Tumor Vascular Interaction in Melanomas and Neurogenesis: A Review

Background: Melanomas arise from multipotent neural crest derivative, the melanocytes. At the tumor-stroma interphase, melanoma cells show organized neural differentiation forming tumor-vascular complexes. The present study explores the sequence of neurogenesis from aurophilic radial glial like cells in relation to angiogenesis, utilizing the tumor-vascular-complex (TVC) as a 3D model. Methods: Serial frozen and paraffin sections stained with HE, reticulin-gold impregnation for aurophilia; dopa oxidase; immunopositivity for: neural differentiation (nestin, Glial Fibrillary Acidic Protein (GFAP), Neural Fibrillary Protein (NFP), synaptophysin); indoleamines: (serotonin and melatonin), catecholamines (dopamine (DA), Noradrenalin (NA), pigment; Dopa Oxidase (DO)); hormones: ((PRL), Prolactin; (HGH), Human Growth Hormone;) and mitosis. The pattern of neural differentiation in tumor-vascular-complexes (TVC) is assessed by positivity in layer1layer 5 and cell counts in each layer of the perimantle zone (PMZ). Statistical Analysis: ANOVA: Kruskal-Wallis One Way Analysis of Variance; All Pairwise Multiple Comparison Procedures [Tukey Test]. [t-test or Mann-Whitney Utest]. Results: A TVC is formed during angiogenic tumor-vascular interaction. Nes +ve angiogenic tubes enter the tumor margins. Periluminar cells show aurophilia, and extend dendritic arbors into the outer layers of the mantle zone. Cells and dendritic processes in layer 1/ layer 2 show Nes, Auro and GFAP positivity. NFP and Syn positivity is seen in layer 4/ layer 5 with a transition zone between layer 2/ layer 3. As two layers accrue, a wave of mitotic activity is seen and cells acquire PRL and HGH and indoleamine positivity. Catecholamine positivity is in layer 4/ layer 5 thus establishing a polarity. Dopamine is positive in layer 3/ layer 4 coinciding with dopa-oxidase which peaks in layer 4 with NA, ACTH positivity in the outer layers is in association with pigmentation in layer 4/ layer 5. Discussion: Thus during tumor-vascular interaction, melanoma cells differentiate into aurophilic radial glial like cells, as during embryonic neurogenesis, Nes, a marker of multi-lineage progenitor cells, identifies them as MASC, which differentiate into neuronal cells. The angiogenic vessel confers polarity and an embryonal microenvironment in the perivascular mantle zone of the TVC, inducing aggressive melanoma cells to function as neuronal stem cells recapitulating neurogenesis of bio-aminergic cells. The cell cycle is orchestrated by the three pituitary hormones, PRL, HGH and ACTH. The expression of PRL and HGH is related to mitotic activity while ACTH and pigment indicate differentiated function.

express genes characteristic of neural cell lineages [9]. Neuronal differentiation with expression of neuron-associated markers such as intermediate filament protein peripherin, neuropeptide substance P, muscarinic acetylcholine receptors, and neuron-specific enolase in primary and metastatic melanomas has been shown in some studies [10,11].
The ability of melanoma cells to undergo proliferation in three dimension is clinically known as the Vertical Growth Phase (VGP). The tumors in the VGP extend into the lower half of the reticular dermis and the net direction of growth tends to be perpendicular to that of the Radial Growth Phase (RGP). It has been observed that tumors having the characteristics of the RGP do not metastasize, while those of the VGP do. VGP melanoma is a highly angiogenic and proliferative lesion. Further genetic changes convert melanoma into an invasive tumor capable of three dimensional growth, increased angiogenesis, and metastasis. The RGP and the VGP are the most important determinants of prognosis in melanoma.
In Vertical Growth Phase (VGP) melanomas there is a definite pattern of neural differentiation in relation to angiogenesis on viewing the tumor as a three dimensional system [12]. Although most human cancers persist in situ for months in a prevascular phase, they require efficient vascularisation, for further growth and metastasis [13]. Production of new blood vessels (angiogenesis) and their remodeling are required for the development of the tumor microcirculation [14]. 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 [15][16][17][18][19]. Angiogenesis is predominant in pigmented melanomas, neovascular channels arising from pre-existing stromal vessels at the invasive margins. In the more aggressive amelanotic melanomas, embryonic vasculogenesis predominates [20].
Recent studies demonstrate reciprocal paracrine interactions between astrocytes, endothelial cells and ependymal cells. The present work has been undertaken to investigate whether a similar interaction occurs in melanomas in response to angiogenesis. This study is a sequel to a recent finding that melanoma cells revert to the embryonic pattern of differentiation, with radial glial like cells and neuronal positive cells during tumor-vascular interaction [12]. A combined reticulin gold impregnation is employed to identify glial cells [21]. The present study assesses the development of the tumor vascular complex as an embryonic microenvironment with identified Multipotent Astrocytic Stem Cells (MASC) during tumor cell-vascular interaction. Neurogenesis and expression of pituitary hormones in various layers of tumor cells in three dimensional tumor vascular complexes (TVC) formed in relation to angiogenesis has been assessed.

Materials and Methods
Nodular melanomas in the Vertical Growth Phase (VGP) were received from the Cancer Surgery Unit of Safdarjung Hospital, New Delhi, fixed in 10% formol glutaraldehyde. The formaldehydeglutardehyde 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. On gross examination the total number of pigmented/amelanotic nodules in each tumor was assessed. 10 nodules were sampled from each of 27 tumors to make a total of 270 blocks, in the ratio of pigmented to poorly pigmented nodules in each tumor ( Figure 1) sampled below the reticular dermis. As the specimen were received and sampled the blocks were arranged in a grid. Pigment positive cells per 1000 cells were counted in random 10 HPF per block, entered into the grid and the percentage of pigment positive cells in the 10 nodules in each of the 4 pigmented, 19 mixed and 4 amelanotic tumors calculated which varied between 7% to 95% positivity.   frozen sections and paraffin sections were cut from each block and maintained under refrigeration at 4°C. These were subjected to routine histochemistry, (HE, PAS, modified reticulin+gold impregnation) [21] enzyme histochemistry (Dopa Oxidase) and Immunohistochemistry using the Avidin/Biotin system [22][23][24][25]. monoclonal antibodies (mAb)used: HMB45, NFP (neuro filament protein), GFAP, Synaptophysin (Syn), (BioGenex) nestin (Nes), (Chemicon) a marker of multi-lineage progenitor cells [26][27][28][29]. The indoleamines: serotonin (SER) & melatonin (MLT), and the catecholamines: dopamine (DA) & noradrenalin (NA), by the avidin-biotin method using Dako Pat kits. localization of ACTH, PRL and HGH by the avidin-biotin method using Dako Pat kits. (HMB-45, PRL, HGH, ACTH) [22][23][24][25] as well as the matrix proteins LN5 and integrin.
As negative control all slides included a serial section stained with no mAb. The same mAb were used simultaneously against known positive sections from human skin as positive controls. 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 is diagnostic of poorly pigmented melanomas. These criteria form the basis of diagnosing each tumor included in this study. Vascular channels are counted at the tumor stroma interphase in each of the 103 blocks to a depth of two high power fields and at a depth of 5 to 6 HPF within the tumor in 10 HPF (1030 HPF marginal and 1030 HPF within tumor). A scatter diagram (Figure 1b) compares the number of vessels at the margin and within the tumor. The number of angiogenic vessels is significantly higher at the margin adjacent to the stroma.

General tumor morphology
General tumor morphology was examined on HE sections as shown in Figure

Tumor/vascular Complex (TVC) Perivascular mantle zone (PMZ)
The interacting tumor cells form a mantle around the angiogenic channels at the stroma/tumor interphase forming spheroidal structures. The sequential formation of the tumor vascular complexes (TVC) around a central angiogenic vessel was assessed by gold impregnation in a total of 897 developing complexes and the sequence of appearance of aurophilia and neural, biogenic amines and hormone marker positivity recorded. Of the 897 TVCs 249 show a single layer of tumor cells, 213 show 2 layers, 217 show 3-4 layers while 218 are fully formed with 5 layers.
Since the sequential positivity is being assessed L1 is present in all 897 TVCs, except for Nes which appears only in the single layer stage, L2 in 648, L3&4 in 435 and L5 in 218 TVCs and the positivity for each marker is collated as percentages for comparison. Positivity for each mAb in each layer is marked out in Camera Lucida (CL) diagrams. The percentage positivity in each layer is mapped onto an aggregate CL figure as well in graphs, in the relation to the angiogenic central vessel.  Quantitation: First, the total number of cells in each layer was counted. This was followed by counts of positive cells in each layer. Since the cell numbers vary in different layers, marker positivity has been presented as percentages of positive cells in the total number of TVCs for comparison.

Morphometry: (Figure 5)
Morphometric analysis of cell and nuclear size and density of the general tumor and TVCs was done using a Semi-automated Zeiss Morphomat, Scion Image of NIH and a Reichert Cytospectrophotometer 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).

Morphology and Morphometry
Morphology and morphometry were assessed on HE sections. Morphometric measurements were done on sections from 2 pigmented and 2 amelanotic random nodules in each case. Size of cells and the nuclear parameters in 50-100 cells in 2 random HPF were measured in each section adding up to 200 counts in pigmented and 200 in amelanotic areas per tumor. Measurements in pigmented and amelanotic section are represented as scatter diagrams for comparison. 100 melanocytes from overlying normal epidermis serve as control ( Figure 5).
The measurements are taken from each of 6 layers in 5 random TVCs in pigmented and amelanotic nodules in all tumors comparing: Pigmented nodules; Amelanotic nodules; General tumor cells around TVCs; TVCs in pigmented nodules; TVCs in poorly pigmented nodules; overlying Epidermal melanocytes. Figure 5: (a) Scatter diagram comparing morphometric parameters in P, A and TVCs from pigment and poorly pigmented nodules. There is a graded reduction in cell, nuclear size and DNA from the pigmented nodules, to pigmented TVCs, P1, P2, P3, amelanotic TVCs, A1, A2 and amelanotic nodules. (b) Table comparing morphometric parameters and differentiation from pigmented to amelanotic areas.
Measurements recorded here are from 5 random TVCs each, from 3 pigmented (P1, P2, P3) and 2 amelanotic (A1, A2) nodules in the same tumor (marked with arrow in Figure 1). Statistical Analysis Anova Analysis: Kruskal-Wallis One Way Analysis of Variance; and Tukey Test: All Pairwise Multiple Comparison Procedures. The epithelioid cells are large with eosinophilic cytoplasm with round to oval vesicular nuclei with prominent nucleoli, arranged in a nested pattern separated by thin or dense connective tissue. Some nuclei are dense and hyperchromatic with an indistinct nucleolus. Occasional tumors show isolated areas with small round cells, scanty cytoplasm and dense nuclei, arranged in a rosettoid pattern mimicking primitive neuroectodermal tumors.

Tumor morphology
Amelanotic areas: show a diffuse pattern of fairly uniform cells ( Figure 2b). Balloon cells are identified by their abundant, clear cytoplasm, relatively little nuclear atypia and scanty pigment; and are seen with epithelial cells. 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.
Mixed tumors are formed of multiple variegated nodules some showing heavy pigmentation, others graded peripheral pigmentation and some amelanotic as arranged in the grid ( Figure 1).
All tumors showed lympho/vascular/perineural invasion with local lymph node metastasis.

Angiogenesis: (Figure 1b)
Invasive margin shows significantly higher blood vessels (bv) counts as compared to that well within the tumor growth. On an average 8.18 bv/hpf are observed within 2hpf of the invasive margins and an average of 1.9 bv/hpf in the depth of tumor. At the margins a maximum of 19 bv/hpf and a minimum of 5 bv/hpf are observed. In the areas of main tumor growth a maximum of 4 bv/hpf and a minimum of 0 bv/ hpf are observed. Thus there is a significant difference between angiogenic vessels at the invasive margins and within the tumor. In a rapidly growing tumor the central portions recede from the margins and are deprived of vascularisation.
The formation of the tumor-vascular complex is best outlined by the reticulin-gold impregnation technique [21]. Within the solid tumor interactions between angiogenic vessels and tumor cells are integrated, interactive phenomenon. The mantle zones of the TVCs are the best if not the only indicators of the in situ responses, since no definite tissue/organ culture methods are available to study this dynamic process. As the vessels grow tumor cells first accrue and then proliferate along the length. Cross sections of these vessels, gives a sequential picture of addition of each layer.
The pattern of neural differentiation and cell morphology is regimented and well defined at the tumor/stroma interphase where the tumor cells interact with the neovascular angiogenic vessels. This pattern is lost within the general tumor away from the margins. Nes positive endothelial buds arising from the normal stromal vessels, canalize and extend towards the tumor margin. Nes positivity is seen in 56.25% in L1 around the lumen. Nes positivity is first seen along the cell membrane as single cells. As the TVC enlarges positive cells increase from 2 to 15 cells and along the length of the vessel. The cells extend nestin positive dendritic processes in 2-3 layered TVCs, and are well outlined (Figure 2 Panel II).
The luminal cells show aurophilia first in the cell membrane followed by the cytoplasm and by the appearance of a single process extending outwards into the proliferating layers of cells. Some cells are seen to extend processes along the circumference of the vessel. Aurophilic cells extend into L2 as the TVC enlarges and further layers are added on. The aurophilic processes elongate outward along with the increasing concentric layers of tumor cells from L3 to L5. These processes branch and arch over to form a complex supporting framework along with reticulin fibers for the proliferating tumor cells.
The cells showing co-expression of aurophilia and nestin/GFAP positivity in L1 & L2 resemble the radial glia seen during neurogenesis, nestin being a marker of multi-lineage progenitor of neuroepithelial stem cells [26]. The tumor cells grow out into concentric layers to form a compact spheroid structure, the TVC, composed of 5 to 6 layers of tumor cells demarcated by slender reticulin fibers but no definite basement membrane. The TVCs are clearly demarcated from the surrounding sheets of tumor cells which are pushed aside by the expansile growth. The outer layers show neural differentiation with NFP and Syn positivity (Figures 3 and 4).

Mitotic counts in TVCs
Mitosis is mainly concentrated in the layers L2 and L3. 87.7%

Discussion
Melanomas provide a mass of cells as one sees in a 3D matrix. Analysis of the growth patterns would be of benefit for the study of embryonic growth patterns as well as for the study of stem cells. The patterns of neuronal differentiation have been detailed in this work including the localization of neural markers (GFAP, NFP and Syn) by tumor cells in relation to pigmentation. There is a distinct difference between the general tumor matrix and areas of angiogenesis where there is a patterned neural expression and melanocyte morphology.
Melanomas are highly angiogenic and proliferative lesions in the vertical growth phase (VGP) [30,31]. Angiogenesis is the sprouting of blood vessels from preexisting ones where endothelial buds grow out towards the tumor margins [32][33][34][35][36]. Reciprocal paracrine interactions between astrocytes, endothelial cells and ependymal cells have been demonstrated in recent studies. Vascular Endothelial Growth Factor (VEGF) is released from both astrocytes and neurons eliciting a burst of mitotic angiogenesis, which is followed by the production of Brain-Derived Neurotrophic Factor (BDNF) by the stimulated microvascular cells [37][38][39]. In foci of concurrent angiogenesis and neurogenesis, neuronal progenitor cells are spatially associated with mitotic endothelial cells, [40][41][42][43].
The cells abutting on the vascular lumen acquire aurophilia, Nes and GFAP positivity first in the cytoplasm, followed by the outward extension of single processes. As concentric layers of tumor cells grow Nestin has been the predominant marker used to describe stem and progenitor cells in the mammalian CNS. It is a protein belonging to class VI of intermediate filaments produced in stem/progenitor cells in the mammalian CNS during development, and is expressed mainly in neuroepithelial stem cells but not in mature elements. During neuroand gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g. neurofilaments and glial fibrillary acidic protein (GFAP). During embryogenesis, nestin is expressed in migrating and proliferating cells, being restricted to areas of regeneration in adult tissues. Its presence in cells indicates multi-potentiality and regenerative potential.
In neural cell cultures during the first week in culture, approximately 10-30% of the total cell population stained for the glial cell marker GFAP, and nearly all co-express nestin. Nestinis expressed in endothelial cells of CNS tumor tissues and of adult tissues that replenish by angiogenesis and so can be used as a marker protein for neovascularization [26][27][28][29]. This fact is well brought out in the present study, since the endothelial cells lining the angiogenic vessels are positive for Nes as they enter the tumor. As the TVC forms, the L1 cells show Nes positivity as well as GFAP/Auro positivity and resemble radial glia the stem and progenitor cells.
Melanoma cells express characteristics of radial glia, on interaction with the endothelial tubes and further proliferate and differentiate into cells positive for neuronal markers and thus resemble MASC which give rise to neuronal differentiation in neurospheres in cultures . At the tumor/stroma interphase the sprouted endothelial tubes cannelise. Initially, a single layer of tumor cells surround the vessels acquire GFAP positivity and extend processes.
Concentric layers of tumor cells grow out from this layer, supported by GFAP positive processes which extend outward through the layers of tumor cells towards the periphery. Where GFAP positivity is absent there is no further proliferation. As the new layers of tumor cells grow out there is a zone where all three markers are co-localized between L2 to L3 followed by NFP and Syn positivity in L4&L5.
It has been shown that on interacting with the angiogenic vessels, MASC give rise to the full gamut of neuronal differentiation as seen in neurospheres in cultures [56][57][58][59][60][61][62][63][64]. Neurofilaments, the neuron-specific intermediate filaments localized by NFP positivity for neuronal differentiation form the dynamic axonal cytoskeleton together with other axonal components such as microtubules to maintain and regulate neuronal cytoskeletal plasticity [59]. During development neuroepithelial cells in the neuronal lineage lose nestin and vimentin [60] to express NF-H when the maturing cells are forming synapses [62]. NFP positivity is seen in differentiated ganglion cells, neoplasms of neuronal or mixed cell origin as well as neuroendocrine tumor cells. Ramirez et al. [63] found rabbit choroidal melanocytes, perivascular and intervascular fibers positive for NFP.
Synaptophysin is a vesicular integral membrane protein specifically expressed in neural tissues [64]. Synaptophysin labels small synaptic like microvesicles (SLMV) present in neuroendocrine cells such as the pituitary and adrenal medulla. Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. These vesicles characterized by synaptophysin contain considerable amounts of the biogenic amines [64,65]. Earlier studies have identified the presence of biogenic amines in melanocytes. These include catechol amines as well as indole amines [2][3][4][5][6][7].
TVCs show organized expression of biogenic amines in relation to the angiogenic vessel in the middle layers (indoleamines) and in outer (catecholamines) coinciding with increasing Syn positivity [66]. On comparing the levels of positivity in the total number of TVC, GFAP positivity is seen in the layers adjacent to the vessel wall (L1-3). NFP is positive in the peripheral layers (L3-5). Both GFAP and NFP are transitional in L3 equivalent to the 'asterons' described by Steindler DA, 2006 [55]. Syn positivity is highest in L4-5 coordinated with NFP positivity.
Presynaptic Syn/synaptobrevin complex (Syp/Syb-complex) appear during neuronal development [67,68]. Thus Syn expression, initiated in the inner layers of the TVC, peaks as the cells differentiate into NFP positive cells. The sequence of progression from radial glia-llke to neuronal positive cells in the TVC simulates the differentiating patterns in in vitro neurospheres and early embryogenesis of the neural tube. The astrocyte-like stem cells have been shown to have the ability to generate neurons [50,56]. Work of others has shown that in differentiating from primary floating neurospheres newly-generated neurons can assume or revert to an astrocytic phenotype. In differentiating primary floating neurospheres neurons can shift into cells with astrocyte characteristics by transiting through an "asteron" (neuron/astrocyte hybrid) morphotype which coexpress a variety of neuron and astrocyte proteins and genes [55].
The percentage of GFAP+vity in L1&L2 correlates with the percentage of NFP/Syn+vity in L4&L5. In the poorly pigmented PMZ the very low GFAP + vity is associated with a low NFP/Syn + vity. NFP does not increase beyond L3. This is in contrast to the pigmented PMZ where high GFAP + vity in L1/L2 is associated with a similar spike in NFP/Syn + vity in L4/L5 suggesting that the neuronal positivity results from the GFAP + vity after passing through a transitional phase. Thus in those areas where the level of differentiation is low as seen by the absence of pigment, the differentiation of the tumor cells into glial cells on interaction with the neovascular channel is low. This in turn results in low neuronal differentiation.
Neurogenesis is a gradual process that transforms undefined neuroepithelial cells into fully differentiated neurons in two phases: an early, premitotic phase when precursor cells proliferate and adopt a more restricted cell fate; and a postmitotic phase, when neurons are fully committed to a specific cell type [69]. During embryogenesis, the serotoninergic and dopaminergic neurones arise from common progenitors. Biogenic amines have common progenitor stem cells with enzymes cat-1 & cat-4 and bas-1 [70][71][72][73]. As development progresses, the serotoninergic cells remain at the brain stem and the post-mitotic dopaminergic cells proceed to the midbrain SNC (substantia nigra pars compacta).
Proliferating biogenic amine progenitor cells are specified by the combined actions of two signaling proteins, sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8). The combined signaling by these two proteins leads to the induction of the progenitor cells [70]. The mesencephalic DA neurons are generated in the immediate vicinity of two organizing centers, the mid/hindbrain boundary (MHB; also called the isthmus organizer) and the floor plate, a specialized cell type that lies along the CNS ventral midline. The secreted molecules of Shh and Fgf8 at the intersection give rise to DA neurons rostrally of the MHB and 5′HT neurons caudally of this boundary. In addition to Shh and Fgf8, 5′HT neurons require signaling by Fgf4, which might be derived from the primitive streak at earlier developmental stages.
Tryptophane is converted to serotonin by MAO and further to melatonin by the light sensitive enzymes NAT and HIOMT during the dark phase [74]. It is observed that the highly dendritic melanocytes in the pigmented proliferative lesions and in melanomas express both serotonin and melatonin in the cytoplasm [75]. These cells show positivity for MAO with tryptamine as substrate. Thus, as in the case of the tyrosine metabolism the melanocytes show the entire machinery for indoleamine metabolism. The conversion of serotonin to melatonin is inhibited on exposure to a pulse of UV indicating the presence of the light sensitive enzymes NAT/HIOMT [5].
The epidermal melanocytes metabolise tryptamine [4] to produce indoleamines on UV exposure in whole skin organ cultures. In the TVC, indoleamines are positive in L2/L3 associated with mitosis. Both serotonin and melatonin are positive in these layers. Conversion of serotonin to melatonin requires photosensitive enzymes HIOMT/NAT which are switched off by light. [74] A high mitotic activity in L2/L3 is a likely source of biophoton emissions of UV. Since this activity is cyclical with light and dark phases, both SER & MLT are expressed in these layers. In turn, the indoleamines modulate the melanocyte cell cycle in response to UV exposure [4][5][6][7].
Tyrosine is metabolised by several cell types which include catecholaminergic neurones in the brain and spinal cord, [76] sympathetic ganglia, adrenal medulla, and melanocytes which are neuroectodermal in origin. The metabolic pathway is through conversion of tyrosine to dopa, by catecholoxidase, a triphasic enzyme which catalyses both wings of dopa metabolism [77]. The catecholaminergic neurones of the locus ceruleus and substantia nigra produce both catecholamines as well as melanin [78]. This feature is observed in sympathetic ganglion cells [79] and occasionally in pigmented lesions of nerve sheath cells and Schwann cells all cells metabolising tyrosine [80]. The dendritic melanocytes in the skin, rich in enzyme positive melanosomes, show tyrosine hydroxylase and dopamine (DA) positivity, Dopamineoxidase (DAO) activity and Noradrenalin (NA) in response to UV exposure [24]. Thus the melanocyte has the enzyme machinery for the conversion of dopa to dopamine and dopamine to noradrenalin. In the skin catecholamines are expressed during S/G2 arrest on UV exposure [81,82]. As with indoleamines, the likely source of UV is the biophoton UV released in L2/L3 by the high mitotic activity with a resultant S/G2 arrest in the postmitotic cells in L4/L5 [83].
Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. Neuroblasts divide asymmetrically to result in apical/basal polarity in mitotic neuroblasts, to generate an apical neuroblast and a smaller basal ganglion mother cell (GMC) which undergoes one subsequent cell division to generate neurons or glia [84][85][86]. Embryonic microenvironments influence development of neuronal stem cells [87][88][89][90]. Similarly multipotent tumor cells, fate and plasticity of adult cell are influenced by the microenvironment [91][92][93][94][95]. 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 [87,91,[96][97][98][99][100][101]. Human metastatic melanoma cells respond to chick embryo NC-rich microenvironment. Highly aggressive human melanoma cells secrete Nodal, (a potent embryonic morphogen), to induce and form a secondary ectopic embryonic axis in zebrafish [102][103][104].
As seen in the present study, during angiogenesis in aggressive melanomas, a microenvironment is created in the mantle zone of TVC around the vessel to induce and recapitulate early embryonal neurogenesis of indoleaminergic and catecholaminergic cells, much in the manner of the neural tube as summarized in (Figure 4). The angiogenic vessel confers a polarity to the interacting tumor cells which wrap themselves around.
PRL, HGH and ACTH are not the exclusive products of the pituitary. Ectopic production is commonly seen in several tumor types. It has been observed that PRL and HGH are produced by tongue tumors [105], and colorectal carcinoma cells [106]. Ectopically produced PRL may act as a major local growth promoter via autocrine and paracrine mechanisms. Similarly, there are roughly two types of ectopic ACTH syndrome (EAS): one associated with overt malignancies and the other with occult neoplasms. The prototype of the first condition is Cushing's syndrome sustained by small-cell lung cancer (SCLC), while bronchial carcinoid tumors are the most common occult sources of ACTH.
PRL, Growth Hormone (GH) and placental lactogen are class I helical cytokines believed to have arisen from a common ancestral gene. Prolactin has a dual function -as a circulating hormone and as a cytokine. This understanding is based on PRL production and distinct regulation in extrapituitary sites, binding to membrane receptors of the cytokine receptor superfamily, and activation of signaling pathways that promote cell growth and survival. One of the major targets of prolactin is the skin where it also stimulates skin melanocyte growth. There is increasing evidence that PRL plays a role in several types of cancer in reproductive and non-reproductive tissues via local production or accumulation.
The effects of PRL and GH are mediated through activation of the PRL receptor [PRLR], which belongs to the cytokine receptor superfamily [107]. Activation of PRLR initiates multiple kinase cascades, the most studied of which is the JAK-STAT pathway. Thus PRL induces transcription of members of Cyclin D family, which associate with CDKs 4 and 6 in early G1 phase of the cell cycle leading to cell cycle progression and proliferation [108,109]. Associated is an increase in cytosolic calcium concentrations with the progression through the cell cycle and exit from quiescence in early G1 phase, the G1/S transition and other checkpoints during S and M phases [110]. Physiological concentrations (0.5-5nM) of PRL result in an increase in the cytosolic free calcium concentration (Ca2+) by stimulating Ca2+ entry (type I) and/ or inducing a mobilization of calcium ions stored in intracellular compartments (type II) [111]. Besides, there is a direct stimulation of calcium and voltage activated potassium channels (Kca) [112].
Melanocytes also express varying levels of MSH/ACTH in response to UV exposure in correlation with repigmentation and depigmentation as seen in the marginal zone in vitiligo [113]. Melanocortins (MSH) and corticotrophins (ACTH) are both derived from pro-opiomelanocortin hormone (POMC), a large precursor glycoprotein of MW 31000, via a series of enzymatic steps in a tissuespecific manner. Melanocytes contain the full machinery for POMC processing and generation of ACTH and α-MSH [114]. In this aspect, the melanocytes display a corticotrophin releasing hormone (CRH)led system, organized similarly to that operating at the systemic level. Melanocytes respond with enhanced production of cortisol and corticosterone, which is dependent on POMC activity. This is a part of the skin's CRH/POMC system that fulfils analogous functions to the hypothalamic-pituitary-adrenal (HPA) stress axis [115][116][117]. Melanocytes respond to stress (UV) through production of stress hormones related to HPA axis by secretion of the POMC-derived MSH and ACTH peptides, which increase cutaneous melanin pigmentation. Pigment is, thus, the characteristic feature of a differentiated melanocyte.
All three pituitary hormones were expressed by the proliferating melanoma cells. HGH and PRL are expressed in the inner three layers, predominantly in the 2nd and 3rd layers. These were the layers showing a large number of mitotic figures. Pigment and ACTH were typical for in the 4th and 5th layers, where few mitotic figures were observed (Figure 3e). Taken together the results show that areas of proliferation and differentiation are clearly demarcated within the TVC, with proliferation being closer to the vessel and differentiation further from it. PRL and HGH were related to activation of the cell cycle as brought out by the presence of PRL/HGH positivity and mitotic figures in the 2nd and 3rd layer.
Pigment, on the other hand, indicated differentiated function. ACTH positivity was associated with pigment in the 4th and 5th layer. ACTH stimulates pigment production during UV exposure in the epidermis. Is there a possible source of UV in the lobule? Earlier observations on melanocyte dendricity in pigmented basal cell carcinomas and seborrheoic keratosis [83] have shown that mitotic cells are a source of biophoton emissions of UV during the separation of the DNA strands. This correlates with the presence of mitotic figures in the middle layer and with expression of ACTH and pigment in the adjacent outer layers. ACTH holds the cell in the S/G2 phase in which the melanocyte produces pigment [118,119]. The absence of abundant mitosis in the outer layers reflects this function of ACTH on the melanocyte cell cycle. In TVC, ACTH/pigment expression limits or pause the growth and enlargement of TVC at the 5th or 6th layer.
It is evident from the above observations that there is a coordinated growth of melanoma cells around neovascular channels, the cell cycle of primitive malignant melanocytes being orchestrated by the expression of the three pituitary hormones -PRL, HGH and ACTH. In the epidermis it is difficult to assign the different roles to each hormone while in the TVC the functions are clearly demarcated. PRL and HGH initiate a wave of mitotic activity releasing biophoton UV, while ACTH holds the cells in S/G2 phase with pigmentation.
The above findings correlate with expression of neural markers as well as biogenic amines in the TVCs [12,21,66]. PRL/HGH positivity associated with mitosis, coincides with the appearance of indoleamines, the presence of melatonin indicating the effect of biophoton emissions during mitosis. Similarly, catecholamine positivity in the outer layers correlates with the S/G2 phase of the cell cycle, presence of ACTH and pigmentation.

Morphometry of TVCs
TVC counts indicate that their numbers are proportionate to pigment levels in association with level of angiogenesis. Thus the pigmented areas show 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.
For comparison, of morphometric parameters three groups of TVCs (P1,P2 & P3) are from pigmented and 2 (A1 & A2) from poorly pigmented areas from the same case from pigmented to amelanotic nodules. The cell & 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 (Table 1). These parameters show a graded change from pigmented to amelanotic suggesting that TVC formation and angiogenesis are linked with melanotic to amelanotic transition. Formation and progression of TVCs has been studied to assess this possibility.
The intriguing features of angiogenesis and TVCs in melanomas are, the presence of normal mitotic figures, the near normal morphometric parameters and a regulated differentiation replicating embryonic neurogenesis of indole and catechol aminergic neurons. The common Nes positive inner layer, continuous with that of endothelial tubes suggests the role of the common origin of neuronal and endothelial stem cells.