Ovarian cancer (OC) is the fifth leading cause of cancer deaths among women in the US, with an estimated 22,240 new cases and 14,070 deaths in 2018 (1). OC alone accounts for 5% of cancer deaths and has frequently disseminated throughout the peritoneal cavity at diagnosis due to asymptomatic early-stage cancer (2). Five-year survival in women diagnosed with distant-stage OC is only 29% (3), urgently requiring a new and better therapeutic strategy. Epithelial OC occurs in 90% of OC, including the following subtypes: serous (68%–71%), endometrioid (9%–11%), clear cell (12%–13%), mucinous (3%), malignant Brenner tumors (1%), and mixed histologies (6%) (4). In particular, high-grade serous ovarian cancer (HGSOC) is highly-aggressive, is diagnosed at the advanced-stage, and has a poor prognosis compared to other subtypes (45). A frequent molecular alteration of HGSOC is closely associated with the genetic mutation in the tumor suppressor protein p53 (TP53) (6). We have shown that the frequent mutation of TP53 in OC enhances proinflammatory chemokines, leading to an inflammatory tumor microenvironment (7). The chemokine network appears to be involved in the progression, metastasis and survival of OC (8910).

Endotoxins are widespread ubiquitously in ambient environmental settings, including air, water, and food. Endotoxins are an integral component of the outer wall of Gram-negative bacteria, and the active component, LPS, is liberated when the cell walls of bacteria break downs (11). LPS, a TLR4 agonist, is a potent inflammatory agent that causes TLR4-mediated signaling, inducing acute and chronic effects, such as fever, chills, septic shock, and respiratory symptoms (11). Despite these adverse effects, epidemiologic, laboratory and limited clinical trial evidence suggest that endotoxins are likely to prevent cancer progression (12). Interestingly, monophosphoryl lipid A, a detoxified endotoxin derivative marketed in Europe for allergy treatment, and approved by the US Food and Drug Administration for use in humans as part of Cervarix^® (a vaccine against human papillomavirus 16 and 18; GlaxoSmithKline, London, UK) (13), reduces the risk of all site cancers (excluding skin) by 25% in both smokers and non-smokers, thereby appearing to be a chemopreventive agent (14). We demonstrated that LPS-induced proinflammatory chemokines in OC cells via TLR4-dependent NF-κB activation (15). Inflammation is a key contributor to OC cell seeding (16) and proinflammatory tumor microenvironment contributes to OC metastasis and chemoresistance (17).

Based on epidemiological studies revealing a decreased cancer risk, due to cumulative exposures of endotoxins and beneficial effects of LPS as a chemopreventive agent in many cancer types despite LPS-induced inflammatory reaction, we investigated how LPS could affect the murine peritoneal dissemination model of OC using SKOV-3 cells as described previously (9).

First, we evaluated epidemiologic links between endotoxins and OC through literature reviews. There were intense studies on relationship between exposure of endotoxins and lung cancer risk. Several occupational groups, including agricultural and cotton textile workers exposed to organic dusts contaminated by endotoxins, showed a reduced lung cancer risk (Fig. 1) (1214202122232425262728293031323334353637383940). On one hand, some reports on lung cancer indicated no change (41), while other reports indicated increased cancer risk (4243). Epidemiologic evidence in OC revealed that cumulative exposures to endotoxins in textile workers reduced the risk of OC (Fig. 1) (202144). Despite these limited data, endotoxins appear to reduce OC progression. We selected LPS, an active component of endotoxins, to clarify inhibitory effects of LPS on the progression of OC using the peritoneal dissemination model of SKOV3Luc OC cells as described (9). No differences in body weight between control and LPS-treated mice were noted (Fig. 2A). LPS-treated mice had significantly increased survival time of more than 116 days compared with 63 days in the control mice (Fig. 2B). Although detoxified Salmonella endotoxins had no effect on tumor growth, extracted fraction of cell-free Propionibacterium acnes, followed by detoxified endotoxins, resulted in long-term survival in a murine OC model (45). Further study requires clarifying whether anti-tumorigenicity between detoxified and toxified endotoxins is differential. Interestingly, there was no tumor burden in three mice out of eight LPS-treated mice, while all control mice showed peritoneal dissemination (Fig. 2C). No significant differences in spleen and tumor weight between control and LPS-treated mice were noted (Fig. 2D and E), which was expected at the terminal stage of OC. As CD-1 mice used in the present study are T cell-deficient, the antitumor effect of LPS is likely to involve directly an innate immune system rather than an adaptive immune system. Alternatively, addition of LPS as an adjuvant for dendritic cell immunotherapy of mouse ID8 OC had no survival benefit in C57BL/6 mice (46). These different results may be due to LPS-TLR4-dependent signaling in OC cells, as ID8 cells showed LPS-induced NF-κB activation, but not in SKOV-3 cells (15). In addition, differential immune response between CD-1 (defected adaptive immune system) and C57BL/6 mice (intact immune system) in OC may be responsible for causing the different LPS-responsive results. We analyzed chemokine signatures in human OC cell lines based on NCBI GEO dataset (GSE29175, GSE34615, GSE43765, and GSE53418). Chemokines, such as CXCL1, CXCL8, and CXCL16, highly expressed in a panel of 76 human OC cell lines (Fig. 3), indicating that proinflammatory tumor microenvironment may occur easily in OC.

We discovered controversial reports in SKOV-3 cells, showing TLR4-positive (47) and -negative expression (15). We investigated LPS-induced chemokine signature in SKOV-3 cells compared with TNFα-induced effects as a positive control. While LPS had no effects on the chemokine signature in SKOV-3 cells (Fig. 4A), TNFα induced CXCL1, CXCL2, CXCL3, and CXCL8 (Fig. 4A), which is consistent with our previous study (18). LPS did not induce NF-κB promoter activity, whereas TNFα induced the activity (Fig. 4B). Promoter activity of CXCR2, one of highly-responsive TNFα-induced chemokines (Fig. 4A), was not affected by LPS but was induced by TNFα (Fig. 4C). Furthermore, we confirmed that TNFα-induced mouse CXCL1 promoter activity was NF-κB-dependent in SKOV-3 cells. Promoter activity of the mouse CXCL1-containing intact NF-κB was induced by TNFα, but its mutant promoter had no effects (Fig. 4D). Differential effects between LPS and TNFα in the chemokine signature may be due to expression levels of their receptors in SKOV-3 cells. Further, we confirmed that SKOV-3 cells expressed low levels of TLR4 but high levels of TNFRSF1A (Fig. 4E), resulting in no LPS-induced, but TNFα-induced, effects on chemokine signature (Fig. 4A). These findings indicate that LPS-induced anti-tumorigenicity in OC cells may be indirect effects through innate immune cells rather than direct effects to OC cells.

As CD-1 mice used in the present study were T cell-deficient, we analyzed LPS-induced chemokine signature in an innate immune system rather than adaptive immune system. High expression of LPS-induced chemokine signature in an innate immune system showed as follows: CCL3, CCL4, and CXCL10 in dendritic cells (DCs); CCL3, CCL4, CCL20, CXCL1, CXCL2, CXCL3, CXCL8, and CXCL10 in macrophages (Mφ); and CCL3, CCL4, CXCL1, and CXCL8 in neutrophils (Fig. 5). These results indicate that LPS may increase proinflammatory chemokines in an innate immune system, appearing anti-tumorigenicity via LPS-enhanced activation of innate immune cells. These LPS-induced proinflammatory chemokines are primarily regulated by IL-1- and TNFα-activated NF-κB in OC (71518). Furthermore, we analyzed the LPS-induced TLR family and proinflammatory cytokine signatures in innate immune cells. DC, Mφ, and neutrophils expressed high levels of TNF, IL1A, and IL1B in response to LPS (Fig. 6A). Interestingly, IL6 was expressed highly in DCs and Mφ, but had low levels in neutrophils (Fig. 6A). These proinflammatory cytokines may be involved in antitumor effects of LPS, but not IFNG and PRF1 (Fig. 6A). As OC cells (Fig. 3), TNFα-treated SKOV-3 cells (Fig. 4A), and LPS-treated innate immune cells (Fig. 5) highly expressed CXCL1 and CXCL8, we investigated whether these chemokines could affect cell viability in SKOV-3 OC and THP-1 monocyte cells. Cell viability assay revealed that CXCL1 and CXCL8 had no effects on SKOV-3 cells but increased cell viability in THP-1 cells. CXCL8 antagonist (G31P) decreased cell viability, and LPS-induced CXCL8 protein, TNF, IL-1B, and IL6 mRNA in THP-1 cells (48), indicating the significant role of CXCL8 in THP-1 cell viability. LPS may activate an innate immune system by potentiating proinflammatory cytokines, such as TNFα, IL-1, and IL-6, which stimulate proinflammatory chemokines, such as CXCL1, CXCL2, and CXCL8, even in LPS-nonresponsive OC cells, and additionally increase CCL3, CCL4, CCL20, and CXCL10 in innate immune cells in an autocrine manner (Fig. 6C). Our current model indicates that the LPS-enhanced innate immunogenicity may exert anti-tumorigenicity to attenuate the peritoneal dissemination of OC in a cytotoxic-related proinflammatory tumor microenvironment.

The present study has several limitations. As CD-1 mice are T cell deficient, antitumor effects of LPS in this study may not reflect the intact immune system, including T cell-mediated immunity, but mainly the innate immune system. In addition, as SKOV-3 cells are human OC and LPS-nonresponsive, antitumor effects of LPS in mice may have different roles in LPS-responsive human OC cells and in human beings. LPS used in this study may activate the cytotoxic immune system to kill OC cells (i.e., M1-like Mφ dominant), which may be disrupted in long-term cancer situation with loss of cytotoxic immune activity (i.e., M2-like Mφ dominant). As LPS induces side effects, such as fever, chills, septic shock, and respiratory symptoms (11), safety issues in using LPS may be a concern, requiring alternative agents, such as detoxified endotoxin derivatives. Further studies and additional data will be warranted to overcome these limitations.

In conclusion, LPS is likely to induce proinflammatory chemokines and cytokines in the innate immune system to establish a cytotoxic-related tumor microenvironment, followed by attenuated peritoneal dissemination of OC, thereby leading to LPS-based therapeutic potentials for OC.