Tthe human gut is flooded in a sea of microbes which quietly ferment fibers, produce vitamins and exchange information with the immune system.1 Today, scientists are giving bacteria another task as they make their way through the digestive system: cancer detection.
An international team of researchers has designed a bacterial biosensor able to identify a DNA mutation associated with cancer, which they published in the journal Science.2 The research team included molecular biologists Robert Cooper And Jeff Hasty from the University of California San Diego and bowel cancer researchers Josephine Wright And Susan Woods at the South Australian Health and Medical Research Institute, and Daniel Worthley at the Colonoscopy Clinic. The study authors hope that this technology will one day facilitate the early diagnosis of colorectal cancer, one of the most common causes of cancer-related deaths worldwide.
While scientists have already engineered bacteria to detect inflammation or bleeding in the intestine, it is the first bacterial biosensor capable of detecting a specific DNA sequence of host tissues. To achieve this feat, scientists exploited Acinetobacter baylyiThe ability of the organism to take up extracellular DNA and integrate these sequences into its own genome.
Taking these naturally competent bacteria, detecting DNA changes, and then using them as a biosensor is a really exciting advancement.
-David Riglar, Imperial College London
“Using live bacteria to detect things in the gut and detect disease is something I find very exciting,” said David Riglar, a microbiome researcher at Imperial College London who was not involved in the study. “Taking these naturally competent bacteria, detecting DNA changes, and then using them as a biosensor is a really exciting advancement.”
In this study, the researchers wanted to design A. baylyi detect a common marker of colorectal cancer: a mutation in codon 12 of KRAS embarrassed. “At the time it seemed like a pretty far-fetched idea,” Worthley recalls. By bringing together an interdisciplinary team with expertise in synthetic biology and animal models of colorectal cancer, the researchers achieved this noble goal.
In their first proof-of-concept experiments, the researchers genetically tinkered with both A. baylyi and the tumor organoids they wanted the bacteria to detect. They engineered tumor cells with a functional copy of the antibiotic resistance gene, KanR, flanked by KRAS homology arm. The bacteria had a match KRAS homology arm, plus two stop codons that prevented expression of kanR. When the bacteria engulfed the donor tumor DNA, the homology arms aligned the DNA sequences and the bacteria integrated the functional sequences. kanR in their own genome, allowing them to grow on plates containing antibiotics.
Create bacteria that specifically detect the mutant KRASthe researchers harnessed the bacteria’s own CRISPR-Cas machinery, directing these molecular scissors to chop up wild-type bacteria, but not mutants. KRAS. This would kill any bacteria that acquired the wild type KRAS.
The researchers then tested these bacteria against colorectal cancer organoids with and without the edited donor DNA. Only bacteria co-cultured with the engineered tumors acquired antibiotic resistance, showing that the sensor bacteria could distinguish between normal and donor tumors.
See also « Mutant RAS proteins combine for oncogenicity«
Next, the researchers tested the biosensors in vivo by administering the bacteria via an enema to three groups: mice without tumors, mice with normal colorectal tumors, and mice with altered colorectal tumors. Again, only biosensors administered to mice with artificial tumors grew in the presence of the antibiotic, confirming that the bacteria could be used to signal the presence of artificial colorectal cancer in mice.
While these data are promising, human colorectal tumors are not designed with an antibiotic resistance gene perfectly placed for bacteria to acquire. Thus, the researchers adjusted their strategy to detect the natural DNA of the tumor using KRAS mutation. This time, they placed a repressor gene inside the KRAS homology arm. This gene prevented the expression of a kanR embarrassed.
When the bacteria exchanged their KRAS DNA for tumor KRAS, the repressor was lost, allowing expression of the antibiotic resistance gene. As before, wild type KRAS was targeted for destruction by the CRISPR-Cas system. In vitro, these new biosensors distinguished between mutants and normals KRAS surviving and becoming resistant to antibiotics only in the presence of the cancer-associated mutation. The team named this technique CATCH for Cellular Analysis for CRISPR-Targeted and Discriminated Horizontal Gene Transfer.
Despite these preliminary successes, Riglar urges caution. “It’s important not to go too far in thinking that these systems are ready to go into the clinic,” he said.
“It’s absolutely not the end point,” Worthley acknowledged.
Researchers are currently working on strategies to improve biosensor sensitivity to natural tumor DNA in the complex environment of the colon. Because of concerns about administering antibiotic-resistant bacteria to humans, they are also developing other ways for biosensors to signal the presence of mutants. KRAS. To be commercially viable, biosensor bacteria must be administered orally, meaning they will need to survive their journey through the digestive system and be able to transmit their findings to the other side.
Since we have designed all the sophistication inside the cell, we don’t need as sophisticated a laboratory outside the cell.
-Daniel Worthley, Colonoscopy Clinic
Ultimately, however, Worthley hopes that these biosensor bacteria will one day be used as point-of-care diagnostics in remote or low-resource areas such as outback Australia. “Since we have designed all the sophistication inside the cell, we don’t need as sophisticated a laboratory outside the cell,” he said.
Researchers also hope for broader applications. Instead of activating an antibiotic resistance gene when they detect tumor DNA, for example, bacteria could be engineered to activate the production of a genotype-specific small molecule therapeutic, delivering treatment precisely there where it is needed. Bacteria could be engineered to detect and respond appropriately to various oncogenic mutations, or even difficult-to-treat infections like Clostridium difficile. Worthley considers this possibility of combining diagnosis and therapy as the major advantage of these modified bacteria.
The references
- Bull MJ, Plummer NT. Part 1: The human gut microbiome in health and disease. Integrated Med (Encinitas). 2014;13(6):17-22.
- Cooper RM et al. Modified bacteria detect tumor DNA. Science. 2023;381(6658):682-686.
Conflict of Interest Statement: JH, DW, and SW own shares in GenCirq Inc., which focuses on cancer treatments. DW, JH, RC, SW and JW have filed a provisional patent application on this technology.
