BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

Vaccination against neoantigens induced in cross-priming cDC1 in vivo.

The conventional type 1 dendritic cells (cDC1) play a pivotal role in protective immunity against pathogens and cancer. However, their low frequency in the blood and tissues limits their use in immune therapy. We have recently described a method to vaccinate against neoantigens that are induced in tumor cells by targeted delivery of a TAP siRNA to dendritic cells using a TLR9 binding CpG oligonucleotide. Since TLR9 is also expressed in immune suppressive myeloid populations TLR9 targeting could reduce the effectiveness of this approach. Here, we describe a modular multivalent antibody platform to target the TAP siRNA to resident Clec9a expressing cDC1 and show that it leads to selective and sustained TAP downregulation in cDC1 and inhibits tumor growth in mice more effectively than CpG targeted siRNA. To induce DC maturation an agonistic CD40 antibody was administered to the siRNA treated mice. To obviate the need for a second drug formulation and reduce the risk of toxicity, we exploited the multivalent nature of this targeting platform to co-deliver the TAP siRNA and a DC maturation agent, a CpG containing oligonucleotide, to cDC1 in vivo and show that it was more effective than Clec9a targeting of TAP siRNA in combination with CD40 antibody. This study describes a way to manipulate the function of cDC1 cells in vivo using a broadly applicable antibody-based targeting platform to deliver multiple biological agents to specific cells in vivo to potentiate (immune) therapy and to probe the biology of specific cell types in their natural settings.

Btk Supports Autoreactive B Cell Development and Protects against Apoptosis but Is Expendable for Antigen Presentation.

Bruton's tyrosine kinase (Btk) propagates B cell signaling, and BTK inhibitors are in clinical trials for autoimmune disease. Although autoreactive B cells fail to develop in the absence of Btk, its role in mature cells is unknown. To address this issue, a model of conditional removal (Btk flox/Cre-ERT2 ) was used to excise Btk from mature transgenic B cells that recognize the pathophysiologic autoantigen insulin. Anti-insulin B cells escape central tolerance and promote autoimmune diabetes, mimicking human autoreactive cells. Lifelong Btk deficiency was previously shown to eliminate 95% of anti-insulin B cells, but in this model, mature anti-insulin B cells survived for weeks after targeted Btk deletion, even when competing with a polyclonal repertoire. BCR-stimulated cells could still signal via Syk, PLCy2, and CD22, but failed to upregulate the antiapoptotic protein Bcl-xL, and proliferation was impaired. Surprisingly, Btk-depleted anti-insulin B cells could still present Ag and activate T cells, a critical function in promoting T cell-mediated islet cell destruction. Thus, pharmacologic targeting of Btk may be most effective by blocking expansion of established autoreactive cells, and preventing emergence of new ones.

Impaired B Cell Apoptosis Results in Autoimmunity That Is Alleviated by Ablation of Btk.

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

Combined Systemic and Intratumoral Administration of Human Papillomavirus Vaccine to Treat Multiple Cutaneous Basaloid Squamous Cell Carcinomas.

Importance: Squamous cell carcinoma (SCC) is the second most common form of skin cancer, and its incidence is increasing. When surgical management is not an option, finding a safe and efficacious treatment is a challenge. Mounting evidence suggests that the human papillomavirus (HPV) is involved in the pathogenesis of some SCCs. Objective: To assess whether the 9-valent HPV vaccine could be an effective treatment strategy for cutaneous SCC. Design, Setting, and Participants: A woman in her 90s with multiple, inoperable cutaneous basaloid SCCs was successfully treated at a university-based outpatient dermatology clinic with a combination of systemic and intratumoral delivery of the 9-valent HPV vaccine from March 17, 2016, through February 27, 2017, and then followed up through May 21, 2018. Main Outcomes and Measures: Reduction in tumor size and number after a combination of systemic and intratumoral administration of the HPV vaccine. Results: All tumors resolved 11 months after the first intratumoral injection of the vaccine. The patient remained free of tumors at the end of follow-up. Conclusions and Relevance: This is the first report, to our knowledge, of complete regression of a cutaneous malignant tumor after combined systemic and direct intratumoral injection of the 9-valent HPV vaccine. This report suggests that the HPV vaccine may have therapeutic utility for SCCs in patients who are poor surgical candidates, have multiple lesions, or defer surgery.

Bruton's Tyrosine Kinase Is Not Essential for B Cell Survival beyond Early Developmental Stages.

Bruton's tyrosine kinase (Btk) is a crucial regulator of B cell signaling and is a therapeutic target for lymphoma and autoimmune disease. BTK-deficient patients suffer from humoral immunodeficiency, as their B cells fail to progress beyond the bone marrow. However, the role of Btk in fully developed, mature peripheral B cells is not well understood. Analysis using BTK inhibitors is complicated by suboptimal inhibition, off-target effects, or failure to eliminate BTK's adaptor function. Therefore a Btkflox/Cre-ERT2 mouse model was developed and used to excise Btk after B cell populations were established. Mice lacking Btk from birth are known to have reduced follicular (FO) compartments, with expanded transitional populations, suggesting a block in development. In adult Btkflox/Cre-ERT2 mice, Btk excision did not reduce FO B cells, which persisted for weeks. Autoimmune-prone B1 cells also survived conditional Btk excision, contrasting their near absence in global Btk-deficient mice. Therefore, Btk supports BCR signaling during selection into the FO and B1 compartments, but is not needed to maintain these cell populations. B1-related natural IgM levels remained normal, contrasting global Btk deficiency, but B cell proliferation and T-independent type II immunization responses were blunted. Thus, B cells have nuanced signaling responses that are differentially regulated by Btk for development, survival, and function. These findings raise the possibility that Btk may also be expendable for survival of mature human B cells, therefore requiring prolonged dosing to be effective, and that success of BTK inhibitors may depend in part on off-target effects.

Corrigendum: Distinct Transcriptomic Features Are Associated with Transitional and Mature B-Cell Populations in the Mouse Spleen.

[This corrects the article on p. 30 in vol. 6, PMID: 25717326.].

Genetic correlations and little genetic variance for reaction norms may limit potential for adaptation to pollution by ionic and nanoparticulate silver in a whitefish (Salmonidae).

For natural populations to adapt to anthropogenic threats, heritable variation must persist in tolerance traits. Silver nanoparticles, the most widely used engineered nanoparticles, are expected to increase in concentrations in freshwaters. Little is known about how these particles affect wild populations, and whether genetic variation persists in tolerance to permit rapid evolutionary responses. We sampled wild adult whitefish and crossed them in vitro full factorially. In total, 2896 singly raised embryos of 48 families were exposed to two concentrations (0.5 µg/L; 100 µg/L) of differently sized silver nanoparticles or ions (silver nitrate). These doses were not lethal; yet higher concentrations prompted embryos to hatch earlier and at a smaller size. The induced hatching did not vary with nanoparticle size and was stronger in the silver nitrate group. Additive genetic variation for hatching time was significant across all treatments, with no apparent environmental dependencies. No genetic variation was found for hatching plasticity. We found some treatment-dependent heritable variation for larval length and yolk volume, and one instance of additive genetic variation for the reaction norm on length at hatching. Our assessment suggests that the effects of silver exposure on additive genetic variation vary according to trait and silver source. While the long-term fitness consequences of low-level silver exposure on whitefish embryos must be further investigated to determine whether it is, in fact, detrimental, our results suggest that the evolutionary potential for adaptation to these types of pollutants may be low.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

Distinct Transcriptomic Features are Associated with Transitional and Mature B-Cell Populations in the Mouse Spleen.

Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principle component analysis. MZ B-cells possessed the most distinct transcriptome including down-regulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7, and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3, and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.

DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers.

UNLABELLED: Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. IMPORTANCE: Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans.

Exosome secretion is enhanced by invadopodia and drives invasive behavior.

Unconventional secretion of exosome vesicles from multivesicular endosomes (MVEs) occurs across a broad set of systems and is reported to be upregulated in cancer, where it promotes aggressive behavior. However, regulatory control of exosome secretion is poorly understood. Using cancer cells, we identified specialized invasive actin structures called invadopodia as specific and critical docking and secretion sites for CD63- and Rab27a-positive MVEs. Thus, inhibition of invadopodia formation greatly reduced exosome secretion into conditioned media. Functionally, addition of purified exosomes or inhibition of exosome biogenesis or secretion greatly affected multiple invadopodia life cycle steps, including invadopodia formation, stabilization, and exocytosis of proteinases, indicating a key role for exosome cargoes in promoting invasive activity and providing in situ signaling feedback. Exosome secretion also controlled cellular invasion through three-dimensional matrix. These data identify a synergistic interaction between invadopodia biogenesis and exosome secretion and reveal a fundamental role for exosomes in promoting cancer cell invasiveness.

B-lymphocyte tolerance and effector function in immunity and autoimmunity.

B-lymphocytes are integral to host defense against microbial pathogens and are associated with many autoimmune diseases. The B-cell receptor implements B-cell self-tolerance based on the antigen specificity, and B-cell-activating factor receptor (BAFF-R) imposes homeostatic control. While shaping the repertoire, the immune tolerance process also culls mature B cells into distinct populations. The activation response of B cells is tailored to the type of pathogen attack and is facilitated by T-cell help via CD40/CD40L interaction and/or innate cell help via toll-like receptors in conjunction with BAFF receptors and ligands. Activated effector B cells not only produce antibodies, but also produce a variety of cytokines to enhance and suppress the immune response. Not surprisingly, B cells play multiple roles in both humoral and cellular immune responses during infection and autoimmune pathogenesis. Here, we discuss how gene expression and signaling networks regulate peripheral B-cell tolerance, B-cell effector functions and emerging therapies targeting B-cell signaling in autoimmune diseases.

MHC class I expression dependent on bacterial infection and parental factors in whitefish embryos (Salmonidae).

Ecological conditions can influence not only the expression of a phenotype, but also the heritability of a trait. As such, heritable variation for a trait needs to be studied across environments. We have investigated how pathogen challenge affects the expression of MHC genes in embryos of the lake whitefish Coregonus palaea. In order to experimentally separate paternal (i.e. genetic) from maternal and environmental effects, and determine whether and how stress affects the heritable variation for MHC expression, embryos were produced in full-factorial in vitro fertilizations, reared singly, and exposed at 208 degree days (late-eyed stage) to either one of two strains of Pseudomonas fluorescens that differ in their virulence characteristics (one increased mortality, while both delayed hatching time). Gene expression was assessed 48 h postinoculation, and virulence effects of the bacterial infection were monitored until hatching. We found no evidence of MHC class II expression at this stage of development. MHC class I expression was markedly down-regulated in reaction to both pseudomonads. While MHC expression could not be linked to embryo survival, the less the gene was expressed, the earlier the embryos hatched within each treatment group, possibly due to trade-offs between immune function and developmental rate or further factors that affect both hatching timing and MHC expression. We found significant additive genetic variance for MHC class I expression in some treatments. That is, changes in pathogen pressures could induce rapid evolution in MHC class I expression. However, we found no additive genetic variance in reaction norms in our study population.

The host-encoded Heme Regulated Inhibitor (HRI) facilitates virulence-associated activities of bacterial pathogens.

Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

Parental influences on pathogen resistance in brown trout embryos and effects of outcrossing within a river network.

Phenotypic plasticity can increase tolerance to heterogeneous environments but the elevations and slopes of reaction norms are often population specific. Disruption of locally adapted reaction norms through outcrossing can lower individual viability. Here, we sampled five genetically distinct populations of brown trout (Salmo trutta) from within a river network, crossed them in a full-factorial design, and challenged the embryos with the opportunistic pathogen Pseudomonas fluorescens. By virtue of our design, we were able to disentangle effects of genetic crossing distance from sire and dam effects on early life-history traits. While pathogen infection did not increase mortality, it was associated with delayed hatching of smaller larvae with reduced yolk sac reserves. We found no evidence of a relationship between genetic distance (W, FST) and the expression of early-life history traits. Moreover, hybrids did not differ in phenotypic means or reaction norms in comparison to offspring from within-population crosses. Heritable variation in early life-history traits was found to remain stable across the control and pathogen environments. Our findings show that outcrossing within a rather narrow geographical scale can have neutral effects on F1 hybrid viability at the embryonic stage, i.e. at a stage when environmental and genetic effects on phenotypes are usually large.

Regulation of late endosomal/lysosomal maturation and trafficking by cortactin affects Golgi morphology.

Cortactin is a branched actin regulator and tumor-overexpressed protein that promotes vesicular trafficking at a variety of cellular sites, including endosomes and the trans-Golgi network. To better understand its role in secretory trafficking, we investigated its function in Golgi homeostasis. Here, we report that knockdown (KD) of cortactin leads to a dramatic change in Golgi morphology by light microscopy, dependent on binding the Arp2/3 actin-nucleating complex. Surprisingly, there was little effect of cortactin-KD on anterograde trafficking of the constitutive cargo vesicular stomatitis virus glycoprotein (VSVG), Golgi assembly from endoplasmic reticulum membranes upon Brefeldin A washout, or Golgi ultrastructure. Instead, electron microscopy studies revealed that cortactin-KD cells contained a large number of immature-appearing late endosomal/lysosomal (LE/Lys) hybrid organelles, similar to those found in lysosomal storage diseases. Consistent with a defect in LE/Lys trafficking, cortactin-KD cells also exhibited accumulation of free cholesterol and retention of the retrograde Golgi cargo mannose-6-phosphate receptor in LE. Inhibition of LE maturation by treatment of control cells with Rab7 siRNA or chloroquine led to a compact Golgi morphology similar to that observed in cortactin-KD cells. Furthermore, the Golgi morphology defects of cortactin-KD cells could be rescued by removal of cholesterol-containing lipids from the media, suggesting that buildup of cholesterol-rich membranes in immature LE/Lys induced disturbances in retrograde trafficking. Taken together, these data reveal that LE/Lys maturation and trafficking are highly sensitive to cortactin-regulated branched actin assembly and suggests that cytoskeletal-induced Golgi morphology changes can be a consequence of altered trafficking at late endosomes.

Absence of mature peripheral B cell populations in mice with concomitant defects in B cell receptor and BAFF-R signaling.

Generation of mature B lymphocytes from early (T1) and late transitional (T2) precursors requires cooperative signaling through BCR and B cell-activating factor receptor 3 (BR3). Recent studies have shown that BCR signaling positively regulates NF-kappaB2, suggesting BCR regulation of BR3 signaling. To investigate the significance of signal integration from BCR and BR3 in B cell development and function, we crossed Btk-deficient mice (btk(-/-)), which are developmentally blocked between the T2 and the mature follicular B cell stage as a result of a partial defect in BCR signaling, and A/WySnJ mice, which possess a mutant BR3 defective in propagating intracellular signals that results in a severely reduced peripheral B cell compartment, although all B cell subsets are present in relatively normal ratios. A/WySnJ x btk(-/-) mice display a B cell-autonomous defect, resulting in a developmental block at an earlier stage (T1) than either mutation alone, leading to the loss of mature splenic follicular and marginal zone B cells, as well as the loss of peritoneal B1 and B2 cell populations. The competence of the double mutant T1 B cells to respond to TLR4 and CD40 survival and activation signals is further attenuated compared with single mutations as evidenced by severely reduced humoral immune responses in vivo and proliferation in response to anti-IgM, LPS, and anti-CD40 stimulation in vitro. Thus, BCR and BR3 independently and in concert regulate the survival, differentiation, and function of all B cell populations at and beyond T1, earliest transitional stage.

Extracellular matrix rigidity promotes invadopodia activity.

Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) [1]. Molecular components of invadopodia include branched actin-assembly proteins, membrane trafficking proteins, signaling proteins, and transmembrane proteinases [1]. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2-4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM-rigidity signals depends on the cellular contractile apparatus [5-7], given that inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia, and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM-rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.

Dependence of invadopodia function on collagen fiber spacing and cross-linking: computational modeling and experimental evidence.

Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells.