Candida albicans White-Opaque Switching Influences Virulence but Not Mating during Oropharyngeal Candidiasis.

The capacity of Candida albicans to switch reversibly between the white phenotype and the opaque phenotype is required for the fungus to mate. It also influences virulence during hematogenously disseminated candidiasis. We investigated the roles of the mating type loci (MTL) and white-opaque switching in the capacity of C. albicans to mate in the oropharynx and cause oropharyngeal candidiasis (OPC). When immunosuppressed mice were orally infected with mating-competent opaque a/a and α/α cells either alone or mixed with white cells, no detectable mating occurred, indicating that the mating frequency was less than 1.6 × 10-6 Opaque cells were also highly attenuated in virulence; they either were cleared from the oropharynx or switched to the white phenotype during OPC. Although there were strain-to-strain differences in the virulence of white cells, they were consistently more virulent than opaque cells. In vitro studies indicated that relative to white cells, opaque cells had decreased capacity to invade and damage oral epithelial cells. The reduced invasion of at least one opaque strain was due to reduced surface expression of the Als3 invasin and inability to activate the epidermal growth factor receptor, which is required to stimulate the epithelial cell endocytic machinery. These results suggest that mating is a rare event during OPC because opaque cells have reduced capacity to invade and damage the epithelial cells of the oral mucosa.

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms.

In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations.

Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis.

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO2 induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO2, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.

Role of Tec1 in the development, architecture, and integrity of sexual biofilms of Candida albicans.

MTL-homozygous ( A: / A: or α/α) white cells form a complex sexual biofilm that exhibits the same architecture as that of MTL-heterozygous ( A: /α) pathogenic biofilms. However, the former is regulated by the mitogen-activated protein (MAP) kinase pathway, while the latter is regulated by the Ras1/cyclic AMP (cAMP) pathway. We previously demonstrated that in the formation of an MTL-homozygous, mature (48 h) sexual biofilm in RPMI 1640 medium, the MAP kinase pathway targets Tec1 rather than Cph1, the latter of which is the target of the same pathway, but for the opaque cell mating response. Here we continued our analysis of the role of Tec1 by comparing the effects of deleting TEC1 on initial adhesion to silicone elastomer, high-resolution confocal microscopy assessments of the stages and cellular phenotypes during the 48 h of biofilm development, human white cell penetration, and biofilm fragility. We show that although Tec1 plays only a minor role in initial adhesion to the silicone elastomer, it does play a major role in the growth of the basal yeast cell polylayer, vertical extension of hyphae and matrix deposition in the upper portion of the biofilm, final biofilm thickness, penetrability of human white blood cells, and final biofilm integrity (i.e., resistance to fluid flow). These results provide a more detailed description of normal biofilm development and architecture and confirm the central role played by the transcription factor Tec1 in the biofilm model employed here.

Nonsex genes in the mating type locus of Candida albicans play roles in a/α biofilm formation, including impermeability and fluconazole resistance.

The mating type locus (MTL) of Candida albicans contains the mating type genes and has, therefore, been assumed to play an exclusive role in the mating process. In mating-incompetent a/α cells, two of the mating type genes, MTLa1 and MTLα2, encode components of the a1-α2 corepressor that suppresses mating and switching. But the MTL locus of C. albicans also contains three apparently unrelated "nonsex" genes (NSGs), PIK, PAP and OBP, the first two essential for growth. Since it had been previously demonstrated that deleting either the a/α copy of the entire MTL locus, or either MTLa1 or MTLα2, affected virulence, we hypothesized that the NSGs in the MTL locus may also play a role in pathogenesis. Here by mutational analysis, it is demonstrated that both the mating type and nonsex genes in the MTL locus play roles in a/α biofilm formation, and that OBP is essential for impermeability and fluconazole resistance.

Alternative mating type configurations (a/α versus a/a or α/α) of Candida albicans result in alternative biofilms regulated by different pathways.

Similar multicellular structures can evolve within the same organism that may have different evolutionary histories, be controlled by different regulatory pathways, and play similar but nonidentical roles. In the human fungal pathogen Candida albicans, a quite extraordinary example of this has occurred. Depending upon the configuration of the mating type locus (a/α versus a/a or α/α), C. albicans forms alternative biofilms that appear similar morphologically, but exhibit dramatically different characteristics and are regulated by distinctly different signal transduction pathways. Biofilms formed by a/α cells are impermeable to molecules in the size range of 300 Da to 140 kDa, are poorly penetrated by human polymorphonuclear leukocytes (PMNs), and are resistant to antifungals. In contrast, a/a or α/α biofilms are permeable to molecules in this size range, are readily penetrated by PMNs, and are susceptible to antifungals. By mutational analyses, a/α biofilms are demonstrated to be regulated by the Ras1/cAMP pathway that includes Ras1→Cdc35→cAMP(Pde2-|)→Tpk2(Tpk1)→Efg1→Tec1→Bcr1, and a/a biofilms by the MAP kinase pathway that includes Mfα→Ste2→ (Ste4, Ste18, Cag1)→Ste11→Hst7→Cek2(Cek1)→Tec1. These observations suggest the hypothesis that while the upstream portion of the newly evolved pathway regulating a/a and α/α cell biofilms was derived intact from the upstream portion of the conserved pheromone-regulated pathway for mating, the downstream portion was derived through modification of the downstream portion of the conserved pathway for a/α biofilm formation. C. albicans therefore forms two alternative biofilms depending upon mating configuration.

Identification of genes upregulated by the transcription factor Bcr1 that are involved in impermeability, impenetrability, and drug resistance of Candida albicans a/α biofilms.

Candida albicans forms two types of biofilm, depending upon the configuration of the mating type locus. Although architecturally similar, a/α biofilms are impermeable, impenetrable, and drug resistant, whereas a/a and α/α biofilms lack these traits. The difference appears to be the result of an alternative matrix. Overexpression in a/a cells of BCR1, a master regulator of the a/α matrix, conferred impermeability, impenetrability, and drug resistance to a/a biofilms. Deletion of BCR1 in a/α cells resulted in the loss of these a/α-specific biofilm traits. Using BCR1 overexpression in a/a cells, we screened 107 genes of interest and identified 8 that were upregulated by Bcr1. When each was overexpressed in a/a biofilms, the three a/α traits were partially conferred, and when each was deleted in a/α cells, the traits were partially lost. Five of the eight genes have been implicated in iron homeostasis, and six encode proteins that are either in the wall or plasma membrane or secreted. All six possess sites for O-linked and N-linked glycosylation that, like glycosylphosphatidylinositol (GPI) anchors, can cross-link to the wall and matrix, suggesting that they may exert a structural role in conferring impermeability, impenetrability, and drug resistance, in addition to their physiological functions. The fact that in a screen of 107 genes, all 8 of the Bcr1-upregulated genes identified play a role in impermeability, impenetrability, and drug resistance suggests that the formation of the a/α matrix is highly complex and involves a larger number of genes than the initial ones identified here.

Candida albicans forms a specialized "sexual" as well as "pathogenic" biofilm.

Candida albicans forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent a/α configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the a/a or α/α configuration, white cells form a biofilm that is architecturally similar to an a/α biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The MTL-homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized MTL-homozygous biofilms facilitate mating. If true, then MTL-homozygous biofilms should have an advantage over MTL-heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque a/a and α/α cells seeded in MTL-homozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in MTL-heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of MTL-heterozygous and MTL-homozygous cells grown on agar at 28°C in air or 20% CO2 and at 37°C. Mating frequencies are negligible in seeded patches of both a/α and a/a cells, in contrast to seeded biofilms. Together, these results support the hypothesis that MTL-homozygous (a/a or α/α) white cells form a specialized "sexual biofilm."

Impact of environmental conditions on the form and function of Candida albicans biofilms.

Candida albicans, like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a/α cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO2, a/a cells make a biofilm in Spider medium similar architecturally to that of a/α cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO2 in the atmosphere, and configuration of the MTL locus.

Tec1 mediates the pheromone response of the white phenotype of Candida albicans: insights into the evolution of new signal transduction pathways.

The way in which signal transduction pathways evolve remains a mystery, primarily because we have few examples of ones that have newly evolved. There are numerous examples of how signal transduction pathways in the same organism selectively share components, most notably between the signal transduction pathways in Saccharomyces cerevisiae for the mating process, the filamentation process, cell wall integrity, ascospore formation, and osmoregulation. These examples, however, have not provided insights into how such pathways evolve. Here, through construction of an overexpression library for 107 transcription factors, and through mutational analyses, we have identified the transcription factor Tec1 as the last component of the newly evolved signal transduction pathway that regulates the pheromone response of the white cell phenotype in Candida albicans. The elucidation of this last component, Tec1, establishes a comprehensive description of the pheromone response pathway in the white cell phenotype of C. albicans, providing a unique perspective on how new signal transduction pathways may evolve. The three portions of this new regulatory pathway appear to have been derived from three different ancestral programs still functional in C. albicans. The upstream portion, including signals, receptors, the trimeric G protein complex, and the MAP kinase cascade, was derived intact from the upstream portion of the opaque pheromone response pathway of the mating process; Tec1, the transcription factor targeted by the MAP kinase pathway, was derived from a filamentation pathway; and the white-specific downstream target genes were derived from an ancestral biofilm process. The evolution of this pheromone response pathway provides a possible paradigm for how such signal transduction pathways evolve.

The "finger," a unique multicellular morphology of Candida albicans induced by CO2 and dependent upon the Ras1-cyclic AMP pathway.

Most experiments exploring the basic biology of pathogenic microbes are performed in vitro under conditions that do not usually mimic those of their host niche. Hence, developmental programs initiated by specific host cues may be missed in vitro. We have tested the effects of growing low-density agar cultures of the yeast pathogen Candida albicans in concentrations of CO(2) found in the gastrointestinal tract. It is demonstrated that in physiological concentrations of CO(2) at 37°C, yeast cells form a heretofore undescribed multicellular "finger" morphology distinct from a previously described stalk-like structure induced by high doses of UV irradiation that kills more than 99.99% of cells. The finger extends aerially, is uniform in diameter, and is visible to the naked eye, attaining lengths of 3 mm. It is composed of a basal yeast cell monolayer adhering to a semispherical crater formed in the agar and connected to a basal bulb of yeast cells at a fragile interface. The bulb extends into the long shaft. We propose that a single, centrally located hypha extending the length of the shaft forms buds at compartment junctions that serve as the source of the yeast cells in the shaft. A mutational analysis reveals finger formation is dependent upon the pathway Ras1→Cdc35→cyclic AMP (cAMP) (PDE2-|)→Tpk2→Tec1. Because of the mechanically fragile interface and the compactness of bulb and shaft, we suggest that the finger may function as a multicellular dispersal mechanism produced in host niches containing high levels of CO(2).

N-acetylglucosamine induces white to opaque switching, a mating prerequisite in Candida albicans.

To mate, the fungal pathogen Candida albicans must undergo homozygosis at the mating-type locus and then switch from the white to opaque phenotype. Paradoxically, opaque cells were found to be unstable at physiological temperature, suggesting that mating had little chance of occurring in the host, the main niche of C. albicans. Recently, however, it was demonstrated that high levels of CO(2), equivalent to those found in the host gastrointestinal tract and select tissues, induced the white to opaque switch at physiological temperature, providing a possible resolution to the paradox. Here, we demonstrate that a second signal, N-acetylglucosamine (GlcNAc), a monosaccharide produced primarily by gastrointestinal tract bacteria, also serves as a potent inducer of white to opaque switching and functions primarily through the Ras1/cAMP pathway and phosphorylated Wor1, the gene product of the master switch locus. Our results therefore suggest that signals produced by bacterial co-members of the gastrointestinal tract microbiota regulate switching and therefore mating of C. albicans.

Fig1 facilitates calcium influx and localizes to membranes destined to undergo fusion during mating in Candida albicans.

Few mating-regulated genes have been characterized in Candida albicans. C. albicans FIG1 (CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In Saccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1 was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca(2+) uptake in mating-competent opaque cells. Uptake was reduced substantially in the fig1Δ null mutant. CaFig1 is therefore involved in Ca(2+) influx and localizes to membranes that are destined to undergo fusion during mating.

Self-induction of a/a or alpha/alpha biofilms in Candida albicans is a pheromone-based paracrine system requiring switching.

Like MTL-heterozygous (a/α) cells, white MTL-homozygous (a/a or α/α) cells of Candida albicans, to which a minority of opaque cells of opposite mating type have been added, form thick, robust biofilms. The latter biofilms are uniquely stimulated by the pheromone released by opaque cells and are regulated by the mitogen-activated protein kinase signal transduction pathway. However, white MTL-homozygous cells, to which opaque cells of opposite mating type have not been added, form thinner biofilms. Mutant analyses reveal that these latter biofilms are self-induced. Self-induction of a/a biofilms requires expression of the α-receptor gene STE2 and the α-pheromone gene MFα, and self-induction of α/α biofilms requires expression of the a-receptor gene STE3 and the a-pheromone gene MFa. In both cases, deletion of WOR1, the master switch gene, blocks cells in the white phenotype and biofilm formation, indicating that self-induction depends upon low frequency switching from the white to opaque phenotype. These results suggest a self-induction scenario in which minority opaque a/a cells formed by switching secrete, in a mating-type-nonspecific fashion, α-pheromone, which stimulates biofilm formation through activation of the α-pheromone receptor of majority white a/a cells. A similar scenario is suggested for a white α/α cell population, in which minority opaque α/α cells secrete a-pheromone. This represents a paracrine system in which one cell type (opaque) signals a second highly related cell type (white) to undergo a complex response, in this case the formation of a unisexual white cell biofilm.

Genes selectively up-regulated by pheromone in white cells are involved in biofilm formation in Candida albicans.

To mate, MTL-homozygous strains of the yeast pathogen Candida albicans must switch from the white to opaque phase. Mating-competent opaque cells then release pheromone that induces polarization, a G1 block and conjugation tube formation in opaque cells of opposite mating type. Pheromone also induces mating-incompetent white cells to become adhesive and cohesive, and form thicker biofilms that facilitate mating. The pheromone response pathway of white cells shares the upstream components of that of opaque cells, but targets a different transcription factor. Here we demonstrate that the genes up-regulated by the pheromone in white cells are activated through a common cis-acting sequence, WPRE, which is distinct from the cis-acting sequence, OPRE, responsible for up-regulation in opaque cells. Furthermore, we find that these white-specific genes play roles in white cell biofilm formation, and are essential for biofilm formation in the absence of an added source of pheromone, suggesting either an autocrine or pheromone-independent mechanism. These results suggest an intimate, complex and unique relationship between switching, mating and MTL-homozygous white cell biofilm formation, the latter a presumed virulence factor in C. albicans.

A Candida albicans-specific region of the alpha-pheromone receptor plays a selective role in the white cell pheromone response.

Candida albicans strains homozygous at the mating type locus can switch from white to opaque, and must do so to mate. Opaque cells then secrete mating pheromones that stimulate opaque cells of opposite mating type to undergo mating. These same pheromones stimulate mating-incompetent white cells to become cohesive and adhesive, and enhance white cell biofilm development, a pathogenic trait. Stimulation is mediated through the same receptor, G protein complex and mitogen-activated protein kinase pathway. Here we present evidence that a C. albicans-specific 55-amino-acid region of the first intracellular loop, IC1, of the alpha-pheromone receptor Ste2p, is required for the alpha-pheromone response of white cells, but not that of opaque cells. This represents a unique regulatory configuration in which activation of a common pathway by the same ligand, the same receptor and the same signal transduction pathway is dependent on a unique region of an intracellular loop of the common receptor in one of the two responding phenotypes.

EFG1 Mutations, Phenotypic Switching, and Colonization by Clinical a/α Strains of Candida albicans.

The transcription factor EFG1 functions as a suppressor of white-to-opaque and white-to-gray switching in a/α strains of Candida albicans In a collection of 27 clinical isolates, 4 of the 17 EFG1/EFG1 strains, 1 of the 2 EFG1/efg1 strains, and all 8 of the efg1/efg1 strains underwent white-to-opaque switching. The four EFG1/EFG1 strains, the one EFG1/efg1 strain, and one of the eight efg1/efg1 strains that underwent switching to opaque did not switch to gray and could not be complemented with a copy of EFG1 Competition experiments in a mouse model for gastrointestinal (GI) colonization confirmed that efg1/efg1 cells rapidly outcompete EFG1/EFG1 cells, and in plating experiments, formed colonies containing both gray and opaque cells. Direct microscopic analysis of live cells in the feces, however, revealed that the great majority of cells were opaque, suggesting opaque, not gray, may be the dominant phenotype at the site of colonization.IMPORTANCE Close to half of a collection of 27 clinical a/α isolates of Candida albicans underwent white-to-opaque switching. Complementation experiments revealed that while approximately half of the a/α switchers were due to EFG1 mutations, the remaining half were due to mutations in other genes. In addition, the results of competition experiments in a mouse GI tract colonization model support previous observations that efg1/efg1 cells rapidly outcompete EFG1/EFG1 strains, but direct microscopic analysis reveals that the major colonizing cells were opaque, not gray.

Roles of the Transcription Factors Sfl2 and Efg1 in White-Opaque Switching in a/α Strains of Candida albicans.

Candida albicans remains the most pervasive fungal pathogen colonizing humans. The majority of isolates from hosts are heterozygous at the mating type locus (MTLa/α), and a third of these have recently been shown to be capable of switching to the opaque phenotype. Here we have investigated the roles of two transcription factors (TFs) Sfl2 and Efg1, in repressing switching in a/α strains. Deleting either gene results in the capacity of a/α cells to switch to opaque en masse under facilitating environmental conditions, which include N-acetylglucosamine (GlcNAc) as the carbon source, physiological temperature (37°C), and high CO2 (5%). These conditions are similar to those in the host. Our results further reveal that while glucose is a repressor of sfl2Δ and efg1Δ switching, GlcNAc is an inducer. Finally, we show that when GlcNAc is the carbon source, and the temperature is low (25°C), the efg1Δ mutants, but not the sfl2Δ mutants, form a tiny, elongate cell, which differentiates into an opaque cell when transferred to conditions optimal for a/α switching. These results demonstrate that at least two TFs, Sfl2 and Efg1, repress switching in a/α cells and that a/α strains with either an sfl2Δ or efg1Δ mutation can switch en masse but only under physiological conditions. The role of opaque a/α cells in commensalism and pathogenesis must, therefore, be investigated.IMPORTANCE More than 95% of Candida albicans strains isolated from humans are MTLa/α, and approximately a third of these can undergo the white-to-opaque transition. Therefore, besides being a requirement for MTL-homozygous strains to mate, the opaque phenotype very likely plays a role in the commensalism and pathogenesis of nonmating, a/α populations colonizing humans.

Reciprocal signaling and direct physical interactions between fibroblasts and breast cancer cells in a 3D environment.

Tumorigenic cells undergo cell aggregation and aggregate coalescence in a 3D Matrigel environment. Here, we expanded this 3D platform to assess the interactions of normal human dermal fibroblasts (NHDFs) and human primary mammary fibroblasts (HPMFs) with breast cancer-derived, tumorigenic cells (MDA-MB-231). Medium conditioned by MDA-MB-231 cells activates both types of fibroblasts, imbuing them with the capacity to accelerate the rate of aggregation and coalescence of MDA-MB-231 cells more than four fold. Acceleration is achieved 1) by direct physical interactions with MDA-MB-231 cells, in which activated fibroblasts penetrate the MDA-MB-231/Matrigel 3D environment and function as supporting scaffolds for MDA-MB-231 aggregation and coalescence, and 2) through the release of soluble accelerating factors, including matrix metalloproteinase (MMPs) and, in the case of activated NHDFs, SDF-1α/CXCL12. Fibroblast activation includes changes in morphology, motility, and gene expression. Podoplanin (PDPN) and fibroblast activation protein (FAP) are upregulated by more than nine-fold in activated NHDFs while activated HPMFs upregulate FAP, vimentin, desmin, platelet derived growth factor receptor A and S100A4. Overexpression of PDPN, but not FAP, in NHDF cells in the absence of MDA-MB-231-conditioned medium, activates NHDFs. These results reveal that complex reciprocal signaling between fibroblasts and cancer cells, coupled with their physical interactions, occurs in a highly coordinated fashion that orchestrates aggregation and coalescence, behaviors specific to cancer cells in a 3D environment. These in vitro interactions may reflect events involved in early tumorigenesis, particularly in cases of field cancerization, and may represent a new mechanism whereby cancer-associated fibroblasts (CAFs) promote tumor growth.

Generating a Battery of Monoclonal Antibodies Against Firefly Luciferase for Dot Blot Analysis, Western Blot Analysis, and Immunostaining of Cells in Culture and Paraffin Sections.

Firefly luciferase (FLuc) is commonly used as a reporter gene PpyLuc1 in bioanalytical assays. We have produced five mouse-derived monoclonal antibodies (mAbs) that recognize FLuc. The mAbs, DSHB-LUC-2, DSHB-LUC-3, DSHB-LUC-9, DSHB-LUC-16, and DSHB-LUC-24, were generated by immunizing mice with purified 6xHIS-tagged FLuc (6xHis-FLuc) in suspension with an adjuvant. All five were validated by dot blots. Four of the mAbs provided strong signals in western blot analysis, and one a weak signal. All five were validated for immunostaining in fixed cell culture. Only one stained cells embedded in paraffin. The five mAbs are available at cost through the Developmental Studies Hybridoma Bank (DSHB), a nonprofit National Resource created by the National Institutes of Health.