Research ArticleExperimental Studies

Tumor-associated Macrophages Facilitate Bladder Cancer Progression by Increasing Cell Growth, Migration, Invasion and Cytokine Expression

CHI-PING HUANG, LIAN-XIU LIU and CHIH-RONG SHYR
Anticancer Research May 2020, 40 (5) 2715-2724; DOI: https://doi.org/10.21873/anticanres.14243

Abstract

Background/Aim: Interactions between stromal and tumor cells in tumor microenvironment contribute to tumor progression. In bladder cancer (BCa), infiltration of macrophages in tumors correlates with cancer progression. Herein, the aim was to study the paracrine effects of tumor-associated macrophages (TAM) on BCa cells. Materials and Methods: The correlation between TAMs and tumor grade and stages was examined in tumor tissue microarrays. In addition, a conditioned media (CM) model was employed to investigate the paracrine effects of macrophages on BCa cell growth, migration, and invasion, as well as on the cytokine profile of each cell line. Results: The correlation of tumor-infiltrating macrophages with high-grade and muscle-invasive BCa was demonstrated in human bladder tumor tissue microarrays. CM from co-cultures of macrophages and BCa cells increased BCa cell growth, migration and invasion. Moreover, higher mRNA and protein expression levels of CCL5 and IL-8 were found in cells and CM from co-cultures, respectively. Conclusion: The paracrine interaction between BCa cells and TAMs led to enhanced BCa cell growth, migration, and invasiveness, and moreover, increased IL-8 and CCL5 cytokine production in tumor microenvironment.

Bladder cancer (BCa) is the 10th most common cancer type worldwide, with an estimated 549,000 new cases and 200,000 deaths in 2018 (1). Ninety percent of bladder tumors are of epithelial origin, arising in the epithelial mucosal lining of the urinary tract, the urothelium, which extends from the renal pelvis, bladder to the urethra bladder (2, 3). Bladder tumors are divided into two groups: non-muscle invasive and usually superficial tumors, which account for the large majority (~75-85%) and have a favorable prognosis; and invasive bladder cancer (~25%) (4, 5). BCa progression is strongly affected by the tumor microenvironment (TME). TME is composed of various cell types, such as cancer-associated fibroblasts and infiltrating immune cells, extracellular matrix, and soluble components as well, including secreted enzymes and cytokines. The interplay between TME and tumor cells plays a key-role in the regulation of cancer cell growth, migration, and invasion (6-8).

Macrophages are highly plastic cells; in response to various environmental cues, these cells are polarized into two different cell types: the classically activated (M1) or the alternative activated (M2) macrophages (9). Infiltrating tumor-associated macrophages (TAMs) in the TME have been shown to closely resemble the M2-polarized and regulate tumor growth, angiogenesis, invasion, and/or metastasis, in various cancer types. Moreover, TAMs suppress antitumor immune responses through releasing cytokines and chemokines such as IL-10 to block T cell activation (9-11). In thyroid and lung cancer patients, high density of TAMs has been associated with poor survival rates (12, 13). In addition, TAMs have been shown to be associated with cancer invasion, angiogenesis, and poor therapeutic outcomes in bladder cancer patients (14). However, the detailed mechanisms through which TAMs affect BCa progression remain elusive.

In the present study, the presence of TAMs positive for the macrophage marker CD68 (15) in bladder tumor tissues and their relationship with tumor grade and stage were explored. In addition, in order to clarify the involvement of infiltrating macrophages in BCa progression, the paracrine effects of macrophages on BCa cellular processes, as well as the changes in cytokine profile after BCa cell and macrophage co-culture were investigated in vitro.

Materials and Methods

Tissue microarrays (TMAs). TMAs were generated using bladder tumor tissue cores from a cohort of patients retrospectively recruited by searching the pathology database of China Medical University Hospital, Taichung, Taiwan. TMAs were prepared by using a semi-automated arraying device (TMArrayer™, Pathology Devices, Westminster, MD, USA). One to three tissue cores (2 mm each) of representative areas from each of the selected formalin-fixed, paraffin-embedded tissue blocks were used to construct the arrays. TAMs in tumor sections were detected by immunohistochemistry (IHC) using a rabbit anti-CD68 antibody (Clone GR021; 1:500) (Genemed, San Francisco, CA, USA) with the Leica Bond III autostainer (Leica microsystems, Mount Waverley, Victoria, Australia) according to the manufacturer's protocol. Slides were then counterstained with hematoxylin.

Cell cultures. Human BCa cell lines J82 and TCCSUP were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both BCa cell lines (J82 and TCCSUP) were cultured in DMEM medium (11965084; Gibco, Grand Island, NY, USA), containing 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (Thermo Fisher, Whaltam, MA, USA). The human acute monocytic leukemia cell line THP-1 (ATCC), was maintained in RPMI-1640 medium (11875127; Gibco) with 10% FBS and 1% penicillin/streptomycin. Cells were cultured at 37°C in an atmosphere of 5% CO2.

Co-cultures and conditioned media. For the mono-cultures, J82 or TCCSUP cells (106 cells) were cultured in DMEM with 10% FBS, on a 10-cm culture dish. For the co-cultures, THP-1 cells (106 cells) were co-cultured with J82 (106 cells) or TCCSUP cells (106 cells) in DMEM medium with 10% FBS, on a 10-cm culture dish. Conditioned media (CM) were collected from mono- and co-cultures; after 24 h of incubation, the collected media were centrifuged 10,000 rpm for 10 min and stored at −80°C for later use.

Colony formation assay. BCa (J82 or TCCSUP cells) cells were plated into 6-well tissue culture plates (104 cells/well) with CM collected from mono- or co-cultures. The plates were further cultured for 10 days in a humidified atmosphere containing 5% CO2 at 37°C. Afterwards, cell monolayers were washed with saline, fixed with 100% methanol, and stained with 0.5% crystal violet. Colonies with more than 50 cells were counted under a Nikon ECLIPSE 80i light microscope (Nikon, Tokyo, Japan).

Wound-healing migration assay. BCa cells were plated into 12-well plates (105 cell/well) and incubated in complete medium until 100% confluency was reached. Wounds were generated by scratching the cells with a pipette tip. Then the cells were washed twice with PBS and treated with CM from BCa cell mono-culture or BCa and THP-1 cell co-culture for another 24 h. After wound generation, images captured at 0 h and 24 h were used to determine cell migration. Wound closure was calculated by the formula: wound closure (%) =(wound at 0 h − wound at 24 h)/wound at 0 h. The relative migration was expressed as fold-change of wound closure over control BCa cells treated with CM from mono-culture.

Transwell invasion assay. In vitro cell invasion assay was performed using 24-well transwell inserts (8-μm pore size; Corning, Corning, NY, USA) coated with a three-dimensional extracellular matrix (Matrigel®, Corning). BCa cells were harvested and resuspended in 200 μl of serum-free medium. Cells (104 cells/well) were plated onto each filter, and 750 μl of CM collected from mono- or co-cultures was added into the lower compartment of invasion chambers. After 24 h, filters were washed with PBS, fixed in 4% paraformaldehyde, and stained with 1% crystal violet. Cells on the upper surface of the filters were removed with cotton swabs. Cells that invaded through the Matrigel®-coated inserts to the lower surface of the filter were counted. Invading cells in 6 random fields were quantified via photomicrograph at ×400 magnification, using a Nikon ECLIPSE 80i light microscope (Nikon, Tokyo, Japan). Results are presented as relative invasion expressed as fold-change over control BCa cells treated with CM from mono-culture (set as 1).

Cytokine array. CM collected from BCa cell and THP-1 cell mono-cultures or BCa and THP-1 cell co-cultures were analyzed by the Human Cytokine Array Panel A (R&D Systems, Minneapolis, MN, USA), which detects 36 human cytokines, chemokines, and acute phase proteins, according to the manufacturer's protocol.

RNA extraction and quantitative real-time PCR (qRT-PCR) analysis. THP-1 and BCa cells were co-cultured in a 6-well transwell plate (105 THP-1 cells in the upper well and 105 BCa cells in the bottom well) for 24 h with 0.45-μM filter, which blocked cell movement but allowed exchange of soluble factors (e.g. chemokines and cytokines) between two wells. THP-1 and BCa cells in mono-culture were also used as controls. Total RNA was extracted by the cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions from. Total RNA (1 μg) was subjected to reverse transcription using Superscript III transcriptase (Invitrogen, Carlsbad, CA, USA). The mRNA expression levels of CCL5 and IL-8 were measured by qRT-PCR that was conducted on a Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA) using KAPA SYBR® fast qPCR kit (Kapa Biosystems, Inc., Woburn, MA, USA) following manufacturer's instructions. Expression levels were normalized to the expression of the housekeeping gene β-actin. The primer sequences were as follows: β-actin, forward primer 5’-TCACCCACACTGTGCCCATCTACGA-3’, and reverse primer 5’-CAGCGGAACCGCTCATTGCCAATGG-3’; CCL5, forward primer 5’-TCTACACCAGTGGCAAGTGCT-3’; reverse primer 5’-TCCCGAACCCATTTCTTCTC-3’; and IL-8, forward primer 5’-TCTGCAGCTCTGTGTGAAGG-3’ and reverse primer 5’-TGAATTCTCAGCCCTCTTCAA-3’. The mRNA expression levels were determined using the 2−(ΔΔCt) method (16).

Enzyme-linked immunosorbent (ELISA) assay. To determine CCL5 and IL-8 protein levels in CM, the supernatants from BCa cell mono-cultures or co-culture of BCa and THP-1 cells in a transwell culture system were collected and analyzed by using the CCL5 and IL-8 ELISA LEGEND MAX™ kits (Biolegend, CA, USA), according to the manufacturer's protocol.

Statistical analysis. Values represent the means±SD for at least three independent experiments. Differences between experimental groups were determined using a two-tailed Student's t-test with SPSS ver. 13.0 (SPSS Inc., Chicago, IL, USA); a p-value <0.05 was considered statistically significant.

Figure 1.
Figure 1.

Infiltrating macrophages in tumors are associated with bladder cancer (BCa) progression. The expression of CD68-positive tumor-associated macrophages (TAMs) was evaluated by immunohistochemistry (IHC) on a tissue microarray (TMA) including 98 cases of primary BCa. (A) Representative images of low and high CD68 staining in bladder tumor tissues are shown. Scale bar, 200 μm. (B) CD68-positive cells were counted and correlated with tumor grade and stage. Results are presented in box plots, in which median is indicated as a horizontal line within the box and whiskers extending from the box represent the 2.5th and 97.5th percentile limit values. Dots represent outliers. **p<0.01, compared to low grade and Ta/1 groups, respectively.

Results

Tumor-infiltrating macrophages were increased in high grade and advanced stages. To study the macrophage-cancer cell interaction in TME and the role of TAMs in BCa progression, we first investigated the expression of the macrophage marker CD68 on BCa biopsies in a 98-patient TMA. The CD68-positive macrophages were mostly observed in tumor and tumor stromal areas (Figure 1A). Moreover, the relationship of tumor-infiltrating CD68-positive macrophage number with tumor grade and stage was investigated. The IHC staining results revealed that the number of CD68-positive macrophages was higher in high-grade tumor samples, compared to low grade samples (12.4/0.1 mm2 vs. 6.6/0.1 mm2, p<0.05). Furthermore, higher number of tumor-infiltrating macrophages was also observed in muscle-invasive T2-T4 tumors as compared to Ta/1 non-muscle invasive tumors (14.0/0.1 mm2 vs. 7.2/0.1 mm2, p<0.05) (Figure 1A and B).

CM from THP-1 cells and BCa cells co-culture enhanced BCa cell growth. The effects of the infiltrating macrophages on BCa cells were then examined using in vitro cell models with the THP-1, J82, and TCCSUP cell lines. The human THP-1 cell line, which is derived from a patient with acute monocytic leukemia is a monocyte-like cell line, resembles primary monocytes and macrophages in morphology and differentiation property (17). In addition, THP-1 cells have previously been characterized as M2-like type macrophages (18). J82 cell line was established from a poorly differentiated, invasive, transitional-cell carcinoma, stage T3 (19). TCCSUP cell line is derived from a poorly differentiated, invasive, and stage T3 transitional-cell carcinoma patient (20). To investigate the functional consequences of the interactions between macrophages and BCa cells, the latter were grown in CM from co-culture of THP-1 and BCa cells or BCa cell mono-culture. The cell growth capacity was evaluated using the colony formation assay. Compared to the CM from BCa cell mono-cultures, CM from co-cultures significantly increased the colony formation number both for J82 (1.3 folds, p<0.05) (Figure 2A) and TCCSUP cells (2.1 folds, p<0.05) (Figure 2B). These results indicate that the secreted factors from the reciprocal interplays between BCa cells and macrophages enhance the tumorigenesis and increase aggressive clinical manifestations of BCa.

Figure 2.
Figure 2.

Conditioned media (CM) from THP1 and BCa cell co-cultures increased BCa cell growth. The J82 and TCCSUP cell lines were treated with CM from J82 or TCCSUP mono- or co-cultures with THP-1 cell. After 7 days, colony formation by J82 or TCCSUP cells was evaluated. Charts show the relative quantification of the colony numbers of J82 (A) and TCCSUP cells (B). Results are presented as fold-change of normalized values relative to the controls (BCa cells treated with CM from BCa cell mono-culture) and are expressed as means±SD from three different experiments. *p<0.05 compared to controls.

Figure 3.
Figure 3.

Conditioned media (CM) from co-cultures of THP1 with bladder cancer (BCa) cells increased BCa cell migration. CM from BCa and THP-1 cell co-culture or BCa cell mono-culture was added in BCa cell culture; cell migration rate was examined by wound healing assay. Relative quantification of the migration capacity of J82 (A) and TCCSUP cells (B) from three different experiments is shown as the mean±SD. *p<0.05, **p<0.01, compared to controls. Scale bar, 200 μm.

The interplay between macrophages and BCa cells enhanced BCa cell migration. The wound-healing assay was used to examine whether BCa cell migration ability is affected by the reciprocal interplay between BCa cells and macrophages. BCa cells were treated for 24 h with CM from BCa cell mono-cultures or BCa and THP-1 cell co-cultures and wound closure was assessed. Increased migration was observed in both J82 (1.4 folds, p<0.05) (Figure 3A) and TCCSUP cells (1.3 folds, p<0.01) (Figure 3B) treated with co-culture CM, compared to those treated with CM from BCa cell mono-cultures.

The interplay between macrophages and BCa cells enhanced BCa cell invasion. The transwell matrigel analysis was used to investigate whether the soluble factors secreted in BCa cells and macrophage co-cultures could enhance BCa cell invasion. The results revealed that CM from THP-1 and J82 or TCCSUP cell co-cultures significantly enhanced invasion ability of both J82 (2.1 folds, p<0.05) (Figure 4A) and TCCSUP cell lines (1.7 folds, p<0.05) (Figure 4B), compared to CM from mono-culture.

Figure 4.
Figure 4.

Conditioned media (CM) from co-cultures of THP1 with BCa cells J82 or TCCSUP cells increased the invasion capacity of BCa cells. BCa cells were treated for 24 h with CM from J82 and THP-1 cell co-culture or BCa cell mono-culture on BCa cells. The effects of CM on BCa cell invasion were examined using the transwell invasion assay. Data are expressed as means±SD from three different experiments. *p<0.05, compared to controls. Scale bar, 100 μm.

The interplay between macrophages and BCa cells changed the immune cytokine profile. The cross-talk between tumor cells and infiltrating macrophages often involves immune cytokines or chemokines secreted from tumors cells or macrophages to affecting cancer progression (21). To identify the cytokines involved in the interaction between BCa cells and macrophages, we applied membrane-based cytokine array analysis (Figure 5A). The cytokine array revealed that CCL5 (J82: 5.3 folds, TCCSUP: 3.4 folds; Figure 5B) and IL-8 (J82: 1.9 folds, TCCSUP: 1.2 folds; Figure 5C) were upregulated in co-culture CM compared to the CM from the respective BCa cell mono-culture.

Co-culture of BCa cells with THP-1 cells increased CCL5 expression in the latter. To further validate the upregulated cytokines in CM from co-cultures and determine the major source of cytokines, we used a transwell culture system, in which THP-1 cells were cultured in the upper well and BCa cells in the lower wells for 48 h. The cells and CM were collected for further investigation. qRT-PCR was performed to determine the CCL5 mRNA level and ELISA assay for CCL5 protein level on the extracted mRNA and the CM, respectively. The results showed that CCL5 mRNA level was the highest in THP-1 cells when co-cultured with J82 (2.37-fold, p<0.01) or TCCSUP cells (1.97-fold, p<0.01) in the transwell system, compared to THP-1 cells in mono-culture. On the other hand, the levels of CCL5 mRNA in J82 and TCCSUP cells, either in mono-cultures or in co-cultures with macrophages, were very low (Figure 6A, B). In line with the mRNA results, the highest CCL5 protein levels were observed in the THP-1 cells in co-cultures with J82 (1.77-fold, p<0.01) or TCCSUP cells (1.54-fold, p<0.01), compared to THP-1 cell mono-cultures (Figure 6C, D). Thus, these results indicate that up-regulation of CCL5 in macrophages may contribute to the cross-talk between BCa cells and macrophages.

Figure 5.
Figure 5.

The cytokine profile was analyzed in conditioned medium (CM) from co-culture of BCa cells and THP-1 cells. The expression of 36 different cytokines, chemokines, and acute phase proteins was examined in CM from THP-1 and BCa cell mono-cultures or THP-1 and BCa cell co-cultures, by using a human cytokine array. Representative images are shown (A). Relative quantification of induced cytokines CCL5 (B) and IL-8 (C) compared co-culture CM to BCa cell mono-culture.

Increased IL-8 RNA and protein expression in BCa-1 cells after the co-culture with THP-1 cells. As described above for CCL5, the same procedure was followed for the investigation of IL-8 levels in THP-1 and BCa cell co-cultures, compared to BCa mono-cultures. The IL-8 mRNA level was significantly higher in both J82 (4.54-fold, p<0.05) and TCCSUP cells (2.81-fold, p<0.05) when co-cultured with macrophages, compared to the corresponding cells in mono-cultures. In macrophages, the levels of IL-8 protein remained low in both mono- and co-cultures (Figure 7A, B). The IL-8 cytokine protein levels were in consistence with the mRNA level results. Specifically, the highest IL-8 protein level was found in the J82 (1.20-fold, p<0.01, compared to J82 cell mono-culture) and the TCCSUP cells (1.40-fold, p<0.05, compared to TCCSUP cell mono-culture) co-cultured with macrophages (Figure 7C, D), while IL-8 protein levels were low in the macrophages from mono- or co-cultures. Therefore, similar to CCL5, up-regulation of IL-8 was shown to contribute to the cross-talk between BCa cells and macrophages. However, IL-8 was up-regulated in BCa cells and not in macrophages.

Figure 6.
Figure 6.

Increased CCL5 expression in THP-1 cells after co-culture with bladder cancer cells. qRT-PCR analysis of CCL5 mRNA level in THP-1 and J82 cells alone or in THP-1 and J82 cell co-culture (A). CCL5 mRNA level in THP-1 and TCCSUP alone or in THP-1 and TCCSUP co-culture (B). ELISA analysis of CCL5 protein level in THP-1 and J82 cells alone or in THP-1 and J82 co-culture (C). ELISA analysis of CCL5 protein level in THP-1 and TCCUP cells alone or in THP-1 and TCCSUP cells co-culture (D). *p<0.05, **p<0.01, compared to mono-culture controls.

Discussion

TME comprises recruited host stromal cells and immune cells which have been shown to play critical roles in tumor progression (22). Among these cells, TAMs are the prominent components of TME. The infiltrating macrophages into tumors as TAMs facilitate tumor initiation, progression, angiogenesis, drugs resistance and metastasis (10, 23-28). Furthermore, studies have demonstrated that high density of TAMs is correlated with poor prognosis such as gastric cancer, breast cancer, and thyroid cancer (29). Regarding BCa, TAM infiltration has also been shown to be significantly associated with poor survival (14); however, the mechanism via which infiltrating macrophages affect cancer cell behaviors is not specifically characterized. In this study, it was revealed that a higher number of tumor infiltrating macrophages was found in specimens from high-grade and advanced stage BCa, indicating that TAMs may affect cancer cell differentiation and capability toward malignant transformation. Furthermore, in vitro macrophage and BCa cell co-culture experiments demonstrated that paracrine interplay between macrophages and BCa cells resulted in increased colony formation, cell migration and invasion ability of BCa cells, as well as altered inflammatory cytokine profile in co-cultures. Our study demonstrates the critical roles of infiltrating macrophages in facilitating BCa cancer development and progression.

Figure 7.
Figure 7.

Increased IL-8 expression in bladder cancer cells after co-culture with THP-1 cells. qRT-PCR analysis of IL-8 mRNA level in THP-1 and J82 cells alone or in THP-1 and J82 cells co-culture (A). IL-8 mRNA level in THP-1 and TCCSUP alone or in THP-1 and TCCSUP co-culture (B). ELISA analysis of IL-8 protein level in THP-1 and J82 cells alone or in THP-1 and J82 co-culture (C). ELISA analysis of IL-8 protein level in THP-1 and TCCUP cells alone or in THP-1 and TCCSUP cells co-culture (D). *p<0.05, **p<0.01, compared to mono-culture controls.

Increased colony formation, cell migration, and invasion rates of BCa cells after treatment with CM from co-cultures with macrophages compared to treatment with CM from mono-cultures suggested that soluble factors secreted in the CM from co-cultures may have altered the characteristics of BCa cells inducing a more “cancer stem-line” phenotype (30). The role of TAMs in activation of cancer stem cells (CSC) has also been demonstrated in breast cancer by promoting CSC-like phenotypes through paracrine signaling pathway between macrophages and breast cancer cells (31). In addition, it has been found that TAMs create a CSC-niche via juxtacrine signaling with CSCs to turn on their cell-biological mesenchymal transition (EMT) program to increase breast cancer cell invasion and migration activities (32). Furthermore, macrophages co-cultured with breast cancer cell lines have led to enhanced invasiveness of the tumor cells in a TNF-alpha- and matrix metalloprotease-dependent manner (33). Therefore, the interplay between macrophages and BCa cells might be similar to those in breast cancer cells.

Since BCa cell- and macrophage-derived cytokines may be responsible for or contribute to the altered cancer cell behaviors related to BCa progression, we evaluated the cytokine profiles in the co-culture medium of macrophage and BCa cells. Two cytokines were identified to be upregulated in co-culture medium, suggesting that these two cytokines play a role in the interaction between BCa cells and macrophages. One of the identified key cytokines was CCL5, also known RANTES (regulated on activation, normal T cell expressed and secreted), which was found overexpressed in macrophages. In previous clinical observations, increased CCL5 in tissue or plasma has been shown to correlate with poor outcomes in breast, prostate, and cervical cancer patients (34-36). CCL5 secreted in TME has been shown to promote macrophage recruitment and influence the tumor cell migration and invasion (26, 37, 38). Blocking of CCL5 signaling pathway has been suggested as a potential therapeutic approach to effectively suppress the mesenchymal stem cell-induced metastasis and cancer cell growth, invasive and metastatic properties in mice (39, 40). Another identified inflammatory cytokine that was identified in the present study is IL-8, which was found overexpressed in BCa cells. IL-8 is a pro-inflammatory CXC chemokine; inappropriate or ectopic release of IL-8 by tumor cells recruits neutrophils to TME and promotes metastasis, assisted by neutrophil chemotaxis and degranulation (41). IL-8 has further been shown to be able to chemoattract myeloid-derived suppressor cells (MDSC), which negatively regulate immune response during tumor progression (42, 43). In preclinical models and in patients with cancer, higher IL-8 serum levels have been correlated with high tumor burden, advanced stage, low objective responses to therapy and poor survival in patients with melanoma, renal cell carcinoma, non–small cell lung cancer and hepatocellular carcinoma (42). In line with these literature reports, increased levels of these two cytokines were observed in co-cultures of TAMs with BCa cells, compared to mono-cultures.

Based on cancer tumor genome sequencing studies, no substantial genetic alterations have been identified to be accounted for the invasive and metastatic properties of cancer cells (44, 45). Thus, the malignant transformation of cancer cells may be driven by paracrine signals released from different stromal cell types within the TME. Dysregulated chemokines, cytokines, and growth factors released into the microenvironment by tumor cells themselves or activated stromal cells like infiltrating macrophages provide molecular guidance cues for cancer cell growth, migration and invasion (8). Collectively, considering these literature data and our findings, the increased number of infiltrating macrophages during BCa progression may reciprocally interact with BCa cells via secretion of cytokines, such as CCL5 and IL-8, to promote cancer cell growth, migration, and invasion. Therefore, infiltrating macrophages in the TME may potentially affect BCa progression. Thus, to improve therapeutic efficacy and prevent therapy resistance, chemotherapeutic agents should be developed to target not only cancer cells, but also the TAMs.

Acknowledgements

This work was supported by China Medical University (grant no. CMU107-S-40), CMUH grant (DMR-CELL-1807) and MOST grant (MOST 108-2320-B-039-012).

Footnotes

  • Authors' Contributions

    Conceptualization was performed by Chi-Ping Huang and Chih-Rong Shyr. Data curation was performed by Chi-Ping Huang, Lian-Xiu Liu and Chih-Rong Shyr. Chi-Ping Huang and Chih-Rong Shyr contributed to writing, reviewing, and editing of the manuscript.

  • Conflicts of Interest

    The Authors disclosed no conflicts of interest.

  • Received January 16, 2020.
  • Revision received March 29, 2020.
  • Accepted March 30, 2020.

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Tumor-associated Macrophages Facilitate Bladder Cancer Progression by Increasing Cell Growth, Migration, Invasion and Cytokine Expression
CHI-PING HUANG, LIAN-XIU LIU, CHIH-RONG SHYR
Anticancer Research May 2020, 40 (5) 2715-2724; DOI: 10.21873/anticanres.14243
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