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Examining molecular biology in humans
 
Alex Parker1, Ian McCaffery2, and Scott D. Patterson2
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Our understanding of disease heterogeneity and complex regulatory processes has benefitted tremendously from the field of genomics, which is advancing at a rapid pace. Already, we are beginning to uncover the spectrum of variability in human physiology through association studies that correlate genomic changes with a specific phenotype (6). DNA sequence–based genomics in particular is advancing exponentially; it is not, therefore, unrealistic to consider complete genomic characterization of individuals and even of multiple specific tissues with respect to sequence, methylation, copy number variations (CNVs), chromosomal rearrangement, and DNA-protein interactions in the coming years. In the infectious-disease context, deep-sequencing methods could be deployed to exhaustively catalog the viral, bacterial, and fungal communities inhabiting various areas of an individual's body (7). Similarly, the analyses of RNA, including splice-variants, and sub-cellular and serum/plasma-based proteomics, are undergoing revolutionary changes that will enable extensive, quantitative characterization of much of the molecular activity within a cell. Although many questions have yet to be studied, what is self-evident from current genomics research is that the information encoded by the genome, transcriptome, and proteome is orders of magnitude more complex than was once thought, illuminating how far we are away from completely grasping biological processes.

The first advance that we believe would have a significant impact is the intersection of multiple high-content’ omic data sets (systems biology), which will help us understand the physiological relevance of genomic variations (SNPs, CNVs, etc.) as they relate to cellular processes. This, in turn, will allow us to focus on key analytes that need to be monitored in humans prior to therapy, for design of personalized therapies, and during treatment to monitor response. This approach would allow the development of probes, or more likely combinations of probes, that could be imaged in vivo to report on the function of specific cell types that are drivers of disease, thereby eliminating one of the existing limitations in tissue-based analyses, which rely on infrequent biopsies or archival tissue samples. Farther in the future, we envision comprehensive genomic and physiological analyses at the single-cell level. Currently, measurement of all of the aforementioned properties of interest in a single cell lies beyond our grasp. The ability to fully characterize the nucleic acids or other analytes contained in a single cell, rather than having to examine the average of many cells sampled together, would revolutionize our understanding of the interrelationships of cells in the context of their tissue [both normal and diseased (e.g., malignant tumor cells)]. Being able to simultaneously interrogate the proteome and phospho-proteome of the cell would allow an unprecedented view of the molecular machinery of life at its most fundamental level, representing a significant breakthrough for both disease research and developmental biology.

Consider a tissue section. At present there are a few methods that enable the measurement of specific analytes while preserving their cellular context. Immuno-histochemistry (IHC) has long been able to achieve this aim, and more quantitative approaches are now in routine research use, [for example, laser scanning cytometry (8)]. These technologies, while powerful, have limitations, including sensitivity, restrictions on the range of analytes that can be measured, and limited multiplexing capabilities. Imaging mass spectrometry—although a higher-content platform that has greater spatial resolution—cannot interrogate the entire proteome (9). Other than in situ hybridization or in situ PCR, most genomic analyses require cells to be removed from their tissue context and lysed prior to analysis.

The next level of technological advance we forsee is the capacity to comprehensively query the individual genomic and physiological profiles of a series of cells while they remain in their tissue-level context. This capability would be of particular benefit in the field of oncology. Tumors are known to exhibit varying degrees of heterogeneity among the tumor cells themselves, driven by variegated genomic alterations within the tumor cell population. This phenomenon is believed to drive the course of the disease and the response of the tumor to therapy, including development of acquired resistance. The examination of tumor heterogeneity has to date been limited by the available technologies as described above (10,11). Although studies similar to these cited clearly demonstrate tumor heterogeneity, it will require knowledge of the full complement of genomic aberrations (and the resulting effects on all nucleic acid and protein species) at the level of individual cells within their in vivo tissue context for complete understanding of the disease process and the effective management of therapy. Development of molecular probes that can be multiplexed to a much higher degree than is presently feasible is one way in which this may be accomplished. Development of existing methods of labeling probes for spatial readouts could exploit the broad range of emission from quantum dots (12) or other chemistries, such as more sophisticated resonant energy transfer systems, or molecular entities whose reporter function can be toggled on or off by the presence of, for example, magnetic or radio frequency fields. These chemistries would undoubtedly require more precise and sensitive imaging instruments or other approaches yet to be developed. The ability to achieve such goals in the future does not seem out of the realm of possibility.

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