The increased number of tumor-associated macrophage is associated with overexpression of VEGF-C, plays an important role in Kazakh ESCC invasion and metastasis

Experimental and Molecular Pathology 102 (2017) 15–21

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The increased number of tumor-associated macrophage is associated with overexpression of VEGF-C, plays an important role in Kazakh ESCC invasion and metastasis Jian Ming Hu a,c, Kai Liu c, Ji Hong Liu c, Xian Li Jiang c, Xue Li Wang c, Lan Yang c, Yun Zhao Chen c, Chun Xia Liu c, Shu Gang Li d, Xiao Bin Cui c, Hong Zou c, Li Juan Pang c, Jin Zhao c, Yan Qi c, Wei Hua Liang c, Xiang Lin Yuan a, Feng Li a,b,c,⁎ a

Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Pathology, Beijing Chaoyang Hospital, Capital Medical University, Beijing, 100020, China c Department of Pathology and Key Laboratory of Xinjiang Endemic and Ethnic Diseases (Ministry of Education), Shihezi University School of Medicine, Shihezi, 832003, China d Department of Preventive Medicine, Shihezi University School of Medicine, Shihezi 832003, China b

a r t i c l e

i n f o

Article history: Received 16 July 2016 and in revised form 19 November 2016 Accepted 6 December 2016 Available online 07 December 2016 Keywords: Tumor associated macrophages CD163 VEGF Esophageal squamous cell carcinoma Kazakh

a b s t r a c t Tumor associated macrophages (TAMs) play an important role in the growth, progression, and metastasis of tumors. The distribution of TAMs in Kazakh esophageal squamous cell carcinoma (ESCC) is not determined. We aimed to investigate the role of TAMs in the occurrence and progression of Kazakh ESCC. CD163 was used as the TAM marker, and immunohistochemistry (IHC) counts were used to quantify the density of TAMs in tumor nest and surrounding stroma. IHC staining was used to evaluate the expression of vascular endothelial growth factor C (VEGF-C) in Kazakh ESCC and cancer adjacent normal (CAN) tissues. The density of TAMs in Kazakh ESCCs tumor nest and stromal was significantly higher than that in CAN tissues. The increased number of CD163-positive TAMs in tumor nest and tumor stromal was positively associated with Kazakh ESCC lymph node metastasis and clinical stage progression. Meanwhile, the expression of VEGF-C in Kazakh ESCCs was significantly higher than that in CAN tissues. Overexpression of VEGF-C in Kazakh ESCCs was significantly associated with gender, depth of tumor invasion, lymph node metastasis and tumor clinical stage. The increased number of TAMs, either in the tumor nests or tumor stroma was positively correlated with the overexpression of VEGF-C, which may promote lymphangiogenesis and play an important role in the invasion and metastasis of Kazakh ESCC. © 2016 Published by Elsevier Inc.

1. Introduction Esophageal carcinoma is one of the 10 most common malignant tumors worldwide. Incidence rates vary between physiographic regions, nations, and races (Parkin et al., 2005). China has high esophageal carcinoma incidence and mortality rates (Chen et al., 2013). The Kazakh national minority (ethnic) living in Xinjiang (northwest of China) is demographic with one of the highest rates of esophageal carcinoma incidence and mortality, its esophageal carcinoma mortality rate has reached 155.9/100,000, which is higher than the average Chinese rate of 15.23/100000 (Zhang, 1988). The 5-year survival rate of esophageal carcinoma is only 10%. Primary reasons for poor prognosis are

⁎ Corresponding author at: Department of Pathology and Key Laboratory of Xinjiang Endemic and Ethnic Diseases (Ministry of Education), Shihezi University School of Medicine, Shihezi, 832003, China. E-mail address: [email protected] (F. Li).

http://dx.doi.org/10.1016/j.yexmp.2016.12.001 0014-4800/© 2016 Published by Elsevier Inc.

associated with early stage cancer cell invasion and high metastasis (Ekman et al., 2008). The tumor microenvironment is important for cancer development and metastasis (Lee et al., 2014; Zhang et al., 2011). It contains a range of inflammatory and immune cells. Macrophages (MØ) are essential immune cells that play a critical role in carcinogenesis and tumor progression (Gwak et al., 2015). Similar to Th1 and Th2 T cells, MØ can be classified into M1 and M2 subtypes (Biswas and Mantovani, 2010). M1 MØ are activated by interferon gamma (IFN-γ) and microorganisms, expressing high levels of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin [IL]-6, IL-12) and major histocompatibility complex class II (MHC class II). M1 MØ are capable of killing pathogens and promoting antitumor immune responses. By contrast, M2 MØ are activated in vitro by IL-4, IL-10 and IL-13, showing reduced MHC class II and IL-12 expression, but increased expression of IL-10 and arginase (Mantovani and Sica, 2010). Most M2 MØ are considered as tumor associated MØ (TAM) and have the effect of promoting tumor angiogenesis, lymphangiogenesis and metastasis (Sica et al.,

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2008). CD163 is a confirmed biomarker of the M2 TAMs that can be used to distinguish M2 from M1 MØ. Vascular endothelial growth factor (VEGF) is an important regulator of the progression of pathological angiogenesis and lymphangiogenesis observed in many different tumors (Eveno and Pocard, 2012; Goel and Mercurio, 2013). VEGF-C is a member of VEGFs family, playing an important role in lymphangiogenesis, which it acts on lymphatic endothelial cells (LECs) primarily via its receptor VEGFR-3, promoting survival, growth and migration (Oh et al., 1997). In addition to its effect on lymphatic vessels, it can also promote the growth of blood vessels and regulate their permeability. TAMs produce many proangiogenic factors and express high levels of VEGF-C, which promotes cancer cell invasion, metastasis, angiogenesis and lymphangiogenesis (Obeid et al., 2013; Skobe et al., 2001). However, the precise role of TAMs in Kazakh esophageal squamous cell carcinoma (ESCC) has yet to be elucidated. We aimed to investigate whether TAMs correlate with VEGF-C levels, promoting the occurrence and progression of Kazakh ESCC. 2. Materials and methods 2.1. Ethics statement All participants were recruited from the Yili Friendship Hospital in Xinjiang, China. Each participant provided written, informed consent before enrolling in this study. Protocols were approved by the institutional ethics committee of Yili Friendship Hospital in accordance with Helsinki Declaration ethical guidelines. 2.2. Study population

DAKO EnVision kit (DAKO, Glostrup, Denmark) following the manufacturer's instructions. Finally, sections were faintly counterstained with hematoxylin and mounted with glycerol gelatin. 2.4. Immunoreactivity evaluation The numbers of CD163-positive MØ were analyzed as described previously (Shabo et al., 2009). The five most representative hot spots were selected from low-power fields (LPFs, 100×) per slide using an Olympus BX51TF microscope (Olympus, Japan). Tumor nest and stroma areas were defined, and the numbers of CD163-positive MØ were counted in high-power fields (HPFs, 400 ×) by two pathologists. When cell counts differed by N 10 cells per HPF, samples would be counted again a week later until recording differences were below 10 counts. The mean number of macrophages per HPF across five hot spots for every sample (tumor nest and tumor stroma) was defined as the TAMs density. VEGF-C immunohistochemistry (IHC) reactivity was evaluated following previously described methods (Hu et al., 2014; J.M Hu et al., 2016). Positive IHC stains were defined as yellow-brown color following the manufacturer's guidelines (Fig. 3). IHC staining slides were scored as positive or negative by percentage and intensity of positive cells, where the scoring percentage of positively stained cells was as follows: 0 = b5%, 1 = 6%–25%, 2 = 26%–50%, 3 = 51%–75%, and 4 = 76%–100%; staining intensity scoring was: 0 = absent, 1 = weak, 2 = moderate, and 3 = strong. A final score was based on multiplying both scores from individual slides (Table 1), where: 0–1 was negative (−), 2–3 was weak positive (1+), 4–6 was moderate positive (2+), and 8–12 was strong positive (3+).

A total of 200 surgically resected and paraffin-embedded human tissues were collected, including 100 Kazakh ESCC tissues and 100 Kazakh cancer adjacent normal tissues (CANs), from the Department of Pathology at Yili Friendship Hospital in Xinjiang, China (collected from 2008 to 2014). Patients were 33–76 years old (68 men and 32 women), all had been diagnosed with ESCC, but none had received radiotherapy or chemotherapy before surgery. CAN group participants were 33–73 years old (68 men and 32 women). All specimens were sectioned into 5 μm slices and subjected to conventional hematoxylin and eosin staining. A diagnosis of ESCC was confirmed by two pathologists following the World Health Organization histological tumor classification criteria (Li and Li, 2011). There were 29 cases of well-differentiated ESCC, 47 cases of moderately differentiated ESCC, and 24 cases of poorly differentiated ESCC. From these, 35 and 65 cases exhibited invasion depths of T1–T2 and T3–T4 respectively. There were 51 cases with lymph node metastasis, 49 without lymph node metastasis, 62 cases in clinical stages I–II, and 38 cases in clinical stages III–IV. CAN specimens, which were sampled N5 cm away from the cancer region, were confirmed to be free of cancer tissue.

2.5. Statistical analysis

2.3. Immunohistochemistry

Table 1 Scoring criteria of immunohistochemistry (IHC) assay with VEGF-C antibody used in this study.

Paraffin-embedded tissue samples were cut into 4 μm-thick sections and mounted on polylysine-coated slides. Samples were dewaxed in xylene and rehydrated using a graded series of ethanol solutions. After deparaffinization, endogenous peroxidase activity was blocked by incubation in a 3% peroxide-methanol solution at room temperature (RT) for 10 min, and then antigen retrieval was performed at 100 °C in an autoclave for 7 min. Samples were then incubated at RT for 30 min. Afterwards, sections were washed with phosphate-buffered saline (PBS) three times for 5 min each time. They were then incubated with mouse anti-human CD163 antigen monoclonal antibody (clone 10D6, Zhongshan Goldenbridge Biotechnology Co., LTD., Beijing, China) and mouse anti-human VEGF-C antigen monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Thorough washing with PBS was then performed, and primary antibody binding was visualized using a

SPSS version 13.0 was used for all statistical analyses. For comparisons of independent samples, t-test and one-way analysis of variance (ANOVA) were used to evaluate correlations between the density of CD163-positive TAM and ESCCs, and clinicopathological features. The ×2 test was adopted for analysis of correlations between VEGF-C and ESCC, and clinicopathological features. Spearman's rank correlation method was used to evaluate the correlations between the CD163 and VEGF-C. p-Values were calculated using the Epi-Info program, and pvalues b 0.05 were considered significant. 3. Results 3.1. Distributions of CD163-positive TAMs in Kazakh ESCC tumor nests, tumor stroma, CAN epithelia, and CAN stroma We used CD163 as a marker to assess TAM distribution. IHC staining for CD163 revealed diffuse staining of TAM membranes and cytoplasm

Staining positive cell

Staining intensity

Final score product

Percent (%)

Score 1

Intensity

Score 2

Score 1 × Score 2

Score 3

b5% 6%–25% 26%–50% 51%–75% 75%–100%

0 1 2 3 4

Absent Weak Moderate Strong

0 1 2 3

0–1 2–3 4–6 8–12

0 (−) 1 (1+) 2 (2+) 3 (3+)

Note: IHC staining slides were scored as positive or negative by the percentage and intensity of positive cells:Scoring the percent of positively stained cells-Score 1 (0 = b5%; 1 = 6%–25%; 2 = 26%–50%; 3 = 51%–75%; and 4 = 76%–100%). Scoring the intensity of the staining-Score 2 (0 = absent; 1 = weak; 2 = moderate; 3 = strong). The final score was based on multiplying both scores from individual slides (Score 1 × Score 2): 0–1 was negative (−), 2–3 was weak positive (1+), 4–6 was moderately positive (2+), and 8–12 was strong positive (3+).

J.M. Hu et al. / Experimental and Molecular Pathology 102 (2017) 15–21

A

17

B

100um

C

100um

D

100um

E

100um

F

100um

100um

Fig. 1. The distribution of CD163-positive TAMs in Kazakh ESCC tumor nest tissue, CAN epithelia, and CAN stroma. A Hematoxylin and eosin staining is shown for ESCC (×200). B Hematoxylin and eosin staining is shown for CANs (×200). C, D, E, F Immunohistochemical staining of CD163, which was used to mark TAMs and to evaluate the density of TAMs in ESCC and CAN tissues (×200). C and E show the distribution of TAMs in CAN epithelia and stroma. A small number of CD163-positive TAMs appear in CAN tissues. D and F show the distribution of TAMs in ESCC tumor nest and stromal tissues, demonstrating that CD163 reveals diffuse staining of membranes and cytoplasm of TAMs, and showing the high density of TAMs located in ESCC tissues (especially in tumor stroma).

(Fig. 1). We found that TAMs are primarily located in the tumor stroma, but a small number of TAMs reside in tumor nests. The density of TAMs in Kazakh ESCC tumor nests (approximately 15/HPF, 0–45) and stroma (approximately 58/HPF, 9–139) were significantly higher than that in CAN epithelia (approximately 2/HPF, 0–10) and stroma (approximately 19/HPF, 3–54) (all p b 0.001, Table 2, Fig. 2).

Table 2 The distribution of CD163-positive TAMs in Kazakh ESCC and CAN tissues. Groups ESCCs CANs

Cases (N) 100 100

Tumor nest Mean ± s.d

p

14.50 ± 12.45 1.62 ± 1.63

0.000⁎

Tumor stroma Mean ± s.d

p

57.81 ± 26.50 19.43 ± 7.82

0.000⁎

Note: ESCCs: Esophageal squamous cell carcinoma tussies. CANs: Cancer adjacent normal tissues. ⁎ p b 0.05.

3.2. Correlation of CD163-positive TAM density in tumor nest and stroma with Kazakh ESCC clinicopathological parameters We evaluated possible correlations between TAMs density and Kazakh clinicopathological parameters, including age, gender, tumor location, histological grade, invasion depth, nodal status and clinical stages (Table 3). The density of CD163-positive TAMs in tumor nests was significantly associated with nodal metastasis and later clinical stages (all p b 0.05). Similar trends were more obvious in tumor stroma (all p b 0.001). No significant correlations were found between TAM distribution and other parameters (p N 0.05). 3.3. VEGF-C expression in Kazakh ESCCs and CANs, and its relationship with ESCC clinicopathological parameters TAMs are reportedly involved in tumor growth and metastasis, and are suspected to be important for tumor angiogenesis and lymphangiogenesis. TAMs could promote tumor angiogenesis and

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Fig. 2. The density of TAMs in tumor nest and stromal tissues was higher than that in CAN epithelia and stroma. A The distribution of CD163-positive TAMs in Kazakh ESCC tumor nest tissue and CAN epithelia. B The distribution of CD163-positive TAMs in Kazakh ESCC and CAN stroma.

lymphangiogenesis by secreting VEGFs, and subsequently promote tumor invasion and metastasis. Lymphangiogenesis and early lymph node metastasis are main reasons for poor prognosis of ESCCs, and VEGF-C is important regulatory factor for lymphangiogenesis. So we decided to investigate a correlation between VEGF-C and TAMs in Kazakh ESCC occurrence and progression. VEGF-C was detected in Kazakh ESCCs and CANs by IHC. As shown in Fig. 3, VEGF-C staining was mainly observed in tumor stromal cells (located in cell membranes and cytoplasm), including MØ and endothelial cells (ECs). Only 4.0% of ESCC tissue cases were negative for VEGF-C antibody staining, and most cases showed strong staining (3+). VEGF-C antibody negative staining was 15.0% in CAN cases and only a few examples of CAN tissues showed strong staining (3+). We compared three categories of VEGF-C positive staining combinations (1+, 2+/3+, and 1+/2+/3+) to VEGF-C negative (0) staining (Table 4). The VEGF-C positive rate (1+/2+/3+) in ESCC was higher than in CAN tissues (96.0% vs 85.0%, p = 0.008). Differences in VEGF-C positive rates from ESCC to CAN tissues were more prominent in strong staining cases (2+/3+, 69.0% vs 39.0%, p b 0.001).

Table 3 Correlation between density of TAMs and clinicopathological parameters in Kazakh ESCCs tumor nest and stroma. Variable Age (y) ≤Median (58 y) NMedian Gender Male Female Tumor location Upper Middle Lower Histologic grade Well Moderate Poor Depth of invasion TI–T2 T3–T4 Nodal status pN− pN+ Clinical stage I–II III–IV

Cases (N)

Tumor nest Mean ± s.d

p

Tumor stroma Mean ± s.d

p

53 47

13.34 ± 9.92 15.81 ± 14.80

0.325

53.40 ± 25.55 62.78 ± 26.93

0.077

68 32

14.40 ± 14.36 14.71 ± 7.00

0.910

59.00 ± 28.05 55.28 ± 23.06

0.516

2 70 28

6.50 ± 2.12 14.20 ± 13.30 15.82 ± 10.45

0.559

53.00 ± 46.67 57.86 ± 25.95 58.04 ± 27.76

0.967

29 47 24

13.07 ± 7.43 12.93 ± 8.46 19.32 ± 20.61

0.093

57.57 ± 18.27 55.08 ± 29.52 63.44 ± 28.72

0.457

35 65

15.09 ± 8.51 14.19 ± 14.18

0.731

56.15 ± 31.20 58.71 ± 23.81

0.674

51 49

11.28 ± 6.65 17.86 ± 15.85

0.009⁎

56.15 ± 31.20 69.45 ± 26.42

0.000⁎

62 38

12.18 ± 6.94 18.45 ± 17.83

0.046⁎

51.39 ± 25.98 68.29 ± 24.17

0.001⁎

Note: pN−, no lymph node metastasis; pN+: node metastasis. ⁎ p b 0.05.

For comparison of VEGF-C expression to Kazakh ESCC clinical parameters, we divided ESCC cases into two categories according to VEGF-C expression level: low expression (−/1+) and high expression (2+/3 +). The expression level of VEGF-C was significantly higher in male compared to female (76.5% vs 53.1%, p b 0.05). Cases with high VEGF-C expression showed strong invasion (T3–T4 vs T1–T2 = 80.0% vs 48.6%, p = 0.003) and metastasis (pN+ vs pN− = 87.8% vs 51.0%, p b 0.001), and were clearly present in advanced ESCC stages (III–IV vs I–II = 94.7% vs 53.2%, p b 0.001) (Table 5). 3.4. Correlation between the density of CD163-positive TAMs and VEGF-C expression in Kazakh ESCCs and CANs To investigate the role of TAMs in tumor lymphangiogenesis and metastasis, the relationship between the CD163 and VEGF-C was analyzed. The density of CD163-positive TAMs, either in the tumor nest or tumor stroma, was positively associated with VEGF-C expression (r = 0.213, p b 0.05 and r = 0.421, p b 0.001, respectively, Table 6). Even in CAN tissues, CD163-positive TAMs in stroma (r = 0.244, p b 0.05), but not in epithelium, were positively associated with the expression of VEGF-C (Table 6). 4. Discussion M2 MØ are prominent stromal cells that play an important role in tumor growth and metastasis to the point that they are also known as TAMs (Qian and Pollard, 2010). Increased numbers of TAMs in tumor stroma and nests have a close relationship with tumor progression and poor prognosis, and this has been confirmed in numerous cancer types (Y Hu et al., 2016; Jensen et al., 2009; Kim et al., 2015; Park et al., 2015). However, the interaction between TAMs and the occurrence or progression of Kazakh ESCC was not elucidated clearly. CD163 is a confirmed marker for the TAM phenotype that can be used to distinguish M2 and M1 MØ. In ESCC tissues, we found that CD163-positive TAMs were primarily located in the tumor stroma, but were also present in the tumor nest. The density of TAMs in the Kazakh ESCC tumor nest and stroma was significantly higher than that in corresponding CAN tissues. The increased number of CD163-positive TAMs in tumor nests was positively associated with Kazakh ESCC metastasis and clinical stage progression, and increased numbers of CD163-positive TAMs in tumor stroma appeared to be more closely correlated with Kazakh ESCC metastasis and progression. Such results are similar to those reported in gastric cancers (Go et al., 2016; Park et al., 2015). Increased CD163-positive TAMs in gastric tumor stroma have been shown to closely correlate with tumor size, invasion depth, TNM stage, lymph node metastasis and lymphovascular invasion (Park et al., 2015). Similar results were also reported in models of oral squamous cell carcinoma

J.M. Hu et al. / Experimental and Molecular Pathology 102 (2017) 15–21

19

Fig. 3. Immunohistochemical staining of VEGF-C in Kazakh ESCC and CAN tissues. VEGF-C staining is primarily observed in tumor stroma (cell membranes and cytoplasm); some ESCC cells also show staining (×200). A Negative VEGF-C staining is shown in CAN tissues (scored as 0). B Weak VEGF-C staining is shown in CAN tissues (scored as 1). C and D show moderate and strong VEGF-C staining in Kazakh ESCC tissues, respectively (scored as 2 and 3, respectively).

(J.M Hu et al., 2016) and endometrial adenocarcinoma (Kubler et al., 2014). Prompt increased numbers of CD163-positive TAMs, especially in tumor stroma, are closely related to the metastasis and progression of Kazakh ESCCs. TAMs promote tumor progression through several mechanisms, including promotion of angiogenesis, lymphangiogenesis and immunosuppression (Sica et al., 2006). Such pro-tumor and pro-angiogenesis activities are thought to be executed by TAMs that secrete VEGFs. Other studies assert that VEGFs also play an important role in the recruitment and polarization of MØ in tumors, induction of MØ into the TAM phenotype, and in tumor metastasis and progression (Tsutsui et al., 2005). To further explore the interaction between TAMs and VEGFs, we evaluated the expression of VEGF-C in Kazakh ESCCs and CANs. We found that VEGF-C was mainly expressed in tumor stromal cells, such as TAMs. Only a small section of tissue showed tumor cell staining, which is consistent with previous findings from models of hepatocellular carcinoma (Zhuang et al., 2013). The data indicates that VEGF-C may be mainly secreted by tumor stromal cells, especially for TAMs. Also we found VEGF-C expression was much higher in ESCC tissues than in CAN tissues (Fig. 3), particularly in the VEGF-C strong positives cases (71.43% in ESCC vs 35.71% in CAN, Table 4). Furthermore, overexpression of VEGF-C in Kazakh ESCCs was significantly associated

with invasion depth, lymph node metastasis and clinical stage (p b 0.05). These results were consistent with previous esophageal carcinoma studies (Juchniewicz et al., 2015), Suggesting that VEGF-C may promote lymphangiogenesis, participate in the invasion and metastasis of Kazakh ESCC. To investigate the interaction between TAMs and VEGF-C, and the function to tumor lymphangiogenesis and metastasis in Kazakh ESCCs, we quantified CD163-positive TAMs and assessed relative levels of VEGF-C expression. The density of CD163-positive TAMs, in either tumor nest or tumor stromal, was positively associated with VEGF-C expression (p b 0.05, Table 6). These results are consistent with previous hepatocellular carcinoma findings (Zhuang et al., 2013), and together with the analysis of clinical parameters, demonstrating VEGF-C may be secreted by TAMs and promote lymphangiogenesis, which are associated with the invasion and metastasis of Kazakh ESCC. One limitation of this study is that the CD163 marker is not highly specific. Although CD163 has been widely used to identify TAMs (Cao et al., 2015; Sugimura et al., 2015), other stromal cells, such as dendritic cells (DCs) and endothelial cells (ECs) can also express CD163, which may increase the number of positive cells as those mixed cells (DCs and ECs). Secondly, evaluation of TAM density exist subjective error to some extent, so the use of image analysis software might be one way

Table 4 The expression of VEGF-C in Kazakh ESCC and CAN tissues. Characteristics

ESCCs CANs

n

100 100

Negative

Positive combination

0 (%)

1 + (%)

4 (4.0%) 15 (15.0%)

27 (27.0%) 46 (45.0%)

×2 1.713

p 0.191

Note: ESCCs: Esophageal squamous cell carcinoma tussies. CANs: Cancer adjacent normal tissues. ⁎ p b 0.05.

2 + 3+ (%) 69 (69.0%) 39 (39.0%)

×2

p

1 + 2 + 3+

×2

p

12.13

0.00⁎

96 (96.0%) 85 (85.0%)

7.037

0.008⁎

20

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Table 5 Correlation between expression of VEGF-C and clinicopathological parameters in Kazakh ESCCs. Variable

Age (y) ≤Median (58y) NMedian Gender Male Female Tumor location Upper Middle Lower Histologic grade Well Moderate poor Depth of invasion TI–T2 T3–T4 Nodal status pN− pN+ Clinical stage I–II III–IV

VEGF low expression 0/1 + (%)

VEGF high expression 2+/3 + (%)

×2

53

17 (32.1%)

36 (67.9%)

0.001

0.976

47

14 (29.8%)

33 (70.2%)

68 32

16 (23.5%) 15 (46.9%)

52 (76.5%) 17 (53.1%)

4.507

0.034⁎

Cases (N)

p

Competing interests

2 70 28

1 (50.0%) 22 (31.4%) 8 (28.6%)

1 (50.0%) 48 (68.6%) 20 (71.4%)

0.421

29 47 24

5 (17.2%) 18 (38.3%) 8 (33.3%)

24 (82.8%) 29 (61.7%) 16 (66.7%)

4.048

0.132

35 65

18 (51.4%) 13 (20.0%)

17 (48.6%) 52 (80.0%)

9.088

0.003⁎

51 49

25 (49.0%) 6 (12.2%)

26 (51.0%) 43 (87.8%)

14.127 0.000⁎

62 38

29 (46.8%) 2 (5.3%)

33 (53.2%) 36 (94.7%)

18.980 0.000⁎

0.810

to reduce subjective error. However, the application of image analysis software could increase the count of mixed cells (DCs and ECs) that are distinguishable by visual analysis, and could result in inaccurate TAM counts. Therefore, the mean of five hot spots counted by two expert pathologists was likely to keep error to a minimum, and may prove to be the preferred method for TAM density evaluation. 5. Conclusions Our findings provide insight into the role of TAMs in the occurrence and progression of Kazakh ESCC. CD163 expression was primarily confined to infiltrating TAMs. Higher densities of TAMs in ESCC tumor nest and stroma compared to that of corresponding CAN tissues.

Table 6 Cross correlation analyses reveal strong relationships among density of TAM in tumor nest, tumor stroma and expression of VEGF-C in Kazakh ESCCs and CANs.

ESCCs TAM density in tumor nest TAM density in tumor stroma VEGF-C CANs TAM density in epithelium TAM density in stroma VEGF-C

The authors declare that they have no competing interests. Acknowledgements This work was supported by Ministry of Science and Technology of China (Nos. 2012AA02A503), National Natural Science Foundation (No. 81460363 and no. 81560399). References

Note: pN−, no lymph node metastasis; pN+: node metastasis. ⁎ p b 0.05.

Characteristics

Furthermore, the increased number of TAMs, either in the tumor nests or tumor stroma was positively correlated with the overexpression of VEGF-C, which may promote lymphangiogenesis and play an important role in the invasion and metastasis of Kazakh ESCC.

TAM density in tumor nest

TAM density in tumor stroma

VEGF-C

1

0.422⁎

0.213⁎

0.422⁎

1

0.421⁎

0.213⁎

0.421⁎

1

1

0.301⁎

0.031

0.301⁎ 0.031

1 0.244⁎

0.244⁎ 1

Note: ESCCs: Esophageal squamous cell carcinoma tussies. CANs: Cancer adjacent normal tissues. The numbers shown in the table are correlation coefficient r values. Spearman rank correlation analysis was used. ⁎ p b 0.05.

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