SDF-1 and MMP2 cross talk in cancer cells and tumor microenvironment in non-small cell lung cancer

Egyptian Journal of Chest Diseases and Tuberculosis (2016) 65, 517–525

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The Egyptian Society of Chest Diseases and Tuberculosis

Egyptian Journal of Chest Diseases and Tuberculosis www.elsevier.com/locate/ejcdt www.sciencedirect.com

ORIGINAL ARTICLE

SDF-1 and MMP2 cross talk in cancer cells and tumor microenvironment in non-small cell lung cancer Nehad M. Osman *, Wesam M. Osman Faculty of Medicine, Ain Shams University, Egypt Received 18 December 2015; accepted 4 January 2016 Available online 15 January 2016

KEYWORDS MMP-2; SDF-1; Immunohistochemistry; NSCLC; Prognosis

Abstract Lung cancer is the cancer killer among men and women worldwide. Attempts have been undertaken to identify potential biomarkers for lung cancer; some of which have a direct impact on tumor behavior. The association between MMPs and SDF-1 was previously studied in other organs yet the current study is conducted to evaluate their immunohistochemical expression in non-small lung cancer (NSCLC) aiming to discover their association with different clinicopathological prognostic parameters. Materials and methods: Bronchoscopy and/or C-T guided biopsy from 72 specimens of NSCLC (27 adenocarcinomas, 23 squamous cell carcinomas, and 12 large cell carcinomas) were immunohistochemically stained with MMPs and SDF-1. Results: Cytoplasmic and stromal expression of MMP-2 was seen in 39 (54.2%) patients. Nuclear SDF-1 staining was observed in 36 cases (50%). High expression of MMP-2 was correlated with tumor size (P = 0.002), lymph node (P = 0.001) and distant metastasis (P = 0.026). Nuclear SDF-1 expression was correlated with the tumor size (P = 0.001) and increased risk of metastasis (P = 0.001). A strong agreement between both markers was detected. Combined expression of MMP-2 and SDF-1 was significantly correlated with high tumor stage. MMP2 and nuclear SDF-1 were significantly independent factors in predicting high tumor stage (III and IV). Conclusion: SDF-1a directed invasion of NSCLC is mediated by crosstalk with MMP-2. The identification of SDF-1a from NSCLC tissues as a potential stimulatory factor of MMP-2 during cancer cell invasion may be involved in aggressiveness of NSCLC. Hence, identification of SDF-1a/ MMP-2 interaction may implicate therapeutic approaches for metastasis from lung cancer. Ó 2016 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).

* Corresponding author. Tel.: +20 1223549008 (N.M. Osman), +20 100639494 (W.M. Osman). E-mail addresses: [email protected] (N.M. Osman), [email protected] (W.M. Osman). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis.

Introduction Lung cancer, the cancer killer among men and women worldwide, is considered to be a deadly illness because of low (15%) 5 years – survival rate. This low survival rate is mainly

http://dx.doi.org/10.1016/j.ejcdt.2016.01.001 0422-7638 Ó 2016 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

518 because most of subjects present with advanced stages at the time of diagnosis. Advances in molecular biology have improved the systematic efforts to identify molecular markers for lung cancer with valuable predictive and prognostic significance [1]. Cancer prognostic markers are patient or tumor characteristics that predict outcome independent of the treatment [2]. The goal of identifying prognostic markers is to define patient subpopulations with significantly different anticipated outcomes and who might benefit from different therapies [3]. Therefore, it is of great significance to make better understanding of the molecular biology of NSCLC invasion and metastasis, and to identify novel molecular markers for the improvement of clinical management of patients with NSCLC. Mounting evidences supports the view that tumor microenvironment plays a critical role in tumorigenesis [4,5]. Although there is a wide range of biological functions of tumor microenvironment in cancer, an important characteristic is the remodeling and stiffening of the extracellular matrix (ECM) [6,7]. Recent experimental studies have revealed that degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is a critical process in progression of malignant tumors, including NSCLC. This is because degradation of the ECM is required in tumor invasion and metastases [8–11]. Among many MMPs that have been identified, MMP-2 (Gelatinase-A) and MMP-9 (Gelatinase-B) are thought to be key enzymes as they degrade type IV collagen, the main component of ECM [10,11]. Clinical studies on MMP-2 in malignant tumors, including NSCLC, have been conducted [12–15], but their clinical significance remains controversial. From the earliest work addressing the role of MMPs and cancer, there has been a clear connection between certain members of the MMP group and local cancer cell invasion [16], but the association with metastatic disease has been less clear. In addition to the ECM, MMPs have additional target proteins which include other proteinases, proteinase inhibitors, clotting factors, chemokines and chemotactic factors, growth factors, a variety of cell surface receptors, and cell matrix adhesion molecules [17]. Stromal cell–derived factor-1 (SDF-1), also called chemokine (CXC) ligand 12 (CXCL12), is a chemotactic factor for T cells, monocytes, hematopoietic progenitor cells, dendritic cells, endothelial cells, and tumor cells [18–20]. One receptor for this chemokine is CXCR4, which was previously called fusin [21]. In addition to its chemotactic functions, SDF-1 has also been shown to regulate tumor cell proliferation, motility, metastasis, and survival through its binding and subsequent activation of CXCR4 [22–24]. In 2001, Muller et al. [25] showed that the SDF-1/CXCR4 pathway mediates human breast cancer metastasis. Several studies followed, providing evidence that blocking of CXCR4 signaling in vivo using a specific antibody [25], selective synthetic polypeptide [26], or small interfering RNA [27] resulted in significant inhibition of breast cancer metastasis to regional lymph nodes and the lung. Similar results have also been reported for colonic adenocarcinoma and non-small cell lung cancer [28]. The association between MMPs and SDF-1 was previously studied in other organs yet the current study is conducted to evaluate the immunohistochemical expression of MMP-2 and SDF-1 in NSCLC; aiming to discover their association with different clinicopathological prognostic parameters.

N.M. Osman, W.M. Osman Materials and methods Patients and tissues This study included an overall 72 specimens of non-small lung cancer (NSCLC) patients. Patients included were presented for bronchoscopy and/or C-T guided biopsy at the chest department, Ain Shams university hospital. The flexible bronchoscopy (Pentax EB-1830T3 videobrochoschope, Asahi Optical Co., Tokyo, Japan) was performed to all patients according to the national and international standards of practice [29,30]. The diagnosis was based on the established pathology in all patients. Staging included full history, physical examination, blood tests and C-T scan (with contrast) of the chest, pelvi- abdominal ultrasound and/or C-T. Bone scintigraphy and brain CT scan were performed only in case of symptoms. None of the patients received radiotherapy or adjuvant chemotherapy. Formalin- fixed, Paraffin embedded tissue samples of 27 adenocarcinomas, 23 squamous cell carcinomas, and 12 cases of large cell carcinomas were included in the current study. Collection of relevant clinical data and approval of the Ethical Committee of Ain Shams University Hospitals were fulfilled before proceeding in this study. The pathologist independently examined the tumor specimens blinded from the patients’ data to confirm the tumor type according to WHO classification [31], tumor stage was revised as well according to the TNM staging system [32]. Immunohistochemistry: All 72 specimens of non-small lung cancer (NSLC) tissue samples were immunohistochemically stained. Paraffin blocks were deparaffinized in xylene and rehydrated through absolute alcohol. Antigen retrieval in citrate buffer (pH9 Lab vision cat#AP9003) after treatment in a microwave at 8° for 5–6 min, then at 3 for 10 min; After cooling for 20 min, peroxidase and protein block was done. Next, incubation overnight with the primary antibodies at room temperature using MMP2 (purified pre diluted, 7 ml, rabbit polyclonal MMP-2, cat. No. E18011, Spring Bioscience, California, USA) and SFD-1 (purified pre diluted, 7 ml, rabbit polyclonal, cat. No. E12541, Spring Bioscience, California, USA), then rinsing in PBS (pH7.6). This was followed by the secondary biotin conjugated antibody for 1 h and finally another hour for the peroxidase conjugated streptavidin. Diaminobenzidine tetrachloride (DAB) was added for 25 min. Then; counterstaining in Harris Hematoxylin, followed by dehydration, clearing and mounting was done. Positive control for MMp-2 antibody was ovarian carcinoma, while that for SDF-1 was human tonsil. Negative controls were done by replacement of the primary antibody with a non-immune antibody. MMP-2 staining: Five different fields (400) were randomly examined. The immunoreactivity score method to evaluate the immunostaining results has been previously described [33]. Stain intensity is as follows: no staining (score 0), weak staining (score 1), moderate staining (score 2), or strong staining (score 3). Staining area is as follows: less than 10% (score 1), 11–50% (score 2), 51–80% (score 3), 81–100% (score 4). Points for staining intensity and percentage of positive tumor cells were added and the overall score was grouped into four categories: negative (610% of tumor cells stained positive, regard-less of intensity), weak expression (score 3), moderate expression (score 4–5), and strong expression (score

SDF-1 and MMP2 cross talk in cancer cells 6–7). Moderate and strong expression was rated as positive, while weak expression was rated as negative for analysis. SDF-1: The intensity of staining (1–3) and percentage of positive cells (1 = <10%, 2 = 10–50%, 3 = >50%) were taken into account to define a composite score. Positive nuclear staining was defined as a nuclear score of 6 or 9 [i.e. any slide with >50% of the cells expressing nuclear staining (3) with intermediate or strong intensity (2 or 3)]. Cells were counted in at least three fields (at 400) in the tumor areas [34].

519 nomas (P = 0.001). Also, association was found between the expression of MMP-2 in tumor cells and tumor size (P = 0.002), But no association was found between the expression of MMP-2 in tumor cells and tumor differentiation (P = 0.556) (Table 1). There was no significant correlation between tumoral MMP-2 status and any other patient characteristic (data not tabulated). Representative images of MMP-2 immunostaining in different types of NSCLC tissues are shown in Figs. 1–3. SDF-1 expression in NSCLC: pattern of expression

Evaluation and statistical analysis Continuous variables are expressed as mean and Standard Deviation. Categorical variables are expressed as frequencies and percents. Student t test was used to assess the statistical significance of the difference between two study group mean. Chi square and Fisher’s exact test was used to examine the relationship between Categorical variables. Logistic regression was used for prediction of high stage tumor based on the presence of tumor markers. A significance level of P < 0.05 was used in all tests. All statistical procedures were carried out using SPSS version 15 for Windows (SPSS Inc, Chicago, IL, USA).

Results

SDF-1 was detected in the nucleus and/or in the cytoplasm of tumor cells. Nuclear staining was observed in 36 cases (50%). Interestingly, tumor cells exhibited also cytoplasmic staining for SDF-1 in 28 cases (38.9%). The intensity of nuclear staining was always intermediate or strong whenever present. A high expression of SDF_1 in tumor cells was correlated with an increased risk for metastasis (P = 0.001), which was also noticed in the subgroup of squamous cell carcinomas (P = 0.004). Also, a positive correlation was found with the tumor size (P = 0.001). In the patients with squamous cell carcinoma, SDF-1expression was correlated with the presence of metastasis (P = 0.001) (Table 2). There was no significant correlation between SDF-1 expression and tumor differentiation .age, gender, or smoking (data not tabulated). Representative images of SDF-1 immunostaining in different types of NSCLC tissues are shown in Figs. 4–6.

The clinical-pathological data MMP-2 Status and SDF-1 Biomarker Seventy-two specimens of NSCLC patients were included in the present study. The mean age was 52.67 years (±7.99 SD), 10 patients (14%) were female. 10 patients (14%) were nonsmokers, and 11 were x-smokers (15.3%). The mean tumor size was 5.80 cm (3.30 ± SD). The adenocarcinoma histologic subtype was present in 27 cases (37.5%), squamous cell carcinoma in 33 cases (45.8%) and 12 cases of large cell carcinoma type (16.7%). The commonest histologic grade was poorly differentiated (33/72; 55%) and the commonest tumor was classified as T1 in (24) patients (33%). Lymph node metastatic involvement was detected in (58) patients (80.6%). Twelve patients (16.7%) developed an evidence of metastatic disease at the time of diagnosis. Hence, AJCC stages at time of biopsy were as follows: 14 (19.5%) stage I, 23 (32%) stage II, 23 (31.9%) stage III and 12 (16.7) stage IV. MMP-2 expression in NSCLC: pattern of expression Expression of MMP-2 was seen mainly in the cytoplasm of tumor cells, and strong MMP-2 expression in tumor cells were seen in 39 (54.2%) patients. Staining in the surrounding stroma was localized in peri-tumoral fibroblasts and inflammatory cells. Interestingly, the tumor stroma showed the same score as that of the tumor cells. Strong MMP-2 expression was more frequent in adenocarcinoma patients (19 of 39, 48.7%) than in squamous cell carcinoma patients (13 of 39, 33.3%) but it didn’t reach a significant difference P_0.054). A high expression of MMP-2 was correlated with lymph node and distant metastasis (P = 0.001, P = 0.026 respectively). This was also noticed in the subgroup of squamous cell carci-

A strong agreement between nuclear SDF_1 and MMP2 was detected (KAPPA = 0.750), as 84.6% of positive MMP2 were positive nuclear SDF_1, and 90.9% of negative MMP2 was negative nuclear SDF_1 (Table 3). Moreover, combined expression of both SDF-1 and MMP2 expression is significantly correlated with high tumor stage. Logistic regression showed that MMP2, and nuclear SDF-1 were significant independent factors predicting cases with high tumor stage (III and IV), as positive MMP2, positive nuclear SDF-1 are associated with 21 and 24 higher risk of Higher tumor stage respectively. Also Nagelkerke R Square (0.788) was highest on using both markers for prediction of higher tumor stage (Table 4). Discussion Many attempts have been undertaken by scientists worldwide to identify potential biomarkers for lung cancer; the world No. 1 among cancer killers. Unfortunately, the acceptance of these markers as part of any of the diagnostic or prognostic procedures in lung cancer patients has been limited [1]. The present era belongs to personalized chemotherapy, and treatment choices based on molecular profiling are being studied intensely worldwide. These molecules are numerous, some of which have conclusively and consistently been shown to directly impact tumor behavior [35]. In the current study we aimed to focus immunohistochemically on two promising molecules; MMP-2 and SDF-1in NSCLC. Cancer invasion and metastasis are multi-factor, multi-step dynamic processes that involve the degradation of extracellular

520 Table 1

N.M. Osman, W.M. Osman Relation between tumor characteristics and MMP2 expression among all tumor cases.

Tumor characteristics

MMP2 Positive

P

Sig

Negative

No.

%

No.

%

T stage

T1 T2 T3 T4

2 14 12 11

5.1 35.9 30.8 28.2

22 9 1 1

66.7 27.3 3.0 3.0

0.001*

HS

LN metastasis

N0 N1 N2 N3 N4

2 7 13 17 0

5.1 17.9 33.3 43.6 0.0

12 19 1 1 0

36.4 57.6 3.0 3.0 0.0

0.001*

HS

Distant Metastasis

M0 M1

29 10

74.4 25.6

31 2

93.9 6.1

0.026*

S

Stage

I II III IV

2 4 23 10

5.1 10.3 59.0 25.6

12 19 0 2

36.4 57.6 0.0 6.1

0.001*

HS

Stage

Stage I/II Stage III/IV

6 33

15.4 84.6

31 2

93.9 6.1

0.001*

HS

Mean 5.99

±SD 2.03

Mean 3.42

±SD 1.82

0.002à

HS

Tumor size à

Student t test. Chi square test.

*

Figure 1 (A) and (B) Large cell carcinoma with positive cytoplasmic MMP-2 immunostaining of the tumor cells as well as the intervening tumor strom. (A) and (B) IHC  200, insets showed higher magnification of tumor cells. (IHC  400).

matrix and basement membrane. MMPs are an important family of proteolytic enzymes that significantly contribute to tumor microenvironment remodeling and associate with tumorigenesis and metastasis [36]. In the current study, MMP-2 was detected in the cytoplasm of tumor cells as well as in the surrounding tumor stroma. This is consistent to previous studies [12,37,38]. The importance of tumor–stroma interactions regulating cancer development has been fully pointed out in some previous studies [39,40]. Additionally, Matrix metalloproteinases (MMPs) are proteolytic enzymes which are proved to enhance tumor invasion and metastasis [39,41]. This was also proved in this study as high expression of MMP-2 was correlated with lymph node

and distant metastasis. Passlick et al. [12]; Kumaki et al. [13] and Ishikawa et al. [38], stated that in lung carcinomas either increased stromal or cancer cell associated with MMP-2 expression has been found to correlate with poor outcome. Furthermore, once a lesion is initiated, cancer associated fibroblasts ‘‘CAFs” have been shown to assist in proliferation and progression of cancer through the production of growth factors and chemotactic factors, angiogenesis factors, and MMPs, allowing invasion and spread of cancer cells i.e. creation of the metastatic niche [38]. Besides, Ma et al. [42] conducted a comparative analysis of gene expression changes and found that the genetic changes of the tumor-associated stroma were similar to those of the tumor

SDF-1 and MMP2 cross talk in cancer cells

521

Figure 2 (A) and (B) Moderately and poorly differentiated adenocarcinoma with positive cytoplasmic of MMP-2 immunostaining of the tumor cells as well as the intervening tumor strom. (A) and (B) IHC  400).

Figure 3 (A) and (B) Moderately and poorly differentiated squamous cell carcinoma with positive cytoplasmic of MMP-2 immunostaining of the tumor cells as well as the intervening tumor strom. (A) and (B) IHC  200).

epithelium. In particular, they found that tumor stroma that had reached the invasive stage expressed an increased level of MMPs, such as MMP-2. Accordingly, these findings support the theory that the tumor mass and its microenvironment have reciprocal relationships with both undergoing genetic expression alterations in cancer [43–45]. Conversely, in other studies the relationship of MMP-2 expression with other clinicopathological parameters predicting tumor aggressiveness has remained controversial [12,46,47]. In the current study, however; the MMP-2 expression was also found to be correlated with tumor size. This may be explained by the abnormal interplay consisting of cell–cell contact and active molecular crosstalk which further drives the cancer stroma phenotype, leading to direct stimulation of tumor cell growth. This unique interplay between the various aspects of the tumor cells and the microenvironment has been the recent target of molecular strategies for tumor treatment [48]. The function of MMPs is adjusted by the action of both different inducers and inhibitors, which regulate their tissue specific expression [40,49]. Chemokines structurally related small (8–14 kD) polypeptide signaling molecules, can bind to and activate a family of seven trans-membrane receptors [50,51]. Chemokines are expressed by many tumor types and can promote mitosis, modulate apoptosis, survival and angiogenesis [28,52]. To date, 19 chemokine receptors have been identified, one of these is SDF1 (also called CXCR12), is expressed in lung, bone and liver [34].

Stromal cell-derived factor (SDF-1) is a member of the superfamily of chemo-attractant chemokines [53]. In the current study, SDF-1 was overexpressed in 50% of NSCLC cases. Overexpression of SDF-1 was associated with poor prognostic parameters of NSCLC. The high expression of SDF_1 in tumor cells was correlated with an increased risk for LN metastasis, tumor size and distant metastasis. Therefore, targeting SDF-1 pathway may offer new potential therapeutic strategies for NSCLCs. This is in concordance with many studies that showed SDF-1 [53–55] expression was reported in a variety of tumor cell lines and human cancer samples, including malignant tumors from breast, brain, lung, kidney, esophagus, and skin [53,55–59]. High levels of SDF-1 expression correlate with metastasis and recurrence in non-small cell lung cancer [60]. Additionally, a previous study – by Mundy (2002) [61], revealed that SDF-1 secreted from bone cells (including osteoblasts and stromal cells) plays a key role in lung cancer cell metastasizing to bone. SDF-1a mediated invasion may involve the activation and secretion of MMP-2 [62,63]. This is in concordance to the current study which declared the direct relationship and strong agreement between both molecules in NSCLC cases. Thus, NSCLC cells have been shown to migrate and invade through ECM components in response to SDF-1a- actions, which is associated with enhanced expressions of MMP-2. This is also in agreement with Hung et al. (2010) [64] and Sze etal. (2011) [65]. However; McQuibban et al. [66], stated that

522 Table 2

N.M. Osman, W.M. Osman Relation between tumor characteristics and nuclear SDF_1 expression in all tumor cases.

Tumor characteristics

SDF-1 nuclear Positive

SDF-1 cytoplasmic Negative

Positive

Mean

±SD

Mean

Mean

±SD

T1 T2 T3 T4

0 13 11 12

0.0 36.1 30.6 33.3

24 10 2 0

66.7 27.8 5.6 0.0

20 6 2 0

71.4 21.4 7.1 0.0

4 17 11 12

9.1 38.6 25.0 27.3

0.001*

HS

LN metast.

N0 N1 N2 N3

0 5 14 17

0.0 13.9 38.9 47.2

14 21 0 1

38.9 58.3 0.0 2.8

10 17 0 1

35.7 60.7 0.0 3.6

4 9 14 17

9.1 20.5 31.8 38.6

0.001*

HS

Distant Metast

M0 M1

24 12

66.7 33.3

36 0

100.0 0.0

28 0

100% 0.0

32 12

72.7 27.3

0.002**

HS

Stage

I II III IV

0 4 20 12

0.0 11.1 55.6 33.3

14 19 3 0

38.9 52.8 8.3 0.0

10 15 3 0

35.7 53.6 10.7 0.0

4 8 20 12

9.1 18.2 45.5 27.3

0.001*

HS

Stage

Stage I/II Stage III/IV

4 32

11.1 88.9

33 3

91.7 8.3

25 3

89.3 10.7

12 32

27.3 72.7

0.001*

HS

Mean 6.18

±SD 1.91

Mean 3.44

Mean 5.68

±SD 2.12

0.001à

HS

±SD 1.83

Mean

Sig

T stage

Tumor size

±SD

P Negative

Mean 3.45

±SD

±SD 1.93

*

Chi square test. Fisher exact test. Student t test.

** à

Figure 4 (A) and (B) Large cell carcinoma with positive nuclear SDF-1 a immunostaining of the tumor cells (A) IHC  200, (B) IHC  400 respectively).

CXCL12/SDF-1 is proteolytically inactivated by MMPs-1, -3, -9 and -14 in vitro. Also, MMPs are proposed to display antimetastatic properties through CXCL12/SDF-1 cleavage [41]. But noticeably this is not true regarding tumors of the lung and MMP-2 where the microenvironment consists of an amalgam of secreted soluble factors, non-cellular solid material, and stromal cells that directly surround the tumor cells. Also these secreted soluble factors from the tumor cells are stored and released when required by the tumor cells [67,68]. Moreover, Logistic regression in the present study showed that MMP2, and SDF-1 nuclear were significant independent factors predicting cases with high tumor stage (3 and 4), as

positive MMP2, nuclear SDF-1 are associated with 21 and 24 higher risk of higher tumor stage respectively. These findings suggest a promising role for the SDF-1a/MMP-2 axis in the metastatic evolution of NSCLC, and its potential use as prognostic markers and drug targets. In conclusion, we present here a novel mechanism of SDF1a directed invasion of lung cancer cells by crosstalk with MMP-2 protein. The identification of SDF-1a from NSCLC tissues as a potential stimulatory factor of MMP-2 during human cancer cell invasion may help understand the mechanisms involved in the aggressive potential of lung cancer cells. In addition, the identification of SDF-1a/MMP-2 interaction

SDF-1 and MMP2 cross talk in cancer cells

523

Figure 5 (A) and (B) Well and moderately differentiated adenocarcinoma with positive nuclear/cytoplasmic SDF-1 immunostaining of the tumor cells (A) and (B) IHC  400).

Figure 6 (A) and (B) Well and moderately differentiated squamous cell carcinoma with positive nuclear SDF-1 immunostaining of the tumor cells respectively (A) and (B) IHC  400).

Table 3

Agreement between MMP2 and SDF-1 nuclear. MMP2 Positive

Nuclear SDF_1

Positive Negative

Kappa

Sig

0.750

0.001

Negative

N

%

N

%

33 6

84.6 15.4

3 30

9.1 90.9

Table 4 Logistic regression to study the effect of MMP and nuclear SDF-1 immunoexpression on tumor stage. OR

MMP2 (1) Nuclear SDF_1

P

Sig

95% C.I. for OR Lower

Upper

21.064

0.002

HS

3.062

144.901

24.229

0.0001

HS

4.061

144.565

Nagelkerke R square

0.788

as an important factor in the invasiveness of human lung cancer cells may implicate potential therapeutic approaches for metastasis from lung cancer.

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