Establishing a mouse model of lung metastases using ultrasound-guided right heart ventricle injection.

We report a technique to generate a murine model of lung metastases by selectively injecting tumor cells into the right heart ventricle under ultrasound guidance. First, we describe cell preparation and reference animal preparation as previously described. We then detail the technique using a previously described 3D-printed instrument stabilization device. Finally, we describe tumor growth surveillance using bioluminescent imaging. For complete details on the use and execution of this protocol, please refer to Labora et al.1.

MEK Inhibition Sensitizes Pancreatic Cancer to STING Agonism by Tumor Cell-intrinsic Amplification of Type I IFN Signaling.

PURPOSE: Stimulator of interferon genes (STING) agonists are currently in development for treatment of solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Response rates to STING agonists alone have been promising yet modest, and combination therapies will likely be required to elicit their full potency. We sought to identify combination therapies and mechanisms that augment the tumor cell-intrinsic effect of therapeutically relevant STING agonists apart from their known effects on tumor immunity. EXPERIMENTAL DESIGN: We screened 430 kinase inhibitors to identify synergistic effectors of tumor cell death with diABZI, an intravenously administered and systemically available STING agonist. We deciphered the mechanisms of synergy with STING agonism that cause tumor cell death in vitro and tumor regression in vivo. RESULTS: We found that MEK inhibitors caused the greatest synergy with diABZI and that this effect was most pronounced in cells with high STING expression. MEK inhibition enhanced the ability of STING agonism to induce type I IFN-dependent cell death in vitro and tumor regression in vivo. We parsed NFκB-dependent and NFκB-independent mechanisms that mediate STING-driven type I IFN production and show that MEK signaling inhibits this effect by suppressing NFκB activation. CONCLUSIONS: Our results highlight the cytotoxic effects of STING agonism on PDAC cells that are independent of tumor immunity and that these therapeutic benefits of STING agonism can be synergistically enhanced by MEK inhibition.

Higher Numbers of Examined Lymph Nodes Are Associated with Increased Survival in Resected, Treatment-Naïve, Node-Positive Esophageal, Gastric, Pancreatic, and Colon Cancers.

BACKGROUND OR PURPOSE: The role of extended lymphadenectomy as part of resection for lymph node (LN)-positive gastrointestinal (GI) malignancies remains controversial with no clear clinical guidance. The purpose of this retrospective study is to determine whether the number of LNs examined as part of GI malignancy resections affects overall survival (OS) among patients with node-positive esophageal, gastric, pancreatic, and colon cancers. METHODS: Participants with LN-positive GI cancers who were diagnosed between 2004 and 2015 and underwent oncologic resections were selected from National Cancer Database (NCDB). The primary predictor was the number of examined LNs categorized in tertiles. The effect on OS was measured by hazard ratio (HR) derived from multivariate Cox regression analyses. RESULTS: From 2004 to 2015, 1877, 10,086, 18,193, and 102,500 patients with LN-positive esophageal, gastric, pancreatic, and colon adenocarcinomas who did not receive neoadjuvant treatment and underwent oncologic tumor resection were registered in the NCDB. Using multivariate Cox proportional hazard modeling, greater LNs examined in surgically resected LN-positive GI cancers were found to be associated with increased OS for all histologies. This association was the strongest (as compared to the lowest tertile) for gastric cancer (middle tertile: HR = 0.91, 95% CI, 0.86-0.96, p = 0.001; highest tertile: HR = 0.73, 95% CI, 0.69-0.78, p < 0.001), followed by colon (highest tertile: HR = 0.86, 95% CI, 0.84-0.88, p < 0.001), esophageal (highest tertile: HR = 0.83, 95% CI, 0.72-0.95, p = 0.01), and pancreatic (highest tertile: HR = 0.93, 95% CI, 0.89-0.98, p = 0.002) cancers. DISCUSSION AND CONCLUSION: In patients with surgically resected node-positive GI malignancies who did not receive neoadjuvant systemic therapy, a higher number of examined LNs is associated with increased OS. This association is the strongest for gastric cancer, followed by colon, esophageal, and pancreatic cancers respectively.

Generation of liver metastases in a mouse model using ultrasound-guided intravenous injection.

Here, we present a protocol to generate a murine model of liver metastasis by directly injecting tumor cells into the portal vein under ultrasound guidance. We describe steps for animal and cell preparation and two techniques for injecting tumor cells. One technique is freehand, while the other technique is device-assisted using a 3D-printed prototype device. Finally, we describe tumor surveillance with bioluminescent imaging.

18F-FDG PET Visualizes Systemic STING Agonist-Induced Lymphocyte Activation in Preclinical Models.

Stimulator of interferon genes (STING) is a mediator of immune recognition of cytosolic DNA, which plays important roles in cancer, cytotoxic therapies, and infections with certain pathogens. Although pharmacologic STING activation stimulates potent antitumor immune responses in animal models, clinically applicable pharmacodynamic biomarkers that inform of the magnitude, duration, and location of immune activation elicited by systemic STING agonists are yet to be described. We investigated whether systemic STING activation induces metabolic alterations in immune cells that can be visualized by PET imaging. Methods: C57BL/6 mice were treated with systemic STING agonists and imaged with 18F-FDG PET after 24 h. Splenocytes were harvested 6 h after STING agonist administration and analyzed by single-cell RNA sequencing and flow cytometry. 18F-FDG uptake in total splenocytes and immunomagnetically enriched splenic B and T lymphocytes from STING agonist-treated mice was measured by γ-counting. In mice bearing prostate or pancreas cancer tumors, the effects of STING agonist treatment on 18F-FDG uptake, T-lymphocyte activation marker levels, and tumor growth were evaluated. Results: Systemic delivery of structurally distinct STING agonists in mice significantly increased 18F-FDG uptake in the spleen. The average spleen SUVmax in control mice was 1.90 (range, 1.56-2.34), compared with 4.55 (range, 3.35-6.20) in STING agonist-treated mice (P < 0.0001). Single-cell transcriptional and flow cytometry analyses of immune cells from systemic STING agonist-treated mice revealed enrichment of a glycolytic transcriptional signature in both T and B lymphocytes that correlated with the induction of immune cell activation markers. In tumor-bearing mice, STING agonist administration significantly delayed tumor growth and increased 18F-FDG uptake in secondary lymphoid organs. Conclusion: These findings reveal hitherto unknown functional links between STING signaling and immunometabolism and suggest that 18F-FDG PET may provide a widely applicable approach toward measuring the pharmacodynamic effects of systemic STING agonists at a whole-body level and guiding their clinical development.

Purine nucleoside phosphorylase enables dual metabolic checkpoints that prevent T cell immunodeficiency and TLR7-associated autoimmunity.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

Respectful and evidence-based birth care in Mexico (or lack thereof): An observational study.

PROBLEM: In Mexico, women are often disrespected and abused during birth, evidence-based practices are seldom used, while outdated and dangerous procedures linger. BACKGROUND: Disrespectful and abusive practices in Mexico have been reported but are not necessarily well-documented; none of the reports so far have relied on direct observation of births. AIM: To describe birth practices and factors associated with respectful and evidence-based care at 15 referral hospitals in Mexico. METHODS: We observed 401 births from 2010-2016. We analysed woman, provider, and hospital characteristics and their association with the performance of 14 evidence-based and 15 respectful birth practices via descriptive statistics and multiple logistic regression models. FINDINGS: Only in four births were all the analysed evidence-based and respectful-birth practices performed. Essential interventions like uterine massage was only given to 46.1% of women and the administration of a uterotonic soon after birth only occurred in 58.3% of births. Professionals who were trained in respectful birth care were more likely to address women by their name (Odds Ratio=3.34, p<0.05), allow consumption of liquids during labour (Odds Ratio=31.6, p<0.05), encourage skin-to-skin contact (Odds Ratio=31.82, p<0.05), and examine the placenta after birth (Odds Ratio=16.55, p<0.01); they were less likely to perform episiotomies (Odds Ratio=0.27, p<0.05). DISCUSSION: This study reveals low rates of evidence-based practices and respectful maternity care but shows training in the topic can have a considerable positive impact. Our results call for further efforts to improve the quality of maternal healthcare, a universal right.

Relationship between body mass index and operative time in women receiving immediate postpartum tubal ligation.

OBJECTIVES: The aim of the study was to (1) assess the relationship between body mass index (BMI) and operative time during immediate postpartum tubal ligation procedures and to (2) determine whether operative time is non-inferior in women with BMI ≥30 versus women with BMI <30 and in women with BMI ≥40 versus women with BMI <40. STUDY DESIGN: We conducted a retrospective cohort study of women who received immediate postpartum tubal ligations following vaginal delivery from 2013 to 2017 at a university hospital. We abstracted demographic information, patient and procedural characteristics, and clinical outcomes. We assessed the relationship between BMI and operative time via linear regression. We also conducted non-inferiority analysis to determine whether the mean operative time in women with BMI ≥30 was non-inferior to the mean operative time in women with BMI <30, within a non-inferiority margin of 10 min. We compared intraoperative and postoperative complications in the two groups. RESULTS: A total of 279 women were included for analysis, among whom N=79 (28%) had a BMI of 25-29.9 and N=171 (61%) had a BMI ≥30. Demographic characteristics were similar in both groups. We found that operative time increased by 35 s for each one-point increase in BMI (p<.01). Although mean operative time was 46.1 min (n=171; 95% CI 43.7, 48.6 min) for women with BMI ≥30 and 40.6 min (n=108; 95% CI 37.9 min, 43.4 min) for women with BMI <30, (p<.01), it was non-inferior within a 10-min margin. There was no difference in rates of intraoperative or postoperative complications, incision length, total anesthesia time, and median length of stay between women with BMI ≥30 and BMI <30. CONCLUSION: There is a small increase in postpartum tubal ligation operative time with increasing BMI. However, among women who received immediate postpartum tubal ligations at our institution, women with BMI ≥30 versus BMI <30 had operative times that were non-inferior within a 10-min margin. IMPLICATIONS: While increasing body mass index slightly increases the operative time for immediate postpartum tubal ligations, this increase in time does not appear to be clinically significant.