Wounding promotes ovarian cancer progression and decreases efficacy of cisplatin in a syngeneic mouse model

Abstract

Background: Primary cytoreductive surgery followed by adjuvant chemotherapy is the standard treatment for advanced epithelial ovarian cancer. The average interval between surgery and chemotherapy initiation is approximately 4-weeks at most centers; however, since surgery may accelerate residual tumor growth, a shorter interval may be more beneficial.

Methods: The murine ID8 cell model of ovarian cancer was used to examine the efficacy of cisplatin treatment administered perioperatively or 7 days after surgical wounding. Luciferase-expressing cells ID8 cells were injected intraperitoneally (i.p.) into female C57/Bl6 mice. Fourteen days post-injection, animals received an abdominal incision or anesthesia alone and received i.p. cisplatin either on the surgical day or 7 days later, or received no chemotherapy. Additional animals received cisplatin 28 days after wounding for comparison.

Results: Abdominal tumor mass increased 2.5-fold in wounded vs. unwounded animals as determined by bioluminescent in vivo tumor imaging. Cisplatin administered on the day of wounding decreased tumor burden by 50%, as compared to 90% in unwounded animals. Cisplatin on day 7 or day 28 decreased tumor burden by 80 and 37% respectively.

Conclusions: Surgical wounding increases ovarian tumor mass and decreases perioperative cisplatin efficacy in this animal model. Administration of cisplatin 1 week after surgery was more effective than cisplatin administered perioperatively or 4 weeks after surgery.

Keywords: Chemoresistance; Cisplatin; ID8 cells; Mouse; Ovarian cancer; Wound healing.

Fig. 1

Bioluminescent luciferase activity and sensitivity to cisplatin of ID8-L11 and ID8-L15 cells in vitro. a Concentration-dependent bioluminesence imaging of both cell sublines compared to non-luciferase expressing parental ID8 cells. Different concentrations of cells were added to a 96-well tissue culture plate and imaged on an IVIS Spectrum In Vivo Imaging System. b Graphical representation of the quantitation of images shown in Panel A (dashed lines). Solid lines indicate a linear fitting of the data. c-e Response of cells to cisplatin. Parental ID8 (c), ID8-L11 (d), or ID8-L15 (e) cells were seeded into 96 well plates. One day later, cells were treated with 0, 10, 25, or 50 μM cisplatin and relative cell viability was determined by a XTT dye-reduction assay. Points represent the mean ± SEM of 8 determinations. Within each time point, points with different letters are statistically different from one another as determined by ANOVA followed by SNK multiple comparison test (p < 0.05)

Fig. 2

In vivo imaging of ID8-L11 cells and effect of serum from wounded mice on growth of parental ID8 cells. a Schematic of the treatment paradigm used in the preliminary study. b Representative bioluminescence images of a mouse not injected with ID8-L11 cells (left) and a mouse injected with 5 million ID8-L11 cells i.p. 13 days earlier (right). c Representative images of the abdominal viscera of mice in each of the four treatment groups at time of necropsy. No evidence of macroscopic seeding was found. d Serum from mice of the different treatment groups did not impact the growth of parental ID8 cells in vitro. Cells were grown in medium supplemented to 10% with serum pooled from within the four treatment groups in the presence or absence of 25 μM cisplatin. XTT dye reduction assay was performed 72 h after initiation of treatment. Bars represent the mean ± SEM of 8 replicates. Two-way ANOVA performed on natural log-transformed data indicated a statistically significant effect of cisplatin but no statistically significant effect of serum from the different treatment groups or interaction

Fig. 3

Study treatment paradigm. a Schematic showing the overall treatment and imaging schedule used in the study. b Schematic showing the stratification of imaged animals to the seven treatment groups and the loss of animals within groups. S = surgery, A = anesthesia, C₀ = cisplatin on the perioperative day, C₇ = cisplatin on postoperative day (POD) 7, C₂₈ = cisplatin on POD 28. All animals were inoculated i.p. with 5 million ID8-L11 cells and were imaged 13 days later

Fig. 4

Change in body weight, incidence of ascites (Panel b) and survival curves for the seven treatment groups. a Change in body weight from the time of cell injection to animal sacrifice is shown for each treatment group. Lines represent group mean. No changes were detected using ANOVA. b Numbers of mice that exhibited signs of ascites development within the treatment groups. No overall effect due to treatment group was detected using a Chi-square analysis. c Kaplan-Meier growth curves obtained for each of the treatment groups using indicators of animal health/distress as a surrogate for survival. An overall effect of treatment was indicated by a Log-Rank test (p = 0.0012)

Fig. 5

Representative bioluminescence images obtained for the seven treatment groups at each of the three imaging sessions

Fig. 6

Compiled results from the imaging of animals at day − 1, 21, and 35 relative to the day of surgical wounding. a Mean levels of abdominal tumor burden measured for each of the seven treatment groups. Bars represent the mean ± SEM. b Statistical results obtained for imaging of animals on day 21. For this analysis, mice assigned to treatment groups S and S + C₂₈ were combined. Data were subjected to a natural log transformation prior to analysis by ANOVA followed by Fishers LSD multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS = not significant. c Statistical results obtained for imaging of animals on day 35. Data were subjected to a natural log transformation prior to analysis by ANOVA followed by Fishers LSD multiple comparison test. d and e Separation of the data shown in (panel a) to highlight the impact of cisplatin in surgically wounded animals (Panel d) and comparing the effect of surgery and cisplatin administered on day 7 to sham-wounded animals (Panel e). Points represent the mean ± SEM