Stratified Tissues Research Articles

3D-bioprinted anisotropic bicellular living hydrogels boost osteochondral regeneration via reconstruction of cartilage–bone interface

Reconstruction of osteochondral (OC) defects represents an immense challenge due to the need for synchronous regeneration of special stratified tissues. The revolutionary innovation of bioprinting provides a robust method for precise fabrication of tissue-engineered OCs with hierarchical structure; however, their spatial living cues for simultaneous fulfilment of osteogenesis and chondrogenesis to reconstruct the cartilage-bone interface of OC are underappreciated. Here, inspired by natural OC bilayer features, anisotropic bicellular living hydrogels (ABLHs) simultaneously embedding articular cartilage progenitor cells (ACPCs) and bone mesenchymal stem cells (BMSCs) in stratified layers were precisely fabricated via two-channel extrusion bioprinting. The optimum formulation of the 7% GelMA/3% AlgMA hydrogel bioink was demonstrated, with excellent printability at room temperature and maintained high cell viability. Moreover, the chondrogenic ability of ACPCs and the osteogenic ability of BMSCs were demonstrated invitro, confirming the inherent differential spatial regulation of ABLHs. In addition, ABLHs exhibited satisfactory synchronous regeneration of cartilage and subchondral bone invivo. Compared with homogeneous hydrogels, the neo-cartilage and neo-bone in ABLHs were augmented by 23.5% and 20.8%, respectively, and more important, a more harmonious cartilage-bone interface was achieved by ABLHs due to their well-tuned cartilage-bone-vessel crosstalk. We anticipate that such a strategy of tissue-mimetic ABLH by means of bioprinting is capable of spatiotemporal cell-driven regeneration, offering insights into the fabrication of anisotropic living materials for the reconstruction of complex organ defects.

The Application of High-frequency Ultrasonic Imaging in Identifying Fillers in the Temporal Region.

This study aimed to evaluate the ultrasonic manifestations of diversified corium fillers in the temporal region and to provide clinicians with suggestions for diagnosis and treatment. The facial ultrasound images of 116 patients, including 110 women and six men, 20-61years of age, were analyzed at the Chinese Academy Medical Sciences and Peking Union Medical College from November 2014 to November 2021. We identified 48 cases of polyacrylamide, 31 cases of fat, 27 cases of hyaluronic acid, seven cases of hydroxyapatite, two cases of silicone oil, and one case of prosthesis. Polyacrylamide mainly showed irregular flocculent hypoechoic or fine punctate hypoechoic in ultrasound; it could be aggregated as a cystic hypoechoic area or diffused in the tissue space, and the probe could be pressurized to see the echo floating or dislocation flow. Fat mainly showed lobulated hypoechoic deposition with some hyperechoic linear intervals. Hyaluronic acid mainly showed an anechoic structure with a clear boundary, uniform internal echo, and no obvious blood flow signal. If diffused in the surrounding tissues, it was mainly characterized by anechoic or low-echo areas in the stratified tissues. Hydroxyapatite mainly showed strongly hyperechoic patch areas with posterior acoustic shadowing artifacts. Silicone oil was mostly found under the epidermis, showing a high echo in the form of clouds and causing unclear displays of deep tissue. Prosthesis showed hypoechoic prosthesis structure. High-frequency ultrasound had a certain significance in the identification of the fillers of the temporal region.

Organic electronic transmembrane device for hosting and monitoring 3D cell cultures.

3D cell models have made strides in the past decades in response to failures of 2D cultures to translate targets during the drug discovery process. Here, we report on a novel multiwell plate bioelectronic platform, namely, the e-transmembrane, capable of supporting and monitoring complex 3D cell architectures. Scaffolds made of PEDOT:PSS [poly(3,4-ethylenedioxythiophene):polystyrene sulfonate] are microengineered to function as separating membranes for compartmentalized cell cultures, as well as electronic components for real-time in situ recordings of cell growth and function. Owing to the high surface area–to–volume ratio, the e-transmembrane allows generation of deep, stratified tissues within the porous bulk and cell polarization at the apico-basal domains. Impedance spectroscopy measurements carried out throughout the tissue growth identified signatures from different cellular systems and allowed extraction of critical functional parameters. This platform has the potential to become a universal tool for biologists for the next generation of high-throughput drug screening assays.

MiRNA induced 3D bioprinted-heterotypic osteochondral interface

The engineering of osteochondral interfaces remains a challenge. MicroRNAs (miRs) have emerged as significant tools to regulate the differentiation and proliferation of osteogenic and chondrogenic formation in the human musculoskeletal system. Here, we describe a novel approach to osteochondral reconstruction based on the three-dimensional (3D) bioprinting of miR-transfected adipose-derived stem cell (ADSC) spheroids to produce a heterotypic interface that addresses the intrinsic limitations of the traditional approach to inducing zonal differentiation via the use of diffusible cytokines. We evaluated the delivery of miR-148b for osteogenic differentiation and the codelivery of miR-140 and miR-21 for the chondrogenic differentiation of ADSC spheroids. Our results demonstrated that miR-transfected ADSC spheroids exhibited upregulated expression of osteogenic and chondrogenic differentiation related gene and protein markers, and enhanced mineralization and cell proliferation compared to spheroids differentiated using a commercially-available differentiation medium. Upon confirmation of the osteogenic and chondrogenic potential of miR-transfected ADSC spheroids, using aspiration-assisted bioprinting, these spheroids were 3D bioprinted into a dual-layer heterotypic osteochondral interface with a stratified arrangement of distinct osteogenic and chondrogenic zones. The proposed approach holds great promise for the biofabrication of stratified tissues, not only for the osteochondral interfaces presented in this work, but also for other composite tissues and tissue interfaces, such as, but not limited to, the bone-tendon-muscle interface and craniofacial tissues.

Dia1 coordinates differentiation and cell sorting in a stratified epithelium.

Although implicated in adhesion, only a few studies address how the actin assembly factors guide cell positioning in multicellular tissues. The formin, Dia1, localizes to the proliferative basal layer of the epidermis. In organotypic cultures, Dia1 depletion reduced basal cell density and resulted in stratified tissues with disorganized differentiation and proliferative markers. Since crowding induces differentiation in epidermal tissues, we hypothesized that Dia1 is essential to reach densities amenable to differentiation before or during stratification. Consistent with this, forced crowding of Dia1-deficient cells rescued transcriptional abnormalities. We find Dia1 promotes rapid growth of lateral cell-cell adhesions, necessary for the construction of a highly crowded monolayer. In aggregation assays, cells sorted into distinct layers based on Dia1 expression status. These results suggest that as basal cells proliferate, reintegration and packing of Dia1-positive daughter cells is favored, whereas Dia1-negative cells tend to delaminate to a suprabasal compartment. This work elucidates the role of formin expression patterns in constructing distinct cellular domains within stratified epithelia.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1–/– skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17–skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1–/– mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

Epidermal Stratification Requires Retromer-Mediated Desmoglein-1 Recycling

Sorting and trafficking transmembrane cargo is essential for tissue development and homeostasis. However, the importance of intracellular trafficking in the development and regeneration of stratified epidermis has not been investigated. Here we identify the interaction between VPS35, an essential component of the retromer endosomal trafficking complex, and the desmosomal cadherin, Desmoglein 1 (Dsg1). Dsg1 is specifically expressed in stratified tissues and when properly localized on the plasma membrane, promotes epidermal stratification. We show that the retromer drives Dsg1 recycling from the endolysosomal system to the plasma membrane to support keratinocyte stratification and differentiation. The retromer enhancing chaperone, R55 promotes the plasma membrane localization of Dsg1 and a Dsg1 mutant associated with Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome, enhancing the ability of SAM-Dsg1 to promote stratification. Our work provides the first evidence for retromer function in epidermal regeneration and identifies it as a potential therapeutic skin disease target.

AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (β-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

Disruption in murine Eml1 perturbs retinal lamination during early development

During mammalian development, establishing functional neural networks in stratified tissues of the mammalian central nervous system depends upon the proper migration and positioning of neurons, a process known as lamination. In particular, the pseudostratified neuroepithelia of the retina and cerebrocortical ventricular zones provide a platform for progenitor cell proliferation and migration. Lamination defects in these tissues lead to mispositioned neurons, disrupted neuronal connections, and abnormal function. The molecular mechanisms necessary for proper lamination in these tissues are incompletely understood. Here, we identified a nonsense mutation in the Eml1 gene in a novel murine model, tvrm360, displaying subcortical heterotopia, hydrocephalus and disorganization of retinal architecture. In the retina, Eml1 disruption caused abnormal positioning of photoreceptor cell nuclei early in development. Upon maturation, these ectopic photoreceptors possessed cilia and formed synapses but failed to produce robust outer segments, implying a late defect in photoreceptor differentiation secondary to mislocalization. In addition, abnormal positioning of Müller cell bodies and bipolar cells was evident throughout the inner neuroblastic layer. Basal displacement of mitotic nuclei in the retinal neuroepithelium was observed in tvrm360 mice at postnatal day 0. The abnormal positioning of retinal progenitor cells at birth and ectopic presence of photoreceptors and secondary neurons upon maturation suggest that EML1 functions early in eye development and is crucial for proper retinal lamination during cellular proliferation and development.

Atopic Dermatitis Studies through In Vitro Models.

Atopic dermatitis (AD) is a complex inflammatory skin condition that is not fully understood. Epidermal barrier defects and Th2 immune response dysregulations are thought to play crucial roles in the pathogenesis of the disease. A vicious circle takes place between these alterations, and it can further be complicated by additional genetic and environmental factors. Studies investigating in more depth the etiology of the disease are thus needed in order to develop functional treatments. In recent years, there have been significant advances regarding in vitro models reproducing important features of AD. However, since a lot of models have been developed, finding the appropriate experimental setting can be difficult. Therefore, herein, we review the different types of in vitro models mimicking features of AD. The simplest models are two-dimensional culture systems composed of immune cells or keratinocytes, whereas three-dimensional skin or epidermal equivalents reconstitute more complex stratified tissues exhibiting barrier properties. In those models, hallmarks of AD are obtained, either by challenging tissues with interleukin cocktails overexpressed in AD epidermis or by silencing expression of pivotal genes encoding epidermal barrier proteins. Tissue equivalents cocultured with lymphocytes or containing AD patient cells are also described. Furthermore, each model is placed in its study context with a brief summary of the main results obtained. In conclusion, the described in vitro models are useful tools to better understand AD pathogenesis, but also to screen new compounds in the field of AD, which probably will open the way to new preventive or therapeutic strategies.

Desmosomal cadherin association with a dynein‐cortactin complex promotes actin rearrangements required for epidermal morphogenesis

The epidermis is a multi‐layered, renewing epithelium that serves as an essential barrier against water loss and environmental insults in vertebrates. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes in which epidermal keratinocytes in the basal layer commit to differentiate, and move towards the skin's surface in a process called stratification. During this process, tissue integrity is ensured by intercellular junctions called desmosomes, which anchor the intermediate filament cytoskeleton to sites of desmosomal cadherin‐mediated adhesion. In contrast to the adherens junction protein, E‐cadherin, which is expressed in all epidermal layers, seven desmosomal cadherins are expressed in a highly patterned differentiation‐dependent manner in epidermis. This genomic complexity and spatial patterning of desmosome components in stratified tissues suggests that they do more than perform textbook functions in mediating cell‐cell adhesion. Here, we reveal an unexpected role for the cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into suprabasal layers through a process of delamination/extrusion. This function requires a newly recognized association between Dsg1 and the dynein light chain, Tctex‐1, and actin scaffolding protein, cortactin. We demonstrate that Tctex‐1, as a part of the dynein complex, controls the proper biochemical compartmentalization and segregation of Dsg1‐containing desmosomes from adherens junctions. Moreover, uncoupling the Dsg1‐Tctex‐1 complex from dynein or abrogation of Dsg1‐Tctex‐1 interactions interfered with the proper positioning of cortactin, which in turn is required to recruit the Arp2/3 complex and promote F‐actin network reorganization. Assembly of this molecular complex at the onset of Dsg1 expression during differentiation in cells already containing E‐cadherin results in decreased tension at adherens junctions, as measured through an E‐cadherin FRET sensor and accumulation of the mechanosensitive molecule vinculin. The proper coordination of this chain of events is required for the efficient delamination of basal cells in an organotypic model of tissue development. Thus, Dsg1 serves as a scaffold that exerts spatial control over mechanical changes necessary to promote early stages of epidermal morphogenesis.Support or Funding InformationSupported by NIH R01AR041836, R37AR043380, R01CA122151, the Dermatology Foundation and J.L. Mayberry Endowment.

Abstract 1084: Clinical significance of p63/miR-138 deregulation in tongue squamous cell carcinoma

Abstract BACKGROUND and AIMS: p63 is critical for the development and maintenance of stratified epithelial tissues. The role of p63 in tumorigenesis remains poorly defined. This study aims to determine the levels of p63 and miR-138 expression both in vitro and in vivo, and further to analyze their correlation with the clinicopathological parameters and prognosis in patients with tongue squamous cell carcinoma (TSCC). METHODS: Levels of p63 and miR-138 in TSCC and paired para-cancerous tissues were investigated by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. The correlations of p63 and miR-138 expression with the clinico- pathological parameters and prognosis of patients with TSCC were analyzed using SPSS 13.0 software package. Expression of p63 and miR-138 in TSCC cell lines (n = 5) was measured using immunoblot analysis and qRT-PCR, respectively. p63 was expressed transgenically, or knocked down with shRNAs in SCC9, SCC25, SCC15 and UM1 cell lines, and effects on cell invasion and migration were evaluated. RESULTS: Elevated level of p63 and reduced level of miR-138 was observed in TSCC tissues in comparison with paired para-cancerous tissues (p < 0.05). In particular, a more elevated expression for p63 appeared in the invasive tumor front compared with superficial or central parts of the primary tissues (p < 0.05). A negative correlation between p63 and miR-138 expression can be observed in the TSCC tissues. Statistical analysis revealed that p63 and miR-138 expression level are associated with cell differentiation, cervical lymph node metastasis status and clinical stage as well in patients with TSCC. COX regression analysis shows that p63 over-expression was correlated with reduced overall survival. P63 can be served as an independent prognostic factor for patients with TSCC. These observations were confirmed in vitro, in which ΔNp63 is the predominant p63 isoform expressed in the TSCC cell lines, and a negative correlation between p63 and miR-138 expression can be observed (p < 0.05). Ectopic transfection of p63 led to reduce miR-138 expression and enhanced cell invasion and migration. On the contrary, knockdown of p63 enhanced miR-138 expression and suppressed cell invasion and migration. Furthermore, transfection of miR-138 partly reversed cell invasion and migration induced by p63 overexpression. CONCLUSION: The dysregulation of p63 and miR-138 are common molecular events in TSCC progression. These findings suggest that p63 and miR-138 may collaboratively play a role in tongue carcinogenesis. Citation Format: Zehang Zhuang, Yun Wang, Cheng Wang, Nan Xie, Yue Wu, Jinsong Hou, Xiqiang Liu, Hongzhang Huang. Clinical significance of p63/miR-138 deregulation in tongue squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1084.

Synthesis of a novel clay-based nanocomposite hydrogel with attractive mechanical properties and its potential application in 3D-Printing

Event Abstract Back to Event Synthesis of a novel clay-based nanocomposite hydrogel with attractive mechanical properties and its potential application in 3D-Printing Xinyun Zhai1, 2*, William Lu1* and Wenguang Liu2* 1 The University of Hong Kong, Department of Orthopaedics and Traumatology, Li Ka Shing Faculty of Medicine, Hong Kong, SAR China 2 Tianjin University, School of Materials Science and Engineering, China Introduction: In our study a novel nanocomposite hydrogel (NC gel) was successfully prepared by in situ free-radical photo-polymerization of the acrylic acid derivatives – 4-Acryloylmorpholine in the presence of exfoliated clay platelets in aqueous systems with different clay contents. Although N-isopropylacrylamide (NIPAM) and N, N-dimethylacrylamide (DAMAA) have already been used as monomers in the clay-based hydrogel, this was the first time to add this kind of acrylic acid derivative into clay system and hydrogel could form without using an organic cross-linker[1],[2]. The obtained hydrogel not only exhibits dramatic improvements in mechanical properties but also has drug loading and release behaviors. Materials and Methods: 4-Acryloylmorpholine (monomer) and 2-Hydroxy-2-methylpropiophenone (1173, initiator) were purchased from Sigma-Aldrich. Synthetic hectorite clay of sol-forming grade LAPONITE XLS (Rockwood Ltd., 92.32 wt % of Mg5.34Li0.66Si8O20(OH)4Na0.66 and 7.68 wt % of Na4P2O7) was used directly. The 4-Acryloylmorpholine/clay nanocomposite hydrogels were synthesized by in situ free-radical photo-polymerization of 4-Acryloylmorpholine in the presence of exfoliated clay. Firstly, clay was dispersed in water under ultrasonication for 1 h. Secondly, 4-Acryloylmorpholine and 1173 were added to the clay suspension. After the solution was mixed well, it was transferred into plastic tubes with about 2 mm inner diameter. Photo-polymerization was carried out for 50 min in a crosslink oven (XL-1000 UV Crosslinker, Spectronics Corporation, NY, USA). The clay content was varied from 1 to 10 wt % with respect to the 4-Acryloylmorpholine weight and the solid content of the nanocomposite hydrogel was varied from 20% to 30%. Tensile tests, compression tests, FTIR, XRD, SEM, TEM, XRD and Raman spectrum were used in our experiment. Results and Discussion: Mechanical tests show that the obtained 4-Acryloylmorpholine clay-based hydrogel has the best tensile strength (about 400 kPa) and excellent stretch ability (higher than 5000%) when clay content and solid content are 5% and 20% respectively. The compression strength of the hydrogel is higher than 8 MPa and can recover to its original shape when compression ratio is less than 80% which will be very attractive for tissue engineering. It is very different from the original physically cross-linked nanocomposite hydrogels that the resulting hydrogel can still stretch up to 2000% in the swollen state. Unlike conventional physically cross-linked hydrogels, this kind of clay-based hydrogel is insoluble in PBS or hot water even the temperature is 80~90 ℃ due to the high hydrogen bonds exist between 4-Acryloylmorpholine polymer and clay which has been confirmed by Raman spectroscopy. Both the XRD and TEM results demonstrate the uniform dispersion of clay in the nanocomposite hydrogel and the formation of the clay-based hydrogel. Furthermore, by using 4-Acryloylmorpholine as the monomer, the hydrogel has drug loading and release behaviors, as the weight ratio of 4-Acryloylmorpholine increases, higher drug release can be observed. Conclusion: Nanocomposite hydrogels composed of 4-Acryloylmorpholine and Laponite XLG clay showed attractive fracture strain up to 5000% and good compression strength (higher than 8 MPa). This kind of hydrogel was synthesized by in situ photo-polymerization of 4-Acryloylmorpholine with the presence of exfoliated Laponite XLG clay. The strong hydrogen bonds between 4-Acryloylmorpholine polymer and clay are suggested to account for the good mechanical properties of the hydrogel. More interestingly, the obtained hydrogel has good biocompatibility and high viscosity before photo-polymerization, which means this kind of hydrogel is a very suitable bioink candidate of the layer-by-layer bioprinting method for the fabrication of stratified tissues[3].

A versatile bioink for three-dimensional printing of cellular scaffolds based on thermally and photo-triggered tandem gelation

Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while HAMA ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.

Invasive leader cells: metastatic oncotarget.

Invasion into surrounding tissues is the first step of metastasis, and an understanding of the molecular basis of the invasive process may suggest novel strategies for anti-metastatic therapy. Breast tumors typically invade as adherent groups of cells, a process known as collective invasion. In vivo, collective invasion occurs within a complex tissue environment and involves interactions among the cancer cells, stromal cells, and the extracellular matrix (ECM). An additional challenge to understanding cancer invasion is that primary breast tumors contain diverse cancer cells with varied molecular phenotypes. It is difficult to determine the relative contribution of these subpopulations to invasion from analysis of histologic sections. We sought to reduce this complexity and to identify the most invasive cancer cells in the primary tumor in an unbiased fashion [1]. To accomplish this goal, we developed a three dimensional (3D) organoid invasion assay (Figure ​(Figure1A).1A). Briefly, we use enzymes and mechanical disruption to isolate organoids from primary breast tumors from mouse models and human patients. Each organoid is composed of several hundred cancer cells and is embedded in an invasion promoting ECM [2] (collagen I). Tumor organoids invade collectively and the cells at the tips of invasion strands are protrusive and highly interactive with the ECM. We refer to the cells at the tips of these strands as invasive leader cells (Figure ​(Figure1B1B). Figure 1 A. Tumor organoids are isolated from fresh primary tumors through a combination of mechanical disruption and enzymatic digestion. Tumor organoids are then embedded in 3D collagen I and become invasive. B. Organoids extend multiple invasive strands into ... We found that the molecular phenotype of invasive leader cells was distinct from the bulk tumor cells and conserved across breast cancer subtypes [1]. Leader cells expressed markers of basal epithelium, which are the cells in normal stratified tissues that interact with the ECM (Figure ​(Figure1C).1C). The basal cytokeratin, keratin-14 (K14), was expressed in ~90% of leader cells. Importantly, K14+ cells led collective invasion in multiple distinct mouse models and in diverse primary human breast tumors. We next used molecular biosensors to demonstrate that K14+ invasive leader cells were generated from non-invasive K14- bulk tumor cells. Importantly, knockdown of either K14 or p63, another leader cell marker, was sufficient to inhibit collective invasion. Our data establish three important concepts. First, we have demonstrated that the invasive behavior of the primary tumor is determined by a subset of specialized cancer cells1. Second, our data suggest a common molecular biology underlying breast cancer metastasis, because K14+ cells led collective invasion in 3D organoid invasion assays from patients with different breast cancer subtypes. In support of this concept, multiple groups have observed that K14 expression in breast tumors correlates with poor patient outcomes, independent of breast cancer subtype [3-5]. Third, since bulk tumor cells can convert into K14+ invasive leaders, we hypothesize that inhibition of the basal molecular program could control progression in patients with metastatic disease. Our 3D organoid invasion assay can also be utilized to identify invasive leader cells in other epithelial cancers. Because basal cells are found in many stratified epithelial tissues, the basal invasive program we identified may be utilized across diverse cancers. In support of this concept, basal cytokeratins are associated with invasion in both lung adenocarcinoma and hepatocellular carcinoma [6,7]. It is now critical to develop the translational framework to leverage our knowledge of invasive leader cells to improve patient outcomes. We are specifically interested to determine whether the abundance of invasive leader cells in the primary tumor will provide independent information about the prognosis of individual patients. Our invasion assays also provide an ideal platform to identify anti-invasive therapeutic compounds. Now that we have the invasive leader cell in our sights, we are working to defeat this new adversary.

An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b

The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.

Abstract 1306: p63 functions as an essential oncogene in squamous cell carcinoma in vivo and is required for tumor maintenance

Abstract Transcription factors mediate diverse functions in normal development and tumorigenesis. The p53-related transcription factor p63 (TP63) plays an essential role in development of stratified epithelial tissues, as germline loss of p63 leads to abnormal proliferation and differentiation of ectoderm-derived precursors. The role of p63 in human cancer, however, is controversial. Loss of p63 is associated with neoplastic progression in some animal models and in a subset of human tumors. Nevertheless, p63 is rarely targeted for somatic mutational inactivation and is indeed overexpressed in squamous cell carcinomas (SCCs), where is has been postulated to function either as a key tumor maintenance factor or as a lineage marker not essential to the malignant process. Here, we show that SCCs are profoundly dependent on p63 expression in vivo, and we identify a key, therapeutically relevant pathway through which p63 mediates tumor maintenance. Conditional genetic ablation of p63 in an endogenous SCC model induces rapid and dramatic tumor regression owing to apoptosis of p63-expressing cells. We identify the FGFR2 oncogene as a direct transcriptional target of p63 that signals through AKT to mediate survival of SCC cells. Finally, we employ the clinical FGFR2 inhibitor AZD4547 to demonstrate the potential therapeutic effect of extinguishing FGFR2 signaling in p63-expressing SCCs. These results define p63 as a key tumor maintenance factor through regulation of FGFR2 signaling in SCC. More broadly, this demonstrates the potential for targeting oncogenic transcription factor pathways in tumors such as SCC that rarely exhibit somatic oncogene-activating mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1306. doi:1538-7445.AM2012-1306

Immunolocalization of lymphatic vessels in human fetal knee joint tissues

We immunolocalized lymphatic and vascular blood vessels in 12- and 14-week-old human fetal knee joint tissues using a polyclonal antibody to a lymphatic vascular endothelium specific hyaluronan receptor (LYVE-1) and a monoclonal antibody to podoplanin (mAb D2-40). A number of lymphatic vessels were identified in the stratified connective tissues surrounding the cartilaginous knee joint femoral and tibial rudiments. These tissues also contained small vascular vessels with entrapped red blood cells which were imaged using Nomarsky DIC microscopy. Neither vascular nor lymphatic vessels were present in the knee joint cartilaginous rudiments. The menisci in 12-week-old fetal knees were incompletely demarcated from the adjacent tibial and femoral cartilaginous rudiments which was consistent with the ongoing joint cavitation process at the femoral-tibial junction. At 14 weeks of age the menisci were independent structural entities; they contained a major central blood vessel containing red blood cells and numerous communicating vessels at the base of the menisci but no lymphatic vessels. In contrast to the 12-week-old menisci, the 14-week meniscal rudiments contained abundant CD-31 and CD-34 positive but no lymphatic vessels. Isolated 14-week-old meniscal cells also were stained with the CD-31 and CD 34 antibodies; CD-68 +ve cells, also abundant in the 14-week-old menisci, were detectable to a far lesser degree in the 12-week menisci and were totally absent from the femoral and tibial rudiments. The distribution of lymphatic vessels and tissue macrophages in the fetal joint tissues was consistent with their roles in the clearance of metabolic waste and extracellular matrix breakdown products arising from the rapidly remodelling knee joint tissues.

Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering

Reconstructing a vascular network is a common task for three-dimensional (3-D) tissue engineering. Three-dimensional stratified tissues were created by stacking cell sheets, and the co-culture with endothelial cells (ECs) in the tissues was found to lead to in vitro pre-vascular network formation and promoted in vivo neovascularization after their transplantation. In this study, to clarify the effect of tissue fabrication process on a pre-vascular network formation, human origin ECs were introduced into the stratified tissue in several different ways, and the behavior of ECs in various positions of the 3-D tissue were compared each other. Human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), and their mixture were harvested as an intact cell sheet from temperature-responsive culture dish at low-temperature (<20 °C). Single mono-culture EC sheet was stacked with two NHDF-sheets in different orders, and 3 co-cultured cell sheets were layered by a cell sheet collecting device. Morphological analyses revealed that pre-vascular networks composing of HUVECs were formed in all the triple layer constructs. Confocal microscope observation showed that the pre-vascular networks formed tube structures like a native microvasculature. These data indicate that the primary EC positioning in 3-D tissues may be critical for vascular formation.

Oxygen deprivation inhibits basal keratinocyte proliferation in a model of human skin and induces regio‐specific changes in the distribution of epidermal adherens junction proteins, aquaporin‐3, and glycogen

It is generally accepted that hypoxia and recovery from oxygen deprivation contribute to the breakdown and ulceration of human skin. The effects of these stresses on proliferation, differentiation and expression of cell-cell adhesion molecules were investigated for the first time in an organotypic model of human skin. Fully stratified tissues were exposed to a time course of oxygen deprivation and subsequent reoxygenation. Regional changes in keratinocyte morphology, glycogen stores and cellular junctions were observed, with more differentiated layers of the epidermis exhibiting the first evidence of oxygen deprivation. Cellular swelling within the granular layer was concurrent with aquaporin-3 depletion. The keratinocyte adherens junction proteins E-cadherin and beta-catenin were dramatically decreased in a regio-specific manner throughout the epidermis following oxygen deprivation. In contrast, P-cadherin and the desmosomal proteins desmoplakin and desmoglein-1 were refractory to oxygen deprivation. Relative to normoxic controls, hypoxic tissues exhibited increased mRNA levels of the transcriptional repressor Slug; however, mRNA levels of the related transcriptional factor Snail were unaffected. All cellular and molecular changes were reversible upon reoxygenation. These results show that oxygen deprivation and reoxygenation exert differential effects on epidermal adhesion proteins and suggest a novel role for cadherins, beta-catenin, and Slug in hypoxia-induced junctional changes occurring in stratified squamous epithelium.