A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma

Abstract

Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC.Ovarian high-grade serous carcinoma (HGSC) and tubal epithelium share similar morphology, immuno-phenotype, and gene and protein expression patterns 1-3 . Transformed human Fallopian tube stem cells give rise to tumors that recapitulate the morphology and gene expression of HGSC after injection into immune-deficient mice 4 . Furthermore, most Fallopian tube cancer lesions (96%), such as serous tubal intraepithelial carcinoma (STIC), are marked by mutant p53, as are their metastatic form HGSC 5,6 . Past research on HGSC has used human tumor cells, but it has been hampered by the inefficiency of platting and sub-culturing of Fallopian tube epithelium, a process that involves a long period of fibroblast contamination reduction 7 . Furthermore, these processes result in the selection of specific cell populations, which lack tumor molecular characteristics, mutations and intra-patient heterogeneity 7-9 . Human HGSC has also been studied using patient-derived xenografts, but these technologies are time and resource-consuming, are poorly suitable for genetic manipulation and drug screening, and experience rapid mouse-specific tumor evolution 7 .Alternatively, knocked-out animals are used, being rodents the most used genetic engineered models of HGSC 10-14 . However, data generated from rodents are not readily translated to the human. First, the mouse does not naturally develop HGSC 15,16 . Secondly, genetically engineered and syngeneic mouse models may not exhibit genetic alterations relevant to human HGSC and, in many instances, may be driven by inappropriate regulatory sequences and promoters 15,16 . Third, cancer phenotypes can vary depending on the mouse strain used to create the cancer model 16 . Fourth, infertility resulting from ovarian tumors has limited the production of transgenic mouse models for ovarian cancer 17 . Nevertheless, the recent advances of the CRISPR-Cas9 system for knocking in and out target genes might offer the same opportunity of gene editing large mammalian species and can specifically increas...

PDMS Curing Inhibition on 3D-Printed Molds: Why? Also, How to Avoid It?

“…In our hands, higher aspect-ratio structures typically required longer UV exposures due to shadowing effects. 18 , 19 Some resins, when exposed to long UV exposure or high-temperature treatments (120 °C), tend to bend and change colors, and molds produced in flexible resins became harder. In the latter case, recipes using a longer thermal treatment at a lower temperature should be preferred.…”

“…Alternatively, PDMS strongly adheres to those molds. 12 , 16 , 17 Therefore, SLA molds must be treated, with, for example, UV post-curing, 11 − 16 , 18 , 19 temperature, 12 , 14 , 17 − 19 solvents, 11 , 13 , 16 sonication, 11 silanization, 12 , 16 , 17 or a coating 11 , 20 (for an overview, see Supporting Information Table S1). However, all these treatments have been reported for one specific resin.…”

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

“…They found that Cas9-induced DSBs lead to a transient, p53-dependent cell cycle arrest at G1 through p53-p21-pRB axis independent of the locus targeted, and inhibition of p53 can improve the HDR efficiency. Furthermore, CRISPR MAX efficiently delivered RNP targeting p53 into dog oviductal epithelia cells cultured in a dynamic microfluidic chip, and successfully created an in vitro model that recapitulated human tubal intraepithelial carcinoma (STIC) 168 . To expand the applications of RNP-mediated genome editing, researchers developed a scaffold-mediated delivery platform for CRISPR/Cas9 genome editing 169 .…”

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