Written by Federico Citterich
Conceived and reviewed by Alessandro Rossetta
Cancer cells exhibit altered metabolic activity, enhancing glycolysis over oxidative phosphorylation. By tracking these metabolic changes, fluorescence lifetime imaging (FLIM) emerges as a powerful tool for improving tumor identification and surgical precision, offering new possibilities for enhanced cancer detection and treatment.
When he switched on the light and entered the room, he realized that his notes were still sitting on the desk. He drew closer, pondering the inscriptions upon the timeworn, yellowed sheet of paper. The data on the sea urchins, which he had previously worked on, had misled him. The rat liver carcinoma did not take up more oxygen than normal liver tissue, as his embryological investigations on sea urchins had led him to hypothesize. Yet, Warburg was still amazed. Something weird was happening. Even in the presence of oxygen, the tumor was producing lactic acid.
Differentiation between normal and cancer cells. Image created using Biorender.
Lactic acid (or lactate) is an organic acid synthesized during glycolysis, one of the main pathways – along with oxidative phosphorylation (OXPHOS) – involved in cellular energy production. Glycolysis is not an efficient process, producing roughly 18 times less energy than OXPHOS. Yet, OXPHOS has a drawback: it absolutely needs oxygen to occur. When oxygen is limited, glycolysis happens instead. The cancer cells investigated by Warburg, however, did not follow this pattern. The production of lactic acid was an indication of glycolysis happening, yet oxygen was not limited. Why were the cells performing aerobic, oxygen-involving glycolysis rather than the far more efficient OXPHOS?
Originally, Warburg proposed that this happened because, in cancer cells, OXPHOS was simply shut down. Now, however, we know that in cancer cells OXPHOS can still happen, and that other factors play a role. First, tumors often have an irregular blood supply, leading to fluctuating oxygen levels. Using glycolysis hence ensures energy production even when oxygen is limited. Second, glycolysis is faster than OXPHOS, allowing cells to meet immediate energy demands. Third, the production of lactate and other acids from glycolysis suppresses anticancer immune effectors and favors tumor invasion. Finally, and most importantly, intermediates from glycolysis further support rapid proliferation of the tumor by contributing to the biosynthesis of essential components such as lipids and nucleotides1.
Differentiation of the different modes of energy production used by normal and cancer cells.
Despite using glycolysis for energy production, inefficient at first glance, cancer cells produce more overall “metabolic fuel” than normal cells. This is because cancer cells alter their metabolic pathway by increasing fuel intake, maximizing biosynthesis, and enhancing glycolysis. Tracking these metabolic shifts is crucial for understanding cancer progression, because these changes allow tight control of key processes that govern growth, maintenance, and survival. However, measuring the presence of metabolites2 in specific organelles or cell compartments is challenging, as it often perturbs the surrounding environment. In addition, most metabolites are present ubiquitously in the cell, but they are spatially regulated inside specific compartments.
In the last decade, fluorescence lifetime imaging (FLIM) has proven to be a strong candidate to face these challenges. FLIM is a non-invasive, all-optical technique that directly targets the metabolites inside the organelles, exploiting a feature named fluorescence lifetime. The fluorescence lifetime of molecules refers to the time a molecule spends in its excited state3 after absorbing light, before returning to its ground state4 by emitting light. Some metabolites, such as NADH and FAD, are intrinsically fluorescent, and they do not necessitate chemical fluorescent labeling. Yet, their fluorescence lifetime changes when they are bound to a protein, meaning FLIM can detect and differentiate when that molecule is in a free state from when it is protein-bound. This is a crucially important factor in the context of NADH. In fact, NADH appears in a free state when produced during glycolysis, but it binds to proteins during OXPHOS. Hence, FLIM-driven detection of higher levels of free NADH is indicative for more glycolytic metabolism, while prevalence of protein-bound NADH denotes more OXPHOS metabolism. In the context of cancer, this implies that FLIM can detect cells performing enhanced glycolysis (high levels of free NADH). If a cell is performing more glycolysis than other cells, then it is probably a tumor cell. Afterall, Warburg got it right, and that’s why the fact that cancer cells mainly rely on glycolysis for energy production is now known as the Warburg effect.
Differentiation between protein-bound and free NADH, found in normal and cancer cells respectively
The main application of FLIM relies in improving the identification of tumor areas and margins, crucial for surgeons in determining where to make incisions during surgery. Currently, the surgical removal of tumors is mainly guided by pre-operative imaging techniques like magnetic resonance imaging (MRI) and computed tomography (CT) scans. These methods are essential for planning surgery, as they help surgeons determine the best place to make an incision, the safest path to the tumor, and which critical structures to avoid. However, this approach relies on mapping pre-operative images onto the surgical field during the operation, which can be challenging. Surgeons often lack real-time feedback and must rely on their own judgment – such as visual inspection and assessing tissue texture – to distinguish between different conditions, like solid tumors versus surrounding tissue or treatment-related damage versus healthy tissue. This subjectivity can sometimes lead to less-than-optimal surgical outcomes. And here is where FLIM comes into play. By detecting fluorescence lifetime shifts and by tracking energy production, FLIM can potentially differentiate between healthy and cancerous tissues directly in the operating room.
But that’s not all. An additional approach by which FLIM can detect surgical margins is through the analysis of tissue composition. Cancerous and normal tissues have distinct fluorescence properties not only due to metabolic changes, but also due to differences in structural and biochemical composition. In other words, tissues contain a variety of naturally fluorescent molecules (e.g., collagen, elastin, lipofuscin, etc.), and the structure and density of these molecules differ between cancerous and normal tissues, causing variations in fluorescence lifetime. In normal tissues, for instance, collagen is highly structured and forms cross-links, leading to a longer fluorescence lifetime. In cancerous tissue, contrarily, collagen is often degraded or rearranged, resulting in shorter fluorescence lifetimes. By detecting these lifetime shifts, FLIM can map out areas with different tissue compositions, helping to identify tumor margins.
The different fluorescence lifetimes of protein-bound and free NADH enable FLIM to recognize cancer cells.
But FLIM goes beyond simply identifying the boundary between tumor and normal tissue, and it does that by capturing subtle variations in fluorescence lifetime within cancerous areas, variations that correspond to gradients in cellular characteristics such as density or metabolic state. This allows FLIM to map the spread and density of tumor cells within the surgical margins, offering a spatial map of how tumor cells are distributed. Since tumors like glioblastoma5 (GBM) are not homogeneous and often have infiltrative edges, FLIM can detect dynamic changes in cell density across different regions of the margin, identifying areas of high or low tumor cell density that might be overlooked by other imaging methods. This capability is crucial for aggressive cancers like GBM, which lack clear boundaries. By pinpointing regions with lower tumor cell density, FLIM helps surgeons determine how much extra tissue to remove while preserving as much healthy tissue as possible, improving surgical outcomes and reducing the risk of recurrence. FLIM has the potential to revolutionize cancer care, but who would have thought that it all started with sea urchin research?
GLOSSARY
- Nucleotides are the building blocks of DNA and RNA.
- Metabolites are products – either final or intermediate – of metabolism.
- The excited state of a molecule is when it has more energy than usual. The molecule stays in this high-energy state for a short time before it releases the energy and returns to the ground state.
- The ground state of a molecule is its normal, stable state where it has the lowest possible energy.
- Glioblastoma is the most aggressive and most common type of brain cancer.
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