Cell-cell communication enhances the capacity of cell ensembles to sense shallow gradients during morphogenesis

Collective cell responses to exogenous cues depend on cell-cell interactions. In principle, these can result in enhanced sensitivity to weak and noisy stimuli. However, this has not yet been shown experimentally, and, little is known about how multicellular signal processing modulates single cell sensitivity to extracellular signaling inputs, including those guiding complex changes in the tissue form and function. Here we explored if cell-cell communication can enhance the ability of cell ensembles to sense and respond to weak gradients of chemotactic cues. Using a combination of experiments with mammary epithelial cells and mathematical modeling, we find that multicellular sensing enables detection of and response to shallow Epidermal Growth Factor (EGF) gradients that are undetectable by single cells. However, the advantage of this type of gradient sensing is limited by the noisiness of the signaling relay, necessary to integrate spatially distributed ligand concentration information. We calculate the fundamental sensory limits imposed by this communication noise and combine them with the experimental data to estimate the effective size of multicellular sensory groups involved in gradient sensing. Functional experiments strongly implicated intercellular communication through gap junctions and calcium release from intracellular stores as mediators of collective gradient sensing. The resulting integrative analysis provides a framework for understanding the advantages and limitations of sensory information processing by relays of chemically coupled cells.Comment: paper + supporting information, total 35 pages, 15 figure

Molecular and cellular regulation of mammary epithelial dissemination

Dissemination of epithelial cancer cells represents the first step in metastatic spread. One postulated mechanism for dissemination is activation of an epithelial to mesenchymal transition (EMT), in which repression of the cell adhesion gene E-cadherin (E-cad) is considered the driving molecular event. We sought to test the sufficiency of single gene perturbations to induce dissemination out of primary mouse mammary epithelium. Deletion of E-cad disrupted simple architecture and morphogenesis but, contrary to expectation, rarely resulted in dissemination. In contrast, expression of the EMT transcription factor Twist1 induced rapid dissemination of cytokeratin+ epithelial cells. A core concept in EMT is that cells lose epithelial characteristics, such as tight cell-cell adhesion, and acquire mesenchymal characteristics to escape. However, we found that Twist1 did not significantly regulate epithelial-specific genes, such as E-cad. Rather, Twist1 induced dramatic transcriptional changes in extracellular compartment and cell-matrix adhesion genes. Surprisingly, Twist1+ cells disseminated with membrane-localized E-cad, and E-cad knockdown strongly inhibited Twist1-induced single cell dissemination. Dissemination can therefore occur through an innately epithelial migratory program. The integrity of the myoepithelium, the outer mammary epithelial layer, is the major diagnostic criterion used to distinguish in situ from invasive breast tumors. Interestingly, we found that constitutive Twist1 expression induced dissemination of both inner luminal and outer myoepithelial cells and abnormal myoepithelial ingression, resulting in gaps in myoepithelial coverage at the basal surface. We next developed mouse models to restrict Twist1 to distinct mammary lineages. Twist1 expression in the myoepithelial compartment induced cell autonomous myoepithelial dissemination. In contrast, Twist1 expression in the luminal compartment rarely resulted in dissemination. Using cell type-specific fluorescent reporters, we observed that normal myoepithelial cells appear to contain Twist1+ luminal cells protruding into the extracellular matrix. Myoepithelial cells display a similar response to invasive E-cad– luminal cell behavior. Taken together, our data supports the hypothesis that normal myoepithelial cells dynamically block luminal cell escape. We now seek to test the molecular basis of myoepithelial barrier function through knockdown of myoepithelial-specific genes important for contractility, cell-cell adhesion, and the intermediate filament network. We expect that perturbation of myoepithelial function will enable E-cad– and Twist1+ luminal cell dissemination

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination.

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1(+) epithelium displayed extensive intercellular junctions, and Twist1(-) luminal epithelial cells could still adhere to disseminating Twist1(+) cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1(+) cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination.

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1(+) epithelium displayed extensive intercellular junctions, and Twist1(-) luminal epithelial cells could still adhere to disseminating Twist1(+) cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1(+) cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination.

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1(+) epithelium displayed extensive intercellular junctions, and Twist1(-) luminal epithelial cells could still adhere to disseminating Twist1(+) cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1(+) cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate

Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia-A Microbiome and Gene Profiling Study and Focused Review.

Oral potentially malignant disorders (OPMDs) are a group of conditions that carry a risk of oral squamous cell carcinoma (OSCC) development. Recent studies indicate that periodontal disease-associated pathogenic bacteria may play a role in the transition from healthy mucosa to dysplasia and to OSCC. Yet, the microbial signatures associated with the transition from healthy mucosa to dysplasia have not been established. To characterize oral microbial signatures at these different sites, we performed a 16S sequencing analysis of both oral swab and formalin-fixed, paraffin-embedded tissue (FFPE) samples. We collected oral swabs from healthy mucosa (from healthy patients), histologically normal mucosa adjacent to dysplasia, and low-grade oral dysplasia. Additionally, FFPE samples from histologically normal mucosa adjacent to OSCC, plus low grade and high-grade oral dysplasia samples were also collected. The collected data demonstrate significant differences in the alpha and beta microbial diversities of different sites in oral mucosa, dysplasia, and OSCC, as well as increased dissimilarities within these sites. We found that the Proteobacteria phyla abundance increased, concurrent with a progressive decrease in the Firmicutes phyla abundance, as well as altered levels of Enterococcus cecorum, Fusobacterium periodonticum, Prevotella melaninogenica, and Fusobacterium canifelinum when moving from healthy to diseased sites. Moreover, the swab sample analysis indicates that the oral microbiome may be altered in areas that are histologically normal, including in mucosa adjacent to dysplasia. Furthermore, trends in specific microbiome changes in oral swab samples preceded those in the tissues, signifying early detection opportunities for clinical diagnosis. In addition, we evaluated the gene expression profile of OSCC cells (HSC-3) infected with either P. gingivalis, T. denticola, F. nucelatum, or S. sanguinis and found that the three periodontopathogens enrich genetic processes related to cancer progression, including skin keratinization/cornification, while the commensal enriched processes related to RNA processing and adhesion. Finally, we reviewed the dysplasia microbiome literature and found a significant decrease in commensal bacteria, such as the Streptococci genus, and a simultaneous increase in pathogenic bacteria, mainly Bacteroidetes phyla and Fusobacterium genus. These findings suggest that features of the oral microbiome can serve as novel biomarkers for dysplasia and OSCC disease progression

Nitazoxanide Inhibits Biofilm Production and Hemagglutination by Enteroaggregative Escherichia coli Strains by Blocking Assembly of AafA Fimbriae▿

Enteroaggregative Escherichia coli (EAEC) strains have emerged as common causes of persistent diarrhea and malnutrition among children and HIV-infected persons. During infection, EAEC typically adheres to the intestinal mucosa via fimbrial adhesins, which results in a characteristic aggregative pattern. In the study described here we investigated whether the broad-spectrum antiparasitic and antidiarrheal drug nitazoxanide (NTZ) might be active against EAEC in vitro. While E. coli strains were resistant to NTZ in rich Luria-Bertani medium (MIC > 64 μg/ml), the drug was slightly inhibitory in a minimal medium supplemented with glucose (MinA-G medium; MIC, ∼32 μg/ml). NTZ also inhibited biofilm production by strain EAEC 042 in both Dulbecco's modified Eagle's medium and MinA-G medium with a 50% inhibitory concentration of ∼12 μg/ml. Immunofluorescence and immunoblot analyses with antibody against the major fimbrial subunit AafA of aggregative adherence fimbriae vaariant II (AAF/II) established that the numbers of AAF/II filaments on bacteria grown in the presence of NTZ were dramatically reduced. Comparative quantitative reverse transcription-PCR and reporter gene fusions (aafA::phoA) indicated that aafA expression was unaffected by NTZ, while aggR transcript levels and aggR::lacZ expression were increased ∼10- and 2.5-fold, respectively, compared with that for the untreated controls. More generally, NTZ inhibited hemagglutination (HA) of red blood cells by the non-biofilm-producing strain JM221 expressing either AAF/I or type I fimbriae. Our findings suggest that the inhibitory action of NTZ on biofilm formation and HA is likely due to inhibition of fimbrial assembly. Antimicrobial agents that inhibit the assembly or function of fimbrial filaments should be good candidates for the prevention of infection

Specific Oral Microbial Differences in Proteobacteria and Bacteroidetes Are Associated with Distinct Sites When Moving from Healthy Mucosa to Oral Dysplasia—A Microbiome and Gene Profiling Study and Focused Review

Oral potentially malignant disorders (OPMDs) are a group of conditions that carry a risk of oral squamous cell carcinoma (OSCC) development. Recent studies indicate that periodontal disease-associated pathogenic bacteria may play a role in the transition from healthy mucosa to dysplasia and to OSCC. Yet, the microbial signatures associated with the transition from healthy mucosa to dysplasia have not been established. To characterize oral microbial signatures at these different sites, we performed a 16S sequencing analysis of both oral swab and formalin-fixed, paraffin-embedded tissue (FFPE) samples. We collected oral swabs from healthy mucosa (from healthy patients), histologically normal mucosa adjacent to dysplasia, and low-grade oral dysplasia. Additionally, FFPE samples from histologically normal mucosa adjacent to OSCC, plus low grade and high-grade oral dysplasia samples were also collected. The collected data demonstrate significant differences in the alpha and beta microbial diversities of different sites in oral mucosa, dysplasia, and OSCC, as well as increased dissimilarities within these sites. We found that the Proteobacteria phyla abundance increased, concurrent with a progressive decrease in the Firmicutes phyla abundance, as well as altered levels of Enterococcus cecorum, Fusobacterium periodonticum, Prevotella melaninogenica, and Fusobacterium canifelinum when moving from healthy to diseased sites. Moreover, the swab sample analysis indicates that the oral microbiome may be altered in areas that are histologically normal, including in mucosa adjacent to dysplasia. Furthermore, trends in specific microbiome changes in oral swab samples preceded those in the tissues, signifying early detection opportunities for clinical diagnosis. In addition, we evaluated the gene expression profile of OSCC cells (HSC-3) infected with either P. gingivalis, T. denticola, F. nucelatum, or S. sanguinis and found that the three periodontopathogens enrich genetic processes related to cancer progression, including skin keratinization/cornification, while the commensal enriched processes related to RNA processing and adhesion. Finally, we reviewed the dysplasia microbiome literature and found a significant decrease in commensal bacteria, such as the Streptococci genus, and a simultaneous increase in pathogenic bacteria, mainly Bacteroidetes phyla and Fusobacterium genus. These findings suggest that features of the oral microbiome can serve as novel biomarkers for dysplasia and OSCC disease progression

ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium.

Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transcriptome analysis of our 3D cultures revealed few ECM-dependent changes in RNA expression. However, we observed many differences between normal and malignant epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene could induce sustained dissemination of nontransformed cells into collagen I. We found that deletion of P-cadherin was sufficient for sustained dissemination, but exclusively into collagen I. Our data reveal that metastatic tumors preferentially disseminate in specific ECM microenvironments. Furthermore, these data suggest that breaks in the basement membrane could induce invasion and dissemination via the resulting direct contact between cancer cells and collagen I