Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death. Despite initial responses to intervention, up to 80% of patient tumors recur and require additional treatment. Retrospective clinical analysis of patients with ovarian cancer indicates antibiotic use during chemotherapy treatment is associated with poor overall survival. Here, we assessed whether antibiotic (ABX) treatment would impact growth of EOC and sensitivity to cisplatin. Immunocompetent or immunocompromised mice were given untreated control or ABX-containing (metronidazole, ampicillin, vancomycin, and neomycin) water prior to intraperitoneal injection with EOC cells, and cisplatin therapy was administered biweekly until endpoint. Tumor-bearing ABX-treated mice exhibited accelerated tumor growth and resistance to cisplatin therapy compared with control treatment. ABX treatment led to reduced apoptosis, increased DNA damage repair, and enhanced angiogenesis in cisplatin-treated tumors, and tumors from ABX-treated mice contained a higher frequency of cisplatin-augmented cancer stem cells than control mice. Stool analysis indicated nonresistant gut microbial species were disrupted by ABX treatment. Cecal transplants of microbiota derived from control-treated mice was sufficient to ameliorate chemoresistance and prolong survival of ABX-treated mice, indicative of a gut-derived tumor suppressor. Metabolomics analyses identified circulating gut-derived metabolites that were altered by ABX treatment and restored by recolonization, providing candidate metabolites that mediate the cross-talk between the gut microbiome and ovarian cancer. Collectively, these findings indicate that an intact microbiome functions as a tumor suppressor in EOC, and perturbation of the gut microbiota with ABX treatment promotes tumor growth and suppresses cisplatin sensitivity. Significance: Restoration of the gut microbiome, which is disrupted following antibiotic treatment, may help overcome platinum resistance in patients with epithelial ovarian cancer. See related commentary by Hawkins and Nephew, p. 4511

The ketogenic diet (KD) is hypothesized to impact tumor progression by altering tumor metabolism. In this study, we assessed the impact of an unrestricted KD on epithelial ovarian cancer (EOC) tumor growth, gene expression, and metabolite concentration in a mouse model. ID8 EOC cells, which were syngeneic with C57Bl/6J mouse strain and transfected with luciferase (ID8-luc), were injectedand monitored for tumor development. Female mice were fed either a strict KD, a high fat/low carbohydrate (HF/LC) diet, or a low fat/high carbohydrate (LF/HC) diet (n = 10 mice per group) ad libitum. EOC tumor growth was monitored weekly, and tumor burden was determined based on luciferase fluorescence (photons/second). At the endpoint (42 days), tumors were collected and processed for RNA sequencing. Plasma and tumor metabolites were evaluated using LC-MS. The KD-fed mice exhibited a statistically significant increase in tumor progression in comparison to the HF/LC- and LF/HC-fed groups (9.1 vs. 2.0 vs. 3.1-fold, respectively, p < 0.001). The EOC tumors of the KD-fed mice exhibited significant enrichment of the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways based on the RNA sequencing analysis when compared to the LF/HC- and HF/LC-fed mice. Thus, unrestricted KD diet enhanced tumor progression in our mouse EOC model. KD was associated with the upregulation of fatty acid metabolism and regulation pathways, as well as enrichment of fatty acid and glutamine metabolites.

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of the interaction region of Cx26 attenuated tumor growth in preclinical models. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. Peptides with high affinity were engineered with a cell-penetrating antennapedia sequence and assessed in functional assays including cell proliferation, tumorsphere formation, and in vivo tumor growth, and downstream signaling changes were measured. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. Here, we provide proof-of-concept that a Cx26 peptide–based strategy inhibits growth and alters NANOG activity specifically in TNBC, indicating the therapeutic potential of this targeting approach.

CEACAM1 regulates endothelial barrier integrity. Because insulin signaling in extrahepatic target tissues is regulated by insulin transport through the endothelium, we aimed at investigating the metabolic role of endothelial CEACAM1. To this end, we generated endothelial cell-specific Ceacam1 null mice (VECadCre+Cc1^fl/fl) and carried out their metabolic phenotyping and mechanistic analysis by comparison to littermate controls. Hyperinsulinemic-euglycemic clamp analysis showed intact insulin sensitivity in VECadCre+Cc1^fl/fl mice. This was associated with the absence of visceral obesity and lipolysis and normal levels of circulating non-esterified fatty acids, leptin, and adiponectin. Whereas the loss of endothelial Ceacam1 did not affect insulin-stimulated receptor phosphorylation, it reduced IRS-1/Akt/eNOS activation to lower nitric oxide production resulting from limited SHP2 sequestration. It also reduced Shc sequestration to activate NF-κB and increase the transcription of matrix metalloproteases, ultimately inducing plasma IL-6 and TNFα levels. Loss of endothelial Ceacam1 also induced the expression of the anti-inflammatory CEACAM1-4L variant in M2 macrophages in white adipose tissue. Together, this could cause endothelial barrier dysfunction and facilitate insulin transport, sustaining normal glucose homeostasis and retaining fat accumulation in adipocytes. The data assign a significant role for endothelial cell CEACAM1 in maintaining insulin sensitivity in peripheral extrahepatic target tissues.

The newly established CD3FLAG-mIR transgenic mouse model on a C57Bl/6 background has a FLAG tag on the mouse Insulin Receptor (mIR), specifically on T cells, as the FLAG-tagged mIR gene was engineered behind CD3 promoter and enhancer. The IR is a chemotactic molecule for insulin and the Flag-tagged mIR T cells in the BL/6-CD3FLAGmIR transgenic mice can migrate into the pancreas, as shown by immunofluorescent staining. While the transgenic mice do not become diabetic, there are phenotypic and metabolic changes in the islets. The transgenic islets become enlarged and disorganized by 15 weeks and those phenotypes continue out to 35 weeks of age. We examined the islets by RT-PCR for cell markers, ER stress markers, beta cell proliferation markers, and cytokines, as well as measuring serum insulin and insulin content in the pancreas at 15, 25, and 35 weeks of age. In transgenic mice, insulin in serum was increased at 15 weeks of age and glucose intolerance developed by 25 weeks of age. Passage of transgenic spleen cells into C57Bl/6 RAG⁻/⁻ mice resulted in enlarged and disorganized islets with T infiltration by 4 to 5 weeks post-transfer, replicating the transgenic mouse studies. Therefore, migration of non-antigen-specific T cells into islets has ramifications for islet organization and function.

Objective: To evaluate intraperitoneal (IP) tumor engraftment, metastasis and growth in a pre-clinical murine epithelial ovarian cancer (EOC) model using both transabdominal ultrasound (TAUS) and bioluminescence in vivo imaging system (IVIS). Methods: Ten female C57Bl/6J mice at six weeks of age were included in this study. Five mice underwent IP injection of 5×10 ⁶ ID8-luc cells (+ D-luciferin) and the remaining five mice underwent IP injection of ID8-VEGF cells. Monitoring of tumor growth and ascites was performed weekly starting at seven days post-injection until study endpoint. ID8-luc mice were monitored using both TAUS and IVIS, and ID8-VEGF mice underwent TAUS monitoring only. Individual tumor implant dimension and total tumor volume were calculated. Average luminescent intensity was calculated and reported per mouse abdomen. Tumor detection was confirmed by gross evaluation and histopathology. All data are presented as mean +/- standard deviation. Results: Overall, tumors were successfully detected in all ten mice using TAUS and IVIS, and tumor detection correlated with terminal endpoint histology/ H&E staining. For TAUS, the smallest confirmed tumor measurements were at seven days post-injection with mean long axis of 2.23mm and mean tumor volume of 4.17mm ³ . However, IVIS imaging was able to detect tumor growth at 14 days post-injection. Conclusions: TAUS is highly discriminatory for monitoring EOC in pre-clinical murine model, allowing for detection of tumor dimension as small as 2 mm and as early as seven days post-injection compared to IVUS. In addition, TAUS provides relevant information for ascites development and detection of multiple small metastatic tumor implants. TAUS provides an accurate and reliable method to detect and monitor IP EOC growth in mouse xenografts.