Embryo analysis and β-galactosidase (lacZ) staining
Time of gestation was calculated using noon on the day of detection of a vaginal plug as E0.5, but also noting and comparing the external appearance of the embryo. Embryos were dissected at E15.5 then fixed in 0.25% glutaraldehyde at 4°C for 45m on a rolling platform. Embryos washed in PBS at 4°C for 15m on a rolling platform, detergent washed (2 mM MgCL2, 0.01% Na-deoxycholate, 0.02% NP-40 in PBS) at room temperature ×3 (30 min, 15 min × 2). β-galactosidase substrate (1M MgCl2, 0.02% NP-40, 0.01% Na-deoxycholate, 0.04% X-Gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6) then added to the embryos which were incubated in darkness overnight. Embryos post-fixed in 4% paraformaldehyde for 2h at 4°C.
For cell number assays, the area used for quantification of cell numbers represented the most proximal point of melanoblast migration from the neural crest which could be accurately assayed. Counting melanoblasts in more distal regions would have introduced a migrational component to the assay which we aimed to avoid.
Mouse treatments, ageing and survival cohorts
80 Tyr::NrasQ61K/°; INK4a−/− and 10 C57BL6 mice were monitored for up to 18 months for development of melanoma and signs of metastasis. All mice checked 3×weekly for development of malignant melanoma or any other pathology. Endpoint criteria were melanomas ≥ 15mm, ulcerating melanomas, cachexia, significant weight loss, or weakness and inactivity. Upon meeting these criteria, mice were euthanized. Mice were examined for the presence of frank metastasis upon dissection, but also visualisation of haematoxylin and eosin (H&E) stained sections for further identification of microscopic metastases. Organs/tumors were removed and fixed in 10% buffered formalin overnight at room temperature. Fixed tissues were paraffin embedded and 5mm sections placed on sialynated/poly-L-lysine slides for immunohistochemical analysis. Lymph node metastases were not included in this analysis due to difficulty in distinguishing them from normal melanocyte populations that can be found in lymphoid tissue.
For TV-injections into C57BL6 mice, cultured cells maintained in antibiotic-free media for 1 week prior to injection. Cells were detached with trypsin, then blocked through suspension in complete culture media supplemented with 10 % foetal bovine serum. Cells subjected to two rounds of washing, involving centrifugation at 100gs for 5m followed by resuspension in 1 × Hanks’ Balanced Salt Solution (HBSS). Cells finally resuspended in HBSS to a concentration of 1 × 107 cells/ml and TV-injected at 1 × 106 cells/animal in a volume of 100µl.
For P-Rex1, serial sections were unmasked in 10 mM citric acid, pH 6.0 with boiling for 30m. Remaining steps were carried out with Thermo ScientificUltraVision LP Detection System (Thermo Fischer Scientific). Primary antibody was incubated in the presence of 5% normal goat serum (Dako). Antigens were developed with Vector Red Alkaline Phosphatase Substrate Kit (Vector Labs). The following antibody dilutions were used: P-Rex1, 1:100; MelanA, 1:100. ‘Positive’ staining refers to any level of staining visualized.
Rabbit polyclonal raised against human P-Rex1 (HPA001927) and mouse monoclonal specific to β-actin (clone AC-15, A1978) were obtained from Sigma-Aldrich limited. Mouse monoclonal specific to Melan-A (clones DT101 + BC199, ab731) was obtained from Abcam Ltd.
A tissue microarray (TMA) was performed on archival paraffin patient samples from St. Vincent’s University Hospital, Dublin, Ireland. Quadruplicate cores from 141 consecutive melanoma patients (1994–2007) were used to construct the TMA. Further samples were received from Radboud University Medical Center, Nijmegen, Netherlands. All patient specimens were used in accordance with institutional and national policies at the respective locations.
Statistical Analyses in mice were carried out using Minitab® version 15 for Windows. Cell migration, invasion, and cell number differences determined using Mann-Whitney test. Distinction of metastasis and primary melanoma incidence was achieved using chi-square testing. Survival differences were determined with log-rank testing. All p values considered significant at p = <0.05. All appropriate value sets tested for normality using a Kolmogorov-Smirnoff normality test.
Normal human melanocytes (NHM) were maintained in Mln254 medium (M-254-500, Cascade Biologics) supplemented with Human Melanocyte Growth Serum (S-002-5, Cascade Biologics) and penicillin/streptomycin at 100 U/ml. Mel224, Mel505, SK-Mel 2, 5, 23, 119, 147 and 187, CHL-1, A375 and WM266.4 were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% foetal calf serum, L-glutamine at 200µM and penicillin/streptomycin at 100 U/ml. SBCL2, Lu1205, WM852, MeWo, Dauv-1, Gerlach, 888mel, 501mel, MNT-1, WM 35, 278, 793, 902b, 1552c and 1789 were maintained in RPMI 1640 supplemented with 10% foetal calf serum and penicillin/streptomycin at 100U/ml.
For melanocyte isolation from mice, pup skin was dissected at P2 then placed in ice cold PBS. Quickly it was cut into pieces and incubated in 1.5ml of collagenase type 1 and 2 at 37°C, 5% CO2 for approximately 25–50m. Contents transferred into 10ml wash buffer (1×HBSS, 1mM CaCl2, 0.005% DNase) and centrifuged at 200gs for 5m at room temperature. Sample resuspended in 2ml dissociation buffer, placed in small Petri dish and incubated at 37°C, 5% CO2 for 10m. Thereafter, sample put through an 18g then 20g needle and transferred into 10ml wash buffer for 10m. Supernatant centrifuged at 200gs for 5m at room temp, pellet resuspended in 2ml PBS, then re-centrifuged at 200gs for 5m. Resuspended and maintained in RPMI 1640 medium supplemented with 10% foetal calf serum, L-glutamine at 200µM, and penicillin/streptomycin at 100 U/ml.
siRNA treatments and RacGEF constructs
Stable cell lines expressing Myc-epitope tagged human P-Rex1 were generated by retroviral infection using the modified Retro-X retroviral expression system (Clontech). An Hpa I restriction site, followed by Kozak consensus translation initiation site was introduced to the 5’ end of the coding sequence of myc-P-Rex113
, myc-P-Rex1 GEF-dead13
, or myc-Tiam1 by PCR (5’ GTTAACCACCATGGAGCAGAAGCTGATC 3’), with a Cla I restriction site introduced to the 3’ end in the same reaction (P-Rex1/P-Rex1 GEF-dead - 5’ CCATCGATTCAGAGGTCCCCATCCACCGG 3’), with pCMV-P-Rex1, pCMV-P-Rex1 GEF-dead or pcDNA3.1-myc-Tiam1 used as template. In each case, the Hpa I-Cla I DNA fragments produced were subcloned into Hpa I and Cla I sites of the pLHCX retroviral expression vector. High-titre, replication-incompetent retroviral particles encoding the RNA of interest were produced in the Phoenix Ampho packaging line (Orbigen), for human target cells, and the Phoenix Eco packaging line (Orbigen) for murine target cells. Subsequent infection of target lines resulted in transfer of the coding region of interest, along with a selectable marker. Pooled cell lines stably expressing the construct of interest were isolated by selection with hygromycin-B (500 µg/ml) over multiple passages. Control lines were infected with retroviral particles expressing an empty pLHCX control vector transcript, and subjected to an identical selection procedure. Expression of the ectopically-introduced proteins of interest was determined by western blot and immunodetection with both epitope-tag specific and protein specific primary antibodies.
Transient knockdown of target proteins was achieved through consecutive rounds of liposome-mediated transfection with the appropriate siRNA oligonucleotides, 48 h apart. Liposomal transfection reagent (301702, HiPerFect), non-targeting control oligonucleotides (1027281, AllStars Negative Control) and P-Rex1 specific oligonucleotides (SI00692405, Hs_PREX1_3; SI03144449, Hs_PREX1_5; SI03246383, Hs_PREX1_6) obtained from Qiagen Ltd.
For inverted Matrigel invasion assays38
, Matrigel protein matrix (BD bioscience) was allowed to polymerise in Transwell permeable inserts (Corning Ltd.) over a period of 60m at 37°C. Inserts were inverted, and cells seeded directly onto the filter surface in complete growth medium. Cells then allowed to adhere over a period of 3h at 37°C, after which both non-adherent cells and residual growth medium were removed with 3×washes in appropriate serum-free medium. Finally, inserts were placed in serum-free tissue culture medium (containing 10% foetal calf serum) above the Matrigel matrix to act as a chemoattractant. In the case of siRNA-mediated transient knockdown experiments, invasion assays were prepared 24 hours after the second round of lipofection. At 72h post-seeding, invasive cells which had entered Matrigel were stained with the fluorescent live-cell dye Calcein-AM, and visualised through confocal microscopy of optical sections obtained in the z-plane at 15 µm intervals. Quantification was with the Area Calculator plugin for ImageJ (http://rsbweb.nih.gov/ij/
For organotypic invasion assays39
/ml primary human fibroblasts were embedded in a three-dimensional matrix of rat tail collagen I. Rat tail tendon collagen solution was prepared by the extraction of tendons with 0.5 M acetic acid to a concentration of ~2 mg/ml. Detached, polymerized matrix (2.5 ml) in 35mm petri dishes was allowed to contract for approximately 6 days in complete media (DMEM, supplemented with 10% FCS, Invitrogen) until the fibroblasts had contracted the matrix to ~1.5 cm diameter. Subsequently, Tyr::NrasQ61K/°; INK4a−/−; P-Rex1−/−
melanocytes, stably expressing either control vector, human P-Rex1 or a GEF-dead mutant of human P-Rex1, were seeded onto the prepared matrix in complete media (4 × 104
cells per assay) and allowed to grow to confluence for 5 days. The matrix was then mounted on a metal grid and raised to the air/liquid interface resulting in the matrix being fed from below with complete media that was changed every 2 days. After 15 days, the cultures were fixed with 4% paraformaldehyde and processed by standard methods for haematoxylin and eosin staining.