NEW YORK (360Dx) – Researchers at Tufts University and their collaborators at the University of California, Irvine, and elsewhere have developed an in vivo imaging method for mitochondria that they plan to develop into a noninvasive label-free diagnostic tool for the early detection of skin and other cancers.
In a proof-of-principle study published in Science Translation Medicine late last year, the team, led by Irene Georgakoudi, a professor in the department of biomedical engineering at Tufts, demonstrated that the approach, which uses multiphoton microscopy, can distinguish between normal and cancerous skin, based on differences in the organization of mitochondria.
The researchers have filed provisional patent applications for their methods and are currently working on a probe-based instrument that would allow them to access other organs besides the skin.
The hope is to be able to detect cancer lesions earlier than doctors can today using conventional imaging — not only skin cancer but cancers of the oral cavity, esophagus, cervix, colon, or bladder.
"Especially the early cancer lesions are very small, and they are not detectable easily by eye or endoscopy," Georgakoudi explained. "There is always a risk that some of these small lesions are not detected until it's too late, the cancer has metastasized, and it's very difficult to treat the patient. So if you have a tool that's able to detect lesions at a much earlier stage, before you have metastases, then in most cases, you can cure the patient of cancer."
Biopsies can discover cancerous changes early but they are invasive and need to be analyzed by a pathologist, which can take days or weeks. This delay could be avoided with an in vivo imaging approach that instantly analyzes the data. "With an approach like this, where you image in situ, you can use algorithms that are entirely automated … so in real time, you get a diagnosis and you can tell the patient," Georgakoudi said.
In some cases, for example cervical lesions, the diagnosis could be followed immediately with treatment, such as the removal of a precancerous lesion, without the need for a second visit. "I think there is definitely good potential to make the whole diagnosis-treatment paradigm quite a bit more efficient and less stressful," she said.
The method her team has developed relies on multiphoton microscopy, a relatively new imaging technique that was first used to look at biological samples about 20 years ago and has only recently been applied in humans.
It can be used directly on patients and generates signals from endogenous chromophores in cells and tissues, so it does not require any staining, and it provides subcellular resolution. The technique requires specialized lasers that emit light in very short pulses, Georgakoudi said, and exploits a nonlinear process where two photons interact with a molecule and excite it. The molecule emits a single photon when it goes back to its initial state. "The use of this multiphoton microscopy technique was essential in enabling us to acquire high-resolution images of the different cell layers of the skin," she said.
JenLab, a spinoff from the University of Jena in Germany, has developed a commercial instrument, called MPTflex, for in vivo multiphoton tomography that was used in the study. The main application of this platform is the early diagnosis of skin diseases, such as malignant melanoma, according to the firm's website. What is unique about the microscope, Georgakoudi said, is that it allows investigators to obtain high-resolution images of thick tissues with multiple layers, as opposed to standard miscroscopy, which can only look at thin tissue samples.
Georgakoudi's collaborators at UC Irvine, who acquired the patient data for the recent study, and JenLab previously published two other studies on the use of in vivo multiphoton microscopy for skin cancer imaging, one focusing on basal cell carcinoma, the other on melanoma.
What distinguishes the Tufts study from these is that it focuses on mitochondria. These organelles, which produce energy in the cell, have been studied extensively, and changes in their shape and organization have been associated with a variety of diseases, including cancer, neurodegenerative, metabolic, and cardiovascular disorders. "They form these beautiful networks inside the cell that are very dynamic," Georgakoudi said.
However, up until now, most studies of mitochondria involved either tissue biopsies or, in living cells, contrast agents that are not approved for use in humans. Multiphoton microscopy has now enabled the group to look at mitochondria without using a label. Specifically, they image co-enzyme NADH, a key player in cellular energy metabolism that has intrinsic fluorescence, which correlates well with the position of mitochondria in the cell.
Using this approach, "we can get a really quantitative measure of how well-organized, or not well-organized, the mitochondria are," Georgakoudi said. "That gives us a sense of the metabolic pathways that the cell is relying on to produce energy. And if something is going wrong in these metabolic pathways, then we can detect it" and correlate it with disease.
For their study, they imaged and analyzed healthy and diseased skin areas from 10 melanoma and basal cell carcinoma patients and four healthy volunteers. They found depth-dependent variations in mitochondrial organization in the healthy tissue but not in the cancer-containing tissue.
To test whether this variation could be used as a quantitative diagnostic biomarker, they developed an algorithm that looked at three tissue metrics to differentiate healthy and diseased skin: the degree of depth-dependent variations in mitochondrial clustering, the median mitochondrial clustering value of the tissue, and the depth-dependent variation of nuclear-to-cytoplasmic ratio. They then applied this classification predictively to three suspicious tissue areas from one of the melanoma patients and classified them correctly, in agreement with the histological findings. "This is very exciting — the dataset is very small, but the initial signs are very positive that [the method] may have some specificity to real lesions," Georgakoudi said.
The next step will be to evaluate the approach in a larger patient cohort, ideally recruited at different clinical centers. In addition, Georgakoudi's group is working with clinicians at Tufts and collaborators at Cornell University to develop a probe-based imaging system that could be used instead of the current microscope, which relies on traditional objectives.
The JenLab instrument has a "fairly bulky" articulating arm, she said, that can be moved around to look at various parts of the skin but not elsewhere in the body. A probe-based system, on the other hand, could access other organs, like the oral cavity or the cervix, and ultimately, through endoscopy, the esophagus, colon, or bladder. Work to develop such probes is pursued by several academic groups, she said, some collaborating with companies.
The approach could eventually be used to diagnose diseases other than cancer that involve mitochondrial dysfunction and changes in mitochondrial organization. For example, Georgakoudi's group recently started working on neurodegenerative diseases, such as Parkinson's.
Another challenge has been that large tissue areas are currently difficult to image, and several investigators are working to come up with smaller, more efficient ways to do high-resolution imaging for large tissue volumes, she said. "I hope in the next couple of years, we'll really have systems in place that will make it feasible to survey large areas."
The cost of imaging instruments is expected to come down over time. The laser used in the current microscope, Georgakoudi said, costs on the order of $100,000, but over the last few years, new lasers that are based on fiber technology have been developed. These are expected to cost on the order of $10,000 to $20,000, which "will make something like this a lot more appealing to a physician, to the hospital that would have to invest in this instrument," she said.
"The other good thing about these types of microscopes is they are really very portable," she added. "You could have an instrument like that in your office; you would not have to go to an imaging suite."
Finally, the time required to conduct the procedure could be further improved. At present, it takes several minutes to acquire images of multiple skin layers, and the analysis takes another few minutes. Better processors and smarter scanning approaches could bring this time down, she said, so the technique can be "implemented near real time."
Her guess is that it will take several years to establish the mitochondrial imaging approach in the clinic. This will require a company to commercialize the instrument and insurance to pay for the procedure. "I hope within the next couple of years, we'll see more broad use of approaches like this in dermatology," she said, and within five years, applications that rely on endoscopy.
In the meantime, the researchers have filed two provisional patent applications related to their work, entitled "Methods and systems for mitochondrial imaging" and "Multifunctional devices for dynamic control of cell culturing conditions." While the IP has not been licensed yet, Georgakoudi has already been approached by an unnamed company interested in the technique. "It will be critical to get commercial interest for this to make it into the clinic," she said.
The imaging device would also require FDA approval. But because it uses neither harmful radiation nor a contrast agent, this is expected to be easier than for some other medical devices, she said.
Ultimately, doctors will need to adopt the technology. "I think dermatologists who really want to push patient care further like these types of approaches because they see the value of having a diagnosis in near real time, and it's much more efficient," Georgakoudi said.
"But you also always have the old-school dermatologist who is doing something in a certain way … and there is always going to be some resistance to change the paradigm," she said. "Pathology and the way we diagnose disease such as cancer is so established, and it has not changed for the past 50 years. The approaches have been so engrained into the culture, it's not easy to say all of a sudden, 'You can do pathology on the patient in situ without needing to take a chunk of tissue.'"