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

Abstract

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.

Keywords: Ascites; High-grade serous ovarian cancer; Maraba virus; Oncolytic virus; Resistance; Tumour heterogeneity.

Fig. 1

Temporal tumour cell biology impacts MRBV oncolytic efficacy in vitro. a. Serum CA-125 concentration (units per mL) for the patient from whom ascites samples were collected to derive new cell lines from October 2008 to March 2012 comprising the complete clinical course of her disease. iOvCA105, iOvCa131, iOvCa142, and iOvCa147 samples were derived from multiple ascites isolated upon first relapse and over a 14-month period (upward arrows). iOvCa105 cells were isolated October 2010 after first relapse with platinum-sensitive disease. iOvCa131, iOvCa142, and iOvCa147 were collected in close succession one year later between October 2011 and December 2011 upon second recurrence and acquisition of platinum resistance. b. Cells from all four cell lines were seeded at 10,000 cells per well of a 96-well plate and infected with different doses of MRBV as indicated, or UV-inactivated MRBV as a control. Cells were assayed for viability using CellTiter-Glo® reagent at 72 h post-infection. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, as determined by one-way ANOVA and Dunnett’s posthoc test)

Fig. 2

Intratumoral heterogeneity impacts MRBV oncolytic efficacy in vitro. a. Seven subcloned derivatives from iOvCa147 were generated through limiting dilution culturing and single-cell expansion to create homogeneous cell lines from the original heterogeneous cell line population. Resultant cells were seeded at 10,000 cells per well of a 96-well plate and infected 24 h post-seeding. Cells were assayed for viability using CellTiter-Glo at 72 h post-infection. (**, p < 0.01; ****, p < 0.0001, as determined by one-way ANOVA and Dunnett’s posthoc test). b. Images of MRBV-infected cells were captured at 72 h post-infection using bright-field and fluorescence microscopy at 50× original magnification

Fig. 3

Differences in LDLR expression can impact MRBV entry. a. iOvCa147-F8 and iOvCa147-G4 cells were seeded at 75,000 cells per well of a 24-well plate. After 24 h, cells were infected with MRBV at an MOI of 1 for 1 h. Supernatant was collected and non-cell associated virus was titrated using Vero cells; supernatant from infections containing no cells were used as a control for total uninfected virus. b. Western blot were performed using lysates collected from iOvCa147-F8 and iOvCa147-G4 cells infected with MRBV at an MOI of 1, or UV-inactivated MRBV and no virus as controls. Actin served as a loading control. The graph represents quantification of LDLR expression performed using Bio-Rad Image Lab software and normalized to actin. c. Two different MRBV-sensitive subclones iOvCa147-F8 and iOvCa147-E2 cells were seeded at 20,000 cells per well of a 48-well plate. Cells were transfected with siNT or siLDLR and 48 h post-transfection, cells were harvested for protein lysates. Western blot for LDLR was performed and actin was used as a loading control. d. iOvCa147- F8 and iOvCa147-E2 cells transfected with siNT or siLDLR were treated with 100 nM of RAP for 1 h at 48 h post-transfection followed by MRBV infection for another 1 h. media was collected for titration of MRBV virus on Vero cells via plaque assay; treatments containing no cells were performed for normalization. e. iOvCa147- F8 and iOvCa147-E2 cells transfected with siNT or siLDLR were treated with 100 nM of RAP for 1 h at 48 h post-transfection followed by MRBV infection for another 1 h. Cells were assayed for viability using CellTiter-Glo at 72 h post-infection. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, as determined by one-way ANOVA and Tukey’s posthoc test)

Fig. 4

Extracellular factors do not impart sensitivity to MRBV. a MRBV-sensitive iOvCa147-F8, and MRBV-resistant iOvCa147-B3 and iOvCa147-G4 cells were seeded at 500,000 cells per well of a 6-well plate. A549 human lung adenocarcinoma cells served as a type I IFN response positive control cell line. The following day, cells were infected with MRBV at an MOI of 1 or UV-inactivated MRBV for 6 h. RNA was isolated to perform qRT-PCR using human-specific IFNB1 primers and SYBR Green detection; GAPDH served as a normalization control. (****, p < 0.0001, as determined by one-way ANOVA and Dunnett’s posthoc test). b Cells were seeded at 10,000 cells per well of a 96-well plate. After 16 h, fresh media was either (i) replaced, (ii) conditioned media was swapped, or (iii) left unchanged between iOvCa147-F8 and iOvCa147-G4 cells. Cells were then immediately infected with MRBV at an MOI of 0.1 for 1 h followed by media change. CellTiter-Glo® assays were performed for cell viability 48 h post-infection. c iOvCa147-F8 and iOvCa-G4 cells were seeded at 10,000 cells per well of a 96-well plate. After 16 h, cells were infected with MRBV at an MOI of 0.1 for 1 h followed by media change. At 12 h, fresh media was either (i) replaced, or (ii) conditioned media was swapped between iOvCa147-F8 and iOvCa-G4 cells, or (iii) left unchanged. CellTiter-Glo® assays were performed for cell viability 48 h post-infection. (Letters indicate whether there is a statistically significant difference (p < 0.05) among conditions as determined by one-way ANOVA and Tukey’s posthoc test)

Fig. 5

MRBV sensitivity can be conferred to resistant cells through direct cell-cell co-culture. a iOvCa147-F8 and iOvCa147-G4 cells were seeded at specific mixtures as indicated to a total of 100,000 cells per well of a 24-well plate. No virus mock-infections at each cell mixture was used as a control to determine relative viability as assessed at 48 h post-infection using CellTiter-Glo®. Expected viability if there was no interaction between subclones was calculated using the data from 100% pure iOvCa147-F8 and 100% pure iOvCa147-G4 MRBV-infected cultures. (**, p < 0.01; ****, p < 0.0001, as determined by paired Student’s t-test) b Fluorescence images of co-cultured cells iOvCa147-F8 (red, DiI-labelled) and iOvCa147-G4 (blue, CMAC-labelled) were captured 16 h post-infection. MRBV-infected iOvCa147-F8 cells appear yellow (GFP and DiI double-positive) whereas MRBV-infected iOvCa147-G4 cells appear teal (GFP and CMAC double-positive). c Double-positive cells were counted for each co-culture concentration and normalized to the total number of iOvCa147-G4 cells (black bars) to determine percent infectivity (100% iOvCa147-F8 served as a control; white bar). (***, p < 0.001, as determined by one-way ANOVA and Dunnett’s posthoc test) d Physical separation of cells was achieved using 0.4-μm Transwell inserts. Media was identical between both upper and lower chambers and 100,000 cells in total were seeded (25,000 cells in the upper chamber and 75,000 cells in the lower chamber). After 24 h, media was changed and cells were infected at 50,000 viral particles (MOI 0.5) of MRBV. After 48 h, viability of the cells in the lower chamber only was measured using CellTiter-Glo® and normalized to uninfected cells. (***, p < 0.001; ****, p < 0.0001, as determined by one-way ANOVA and Tukey’s posthoc test)