Association between constitutive TNF-α production and release of other inflammatory mediators by ovarian cancer cells
In our first experiments, we used four ovarian cancer cell lines with differing constitutive production of TNF-α. After 48 h of culture, tissue culture medium from TOV112D and SKOV-3 cells did not contain measurable TNF-α, whereas TOV21G and IGROV-1 cells reproducibly released 15 to 20 pg/mL (; P
< 0.0001). We then measured the production by these cell lines of six different factors known to be present in ovarian cancer biopsies and thought to be associated with ovarian cancer growth and spread: chemokines CCL2 (14
) and CXCL12 (15
); the angiogenic factor VEGF (16
); and the cytokines IL-6 (17
) and MIF (18
). We also measured release of the growth factor fibroblast growth factor (FGF) 2 (19
Figure 1 TNF-α release by ovarian cancer cell lines is associated with expression of other protein mediators. Proteins secreted by ovarian cancer cell lines were measured by ELISA after 48 h. Data are representative of three independent experiments done. (more ...)
The TNF-α-producing TOV21G and IGROV-1 cells produced higher amounts of CCL2 (P
= 0.017), CXCL12 (P
< 0.0001), VEGF (P
< 0.0001), IL-6 (P
< 0.0001), and MIF (P
= 0.002) compared with TOV112D and SKOV-3 cells that did not release measurable TNF-α (). IGROV-1 and TOV21G cells also had higher expression of CXCR4 as reported previously (8
). In contrast, there was no association between TNF-α production and levels of FGF2 in the tissue culture medium; indeed, SKOV-3 was the only cell line that produced detectable amount of FGF2 (67 pg/mL) after 48 h (data not shown).
To investigate the hypothesis that these differences in endogenous cytokine network would influence growth and spread of the tumor cells, we injected the cell lines i.p. into nude mice and studied patterns of spread.
Association between tumor invasiveness and TNF-α production
The TOV112D and SKOV-3 cell lines that did not produce TNF-α grew as well-defined encapsulated peritoneal masses, whereas the two cell lines that produced TNF-α, TOV21G, and IGROV-1 showed more widely dispersed tumors (, arrows). This was confirmed by histologic examination of mouse organs at the survival end point (20% increase in abdominal girth). The distribution of both intraperitoneal and extraperitoneal deposits from the cell lines that did not produce TNF-α was much less than those that produced TNF-α (). The TNF-α-producing ovarian cancer cells were capable of colonizing at least 12 different sites after i.p. injection into nude mice, whereas tumor colonies from those cell lines that did not produce TNF-α were only detected in five different sites.
Figure 2 Appearance and location of tumor deposits in nude mice injected with ovarian cancer xenografts. A, gross patterns of peritoneal tumor spread of TOV112D, SKOV-3, TOV21G, and IGROV-1. B, location of tumor deposits in various organs as determined by histologic (more ...)
If endogenous TNF-α was involved in tumor dissemination, we reasoned that TNF-α knockdown would inhibit this. We therefore established stable inhibition of TNF-α mRNA in TNF-α-producing IGROV-1 cells using RNAi technology.
Knockdown of TNF-α reduces production of other mediators by ovarian cancer cells
We transfected IGROV-1 cells with short hairpin RNA (shRNA) to TNF-α and established two clones of the IGROV-1 cell line with stable knockdown of TNF-α RNA (RNAi TNF-α I and II). The shRNA transfection resulted in reproducible, significant, and stable decreases in TNF-α release of 71% and 74% in two different TNF-α RNAi clones after 72 h (; P
= 0.0051 and 0.0034 compared with mock transfected). When supernatants from RNAi TNF-α cells were compared with those from cells transfected with shRNA with limited homology to any known sequences in the human, mouse, and rat genome (scrambled RNA, IGROV-Mock cells) release of CCL2, CXCL12, VEGF, IL-6, and MIF () was lower in the knockdown cells compared with mock-transfected cells. Silencing endogenous TNF-α decreased production of CCL2 by 86% and 90% in the two different TNF-α RNAi clones (P
< 0.0001 and 0.0001 compared with mock transfected) and of VEGF by 40% and 29% (P
= 0.016 and 0.008). IL-6 was reduced by 88% and 89% (P
< 0.0001 and 0.0001) and MIF by 59% and 70% (P
= 0.0015 and 0.0012). CXCL12 protein expression was completely inhibited in both the TNF-α RNAi IGROV clones. As described before, CXCR4 expression was also reduced by RNAi to TNF-α (8
). None of the IGROV-1 cells released FGF nor did the cells release any IFN-γ whether they expressed shRNA constructs.
Figure 3 In vitro effects of stable expression of shRNAto TNF-α. In all experiments, IGROV-Mock cells are compared with two independently isolated TNF-α shRNA clones RNAi TNF-α (I) and RNAi TNF-α (II). A, TNF-α protein secretion (more ...)
Knockdown of TNF-α and growth of cells in vitro
TNF-α did not, however, seem to be a growth factor for the ovarian cancer cells in tissue culture. The TNF-α RNAi cells grew at similar rates to IGROV-Mock cells and wild-type cells. Viable cell counts were assessed by trypan blue staining over 4 days of culture (data not shown) or when the WST-1 assay was used (). Apoptosis was also assessed by fluorescence-activated cell sorting analysis using Annexin V and propidium iodide. Neither of these assays showed any difference in apoptosis between IGROV-Mock and TNF-α RNAi cells (data not shown). Addition of exogenous TNF-α at doses of 1, 10, and 100 ng did not alter growth rates of any of the lines (data not shown).
We therefore concluded that reduction of constitutive TNF-α production in ovarian cancer cells did not affect their ability to proliferate in vitro. However, knockdown of TNF-α was able to down-regulate several other soluble factors that could be important in growth and dissemination in vivo. To investigate this, we generated peritoneal xenografts of the cell lines, in which TNF-α production had been reduced.
Knockdown of TNF-α influences the growth and distribution of ovarian cancer xenografts
The influence of TNF-α knockdown on ovarian cancer xenograft growth and spread was measured in several ways. First, we measured the in vivo growth of IGROV-1 cells that expressed luciferase and were transfected with shRNA constructs. We selected representative mock-transfected and TNF-α RNAi clones that showed similar in vitro luciferase activity (46,000 ± 6,000 and 47,000 ± 2,000 RLU per 1 × 104 cells, respectively). Cohorts of mice were investigated 14, 28, and 42 days after i.p. tumor cell injection. Both the tumor burden and the distribution were reduced in IGROV TNF-α RNAi cells compared with IGROV-Mock. Six weeks after IGROV-1 cell injection, IGROV-Mock cells were distributed widely in all areas of the peritoneum forming invasive masses in abdominal organs, but the IGROV RNAi TNF-α cells only localized to the peritoneal surface, spleen capsule, and uterine serosa (). Quantitation of luciferase activity confirmed these imaging results (; P < 0.0001).
Figure 4 Effects of TNF-α knockdown on growth of cells in vivo. A, a, representative bioluminescence imaging in vivo of IGROV-Mock and RNAi TNF-α IGROV 42 d after i.p. injection. Bioluminescence is presented as a pseudocolor scale: red, highest (more ...)
In a separate experiment, we weighed all dissectable peritoneal tumor 6 weeks after mice had been injected with IGROV-Mock and RNAi TNF-α IGROV cell lines (). The tumor burden was again significantly decreased when TNF-α production was reduced (P = 0.0033).
When organs from mice injected with mock or TNF-α RNAi cells were subjected to detailed histologic analysis 6 weeks after tumor cell injection, we found that knockdown of TNF-α significantly reduced the extent and invasiveness of tumor (). There were tumor deposits in diaphragm, liver, uterus/fallopian tube, and spleen in 80% mice bearing IGROV-Mock–transfected cells in contrast to deposits 62.5%, 25%, 12.5%, and 37.5%, respectively, in the RNAi TNF-α–bearing mice. The pancreas was involved in all mice bearing IGROV-Mock tumors but in only 25% RNAi TNF-α-bearing mice. Involvement of bowel serosa (colon), heart, and lungs as well as pleura and mediastinum (data not shown) was only seen in mice bearing IGROV-Mock cells. The distribution of tumor deposits of the IGROV-Mock cells closely resembled that seen for the original IGROV cells at the survival end point (). These differences at 6 weeks were also reflected in the times at which the mice had to be killed because they had reached ethical limits of abdominal swelling (20% increase in abdominal girth). Combining results from two separate experiments, injection of IGROV-Mock cells led to a median survival of 46 days (range, 39–62; n = 13) compared with 95 days (range, 54–272; n = 19) in mice bearing RNAi TNF-α I tumors and 210 days (range, 127–300; n = 13) in mice bearing RNAi TNF-α II tumors (P < 0.0001). TNF-α knockdown was sustained in vivo as measured by ELISA of tumor cell lysates at the survival end point. Lysates from IGROV-Mock tumors contained an average of 30 pg TNF-α/100 μg protein compared with 4 pg TNF-α/100 μg protein and 6 pg TNF-α/100 μg protein in lysates from RNAi TNF-α I and II tumors (P = 0.036 and 0.048, Mock compared with RNAi).
TNF-α knockdown influences tumor invasiveness
Tumors formed by IGROV-Mock cells were classified as invasive or invasive attached; none were well-circumscribed. In contrast, tumors derived from RNAi TNF-α cells were all classified as noninvasive and well-circumscribed tumors, although some were attached to the outer surface of abdominal organs. These differences in invasive patterns were statistically significant (P = 0.0038 comparing results from IGROV-Mock–injected mice to mice injected with RNAi TNF-α cells). Lymphovascular space invasion was present in all IGROV-Mock tumors but not in the RNAi TNF-α I and II tumors ().
There was no difference in tumor differentiation between the lines in terms of grade or papillary versus solid pattern. All tumors had abnormal mitotic activity.
However, clusters of apoptotic cells were prominent in all tumors developing from the RNAi TNF-α lines even when the tumors were very small. In IGROV-Mock tumors, apoptotic activity invariably was low and seen only in isolated single cells ().
TNF-α/CXCR4/CXCL12 and tumor dissemination
We have shown previously that knockdown of TNF-α in IGROV-1 cells results in down-regulation of CXCR4 (8
). Therefore, one explanation for the reduction in tumor growth and dissemination could be the concurrent down-regulation of CXCR4 and its ligand CXCL12 () in these RNAi TNF-α lines. Expression of this chemokine receptor:ligand pair is a feature of malignant ovarian surface epithelium and has been implicated in cancer growth and spread (15
). We therefore studied the ability of the cell lines to migrate to CXCL12. As shown in , TNF-α knockdown abolished the ability of the IGROV-1 cells to migrate to CXCL12. The level of migration of the IGROV-Mock cells was essentially the same as the parental line (data not shown).
TNF-α knockdown influences tumor angiogenesis
A further explanation for differences in apoptosis, tumor size, and tumor dissemination could be that the RNAi TNF-α lines were not efficient in stimulating neovascularization. VEGF knockdown alone could have a profound effect on generation of blood vessels. In addition, recent data indicate that CXCL12 is important in attracting hemangiocytes from the bone marrow (20
). We had noticed previously that angiogenesis was less in the SKOV-3-derived tumors than in IGROV-1-derived tumors, which suggested that TNF-α might be involved in the process. In addition, ascitic fluid never accumulated in the mice injected with TNF-α knockdown cells; however, it was a feature of animals injected with IGROV-1 parental and Mock-transfected cells. To investigate this further, we used confocal microscopy of peritoneal deposits to visualize and quantitate tumor blood vessels.
Silencing TNF-α in IGROV-1 cells markedly reduced tumor vascular area (). For each group, mean vascular area of five tumors of matched size, taken from two different experiments, was assessed. IGROV-Mock tumors had a mean vascular area of 18% (range, 6–25%) compared with 5% (range, 1–17%) and 6% (range, 5–8%) for tumors from TNF-α RNAi clones I and II, respectively (; P = 0.0027).
Figure 5 TNF-α knockdown influences tumor angiogenesis. In vivo angiogenesis was evaluated in mice 42 d after tumor cell injection. A, confocal images (magnification, ×20) of representative sections from (I) IGROV-Mock, (II) RNAi TNF-α (more ...)
To provide further proof that the network of soluble factors stimulated by TNF-α could stimulate tumor angiogenesis, we studied the influence of culture supernatants on endothelial cell growth in vitro. Culture supernatants from the original IGROV-1 cells and IGROV-Mock cells strongly stimulated the growth of primary mouse lung endothelial cells in vitro. In contrast, supernatants from the two clones of TNF-α RNAi cells had no such activity (; P = 0.024 on day 3 and P = 0.037 on day 4).