Patient-derived ovarian cancer explants: preserved viability and histopathological features in long-term agitation-based cultures

Ovarian carcinoma (OvC) remains a major therapeutic challenge due to its propensity to develop resistance after an initial response to chemotherapy. Interactions of tumour cells with the surrounding microenvironment play a role in tumour survival, invasion capacity and drug resistance. Cancer models that retain tissue architecture and tumour microenvironment components are therefore essential to understand drug response and resistance mechanisms. Herein, our goal was to develop a long-term OvC patient-derived explant (OvC-PDE) culture strategy in which architecture and cell type heterogeneity of the original tumour would be retained. Samples from 25 patients with distinct OvC types and one with a benign tumour, were cultured for 30 days in agitation-based culture systems with 100% success rate. OvC-PDE cultures retained the original tumour architecture and main cellular components: epithelial cells, fibroblasts and immune cells. Epithelial cells kept their original levels of proliferation and apoptosis. Moreover, the major extracellular components, such as collagen-I and -IV, were retained in explants. OvC-PDE cultures were exposed to standard-of-care chemotherapeutics agents for 2 weeks, attesting the ability of the platform for drug assays employing cyclic drug exposure regimens. We established an OvC-PDE dynamic culture in which tumour architecture and cell type heterogeneity were preserved for the different OvC types, replicating features of the original tumour and compatible with long-term drug exposure for drug efficacy and resistance studies.


Results
OvC samples were obtained after surgical resection of ovarian tumours with distinct pathologies. Table 1 lists the pathological types and grades of the 25 OvC employed in this study: 12 high-(HGSC) and 1 low-grade serous carcinomas (LGSC), 1 mucinous, 3 mucinous borderline, 4 endometrioid, 2 clear cell carcinomas, 1 undifferentiated tumour, 1 carcinosarcoma (in the sample collected for culture, the epithelial component presented HGSC architecture); a fibroma (benign) was also utilised (Table 1; Supplementary Table S1). The majority of the OvC (48%) were HGSC, consistent with the high frequency of this OvC type 28 .
All samples were processed for OvC-PDE culture, as described in detail in the methods section (Fig. 1). Immediately after dicing the tumour, explants were incubated with fluorescein diacetate (FDA) and propidium iodide (PI) to assess viable and dead cells, respectively. Typically, dead cells were observed in the outer cell layer, probably a consequence of the mechanical processing. However, over culture, these cells dissociated from the explant, thus obtaining highly viable explants throughout one month of culture ( Supplementary Fig. S1A,  Fig. S2A). OvC-PDE cultures derived from serous carcinoma and carcinosarcoma (of which the epithelial component was exclusively composed of HGSC) had an average explant area of 1.0 ± 0.5 mm 2 (OVC12, OVC14, OVC15, OVC16, OVC17, OVC20 and OVC23, Supplementary Fig. S1B), whereas OvC-PDE cultures derived from mucinous tumours and clear cell carcinomas were composed of larger explants, with a more heterogeneous size distribution (2.9 ± 1.0 mm 2 , OVC11, OVC13, OVC21 and OVC25, Supplementary Fig. S1B). After 10 days

FIGO staging Number of cases (N = 25) Percentage (%)
Stage I 8 32 Stage II 1 4 Stage III 16 64 Scientific Reports | (2020) 10:19462 | https://doi.org/10.1038/s41598-020-76291-z www.nature.com/scientificreports/ in culture, we observed a significant decrease in explant size (0.7 ± 0.3-fold relative to the initial size at day 0), probably corresponding to the dead outer layer that dissociated from each explant. By day 30 of culture, average explant size was 0.3 ± 0.1 times the initial size (p < 0.0001 between day 0 and day 30 of culture, Supplementary  Fig. S2B) and with higher homogeneity as indicated by the different variance of the samples (F test, p < 0.05). Additionally, explant concentration increased overtime, reaching a 2.8 ± 1.5-fold increase relative to the initial concentration ( Supplementary Fig. S2C). All OvC types and the fibroma maintained high cell viability during the culture period. By live/death analysis, we observed homogenous green fluorescence in OvC-PDE cultures, indicating high cell viability ( Supplementary  Fig. S2A). After 30 days in culture, OvC-PDE cultures remained metabolically active, as assessed by resazurin reduction capacity (0.8 ± 0.5-fold change relative to day 0 of culture, Fig. 2A), and maintained the original apoptosis levels (1.42 ± 0.89-fold change in cleaved caspase-3+ cells relative to day 0 of culture, Fig. 2C). Importantly, after 20 days in culture, no statistical significant difference was observed in terms of proliferation (0.65 ± 0.35-fold change in Ki67 + cells relative to day 0 of culture, Fig. 2D). After 30 days, OvC-PDE cultures maintained a high cell proliferation rate, despite the slight decrease observed (0.53 ± 0.28-fold change in Ki67 + cells relative to day 0 of culture, Fig. 2D). Curiously, there was a transient increase in cell death (necrosis and apoptosis) up to day 10 of culture, detected by a peak in LDH activity in the culture supernatant (Fig. 2B) and a twofold increase of cleaved caspase-3 (Fig. 2C), respectively and then the levels returned to values similar to day 0 of culture up to 30 days of culture.
Moreover, we compared the antigen profile of OvC-PDE cultures with the original tumour. Detection of the OvC marker Cancer Antigen 125 (CA125) 29 was maintained in HGSC-and LGSC-derived OvC-PDE cultures (Fig. 3A,B, respectively). PAX8 and WT1, HGSC markers 30 , were also detected (Fig. 3A,B). For OVC3, p53 was not detected in both the original tumour and the OvC-PDE, suggesting that this particular tumour was a p53 null expression (nonsense mutation of TP53, Fig. 3A). In clear cell carcinoma, the patterns of detection of oestrogen receptor (ER), HNF1β and WT1 were also similar to the original tumour, despite variations in intensity (Fig. 3C).
Epithelial cells and fibroblasts were identified after one month of OvC-PDE culture of all the tumours employed in the study, by morphologic inspection of H&E staining (Fig. 4A). Importantly, the ratio of the two cellular compartments was also maintained in all samples analysed (Fig. 4B). Furthermore, OvC-PDE derived from tumours with immune infiltrate preserved the tumour infiltrating lymphocytes (TILs), namely CD4+ and CD8+ T cells (Fig. 5). The immune infiltrate of the OVC16 tumour contained B cells and macrophages; these were also detected in OvC-PDE culture, by IHC for CD20 and CD68, respectively ( Supplementary Fig. S3A). Moreover, in one of the OvC-PDE cultures we evaluated collagen I and IV, two of the major extracellular matrix (ECM) components in OvC, as well as integrins β1 and β4, the cell surface receptors for collagen-I and laminin adhesion 31,32 . Labelling was similar in the original tumour and in OvC-PDE cultured for 30 days ( Supplementary  Fig. S3B). Sequential series of cross-sections of several cases were analysed by H&E; the phenotype and morphology within each individual OvC-PDE was homogeneous across its depth, suggesting that OvC-PDE did not present necrotic cores nor induced regionalization of specific cell types ( Supplementary Fig. S4).
Overall, the data showed that OvC-PDE of approximately 1 mm 2 can be maintained in agitation-based culture systems for at least 30 days, retaining features of the original tumour, such as architecture, cellularity (epithelial, stromal and immune infiltrate), as well as subtype-specific antigen profile, proliferation and apoptotic indexes.
As a proof-of-concept of the applicability of our OvC-PDE culture system to test repeated drug exposure, we challenged OvC-PDE cultures with standard-of-care chemotherapeutic agents (Figs. 1, 5). Resazurin reduction capacity of OvC-PDE challenged with carboplatin or paclitaxel, at the reported physiological peak plasma concentrations 33,34 , remained similar to the control after the 1st cycle of treatment (Fig. 6A). Only after the 2nd cycle, a significant reduction was observed relative to untreated control cultures, with a decrease in viability of 50 ± 19% (p < 0.01) for carboplatin and 58 ± 21% (p < 0.05) for paclitaxel (Fig. 6A). Drug-induced cell death was confirmed by immunohistochemistry analysis: we observed a decrease in epithelial cell content after 2 cycles of treatment, with lower levels of proliferative Ki67 + cells (0.78-fold change with carboplatin and 0.42-fold change with paclitaxel, relative to control, Fig. 6B, Supplementary Fig. S5A), although the levels of apoptotic cleaved caspase-3+ cells were similar to the control condition (1.15-fold change with paclitaxel, Fig. 6C, Supplementary  Fig. S5A). Moreover, 2 additional OvC-PDE were exposed to the same drug exposure regimen, at the same drug concentrations and 10 times higher ones. At the higher drug concentrations, reazurin reduction capacity Resazurin reduction capacity of the OvC-PDE along culture time relative to day 0. Data is presented as mean ± SD (N ≥ 7). Two-way ANOVA statistical test (Tukey's multiple comparison test) was applied to compare the mean values of resazurin reduction capacity along culture period relative to day 0 (*) and between timepoints (#). Statistical analysis was carried out using GraphPad Prism 6 Software; **, ## (p < 0.01) and ### (p < 0.005); (B) LDH activity in the OvC-PDE culture supernatants. Data is presented as mean ± SD (N = 5). One-way ANOVA statistical test was applied to compare LHD activity along culture period. Statistical analysis was carried out using GraphPad Prism 6 Software. ## (p < 0.01); ### (p < 0.001); (C) quantification of apoptosis (cleaved caspase 3 + cells) by immunohistochemistry (N = 15) and representative images (OVC15) of OvC-PDE cultures collected at days 0, 10, 20, and 30. Scale bars represent 100 µm. One-way ANOVA statistical test was applied to compare cleaved caspase 3 + cells along culture time versus day 0. Statistical analysis was carried out using GraphPad Prism 6 Software. *(p < 0.05); n.s. not significant; (D) quantification of proliferation (Ki67 + cells) by immunohistochemistry analysis (N = 13) and representative imagens (OVC15) of OvC-PDE maintained in agitation-based cultures and collected at days 0, 10, 20, and 30. Scale bars represent 100 µm. One-way ANOVA statistical test was applied to compare Ki67 + cells along culture time versus day 0. Statistical analysis was carried out using GraphPad Prism 6 Software. *(p < 0.05); n.s. not significant.

Discussion
OvC remains a major therapeutic challenge due to its propensity to develop resistance after an initial response to chemotherapy 35 . Interactions of tumour cell with the surrounding microenvironment play a role in tumour survival, invasion capacity and drug resistance 36,37 . Cancer cell models that retain tissue architecture and tumour microenvironment components are therefore essential to understand drug response and resistance mechanisms.  www.nature.com/scientificreports/ Herein, we describe a novel culture strategy that improves the longevity and preserves the histopathological features of OvC explants, by taking advantage of agitation-based culture systems 38 . Our platform sustained high cell viability levels and maintained the original tumour phenotype for at least 30 days with 100% success rate. Importantly, this strategy was successful for the culture of eight subtypes of OvC, from type I, type II and borderline tumours 39,40 . Interestingly, the methodology also worked for a non-malignant tumour (fibroma), with the preservation of stromal cells.
OvC-PDE cultivated up to one month retained metabolic activity, proliferation rates and apoptotic levels similar to the original tumour without formation of hypoxia gradient and necrotic cores. The 30 days of culture reported herein are an improvement over the commonly reported 2 to 7 days 18,20,21,41 . The transient increase in cell apoptosis observed during the first 10 days of culture suggests a period of adaptation to culture conditions after the sample's mechanical processing. We explored agitation-based systems, reported to improve the diffusion of oxygen and soluble factors (nutrients, metabolic waste products, soluble factors and cytokines) 38,[42][43][44] . Dynamic culture systems have been previously proposed for culture of ex vivo cancer models 19,38,45,46 . These studies report improvements in cellular physiology and viability 47 over static systems. For instance, Van der Kuip et al. described a strategy for the culture of breast cancer precision cut thin slices based on orbital shaking, in which cells remained viable and proliferative for at least 4 days 48 . Naipal et al. used a similar strategy and found that the continuous movement promoted nutrient exchange, leading to a higher percentage of proliferative cells and extended culture viability (7 days), in comparison to stationary conditions 21 . We generated OvC-PDE of an average size of 1 mm 2 . For mucinous and clear cell carcinoma, OvC-PDE were larger and had a more heterogeneous size distribution in comparison with their counterparts derived from serous carcinomas and a carcinosarcoma with serous component. This could be correlated with the presence of mucus that created thread-like structures. Naipal et al. reported difficulties in homogeneous processing of soft, mucinous and fibrous tissue 21 .
We hypothesised that thick OvC-PDE could have advantages over thin slices by retaining endogenously secreted soluble factors and sustaining retention of ECM, contributing to sustain cellular crosstalk within the tissue 49,50 . ECM components have been gaining attention due to their important role in tumour growth and progression 51 . Tumour architecture and stroma composition, but also their secreted factors, have been recognised as major players in the establishment and progression of cancer cells 49,50 . Specifically, in the OvC context, CAFs are known to express and secrete high levels of chemokine (CXC motif) ligand CXCL-1, which binds to its receptor CXCR2, highly expressed on OvC cells, thus promoting cell proliferation 52 . CAFs also secrete high levels of Interleukin 6 (IL-6) and cyclooxygenase 2 (COX-2), factors correlated with inflammation, angiogenesis and proliferation support, this way circumventing apoptosis and promoting tumour progression 52,53 . In addition, OvC CAFs are the main source of ECM components, such as collagens type I, III and V and fibronectin and matrix metalloproteinases (MMPs) 54 .
In fact, our culture system allowed the retention of epithelial, stromal and immune compartments of the original tumours, by applying a common strategy and a basal medium composition to the different subtypes. www.nature.com/scientificreports/ Interestingly, when ECM components were analysed in one of the OvC-PDE, collagen-I and -IV, as well as the ECM receptors β1 and β4 integrin 55 were strongly detected after one month of culture. To our knowledge, this is the first long-term ex vivo study reporting the maintenance of these OvC microenvironment components.
Recently, Hill et al. 56 and Kopper et al. 27 reported short and long-term organoid platform for OvC, respectively. Organoids recapitulate subtype-specific histological and genomic features and allow for the expansion of tumour cells. However, these models are derived from malignant epithelial cells, lacking the tumour microenvironment 27,56 .
A long-term model of OvC that retains the tumour microenvironment and patient-specific features allows to explore unaddressed disease mechanisms, mainly related with OvC tumour progression and metastasis formation, as well as to perform resistance studies through the evaluation of cyclic drug treatments. To demonstrate the potential use of our model as a drug assay platform, we challenged OvC-PDE with two cycles of standard-of-care chemotherapeutic agents. Exposure to the reported physiological peak plasma concentrations of carboplatin and paclitaxel 33,34 led to a significant reduction in cell viability only after the second cycle of treatment. A tenfold higher drug concentration led to total cell death, highlighting the potential influence of the drug exposure regimen in drug efficacy. We also assessed proliferation and apoptosis after the drug challenge by immunohistochemistry (IHC), as the latter is broadly used in the clinics as a readout of drug response. Although not significant, both drugs tended to reduce the epithelial compartment and the remaining epithelial cells decreased their proliferation. Additionally, we observed an increased apoptosis upon the second cycle. These results are aligned with previous reports, where carboplatin was used in the same concentration range, and with clinical response; in neoadjuvant therapy at least three cycles are preconized 57,58 .
In the long-term perspective, one can envision the utilisation of the OvC-PDE to assess patient chemosensitivity, to assist in therapeutic decisions. Nonetheless, this implies the previous evaluation of the degree of correlation between patient response/clinical outcome and the ex vivo response, which requires longer follow-up time, as well as increased cohort size. Some correlations studies using ex vivo platforms are already described. For example, a platform based machine-learning algorithms that combined the results in ex vivo models with clinical information (patient history, tumour stage and pathology from biopsies) to predict the clinical outcome after treatment 16 .
Our model can also be used to explore and evaluate the efficiency of new compounds in preclinical phase or assessing drug combinations, although it cannot accommodate high-throughput screening campaigns. The www.nature.com/scientificreports/ fact that proliferation and apoptosis rates within OvC-PDE are similar to the original tumour rates impairs propagation of the material. Currently, the gold standard in cancer drug discovery is the xenograft mouse model 20,[59][60][61] . Although this model preserves tumour cell viability and architecture 62 , patient-derived xenografts (PDX) present a low engraftment rate 63,64 and along passages the human tumour microenvironment is replaced by mouse components [65][66][67] . Moreover, OvC PDX have been reported to exhibit a significant loss of steroid hormone receptors and altered expression of immunoresponsive genes 68 . PDX are mostly generated in immunocompromised mice, which lack the contribution of the immune system 69 . For evaluation of immunotherapies, fresh PDX which retain patient's immune cells 70 , and humanised mice have been developed 71 . Nevertheless, some limitations remain such as missing cross-reactivity of cytokines and growth factors between species, resulting in graft-versus-host disease (GVHD) typically within 4 weeks 72 . In addition, these models demand considerable costs and are extremely laborious 73 . OvC-PDE retained viable tumour infiltrating lymphocytes (TILs), presenting both CD4+ and CD8+ T cells. In accordance, in a study of 186 samples of advanced-stage OvC, it was found that around half of the patients had CD3+ TILs, presenting both CD4+ and CD8+ T cell populations 74 . In the future, it will be interesting to evaluate the functionality and immunosuppressive status of the TILs present within OvC-PDE cultures to evaluate their real potential for immunoncology studies.

Conclusions
In this work, we provided experimental evidence of the feasibility to culture OvC-PDE in agitation-based culture systems for at least 1 month. The main OvC types were successfully cultured as OvC-PDE and we could establish and maintain these cultures up to 30 days with high cell viability and with proliferation and apoptosis levels similar to the original tumour. OvC-PDE cultures preserved the histopathological features of original tumours, with maintenance of the epithelial and stromal components, as well as the immune infiltrate. As a proof-of-concept of the applicability of the model for repeated-dose drug assays, OvC-PDE cultures were challenged weekly with standard-of-care chemotherapy. To sum up, this is the first report of one-month long ex vivo patient-derived OvC model with preservation of microenvironment features, compatible with cyclic drug challenge. With such characteristics, this model system can contribute to fundamental research of OvC but also for precision medicine approaches.

Materials and methods
Sample collection and processing of tumour tissue. Fresh human tumours were collected from patients with signed informed consent that underwent surgery at the Instituto Português de Oncologia de Lisboa, Francisco Gentil (IPOLFG). Samples were named chronologically from OVC1 to OVC27 (Supplementary  Table S1); sample OVC22 was not included in the study since it was diagnosed as a breast cancer metastasis.

Establishment of patient-derived explant cultures (OvC-PDE).
OvC-PDE cultures were maintained at 5 explants/mL, in 20 mL culture medium (DMEM supplemented with 10% FBS and 1% P/S), in 125 mL regular Erlenmeyer shake flasks (Corning). Cultures were kept under orbital shaking (IKA KS 260 basic) at 100 rpm, to prevent adhesion and increase oxygen and nutrients diffusion, in an incubator (Nuaire US Autoflow) at 37 °C, 5% CO 2 in air. Cultures were maintained up to 30 days. Culture medium was renewed once a week (50% of the total volume). OvC-PDE were collected at day 0 (surgery day, after sample processing), 10, 20 and 30 of culture. Due to restriction of primary material, and the destructive nature of several endpoints, not all OvC-PDE could be used for all read-outs, at all timepoints: all OvC-PDE cultures were evaluated by H&E in all timepoints; as for the remaining read-outs, they were selected taking into account the work phase and availability of material, as identified in each method.
Live/dead assay. The

Resazurin reduction capacity. Resazurin reduction capacity of cells present in the explants was assessed
using the PrestoBlue Cell Viability Reagent (A13262, Invitrogen). The active ingredient of PrestoBlue reagent (resazurin) is a non-toxic and non-fluorescent dye, that when in contact with a viable cell is reduced, becoming red-fluorescent resorufin 76 . At days 0, 10, 20 and 30 of each OvC-PDE culture analysed (N = 7-8), 1 mL of culture suspension (on average, 5 explants) was collected in triplicate, and incubated with PrestoBlue reagent (diluted 1:10) during 1 h at 37 °C. After this, supernatants of triplicates were collected to a 96-well black fluo-  www.nature.com/scientificreports/ mL for carboplatin 33 and 10 µg/mL for paclitaxel 34 ). OvC-PDE were exposed to 2 cycles of treatment (Fig. 1), with a one-week interval between exposures; each treatment lasted 24 h (100% medium exchange after 24 h), as the drugs are reported to be catabolised and eliminated from the body in 24 h 78 ; in particular, 77% of cumulative urinary platinum is secreted in 24 h 79 . Evaluation of chemotherapy efficacy was assessed over time by resazurin reduction capacity (at day 14 and 21 of culture, i.e., DC7 and DC14, respectively). Additionally, morphology, proliferation and apoptosis status were assessed by histology and IHC, also at DC7 and DC14. In addition, OvC-PDE from OVC26 and OVC27 samples (N = 2) were challenged with 25 or 250 µg/mL carboplatin and 10 or 100 µg/mL paclitaxel (the reported peak plasma concentrations and 10× those concentrations). Evaluation of chemotherapy efficacy was assessed at day 7 (DC0) and 21 of culture (DC14) by resazurin reduction capacity.
Statistical analysis. One-way ANOVA statistical test or two-way ANOVA statistical test, followed by the Tukey's multiple comparison test, were used for comparisons between more than two groups. p value were obtained by t test using Holm-Sidak method (95% confidence and statistical significance is defined using an α = 0.05). Data are shown as mean ± standard deviation of the means of N (indicated in each figure legend). Statistical analysis was carried out using GraphPad Prism 6 software for Windows (GraphPad Software, La Jolla California USA, www.graph pad.com).

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.