Ovarian Cancer is the most lethal gynecological malignancy in the western world. Despite efforts to find biomarkers for early diagnosis, patients often present with advanced disease with widespread peritoneal tumor load and malignant ascites. Recently, we described two different modes of peritoneal tumor spread in high grade serous ovarian cancer patients (HGSOC): miliary, characterized by small, millet-like lesions, scattered all over the peritoneal surface and leading to a worse overall survival compared to non-miliary, defined by only few but bigger metastases in the peritoneum (Auer et al. Oncotarget (2015) 6:17261). Although we discovered differences in the tumor cell transcriptome between miliary and non-miliary, the impact of the immune system on the tumor development remained unclear. Therefore, we collected peripheral blood, ascites and tumor tissues from 41 HGSOC patients and analyzed them with respect to tumor spread types.
The local and global immune system was assessed by flow cytometry. Activated NK cells and naïve B-cells were significantly elevated in miliary blood. In ascites, reduced levels of cytotoxic T-cells and activated NKT cells as well as higher frequencies of NK cells and B-cells could be observed. As monocytes could not be assessed from cryopreserved specimen, CD14+ and/or CD16+ monocytes in ascites were measured by fluorescence staining and quantification. CD14+ monocytes (FDR 0.03) as well as the pro-inflammatory subset of CD14+CD16+ monocytes (trend) were enriched in non-miliary ascites.
Transcriptomes of immune cells were assessed by RNA-sequencing of CD45 enriched floating ascites cells and digested primary and metastatic tumors. In ascites, 26 genes were significantly differentially expressed (FDR<0.05), all upregulated in miliary, and in ovarian and peritoneal tumors, 585 genes were significantly differentially expressed between miliary and non-miliary, 583 of them upregulated and two downregulated in miliary. However, pathway analysis revealed an inhibition of cell cycle in ascites and WNT signaling in ascites and tissue immune cells.
Cytokines were measured in serum and ascites with a multiplexed immunoassay. In accordance with elevated numbers of B-cells, the B-cell attractant CXCL13 was enriched in miliary ascites, as well as the anti-inflammatory cytokine IL-10. In non-miliary ascites prominent pro-angiogenic factors (FGFbasic, MIF, sVEGFR1), the hormone prolactin, and CCL25, shown to play a role in chemotaxis of thymocytes and ovarian carcinoma cells, were elevated significantly.
Taking together these comprehensive results shed some light on the impact of the immune system on the tumor spread in HGSOC: patients with only few, big metastases (non-miliary type) show a more inflamed situation with higher levels of monocytes, cytotoxic T-cells, thymocyte attractants and pro-inflammatory cytokines in the peritoneal cavity as compared to the miliary type. It is conceivable that tumor cells that survive the specific milieu of the ascites (hypoxia, inflammation) or use other routes to metastasize (i.e. the haematolymphatic system) are capable of forming new lesions on the peritoneal surface only in limited numbers and these grow into big, vascularized tumors.
On the other hand miliary is characterized by less inflammation in the peritoneal cavity, elevated levels of naïve B-cells and IL-10 in the ascites and inhibited WNT and cell cycle pathways of immune cells. The tumor cells of this subtype probably adhere to the peritoneal wall more easily and grow into small nodules with impaired ability to induce neoangiogenesis.
We conclude that these two types of tumor spread are not only driven by the tumor itself but rather by a complex interplay with the microenvironment, especially the local immune system.
Citation Format: Katharina Auer, Anna Bachmayr-Heyda, Nyamdelger Sukhbaatar, Stefanie Aust, Christoph Grimm, Reinhard Horvat, Dietmar Pils. Peritoneal tumor spread in high-grade serous ovarian cancer: Differences in the immune response. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B55.