Background
Angiogenesis inhibition [
1] has become a clinically accepted treatment of numerous malignant diseases (
www.cancer.gov/cancertopics/factsheet/Therapy/angiogenesis-inhibitors). The primary targets of anti-angiogenic therapy so far have been the main angiogenic factor VEGF (vascular endothelial growth factor) or its receptor VEGFR-2 [
2,
3]. Despite these advances, this therapeutic regimen is clearly not as straightforward as initially thought and certain reports suggest that some tumors may become more aggressive showing increased invasiveness and metastatic spread as a consequence of anti-angiogenic treatment [
4,
5].
SHB (Src homology domain containing protein B) is an adaptor protein [
6] operating downstream of VEGFR-2 [
7], the receptor active in VEGF’s angiogenic response [
8]. The
Shb knockout mouse phenotype was found to be pleiotropic with aberrations in female reproduction [
9,
10], glucose homeostasis [
11], the T lymphocyte response to T cell receptor stimulation [
12,
13] and the vasculature [
14-
16]. In particular, the vasculature displayed reduced angiogenesis and vascular permeability in response to VEGF [
14,
16]. Consequently, absence of one
Shb-allele conferred a restriction on tumor growth of Lewis lung carcinoma and T241 fibrosarcoma cells [
14] and of inheritable RT2 (rat insulin promoter-SV 40 T antigen) insulinomas [
15]. In addition to the ameliorating effects of
Shb-deficiency on pathological angiogenesis,
Shb knockout mice displayed vascular abnormalities that resulted in impaired recovery after ischemic injury [
16].
Shb knockout endothelial cells show reduced responsiveness to VEGF-stimulation with respect to ERK (extracellular-signal regulated kinase), Akt, FAK (focal adhesion kinase), Rac1 and myosin light chain kinase [
14,
17]. In concert, this abnormal signaling signature affects endothelial cell migration and adherens junction dissolution in response to VEGF [
14,
16,
17], explaining the vascular dysfunction
in vivo.
Melanomas are highly invasive cancers that metastasize at an early stage [
18,
19]. Since
Shb-deficiency appears to reduce tumor growth by restricting the angiogenic expansion of the vasculature, the question of whether this will cause increased tumor invasiveness and metastasis or not remains unanswered. In the RT2 insulinoma model, no evidence for increased liver metastasis was obtained [
15]. However, melanomas have a high inherent propensity for metastasis and for that reason, B16F10 melanoma cells were grown in
Shb-deficient mice and the numbers of lung metastases determined. Indeed, it was observed that melanoma metastasis was increased in
Shb-deficient mice because of a defective vasculature showing elevated vascular permeability and diminished recruitment of CD8+ cells to vascular structures.
Methods
Animals
Shb +/+ and +/− mice were bred on the C57Bl6 background for 8 generations (F8). Alternatively,
Shb −/− and +/+ mice bred for four generations (F4) on that strain of mice were used. It was previously shown that
Shb −/− mice cannot be obtained after breeding for more than 4 generations onto the C57Bl6 background [
10]. All animal experiments had been approved by the local animal ethics committee at the Uppsala County Court.
Tumor cell injections
B16F10 melanoma cells (2 x 105) were injected subcutaneously in the subscapular region. When the tumor reached a size of 0.5 – 1 cm3 (determined by a caliper) the tumor was resected under anesthesia. Excised tumors were weighed for size determination. The mice were housed for an additional 10–19 days (commonly, but not always, there was a tumor relapse deciding the end-point of the experiment) after which the mice were sacrificed. Some of the mice were injected with 2 mg/kg FITC-conjugated Dextran-70000 (46945, Sigma, St. Louis, MO, USA) 30 minutes before sacrifice into the tail vein in order to determine blood vessel permeability. For lung seeding, 200000 B16F10 cells were injected in the tail vein and the mice maintained for three weeks before sacrifice. Lungs were excised and macroscopically visible metastases counted. The area was also inspected carefully for lymph node metastases but none were detected. The resected primary tumor was frozen on dry ice for immunofluorescence staining or stored in RNA-later (Quiagen, Hilden, Germany) for subsequent RNA preparation.
Immunofluorescence
Excised tumors were sectioned (5 μm) and subjected to immunofluorescence staining for CD31 (553370, BD Pharmingen, Franklin Lakes, NJ, USA), VE-cadherin (vascular endothelial-cadherin) (AF1002, R&D Systems, Minneapolis, MN, USA), desmin (ab6322, Abcam, Cambridge, UK) and fibrin/fibrinogen (GAM/Fbg/7S, Nordic Immunological Laboratories, Eindhoven, the Netherlands) as previously described [
15].
At least five pictures were taken randomly of each tumor using a Nikon fluorescence and confocal C-1 microscope (Nikon, Japan). The area, diameter, perimeter of blood vessels, the fibrin spread area and pericyte covered length were measured with Image J software. Quantification of blood vessel permeability of FITC-conjugated Dextran was performed using Photoshop software.
Isolation of vascular fragments
Microvascular fragments were isolated from B16F10 melanomas grown on
Shb +/− and control mice as previously described [
20]. Briefly, tumors (0.5-1.0 cm
3) were perfused with Hanks’ salt solution under anesthesia and then excised. They were then cut into small pieces and digested in 1.5 ml of 5 mg/ml Collagenase A (#103586, Roche Diagnostics, Basel, Switzerland) and 100 U/ml DNaseI (Invitrogen, Carlsbad, CA) Hanks’ solution per tumor for 15 min at 37°C. The tumor suspension was pipetted, filtered through a 70 μm diameter cell strainer (BD Bioscience, Franklin Lakes, NJ), washed and filtered a second time with a 40 μm cell strainer. After washing, the cells were incubated with CD31-coated Dynabeads. The magnetic beads (with the captured vascular fragments) were collected using a magnetic rack, washed extensively after which RNA was prepared from the captured cells using the Quiagen RNeasy Mini Kit (Quiagen, Hilden, Germany). Endothelial cells were isolated as described [
14].
Gene expression
Total RNA of tumor was extracted according to RNeasy mini kit (74104; Qiagen) with RNase-Free DNase set (79254,Qiagen). One-step quantitative real-time RT-PCR was performed with QuantiTect™ SYBR®Green RT-PCR-kit (204243,Qiagen) on a LightCycler™ real-time PCR machine (lightcycler 2.0; Roche, Mannheim, Germany). Cycle threshold (Ct) values were determined with the LightCycler Software v3.5 (Qiagen). Gene expression was normalized for differences in RNA by subtracting the corresponding β-actin Ct-value. Statistical comparisons were made on normalized Ct-values. TaqMan qPCR gene expression analysis (Taqman, Life technologies, Carlsbad, CA) was used for analysis of PDGF-D (platelet-derived growth factor), CSFR2 (colony stimulating factor receptor 2), CXCL12 (chemokine C-X-C motif ligand), CXCR-4 (CXCL receptor) and CXCR-7.
Microarray analysis
High quality vascular fragment RNA from five tumors of each genotype was analyzed with Affymetrix 1.0 ST chips at the microarray core facility at Uppsala University Hospital (Uppsala Array Platform, Department of Medical Science, Science for Life Laboratory, Uppsala University Hospital, Sweden). Ingenuity software (Quiagen, Hilden, Germany) was used to perform pathway analysis on the microarray samples.
Bone marrow transplantation for generating chimeric mice
Iliac bones, femurs and tibias were collected from 8 to 10 week-old C57Bl/6
Shb +/+ and
Shb +/− donor mice and processed as described [
21,
22]. Cell numbers were determined and 1.5 x10
6 cells were transplanted into congenic
Shb +/+ or
Shb +/− recipients by retro-orbital injection. Prior to the bone marrow transfer, the recipients were irradiated [
22] with a split dose separated by two hours of 10 Gy. Peripheral blood chimerism in the recipient mice was determined 6 weeks post-transplantation by bleeding 100–200 μl blood in 0.05 mM EDTA. Total RNA was isolated from the remaining leukocytes with the RNeasy mini kit (74104; Qiagen). The
Shb gene expression in the chimeric mice was compared between different
Shb genotypes, following the real-time RT-PCR procedure.
In separate bone marrow transplantation experiments we assessed chimerism after bone marrow transfer by CD45.1 and CD45.2 staining after transfer of CD45.2-positive bone marrow to CD45.1-positive recipients. In such experiments, the donor bone marrow repopulated the host with more than 75% efficiency (results not shown).
Statistics
All values are given as means ± SEM. Probabilities (P) of chance differences between the groups were calculated with Mann–Whitney rank sum test (tumor metastases) or Students’ t-test (all other comparisons). Relative frequencies of metastasis were determined as follows: each mouse was categorized with category 0 having 0 metastases, category 1 having 1–5 metastases and category 2 having >5 metastases.
Discussion
The current study elucidates the metastatic properties of melanoma cells in relation to the absence of the
Shb gene. This cancer has been extensively investigated and tumor progression follows a characteristic pattern [
19]. The driver mutations in melanomas have recently been mapped [
26] and revealed a limited number of genetic changes in this cancer. Melanoma growth is dependent on angiogenesis [
18,
27,
28] and numerous angiogenic factors have been linked to melanoma growth [
29,
30] although the role of VEGF appears modest [
31]. Alternative candidates for support of melanoma growth and metastasis are inflammatory cytokines and chemokines [
32]. Factors of particular importance in this context are IL-8 [
29], CCL19/21 [
33], CXCL12 [
34], IL-6 and macrophage migration inhibitory factor [
35,
36]. The combined data suggest a scenario that utilizes multiple factors for melanoma growth and angiogenesis.
The B16F10 melanoma cell line is a useful model for studying melanoma metastasis
in vivo [
23] and consequently, we tested tumor metastasis in
Shb deficient mice. The increased rate of lung metastasis observed in
Shb deficient mice may have several explanations. One possibility is increased vascular permeability due to reduced pericyte coverage. However, gene expression profiling showed less expression of various markers for cytotoxic CD8+ lymphocytes in the
Shb +/− vasculature and this may offer an alternative suggestion. Due to some yet unknown feature of the
Shb deficient vasculature, passage of CD8+ cells over the vascular barrier into the tumor is specifically hampered thus causing less CD8+ cell infiltration into the tumor. This may contribute to increased metastasis since it is well established that CD8+ cytotoxic T cells combat melanoma growth and metastasis [
37-
39]. Indeed, treatment with an activator of CD8+ T cells, Ipilimumab (anti-CTLA-4), together with an inhibitor of VEGF signaling (Bevacizumab) causes perivascular CD8+ cell accumulation [
40], thus confirming the relevance of the vasculature for tumor-infiltration of cytotoxic T cells. The bone marrow transplantation experiments further support this notion, since the metastatic phenotype followed the recipient genotype,
i.e. more metastasis was seen in
Shb +/− recipients with a
Shb deficient vasculature despite these having a wild type bone marrow producing wild type blood cells. The decrease in tumor infiltration of CD8+ cells was indeed surprising, considering that vascular permeability was increased these conditions. Apparently, specific mechanisms operating in the vascular component control lymphocyte endothelial transmigration. Lymphocyte extravasation depends on numerous endothelial processes but selective mechanisms operating for lymphocytes and in particular for CD8+ lymphocytes are poorly defined.
The microarray analysis provides no obvious explanation for leaky vascular phenotype, although changes in the expression of numerous cytokines/chemokines could contribute to this. PlGF is one factor that has been suggested to increase vascular permeability [
41-
43] but more recent studies suggest a modest role, if any, of PlGF for vascular permeability [
44,
45]. Reduced PDGFRA expression could be explained as downregulation of its expression in a subset of pericytes, the B-pericyte [
25]. Apart from the reduction in PDGFRA expression, our study shows decreased perivascular desmin staining, a feature shared by these B-pericytes. Alternatively, reduced PDGFRA expression might indicate a reduction in myofibrillar cells. PDGFRA-expressing cells can be found on tumor-associated fibroblasts infiltrating B16 melanomas [
46] but their association with the vasculature has not been investigated. It is likely that in certain malignancies, fibroblasts might be found in the vicinity of the vasculature [
47], but this occurs normally in conjunction with alpha-smooth muscle actin expression, conferring these cells a myofibroblast phenotype. In our study, as analyzed by microarray, we did not detect significant differences in alpha smooth muscle actin expression, indicating that the decrease in PDGFRA expression may indeed reflect down-regulation of the B-type subset of pericytes. It is tempting to speculate that the shift towards an increase in type-A pericytes could confer the microvascular environment with features that enhance malignant cell intravasation. Increased expression of
Pten is, however, likely to have profound effects on endothelial function since this gene reduces phosphatidyl-3’-inositol levels and thus suppresses PI3K and Akt activities. These play major roles for angiogenesis and vascular permeability [
48] and thus a reduction in these would be predicted to be deleterious for vascular integrity.
The compromised vasculature in
Shb deficient mice increases the risk of intravasation of melanoma cells allowing them to disseminate in blood and infiltrate target tissues such as lung. Apparent is the fact that lung seeding of metastases after tail vein injections was not different between the genotypes, further implicating local vascular changes in the primary tumors as responsible for the effects. We were unable to detect increased metastasis to the liver of
Shb +/− insulinomas [
15] and the discrepancy between those findings and the current may lie in the difference in the local angiogenic milieu, which is probably dependent on a multitude of factors in melanomas whereas RIP-Tag2 insulinomas are highly dependent on VEGF and FGF-2 [
49].
Acknowledgements
We are grateful to Xin Wang for help in performing real-time RT-PCR, Anna Bereza-Jarocinska for immune staining and Ross Smith for tail vein injections. The study was supported by grants from the Swedish Cancer Foundation (130618, 120831, 2013–0782), Swedish Research Council (54x-10822), Swedish Diabetes fund (DIA 2012) and Family Ernfors fund. The microarrays were performed by the Uppsala Array Platform, Department of Medical Science, Science for Life Laboratory, Uppsala University, Entrance 61, 3rd floor, Uppsala University Hospital 751 85 Uppsala.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors participated in experimental design, in performing the experiments and in analysis and interpretation of the data. The specific contributions were: tumor growth and metastasis (GZ), tumor morphology (GZ, MJ, MW), bone marrow transplantation (KG), isolation of vascular fragments or endothelial cells (KG, GG, JH), RNA isolation (GZ, KG, MJ, GG, JH), qPCR (GZ, KG, MJ, JH) and Ingenuity analysis (GG, MW). MW wrote the paper and all authors provided comments. All authors read and approved the final manuscript.