Three-dimensional Cell Clusters Research Articles

Remotely operated robotic microscopy for rapid diagnosis of bronchoscopic cytology specimens.

Obtaining a diagnosis and treating pulmonary malignancies during the same anesthesia requires either an on-site pathologist or a system for remotely evaluating microscopic images. Cytology specimens are challenging to remotely assess given the need to navigate through dispersed and three-dimensional cell clusters. Remote navigation is possible using robotic telepathology, but data are limited on the ease of use of current systems, particularly for pulmonary cytology. Air dried modified Wright-Giemsa stained slides from 26 touch preparations of transbronchial biopsies and 27 smears of endobronchial ultrasound guided fine needle aspirations were scored for ease of adequacy assessment and ease of diagnosis on robotic (rmtConnect Microscope) and non-robotic telecytology platforms. Diagnostic classifications were compared between glass slides and the robotic and non-robotic telecytology assessments. Compared to non-robotic telecytology, robotic telecytology had a greater ease of adequacy assessment and non-inferior ease of diagnosis. The median time to diagnosis using robotic telecytology was 85 s (range 28-190 s). Diagnostic categories were concordant for 76% of cases in robotic versus non-robotic telecytology and 78% of cases in robotic telecytology versus glass slide diagnosis. Weighted Cohen's kappa scores for agreement in these comparisons were 0.84 and 0.72, respectively. Use of a remote-controlled robotic microscope improved the ease of adequacy assessment compared to non-robotic telecytology and enabled strongly concordant diagnoses to be expediently rendered. This study provides evidence that modern robotic telecytology is a feasible and user-friendly method of remotely and potentially intraoperatively rendering adequacy assessments and diagnoses on bronchoscopic cytology specimens.

Evaluation of chorionic girdle organoids for ultrastructure and equine Chorionic Gonadotropin production

The equine chorionic girdle (CG) is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (Day 0). The CG cells differentiate into binucleate endometrial cup cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG), which has LH-like activity in the horse but variable LH- and FSH-like activity in other species. eCG has been utilized for these properties both in vivo for estrous cycle manipulation and in vitro. To collect this hormone, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare. Attempts to produce eCG in vitro using long-term culture of CG explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and remain genetically and phenotypically stable throughout long-term (i.e. months) culture. Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin long-term. The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. CG was collected from Day 33 equine conceptuses (n=3). CG was isolated manually using a dissection microscope and then enzymaticallydigested and cultured as organoids using human trophoblast organoid medium. A monolayer culture was performed as a control 2D culture model for comparison. CG organoids were assessed morphologically using brightfield microscopy, immunohistochemistry (IHC) with an anti-horse trophoblast antibody, and transmission electron microscopy (TEM), and all cultures were assessed physiologically by quantifying equine LH (as a proxy for eCG) via radioimmunoassay of conditioned media. The dense, trophoblast organoid phenotype, as described for human trophoblast organoids, was maintained for 5 passages, a period of 6 weeks. TEM of organoids revealed characteristic binucleate cells in both passages 1 and 4 and exhibited the presence of girdle cell processes, which have been described previously in endometrial cups in vivo. Furthermore, eCG was secreted at high concentrations (up to 67,000 ng/mL) up to passage 4 in organoids but only for passage 0 in monolayer cultures (up to 1,100 ng/mL). Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for early equine pregnancy. Funding was provided by Foundation for the Horse.

Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

Standardization of organoid culture in cancer research.

Establishing a valid in vitro model to represent tumor heterogeneity and biology is critical but challenging. Tumor organoids are self-assembled three-dimensional cell clusters which are of great significance for recapitulating the histopathological, genetic, and phenotypic characteristics of primary tissues. The organoid has emerged as an attractive in vitro platform for tumor biology research and high-throughput drug screening in cancer medicine. Organoids offer unique advantages over cell lines and patient-derived xenograft models, but there are no standardized methods to guide the culture of organoids, leading to confusion in organoid studies that may affect accurate judgments of tumor biology. This review summarizes the shortcomings of current organoid culture methods, presents the latest research findings on organoid standardization, and proposes an outlook for organoid modeling.

Human enteroids as a tool to study conventional and ultra-high dose rate radiation.

Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.

Magnetic levitation for non-contact manipulation and measurement of cells

Non-contact manipulation and measurement of cells are the major concerns in the biomedical field. However, the existing methods are limited by the physical properties of cells, and may cause damages to the cells. This paper proposes an original non-contact manipulation and measurement method by magnetic levitation. Based on the configuration of two ring magnets with like poles facing each other, the cells can aggregate and levitate in the paramagnetic medium. The mathematical model was established to analyze the distributions of magnetic fields and movements of cells for the first time. Theoretical and experimental results show that the magnetic levitation method can be adaptive to the cells by tuning the size of the configuration. The three-dimensional cell clusters were formed and the average densities of 3T3, MDA, B16 and 4T1 cells were measured as 1.074 ± 0.005 g/cm3, 1.084 ± 0.003 g/cm3, 1.079 ± 0.002 g/cm3 and 1.124 ± 0.003 g/cm3 respectively. Moreover, the dead cells and living cells were distinguished based on the levitating heights. The results indicate that the proposed magnetic levitation method is promising for the three-dimensional culture and rapid measurement of cells.

Vascularized Tumor Spheroid-on-a-Chip Model Verifies Synergistic Vasoprotective and Chemotherapeutic Effects.

Prolyl hydroxylases (PHD) inhibitors have been observed to improve drug distribution in mice tumors via blood vessel normalization, increasing the effectiveness of chemotherapy. These effects are yet to be demonstrated in human cell models. Tumor spheroids are three-dimensional cell clusters that have demonstrated great potential in drug evaluation for personalized medicine. Here, we used a perfusable vascularized tumor spheroid-on-a-chip to simulate the tumor microenvironment in vivo and demonstrated that the PHD inhibitor dimethylallyl glycine prevents the degradation of normal blood vessels while enhancing the efficacy of the anticancer drugs paclitaxel and cisplatin in human esophageal carcinoma (Eca-109) spheroids. Our results point to the potential of this model to evaluate anticancer drugs under more physiologically relevant conditions.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

SummaryGenetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

Generation and Characterization of Murine Oral Mucosal Organoid Cultures.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

Homotypic aggregates contribute to heterogeneity in B cell fates due to an intrinsic gradient of stimulant exposure

Monocultures of several cell types result in the formation of robust clusters called homotypic aggregates (HAs). How this physical aggregation affects cell fates in immune cell cultures, is poorly understood. We studied anti-CD40-stimulated primary B cell cultures, where cells assembled into large three-dimensional LFA1-driven HAs by 72 h. The dense packing in these aggregates restricts the infiltration of stimulants, such as antibodies, to cells inside the clusters. This creates a concentration gradient of stimulant availability across the cross-section of HAs. We describe a method to retain this positional information even after the disruption of HAs, for analysis by flow cytometry. Comparison of stage-specific cell-surface markers showed that the extent of stimulant-binding affected multiple fates non-uniformly. While germinal center and lineage markers were moderately upregulated, immunoglobulins and markers associated with memory were more than doubled in the peripheral cells binding more anti-CD40. These cells also experienced a strong repression of the plasma cell regulator Prdm1 and an upregulation of the oncogene Myc. Thus, cells at different locations in HAs are subjected to unequal doses of stimulants, leading to a hitherto unreported source of heterogeneity in cell fates. These findings can be extrapolated to understand the dose-dependent effects of stimulants in other three-dimensional cell clusters.

Decellularized Matrix Produced by Mesenchymal Stem Cells Modulates Growth and Metabolic Activity of Hepatic Cell Cluster.

Miniature organlike three-dimensional cell clusters often called organoids have emerged as a useful tool for both fundamental and applied bioscience studies. However, there is still a great need to improve the quality of organoids to a level where they exhibit similar biological functionality to an organ. To this end, we hypothesized that a decellularized matrix derived from mesenchymal stem cell (MSC) could regulate the phenotypic and metabolic activity of organoids. This hypothesis was examined by culturing cells of interest in the decellularized matrix of MSCs cultured on a 2D substrate at confluency or in the form of spheroids. The decellularized matrix prepared with MSC spheroids showed a 3D porous structure with a higher content of extracellular matrix molecules than the decellularized matrix derived from MSCs cultured on a 2D substrate. HepG2 hepatocarcinoma cells, which retain the metabolic activity of hepatocytes, were cultured in these decellularized matrices. Interestingly, the decellularized matrix from the MSC spheroids served to develop the hepatic cell clusters with higher levels of E-cadherin-mediated cell-cell adhesion and detoxification activity than the decellularized matrix from the MSCs cultured on a 2D substrate. Overall, the results of this study are useful in improving biological functionality of a wide array of organoids.

Emergent versus Individual-Based Multicellular Chemotaxis.

Multicellular chemotaxis can occur via individually chemotaxing cells that are mechanically coupled. Alternatively, it can emerge collectively, from cells chemotaxing differently in a group than they would individually. Here we consider collective movement that emerges from cells on the exterior of the collective responding to chemotactic signals, whereas bulk cells remain uninvolved in sensing and directing the collective. We find that the precision of this type of emergent chemotaxis is higher than that of individual-based chemotaxis for one-dimensional cell chains and two-dimensional cell sheets, but not three-dimensional cell clusters. We describe the physical origins of these results, discuss their biological implications, and show how they can be tested using common experimental measures such as the chemotactic index.

Synergistic Therapeutic Effect of Three-Dimensional Stem Cell Clusters and Angiopoietin-1 on Promoting Vascular Regeneration in Ischemic Region.

Peripheral artery disease (PAD) is an ischemic disease characterized by reduced blood flow to the legs, feet, and hands. Human mesenchymal stem cells are an attractive cell source to treat PAD in regenerative medicine. However, in clinical applications, the use of adult stem cells has several limitations, such as low cell viability and low therapeutic efficiency. In this study, we described an innovative method of culturing three-dimensional stem cell clusters (Angiocluster™ [AC]), demonstrated the potential for ACs to differentiate into vascular cells, and assessed the synergistic effects of ACs and angiopoietin-1 (Ang-1) on angiogenesis in ischemic animal models. ACs were formed by culturing human adipose-derived stem cells (hASCs) on a maltose-binding protein-linked basic fibroblast growth factor-immobilized polystyrene surface. ACs released various angiogenic factors, such as vascular endothelial growth factor and interleukin-8, and could differentiate into endothelial lineage cells. However, ACs did not secrete Ang-1, which is an essential component of vascular maturation and anti-inflammation. ACs were combined with Ang-1 and were transplanted into the ischemic lesions of mice for 28 days. Most of the mice receiving the AC + Ang-1 treatment exhibited limb salvage and exhibited similar blood perfusion ratio compared to normal limb. The combination therapy of AC and Ang-1 enhanced angiogenic efficacy by increasing blood vessel regeneration and facilitating the implantation of stem cells into host vessels. Importantly, fibrotic collagen was observed in most of the groups after 28 days of treatment, except for the AC + Ang-1 group. This indicates that the combination therapy is synergistic in minimizing ischemic fibrosis and muscle degeneration. Our results demonstrate that the combination therapy significantly enhanced tissue regeneration and angiogenic efficacy of hASCs and may have wide applications in regenerative medicine.

Superiority of three-dimensional stem cell clusters over monolayer culture: An archetype to biological application

Mesenchymal stem cells (MSCs) are attractive and potential alternatives to cell-based therapeutics in the field of regenerative medicine. Various clinical trials are being conducted to investigate the therapeutic efficacy of MSCs in a variety of diseases. Many studies show three-dimensional (3D) aggregates as more physiologically relevant than conventional two-dimensional (2D) cultures. Cell morphology, viability, functionality, as well as various gene expressions at the molecular level are drastically changed after 3D culture. MSCs as 3D aggregates provide enhanced differentiation potential and upregulation of pluripotency markers that subsequently result in stemness of MSCs. Furthermore, other biological properties like angiogenic effect, anti-inflammatory effect, regenerative effect, and survival after transplantation are also significantly enhanced in spheroids. Thus, 3D culture is a novel approach in cell-based therapeutics applicable in the field of regenerative medicine and tissue engineering, immunomodulatory diseases and organ transplantation for improved therapeutic outcomes. Various mechanism and techniques have been described for the formation of spheroids in different studies, some of which are discussed here. However, the molecular mechanism underlying the improved functionality and other changes is still unclear and requires further research. Similarly, clinically relevant studies are required to establish the clinical applicability.

Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters.

Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.

Cytologic features of pancreatic adenocarcinoma with “vacuolated cell pattern.” report of a case diagnosed by endoscopic ultrasound‐guided fine‐needle aspiration

The "vacuolated cell pattern" has only been recently described as a distinct morphologic variant of pancreatobiliary adenocarcinoma. Herein, we report the endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) cytologic features of a case of pancreatic adenocarcinoma with "vacuolated cell pattern" occurring in a 60-year-old man. The aspirate smears and cell block sections from the EUS-FNA of a 23.5 mm hypoechoic pancreatic head mass were highly cellular, showing variably-sized crowded three-dimensional cell clusters, flat sheets, and numerous highly atypical single cells. The background was bloody and showed necrotic debris, but no discernible mucus. The most striking feature of the aspirate was the presence of numerous very large (20-50 µm) vacuoles, occupying the entire cytoplasm, pushing the nuclei to the side and indenting them, that imparted a cribriform appearance to the sheets of neoplastic cells. The non-vacuolated neoplastic cells were large, had abundant dense (squamoid) cytoplasm, irregularly contoured hyperchromatic nuclei, and prominent macronucleoli. Histologic evaluation of the pancreatectomy specimen showed a "vacuolated cell pattern" adenocarcinoma composed of poorly formed glands, solid sheets, and infiltrating single cells with pleomorphic nuclei and large cytoplasmic vacuoles. To our knowledge, this is the first report describing the cytologic features of this rather uncommon morphologic variant of pancreatic adenocarcinoma. Recognition of this morphologic variant of pancreatic adenocarcinoma in ESU-FNA samples allows its differentiation from primary and metastatic signet-ring cell carcinomas.

Towards a bioengineered liver

A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interac- tions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver- specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demon- stration of organ-bud transplantation provides a promising new approach to study regenerative medicine. 2013 European Association for the Study of the Liver. Published

The Effects of irridation on glioblastoma cell lines: a potential tool to study the mechanism of tumor resistance

Glioblastoma Multiforme (GBM) is one of the most devastating brain cancers. Here, we sequentially irradiated rat and human GBM cell lines to examine cell injury and cell death in pre‐ and post‐irradiated cells. We have used TUNEL, flow cytometry, western blotting and immunocytochemistry methods to study the effects of radiation on cell death and cell cycle and to exam various proteins expression in post‐irradiated radio‐resistant cells. The results showed that radiation induces apoptotic cell death, and the mortality rate on post‐irradiated GBM cells and their vimentin expression have a negative correlation. Interestingly, vimentin not only expressed in the cytoplasm(s) of post‐irradiated cells but also formed aggregates on the nuclei. Post‐irradiated cells also formed three‐dimensional cell clusters and expressed desmin and myogenin. The radiation affected the expression of p53, Cdk‐2, and Cdk‐4. Correlation analysis of p53 and Cdk‐2 proteins expression on day 3 after 4 Gy radiation suggested that their expression may regulate through p21 in the cell cycle. Control cells and post‐irradiated cells do not express E‐cadherin but have different protein expression patterns for fibronectin and vimentin. This indicates that EMT may occur in these post‐irradiated cell lines. This may indicate that EMT occurs in this post‐irradiated cell lines.

Cytology of metastatic cervical squamous cell carcinoma in pleural fluid: Report of a case confirmed by human papillomavirus typing

Cervical squamous cell carcinomas are rarely the cause of malignant effusions. Their identification can be relatively easy when keratinizing atypical squamous cells are present, but may be very difficult when only nonkeratinizing malignant cells are present. We present the case of a 47-year-old woman who presented with a large left pleural effusion after having recently completed chemoradiation therapy for stage IIB cervical squamous cell carcinoma. Cytologic examination of the fluid showed a uniform population of single atypical cells with finely vacuolated cytoplasm, ectoendoplasmic demarcation, cell-in-cell arrangements, and short rows of cells with intervening "windows," all features reminiscent of mesothelial cells. No keratinization or three-dimensional cell clusters were identified. A panel of immunohistochemical stains was performed on the cell block material, and the atypical cells were positive for cytokeratin 5/6, p63, and p16 but not for cytokeratin 7, calretinin, WT1, or Ber-EP4 or TTF1. These findings were consistent with metastatic squamous cell carcinoma. HPV DNA determination and typing by PCR confirmed the presence of HPV16 in an aliquot of pleural fluid. This is to our knowledge the first reported case of pleural fluid involved by metastatic squamous cell carcinoma where HPV DNA testing was used to confirm the origin of the metastasis. Despite its rarity, metastatic nonkeratinizing squamous cell carcinoma should be considered when a single cell population of large atypical cells is found in effusions. Immunoperoxidase stains and HPV testing can be performed to establish the diagnosis and confirm the origin from a cervical primary.

A three-dimensional assay for measurement of viral-induced oncolysis

Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/3-Delta24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-Delta24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.