Flow Cytometry-based Method for Efficient Sorting of Senescent Cells.

Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-ß-galactosidase (SA-ß-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-ß-D-galactopyranoside (C12FDG), a fluorigenic substrate of ß-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents. Graphical abstract.

Macrophage-Colony-Stimulating Factor Receptor Enhances Prostate Cancer Cell Growth and Aggressiveness In Vitro and In Vivo and Increases Osteopontin Expression.

Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of spp1 transcript encoding for osteopontin, a key player in cancer development and metastasis. Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.

Suppression of mucosal Th17 memory responses by acellular pertussis vaccines enhances nasal Bordetella pertussis carriage.

Pertussis has made a spectacular rebound in countries that have switched from whole-cell (wPV) to acellular pertussis vaccines (aPV). Here, we show that, unlike wPV, aPV, while protective against lung colonization by Bordetella pertussis (Bp), did not protect BALB/c mice from nasal colonization, but instead substantially prolonged nasal carriage. aPV prevented the natural induction of nasal interleukin-17 (IL-17)-producing and interferon-γ (IFN-γ)-producing CD103+ CD44+ CD69+ CD4+-resident memory T (TRM) cells. IL-17-deficient, but not IFN-γ-deficient, mice failed to clear nasal Bp, indicating a key role of IL-17+ TRM cells in the control of nasal infection. These cells appeared essential for neutrophil recruitment, crucial for clearance of Bp tightly bound to the nasal epithelium. Transfer of IL-17+ TRM cells from Bp-infected mice to IL-17-deficient mice resulted in neutrophil recruitment and protection against nasal colonization. Thus, aPV may have augmented the Bp reservoir by inhibiting natural TRM cell induction and neutrophil recruitment, thereby contributing to the pertussis resurgence.

Expression of CD300lf by microglia contributes to resistance to cerebral malaria by impeding the neuroinflammation.

Genetic mapping and genome-wide studies provide evidence for the association of several genetic polymorphisms with malaria, a complex pathological disease with multiple severity degrees. We have previously described Berr1and Berr2 as candidate genes identified in the WLA/Pas inbreed mouse strain predisposing to resistance to cerebral malaria (CM) induced by P. berghei ANKA. We report in this study the phenotypic and functional characteristics of a congenic strain we have derived for Berr2WLA allele on the C57BL/6JR (B6) background. B6.WLA-Berr2 was found highly resistant to CM compared to C57BL/6JR susceptible mice. The mechanisms associated with CM resistance were analyzed by combining genotype, transcriptomic and immune response studies. We found that B6.WLA-Berr2 mice showed a reduced parasite sequestration and blood-brain barrier disruption with low CXCR3+ T cell infiltration in the brain along with altered glial cell response upon P. berghei ANKA infection compared to B6. In addition, we have identified the CD300f, belonging to a family of Ig-like encoding genes, as a potential candidate associated with CM resistance. Microglia cells isolated from the brain of infected B6.WLA-Berr2 mice significantly expressed higher level of CD300f compared to CMS mice and were associated with inhibition of inflammatory response.

Pyrrolomycins Are Potent Natural Protonophores.

The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.

s-SHIP Promoter Expression Identifies Mouse Mammary Cancer Stem Cells.

During normal mammary gland development, s-SHIP promoter expression marks a distinct type of mammary stem cells, at two different stages, puberty and early mid-pregnancy. To determine whether s-SHIP is a marker of mammary cancer stem cells (CSCs), we generated bitransgenic mice by crossing the C3(1)-SV40 T-antigen transgenic mouse model of breast cancer, and a transgenic mouse (11.5kb-GFP) expressing green fluorescent protein from the s-SHIP promoter. Here we show that in mammary tumors originating in these bitransgenic mice, s-SHIP promoter expression enriches a rare cell population with CSC activity as demonstrated by sphere-forming assays in vitro and limiting dilution transplantation in vivo. These s-SHIP-positive CSCs are characterized by lower expression of Delta-like non-canonical Notch ligand 1 (DLK1), a negative regulator of the Notch pathway. Inactivation of Dlk1 in s-SHIP-negative tumor cells increases their tumorigenic potential, suggesting a role for DLK1 in mammary cancer stemness.

s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells.

Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin- CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells.

Enrichment of Human Stem-Like Prostate Cells with s-SHIP Promoter Activity Uncovers a Role in Stemness for the Long Noncoding RNA H19.

Understanding normal and cancer stem cells should provide insights into the origin of prostate cancer and their mechanisms of resistance to current treatment strategies. In this study, we isolated and characterized stem-like cells present in the immortalized human prostate cell line, RWPE-1. We used a reporter system with green fluorescent protein (GFP) driven by the promoter of s-SHIP (for stem-SH2-domain-containing 5'-inositol phosphatase) whose stem cell-specific expression has been previously shown. We observed that s-SHIP-GFP-expressing RWPE-1 cells showed stem cell characteristics such as increased expression of stem cell surface markers (CD44, CD166, TROP2) and pluripotency transcription factors (Oct4, Sox2), and enhanced sphere-forming capacity and resistance to arsenite-induced cell death. Concomitant increased expression of the long noncoding RNA H19 was observed, which prompted us to investigate a putative role in stemness for this oncofetal gene. Targeted suppression of H19 with siRNA decreased Oct4 and Sox2 gene expression and colony-forming potential in RWPE-1 cells. Conversely, overexpression of H19 significantly increased gene expression of these two transcription factors and the sphere-forming capacity of RWPE-1 cells. Analysis of H19 expression in various prostate and mammary human cell lines revealed similarities with Sox2 expression, suggesting that a functional relationship may exist between H19 and Sox2. Collectively, we provide the first evidence that s-SHIP-GFP promoter reporter offers a unique marker for the enrichment of human stem-like cell populations and highlight a role in stemness for the long noncoding RNA H19.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123(low)) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123(high) progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123(low) cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123(low) cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123(high) cells to the Rh123(low) phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123(low) cells. Moreover, the Rh123(low) cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123(low) melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123(low) G0 cells promotes the exit of cycling cells from the stem cell compartment.

Asymmetric distribution of epidermal growth factor receptor directs the fate of normal and cancer keratinocytes in vitro.

Cancer cells are unequal in a tumor mass and in established cultures. This is attributable to cancer stem cells with the unique ability to self-renew and to generate differentiating progeny. This ability is controlled at the level of asymmetric division by mechanisms that are yet not well defined. We found that normal and cancer keratinocyte fate was linked to the asymmetric distribution of epidermal growth factor receptor (EGFR) during mitosis. Although essential for epithelial cell proliferation, differentiation, and survival, this receptor was not present on the surface of cells satisfying criteria for stem cells such as quiescence, competence to produce functionally distinct daughters, high proliferative and clonogenic potential, sphere formation ability, and expression of stem cell markers. In contrast, keratinocytes displaying EGFR acquired a more differentiated phenotype, suggesting that EGFR may be involved in a switch from stem to transient amplifying cell fate. This switch was associated with changes in the expression profile of cell cycle, survival, and mitochondria controlling proteins that varied between normal and cancer cells. In conclusion, it appears that an unequal distribution of EGFR at mitosis controls keratinocyte fate by balancing quiescence and cycling of EGFR(-) cells, clearly malfunctioning in cancer. We believe that our findings provide mechanistic insights into the development of resistance to anti-EGFR therapies.