Lymphatic vasculature in ovarian cancer

Abstract

Ovarian cancer (OVCA) is the second most common gynecological cancer and one of the leading causes of cancer related mortality among women. Recent studies suggest that among ovarian cancer patients at least 70% of the cases experience the involvement of lymph nodes and metastases through lymphatic vascular network. However, the impact of lymphatic system in the growth, spread and the evolution of ovarian cancer, its contribution towards the landscape of ovarian tissue resident immune cells and their metabolic responses is still a major knowledge gap. In this review first we present the epidemiological aspect of the OVCA, the lymphatic architecture of the ovary, we discuss the role of lymphatic circulation in regulation of ovarian tumor microenvironment, metabolic basis of the upregulation of lymphangiogenesis which is often observed during progression of ovarian metastasis and ascites development. Further we describe the implication of several mediators which influence both lymphatic vasculature as well as ovarian tumor microenvironment and conclude with several therapeutic strategies for targeting lymphatic vasculature in ovarian cancer progression in present day.

Introduction:

Almost every organ system in vertebrates is composed of two circulatory systems, blood and lymphatic vascular system. While the blood vasculature transports nutrients, provides gaseous exchange to the tissues, lymphatic vasculature maintains the homeostatic fluid balance, transport large macromolecules and traffic immune cells from the tissue parenchyma to the regional lymph node and brings back to the systemic blood circulation (K. Alitalo et al., 2005; Oliver et al., 2020). Lymphatic system as an open loop circulatory system, its association has been well established in the metastatic spread of cancer, (Sleeman & Thiele, 2009) however, the significance of organ specific lymphatic vascular network in tumor cell invasion, microcirculation of molecular cues in organ parenchymal microenvironment and engagement of immune responses exploiting lymphatic system is still an unexplored area (Augustin & Koh, 2017; Hampton & Chtanova, 2019; Niec et al., 2022; Pal, Gasheva, et al., 2020).

Similar to other organs, ovarian parenchyma is perfused with lymph through network of lymphatic capillaries and collecting vessels (Brown & Russell, 2014; Karaman & Detmar, 2014). However, with an added complexity, ovary undergoes cyclical changes in its microenvironment due to periodic ovulation, where hundreds of growing follicles secrete hormones, drives high metabolic demand and facilitate active parenchymal remodeling (Jensen et al., 2017; Kinnear et al., 2020; Liu et al., 2022). In this dynamic environment how the resident lymphatic network compensates this cyclical ovarian morphogenesis, and metastasis from primary ovarian tumor site how upregulated lymphangiogenesis facilitate ascites development and alters ovarian immune landscape and macromolecular transport is still not known (Migone et al., 2016; Sangoi et al., 2008). The first part of the review we focused on general ovarian cancer pathology, present epidemiological relevance and ovarian lymphatic architecture. Subsequently we discussed the role of lymphangiogenesis and its metabolic basis in ovarian carcinogenesis, development of ascites which is a typical hallmark of ovarian cancer pathology and adaptive immune cell engagement in OC pathophysiology. We have demonstrated several critical mediators such as VEGFC, TNF alpha, Ephrin, Histamine which share crossroad functions regulating both lymphatic vasculature as well as ovarian tumor microenvironment along with their signaling axis. Finally, we conclude by discussing present therapeutic modalities of OVCA.

Present epidemiological landscape of Ovarian Cancer:

Ovarian cancer (OC) constitutes one of the deadliest forms of cancer among women (Coburn et al., 2017) and continues to be a critical public health challenge, globally. Although OC is the most common gynecological cancer worldwide (Siegel et al., 2021; Zhou et al., 2021), its incidence displays great geographical variation (Lowe et al., 2013; Momenimovahed et al., 2019). The highest incidence of OC has been observed in countries from developed regions, including North America and Central and Eastern Europe, with lower rates present in Asia and Africa (Brett M. et al., 2017). As OC is difficult to diagnose, and the majority of cases are detected in the advanced stages, this results in poor prognosis (Siegel et al., 2017). However, the incidence and mortality of OC is expected to rise in the coming decades (Bray et al., 2018; Y. Zhang et al., 2019) due to an increased disease recurrence, disproportionate disease occurrence among the races. Prevalent factors such as lack of access to proper health care and diagnostic tools increase OC mortality rate with highest mortality rates present in African populations (Chornokur et al., 2013; Momenimovahed et al., 2019). Recent data also predicts increased OC mortality in certain Latin American countries (Carioli et al., 2020). In terms of race/ethnic demographics, the incidence of ovarian cancer is known to differ greatly across racial groups (Siegel et al., n.d.). OC is more prevalent in white women, followed by Hispanics, African Americans, and Asians (Torre et al., 2018). However, the same is not true in terms of mortality and survival rates. African American women are much more prone to be affected by this malignancy, with a considerably lower survival rates, higher mortality rate when compared to white women (C. Chen et al., 2018; Collins et al., 2014). However, the underlying causes of these prominent racial-based disparities remain poorly understood (Peres et al., 2018). Although social and cultural factors are known to negatively influence these differences (Long et al., 2015), biological factors have been understudied and are only emerging as significant factors underlying ovarian cancer-related racial disparities (Manichaikul et al., 2020).

Ovarian Carcinoma Pathology

Pathologically, malignant ovarian tumors can be classified into epithelial (carcinoma), sex-cord stromal, and germ cell tumors, with carcinomas comprising the large majority (over 90%) of all OC cases (V. W. Chen et al., 2003; de Leo et al., 2021).The 2020 fifth edition of the WHO Classification of Female Genital Tumors classifies ovarian carcinoma into five principal groups namely, high-grade serous carcinoma (HGSC), low-grade serous carcinoma (LGSC), endometrioid carcinoma (EC), clear cell carcinoma (CC), and mucinous carcinoma (MC) based on their distinct morphologic and immunohistochemical characteristics [Figure 1].

Figure 1:

A. High-grade serous carcinoma showing papillary architecture with hierarchical branching and high-grade nuclear atypia (H&E 200X). B. Low-grade serous carcinoma showing solid/cribriform pattern of growth intermixed with small papillae lined by cells with low-grade nuclear atypia (H&E 200X). C. Endometrioid adenocarcinoma of the ovary showing well-formed back-to-back arranged glands lined by columnar epithelial cells with pseudostratified polarized nuclei with moderate degree of atypia (H&E 200X). D. Clear cell carcinoma of the ovary showing papillary formations with hyalinized cores lined by single layer of clear cells with high-grade nuclear atypia and hob nailing (H&E 200X). E. Mucinous carcinoma of the ovary showing columnar cells with variable amounts of apical mucinous cytoplasm and severe nuclear atypia (H&E 200X)

These morphologic subtypes are subsequently further sub-classified based on molecular alterations (Köbel & Kang, 2022).

Histopathologically, HGSC comprises of papillary, cribriform, or glandular structures with high-grade nuclear atypia and prominent nucleoli and association with tumor infiltrating lymphocytes. Immunohistochemically, HGSCs show immunoreactivity to Wilms tumor 1 (WT1), p16, and mutation-type pattern of p53 (Lisio et al., 2019).

In contrast to HGSC, LGSC shows smaller papillary formations, less nuclear pleomorphism and a wild type p53 upon immunohistochemistry (de Leo et al., 2021). EC shows similar histopathologic features to its endometrial counterpart including glandular, cribriform, and villoglandular patterns. The high-grade EC reveals solid growth pattern with high-grade nuclear atypia. The characteristic features such as metaplastic features, association with endometriosis, or presence of synchronous uterine endometrioid neoplasm provide support for the diagnosis of EC in such cases (Matias-Guiu & Stewart, 2018). Diffuse positive immunohistochemical staining for estrogen receptor (ER) and progesterone receptor and negative staining for WT1, mutant p53, and p16 help in differentiating such cases from HGSC (de Leo et al., 2021). CC shows clear cells with abundant glycogen content and hobnailing pattern arranged in tubulocystic, solid, or papillary patterns. Unlike both HGSC and EC, CC is immunonegative for both ER/PR and WT1. Hepatocyte nuclear factor-1β (HNF-1β) is a reliable immunohistochemical marker for CC (Fujiwara et al., 2016). MC of the ovary can be of two major types: intestinal and endocervical based on the morphology of the lining epithelium (Ramalingam, 2016). Lymphovascular space invasion (LVSI) has been described as an independent predictor of lymph nodal metastases, overall survival (OS) and recurrence-free survival (RFS) in both LGSC and HGSC cases (Durmuş et al., 2022; Lorenzini et al., 2022). The presence of LVSI also impacts overall survival (OS) and recurrence-free survival (RFS) in the non-serous ovarian carcinomas of FIGO stage I-IIa (Delvallée et al., 2021).

Ovarian lymphatic architecture and functional role in ovarian physiology:

The major ovarian artery originates from abdominal aorta and the major ovarian veins, left and right, anastomose to left renal vein and inferior vena cava. Both arterial and venous supply divided into extrinsic (hilar region) and intrinsic (cortex and medulla) regions however arteries are much more coiled and tortuous in nature, forms discrete vascular arcade regions compared to veins (Hossain & O’Shea, 1983). However, the role and the function of the ovarian lymphatics in health and its association in clinical scenarios such as ovarian cancer or prevalent ovarian pathophysiology such as polycystic ovarian syndrome has been largely understudied. Recent animal studies using lymphatic drainage tracing experiments (Burchill et al., 2021; Goldberg et al., 2022) suggest there are three routes of lymphatic drainage 1. Lymphatic vessels through ovarian ligament drains into the lymph nodes near iliac artery 2. Lymphatic vessels through suspensory ligament drains towards paracaval and paraaortic lymph nodes and 3. Lymphatic vessels through the round ligament drains into the inguinal nodes (Kleppe et al., 2015). In the context of intra ovarian lymphatic architecture there is a species variability however studies in primates, rabbit and sheep have demonstrated the presence of diffused network of lymphatic capillaries in corpus luteum in addition studies have shown that there is also periodic variation of lymphatic density based on the ovarian luteal phase (Otsuki et al., 1987; F. Xu & Stouffer, 2009). Figure 2 represents a spatial organization of the ovary draining lymphatic node in healthy and ovarian cancer pathophysiology.

Figure 2:

Lymphatic drainage of the female reproductive tract and functions of the lymphatic system in ovarian cancer progression. Lymphatic architecture of non-diseased female reproductive tract. Lymphatic spread of the ovarian tumor and multifunctional role of lymphatics in ovarian tumorigenesis.

The organ level lymphatic architecture in relation to ovarian physiology is under studied. However based on the recently explored lymphatic networks in several organ systems (Burchill et al., 2021; Goldberg et al., 2022; Louveau et al., 2015), (Baranwal et al., 2021; Creed & Rutkowski, 2021; Donnan et al., 2021), it is evident that ovarian lymphatic circulation can potentially play critical roles through ovarian parenchymal fluid homeostasis, transporting macromolecules such as growth factors and trafficking immune cells (Pal et al., 2017; Ye et al., 2016). Figure 3 represents a typical lymphatic network of collecting lymphatic vessels.

Figure 3:

Lymphatic architecture in a typical tissue niche. 3 A. Represents the network of lymphatic vessel surrounded by blood vessels and adipose tissue. A1. Lymphatic vessel A2. Blood vasculature 3B. B.1 Architecture of a typical collecting lymphatic vessel immune-stained with CD31(PECAM 1), which also shows a bi-cuspid valve, typical characteristics of a collecting lymphatic vessel which helps to propel unidirectional flow of lymph in the tissue microenvironment

(Stefańczyk-Krzymowska & Krzymowski, 2002) After puberty ovary cyclically goes through pre ovulatory and post ovulatory periodic changes such as follicular maturation, progression, degeneration, and regeneration of corpus luteum. Studies in pig and sheep animal models have shown these changes can potentially affect ovarian lymph flow as well as ovarian lymph contents Click or tap here to enter text.(Stefańczyk-Krzymowska & Krzymowski, 2002). , 2002).

Therefore, the ovarian physiology specific factors such as hormones, cytokines, growth factors and their specific contribution in ovarian lymph flow and lymph volume and, how these factors periodically regulate ovarian lymphatic vasculature remodeling such as lymphatic vessel sprouting or pruning need in depth investigation.

Lymphatic lineage and lymphangiogenesis in ovarian tumor microenvironment:

The development of lymphatic vasculature starts during early embryonic development, mainly differentiating from veinous origin. Lineage tracing experiments show progenitor cells expressing COUP-TFII induces the expression of transcription factor prospero related homeobox 1 (Prox-1) which further drives the expression of vascular endothelial growth factor 3 in the lymphatic progenitor cells. Prox 1 + Vegfr 3+ lymphatic progenitor cells subsequently undergo downregulation of blood vessel lineage specific genes and starts to express lymphatic lineage specific markers such as Podoplanin, Lyve-1 in initial lymphatics. Initial lymphatics further starts to develop lymphatic capillaries which essentially form lymphatic collectors. The organ specific lymphatic network consisting lymphatic capillaries, collectors in a tissue environment dependent manner express relevant lymphatic lineage genes such as Foxc2, Connexin 37, VE cadherin, PECAM. However, in-depth studies on lymphatic development, growth and maturation can be found somewhere else (Oliver et al., 2020) as this is beyond the present focus of the topic.

A recent clinical finding by Sundar et al demonstrates significant expansion and density of lymphatic vessels (Abouelkheir et al., 2017; Sundar et al., 2006) at primary ovarian tumor site. This tumor associated lymphangiogenesis can be corelated with higher metastasis and decreased survival rate which is often observed in ovarian cancer patients. The growth of lymphatic capillaries in tumor site plays a critical role in metastasizing tumor cells from tumor foci to draining lymph node and subsequently egression to systemic circulation via nodal lymphatic or blood vessels. In this multistep process the newly formed lymphatic capillaries not only support inflammatory microenvironment of the tumor, but also bidirectionally provides signals for lymphangiogenic growth. Tumor cells in the primary tumor site, cancer associated fibroblasts (Erez et al., 2013; Nishida et al., 2006) can secrete lymphangiogenic factors such as VEGFC, COX-2, HIF1 alpha (Gomez-Roman et al., 2016; Timoshenko et al., 2007) chemokines such as CCL21, CXCL12 (Zhuo et al., 2012) which affect lymphatic endothelial cell permeability, expression of adhesion molecules, migration ability of lymphatic endothelial cells for nascent capillary tube formation as well as remodeling of extracellular matrix (ECM) of discontinuous basement membrane in peri and intra tumor lymphatic vessels. The lymphangiogenic signals originated from the tumor microenvironment often accompany with alteration in interstitial fluid volume which results into increased intra tumoral fluid pressure. The alteration of tissue fluid dynamics in the tumor niche affects luminal shear stress, lymphatic vessel diameter as well as lymph flow. As a consequence long term vascular remodeling pathways is ensued for example YAP-TAZ signaling cascade (Frye et al., 2018; Grimm et al., 2019) which subsequently orchestrate changes in bio-physical properties of lymphatic vessel such as vascular compliance, lymphatic tone, ejection fraction, as well as other molecular features such as endothelial junctional, lymphatic muscle cell investiture, permeability of peritumoral lymphatic capillaries, lymphatic vessel carrying capacity inside of a the primary tumor mass.

Recent study using cancer stem cells (CSC) isolated from the ascites of high-grade serous adenocarcinoma patients shows in xenografts as well as in spheroid, differentiated cells contribute to form tube like structures showing higher expression of lymphatic endothelial cell markers. Suggesting positive association of lymphangiogenesis and ovarian cancer progression (Krishnapriya et al., 2019). Furthermore, severity of ovarian carcinoma in postmenopausal women shown to be related with decreased level of estrogen secretion and increased level of luteinizing hormone (LH) and follicle stimulating hormone (FSH). In line with these observations study by Sapoznik et al in an ovariectomy mice model have shown that LH and FSH can potentially induce the expression of VEGF-C in a p75 dependent manner augmenting ovarian tumor lymphangiogenesis (Sapoznik et al., 2009).

Role of fatty acid metabolism in lymphangiogenesis in the course of ovarian cancer:

About 20 to 25 percent of women diagnosed with ovarian cancer have a hereditary tendency to develop the disease. Although, numerous genes are implicated in this phenomenon, patients who carry BRCA (Breast Cancer gene) - BRCA 1 and BRCA 2-gene mutations are significantly at increased risk and make up about 17 percent of the approximately 25 percent of inherited OVCA. Recent advancements have been made in understanding the relationship between the genes and fatty acid receptors which are specifically upregulated in ovarian cancer progression and metastasis. Other than germline BRCA mutations, mutated inherited genes like TP53 and CHEK2 could increase the risk of OVCA (Matulonis et al., 2016). Unlike non-epithelial tumors, which make up about 5 to 10 percent of OVCA, High-grade serious ovarian cancers are mostly caused by mutations in the genes that code for p53 and BRCA (Tomczak et al., 2015). Previous work indicating that adipocytes may be an energy source for augmenting cancer cell proliferation supports the hypothesis of lipid metabolism being an essential factor is OVCA progression (R. R. Chen et al., 2019). The most common fatty acid receptors implicated in OVCA progression are: Low density lipoprotein (LDLR) and Lysophosphatidic Acid Receptor (LPAR). LDLRs-lipid molecules that regulate the uptake of fatty acids (X.-L. Chang et al., 2017) and are mostly connected to enhanced chemoresistance in OVCA. Overexpression of LDLRs independently and LDLR tagged with SREBP2 increases chemoresistance while knockdown of LDLRs increased sensitivity to platinum-derived drugs (Ji et al., 2020). LDLRs promote cell proliferation via binding to G protein coupled receptor (Tsujiuchi et al., 2014; P. Wang et al., 2007). LPAR, grouped into LPA 1–3 and LPA 4–6, are actively involved in cancer migration and oncogenic signaling pathways via metastasis as evidenced in Xu et. Al’s findings of TRIP6-thyroid receptor interference protein 6-influencing “LPA-induced cell migration by binding to LPA2 receptor (J. Xu et al., 2004).

The mutually benefiting OVCA-Adipocyte’s relationship has also shown to be essential in OVCA metastasis where CD36, a lipid uptake regulator, FABP4-fatty acid transport protein, and SCD1-fatty acid desaturase-being major players implicated in cancer migration (Ladanyi et al., 2018a; Nagaraja et al., 2016). The lipid rich environment in ovarian tissue supports increased metabolic demand of OVCA through lipogenesis and fatty acid uptake and contributes towards metastasize and survival of cancer stem cells survival (CSCs) in OVCA niche (Ladanyi et al., 2018b; Shibue & Weinberg, 2017). In addition to CD36, fatty acid metabolism is regulated by fatty acid binding proteins (FABPs), transport proteins (FATPs) and translocases which are frequently shown to be transcriptionally upregulated in multiple cancer cells to meet increased metabolic demand for their proliferation and metastasis (Zhao et al., 2019). For example, overexpression of FABP3 and FABP7 resulted in increased lipid uptake in a VEGF inhibitor-resistant xenograft mouse model of glioblastoma (Bensaad et al., 2014) while downregulated FABP5 in prostate cancer showed a decrease in lipid uptake implicated in the metastatic ability of the cancer cells (Carbonetti et al., 2019). Furthermore, recent studies done in vivo orthotopic mouse models have shown the role of FABP4 in mediating OVCA metastasis in vitro with the downregulation of FABP4 correlating with reduced OVCA cell migration and treating OVCA cells using tamoxifen-FABP4 inhibitor-showed a dose-dependent reduction in free fatty acid synthesis (Gharpure et al., 2018). Out of many, these are few examples suggesting close associations of altered lipid metabolism and metastasis regulated by FABPs, FATPs which are also often upregulated in OVCA, contributing towards late diagnosis and poor prognosis (Ladanyi et al., 2018b).

In addition to OVCA progression, upregulated fatty acid metabolism was also shown to have a positive effect towards lymphangiogenesis. Increased lymphatic vessel density due to enhanced lymphangiogenesis facilitates aggressive metastatic spread of ovarian tumor cells as well as support tumor microenvironment. As LECs compose the inner lining of the lymphatic vessel and capillary lumen, therefore, they are constantly exposed to circulating lymph which is enriched with fatty acid content. The abundance of FAs in lymph allows enhanced transcellular FA transport through expressed FABPs and FATPs by LECs. Exported fatty acids or circulating oxidized LDL, especially long carbon chain ones contribute to the supply of energy demands during lymphangiogenesis and LEC specific differentiation though fatty acid oxidation (FAO) (Neiman et al., 2011). Increased FAO enhances LEC lineage specific transcription factor PROX1 mediated expression of mitochondrial enzyme CPT1A, which contributes to the transfer of acyl groups and upregulate the synthesis of acetyl co enzyme A in fatty acid oxidation. Acetyl co enzyme A further contributes to histone acetylation enhancing the transcription of lymphangiogenic genes such as Prox-1, Vefr3, integrin alpha 9. This feed forward loop results not only upregulation of lymphangiogenesis and subsequent metastatic spread but also the morphology of lymphatic capillary such as permeability is altered. Through VEGFR3-VE Cadherin signaling axis increase zipper like junctions in capillary lumen causing decreased uptake of interstitial tissue fluid through lymphatic microcirculation, in turn aggravating tissue inflammatory environment (Wong et al., 2017). Figure 4 represents fatty acid metabolism in relation to lymphangiogenesis in the context of ovarian cancer.

Figure 4.

Schematic of Fatty acid metabolism and lymphangiogenesis in OVCA pathophysiology

Role of lymphatics in ascites development:

The major hallmarks of the terminal ovarian cancer pathology, accounting approximately 38%, is the formation of malignant ascites in the peritoneal cavity (Kipps et al., 2013). Besides ascites served to be the prognostic factor for the stage 3 and 4 diagnoses, one of the major recurring problems in OVCA pathogenesis is ascites, which are also contributing factors such as severe pain, anorexia, respiratory distress (Tan et al., 2006). Ascites is mainly characterized by accumulation of protein rich fluid, malignant and nonmalignant cellular composition present in the peritoneal cavity. Pathogenesis of the ascites is complex and often intractable ascites is the major contributing factor for the ovarian cancer recurrence and chemo resistance after combination therapy (Ayantunde & Parsons, 2007).

One of the major factors for ascites formation is, when the generation of the peritoneal fluid production (due to increased capillary permeability) is greater than its drainage capacity (Chung & Iwakiri, 2013) (due to decreased peritoneal fluid clearance) ascites tend to build up. One of the critical factors for decreased peritoneal fluid drainage is reduced oncotic pressure in portals, named as lymphatic stomata, which circulates macromolecules, protein rich lymph to the omental and subdiaphragmatic surfaces. Obstruction of these lymphatic stomata by cancer cells lead to an increase in interstitial fluid pressure (IFP) which alters lymphatic microvasculature drainage capacity. However, the specific mechanism/s of how lymphatic microvasculature or capillary function is disrupted still requires in depth elucidation. Based on the present knowledge there could be multiple possibilities. Functionality of the lymphatic capillaries is dependent upon lymphatic endothelial cell junctional permeability as well as anchoring filaments which attaches the lymphatic lumen with the adjacent tissue connective tissue. Elevation of IFP can alter structural organization of the anchoring filaments which can disrupt lymphatic lumen patency and as a result obstructing lymphatic fluid flow and lymph drainage (Chung & Iwakiri, 2013). Furthermore, chronic inflammatory environment can potentially induce LEC junctional architecture, inducing zipper like pattern from button like pattern, affecting interstitial fluid permeability to enter into the lymphatic lumen, overall decreasing the capability of fluid drainage from peritoneum. Recent studies have also shown that overexpression of VEGF is associated with formation of malignant ascites and it accumulates in the ascitic fluid over time (Barton et al., 1997; Kipps et al., 2013). VEGF can induce lymphangiogenesis, as a consequence enhance metastatic spread (Neufeld et al., 1999). Therefore, drainage of peritoneal ascitic fluid can potentially promote lymphatic vasculature pruning, downregulate lymphangiogenic signaling (Mesiano et al., 1998; Yukita et al., n.d.) which can result into inhibition of metastatic spread.

Lymphatic vasculature in ovarian cancer metastasis:

In tumor microenvironment (TME), a fraction of cellular composition is tumor cells. However, the major cell types are stromal cells, immune cells such as dendritic cells, T cells, tumor associated macrophages, cancer associated fibroblasts. This complex composition of ovarian tumor not only alters ovarian parenchymal ECM stiffness (Heerma van Voss et al., 2010), elasticity, organizational microstructures but also their secreted biochemical cues alter tumor progression, evolution of tumor pathology as well as intra-organ trafficking of immune cells and cancer cells. As we discussed earlier, one of the integral functions of the lymphatic vascular network is to traffic immune cells. Generation of unidirectional lymph flow and the composition of the lymph provides a cue for the migrating immune cells in the parenchyma to enter into the lymphatic lumen through endothelial junctions. However, unlike blood vascular endothelial junctions, which are mainly tight or adherent in nature, lymphatic endothelial junctions have different structural organizations. In lymphatic capillaries lymphatic endothelial cells form button like junctions which are discontinuous, highly permeable in nature, allows entry of immune cells and macro molecules on the other hand, the junctions in the collecting lymphatic vessels are continuous and zipper like in nature (F. Zhang et al., 2020), has low permeability, prevents lymph leakage contributes towards movement of lymph towards draining lymph nodes. The cascades of events are represented in Figure 4.

The association between lymphatic vasculature with different ovarian carcinoma types is still unexplored. Based on the origin of the ovarian cancers, mostly they are epithelial, among them high-grade serous carcinomas (HGSC) which shows most aggressive and high metastatic potential. The metastatic behavior of HGSC ovarian cancer type is distinctly different than cancers such as lung, liver or colorectal due to its ovary’s adjacent anatomical location to the peritoneal cavity. Therefore, there is less anatomical barrier for migration of the ovarian cancer cells to cross from the primary tumor site to the milieu of omentum or intraperitoneal cavity (Lengyel, 2010) through direct transcoelmic or vascular route (Pradeep et al., 2014). However, the contribution of vascular route in metastatic progression ovarian cancer is still not clearly defined. Multiple recent studies from several cancer pathologies suggest that in addition to the spread to the lymph node newly formed peritumoral lymphatic vessels are closely associated with primary tumor site (A. Alitalo & Detmar, 2012). These nascent lymphatic vessels can serve as a conduit for metastatic dissemination of the tumor cells from tumor site and contribute towards the recirculation of tumor derived metabolites to the systemic lymphatic circulation.

This eventually facilitate towards creation of omental metastatic niche for the growth of the ovarian cancer. The mechanisms of the entrapment, adhesion and seeding of the circulating tumor cells from primary sites through lymphatic vasculature specifically in ovarian cancer phenotypes is still far from mature understanding. However, in general, cancer cell derived factors for example TNF alpha, histamine, VEGF,1/2, and secreted vesicles such as exosomes (Johnson et al., 2016; Joyce & Pollard, 2009; Minciacchi et al., 2015; Santin et al., 1999) play critical roles through the downstream signaling in peritumoral lymphatic endothelial cells. For example, in a study with by Santin et al reported that patients with ovarian cancer shown to have high level VEGF in ascitic fluids as well as in plasma (Santin et al., 1999). This upregulation of VEGF by the ovarian tumor cells can induce the expression of adhesion molecules such as ICAM1, VCAM-1 in lymphatic endothelial cells in a NFKB dependent manner (Teijeira et al., 2013). Furthermore, upregulation of transforming growth factor beta 1, which Is also shown to be associated with epithelial ovarian carcinoma (J.-C. Cheng et al., 2012), can upregulate the expression of CCL21 in LECs. Cancer cells through CCR7/CCL21 or CXCL12/CXCR4 axis (Shayan et al., 2013) can subsequently influence the trans lymphatic endothelial associated distant migration of ovarian cancer from its primary sites.

Lymphatic vasculature in immune cell trafficking and immune tolerance in ovarian cancer:

From the recent studies it is being evident that the evolution of organ specific primary cancer cells and its metastatic potential has a complex non-linear dynamics . It varies from primary tumor site to the distant seeding sites due to many factors, such as, clonal variations, acquired mutations during colonization in the seeding organ’s parenchymal microenvironment, bidirectional interactions between invading cancer cells with immune cells as well as with blood and lymphatic vessels in the time of trafficking (Naxerova et al., 2017; Pal, Nath, et al., 2020). Besides lymphatic vasculatures contribution of cancer cells’ migration, infiltration of migratory immune cells such as T cells, antigen presenting calls and cells near perilymphatic region into lymphatic lumen engage interaction with lymphatic endothelial cells. However, the immunomodulatory role of lymphatic endothelial cells whether influence the progression of cancer or inhibits tumor growth appears to be dichotomous. To render a better insight some of the recent studies is discussed here. During tissue inflammation antigen presenting cells such as macrophages have shown to be recruited towards lymphatic vessels (Kuan et al., 2015). Recruitment of macrophages can negatively regulate lymphatic function as higher expression of iNOS by macrophages increases NO bioavailability which reduces lymphatic vessel tone, inhibits self-contractility thereby pumping of lymph by collecting lymphatics causing lymph stasis which eventually induces immune suppression (Liao et al., 2011) in that tissue niche. Similarly in a recent study where diphtheria toxin mediated specific depletion of lymphatic endothelial cell in breast cancer model shows enhanced tumor PD-L1 expression, an inhibitory check point molecule, increased inflammatory cell accumulation and significant reduction in cytotoxic T cells population aggravating tumor pathology (Kataru et al., 2019). Furthermore, lymphangiogenic signals such as VEGFR-3/VEGFC signaling in LEC can upregulate chemokine ligand 21 (CCL21) which enhances CCR7 dependent dendritic cell trafficking to lymphatic vessels. Increased infiltration of DC into the tumor site through lymphatic capillaries can alter TME, enhance immune activation through engaging effector cells such as CD4+ or CD8+ T cells, which essentially mitigate disease progression. For example, in a study by Zhang et al, in human epithelial ovarian cancer patients have shown that there is a significant difference in five-year overall survival rate (73.8 %) with patients with ovarian tumors having infiltration of T cells in comparisons with patients no to less intratumoral T cell infiltration (11.9%) (L. Zhang et al., 2003). Altogether these studies suggest the beneficial immunomodulatory and inhibitory role lymphatic vasculature in progression of cancer pathology. In the contrary there are multiple reports also suggest that increased lymphatic vessel density is associated poorer outcome and fuels advanced cancer progression (Abouelkheir et al., 2017). However, the bidirectional communication between lymphatic vasculature and cancer tissue is one of the key rate determining factor and in this process constant crosstalk through several mediators or their receptors expressed by both lymphatics of ovarian tumor plays a critical role in the evolution of ovarian cancer pathophysiology.

Non codi ng RNAs in regulat ion of ovarian cancer and in lymphatic vasculature:

In recent years noncoding genome has significantly shown to be related with regulation of physiological homeostasis as well as different disease pathologies. Different non-coding RNA classes such as micro RNA, long non coding RNA, siRNA, piwi interacting RNA, how they are involved in the growth and maintenance of lymphatic vasculature in the ovary or ovarian cancer remains to be elucidated. Here are some of the non-coding RNA functions (Table-2) which have been reported in the regulation of lymphatic vasculature at the same time shown to be associated with ovarian cancer pathology.

Table-2.

Non-coding RNA in context of ovarian cancer and lymphangiogenesis

Noncoding RNA	Lymphatic context	Ovarian cancer context	References
Micro RNA 31	Downregulation of lymphatic sprouting through Prox −1 down regulation.	Reduction of expression is associated with increase in chemo resistance through receptor tyrosine kinase MET activation	Pedrioli et al., 2010 Mitamura et al., 2013
Micro RNA 181 a	Bind with 3’UTR of Prox-1 causing translation inhibition and lymphangiogenesis	TGF beta mediated epithelial to mesenchymal transition via SMAD 7 in high grade serous ovarian cancer, increase tumor burden and drug resistance	Kazenwadel et al., 2010 Parikh et al., 2014
Micro RNA 126	Induce lymphangiogenesis through Vegfr3 signaling and VE Cadherin expression.	Association of miRNA 126 with BRCA1 methylation positive OC patients	Chen et al., 2016 Kontarakis et al., 2018 Al-Showimi et al., 2022
Micro RNA 182	Attenuates FOXO1 expression and thereby vascular development	Associated with higher invasion, and overexpressed in high serous ovarian carcinoma	Kiesow et al., 2015 Liu et al., 2012
Micro RNA 128	Inhibits the expression of Vegfr3/Vegfc expression	Inhibition of expression associated with cis platin resistant ovarian cancer	Zhou et al., 2018 Li et al., 2014
Inc RNA-LETR1	Modulate lymphatic specific genes through KLF4, SEMA3	Yet to be elucidated	Ducoli et al., 2021

Therapeutics of OVCA

Lack of early detection makes ovarian cancer (OC) a silent killer, such that OC biomarkers are very crucial for better outcome. To date, carbohydrate antigen 125 (CA 125) is one such clinically used biomarker for OC screening, but it may not be sufficiently reliable for early stage detection, as many non-cancerous conditions may also increase CA 125 level in the blood (M. Zhang et al., 2021). For such reasons, directing and understanding OC treatment courses is pedantic for a patient’s survival especially in advanced stages. Conventional treatment/remedy for OC offers/include primary debulking surgery with intravenous chemotherapy (mainly platinum and taxane based), for instance, carboplatin, cisplatin, and paclitaxel. Besides, in some cases of distant OC metastasis of high-grade advance stage epithelial OCs, cytoreductive surgery followed by HIPEC (Hyperthermic intraperitoneal chemotherapy) is given (Cianci et al., 2020; Riggs et al., 2020). This approach resulted in better patient outcomes with delayed recurrence-free survival. However, HIPEC may sometimes show life-threatening complications post-treatment, which include hematological toxicity, pleural effusion, kidney damage, or failure (Cianci et al., 2020). Due to such adverse post-treatment complicacy, late diagnosis, and platinum-resistance, or refractory cancer (which accounts for less than 30% 5 years survival rate), novel approaches to improve patient outcomes are much needed.

An extension to IP, a newer approach to treat OC patients showing peritoneal carcinomatosis is administered with PIPAC (Pressurized Intraperitoneal Aerosol Chemotherapy). Unlike HIPEC, PIPAC makes use of the aerosolized form of chemotherapeutic drugs instead of heated drugs spread over the abdominal cavity during open surgery, as a result, it assures depth penetration of the drug. PIPAC is in various clinical trials with several drugs, for instance, PIPAC Nab-pac (albumin-bound nanoparticle paclitaxel) is in phase II trial for stage IIIB, IIIC, IV OC, breast, stomach, and pancreas cancer (Coleman et al., 2011). For plantin-resistant OC (rPROC) patients PIPAC-OV3 is designed which uses cisplatin and doxorubicin with PIPAC (PIPAC C/D) (Bakrin et al., 2018). The phase II trial has already reported enhanced tumor regression with comparatively low systemic toxicity. PIPAC-OV3 is now under clinical trial phase III, which evaluates the effectiveness of PIPAC C/D for PFS compared to the standard anti-cancer treatment (Bakrin et al., 2018). A highlight of OC is frequent early relapse of the disease, most OC patients show relapse after 18 months with refractory cancer or rPROC, and eventually die from disease (Schmid & Oehler, 2014; Yap et al., 2009). Therefore, one very crucial segment of OC treatment is maintenance therapy especially for advance stages. The current understanding of the underlying biology of OC has led to the development of different targeted therapies, some of them are under clinical trials and represents a rational strategy OC cure and progression-free survival (PFS) for the patients (Áyen et al., 2018; Schmid & Oehler, 2014). One such therapy targets angiogenesis because of its extensive involvement in outgrow of tumor and metastasis (Burger, 2011; Folkman, 1972). Vascular endothelial growth factor (VEGF) and its tow receptor VEFR receptor-1 (Flt-1) and VEGF receptor-2 (KDR) are the key angiogenic factor that helps solid tumor growth. Because of this VEGF inhibition is essential to reduce the blood supply and starve the tumor cells. A humanized recombinant antibody, Bevacizumab (BEV) targets VGF and decreases the angiogenic potency of cancerous cells (Grunewald & Ledermann, 2017). BEV is explored as a single agent for targeted anti-angiogenic therapy. Over the last few years, BEV have been in various clinical trials and till date there has been five phase III trials. Among these, recently the open-label phase III AURELIA (Avastin Use in Platinum-Resistant Epithelial Ovarian Cancer) trial, tested BEV in combination with PLD, paclitaxel, or topotecan in OC patients who have completed their four cycles of platinum based chemotherapy and showed recurrence within 6 months (Lyon & Huang, 2020; Pujade-Lauraine et al., 2014). Results of this trial revealed promising PFS benefit in rPROC, and thus, it has been approved to treat rPROC patients by the United States and the European Commission (Lyon & Huang, 2020). However, AURELIA had a limited patient eligibility, that’s why REBECA (Real-world effectiveness of BEV based on AURELIA in platinum-resistant recurrent ovarian cancer), an observational study is created to analyze the efficacy of AURELIA (Lee et al., 2019). (The PFS of AURELIA is only 6.7 months from the onset of second-line chemotherapy). Next to angiogenic inhibition, another important maintenance therapy for OC focuses on PARP inhibition (Grunewald & Ledermann, 2017; Q. Wang et al., 2020). PARP inhibitors (PARPi) recently have been under many clinical trials, and both BRCA mutants (BRCAm) and non-BRCAm OC patients been beneficiaries (Q. Xu & Li, 2021). In this regard, SOLO-1 phase III trial was done to investigate olaparib (PARPi) as a maintenance monotherapy for BRCAm advance epithelial OC patients (Walsh, 2020). Later on, PAOLA-1 phase III study evaluated the combination effect of olaparib with BEV in OC patients regardless of BRCA status, and it was the first phase III clinical trial for PARPi combination regimen (Ray-Coquard et al., 2019). Both of them are showed promising survival benefit and prolonged PFS, they have also been approved by FDA for OC treatment in clinical practice (Q. Xu & Li, 2021).

In addition, various treatment strategies are being explored for OC gene therapy (GT): i) tumor-suppressor genes for either altering the gene or compensating the mutation. ii) onco-factor inactivation approach, i.e. inhibiting dominant oncogene or growth factors. Here in, both oncogene and signaling pathways are targeted. One of the most studied oncogene is EGFR as it is overexpressed in 35–70% OC cases. Secondly PI3K/AKT/mTOR signaling pathway, which is frequently activated in most OCs as well as mutated PI3K and/or AKT, mTOR oncogenes occurs in good percentage of OC cases (Li et al., 2014). Some PI3K/AKT/mTOR inhibitors have been tested, for instance, an AKT inhibitor Afuresertib with chemotherapy provided an acceptable safety prolife in rPROC patients in phase IB trial (Blagden et al., 2019). A phase II study of it combined with weekly paclitaxel in same patient group is under way. EGFR silencing has also been in practice, however it has not been evaluated in vivo or approved by FDA. iii) anti-angiogenic GT which involves refining vascularity of tumor. iv) multi-drug resistance (MDR) associated gene treatment (like PRP-4 knockdown and survivin). v) suicide GT also called molecular chemotherapy, employs a prodrug system to deliver intra-tumoral genes which encodes for toxic anti-metabolites to kill tumor cells like HSV-1 TK (herpes simplex virus thymidine kinase) gene. vi) oncolytic-virotherapy, tumor-specific competent replicating viruses are engineered that destroy or eliminate cancerous cells and stimulate anti-tumor immunity. vii) tumor immunopotentiation, which implicates the transfer of cytokine and interleukin at the site of tumor or expression of tumor antigen or strengthens immunity against cancer cells (Áyen et al., 2018; Kaur et al., 2009; Q. Wang et al., 2020).

Since most OC cases are diagnosed in later stages and by that time cancerous cells have metastasized to peritoneal cavity and pelvic or para-aortic lymph nodes, OC regarded lymphatic system management may provide a prognostic benefit. On this point, systematic lymphadenectomy was initiated in patients with advance OC. LION (Lymphadenectomy in Ovarian Neoplasm) trial was practiced, however this study did not show any profitable outcome in the patients (Harter et al., 2019). Moreover, splenectomy revealed promising results for both early and advance stage disease (Macciò et al., 2021). In addition, for epithelial OC that have spread extra-abdominally (very rare) cardiophrenic lymph nodes plays a vital role in preferably stage IV, and its resection may contribute to longer PFS. Until now conclusive report on long-term outcomes is still under investigation (Boria & Chiva, 2021; Larish et al., 2020).

Conclusion

Lymphatic vascular network and lymphangiogensis associated with inflammation have recently emerged as signaling platform for tumor pathogenesis. The lymphangiocrine signals from lymphatic endothelial cells not only influence tumor microenvironment by inducing migration, invasion and trafficking of cancer cells through lymphatic vessels but also changes immune landscape by transporting tissue resident and peripheral immune cells to the tumor site. Despite the growing evidence of the involvement of lymphatic vasculature in ovarian cancer pathogenesis, such as formation of ascites, co-relation of lymphatic vessel density with metastasis, and chemoresistance, still there is much mechanistic insights are still undefined. In this article discussing ovarian cancer pathogenesis from the context of lymphatic circulation makes the case that basic understanding lymphatic vascular network in the ovary is still significantly to be explored and likely to open new direction in OVCA therapeutic approach.

Figure 5.

Contribution of the lymphatics in the ovarian tumor growth and metastasis.

Table 1.

Mediators influencing both lymphatic vasculature and ovarian tumor microenvironment

Mediator names	Cellular	Function	Signaling axis	Reference
	Source
VEGFC	Tumor associated myeloid cells; Tumor associated macrophages	>initiate lymphangiogenesis which promotes metastases through lymphatic route. >Upregulate of BRCA1 and 2 gene expression	1. VEGFC-VEGFR3 > BRCA1/2 upregulation 2. VEGFC-VEGFR3>MEK>ERK>Sprouting	(Lim et al., 2014; Zheng et al., 2014) (D. Cheng et al., 2013; Decio et al., 2014),
CCL21	High endothelial venule, lymphatic endothelial cells, secondary lymphoid tissue	>migration and invasion of dendritic and T cells through lymphatic vasculature at ovarian tumor site.	CCL21/CCR7 chemokine axis TGF beta mediated activation of CCR7/CCL21 mediated chemotaxis.	(Marcovecchio et al., 2017; Yin et al., 2013) (Karlsen et al., 2017; Tutunea-Fatan et al., 2015)
TNF alpha	Epithelial ovarian cancer cells, Ovarian cancer ascites, minority of the cells in the corpus luteum	>Inflammatory cytokine which promotes cancer cell growth and dissemination >aberrant lymphangiogenesis >Collecting lymphatic vessel contractile dysfunction	ERK1,2>AP-1>VEGF D TNF a/TNFR>NFkB NFkB – iNOS signaling pathway in lymphatic muscle cells.	(Gupta et al., 2016; Hong et al., 2016) (Y. Chen et al., 2017; Sipak-Szmigiel et al., 2017) (Kulbe et al., 2007; Naylor et al., 1993)
Angiotensin 2	Ovarian cancer cells, Ascitic fluid	>metastasis by upregulating lipid desaturation promoting procancer microenvironment >Promotes lymphangiogenesis	ANGII /AGTR1 pathway ANGII>MAPK>EGFR>AKT ANGII >Src>ERK1/2	(Lin et al., 2020; Q. Zhang et al., 2019) (Ino et al., 2006; Touyz et al., 2002)
Ephrin B2, B4	Ovarian cancer cells, endothelial cells	>Tumor advancement by cell adhesion and promoting cellular movement >Lymphatic vasculature remodeling	in OC, perspective yet to be elucidated	(Alam et al., 2008; Mäkinen et al., 2005) (Herath et al., 2006; Jukonen et al., 2021)
Histamine	Lymphatic endothelial cells, ovarian mast cells.	>induces release of inflammatory cytokines through activation of Histamine receptors. >Induce lymphatic leakage, decrease lymphatic contractility through	Histamine>NFkB>inflammatory cytokine Histamine> Histamine Receptor> RhoA-ROCK signaling pathway>barrier and lymphatic muscle cell contractile function	(Krishna et al., 1989; Mikelis et al., 2015) (Gasheva et al., 2019) (Pal, Gasheva, et al., 2020)(Pal, Gasheva, et al., 2020) (Pal et al., 2022; Pal, Nath, et al., 2020) (Akdis & Blaser, 2003; Pal et al., 2017)
Estrogen	Ovarian surface epithelium, theca interna cells of ovary	>OC proliferation, transcriptional activation of ER responsive genes >ER alpha induce	Estrogen> Estrogen receptor>ERK>PI3K ERapha>transcription of lymphangiogenic genes such as VEGFR3, Lyve 1	(Ho, 2003; Morfoisse et al., 2018)
		lymphangiogenesis
Thrombospondin 1	Ovarian surface epithelium, small ovarian follicles	>promote lymphangiogenesis >High level of TSP1 in epithelial ovarian cancer	TSP1>CD36 >SHP1>VEGF TSP1>p53 Expression of TSP1 is inversely proportional with VEGF	(Cursiefen et al., 2011; Russell et al., 2015) (Alvarez et al., 2001; Carpino et al., 2021)
PDGF BB	Epithelial ovarian cancer cells	>Lymphatic metastasis, Lymphangiogenesis	PDGF BB>MMP2/9 PDGF>PDGFRB>VEGF Secretion PDGFBB>MAPK	(Cao et al., 2004; Y. Wang et al., 2011) (Avril et al., 2017; Vincent & Rafii, 2004) (Matei et al., 2007)
FSH	Anterior pituitary	>induce Lymphangiogenesis	FSH>VEGFR3	(Brown & Russell, 2014) (McSorley et al., 2009)
DLL4	Endothelial cells	>lymphatic vessel valve formation. >Inhibits lymphatic sprouting in the presence of luminal flow >overexpressed in ovarian cancer	DLL4>Notch>VEGFR3 DLL4>Notch> NCID Tip cell activation during lymphangiogenesis	(Choi et al., 2017; Liao et al., 2010) (Geng et al., 2020; J. Huang et al., 2016)
IL6	Ovarian carcinoma cells	>inflammatory cytokine production >Lymphangiogenesis	IL6>Src>JAK/STAT3>metastasis IL6>IL6R>STAT3>VEGFR3	(Coward et al., 2011; Marcus, 2015) (Y.-H. Huang et al., 2016; Shinriki et al., 2011) (Nilsson et al., 2005)
TGF beta	Ovarian somatic cells, oocytes	> Epithelial mesenchymal transition > Systemic immune suppression > TGFB regulate lymphangiogenesis.	TGF b>SMAD3 TGF b> co-expression of adhesion molecule	(Knight & Glister, 2006; Yang et al., 2010) (James et al., 2013)
FGF2	Ovarian tumor cells,	>Lymphatic growth and identity >tumor cell proliferation, invasion	FGF2>RAS/MAPK>Lymphatic identity FGF2>LYVE1 and VEGFC expression FGF2>PI3K>Akt>mTOR	(Ichise et al., 2014; Platonova et al., 2013) (Cao et al., 2012; Lau et al., 2013) (L. K. Chang et al., 2004)
Prostaglandin E2,	Ovarian stromal cells, ovarian tumor cells	>proliferation, invasion of cancer cells >induce metastatic spread via lymphatics >induces lymphangiogenesis and lymphatic vessel development	COX 1/2> PGE2 pathway PGE2>VEGFC>VEGFR3	(Nagaraja et al., 2016; Rask et al., 2006) (Lala et al., 2018; Rask et al., 2006) (Iwasaki et al., 2019; Kashiwagi et al., 2011)
IL8	Ovarian cancer cell, ovarian cyst fluid	>proliferation of lymphatic endothelial cells >Cancer cell proliferation	IL8/L8R>RAF/MEK/ERK in OVC patient IL8/IL8R>MMP2/MMP9 in OVC patient	(Shi et al., 2015; Y. Wang et al., 2012)