Triple-Decker Sandwich Cultures of Intestinal Organoids for Long-Term Live Imaging, Uniform Perturbation, and Statistical Sampling.

Three-dimensional organoid cultures enable the study of stem cell and tissue biology ex vivo, providing improved access to cells for perturbation and live imaging. Typically, organoids are grown in hydrogel domes that are simple to prepare but that lead to non-uniform tissue growth and viability. We recently developed a simple alternative culture method to embed intestinal organoids in multilayered hydrogels, called "triple-decker sandwiches," that align organoids in a common z-plane with uniform access to medium. This culture configuration improves the growth and survival of organoids over a wide working area and facilitates long-term confocal imaging and molecular perturbation. Here, we present protocols for preparing organoids in triple-decker sandwich cultures and using them for live imaging, immunostaining, and single-cell RNA sequencing. We have tested our methods on mouse and human intestinal organoids and expect them to be useful for other highly proliferative three-dimensional cell cultures. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Pre-coating plates with PolyHEMA to prepare them for triple-decker sandwich culture Support Protocol 1: Preparing PolyHEMA solution to coat glass-bottom dishes Basic Protocol 2: Embedding intestinal organoids in triple-decker sandwiches Alternate Protocol 1: Seeding single cells or organoids at low density in triple-decker sandwiches Support Protocol 2: Embedding intestinal organoids in hydrogel domes Support Protocol 3: Production of Wnt3a-conditioned medium Support Protocol 4: Production of Rspo1-conditioned medium Basic Protocol 3: Live imaging of mouse intestinal organoids in triple-decker sandwich cultures Alternate Protocol 2: Live imaging of vital dye-treated mouse intestinal organoids in triple-decker sandwich cultures Basic Protocol 4: Immunofluorescence imaging of mouse organoids liberated from triple-decker sandwich cultures Alternate Protocol 3: Liberating and fixing mouse intestinal organoids from dome cultures Support Protocol 5: Measuring cell proliferation by EdU staining of mouse intestinal organoids Basic Protocol 5: Single-cell RNA sequencing and analysis of mouse intestinal organoids.

Inflation-collapse dynamics drive patterning and morphogenesis in intestinal organoids.

How stem cells self-organize to form structured tissues is an unsolved problem. Intestinal organoids offer a model of self-organization as they generate stem cell zones (SCZs) of typical size even without a spatially structured environment. Here we examine processes governing the size of SCZs. We improve the viability and homogeneity of intestinal organoid cultures to enable long-term time-lapse imaging of multiple organoids in parallel. We find that SCZs are shaped by fission events under strong control of ion channel-mediated inflation and mechanosensitive Piezo-family channels. Fission occurs through stereotyped modes of dynamic behavior that differ in their coordination of budding and differentiation. Imaging and single-cell transcriptomics show that inflation drives acute stem cell differentiation and induces a stretch-responsive cell state characterized by large transcriptional changes, including upregulation of Piezo1. Our results reveal an intrinsic capacity of the intestinal epithelium to self-organize by modulating and then responding to its mechanical state.

Site occupancy calibration of taxane pharmacology in live cells and tissues.

Drug receptor site occupancy is a central pharmacology parameter that quantitatively relates the biochemistry of drug binding to the biology of drug action. Taxanes and epothilones bind to overlapping sites in microtubules (MTs) and stabilize them. They are used to treat cancer and are under investigation for neurodegeneration. In cells, they cause concentration-dependent inhibition of MT dynamics and perturbation of mitosis, but the degree of site occupancy required to trigger different effects has not been measured. We report a live cell assay for taxane-site occupancy, and relationships between site occupancy and biological effects across four drugs and two cell lines. By normalizing to site occupancy, we were able to quantitatively compare drug activities and cell sensitivities independent of differences in drug affinity and uptake/efflux kinetics. Across all drugs and cells tested, we found that inhibition of MT dynamics, postmitotic micronucleation, and mitotic arrest required successively higher site occupancy. We also found interesting differences between cells and drugs, for example, insensitivity of the spindle assembly checkpoint to site occupancy. By extending our assay to a mouse xenograft tumor model, we estimated the initial site occupancy required for paclitaxel to completely prevent tumor growth as 80%. The most important cellular action of taxanes for cancer treatment may be formation of micronuclei, which occurs over a broad range of site occupancies.

Off-patent drugs at brand-name prices: a puzzle for policymakers.

In August 2015, Turing Pharmaceuticals acquired the marketing rights to Daraprim (pyrimethamine), a drug used to treat parasitic infections like malaria and toxoplasmosis. Soon after, Turing caused an uproar when it announced that it would raise the price per tablet of Daraprim from [Formula: see text], a 5500% price hike for a drug that has been on the market for over 60 years and off patent since the 1970s. Old, off-patent drugs are becoming increasingly expensive; Daraprim is the archetypal example. Turing had the power to set a high price for Daraprim because the drug's limited patient population, the absence of competing manufacturers, and a lack of therapeutic alternatives all created an effective monopoly. Similar forces have driven up the prices of other off-patent drugs that treat diseases as diverse as heart failure and multi-drug-resistant tuberculosis. Thus, policymakers will have to consider how the high cost of off-patent drugs impacts public health as well as public spending. In this Note I outline the extent of the high-cost off-patent drug problem, drawing special attention to the problem's negative effects on both health outcomes and government budgets. After discussing some of the problem's underlying causes, I present several solutions to the problem that policymakers could consider, with a focus on proposals like reference pricing and expanded compounding that have received relatively little media attention.

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT.

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.

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.