Feature: Microscopy in the third dimension

This feature appeared in the September/October 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Dr Nicole Bryce is the only cell biologist among a swag of chemists in her position at the University of Sydney. She is applying her experience in 3-D imaging to see just how far fluorescent drugs can penetrate into live tumour-cell masses, or spheroids.

Many current cancer-killing drugs penetrate through four or five cell layers in a mass before stopping, meaning that only the very outside layers of the mass are affected. Bryce and her colleagues aim to identify those chemotherapeutic agents that will access all of the cells in a solid tumour and destroy them.

Bryce is using a spheroid tumour model for this work with the ultimate aim of developing a high-throughput microscopy-based drug screen. Cultured cells are grown in 96-well plates over a few days until they ball up into a tight aggregate, or cell mass, which can grow up to 700 μm in diameter.

The cells are usually of breast, ovarian or colon cancer origin, and number up to 25,000 in one spheroid. As the resident microscopist, Bryce was set the not-so-easy task of tracking selected fluorescent drug compounds through up to 200 μm of these solid cell masses. The key to this aim is that many chemotherapeutic drugs have a fluorescent moiety as part of their natural or engineered structure.

“What we really hope to do is modify existing drugs with known activity such that they reach most of the cells in a tumour, but also still be active to kill the cells that normally would not be as responsive. To do both is the trick. Actually even to do one of those things would be great,” she says.

The inherent or engineered fluorescence of chemotherapeutics drugs has not been exploited before in this way, and Bryce hopes that their work will have clinical consequences down the line.

“We are using conventional confocal microscopy in this preliminary work, but following the diffusion of a fluorescent drug into a living spheroid or cell mass and being able to image usefully deep into that mass is the real challenge.”

The imaging part takes a lot of care and patience, says Bryce. “Our main limitation is focal length of a microscope objective versus the limitations of optical resolution. We can only use the lowest-power: 10X objective. We can determine individual cells and distinguish between nuclear and cytosolic regions, but that is about it resolution-wise.”

Sacrificing subcellular detail is a reality of tracking fluorescence through such a large depth of cells, up to 200 μm. “If intracellular localisation studies need to be done, we do them first in 2-D with the higher magnification objectives, and then just do the drug diffusion studies in 3-D.”

The painstaking work is yielding some very promising and important findings. In work published earlier this year, Bryce and colleagues compared three drugs for uptake into spheroids with unexpected and far-reaching results. It seems the drug that was least taken up by the cells in 2-D imaging actually penetrated deepest into the spheroid, and vice versa. Drug screening studies often use accumulation rate into cells as a measure of drug success in addition to activity.

“The drugs of choice for clinical use are deemed to be those that go into a cell the fastest and stay there in the highest concentrations, thus the patient would need less of the drug overall,” Bryce explains. “However, we have shown that these might not be the best agents for use on solid tumours as they do not penetrate in very far, so not as many cells in the mass would be affected.”

The issue seems to be diffusion rate, according to Bryce. “What we saw in 3-D images of living spheroids was these fast-accumulating drugs getting very quickly into the outer two to three layers of cells and then stopping. Those cells effectively could not pass anything else through. But what you want is something that can pass through at a more measured pace and be able to go around cells via the intracellular space by diffusion to be taken up by cells further into the mass at a more even rate.”

What this work highlights is that, as with almost everything, “you have to find the balance, and if you can’t get it to the majority of cells, there is not much point in giving it”.