The role of tumor-associated macrophages in the radioresistance of esophageal cancer cells via regulation of the VEGF-mediated angiogenic pathway

Tumor-associated macrophages (TAMs) are known to promote tumor growth, invasion, metastasis, and protumor angiogenesis, but the role of TAMs in the radiotherapy of esophagus cancer remains unclear. In this study, we �rst induced TAMs from human monocytes (THP-1) and identi�ed using immuno�uorescence and Western blotting assays. We then co-cultured them with human esophageal cancer cell lines. CCK-8, colony formation, Transwell, scratch test, and TUNEL assays showed that TAMs could promote proliferation, survival rate, invasion, migration, and radio-resistance and could inhibit apoptosis of the esophageal squamous carcinoma cell lines KYSE-150 and TE-1 before and after radiotherapy both in vivo and in vitro. Using LV-VEGFA-RNAi lentiviral vectors, we also found that TAMs could increase the expression of VEGFA and that inhibition of VEGFA could inhibit the biological function caused by TAMs. Finally, a Western blotting assay was used to evaluate the expression of various factors underlying the mechanism of TAMs. VEGFA, MAPK, P-MAPK, BCL-2, and Snail proteins were found to be overexpressed in co-cultured groups, whereas after VEGFA inhibition, MAPK, P-MAPK, BCL-2, and Snail proteins were found to be signi�cantly down-regulated in the radiotherapy group. These study results offer important information regarding the mechanism of radio-resistance in esophageal cancer.


Introduction
Esophageal carcinoma is the eighth leading cause of morbidity and the sixth leading cause of mortality worldwide (1).There are two main histological types of esophageal carcinoma: Adenocarcinoma and squamous cell carcinoma are main histological types of esophageal carcinoma (2).Because the symptoms of esophageal carcinoma are non-speci c, most cases are diagnosed late in the disease process, leading to a poor prognosis.Radiotherapy is one of the main treatments for esophageal cancer; however, owing to distant metastatic and local recurrence, the 5-year survival rate after radiotherapy is only 10-30%.Radio-resistance is a key factor affecting the e cacy of this treatment (3).
Recent studies have shown that tumor-associated microphages (TAMs), which are present in various tumor types, are in uenced by the tumor microenvironment (TME).TAMs regulate various processes during tumor development and progression, such as metastasis, proliferation, immunosuppression, and angiogenesis (4)(5)(6)(7).TAMs are present in the TME, which is mainly derived from monocytes of hemopoietic stem cells and yolk sac tissue from the embryonic period (8, 9).TAMs are classi ed as M1type or M2-type macrophages depending on the state they activate, the functions they perform, and the factors they secrete.In the TME, they are characterized by strong plasticity, and interconversion between M1 and M2 types will occur when the TME is locally changed.M2-type macrophages have been shown to be involved in tumor cell invasion, metastasis, and promotion of angiogenesis (10)(11)(12)(13).
Vascular endothelial growth factor (VEGF) is a powerful factor that promotes the growth of blood vessels and is involved in the development and spread of tumors.Research has shown that VEGFmediated angiogenesis plays an important role in most tumors (14), with one study demonstrating that the risk of death was increased by 1.82 in patients with esophageal cancer with high VEGF expression (2).In previous research, we also found that a high expression level of VEGF is closely related to radioresistance and poor prognosis in cases of esophageal cancer (15,16).
Although the conventional view is that macrophages are intrinsic immune cells whose role is to promote in ammatory responses and thus inhibit tumor growth, there is increasing experimental and clinical evidence that TAMs can promote tumor growth, invasion, metastasis, and radiation resistance through VEGF-stimulated angiogenesis and that the malignancy of tumors is positively correlated with the level of TAMs (17,18).In the present study, we aimed to observe the effect of TAMs on the radio-resistance of esophageal cancer cells and to investigate the interaction between VEGF and TAMs after radiotherapy for esophageal cancer.

Cell culture
The human esophageal squamous cancer cell lines: KYSE-150 was procured from the Shanghai Hongshun Biotechnology Co. LTD, TE-1 was procured from Gene Co., and human mononuclear leukemia cell THP-1 was procured from the Geman Biotechnology Center.The cell lines were grown in RPMI1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scienti c) and 1% penicillin-streptomycin and were cultured in a humidi ed atmosphere at 37°C and 5% CO 2 .

TAM induction
Once THP-1 cells reached the logarithmic growth phase, they were induced with PMA (80 ng/L) for 48 hours.THP-1 cells were then differentiated into macrophages, and the suspension-grown cells were transformed into adherent cells under microscopy.The adherent macrophages were induced with IL-4 (20 ng/mL) for 48 hours.The macrophages were then differentiated into M2-type TAMs.

Construction of lentiviral vectors
To establish stable VEGFA knockdown esophageal cancer cells, we constructed a lentiviral-based siRNA targeting VEGFA vector.The primers were designed by Gene Co., which contained 2 restriction sites, AgeI and EcoRI (siRNA F:ccgggaGCTTCCTACAGCACAACAAttcaagagaTTGTTGTGCTGTAGGAAGCtctttttg, R: aattcaaaaagaGCTTCCTACAGCACAACAAtctcttgaaTTGTTGTGCTGTAGGAAGCtc).The primers were designed to anneal and form double-stranded DNA.The sequences were cloned into the GV493 vector based on the manufacturer's instructions, and the viruses were then packaged using 293 T cells according to standard protocol.

Lentivirus transfection and stable cell screening
The validated VEGFA knockdown lentiviral vector and empty vector were co-transfected into KYSE-150 and TE-1 cells.After screening with puromycin for 2 weeks, VEGFA knockdown cells were veri ed in RNA and protein level.Stably VEGFA knockdown cells were selected for subsequent experiments.
Establishment of in vitro co-culture system and irradiation treatment The KYSE-150 and TE-1 cell lines were cultured and maintained in the lower Transwell (Corning) cell coculture chambers (3×10 5 cells/mL) in RPMI1640 medium with 10% FBS.The induced differentiated TAMs were inoculated in the upper Transwell cell co-culture chambers (6×10 5 cells/mL).After noncontact co-culture at 37°C and 5% CO 2 for 48 hours, the cells were treated with irradiation at a dose of 4 Gy.Subsequent experiments were performed 24 hours later.

Immuno uorescence
Cells (THP-1, M0 macrophages, and M2-type macrophages) were grown on 35-mm glass-bottom culture dishes (NEST; laser confocal special) until the experimental endpoint.Then, cells were xed with 4% paraformaldehyde and washed 3 times with PBS, and afterwards probed with primary antibody against CD68 (ab213363) and CD163 (ab87099) at 4℃ overnight.Secondary antibody conjugated with uorescence (Invitrogen) were then incubated with the cells for 1 hour.After washing with PBS, cells were stained by DAPI (Life Technology) for 15 minutes at room temperature.Finally, the stained cells were photographed using an inverted uorescence microscope.

CCK-8 assay
A CCK-8 assay was applied to assess cell proliferation of esophageal cancer cells.Cells were seeded in a 96-well plate at a density of 2,000 cells/well and were incubated at 37°C and 5% CO 2 .After 24, 48, 72, and 96 hours, the culture medium was discarded and 10% CCK-8 serum-free medium was added.Then, a microplate reader (Epoch) was used to estimate cell proliferation rate after incubating 2 hours.

Transwell assay
A Transwell assay was used to assess cell invasion.The upper chamber of a Transwell insert (8-mm pore size) was coated with Matrigel (BD Biosciences) and seeded with 9×10 4 cells.20% FBS medium was contained in the lower chamber.After incubating at 37°C and 5% CO 2 for 48 hours, the chamber was taken out and washed with calcium-free PBS for 3 times.The cells in the lower chamber were xed with 4% paraformaldehyde and then stained with 0.1% crystal violet.The lower migrated cells were counted under microscopy.

Wound-healing assay
Cell migration was detected using a wound-healing assay.The cells were seeded in a 6-well plate and cultured.After the cells growed to the density of 100%, the cell layer was scratched with a 20-µL pipette tip and a serum-free medium was replaced.After 0, 24, and 48 hours, images of the cells were captured under microscopy.

TUNEL assay
A TUNEL assay was used to investigate cell apoptosis.Cells in each group were treated using the TUNEL kit, and the uorescence results were observed and photographed using an inverted uorescence microscope.

Animal experiment
The animals used in the experiment were 6-week-old SPF-grade male BALB/c nude mice weighing approximately 20 g.KYSE-150 (1×10 6 ) or KYSE-150 (1×10 6 ) mixed with TAMs (3×10 6 ) in serum-free RPMI1640 medium were subcutaneously injected into the right haunch of 10 mice.The tumor volume in the mice was then measured every 5 days using the formula V = 1/2*L 2 (V: volume, L: length, W: width), and a correlation function graph was plotted.After 20 days (when the tumor diameter had reached 10 mm), the mice were irradiated with a single X-ray of 10 Gy.After 7 days of irradiation, the mice were euthanized by cervical dislocation, and the tumors were removed, photographed, and weighed.The tumors were then soaked in formalin for H&E staining and immunohistochemistry analysis.The animal experiments were approved by the Institutional Committee on Animal Care at Nanjing Medical University.

Statistical analysis
All statistical analysis was performed using GraphPad Prism 9 software.The data were expressed as mean ± standard deviation (SD).Two group comparisons were analyzed by Student's t-test and multiple group comparisons were conducted by one-way ANOVA.*P values < 0.05 were considered statistically signi cant.

TAM induction
CD68 is a marker of macrophages in normal esophagus tissues and human esophageal cancer (19), and CD163 ( 20) is a speci c marker of M2 macrophages.We used PMA to induce M0 polarization, and we used PMA and IL-4 macrophages to induce M2 polarization.On immuno uorescence staining, CD68 was positive after PMA treatment and CD163 was positive after PMA and IL-4 treatment (Fig. 1A), suggesting THP-1 cells were successfully transformed to M0 and M2 macrophages.Additionally, Western blotting assays demonstrated that CD68 was highly expressed in M0 and M2 macrophages, and M2 macrophages showed a high expression of CD163, indicating successful induction of TAMs (Fig. 1B).

TAMs promote the proliferation of esophageal cancer cells after radiotherapy
To investigate the role of TAMs in the radiosensitivity of esophageal cancer, we co-cultured TAMs with KYSE-150 and TE-1 cells.A CCK-8 assay demonstrated that the proliferation rate of cells in the cocultured group was signi cantly elevated at all time periods (24, 48, 72, and 96 hours) when compared with the rate in the control group; the proliferation rate was also signi cantly increased after radiotherapy (Fig. 2A).
A colony formation assay demonstrated that with an increase in radiation dose, the cloning e ciency of cells was gradually weakened.The clonogenic ability of the co-cultured group was stronger than that of the control group.The mean lethal dose (D 0 ), quasi-threshold dose (D q ), and cell survival fraction after irradiation with 2 Gy (SF 2 ) were obtained based on the plot of cell survival fraction tted using a singlemachine multitarget model.The sensitizer enhancement ratio (SER Do ) was de ned as D 0 of the control group/D 0 of the co-cultured group (Fig. 2B).The values of D 0 , D q , and SF 2 were higher in the co-cultured group than in the control group.The SER Do and SER Dq of KYSE-150 cells were 0.93 and 0.92, respectively; those of TE-1 cells were 0.90 and 0.87, respectively (Table 1).A TUNEL assay showed that the apoptosis rate in the co-cultured group was decreased compared with the rate in the control group (Fig. 2C).
To verify whether tumor growth in vivo was the same as in vitro, co-cultured KYSE-150 cells were subcutaneously xenografted into mice, and after 20 days, a 10-Gy X-ray was used to irradiate the tumors in the radiation group.Consistent with our in vitro results, before and after irradiation, the tumor volume and weight of the co-cultured groups were signi cantly higher than those of the other groups (Fig. 2D-F).H&E staining of the transplanted tumor specimens demonstrated that the morphology and size of cells in each group were varied.We found that necrotic tumor tissues appeared in the radiation group; however, in the co-cultured group, the formation of necrotic tissue was lessened, indicating that TAMs had a certain radiation resistance in KYSE-150 cells (Fig. 2G).Ki67 protein was expressed in the nucleus, appearing as a brownish yellow stain.The expression of Ki67 protein in the tumor tissues of the cocultured group was higher than in that of the other groups before and after irradiation (Fig. 2H).

TAMs promote the migration and invasion of esophageal cancer cells after radiotherapy
To determine the in uence of TAMs on the migration and invasion of esophageal cancer cells, we performed a Transwell assay and scratch test.As shown in Fig. 3A, the number of esophageal cancer cells penetrating the basement membrane of the Transwell chamber was increased after co-culturing with TAMs.The wound-healing assay demonstrated that the wound-healing width of esophageal cancer cells was narrower in the co-cultured group than in the control group after 48 hours of incubation (Fig. 3B).
TAMs increase the expression of VEGFA in esophageal cancer cells before and after radiotherapy Through qRT-PCR and Western blotting assays, we found that VEGFA mRNA and protein were signi cantly upregulated in cells co-cultured with TAMs before and after radiation (Fig. 4A, B).
As described earlier, TAMs play an important role in the radio-resistance of esophageal cancer cells, and the mechanism may be related to the up-regulation of VEGFA.To further verify the effect of TAMs and VEGFA on the radiosensitivity of EC, we constructed VEGFA decreased expression cells using lentivirus transfection.The decreased VEGFA mRNA and protein levels were then veri ed by qRT-PCR and Western blot analysis in KYSE-150 and TE-1 cells.VEGFA mRNA expression and protein level in the transfection group were signi cantly inhibited when compared with the control and negative control (nc) group (Fig. 4C, D).
We co-cultured TAMs with VEGFA-nc cells and VEGFA-si cells and then applied radiotherapy.
CCK-8 assay showed that in the VEGFA-si group, cell proliferation was signi cantly reduced after radiotherapy when compared with the VEGFA-nc group (Fig. 5A).Clonogenic ability was also signi cantly decreased in the VEGFA-si group with increased dose of irradiation (Fig. 5B).The values of D 0 , D q , and SF 2 were decreased after VEGFA knockdown.The SER Do and SER Dq of KYSE-150 cells were 1.18 and 1.18, respectively, and those of TE-1 cells were 1.21 and 1.22, respectively (Table 2).On the TUNEL assay, the apoptosis rate was signi cantly increased in the VEGF-si group (Fig. 5C).The number of cells penetrating the basement membrane of the Transwell chamber was signi cantly reduced (Fig. 5D) and the scratch width was signi cantly increased in the VEGFA-si group when compared with the co-cultured with control group (Fig. 5E).In the experiments, we described TAMs acting as negative regulators in radiotherapy of esophageal cancer by up-regulating VEGFA protein.To further con rm this theory, we used Western blotting assay to assess how TAMs and VEGFA affected the expression of MAPK, BCL-2, and Snail.
Compared with the control group, the group co-cultured with TAMs was found to promote the activation of VEGFA, MAPK, P-MAPK, BCL-2, and Snail protein.However, after inhibition of VEGFA expression, the expression of VEGFA, MAPK, P-MAPK, and BCL-2 decreased signi cantly (Fig. 6).

Discussion
Esophageal cancer is a relatively common type of cancer and has a high morbidity rate.Most cases are too advanced to be treated with surgery when they are initially diagnosed and are instead treated with radiotherapy.Radiotherapy is one of the main treatments for solid tumors, but patients undergoing this treatment may have a poor prognosis due to tumor recurrence (21,22).Different kinds of radiotherapy can reprogram TAMs in different tumor types, which further affects the e cacy of radiotherapy and leads to resistance to radiotherapy and tumor recurrence (23).Therefore, it is important to understand the role of TAMs in radiotherapy so that treatment can be planned and adjusted accordingly.
TAMs play an important role in tumorigenesis and progression, as well as in angiogenesis.Previous studies have shown that radiation can promote TAM in ltration in tumors through the VEGF/VEGF receptor (VEGFR) signaling pathway; this in turn affects the e cacy of radiotherapy (24,25).Therefore, targeting pro-angiogenic-related factors released by TAMs may be an important tool in antitumor therapy.VEGF is the main pro-angiogenic factor.VEGFA, a member of the VEGF family, has elevated expression in most tumors and is closely associated with the progression of tumors.VEGFA binds mainly through its receptors, activating downstream signaling pathways and causing a series of biological effects (26-28).
In this study, we successfully induced the M2-type macrophages and co-cultured them with esophageal cancer cell lines KYSE-150 and TE-1.In vivo and in vitro experiments showed that TAMs could promote the proliferation, invasion, and migration of esophageal cancer cells before and after radiotherapy, which indicates that TAMs may cause radio-resistance in esophageal cancer.We also found that VEGFA was signi cantly elevated in co-cultured and irradiated esophageal cancer cells.In addition, VEGFA knockdown was found to reverse the biological response induced by TAMs.
We also assessed the of MAPK and the phosphorylation level of MAPK in esophageal cancer cells and found that the tendency was the same as VEGFA.This suggest that TAMs may act via the VEGFA/MAPK pathway.
A TUNEL assay demonstrated that TAMs could inhibit the apoptosis rate of esophageal cancer cells before and after radiotherapy, whereas inhibition of VEGFA led to an increase in apoptosis rate.BCL-2 is known as an anti-apoptosis protein.Previous research has shown that BCL-2 overexpression in cell lines may promote cell growth and proliferation (14), which is consistent with the results we observed in this study.
Snail is a major transcriptional factor that induces Epithelial-mesenchymal transition (EMT) by repressing E-cadherin protein and also promotes tumor-initiating while enhancing tumorigenicity, tumor invasion, metastasis and therapeutic resistance (29).In this study, we found that Snail protein was overexpressed in the co-cultured group; however, in the VEGF-si group, Snail expression was inhibited, indicating that TAMs promote the invasion of esophageal cancer cells.
Although we have con rmed that TAMs could promote radio-resistance of esophageal cancer cells via angiogenic pathway, there are few evidence in clinical application.Further researches on clinical samples are urgent to nd the role of TAMs in the progression of esophageal cancer patients.
In conclusion, our research con rmed that TAMs can promote the proliferation, invasion, and migration and inhibit the apoptosis of esophageal cancer cells by up-regulating VEGFA expression, thus resulting in radio-resistance.This study provides new information regarding radio-resistance in esophageal cancer and may lead to improvements in the e cacy of radiotherapy in affected patients.Legend not included with this version.
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Table 1
The radiosensitization parameters and sensitization ratio after co-cultivation of KYSE-150 and TE-1 cells

Table 2
The radiosensitization parameters and sensitization ratio after down-regulation of VEGFA of KYSE-150 and TE-1 cells