3

Abstract: Ovarian cancer is a leading cause of death among women in most developed countries. This malignancy is characterized by rapid growth and spread of intraperitoneal tumors, leading to ascites, which is accumulation of fluid in the peritoneum. Despite proof that the accumulation of peritoneal fluid signifies the poorest outcome for cancer patients, the role of malignant ascites in promoting metastasis and therapy resistance remains poorly understood. Malignant ascites presents a unique tumor microenvironment to the tumor cells, non-tumor cells, and various biofactors such as growth factors, cytokines, and lipids. Interest in the characterization of the components of the microenvironment of malignant ascites and their role in the progression of ovarian cancer has increased over the years. In this chapter, we summarize the role of malignant ascites as a liquid tumor microenvironment in the development and progression of ovarian cancer.

Keywords: classification of ovarian cancer; immunology of ovarian cancer; malignant ascites in ovarian cancer; ovarian cancer ascites; tumor microenvironment in ovarian cancer

INTRODUCTION

Ovarian cancer is a leading cause of death among women in most developed countries (1). This malignancy is characterized by rapid growth and the spread of intraperitoneal metastasis (2). Ovarian cancer is distinct from other malignancies in some specific characteristics: (i) the origin of primary tumors can be from multiple sites, such as, the ovarian epithelium, the Fallopian tubes, the endometrium, or the peritoneum; (ii) tumor cells can disseminate by exfoliation from the ovaries (or the tubes) and migrate through the peritoneum; and (iii) secondary tumors do not have additional genetic mutations from that of the primary tumors (3). The World Health Organization classifies ovarian tumors as epithelial (~90% of the cases), germ cell (~3%) and sex cord-stromal (~2%) (4). Epithelial ovarian carcinoma (EOC) comprises five main types (Figure 1) based on its histopathology, immune, and molecular profile: (i) high-grade serous carcinoma (HGSC, 70%); (ii) low-grade serous carcinoma (LGSC, 5%); (iii) endometrioid carcinoma (10%); (iv) clear cell carcinoma (6%); and (vi) mucinous carcinoma (3–4%) (4). These subtypes are distinct but are clinically managed as a single entity, i.e., cytoreductive surgery followed by platinum-taxane combination chemotherapy. The response rate to first-line therapy is around 80–90%, but most patients relapse and develop chemotherapy resistance contributing to a poor 5-year survival rate of <35% (5, 6). Heterogeneity is a key feature of these tumors, explaining, in part, the lack of successful treatment. With the development of molecular tools such as deep sequencing, along with RNA sequencing, epigenomics, proteomics, and immunologic studies, we are gaining further insight into the complexity of heterogeneity within these subtypes and within individual patient tumors (1).

Figure 1 Origins of ovarian tumors. The EOC type comprises several subtypes. Created by Biorender.

CLASSIFICATION OF OVARIAN CANCER

LGSC and HGSC represent two separate tumor types with different morphology, pathogenesis, molecular events, and prognosis (4). HGSC usually occurs in older patients, is detected at an advanced stage, and is responsible for most ovarian cancer deaths. Morphologically, HGSCs are composed of ciliated, columnar cells that form papillae, solid masses, or slit-like spaces with high-grade nuclear atypia. Immunohistochemistry is positive for cytokeratin 7 (CK7), paired box gene 8 (PAX8), Wilms tumor gene product (WT1), but negative for cytokeratin 20 (CK20). The cell cycle checkpoint p53 is generally mutated, resulting in overexpression, or null mutation which translates to a negative immunohistochemistry result. The genomic analysis of HGSC demonstrated a few recurrently mutated genes, such as TP53 (96% of the cases) and BRCA1/BRCA2 (22% of the cases) (7). Most of these carcinomas arise from the distal fimbrial end of the Fallopian tube from a precursor lesion known as serous tubal intraepithelial carcinoma (STIC) (4). Primary peritoneal HGSCs are extremely rare (4).

LGSC are uncommon, comprising 2% of all ovarian carcinomas, and are more frequently found in younger women (median 43 years) (4). These tumors are slow-growing, arising from benign and borderline serous tumors (4), and have a 10-year survival rate of 50% (8). Morphologically, these carcinomas seem like HGSC but with less atypia and an immunohistochemical profile also similar to HGSC (positive for CK7, WT1) however, the expression of p53 is normal-like. LGSCs are genomically stable and display somatic mutations in KRAS and BRAF in approximately half of the cases; these mutations are mutually exclusive (8, 9).

Endometrioid carcinoma usually presents as unilateral solid masses, low grade, and associated with a good prognosis (10). Most of these tumors arise from transformed ovarian endometriosis or benign and borderline tumors (4). Histologically, they are composed of glands resembling endometrial epithelium and typically exhibit a glandular architecture with squamous differentiation, but solid areas can be seen. The immunohistochemistry profile shows positivity for CK7, PAX8, and hormone receptors, and negativity for WT1 and CK20 (10). Endometrioid carcinoma displays somatic mutations of CTNNB1, PI3KCA, PPP2R1A, PTEN, and ARID1A genes (11, 12). Based on its analogous molecular features, seromucinous carcinoma is considered a subtype of endometrioid carcinoma (4).

Clear cell carcinoma is quite uncommon, and some studies show that it has the worst prognosis of all EOCs subtypes (13). These carcinomas occur at a younger age and have a clear association with endometriosis (14–16). The pattern of growth is in the form of a large pelvic mass; it is rarely bilateral, and associated with thromboembolic complications, hypercalcemia, and lymph node metastases (17–19). Histologically, they are composed of glycogen-laden, large, cuboidal, hob-nailed, or flattened clear cells and display an admixture of growth patterns including solid, tubulocystic, or papillary (20). The immunohistochemistry profile of clear cell carcinoma is characterized by the expression of napsin A and the absence of WT1, p53, and ER expression (21, 22). Some studies show that the tumor suppressor ARID1A is mutated in most clear cell carcinoma cases (12, 21). PI3KCA exhibit activating mutations (22). Recent studies showed that clear cell carcinoma is resistant to platinum-based chemotherapy, but, despite this, its management is similar to the rest of EOC (18).

Mucinous carcinomas are rare, and patients are usually diagnosed at an early stage with an excellent prognosis after surgery. However, when patients have relapses (or metastatic mucinous carcinoma) they have a worse prognosis (23, 24). Usually, these type of EOC are unilateral, large, multicystic tumors filled with mucus and frequently containing solid areas. Morphologically, mucinous carcinoma is composed of cysts and glands of variable size with a confluent pattern and back-to-back glands. The cells are tall, columnar, and stratified, with a large cytoplasm containing mucin (25, 26). Immunohistochemistry of mucinous carcinoma shows CK7 and CK20 positivity but are usually negative for hormone receptors and WT1. These carcinomas seem to arise from borderline mucinous neoplasms and show a heterogeneous pattern with coexisting mucinous, benign, borderline, and adenocarcinoma areas (27). The most common molecular alterations are KRAS and TP53 mutations (both 64%) (4), which have been identified in benign and borderline areas as well as in adjacent carcinomas (28–30). HER2 amplification is also found in around 20% of mucinous carcinoma, as well copy-number loss of CDKN2A (76% of cases) (4). These genomic abnormalities are mutually exclusive (31).

MALIGNANT ASCITES AS A LIQUID TUMOR MICROENVIRONMENT

Several studies associate different ovarian cancer characteristics with the intrinsic properties of tumors and their microenvironment (32–34). In ovarian cancer, most patients are diagnosed at advanced stages (stage III/IV), presenting metastasis throughout the pelvic and peritoneal cavities, and by the accumulation of a large volume of peritoneal fluid (malignant ascites, MA) (35). The role of MA is to facilitate the spread of tumor cells to other pelvic and peritoneal organs, serving as a vehicle for tumor cells (36). This form of transcoelomic dissemination is crucial to the adhesion of tumor cells to the omentum and serous membranes lining the peritoneal organs, leading to metastatic lesions in the peritoneal cavity, instead of invading the lamina propria like the majority of other solid tumors (37). Ovarian cancer cells disseminate into peritoneal sites such as the hepatic, omentum, spleen, uterus, etc, using the MA flux. MA comprises not only tumor cells, but also many other non-tumor cell types (Figure 2), which produce a unique microenvironment that can modify the neoplastic properties of tumor cells (38).

Figure 2 Scheme of cellular and acellular components of ascites. Ascites is composed by tumor cells (single cells and spheroids), and non-tumoral cells, including fibroblasts, mesothelial cells, endothelial cells, adipocytes, and immunologic cells. These types of cells communicate with each other through acellular factors, including cytokines, proteins, metabolites, and exosomes. Created by Biorender.

The peritoneum is lined by mesothelial cells that cover and protect the viscera and the stroma that contains a collagen-based matrix, activated fibroblasts, blood vessels, and lymphatics vessels. This conjugation creates a unique milieu full of factors secreted by all tumor cellular components that support metastatic seeding and tumor proliferation (39). MA is an exudative fluid composed by a cellular fraction with highly tumorigenic cancer cells (40), immune cells, including different types of T cells (41), tumor-associated macrophages (42), and other host cells. The acellular fraction contains tumor-promoting soluble factors, bioactive lipids, cytokines, and extracellular vesicles (43).

Several studies have demonstrated an “activated” phenotype of the peritoneal environment associated with ovarian cancer, as opposed to its quiescent state in benign conditions (44). The pro-inflammatory signature, associated with cancer, promotes angiogenesis, and exerts chemotactic and protective effects on cancer cells. Chemokines, cytokines, and growth factors commonly secreted in the tumor microenvironment include the stromal cell-derived factor, interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1, Chemokine (C-C motif) ligand 5 and 7 (CCL5 and CCL7), transforming growth factor- β1, tumor necrosis factor α (TNFα), fibroblast growth factor, and others (44–46). While tumor cells play a role in the secretion of factors that modulate angiogenesis, non-transformed tumor-infiltrating cells such as fibroblasts, myeloid cells, immune cells, and endothelial precursors also play a crucial role in modulating neo-vascularization (47). All these factors present in the MA microenvironment induce tumor cell proliferation, progression, chemoresistance, and immune evasion (3) unveiling a key role of this serous liquid in the development and progression of ovarian cancer (48).

The cellular components of malignant ascites

Cancer cells in MA can be found as single cells with adherent properties or multicellular spheroids with no-adherent properties (49), being the major contributors to the peritoneal dissemination (50). The multicellular spheroids are key mediators of peritoneal dissemination since they have low expression levels of E-cadherin (49) and allow ovarian cancer cells to resist anoikis and apoptosis, including that induced by chemotherapeutic agents, since drugs do not penetrate in such multicellular structure (35, 51, 52).

The stromal cells, such as fibroblasts, endothelial or mesothelial cells, adipocytes, adipose tissue-derived stromal cells, bone marrow-derived stem cells and immune cells (53, 54), can regulate the extracellular matrix composition and produce molecules that attract ovarian cancer cells to specific sites (55, 56). These tumors are typically highly vascularized, because some of these cells show abnormal activities, like the stimulation of growth and angiogenesis (57, 58), which correlates with a poor prognosis and contributes to tumor development (38, 59) (Figure 3).

Figure 3 Cancer-associated changes in stromal cells. IL-10, interleukin 10; IL-1β, interleukin 1β; IL-6, interleukin 6; M-CSF, macrophage colony-stimulating factor; TGFβ, transforming growth factor β; TNFα, tumor necrosis factor α. VEGF, vascular endothelial growth factor. Created by Biorender.

The malignant role of cancer-associated fibroblasts is to promote proliferation, migration, and invasion of cancer cells. Cancer-associated fibroblasts secrete factors that can transduce signals to cancer cells as well as to themselves, establishing reciprocal reinforcement of growth and migration signals and contributing to chemoresistance (60). Mesothelial cells lining the peritoneum are also important for tumor progression (57), as they secrete factors that promote tumor growth. Lysophosphatidic acid is produced by immortalized peritoneal mesothelial cells and it was shown to improve adhesion, migration, and invasion of ovarian cancer cells (61). In addition, mesothelial cells produce dipeptidyl peptidase IV and vascular endothelial growth factor (VEGF) in response to MA environmental exposure (62, 63).

The complex immune suppression system that efficiently neutralizes antitumor immunity is one of the reasons for disease progression and treatment failure (64) as cancer cells are able to subvert the natural purpose of immune cells for their own benefit. The equilibrium between these immune reactive and immune suppressive cells defines the immunosuppressive and pro-tumoral properties of MA microenvironment (38, 39) (Figure 4).

Figure 4 Immune cells in the tumor microenvironment. The left panel represents the immune cells that act as tumor killers by the production of cytokines that destroy tumor cells. The right panel represents the cells that contribute to immune suppression. CD8⁺T, CD8⁺ T cells; DC, dendritic cell; M1, macrophage type 1; M2, macrophage type 2; MDSc, myeloid-derived suppressor cells; N1, neutrophil type 1; N2, neutrophil type 2; NK, natural killer; TApDC, tumor-associated plasmacytoid dendritic cell; Th1, T “helper” 1 cell; Th2, T “helper” 2 cell; TIL, tumor-infiltrating lymphocytes; Treg, egulator T cell. Created by Biorender.

The immune reactive cells include cytotoxic T lymphocytes and activated CD4⁺ T cells. The immune-suppressive cells are myeloid-derived suppressor cells, tumor-associated macrophages (especially M2 subtype), dendritic cells, lymphocyte T helper cells (Th2 subtype), and T regulatory cells (Tregs). The presence of CD3⁺ tumor-infiltrating lymphocytes (TILs) in ovarian cancer is associated with increased survival (65). It was shown that, in patients whose tumors contained T cells, the 5-year overall survival was 38% compared to 4.5% in patients with tumors lacking T cells. In addition, a strong correlation between the presence of CD8⁺ TILs and favorable clinical outcomes of HGSC (66–68) has been demonstrated. The ratio of CD8⁺ T cells/Tregs cells is also related to increased survival of ovarian cancer patients (67). A positive correlation between the presence of oligoclonal expanding T cells and the regression or stabilization of metastases also demonstrates the value of the tumor immune microenvironment in the outcome of ovarian cancer patients (69). There is increasing evidence that non-tumoral cells in the tumor microenvironment have a key regulatory role in ovarian cancer and should be evaluated for diagnostic and treatment purposes (39).

The acellular components of malignant ascites

The cellular components of MA communicate with each other through soluble factors, including cytokines, proteins, metabolites, and the secretion and exchange of exosomes (2).

The cytokine profiles of ascites can be pro-tumorigenic or anti-tumorigenic (70–73). The pro-tumorigenic cytokines are regulated by Th2, such as, IL-4, IL-6, IL-8, IL-10, IL-13, IL-15, CCL2 and VEGF and the anti-tumorigenic are regulated by Th1, such as, IL-2, IL-3, IL-5, IL-7, IL-17, CXCL-10, CCL4, INFγ, and TNFα (74, 75). These cytokines contribute to the creation of a pro-inflammatory and immunosuppressive tumor microenvironment (73).

The metabolome profiling of ascites has demonstrated important differences in fatty acids, cholesterol, ceramide, glycerol-3-phosphate, glucose, and glucose-3-phosphate. The MA present low levels of 2-hydroxyisovalerate, although glucose-1-phosphate is present in high levels in this liquid microenvironment. 2-hydroxyisovalerate is the result of breakdown of branched-chain amino acids (76) and is found in the urine of patients with lactic and ketoacidosis, which indicates an increase in amino acid catabolism (77). The glucose-1-phosphate is a product of glycogenolysis which is correlated with the increased use of glucose by the tumor cells in the MA microenvironment (78). The glucose transporter 1 or 3 and glycolytic enzymes, such as hexokinase II, are overexpressed in ovarian cancer, and are indicators of poor prognosis (36), as they are associated with chemoresistance and poorer progression-free survival (37). In addition, glycolate, glucose, furanose and fructose are found in low levels, while glycerol-3-phosphate, cholesterol, ceramide and monoacylglycerol are elevated in ovarian cancer patient-derived MA (38).

Proteomics of ascites has revealed the presence of over 2000 different proteins (79, 80). Examples of proteins found abundantly in MA are pyruvate kinase isozymes M1/M2, glyceraldehyde phosphate dehydrogenase and mesothelin (81). Moreover, the most abundant proteins are related to the components associated with RNA splicing (79). Exosomes were also detected in ovarian cancer MA. These nano-sized microvesicles (30–100nm of diameter) are membrane-bound extracellular vesicles that are produced in the endosomal compartment of most eukaryotic cells and carry various lipids, proteins and nucleic acids, within the membrane-covered vesicles (82). These structures have the molecular signature of donor cells and circulate in the organism, with the objective of transporting information between cells to change the gene expression of receptor cells (83). Exomes have disease-specific biomarkers in ovarian cancer, such as miR-200c, miR-214, CA125, Mucina-1 and CD24 (82, 84).

CONCLUSION

Metastatic ovarian cancer is a deadly disease. The mechanism of tumor dissemination in the peritoneal cavity leads to the formation of MA. MA constitute an easily accessible source of cancer cells and cancer-associated factors. The presence of this liquid tumor microenvironment is correlated with a poor prognosis in ovarian cancer patients but its association with chemoresistance is poorly understood. Further studies supported by technological advances are needed to better explore the multidimensional potential of this unique tumor microenvironment that supports ovarian cancer cell growth, progression, and metastatic outgrowth. The actual challenge is to understand the complexity of the multiple interactions between ovarian cancer ascites components and to develop new drugs to abrogate these tumor microenvironment communications routes.

Acknowledgement: This work was developed at i3S/IPATIMUP, an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education (MCTES), and partially supported by Fundação para a Ciência e a Tecnologia (FCT)/MCTES. Diana Nunes acknowledges to FCT/MCTES and UE for financial support through a PhD fellowship (2021.05081.BD) co-sponsored by Fundo Social Europeu (FSE) through Programa Operacional Regional Norte (Norte 2020).

Conflict of Interest: The authors declare no potential conflict of interest with respect to research, authorship and/or publication of this chapter.

Copyright and Permission Statement: The authors confirm that the materials included in this chapter do not violate copyright laws. Where relevant, appropriate permissions have been obtained from the original copyright holder(s), and all original sources have been appropriately acknowledged or referenced.

REFERENCES

1. Kossaï M, Leary A, Scoazec JY, Genestie C. Ovarian Cancer: A Heterogeneous Disease. Pathobiology. 2018;85(1–2):41–9. https://doi.org/10.1159/000479006
2. Kim S, Kim B, Song YS. Ascites modulates cancer cell behavior, contributing to tumor heterogeneity in ovarian cancer. Cancer Science. 2016;107(9):1173–8. https://doi.org/10.1111/cas.12987
3. Vaughan S, Coward JI, Bast RCJ, Berchuck A, Berek JS, Brenton JD, et al. Rethinking ovarian cancer: recommendations for improving outcomes. Nat Rev Cancer. 2011 Sep;11(10):719–25. https://doi.org/10.1038/nrc3144
4. de Leo A, Santini D, Ceccarelli C, Santandrea G, Palicelli A, Acquaviva G, et al. What is new on ovarian carcinoma: Integrated morphologic and molecular analysis following the new 2020 world health organization classification of female genital tumors. Diagnostics. 2021;11(4):1–16. https://doi.org/10.3390/diagnostics11040697
5. Herzog TJ. Recurrent ovarian cancer: how important is it to treat to disease progression? Clinical cancer research : an official journal of the American Association for Cancer Research. 2004 Nov;10(22):7439–49. https://doi.org/10.1158/1078-0432.CCR-04-0683
6. Herzog TJ, Pothuri B. Ovarian cancer: a focus on management of recurrent disease. Nat Clin Pract Oncol. 2006 Nov;3(11):604–11. https://doi.org/10.1038/ncponc0637
7. Malpica A, Deavers MT, Lu K, Bodurka DC, Atkinson EN, Gershenson DM, et al. Grading ovarian serous carcinoma using a two-tier system. Am J Surg Pathol. 2004 Apr;28(4):496–504. https://doi.org/10.1097/00000478-200404000-00009
8. Jones S, Wang TL, Kurman RJ, Nakayama K, Velculescu VE, Vogelstein B, et al. Low-grade serous carcinomas of the ovary contain very few point mutations. J Pathol. 2012 Feb;226(3):413–20. https://doi.org/10.1002/path.3967
9. Mayr D, Hirschmann A, Löhrs U, Diebold J. KRAS and BRAF mutations in ovarian tumors: a comprehensive study of invasive carcinomas, borderline tumors and extraovarian implants. Gynecol Oncol. 2006 Dec;103(3):883–7. https://doi.org/10.1016/j.ygyno.2006.05.029
10. Lu FI, Gilks CB, Mulligan AM, Ryan P, Allo G, Sy K, et al. Prevalence of loss of expression of DNA mismatch repair proteins in primary epithelial ovarian tumors. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2012 Nov;31(6):524–31. https://doi.org/10.1097/PGP.0b013e31824fe2aa
11. Geyer JT, López-García MA, Sánchez-Estevez C, Sarrió D, Moreno-Bueno G, Franceschetti I, et al. Pathogenetic pathways in ovarian endometrioid adenocarcinoma: a molecular study of 29 cases. Am J Surg Pathol. 2009 Aug;33(8):1157–63. https://doi.org/10.1097/PAS.0b013e3181a902e1
12. Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, Zeng T, et al. ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med. 2010 Oct;363(16):1532–43. https://doi.org/10.1056/NEJMoa1008433
13. Lee YY, Kim TJ, Kim MJ, Kim HJ, Song T, Kim MK, et al. Prognosis of ovarian clear cell carcinoma compared to other histological subtypes: a meta-analysis. Gynecol Oncol. 2011 Sep;122(3):541–7. https://doi.org/10.1016/j.ygyno.2011.05.009
14. Gounaris I, Charnock-Jones DS, Brenton JD. Ovarian clear cell carcinoma--bad endometriosis or bad endometrium? J Pathol. 2011 Oct;225(2):157–60. https://doi.org/10.1002/path.2970
15. Bounous VE, Ferrero A, Fuso L, Ravarino N, Ceccaroni M, Menato G, et al. Endometriosis-associated Ovarian Cancer: A Distinct Clinical Entity? Anticancer Res. 2016 Jul;36(7):3445–9.
16. Kobayashi H, Kajiwara H, Kanayama S, Yamada Y, Furukawa N, Noguchi T, et al. Molecular pathogenesis of endometriosis-associated clear cell carcinoma of the ovary (review). Oncol Rep. 2009 Aug;22(2):233–40. https://doi.org/10.3892/or_00000429
17. Anglesio MS, Carey MS, Köbel M, Mackay H, Huntsman DG. Clear cell carcinoma of the ovary: a report from the first Ovarian Clear Cell Symposium, June 24th, 2010. Gynecol Oncol. 2011 May;121(2):407–15. https://doi.org/10.1016/j.ygyno.2011.01.005
18. Sugiyama T, Kamura T, Kigawa J, Terakawa N, Kikuchi Y, Kita T, et al. Clinical characteristics of clear cell carcinoma of the ovary: a distinct histologic type with poor prognosis and resistance to platinum-based chemotherapy. Cancer. 2000 Jun;88(11):2584–9. https://doi.org/10.1002/1097-0142(20000601)88:11<2584::AID-CNCR22>3.0.CO;2-5
19. Takeshima N, Hirai Y, Umayahara K, Fujiwara K, Takizawa K, Hasumi K. Lymph node metastasis in ovarian cancer: difference between serous and non-serous primary tumors. Gynecol Oncol. 2005 Nov;99(2):427–31. https://doi.org/10.1016/j.ygyno.2005.06.051
20. McCluggage WG. My approach to and thoughts on the typing of ovarian carcinomas. J Clin Pathol. 2008 Feb;61(2):152–63. https://doi.org/10.1136/jcp.2007.049478
21. Jones S, Wang TL, Shih IM, Mao TL, Nakayama K, Roden R, et al. Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science. 2010 Oct;330(6001):228–31. https://doi.org/10.1126/science.1196333
22. Kuo KT, Mao TL, Jones S, Veras E, Ayhan A, Wang TL, et al. Frequent activating mutations of PIK3CA in ovarian clear cell carcinoma. Am J Pathol. 2009 May;174(5):1597–601. https://doi.org/10.2353/ajpath.2009.081000
23. Rodríguez IM, Prat J. Mucinous tumors of the ovary: a clinicopathologic analysis of 75 borderline tumors (of intestinal type) and carcinomas. Am J Surg Pathol. 2002 Feb;26(2):139–52. https://doi.org/10.1097/00000478-200202000-00001
24. Ludwick C, Gilks CB, Miller D, Yaziji H, Clement PB. Aggressive behavior of stage I ovarian mucinous tumors lacking extensive infiltrative invasion: a report of four cases and review of the literature. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2005 Jul;24(3):205–17. https://doi.org/10.1097/01.pgp.0000159935.38913.57
25. Lee KR, Scully RE. Mucinous tumors of the ovary: a clinicopathologic study of 196 borderline tumors (of intestinal type) and carcinomas, including an evaluation of 11 cases with “pseudomyxoma peritonei”. Am J Surg Pathol. 2000 Nov;24(11):1447–64. https://doi.org/10.1097/00000478-200011000-00001
26. Hart WR. Mucinous tumors of the ovary: a review. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2005 Jan;24(1):4–25. https://doi.org/10.1097/01.pgp.0000166997.57196.82
27. Kurman RJ, Shih IM. Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer--shifting the paradigm. Hum Pathol. 2011 Jul;42(7):918–31. https://doi.org/10.1016/j.humpath.2011.03.003
28. Garrett AP, Lee KR, Colitti CR, Muto MG, Berkowitz RS, Mok SC. k-ras mutation may be an early event in mucinous ovarian tumorigenesis. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists. 2001 Jul;20(3):244–51. https://doi.org/10.1097/00004347-200107000-00007
29. Gurung A, Hung T, Morin J, Gilks CB. Molecular abnormalities in ovarian carcinoma: clinical, morphological and therapeutic correlates. Histopathology. 2013 Jan;62(1):59–70. https://doi.org/10.1111/his.12033
30. Ichikawa Y, Nishida M, Suzuki H, Yoshida S, Tsunoda H, Kubo T, et al. Mutation of K-ras protooncogene is associated with histological subtypes in human mucinous ovarian tumors. Cancer Res. 1994 Jan;54(1):33–5.
31. Anglesio MS, Kommoss S, Tolcher MC, Clarke B, Galletta L, Porter H, et al. Molecular characterization of mucinous ovarian tumours supports a stratified treatment approach with HER2 targeting in 19% of carcinomas. J Pathol. 2013 Jan;229(1):111–20. https://doi.org/10.1002/path.4088
32. Castells M, Thibault B, Delord JP, Couderc B. Implication of tumor microenvironment in chemoresistance: tumor-associated stromal cells protect tumor cells from cell death. Int J Mol Sci. 2012;13(8):9545–71. https://doi.org/10.3390/ijms13089545
33. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell [Internet]. 2011;144(5):646–74. Available from: http://dx.doi.org/10.1016/j.cell.2011.02.013https://doi.org/10.1016/j.cell.2011.02.013
34. Hanahan D, Coussens LM. Accessories to the Crime: Functions of Cells Recruited to the Tumor Microenvironment. Cancer Cell [Internet]. 2012;21(3):309–22. Available from: http://dx.doi.org/10.1016/j.ccr.2012.02.022https://doi.org/10.1016/j.ccr.2012.02.022
35. Matte I, Legault CM, Garde-Granger P, Laplante C, Bessette P, Rancourt C, et al. Mesothelial cells interact with tumor cells for the formation of ovarian cancer multicellular spheroids in peritoneal effusions. Clinical and Experimental Metastasis. 2016;33(8):839–52. https://doi.org/10.1007/s10585-016-9821-y
36. Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010 Sep;177(3):1053–64. https://doi.org/10.2353/ajpath.2010.100105
37. Steinkamp MP, Winner KK, Davies S, Muller C, Zhang Y, Hoffman RM, et al. Ovarian tumor attachment, invasion, and vascularization reflect unique microenvironments in the peritoneum: insights from xenograft and mathematical models. Front Oncol. 2013;3:97. https://doi.org/10.3389/fonc.2013.00097
38. Thibault B, Castells M, Delord JP, Couderc B. Ovarian cancer microenvironment: Implications for cancer dissemination and chemoresistance acquisition. Cancer and Metastasis Reviews. 2014;33(1):17–39. https://doi.org/10.1007/s10555-013-9456-2
39. Nwani NG, Sima LE, Nieves-Neira W, Matei D. Targeting the microenvironment in high grade serous ovarian cancer. Cancers (Basel). 2018;10(8):1–22. https://doi.org/10.3390/cancers10080266
40. Latifi A, Luwor RB, Bilandzic M, Nazaretian S, Stenvers K, Pyman J, et al. Isolation and characterization of tumor cells from the ascites of ovarian cancer patients: molecular phenotype of chemoresistant ovarian tumors. PLoS One. 2012;7(10):e46858. https://doi.org/10.1371/journal.pone.0046858
41. Preston CC, Goode EL, Hartmann LC, Kalli KR, Knutson KL. Immunity and immune suppression in human ovarian cancer. Immunotherapy. 2011;3(4):539–56. https://doi.org/10.2217/imt.11.20
42. Reinartz S, Schumann T, Finkernagel F, Wortmann A, Jansen JM, Meissner W, et al. Mixed-polarization phenotype of ascites-associated macrophages in human ovarian carcinoma: Correlation of CD163 expression, cytokine levels and early relapse. International Journal of Cancer. 2014;134(1):32–42. https://doi.org/10.1002/ijc.28335
43. Kulbe H, Chakravarty P, Leinster DA, Charles KA, Kwong J, Thompson RG, et al. A dynamic inflammatory cytokine network in the human ovarian cancer microenvironment. Cancer Research. 2012;72(1):66–75. https://doi.org/10.1158/0008-5472.CAN-11-2178
44. Freedman RS, Deavers M, Liu J, Wang E. Peritoneal inflammation - A microenvironment for Epithelial Ovarian Cancer (EOC). J Transl Med. 2004 Jun;2(1):23. https://doi.org/10.1186/1479-5876-2-23
45. Said N, Socha MJ, Olearczyk JJ, Elmarakby AA, Imig JD, Motamed K. Normalization of the ovarian cancer microenvironment by SPARC. Mol Cancer Res. 2007 Oct;5(10):1015–30. https://doi.org/10.1158/1541-7786.MCR-07-0001
46. Gopinathan G, Milagre C, Pearce OMT, Reynolds LE, Hodivala-Dilke K, Leinster DA, et al. Interleukin-6 Stimulates Defective Angiogenesis. Cancer Res. 2015 Aug;75(15):3098–107. https://doi.org/10.1158/0008-5472.CAN-15-1227
47. Nozawa H, Chiu C, Hanahan D. Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis. Proc Natl Acad Sci U S A. 2006 Aug;103(33):12493–8. https://doi.org/10.1073/pnas.0601807103
48. Cohen M, Pierredon S, Wuillemin C, Delie F, Petignat P. Acellular fraction of ovarian cancer ascites induce apoptosis by activating JNK and inducing BRCA1, Fas and FasL expression in ovarian cancer cells. Oncoscience. 2014;1(4):262–71. https://doi.org/10.18632/oncoscience.31
49. Wintzell M, Hjerpe E, Åvall Lundqvist E, Shoshan M. Protein markers of cancer-associated fibroblasts and tumor-initiating cells reveal subpopulations in freshly isolated ovarian cancer ascites. BMC Cancer. 2012 Aug;12:359. https://doi.org/10.1186/1471-2407-12-359
50. Liao J, Qian F, Tchabo N, Mhawech-Fauceglia P, Beck A, Qian Z, et al. Ovarian cancer spheroid cells with stem cell-like properties contribute to tumor generation, metastasis and chemotherapy resistance through hypoxia-resistant metabolism. PLoS One. 2014;9(1):e84941. https://doi.org/10.1371/journal.pone.0084941
51. Tannock IF, Lee CM, Tunggal JK, Cowan DSM, Egorin MJ. Limited penetration of anticancer drugs through tumor tissue: a potential cause of resistance of solid tumors to chemotherapy. Clinical cancer research : an official journal of the American Association for Cancer Research. 2002 Mar;8(3):878–84.
52. Fayad W, Brnjic S, Berglind D, Blixt S, Shoshan MC, Berndtsson M, et al. Restriction of cisplatin induction of acute apoptosis to a subpopulation of cells in a three-dimensional carcinoma culture model. Int J Cancer. 2009 Nov;125(10):2450–5. https://doi.org/10.1002/ijc.24627
53. Wels J, Kaplan RN, Rafii S, Lyden D. Migratory neighbors and distant invaders: tumor-associated niche cells. Genes Dev. 2008 Mar;22(5):559–74. https://doi.org/10.1101/gad.1636908
54. Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation and progression. Nature. 2004 Nov;432(7015):332–7. https://doi.org/10.1038/nature03096
55. Rieppi M, Vergani V, Gatto C, Zanetta G, Allavena P, Taraboletti G, et al. Mesothelial cells induce the motility of human ovarian carcinoma cells. Int J Cancer. 1999 Jan;80(2):303–7. https://doi.org/10.1002/(SICI)1097-0215(19990118)80:2<303::AID-IJC21>3.0.CO;2-W
56. Touboul C, Lis R, al Farsi H, Raynaud CM, Warfa M, Althawadi H, et al. Mesenchymal stem cells enhance ovarian cancer cell infiltration through IL6 secretion in an amniochorionic membrane based 3D model. J Transl Med. 2013 Jan;11:28. https://doi.org/10.1186/1479-5876-11-28
57. Pasquet M, Golzio M, Mery E, Rafii A, Benabbou N, Mirshahi P, et al. Hospicells (ascites-derived stromal cells) promote tumorigenicity and angiogenesis. Int J Cancer. 2010 May;126(9):2090–101. https://doi.org/10.1002/ijc.24886
58. Matte I, Lane D, Bachvarov D, Rancourt C, Piché A. Role of malignant ascites on human mesothelial cells and their gene expression profiles. BMC Cancer. 2014 Apr;14:288. https://doi.org/10.1186/1471-2407-14-288
59. Duncan TJ, Al-Attar A, Rolland P, Scott I v, Deen S, Liu DTY, et al. Vascular endothelial growth factor expression in ovarian cancer: a model for targeted use of novel therapies? Clinical cancer research : an official journal of the American Association for Cancer Research. 2008 May;14(10):3030–5. https://doi.org/10.1158/1078-0432.CCR-07-1888
60. Paraiso KHT, Smalley KSM. Fibroblast-mediated drug resistance in cancer. Biochem Pharmacol. 2013 Apr;85(8):1033–41. https://doi.org/10.1016/j.bcp.2013.01.018
61. Ren J, Xiao Y jin, Singh LS, Zhao X, Zhao Z, Feng L, et al. Lysophosphatidic acid is constitutively produced by human peritoneal mesothelial cells and enhances adhesion, migration, and invasion of ovarian cancer cells. Cancer Res. 2006 Mar;66(6):3006–14. https://doi.org/10.1158/0008-5472.CAN-05-1292
62. Kajiyama H, Kikkawa F, Maeda O, Suzuki T, Ino K, Mizutani S. Increased expression of dipeptidyl peptidase IV in human mesothelial cells by malignant ascites from ovarian carcinoma patients. Oncology. 2002;63(2):158–65. https://doi.org/10.1159/000063801
63. Stadlmann S, Amberger A, Pollheimer J, Gastl G, Offner FA, Margreiter R, et al. Ovarian carcinoma cells and IL-1beta-activated human peritoneal mesothelial cells are possible sources of vascular endothelial growth factor in inflammatory and malignant peritoneal effusions. Gynecol Oncol. 2005 Jun;97(3):784–9. https://doi.org/10.1016/j.ygyno.2005.02.017
64. Thibault B, Castells M, Delord JP, Couderc B. Ovarian cancer microenvironment: implications for cancer dissemination and chemoresistance acquisition. Cancer Metastasis Rev. 2014 Mar;33(1):17–39. https://doi.org/10.1007/s10555-013-9456-2
65. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med. 2003 Jan;348(3):203–13. https://doi.org/10.1056/NEJMoa020177
66. Clarke B, Tinker A v, Lee CH, Subramanian S, van de Rijn M, Turbin D, et al. Intraepithelial T cells and prognosis in ovarian carcinoma: novel associations with stage, tumor type, and BRCA1 loss. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2009 Mar;22(3):393–402. https://doi.org/10.1038/modpathol.2008.191
67. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A. 2005 Dec;102(51):18538–43. https://doi.org/10.1073/pnas.0509182102
68. Santoiemma PP, Reyes C, Wang LP, McLane MW, Feldman MD, Tanyi JL, et al. Systematic evaluation of multiple immune markers reveals prognostic factors in ovarian cancer. Gynecol Oncol. 2016 Oct;143(1):120–7. https://doi.org/10.1016/j.ygyno.2016.07.105
69. Bösmüller HC, Wagner P, Peper JK, Schuster H, Pham DL, Greif K, et al. Combined Immunoscore of CD103 and CD3 Identifies Long-Term Survivors in High-Grade Serous Ovarian Cancer. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society. 2016 May;26(4):671–9. https://doi.org/10.1097/IGC.0000000000000672
70. Yigit R, Figdor CG, Zusterzeel PLM, Pots JM, Torensma R, Massuger LFAG. Cytokine analysis as a tool to understand tumour-host interaction in ovarian cancer. Eur J Cancer. 2011 Aug;47(12):1883–9. https://doi.org/10.1016/j.ejca.2011.03.026
71. Lane D, Matte I, Rancourt C, Piché A. Prognostic significance of IL-6 and IL-8 ascites levels in ovarian cancer patients. BMC Cancer. 2011 May;11:210. https://doi.org/10.1186/1471-2407-11-210
72. Matte I, Lane D, Laplante C, Rancourt C, Piché A. Profiling of cytokines in human epithelial ovarian cancer ascites. Am J Cancer Res. 2012;2(5):566–80.
73. Giuntoli RL 2nd, Webb TJ, Zoso A, Rogers O, Diaz-Montes TP, Bristow RE, et al. Ovarian cancer-associated ascites demonstrates altered immune environment: implications for antitumor immunity. Anticancer Res. 2009 Aug;29(8):2875–84.
74. Mesiano S, Ferrara N, Jaffe RB. Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization. Am J Pathol. 1998 Oct;153(4):1249–56. https://doi.org/10.1016/S0002-9440(10)65669-6
75. Giuntoli RL, Webb TJ, Zoso A, Rogers O, Diaz-Montes TP, Bristow RE, et al. Ovarian Cancer-associated Ascites Demonstrates altered immune environment-2009. Anticancer Research. 2009;29(8):2875–84.
76. McMillan A, Rulisa S, Sumarah M, Macklaim JM, Renaud J, Bisanz JE, et al. A multi-platform metabolomics approach identifies highly specific biomarkers of bacterial diversity in the vagina of pregnant and non-pregnant women. Sci Rep. 2015 Sep;5:14174. https://doi.org/10.1038/srep14174
77. Landaas S, Jakobs C. The occurrence of 2-hydroxyisovaleric acid in patients with lactic acidosis and ketoacidosis. Clin Chim Acta. 1977 Aug;78(3):489–93. https://doi.org/10.1016/0009-8981(77)90082-1
78. Finley LWS, Carracedo A, Lee J, Souza A, Egia A, Zhang J, et al. SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization. Cancer Cell. 2011 Mar;19(3):416–28. https://doi.org/10.1016/j.ccr.2011.02.014
79. Shender VO, Pavlyukov MS, Ziganshin RH, Arapidi GP, Kovalchuk SI, Anikanov NA, et al. Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication. Mol Cell Proteomics. 2014 Dec;13(12):3558–71. https://doi.org/10.1074/mcp.M114.041194
80. Gortzak-Uzan L, Ignatchenko A, Evangelou AI, Agochiya M, Brown KA, St Onge P, et al. A proteome resource of ovarian cancer ascites: integrated proteomic and bioinformatic analyses to identify putative biomarkers. J Proteome Res. 2008 Jan;7(1):339–51. https://doi.org/10.1021/pr0703223
81. Elschenbroich S, Ignatchenko V, Clarke B, Kalloger SE, Boutros PC, Gramolini AO, et al. In-depth proteomics of ovarian cancer ascites: combining shotgun proteomics and selected reaction monitoring mass spectrometry. J Proteome Res. 2011 May;10(5):2286–99. https://doi.org/10.1021/pr1011087
82. Guo L, Guo N. Exosomes: Potent regulators of tumor malignancy and potential bio-tools in clinical application. Crit Rev Oncol Hematol. 2015 Sep;95(3):346–58. https://doi.org/10.1016/j.critrevonc.2015.04.002
83. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007 Jun;9(6):654–9. https://doi.org/10.1038/ncb1596
84. Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol. 2008 Jul;110(1):13–21. https://doi.org/10.1016/j.ygyno.2008.04.033