Organotypic Raft Cultures Research Articles

An in vitro model and the underlying pathways of sinonasal inverted papilloma development

Recently, the specific association between Sinonasal inverted papilloma (SIP) and EGFR exon 20 mutations has been reported. To investigate the link between specific EGFR mutations and SIP development, we established organotypic raft culture system using nasal polyp-derived immortalized NP2 (iNP2) cells expressing EGFR exon 20 mutants or an exon 19 mutant, and SIP-derived iIP4 cells harboring P772_H773insPYNP mutation. In the raft culture, iIP4 cells showed the inverted growth pattern characteristic to SIP. Interestingly, iNP2 cells expressing EGFR exon 20 duplication mutants, S768_D770dup and N771_H773dup, but not of EGFR exon 19 mutant, E746_A750del, showed the inverted growth pattern. Enhanced activation of the PI3K/AKT signaling pathway was observed in iNP2_S768_D770dup and iIP4 cells, while increased MAPK signaling was found in iNP2_N771_H773dup. Increased cell migration and invasion were found in all cells carrying EGFR mutations when compared to iNP2 cells, and this effect was inhibited by either PI3K or MEK inhibitor. Notably, iNP2 cells expressing the N771_H773dup mutant showed the highest migration and invasion abilities. These results suggest that specific mutations in EGFR exon 20 play a crucial role in SIP development, partially though hyper-activation of the PI3K/AKT and MAPK signaling pathways. This study presents the first in vitro model for SIP development, which could facilitate further investigations into SIP pathogenesis and preclinical studies for new therapeutic agents.

Human Papillomavirus Genome Copy Number Is Maintained by S-Phase Amplification, Genome Loss to the Cytosol during Mitosis, and Degradation in G1 Phase.

The current model of human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. The HPV genome copy number remains constant during the maintenance stage. The differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that the viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. The nuclear viral genome copy number is reset to pre-S-phase levels during mitosis. The majority of the viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genomes gradually decrease during cell cycle progression. The loss of cytosolic genomes is blocked in the presence of NH4Cl or other drugs that interfere with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genomes were not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genomes as well. We conclude that HPV maintains viral genome copy numbers by balancing viral genome amplification during S phase with the loss of viral genomes to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes reset genome copy numbers in each cell cycle. IMPORTANCE HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes. Here, we demonstrate that the HPV genome is amplified in each S phase. The nuclear genome copy number is reset during mitosis by a failure of the majority of the genomes to tether to mitotic chromosomes. Rather, HPV genomes accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is degraded in G1 in a lysosome-dependent manner, contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contribute to viral genome maintenance.

Retinoblastoma Protein Is Required for Epstein-Barr Virus Replication in Differentiated Epithelia.

Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.

FOXP4 inhibits squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre‐existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4‐positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1‐derived HPV 16‐positive W12 cells via an ELF3‐dependent pathway. In organotypic raft cultures, FOXP4‐downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte‐derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3‐dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.

847 Modeling basal cell carcinoma in 3D organotypic raft cultures

Basal cell carcinoma (BCC) is the most-commonly diagnosed cancer worldwide with limited availability for non-surgical treatments. The Hedgehog signaling cascade drives tumorigenesis, but efforts to develop effective chemotherapeutics have been thwarted by chemoresistant tumor recurrence. A contributing factor to this is an overall lack of human-based preclinical models. We endeavored to develop 3D organotypic raft models that mimic characteristics of human BCC by combining primary normal human epidermal keratinocytes (NHEK) with established murine BCC cells.

Organotypic Epithelial Raft Cultures as a Three-Dimensional In Vitro Model of Merkel Cell Carcinoma.

Simple SummaryMerkel cell carcinoma (MCC) is an extremely aggressive type of skin cancer. Nevertheless, the development of new therapeutic approaches to treat this malignancy is hampered by the absence of in vitro culture models. Furthermore, prospective clinical trials are scarce due to the rarity of the disease. Thus, the aim of the present study was to generate organotypic epithelial raft cultures (OERCs) of MCC by growing primary human keratinocytes and MCC cell lines on artificial dermal equivalents. Histological and immunohistochemical analyses confirmed the proliferation of MCC cell lines. In addition, gene expression analysis showed genes that were differently expressed in the tumor cells and the keratinocytes. In brief, we developed a three-dimensional cell culture model of MCC that can potentially be used for evaluating the efficacy and selectivity of new drug candidates against this disease.Merkel cell carcinoma (MCC) is a rare type of skin cancer for which an in vitro model is still lacking. MCC tumorigenesis is associated either with the integration of Merkel cell polyomavirus into the host genome, or with the accumulation of somatic mutations upon chronic exposure to UV light. Transgenic animals expressing the viral oncoproteins, which are constitutively expressed in virus-related MCC, do not fully recapitulate MCC. Although cell-line-derived xenografts have been established for the two subtypes of MCC, they still present certain limitations. Here, we generated organotypic epithelial raft cultures (OERCs) of MCC by using primary human keratinocytes and both virus-positive and virus-negative MCC cell lines. The primary human keratinocytes and the tumor cells were grown on top of a dermal equivalent. Histological and immunohistochemical examination of the rafts confirmed the growth of MCC cells. Furthermore, gene expression analysis revealed differences in the expression profiles of the distinct tumor cells and the keratinocytes at the transcriptional level. In summary, considering the limited availability of patient samples, OERCs of MCC may constitute a suitable model for evaluating the efficacy and selectivity of new drug candidates against MCC; moreover, they are a potential tool to study the oncogenic mechanisms of this malignancy.

CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle.

ABSTRACTDuring the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication, and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A (E2 with serine 23 mutated to alanine) activates and represses transcription identically to E2-WT (wild-type E2), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Remarkably, S23A cells had a disrupted viral life cycle in organotypic raft cultures, with a loss of E2 expression and a failure of viral replication. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 and that this interaction is critical for the viral life cycle.

Aberrant activation of the Hedgehog signalling pathway in squamous cell carcinoma of the vulva as a potential target for cancer therapy

In a previous study, we showed that the Hedgehog (Hh) signalling pathway is aberrantly activated in vulval squamous cell carcinoma (VSCC). In this study, we further validated our findings on a prospective cohort of primary VSCC cases, where immunohistochemical staining confirmed that key Hh pathway components were overexpressed in VSCC compared to normal vulval epithelium. We also undertook a series of in vitro studies to determine the extent of Hh pathway activation in VSCC-derived cell lines, and examine the consequences of pathway inhibition on the growth of these cells. We found that of six cell lines tested, four displayed elevated baseline Hh pathway activity that was dependent on SHH ligand, or in one case, a PTCH1 gene mutation. Hh signalling appeared necessary to sustain cell growth, as SHH ligand depletion with Robotikinin or SMO inhibition, either with chemical inhibitors (Itraconazole or LDE-225) or SMO-specific siRNA, attenuated GLI1 activity and cell proliferation in both monolayer and organotypic raft culture. Furthermore, treatment of Hh-dependent cell lines with SMO inhibitors sensitised cells to Cisplatin. Findings from our study offer us the opportunity to explore further the development of targeted chemotherapy for women with VSCC driven by aberrant Hh activation.

The Major Constituent of Green Tea, Epigallocatechin-3-Gallate (EGCG), Inhibits the Growth of HPV18-Infected Keratinocytes by Stimulating Proteasomal Turnover of the E6 and E7 Oncoproteins.

Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.

HIV-1 Protease Inhibitors Slow HPV16-Driven Cell Proliferation through Targeted Depletion of Viral E6 and E7 Oncoproteins.

Simple SummaryHigh-risk strains of human papillomavirus (HPV) such as HPV16 cause anogenital and oropharyngeal cancers, with HPV-associated cervical cancer being the fourth most common cancer worldwide and a major cause of death for women. Anti-HPV vaccines are available but coverage is low, and while the vaccines prevent infection they are not therapeutic for treating cancers in individuals already infected with high-risk HPV. Instead, current treatments for HPV-associated cancers include surgery, chemotherapy, and radiation, and affect diseased and healthy tissues alike. Accordingly, there is a dire need for better, molecularly targeted strategies. Herein, we report the novel finding that at least four protease inhibitor drugs currently used to treat HIV/AIDS—lopinavir, ritonavir, nelfinavir, and saquinavir—cause marked and selective depletion of HPV16 E6 and E7 oncoproteins in HPV-positive cancer cells and a 3-D cell culture model for HPV16-positive epithelial pre-cancer (NIKS16 cells). E6 and E7 depletion correlated with anticancer phenotypes including increased p53 protein levels and induction of cell growth arrest in CaSki cells as well as slowed epithelium growth and reversal of HPV16-driven phenotypes consistent with inhibition of the cellular autophagy pathway in the NIKS16 model. Based on this progress in vitro, we propose that HIV protease inhibitors, already known to have anticancer properties, be studied further in the context of developing a targeted therapy for HPV16-driven cancers and pre-cancers.High-risk human papillomavirus strain 16 (HPV16) causes oral and anogenital cancers through the activities of two viral oncoproteins, E6 and E7, that dysregulate the host p53 and pRb tumor suppressor pathways, respectively. The maintenance of HPV16-positive cancers requires constitutive expression of E6 and E7. Therefore, inactivating these proteins could provide the basis for an anticancer therapy. Herein we demonstrate that a subset of aspartyl protease inhibitor drugs currently used to treat HIV/AIDS cause marked reductions in HPV16 E6 and E7 protein levels using two independent cell culture models: HPV16-transformed CaSki cervical cancer cells and NIKS16 organotypic raft cultures (a 3-D HPV16-positive model of epithelial pre-cancer). Treatment of CaSki cells with some (lopinavir, ritonavir, nelfinavir, and saquinavir) but not other (indinavir and atazanavir) protease inhibitors reduced E6 and E7 protein levels, correlating with increased p53 protein levels and decreased cell viability. Long-term (>7 day) treatment of HPV16-positive NIKS16 raft cultures with saquinavir caused epithelial atrophy with no discernible effects on HPV-negative rafts, demonstrating selectivity. Saquinavir also reduced HPV16′s effects on markers of the cellular autophagy pathway in NIKS16 rafts, a hallmark of HPV-driven pre-cancers. Taken together, these data suggest HIV-1 protease inhibitors be studied further in the context of treating or preventing HPV16-positive cancers.

Delta-Like Ligand-Notch1 Signaling Is Selectively Modulated by HPV16 E6 to Promote Squamous Cell Proliferation and Correlates with Cervical Cancer Prognosis.

Human papillomavirus (HPV) drives high-grade intraepithelial neoplasia and cancer; for unknown reasons, this occurs most often in the cervical transformation zone. Either mutation or HPV E6-driven inhibition of Notch1 can drive neoplastic development in stratified squamous epithelia. However, the contribution of Notch1 and its Delta-like ligands (DLL) to site susceptibility remains poorly understood. Here, we map DLL1/DLL4 expression in cell populations present in normal cervical biopsies by immunofluorescence. In vitro keratinocyte 2D monolayer models, growth assays, and organotypic raft cultures were used to assess the functional role of DLL-Notch signaling in uninfected cells and its modulation by HPV16 in neoplasia. An RNA sequencing-based gene signature was used to suggest the cell of origin of 279 HPV-positive cervical carcinomas from The Cancer Genome Atlas and to relate this to disease prognosis. Finally, the prognostic impact of DLL4 expression was investigated in three independent cervical cancer patient cohorts. Three molecular cervical carcinoma subtypes were identified, with reserve cell tumors the most common and linked to relatively good prognosis. Reserve cells were characterized as DLL1-/DLL4+, a proliferative phenotype that is temporarily observed during squamous metaplasia and wound healing but appears to be sustained by HPV16 E6 in raft models of low-grade and, more prominently, high-grade neoplasia. High expression of DLL4 was associated with an increased likelihood of cervical cancer-associated death and recurrence. Taken together, DLL4-Notch1 signaling reflects a proliferative cellular state transiently present during physiologic processes but inherent to cervical reserve cells, making them strongly resemble neoplastic tissue even before HPV infection has occurred. SIGNIFICANCE: This study investigates cervical cancer cell-of-origin populations and describes a DLL-Notch1 phenotype that is associated with disease prognosis and that might help identify cells that are susceptible to HPV-induced carcinogenesis.

A Novel In Vitro Culture Model System to Study Merkel Cell Polyomavirus-Associated MCC Using Three-Dimensional Organotypic Raft Equivalents of Human Skin.

Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro using an organotypic (raft) culture system that is traditionally used to recapitulate the dermal and epidermal equivalents of skin in three dimensions (3D). In the optimal culture condition, MCPyV+ MCC cells were embedded in collagen between the epidermal equivalent comprising human keratinocytes and a dermal equivalent containing fibroblasts, resulting in MCC-like lesions arising within the dermal equivalent. The presence and organization of MCC cells within these dermal lesions were characterized through biomarker analyses. Interestingly, co-culture of MCPyV+ MCC together with keratinocytes specifically within the epidermal equivalent of the raft did not reproduce human MCC morphology, nor were any keratinocytes necessary for MCC-like lesions to develop in the dermal equivalent. This 3D tissue culture system provides a novel in vitro platform for studying the role of MCPyV T antigens in MCC oncogenesis, identifying additional factors involved in this process, and for screening potential MCPyV+ MCC therapeutic strategies.

Zoledronic acid impairs oral cancer stem cells by reducing CCL3.

Cancer stem‑like cells (CSCs; also referred to as tumor‑initiating cells) play crucial roles in tumor progression and aggressiveness. Recent studies have demonstrated the antitumor activity of zoledronic acid (ZA), a third‑generation bisphosphonate, in various types of human cancer. However, its effect on oral CSCs and the underlying mechanism remain obscure. The present study demonstrated that ZA suppresses the growth and stemness properties of oral/oropharyngeal squamous cell carcinoma (OSCC) cells. ZA inhibited the malignant characteristics of OSCC cells, such as anchorage‑independent growth and epithelial thickening in organotypic raft cultures. Moreover, ZA treatment resulted in suppression of self‑renewal capacity, a key feature of CSCs. ZA also inhibited important CSC properties, such as migration and chemo‑radioresistance. Mechanistically, ZA exposure significantly decreased chemokine (C‑C motif) ligand 3 (CCL3) expression in OSCC cells. It was further demonstrated that CCL3 signaling via its receptor is crucial for supporting the CSC phenotype in OSCC cells. Moreover, an antagonist of the CCL3 receptor reversed the effect of CCL3 on CSC properties, and exogenous CCL3 rescued the suppressaed CSC phenotype in ZA‑treated OSCC cells. These results demonstrated that ZA suppresses the CSC phenotype in OSCC cells by reducing CCL3 expression, suggesting that ZA may be an effective therapeutic agent for oral cancer by targeting CSCs.

Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets.

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.

In Vitro Organotypic Systems to Model Tumor Microenvironment in Human Papillomavirus (HPV)-Related Cancers.

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

Generation of esophageal organoids and organotypic raft cultures from human pluripotent stem cells.

The human and murine esophagus have some substantial differences that limit the utility of mouse as a model to study human esophagus development and disease. Due to these limitations several recent reports describe the development of methods to generate human esophageal tissues via the directed differentiation of pluripotent stem cells. Methods for differentiation are based on knowledge of years of studying embryonic development of the esophagus in vertebrate animal models. Esophageal tissues derived from human pluripotent stem cells have been used to study both development and diseases affecting the esophagus. Here, we provide a detailed protocol for the directed differentiation of human pluripotent stem cells into human esophageal organoids and organotypic raft cultures, that are highly similar, morphologically and transcriptionally, to the human esophagus epithelium. We discuss limitations of the current esophageal models and the importance of engineering more complex tissue models with muscle and enteric nerves. Moving forward, these models might be utilized for the development of personalized treatments, as well as other therapeutic solutions.

Application of human organotypic skin raft cultures for analysis of retinoid metabolism, retinoic acid signaling, and screening of bioactive rexinoids.

Several human enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins exhibit catalytic oxidoreductive activity toward retinoid substrates in vitro. For some retinoid-active enzymes, their physiological significance for retinoid metabolism is supported by phenotypes linked to naturally occurring mutations in human genes or by targeted gene knockout studies of their murine homologs. However, for those enzymes that are not well conserved or display properties different from their murine counterparts, evaluation of their physiological roles can be challenging. Here, we describe the adaptation of stratified organotypic culture of human epidermis for evaluating the contribution of human putative SDR retinol dehydrogenases to biosynthesis of all-trans-retinoic acid in a three-dimensional cellular model highly sensitive to the levels of all-trans-retinol and all-trans-retinoic acid. In addition to providing a valuable readout of metabolic changes occurring in the presence or absence of the enzyme of interest, this model allows for evaluation of the effects of various retinoid and rexinoid therapeutic compounds on retinoic acid signaling, cell growth and differentiation.

Targeting DNA Damage Response as a Strategy to Treat HPV Infections.

Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90–99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.

Small Molecule and Pooled CRISPR Screens Investigating IL17 Signaling Identify BRD2 as a Novel Contributor to Keratinocyte Inflammatory Responses.

Interleukin-17A (IL17A) plays a critical role in the development of numerous autoimmune diseases, including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of ∼22000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format by utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest-ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins and bromodomain-containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF-mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2-deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2-deficient keratinocytes. In IL17A/TNF/IL22 stimulated three-dimensional organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling.

Effect of Productive Human Papillomavirus 16 Infection on Global Gene Expression in Cervical Epithelium.

Human papillomavirus (HPV) infection is the world's most common sexually transmitted infection and is responsible for most cases of cervical cancer. Previous studies of global gene expression changes induced by HPV infection have focused on the cancerous stages of infection, and therefore, not much is known about global gene expression changes at early preneoplastic stages of infection. We show for the first time the global gene expression changes during early-stage HPV16 infection in cervical tissue using 3-dimensional organotypic raft cultures, which produce high levels of progeny virions. cDNA microarray analysis showed that a total of 594 genes were upregulated and 651 genes were downregulated at least 1.5-fold with HPV16 infection. Gene ontology analysis showed that biological processes including cell cycle progression and DNA metabolism were upregulated, while skin development, immune response, and cell death were downregulated with HPV16 infection in cervical keratinocytes. Individual genes were selected for validation at the transcriptional and translational levels, including UBC, which was central to the protein association network of immune response genes, and top downregulated genes RPTN, SERPINB4, KRT23, and KLK8 In particular, KLK8 and SERPINB4 were shown to be upregulated in cancer, which contrasts with the gene regulation during the productive replication stage. Organotypic raft cultures, which allow full progression of the HPV life cycle, allowed us to identify novel gene modulations and potential therapeutic targets of early-stage HPV infection in cervical tissue. Additionally, our results suggest that early-stage productive infection and cancerous stages of infection are distinct disease states expressing different host transcriptomes.IMPORTANCE Persistent HPV infection is responsible for most cases of cervical cancer. The transition from precancerous to cancerous stages of HPV infection is marked by a significant reduction in virus production. Most global gene expression studies of HPV infection have focused on the cancerous stages. Therefore, little is known about global gene expression changes at precancerous stages. For the first time, we measured global gene expression changes at the precancerous stages of HPV16 infection in human cervical tissue producing high levels of virus. We identified a group of genes that are typically overexpressed in cancerous stages to be significantly downregulated at the precancerous stage. Moreover, we identified significantly modulated genes that have not yet been studied in the context of HPV infection. Studying the role of these genes in HPV infection will help us understand what drives the transition from precancerous to cancerous stages and may lead to the development of new therapeutic targets.