Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential

Background: Epithelial ovarian cancer exhibits extensive interpatient and intratumoral heterogeneity, which can hinder successful treatment strategies. Herein, we investigated the efficacy of an emerging oncolytic, Maraba virus (MRBV), in an in vitro model of ovarian tumour heterogeneity. Methods: Four ovarian high-grade serous cancer (HGSC) cell lines were isolated and established from a single patient at four points during disease progression. Limiting-dilution subcloning generated seven additional subclone lines to assess intratumoral heterogeneity. MRBV entry and oncolytic efficacy were assessed among all 11 cell lines. Low-density receptor (LDLR) expression, conditioned media treatments and co-cultures were performed to determine factors impacting MRBV oncolysis. Results: Temporal and intratumoral heterogeneity identified two subpopulations of cells: one that was highly sensitive to MRBV, and another set which exhibited 1000-fold reduced susceptibility to MRBV-mediated oncolysis. We explored both intracellular and extracellular mechanisms influencing sensitivity to MRBV and identified that LDLR can partially mediate MRBV infection. LDLR expression, however, was not the singular determinant of sensitivity to MRBV among the HGSC cell lines and subclones. We verified that there were no apparent extracellular factors, such as type I interferon responses, contributing to MRBV resistance. However, direct cell-cell contact by co-culture of MRBV-resistant subclones with sensitive cells restored virus infection and oncolytic killing of mixed population. Conclusions: Our data is the first to demonstrate differential efficacy of an oncolytic virus in the context of both spatial and temporal heterogeneity of HGSC cells and to evaluate whether it will constitute a barrier to effective viral oncolytic therapy

Exploring Myxoma Virus Oncolytic Virotherapy in Combination with Carboplatin for the Treatment of Epithelial Ovarian Cancer

Embargoed paper. Medical Sciences Overall Winner Abstract Epithelial ovarian cancer (EOC) is the most lethal of the gynaecologic malignancies as 75% of cases are diagnosed after extensive metastasis. EOC metastasis involves shedding of cancer cells from the primary tumor directly into the peritoneal cavity where EOC cells form multicellular aggregates or spheroids that implant in and invade the peritoneal mesothelium to initiate secondary tumors. EOC spheroids undergo dormancy in suspension to promote survival and in turn demonstrate resistance to standard chemotherapeutics such as carboplatin. In an in vitro model of EOC metastasis, we have recently demonstrated that Myxoma virus (MYXV) effectively kills adherent EOC cells and reduces spheroid reattachment. In order for MYXV to advance toward clinical use as a first-line therapy for ovarian cancer, its compatibility with current chemotherapeutics must be assessed. We propose that combination treatment using MYXV and carboplatin will decrease the viability of EOC spheroids. Herein, we show that carboplatin does not impede MYXV replication or oncolytic potential in EOC cells in monolayer or spheroid culture. Administration of different sequential regimens of carboplatin and virus does not enhance sensitivity to oncolysis of EOC cell lines in monolayer culture. Furthermore, co-treatment of MYXV and carboplatin did not consistently induce oncolysis in spheroids generated from EOC cell lines or patient ascites. However, upon reattachment, spheroids treated with various combination regimens revealed additive cytopathic effects. Remarkably, we also encountered a patient sample which responded to MYXV only in the presence of carboplatin suggesting that chemotherapy-sensitive primary EOC spheroid cells are possibly eliminated by MYXV-mediated oncolysis during co-treatment. We hope to include more patient sample analyses that allow for strong pre-clinical evaluation of this combination treatment

A Case of Brevibacillus brevis Meningitis and Bacteremia

Brevibacillus species are environmental organisms that are rarely implicated as human pathogens. We present the case of postsurgical Brevibacillus brevis bacterial meningitis and an associated bacteremia after debulking surgery for a newly diagnosed pilocytic astrocytoma in a 19-year-old woman. The patient experienced clinical cure with a 4-week course of vancomycin, but her postinfectious course was complicated by the development of a pseudomeningocele that required surgical repair. To our knowledge, this is the first described case of a central nervous system infection caused by Brevibacillus brevis in the literature

Additional file 3: Figure S3. of Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential

Validation of LDLR knockdown using two independent siRNAs. a. iOvCa147-F8 cells were seeded at 20,000 cells per well of a 48-well dish, then transfected with each siLDLR siRNA or siNT control for 48 h. Transfected cells were harvested for protein lysis to perform western blotting for LDLR expression. b. Cells transfected with siLDLR-1, siLDLR-2, or siNT, were infected with MRBV at an MOI of 0.05 for 48 h and viability was measured using CellTiter-Glo®. (PPTX 85 kb

MD/PhD Training in Canada: Results from a national trainee and program director review

Purpose: There has been limited examination of clinician scientist training in Canada, particularly regarding training integration and funding. This study assessed program structure, funding, tuition and mentorship structures available at Canadian MD/PhD programs. Methods: Clinician Investigator Trainee Association of Canada administered an anonymous survey to current trainees and program directors that captured program structure, trainee funding, tuition and mentorship opportunities and needs across institutions. Results: In June 2015, 101/228 (44%) trainees and 9/13 (69%) program directors completed the online survey. In all programs, students completed the PhD degree prior to clerkship training. Seven programs offered research training upon completion of pre-clerkship, four offered concurrent clinical and research training, and three offered alternative structures. Nine held seminars exposing students to clinical and research integration and two offered clinician scientist skills courses. Stipend funding and tuition varied, especially during clinical training years. Regarding mentorship, all programs held regular meetings, though eight programs do not have formal mentorship opportunities. Both trainees and program directors identified the need for further career planning and development support as a student priority. Conclusion: MD/PhD programs varied by program structure, funding, tuition and mentorship opportunities. Mechanisms to share and spread program innovations should be instated. Students may benefit from concurrent research and clinical training as well as courses specific to clinician scientist skill development. Decreasing debt burden may attract and retain trainees in this demanding path. To ensure mentorship programs align with trainee priorities, program directors should directly collaborate with students in their development and evaluation

Additional file 1: Figure S1. of Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential

Highest alteration frequency of the LDLR gene in serous ovarian cancers. a. Mutation and gene copy-number status for LDLR across human cancers as determined using The Cancer Genome Atlas (TCGA) provisional datasets accessed from cBioPortal as of November 12, 2015. Ovarian cancer (serous adenocarcinoma) has the highest prevalence of LDLR gene alterations, particularly amplifications, as compared with all other malignancies in the data set. b. Oncoprint of serous ovarian tumours harbouring LDLR mutations, copy-number changes, and gene expression changes (z-score > 2) from the TCGA provisional dataset. Only tumour samples with alterations (13%; 78/599 samples) are displayed for clarity. c. LDLR mRNA expression as compared with gene copy-number status among all serous ovarian tumours from the TCGA provisional dataset. (PPTX 240 kb

Additional file 2: Figure S2. of Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential

Reduced LDLR expression in spheroids decreases MRBV-mediated oncolysis. a. Cells were seeded into standard tissue culture plates or ultra-low attachment (ULA) plates to form spheroids and harvested for protein lysis 24 h after seeding. Western blotting was performed for LDLR expression and actin served as a loading control. LDLR expression is reduced in iOvCa147-F8 spheroids to similar levels seen in iOvCa147-G4 cells and spheroids. b. iOvCa147-F8 and -G4 cells were seeded at 50,000 cells per well of a 24-well ULA plate and spheroids were formed over 72 h. Spheroids were then infected with MRBV at an MOI of 0.1 for 48 h and viability was assessed using CellTiter-Glo®; MRBV-infected adherent cells were used for comparison. (PPTX 65 kb