NanoString are one of the most innovative technology companies out there. They’ve built a great reputation for understanding the limitations of existing technologies and developing platforms that help researchers ask more powerful questions. Ahead of this year’s Association for Molecular Pathology meeting in San Antonio we caught up with Niro Ramachandran, Senior Director of Product Management for 3D Biology Products, to talk about their latest revolution the GeoMx Digital Spatial Profiler. If you’ve been part of the Technology Access Program, then you’ll already know why this is so exciting. If you haven’t heard about it yet, then you should make sure you’re sitting down – yes it really is that exciting.

Niro Ramachandran PhD is currently the Senior Director of Product Management for 3D Biology Products at Nanostring.

FLG: As it’s not commercially available yet, could you talk us through what the GeoMx Digital Spatial Profiler is?

NR: The system integrates two very conventional technologies and creates something unique out of them. Essentially what we’ve done is integrated standard 4-color imaging technology with standard barcoding chemistry that allows us to perform highly multiplexed expression profiling. The combination offers some unique capabilities that allow scientists to probe very specific regions of the tissue, guided by the imaging technology. It then allows them to layer on the profiling capabilities to understand the biology in specific regions of tissues that contain heterogeneity. This ability uniquely defines what Digital Spatial Profiling (DSP) is.

 

FLG: It’s a very different approach to traditional methods – what was the thinking behind the instrument during development?

NR: The problem we are trying to solve is sample heterogeneity. Heterogeneity within a sample or within a patient population may predict if a patient responds to a particular therapy.  If heterogeneity holds the key, then what is the best approach to unlock the biological insight?

There are a number potential approaches. One could imagine using purely an imaging-based solution or a bulk profiling solution. The challenge with multiplexed imaging is that you generate a series of analog images that need be to converted to numbers in order to quantify sample heterogeneity. This can often be error prone and limited to a few analytes given the time-consuming effort of performing highly multiplexed imaging experiments. Conversely, digital bulk expression profiling can rapidly generate quantitative data across many samples but fails to capture the sample heterogeneity at the same resolution.  Emerging approaches rely on dissociating cells from tissues and mapping heterogeneity by sequencing RNA from sub populations of single cells. Dissociating cells and profiling one cell at a time may be able to more accurately estimate sample heterogeneity, but the approach is often limited to fresh tissue samples and does not reveal where those specific cells are located in the tissue.

A combined approach that relies on imaging chemistry for resolution and profiling chemistry for scale may ideally address this dilemma of accurately assessing sample heterogeneity. Imaging will enable the visual identification of regions of interest that are likely to be heterogenous. The profiling enables those same heterogenous regions to be deeply characterized. Thus, truly mapping the heterogeneity within a tissue. This can be achieved at reasonable level of throughput to support translational research.

 

FLG: Researchers have been able to work with the output of the GeoMx DSP through your Technology Access Program. I know you guys work very closely with your customers – what’s the main feedback been so far, and what are people typically using it for?

NR: GeoMx DSP can be applied to discover biomarkers, understand mechanism of action and/or discovery new therapeutic targets. Across all of these application categories, the basic question has been sample heterogeneity – “How can I use GeoMx DSP to map out sample heterogeneity in a way that allows me to discover and explain new biology?” Today, through the Digital Spatial Profiling Technology Access Program (TAP) we’ve done over 60 projects with over a thousand samples. One way to measure the productivity of the Technology Access Program is based on publications. Recently, work done by two independent academic centers, Netherlands Cancer Institute and MD Anderson Cancer Center (MDACC), were published in Nature Medicine. We expect to see numerous publications in 2019 that have been driven by the work done through DSP TAP.

 

The DSP TAP provides a unique experience to customers. There are no platforms out there that combine imaging and profiling in one instrument or one solution that allows researchers to ask biological questions in a new way. Most customers who have experienced GeoMx DSP through the TAP program will find that it truly stimulates the scientific curiosity in any researcher. Witnessing each user explore and evolve their thinking has been rewarding.  From this experience, we have identified 5 modalities in which one could use GeoMx DSP to assess sample heterogeneity.

  1. Geometric profiling: Regions of interest can be highlighted using standard shapes like circles, squares etc of varying sizes to assess sample heterogeneity.
  2. Segment profiling: Homogeneous regions within a tissue can be teased apart from one another based on fluorescent staining and profiled with nearly 100% cellularity. For example, the tumor and immune regions though interwoven in the tissue could be separately profiled.
  3. Rare Cell profiling: Rare cells that may disproportionately contribute to biology could be identify by fluorescent staining and profiled.
  4. Contour profiling: Radiating regions with fixed or variable distance from feature of interest could be identified and profiled. For example, the invasive immune margin could be profiled with respect to distance from the tumor region.
  5. Gridded profiling: An unbiased approach of gridding and systematically sampling the tissue.

 

FLG: You mentioned a couple of papers in Nature Medicine?

NR: The two publications that came out in Nature Medicine were seminal publications for DSP and convey truly the power of DSP in multiple fronts. These publications came from the Netherlands Cancer Institute by Christian Blank and the second study was done by Jennifer Wargo, James Allison, and others at MD Anderson Cancer Center. These were two independent studies performed on melanoma clinical cohorts focused on understanding the impact of combination immuno-therapy in adjuvant and/or neoadjuvant settings. This study revealed two unmet clinical needs that are well served by GeoMx DSP. First, the biopsies collected from these patients are of limited quantity therefore it becomes increasingly important to rely on highly multiplexed methodologies that only require a single 5-micron section. Second, these early stage patients are less tolerant of toxicities, therefore discovering highly predictive markers becomes that much more important.

Surprisingly, across these two independent studies, GeoMx revealed similar biomarkers demonstrating showing biological congruence that can be captured by the precision of a digital platform. Moreover, the markers discovered by GeoMx DSP revealed an unexpected connection that B cell biology may play a role in response to therapy.  

 

 

FLG: The importance of quantitative data is fairly well understood. What are the big advantages of having the spatial context to go with it?

NR: It is becoming increasingly clear that the context in which a cell exists influences the cells behaviour. The growing body of research in immuno-oncology continues to emphasize the importance of spatial information. Fundamental questions in immuno-oncology stem from understanding how the various immune cells organize themselves and respond to tumor cells. There are number of spatial factors that affect cellular function and response to therapy. For example, the composition of immune cell population in the tissue may predict how responsive the patient might be to immunotherapy; moreover, the distance between the immune cells and the tumor cells may also impact how responsive the immune cells are the tumor cells.

This phenomenon is not limited to immuno-oncology as you see similar effects anytime a tissue is embedded with a complex milieu of cell types. Large initiatives like the Human Cell Atlas are designed to capture complex biology that arise from cell networks that are often lost in traditional profiling assays.

 

FLG: One of the remarkable things about the system is the ability to reuse the sample. How big of a bonus is that? 

NR: Since GeoMx DSP uses light to interrogate the sample; the sample is not consumed during the experiment. This presents an opportunity for two downstream use cases for the preserved sample. First, users can perform sequential probing of the same tissue with different panels to increase the scope of their discovery study.  Though GeoMx DSP allows you to probe large number of targets in a single cycle, there may be use cases where a sequential processing maybe required for follow on discovery studies. Second, this feature lends itself nicely to perform orthogonal studies. One could follow up on GeoMx DSP experiment by performing a H&E stain on the same smapl or perhaps perform a sequencing run to determine the genotype of this particular sample. This capability provides a lot of options for the end user.

 

FLG: NanoString are developing a great reputation for producing technologies that allow researchers to ask questions of their samples that just weren’t possible before. When it comes to Immuno-Oncology and Neuroscience, what are some of the questions that Digital Spatial Profiling makes possible and what will their answers mean for translational science?

NR: Functionally speaking, with GeoMx DSP, there are three experimental variables to consider depending on what biological question you are asking. First is to identify the morphology (imaging) reagents that dictate which part or parts of the tissue are of interest to the biological question at hand. Second is the Region of Interest (ROI) selection strategy.  Selecting among one of the 5 ROI selection modalities (Geometric, Segment, Rare cell, Contour or Gridding) will reveal the resolution of sampling that is required to assess sample heterogeneity. Lastly, target selection for profiling. Selecting analyte class (RNA and/or protein) and biological relevance (Neuroscience, Oncology, Immunology etc) will reveal the last piece of the puzzle with respect to what biological question is being asked and answered.

The most desired application in immuno-oncology has been to stain tissue with tumor markers (PanCk) and immune markers (CD45) and perform Segment profiling with highly multiplexed RNA (>80 plex) and protein IO (>40 plex) panel.  With segmentation profiling, a mask is applied to the tumor region and different mask is applied to the tumor micro-environment. Using these masks, the tumor region can be profiled separately from the tumor micro-environment to maximize cellularity. This allows for high resolution mapping of unique tumor and tumor microenvironment molecular profiles across many patient samples.

The most desired application in neuroscience has been to profile the amyloid plaque microenvironment in brain tissue using Contour profiling with the plaque as a central structure. The plaque is identified (pan amyloid beta), and concentric regions are mapped and profiled at various distance radiating out from the plaque. This allows users to understand the plaque microenvironment and how the immune response changes in distinct cohorts of patients.

 

FLG: You’ll be showing off the GeoMx Digital Spatial Profiler at AMP next month, in San Antonio. What do you guys have planned for the audience there?

NR: There are three studies that we will highlight at AMP. First, we will focus on the process for validating antibodies to be used on the GeoMx DSP platform. Antibody validation has been a topic of discussion for many decades, however in spatial analysis, the challenges with antibody validation are exacerbated. There are no established standards for validating antibodies for highly multiplexed spatial analysis. Therefore, we have chosen to establish a process and standards by which antibodies are qualified for the assay. We expect this to start a dialogue on how this can be best done as a scientific community. Second, we will highlight our flagship application, segment profiling, where the tumor and tumor micro-envirment are optically dissected and analysed with near 100% cellularity. Lastly, Dr. Bernard Fox from Providence, in collaboration with Nanostring and Definiens, will highlight the power of leveraging state of the art image analysis algorithms with GeoMx DSP to increase the biological insights that can be uncovered. This case study will focus on samples collected from patents with mesothelioma to show how GeoMx DSP and Definiens’ Immuno-Oncology Profiling can be combined to spatially characterise the interface between mesothelioma cells, stroma and immune cells in the tumor micro-environment in a high-plex, digital capacity. This allows us to harvest the know-how and the technologies of the field to really be able to amplify the type of biology that can be discovered on a platform like this.

 

Niro Ramachandran

Niro Ramachandran, PhD, is currently the Senior Director of Product Management for 3D Biology Products at Nanostring. Prior to this, Niro led product development for the protein analysis portfolio of consumables, devices and bench-top instrumentation at Life Technologies. Niro has a strong interest in developing platform technologies for protein biomarker discovery. He is the inventor of in situ planar protein arrays and has developed a variety of high content protein products on planar arrays, bead arrays and label free platforms. Niro is a graduate of the University of Toronto and received his PhD from the University of Windsor in Canada. Niro completed his post doctorate at the Harvard Institute of Proteomics, Harvard Medical School.