Bacteria generally have a bad reputation, as people first think of certain strains that can cause serious illnesses like pneumonia or meningitis.
University of Cincinnati researchers have now engineered a probiotic designed to target and break down cancer cell defenses, giving therapies an easier way inside to kill tumors. The findings were recently published in the journal Advanced Healthcare Materials.
Nalinikanth Kotagiri, Ph.D., the senior author of this study, an assistant professor in UC’s James L. Winkle College of Pharmacy and a UC Cancer Center member, studies “solid cancers” or those defined as abnormal cellular growths in “solid” organs such as the breast or prostate, as opposed to leukemia, a cancer affecting the blood.
Kotagiri explains many solid cancers have an extracellular matrix made up of collagen and hyaluronic acid. The matrix forms a barrier around the cells and makes it harder for antibodies and immune cells to reach the tumors.
Shindu Thomas, the first author of this study and a graduate student in the Kotagiri lab, worked with E. coli Nissle, a bacteria that has been used as a probiotic for around 100 years and is different from E. coli strains that cause sickness. Through new technology, any protein or enzyme can be manufactured on the E. coli Nissle bacteria.
In this case, the bacteria was engineered to secrete an abundance of smaller structures called outer membrane vesicles on the outer edge of cells. The vesicles carry the same materials present on the bacteria itself, so researchers designed the bacteria to carry an enzyme that breaks down cancers’ extracellular matrix.
Kotagiri said bacteria tend to thrive in low-oxygen and immunodeficient environments, two characteristics found in solid cancers. Because of this, the specially designed bacteria are naturally drawn to these cancers.
“We took advantage of this unique feature of E.coli Nissle to home and localize into these tumors,” said Kotagiri. “And then once bacteria are lodged there, they start making nanoscale vesicles which carry the enzyme much deeper into the tumor matrix.”
After creating the new probiotic, researchers studied the bacteria’s effect on animal models of breast and colon cancer. The bacteria is delivered intravenously about four or five days prior to the cancer treatment, allowing the bacteria time to populate and break down the cancer’s defenses and prepare it to take to the treatment.
After administering the bacteria and then subsequent doses of either immunotherapy or another pharmaceutical, drugs used in targeted therapy, Kotagiri said mice survived twice as long compared to those given the cancer therapy alone. Imaging showed the bacteria and enzyme were effective at breaking down the extracellular matrix and allowing the therapy to reach the cancer cells.
The study found the bacteria affected the tumors but was not attacking healthy cells in other organs like the heart, lungs, liver and brain. Kotagiri said this shows the bacteria can be safe and will not cause infection in other parts of the body, but more research needs to be done to examine its safety in large animal models and potentially humans, particularly in immunodeficient environments.
“This always comes with a word of caution as to how you can utilize this strategy without causing any sepsis or any overt infections in the body,” he said.
Kotagiri said his lab began to look more closely at how bacterial probiotics can address biomedical problems around 2018, as there are about one to two times as many bacterial cells than human cells in your body at any given time.
“There’s bacteria in the gut, on the skin, inside your lungs, inside your mouth, even inside tumors,” Kotagiri said. “So why not take advantage of that and find interesting ways to make them a bit more proactive?”
If the engineered bacteria continues to prove itself safe and effective, Kotagiri said there are a wide variety of ways to engineer the bacteria for different uses, including potentially using the bacteria to treat disease conditions in the gut, mouth and skin. There is also potential to engineer the bacteria armed with multiple proteins and molecules to make a monotherapy platform (or therapy that uses one type of treatment) rather than just facilitating combination therapy, he said.
“So the bacteria can essentially serve as a mothership that would carry the necessary therapeutic payload to unique niches in the body and from there it’s a self-sustaining entity,” Kotagiri said. “While the possibilities are endless there are also significant challenges. We have to be good stewards of making that kind of evidence possible for the community to understand what are the limits and what can be done.”
The microenvironment surrounding tumor tissues provides a favorable niche for bacteria to inhabit. Bacteria including Bifidobacterium5, Clostridium6, Salmonella7, and Escherichia8 have been illustrated to preferentially colonize in tumors after being administrated in mice. Following bloodstream clearance mediated by inflammation, bacteria are generally entrapped in the tumor vasculature.
Obligate anaerobes such as Bifidobacterium and Clostridium survive in the anoxic region. In addition, the presence of available nutrients in necrotic tumor tissues attracts facultative anaerobes like Salmonella and Escherichia to the cancerous site via chemotaxis.
Consequently, they thrived in the hypoxic/necrotic regions of tumors to evade clearance by the immune system. Bacterial therapy is not new9, and its implementation for tumor treatment has been recently acknowledged by the advent of synthetic biology. In general, the tumor-seeking bacteria are tailored to synthesize a variety of therapeutic agents12.
By administration locally or systemically, the engineered bacteria target tumors where they reside, replicate, and continuously produce the payloads on site. It enables in situ delivery of the produced bioactive molecules to tumor site, which improves the therapeutic efficacy.
The tumor-targeting bacteria have been genetically instructed to deliver a variety of bioactive payloads, notably involving prodrugs-converted enzymes10, short hairpin RNA11, cytokines12, antigens13, antibodies14, and bacterial toxins15. These approaches generally show encouraging results. Nevertheless, they have intrinsic limitations that most of the produced payloads are restricted to proliferating cells or/and afflicted with tumor penetration. Hemolysin appears to be a promising protein payload.
It is naturally produced in bacteria and displays a pore-forming activity that lyses mammalian erythrocytes16. As illustrated previously, Staphylococcus aureus α-hemolysin (SAH) was expressed in E. coli17. Recombinant SAH was shown to penetrate into tumor tissue and eradicate cancer cells.
As a result, in situ delivery of SAH by E. coli reduced the volume of MCF7 tumor by 41%. Like SAH, hemolysin E (HlyE) is a pore-forming protein which naturally appears in E. coli18, S. enterica19, and Shigella flexneri20. HlyE is cytotoxic to cultured mammalian cells and macrophages21. It causes the formation of transmembrane pore on the host cell. The damaged cell membrane in turn induces cell apoptosis.
The application of the HlyE-mediated cell lysis for cancer treatment was investigated in the later work. As first exemplified by 4T1 tumor, the administration of HlyE-expressing S. typhimurium significantly decreased the tumor volume15.
Colorectal cancer ranks the third most common malignant tumor and is marked with a low 5-year survival rate. The number of patients who newly contracted this disease accounts for almost 10% of new cancer cases worldwide22. It appears necessary to further explore a potential method for medical intervention of colorectal cancer.
Probiotic bacteria have emerged as the most promising chassis for living therapeutics23. E. coli Nissle 1917 (EcN) is a probiotic strain free of enterotoxins and cytotoxins and is used for the conventional treatment of various gastrointestinal illnesses24. EcN that produces the therapeutic proteins has been illustrated for cancer therapy in the murine tumor model. Azurin is a cytotoxic protein which induces cancer cell apoptosis.
The administration of azurin-producing EcN suppressed the growth of B16 melanoma and 4T1 breast cancer while prolonged the survival of tumor-bearing mice25. EcN with azurin also enabled to restrain pulmonary metastasis developed by 4T1 cancer cells. EcN has innate prodrug-converting enzymes. By intratumoural injection, EcN caused a significant reduction in tumor growth and an increase in survival of the CT26 colon cell-bearing mice after prodrugs were administrated26.
In the human tumor model, tumor growth was suppressed by engineered EcN which produced cytotoxic compounds27. In this study, the issue was addressed by development of bacterial cancer therapy (BCT) based on HlyE-producing EcN. To approach the goal, HlyE was expressed under the control of the araBAD promoter (PBAD).
The strategy of metabolic engineering was applied to EcN for the temporal and spatial control of the HlyE expression. As a result, the engineered EcN preferred colonization in tumor tissues and expressed HlyE that effectively caused tumor regression in mice xenografted with human colorectal cancer cells.
reference link : https://www.nature.com/articles/s41598-021-85372-6
More information: Shindu C. Thomas et al, Engineered Bacteria Enhance Immunotherapy and Targeted Therapy through Stromal Remodeling of Tumors, Advanced Healthcare Materials (2021). DOI: 10.1002/adhm.202101487