Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.National Science Foundation (U.S.). Graduate Research FellowshipNational Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)Janssen Pharmaceutical Ltd.Klarman Family Foundatio

Functional odor classification through a medicinal chemistry approach

Crucial for any hypothesis about odor coding is the classification and prediction of sensory qualities in chemical compounds. The relationship between perceptual quality and molecular structure has occupied olfactory scientists throughout the 20th century, but details of the mechanism remain elusive. Odor molecules are typically organic compounds of low molecular weight that may be aliphatic or aromatic, may be saturated or unsaturated, and may have diverse functional polar groups. However, many molecules conforming to these characteristics are odorless. One approach recently used to solve this problem was to apply machine learning strategies to a large set of odors and human classifiers in an attempt to find common and unique chemical features that would predict a chemical’s odor. We use an alternative method that relies more on the biological responses of olfactory sensory neurons and then applies the principles of medicinal chemistry, a technique widely used in drug discovery. We demonstrate the effectiveness of this strategy through a classification for esters, an important odorant for the creation of flavor in wine. Our findings indicate that computational approaches that do not account for biological responses will be plagued by both false positives and false negatives and fail to provide meaningful mechanistic data. However, the two approaches used in tandem could resolve many of the paradoxes in odor perception

Applying medicinal chemistry strategies to understand odorant discrimination

Associating an odorant’s chemical structure with its percept is a long-standing challenge. One hindrance may come from the adoption of the organic chemistry scheme of molecular description and classification. Chemists classify molecules according to characteristics that are useful in synthesis or isolation, but which may be of little importance to a biological sensory system. Accordingly, we look to medicinal chemistry, which emphasizes biological function over chemical form, in an attempt to discern which among the many molecular features are most important for odour discrimination. Here we use medicinal chemistry concepts to assemble a panel of molecules to test how heteroaromatic ring substitution of the benzene ring will change the odour percept of acetophenone. This work allows us to describe an extensive rule in odorant detection by mammalian olfactory receptors. Whereas organic chemistry would have predicted the ring size and composition to be key features, our work reveals that the topological polar surface area is the key feature for the discrimination of these odorants

Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.Janssen Pharmaceutical Ltd.National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Science Foundation (U.S.). Graduate Research FellowshipSingapore. Agency for Science, Technology and ResearchSwiss National Science Foundation (Fellowship for Advanced Researchers PA00P3 139659

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity, using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two prostate cancer patients including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were observed in matched tissue. Moreover, we identified 10 early-trunk and 56 metastatic-trunk mutations in the non-CTC tumor samples and found 90% and 73% of these, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic

Applying medicinal chemistry principles to the Olfactory Code

The mammalian olfactory system is capable of decoding complex mixtures of volatile chemical odorants into identifiable percepts. While the general mode of peripheral signal transduction is largely known, the mechanism relies on a rather complicated combinatorial “olfactory code”, where each of the hundreds of expressed odorant receptors (ORs) detects multiple odorants, and a given odorant in turn activates multiple ORs (Malnic, Hirono et al. 1999). Since the first identification of mammalian ORs in 1991, the deorphanization, i.e. solving of the substrate, of ORs has proven to be a challenge. Many attempts at systematic monitoring of the olfactory code have seen marginal successes for a number of reasons. First of all, there are still no solved structures of mammalian ORs to be used for high throughput computational modeling. Second, experimental validation methods such as heterologous expression still face considerable challenges. Lastly, primary chemical features of odors that allow for OR tuning are not yet defined. The traditional organic chemistry-based classification of odorants fails to predict biological activity, while percept-based computational analyses isolate esoteric descriptors that are difficult to chemically manipulate. Receptor level structure-activity analysis can provide a missing context to the odorant discrimination in the peripheral olfactory system. A critical finding by Manic et al (1999) indicates that each mature olfactory sensory neuron (OSN) only expresses one type of OR, allowing for high throughput screening of carefully crafted odorant panels using dissociated OSN calcium imaging. A few bioisosteric substitutions widely utilized in medicinal chemistry were used to construct odorant panels, showing greater success in defining odorant-OR interaction than previously used organic chemistry-based clustering methods. Among classical substitutions used by medicinal chemists, heteroaromatic ring exchanges are especially well tolerated when heteroatoms with a similar topological polar surface area (TPSA) are used as replacements. Among odorants with differing TPSA, it is likely that an OR activated by analogous odorants at two extremes of the TPSA spectrum will be activated by an odorant with an intermediate TPSA. Flipping of a polar functional group, which is often used with amides in drug target replacements, is well tolerated by the ORs in esters. Furthermore, there is a predictable activation pattern relative to number of carbons in a hydrophobic chain uninterrupted by polar epitopes. Using binary mixtures, the OR activity can be further surveyed through enhancement or inhibition of OSN activation signals. Odorants activating a smaller subset of an OR population may also be binding to a larger subset of ORs, resulting in mixture inhibition. Specifically, this work indicates that extracted odorant fragments may be binding but not activating some of the OR repertoire of the original odorant. The concept of non-classical bioisosteres is applied to the OR repertoire using aliphatic and aromatic aldehydes. It appears that the specialized electronics of a fully conjugated benzene ring can in fact be dispensable, only acting as conformational restrictor of the odorant in most cases. Not only do analogous non-conjugated systems substitute well for benzaldehyde, but so do non-cyclic odorants possessing tiglic moieties. Conformationally restricted extractions act as more faithful replacements for larger molecules in a subset of ORs. While the dissociated OSN results alone have broad implications for binding patterns of GPCRs in general, simple behavioral tests in mice using the same odorant panels indicate concrete perceptual links to medicinal chemistry-based odorant discrimination. The results from the behavioral data suggest that there may be a maximum constraint for percent OSN activation for two sequentially presented odors to be interpreted as the “same”. The results open a window to exploring other medicinal chemistry-based substitutions. Furthermore, many methodological improvements have been made over the past decade to allow for increased efficiency of deorphanization and validation of ORs