Background
Uveal Melanoma (UM) is the most common intraocular malignancy in adults [
1,
2]. Despite effective control of the primary tumor, about 50% of UM patients develop metastatic disease. The mortality rate of these patients has not significantly changed in the last four decades due to the lack of an effective clinical treatment against metastatic disease [
2]. The liver is the main site of primary metastasis in over 75% of cases, as UM tumor cells disseminate hematogenously [
1,
3], and the CXCR4 [
4‐
6] and c-Met [
7,
8] receptors, which are present in UM, mediate migration toward ligand gradients produced by the liver. Studies aiming to understand the mechanisms and patterns of UM dissemination within the liver are limited. We recently identified two types of metastatic growth pattern through
post-mortem analyses in human livers of metastatic UM patients: nodular and infiltrative [
9]. The nodular pattern is characterized for growing adjacent to the portal venule effacing the surrounding hepatic parenchyma. The hepatocytes are pushed aside destroying the pre-existing liver architecture. These hepatocytes are separated from the tumor cells by a thin layer of reticulin fibers. In contrast, the infiltrative pattern shows invasion of the hepatic lobule, replacing healthy hepatocytes [
9]. The metastatic cells invade the liver parenchyma without disturbing the pre-existing liver structure at the interface [
10].
The landscape of metastatic progression is of great importance for the understanding of the interplay between tumor cells and the microenvironment. Niederkorn and colleagues [
11] initially reported NK cell activity in the eye promoted the growth of UM. A follow up in vitro study by these authors suggested macrophage migration inhibitory factor (MIF) production by UM cells protects against NK cell-mediated killing [
12]. Our group developed an ocular melanoma murine model and we formally demonstrated NK cells are pivotal for the control of hepatic metastases [
13].
As the role of PEDF in suppression of ocular neovascularization was elucidated, our group discovered that the ratio of vascular endothelial growth factor (VEGF) to PEDF played a role in the migration of UM cells and hepatic metastases [
14]. Furthermore, we recently demonstrated the role of PEDF as an anti-angiogenic and anti-stromagenic factor in UM [
15]. Still, the role on NK cells and PEDF in the development of metastatic growth patterns and immune polarization is not understood. To evaluate the roles of NK cells and PEDF in the tumor microenvironment in metastatic UM in the liver, we compared the tumor microenvironment relative to metastatic UM growth using our established orthotopic murine model of ocular melanoma. Our results suggest a role for NK cells in the development of the infiltrative metastatic growth pattern and a role for PEDF in the nodular growth. We measured a reduction in the myeloid lineage within the metastatic liver and discovered the expression of both pro-inflammatory and anti-inflammatory genes.
Methods
Tumor and cell culture conditions
The mouse melanoma cell line B16-LS9 was kindly provided by Dario Rusciano at the Friedrich Miescher Institut, Basel, Switzerland. The complete culture medium included RPMI1640 with HEPES, L-glutamine, 10% FBS, 1% nonessential amino acids, 1% sodium pyruvate solution, 1% MEM vitamin solution, and a 1% antibiotic-antimycotic solution and incubated at 37 °C/5%CO2. Cells were grown to 90% confluence prior to harvest, washed with Hank’s solution, trypsinized and expanded for experiment use.
Mice
Eight-week old female C57BL/6 J mice were purchased from Jackson Laboratories (Bar Harbour, ME).
PEDF−/− mice were generated on a C57BL/6 background as previously described [
16] and provided courtesy of Dr. Susan Crawford (St. Louis University, St. Louis, MO). All experiments were performed according to the Declaration of Helsinki, in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in Ophthalmic and Visual Research and with the Institutional Animal Care and Use Committee policies and procedures from Emory University.
A cohort of 15 female C57BL/6 J mice was treated with anti-asialo GM1 serum to deplete NK cells. Anti-asialo GM1 (Rabbit; Wako Pure Chemical Industries, Osaka, Japan) was diluted to 1:4 in distilled water prior to injection. To achieve NK cell depletion mice were injected intraperitoneally (i.p.) with 100 μL anti-asialo GM1 every 3 days, beginning two weeks prior to cell line inoculation until they were euthanized [
17].
Murine model of ocular melanoma
Eight-week old female C57BL/6 J, C57BL/6 PEDF−/−, and C57BL/6 J anti-asialo GM1-treated mice were inoculated in the posterior compartment of the right eye with 5 × 105 B16-LS9 melanoma cells in a 2.5 μL final volume using a transcorneal technique with a 30-gauge needle under guidance of a dissection microscope. The mice were anesthetized by intraperitoneal injection (i.p.) of 100 mg/kg ketamine and 12 mg/kg xylazine mixture in PBS. The inoculated eyes were enucleated after one-week. Mice were euthanized in a CO2 chamber 28 days post inoculation. Liver tissue were collected and processed for histological and cellular evaluation.
Histology assessment
Excised livers were grossly examined and submerged in 4% neutral buffered formaldehyde for routine processing. Formalin fixed-paraffin embedded slides were stained with hematoxylin and eosin (H&E) and microscopically evaluated (Olympus, Waltham, MA) for metastases. Three sections through the center of each liver were evaluated for the number of metastases, and the average number per section was determined, as previously described [
17,
18]. To determine relative metastasis area the sizes of all metastases in a single liver section were added to calculate a total metastasis area (μm
2) and averaging this value per mouse. Metastases were separated by size into stage 1 (< 50 μm in diameter), stage 2 (50–200 μm), and stage 3 macrometastases (> 200 μm). Measurements were performed in triplicate and averaged per mouse as previously described [
19].
Evaluation of tumor vascular density
The number of blood vessels per 40x high-powered field (HPF) was calculated and averaged to mean vascular density (MVD). An individual vessel was defined as an area of lumen lined with endothelial cells, while tracts and branches were counted as separate vessels. C57BL/6 J, n = 9; C57BL/6 PEDF−/−, n = 9; and C57BL/6 J anti-asialo GM1-treated mice, n = 8.
Cell suspension from liver tissue
Murine livers were isolated by maceration using a pestle (MidSci, St. Louis, MO) on a 70 μm nylon strainer (BD Biosciences, San Jose, CA). Cell suspension was washed and red blood cell contaminants were eliminated using 1x RBC Lysis Buffer (BioLegend, San Diego, CA) following manufacturers instructions. Cells were resuspended in PBS/1% FBS and kept on ice until ready to use.
Cell surface labeling for flow cytometry analyses
Cell suspension was labeled with the following antibodies: anti-CD11b (clone M1–70) PE, anti-Ly-6C (clone HK1.4) FITC, anti-Ly-6G (clone 18A) APC, anti-Gr-1 (clone RB6-8C5) FITC, and anti-F4/80 (clone BM8) PE. All anti-mouse antibodies were purchased from BioLegend. We labeled each sample with 1 μg of each antibody and incubated on ice for 30 min in dark. Samples were washed 3x in PBS/1% FBS. Acquisition strategy: CD11b
+ cells (myeloid lineage): we gated FSC versus SSC to eliminate debris (Gate 1), followed by plotting of CD11b versus SSC to acquire CD11b
+ cells; CD11b-subsets: cells from Gate 1 were plotted CD11b versus Ly6C, or Ly6G, or Gr-1; Kupffer cells: F4/80 versus SSC. Immunopositivity was determined by using isotype controls of each antibody and unlabeled samples. Single label controls were set up using The AbC™ Total Antibody Compensation Bead Kit (Thermo Fisher Scientific, Waltham, MA). Data acquisition was done in BD Biosciences LSRII Flow Cytometer and data analyses performed using FlowJo vX.0.0.6 (FlowJo, LLC, Ashland, OR), as before [
20].
Gene expression analyses
RNA was extracted from cells or tissue using RNeasy Mini Kit (Qiagen Inc., Valencia, CA) following manufacturer’s conditions. We used 100 ng of RNA material for cDNA synthesis and further pre-amplification prior to assay set up to increase test sensitivity. Samples were run in Roche® LightCycler 480 and analyzed using the Comparative ∆C
T Method as before [
21]. We used the following assays (Thermo Fisher Scientific):
Tnfa: Mm00443258_m1;
Socs3: Mm00545913_s1;
Arg1: Mm00475988_m1;
Tgfb1: Mm01178820_m1;
Gapdh: Mm99999915_g1;
Casp3: Mm01195085_m1;
Trp53: Mm01731290_g1;
Cd68:Mm03047340_m1.
Statistical analysis
A one-way ANOVA or two-way ANOVA with Tukey’s post-test was performed to determine statistical significance. Experiments comparing the immune cell populations used The Holm-Sidak test. The value p < 0.05 was used to define statistical significance for all assays. All data was reported as mean ± SEM using Prism Graph Pad (GraphPad Software, La Jolla, CA).
Discussion
Our findings suggest NK cells and PEDF play a role in the growth pattern of metastatic UM. Hepatic metastases are more abundant in the absence of NK cells and PEDF. Moreover, we characterized for the first time the growth patterns of metastatic UM in an orthotopic murine model of ocular melanoma. The WT group shows a higher ratio of infiltrative to nodular metastatic pattern similar to that observed in post mortem human livers from metastatic UM patients [
9]. The ratio of infiltrative to nodular is reduced in the absence of NK cells and PEDF. Divergence among these two was observed in the studies characterizing the microenvironment and in the tumor vascular supply. NK cells play a role in the microenvironment of the hepatic metastases while PEDF is more critical for the angiogenesis.
We have previously demonstrated that the hepatic sinusoidal space contains resident NK cells [
9]. However, these NK cells are in an immature state. Recent investigations from our group showed the Toll-like receptor-5 (TLR-5) agonist entolimod enhanced anti-metastatic activity against hepatic metastases by mobilization of NK cells to the liver, and stimulate the maturation, differentiation and activation of the resident ones [
13]. Because of the presence of NK cells in the sinusoidal space within the infiltrative pattern of metastatic growth we hypothesized that depletion of this population will shift the ratio of infiltrative to nodular, increasing the nodular pattern. Our results supported our hypothesis as it increased the number of metastases following the nodular pattern. This increase in nodular pattern shifted the ratio to a 1:1, showing a similar distribution of infiltrative and nodular patterns. The staging or stratification of the hepatic metastases also differed in the absence of NK cells. In both the infiltrative and nodular patterns the hepatic metastases followed the patter of higher stage 2 > stage 1 > stage 3, compared to the WT group, which followed stage 1 > stage 2 > stage 3. Collectively, our work supports a critical role for NK cells in the control of hepatic metastases.
Multiple groups, including ours, have investigated M1/M2 polarization as predictors of outcome in UM patients [
26‐
33]. A number of molecular markers and cytokines are used to classify macrophages into M1 and M2. Classically, M1 are considered the destroyers of tumor cells. These M1 macrophages present antigen to T cells, produce high levels of pro-inflammatory cytokines, and express both nitric oxide synthase and reactive oxygen species [
34,
35]. In contrast, M2 suppress type-1 immune responses, promote tissue remodeling and wound healing, angiogenesis via VEGF and promote tumor development [
34,
36]. We examined the genetic expression of two markers associated with M1 and M2 polarization. The absence of NK cells measured similar expression of
Socs3 mRNA to WT groups, associated with tumor killing, but base levels of
Tnf mRNA, a key pro-inflammatory cytokine in anti-tumor responses. Similar
Arg1 and
Tgfb mRNA expression were observed in the absence of NK cells compared to WT groups. This raises multiple questions for further investigations on the plasticity of these macrophages and their contribution in angiogenesis and disease progression [
37].
Tumor cell survival is associated with a decreased capacity of tumor suppressors, such as p53, to execute their function. P53-dependent apoptosis is induced by activation of effector caspases, such as caspase-3 [
38]. In our investigation we measured reduction of both
Casp3 and
Trp53 mRNA in NK-depleted animals compared to WT. This data confirms caspase-3 is an important effector molecule in NK cell-mediated apoptosis in tumors. The
PEDF−/− animals measured a stronger effect on downregulation of both
Casp3 and
Trp53 mRNA. Work from Takenaka and colleagues [
39] recently demonstrated activation of caspase-3 and induction of apoptosis by PEDF using the MG63 human osteosarcoma cells. These results suggest PEDF, in addition to decreasing hepatic metastases by eliciting anti-angiogenic responses [
15,
19] also regulates caspase-3 activation. Based on the work performed in the human livers from metastatic UM patients [
9] we hypothesized that the nodular pattern is controlled by PEDF in the periportal area, thus, absence of PEDF will increase the ratio of nodular to infiltrative growth. Our results supported our hypothesis as the
PEDF−/− group had a significant increase in the metastatic growth following the nodular pattern compared to WT and NK depleted groups. Nodular metastases in human livers express MMP-9, VEGF, and contain CD31
+ vascular channels [
9]. Therefore, we can speculate PEDF from hepatocytes inhibit the VEGF from the tumor microenvironment. Here, we provide evidence of the anti-angiogenic role of PEDF in the hepatic metastases as the
PEDF−/− grafts showed a significant increase in MVD compared to WT.
Characterization of the myeloid-derived cells in the liver microenvironment suggests that dysfunction of multiple cells types can lead to failure to control hepatic metastases. Our results showed a reduction in CD11b
+Ly6C
+, CD11b
+Ly6G
+, and CD11b
+Gr-1
+ cells in the NK-depleted and
PEDF−/− groups. Both groups failed to control the growth of hepatic metastases in our orthotopic ocular melanoma model. These results are consistent with the concept that the absence of tissue infiltrating myeloid-derived cells modifies the tumor microenvironment, leading to failure to control tumorigenesis [
40,
41]. Neutrophils are considered of great importance in the control or the progression of different cancers [
42‐
44]. However, the dual roles of neutrophils, as an anti-tumor and pro-tumor functions, needs to be further studied [
45‐
49]. Recent work from Sadegh et al. [
50], demonstrated the development of hepatic metastases to be associated with the upregulation of IL-10 in the liver microenvironment and its receptor on NK cells. The source of IL-10 was confined to bone marrow-derived cells that are not MDSCs. Sadegh et al., used Gr-1-depletion to investigate if MDSCs were the source of IL-10. It has been demonstrated Gr-1
+ is also a neutrophil marker; therefore, in our investigations we characterized cells based on Gr-1- and Ly6C/G-positivity. MDSCs have been considered pro-tumoral at the systemic level. Our work suggests that the presence of these cells have an anti-tumoral effect, as their percentages are reduced in those groups with elevated number of metastases. It is tempting to speculate their effector function is locally within the liver microenvironment.
In this investigation we demonstrated the two distinctive metastatic growth patterns in hepatic metastases of an orthotopic murine model of ocular melanoma. We demonstrated NK cells play a critical role in the control of hepatic metastases following an infiltrative growth pattern. In addition, we demonstrated the importance of PEDF in the control of angiogenesis in this animal model, which also contributes to the generation of large nodular growth. Together, our work provides a novel understanding of the infiltrative and nodular patters of metastases and on the role of myeloid-derived suppressor cells in metastatic UM.