Researchers have engineering a strain of non-pathogenic bacteria that can colonize solid tumors and safely deliver potent immunotherapies

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The emerging field of synthetic biology – designing new biological components and systems – is revolutionizing medicine.

Through the genetic programming of living cells, researchers are creating engineered systems that intelligently sense and respond to diverse environments, leading to more specific and effective solutions in comparison to current molecular-based therapeutics.

At the same time, cancer immunotherapy – using the body’s immune defenses to fight cancer – has transformed cancer treatment over the past decade, but only a handful of solid tumors have responded, and systemic therapy often results in significant side effects.

Designing therapies that can induce a potent, anti-tumor immune response within a solid tumor without triggering systemic toxicity has posed a significant challenge.

Researchers at Columbia Engineering and Columbia University Irving Medical Center (CUIMC) announced today that they are addressing this challenge by engineering a strain of non-pathogenic bacteria that can colonize solid tumors in mice and safely deliver potent immunotherapies, acting as a Trojan Horse that treats tumors from within.

The therapy led not only to complete tumor regression in a mouse model of lymphoma, but also significant control of distant, uninjected tumor lesions.

Their findings are published today in Nature Medicine.

“Seeing untreated tumors respond alongside treatment of primary lesions was an unexpected discovery.

Histology image of bacteria growing within necrotic regions of lymphoma tumors (LEFT). Bacteria are programmed to undergo waves of growth and self-destruction leading to immunotherapeutic release (RIGHT). Credit: Hasty Lab/UCSD

It is the first demonstration following a bacterial cancer therapy of what is termed an ‘abscopal’ effect,” says Tal Danino, assistant professor of biomedical engineering.

“This means that we’ll be able to engineer bacteria to prime tumors locally, and then stimulate the immune system to seek out tumors and metastases that are too small to be detected with imaging or other approaches.”

The study was led in collaboration with Nicholas Arpaia, assistant professor of microbiology & immunology at CUIMC, and co-senior author on the publication.

The team combined their expertise in synthetic biology and immunology to engineer a strain of bacteria able to grow and multiply in the necrotic core of tumors.

When bacteria numbers reach a critical threshold, the non-pathogenic E. coli are then programmed to self-destruct, allowing for effective release of therapeutics and preventing them from wreaking havoc elsewhere in the body.

Subsequently, a small fraction of bacteria survive lysis and reseed the population, allowing for repeated rounds of drug delivery inside treated tumors.

The proof of concept in programming the bacteria in this way was originally developed a few years ago (Din & Danino et al. Nature 2016).

In the current study, the authors chose to release a nanobody that targets a protein called CD47.

CD47, a “don’t-eat-me” signal, protects cancer cells from being eaten by innate immune cells such as macrophages and dendritic cells.

It is found in abundance on a majority of human solid tumors and has recently become a popular therapeutic target.

“But CD47 is present elsewhere in the body, and systemic targeting of CD47 results in significant toxicity as evidenced by recent clinical trials.

To solve this issue, we engineered bacteria to target CD47 exclusively within the tumor and avoid systemic side-effects of treatment,” adds Sreyan Chowdhury, the paper’s lead author and a Ph.D. student co-mentored by Arpaia and Danino.

The combined effect of bacterially induced local inflammation within the tumor and the blockade of CD47 leads to increased ingestion, or phagocytosis of tumor cells and subsequently to enhanced activation and proliferation of T cells within the treated tumors.

The team found that treatment with their engineered bacteria not only cleared the treated tumors but also reduced the incidence of tumor metastasis in multiple models.

“Treatment with engineered bacteria led to priming of tumor-specific T cells in the tumor that then migrated systemically to also treat distant tumors,” Arpaia says.

“Without both live bugs lysing in the tumor and the CD47 nanobody payload, we were not able to observe the therapeutic or abscopal effects.”

The team is now performing further proof-of-concept tests, as well as safety and toxicology studies, of their engineered immunotherapeutic bacteria in a range of advanced solid tumor settings in mouse models.

Positive results from those tests may lead to a clinical trial in patients.

They are also collaborating with Gary Schwartz, CUIMC’s chief of hematology/oncology and deputy director of the Herbert Irving Comprehensive Cancer Center, on clinical translation aspects of their work, and have started a company to translate their promising technology to patients.


Despite the advancement in cancer treatment and detec- tion, cancer has remained a major health  problem  and one of the leading causes of deaths worldwide.

About 600,000 cancer deaths were projected to occur in the United States in 2016, out of 1.6 million newly reported cancer cases [1].

Similarly, about 3million cancer deaths were estimated to occur in China in 2015, out of 4 mil- lion reported cancer cases [2].

The conventional chemotherapeutic agents  used  for the treatment of cancer possess non-specific toxicity to- ward normal body cells.

Also the body cells that are ex- posed to chemotherapy often become resistant to drugs because of enhanced capability to repair DNA defects in cellular machinery which intervenes apoptosis.

The exposed cells increase the production of enzymes  that  cause detoxification of drug and drug delivery services [3].

These inherent complications of chemotherapy, which include drug resistive mechanism of  cells caused by chemotherapy, have caused scientists to focus on examining the potential of using bacteria and their com- pounds for anti-cancer therapy [3].

Bacteria are carcinogens and tumor promoters [4].

Bacteria produce toxins that disrupt the cellular signal thus perturbing the regulation of cell growth.

Also, they are potential tumor promoters through inducing inflammation.

Some bacteria strains notable for causing cancer are preented in Fig. 1.

The strains include Helicobacter pylori, which is associated with gastric cancer [5], Salmonella typhi which is associated with hepatobiliary carcinoma [6], Cam- pylobacter Jejuni which is associated with small intestinal lymphomas [7], Chlamydia psittaci which is associated with ocular lymphomas [8], Mycobacte-rium  tuberculosis  which is associated with lung cancer [9], and Citrobacter roden- tium, which is associated with human colorectal cancer [10].

In addition, the enzymes produceed by bacteria are potential carcinogens, such as peptidyl arginine deaminase (PAD) enzymes that are found in oral bacteria and associated with pancreatic cancer [11, 12].

Also, quorum sensing peptides, such as PhrG from Bacillus subtilis, competence stimulating peptide (CSP) from Streptococcus mitis and extracellular death factors (EDF) from Escherichia coli together with their tripeptide analogue, are reported to promote tumor cell in- vasion and angiogenesis through type I collagen extracellular matrix, which influences tumor metastasis [13].

The quorum sensing peptides down-regulate  microRNA-222  and initiate angiogenesis which promotes neovascularization and results in tumor metastasis [14].

Conversely, bacteria have shown great potential for cancer therapy.

Bacteria of many species  demonstrate  the surprising ability to invade and colonize solid tumors, which often results in neoplasm growth retard- ation, and in some instances, complete tumor clearance [15].

Different strains of Clostridia, Bifidobacteria and Salmonella are capable of colonizing the hypoxic area of the tumor and destroy the tumor cells.

Therefore, they    are potential strains for selective tumor targeting therapy [1621] (Fig. 1).

Bacteria create anti-tumor effects through the deple- tion of nutrients required for cancer  cell  metabolism [22].

The tumor tissues that  are  deoxygenated  nurture the accumulation of obligate anaerobic bacteria – which only survive in the anoxic region [23].

Observation has shown that the systemic administration of Salmonella bacteria flushed into the solid tumor through severe haemorrhaging area, the area which leads to necrotic re- gions in which bacteria proliferate [24], colonized the tumor and decreased the proliferation of the tumor.

The necrotic regions are formed because of the reduction of oxygen and nutrient supply, which leads to the breaking down of blood vessels in the hemorrhagic area.

This causes the tumor cells in the center of the tumor to die from starvation and suffocation [24]. The tumor micro- environment may be conducive to bacterial survival and
growth, as it may provide protection from the host im- mune system and nutrients [25].

Bacteria mediated tumor therapy (BMTT) has been demonstrated for centuries. However, the associated adverse side effects hinder its development.

Adequate balance between the control of infection and the therapeutic benefit of bacteria is an essential require- ment for a successful BMTT, but this can only be achieved via the heat-inactivation method [26].

Recently, state-of-art genetic  engineering  has  increased the ability to alter bacterial strains  [27]. 

Such  alteration reduces bacteria side  effects  while  increasing their therapeutic benefits. For example, the well- established bacillus  Calmette–Guerin  (BCG)  vaccine for the treatment of  human  bladder  cancer  is  argu-  ably superior to intravesical chemotherapy for  superficial disease.

The therapy is commonly used as the first-line adjuvant treatment [28].

Similarly, tumor-detecting bacteria provide a sensitive and minimally invasive method to detect tumor recurrence, monitor treatment efficacy, and identify the onset of metastatic disease [29].

For example, probiotic Escherichia coli Nissle 1917 has been used to develop the orally administered diagnosis that can noninvasively indicate  the presence of liver metastasis by producing easily detectable signals in urine [30].

Such genetic engin- eering approaches have paved the way for further development of promising bacteria-based cancer  therapy.

Bacteria as anti-cancer agents through enhancing human immunity

An important factor that applies to the spontaneous re- gression of cancer is the duality of the immune system [31]. Bacteria interact with the host as either pathogen or normal flora. The pathogenic interaction of bacteria enhances the immune system of the host in different ways.

Activating inflammasome pathways

The ΔppGpp Salmonella typhimurium strain activates inflammasome pathways by damaging the signals released from cancer cells.

This phenomenon significantly increases the amount of inflammatory cytokine IL-1β, TNF-α and Il- 18 in tumors, which results in drastic tumor growth sup- pression [32].

IL-1β is the proinflammatory cytokine that plays a pivotal role in immunity against pathogens  [33].

The IL-1β is secreted by LPS (lipopolysaccharides) during the activation of toll like receptor (TLR4) and inflamma- somes, which then causes the damage to cancer cells [32]. The cancer cells are also damaged when bacteria activate inflammasomes in BMDM, which is involved in phagocyt- osis of damaged cancer cells by macrophages [32].

Therefore, the ΔppGpp Salmonella typhimurium shows therapeutic efficacy for cancer through involving the inflammasome pathway [32].

CD4, CD25 and CD8 anti-tumor effectors T cell responses Anaerobic bacteria such as E. Coli, which are capable of engulfing the solid tumors, are indirectly involved in clearance of some tumor cells (e.g. CT26) through infectious-defense mechanism.

Once these bacteria in- vade the host, they stimulate the initiation of the defense mechanism of the host, which results in the production   of lymphocytes T cells.

The produced lymphocytes T  cells are significantly involved in anti-tumor activity.

During the induction phase of bacterial  infection,  CD8+ T cells are the only effectors responsible for tumor clear- ance; whereas, in the memory phase the clearance also involves CD8+ and CD4+T cells [34].

CD8+ T is reported to take part in the clearance of the original tumor after bacterial infection [34].

Similarly, the anti-tumor effec- tors T cells (CD4+ and CD8+) have the potential to block the formation of a new set of tumors. The CD8+ T cell is said to have the additional ability to eradicate even the already established tumors [34].

Furthermore, the lymphocytes (CD4 and CD25) and cytokines are reported to be the novel therapies for colon cancer in humans [35].

Their role in host immune response on carcinogenesis is invaluable [34, 35].

The regulatory cells (like CD4 and CD25) are capable of reducing the severity of inflamma- tory bowels and lower the risk of colon cancer [35]. In addition, the introduction of regulatory cells into chron- ically infected mice with established cancer showed the

reduction of the severity of colitis, epithelial dysplasia, and cancer [35].

The TNF-α innate immune system in bacteria-based tumor necrosis

The tumor necrosis factor (TNF- α) has the potential to damnify the vascular endothelial cells. TNF-α plays the significant part in the formation of the large haemorrha- ging area that appears within the tumor.

Systematic administering of Salmonella enterica serovar Typhimurium to mice models indicated that haemorrhaging increases the flushing of bacteria into the solid tumor resulting in necrosis [24].

Moreover, the activeness of the innate immune system of the host, especially neutrophilic granulo- cytes, is proportional to the area of necrotic.

Neutrophiles function to separate the bacteria-containing necrotic re- gion from the bacteria that migrate into the tumor from viable tumor cells. The depletion of host neutrophils in- creases the number of bacteria in the tumor and increases the ability of bacteria to migrate into vital tumor tissue [36].

Thus, the complete eradication of the established tu- mors could be attained with the increasing size of necro- sis. Similarly, the depletion of host neutrophils amplifies the bacteria-mediated tumor therapy.

Bacteria as anti-cancer agents through released substances

Some substances secreted by bacteria, such as enzymes, can inhibit the growth of tumors. Several experimental studies discovered the therapeutic potential of different substances released from bacteria for treating cancer cell lines.

Bacteriocins

Bacteriocins are cationic peptides that are synthesized by almost all groups of bacteria ribosomally.

Bacteriocins are non-immunogenic, biodegradable and contain cancer cell-specific toxicities.

The bacteriocins have the potential to serve as synergistic agents to conventional cancer drugs [37].

Cancer cell membranes predominantly carry negative charge; thus bacteriocins preferentially bind to cancer cell membranes than to the normal cell  membranes,  which are neutral in charge and selective for binding of bacteria [38].

Colicins, the bacteriocin secreted from Enterobacteria- ceae such as Escherichia coli (E.coli), are known to have anti-cancer activities against a variety of human tumor cell lines in vitro, including breast cancer, colon cancer, bone cancer and uteri cell line HeLa (human cervical adenocar- cinoma) [38].

Microcin E492, part of Colicins from Klebsi-ella pneumoniae, was found to induce apoptosis in some human malignant cell lines such as HeLa, Jurkat (T cell derived from acute T cell leukemia), RJ2.25 (a variant of Burkitt’s lymphoma), and colorectal carcinoma cells, with no effect on normal cells [17].

Pediocin isolated from Pediococcus acidilactici K2a2-3 was reported to have cyto-toxic activities against HT29 (human colon adenocarcinoma) and HeLa cell lines [39].

Similarly, Nisin (the bacteriocins from Lactobacillus lactis) possesses cytotoxic effect on MCF-7 (human breast adenocarcinoma cell line) [40], HepG2 (liver hepatocellular carcinoma) [41], and HNSCC (head and neck squamous cell carcinoma) [42], both in vitro and in vivo. Conversely, Nisin is non- toxic and safe to humans, WHO has approved it for hu- man consumption.

Furthermore, partially purified bacteriocins produced  by certain bacteria such as Pseudomonas  aeruginosa have shown anti-cancer activities [43].

Pyocin, the bacteriocin produced by more than 90% of Pseudomonas aeruginosa strains [4446], showed lethal effect on the L6OT mice fibroblast cell line [47].

Likewise, purified and partially purified pyocin S2 showed the cytotoxicity effect on tumor cell line HepG2 and Im9 (Human immunoglobulin-secreting cell line derived from multiple myeloma) with no effect on normal cell line HFFF (Human fetal foreskin fibroblast) [48].

Phenazine 1,6-di-carboxylic acid (PDC)

Multiple phenazine metabolites such as phenazine 1- carboxylic acid (PCA) and Phenazine 1,6-di-carboxylic acid (PDC) are derived from bacteria strains (e.g. Pseudo-monus aeruginosa).

The PDC phenazine was first isolated from Streptomyces species; it was demonstrated to be the potential agent for controlling metabolism and biofilm formation in Candida albicans [49, 50].

Compared to other phenazine metabolites, PDC showed a substantially broader spectrum of cytotoxicity effect towards a number of cancer cells of different origins, including HT29, HeLa, and MCF7 cell lines, with less activity on DU145 (Human prostate cancer cell lines) [51].

Bacteria as anti-cancer agents through biofilms Biofilm is a primitive form of multicellular life that provides bacteria with tolerance strength against antibiotics and host defense mechanisms [52].

Biofilms are common to opportunistic bacterial pathogens such as Salmonella tyhimurium, and they (the biofilms) are decisive in the pathogenesis of chronic infectious diseases [53].

Salmonella tyhimurium and some other pathogens are known to cause severe haemorrhage within the tumor. Once the haemorrhae is activated, it induces the produc- tion of T cells that are very significant in biofilm induc- tion [53].

Notwithstanding the etiopathogenesis of biofilms and its protective role that allows bacteria to es- cape from the host defense system [53], recent discover- ies have revealed the potential ability and efficacy of biofilms in cancer therapy.

Anti-cancer drugs cause the induction of biofilm for- mation during cancer treatment, which results in metas- tasis distraction [54, 55].

Similarly, the formation of bacteria biofilm on cancer cells during the SOS response results in metastasis disruption. T

hus bacteria biofilm shows potential usefulness in cancer treatment [56]. Bacteria biofilm can affect colon cancer development and progression through modifying cancer metabolome to produce a regulator of cellular proliferation [56].

Also, the bacterial macromolecules necessary for biofilm formation such as proteins and DNA coat cancer cells to block metastasis [57].

For example, polysaccharides released by Streptococcus agalactiae inhibit adhesion of cancer cells to endothelial cells, an essential step in cancer metastasis [58].

Furthermore, [59] demonstrates the potential application of iron oxide nanowires from a biofilm waste produced by bacteria (Mariprofundus ferroxydans) as a new multifunctional drug carrier for cancer therapy and cancer hyperthmia.

While the above hypotheses avow for the potential of bacterial biofilm in cancer therapy, the evidence is rela- tively insufficient to build-up the case.

However, the effi- cacy of  bacteria biofilm for metastasis distraction calls  for the further examination and investigation of the anti- cancer ability of bacteria biofilm.

Bacteria as a carrier for cancer therapeutic agents Apart from their direct anti-cancer effect, tumor- targeting bacteria can also be used as carriers for cancer therapeutic agents in cancer treatments. Recent studies have revealed that bacteria are capable of targeting both primary tumors and metastasis [6063].

Bacteria-mediated anti-angiogenesis therapy

The growth and metastasis of solid tumors depend on the formation of new blood vessels (angiogenesis).

Thus blocking tumor angiogenesis can be a reasonable approach to treat solid tumors. Jia et al. [64] applied the combination therapy of a low dose of Salmonella (attenu- ated, auxotrophic) and rhEndostatin in a murine model of malignant melanoma which resulted in the reduction of the tumor growth.

The therapy is argued to be safer and effective, but also economically desirable – it decreases possible side effects and lowers the therapeutic expense. Moreover, Li et al. [65] used Bifidobacterium adolescentis (non-pathogenic) as a vector for the expression of endo- statin within tumors.

Their findings showed that Bifido- bacterium adolescentis strongly inhibit the angiogenesis and significantly inhibit local tumor growth. Bifidobacterium longum efficiently delivered the anti-angiogenic protein (endostatin) to murine liver tumors and induced anti-tumor activity [66, 67].

In addition, the oral anti- angiogenic bacterial vaccines directed against vesicular endothelial growth factor receptor 2 (VEGFR-2) were

proven efficacious in animal models of malignant melan- oma, colorectal carcinoma and lung cancer [68].

The combined therapy of bacteriolytic and anti- angiogenic using tumor-targeting bacteria has also shown promising results.

Bacteria are capable of invading the poorly perfuse tumor areas (which are not accessible by systemically administered agents) using their unique metabolic features.

They cause the inhibition of angiogenisis to kill residual tumor cells, and hence significantly increase the chance for tumor eradication [69].

In addition, the anti-tumor effect can be enhanced through coadministration of tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) and endostatin [70].

The combined treatment  of  bacteria  and  viruses Oncolytic viruses (OVs) have shown positive outcomes for cancer treatment through their tumor-selective repli- cation and multi-modality attack against cancers [71]. 

The viral-mediated oncolysis cancer therapy approach is potentially more effective and less toxic than current treatment regimes.

While bacteria are arguably better at targeting the tumor, viruses are argued to possess unpre- cedented abilities to kill cancer cells.

Apart from the generic effective killing mechanisms, certain virus mu- tants have the ability to selectively kill cancer cells [72].

Bacteria have the ability to disseminate the virus inside the tumor and induce a strong immune response against tumor  antigens  [73].  In  their  experiment,  Cronin  et al.

[74]  found  that  non-pathogenic  bacteria (Escherichia coli) expressing B18R enhanced the oncolytic potential  of the vesicular stomatitis virus (VSVΔ51) to reduce tumor growth, and thus prolonged the survival of an ag- gressive tumor model.

Bacteria-based microrobot (Bacteriobot)

The bacteriorobot technique is a new innovative thera- nostic methodology of bacteria-based fabrication for tumor therapy.

The technique uses bacteria as microac- tuators and microsensors to deliver microstructures for targeting and treating solid tumors [75].

Various types of biomedical microrobots have been invented through the convergence of technologies from micro electromechanical system (MEMS) with nano- and bio-technologies  [76, 77].

In an attempt to develop microrobot therapy, Park et al. [75] encapsulated therapeutic bacteria (Salmonella typhimurium) in biocompatible/biodegradable alginate microbeads and attached flagellated bacteria (Salmonella typhimurium) on the microbead to fabricate a bacteria-based microrobot in the targeted tumor re- gion.

The bacteria-encapsulated delivery system protects the bacteria from being attacked by the immune system, which is safer than the direct inoculation  of  bacteria [78].


More information: “Programmable bacteria induce durable tumor regression and systemic antitumor immunity.” Nature Medicine (2019). DOI: 10.1038/s41591-019-0498-z

ournal information: Nature Medicine , Nature
Provided by Columbia University School of Engineering and Applied Science

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