Human Ovarian Cancer Tumor Formation in Severe Combined Immunodeficient (SCID) Pigs

Ovarian cancer (OvCa) is the most lethal gynecologic malignancy, with two-thirds of patients having late-stage disease (II-IV) at diagnosis. Improved diagnosis and therapies are needed, yet preclinical animal models for ovarian cancer research have primarily been restricted to rodents, for data on which can fail to translate to the clinic. Thus, there is currently a need for a large animal OvCa model. Therefore, we sought to determine if pigs, being more similar to humans in terms of anatomy and physiology, would be a viable preclinical animal model for OvCa. We injected human OSPC-ARK1 cells, a chemotherapy-resistant primary ovarian serous papillary carcinoma cell line, into the neck muscle and ear tissue of four severe combined immune deficient (SCID) and two non-SCID pigs housed in novel biocontainment facilities to study the ability of human OvCa cells to form tumors in a xenotransplantation model. Tumors developed in ear tissue of three SCID pigs, while two SCID pigs developed tumors in neck tissue; no tumors were detected in non-SCID control pigs. All tumor masses were confirmed microscopically as ovarian carcinomas. The carcinomas in SCID pigs were morphologically similar to the original ovarian carcinoma and had the same immunohistochemical phenotype based on expression of Claudin 3, Claudin 4, Cytokeratin 7, p16, and EMA. Confirmation that OSPC-ARK1 cells form carcinomas in SCID pigs substantiates further development of orthotopic models of OvCa in pigs

MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function

MicroRNA21 (MIR21) abundance in porcine oocytes and cumulus cells increases during in vitro maturation. The mechanism by which MIR21 regulates oocyte maturation and the effect on the developmental competence of subsequent embryos remains unclear. The objective of this study was to assess the function of MIR21 during porcine oocyte maturation and its effect on embryonic development. Treatment with peptide nucleic acid MIR21 inhibitor (MIR21-PNA), designed to specifically bind to and prevent MIR21 activity during in vitro oocyte maturation, decreased cumulus cell expansion, and the oocyte ability to achieve metaphase II maturation stage when compared to control groups. Following parthenogenetic activation, the cleavage rate at 48 h in the MIR21-PNA group was decreased (p ≤ 0.03) relative to the control groups. Additionally, liquid chromatography-mass spectrometry (LC-MS/MS) of oocyte and cumulus cell total protein following MIR21-PNA treatment during in vitro maturation identified changes in signaling pathways with primary involvement of glucose metabolism (GM) pathways. Furthermore, there was no difference (p = 0.21) in oocyte maturation of control and MIR21-PNA treated oocytes when cultured in pyruvate lacking medium. Finally, MIR21-PNA treatment decreased (p = 0.04) glutathione and increased (p = 0.07) reactive oxygen species production in the oocyte. These data suggest that MIR21 influences porcine oocyte maturation by regulating GM pathways in the cumulus–oocyte complex.This article is published as Li, Yunsheng, Malavika K. Adur, Steven M. Lonergan, Aileen F. Keating, and Jason W. Ross. "MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function." Molecular Reproduction and Development (2022). doi:10.1002/mrd.23641. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made

Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

Heat stress (HS) deleteriously affects multiple components of porcine reproduction and is causal to seasonal infertility. Environment-induced hyperthermia causes a HS response (HSR) typically characterized by increased abundance of intracellular heat shock proteins (HSP). Gilts exposed to HS during the peri-implantation period have compromised embryo survival, however if (or how) HS disrupts the porcine endometrium is not understood. Study objectives were to evaluate the endometrial HSP abundance in response to HS during this period and assess the effect of oral progestin (altrenogest; ALT) supplementation. Postpubertal gilts (n = 42) were artificially inseminated during behavioral estrus (n = 28) or were kept cyclic (n = 14), and randomly assigned to thermal neutral (TN; 21 ± 1 °C) or diurnal HS (35 ± 1 °C for 12 h/31.6 ± 1 °C for 12 h) conditions from day 3 to 12 postestrus (dpe). Seven of the inseminated gilts from each thermal treatment group received ALT (15 mg/d) during this period. Using quantitative PCR, transcript abundance of HSP family A (Hsp70) member 1A (HSPA1A, P = 0.001) and member 6 (HSPA6, P < 0.001), and HSP family B (small) member 8 (HSB8, P = 0.001) were increased while HSP family D (Hsp60) member 1 (HSPD1, P = 0.01) was decreased in the endometrium of pregnant gilts compared to the cyclic gilts. Protein abundance of HSPA1A decreased (P = 0.03) in pregnant gilt endometrium due to HS, while HSP family B (small) member 1 (HSPB1) increased (P = 0.01) due to HS. Oral ALT supplementation during HS reduced the transcript abundance of HSP90α family class B member 1 (HSP90AB1, P = 0.04); but HS increased HSP90AB1 (P = 0.001), HSPA1A (P = 0.02), and HSPA6 (P = 0.04) transcript abundance irrespective of ALT. ALT supplementation decreased HSP90α family class A member 1 (HSP90AA1, P = 0.001) protein abundance, irrespective of thermal environment, whereas ALT only decreased HSPA6 (P = 0.02) protein abundance in TN gilts. These results indicate a notable shift of HSP in the porcine endometrium during the peri-implantation period in response to pregnancy status and heat stress.This article is published as Adur, Malavika K., Jacob T. Seibert, Matthew R. Romoser, Katie L. Bidne, Lance H. Baumgard, Aileen F. Keating, and Jason W. Ross. "Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period." Journal of Animal Science 100, no. 7 (2022): skac129. doi:10.1093/jas/skac129. Posted with permission. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited

Progesterone Alleviates Endometriosis via Inhibition of Uterine Cell Proliferation, Inflammation and Angiogenesis in an Immunocompetent Mouse Model.

Endometriosis, defined as growth of the endometrial cells outside the uterus, is an inflammatory disorder that is associated with chronic pelvic pain and infertility in women of childbearing age. Although the estrogen-dependence of endometriosis is well known, the role of progesterone in development of this disease remains poorly understood. In this study, we developed a disease model in which endometriosis was induced in the peritoneal cavities of immunocompetent female mice, and maintained with exogenous estrogen. The endometriosis-like lesions that were identified at a variety of ectopic locations exhibited abundant blood supply and extensive adhesions. Histological examination revealed that these lesions had a well-organized endometrial architecture and fibrotic response, resembling those recovered from clinical patients. In addition, an extensive proliferation, inflammatory response, and loss of estrogen receptor alpha (ERα) and progesterone receptor (PR) expression were also observed in these lesions. Interestingly, administration of progesterone before, but not after, lesion induction suppressed lesion expansion and maintained ERα and PR expressions. These progesterone-pretreated lesions exhibited attenuation in KI67, CD31, and pro-inflammatory cytokine expression as well as macrophage infiltration, indicating that progesterone ameliorates endometriosis progression by inhibiting cell proliferation, inflammation and neovascularization. Our studies further showed that suppression of global DNA methylation by application of DNA methyltransferase inhibitor to female mice bearing ectopic lesions restrained lesion expansion and restored ERα and PR expression in eutopic endometrium and ectopic lesions. These results indicate that epigenetic regulation of target gene expression via DNA methylation contributes, at least in part, to progesterone resistance in endometriosis

Interleukin 16- (IL-16-) Targeted Ultrasound Imaging Agent Improves Detection of Ovarian Tumors in Laying Hens, a Preclinical Model of Spontaneous Ovarian Cancer

Limited resolution of transvaginal ultrasound (TVUS) scanning is a significant barrier to early detection of ovarian cancer (OVCA). Contrast agents have been suggested to improve the resolution of TVUS scanning. Emerging evidence suggests that expression of interleukin 16 (IL-16) by the tumor epithelium and microvessels increases in association with OVCA development and offers a potential target for early OVCA detection. The goal of this study was to examine the feasibility of IL-16-targeted contrast agents in enhancing the intensity of ultrasound imaging from ovarian tumors in hens, a model of spontaneous OVCA. Contrast agents were developed by conjugating biotinylated anti-IL-16 antibodies with streptavidin coated microbubbles. Enhancement of ultrasound signal intensity was determined before and after injection of contrast agents. Following scanning, ovarian tissues were processed for the detection of IL-16 expressing cells and microvessels. Compared with precontrast, contrast imaging enhanced ultrasound signal intensity significantly in OVCA hens at early (P<0.05) and late stages (P<0.001). Higher intensities of ultrasound signals in OVCA hens were associated with increased frequencies of IL-16 expressing cells and microvessels. These results suggest that IL-16-targeted contrast agents improve the visualization of ovarian tumors. The laying hen may be a suitable model to test new imaging agents and develop targeted anti-OVCA therapeutics

P₄ suppresses E₂-dependent inflammatory responses in the ectopic lesions.

<p>Ectopic lesions were harvested from E₂ or E₂ plus P₄-treated recipients (n = 6) by 16 days after induction. (A) Representative images (20X) showing IHC analysis using antibodies against pan-macrophage biomarker (F4/80), inflammatory M1 (CCR7), anti-inflammatory M2 (CD206) macrophages or Treg cell biomarker (FOXP3), respectively. (B) The relative level of mRNA expression corresponding to <i>Ccl2</i>, <i>Ccl5</i>, <i>Il1b</i>, <i>Il6</i>, <i>Tnfa</i>, and <i>Tgfb</i> was analyzed by qPCR after moralization to the internal control gene, <i>36B4</i>. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.</p

Immunocompetent mouse model of endometriosis.

<p>6–8 weeks-old CD1 female mice were primed with PMSG for 48 hours. Uterine tissues were then harvested and minced into tiny cell aggregates after myometrial removal. Female mice with the same genetic background were subjected to ovariectomy and served as recipients. 2 weeks after surgery, equal volumes of uterine cell aggregate suspension were transferred into the peritoneal cavities of recipients. Endometriosis was maintained by subcutaneous administration of 100 ng of E₂ once every 4 days until tissue collection. For the studies of the role of P₄ in endometriosis, 1 mg of P₄ (pre-P₄) was administrated along with E₂ beginning at 4 days before transplantation. For P4-resistance experiments (Post-P₄), 1 mg of P₄ was administration along with E2 beginning at 4 days after transplantation. The ectopic lesions were assessed under a dissecting microscope at different days after endometrial cell transplantation (n = 6 per treatment group).</p

P₄ alleviates E₂-dependent establishment and growth of ectopic lesions.

<p>Ectopic lesions were established in the peritoneal cavities of immunocompetent female mice and treated with E₂ or P₄ along with E₂ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165347#pone.0165347.g001" target="_blank">Fig 1</a> (N = 6). (A) Locations of ectopic lesions (Dashed circles). (B) Histology of ectopic lesions. Dashed lines indicate the contact sites of the ectopic lesions with adjacent peritoneum. Representative images of ectopic lesions harvested at four weeks after induction are shown (40X). (C) Time-course progression of ectopic lesions. The average volumes of the ectopic lesions at the indicated time points are shown. (D) The average numbers of ectopic lesions identified in each treatment group are shown. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as ^#: p < 0.05, *: p<0.01.</p

P₄ inhibits E₂-dependent cell proliferation and angiogenesis in ectopic lesions.

<p>Sections of the ectopic lesions collected from E₂ or E₂ plus P₄-treated recipients (D16, n = 6) were subjected to histological examination. (A) Representative images (20X) showing H&E and Trichrome staining, or IHC analysis using antibody against myofibroblast biomarker αSMA, uterine epithelial biomarker KRT11, or uterine stromal biomarker VIM, respectively. (B) Representative images (20X) showing IHC analysis using antibodies against cell proliferation biomarker KI67, smooth muscle biomarker αSMA, endothelial cells CD31, or an angiogenetic regulator CCN1, respectively. The numbers of KI67-positive cells, the perimeters of the supporting blood vessels, and the immunostaining intensities of CCN1 and CD31 were analyzed by ImageJ software. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.</p

Loss of ERα/PR-mediated signaling contributes to P₄-resistance in this mouse model of endometriosis.

<p>Endometriosis was induced and maintained with E₂ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165347#pone.0165347.g001" target="_blank">Fig 1</a>. The host females were then treated with P₄ beginning at 4 days before (Pre-) or 4 days after (Post-) endometrial cell transplantation until tissue collection (n = 6). Donor uterine tissue (D0) and ectopic lesions were subjected to IHC analysis (A) for ERα, PR and HAND2 protein expression (20X) or qPCR analysis (B) to assess expression level of mRNA corresponding to <i>Esr1</i>, <i>Pgr</i>, <i>Hand2</i>, and <i>Hoxa10</i>, respectively. (C) Lesion volumes were quantitated by 16 days after induction. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.</p