Mechanisms of Nodular and Infiltrative Pattern of Uveal Melanoma in the Liver Open Access

Jones, Nyasia Monet (2016)

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The goal of this research is to gain more insight into the mechanisms by which uveal melanoma (UM) metastasizes to the liver and use this understanding to identify potential targets for therapy. There are two different metastatic growth patterns in the liver: the nodular pattern in which tumor cells surround the portal triad and the infiltrative pattern in which tumor cells invade the sinusoidal space. Pigment epithelium derived factor (PEDF) and natural killer (NK) cells have been implicated by previous research as protective mechanisms against progression of metastatic disease. NK cell depletion in a murine model of UM increased the number of hepatic metastases. Likewise, implanting murine melanoma cells in the uvea of PEDF KO mice resulted in dramatically larger angiogenic metastatic foci. Based on these observations, we hypothesized that knock out of PEDF would result in predominantly nodular pattern while depletion of NK cells would result in the infiltrative pattern of growth predominating. The wild-type model would have equal distribution of both patterns. To test this, we developed three murine models of uveal melanoma in mice with C57BL/6 background that form hepatic metastases: a NK cell deficient model using anti-asialo GM1 antibody, a PEDF KO model, and a model in wild-type mice. The right eyes of mice were inoculated with B16LS9 melanoma cells and at 7 days the eyes were enucleated. Three weeks later, the mice were euthanized and livers were routinely processed for histological evaluation. PEDF KO mice showed increased nodular to infiltrative pattern in comparison to wild type and NK cell deficient mice. Interestingly, NK cell deficient mice did not show increased infiltrative pattern, however NK cell deficiency resulted in significant increase in the number of hepatic metastases and significant increase in stage 2 nodular and infiltrative metastases. Finally, we investigated how loss of the protective functions of PEDF and NK cells affected infiltration of host protective immune cells in the liver. FACS data evidenced an increase in myeloid derived suppressor cells (MDSCs) and tumor associated dendritic cells, suggesting that NK cell or PEDF loss promote an immunosuppressed tumor microenvironment that facilitates tumor growth.

Table of Contents

Introduction. 1

Epidemiology of Uveal Melanoma. 1

The Biology of the Primary Tumor. 1

Diagnosis, Treatment, and Prognosis. 3

Metastatic Disease and Dissemination to the Liver.6

Liver Microniche in Progression from Micrometastases to Macrometastases.10

Nodular and Infiltrative Growth Patterns. 11

Intrinsic Defense of the Hepatic Sinusoid. 14

Role of the Immune Response and Natural Killer Cells in Tumor Progression. 14

Anti-Asialo GM1 induced NK deficiency. 17

VEGF: PEDF Ratio Mediates Tumor Progression. 18

Central Hypothesis and Project Focus. 20

Methods and Materials. 22

Cell Culture. 22

Mouse Model. 22

Inoculation of Melanoma Cells into the Posterior Compartment of the Eye. 23

Anti-Asialo Gm1-mediated NK Cell Depletion. 23

Metastasis, Frequency, and Stage Assays. 23

Evaluation of tumor vascular density. 24

Liver Processing for FACS. 24

Cell surface labeling. 25

Results. 26

[A]. Effects of NK cell depletion and PEDF KO on metastasis frequency and size. 26

[B]. Effects of NK cell depletion and PEDF KO on ratio of infiltrative: nodular pattern. 30

[C]. Effect of NK cell depletion and PEDF KO in mean vascular density of metastases. 34

[D.] FACS analysis of NK cell population in mouse models. 36

[E.] FACS analysis of T cells. 38

[F.] FACS analysis of B cell populations in mouse models. 39

Discussion. 43

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