Nearly half of American adults suffer from gum disease, including mild inflammation of gingival tissue, known as gingivitis. Currently, advances in therapeutic treatments are hampered by a lack of mechanistic understanding of disease progression in physiologically relevant vascularized tissues. To address this, we present a high-throughput microfluidic organ-on-chip model of human gingival tissue containing keratinocytes, fibroblast and endothelial cells. We show the triculture model exhibits physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion over several weeks. Through inflammatory cytokine administration, we demonstrate the induction of inflammation measured by changes in barrier function and cytokine secretion. These states of inflammation are induced at various time points within a stable culture window, providing a robust platform for evaluation of therapeutic agents. These data reveal that the administration of specific small molecule inhibitors mitigates the inflammatory response and enables tissue recovery, providing an opportunity for identification of new therapeutic targets for gum disease with the potential to facilitate relevant preclinical drug efficacy and toxicity testing.

The human fallopian tube epithelium (hFTE) is the site of fertilization, early embryo development, and the origin of most high-grade serous ovarian cancers (HGSOCs). Little is known about the content and functions of hFTE-derived small extracellular vesicles (sEVs) due to the limitations of biomaterials and proper culture methods. We have established a microfluidic platform to culture hFTE for EV collection with adequate yield for mass spectrometry-based proteomic profiling, and reported 295 common hFTE sEV proteins for the first time. These proteins are associated with exocytosis, neutrophil degranulation, and wound healing, and some are crucial for fertilization processes. In addition, by correlating sEV protein profiles with hFTE tissue transcripts characterized using GeoMx^® Cancer Transcriptome Atlas, spatial transcriptomics analysis revealed cell-type-specific transcripts of hFTE that encode sEVs proteins, among which, FLNA, TUBB, JUP, and FLNC were differentially expressed in secretory cells, the precursor cells for HGSOC. Our study provides insights into the establishment of the baseline proteomic profile of sEVs derived from hFTE tissue, and its correlation with hFTE lineage-specific transcripts, which can be used to evaluate whether the fallopian tube shifts its sEV cargo during ovarian cancer carcinogenesis and the role of sEV proteins in fallopian tube reproductive functions.

Fallopian tube epithelium (FTE) plays a critical role in reproduction and can be the site where High Grade Serous Ovarian Carcinoma (HGSOC) originates. Tumorigenic oviductal cells, which are the murine equivalent of human fallopian tube secretory epithelial cells (FTSEC), enhance testosterone secretion by the ovary when co-cultured with the ovary, suggesting that testosterone is part of the signaling axis between the ovary and FTSEC. Furthermore, testosterone promotes proliferation of oviductal cells. Oral contraceptives, tubal ligation, and salpingectomy, which are all protective against developing ovarian cancer, also decrease circulating levels of androgen. In the current study, we investigated the effect of increased testosterone on FTE and found that testosterone upregulates wingless-type MMTV integration family, member 4 (WNT4) and induces migration and invasion of immortalized human fallopian tube cells. We profiled primary human fallopian tissues grown in the microfluidic system SOLO-microfluidic platform –(MFP) by RNA sequencing and found that p53 and its downstream target genes, such as paired box gene 2 (PAX2), cyclin-dependent kinase inhibitor 1A (CDK1A or p21), and cluster of differentiation 82 (CD82 or KAI1) were downregulated in response to testosterone treatment. A microfluidic platform, the PREDICT-Multi Organ System (PREDICT-MOS) was engineered to support insert technology that allowed for the study of cancer cell migration and invasion through Matrigel. Using this system, we found that testosterone enhanced FTE migration and invasion, which was reversed by the androgen receptor (AR) antagonist, bicalutamide. Testosterone also enhanced FTSEC adhesion to the ovarian stroma using murine ovaries. Overall, these results indicate that primary human fallopian tube tissue and immortalized FTSEC respond to testosterone to shift expression of genes that regulate invasion, while leveraging a new strategy to study migration in the presence of dynamic fluid flow.

Influenza and other respiratory viruses represent a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as the current COVID-19 crisis. One of the greatest barriers to the development of effective therapeutic agents to treat influenza, coronaviruses, and many other infections of the respiratory tract is the absence of a robust preclinical model. Preclinical studies currently rely on high-throughput, low-fidelity in vitro screening with cell lines and/or low-throughput animal models that often provide a poor correlation to human clinical responses. Here, we introduce a human primary airway epithelial cell-based model integrated into a high-throughput platform where tissues are cultured at an air-liquid interface (PREDICT96-ALI). We present results on the application of this platform to influenza and coronavirus infections, providing multiple readouts capable of evaluating viral infection kinetics and potentially the efficacy of therapeutic agents in an in vitro system. Several strains of influenza A virus are shown to successfully infect the human primary cell-based airway tissue cultured at an air-liquid interface (ALI), and as a proof-of-concept, the effect of the antiviral oseltamivir on one strain of Influenza A is evaluated. Human coronaviruses NL63 (HCoV-NL63) and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for protein priming, and we confirm expression of both in our ALI model. We also demonstrate coronavirus infection in this system with HCoV-NL63, observing sufficient viral propagation over 96 hours post-infection to indicate successful infection of the primary cell-based model. This new capability has the potential to address a gap in the rapid assessment of therapeutic efficacy of various small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

Serous tubal intraepithelial carcinomas (STIC lesions) in the human fallopian tube epithelium (hFTE) are theorized to give rise to high grade serous ovarian cancers (HGSOC). Small extracellular vesicles (sEVs) are known to mediate key signaling in both normal and cancerous tissues, but few ex vivo systems exist for studying sEV impact on hFTE tissue. Here, we present a microfluidic tissue culture platform with combined spatial transcriptomic and proteomic readouts that allows us to profile dual responses in tissue exposed to sEV “messages”—capturing both short-term transcriptomic shifts in the tissue and long-term changes in protein cargo of secreted EVs (the “reply”). Using spatial transcriptomics, we show that the short-term 1-day exposure to ovarian cancer-derived sEVs alters expression of 68 transcripts in secretory cells, the progenitor of HGSOC, notably upregulating immune-related mRNA, including CXCL family chemokines, VCAM1, and pro-inflammatory mediators (NFKB1, IL1B, IFNA7/17). Additionally, we observed the long-term 14-day exposure to sEVs alters the expression of 7 transcripts and 25 EV cargo proteins of fallopian tube derived EVs (“secondary release EVs”) following stimulus from cancer EVs. Together, tissue transcriptomics and tissue-derived EV proteomics indicate that ovarian cancer derived sEVs rewire target cell signaling to modify the tubal immune landscape. This study provides insights into the early molecular changes associated with the pathogenesis of ovarian cancer in its tissue of origin, providing a platform to study EV-tissue interactions and identify how sEVs drive cell signaling reprogramming in hFTE.

GeoMx DSP quality control analysis.