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Introduction

Pancreatic cancer is a very aggressive disease with a median survival of 6 months and a dismal 5-year survival rate of 5% (1–3). Surgical resection remains the only curative option for pancreatic cancer (4–6). However, only 15–20% of patients undergo potentially curative surgery, as the majority of cases are unresectable at the time of diagnosis due to local major vessel invasion or remote metastases (7). Moreover, the median survival rate for those with clean microscopic surgical margins is approximately 2 years, with a 5-year survival of 15–20% (8). The malignancy of pancreatic cancer is strongly associated with its local recurrences and systemic metastases, even after curative resection for early stage pancreatic cancer (7,9,10).

As one of the most important routes for recurrence and metastases in pancreatic cancer, lymphatic metastases significantly correlate with poor prognosis (6,11–13). The number of metastatic lymph nodes and the ratio of metastatic lymph nodes to total number of examined lymph nodes have both been reported to significantly correlate with survival after surgical resection of pancreatic cancer (12,14). Lymphatic metastases also play important roles in systemic dissemination of cancer cells, as tumor cells in lymph nodes may provide a reservoir of cells leading to distant, lethal metastases (15–17). Moreover, the lymph node may also promote metastasis formation in distant organs by lymphatic-venous communications (18). However, the mechanisms of lymphatic metastasis in pancreatic cancer have not been fully studied for the lack of animal models that faithfully replicate the process of lymphatic metastasis in humans.

In the present study, we developed a BxPC-3-LN subline from a BxPC-3 pancreatic cancer cell line by serial in vivo selection. Subsequently, we characterized the phenotypes of the BxPC-3-LN cells by comparing them to the parental cells. Furthermore, to investigate the molecular mechanism in lymphatic metastasis, a comparative analysis of differential gene expression profiles in these two cell lines was performed by cDNA microarray.

Materials and methods

Cell culture and laboratory animals

The human pancreatic cancer cell line BxPC-3 was obtained from the American Type Culture Collection (Rockville, MD). Monolayer cultures of BxPC-3 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Seven to eight-week old immunodeficient male mice (BALB/c nu/nu) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., China, and housed in laminar-flow cabinets under specific pathogen-free conditions. All animal procedures were approved by the institutional animal care committee, Fudan University, China.

Establishment of new cell subline

The BxPC-3 cell subline was established by a serial in vivo selection process performed as previously described with some modifications (19,20) (Fig. 1). Each male nude mouse (n=6) was injected subcutaneously in the left hind footpad with 0.1 ml of BxPC-3 cells (1×107/ml). The mice were sacrificed at 8 weeks after tumor implantation. The possible metastatic lymph nodes, including homolateral popliteal, inguinal, iliac, para-aortic, para-renal, and mesenteric lymph nodes, were dissected and dissociated sterilely. Metastatic lymph nodes were confirmed by hematoxylin and eosin staining. The suspension was filtered using a 200-metal mesh strainer (Xinrui Biotechnology, Shanghai, China) and centrifuged at 1000 rcf/ min for 5 min at 4°C. The cell pellet was resuspended in culture medium and cultivated until stable populations of human tumor cells were established under the same conditions as detailed above. Cultured tumor cells were then serially re-inoculated into new recipient mice. After five rounds of in vivo selection, a variant of the BxPC-3 human pancreatic cancer cell line (BxPC-3-LN) with high lymphatic metastasis capacity was established.

Figure 1. Establishment of a highly lymphatic metastatic subline via in vivo selection. BxPC-3 cells were injected subcutaneously in the left hind footpad of male nude mice. The tumor diameter reached approximately 10-15 mm in 8-weeks. Autopsy was done to remove the possible homolateral metastatic lymph nodes. Metastatic lymph nodes were confirmed by pathological confirmation. Lymph nodes were then dissected, minced, dissociated and filtered using a 200-metal mesh strainer. Cells were plated, expanded and repassaged in the animals for additional rounds.

Cell morphology and ultrastructure

Cell morphology was viewed and photographed under a light microscope (Estativo Eclipse TE 2000S, Nikon, Spain). For ultrastructural observation, cells were fixed with 2.5% glutaraldehyde in 0.1 M PBS for 2 h at room temperature. For observation under scanning electron microscope (SEM), the specimens were immersed in isoamyl acetate, air-dried, and sputter-coated with gold before examination under the Hitachi S-520 microscope (Hitachi Ltd., Tokyo, Japan). For transmission electron microscopy (TEM), the specimens were immersed twice in absolute propylene oxide and embedded in Quetol 812. Following staining with uranyl acetate and lead citrate, the specimens were observed with a Jeol JEM-1230 electron microscope (Jeol, Tokyo, Japan) at 80 kV.

Cell cycle analysis

Cells were harvested, fixed by resuspension in 10 ml of 70% ethanol for 30 min, and washed in ice-cold PBS. They were incubated, washed and resuspended in DNA staining solution containing 20 μg/ml propidium iodide (Sigma-Aldrich) and 100 μg/ml RNase (Invitrogen). DNA content was determined by flow cytometry (BD FACS Aria, USA).

In vitro growth rate assay

To evaluate in vitro growth kinetics, growth curves were prepared for each cell line. A total of 2×103 cells/well were plated in triplicate in a 96-well plate and grown for 1–7 days. Cells were then incubated at 37°C for 4 h with 20 μl of MTS (Promega) per well, and absorbance was read at 492 nm. The number of viable cells was counted at the indicated time-points in triplicate.

Cell adhesion assay

The cell adhesion assay was performed as previously described with a slight change (21). Twenty-four-well plates were coated with 10 μg/ml of rat tail type IV collagen (BD Biosciences, Bedford, MA). The wells were blocked with 1% BSA for 1 h at 37°C. After the BSA solution was aspirated, 1×106 cells in 0.1% BSA were added to the wells in triplicate. At 15 and 30 min, the wells were washed to remove unattached cells, incubated at 37°C for 4 h in 100 μl MTS and measured for absorbance at 492 nm. The assay was repeated at least 3 times.

Cell migration assay

The cell migration assay was performed using a 24-well transwell chamber (Corning Costar, Corning, NY) migration assay as previously described with some change (22). Briefly, exponentially growing cells were trypsinized and washed in RPMI-1640/0.1% BSA medium twice. Cells (∼1×106) were seeded in the upper chamber of each transwell unit, which contained a polycarbonate 8-μm pore membrane pre-coated with 50 μl of 10 μg/ml fibronectin (BD Biosciences) overnight. The cells were allowed to migrate at 37°C and 5% CO2 for 24 h. The unattached cells were rinsed off with PBS and the membranes containing attached cells were fixed in 10% formalin for 30 min and washed with PBS. The cells were stained with crystal violet for 20 min and rinsed with water. Cells on the unmigrated side were gently wiped off with a wet cotton tip applicator and the membrane was rinsed with water. The membranes containing the migrated cells were dried and mounted onto slides with permount. The number of migrated cells per high power field (hpf) was determined by averaging 20 randomly counted hpfs. The assays were performed in triplicate and were reproducible in at least three batches of independent experiments.

Cell invasion assay

Transwell chamber inserts (Corning Costar) with filter membrane pore size of 8 μm were coated with 0.5 mg/ ml Matrigel (BD Biosciences) overnight. The membranes were rehydrated and 1×105 cells in serum-free RPMI-1640 medium were placed onto the inner chamber of each insert unit. Media with 10% FBS was placed into the outer chamber as a chemoattractant. The cells were allowed to invade at 37°C and 5% CO2 for 24 h. Cells were fixed in 10% formalin and washed with PBS. The cells were stained with crystal violet and rinsed with water. Cells on the unmigrated side were gently wiped off with a wet cotton tip applicator and the membrane was rinsed with water. The membranes containing the invaded cells were dried, and mounted onto slides with permount. The number of migrated cells per hpf was determined by averaging 20 randomly counted hpfs. The assays were performed in triplicate and were reproducible in at least three batches of independent experiments.

Drug cytotoxicity assay

Cells were plated at a density of 104 cells per well into 96-multiwell plates and were allowed to grow for 24 h before the experiment. The different concentrations (from 0 to 104 nM) of gemcitabine resuspended in 100 μl RPMI-1640 medium were added to the cells, which were then incubated for 24 h at 37°C. After initial incubation, cells were incubated at 37°C for 4 h with 20 μl of MTS (Promega), and absorbance was read at 492 nm. Chemosensitivity was expressed as the drug concentration that inhibited cell proliferation by 50% (IC50 values). The number of viable cells was counted at the indicated time-points in triplicate. Expression of chemoresistance genes related to gemcitabine, including ribonucleotide reductase M1 (RRM-1), ribonucleotide reductase M2 (RRM-2), human equilibrative nucleoside transporter-1 (hENT-1), and deoxycytidine kinase (dCK), were examined by reverse-transcription polymerase chain reaction (RT-PCR) as previously described (23).

In vivo tumor growth and metastatic potential

Eight-week old mice were injected with 1×106/ml BxPC-3 and BxPC-3-LN cells into the footpad (n=6). The primary tumors were allowed to form and the weight was measured every week. Tumor volume was calculated according to the formula: (length in centimeters x (width)2)/2. Six mice in each group were sacrificed separately at 8-week end-points to examine lymphatic involvement. The possible metastatic lymph nodes were divided into three groups: N1 (popliteal, inguinal), N2 (iliac, para-aortic), N3 (para-renal). At end-point, animals were euthanized and autopsies were performed. Organs (including lymph nodes, lungs, liver, spleen, pancreas, kidneys, ovaries, and any other tissues of abnormal appearance) were examined superficially for evidence of metastases using a dissecting microscope. Upon sacrifice, the tumors, metastatic lymph nodes, and other organs were fixed for hematoxylin and eosin staining.

cDNA microarray

Total RNA was extracted from BxPC-3 and BxPC-3-LN cells, respectively, with TRIzol (Life Technologies, USA) and dissolved in Milli-Q H2O. The RNA from BxPC-3 was labeled with Cy3-dUTP and that from BxPC-3-LN with Cy5-dUTP. Fluorescent probe mixtures were denatured at 95°C for 5 min, and applied to the prehybridized chip (Affymetrix, Inc., USA) under a cover glass, which was hybridized at 42°C for 15–20 h. After washing, the microarray was scanned on a scanner (Agilent, G2565BA) and the image was analyzed using Feature Extraction software (Agilent). The signal intensity of each spot was calibrated by subtraction from the intensity of the negative control. The assay was performed twice and only genes showing the same tendency of differential expression were eligible for the final-result analysis.

Statistical analysis

Fisher’s exact test and Student’s t-test were used for comparisons of enumeration data and to measure mean t data, respectively. The statistical analysis software package Stata 10.0 was used for the tests, and P<0.05 was considered statistically significant.

Results

Morphology and ultrastructure

Under a light microscope, the morphology of the BxPC-3-LN cell line was similar to the parental BxPC-3 cell line observed in culture. Both of them demonstrated polygonal epithelial morphology with large nuclei and 3–6 conspicuous nucleoli (Fig. 2A1 and A2). In addition, under SEM, in comparison to the parental cells, the BxPC-3-LN cells exhibited discernible aggressive morphological alterations such as filopodia and lamellipodia, protrusions emerging from the cellular membrane (Fig. 2B1 and B2). Under TEM, compared to the parental cells, the BxPC-3-LN cells contained more secretory granules (∼50–400 nm in diameter) (Fig. 2C1 and C2).

Figure 2. Morphology and ultrastructure of BxPC-3 and BxPC-3-LN cell lines. The morphology of BxPC-3-LN cells was similar to the parental BxPC-3 cells under light microscopy (A1 and A2). However, under SEM and TEM, the BxPC-3-LN cells exhibited discernible aggressive morphological alterations, such as filopodia and lamellipodia, and an increase in the number of secretory granules (B1, B2, C1 and C2).

In vitro cell growth

Cell cycle analysis by flow cytometry revealed that the percentage of BxPC-3-LN cells in S phase (13.3%) was similar to that of the parental cells (12.2%) (Fig. 3A). Growth curves were employed to evaluate the in vitro growth kinetics through MTS assay. BxPC-3-LN cells exhibited similar in vitro proliferation to BxPC-3 cells (Fig. 3B).

Figure 3. In vitro cell growth examined by flow cytometry, MTS assay and cell adhesion assay. The in vitro proliferation and proportion of cells in S phase was similar in BxPC-3-LN cells compared to parental cells (A and B). At the 15- and 30-min time-points, cell adhesion was significantly lower in BxPC-3-LN cells when compared to the BxPC-3 cells, ranging from 36.0 to 57.0% [(C) *,**P<0.05].

Cell adhesion, migration, and invasion assay

As shown in Fig. 3C, the highly metastatic BxPC-3-LN cells had decreased relative adherence compared to the parental BxPC-3 cells. At the 15- and 30-min time-points, the decrease in relative adherence ranged from 36.0 to 57.0% (P<0.05). Conversely, BxPC-3-LN cells demonstrated increased cell migration and invasion compared to the parental BxPC-3 cells (Fig. 4). In fact, when the migrated cells were counted, there was more than twice the number of cells migrated per high power field in BxPC-3-LN samples than in BxPC-3 samples (Fig. 4B1, B2 and B3).

Figure 4. In vitro migration and invasion ability assay. (A and B) BxPC-3-LN cells demonstrated increased cell migration and invasion compared to the parental BxPC-3 cells. (A2, A3, B2 and B3) Representative sample of a high power field of the migrated and invaded cells for the two cell lines (x400).

Drug cytotoxicity assay

We examined the cellular sensitivity of BxPC-3 cells and BxPC-3-LN cells to gemcitabine in vitro using the proliferation assay. Cells were treated with increasing concentrations of gemcitabine (50–300 nM) (Fig. 5A). BxPC-3-LN cells exhibited native resistance to gemcitabine and the IC50 value of BxPC-3-LN cells (210.5 nM) was higher than that of BxPC-3 cells (125.3 nM) (P<0.05). In addition, expression of chemoresistance genes related to gemcitabine was examined by RT-PCR. RRM-1, RRM-2 and hENT-1 expression was higher in BxPC-3-LN cells compared to BxPC-3 cells (Fig. 5B).

Figure 5. Drug cytotoxicity assay. (A) Cell proliferation following 24-h gemcitabine treatment was estimated by MTS assay. Quantitative values are means ± SEM from 3 independent experiments performed in quadruplicate. (B) Expression of chemoresistance genes related to gemcitabine examined by RT-PCR. RRM-1, RRM-2 and hENT-1 expression was higher in BxPC-3-LN cells compared with that in BxPC-3 cells.

In vivo spontaneous lymphatic metastasis via footpad injection

After five rounds of consecutive in vivo selection, the lymphatic metastases of BxPC-3-LN cells were obvious. When injected into nude mice, BxPC-3-LN cells had a >12-fold increase in presence of lymphatic metastases compared to the parental BxPC-3 cells (Fig. 6 and Table I). BxPC-3-LN cells also formed larger primary tumors when compared to the parental BxPC-3 cells (Fig. 6C). Total lymph node and total animal metastatic ratio of the BxPC-3-LN cells was 71.1 and 100.0%, which was higher than that of the parental BxPC-3 cells (5.6 and 16.7%, respectively) (Table I). No metastases were found in the liver, other abdominal viscera or the lungs.

Figure 6. In vivo spontaneous lymphatic metastasis via footpad injection. (A and B) Metastatic lymph nodes (A1 and B1) confirmed by pathological examination through hematoxylin and eosin staining (A2, A3, B2 and B3) (x100). (C) A million cells were injected into the footpad of nude mice and the dimensions and volumes of the tumor were measured and calculated every week.

Table I. Lymphatic metastasis of each lymph node group.

Table I.

Lymphatic metastasis of each lymph node group.

Cell lines	N1 (M-LNs/T-LNs) (%)	N2 (M-LNs/T-LNs) (%)	N3 (M-LNs/T-LNs) (%)	All lymph nodes (M-LNs/T-LNs) (%)	Total animals (M /T) (%)
BxPC-3	1/12 (8.3)	1/12 (8.3)	0/12 (0.0)	2/36 (5.6)	1/6 (16.7)
BxPC-3-LN	12/12 (100.0)	10/14 (71.4)	5/12 (41.7)	27/38 (71.1)	6/6 (100.0)

[i] M, metastatic; T. total; LNs, lymph nodes; N1, popliteal, inguinal lymph nodes; N2, iliac, para-aortic lymph nodes; N3, para-renal lymph nodes.

Comparison of gene expression between BxPC-3 and BxPC-3-LN cell lines

The differences in the gene expression levels between the two cell lines were assessed by measuring the ratios of their expression. Genes were identified as differentially expressed if the absolute value of the natural logarithm of the ratios was <0.3 or >2.0. Thirty-six genes were identified as being differentially expressed, including 6 with decreased expression and 30 with increased expression in BxPC-3 vs BxPC-3-LN (Fig. 7).

Figure 7. Comparison of gene expression by cDNA microarray. (A) BxPC-3; (B) BxPC-3-LN. Thirty-six metastasis-related genes were identified as being differentially expressed, including 6 with decreased expression and 30 with increased expression.

Discussion

Several human pancreatic cancer cell lines have been employed in studying the molecular mechanisms of pancreatic cancer lymphatic metastasis (24,25). Although these cell lines show different phenotypes and genotypes that are representative of pancreatic cancer, few of them have the capability for spontaneous lymphatic metastasis in immunodeficient mice (26,27). It is critical to establish appropriate cell lines and animal models to interpret the molecular mechanisms of lymphatic metastasis for pancreatic cancer.

The parental BxPC-3 cell line was first established from the body of the pancreatic adenocarcinoma of a 61-year-old female patient reported in 1986 (27). It is one of the most commonly referenced cell lines of pancreatic cancer (28). Compared to other pancreatic cancer cell lines, BxPC-3 shows moderate propensities of adhesion, migration, invasion and tumorigenicity (27). Evidence of lymphatic metastasis was rarely found to be associated with BxPC-3 (27). Therefore, it could serve as an ideal parental cell line from which to select highly lymphatic metastasis cell lines.

We used an in vivo selection process by injecting tumor cells into the footpad of nude mice and then examining and culturing the draining metastatic lymph nodes to establish the subline. This method was first developed by Carr et al in 1976 (19), and later confirmed as a useful and efficient method to develop lymphatic metastatic animal models (20,29). The basic model is to inject tumor cells into the footpad and then to examine the draining popliteal lymph node histologically at varying times thereafter (29). We have made some modifications from the previous methods. First, a repetitive in vivo selection process was used to establish the high lymphatic metastasis subline. Second, lymph nodes in the lymph draining route, including popliteal, inguinal, iliac, para-aortic, or para-renal lymph nodes, were removed and pathologically confirmed. Last, but most importantly, we improved this technique by using a metal mesh strainer to get rid of major non-tumor cells.

After five rounds of in vivo selection, a variant of the BxPC-3 human pancreatic cancer cell line with high lymphatic metastatic potential, BxPC-3-LN, was established. Although the morphology of BxPC-3-LN cells was similar to the parental cells by light microscopy, the ultrastructure of BxPC-3-LN cells observed under SEM and TEM showed a more aggressive change, with the presence of filopodia, lamellipodia and a greater number of secretory granules. In addition, our results demonstrated that the BxPC-3-LN cells exhibited decreased cell adhesion, and increased migration, invasion, drug resistance, and spontaneous lymphatic metastases in vivo, indicating the aggressive phenotype of the BxPC-3-LN cells.

BxPC-3-LN cells can be used as a valuable tool to study the molecular mechanisms of lymphatic metastasis for pancreatic cancer. For example, lymphatic metastatic related genes and proteins can be identified by comparing BxPC-3 and BxPC-3-LN cells. In the present study, we compared the gene expression between BxPC-3 and BxPC-3-LN by cDNA array assay. As expected, altered expressions of numerous genes related to invasion, motility and tumorgenicity in these cells were revealed. Metastasis-related gene alteration including upregulation of MMP14, MMP24, MIF, and ADRM1, and downregulation of TGFB2 and ROBO1 were observed. Our study demonstrated that gene expression patterns undergo complex alterations during the course of lymphatic metastasis of pancreatic cancer. In addition, this model may serve as an ideal platform to study potential therapeutic approaches for lymphatic metastasis of pancreatic cancer. For example, it can be used as a model to test drugs that may potentially inhibit lymphatic metastasis and eradicate tumor cells.

In conclusion, we have developed and characterized a highly lymphatic metastatic human pancreatic cancer cell line that could serve as a useful platform for the study of pancreatic cancer lymphatic metastasis and potential therapeutics.

Acknowledgements

This study was supported in part by the National Science Foundation of China (grant nos. 81101807, 81001058, 30901435 and 30972905), the Shanghai Science and Technology Commission (10JC1418900), the MacDonald Research Fund, and the William and Ella Owens Medical Research Foundation.

References