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Abstract

Matrix metalloproteinase-12 (MMP12) is involved in many pathological processes including cancer. The expression and function of MMP12 in lung adenocarcinoma (LAC) remain unclear. The present study aimed to investigate the correlation of MMP12 expression with LAC patients and clarify its role in growth and invasion of LAC cells. The expression of MMP12 in human LAC was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was used for observing the effects of lentiviral vector-mediated MMP12 shRNA (shMMP12) on cell growth and invasion in LAC cell lines (A549), indicated by MTT and Transwell assays. We found that the expression of MMP12 protein was significantly increased in LAC tissues compared with that in adjacent non-cancerous tissues (ANCT) (57.69% vs. 32.69%, P = 0.019), and was closely correlated with the pathological stage and lymph node metastasis of LAC patients (P = 0.01; P = 0.003). Knockdown of MMP12 inhibited proliferation and invasion of LAC cells followed by the downregulation of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF). In conclusion, our findings show that high expression of MMP12 is correlated with the pathological stage and tumor metastasis of LAC patients, and knockdown of MMP12 suppresses the development of LAC cells, suggesting that MMP12 may be a promising therapeutic target for the treatment of LAC.

Keywords

growth invasion lung adenocarcinoma MMP12

Lung cancer is the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) subtypes, including adenocarcinoma (LAC), account for more than 80% of all lung cancers.¹ Although tremendous efforts have been devoted to improving treatment procedures through novel chemotherapies combined with targeted agents, the overall survival rate remains low.² Aberrant epigenetic landscape has been documented in lung cancer at early and late stages of carcinogenesis.³ Therefore, it is required for identifying survival predictors and prognostic biomarkers for LAC in the future.

The matrix metalloproteinases (MMPs) cause degradation of the extracellular matrix and basement membranes, and contribute to the pathogenesis of tissue destructive processes in a wide variety of diseases.⁴ MMP-12 is expressed in aortic dissection patients as a clinically diagnostic tool for vascular injury,⁵ and regulates adipose tissue expansion, insulin sensitivity, and expression of inducible nitric oxide synthase.⁶ MMP12 is overexpressed in cervical scrape cells from cervical precursor lesions,⁷ and contributes to the pathogenesis of cervical cancer.⁸ In addition, MMP-12 polymorphism is connected with a higher risk of disseminated colorectal cancer (CRC)⁹ and lung cancer,¹⁰ and indicates a poor prognosis in breast cancer¹¹ and esophageal adenocarcinoma.¹² Functionally, MMP12-induced myeloid cell autonomous defect leads to abnormal myelopoiesis, immune suppression, and tumorigenesis of LAC.¹³ MMP-12 promotes glioma invasiveness through interaction with tenascin-C.¹⁴ These studies show that MMP12 may be a critical regulator implicated in the development and progression of malignancies.

Few studies report that MMP12 expression correlates with the invasiveness of squamous cell carcinoma of the vulva but macrophage-derived MMP-12 predicts better outcome.¹⁵ To further verify the significance and function of MMP12 in cancer, we examined the expression of MMP12 in LAC tissues through immunohistochemical assay and investigated the function of MMP12 in cell growth and invasion of LAC cells. We found that high expression of MMP12 was correlated with the pathological stage and lymph node metastasis of LAC patients, and knockdown of MMP12 suppressed the development of LAC cells, suggesting that MMP12 may be a promising therapeutic target for the treatment of LAC.

Materials and methods

Materials

LAC cell line (A549) used for our experiments was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, PR China). Lentivirus-mediated MMP12 shRNA (shMMP12), negative control vector, and virion-packaging elements were purchased from Genechem (Shanghai, PR China). Human LAC tissues and the corresponding ANCT were collected from the Department of Thoracic Surgery of Huadong Hospital. The tissue microarray of LAC was made by Shanghai Outdo Biotech CO.LTD (Shanghai, PR China). All the antibodies were purchased from Cell Signaling Technologies (Boston, MA, USA). MMP12 primer was synthesized by ABI (Framingham, MA, USA).

Drugs and reagents

Dulbecco’s Modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA), TRIzol Reagent and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA,USA), M-MLV Reverse Transcriptase was purchased from Promega (Madison, WI, USA), SYBR Green Master Mix was obtained from Takara (Otsu, Japan), and the ECL Plus Kit was obtained from GE Healthcare (Piscataway, NJ, USA).

Clinical samples and data

Tissue microarray was prepared for IHC test. Human LAC tissues and the corresponding ANCT were obtained from biopsy in a total of 52 consecutive LAC cases admitted in our hospital from January 2009 to December 2011. The baseline characteristics of the patients before neo-adjuvant chemotherapy were summarized. The study was approved by the Medical Ethics Committee of Shanghai Fudan University and written informed consent was obtained from the patients or their parents before sample collection. Two pathologists respectively reviewed all of the cases.

Tissue microarray

The advanced tissue arrayer (ATA-100, Chemicon International, Tamecula, CA, USA) was used to create holes in a recipient paraffin block and to acquire cylindrical core tissue biopsies with a diameter of 1 mm from the specific areas of the ‘donor’ block. The tissue core biopsies were transferred to the recipient paraffin block at defined array positions. The tissue microarrays contained tissue samples from 52 formalin-fixed paraffin-embedded cancer specimens with known diagnosis, and corresponding ANCT from these patients. The block was incubated in an oven at 45°C for 20 min to allow complete embedding of the grafted tissue cylinders in the paraffin of the recipient block, and then stored at 4°C until microtome sectioning.

Immunohistochemical staining

Tissue microarray sections were processed for IHC analysis of MMP12 protein as follows. Immunohistochemical examinations were carried out on 3 mm thick sections. For anti-MMP12 immunohistochemistry, unmasking was performed with 10 mM sodium citrate buffer, pH 6.0, at 90°C for 30 min. For anti-MMP12 immunohistochemistry, antigen unmasking was not necessary. Sections were incubated in 0.03% hydrogen peroxide for 10 min at room temperature, to remove endogenous peroxidase activity, and then in blocking serum (0.04% bovine serum albumin, A2153, Sigma-Aldrich, Shanghai, PR China and 0.5% normal goat serum X0907, Dako Corporation, Carpinteria, CA, USA, in PBS) for 30 min at room temperature. Anti-MMP12 antibody was used at a dilution of 1:200. The antibody was incubated overnight at 4°C. Sections were then washed three times for 5 min in PBS. Non-specific staining was blocked with 0.5% casein and 5% normal serum for 30 min at room temperature. Finally, staining was developed using diaminobenzidine substrate, and sections were counterstained with hematoxylin. Normal serum or PBS was used to replace anti-MMP12 antibody in negative controls.

Quantification of protein expression

The expression of MMP12 was semi-quantitatively estimated as the total immunostaining scores, which were calculated as the product of a proportion score and an intensity score. The proportion and intensity of the staining was evaluated independently by two observers. The proportion score reflected the fraction of positive staining cells (0, none; 1, ⩽10%; 2, 10% to ⩾25%; 3, >25% to 50%; 4, >50%), and the intensity score represen-ted the staining intensity (0, no staining; 1, weak; 2, intermediate; 3, strong). Finally, a total expression score was given in a range of 0–12. Based on the analysis in advance, MMP12 expression was categorized into two groups: negative expression and positive expression. The scoring was independently assessed by two pathologists.

Cell culture and transfection

LAC cell lines were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Cells in this medium were placed in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were subcultured at a 1:5 dilution in medium containing 300 µg/mL G418 (an aminoglycoside antibody, commonly used stable transfection reagent in molecular genetic testing). On the day of transduction, LAC cells were replated at 5×10⁴ cells/well in 24-well plates containing serum-free growth medium with polybrene (5 mg/mL). When reached 50% conﬂuence, cells were transfected with recombinant experimental virus or control virus at the optimal MOI (multiplicity of infection) of 50, and cultured at 37°C and 5% CO₂ for 4 h. Then supernatant was discarded and serum containing growth medium was added. At 4 days post transduction, transduction efﬁciency was measured by the frequency of green ﬂuorescent protein (GFP)-positive cells. Positive and stable transfectants were selected and expanded for further study. The shMMP12 vector-infected clone, the negative control vector-infected cells and untreated cells were named as Lv-shMMP12 group, NC group, and control (CON) group.

Quantitative real-time PCR

To quantitatively determine the mRNA expression level of MMP12 in LAC cells, real-time PCR was performed. Total RNA was extracted for each clone using TRIzol according to the manufacturer’s protocol. Reverse transcription was carried out using M-MLV and cDNA amplification was performed using the SYBR Green Master Mix kit according to the manufacturer’s guidelines. Data were analyzed using the comparative Ct method (2^−∆∆Ct). Three separate experiments were performed for each clone.

Western blot assay

LAC cells were harvested and extracted using lysis buffer (Tris-HCl, SDS, mercaptoethanol, and glycerol). Cell extracts were boiled for 5 min in loading buffer, and then an equal amount of cell extracts was separated on 15% SDS-PAGE gels. Separated protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes, which were subsequently blocked in 5% skim milk powder. Primary antibodies against MMP12, PCNA, and VEGF were diluted according to the manufacturer’s instructions and incubated overnight at 4°C. Subsequently, horseradish peroxidase-linked secondary antibodies were added at a dilution of 1:1000 and incubated at room temperature for 2 h. The membranes were washed three times with PBS, and the immunoreactive bands were visualized using the ECL Plus Kit according to the manufacturer’s instructions. The relative protein levels in different cell lines were normalized to the concentration of GAPDH. Three separate experiments were performed for each clone.

Cell proliferation assay

Cell proliferation was analyzed using the MTT assay. Briefly, cells infected with shMMP12 virus were incubated in 96-well-plates at a density of 1×10⁵ cells per well with DMEM medium supplemented with 10% FBS. Cells were treated with 20 μL of MTT dye at 0, 24 h, 48 h, 72 h, and subsequently incubated with 150 μL of DMSO for 5 min. The color reaction was measured at 570 nm using an Enzyme Immunoassay Analyzer (Bio-Rad, Hercules, CA, USA). The proliferation activity was calculated for each clone.

Transwell invasion assay

Transwell filters were coated with Matrigel (3.9 µg/µL; 60–80 µL) on the upper surface of a polycarbonate membrane (diameter, 6.5 mm; pore size, 8 µm). After incubating at 37°C for 30 min, the Matrigel solidified and served as the extracellular matrix for analysis of tumor cell invasion. Harvested cells (1×10⁵) in 100 µL of serum-free DMEM were added into the upper compartment of the chamber. A total of 200 µL of conditioned medium derived from NIH3T3 cells was used as a source of chemoattractant, which was placed in the bottom compartment of the chamber. After 24 h of incubation at 37°C with 5% CO₂, the medium was removed from the upper chamber. The non-invaded cells on the upper side of the chamber were scraped off with a cotton swab. Cells that had migrated from the Matrigel into the pores of the inserted filter were fixed with 100% methanol, stained with hematoxylin, then mounted and dried at 80°C for 30 min. The number of cells invading through the Matrigel was counted in three randomly selected visual fields from the central and peripheral portion of the filter by using an inverted microscope (200× magnification). Each assay was repeated three times.

Statistical analysis

SPSS 20.0 was used for the statistical analysis. Kruskal-Wallis H test and Chi-squared test were used to analyze the expression rate in all groups. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. The LSD method of multiple comparisons was used when the probability for ANOVA was statistically significant. Statistical significance was set at P <0.05.

Results

Expression of MMP12 in LAC tissues

The expression of MMP12 protein was examined by IHC staining. The positive expression of MMP12 protein was detected in LAC and ANCT tissues (Figure 1). Positive MMP12 immunostaining was mainly localized in the cytoplasm of LAC cells. According to the MMP12 immunoreactive intensity, the positive expression of MMP12 was significantly increased in LAC tissues compared with the ANCT (57.69% vs. 32.69%, P = 0.019) (Table 1).

Figure 1.

The expression of MMP12 in LAC tissues (× 200). The expression of MMP12 protein was increased in LAC tissues but decreased in ANCT.

Table 1.

Expression of MMP12 proteins in LAC tissues.

Target	Group	Total	n				Positive rate (%)	χ²	P
			−	+	++	+++	Positive rate (%)
MMP12	LAC	52	22	16	10	4	57.69
	ANCT	52	35	8	7	2	32.69	5.487	0.019

ACT, adjacent non-cancerous tissues; LAC, lung adenocarcinoma.

Correlation of MMP12 expression with clinicopathologic parameters

The association between MMP12 expression and clinical and pathologic features was analyzed. As shown in Table 2, increased expression of MMP12 was closely correlated with pathological stage and lymph node metastases of LAC (P = 0.01; P = 0.003). However, no significant link was found between MMP12 expression and other factors including age, gender of the patients, and tumor size and degree of differentiation of LAC (P >0.05, respectively).

Table 2.

The correlation of MMP12 expression with clinicopathologic characteristics of LAC patients.

Variables	Cases (n)	MMP12 expression		P value
		−	+
Total	52	22	30
Age (years)
⩾60	30	12	18
<60	22	10	12	0.697
Sex
Male	37	14	23
Female	15	8	7	0.310
Tumor size (cm)
⩾5	42	17	25
<5	10	5	5	0.587
Degree of differentiation
Well/Moderately	12	7	5
Poorly	40	15	25	0.205
Pathological stage
I + II	18	12	6
III + IV	34	10	24	0.01
Lymph node metastasis
Negative	23	15	8
Positive	29	7	22	0.003

Effect of MMP12 knockdown on the expression of PCNA and VEGF

After Lv-shMMP12 was transfected into LAC cell line (A549) for 24 h, the expression level of MMP12 mRNA (Figure 2A) and those of MMP12, PCNA, and VEGF proteins (Figure 2B) were detected by real-time PCR and western blot assays, respectively, which indicated that the expression of MMP12, PCNA, and VEGF was downregulated in shMMP12 group compared with the CON and NC groups in LAC cells (**P <0.01).

Figure 2.

The effect of MMP12 knockdown on PCNA and VEGF expression. (A) The mRNA expression of MMP12 indicated by real-time PCR assay was decreased in shMMP12 group compared with the CON and NC groups in LAC cells (**P <0.01). (B) The protein expression of MMP12, PCNA, and VEGF indicated by western blot assay was reduced in shMMP12 group compared with the CON and NC groups in LAC cells.

Effect of MMP12 knockdown on cell proliferation

To verify the effect of MMP12 on cell growth, we evaluated cell proliferative activities by MTT assay. The results showed that knockdown of MMP12 could reduce the proliferative activities of LAC cells in a time-dependent manner compared to the CON and NC groups (**P <0.01) (Figure 3).

Figure 3.

The effect of MMP12 knockdown on cell proliferation. MTT assay was used to assess the growth of LAC cells. Knockdown of MMP12 significantly inhibited the proliferative activities of LAC cells in a time-dependent manner compared with the CON and NC groups (**P <0.01).

Effect of MMP12 knockdown on cell invasion

To determine the effect of MMP12 on cell invasion, a Transwell assay was performed. The invasive potential of tumor cells in Transwell assay was determined by the ability of cells to invade a matrix barrier containing laminin and type IV collagen, the major components of the basement membrane. Representative micrographs of Transwell filters can be seen in Figure 4A. The invasive potential of LAC cells was markedly weakened in shMMP12 group compared to the CON and NC groups (**P <0.01) (Figure 4B).

Figure 4.

The effect of MMP12 knockdown on cell invasion. (A) The invasive potential assessed by Transwell assay was determined on the basis of the ability of cells to invade a matrix barrier containing laminin and type IV collagen. (B) The invasive capabilities of LAC cells were decreased in shMMP12 group compared with the CON and NC groups (**P <0.01).

Discussion

The alterations in genomic content and changes in gene expression levels are central features of tumors and are pivotal to the tumorigenic process. It is verified that the changes in expressions of MMP12 contribute to the tumor development and progression of NSCLC.¹⁶ MMP12 as a potent pro-inflammatory and oncogenic molecule promotes the emphysema to LAC transition that is facilitated by inflammation.¹⁷ MMP-12 is also a valuable prognostic marker for overall survival and tumor recurrence of hepatocellular carcinoma (HCC) patients after liver resection,¹⁸ and may serve as an unfavorable prognostic marker of non-invasive oral carcinoma.¹⁹ Few studies show that MMP1 and MMP10 but not MMP12 are potential oral cancer markers.²⁰ To further clarify the significance of MMP12 in LAC, our present study indicated that MMP12 was highly expressed in LAC tissues compared to the ANCT, and was closely correlated with the pathological stage and lymph node metastasis of LAC patients. Additionally, Hofmann et al.²¹ found that MMP12 is also correlated with local recurrence and metastatic disease. Multivariate Cox regression analysis reveals MMP-12 expression to be an independent prognostic factor for tumor relapse-free interval in NSCLC. These findings suggest MMP12 is a valuable therapeutic target for LAC.

MMPs play an important role in tumor invasiveness and metastasis. As a molecular marker, MMP12 has been reported involved in the metastatic nodal diseases including LAC²¹ and head and neck squamous cell carcinomas.²² Increased expression of MMP12 by S100A8/A9 promotes cell migration and invasion through p38 MAPK-dependent NF-κB activation in gastric cancer.²³ Downregulation of MMP12 by siRNA or specific inhibitor PF-356231 can significantly inhibit the migration and invasion of nasopharyngeal carcinoma cells.²⁴ We further evaluated the function of MMP12 in LAC cells and found that knockdown of MMP12 also decreased cell proliferation and invasion of LAC cells. MMP12-engineered endothelial progenitor cells (EPCs) inhibit tumor growth, tumor angiogenesis, and development of lung metastasis.²⁵ These studies provide a new therapeutic approach to control tumor progression and metastasis.

PCNA is a simple and clinically useful method for assessing cell proliferation in formalin-fixed and paraffin-embedded NSCLC tissue.²⁶ Tumor growth is dependent on new blood vessel formation. Inhibition of vascular endothelial growth factor (VEGF) is sufficient to prevent tumor growth and dissemination.²⁷ Overexpression of MMP12 controls uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.²⁸ In the present study, we found MMP12 knockdown downregulated the expression of PCNA and VEGF in LAC cells, suggesting that MMP12 might be involved in the development of LAC cells through the PCAN and VEGF expression.

In conclusion, our findings show that high expression of MMP12 is correlated with the pathological stage and tumor metastasis of LAC patients, and knockdown of MMP12 suppresses the development of LAC cells, suggesting that MMP12 may be a promising therapeutic target for the treatment of LAC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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