Mohamed I. Gatie

Plasticity transitions during carcinoma progression generate fetal-like progenitor states with metastatic capacity. How these progenitors emerge and persist during tumor progression remains unclear. Here, we elucidate a process that drives the emergence of SOX2⁺ metastatic progenitors in lung adenocarcinomas (LUAD). LUAD cells at the tumor invasive front and distant metastases express the cell adhesion molecule L1CAM, a marker of regenerative epithelial progenitors and a mediator of cell-basement membrane and cell-cell interactions, as well as the proliferation of extravasated micrometastatic cells. We now identify a distinct and broader role of L1CAM as promoter of the SOX2⁺ LUAD progenitor state. We show that L1CAM at cell-cell interfaces promotes the assembly of the planar cell polarity (PCP) complex in metastatic LUAD progenitors. L1CAM-dependent PCP acting through a non-canonical WNT signaling activates c-Jun, which cooperates with the chromatin remodeling factor CHD1 to drive SOX2 expression and metastatic activity. This axis sustains the tumor-initiating and regenerative capacity of LUAD progenitor cells. By illuminating the role of L1CAM and PCP signaling in the generation of SOX2⁺ LUAD progenitors, our findings identify potential new targets to treat metastatic cancer.

Tissue-resident macrophages (TRM) are critical for mammalian organismal development and homeostasis. Here we report that with-no-lysine 1 (WNK1) controls myeloid progenitor fate, with Csf1r ^iCre-mediated Wnk1 deletion in mice (WNK1-deficient mice) resulting in loss of TRMs and causing perinatal mortality. Mechanistically, absence of WNK1 or inhibition of WNK kinase activity disrupts macrophage colony-stimulating factor (M-CSF)-stimulated macropinocytosis, thereby blocking mouse and human progenitor and monocyte differentiation into macrophages and skewing progenitor differentiation into neutrophils. Treatment with PMA rescues macropinocytosis but not macrophage differentiation of WNK-inhibited progenitors, implicating that M-CSF-stimulated, macropinocytosis-induced activation of WNK1 is required for macrophage differentiation. Finally, M-CSF-stimulated macropinocytosis triggers WNK1 nuclear translocation and concomitant increased protein expression of interferon regulatory factor (IRF)8, whereas inhibition of macropinocytosis or WNK kinase activity suppresses IRF8 expression. Our results thus suggest that WNK1 and downstream IRF8-regulated genes are important for M-CSF/macropinocytosis-mediated regulation of myeloid cell lineage commitment during TRM development and homeostasis.

A key challenge of analyzing data from high-resolution spatial profiling technologies is to suitably represent the features of cellular neighborhoods or niches. Here we introduce the covariance environment (COVET), a representation that leverages the gene–gene covariate structure across cells in the niche to capture the multivariate nature of cellular interactions within it. We define a principled optimal transport-based distance metric between COVET niches that scales to millions of cells. Using COVET to encode spatial context, we developed environmental variational inference (ENVI), a conditional variational autoencoder that jointly embeds spatial and single-cell RNA sequencing data into a latent space. ENVI includes two decoders: one to impute gene expression across the spatial modality and a second to project spatial information onto single-cell data. ENVI can confer spatial context to genomics data from single dissociated cells and outperforms alternatives for imputing gene expression on diverse spatial datasets.

The regulation of proteins through the addition and removal of O-linked β-N-acetylglucosamine (O-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripotency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through O-GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global O-GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global O-GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding O-GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low O-GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high O-GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an O-GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global O-GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global O-GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage.

Metabolism plays a crucial role for cell survival and function; however, recent evidence has implicated it in regulating embryonic development. In the embryo, the inner cell mass undergoes orchestrated cellular divisions resulting in the formation of pluripotent epiblast stem cells and primitive endoderm cells. However, both lineages can be captured in vitro as embryonic stem (ES) cells and extraembryonic endoderm (XEN) cells. Concomitantly, changes in the metabolic profile occurs during development, and are well documented in the embryonic lineages. However, a comprehensive multi-omic analysis of these features in XEN cells remains lacking. We observed that mouse XEN cells exhibited high sensitivity to glycolytic inhibition in addition to maintaining elevated intra- and extracellular lactate levels in vitro. Extraembryonic endoderm cells maintain high lactate levels by increased LDHA activity, and re-routing pyruvate away from the mitochondria resulting in reduced mitochondrial activity due to disruptions in electron transport chain stoichiometry. Importantly, exogenous lactate supplementation or promoting intracellular lactate accumulation enhances XEN differentiation in vitro. These results highlight how lactate contributes to XEN differentiation in vitro and may serve to enhance reprogramming efficiency of cells used for regenerative medicine.

Summary: Metabolism plays a crucial role for cell survival and function; however, recent evidence has implicated it in regulating embryonic development. The inner cell mass undergoes orchestrated cellular divisions resulting in the formation of embryonic stem cells and extraembryonic endoderm (XEN) cells. Concomitantly, changes in the metabolic profile occurs during development and are well-documented in the embryonic lineages. However, a comprehensive multi-omics analysis of these features in XEN cells remains lacking. We observed that feeder-free XEN cells exhibited high sensitivity to glycolytic inhibition in addition to maintaining elevated intra- and extracellular lactate levels. XEN cells maintain high lactate levels by increased LDHA activity and re-routing pyruvate away from the mitochondria. Importantly, exogenous lactate supplementation or promoting intracellular lactate accumulation enhances XEN differentiation in vitro . Our results highlight how lactate contributes to XEN differentiation in the mammalian embryo and may serve to enhance reprogramming efficiency of cells used for regenerative medicine. Highlights: Feeder-free XEN cells exhibit high sensitivity to glycolytic inhibition Distinct transcriptomic, proteomic and metabolomic profile exists between feeder-free ES and XEN cells Elevated intracellular and extracellular lactate is observed in feeder-free XEN cells Lactate enhances feeder-free XEN differentiation in vitro

PARP2 belongs to a family of proteins involved in cell differentiation, DNA damage repair, cellular energy expenditure, chromatin modeling and cell differentiation. In addition to these overlapping functions with PARP1, PARP2 participates in spermatogenesis, T-cell maturation, extraembryonic endoderm formation and adipogenesis. The function(s) of PARP2 is far from complete, and the mechanism(s) by which the gene and protein are regulated are unknown. In this study, we found that two different mechanisms are used in vitro to regulate PARP2 levels. In the presence of serum, PARP2 is degraded through the ubiquitin-proteasome pathway, however, when serum is removed, PARP2 is rapidly sequestered into an SDS- and urea-insoluble fraction. This sequestration is relieved by serum in a dose-dependent manner, and again PARP2 is detected by immunoblotting. Furthermore, and despite the presence of a putative serum response element in the PARP2 gene, transcription is not affected by serum deprivation. These observations that PARP2 is tightly regulated by distinct pathways highlights the critical roles PARP2 plays under different physiological conditions.

Glucose metabolism has a crucial role for providing substrates required to generate ATP and regulate the epigenetic landscape. We reported that F9 embryonal carcinoma stem-like cells require cytosolic reactive oxygen species to differentiate into extraembryonic endoderm; however, mitochondrial sources were not examined. To extend these studies, we examined the metabolic profile of early and late-passage F9 cells, and show that their ability to differentiate is similar, even though each population has dramatically different metabolic profiles. Differentiated early-passage cells relied on glycolysis, while differentiated late-passage cells transitioned towards oxidative phosphorylation (OXPHOS). Unexpectedly, electron transport chain protein stoichiometry was disrupted in differentiated late-passage cells, whereas genes encoding mitofusion 1 and 2, which promote mitochondrial fusion and favor OXPHOS, were upregulated in differentiated early-passage cells. Despite this, early-passage cells cultured under conditions to promote glycolysis showed enhanced differentiation, whereas promoting OXPHOS in late-passage cells showed a similar trend. Further analysis revealed that the distinct metabolic profiles seen between the two populations is largely associated with changes in genomic integrity, linking metabolism to passage number. Together, these results indicate that passaging has no effect on the potential for F9 cells to differentiate into extraembryonic endoderm; however, it does impact their metabolic profile. Thus, it is imperative to determine the molecular and metabolic status of a stem cell population before considering its utility as a therapeutic tool for regenerative medicine.

Mouse F9 cells differentiate into primitive extraembryonic endoderm (PrE) when treated with retinoic acid (RA), and this is accompanied by an up-regulation of Gata6. The role of the GATA6 network in PrE differentiation is known, and we have shown it directly activates Wnt6. Canonical Wnt/β-catenin signaling is required by F9 cells to differentiate to PrE, and this, like most developmental processes, requires input from one or more additional pathways. We found both RA and Gata6 overexpression, can induce the expression of Indian Hedgehog (Ihh) and a subset of its target genes through Gli activation during PrE induction. Chemical activation of the Hh pathway using a Smoothened agonist (SAG) also increased Gli reporter activity, and as expected, when Hh signaling was blocked with a Smoothened antagonist, cyclopamine, this RA-induced reporter activity was reduced. Interestingly, SAG alone failed to induce markers of PrE differentiation, and had no effect on Wnt/β-catenin-dependent TCF-LEF reporter activity. The expected increase in Wnt/β-catenin-dependent TCF-LEF reporter activity and PrE markers induced by RA was, however, blocked by cyclopamine. Finally, inhibiting GSK3 activity with BIO increased both TCF-LEF and Gli reporter activities. Together, we demonstrate the involvement of Hh signaling in the RA-induced differentiation of F9 cells into PrE, and while the activation of the Hh pathway itself is not sufficient, it as well as active Wnt/β-catenin are necessary for F9 cell differentiation.