Intestinal dysbiosis promotes epithelial-mesenchymal transition by activating tumor-associated macrophages in ovarian cancer

ABSTRACT

We aimed to investigate the relationship of intestinal dysbiosis (IDB) and ovarian cancer progression, and understand its underlying signaling mechanisms. IDB was induced with high dose antibiotics. The xenograft mouse model was used to assess the tumor progression. Real-time polymerase chain reaction and immunoblotting are commonly used quantitative methods, and they were used to quantify epithelial-mesenchymal transition (EMT) markers in this paper. Meanwhile, cellular proliferation was also measured. First, IDB could promote the growth of xenograft tumors and induce the EMT. Serum levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 also increased remarkably. In addition, the production and secretion of TNF-α and IL-6 in macrophages isolated from IDB model mice were observably higher. In vitro, conditioned medium could significantly stimulate the development of EMT in ovarian cancer cells. Loss of macrophages completely offset the pro-tumor effects of IDB. IDB can stimulate the activation of tumor-associated macrophages in ovarian cancer, which is achieved by secreting pro-inflammatory cytokines IL-6 and TNF-α, and ultimately induces the development of EMT.

intestinal dysbiosis, tumor-associated macrophage, epithelial-mesenchymal transition, ovarian cancer, cytokines

INTRODUCTION

Ovarian cancer is one of the most commonly occurring gynecological tumors around the world, with high morbidity and mortality among women. It's estimated that 239 000 new cases were diagnosed and 161 000 deaths claimed by ovarian cancer in 2015 (Siegel, Miller and Jemal 2016). Epidemiological assessments have reported more cancer-related deaths in Europe and North America than in Asia and Africa. Risk factors correlated with ovarian cancer include over-ovulation, fertility medication, hormone usage and obesity (Jayson et al. 2014). Inherited genetic abnormalities also constitute approximately 10% of ovarian cancer cases, reporting mutations in BRCA1 and BRCA2 genes (Toss et al. 2015). Diagnosis of ovarian cancer in clinic is usually achieved with a tissue biopsy acquired during surgery. Nowadays, reliable early diagnostic methods for ovarian cancer are still lacking. At the same time, screening of ovarian cancer is also not favorable because there has been a high false-positive rate and little evidence supporting reduction in death (Menon, Griffin and Gentry-Maharaj 2014). Therefore, non-invasive and precise early diagnostic methods are still urgently needed.

Increasing evidence has demonstrated that intestinal microorganisms play a fundamental part in cancer biology (Van Raay and Allen-Vercoe 2017). New models have shown that intestinal dysbiosis (IDB) can stimulate immune response, and then causes local chronic inflammation, which has adverse effects and eventually leads to tumorigenesis. In order to clearly explain the interaction between microorganisms, immune response and malignant transformation, some hypotheses have been put forward. For example, Sears and Pardoll have proposed the alpha-bug hypothesis for colon cancer, which states that tumor is caused chiefly by microorganisms with specific toxicity determinants (Sears and Pardoll 2011). Tjalsma et al. proposed the hypothesis of driver passenger, that is, tumors caused by driver microorganisms may lead to fundamental changes in the local infectious microenvironment, which conversely leads to the loss of the sponsor through competition from other symbiotic bacteria (passengers) (Tjalsma et al. 2012). Although it is known that certain microbial infections are closely related to human cancers, the microorganism-related etiology of ovarian cancer is still under study.

On one hand, it's universally known that chronic inflammation can change the local conductive environment, and then cause the formation and development of tumors. Tumor-associated macrophages (TAMs) are a group of cells composed of many kinds of cells. These cells come from peri-tumoral tissues or bone marrows that can be divided into two main subtypes: M1 and M2. In the early stage of tumorigenesis, M1 and M2 play opposite roles, that is, M1 inhibits the progress of tumor, whereas M2 mainly promotes the growth of tumors (Noy and Pollard 2014; Cassetta and Kitamura 2018). High density infiltrating macrophages have been reported to exhibit a correlation with advanced tumor stages and adverse outcome (Biswas, Allavena and Mantovani 2013; Zhong, Chen and Yang 2018). Many cytokines and soluble factors produced and secreted by TAMs are involved in various biological processes, including cell proliferation, cell survival, angiogenesis, epithelial to mesenchymal transition (EMT) and cancer stem cell etiology, during the initiation and progression of tumor (Zhang et al. 2015). EMT is one of the basic steps that mediate local tumor cell invasion and metastasis, and it's often observed in patients with advanced malignant tumors and is highly correlated with tumor advancement and therapy tolerance. Only when cancer cells interact with other parts of the local microenvironment can EMT be activated. Therefore, it is particularly important to understand the signaling pathways connecting TAMs and EMT cascades, which may provide a valuable opportunity for the development of promising therapeutic drugs targeting cancer metastasis.

In this paper, the interactions between IDB and TAM activation during the development of ovarian cancer in xenograft mice were investigated systematically. The IDB mouse model was established after oral administration of antibiotics (Kanwal et al. 2018). And the clodronate-mediated depletion of TAMs further confirmed the dominant role of macrophages in the progression of tumor in IDB mice (Carron et al. 2017). Our study has, for the first time, demonstrated that IDB stimulates macrophages, resulting in increased circulating levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α, thus promoting the progression of ovarian cancer EMT.

MATERIALS AND METHODS

IDB mouse model

Eight-week-old female C57BL/6 mice were obtained from Vital River Laboratory Animal Technology (Beijing, China). For the accuracy of the results, they were fed adaptively for one week. A standard pathogen-free environment was used to house these animals. The Animal Use and Care Committee of Heze Municipal Hospital of Shandong Province approved this study. The IDB model was established based on the previously published method (Aguilera, Cerda-Cuellar and Martinez 2015). Briefly, ampicillin, vancomycin, neomycin and metronidazole were dissolved in drinking water and provided to the mice for four weeks, in specific doses as shown in Table 1. Considering the poor stability of antibiotics, they were freshly replaced in drinking water every day for the accuracy of the test. After four weeks, colonic feces were analyzed to determine whether the flora was completely depleted.

Table 1.

Dosage of antibiotics in drinking water.

Antibiotics	Dosage	Source
Ampicillin	1 g/L	Sigma, St. Louis, MO
Vancomycin	0.5 g/L	Abbott Labs, Abbott Labs
Neomycin	1 g/L	Pharmacia, Abbott Labs
Metronidazole	1 g/L	Sidmack Labs, Abbott Labs

Ovarian cancer cell lines

Ovarian cancer cell line SKOV3 was ordered from the America Type Culture Collection (ATCC, Manassas, VA, USA), which passed short tandem repeat analysis and was cultured under the conditions as suggested by the ATCC. RPMI medium was used to culture cells, containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were placed in 96-well plates and then cultured at 37°C in a humidified incubator containing 5% CO₂. Cells with exponential growth were collected and subsequently analyzed.

Xenograft mouse model

When SKOV3 cells grew logarithmically, they were digested with trypsin, then the cells were placed on ice and treated with HEPES buffer to prepare single cell suspension. Cells were mixed in suspension with isovolumetric matrix glue (BD BioSciences, Franklin Lakes, NJ, USA). Next, 5 × 10⁶ cells/200 μL of the resulting mixture was hypodermically injected into the lower abdomen of mice. The size of xenograft tumors was surveyed regularly, using the formula: volume = (width)² × length/2 to calculate the size of xenograft tumors.

Real-time polymerase chain reaction (PCR)

Trizol reagent was used to extract RNA from cells. In order to ensure the accuracy of subsequent experiments, the quantity and purity of isolated RNAs were detected with a Bioanalyzer (Agilent, Santa Clara, CA, USA). This experiment used the cDNA reverse transcription kit (ThermoFisher, Waltham, MA, USA) for reverse transcription. The PCR reaction was conducted with the PowerUp SYBR Green Master Mix (ThermoFisher). The 2^−∆∆C method was used for calculating relative expression and normalizing gene expression to GAPDH. Primers used in this study were designed according to the sequence of human genes, which are all listed in Table 2.

Table 2.

List of primers used in this study.

E-cadherin, forward	5΄-GTCGAGGGAAAAATAGGCTG-3΄
E-cadherin, reverse	5΄-GCCGAGAGCTACACGTTCAC-3΄
N-cadherin, forward	5΄-GGCATACACCATGCCATCTT-3΄
N-cadherin, reverse	5΄-GTGCATGAAGGACAGCCTCT-3΄
Vimentin, forward	5΄-GCAAAGATTCCACTTTGCGT-3΄
Vimentin, reverse	5΄-GAAATTGCAGGAGGAGATGC-3΄
GAPDH, forward	5΄-GGAGCGAGATCCCTCCAAAAT-3΄
GAPDH, reverse	5΄-GGCTGTTGTCATACTTCTCATGG-3΄

Western blot

Radioimmunoprecipitation assay buffer was used to lyse the cells. Then the cell debris was removed by freeze centrifugation. The concentration of proteins was then quantified by measuring with a BCA kit (ThermoFisher). The same quantity of proteins obtained was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transferring onto polyvinylidene difluoride (PVDF) membranes under the condition of an ice bath. This was followed by a blocking operation by Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% skim milk at 4°C overnight to hybridize respective primary antibodies from Cell Signaling Technology: anti-human-E-cadherin, 1:1000, #14 472; anti-human-Vimentin, 1:1000, #3390; anti-human-N-cadherin, 1:1000, #14 215; anti-mouse-TNF-α, 1:1000, #11 948; anti-mouse-IL-6, 1:1000, #12 912; anti-GAPDH, 1:1000, #2118. The next day, PVDF membrane was washed several times with TBST to remove the unbounded antibodies and was incubated at 25°C for 1 h in a solution containing specific secondary antibodies: anti-mouse IgG, HRP-linked antibody, 1:5000; anti-rabbit IgG, HRP-linked antibody, 1:5000. Finally, for ease of observation, the membrane was washed using TBST for 30 min, and protein bands were developed with an ECL kit (Millipore, Billerica, MA, USA). GAPDH protein was used as control for equal loading.

Enzyme-linked immunosorbent assay (ELISA)

Blood samples of experimental mice were acquired and centrifuged to prepare serum. Levels of IL-6 and TNF-α in serum were measured by ELISA kits (ab46042 and ab46042, respectively; Abcam, MA, USA). Briefly, 50 μL biotinylated antibody solution and 100 μL sample were added into each well and incubated at 25°C for 3 h. After washing, 100 μL streptavidin-horseradish peroxidase solution was added to all the wells, and the plates were incubated at room temperature for 30 min. Then 100 μL TMB solution was added to the reaction system at room temperature and placed in darkness for 15 min. After removal, 100 μL stop reagent was added to the reaction system. Spectra Max Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used to determine the absorbance of the solution at 450 nm.

Mouse macrophage isolation

The C57BL/6 mice were cervically dislocated, followed by intraperitoneal instillation of 7 ml sterile ice-cold phosphate-buffered saline (PBS). Gentle massage with fingers was performed on the abdomen of mice for 3 min to draw and absorb ascites. Ascites were centrifuged to collect macrophages, and washed twice with Durbeco's Modified Eagle medium containing 10% FBS, followed by standard cell culture. Flow cytometry was used to examine each isolated cell population to confirm that before the experiment macrophages were the dominant group.

Proliferation assay

Cell counting kit-8 (CCK-8; Dojindo, Dalian, China) was used to measure cell proliferation. The exponentially grown cells were seeded into 96-well plates, and incubated in a humid CO₂ thermostat for 24 h. Then 10 μL CCK-8 solution was applied into all wells and then incubated for 1 h at 37°C. The absorbance was measured at 450 nm by enzyme labeling and the viability of cells was computed.

Mouse macrophage depletion

SKOV3 xenografts were established in control and IDB mice according to the previous method (Carron et al. 2017). Two weeks after successful establishment, the tumor-bearing IDB mice were randomly divided into two groups. One group received 100 μL chloro-phosphonate liposome solution (5 mg/10 g body weight, chloroform LIP B.V.) intravenously every 3 d, and the other group received normal saline with the same method. In the liver, bone marrow, spleen and precursor blood mononuclear cells, there was successful depletion of macrophages. In order to know the incubation period of tumor clearly, a digital calliper was used every other day to measure the size of tumor.

Statistical analysis

All experiments were conducted with at least three independent biological repeats. Data were processed and analyzed by SPSS software version 23.0 (SPSS, Chicago, IL). One-way ANOVA was adopted for statistical comparison among different groups. P-value < 0.05 was considered to be statistically different.

RESULTS

IDB promotes ovarian xenograft tumor growth

First of all, mixed antibiotics were freshly supplied in drinking water on a daily basis to induce IDB in the mice so that their intestinal flora was completely depleted (Fig. 1). To investigate whether IDB was associated with tumor progression, we established SKOV3 xenograft tumor in the IDB mouse model. The tumor size was surveyed until 3 wk post-inoculation. The xenograft tumor in IDB mice was evidently larger than in control (Fig. 2A). In addition, the mice were sacrificed and the weight of the tumor was measured at end point. The results showed that the tumor weight in the IDB group increased remarkably compared with the control group (Fig. 2B). Our outcome suggested that IDB promoted tumor progression in vivo.

Figure 1.

Intestinal flora is depleted in the intestinal dysbiosis (IBD) mouse model. Colonic fecal matter from both control and IDB mice was cultured aerobically and anaerobically, and commensal bacteria were identified and quantified using biochemical analysis, morphologic appearance and Gram-staining. Bacterial counts in the IDB mouse group were too small to appear on the bar graph.

Figure 2.

Intestinal dysbiosis (IDB) promotes the growth of xenograft tumor from inoculated ovarian cancer cells. (A) Ovarian cancer cell SKOV3 was inoculated into control and IDB mice, respectively (n = 12), followed by measurements of tumor size on indicated days after cell inoculation. (B) On day 21, mice from both groups were sacrificed to extract the xenograft and weigh the tumor. Data are shown as mean ± SD (n = 12 each). * P < 0.05, compared with control.

IDB promotes EMT in xenograft ovarian tumors

The outcomes observed above showed that IDB could remarkably accelerate the growth of xenograft tumors, and next we aimed to search for changes at the molecular level to explain this phenomenon. Tumor tissue specimens from two groups of mice were collected and specific EMT markers were determined. E-cadherin, an epithelial marker, was observably lower (Fig. 3A), while mesenchymal markers Vimentin and N-cadherin were increased significantly (Fig. 3B). Western blotting further validated our speculations at the protein level. Accordingly, the protein level of E-cadherin was lowered, while the results of N-cadherin and Vimentin were opposite (Fig. 3C). Our experimental data suggested that EMT was evidently induced in IDB mice.

Figure 3.

Intestinal dysbiosis (IBD) promotes epithelial-mesenchymal transition in xenograft tumor from inoculated ovarian cancer cells. Ovarian cancer cell SKOV3 was inoculated into control and IDB mice, respectively (n = 12). On day 21, mice from both groups were sacrificed to extract the xenograft tumor, followed by analyses of (A) E-cadherin mRNA, (B) N-cadherin and Vimentin mRNA, and (C) their protein levels (numbers below bands indicate relative level to loading control). Data are shown as mean ± SD (n = 12 each). ** P < 0.01, compared with control.

IDB increases serum levels of TNF-α and IL-6

We also made efforts to observe and evaluate the immune response in the IDB model mice. At the end point, the serum of mice in every group was collected. The relative serum levels of TNF-α and IL-6 were detected, which were significantly higher in IDB mice (Fig. 4A, B). These results suggested that the peripheral blood of IDB mice with xenograft tumors exhibited obvious inflammatory reaction.

Figure 4.

Intestinal dysbiosis (IDB) increases serum concentrations of cytokines IL-6 and TNF-α in experimental mice. Serum was collected from both control and IDB mice, respectively, followed by analyses of IL-6 and TNF-α concentrations by ELISA. Data are shown as mean ± SD (n = 12 each). * P < 0.05, ** P < 0.01, compared with control.

IDB increases production of TNF-α and IL-6 in mouse macrophages

Subsequently, we tried to evaluate secreted TNF-α and IL-6 in the IDB mouse model. Firstly, macrophages were isolated from experimental mice by intraperitoneal perfusion with PBS solution. The obtained macrophages were cultured in vitro, and the conditioned media were collected, followed by analysis of TNF-α and IL-6 by Western blot (Fig. 5A) and ELISA (Fig. 5B), respectively. The results showed that the levels of TNF-α and IL-6 in conditioned media of IDB mouse macrophages were significantly higher than those in the control group, which was consistent with those results observed in peripheral blood. The above outcome manifested that macrophages from IDB mice were more likely to promote the production of inflammatory cytokines such as TNF-α and IL-6.

Figure 5.

Intestinal dysbiosis (IDB) increases expression and secretion of cytokines IL-6 and TNF-α in the macrophages isolated from experimental mice. Macrophages were isolated from both control and IDB mice, respectively, followed by analyses of (A) IL-6 and TNF-α protein expressions by Western blot (numbers below bands indicates relative level to loading control) and (B) their secretions into the media by ELISA. Data are shown as mean ± SD from at least three independent experiments. * P < 0.05, ** P < 0.01, compared with control.

Conditioned medium from isolated IDB mouse macrophages promotes proliferation and EMT of SKOV3 cells invitro

We next conducted a series of experiments to further explore the potential effects of macrophages isolated from IDB on SKOV3 malignant behavior. Conditioned medium was applied to in vitro SKOV3 cell culture, and cell proliferation and EMT were assessed after a period of time. Compared with the control group, conditioned medium could prominently stimulate cell viability (Fig. 6A). In addition, consistent with our in vivo observations, E-cadherin, an epithelial marker, was inhibited, whereas Vimentin and N-cadherin, two mesenchymal markers, were elevated (Fig. 6B). The above outcomes manifested that the cytokines secreted by macrophages isolated from IDB mice were remarkably conducive to EMT and proliferation of ovarian cancer cells.

Figure 6.

Conditional medium from macrophages isolated from intestinal dysbiosis (IDB) mice promotes proliferation and epithelial-mesenchymal transition of SKOV3 cells in vitro. Macrophages were isolated from both control and IDB mice, and cultured in vitro to collect their respective conditional media. SKOV3 cells were then cultured with these two types of collected conditional media, respectively, followed by assessments of (A) proliferation and (B) protein levels of E-cadherin, N-cadherin and Vimentin (numbers below bands indicate relative level to loading control). Data are shown as mean ± SD from at least three independent experiments. ** P < 0.01, compared with control.

Macrophages are required for the growth promotion of xenograft tumors by IDB

We attempted to evaluate the effect of macrophages isolated from IDB in the growth of tumor in vivo. In order to achieve this goal, we gave drinking water containing large doses of combination antibiotics to mice, so that the macrophages of SKOV3 tumor-bearing mice were completely depleted. The tumor size was surveyed and compared among control, IDB mice and IDB + macrophage-depletion mice. Xenograft tumor in IDB mice progressed significantly faster than those in the control group, but this situation was restored by depletion of macrophages (Fig. 7A). Similar results were acquired in terms of tumor weight at end point. Compared with the normal group, the weight of xenograft tumor was increased in IDB mice and decreased after macrophage depletion in IDB mice (Fig. 7B). The above data authenticated the requisite function of macrophages in promoting in vivo xenograft growth in IDB mice.

Figure 7.

Growth promotion of xenograft tumor by intestinal dysbiosis (IDB) requires macrophages. (A) Ovarian cancer cell SKOV3 was inoculated into control + sham, IDB + sham and IDB-macrophage mice, respectively (n = 12), followed by measurements of tumor size on indicated days after cell inoculation. (B) On day 21, mice from all groups were sacrificed to extract the xenograft and weigh the tumor. Data are shown as mean ± SD (n = 12 each). * P < 0.05, compared with control + sham. # P < 0.05, compared with IDB + sham.

Macrophages are necessary for EMT of xenograft tumor promoted by IDB

Our previous research data have confirmed that the promotional role of IDB in ovarian cancer requires the involvement of macrophages. Next, we aimed to find out its effects on EMT-related molecules, which is fundamentally related to the progress of malignant tumors. The expressions of E-cadherin, Vimentin and N-cadherin were checked at transcriptional and protein levels, respectively. E-cadherin transcript was evidently reduced in the IDB mice compared with the control, and then recovered upon macrophage depletion (Fig. 8A). The results of Vimentin and N-cadherin were opposite to those of E-cadherin: in the IDB group, both of them were induced and increased, whereas when macrophages were deficient, they were inhibited (Fig. 8B). Western blotting further validated the above changes (Fig. 8C).

Figure 8.

EMT promotion of xenograft tumor by intestinal dysbiosis (IDB) requires macrophages. Ovarian cancer cell SKOV3 was inoculated into control + sham, IDB + sham and IDB-macrophage mice, respectively (n = 12). On day 21, mice from all groups were sacrificed to extract the xenograft tumor, followed by analyses of (A) E-cadherin mRNA, (B) N-cadherin and Vimentin mRNA, and (C) their protein levels (numbers below bands indicate relative level to loading control). Data are shown as mean ± SD (n = 12 each). * P < 0.05, ** P < 0.01, compared with control + sham. ## P < 0.01, # P < 0.05, compared with IDB + sham.

DISCUSSION

IDB is a risk factor correlated to a great deal of human illnesses, including inflammatory bowel disease, periodontal disease, chronic fatigue syndrome, bacterial vaginosis, obesity, colitis and cancers (Petersen and Round 2014). Repeated and inappropriate use of antibiotics, alcohol abuse and inappropriate diet can lead to malnutrition (Butto and Haller 2016). Owing to the complex microenvironment and symbiotic microorganism relationship of ovarian cancer, this study focused on the etiology of ovarian cancer. Drinking water with excessive antibiotic mixtures was given to mice. Our experiments not only demonstrated that IDB could significantly promote the development of xenograft ovarian tumor, but also stimulate EMT. After the establishment of IDB, the peripheral serum levels of IL-6 and TNF-α were observably increased. The amounts of IL-6 and TNF-α produced and secreted by macrophages in the model group were evidently elevated compared with the normal control group. Furthermore, with a co-culture system, our results indicated that conditioned medium markedly stimulated EMT in SKOV3 ovarian cancer cells. In our macrophage-depletion experiment, macrophages played a unique role in the preneoplastic effect of IDB, in which growth and EMT processing of xenograft tumors were nearly eliminated.

EMT is an extremely complex process, where epithelial cells first lose cell polarity and cell-to-cell attachments, then migrate and have the ability to invade other cells, eventually transforming into mesenchymal stem cells (Kalluri and Weinberg 2009). EMT is an important physiological process, which is indispensable in the formation of mesoderm and neural tube, and has proven to be implicated in organ fibrosis, wound healing and the inception of tumor metastasis (Micalizzi, Farabaugh and Ford 2010). The depletion of E-cadherin is a basic change in EMT, after which expressions of N-cadherin, Vimentin and other mesenchymal markers are initiated. Recent studies have shown that cancer-associated inflammation is characterized by stimulation of EMT. For example, the inflammatory cytokine TNF-α could induce migration and EMT in head and neck squamous cell carcinoma cells (Liu et al. 2018). Zhang et al. conducted a series of experiments and found that EMT induced by TNF-α could increase renal cell carcinoma cell stemness (Zhang et al. 2014). In terms of IL-6, Rokavec et al. attested that the feedback loop of IL-6/STAT3/microRNA-34a promoted invasion and metastasis of colon cancer, which was mediated by EMT (Rokavec et al. 2014). Castellana et al. confirmed YB-1 and IL-6 may interact and further promote the metastatic phenotypes of breast cancer (Castellana et al. 2015). Lee et al. also certified IL-6 advanced EMT of CD133 positive cells in non-small cell lung cancer (Lee et al. 2016). Chen et al. confirmed that Helicobacter pylori TNF-α-inducible protein could induce EMT in gastric cancer cells, which was mainly achieved by activating the IL-6/STAT3 signaling cascade (Chen et al. 2017).

Consistent with these previous results, our experiments confirmed that the elevated levels of IL-6 and TNF-α in the IDB mouse model of xenograft ovarian tumor were positively associated with the EMT process, and conditioned medium containing these two cytokines observably provoked the EMT phenotype of SKOV3 cells in vitro. It is worth noting that the specific molecular signal transduction pathway of EMT markers regulated by IL-6/TNF-α is still unclear, and which needs further research to confirm.

TAMs play irreplaceable roles in the initiation, development and metastasis as well as therapeutic resistance of tumor biology. Precise TAM depletion presents a trending approach for therapeutic exploitations. For instance, Wu et al. showed that the reduction of M2-like TAMs could delay further in vivo development of cutaneous T-cell lymphoma (Wu et al. 2014). Patwardhan et al. thought that PLX3397 and rapamycin could continuously inhibit the loss of receptor tyrosine kinase and macrophages, which would be a potential new method for the remedy of MPNST (Patwardhan et al. 2014). By establishing a spontaneous mouse model of melanoma, Tam et al. reported macrophage depletion could effectively reduce the post-operative recurrence and metastasis of the tumor (Tham et al. 2015). Zhang et al. suggested that the TAM depletion improved the role of sorafenib in metastasis of hepatocellular carcinoma through its anti-metastasis and anti-angiogenesis properties (Zhang et al. 2010). Furthermore, results also showed that TAM depletion improved anti-tumor immunity induced by toll-like receptor agonist binding peptides (Shen et al. 2014). Shashkova et al. affirmed that the consumption of macrophages increased the treatment window of systemic therapy for oncolytic adenovirus by combining with anti-coagulant therapy (Shashkova et al. 2008). Our results are consistent with other studies, in which clodronate and liposome-mediated depletion of macrophages significantly repressed tumor progression and EMT in the IDB xenograft mouse model. Considering the abnormal activation of TAMs in IDB mice, our data highlight the effectiveness of immunotherapy based on TAM depletion for IDB. However, it should be noted that in order to get rid of the potential effects of host transplant rejection, we will further consolidate the main observations in immunodeficiency animal models in the future. Furthermore, metronidazole was suggested to cause immuno-suppression in BALB/C mice, resulting in reduced macrophages and decreased TNF-α concentration (Fararjeh et al. 2008), hinting that the immune responses may be different due to the different genetic backgrounds of murine strains, as mentioned above (Sellers et al. 2012). As a result, experiments using different mouse strains and different methods of inducing IDB are needed to demonstrate the universality of our outcomes.

CONCLUSIONS

In summary, through the experiments described above, we have reached the conclusion that IDB can significantly stimulate the activity of macrophages, which conversely promotes the secretion of TNF-α and IL-6. The increased levels of these cytokines in the peripheral blood can furtherance the EMT process of ovarian cancer, and the result is to promote the development and metastasis of ovarian cancer. This investigation emphasizes the vital function of macrophages and cytokines in ovarian tumor progression in the IDB mouse model, which represents a probable route for intervention and therapeutic exploitations by employing drugs that could potentially antagonize IDB symptoms.

ACKNOWLEDGEMENTS

Not applicable.

FUNDING

None.

Conflict of interest. None declared.

Author notes

These authors contributed equally to this work