Lymphatic vasculature in ovarian cancer.

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.

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.

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. 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. ( 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.

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 ).

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.

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.

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.

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 ).