Custom Probe-Based Spatial Transcriptomics Enables Microbiome Detection in FFPE Colorectal Cancer Tissue

Inside every tumor, there is a hidden world. For years, scientists have known that bacteria live within human cancers, not just as accidental passengers but as active participants that can influence how a disease grows, how the immune system reacts, and even how well a patient responds to treatment. These microscopic communities are not scattered randomly; they form specific neighborhoods within the tissue, interacting with human cells in complex ways. To understand these interactions, researchers need to see where the bacteria are located relative to the human cells, much like a map that shows both the houses and the people living inside them. For a long time, this kind of detailed mapping was only possible with fresh tissue samples, which are rare and difficult to collect. The vast majority of medical records, however, consist of tissue that has been preserved in wax blocks, a method that has been the standard for decades but was previously thought too degraded for this kind of high-resolution bacterial detective work.

A team of researchers has now bridged this gap, developing a new way to map bacteria inside these preserved tissue samples. By adapting a technology designed to read human genes, they created a custom set of molecular tools that can also find and locate specific types of bacteria. They tested this approach on samples from colorectal cancer, a disease where the relationship between bacteria and tumors is particularly important. Their work proves that it is possible to see the spatial arrangement of microbes in old, archived medical specimens without losing the ability to read the human genetic code alongside them. This opens the door to studying decades of stored patient samples to understand how bacteria influence cancer in ways that were previously invisible.

The researchers began by designing a new set of molecular probes, which are essentially tiny, custom-made tags that stick to specific genetic sequences. In this case, they targeted the genetic code of bacteria, specifically the variable regions of a molecule called 16S rRNA, which acts as a unique identifier for different bacterial groups. They built these probes to work with a modern imaging platform called Visium CytAssist, a system that captures genetic information from thin slices of tissue while keeping their original shape and location intact. The challenge was to make these bacterial probes work alongside the standard probes used to read human genes, ensuring that the two systems would not interfere with each other. They created a pool of 195 pairs of these probes, targeting 15 different groups of bacteria known to be relevant to colorectal cancer, and mixed them into the standard testing kit.

To test if this new method worked, the team applied it to six samples of colorectal tissue that had been preserved in wax blocks. Three of these samples were from tumors, and three were from healthy tissue taken from the same patients near the tumor. The process involved slicing the tissue, treating it with the custom probe mixture, and then using a machine to read the genetic signals. The results were immediate and clear: the system successfully detected both human genes and bacterial signals in the same tissue sections. Crucially, the presence of the bacterial probes did not disrupt the reading of human genes. When the researchers analyzed the human genetic data, the cells organized themselves into the expected patterns, with distinct groups for the lining of the colon, the muscle layers, and immune cells, just as they do in fresh tissue. This confirmed that the new method was safe to use and did not damage the delicate information stored in the preserved samples.

The most exciting discovery came when the researchers looked at where the bacteria were located. In the healthy tissue samples, the bacterial signals were mostly found at the very surface, lining the inside of the colon, which matches what is known about how bacteria naturally live in a healthy gut. However, the tumor samples told a more complex story. In most of the tumor samples, the bacteria were found in small, concentrated clusters. But one specific tumor sample stood out dramatically. It contained a much higher amount of bacterial signal than the others, and these bacteria were not just sitting on the surface; they were deeply embedded within the tumor tissue itself.

When the researchers looked closer at the types of bacteria in this high-signal tumor, they found two main groups: one related to a genus called Porphyromonas and another related to Bacteroides-Phocaeicola. While both groups were present throughout the tumor, they followed different paths as they moved deeper into the tissue. One group seemed to penetrate the tumor in a different direction or at a different rate than the other. This suggests that different types of bacteria might be invading the tumor in unique ways, potentially interacting with the human cells around them differently. The ability to see these distinct patterns in a single sample is something that older methods could not achieve, as those methods would have mixed all the bacteria together, losing the information about where each type was actually living.

This study does not claim to have solved the mystery of how bacteria cause cancer, nor does it prove that these specific bacteria are the sole drivers of the disease. Instead, it establishes a new capability. It shows that scientists can now take the vast archives of preserved tissue samples from hospitals around the world and begin to map the bacterial communities inside them with high precision. The researchers noted that because their method uses specific probes, they can only find the bacteria they designed the probes for, meaning they cannot discover entirely new, unknown species with this specific setup. However, the system is flexible enough to be expanded with more probes in the future.

The findings also highlight the importance of looking at the spatial arrangement of microbes. The fact that different bacteria showed different distribution patterns within the same tumor suggests that their location matters. Some might be lurking on the surface, while others are actively infiltrating the core of the cancer. This level of detail was previously impossible to obtain from the most common type of medical specimen. By proving that this technique works on formalin-fixed, paraffin-embedded tissue, the researchers have provided a general framework that can be applied to other diseases and other types of preserved samples. It transforms the way scientists can look back at medical history, allowing them to ask new questions about the invisible microbial world that lives within our bodies and how it shapes our health.