In ovarian cancer, detached single and tumor cell aggregates (spheroids) from the primary tumor that persist in the peritoneal fluid represent the main source of intraperitoneal metastasis (14). Specially, tumor cells within the spheroids exhibit a survival benefit and may represent a key element of chemotherapy-sensitive recurrence. In the present study we were able to identify and enrich this tumorigenic subpopulation within the ascites of ovarian cancer patients and further elucidate via RNAseq analysis unique molecular characteristics of these cellular structures.
Malignant ascites itself constitutes a favorable milieu for tumor cells to progress. It contains soluble factors such as cytokines, chemokines, growth factors, and extracellular matrix fragments as well as a complex mixture of cells including tumor, stromal cells and infiltrating immune cells [2]. The cellular part of ascites includes single cells and cell aggregates, so-called floating spheroids [5, 6]. In the present study, we could show that the single cell population includes some tumor cells, but it is principally composed of immune cells and to a less extent of a mesenchymal-like cell population, defined in our analysis by a CD90 positivity. In contrast, the cell aggregates found in most ovarian cancer samples contained a much higher (> 80%) percentage of tumor cells showing a strong EpCAM expression or combined EpCAM and CD24 positivity. Here, the tumor purity as well as the protective environment created by these structures may explain the high rate of successful tumor development observed, when injecting tumor-spheroids intraperitoneally in immunodeficient mice, in contrast with single cell tumor injections. Moreover, the interaction between tumor cells and other cellular components within the spheroids seems to be essential in order to keep their compact structure, but it also enhances the survival ability and invasive potential of the tumor cells. In this context, different cell populations such as cancer-associated fibroblasts (CAFs) or tumor-associated macrophages (TAMs) have been described as key players for the aggregation as well as for the adhesive and invasive properties of these tumor cell structures, thereby potentiating their malignant phenotype and facilitating the peritoneal metastatic process (15–18). Our FACs and RNAseq analysis on purified tumor spheroids are in line with this findings. We could frequently detect CD45-positive and CD90-positive cells within the spheroids, although both represented a small fraction within the spheroids, and further two macrophage-associated genes (CD163 and MARCO) were found to be highly up-regulated in the tumor-spheroids in comparison with the solid tumor tissue. CD163 is a characteristic marker of M2 macrophages, which are the most predominantly TAM subtype found in ovarian cancer, and are associated with tumor invasion, angiogenesis, metastatic disease and early recurrence (18–20). MARCO is a class A scavenger receptor expressed by immune suppressive tumor-associated macrophages and has been linked to poor prognosis in breast cancer (21, 22). Interestingly, targeting MARCO-positive TAMs with a specific antibody reduces tumor growth and metastasis in breast, colon and melanoma mouse models (23). In glioblastoma, MARCO-expressing TAMs induce a phenotypic shift towards mesenchymal cellular state of glioma stem cells, promoting both invasive and proliferative activities, as well as therapeutic resistance to irradiation (24). Additional analysis are required in order to elucidate the impact of MARCO-positive TAMs on spheroid tumor cells and whether this interaction might further influence disease progression of ovarian cancer patients.
One characteristic feature of tumor spheroids is their low chemosensitivity, in part attributed to a low proliferative profile (3). The standard chemotherapy for ovarian cancer patients, consisting in a paclitaxel and carboplatin combination, selectively targets and eliminates highly proliferative tumor cells (25). In poorly vascularized tumor areas however, cells become quiescent (26) and in turn less responsive to therapy. A recent study has estimated that in ovarian cancer spheroids more than 60% of the cells are quiescent (4). Moreover, it has been described that quiescent tumor cells use preferentially the mitochondrial OXPHOS pathway for their ATP production (27). In line with this data, our RNAseq analysis revealed a significant up-regulation of the OXPHOS pathway in the tumor-spheroids isolated from the ascites of ovarian cancer patients compared to corresponding solid tumor tissue samples. Thus, OXPHOS pathway inhibition opens an attractive therapeutic window for the specific target of tumor spheroids, as the major vehicle of peritoneal metastasis in OvCa. By using OXPHOS inhibitors, spheroid tumor cells might not be able to cover their high ATP demand. Contrary to normal cells that can activate glycolysis in response to OXPHOS inhibition, quiescent tumor cells within the spheroids have no access to sufficient glucose in order to compensate the loss of ATP production and might die (28). Interestingly, several drugs, including metformin, that have been used clinically for non-oncologic indications have emerged as effective OXPHOS inhibitors (29). Several cohort studies have described a protective effect and an association of metformin with longer overall survival in ovarian cancer patients (30–33). Also, two in vitro studies have found reduced ovarian cancer cell proliferation, migration and increased apoptosis (34) as well improved sensitivity in drug-resistant ovarian cancer cell lines (35) after metformin treatment. In contrast, a recent meta-analysis that excluded studies considered to have the potential for immortal time bias, suggested no overall survival benefit associated with use of metformin (36). A recent pilot study evaluating the efficacy of metformin plus first-line chemotherapy versus chemotherapy alone in a small cohort of ovarian cancer patients found no effects of metformin, neither(37) Our in vitro analyses might explain these contradictory findings. Here, ascites-derived tumor-spheroids from different ovarian cancer patients showed in the majority of the samples a strong effect of metformin alone as well as in combination with cisplatin treatment, on the tumor cell viability, including a cisplatin-resistant tumor from a recurrent ovarian cancer patient. One sample, however, showed no response to metformin even at high concentrations, indicating that inhibition of the OXPHOS pathway might be not a universal target for all ovarian cancer patients. Ongoing analyses in our group aim the identification of the specific molecular features that might discriminate metformin responders from non-responders.
Our RNAseq data has further revealed several factors up-regulated in spheroids that are linked to cell chemoresistance. The transglutaminase 1 (TGM1), an enzyme that is mainly found in the epidermis, catalyzes protein bonds, so-called cross-linking, which give the tissue strength and stability. In gastric carcinoma TGM1 has been shown to promote the stem cell character and chemoresistance of tumor cells via modulation of the Wnt/beta-catenin signalling pathway. Further, several members of the heat shock protein 70 family (Hsp70) were found to be significantly up-regulated in the tumor spheroids compared with the tumor tissue. The human Hsp70 family consists of eight highly homologous members of chaperone molecules that differ in their intracellular localization and expression pattern. Specially, HSPA1A/1B and HSPA6, which code for the proteins Hsp70 and Hsp70-6, respectively are only expressed at low or undetectable levels under physiological conditions, but are rapidly induced by cellular stress (38). In cancer cells, the effect of Hsp70 has been not only related to its chaperone activity, but rather to its antiapoptotic role and the regulation of cell signaling. In ovarian cancer, increased Hsp70 expression was found in chemoresistant cells. Here, Hsp70 proteins block the translocation of Bax into the mitochondria and the release of mitochondrial proteins into the cytosol (39). Additionally, three metallothioneins (MT1E, MT1M and MT1X) were highly up-regulated in the spheroids. MTs are small cysteine-rich proteins with a key role in metal homeostasis and protection against heavy metal toxicity. Consequently, a drug resistance function has been described in the context of cancer (40), though specifically in ovarian cancer no difference between MT expression in tumors from chemotherapy-treated vs. untreated patients could be found (41). Still, MT expression has been negatively associated with survival time in primary ovarian carcinomas (42).
Besides the low proliferative rate and chemosensitivity, tumor cells within the spheroids might acquire specific adhesive characteristics that support a protective and compact cellular aggregation structure (43). In this context, our RNAseq analysis revealed high mRNA levels of integrin α3 (ITGA3), claudins 4 and 7 (CLDN4 / 7), desmosome proteins plakophilin 3 (PKP3) and periplakin (PPL) as well as the barrier protein filaggrin (FLG), the last showing an aprox. 10fold up-regulation in the ascites-derived spheroids compared with tumor tissue. Interestingly, recent data raised the possibility that molecules with mechanical barrier function may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction. Here, authors identified eight genes, including PPL and PKP3, whose increase expression in human melanoma metastases and ovarian cancers was associated with a lack of Th1 immune signatures and further strongly correlated with shorter overall survival (44).
The in vitro and in vivo behavior of ascites-derived cells has been reported by other groups before (2, 43, 45, 46). In our study we showed similar results as previously described, namely in the majority of the samples ascites tumor spheroids from ovarian cancer patients showed a quiescent and non-adhesive phenotype when cultured in vitro, whereas the single cells gave rise to an adherent and highly proliferative population. In contrast, intraperitoneal injection in immunodeficient mice showed just the opposite picture; namely tumor development was observed in most of the spheroid samples, but none of the ADs developed carcinosis. FACS and ICC analyses revealed an explanation for this contrary behavior, showing that the spheroids consist principally of tumor cells population, whereas the single cells were mainly of non-epithelial origin. We assume the lack of an adequate stimulus in vitro prevent tumor spheroids to attach and further spread, thereby highlighting the key role of the intraperitoneal environment for tumor progression in ovarian cancer. Thus, the key role of fibroblasts, immune, adipocytes, mesothelial and endothelial cells for disease progression has been broadly described in the last years (47, 48).
Remarkably, two samples showed a totally different pattern regarding their cellular distribution and in vitro behavior. Here, the AD populations included a high percentage of tumor cells, as they showed a strong staining for EpCAM. These results emphasizes the high heterogeneity of “ovarian cancer” and the need to decipher the different biological subtypes behind this entity, in order to develop specific and targeted therapies. In this context, the ascites-derived tumor spheroids might represent a suitable model to address this question. In the present study we could show that ascites-derived spheroids from ovarian cancer patients clearly depict the biology of the individual disease, especially if we consider that their dissemination pattern in the mice clearly mimic the one observed in the patient.
Moreover, we could identify several molecular players that might help us to better understand the biology of ascites-derived tumor spheroids. Ongoing analysis in our group aim to prove the functionality of these markers and to explore their role as potential therapeutic targets.