Medicine is moving from very blunt instruments to molecules that are “the finest scalpel you could ever have.” So says George Church, PhD, professor of genetics at Harvard Medical School (HMS), who has a pretty decent track record when it comes to appraising new genomics technologies. This evolution, Church notes in a Wyss Institute video, has created the need for observational tools that allow researchers to “see at that high level of resolution and comprehensiveness.”

Church is referring to the recent development of spatial transcriptomic technology (known simply as “spatial”). Until now, single-cell sequencing techniques, such as RNA sequencing (RNA-seq), have been limited to tissue-dissociated cells, that is, cells extracted from ground-up tissue. Such cells lose all spatial information.

Spatial transcriptomics gives a rich, spatial context to gene expression. By marrying imaging and sequencing, spatial transcriptomics can map where particular transcripts exist on the tissue, indicating where particular genes are expressed.

The understanding of the spatial context of biology in human specimens has always been critical to our understanding of human disease, notes Church’s HMS colleague David Ting, MD, assistant professor in medicine. Interpretation of histology, he adds, is still an art that makes the anatomic pathologist indispensable for the accurate diagnosis of disease. But these new spatial technologies will help unlock a deeper understanding of what is happening in tissue, which will be applicable to most areas of biomedical research.

Elana J. Fertig, PhD

With spatial transcriptomics, “not only can you characterize what is there, but now you can go one step further to see how cells are interacting,” notes Elana J. Fertig, PhD, associate professor of oncology, applied mathematics and statistics, and biomedical engineering, Johns Hopkins University.

“We used to attempt to do this in cancer biology, using laser capture microdissection and bulk RNA-seq data,” Fertig adds, “by cutting out regions and profiling them.” But those expedients still averaged gene expression data from individual cells, losing fine-grained information about cell-state transitions that could have been used to clarify cell-to-cell interactions.

Now, contextual information that used to slip away can be captured with spatial transcriptomics. “We see not only where the immune cells are,” Fertig explains, “but what states they are in, and the interaction between the tumor and immune system.” It is a true combination of cellular data and molecular genomics which, she insists, is needed to improve outcomes in cancer biology.

No stranger to the development of new sequencing technologies, Joe Beechem, PhD, the chief scientific officer and senior vice president of research and development at NanoString Technologies, moved from a substantial academic career to industry about 20 years ago. A pioneer of some of the first next-generation sequencing (NGS) instruments, Beechem tells GEN that it is fun to see biology continually reinventing itself in these new ways.

He maintains the way the spatial transcriptomics field is breaking feels “almost identical” to the rush of building the first NGS instruments. “This time,” he declares, “is as big—if not bigger.”

Spatial, meet COVID-19

Researchers are diving into COVID-19 research head first, working at full throttle to elucidate the effect of the SARS-CoV-2 virus on its human host, with many focused naturally on the lung. To understand changes in discrete areas of host tissue, some researchers are turning to spatial transcriptomics.

For example, a group of researchers at Massachusetts General Hospital, including Ting, used spatial transcriptomics, among other techniques, to analyze autopsy specimens from 24 patients who succumbed to COVID-19. The work, notes Ting, has expanded our understanding of SARS-CoV-2 infection “through the lens of histological findings identified by pathologists.”

First, Ting’s group pinpointed the location of the virus using RNA in situ hybridization in lung tissue. Using NanoString’s GeoMx Digital Spatial Profiler, the group was able to analyze the transcriptional and proteomic changes in these areas. The findings corresponded to distinct spatial expression of interferon response genes and immune checkpoint genes, demonstrating the intrapulmonary heterogeneity of SARS-CoV-2 infection.

According to Ting, the data revealed a tremendous interferon response specifically to the regions containing SARS-CoV-2 viral RNA, indicating that this is the dominant immunological response to the virus. In addition, protein analysis in this same region showed upregulation of immune regulatory molecules including PD-L1, CTLA4, and IDO1, which are known to be T-cell suppressive in the context of cancer.

The results have been posted as a preprint, “Temporal and Spatial Heterogeneity of Host Response to SARS-CoV-2 Pulmonary Infection,” on the medRxiv preprint server.

Sarah Warren, PhD, NanoString’s director of translational science, tells GEN that GeoMX was well positioned to support COVID-19 research because the platform had already been established to work with formalin-fixed paraffin embedded (FFPE) specimens—samples of the type being provided by COVID-19 autopsies. Warren asserts that spatial can be implemented to understand the diversity of ways in which SARS-CoV-2 is impacting the different organ systems, which is, she emphasizes, “something that cannot be done with any other platform.”

Another group of researchers is utilizing spatial transcriptomics to probe COVID-19 patient lung tissue. A recent talk from the NIH COVID-19 Scientific Interest Group (SIG) given by Aviv Regev, PhD, former core member at the Broad Institute and recently appointed head of San Francisco–based Genentech Research and Early Development (gRED), highlighted her laboratory’s work to understand the relationship between the virus and host by performing spatial transcriptomics.

Regev presented data from FFPE specimens from the trachea and left upper lobe from a COVID-19 patient. By using the GeoMx to analyze 1,800 RNA targets, including key genes for cells that are infected by the virus, her team was able to analyze and compare the RNA expression of SARS-CoV-2-infected cells versus neighboring, uninfected cells.

According to Regev, images of the FFPE specimens are “terrifying” because they depict how aggressively the virus proliferates and ravages the lungs. But she also noted that the infection “is specific to some lung regions, while other lung regions remain untouched.” Such patterns offer clues to SARS-CoV-2 infection that may help in the battle to stop the pandemic.

A consortium and the next Chromium

When 10x Genomics rolled out its single-cell sequencing platform, the Chromium, the company kept asking its customers what the single-cell world needed. It was by talking to customers at Human Cell Atlas meetings that 10x Genomics fully realized the excitement behind spatial transcriptomics technology.

Ben Hindson, PhD

Ben Hindson, PhD, chief scientific officer and president of 10x Genomics, tells GEN that customer feedback led the company to acquire Spatial Transcriptomics, a Swedish company that took what Hindson describes as a nice, scalable approach. 10x Genomics then incorporated Spatial Transcriptomics’ technology into the Visium Spatial Gene Expression Solution, a product that began shipping in November 2019. Hindson says that adoption has been tremendous since then, and that 10x Genomics is enhancing Visium so that it may use fluorescently labeled antibodies and detect proteins.

A nice thing about Visium, notes Hindson, is that a lot of big equipment is unnecessary. It’s just a microscope slide and a reagent kit. The slide has 5,000 regions, each of which can capture RNA from 1–10 cells at a time. The Visium takes an unbiased approach to profiling the cells, something that Johns Hopkins’ Fertig really appreciates.

Fertig, who uses Visium in her cancer research lab, is a member of the 10x Genomics Visium Clinical Translational Research Network (CTRN). The newly formed group (which has yet to have its first Zoom meeting) has been formed across different disciplines. Fertig says it is really exciting to have researchers from different fields focused on implementing this technology in the clinic.

She explains that the commonalities that exist across fields enable “team science” across disciplines. The CTRN, she adds, gives the researchers a “chance to think in a similar headspace” and suggests “new ways to translate biological discoveries into therapies.” She emphasizes that the CTRN fosters a new research model. Ordinarily, research consortia are centered around a common disease. The CTRN, however, is focused on how a common technology may drive progress against multiple diseases.

Teamwork makes the dream work

The GeoMX and Visium platforms may make spatial transcriptomic experiments more accessible for researchers, but challenges still exist. The first challenge is knowing which areas to study. Without some direction into the region or cells to study, it is nearly impossible, Ting tells GEN, to determine if differences are based on the same cell types in different locations or if they are just driven by the wide variety of cell types inherent to human tissue.

The second challenge is interpreting the increasing amounts of transcriptional and proteomic data. Here, again, dealing with the challenge is a matter of knowing where to look—or asking the right question. “It’s more about knowing what constellations you are looking for in the sky,” Ting explains, “rather than staring at the entire night sky and trying to understand the entire galaxy.”

Yet another challenge is the diversity of skills needed to run a successful spatial transcriptomics experiment. One kind of expertise is needed to obtain the best sample from the most appropriate portion of the tumor; another is needed to implement the long protocol; and yet another is needed to interpret the data.

Coordinating all these kinds of expertise can realize the team science approach described by Fertig. She admits that if scientific teams are to succeed, significant investments in time and effort are needed because “different layers of skills” are required. She adds, however, that the investments can be worthwhile. Not only can the team approach be a very effective way to do science, it can be critical, she stresses, for favorable outcomes.

Neither first nor last

Several spatial transcriptomics technologies are in development. One iteration is known as fluorescent in situ sequencing (FISSEQ). It was first proposed in 2003 by the Church lab, which published a realization of the FISSEQ concept in a 2014 Science paper. The technology was further developed at the Wyss Institute and is now being commercialized by the startup company Readcoor, which provides FISSEQ-based instruments, kits, and software.

A fluorescent in situ sequencing (FISSEQ) technology developed at the Wyss Institute can present 3D images of molecular targets in tissue or cell samples. The technology allows mRNA to stay fixed in its original location while it is converted into DNA amplicons. Then, DNA sequences are revealed using fluorescent dyes and a super-resolution microscope. The technique allows for panomic spatial sequencing that captures cell morphology.

Last February, Readcoor staged a splashy launch of its FISSEQ platform at the 2020 Advances in Genome Biology and Technology meeting. Company representatives announced a Select Release Program and even distributed T-shirts displaying George Church’s face. Since then, the platform’s rollout has been overtaken by events, namely, the disruptions due to the COVID-19 pandemic.

Readcoor tells GEN that the pandemic has “thrown a wrench” into the rollout by delaying or otherwise complicating the company’s installation plans. The size of the wrench is unclear, as Readcoor did not reveal how many customers are still waiting on installation, or even to speak to a more general point, namely, the number of customers that agreed to participate in the Select Release Program. Another area where Readcoor is quiet is on the cost of its platform. By comparison, NanoString has sold over 125 GeoDX systems (with just over 70 installed) at $300,000 per system.

But Evan Daugharthy, PhD, ReadCoor’s vice president of science, is optimistic that the company will “turn a corner soon and be able to deliver our instruments to our customers.” Readcoor affirms that it is on track for full commercial launch of the platform in 2021.

Readcoor is not the first company out of the gate, and it will certainly not be last. Rest assured, Beechem says, there will be multiple new companies every year—“ankle biters,” he calls them—trying to gain a foothold in this space. But Beechem warns that it takes a long time to build a platform. You not only have to have a chemistry that works to create a spatial platform, you also need to marry an imaging platform and a sequencer, which is a multidimensional challenge. Five years ago, he explains, he was spending 90% of his time building the technology.

Regardless of whether the spatial pioneers or the new and emerging companies mentioned in this article are successful, the promise of spatial transcriptomics is profound. This “could lead to a new era,” asserts Church, because it affords investigation into “comprehensive expression and relationships among cells over vast spatial distances.” When you look at the whole set, he notes, you find that the “the place where you didn’t think to look” is way out of proportion. Through comprehensive transcript surveys, there is a better chance of finding the one transcript that is causative—a potential weak point—where a therapeutic might just work.

Two decades after we celebrated our first detailed map of the human genome, it seems that spatial is the next exciting frontier for genomics to conquer.

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