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

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 αß 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.

Patient-derived and mouse endo-ectocervical organoid generation, genetic manipulation and applications to model infection.

The cervix is the gateway to the upper female reproductive tract, connecting the uterus and vagina. It plays crucial roles in fertility and pregnancy maintenance from onset until delivery of the fetus, and prevents pathogen ascension. Compromised functionality of the cervix can lead to disorders, including infertility, chronic infections and cancers. The cervix comprises two regions: columnar epithelium-lined endocervix and stratified squamous epithelium-lined ectocervix, meeting at the squamocolumnar transition zone. So far, two-dimensional cultures of genetically unstable immortalized or cancer cell lines have been primarily used to study cervix biology in vitro. The lack of an in vitro system that reflects the cellular, physiological and functional properties of the two epithelial types has hampered the study of normal physiology, disease development and infection processes. Here we describe a protocol for cell isolation, establishment, long-term culture and expansion of adult epithelial stem cell-derived endocervical and ectocervical organoids from human biopsies and mouse tissue. These two organoid types require unique combinations of growth factors reminiscent of their in vivo tissue niches and different culturing procedures. They recapitulate native three-dimensional tissue architecture and patterning. The protocol to generate these organoids takes 4-6 weeks. We also describe procedures to introduce human papillomavirus oncogenes into the cervical stem cells by genetic manipulation to model cervical cancer and infection of the organoids with the highly prevalent sexually transmitted bacterial pathogen Chlamydia trachomatis. These organoid systems open new possibilities to study cervix biology, infections and cancer evolution, and have potential applications in personalized medicine, drug screening, genome editing and disease modeling.

Modelling Chlamydia and HPV co-infection in patient-derived ectocervix organoids reveals distinct cellular reprogramming.

Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modeled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renewal capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrates the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression.

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.

High-intensity focused ultrasound for the targeted destruction of uterine tissues: experiences from a pilot study using a mobile HIFU unit.

BACKGROUND: High intensity focused ultrasound (HIFU) is a novel method which offers the non-invasive ablation of tissues without harming overlying organs or skin. It has been introduced successfully in urology for the ablation of prostatic hyperplasia and seems to be promising in the treatment of uterine fibroids. In this study we aimed to examine the feasibility and possible side effects of HIFU treatment of uterine tissues using an experimental mobile HIFU unit with ultrasound guidance. METHODS: For these experiments, a 1.07 MHz ultrasound source was used which allows treatment depths between 0 and 10 cm. In 12 patients scheduled to have abdominal hysterectomy, 5-60 impulses of HIFU were applied through the intact skin upon uterine tissues directly prior to the surgical procedure. Tissue intensities lay between 3,200 and 6,300 W/cm(2) and a fixed pulse length of 4 s was used. RESULTS: No side effects were encountered other than one first-degree skin burn and the treatment was well tolerated. Histology showed clearly demarcated coagulative necrosis in the targeted tissues. Treatment was concluded in less than 45 min for each patient. CONCLUSION: Focused ultrasound is an effective method to selectively destroy tissue within the uterus and the transabdominal access route is very feasible. This study shows that a mobile ultrasound source can be used safely and effectively to destroy uterine tissues, such as fibroids, without major side effects.

Conception and spontaneous delivery after total hypophysectomy.

OBJECTIVE: To report the first case of pregnancy and spontaneous delivery after total hypophysectomy. DESIGN: A case report. SETTING: University hospital. PATIENT(S): A 34-year-old woman with panhypopituitarism after hypophysectomy in childhood for craniopharyngioma. INTERVENTION(S): Successful fertility treatment followed by sufficient hormone substitution therapy during pregnancy. MAIN OUTCOME MEASURE(S): Clinical variables. RESULT(S): The patient had an uncomplicated spontaneous delivery without any substitution of oxytocin before, during, or after delivery. CONCLUSION(S): Normal pregnancy and delivery are possible in women with lack of pituitary function. Maternal pituitary oxytocin release seems to play a limited role in the onset and progression of labor.