Tissue-Pixelation Chip Measures Spatial RNA Expression in Tumor Samples

Scientists have developed a microchip-based RNA analysis technology that can directly measure gene expression spatially across samples of cancer tissue within just a couple of hours. The chip’s developers, led by Rashid Bashir, Ph.D., and Farhad Kosari, Ph.D., at the Mayo-Illinois Alliance for Technology Based Healthcare, suggest that their “tissue pixelation” spatial amplification technology will provide greater insight into the expression profiles of patients’ tumors than current protein-based approaches, as well as represent a valuable research tool.  “Our method can detect messenger RNA (mRNA) expression, which can provide additional insight than just the final protein concentration,” Prof. Bashir comments.

In their published paper in Nature Communications (“Pixelated Spatial Gene Expression Analysis from Tissue”), the scientists report using the technology to directly amplify and analyze the mRNA of the prostate cancer marker, topoisomerase II alpha (TOP2A), in frozen human prostate tissue xenografts grown in mice. “Our technique, which can be easily performed in routine practice, has many important clinical and biological applications, such as understanding tumor heterogeneity, predicting patient outcomes, and postoperative characterization of surgical margins,” they conclude. “As such, we predict that microchip amplification will find a wide range of applications in clinical and research settings.”

Spatial gene expression can provide key information on tissue organization and function, as well as on pathological changes, but maintaining spatial organization in tissue sections that are used for routine analysis is challenging, the researchers point out. Current techniques, including amplification-based gene expression analysis, and direct probe-based hybridization techniques, such as single-molecule fluorescence in situ hybridization (FISH), all have drawbacks that hinder their use in routine research or clinical practice. “They are cumbersome and slow, taking hours or even many days to do the analysis across just one tissue sample,” notes Dr. Bashir, who is a Grainger Distinguished Professor of Bioengineering and the Carle Illinois College of Medicine executive associate dean. “Our technique does the entire analysis across the tissue slice in two hours or less.”

The new technology is constructed on a fingernail-sized silicon chip that contains an array of more than 5000 pyramid-shaped wells with “knife-like” sharp edges. When a cancer tissue sample is placed on the chip, it is sliced up into thousands of pieces, which are analyzed in parallel using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). The method allows thousands of independent, picoliter-scale LAMP reactions to be performed in each well, without any need for purification. “Loading the cryopreserved tissue section onto the chip and slicing it into the tiny tissue pixels takes less than 2 minutes. “This is followed by tissue fixation (10 min), permeabilization (30 min), loading of wells with amplification reagents (2 min), and finally on-chip RT-LAMP reaction on a hot plate (45 min),” the authors state. 

“Laser capture microdissection (LCM) followed by downstream purification and amplification has been used in the past to look at specific regions of stained tissue samples and analyze the heterogeneity within the sample,” comments Dr. Kosari, who is an assistant professor of biochemistry and molecular biology at Mayo Clinic. “Our technique is similar to performing more than 5000 LCM steps, with the downstream amplification in a single step on a microchip.”

The researchers compared their spatial amplification technology with an mRNA FISH technology for analyzing tissue sections from the frozen xenograft samples. The results suggested that the spatial RT-LAMP had “a distinct advantage over mFISH in that it provided quantitative expression values spanning over four orders of magnitude in only 2 hours (including the amplification time),” they write. This compared with the 2 days required to perform the mFISH experiments, “and that is without taking into account the considerable time that was required to capture and process mFISH images,” they authors add. “These important advantages make microchip amplification a suitable technique for a wide range of research and clinical applications.”

The researchers envisage that their technology will help clinicians study spatial gene expression in clinical samples, improve surgery by helping to determine tumor margins, and help to understand how tumors grow. “If you were studying tumor microenvironments, you'd want to know what genes are expressed at a specific location of the tumor,” Kosari points out. “This ability to look at localized expression of genes is currently done with FISH, but our technique is a lot faster and more quantitative.”

“The technique presented here can be tuned to perform quantitative spatially mapped nucleic acid analysis of any tissue sample type on a simple hot plate and a fluorescence reader,” the authors write. They project that the technology could also be integrated into a portable setup using a smartphone and in-built heater. “We anticipate that this technique, with its ease of use, fast turnaround, and quantitative molecular outputs, would become an invaluable tissue analysis tool for researchers and clinicians in the biomedical arena.”