS Pathog
2012;8. e1002810.
53. Kubori T, Galan JE. Temporal regulation of Salmonella virulence
effector function by proteasome-dependent protein degradation. Cell
2003;115:333e42.
54. Hardt WD, Chen LM, Schuebel KE, Bustelo XR, Galan JE. S. typhi-
murium encodes an activator of Rho GTPases that induces membrane
ruffling and nuclear responses in host cells. Cell 1998;93:815e26.
55. LaRock DL, Chaudhary A, Miller SI. Salmonellae interactions with
host processes. Nat Rev Microbiol 2015;13:191e205.
56. Galan JE. Salmonella typhimurium and inflammation: a pathogen-
centric affair. Nat Rev Microbiol 2021;19:716e25.
57. Fensterle J, Bergmann B, Yone CL, Hotz C, Meyer SR, Spreng S, et al.
Cancer immunotherapy based on recombinant Salmonella enterica
serovar typhimurium aroA strains secreting prostate-specific antigen and
cholera toxin subunit B. Cancer Gene Ther 2008;15:85e93.
58. Avogadri F, Martinoli C, Petrovska L, Chiodoni C, Transidico P,
Bronte V, et al. Cancer immunotherapy based on killing of Salmo-
nella-infected tumor cells. Cancer Res 2005;65:3920e7.
59. Nishikawa H, Sato E, Briones G, Chen LM, Matsuo M, Nagata Y, et al.
In vivo antigen delivery by a Salmonella typhimurium type III secretion
system for therapeutic cancer vaccines. J Clin Invest 2006;116:1946e54.
60. Rüssmann H, Shams H, Poblete F, Fu Y, Galán JE, Donis RO. De-
livery of epitopes by the Salmonella type III secretion system for
vaccine development. Science 1998;281:565e8.
61. Moreno M, Kramer MG, Yim L, Chabalgoity JA. Salmonella as live
trojan horse for vaccine development and cancer gene therapy. Curr
Gene Ther 2010;10:56e76.
1025
62. Carleton HA, Lara-Tejero M, Liu X, Galan JE. Engineering the type
III secretion system in non-replicating bacterial minicells for antigen
delivery. Nat Commun 2013;4:1590.
63. Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M, et al. Lipid A
mutant Salmonella with suppressed virulence and TNFalpha induc-
tion retain tumor-targeting in vivo. Nat Biotechnol 1999;17:37e41.
64. Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP,
Schwartzentruber DJ, et al. Phase I study of the intravenous
administration of attenuated Salmonella typhimurium to patients with
metastatic melanoma. J Clin Oncol 2002;20:142e52.
65. Cantor JR, Sabatini DM. Cancer cell metabolism: one hallmark,
many faces. Cancer Discovery 2012;2:881e98.
66. O’Sullivan D, Sanin DE, Pearce EJ, Pearce EL. Metabolic in-
terventions in the immune response to cancer. Nat Rev Immunol
2019;19:324e35.
67. Pearce EL, Poffenberger MC, Chang CH, Jones RG. Fueling im-
munity: insights into metabolism and lymphocyte function. Science
2013;342:1242454.
68. Geiger R, Rieckmann JC, Wolf T, Basso C, Feng Y, Fuhrer T, et al.
L-Arginine modulates T cell metabolism and enhances survival and
anti-tumor activity. Cell 2016;167:829e42.
69. Yang D, Park SY, Park YS, Eun H, Lee SY. Metabolic engineering of
Escherichia coli for natural product biosynthesis. Trends Biotechnol
2020;38:745e65.
70. Ginesy M, Belotserkovsky J, Enman J, Isaksson L, Rova U. Meta-
bolic engineering of Escherichia coli for enhanced arginine biosyn-
thesis. Microb Cell Factories 2015;14:29.
71. Canale FP, Basso C, Antonini G, Perotti M, Li N, Sokolovska A,
et al. Metabolic modulation of tumours with engineered bacteria for
immunotherapy. Nature 2021;598:662e6.
72. Kurtz CB, Millet YA, Puurunen MK, Perreault M, Charbonneau MR,
Isabella VM, et al. An engineered E. coli Nissle improves hyper-
ammonemia and survival in mice and shows dose-dependent expo-
sure in healthy humans. Sci Transl Med 2019:11.
73. Duong MT, Qin Y, You SH, Min JJ. Bacteria-cancer interactions:
bacteria-based cancer therapy. Exp Mol Med 2019;51:1e15.
74. Branger CG, Torres-Escobar A, Sun W, Perry R, Fetherston J,
Roland KL, et al. Oral vaccination with LcrV from Yersinia pestis
KIM delivered by live attenuated Salmonella enterica serovar
typhimurium elicits a protective immune response against challenge
with Yersinia pseudotuberculosis and Yersinia enterocolitica. Vaccine
2009;27:5363e70.
75. Xiong G, Husseiny MI, Song L, Erdreich-Epstein A, Shackleford GM,
Seeger RC, et al. Novel cancer vaccine based on genes of Salmonella
pathogenicity island 2. Int J Cancer 2010;126:2622e34.
76. Yu B, Yang M, Shi L, Yao Y, Jiang Q, Li X, et al. Explicit hypoxia
targeting with tumor suppression by creating an “obligate” anaerobic
Salmonella typhimurium strain. Sci Rep 2012;2:436.
77. Jellbauer S, Panthel K, Hetrodt JH, Russmann H. CD8 T-cell in-
duction against vascular endothelial growth factor receptor 2 by
Salmonella for vaccination purposes against a murine melanoma.
PLoS One 2012;7. e34214.
78. Jones S, Portnoy DA. Characterization of Listeria monocytogenes
pathogenesis in a strain expressing perfringolysin O in place of lis-
teriolysin O. Infect Immun 1994;62:5608e13.
79. Camilli A, Tilney LG, Portnoy DA. Dual roles of plcA in Listeria
monocytogenes pathogenesis. Mol Microbiol 1993;8:143e57.
80. O’Riordan M, Moors MA, Portnoy DA. Listeria intracellular growth and
virulence require host-derived lipoic acid. Science 2003;302:462e4.
81. Bakardjiev AI, Stacy BA, Fisher SJ, Portnoy DA. Listeriosis in the
pregnant Guinea pig: a model of vertical transmission. Infect Immun
2004;72:489e97.
82. Blattner FR, Plunkett 3rd G, Bloch CA, Perna NT, Burland V,
Riley M, et al. The complete genome sequence of Escherichia coli
K-12. Science 1997;277:1453e62.
83. Jensen KF. The Escherichia coli K-12 “wild types” W3110 and MG1655
have an rph frameshift mutation that leads to pyrimidine starvation due to
low pyrE expression levels. J Bacteriol 1993;175:3401e7.
1026
84. Krzywinska E, Stockmann C. Hypoxia, metabolism and immune cell
function. Biomedicines 2018;6:56.
85. Ryan RM, Green J, Williams PJ, Tazzyman S, Hunt S, Harmey JH,
et al. Bacterial delivery of a novel cytolysin to hypoxic areas of solid
tumors. Gene Ther 2009;16:329e39.
86. McNerney MP, Doiron KE, Ng TL, Chang TZ, Silver PA. Thera-
nostic cells: emerging clinical applications of synthetic biology. Nat
Rev Genet 2021;22:730e46.
87. Galan JE, Nakayama K, Curtiss 3rd R. Cloning and characterization
of the asd gene of Salmonella typhimurium: use in stable mainte-
nance of recombinant plasmids in Salmonella vaccine strains. Gene
1990;94:29e35.
88. Huber V, Camisaschi C, Berzi A, Ferro S, Lugini L, Triulzi T, et al.
Cancer acidity: an ultimate frontier of tumor immune escape and a novel
target of immunomodulation. Semin Cancer Biol 2017;43:74e89.
89. Singer K, Cheng WC, Kreutz M, Ho PC, Siska PJ. Immunometab-
olism in cancer at a glance. Dis Model Mech 2018:11.
90. Flentie K, Kocher B, Gammon ST, Novack DV, McKinney JS,
Piwnica-Worms D. A bioluminescent transposon reporter-trap iden-
tifies tumor-specific microenvironment-induced promoters in Sal-
monella for conditional bacterial-based tumor therapy. Cancer
Discovery 2012;2:624e37.
91. Carr EL, Kelman A, Wu GS, Gopaul R, Senkevitch E, Aghvanyan A,
et al. Glutamine uptake and metabolism are coordinately regulated
by ERK/MAPK during T lymphocyte activation. J Immunol 2010;
185:1037e44.
92. Blagih J, Coulombe F, Vincent EE, Dupuy F, Galicia-Vazquez G,
Yurchenko E, et al. The energy sensor AMPK regulates T cell
metabolic adaptation and effector responses in vivo. Immunity 2015;
42:41e54.
93. Leone RD, Powell JD. Metabolism of immune cells in cancer. Nat
Rev Cancer 2020;20:516e31.
94. Warburg O, Wind F, Negelein E. The metabolism of tumors in the
body. J Gen Physiol 1927;8:519e30.
95. Anderson KG, Stromnes IM, Greenberg PD. Obstacles posed by the
tumor microenvironment to T cell activity: a case for synergistic
therapies. Cancer Cell 2017;31:311e25.
96. Panteli JT, Forbes NS. Engineered bacteria detect spatial profiles in
glucose concentration within solid tumor cell masses. Biotechnol
Bioeng 2016;113:2474e84.
97. Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of
proteins. J Mol Biol 1961;3:318e56.
98. Monod J, Jacob F. Teleonomic mechanisms in cellular metabolism,
growth, and differentiation. Cold Spring Harbor Symp Quant Biol
1961;26:389e401.
99. English MA, Gayet RV, Collins JJ. Designing biological circuits:
synthetic biology within the operon model and beyond. Annu Rev
Biochem 2021;90:221e44.
100. Wen M, Zheng JH, Choi JM, Pei J, Li CH, Li SY, et al. Genetically-
engineered Salmonella typhimurium expressing TIMP-2 as a thera-
peutic intervention in an orthotopic glioma mouse model. Cancer
Lett 2018;433:140e6.
101. Nguyen VH, Kim HS, Ha JM, Hong Y, Choy HE, Min JJ. Genetically
engineered Salmonella typhimurium as an imageable therapeutic
probe for cancer. Cancer Res 2010;70:18e23.
102. Jeong JH, Kim K, Lim D, Jeong K, Hong Y, Nguyen VH, et al. Anti-
tumoral effect of the mitochondrial target domain of Noxa delivered
by an engineered Salmonella typhimurium. PLoS One 2014;9:
e80050.
103. St Jean AT, Swofford CA, Panteli JT, Brentzel ZJ, Forbes NS. Bac-
terial delivery of Staphylococcus aureus alpha-hemolysin causes
regression and necrosis in murine tumors. Mol Ther 2014;22:
1266e74.
104. Hong H, Lim D, Kim GJ, Park SH, Sik Kim H, Hong Y, et al. Tar-
geted deletion of the ara operon of Salmonella typhimurium enhances
L-arabinose accumulation and drives PBAD-promoted expression of
anti-cancer toxins and imaging agents. Cell Cycle 2014;13:3112e20.
Zhuo Feng et al.
105. Anderson JC, Clarke EJ, Arkin AP, Voigt CA. Environmentally
controlled invasion of cancer cells by engineered bacteria. J Mol Biol
2006;355:619e27.
106. Jiang SN, Phan TX, Nam TK, Nguyen VH, Kim HS, Bom HS, et al.
Inhibition of tumor growth and metastasis by a combination of
Escherichia coli-mediated cytolytic therapy and radiotherapy. Mol
Ther 2010;18:635e42.
107. Kim JE, Phan TX, Nguyen VH, Dinh-Vu HV, Zheng JH, Yun M,
et al. Salmonella typhimurium suppresses tumor growth via the pro-
inflammatory cytokine interleukin-1beta. Theranostics 2015;5:
1328e42.
108. Danino T, Prindle A, Kwong GA, Skalak M, Li H, Allen K, et al.
Programmable probiotics for detection of cancer in urine. Sci Transl
Med 2015;7:289.
109. Jiang SN, Park SH, Lee HJ, Zheng JH, Kim HS, Bom HS, et al.
Engineering of bacteria for the visualization of targeted delivery of a
cytolytic anticancer agent. Mol Ther 2013;21:1985e95.
110. Baron U, Freundlieb S, Gossen M, Bujard H. Co-regulation of two
gene activities by tetracycline via a bidirectional promoter. Nucleic
Acids Res 1995;23:3605e6.
111. Royo JL, Becker PD, Camacho EM, Cebolla A, Link C, Santero E,
et al. In vivo gene regulation in Salmonella spp. by a salicylate-
dependent control circuit. Nat Methods 2007;4:937e42.
112. Becker PD, Royo JL, Guzman CA. Exploitation of prokaryotic
expression systems based on the salicylate-dependent control circuit
encompassing nahR/P(sal)::xylS2 for biotechnological applications.
Bioeng Bugs 2010;1:244e51.
113. Medina C, Camacho EM, Flores A, Mesa-Pereira B, Santero E.
Improved expression systems for regulated expression in Salmonella
infecting eukaryotic cells. PLoS One 2011;6. e23055.
114. Cheng K, Zhao R, Li Y, Qi Y, Wang Y, Zhang Y, et al. Bioengineered
bacteria-derived outer membrane vesicles as a versatile antigen
display platform for tumor vaccination via Plug-and-Display tech-
nology. Nat Commun 2021;12:2041.
115. Williams KJ, Joyce G, Robertson BD. Improved mycobacterial
tetracycline inducible vectors. Plasmid 2010;64:69e73.
116. Cebolla A, Sousa C, de Lorenzo V. Rational design of a bacterial
transcriptional cascade for amplifying gene expression capacity.
Nucleic Acids Res 2001;29:759e66.
117. Ganai S, Arenas RB, Forbes NS. Tumour-targeted delivery of TRAIL
using Salmonella typhimurium enhances breast cancer survival in
mice. Br J Cancer 2009;101:1683e91.
118. Nuyts S, Van Mellaert L, Theys J, Landuyt W, Bosmans E, Anne J,
et al. Radio-responsive recA promoter significantly increases
TNFalpha production in recombinant clostridia after 2 Gy irradiation.
Gene Ther 2001;8:1197e201.
119. Nuyts S, Van Mellaert L, Theys J, Landuyt W, Lambin P, Anné J. The
use of radiation-induced bacterial promoters in anaerobic conditions:
a means to control gene expression in clostridium-mediated therapy
for cancer. Radiat Res 2001;155:716e23.
120. Abedi MH, Yao MS, Mittelstein DR, Bar-Zion A, Swift MB, Lee-
Gosselin A, et al. Ultrasound-controllable engineered bacteria for
cancer immunotherapy. Nat Commun 2022;13:1585.
121. Stirling F, Bitzan L, O’Keefe S, Redfield E, Oliver JWK, Way J, et al.
Rational design of evolutionarily stable microbial kill switches. Mol
Cell 2018;72:395.
122. Piraner DI, Abedi MH, Moser BA, Lee-Gosselin A, Shapiro MG.
Tunable thermal bioswitches for in vivo control of microbial thera-
peutics. Nat Chem Biol 2017;13:75e80.
123. Young KD, Young R. Lytic action of cloned phi X174 gene E. J Virol
1982;44:993e1002.
124. Whitehead NA, Barnard AM, Slater H, Simpson NJ, Salmond GP.
Quorum-sensing in gram-negative bacteria. FEMS Microbiol Rev
2001;25:365e404.
125. Fuqua C, Parsek MR, Greenberg EP. Regulation of gene expression
by cell-to-cell communication: acyl-homoserine lactone quorum
sensing. Annu Rev Genet 2001;35:439e68.
Bacterial therapeutics in cancer
126. Papenfort K, Bassler BL. Quorum sensing signal-response systems in
Gram-negative bacteria. Nat Rev Microbiol 2016;14:576e88.
127. Din MO, Danino T, Prindle A, Skalak M, Selimkhanov J, Allen K,
et al. Synchronized cycles of bacterial lysis for in vivo delivery.
Nature 2016;536:81e5.
128. Chowdhury S, Castro S, Coker C, Hinchliffe TE, Arpaia N,
Danino T. Programmable bacteria induce durable tumor regression
and systemic antitumor immunity. Nat Med 2019;25:1057e63.
129. Wang Y, Zhao C, Liu Y, Wang C, Jiang H, Hu Y, et al. Recent ad-
vances of tumor therapy based on the CD47eSIRPalpha axis. Mol
Pharm 2022;19:1273e93.
130. Gurbatri CR, Lia I, Vincent R, Coker C, Castro S, Treuting PM, et al.
Engineered probiotics for local tumor delivery of checkpoint
blockade nanobodies. Sci Transl Med 2020;12:eaax0876.
131. Stapels DAC, Hill PWS, Westermann AJ, Fisher RA, Thurston TL,
Saliba AE, et al. Salmonella persisters undermine host immune de-
fenses during antibiotic treatment. Science 2018;362:1156e60.
132. Personnic N, Barlocher K, Finsel I, Hilbi H. Subversion of retrograde
trafficking by translocated pathogen effectors. Trends Microbiol
2016;24:450e62.
133. Wang CZ, Kazmierczak RA, Eisenstark A. Strains, mechanism, and
perspective: Salmonella-based cancer therapy. Internet J Microbiol
2016;2016:5678702.
134. Cress BF, Englaender JA, He W, Kasper D, Linhardt RJ, Koffas MA.
Masquerading microbial pathogens: capsular polysaccharides mimic
host-tissue molecules. FEMS Microbiol Rev 2014;38:660e97.
135. Nzakizwanayo J, Kumar S, Ogilvie LA, Patel BA, Dedi C,
Macfarlane WM, et al. Disruption of Escherichia coli Nissle 1917 K5
capsule biosynthesis, through loss of distinct kfi genes, modulates
interaction with intestinal epithelial cells and impact on cell health.
PLoS One 2015;10:e0120430.
136. Griffiths G, Cook NJ, Gottfridson E, Lind T, Lidholt K, Roberts IS.
Characterization of the glycosyltransferase enzyme from the Escher-
ichia coli K5 capsule gene cluster and identification and character-
ization of the glucuronyl active site. J Biol Chem 1998;273:11752e7.
137. Leroux M, Priem B. Chaperone-assisted expression of KfiC glucur-
onyltransferase from Escherichia coli K5 leads to heparosan pro-
duction in Escherichia coli BL21 in absence of the stabilisator KfiB.
Appl Microbiol Biotechnol 2016;100:10355e61.
138. Echols H. Lysogeny: viral repression and site-specific recombination.
Annu Rev Biochem 1971;40:827e54.
139. Xu Z, Thomas L, Davies B, Chalmers R, Smith M, Brown W. Ac-
curacy and efficiency define Bxb1 integrase as the best of fifteen
candidate serine recombinases for the integration of DNA into the
human genome. BMC Biotechnol 2013;13:87.
140. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ,
Mai QA, et al. Precise and reliable gene expression via standard transcrip-
tion and translation initiation elements. Nat Methods 2013;10:354e60.
141. Heimann DM, Rosenberg SA. Continuous intravenous administration
of live genetically modified Salmonella typhimurium in patients with
metastatic melanoma. J Immunother 2003;26:179e80.
1027
142. Janku F, Zhang HH, Pezeshki A, Goel S, Murthy R, Wang-Gillam A,
et al. Intratumoral injection of clostridium novyi-NT spores in pa-
tients with treatment-refractory advanced solid tumors. Clin Cancer
Res 2021;27:96e106.
143. Laheru D, Lutz E, Burke J, Biedrzycki B, Solt S, Onners B, et al.
Allogeneic granulocyte macrophage colony-stimulating factor-
secreting tumor immunotherapy alone or in sequence with cyclo-
phosphamide for metastatic pancreatic cancer: a pilot study of safety,
feasibility, and immune activation. Clin Cancer Res 2008;14:
1455e63.
144. Le DT, Picozzi VJ, Ko AH, Wainberg ZA, Kindler H, Wang-
Gillam A, et al. Results from a phase IIb, randomized, multi-
center study of GVAX pancreas and CRS-207 compared with
chemotherapy in adults with previously treated metastatic
pancreatic adenocarcinoma (ECLIPSE study). Clin Cancer Res
2019;25:5493e502.
145. Hassan R, Alley E, Kindler H, Antonia S, Jahan T, Honarmand S,
et al. Clinical response of live-attenuated, listeria monocytogenes
expressing mesothelin (CRS-207) with chemotherapy in patients
with malignant pleural mesothelioma. Clin Cancer Res 2019;25:
5787e98.
146. Ali MK, Liu Q, Liang K, Li P, Kong Q. Bacteria-derived minicells
for cancer therapy. Cancer Lett 2020;491:11e21.
147. MacDiarmid JA, Mugridge NB, Weiss JC, Phillips L, Burn AL,
Paulin RP, et al. Bacterially derived 400 nm particles for encapsu-
lation and cancer cell targeting of chemotherapeutics. Cancer Cell
2007;11:431e45.
148. Sagnella SM, Yang L, Stubbs GE, Boslem E, Martino-Echarri E,
Smolarczyk K, et al. Cyto-immuno-therapy for cancer: a pathway
elicited by tumor-targeted, cytotoxic drug-packaged bacterially
derived nanocells. Cancer Cell 2020;37:354e70.
149. MacDiarmid JA, Amaro-Mugridge NB, Madrid-Weiss J, Sedliarou I,
Wetzel S, Kochar K, et al. Sequential treatment of drug-resistant
tumors with targeted minicells containing siRNA or a cytotoxic
drug. Nat Biotechnol 2009;27:643e51.
150. Yue Y, Xu J, Li Y, Cheng K, Feng Q, Ma X, et al. Antigen-bearing
outer membrane vesicles as tumour vaccines produced in situ by
ingested genetically engineered bacteria. Nat Biomed Eng 2022;7:
898e909.
151. Deng X, Yang W, Shao Z, Zhao Y. Genetically modified bacteria for
targeted phototherapy of tumor. Biomaterials 2021;272:120809.
152. Engelhardt R, Mackensen A, Galanos C. Phase I trial of intrave-
nously administered endotoxin (Salmonella abortus equi) in cancer
patients. Cancer Res 1991;51:2524e30.
153. Kramer MG, Masner M, Ferreira FA, Hoffman RM. Bacterial therapy
of cancer: promises, limitations, and insights for future directions.
Front Microbiol 2018;9:16.
154. Nejman D, Livyatan I, Fuks G, Gavert N, Zwang Y, Geller LT, et al.
The human tumor microbiome is composed of tumor type-specific
intracellular bacteria. Science 2020;368:973e80.