The chemokine CX3CL1 promotes intraperitoneal tumour growth despite enhanced T-cell recruitment in ovarian cancer

Abstract

T-cell recruiting chemokines are required for a successful immune intervention in ovarian cancer, and also for the efficacy of modern anticancer agents such as PARP inhibitors. The chemokine CX3CL1 recruits tumour-suppressive T-cells into solid tumours, but also mediates cell-cell adhesions, e.g. of tumour cells, through its membrane-bound form. So far, its role in ovarian cancer has only been rudimentarily addressed. We show that high CX3CL1 expression significantly correlates with worsened survival in human high-grade serous ovarian cancer (n=219). In preclinical ovarian cancer, CX3CL1 plays a dual role, as it enhances the adaptive anti-tumour response, but overall still promotes tumour growth, the latter as a feature of the intraperitoneal environment. Moreover, PARP inhibitors are able to increase CX3CL1 release from human ovarian cancer cells. Collectively, our study shows that CX3CL1 is a driver of intraperitoneal tumour growth in ovarian cancer, a feature that may compromise the anticancer effect of CX3CL1-inducing PARP inhibitors.

Keywords: CX3CL1; Chemokines; Mouse model; PARP inhibition; Tumour-infiltrating lymphocytes.

Fig. 1

CX3CL1 is an unfavourable prognostic marker in high-grade serous ovarian cancer (HGSOC) patients. A, Representative immunohistochemistry (IHC) images of high or low CX3CL1 expression in a patient cohort of high-grade serous ovarian cancer (scale bars represent 50 µm). B, Digitally analysed and quantified CX3CL1 IHC staining intensity in omental (n = 122) and peritoneal (n = 126) metastases (mean values were normalised to TMA control tissue). C, Kaplan Meier curves showing progression free survival (left) and overall survival (right) in CX3CL1 high vs. low expressing groups of HGSOC.

Fig. 2

CX3CL1 expression and regulation in human and murine ovarian cancer cell lines. OV-MZ-6 cells (A, B), OVCAR-3 cells (C), or ID8-Trp53^−/− cells (D) were stimulated for 24 h ± TNF-α (50 ng/mL) and ± TAPI-2 (50 µM) or ± GI254 (5 µM) or the corresponding solvent controls as indicated. Bar graphs on the left show soluble CX3CL1 in cell supernatant measured after 24 h by ELISA (mean ± SEM). The plots on the right show membrane-bound CX3CL1 as determined via FACS analysis. B, Immunocytochemical staining of CX3CL1 in OV-MZ-6 cells stimulated with or without TNF-α in the presence or absence of TAPI-2, visualised by confocal laser scanning microscopy (CLSM) using an Alexa 488-conjugated secondary antibody.

Fig. 3

Cx3cl1 accelerates intraperitoneal tumour growth while enhancing immune infiltration. A, Expression of murine Cx3cl1 was verified in ID8-Trp53^-/-empty vector (Ctrl) and ID8-Trp53^-/-Cx3cl1⁺ (Cx3cl1⁺) cells via cell supernatant ELISA measurement of untreated or TNF-α stimulated cells (24 h, 25 ng/mL). Bars represent mean ± SEM. B, Proliferation rates of ID8-Trp53^-/-Ctrl and Cx3cl1⁺ cells were measured via MTT assay and normalised to the baseline value at 4 h. C, Representative pictures of ascites accumulation (left) as well as tumour load on the mesentery (upper right) and diaphragm (lower right) at the time of finalization (black arrows indicate exemplary tumour nodes). D, Kaplan–Meier curves showing ascites-free (left) and overall survival (right) of C57BL/6 mice intraperitoneally injected with 1×10⁷ ID8-Trp53^-/-Ctrl or Cx3cl1⁺ cells (differences in median survival are indicated as Δ days). E, Relative Cx3cl1 mRNA expression in mesenteric tumour tissue of ID8-Trp53^-/-Ctrl or Cx3cl1⁺ tumours at the end of the experiment. F, Quantification of tumour-infiltrating immune cell subsets (CD3, CD8, granzyme B, F4/80, CD206) by immunohistochemical stainings of ID8-Trp53^-/-Ctrl or Cx3cl1⁺ mesenteric tumour tissues. DAB positive immune cell markers (CD3, CD8, granzyme B) were manually counted in intratumoural report images. Positive macrophage subset cells (F4/80, CD206) were digitally evaluated as percentage of all cells. G, Ki67-positive proliferation status was digitally assessed as percentage of total cells. H, 1×10⁷ ID8-Trp53^−/−Ctrl or Cx3cl1⁺ cells were implanted intraperitoneally into athymic nude mice, and onset of ascites (left) and overall survival (right) was determined and shown as Kaplan–Meier curves (differences in median survival are indicated as Δ days). I, F4/80 macrophage marker was immunohistochemically stained in mesenteric tumour tissue from athymic nude mice, and positive cells were calculated as percentage of all cells. Horizontal lines in E-G and I indicate the mean, each dot indicates data from one individual mouse. Mouse experiments were conducted with at least 7 mice per group.

Fig. 4

Cx3cl1 inhibits tumour growth in the subcutaneous ID8 ovarian cancer model. A, ID8luc-Trp53^-/-Control (Ctrl) and ID8luc-Trp53^-/-Cx3cl1⁺ (Cx3cl1⁺) cells were tested for Cx3cl1 protein secretion into the cell supernatant of untreated or TNF-α (24 h, 50 ng/mL) stimulated cells by ELISA. Bars represent mean ± SEM. B, Cell proliferation rates of ID8luc-Trp53^-/-Ctrl and Cx3cl1⁺ cells were compared via manual counting with a Neubauer counting chamber and normalised to the baseline value at 4 h. C-G, 1×10⁷ ID8luc-Trp53^-/-Ctrl or Cx3cl1⁺ cells were subcutaneously inoculated into the flanks of C57BL/6 mice (n = 6 each group). C, Graph shows caliper measurement of maximal tumour diameter (mm) once a week until the first mouse reached predefined endpoints. D, Weekly flux (photons/sec) measurement of luciferase expressing tumour cells upon intraperitoneal luciferin substrate injection. E, Representative pictures of each group indicate bioluminescence signals (day 93). The pseudocolors represent the average radiance in the unit of p/s/cm²/sr with a maximum radiance of 5×10⁵ depicted as red color. F, Representative pictures of a subcutaneously grown tumour with a reached finalization endpoint of 1.5 cm diameter (in vivo). G, Kaplan–Meier plot showing survival of tumour mice (differences in median survival are indicated as Δ days). H, Cx3cl1 expression at mRNA level was measured in tumour tissue via qRT-PCR and normalised to the housekeeping control. I, Intratumoural cell proliferation was assessed via digital Ki67 marker IHC analysis, and positive cells were are given as percentage of total cells. J-K, Tumour-infiltrating CD3⁺ T-cells and CD8⁺ cytotoxic T-cells (J) and Foxp3⁺ regulatory T-cells (K) were determined immunohistochemically. DAB positive cells were manually counted in five report images. L, Intratumoural F4/80-positive macrophages were immunohistochemically stained and digitally analysed as percent of total cells. Horizontal lines in H-L indicate the mean, each dot indicates data from one individual mouse.

Fig. 5

PARP inhibition enhances CX3CL1 secretion from human ovarian cancer cell lines. Human ovarian cancer cell lines OV-MZ-6, CAOV-3 and OVCAR-3 were stimulated with 10 µM olaparib, 10 µM niraparib or corresponding DMSO solvent control in the absence or presence of TNF-α (10 ng/mL). After 48 h, cell supernatants were harvested, and soluble CX3CL1 concentrations were assessed via ELISA.