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The lymphatic vasculature is a key player in progression of many cancers, with lymphangiogenesis at the primary tumour and tumour-draining lymph nodes (TDLNs) associated with poor patient prognosis. As well as providing a highway for metastatic tumour cells, recent reports propose lymphatics as modulators of immunity, highlighting a need for greater understanding of immune regulation by lymphatics. The specific role of lymphatic endothelial cells (LECs) in this context, particularly in TDLNs, is unknown. As TDLNs are immune hubs, yet anti-tumour immune responses are often ineffective, this thesis aimed to investigate functional changes to lymphatics in TDLNs and the role of TDLN-derived LECs in anti-tumour immunity. I hypothesised that factors from the tumour microenvironment alter functionality of TDLN-LECs from early stages of tumour development. I further hypothesised that these changes would promote immune tolerance, with this thesis exploring specific impact on dendritic cell (DC) mediated immunity. Using the B16-F10 melanoma model, this work confirmed expansion of TDLN-LECs prior to metastasis and demonstrated transcriptional reprogramming of immune-associated pathways in LECs isolated from early TDLNs. This was accompanied by differentially localized migratory DCs, clustered at lymphatic subcapsular sinuses. In vitro using co-culture assays revealed mature DCs undergo prolonged interactions with LECs conditioned with B16-F10 tumour-conditioned media, suggesting a change in the physical interactions occurring in vivo in early TDLNs. Additionally, we investigated possible mechanistic contributors, demonstrating using in vitro and in vivo blockade and knockout models, a role for lymphatic expressed Podoplanin in DC interactions and migration. Prolonged physical interactions were further found to facilitate antigen transfer from ovalbumin-loaded LECs to DCs yet inhibit DC priming of T-cells, with DCs found capable of acquiring TDLN-LEC archived antigen in vivo. These results show that in lymph nodes conditioned by factors derived from the tumour microenvironment, prolonged physical interactions between LECs and DCs impact DC migration and T-cell priming. As immune tolerance is a key feature of the tumour microenvironment, this work has highlighted lymphatics as key modulators of the anti-tumour immune response. Furthermore, this work provides new insight into lymphatic involvement during tumour development, identifying lymphatics as a potential target for early intervention therapies.

A collection of papers (D.Sc.1971) from the Dept. of Zoology, Univof Adelaide / 1v. various pagings / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D.Sc.)--University of Adelaide, 1971

The lymphatic system acts to return fluid from the interstitial space back into the blood circulation. In normal conditions, lymphangions, the segment of lymphatic vessel in between valves, cyclically contract and can pump lymph from low pressure tissues to the higher-pressure veins of the neck. With edema, however, this pressure gradient can reverse, and the role of contraction is less clear. Like ventricles, lymphangions are sensitive to both preload and afterload. Unlike ventricles, lymphangions are arranged in series, so that the outlet pressure of one lymphangion becomes the inlet pressure of another. Anything that alters the relative timing and frequency of adjacent lymphangions alters both preload and afterload of each lymphangion and thus mean lymph flow. To explore the effect of timing and frequency of contraction on lymph flow, we developed a computational model of a lymphatic vessel with lymphangions described by the classic description of time-varying elastance. When pumping up a pressure gradient, as in normal conditions, or when pumping down a pressure gradient, as in some cases of edema, we found that flow was optimized when the lymphangions in the vessel were pumping with a very short time delay between their cycles, and the flow was reduced when the time delay between the contractions was reduced to zero. However, a difference in frequency between adjacent lymphangions alters instantaneous flow but does not affect mean flow. These results suggest an important role for the timing of the contraction in optimizing lymph flow. However, a difference in frequencies between adjacent lymphangions has little effect on altering lymph flow, suggesting that tight control of lymphangion coordination may not be critical for lymphatic function.

The lymphatic system acts to return fluid from the interstitial space back into the blood circulation. In normal conditions, lymphangions, the segment of lymphatic vessel in between valves, cyclically contract and can pump lymph from low pressure tissues to the higher-pressure veins of the neck. With edema, however, this pressure gradient can reverse, and the role of contraction is less clear. Like ventricles, lymphangions are sensitive to both preload and afterload. Unlike ventricles, lymphangions are arranged in series, so that the outlet pressure of one lymphangion becomes the inlet pressure of another. Anything that alters the relative timing and frequency of adjacent lymphangions alters both preload and afterload of each lymphangion and thus mean lymph flow. To explore the effect of timing and frequency of contraction on lymph flow, we developed a computational model of a lymphatic vessel with lymphangions described by the classic description of time-varying elastance. When pumping up a pressure gradient, as in normal conditions, or when pumping down a pressure gradient, as in some cases of edema, we found that flow was optimized when the lymphangions in the vessel were pumping with a very short time delay between their cycles, and the flow was reduced when the time delay between the contractions was reduced to zero. However, a difference in frequency between adjacent lymphangions alters instantaneous flow but does not affect mean flow. These results suggest an important role for the timing of the contraction in optimizing lymph flow. However, a difference in frequencies between adjacent lymphangions has little effect on altering lymph flow, suggesting that tight control of lymphangion coordination may not be critical for lymphatic function.

The ovary provides a niche environment where female germ cells (or oocytes) are generated, stored within follicles and later matured in preparation for use during reproductive life. Following an extensive period of quiescence, which in human, may be up to forty years; the follicle surrounding the oocyte begins to grow, promoting maturation of the oocyte, and culminating in the expulsion of a fully mature oocyte in preparation for fertilisation. These events occur cyclically as part of the menstrual (or estrus) cycle and involve extensive remodelling of both the follicle and its surrounding extracellular tissue. Cyclic follicle activation and growth is also associated with concurrent remodelling of the blood vasculature within the ovary, specifically the vessels surrounding the growing follicle. These blood vascular remodelling changes are well explored and have been demonstrated to be necessary for follicle growth, hormone synthesis, ovulation and for the development and function of the corpus luteum. Physiologically, the lymphatic vasculature is known to closely interact with the blood vasculature and plays a number of important physiological roles including the return of extra-vascular fluid to the blood circulation, and in turn, maintenance of systemic fluid homeostasis. Additionally the lymphatic network is required for the trafficking of immune cells from the periphery to lymph nodes; during the initiation of an immune response and for the absorption of lipids and lipid soluble vitamins in the gastrointestinal tract. The lymphatic vasculature develops and functions concomitantly with the blood vasculature; however unlike the blood vasculature, the aetiology of lymphatic vasculature within the ovary is unknown. It is unclear whether lymphatic remodelling events occur in association with folliculogenesis and are necessary for fertility, as is seen with the blood vasculature. To elucidate the mechanisms involved in the establishment and remodelling of the lymphatic vasculature within the ovary, I undertook expansive characterisation of its development and hormonal regulation. I exploited both hormonal manipulation and a known model of disrupted ovarian lymphatic development, the Adamts1 null mouse line, to examine the mechanisms controlling ovarian lymphangiogenesis. Quantitative morphometric analysis of vessel size and number in postnatal mouse ovary revealed that the ovarian lymphatic vasculature develops postnatally between day 8.5 and 12.5, and in synchrony with induction of ovarian CYP19a1 (Aromatase); the time when secondary follicles become FSH-responsive and estrogenic. The establishment of the lymphatic vasculature was also associated with the induction of pro-lymphangiogenic growth factors Vegfc and Vegfd, and their receptor, Vegfr3. Formation of ovarian lymphatics required the matrix-remodelling protease Adamts1, since ovaries from Adamts1⁻/⁻ mice failed to undergo normal lymphatic vascular development. FSH promoted remodelling of the existing lymphatic vascular maturation by increasing lymphatic vessel size in normal (Adamts1⁺/⁻) ovaries, and promoted the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. These vessel changes were also associated with the induction of prolymphangiogenic factors, Vegfc and Vegfd, as well as their receptor, Vegfr3 providing a mechanistic explanation for the hormonal mediated lymphangiogenesis. The concurrent establishment of the lymphatic vasculature with the first postnatal induction of ovarian Aromatase, and the hormone-regulated lymphangiogenesis suggests that a hormonal communication, likely estrogen, may synchronise lymphangiogenesis with folliculogenesis. Like FSH, exogenous estradiol was able to promote the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. Additionally, FSH-analog eCG was able to enhance ovarian lymphangiogenesis during the window of lymphatic establishment (postnatal development of Adamts1 null), whilst inhibition of the production of estradiol using the Aromatase inhibitor Letrozole, during this same window failed to have any effect. This study is the first to investigate the relationship between ovarian lymphatic development and remodelling and folliculogenesis. The present studies reveal that the ovary undergoes lymphatic vascular remodelling, described elsewhere as adult or secondary lymphangiogenesis and that this process involves hormonal contributions from FSH and estradiol, as well as the extracellular matrix protease, Adamts1. This work provides the first evidence of a malleable lymphatic system and a model for regulation of normal adult lymphangiogenesis, and may one day be used to explore ways in which to regenerate damaged vessels to cure lymphatic diseases and disorders. These results also significantly advanced the understanding of the tightly regulated processes controlling fluid dynamics within the ovary. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1454847 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2009

The ovary provides a niche environment where female germ cells (or oocytes) are generated, stored within follicles and later matured in preparation for use during reproductive life. Following an extensive period of quiescence, which in human, may be up to forty years; the follicle surrounding the oocyte begins to grow, promoting maturation of the oocyte, and culminating in the expulsion of a fully mature oocyte in preparation for fertilisation. These events occur cyclically as part of the menstrual (or estrus) cycle and involve extensive remodelling of both the follicle and its surrounding extracellular tissue. Cyclic follicle activation and growth is also associated with concurrent remodelling of the blood vasculature within the ovary, specifically the vessels surrounding the growing follicle. These blood vascular remodelling changes are well explored and have been demonstrated to be necessary for follicle growth, hormone synthesis, ovulation and for the development and function of the corpus luteum. Physiologically, the lymphatic vasculature is known to closely interact with the blood vasculature and plays a number of important physiological roles including the return of extra-vascular fluid to the blood circulation, and in turn, maintenance of systemic fluid homeostasis. Additionally the lymphatic network is required for the trafficking of immune cells from the periphery to lymph nodes; during the initiation of an immune response and for the absorption of lipids and lipid soluble vitamins in the gastrointestinal tract. The lymphatic vasculature develops and functions concomitantly with the blood vasculature; however unlike the blood vasculature, the aetiology of lymphatic vasculature within the ovary is unknown. It is unclear whether lymphatic remodelling events occur in association with folliculogenesis and are necessary for fertility, as is seen with the blood vasculature. To elucidate the mechanisms involved in the establishment and remodelling of the lymphatic vasculature within the ovary, I undertook expansive characterisation of its development and hormonal regulation. I exploited both hormonal manipulation and a known model of disrupted ovarian lymphatic development, the Adamts1 null mouse line, to examine the mechanisms controlling ovarian lymphangiogenesis. Quantitative morphometric analysis of vessel size and number in postnatal mouse ovary revealed that the ovarian lymphatic vasculature develops postnatally between day 8.5 and 12.5, and in synchrony with induction of ovarian CYP19a1 (Aromatase); the time when secondary follicles become FSH-responsive and estrogenic. The establishment of the lymphatic vasculature was also associated with the induction of pro-lymphangiogenic growth factors Vegfc and Vegfd, and their receptor, Vegfr3. Formation of ovarian lymphatics required the matrix-remodelling protease Adamts1, since ovaries from Adamts1⁻/⁻ mice failed to undergo normal lymphatic vascular development. FSH promoted remodelling of the existing lymphatic vascular maturation by increasing lymphatic vessel size in normal (Adamts1⁺/⁻) ovaries, and promoted the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. These vessel changes were also associated with the induction of prolymphangiogenic factors, Vegfc and Vegfd, as well as their receptor, Vegfr3 providing a mechanistic explanation for the hormonal mediated lymphangiogenesis. The concurrent establishment of the lymphatic vasculature with the first postnatal induction of ovarian Aromatase, and the hormone-regulated lymphangiogenesis suggests that a hormonal communication, likely estrogen, may synchronise lymphangiogenesis with folliculogenesis. Like FSH, exogenous estradiol was able to promote the expansion of a new lymphatic vascular network by increasing vessel number and size in Adamts1⁻/⁻ ovaries. Additionally, FSH-analog eCG was able to enhance ovarian lymphangiogenesis during the window of lymphatic establishment (postnatal development of Adamts1 null), whilst inhibition of the production of estradiol using the Aromatase inhibitor Letrozole, during this same window failed to have any effect. This study is the first to investigate the relationship between ovarian lymphatic development and remodelling and folliculogenesis. The present studies reveal that the ovary undergoes lymphatic vascular remodelling, described elsewhere as adult or secondary lymphangiogenesis and that this process involves hormonal contributions from FSH and estradiol, as well as the extracellular matrix protease, Adamts1. This work provides the first evidence of a malleable lymphatic system and a model for regulation of normal adult lymphangiogenesis, and may one day be used to explore ways in which to regenerate damaged vessels to cure lymphatic diseases and disorders. These results also significantly advanced the understanding of the tightly regulated processes controlling fluid dynamics within the ovary. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1454847 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2009

A collection of papers (D.Sc.1971) from the Dept. of Zoology, Univof Adelaide / 1v. various pagings / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D.Sc.)--University of Adelaide, 1971

Thesis (D.Sc.) -- University of Adelaide, 1971. / A collection of papers (D.Sc.1971) from the Dept. of Zoology, Univof Adelaide.