Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression, conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.

Summary Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor (mTORki) resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations.

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called “Nucleic Acid Cell Sorting (NACS)”, single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network.

Surface chemistry meets tissue engineering: A novel surface‐patterning approach for creating arrays of DNA squares is combined with a unique method for DNA‐encoding of cells to construct dense arrays of distinct single cells. The cell patterns can be transferred from the substrate surface into thin hydrogel films, and these layers can be stacked to form 3D tissue constructs.

We report on a radiopharmaceutical imaging platform designed to capture the kinetics of cellular responses to drugs. Methods A portable in vitro molecular imaging system, comprised of a microchip and a beta-particle imaging camera, permits routine cell-based radioassays on small number of either suspension or adherent cells. We investigate the response kinetics of model lymphoma and glioblastoma cancer cell lines to [18F]fluorodeoxyglucose ([18F]FDG) uptake following drug exposure. Those responses are correlated with kinetic changes in the cell cycle, or with changes in receptor-tyrosine kinase signaling. Results The platform enables radioassays directly on multiple cell types, and yields results comparable to conventional approaches, but uses smaller sample sizes, permits a higher level of quantitation, and doesn’t require cell lysis. Conclusion The kinetic analysis enabled by the platform provides a rapid (~1 hour) drug screening assay.

The ability to arrange distinct cells in specific, predefined patterns at single-cell resolution could have broad applications in tissue engineering, cell-based assays, cell and tissue cocultures, and fundamental studies of cell-cell interactions. In particular, the proper functioning of engineered constructs for tissue and organ transplantation requires positioning different cell types in anatomically precise arrangements that mimic their microarchitectural configurations in native tissues. Existing methods for micrometer-scale cell patterning include microcontact printing, [1] inkjet printing, [2,3] elastomeric stencils, [4] dielectrophoresis, [5,6] and others. [7,8] The major limitation of most existing platforms, however, is that either they cannot be straightforwardly scaled for highly multiplexed cell patterning, or they do not offer single-cell patterning precision and control, or both.We report on the microfluidic-guided flow-patterning of surfaces as an avenue for constructing tissues. While micro-fluidic channels have been previously utilized to spatially control the deposition of biomolecules onto a surface as one-dimensional stripes, [9] patterns of arbitrary, two-dimensional complexity at single cell resolution are more challenging. We report on three advances to achieve such patterns. First, we execute two sequential steps of microfluidic flow patterning, one at right angles with the other, such that the intersections of the flow patterned DNA stripes constitute an n × m array of unique elements, where n and m are the respective numbers of microchannels utilized for each of the two flow patterning steps. Second, the set of ssDNA oligomers used in the second flow-patterning step is designed so that each of the resulting n × m array elements has a unique molecular identity.Third, we introduce a cell-surface marker-independent chemistry for labeling cells to enable specific cells to be captured at specific spots within the n × m array (Figure 2). These advances, when integrated together, enable the construction of tissues with architectural precision at the single cell level.

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.