Science often starts with a simple observation, a curiosity that refuses to be ignored. In our case, it was the nagging realization that despite nerves being widely acknowledged as important in pancreatic cancer (PDAC), they were somehow missing from all worldwide reported transcriptomic datasets available from sequencing tumor samples. It was bizarre, neuronal innervation was right there with their axons infiltrating tumors, but yet they were invisible in the molecular landscape. This is due to the fact  that their cell bodies are located far outside the tumors in peripheral ganglia and thus are missed in any sequencing effort. The literature hinted at their significance, but they were always a footnote, never a main character. So, we set out to change that.

Step One: The Tracing Nightmare

It turns out there was a reason nobody had done this before. Tracing and isolating neurons in tumors is highly challenging. Every step in the process was a potential disaster, and we were basically stumbling through the dark. The first hurdle? How do you even find these neurons?

We started by using retrograde tracing—injecting fluorescent dyes or AAVs into the tumor to label the neurons that innervate it. Sounds simple, right? It wasn’t. We never knew:

• Was the tracer working at all?
• Was the tracing time too short? Too long?
• Were we even dissecting the right structures? (It turns out ganglia are much harder to find than textbooks suggest, especially in small mice.)
• Did we digest correctly? (Spoiler: We tried over 30 different digestion methods, most of them killed the neurons)

It turns out, neurons are drama queens. They are extremely stable in vivo but as soon as you try to isolate them, they fall apart like overcooked spaghetti. Most digestion mixes wiped them out.  We couldn’t identify them in standard live-cell FACS sorting using staining antibodies. And even when we did manage to sort them, FACS sorting itself killed them. Fun times.

It was a constant cycle of trial and error, back and forth, rinse and repeat. We spent months failing at every single step, but every tiny breakthrough felt monumental. And then, one day, everything clicked. We figured it out. Trace-n-Seq was born.

Fig. 1: Characterization of pancreas and PDAC innervating neurons via Trace-n-Seq

Step Two: Cracking Open the Neural Code

Once we had a working protocol, it felt like turning on a light in a dark room. Suddenly, we weren’t just looking at neurons; we were seeing how they behaved in cancer, what genes they expressed, and how they interacted with the tumor and the cells of the tumor microenvironment (TME). 

Indeed, neurons weren’t just passive passengers in the TME. They were active players. The tumor wasn’t just infiltrated by nerves - it was hijacking and reprogramming them.  Making them grow and sprout, communicate, and even help its own survival by direct communication but also by influencing the TME and these cells. These persistent changes in the TME even enhanced relapse after primary tumor resection and re-seeding the tumor.

We realized that neurons actively communicate with cancer cells and the tumor microenvironment, specifically the tumor associated fibroblasts, another difficult and abundant player in PDAC. It was as if we had stumbled upon a hidden network that had been shaping PDAC all along, but no one had been able before to fully capture it.

Step Three: Functional Proof and a Few Surprises

New data sets are great, but do the neurons actually matter for the tumor? We had to find out in more detail.

So we went back to the lab and ran experiments to silence these nerves, chemically, surgically, in every way we could think of. We found that shutting down sympathetic or sensory nerves slowed tumor growth. It also disrupted the immune landscape, activating immune cells by reducing suppressive stromal cells. Nab-Paclitaxel, a standard chemotherapy, didn’t just hit cancer cells—it also wiped out nerve fibers, especially sensory ones. Knowing that patients showing stronger neuropathy side effects after Paclitaxel treatment showed better survival rates, suggested that nerve fiber infiltration could have real clinical significance. Therefore, we weren´t surprised that the biggest effect was achieved by combining nerve-targeting approaches with chemotherapy - it was like cutting off the tumor’s escape routes while hitting it directly.

Step Four: The Beauty of the Beast

No matter how frustrating this process was—one thing was undeniable: These cells are beautiful.

Our 3D light-sheet microscopy revealed by imaging fully cleared healthy organs and pancreatic tumors with a breathtaking neuronal network. The nerves grew into the tumor like trees grow in the spring, and every new image or video was a tiny piece of art. While the lab work was often exasperating, we had incredible fun creating and analyzing these images.

Fig. 2: Light sheet microscopy of pancreas and PDAC.

Why This Matters

The key take home message? Neurons are not just bystanders in cancer—they are architects of the tumor microenvironment and the tumor itself. If we ignore them, we miss a major piece of the tumor ecosystem puzzle.

With Trace-n-Seq, we can finally capture and understand this hidden world in great detail. We hope our work advance the emerging field of Cancer Neuroscience unlocking new possibilities for treating PDAC.  Sometimes, the most groundbreaking discoveries are hiding in plain sight— waiting for the right tools to reveal them.

This project was one of the most challenging, rewarding, and undeniably fascinating we have ever undertaken. In the end, the neurons didn’t just emerge in our transcriptomic data—they reshaped how we “think” about PDAC. That’s the beauty of science.