3D spheroid models to study tumor colonizing anaerobic bacteria: a bridge from in vitro to in vivo

Inside the body's most stubborn tumors, a strange and dangerous landscape often forms. As cancer cells multiply with reckless speed, they outgrow their own blood supply, creating pockets of deep starvation where oxygen cannot reach. These areas die off, turning into necrotic tissue, a hallmark of aggressive disease that usually signals a poor outlook for the patient. Yet, this dead zone holds a unique secret: it is the only place where certain types of bacteria, which cannot survive in oxygen, can thrive. Scientists have long known that these anaerobic bacteria naturally seek out and colonize these dying tumor cores. This discovery has sparked a decades-long effort to turn these microscopic invaders into allies, engineering them to deliver drugs or trigger the immune system to attack the cancer from the inside.

However, before these living medicines can be tested in people, researchers must prove they work safely and effectively. Traditional lab tests using flat layers of cells in a dish fail to recreate the dead, oxygen-starved center of a tumor, making it impossible to see if the bacteria will actually find their target. Conversely, testing directly in animals raises serious ethical concerns and is often too complex to screen many different bacterial strains quickly. To bridge this gap, a team of researchers at Maastricht University has developed a new, three-dimensional model that mimics the real conditions inside a tumor, allowing them to watch how these bacteria behave in a setting that closely resembles the human body.

The researchers started by growing tiny, ball-shaped clusters of cancer cells from three different sources: two types of mouse cancer and one type of human colon cancer. They let these clusters grow until they developed their own internal dead zones, just like real tumors do. Once these necrotic cores formed, the team introduced spores of Clostridium sporogenes, a non-harmful bacterium that is safe for humans but loves to live in oxygen-free environments. They observed that the bacteria successfully hatched from their spores and moved deep into the dead centers of the cell balls. In the mouse cancer models, the bacteria grew so vigorously that they eventually caused the entire tumor ball to fall apart, while in the human cancer models, the bacteria settled in and remained stable, mirroring what happens in living animals.

With the bacteria successfully colonizing these models, the team turned to safety. A major concern with using bacteria as medicine is the ability to stop them if they cause problems or if the treatment needs to end. The researchers tested several common antibiotics to see which ones could clear the bacteria from inside the tumor balls. They found that some drugs, like tigecycline, only paused the bacteria's growth; once the drug wore off, the bacteria started multiplying again. However, other antibiotics, specifically augmentin and vancomycin, acted as true killers, wiping out the bacterial population completely and leaving the tumor balls intact and free of infection. This distinction is vital, as it proves the model can tell the difference between drugs that merely slow bacteria down and those that can eliminate them entirely.

Finally, the team tested a specific cancer therapy called Clostridium-Directed Enzymatic Prodrug Therapy. In this approach, the bacteria are genetically modified to carry a special tool that converts a harmless drug into a poison, but only inside the tumor where the bacteria live. The researchers added these engineered bacteria and the harmless drug to their tumor models. In the mouse cancer models, the combination caused the tumor balls to shrink significantly more than when either the bacteria or the drug was used alone. The human cancer models showed a similar trend of growth inhibition, though the effect was less pronounced. These results matched what scientists had previously seen in living animals, confirming that this simple, three-dimensional ball of cells can accurately predict how a complex bacterial therapy will perform in a living body.

By creating a system that captures the dead, oxygen-starved heart of a tumor, the researchers have provided a powerful new tool for the future of cancer treatment. This model allows scientists to screen dozens of bacterial strains and drug combinations quickly and ethically, filtering out the ones that won't work before ever testing them on an animal or a person. It offers a clear path forward for refining these living medicines, ensuring that when they eventually reach the clinic, they are safe, effective, and ready to target the most dangerous parts of cancer.