Background
Compartmentalization of eukaryotic cells requires transport of lipids and proteins between distinct membrane-bound organelles. Rab GTPases, which belong to the Ras superfamily of small GTP-binding proteins, are key regulators of membrane trafficking in eukaryotic cells. Up to now, more than 60 different human Rab proteins have been identified[
1,
2]. The functions of Rab GTPases depend on their ability to alternate between two conformational states, the inactive (GDP-bound) and the active (GTP-bound) state. Furthermore, their function depends on their capacity to reversibly associate with specific membrane compartments[
3,
4].
A growing number of Rab and Rab-related proteins has been functionally characterized [for a review see 1]. Rab31, also known as rab22B, is a 194 amino acid protein (Mr = 22,000), which shares highest homology with rab22A (71% sequence identity). Rab31 is mainly localized to the trans-Golgi, the trans-Golgi network (TGN) and to endosomes[
4,
5] and is involved in the vesicle transport from the Golgi apparatus to early and late endosomes,
e.g. of mannose-phosphate receptors[
6]. Furthermore, rab31 was shown to modulate epidermal growth factor receptor (EGFR) internalization in the epidermoid carcinoma cell line A431[
7]. Interaction with GDP/GTP exchange factors (GEFs) such as Gapex-5, EEA-1, and RIN proteins was demonstrated[
8,
9]. Additionally, direct interaction with the mRNA-binding protein HuR[
10] and the phosphatidyl-inositol(PI)-4,5-diphosphate-5-phosphatase ORCL-1[
11] was observed. ORCL-1 plays a key role in the regulation of the levels of PI(4)P and PI(4,5)P
2, two signaling molecules involved in membrane trafficking and Golgi/TGN organization[
11].
Dysfunctions in Rab pathways can lead to immunodeficiencies and neurological disorders. Also, dysregulation of Rab expression was shown to affect cancer progression[
12,
13]. Rab31 was identified as one out of 11 genes that are overexpressed in estrogen receptor (ER)-positive breast cancer patients[
14]. Recently, we could show, that elevated rab31 mRNA levels are significantly associated with shorter distant metastasis-free and overall survival of untreated, lymph node-negative breast cancer patients[
15]. More extensively characterized Rab proteins such as rab25 and rab21 were shown to be associated with increase in tumor cell proliferation and are required to promote cancer cell invasion[
13,
16]. High expression levels of rab25 are present in breast and ovarian cancer tissues and are associated with poor outcome of the patients[
17]. Conversely, rab25 overexpression in cancer cell lines substantially suppressed cell invasion
in vitro and tumor formation
in vivo[
18]. This indicates that dysregulation of Rab expression may exert either tumorigenic or tumor suppressive effects[
12].
However, little is known about the tumor biological effects of rab31 expression in breast cancer. Therefore, in the present study we aimed at analyzing the impact of differential rab31 expression in breast cancer cells on important aspects of tumor progression in vitro and in vivo. Using proliferation, adhesion, and invasion assays, we first characterized the phenotype of the cell transfectants with different rab31 expression levels in vitro, and then monitored the impact of rab31 expression on experimental metastasis in a xenograft tumor model in mice. Increased rab31 protein levels were associated with enhanced proliferation of breast cancer cells, led to a reduced adhesion of cells towards extracellular matrix proteins and decreased invasive capacity through MatrigelTM. In addition, we observed a significantly impaired metastatic dissemination of rab31 overexpressing MDA-MB-231 cells to the lung using a xenograft mouse model. Taken together, our results demonstrate that rab31 overexpression leads to a switch from an invasive to a proliferative phenotype as indicated by an increased cell proliferation, reduced cell adhesion, and decreased cellular invasion in vitro and in vivo.
Discussion
Rab proteins constitute the largest family of monomeric small GTPases. Numerous studies have established that Rab proteins are distributed to distinct intracellular compartments and regulate transport between organelles. Mutations in Rab proteins as well as altered GTPase expression or activity were shown to be involved in neurologic and neurodegenerative diseases, lipid storage disorders and cancer[
24]. A growing number of Rab proteins such as rab5, rab11, rab21, rab25, and rab27B have been shown to be associated with tumor growth/behavior and prognosis of breast cancer patients[
16,
25,
26]. However, still few data are available on rab31 expression in cancer in general or its impact on disease progression in particular[
10,
27]‒[
30]. Abba et al.[
14] described that rab31 mRNA is overexpressed in ERα-positive breast cancer tissue. We recently reported that elevated rab31 mRNA expression in primary tumor tissue is associated with poor prognosis of lymph node-negative breast cancer patients[
15].
In the present study, antibodies were raised against purified recombinant human rab31 and tested for cross reactivity with other, closely related Rab proteins, rab5 and rab22A. Because the generated polyclonal antibodies turned out to be highly specific for rab31, rab31 protein expression was verified in breast cancer tissue by immunohistochemistry. We detected a weak to moderate cytoplasmic staining for rab31 and, occasionally, strong perinuclear and/or nuclear staining of cancer cells, whereas stromal cells were less frequently stained. This cellular staining pattern is in accordance with data published in the human protein atlas (see
http://www.proteinatlas.org) in normal as well as tumor tissue. Moreover, in immunocytochemistry, we occasionally observed a pronounced perinuclear staining in MDA-MB-231 cells overexpressing rab31 which is very similar to the results of published immunocytochemical studies showing in different cell types a perinuclear patch of rab22B/rab31 in the trans-Golgi region [5,7,8; Additional file
1: Figure
S1].
To analyze possible cellular effects of variable rab31 protein expression, we stably transfected breast cancer cell lines with the eukaryotic expression plasmid pRcRSV harboring the rab31 cDNA sequence. In general, data based on unphysiologically high protein overexpression should be interpreted with caution. However, applying Western blot analysis and, in particular, a newly developed sensitive ELISA for quantitation of rab31, we found that for instance in MDA–MB-231 cells rab31 protein was increased up to about 5-fold only in the high expressing clones (≈ 2 ng/mg total protein) as compared to vector control cells (≈ 0.4 ng/mg total protein). In a study by Bao et al.[
31], the physiological rab31 content in platelets was estimated to be approximately 5 ng/mg total protein. Thus, it can be concluded that in the present study modulation of rab31 levels was within the physiologic range.
On one hand, overexpression of rab31 caused enhanced cell growth of breast cancer cells. These effects are not related to an increase in apoptosis in vector control cells if compared to rab31 overexpressing cells. Interestingly, using cell lines with a basic level of endogenous rab31 expression (MDA-MB-231), high levels of rab31 were necessary to induce a significant increase in cell proliferation. In opposite, already moderate levels of rab31 significantly elevated proliferation in cell lines not displaying any detectable endogenous rab31 expression (CAMA-1 and MDA-MB-435). On the other hand, high levels of rab31 in breast cancer cells resulted in significantly reduced adhesion towards ECM proteins as well as decreased invasive capacity. Likewise, the effects on cell adhesion and invasion were dose-dependent since effects monitored in low rab31 expressing cell clones differed significantly from those observed in rab31 high expressing cell clones. Comparing cell lines with differential endogenous rab31 levels, again the strongest effects were detected in breast cancer cell lines not showing any detectable endogenous rab31 expression (CAMA-1 and MDA-MB-435). In line with its reduced invasive capacity in Matrigel
TM invasion assays, we observed a significantly reduced number of lung metastases in immunologically compromised mice injected with rab31 overexpressing MDA-MB-231 cells compared to mice injected with vector control cells. Since development and growth of tumor metastases require that neoplastic cells must either have the potential to shift between proliferative and invasive phenotypes or simply express both simultaneously[
32], our results suggest that overexpression of rab31 may lead to a proliferative rather than an invasive phenotype in breast cancer cells (Table
2).
Table 2
Characteristics of MDA-MB-231 cells expressing different levels of rab31
Proliferation | + | ++ |
Adhesion | ++ | + |
Invasion | ++ | + |
Lung colonization | ++ | - |
To verify that the observed cellular effects are not artefacts due to overexpression of a protein
per se, we also analyzed overexpression of two rab31 mutants in MDA–MB-231 cells: rab31-Q64L is equivalent to constitutively activated GTPase-deficient mutants in other Rab proteins[
33], whereas rab31-ΔCC lacks the two C-terminal cysteine residues obligatory for prenylation (responsible for membrane association) and, in consequence, is inactive. In fact, overexpression of rab31-Q64L in MDA-MB-231 cells, similar to wild-type rab31 overexpression, led to significantly increased cell proliferation and decreased adhesion to ECM proteins, whereas cells overexpressing the inactive rab31-ΔCC behaved like the vector control cells (Additional file
2: Figure
S2).
Recently, it was shown that rab31 levels are regulated by the mRNA binding protein HuR[
10]. HuR is aberrantly expressed in early stages of breast carcinogenesis and high cytoplasmic expression is associated with reduced patients' survival in breast cancer[
34,
35]. Through its post-transcriptional influence on specific target mRNAs, HuR modulates cellular response to proliferative, apoptotic, differentiation and other stimuli [reviewed in[
36]. By enhancing the stability of rab31 transcripts and regulation of their translation, HuR may cause higher rab31 levels in tumor cells. In fact, when HuR expression was silenced in epithelial 184B5Me breast cancer cells, a significant reduction of rab31 mRNA expression was observed[
10]. Consistent with published results for other cancer types, HuR overexpression in MDA-MB-231 breast cancer cells (also used in the present study) resulted in increasing cellular growth rates and alterations in cell cycle kinetics
in vitro[
37]. Surprisingly, however, HuR overexpression significantly supressed tumor growth
in vivo whereas vector control and wild-type MDA-MB-231 cells grew similarly and resulted in much larger tumors than those formed by HuR overexpressing cells. This is very similar to our observation, indicating a strongly reduced metastatic capacity of rab31 overexpressing cancer cells in a xenograft mouse model despite the fact that overexpression of rab31 leads to increased proliferation of MDA-MB-231 cells
in vitro and high rab31 mRNA levels in (highly proliferating) primary tumor tissue are associated with a poor prognosis in breast cancer patients[
15].
In contrast to other Rab proteins[
38], the molecular mechanism of rab31 function is still unclear, in spite of identification of cytoskeletal participation and several interacting proteins. Immunocytochemistry analyses indicate that a large fraction of rab31 protein is localized to the perinuclear region, in the TGN and in endosomes[
4,
7,
8]. Recently, it has been demonstrated that rab31 is involved in the transport of mannose 6-phosphate (Man-6-P) receptors (MPRs), especially of the cation-dependent (CD-)MPR, from the TGN to endosomes[
6,
9].
In eukaryotic cells, MPRs are key components of the lysosomal enzyme targeting system that bind newly synthesized Man-6-P-containing acid hydrolases and function as efficient cargo transporters from the TGN to endosomal/lysosomal compartments[
39,
40]. The two distinct MPRs, the 46 kDa CD-MPR and the 300 kDa cation-independent (CI-)MPR are the sole members of the P-type lectin family [reviewed in[
41]. Although CD-MPR and CI-MPR bind the same array of proteins, the respective affinity of each MPR for different phosphorylated glycoproteins varies. This provides a biochemical mechanism, which, in part, may explain the interaction of the two MPRs with overlapping yet distinct subsets of ligands
in vivo[
42]. The CI-MPR has been described to bind proteins bearing the Man-6-P recognition marker as well as the peptide hormone IGF-II and to be implicated in numerous cellular processes, including cell growth, apoptosis, and cell migration[
43,
44]. In addition, CI-MPR has been shown to interact also with a number of proteins that do not contain Man-6-P such as urokinase-type plasminogen activator receptor (uPAR)[
45]‐[
47]. The uPAR binding epitope on CI-MPR is different from those binding Man-6-P and IGF-II. Binding of uPAR to MPRs seems to be specific for CI-MPR and independent of its ligand, the serine protease uPA. CI-MPR was proposed to be involved in the turnover of uPAR and to regulate the cell surface concentration of uPAR by directing uPAR to lysosomes as well as internalizing uPAR when it interacts with uPA[
45]. The detection of uPAR in endosomes agrees well with the primary function of MPRs, which is the transport of ligands from the TGN or cell surface to the endosome. Interestingly, both uPA and uPAR were upregulated in CI-MPR knockdown cells[
48].
Several splice variants of human uPAR have been described and their expression was analyzed in breast cancer cells and tissues. One of these splice variants, uPAR-del4/5, encodes a molecular form of uPAR which lacks domain DII of uPAR and does not interact with its ligand uPA[
21,
22]. Quantification of mRNA levels of uPAR-del4/5 in tumor tissues revealed that higher uPAR-del4/5 expression - similar to rab31 - is associated with shorter disease-free survival of breast cancer patients[
15,
21,
49]. Since uPAR as well as rab31 mRNA are known to be regulated by the RNA binding protein HuR both
in vitro and
in vivo[
10,
50], the observed elevated uPAR-del4/5 as well as rab31 expression in metastasizing breast cancer may be due to increased HuR expression in these tumors and points to a possible link between rab31 and the uPA/uPAR system in cancer cells. All three factors, HuR, uPAR-del4/5, and rab31, have been demonstrated to modulate cellular processes such as proliferation, adhesion and/or invasion. Strikingly, although high expression levels of these three factors (measured in highly proliferating primary tumor tissue) are clearly associated with poor prognosis of breast cancer patients, in experimental animal xenograft tumor models - using the same breast cancer cell line MDA-MB-231 - overexpression in each case unexpectedly leads to a seemingly reduced tumorigenicity. Tumor progression is known to be a multi-step process, including transitions of the malignant phenotype of tumor cells from a predominantly proliferative to a mainly invasive phenotype as well as interactions between tumor cell and tumor-associated stromal cells. The results obtained with the xenograft animal models, therefore, may not directly mirror the malignant features of rab31-, uPAR-del4/5- or HuR overexpressing tumors in human breast cancer. With regard to the plasticity of tumor cells in reliance of rab31 expression, our results, however, demonstrate that rab31 expression is implicated in modulation of tumor-relevant biological processes.
Methods
Cell culture and cell transfection
The human breast adenocarcinoma cell lines MDA-MB-231 and CAMA-1 (American Type Culture Collection [ATCC], Manassas, VA) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Eggstein, Germany) supplemented with 10% (v/v) fetal calf serum (FCS) (Gibco BRL), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gibco BRL), 0.55 mM L-arginine, and 0.272 mM L-asparagine (Sigma-Aldrich, Saint-Louis, MO). In some experiments, the human MDA-MB-435 cell line (ATCC) was used as well. Cells were routinely checked by polymerase chain reaction (PCR) to be free of mycoplasma infection.
Human full-length rab31 cDNA was obtained by real-time PCR (RT-PCR) using mRNA from human breast cancer tissue as a template. The cDNA was directionally subcloned into the pRcRSV plasmid using 5´-HindIII and 3´-XbaI restriction sites, which were introduced into the cDNA by PCR. The sequence was verified by sequencing and was found to be identical to that of the Genbank entry NM_006868.3.
Cells were transfected using Lipofectin® (Invitrogen, Karlsruhe, Germany). Cell transfectants were selected by addition of 1 mg/ml G418 (Gibco BRL) to the cell culture medium (Gibco BRL). In an independent transfection experiment, cell clones with a homogenous high, medium, or low expression of rab31 were isolated by limited dilution.
Generation of polyclonal antibodies directed against rab31
Recombinant rab31, harboring an N-terminal histidine (His)
6-tag, was expressed in
Escherichia coli and purified by nickel-nitrilotriacetic acid agarose affinity chromatography (Qiagen, Hilden, Germany) under denaturing/slightly reducing conditions as described previously[
51]. The purified recombinant rab31 protein, dialyzed against phosphate-buffered saline (PBS), pH 7.4, containing 1 mM dithiothreitol (DTT), was used as antigen for immunization of chickens and rabbits (Pineda Antibody Service, Berlin, Germany). Chicken IgY antibodies were purified from egg yolk as previously described[
52]. The IgG fraction of polyclonal rabbit antibodies was isolated from hyperimmune serum of rabbits by protein A affinity chromatography (Pineda Antibody Service).
One-sided ELISA for testing the reactivity of rab31-directed polyclonal antibodies
Purified antibodies were characterized using a ‘one-sided ELISA’ assay in which the specific antigen or an irrelevant protein were coated onto the wells of polystyrene microtiter plates as described[
19]. Briefly, test antigens diluted to 1 μg/ml in coating buffer (67 mM Na
2CO
3, 100 mM NaHCO
3, pH 9.6) were adsorbed to the wells of 96-well microtiter plates (Thermo Scientific, Nunc, Rochester, NY) and incubated overnight at 4°C. Following three washing steps with Tris-buffered saline (TBS), pH 7.4, containing 0.05% (v/v) Tween-20 (TBS-T), plates were blocked using blocking buffer (TBS-T containing 2% [w/v] bovine serum albumin, albumin [BSA], Sigma-Aldrich) for 1 h at room temperature (RT). After washing, polyclonal rabbit or chicken antibodies to rab31 (diluted 1:5,000 in TBS-T containing 0.5% [w/v] BSA) were applied to the wells for 1 h at RT. Subsequently, incubation with a secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA) or an HRP-labeled rabbit anti-chicken IgY (Sigma-Aldrich) as detection antibodies was carried out for 2 h at RT. Antigen binding was visualized using 3,3’5,5’-tetramethylbenzidine (TMB) / H
2O
2 as substrate (Thermo Scientific, Pierce, Stonehouse, UK) for 20 min at RT. After color development, the reaction was stopped by addition of 0.5 M H
2SO
4. The optical density of the resulting color was determined at 450 nm using an automated ELISA plate reader.
Cultured MDA-MB-231 and CAMA-1 cells were washed with PBS, pH 7.4 (Invitrogen), and cell pellets disrupted by two freezing and thawing cycles, followed by solubilization of rab31 antigen in extraction buffer (20 m M Tris-HCl, 125 mM NaCl, pH 7.6, containing 1% [v/v] Triton X-100, and the “Complete” protease inhibitor cocktail, Sigma-Aldrich). Cell lysates were centrifuged at 13,000 x g for 10 min at 4°C and the supernatant collected. The protein content was quantified using the Pierce Micro BCATM protein assay reagent kit (Thermo Scientific, Pierce).
Western blot
Proteins were separated by electrophoresis on 12% (w/v) polyacrylamide gels (SDS-PAGE), and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MA) in a semi-dry transfer device (Biometra, Göttingen, Germany). Membranes were incubated for 60 min in TBS, pH 7.4, containing 0.1% (v/v) Tween-20 (TBS-T) and 5% (w/v) dried skimmed milk, followed by an overnight incubation with the polyclonal rab31-directed chicken or rabbit antibodies, diluted in TBS-T supplemented with 5% (w/v) dried skimmed milk. After washing with TBS-T, binding of the antibodies was visualized by incubation of the membranes with a secondary HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Lab) or an HRP-labeled rabbit anti-chicken IgY (Sigma-Aldrich), followed by chemiluminescent reaction using ECL (Thermo Scientific, Pierce).
ELISA for quantitative assessment of rab31
For detection of rab31 in cell lysates, a sandwich ELISA format was developed using a commercial monoclonal antibody (mAb) to rab31 (mAb M01, Novus Biologicals, Inc., Littleton, CO) as catcher antibody and own polyclonal antibody (pAb) from rabbit #3 (RT3-IgG) as detecting antibody. Recombinant, purified rab31-His protein, dialyzed against PBS, pH 7.4, containing 1 mM DTT, was used as the reference antigen (stock 20 μg/ml). Ninety-six-well polystyrene plates (MaxiSorpTM; Thermo Scientific, Nunc) were coated overnight at 4°C with mAb M01 (clone 1C6; Novus Biologicals) diluted in coating buffer (15 mM Na2CO3, 33 mM NaHCO3, pH 9.6). After washing of the plates twice with washing buffer (PBS, containing 0.5% [v/v] Tween 20, pH 7.6), wells were blocked with blocking solution (washing buffer containing 2% [v/v] neonatal calf serum; Gibco BRL) for 30 min at 37°C. Thereafter, plates were incubated with cell lysates diluted in sample buffer (50 mM Tris-HCl, 100 mM NaCl, containing 0.2% [v/v] Triton X-100, and 0.2% [w/v] BSA, pH 7.6) for 90 min at 37°C. Two-fold serial dilutions of recombinant rab31-His protein in sample buffer, covering a concentration range of 0.15 to 5 ng/ml, were employed to construct a standard curve. After washing, wells were incubated with pAb RT3-IgG for 90 min at 37°C followed by incubation with secondary HRP-labeled goat anti-rabbit IgG (Novus Biologicals) for 60 min at 37°C. Finally, plates were washed and the peroxidase reaction initiated by addition of TMB / H2O2 (K & P Laboratories, Gaithersburg, MD) as the peroxidase substrate. After 20 min at RT, the reaction was stopped by addition of 0.5 M H2SO4. The optical density of the resulting color was determined at 450 nm (reference wavelength 620 nm) using an automated multichannel ELISA reader. Rab31 antigen levels are given as ng per mg of total protein.
Immunohistochemistry
Full-face sections from invasive ductal breast cancer tissue specimens were selected for immunostaining with pAb RT3-IgG and pAb from animal #4 (RT4-IgG), respectively. Formalin-fixed, paraffin-embedded breast cancer tissue specimens were obtained from archival material of the Institute of Pathology, Dresden University of Technology. The study adhered to national regulations on ethical issues and was approved by the local ethics committee at the Dresden University Medical Center. Immunohistochemical staining was performed as described previously with minor modifications[
19,
53]. Briefly, tissue sections were dewaxed, rehydrated, and treated for antigen retrieval by pressure cooking (15 min at 120°C) in 100 mM citrate buffer, pH 6.0. After several washes with PBS, sections were treated with 0.3% H
2O
2 for 10 min at RT to block endogenous peroxidase activity. Normal serum diluted in PBS was applied for 45 min at RT to block nonspecific antibody binding. Subsequently, primary antibodies (dilution: 1:2,000) were allowed to react overnight at 4°C followed by incubation with biotinylated anti-rabbit IgG (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA) for 50 min at RT. After washing, the Vectastain Elite ABC-reagent was applied for 50 min at RT, and the washing steps were repeated. The peroxidase reaction was developed with 3,3’-diaminobenzidine (Sigma-Aldrich) for 10 min at RT. Finally, counterstaining of nuclei was performed with hematoxylin. As a negative control, the primary antibody was omitted and replaced by PBS or by an irrelevant antibody.
Cell-based in vitro assays
Proliferation assays
Transfected MDA-MB-231 (and MDA-MB-435) cells were seeded in 24-well microtiter plates at a density of 20,000 cells per well, CAMA-1 cells at a density of 30,000 cells per well. All cell lines were incubated for 24, 48, 72 or 96 h at 37°C. After incubation, cells were detached by PBS plus 0.05% (w/v) EDTA (Biochrom AG, Berlin, Germany). Living cells were identified by trypan blue exclusion and counted in a hemocytometer under a light microscope.
Cell adhesion assays
Ninety-six-well plates were coated for 1 h at RT with vitronectin (2 μg/ml) or fibronectin (5 μg/ml) (BD Biosciences, Franklin Lakes, NJ), or for 3 h at 37°C with 5 μg/well of laminin, collagen type I or collagen type IV (Sigma-Aldrich), all diluted in PBS. Cells were resuspended in culture medium containing 0.5% (w/v) BSA and HEPES (Invitrogen), were seeded at a density of 20,000 cells/well (MDA-MB-231 and MDA-MB-435) or 30,000 cells/well (CAMA-1), and allowed to adhere to the extracellular matrix proteins for 2 h at 37°C. Subsequently, non-adherent cells were removed by washing three times with PBS. The number of adherent cells was quantified by a hexosaminidase activity assay. For this, cells were incubated with p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) diluted to 15 mM in 100 mM sodium citrate, pH 5.0, containing 0.5% (v/v) Triton X-100 for 90 min at 37°C. The reaction was terminated by the addition of stop buffer (0.2 M NaOH, 5 mM EDTA) and the optical density recorded at 405 nm using an automated ELISA reader.
Cell invasion assays
Invasion assays were performed using transwell inserts (8 μm pore size; Corning Costar, Amsterdam, The Netherlands). Thirty μg of basement membrane complex growth factor reduced MatrigelTM (BD Biosciences) was diluted in 100 μl cold FCS-free DMEM, containing 0.1% (w/v) BSA and applied to the upper side of the insert. After 3 h incubation at 37°C, followed by overnight incubation in a laminar hood at RT, inserts were rehydrated with 200 μl FCS-free DMEM, containing 0.1% (w/v) BSA for 2 h at 37°C. Cells suspended in culture medium were seeded into the upper chamber of the device at a density of 40,000 cells/chamber. The lower chambers were filled with 600 μl DMEM supplemented with 10% (v/v) FCS as the chemoattractant. After 24 h incubation at 37°C, MatrigelTM and non-invaded cells, located on the upper side of the insert, were removed with a Q-tip, whereas invaded cells on the lower side of the insert were fixed, stained using Hemacolor (Merck, Darmstadt, Germany), and counted under a light microscope.
MDA-MB-231 cells are tumorigenic and invasive cells, which upon intravenous injection in mice colonize to the lungs[
54]. Transfected MDA-MB-231 cells were genetically tagged with the
lacZ- gene, allowing X-Gal staining, as reported previously[
55]. Pathogen-free, 8 to 10 weeks old female athymic (nu/nu) mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice were allocated to two groups and 1 × 10
6 control MDA-MB-231-vector cells (n = 7) or pRcRSV-rab31-transfected MDA-MB-231 (batch) cells (n = 7) intravenously inoculated into the tail vein. At day 35 post-injection animals were sacrificed, lungs isolated and the
lacZ-tagged tumor cells stained with 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-Gal) (Roche Diagnostics, Mannheim, Germany) as described[
56]. Thereafter, blue stained cellular foci were counted as metastases. All animal experiments were done in compliance with the guidelines and with confirmed ethical approval of the
Tierschutzgesetz des Landes Bayern (Regierung von Oberbayern).
Statistical analyses
Group differences and p-values were calculated using the Mann-Whitney U-test. P values < 0.05 were considered as statistically significant.
Acknowledgements
This study was supported in part by grants provided by the Deutsche Krebshilfe e.V., Germany, Grant No. 108 050 (to MK) and Grant No. 107 965 (to VM), and by Framework Programme 7 project HEALTH-2007-201279, Microenvimet (to AK). We are grateful to Sabine Creutzburg, Antje Zobjack and Jörg Hofmann for excellent technical assistance.
Author details
1Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University of Munich, Ismaninger Str. 22, Munich 81675, Germany. 2Institute of Experimental Oncology and Therapy Research, Technical University of Munich, Ismaninger Str. 22, Munich 81675, Germany. 3Medizinisches Labor Ostsachsen, Töpferstr. 17, Bautzen, 02625, Germany. 4Institute of Pathology, Dresden University of Technology, Fetscherstr. 74, Dresden 01307, Germany.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
VM conceived the study, made substantial contributions to the conception and design of the study, and drafted the manuscript. BG participated in the design of the study, generated rab31 overexpressing cell lines, isolated cell clones, carried out cell-based in vitro assays, participated in the characterization of antibodies and in the in vivo experiment, and has been substantially involved in drafting the manuscript. SSö generated rab31(-mutant) overexpressing cell lines and carried out cell-based in vitro assays. SSch produced and purified the rab31 immunogen and was involved in the characterization of antibodies by Western blot and microtiter plate-based assays. BS performed the experimental in vivo metastasis assay. TK participated in the characterization of antibodies and carried out the immunoassay experiments. TL and MK were involved in the design and coordination of the study, participated in the characterization of antibodies and its application in immunoassays and immunohistochemistry, and have been substantially involved in drafting the manuscript. AK supervised the in vivo experiment and has been involved in drafting and critically revising the manuscript. GB and MS participated in the design of the study and have been involved in drafting and critically revising the manuscript. All authors have read and approved the final version of the manuscript.