Dr. Joshua Snyder wants to know what happens on the way to cancer. “What is a pre-cancer — what makes it a pre-cancer?,” the Duke University Assistant Professor of Surgery and Assistant Professor of Cell Biology asks. What gives it a trajectory towards invasive disease? What is the risk that it will develop into an invasive cancer? And ultimately, to translate this research to the clinic: “If we can identify those pre-cancers that develop into an invasive cancer, how do we treat them?”
His lab relies on a multiplicity of techniques to create and study mouse models of human cancers such as colon and breast. For many experiments, they engineer oncogenes into XFP-expressing stem cells which, when introduced into mice, allows clonal expansion and spread to be tracked. A cell-type-specific inducible promoter system allows the team to “control precisely the oncogenes that are expressed, when in time they’re expressed, and then we can visualize their effects on the evolution of disease,” Snyder explains.
The XFP barcoded “Crainbow” (cancer rainbow) mice allow cells expressing a particular oncogene to fluoresce a particular color. The oncogenic proteins themselves are also epitope tagged, facilitating localization within the cell by immunohistochemistry and confocal microscopy, often when a palpable tumor is not yet detectable.
Not surprisingly, different mutations manifest themselves differently, and at different stages of development. For example, in results reported in Nature Communications in December 2019, introducing mutant isoforms to intestinal stem cells of genes thought to increase stem cell fitness leads to clonal spread of the pre-malignant cells throughout the intestine during perinatal development, but not in adults.1 Such fields can grow and spread without being detected, and can (sometimes harmlessly) lay in wait. Yet the process, called “field cancerization”, leaves the animal dramatically more susceptible to future cancers.
On the other hand, oncogenic mutations such as those that extrinsically disrupt the stem cell microenvironment itself — for example by increasing intestinal crypt fission and inhibiting crypt fixation — can spread like wildfire in adult animals. They term this restructuring of the microenvironment and ectopic expansion of proliferative stem cells “microenvironmental oncogenesis”.
To get a handle on what cell types are involved, the researchers relied on Advanced Cell Diagnostics RNAscope® 4-Plex RNA fluorescent in situ hybridization (RNA FISH) to molecularly map the stem cell compartment on paraffin slices of colon. RNA markers for differentiated cells and transiently amplifying cells, for example, were identified fluorescently on the slides and could be co-registered with XFP and immunohistochemical data to provide a more robust understanding of the lesions.
Independent of your genes of interest, RNAscope HiPlex in situ hybridization assays use common reagents and protocols, providing universal assay conditions. Assays allow simultaneous detection of up to 12 different RNA targets per slide-mounted sample.
In other work, the RNAscope HiPlex assay, capable of detecting up to 12 RNA targets – of which Snyder’s lab was an early adopter — was used to molecularly phenotype lesions, with the aim of developing a panel that can ultimately be used prognostically and as a tool for stratification of treatment.
They began by taking single cells from the lesions and subjecting them to RNA sequencing. “We have found in some of our mouse models oncogenic drivers that can help us understand which precancerous [lesions] will become invasive quite quickly,” Snyder says. “We’re looking for cell molecular signatures of these lesions.”
They used the expression profiles of the cells to identify candidate genes affected in these fields by the mutant genes. The idea is to develop a short-list of probes to rely on for screening mouse tissue, “to prove that these molecular signatures are valid for identifying those pre-cancers that will progress into invasive cancer” in the mice, he says. “But then we can hopefully build similar panels … to study these molecular signatures in humans.”
Snyder likens using HiPlex to using TaqMan probes “back in the old days” for real-time PCR, “where we can just pick our favorite genes, send them off, get the probes made, and know that they’re going to work. So it takes any of the design phase out of it.”
Working with a HiPlex panel is technically no different from running the 4-Plex – “it’s very easy to use; it’s very straightforward,” says Snyder. “We’ve been quite happy with it because we can just take the probes, we run them, we scan, we do the imaging, and we move on.”
What the Snyder lab is moving on to “is all about the ability to perform in situ transcriptomics on tumor specimens.” They want to work out the kinks in the mouse models and then build a pipeline that lets them translate what they find to the clinic.
“[The future] is all about the ability to perform in situ transcriptomics on tumor specimens.”.
“Once we identify the signatures that can be used to stratify patients that are at risk of developing invasive disease, we’d like to use those in clinical specimens for patients that have had precancerous lesions resected, and help us understand the likelihood that this will recur,” Snyder explains. That, in turn, may help to answer what kind of therapy they need to receive and how aggressive it needs to be.
“Our technique allows us to model how premalignant cells compete and expand within a field by simple fluorescent imaging, potentially leading to earlier diagnosis and treatment,” Snyder concludes.
1 Boone PG, Rochelle LK, Ginzel JD, Lubkov V, Roberts WL, Nicholls PJ, Bock C, Flowers ML, von Furstenberg RJ, Stripp BR, Agarwal P, Borowsky AD, Cardiff RD, Barak LS, Caron MG, Lyerly HK, Snyder JC. Nat Commun. 2019
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