Biosensor for colorectal cancer | Scientist Magazine®

TThe human internal organs are overflowing in the ocean. microorganisms It gently ferments fiber, produces vitamins, and exchanges information with the immune system.¹ Now, scientists are tasking the bacteria that explore the digestive system with yet another task: detecting cancer.

Designed by an international team of researchers bacterial biosensor We were able to identify a DNA mutation associated with cancer and published it in an academic journal. science.² The research team also included molecular biologists. robert cooper and jeff hasty Professor and colorectal cancer researcher at the University of California, San Diego Josephine Wright and susan woods at the South Australian Health and Medical Research Institute, and Daniel Worsley At the colonoscopy clinic. The study authors hope that this technology could one day help in the early diagnosis of colorectal cancer, one of the most common causes of cancer-related death worldwide.

Scientists have previously genetically engineered bacteria to detect inflammation and bleeding in the intestines, but this is the first bacterial biosensor to detect specific DNA sequences in host tissue. To accomplish this feat, scientists utilized: Acinetobacter baileyThe ability to take up extracellular DNA and incorporate these sequences into its own genome.

Harvesting these naturally competent bacteria, detecting DNA changes, and using them as biosensors is an incredibly exciting advance.
-David Rigler, Imperial College London

“Using live bacteria to sense things in the gut and detect disease is something that I think is very interesting,” he said. david rigler, a microbiome researcher at Imperial College London, was not involved in the study. “Taking these naturally competent bacteria, detecting DNA changes, and using them as biosensors is a really exciting advance.”

For this study, the researchers wanted to do some engineering. A. Bailey How to detect mutations in codon 12 of a common colorectal cancer marker: colorectal cancer class gene. “At the time, it seemed like a pretty far-fetched idea,” Worsley recalls. The researchers achieved this lofty goal by bringing together an interdisciplinary team with expertise in synthetic biology and animal models of colorectal cancer.

In early proof-of-concept experiments, researchers tinkered with both genetically. A. Bailey and the tumor organoids they wanted the bacteria to detect. They engineered tumor cells with functional copies of antibiotic resistance genes. KanR, on both sides class homology arm.bacteria matched class homology arm and two stop codons that prevent expression Can R. When the bacteria engulfed the donor tumor’s DNA, the homology arms aligned the DNA sequences and the bacteria integrated a functional DNA sequence. Can R It becomes integrated into its own genome and can be grown on plates containing antibiotics.

Create bacteria that specifically detect mutant strains classThe researchers harnessed the bacteria’s own CRISPR-Cas machinery and instructed these molecular scissors to cut up the mutant rather than the wild type. class. This kills any bacteria that have acquired the wild type. class.

The researchers then tested these bacteria against colorectal cancer organoids with and without engineered donor DNA. Only bacteria co-cultured with genetically engineered tumors acquired antibiotic resistance, demonstrating that the sensor bacteria can discriminate between normal and donor tumors.

See also: Mutant RAS proteins collaborate for oncogenic potential

The researchers then tested the bacteria in vivo by administering the bacteria via enema to three groups: mice without tumors, mice with normal colorectal tumors, and mice with engineered colorectal tumors. I tested the sensor. Again, only the biosensor administered to mice with genetically engineered tumors grew in the presence of antibiotics, confirming that this bacterium can be used to signal the presence of genetically engineered colorectal cancer in mice. It was done.

Although these data were encouraging, human colorectal tumors do not contain perfectly positioned antibiotic resistance genes for bacteria to acquire. So the researchers tailored their strategy to detect natural tumor DNA. class mutation. This time, they placed a repressor gene inside the body. class homology arm.This gene prevented the expression of downstream genes Can R gene.

when bacteria are replaced class tumor DNA class, the repressor is lost, allowing antibiotic resistance genes to be expressed.As before, wild type class Targeted for destruction by CRISPR-Cas system.These new biosensors discriminated between mutants and normals in vitro class Survive only if cancer-associated mutations are present, by acquiring antibiotic resistance. The research team named the technology CATCH, which stands for Targeted CRISPR Discriminated Horizontal Gene Transfer Cellular Assay.

Despite these preliminary successes, Rigler urged caution. “It’s important not to get too ahead of ourselves in terms of thinking these systems are ready to be introduced into clinics,” he said.

“This is by no means the end point,” Worsley agreed.

Researchers are currently working on strategies to improve the sensitivity of biosensors to natural tumor DNA in the complex environment of the colon. Because of concerns about introducing antibiotic-resistant bacteria into humans, they are also developing other ways for biosensors to signal the presence of mutants. class. To be commercially viable, biosensor bacteria must be delivered orally. This means that the bacteria must be able to survive through the digestive system and report its results on the other side.

We have engineered all of our sophisticated functions inside the cell, so we don’t need such a sophisticated laboratory outside the cell.
-Daniel Worsley, Colonoscopy Clinic

But ultimately, Worsley hopes these biosensor bacteria will one day be used as point-of-care diagnostics in remote and low-resource areas, such as the Australian outback. “We engineered all the advanced functionality inside the cell, so we don’t need such a sophisticated laboratory outside the cell,” he said.

Researchers hope for broader applications. For example, instead of turning on antibiotic resistance genes when bacteria sense tumor DNA, they could turn on the production of genotype-specific small molecule therapeutics to deliver treatment precisely where it is needed. It may be possible to manipulate bacteria. Bacteria can be genetically engineered to detect and respond appropriately to various cancer-causing mutations and even difficult-to-treat infections, such as: clostridium difficile. Worsley sees the potential for linking diagnosis and treatment as a major advantage of these genetically engineered bacteria.

References

1. Bull MJ, Plummer NT. Part 1: Human gut microbiota in health and disease. Integration Med (Encinitas). 2014;13(6):17-22.
2. Cooper RM et al. Genetically engineered bacteria detect tumor DNA. science. 2023;381(6658):682-686.

Conflict of Interest Statement: JH, DW, and SW own stock in GenCirq Inc., which focuses on cancer drugs. DW, JH, RC, SW, and JW have filed a provisional patent application for this technology.