Organoids of the female reproductive tract.

Funder: Centre for Trophoblast ResearchHealthy functioning of the female reproductive tract (FRT) depends on balanced and dynamic regulation by hormones during the menstrual cycle, pregnancy and childbirth. The mucosal epithelial lining of different regions of the FRT-ovaries, fallopian tubes, uterus, cervix and vagina-facilitates the selective transport of gametes and successful transfer of the zygote to the uterus where it implants and pregnancy takes place. It also prevents pathogen entry. Recent developments in three-dimensional (3D) organoid systems from the FRT now provide crucial experimental models that recapitulate the cellular heterogeneity and physiological, anatomical and functional properties of the organ in vitro. In this review, we summarise the state of the art on organoids generated from different regions of the FRT. We discuss the potential applications of these powerful in vitro models to study normal physiology, fertility, infections, diseases, drug discovery and personalised medicine

Mechanism of cell death in Burkitt lymphomas

Apoptoseresistenz ist einer der Gründe für ein Versagen von Chemotherapie bei vielen Krebserkrankungen, darunter das Burkitt Lymphom. Um die molekularen Mechanismen der Apoptoseresistenz aufzuklären, wurde die Apoptoseinduktion in 15 Burkitt-Lymphom-Zelllinien nach Behandlung mit den Spindelgiften Taxol (Paclitaxel), Nocodazol und Vincristin untersucht. Interessanterweise entwickelten Zellen, die sich als resistent gegenüber Taxol- und Nocodazol-induzierter Apoptose erwiesen, nach Behandlung eine Polyploidie (>4N DNA), was eine inverse Relation von Apoptose und Polyploidie aufzeigt. In den sensitiven Zelllinien war die Taxol- und Nocodazol-induzierte Apoptose von Caspase-Aktivierung, Bid-Spaltung und Herunterregulation von Mcl-1 begleitet. Im Gegensatz zu den sensitiven Zelllinien wiesen die meisten apoptoseresistenten Zellen einen Verlust von Bax und Bak auf und waren durch einen anhaltenden mitotischen Arrest mit Auftreten eines >4N DNA-Gehalts nach Behandlung charakterisiert. Um weitere Einblicke in den Mechanismus der Spindelgift-induzierten Apoptose zu erhalten, wurde die Rolle der mitotische Kinase PLK1 (polo-like kinase) näher untersucht. Eine dominant-negative PLK1-Mutante induziert Apoptose. Allerdings zeigte eine zusätzliche Behandlung mit Spindelgiften keinen synergistischen Effekt, was darauf schließen lässt, dass sowohl Inhibierung von PLK1 als auch Mikrotubuli-destabilisierende Agenzien den gleichen Stress-Signalweg aktivieren. Andererseits unterstützte Überexpression von Wildtyp-PLK1 in Taxol behandelten Zellen die Zellzyklus-Progression. Dies deutet auf eine Verbindung zwischen Zelltodresistenz und genetischer Instabilität (Aneuplodie) hin. Inhibition von Apoptose in sensitiven Zelllinien durch Caspase-Inhibierung förderte Polypoidie, welche die inverse Relation bestätigte. Medikamente, welche die Caspase-Aktivierung unabhängig von Bax und Bak induzieren, könnten eine weitere Möglichkeit zur Behandlung von resistenten Burkitt-Lymphomen darstellen.Apoptosis resistance is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphomas (BL). To elucidate molecular mechanisms regulating the development of apoptosis resistance, a panel of 15 BL cell lines was investigated for apoptosis induction upon treatment with microtubule inhibitors taxol, nocodazole and vincristine. Significant differences were observed in the extent of apoptosis induction among BL cell lines examined. Interestingly, cell lines exhibiting resistance to taxol- or nocodazole-induced apoptosis, showed development of polyploidy (>4N) and vice versa, displaying an inverse relationship between apoptosis and polyploidy induction. Further, in sensitive cell lines taxol-induced apoptosis was accompanied by caspase activation, Bid cleavage and Mcl-1 down-regulation. In contrast, most apoptosis resistant cell lines exhibited a loss of Bax and Bak expression and showed prolonged mitotic arrest with >4N DNA content upon treatment. To gain mechanistic insights into microtubule inhibitor-induced cell death, the role of the mitotic kinase PLK1 was addressed. Dominant negative PLK1 mutant induced apoptosis, however, failed to show synergism in induction of apoptosis in combination with microtubule inhibitors. This indicates that PLK1 inhibition and spindle toxins might trigger a similar mitotic stress pathway. Conversely, overexpression of wildtype PLK1 promoted cell cycle progression in cells treated with taxol. Remarkably, inhibition of apoptosis in sensitive cell lines by caspase inhibition promoted polyploidy confirming the inverse relationship between apoptosis and polyploidization. Considering targets to induce Bax/Bak independent caspase activation would be of great importance to avoid undesirable events leading to chromosomal imbalances in treating resistant cancers

Chlamydia trachomatis Inhibits Homologous Recombination Repair of DNA Breaks by Interfering with PP2A Signaling

Chlamydia trachomatis induces DNA double-strand breaks in host cells but simultaneously inhibits proper DNA damage response and repair mechanisms. This may render host cells prone to loss of genetic integrity and transformation. Here we show that C. trachomatis prevents activation of the key DNA damage response mediator ATM by preventing the release from PP2A, leading to a complete absence of homologous recombination repair in host cells.Cervical and ovarian cancers exhibit characteristic mutational signatures that are reminiscent of mutational processes, including defective homologous recombination (HR) repair. How these mutational processes are initiated during carcinogenesis is largely unclear. Chlamydia trachomatis infections are epidemiologically associated with cervical and ovarian cancers. Previously, we showed that C. trachomatis induces DNA double-strand breaks (DSBs) but suppresses Ataxia-telangiectasia mutated (ATM) activation and cell cycle checkpoints. The mechanisms by which ATM regulation is modulated and its consequences for the repair pathway in C. trachomatis-infected cells remain unknown. Here, we found that Chlamydia bacteria interfere with the usual response of PP2A to DSBs. As a result, PP2A activity remains high, as the level of inhibitory phosphorylation at Y307 remains unchanged following C. trachomatis-induced DSBs. Protein-protein interaction analysis revealed that C. trachomatis facilitates persistent interactions of PP2A with ATM, thus suppressing ATM activation. This correlated with a remarkable lack of homologous recombination (HR) repair in C. trachomatis-infected cells. Chemical inhibition of PP2A activity in infected cells released ATM from PP2A, resulting in ATM phosphorylation. Activated ATM was then recruited to DSBs and initiated downstream signaling, including phosphorylation of MRE11 and NBS1 and checkpoint kinase 2 (Chk2)-mediated activation of the G2/M cell cycle checkpoint in C. trachomatis-infected cells. Further, PP2A inhibition led to the restoration of C. trachomatis-suppressed HR DNA repair function. Taking the data together, this study revealed that C. trachomatis modulates PP2A signaling to suppress ATM activation to prevent cell cycle arrest, thus contributing to a deficient high-fidelity HR pathway and a conducive environment for mutagenesis

Integrated Phosphoproteome and Transcriptome Analysis Reveals Chlamydia-Induced Epithelial-to-Mesenchymal Transition in Host Cells

Summary: Chlamydia trachomatis (Ctr) causes a range of infectious diseases and is epidemiologically associated with cervical and ovarian cancers. To obtain a panoramic view of Ctr-induced signaling, we performed global phosphoproteomic and transcriptomic analyses. We identified numerous Ctr phosphoproteins and Ctr-regulated host phosphoproteins. Bioinformatics analysis revealed that these proteins were predominantly related to transcription regulation, cellular growth, proliferation, and cytoskeleton organization. In silico kinase substrate motif analysis revealed that MAPK and CDK were the most overrepresented upstream kinases for upregulated phosphosites. Several of the regulated host phosphoproteins were transcription factors, including ETS1 and ERF, that are downstream targets of MAPK. Functional analysis of phosphoproteome and transcriptome data confirmed their involvement in epithelial-to-mesenchymal transition (EMT), a phenotype that was validated in infected cells, along with the essential role of ERK1/2, ETS1, and ERF for Ctr replication. Our data reveal the extent of Ctr-induced signaling and provide insights into its pro-carcinogenic potential. : Zadora et al. performed an integrated global phosphoproteomic and transcriptomic analysis, revealing a comprehensive map of Chlamydia-induced host cell signaling and identifying transcription factors ETS1 and ERF, which drive epithelial-to-mesenchymal transition. These insights provide mechanistic clues to Chlamydia pathogenesis and serve as an important resource for future studies. Keywords: Chlamydia trachomatis, signaling, human papillomavirus, transcription factors, cervical cancer, ovarian cancer, human primary cell

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages

DataSheet_1_γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids.docx

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.</p

Table_1_γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids.xlsx

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.</p