Therapy-Induced Evolution of Human Lung Cancer Revealed by Single-Cell RNA Sequencing.

Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes.

Lineage recording in human cerebral organoids.

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

Proteomic changes associated with predator-induced morphological defences in oysters.

Inducible prey defences occur when organisms undergo plastic changes in phenotype to reduce predation risk. When predation pressure varies persistently over space or time, such as when predator and prey co-occur over only part of their biogeographic ranges, prey populations can become locally adapted in their inducible defences. In California estuaries, native Olympia oyster (Ostrea lurida) populations have evolved disparate phenotypic responses to an invasive predator, the Atlantic oyster drill (Urosalpinx cinerea). In this study, oysters from an estuary with drills, and oysters from an estuary without drills, were reared for two generations in a laboratory common garden, and subsequently exposed to cues from Atlantic drills. Comparative proteomics was then used to investigate molecular mechanisms underlying conserved and divergent aspects of their inducible defences. Both populations developed smaller, thicker, and harder shells after drill exposure, and these changes in shell phenotype were associated with upregulation of calcium transport proteins that could influence biomineralization. Inducible defences evolve in part because defended phenotypes incur fitness costs when predation risk is low. Immune proteins were downregulated by both oyster populations after exposure to drills, implying a trade-off between biomineralization and immune function. Following drill exposure, oysters from the population that co-occurs with drills grew smaller shells than oysters inhabiting the estuary not yet invaded by the predator. Variation in the response to drills between populations was associated with isoform-specific protein expression. This trend suggests that a stronger inducible defence response evolved in oysters that co-occur with drills through modification of an existing mechanism.

Transcriptomic responses to extreme low salinity among locally adapted populations of Olympia oyster (Ostrea lurida).

The Olympia oyster (Ostrea lurida) is a foundation species inhabiting estuaries along the North American west coast. In California estuaries, O. lurida is adapted to local salinity regimes and populations differ in low salinity tolerance. In this study, oysters from three California populations were reared for two generations in a laboratory common garden and subsequently exposed to low salinity seawater. Comparative transcriptomics was then used to understand species-level responses to hyposmotic stress and population-level mechanisms underlying divergent salinity tolerances. Gene expression patterns indicate Olympia oysters are sensitive to hyposmotic stress: All populations respond to low salinity by up-regulating transcripts indicative of protein unfolding, DNA damage and cell cycle arrest after sub-lethal exposure. Among O. lurida populations, transcriptomic profiles differed constitutively and in response to low salinity. Despite two generations in common-garden conditions, transcripts encoding apoptosis modulators were constitutively expressed at significantly different levels in the most tolerant population. Expression of cell death regulators may facilitate cell fate decisions when salinity declines. Following low salinity exposure, oysters from the more tolerant population expressed a small number of mRNAs at significantly higher levels than less tolerant populations. Proteins encoded by these transcripts regulate ciliary activity within the mantle cavity and may function to prolong valve closure and reduce mortality in low salinity seawater. Collectively, gene expression patterns suggest sub-lethal impacts of hyposmotic stress in Olympia oysters are considerable and that even oysters with greater low salinity tolerance may be vulnerable to future freshwater flooding events.

Maya children working in the streets: Value mismatches from the village to the street setting.

In the highlands of Chiapas, Mexico, a large number of indigenous Maya people have relocated from their villages to an urban centre where many families, especially mothers and children, work as street vendors. We were interested in the typical cultural expectations for the development of Maya girls, the ways that these expected patterns were interrupted by street work, and the ways that girls and families dealt with this novel situation. In order to provide a more complete picture, we compared the daily experiences of girls who work on the street with those of their male counterparts and with the routines of girls who lived in traditional, rural settings. Our data include ethnographic observations and a census (N = 250-300), informal chats (N = 250-300), and semi-structured interviews with children (N = 51) and adults (N = 25). Using Greenfield's theory of social change and development, we found a coexistence of value matches and mismatches. These addressed adherence to tradition versus embracing ethnic variety and innovation valued in money-based market economies and collective responsibility versus individual choice and expression. This mix of values occurred in the domains of gender roles for work, motivation for street work, leisure time, and schooling.

Developmental effects of economic and educational change: cognitive representation in three generations across 43 years in a Maya community.

We studied the implications of social change for cognitive development in a Maya community in Chiapas, Mexico, over 43 years. The same procedures were used to collect data in 1969-1970, 1991, and 2012-once in each generation. The goal was to understand the implications of weaving, schooling and participation in a commercial economy for the development of visual pattern representation. In 2012, our participants consisted of 133 boys and girls descended from participants in the prior two generations. Procedures consisted of placing colored sticks in a wooden frame to make striped patterns, some familiar (Zinacantec woven patterns) and some novel (created by the investigators). Following Greenfield (2009), we hypothesised that the development of commerce and the expansion of formal schooling would influence children's representations. Her theory postulates that these factors move human development towards cognitive abstraction and skill in dealing with novelty. Furthermore, the theory posits that whatever sociodemographic variable is changing most rapidly functions as the primary motor for developmental change. From 1969 to 1991, the rapid development of a commercial economy drove visual representation in the hypothesised directions. From 1991 to 2012, the rapid expansion of schooling drove visual representation in the hypothesised directions.

Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma.

The neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed an in vivo CRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

Single-cell analyses of axolotl telencephalon organization, neurogenesis, and regeneration.

Salamanders are tetrapod models to study brain organization and regeneration; however, the identity and evolutionary conservation of brain cell types are largely unknown. We delineated the cell populations in the axolotl telencephalon during homeostasis and regeneration using single-cell genomic profiling. We identified glutamatergic neurons with similarities to amniote neurons of hippocampus, dorsal and lateral cortex, and conserved γ-aminobutyric acid-releasing (GABAergic) neuron classes. We inferred transcriptional dynamics and gene regulatory relationships of postembryonic, region-specific neurogenesis and unraveled conserved differentiation signatures. After brain injury, ependymoglia activate an injury-specific state before reestablishing lost neuron populations and axonal connections. Together, our analyses yield insights into the organization, evolution, and regeneration of a tetrapod nervous system.

Human melanocyte development and melanoma dedifferentiation at single-cell resolution.

In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy.

Ageing compromises mouse thymus function and remodels epithelial cell differentiation.

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance.

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers.

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We defined new pathways limiting EGFR-inhibitor response, including WNT/ß-catenin alterations and cell-cycle-gene (CDK4 and CDK6) mutations. Tumor genomic complexity increases with EGFR-inhibitor treatment, and co-occurring alterations in CTNNB1 and PIK3CA exhibit nonredundant functions that cooperatively promote tumor metastasis or limit EGFR-inhibitor response. This study calls for revisiting the prevailing single-gene driver-oncogene view and links clinical outcomes to co-occurring genetic alterations in patients with advanced-stage EGFR-mutant lung cancer.

Burdens and difficulties experienced by caregivers of children and adolescents with schizophrenia-spectrum disorders: a qualitative study.

AIM: The purpose of this qualitative study was to investigate the burdens and difficulties associated with the experience of caring for youth with schizophrenia-spectrum disorders. METHODS: Ten caregivers participated in a modified version of the Knowledge about Schizophrenia Illness interview. RESULTS: The most common areas of general difficulties reported by caregivers were emotional burdens and the everyday practical demands and sacrifices required in caring for their dependents. RESULTS also suggested high levels of burden for caregivers concerning difficulties with mental health services. CONCLUSION: Additional work is needed to learn more about the challenges that caregivers of youth with schizophrenia-spectrum disorders are facing, as well as to develop empirically based strategies for helping these caregivers and their dependents.

Cultural teaching: the development of teaching skills in Maya sibling interactions.

Psychology has considered the development of learning, but the development of teaching in childhood has not been considered. The data presented in this article demonstrate that children develop teaching skills over the course of middle childhood. Seventy-two Maya children (25 boys, 47 girls) ranging in age from 3 to 11 years (M = 6.8 years) were videotaped in sibling caretaking interactions with their 2-year-old brothers and sisters (18 boys, 18 girls). In the context of play, older siblings taught their younger siblings how to do everyday tasks such as washing and cooking. Ethnographic observations, discourse analyses, and quantification of discourse findings showed that children's teaching skills increased over the course of middle childhood. By the age of 4 years, children took responsibility for initiating teaching situations with their toddler siblings. By the age of 8 years, children were highly skilled in using talk combined with manual demonstrations, verbal feedback, explanations, and guiding the body of younger learners. Children's developing competence in teaching helped their younger siblings increase their participation in culturally important tasks.

Cultural pathways through universal development.

We focus our review on three universal tasks of human development: relationship formation, knowledge acquisition, and the balance between autonomy and relatedness at adolescence. We present evidence that each task can be addressed through two deeply different cultural pathways through development: the pathways of independence and interdependence. Whereas core theories in developmental psychology are universalistic in their intentions, they in fact presuppose the independent pathway of development. Because the independent pathway is therefore well-known in psychology, we focus a large part of our review on empirically documenting the alternative, interdependent pathway for each developmental task. We also present three theoretical approaches to culture and development: the ecocultural, the sociohistorical, and the cultural values approach. We argue that an understanding of cultural pathways through human development requires all three approaches. We review evidence linking values (cultural values approach), ecological conditions (ecocultural approach), and socialization practices (sociohistorical approach) to cultural pathways through universal developmental tasks.