Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effects on the migration of T-cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected Treg recruitment. These data suggest while age-related physical changes in the ECM can promote tumor cell motility, they may adversely impact the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older melanoma patients.

Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and non-specific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400x) signal amplification. ClampFISH probes form a “C” configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bioorthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples.

Even amongst genetically identical cancer cells, therapy resistance often only emerges from a very small subset of those cells. Much effort has gone into uncovering the molecular differences in rare individual cells in the initial population that may allow certain cells to become therapy resistant; however, comparatively little is known about variability in the resistant outcomes themselves. Here, we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically, and functionally distinct fate types. These different resistant types are largely predetermined by molecular differences between cells before addition of drug and not by extrinsic cell-specific microenvironmental factors. Changes in dose and kind of drug can, however, switch the resistant fate type of an initial cell, even resulting in the generation and elimination of certain fate types. Diversity in resistant fates was observed across several single-cell-derived cancer cell lines and types treated with a variety of drugs. Cell fate diversity as a result of variability in intrinsic cell states may be a generic feature of response to external cues.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.genome engineering | conditional-null

No abstract