Colour coding what's happening in the tumour microenvironment

In the operating room, a removed tumour can look deceptively uniform — like any other piece of tissue. But within it lies a bustling city of cancer cells, blood vessels, and connective tissue, invisible to the naked eye.

Dr. Ralph DaCosta's research team at UHN's Princess Margaret Cancer Centre turns this hidden world into vivid colour, revealing cancer's behaviour in real time.

His team discovered a dynamic, constantly changing tumour microenvironment surrounding pancreatic cancer cells (published in Science Advances), that could help oncologists tailor treatment schedules for patients who have one of Canada's deadliest cancers.

"We saw pockets of pancreatic cancer cells that are inherently hypoxic — in other words, they have low oxygen levels. They are enveloped by very dense layers of connective tissue called collagen fibers, preventing blood vessels from getting inside," says Dr. DaCosta, an Allan Slaight Scientist.

To detect pockets of oxygen-starved (hypoxic) cancer cells, the team, led by former doctoral student Dr. Timothy Samuel, used a sophisticated combination of fluorescent labelling and advanced optical microscopy in living models of cancer to light up different components in the tumour microenvironment.

Cancer cells in preclinical models were engineered to glow red (DsRed) at all times, while a built-in hypoxia sensor made them glow green (GFP) only under low-oxygen conditions. Blood vessels were highlighted in cyan using a fluorescent antibody, allowing the team to measure oxygen access relative to blood supply.

Meanwhile, collagen fibers — key components of the tumour's stiff, fibrotic structure — were visualized using second harmonic generation microscopy, a special form of laser light that makes collagen fibers shine like white snow under a spotlight.

These rigid, oxygen-starved pockets of cancer emerge, persist, and can contribute to tumour progression: They may fuel a vicious cycle of tumour growth and spread by cutting off oxygen, reshaping collagen and the microenvironment over time, and helping cancer cells evade treatment.
 

In a follow-up study published in Scientific Reports, the team used this imaging platform to track how patient pancreatic tumour cells responded to radiation. After radiation treatment, fewer cells continued to glow red, indicating reduced viability of cancer cells, and there was a reduction in green fluorescence, signalling less hypoxia.

The presence of DNA damage markers and slowed cell growth confirmed that the treatment was effective. Through quantitative analysis, the study also offered insights into optimizing treatment timing and dosing to improve treatment outcomes.

This imaging system gained widespread recognition that led to a collaboration with Drs. Mark Minden, John Dick, Stephanie Xie, and Tak Mak (and collaborators abroad), to illuminate how blood cancers interact with the immune system and respond to immunotherapy. This time, the team needed ways to visualize whether cancer cells were alive or dead to better track treatment response, as the bone marrow microenvironment is remodelled during disease progression.

They developed a new marker for tumour cell death — a pink fluorescent signal that is emitted when the treatment kills a leukemia cell.

"We have also found ways to visualize different immune cell populations in the bone marrow where leukemia cells develop," says Dr. DaCosta. "It will let us see an entirely different cellular landscape with complex microarchitecture, and the interaction between leukemia cells and immune cells. I'm very excited by this new frontier where imaging can play a key role in improving our understanding of tumour biology. This type of work has never been done before."