Spatial biology is the study of tissues—lymph nodes, intestinal crypt, or diseased organs—in the context in which they are found. Cells do not operate in a vacuum, but rather they interact with each other and their environment in two- and three-dimensions. When we dissociate cells for traditional high-parameter interrogation, studying cells in bulk or even individually, we lose the context in which those interactions occur, and thus we are unable to unlock the wealth of information spatial context can provide.
By simultaneously querying multiple biomarkers in intact tissue, we can individuate distinct cell types, sub-types, and even organelles. Activation states, gene expression, and secreted and captured proteins can be surveyed, and the cells’ interactions and the tissue’s organization can be dissected.
Spatial biology can benefit researchers in a host of fields, whether it is the study of basic cell biology, the tumor microenvironment in immuno-oncology, neuroscience, or developmental biology. It allows them to enhance their research by bringing in situ evidence into their program.
Spatial biology is largely defined by the ability to capture and analyze multiple parameters of a single tissue, in situ, in a single data set—termed highly multiplexed tissue imaging (HMTI). One stumbling block to implementing most HMTI technologies is the need for specialized reagents, as well as the steep learning curve associated with adopting a novel technology, thereby limiting their applicability to many research questions.
Imaging mass spectrometry, for example, utilizes multiple isotope-labeled antibodies to stain the tissue. An ion beam then micro-dissects the sample—destroying it in the process—and the microparticles are fed into a mass spectrometer, with subsequent analysis reconstructing an image of the original stained tissue with the location of the targeted antigens.
Another popular technique to examine the distribution of proteins in a tissue sample uses DNA barcode-tagged antibodies to stain the tissue, in a manner similar to multiplexed immunohistochemistry (IHC), followed by a sequencing-based decoding. Here the resolution is at the level of regions of interest, rather than being able to localize the signal to a single cell.
Immunofluorescence (IF)-based HMTI techniques have also been developed. In one of these, barcode-tagged antibodies are detected using fluorescently labeled oligonucleotides complementary to the barcode. In others, commonly available fluorescently labeled primary (or secondary) antibodies are used to stain the tissue. After the tissue is imaged, the sample can be chemically bleached, allowing additional markers to be queried by iterative rounds of staining, imaging, and bleaching. ChipCytometry builds on this, using a less harsh photobleaching to prepare for the next staining cycle, and adding high dynamic range (HDR) imaging to allow data from both dim and bright signals (lowly and highly expressed antigens, respectively) to be captured and quantified simultaneously.
The same fluorescent antibodies that are used for flow cytometry, or IHC, or other IF-based assays can be used for techniques like ChipCytometry as well. The only requirements are that they can be excited in the UV-vis range, that they emit in the UV-vis range, and that they can be easily photobleached—making many of the early generation photolabile fluors, like FITC, PE, and PerCP, ideal. Given the large and growing number of fluorescently labeled antibodies available from trusted vendors, it is often possible to find multiple validated pre-conjugated antibodies against a researcher’s biomarkers of interest, making it easier to add additional targets.
Another advantage of using antibodies common to other gold standard methods like single- and low-plex IF microscopy is that it greatly reduces the effort required to validate spatial biology results, allowing the researcher to build upon prior work. There is an almost direct correlation between the specificity demonstrated by an antibody in single- or low-plex IHC staining and that shown by each individual antibody interrogating ChipCytometry markers sets.
There is a wealth of information to be discovered by understanding the context in which cells interact with each other and their surroundings—knowledge that cannot otherwise be gleaned from studying either bulk tissue or single cells in isolation. By multiplexing the interrogation of several markers simultaneously, HTMI allows for the differentiation of numerous cell types and sub-types, and an understanding of their relationships to each other.
Using techniques and reagents already familiar to users of IHC, and analyses similar to flow cytometry, HTMI platforms like ChipCytometry afford labs an entry into multiplexing and spatial biology, without the need to specialize in either.