Angiogenesis Related Remodeling and Reprogramming in Melanomas

Introduction: Melanomas show a wide variation in pigmentation. The present study evaluates the role of angiogenesis in reprogramming/remodeling of the tumor through tumor vascular interactions. Methods: Paraffin and frozen sections from melanomas in vertical growth phase, were subjected to routine, enzyme/Immunohistochemistry, and electron microscopy. Quantitation was done by morphometric analysis of cell/nuclear size and density and mitotic counts to compare pigmented, amelanotic areas and tumor-vascular-complexes[TVCs]. Progression and fate of TVCs was assessed in 3D constructs of angiogenic vessels. Results: Pigmented areas show marked variation in cell/nuclear size, nuclear content and abnormal mitosis. Cells in amelanotic areas are uniform, with near normal morphometric values and synchronization of mitosis. Vasculogenesis is evident in amelanotic zones. Angiogenic tubes interact with tumor, forming 3D tumor-vascular-complexes [TVCs] with a central vessel and a perivascular mantle of cells, showing normal mitosis and regulated neural differentiation. Morphometric parameters and differentiation are graded proportionate to level of pigmentation. Discussion: TVCs grow into expansile nodules with peripheral pigmented layers which elicit further angiogenesis at the stromal margin. A cyclical process results in amelanotic nodules Angiogenesis from normal vasculature gives polarity and creates an embryonal microenvironment with reprogramming of pleomorphic pigmented to amelanotic near normal cells with embryonic characteristics. Clinically, the therapeutic strategies have to target both angiogenesis as well as the self propagating vascular networks. Corresponding author: Bhanu Iyengar. Pigment Cell Center, New Delhi, India. E-mail: bhanu_i@yahoo.com Received Date: April 18, 2015 Accepted Date: May 07, 2015 Published Date: May 13, 2015 Citation: Iyengar, B. Angiogenesis related Remodeling and Reprogramming in Melanomas. (2015) J Dermatol Skin Biol 1(1): 1-10. J Dermatol Skin Biol | Volume 1: Issue 1 www.ommegaonline.com


Introduction
Melanocytes are derived from the multipotent neural crest cells [NC] during embryogenesis of the neural tube [1] . The NC cells arise from the neural fold which lies at the confluence of the neuroepithelium and the general epidermis. These cells differentiate into neuronal, epidermal as well as pigment cells. The melanocytes actively produce catecholamines as well as indoleamines [2][3][4] . Melanomas are highly malignant tumors arising from the melanocytes in the basal layer of the epidermis, uveal tract of the eyes, inner ear, mucous membrane, genital organs, anus and leptomeninges.
Human cutaneous melanomas show molecular plasticity and often express genes characteristic of neural cell lineages [5] . In vertical growth phase[VGP] melanomas there is a definite pattern of neural differentiation in relation to angiogenesis on viewing the tumor vascular interaction as a three dimensional system [6] .
Human cancers which persist in situ for months in a prevascular phase, require efficient vascularisation, for further growth and metastasis. Angiogenesis and remodeling are required for the development of the tumor microcirculation [7] . Neoangiogenesis results from the proliferation, sprouting, and migration of endothelial cells within normal tissues adjacent to the tumor. Studies have shown that varying types of vascularisation are evident in melanomas [8] . Angiogenesis is predominant in pigmented melanomas, neovascular channels arising from pre-existing stromal vessels at the invasive margins. In the aggressive amelanotic melanomas, embryonic vasculogenesis predominates [9] .
Melanomas show a wide variation in pigmentation, some tumors showing heavy pigmentation, while some are 2 amelanotic and others show nodules varying from deeply pigmented to amelanotic. The present study evaluates whether this variation is due to a clonal difference or due to a reprogramming of the component tumor cells and the possible role of angiogenesis and tumor vascular interactions.

Morphologic Patterns General tumor
Pigmented areas show marked anisocytosis and anisonucleosis[ Figure 1a]. Pleomorphism is prominent with cells varying in shape from spindloid to rhabdoid to tadpole shaped. The epithelioid cells are large cells with polygonal outlines, eosinophilic cytoplasm with round to oval vesicular nuclei arranged in a nested pattern. The fascicles and nests are separated by thin or dense connective tissue. The nuclear appearance too varies with some nuclei being dense and hyperchromatic with an indistinct nucleolus. Occasional tumors show areas with small round cells with scanty cytoplasm and dense nuclei are arranged in a rosettoid pattern mimicking primitive neuroectodermal tumors.
Amelanotic areas show a diffuse pattern of continuous sheets of fairly uniform cells [ Figure 1b]. Balloon cells are identified by their abundant, clear cytoplasm, relatively little nuclear atypia and scanty pigment; and are seen with epithelial cells [10][11][12][13] . The nucleus is large and vesicular with chromatin scattered in irregular clumps. This type of nucleus is mostly associated with a large and prominent nucleolus.

Morphometry
Morphometry highlights the variations in pigmented and amelanotic tumors. The cell and nuclear areas, and the DNA quantum have been compared with epidermal melanocytes [Figure 1, Table 1]

Amelanotic Areas
In contrast the values in the amelanotic tumors are closer to the normal epidermal values with near normal variation in cell and nuclear size and DNA content. Cells vary from a minimum of 25.8µ² to 380.6µ², a range of 354.8µ², Nuclear areas vary from 25.8µ² to 193.5µ², a range of 167.7µ² and nuclear DNA varies from 0.0504 -0.5209: range 0.4705.

Mitotic Activity [Figure 4]
All counts were done on serial PCNA and Ki67 stained sections.

Patterns of Differentiation [Figure 5, Table 2, 3]
The expression of neural markers, pituitary hormones; indoleamines; catecholamines and matrix proteins by melanocytes in association with pigmentation and morphological variation has been examined in this section.  [1] ; ACTH, PRL & HGH in [2] ; Serotonin, Melatonin & HMB45 in [3] . The attached scatter diagram shows high positivity of all markers in pigmented areas and very low positivity in amelanotic areas. Positive Cell Counts [ Figure 5, Table 2, 3] All pigmented areas were positive for the neural markers, the three neurohormones, catecholamines, indoleamines as well as the matrix proteins. Positive cell counts showed a range of 698-1299 positive cells per block in 84 blocks from pigmented nodules as shown in the scatter diagram and values in [ Table  3]. Positivity for all the markers is low or absent in amelanotic areas. Positive cells counted in 83 blocks from amelanotic nodules showed a range of 0-649 cells per block. Thus the total range of immune positivity is 70% to 100% in pigmented areas and 0% to 65% in amelanotic areas.
Differentiation in pigmented and amelanotic nodules were assessed and compared in purely pigmented and amelanotic tumors and mixed tumors.
In the Polar Group [ Figure   Thus the pigmented and amelanotic areas form polar groups with extreme divergence in morphology, morphometric parameters and differentiation.

Incorporated Vessels [INC] [Figure 6a]
General pigmented areas show incorporated vessels. Blood vessels incorporated within the fibrous tissue of the tumor show sprouting and branching. Angiogenic vessels seen at the marginal interphase is not apparent. The surrounding tumor cells remain chaotic with no definite organization as seen with the marginal tumor vascular interaction.
Angiogenesis from normal stromal vessels at the advancing margin of tumor: Vascular counts [ Figure 6c] The margins show a maximum of 20 bv/hpf and a minimum of 5 bv/HPF while areas of main tumor growth show a maximum of 6 bv/HPF and a minimum of 0 bv/HPF are observed. Thus angiogenesis is significantly higher at the margins as compared to that within the tumor [5hpf].

Tumor Vascular Interaction
The adjacent pre-existing blood vessels within the stroma extend endothelial tubes which cannelise and enter the tumor margins. The tumor cells interact with the angiogenic vessels to a depth of 2hpf to form a tumor-vascular-complex [TVC] with a perivascular mantle zone [PMZ] of 5 to 6 cell layers labeled L1-L5. [ Figure 6b] [6] Distribution of TVCs [ Figure 2, 3a] The TVCs from both pigmented and amelanotic nodules are counted in 5 random hpf along the tumor margin in contact with the surrounding stroma as given in the grid. Serial www.ommegaonline.com Reprogramming in Melanomas sections are treated as given in Methods.

Pigment vs Angiogenesis
Grid shows the number of TVCs in pigmented [P] vs amelanotic [A] nodules at the tumor/stroma interphase the [Figure 3a] showing the percentage in each case for comparison. Each hpf at the marginal zone has a potential for 20 TVCs, and 100 in 5hpf expressed as a percentage [14] .
There  Thus there is a regimented neurogenesis in the TVCs, initiated by endothelial tubes interacting with tumor cells beginning with glial differentiation followed by indoleamines and HGH/PRL in the middle layers and further by catecholamines and ACTH in the outer layers. Differentiation is proportionate to the level of pigmentation.
All markers are higher in pigmented TVCs as compared to those from poorly pigmented areas as illustrated in [Table 4 Table 5] Since mitosis appears near normal in poorly pigmented areas and in TVCs, morphometry was done to compare the morphometric parameters of pigmented, poorly pigmented from general areas and the mantle zone of TVCs with normal epidermal melanocytes. 5 random TVCs were taken from three pigmented [P1, P2 & P3] and 2 poorly pigmented areas [A1 & A2] from the same case for comparison [ Table 5].   The cell and nuclear sizes and range show a gradual reduction from pigmented to amelanotic TVCs as can be seen in [ Figure 7a] in the scatter diagram and [ Table 5], the parameters being near normal in those from poorly pigmented areas. The component cells of the mantle zone of TVCs are more uniform matching normal epidermal melanocytes, those from pigmented nodules showing a greater scatter due the presence of glial cells. This is associated with a similar reduction in differentiation as depicted in the comparative graph 7c.

Progression and Fate of TVCs [Figure 8]
The progression and fate of the TVCs was studied in depth in view of the gradation seen in the differentiation and morphometric parameters from pigmented to amelanotic nodules. Serial sections are used to follow the progression and growth of TVCs beyond 2 hpf from the stromal margins. Stacks of serial sections are used for 3D constructs and are drawn out as a collage [ Figure  8] to follow the growth and fate of the TVCs and to study vascular interactions within and around the growing tumor.

Reprogramming and Remodeling [Figure 8 Table 5]
Angiogenesis and TVCs are directly proportionate to the level of pigmentation [ Figure 6b]. Contact of pigmented tumor margins with normal stroma elicits angiogenesis from the adjoining pre-existing normal stromal vessels. As the angiogenic vessel grows beyond 2 hpf the mantle layers continue to increase to form enlarging nodules with pigmented peripheral layers, the overall pigment being less than the general tumor. When these contact the stromal margin angiogenesis and TVC formation is elicited in proportion to the pigment level. There is a reduction in pigment at each cycle. This cycle repeats itself until differentiated pigmented cells are depleted. With the absence of angiogenesis in the poorly pigmented TVCs the organized growth of the layers is lost and sheets of characteristic amelanotic cells are formed.

Discussion
The above study highlights the role of angiogenesis in remodeling and reprogramming in melanomas. Nodules of varying sizes and pigmentation are seen side by side in mixed tumors. The general melanotic and amelanotic areas are in sharp contrast forming polar groups [ Figure 1, 5]. The pigmented areas show prominent pleomorphism, marked variation in cellular and nuclear size, high levels of chaotic differentiation, and prominent abnormal mitosis. In contrast, amelanotic areas show uniformity in cell morphology, cell and nuclear size tend to be near normal epidermal parameters, differentiation being low with near normal synchronized mitosis [ Figure 4].
The remodeling of preexisting vessels surrounding the growing tumor active sprouting and branching of these vessels helps meet the metabolic demands of tumor cells [15] . Angiogen-7 Iyengar, B Reprogramming in Melanomas esis begins by focal reduction of vascular intercellular interactions in preexisting ones and increased interactions between vascular cells and the extracellular matrix [ECM]. The tumor cells at the advancing edge produce LN5 and integrin [α 5 β 1 ] both of which are involved in the dissolution of the local ECM to allow the cells to move forth further [9] . Continuous stimulation of vessel outgrowth expands the vascular compartment of the growing tumor.
Melanomas show incorporated vessels [INC], vasculogenic mimicry [VM], and angiogenesis [9] . Angiogenesis is seen at the tumor-normal stroma interphase and is proportionate to the pigment levels, very low angiogenesis being elicited in amelanotic areas. Tumor induced angiogenic tubes enter the margins, interact with the tumor cells, and form 3D tumor-vascular-complexes[TVCs] consisting of a central vessel and a perivascular mantle of cells [PMZ]. The component cells are more uniform, with normal mitotic figures and regulated organized, neural differentiation.
There is a reciprocal paracrine interaction between astrocytes, endothelial cells and ependymal cells. Astrocytes and neurons release vascular endothelial growth factor [VEGF165] to elicit a burst of mitotic angiogenesis. The activated microvascular cells produce brain-derived neurotrophic factor BDNF [27,28] . Neuronal progenitor and mitotic endothelial cells are spatially associated in foci of concurrent angiogenesis and neurogenesis [29] .
The steps of progression, from radial glial to neuronal cells, in the peri-mantle zone [PMZ] is similar to the differentiation in invitro neurospheres and early neuro-embryogenesis. The newly-generated neurons from astrocyte-like stem cells, can assume or revert to an astrocytic phenotype [20,23] . Neurons transit through an "asteron" (neuron/astrocyte hybrid) to shift into cells with astrocyte characteristics in differentiating primary floating neurospheres. These asterons coexpress a variety of neuron and astrocyte proteins and genes.
Earlier studies have identified the presence of biogenic amines in melanocytes [2][3][4] . Synaptophysin labels small synaptic like microvesicles (SLMV), which are positive for biogenic amines. SLMVs are present in neuroendocrine cells such as the pituitary and adrenal medulla. During embryogenesis, the serotoninergic and dopaminergic neurones arise from common progenitors stem cells with enzymes cat-1 & cat-4 and bas-1 [30][31] . As development progresses, the serotoninergic cells remain at the brain stem and the post-mitotic dopaminergic cells proceed to the midbrain SNC. This feature is replicated in the TVC, with indoleamines positivity in middle layers and catecholamines in the outer layers. All three pituitary hormones are expressed by the proliferating melanoma cells. HGH and PRL are expressed in the middle layers associated with mitosis, while ACTH is positive in the outer layers [32] .
Mitosis is mainly concentrated in the middle layers of the TVCs. Outer layers do not show significant numbers of mitotic figures. The mitotic figures in the TVCs are predominantly bipolar and normal in appearance as compared to the abnormal mitotic figures seen in the surrounding tumor. Polarity and asymmetric mitosis are evidenced by the generation of indoleaminergic and catecholaminergic cells in different layers of the TVCs in response to angiogenesis. Cell diversity is generated by essential cell polarity required for the proper function of most differentiated cell types. Apical/basal polarity in mitotic neuroblasts, results from asymmetrical division to generate an apical neuroblast and a smaller basal ganglion mother cell (GMC). The GMC undergoes one subsequent cell division to generate neurons or glia [33,34] .
TVC counts are proportionate to pigment levels in association with level of angiogenesis, the pigmented areas showing higher counts as compared to poorly pigmented areas. TVCs taken from a single tumor from pigmented to amelanotic nodules shows a gradation of neurogenesis, the pigmented showing prominent differentiation, with the poorly pigmented showing very low neurogenesis [ Figure 3b].
The cell and nuclear size show a gradual reduction from pigmented to amelanotic TVCs, parameters being near normal in those from poorly pigmented areas. The component cells of the mantle zone of TVCs are more uniform matching normal epidermal melanocytes, those from pigmented nodules showing a greater scatter due the presence of glial cells [ Figure 7 Table 5]. These findings suggest that TVC formation and angiogenesis are linked with melanotic to amelanotic transition. Formation and progression of TVCs has been studied to assess this possibility [ Figure 8].
The formation and fate of TVCs is elucidated by 3D constructs [ Figure 8] by tracing individual vessels beyond marginal 2 hpf through serial and contiguous sections. The outer layers of the enlarging TVCs continue to have pigment at the margins but less within the core around the vessel. Nodules of different sizes are formed with varying levels of pigment. When these come into contact with the stroma a further wave of angiogenesis occurs, elicited by the peripheral pigmented cells this time forming TVCs within the margins of the TVC generated nodules.
This cyclical process continues with differentiation and pigment diminishing with every cycle. This is well illustrated by the comparative differentiation within TVCs in pigmented and poorly pigmented zones within the same tumors. At this stage the cells continue to proliferate and enlarge into amelanotic nodules and further into sheets of amelanotic cells replacing the pigmented areas. This brings out interesting features of TVC progression leading to a reprogramming and remodeling of the tumor, which is gradually replaced by amelanotic cells.
Vasculogenesis, occurs with the formation of blood islands in the extra embryonic yolk sac during embryogenesis [7] . An inner layer of primitive hematopoietic cells and a peripheral population of angioblasts differentiate into endothelial cells. A similar process occurs in the amelanotic melanoma giving rise to hemopoeitic cells that normally derive from germ layers other than the neuroectoderm and neuralcrest [44][45][46] .
Embryonic microenvironments influence development of neuronal stem cells [47] . Similarly multipotent tumor cells, fate and plasticity of adult cell are influenced by the microenvironment [48] . Multiple cell types which originate from the highly invasive neural crest cell population of the vertebrate embryo can be reprogrammed by simulating a natural environment in 3D organ culture [47][48][49][50][51] .
The present observations indicate that an embryonic microenvironment is created in the mantle zone of TVC around the angiogenic vessel, arising from pre-existing normal stromal vessels, to induce and recapitulate early embryonal neurogenesis of indoleaminergic and catecholaminergic cells. The angiogenic vessel confers a polarity to the interacting tumor cells resulting in a stepwise manner replacing the highly pleomorphic pigmented melanotic tumor cells by uniform, amelanotic tumor cells which behave like embryonal stem cells as is evidenced by the development of vasculogenic spaces [9] .

Clinical and Therapeutic Significance
The present study outlines the role of angiogenesis in remodeling/reprogramming in melanomas. Anti-angiogenesis therapy has been used successfully in the treatment of a significant number of melanomas. It has been demonstrated by Griffioen and colleagues [52][53][54][55][56] , that harmful endothelial cell anergy can be counteracted by inhibitors of angiogenesis. In the early phases of tumor growth angiogenic TVC result in the replacement of pigmented nodules by amelanotic nodules as described above. This can be inhibited by anti-angiogenesis therapy. The present study highlights the importance of anti-angiogenesis therapy before extensive amelanotic change occurs, capable of vasculogenesis resulting in resistance to anti-angiogenesis therapy. In view of the varying types of vascularisation seen in melanomas, the therapeutic strategies have also to target the self propagating vascular networks within the tumor, not involving the angiogenic pathways.

Material and Methods
Nodular melanomas in the vertical growth phase [VGP] were received from the Cancer Surgery Unit of Safdarjung Hospital, New Delhi, fixed in cold [4°C] 10% formol glutaraldehyde. The formaldehyde-glutardehyde cold fixation retains the morphology, gives crisp staining and efficient immunohistochemical staining both in frozen as well as paraffin sections. The same blocks can be subjected to electron microscopy as well. 10 nodules were sampled from each of 27 tumors, to make a total of 270 blocks, in the ratio of pigmented to poorly pigmented areas in each tumor. As the specimens were received and sampled the blocks were arranged in a grid, according to the pigment level which varied between 7% -95% [ Figure 2]. 20 to 40, 5μm thick serial frozen and paraffin sections of each block were maintained under refrigeration at 4°C for the following: Histochemistry: HE, PAS, combined reticulin+gold impregnation for aurophilia [10] . Gold chloride sublimate specifically and exclusively stains astrocytes [confirmed by GFAP and nestin positivity] which stain intensely black. Reticulin is seen as a network of fine fibrils and the vascular basement membrane; Enzyme histochemistry: dopa oxidase, tyrosinase and monoamino oxidase methods; Immunohistochemistry: using the Avidin/ Biotin system [11][12][13] .
The amelanotic melanocyte has been identified by the specific melanocyte markers HMB-45 and S-100 protein.

Morphology and Morphometry
Morphology and morphometry were assessed on HE sections. Morphometric analysis of cell and nuclear size and nuclear density was done using a Semi-automated Zeiss Morphomat, Scion Image of NIH and a Reichert Cyto spectrophotometer to compare the size and the nuclear parameters of tumor cells with normal epidermal melanocytes. The nuclear content was assessed as follows: Area of nucleus x optical density x 5 (tissue thickness) = DNA [Q] Electron microscopy: routine and after en bloc stain for dopa. The above methods were applied to: a. General tumor areas and b. Tumor Vascular Complexes [TVCs]. a. General Tumour Areas [ Figure 2] 167 blocks, 84 pigmented and 83 poorly pigmented and amelanotic nodules have been included.
In Group I, 4 cases are pigmented and 4 amelanotic to include 40 pigmented and 40 amelanotic nodules/ blocks.
In Group II 19 cases are mixed showing both pigmented and amelanotic general nodules composed of 87 blocks, 44 melanotic and 43 amelanotic.
All counts were double blind observations made by two unrelated observers and the author. Counts and measurements were repeated three times at 6 monthly intervals to remove any bias.

Quantitation by Morphometric Analysis
Morphometric measurements of cell size and the nuclear parameters were done in 200 cells on random pigmented and amelanotic nodules in each tumor. Measurements are represented as scatter diagrams for comparison. 100 melanocytes from overlying normal epidermis serve as control [ Figure 1, Table1]. Morphometric counts were done blinded to the category of the tissue block (pigmented or amelanotic or mixed). Mitotic Activity and morphology was assessed in pigmented amelanotic areas and TVCs [ Figure 4]. Mitotic counts were done and compared in these areas on sections stained with PCNA and Ki67 both specific for mitotic cells. www.ommegaonline.com

Reprogramming in Melanomas
Immunohistochemistry [ Figure 5] Positive cell counts, of each marker were entered into the grid. Positive cells per 1000 cells were counted in random 10 HPF per block for each marker in serial sections. The positive counts are presented as scatter diagrams, comparing pigmented and amelanotic areas in: Group I. [  Table 3]. Expression of various markers in these two groups has been compared.

Tumor Vasculature
Incorporated Vessels [INC] are seen in 84 general pigmented nodules away from the tumor stroma interphase. [ Figure 6].

Angiogenesis from Normal Stromal Vessels at the Advancing Margin of Tumor
103 blocks from mixed tumors, 51 blocks from pigmented and 52 from poorly pigmented nodules [ Figure 2] at the interphase between the tumor and surrounding preexisting stroma, have been included for the study. A large number of angiogenic vessels are seen within 2hpf of the tumor margin with a maximum of 20 vessels per HPF associated with TVCs. [ Figure  6]. Vascular channels are counted at the tumor margins in each of the 103 blocks to a depth of two high power fields [HPF] and at a depth of 5 to 6 HPF within the tumor in 10 HPF [1030 HPF marginal and 1030 HPF within tumor]. Vessel counts were done on sections stained with nestin, PAS and reticulin stain.

Tumor-Vascular-Complexes [TVCs]
Endothelial tubes from normal stromal vessels interact with tumor cells to form tumor-vascular-complexes [TVCs]. The TVCs from both pigmented and amelanotic nodules are counted in 5 random HPF in each block along the tumor margin at the tumor-stromal interphase [out of a possible maximum of 100 vessels]. A total of 1030 TVCs formed around vessels in cross section were included in the study [ Figure 3].

Immunohistochemistry [Figure 3c]
Neural marker positivity has been examined in the lay-  Table 5].

Progression and Fate of TVCs [Figure 8]
Stacks of serial and contiguous HE, mAb, reticulin stained and gold impregnation sections, are used to make 3D reconstructs of angiogenic vessels arising from pre-existing normal stromal vascular channels showing interaction with the advancing tumor. These channels are traced beyond the marginal 9 2hpf to follow the growth of the TVCs and to study vascular interactions within and around the growing tumor. Presence of pigment, a positive DOPA reaction, and HMB-45 positivity are criteria for diagnosis. In the absence of pigment a positive dopa reaction, HMB45 positivity and the presence of premelanosomes on electron microscopy [EM] is diagnostic of amelanotic melanomas. These criteria form the basis of diagnosing each tumor included in this study.
Statistical Analysis: Anova Analysis: Kruskal-Wallis One Way Analysis of Variance; and Tukey Test: All Pairwise Multiple Comparison Procedures. All procedures were performed with the informed consent of patients, in full compliance with the Helsinki Declaration.