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PARP inhibitors (PARPi) have changed the management of patients with ovarian cancer and their effectiveness has been demonstrated especially in homologous recombination repair–deficient tumors. These first-generation drugs target PARP1, but also PARP2 and other family members potentially responsible for adverse effects that limit their therapeutic potential and restrict their use in combination with chemotherapeutic agents. We investigated ovarian cancer patient-derived xenografts (OC-PDXs) to assess whether malignant progression could be impaired by a novel inhibitor selective for PARP1 (AZD5305) and to assess the potential of its combination with carboplatin (CPT), the standard-of-care for patients with ovarian cancer. In BRCA-mutated OC-PDXs, AZD5305 achieved greater tumor regressions and longer duration of response as well as a superior impairment of visceral metastasis and improved survival benefit compared with the first-generation dual PARP1/2 inhibitors. The combination of AZD5305 plus CPT was more efficacious than single agents. Subcutaneously growing tumors experienced regression that persisted after therapy stopped. Combination efficacy was greater against tumors that did not respond well to platinum, even at a dose at which AZD5305 monotherapy was ineffective. The combination therapy impaired metastatic dissemination and significantly prolonged the lifespan of mice bearing OC-PDXs in their abdomen. This combination benefit was evident even when CPT was used at suboptimal doses, and was superior to full-dose platinum treatment. These preclinical studies demonstrate that the PARP1-selective inhibitor AZD5305 retains and improves the therapeutic benefit of the first-generation PARPi, providing an opportunity to maximize benefits for this class of anticancer agents. Significance: Selective PARP1i AZD5305 can exceed the efficacy of first-generation PARPi, which target both PARP1 and PARP2, and potentiates the efficacy of CPT when given in combination. AZD5305 alone or in combination with platinum delayed visceral metastasis, ultimately extending the lifespan of OC-PDX–bearing mice. These preclinical models mimic the progression of the disease occurring in patients after debulking surgery, and are translationally relevant.

MALDI MSI images from the tumor models described in Fig. 4 and 5 of the Results and Discussion, were quantified for ion signal distribution.

MALDI MSI images from A2780-1A9 subcutis tumors described in Fig. 2, of the Results and Discussion, were quantified for ion signal distribution.

PDF - PDF 109K, Effect of sunitinib on metastatic Renca-luc tumors, orthotopic injection, neoadjuvant setting.

PDF - PDF 154K, Effect of sunitinib and sorafenib on metastatic Lewis Lung carcinoma.

PDF - 35K, Table 1: The effect of sunitinib combined with different chemotherapeutic agents on LLC primary tumors and metastasis; Supplementary Table 2: The effect of metronomic topotecan alone or combined with sunitinib on LLC primary tumor and metastasis.

Materialds and Methods details concerning MALDI MSI, Immunihistochemistry and DCE-MRI

Supplementary Data from PGC1α/β Expression Predicts Therapeutic Response to Oxidative Phosphorylation Inhibition in Ovarian Cancer

Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1β. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1β (PGC1α/β) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling and ATP depletion that consequently delayed malignant progression and prolonged the lifespan of high PGC1α/β-expressing OC-PDX-bearing mice. Conversely, low PGC1α/β OC-PDXs were not affected by IACS-010759, thus pinpointing a selective antitumor effect of OXPHOS inhibition. The clinical relevance of these findings was substantiated by analysis of ovarian cancer patient datasets, which showed that 25% of all cases displayed high PGC1α/β expression along with an activated mitochondrial gene program. This study endorses the use of OXPHOS inhibitors to manage ovarian cancer and identifies the high expression of both PGC1α and β as biomarkers to refine the selection of patients likely to benefit most from this therapy.Significance:OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/β expression and offers an effective approach to manage patients on the basis of PGC1α/β expression.

Supplementary Figure 1. Histologic characteristics of the original patient tumors and corresponding EOC-xenografts.

Figure S1 shows absence of drug-related toxicity

Supplementary Figure 2. Comparative immuno-histologic analysis of patient tumor and the corresponding xenograft.

Supplementary Figure 4. Gene copy number in patient and xenograft

Supplementary Information

PARP inhibitors (PARPi) have changed the management of patients with ovarian cancer and their effectiveness has been demonstrated especially in homologous recombination repair–deficient tumors. These first-generation drugs target PARP1, but also PARP2 and other family members potentially responsible for adverse effects that limit their therapeutic potential and restrict their use in combination with chemotherapeutic agents.We investigated ovarian cancer patient-derived xenografts (OC-PDXs) to assess whether malignant progression could be impaired by a novel inhibitor selective for PARP1 (AZD5305) and to assess the potential of its combination with carboplatin (CPT), the standard-of-care for patients with ovarian cancer.In BRCA-mutated OC-PDXs, AZD5305 achieved greater tumor regressions and longer duration of response as well as a superior impairment of visceral metastasis and improved survival benefit compared with the first-generation dual PARP1/2 inhibitors.The combination of AZD5305 plus CPT was more efficacious than single agents. Subcutaneously growing tumors experienced regression that persisted after therapy stopped. Combination efficacy was greater against tumors that did not respond well to platinum, even at a dose at which AZD5305 monotherapy was ineffective. The combination therapy impaired metastatic dissemination and significantly prolonged the lifespan of mice bearing OC-PDXs in their abdomen. This combination benefit was evident even when CPT was used at suboptimal doses, and was superior to full-dose platinum treatment.These preclinical studies demonstrate that the PARP1-selective inhibitor AZD5305 retains and improves the therapeutic benefit of the first-generation PARPi, providing an opportunity to maximize benefits for this class of anticancer agents.Significance:Selective PARP1i AZD5305 can exceed the efficacy of first-generation PARPi, which target both PARP1 and PARP2, and potentiates the efficacy of CPT when given in combination. AZD5305 alone or in combination with platinum delayed visceral metastasis, ultimately extending the lifespan of OC-PDX–bearing mice. These preclinical models mimic the progression of the disease occurring in patients after debulking surgery, and are translationally relevant.

Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1β. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1β (PGC1α/β) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling and ATP depletion that consequently delayed malignant progression and prolonged the lifespan of high PGC1α/β-expressing OC-PDX-bearing mice. Conversely, low PGC1α/β OC-PDXs were not affected by IACS-010759, thus pinpointing a selective antitumor effect of OXPHOS inhibition. The clinical relevance of these findings was substantiated by analysis of ovarian cancer patient datasets, which showed that 25% of all cases displayed high PGC1α/β expression along with an activated mitochondrial gene program. This study endorses the use of OXPHOS inhibitors to manage ovarian cancer and identifies the high expression of both PGC1α and β as biomarkers to refine the selection of patients likely to benefit most from this therapy. Significance: OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/β expression and offers an effective approach to manage patients on the basis of PGC1α/β expression.

Endoplasmic reticulum (ER) stress triggers an adaptive response in tumours which fosters cell survival and resilience to stress. Activation of the ER stress response, through its PERK branch, promotes phosphorylation of the α-subunit of the translation initiation factor eIF2, thereby repressing general protein translation and augmenting the translation of ATF4 with the downstream CHOP transcription factor and the protein disulfide oxidase, ERO1-alpha EXPERIMENTAL APPROACH: Here, we show that ISRIB, a small molecule that inhibits the action of phosphorylated eIF2alpha, activating protein translation, synergistically interacts with the genetic deficiency of protein disulfide oxidase ERO1-alpha, enfeebling breast tumour growth and spread.ISRIB represses the CHOP signal, but does not inhibit ERO1. Mechanistically, ISRIB increases the ER protein load with a marked perturbing effect on ERO1-deficient triple-negative breast cancer cells, which display impaired proteostasis and have adapted to a low client protein load in hypoxia, and ERO1 deficiency impairs VEGF-dependent angiogenesis. ERO1-deficient triple-negative breast cancer xenografts have an augmented ER stress response and its PERK branch. ISRIB acts synergistically with ERO1 deficiency, inhibiting the growth of triple-negative breast cancer xenografts by impairing proliferation and angiogenesis.These results demonstrate that ISRIB together with ERO1 deficiency synergistically shatter the PERK-dependent adaptive ER stress response, by restarting protein synthesis in the setting of impaired proteostasis, finally promoting tumour cytotoxicity. Our findings suggest two surprising features in breast tumours: ERO1 is not regulated via CHOP under hypoxic conditions, and ISRIB offers a therapeutic option to efficiently inhibit tumour progression in conditions of impaired proteostasis.

Background: BRCA1/2 mutant tumors cells are deficient for homologous recombination (HR)-mediated DNA repair and are particularly sensitive to PARP inhibitors (PARPi). PARPi have proved efficacy in breast, ovarian, prostate, and pancreatic cancers, particularly in HR-deficient tumors, while their activity is limited in HR-proficient tumors. However, PARPi resistance is inevitable and therapeutic resistance resulting from restoration of HR repair is a pressing clinical problem. Identifying combination treatments to sensitize tumors cells to PARPi and/or overcome PARPi resistance is critical to expand the benefit of these therapies. The Polo-like kinase 1 (PLK1), a serine threonine kinase, is a master regulator of mitosis, overexpressed in many cancers. PLK1 is also involved in the DNA damage response through the promotion of HR-mediated DNA repair and the recovery from the G2/M checkpoint. PLK1 roles in HR repair suggest that PLK1 inhibition may reverse PARPi resistance. Methods: To test the effect of PLK1 and PARP inhibitors combination, we used onvansertib, a highly selective, ATP-competitor PLK1 inhibitor currently in clinical development and the FDA-approved PARPi olaparib. The antitumor effect of the single and combined drug treatments was tested in 2 BRCA1 mutated high-grade serous ovarian cancer (HGSOC) patient derived (PDX) models resistant to olaparib. Orthotopically PDX transplanted mice were treated for 4 weeks and followed for survival. Results: The combination of onvansertib and olaparib was well tolerated and showed strong anti-tumor activity in both PDX models. The combination significantly increased mice survival in comparison to vehicle, olaparib and onvansertib, and showed that onvansertib can re-sensitize PARPi-resistant tumors to olaparib. Median survival increased by 2.7-fold and 8.1-fold respectively in the 2 PDX models in the combination group versus vehicle and the Kaplan Meyer survival curves of mice treated with the combination showed a statistically survival advantage versus control and single agent treated mice. Pharmacodynamic analyses showed an increase in mitotic, apoptotic and DNA damage markers in tumors treated with the combination versus vehicle. Conclusions: The combination of the PLK1 inhibitor onvansertib and the PARPi olaparib showed potent anti-tumor activity in olaparib-resistant BRAC1 mutant HGSOC PDX models. Additional studies are ongoing to further assess the potential of the combination in BRCA wild-type and mutant ovarian, prostate, pancreatic and breast cancer preclinical models. Citation Format: Michela Chiappa, Federica Guffanti, Alessandra Decio, Alessandro Aliverti, Francesca Ricci, Eugenio Scanziani, Federica Camin, Ilaria Craparotta, Maria Chiara Barbera, Marco Bolis, Maya Ridinger, Giovanna Damia. Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3237.

Background Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. Methods The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. Results We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. Conclusions Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

SummarySolid tumors are often characterized by a hypoxic microenvironment which contributes, through the hypoxia-inducible factor HIF-1, to the invasion-metastasis cascade. Endoplasmic reticulum (ER) stress also leads tumor cells to thrive and spread by inducing a transcriptional and translational program, the Unfolded Protein Response (UPR), aimed at restoring ER homeostasis. We studied ERO1 alpha (henceforth ERO1), a protein disulfide oxidase with the tumor-relevant characteristic of being positively regulated by both ER stress and hypoxia. Analysis of the redox secretome indicated that pro-angiogenic HIF-1 targets, were blunted in ERO1-devoid breast cancer cells under hypoxic conditions. ERO1 deficiency reduced tumor cell migration and lung metastases by impinging on tumor angiogenesis, negatively regulating the upstream ATF4/CHOP branch of the UPR and selectively impeding oxidative folding of angiogenic factors, among which VEGF-A. Thus, ERO1 deficiency acted synergistically with the otherwise feeble curative effects of anti-angiogenic therapy in aggressive breast cancer murine models and it might be exploited to treat cancers with pathological HIF-1-dependent angiogenesis. Furthermore, ERO1 levels are higher in the more aggressive basal breast tumors and correlate inversely with the disease- and metastasis-free interval of breast cancer patients. Thus, taking advantage of our in vitro data on ERO1-regulated gene products we identified a gene set associated with ERO1 expression in basal tumors and related to UPR, hypoxia, and angiogenesis, whose levels might be investigated in patients as a hallmark of tumor aggressiveness and orient those with lower levels toward an effective anti-angiogenic therapy.

The efficacy of therapeutic regimens incorporating weekly or every-3-weeks paclitaxel (PTX) for ovarian cancer is debated. We investigated the addition of bevacizumab in regimens of chemotherapy with different PTX doses and schedules in preclinical models. Treatments were cisplatin (DDP) with weekly PTX (conventional), or dose-dense-equi (every other day to the conventional cumulative dose), or dose-dense-high (total dose 1.5 times higher), with or without bevacizumab. Treatment efficacy was evaluated analyzing tumor growth in different time-windows in two patient-derived ovarian cancer xenografts with different sensitivity to cisplatin. Tumor progression, metastasis and survival were studied in ovarian cancer models growing orthotopically and disseminating in the mouse peritoneal cavity. Short-term effects on cell cycle, tumor cell proliferation/apoptosis and vasculature were evaluated by flow cytometry and immunohistochemistry. PTX dose-dense (with/without DDP) was superior to the conventional scheme in a dose-dependent manner; the high efficacy was confirmed by the lower ratio of tumor to normal cells. All schemes benefited from bevacizumab, which reduced tumor vessels. However, DDP/PTX dose-dense-high (only chemotherapy) was at least as active as DDP/PTX conventional plus bevacizumab. DDP/PTX dose-dense-high plus bevacizumab was the most effective in delaying tumor progression, though it did not prolong mouse survival and the continuous treatment with bevacizumab was associated with a malignant disease. These findings indicate that the effect of bevacizumab in combination with chemotherapy may depend on the schedule-dose of the treatment and help to explain the unclear benefits after bevacizumab.

Since the approval of poly (ADP-ribose) polymerase inhibitors (PARPi) for BRCA-mutated ovarian cancer patients, different PARPi have been developed and have shown efficacy in homologous recombination deficient (HRD) tumours. These first-generation drugs inhibit both PARP1 and PARP2 (as well as other PARP family members) and present undesirable adverse effects, such as haematological and intestinal toxicity, that restricted their use especially in combination with already poorly tolerated chemotherapeutic agents. Here, patient-derived ovarian cancer xenografts (OC-PDXs) were used i) to evaluate the dose response efficacy of AZD5305, a next generation, potent and selective PARP1 inhibitor and ii) to investigate the effect of the combination with carboplatin, a standard-of-care treatment for ovarian cancer patients. AZD5305 was administered orally (po) once daily (QD) for 8 weeks as monotherapy (0.1, 1 and 10 mg/kg) and in combination with carboplatin (CPT) dosed intravenously (35 mg/kg; Q7x4). Two BRCA1 mutated (HOC106 and HOC107) and a BRCA wild-type (HOC84) OC-PDXs (all TP53 mutated) were implanted subcutis and the effect of treatments on tumour growth evaluated. AZD5305 dosed at 0.1, 1 and 10 mg/kg to mice bearing HOC106 tumours, inhibited tumours growth in a dose dependent manner. Administered in combination at 0.1 and 1 mg/kg, AZD5305 potentiated the effect of carboplatin, causing a significant and sustained regression of HOC106 tumours. The anti-tumour efficacy was also evident against the PARPi-resistant OC-PDX HOC107, whereby the tumour growth was inhibited by AZD5305 at 1 and 10 mg/kg dose. Combined with carboplatin, AZD5305 stabilized the growth of HOC107 tumours at doses as low as 0.1 mg/kg that on its own was not effective. As expected, none of the treatments affected the growth of the BRCA wild-type HOC84 (PARPi and carboplatin resistant OC-PDX). Overall, AZD5305 a next generation PARP1-selective inhibitor and trapper, shows improved efficacy and tolerability in combination with chemotherapy compared to first generation, PARP1/2 inhibitors, making it a promising clinical candidate for the treatment of ovarian cancer. Citation Format: Giulia Dellavedova, Alessandra Decio, Anna Staniszewska, Elisabetta Leo, Raffaella Giavazzi, Maria Rosa Bani. The next generation PARP inhibitor AZD5305 is active in a broad range of pre-clinical models of ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P217.

Platinum resistance is an unmet medical need in ovarian carcinoma. Molecular biomarkers to predict the response to platinum-based therapy could allow patient stratification and alternative therapeutic strategies early in clinical management. Sensitivity and resistance to platinum therapy are partially determined by the tumor&rsquo, s intrinsic DNA repair activities, including nucleotide excision repair (NER) and base excision repair (BER). We investigated the role of the NER proteins&mdash, ERCC1, XPF, ERCC1/XPF complex&mdash, and of the BER protein DNA polymerase &beta, as possible biomarkers of cisplatin (DDP) response in a platform of recently established patient-derived ovarian carcinoma xenografts (OC-PDXs). ERCC1 and DNA polymerase &beta, protein expressions were measured by immunohistochemistry, the ERCC1/XPF foci number was detected by proximity ligation assay (PLA) and their mRNA levels by real-time PCR. We then correlated the proteins, gene expression and ERCC1/XPF complexes with OC-PDXs&rsquo, response to platinum. To the best of our knowledge, this is the first investigation of the role of the ERCC1/XPF complex, detected by PLA, in relation to the response to DDP in ovarian carcinoma. None of the proteins in the BER and NER pathways studied predicted platinum activity in this panel of OC-PDXs, nor did the ERCC1/XPF foci number. These results were partially explained by the experimental evidence that the ERCC1/XPF complex increases after DDP treatment and this possibly better associates with the cancer cells&rsquo, abilities to activate the NER pathway to repair platinum-induced damage than its basal level. Our findings highlight the need for DNA functional assays to predict the response to platinum-based therapy.

Mass spectrometry imaging is a valuable tool for visualizing the localization of drugs in tissues, a critical issue especially in cancer pharmacology where treatment failure may depend on poor drug distribution within the tumours. Proper preprocessing procedures are mandatory to obtain quantitative data of drug distribution in tumours, even at low intensity, through reliable ion peak identification and integration. We propose a simple preprocessing and quantification pipeline. This pipeline was designed starting from classical peak integration methods, developed when “microcomputers” became available for chromatography, now applied to MSI. This pre-processing approach is based on a novel method using the fixed mass difference between the analyte and its 5 d derivatives to set up a mass range gate. We demonstrate the use of this pipeline for the evaluating the distribution of the anticancer drug paclitaxel in tumour sections. The procedure takes advantage of a simple peak analysis and allows to quantify the drug concentration in each pixel with a limit of detection below 0.1 pmol mm-2 or 10 μg g−1. Quantitative images of paclitaxel distribution in different tumour models were obtained and average paclitaxel concentrations were compared with HPLC measures in the same specimens, showing github.com/FrancescaFalcetta/Imaging_of_drugs_distribution_and_quantifications.git .

Cediranib is a pan-vascular endothelial growth factor receptor tyrosine kinase inhibitor that affects tumor angiogenesis and is under investigation in clinical studies on ovarian cancer. Using a panel of eleven patient-derived ovarian cancer xenografts (EOC-PDX) growing orthotopically in the peritoneal cavity of nude mice we investigated the effect of cediranib as monotherapy or in combination with chemotherapy on overall survival (primary endpoint, at euthanasia), and tumor dissemination and metastasis in the peritoneal cavity (secondary endpoint, interim analysis). The response of EOC-PDX to cediranib varied (increment of lifespan, ILS between 12 and 85 %) in the different EOC-PDX, independently from tumor responsiveness to cisplatin (DDP). Cediranib combined with DDP and in maintenance regimen prolonged the survival of mice bearing EOC-PDX with different responsiveness to DDP (ILS between 34 and 224 % with only DDP and between 135 and 337 % with DDP plus Cediranib); survival was extended with the addition of paclitaxel to chemotherapy (50-77 % complete remissions). Cediranib reduced ascites of advanced EOC-PDX, but had limited effect on tumor dissemination; only combined with chemotherapy, ascites and metastases were both reduced. The reduction of tumor dissemination was associated to the increase of overall survival. In conclusion, the response to cediranib differs in the various EOC-PDX, reproducing the heterogeneous response of cancer patients to angiogenesis inhibitors. Cediranib potentiated chemotherapy, significantly inhibiting tumor progression and dissemination to metastatic organs, even in tumors poorly responsive to DDP. EOC-PDX preclinical models with different responsiveness to Cediranib may help in identifying determinants of response to cediranib and mechanisms of adaptation to antiangiogenic treatments.

Tumor endothelial cells (TEC) differ from the normal counterpart, in both gene expression and functionality. TEC may acquire drug resistance, a characteristic that is maintained in vitro. There is evidence that TEC are more resistant to chemotherapeutic drugs, substrates of ATP-binding cassette (ABC) transporters. TEC express p-glycoprotein (encoded by ABCB1), while no difference in other ABC transporters was revealed compared to normal endothelia. A class of tyrosine kinase inhibitors (TKI), used as angiostatic compounds, interferes with the ATPase activity of p-glycoprotein, thus impairing its functionality. The exposure of ovarian adenocarcinoma TEC to the TKIs sunitinib or sorafenib was found to abrogate resistance (proliferation and motility) to doxorubicin and paclitaxel in vitro, increasing intracellular drug accumulation. A similar effect has been reported by the p-glycoprotein inhibitor verapamil. No beneficial effect was observed in combination with cytotoxic drugs that are not p-glycoprotein substrates. The current paper reviews the mechanisms of TEC chemoresistance and shows the role of p-glycoprotein in mediating such resistance. Inhibition of p-glycoprotein by anti-angiogenic TKI might contribute to the beneficial effect of these small molecules, when combined with chemotherapy, in counteracting acquired drug resistance.

Epithelial ovarian cancer (EOC) is the fifth commonest cancer-related cause of female death in the developed world. In spite of current surgical and chemotherapeutic options the vast majority of patients have widely metastatic disease and the survival rate has not much changed over the last years. The anti-angiogenic drugs are driving the field of agents targeting the tumor microenvironment in ovarian cancer. Preclinical models that accurately reproduce the molecular and biological features of ovarian cancer patients are a valuable means of producing reliable data on personalized medicine and predicting the therapeutic response in clinical trials.In this methodological chapter we describe the orthotopic model of ovarian cancer implanted under the ovarian bursa of mice. In spite of anatomical differences between the rodent and human bursa-fallopian tube, the appropriate primary tumor microenvironment at the site of the implant allows investigation of tumor-stroma interactions (e.g., angiogenesis), and is well suited for studying the tumor dissemination and metastasis typical of this disease.This model-although fairly labor intensive-may be useful for assessing novel, more selective therapeutic interventions and for biomarker discovery, reflecting the behavior of this disease.

The penetration of anticancer drugs in solid tumors is important to ensure the therapeutic effect, so methods are needed to understand drug distribution in different parts of the tumor. Mass spectrometry imaging (MSI) has great potential in this field to visualize drug distribution in organs and tumor tissues with good spatial resolution and superior specificity. We present an accurate and reproducible imaging method to investigate the variation of drug distribution in different parts of solid tumors. The method was applied to study the distribution of paclitaxel in three ovarian cancer models with different histopathological characteristics and in colon cancer (HCT116), breast cancer (MDA-MB-231) and malignant pleural mesothelioma (MPM487). The heterogeneous drug penetration in the tumors is evident from the MALDI imaging results and from the images analysis. The differences between the various models do not always relate to significant changes in drug content in tumor homogenate examined by classical HPLC analysis. The specificity of the method clarifies the heterogeneity of the drug distribution that is analyzed from a quantitative point of view too, highlighting how marked are the variations of paclitaxel amounts in different part of solid tumors.

Epithelial ovarian cancer (EOC) is the fifth commonest cancer-related cause of female death in the developed world. In spite of current surgical and chemotherapeutic options the vast majority of patients have widely metastatic disease and the survival rate has not much changed over the last years. The anti-angiogenic drugs are driving the field of agents targeting the tumor microenvironment in ovarian cancer. Preclinical models that accurately reproduce the molecular and biological features of ovarian cancer patients are a valuable means of producing reliable data on personalized medicine and predicting the therapeutic response in clinical trials.In this methodological chapter we describe the orthotopic model of ovarian cancer implanted under the ovarian bursa of mice. In spite of anatomical differences between the rodent and human bursa-fallopian tube, the appropriate primary tumor microenvironment at the site of the implant allows investigation of tumor-stroma interactions (e.g., angiogenesis), and is well suited for studying the tumor dissemination and metastasis typical of this disease.This model-although fairly labor intensive-may be useful for assessing novel, more selective therapeutic interventions and for biomarker discovery, reflecting the behavior of this disease.

Epithelial ovarian carcinoma (EOC) is an aggressive neoplasm, which mainly disseminates to organs of the peritoneal cavity, an event mediated by molecular mechanisms that remain elusive. Here, we investigated the expression and functional role of neural cell adhesion molecule (NCAM), a cell surface glycoprotein involved in brain development and plasticity, in EOC. NCAM is absent from normal ovarian epithelium but becomes highly expressed in a subset of human EOC, in which NCAM expression is associated with high tumour grade, suggesting a causal role in cancer aggressiveness. We demonstrate that NCAM stimulates EOC cell migration and invasion in vitro and promotes metastatic dissemination in mice. This pro-malignant function of NCAM is mediated by its interaction with fibroblast growth factor receptor (FGFR). Indeed, not only FGFR signalling is required for NCAM-induced EOC cell motility, but targeting the NCAM/FGFR interplay with a monoclonal antibody abolishes the metastatic dissemination of EOC in mice. Our results point to NCAM-mediated stimulation of FGFR as a novel mechanism underlying EOC malignancy and indicate that this interplay may represent a valuable therapeutic target. © 2011 EMBO Molecular Medicine.

Introduction and Aim Metabolic re-programming is an emerging hallmark of cancer. Alterations of the metabolism of glucose (i.e. glycolysis) and oxidative phoshorylation (OxPhos) are frequently observed in cancer cells. Ovarian adenocarcinoma (OC) is the third most common gynecologic malignancy in the developed countries and the most lethal one, which has shown little improvement in overall survival for the last 20 years. OC is a widely metastatic disease disseminating to the organs of the abdomen through ascitic effusion. Understanding the mechanisms facilitating metastatic spread and disease recurrence is needed to develop therapeutic strategies.Our aim is the comprehension of the metabolic perturbations favoring the aggressiveness and metastatic dissemination of OC. Experimental methods We characterized a panel of OC-patient derived xenografts (OC-PDX) for their malignant behavior (ascites production, abdominal metastasis formation and survival time). Their metabolic features were depicted as i) glucose and lactate levels measured in ascites, ii) the monocarboxyate transporters MCT1 and MCT4 of the abdominal tumor masses (by IHC), iii) glycolysis- and OxPhos- related enzymes, as well as glucose and lactate transporters (by gene expression) in abdominal masses and ascites. Results We found a direct correlation between the amount of lactate and the expression of its transporters, thus OC-PDX showing higher expression of MC1/MCT4 and higher amount of lactate were categorized as “high glycolytic” and those with lower MCT1/MCT4 and lactate “low glycolytic”. The low glycolytic OC-PDXs developed more abdominal metastasis than the highly glycolytic ones, indicating an inverse correlation between the glycolytic phenotype and the capability of the OC-PDX to disseminate, invade and grow into the organs of the peritoneal cavity. Interestingly, we found that the abdominal masses express lower levels of the glucose transporters (i.e. GLUT1), glycolysis-related enzymes (i.e. HK, LDHA) and MCT4 compared to cell aggregates from abdominal effusion; suggesting that ovarian cancer cells are able to modulates their metabolism (glycolysis/OxPhos) to best fit the needs favoring metastatic spread. Further studies are needed to fully understand this association. Conclusions Nowadays, metabolism is potentially "targetable”. Our results strongly indicate that perturbing tumor's metabolic re-programming might affect metastatic dissemination. M R B and C G contributed equally. Supported by Italian Association for Cancer Research (AIRC) IG2016 18853 to RG. Citation Format: MariaRosa Bani, Carmen Ghilardi, Alessandra Decio, Alessia Anastasia, Martina Verza, Stefano Indraccolo, Raffaella Giavazzi. Metabolic phenotype and metastasis in patient-derived ovarian cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2406.

The neuroprotective potential of cannabinoids has been examined in rats with striatal lesions caused by 3-nitropropionic acic (3NP), an inhibitor of mitochondrial complex II. We used the CB1 agonist arachidonyl-2-chloroethylamide (ACEA), the CB2 agonist HU-308, and cannabidiol (CBD), an antioxidant phytocannabinoid with negligible affinity for cannabinoid receptors. The administration of 3NP reduced GABA contents and also mRNA levels for several markers of striatal GABAergic projection neurons, including proenkephalin (PENK), substance P (SP) and neuronal-specific enolase (NSE). We also found reductions in mRNA levels for superoxide dismutase-1 (SOD-1) and -2 (SOD-2), which indicated that 3NP reduced the endogenous antioxidant defences. The administration of CBD, but not ACEA or HU-308, completely reversed 3NP-induced reductions in GABA contents and mRNA levels for SP, NSE and SOD-2, and partially attenuated those found in SOD-1 and PENK. This indicates that CBD is neuroprotective but acted preferentially on striatal neurons that project to the substantia nigra. The effects of CBD were not reversed by the CB1 receptor antagonist SR141716. The same happened with the TRPV1 receptor antagonist capsazepine, in concordance with the observation that capsaicin, a TRPV1 receptor agonist, failed to reproduce the CBD effects. The effects of CBD were also independent of adenosine signalling as they were not attenuated by the adenosine A2A receptor antagonist MSX-3. In summary, this study demonstrates that CBD provides neuroprotection against 3NP-induced striatal damage, which may be relevant for Huntington's disease, a disorder characterized by the preferential loss of striatal projection neurons. This capability seems to be based exclusively on the antioxidant properties of CBD.

Cannabinoids have been proposed as clinically promising neuroprotective molecules, as they are capable to reduce excitotoxicity, calcium influx, and oxidative injury. They are also able to decrease inflammation by acting on glial processes that regulate neuronal survival and to restore blood supply to injured area by reducing the vasoconstriction produced by several endothelium-derived factors. Through one or more of these processes, cannabinoids may provide neuroprotection in different neurodegenerative disorders including Parkinson's disease and Huntington's chorea, two chronic diseases that are originated as a consequence of the degeneration of specific nuclei of basal ganglia, resulting in a deterioration of the control of movement. Both diseases have been still scarcely explored at the clinical level for a possible application of cannabinoids to delay the progressive degeneration of the basal ganglia. However, the preclinical evidence seems to be solid and promising. There are two key mechanisms involved in the neuroprotection by cannabinoids in experimental models of these two disorders: first, a cannabinoid receptor-independent mechanism aimed at producing a decrease in the oxidative injury and second, an induction/upregulation of cannabinoid CB2 receptors, mainly in reactive microglia, that is capable to regulate the influence of these glial cells on neuronal homeostasis. Considering the relevance of these preclinical data and the lack of efficient neuroprotective strategies in both disorders, we urge the development of further studies that allow that the promising expectatives generated for these molecules progress from the present preclinical evidence till a real clinical application.

The antitumor activity of angiogenesis inhibitors is reinforced in combination with chemotherapy. It is debated whether this potentiation is related to a better drug delivery to the tumor due to the antiangiogenic effects on tumor vessel phenotype and functionality. We addressed this question by combining bevacizumab with paclitaxel on A2780-1A9 ovarian carcinoma and HT-29 colon carcinoma transplanted ectopically in the subcutis of nude mice and on A2780-1A9 and IGROV1 ovarian carcinoma transplanted orthotopically in the bursa of the mouse ovary. Paclitaxel concentrations together with its distribution by MALDI mass spectrometry imaging (MALDI MSI) were measured to determine the drug in different areas of the tumor, which was immunostained to depict vessel morphology and tumor proliferation. Bevacizumab modified the vessel bed, assessed by CD31 staining and dynamic contrast enhanced MRI (DCE-MRI), and potentiated the antitumor activity of paclitaxel in all the models. Although tumor paclitaxel concentrations were lower after bevacizumab, the drug distributed more homogeneously, particularly in vascularized, non-necrotic areas, and was cleared more slowly than controls. This happened specifically in tumor tissue, as there was no change in paclitaxel pharmacokinetics or drug distribution in normal tissues. In addition, the drug concentration and distribution were not influenced by the site of tumor growth, as A2780-1A9 and IGROV1 growing in the ovary gave results similar to the tumor growing subcutaneously. We suggest that the changes in the tumor microenvironment architecture induced by bevacizumab, together with the better distribution of paclitaxel, may explain the significant antitumor potentiation by the combination. Mol Cancer Ther; 15(1); 125–35. ©2015 AACR.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. On the basis of its histopathology and molecular-genomic changes, ovarian cancer has been divided into subtypes, each with distinct biology and outcome. The aim of this study was to develop a panel of patient-derived EOC xenografts that recapitulate the molecular and biologic heterogeneity of human ovarian cancer. Thirty-four EOC xenografts were successfully established, either subcutaneously or intraperitoneally, in nude mice. The xenografts were histologically similar to the corresponding patient tumor and comprised all the major ovarian cancer subtypes. After orthotopic transplantation in the bursa of the mouse ovary, they disseminate into the organs of the peritoneal cavity and produce ascites, typical of ovarian cancer. Gene expression analysis and mutation status indicated a high degree of similarity with the original patient and discriminate different subsets of xenografts. They were very responsive, responsive, and resistant to cisplatin, resembling the clinical situation in ovarian cancer. This panel of patient-derived EOC xenografts that recapitulate the recently type I and type II classification serves to study the biology of ovarian cancer, identify tumor-specific molecular markers, and develop novel treatment modalities. Cancer Res; 74(23); 6980–90. ©2014 AACR.

Vascular endothelial growth factor C (VEGFC) has been reported to promote tumor progression in several tumor types, mainly through the stimulation of lymphangiogenesis and lymphatic metastasis. However, the expression and biological significance of the VEGFC/VEGF receptor (VEGFR)-3 pathway in ovarian cancer growth and dissemination are unclear, and have been investigated in this study. Soluble VEGFC was detected in the plasma and ascites of patients with ovarian carcinoma, and VEGFR3 expression was found in their tumor tissues. In human ovarian carcinoma xenograft models, high levels of soluble VEGFC in ascites and serum were detected, in association with disease progression, tumor burden, and volume of ascites. Peak VEGFC expression preceded para-aortic lymph node infiltration by HOC8 neoplastic cells. Histological detection of tumor cells in blood and lymphatic vessels indicated both hematogenous and lymphatic dissemination. Overexpression of VEGFC in the VEGFR3-positive and luciferase-expressing IGROV1 cells promoted carcinoma dissemination after orthotopic transplantation in the ovary of immunodeficient mice. In vitro , VEGFC released by the tumor cells stimulated tumor cell migration in an autocrine manner. Cediranib, an inhibitor of VEGFR1-3 and c-kit, inhibited in vivo metastasis of VEGFC-overexpressing IGROV1 and in vitro autocrine effects. These findings suggest that the VEGFC/VEGFR3 pathway acts as an enhancer of ovarian cancer progression through autocrine and paracrine mechanisms, hence offering a potential target for therapy.

The murine B16 melanoma is one of the most used tumor models, its application having been used to determine the mechanisms associated with the metastatic process and the development of anticancer therapies. The B16 melanoma was originally established by Fidler and collaborators as a tumor line metastasizing to the lung. Since that time a variety of cell lines have been derived, in vitro or in vivo, having different metastatic behaviors.The methods used to obtain artificial metastases to the lung through the intravenous injection of B16 melanoma cells and spontaneous metastasis formation following cancer cell growth in the footpad are described in this chapter.

The murine B16 melanoma is one of the most used tumor models, its application having been used to determine the mechanisms associated with the metastatic process and the development of anticancer therapies. The B16 melanoma was originally established by Fidler and collaborators as a tumor line metastasizing to the lung. Since that time a variety of cell lines have been derived, in vitro or in vivo, having different metastatic behaviors.The methods used to obtain artificial metastases to the lung through the intravenous injection of B16 melanoma cells and spontaneous metastasis formation following cancer cell growth in the footpad are described in this chapter.

The development of resistance and progressive disease after treatment with angiogenesis inhibitors is becoming a controversial issue. We investigated the experimental conditions that cause multireceptor tyrosine kinase inhibitors (RTKI) to augment metastasis and whether opportune combinations with chemotherapy could counteract this effect. The renal Renca-luc tumor was transplanted orthotopically in the kidney of Balb/c mice, which then were or were not nephrectomized. The Lewis Lung carcinoma (LLC) was transplanted in the tibial muscle of C57/Bl6 mice. Treatment with the RTKI sunitinib started at different stages of tumor progression, mimicking neoadjuvant or adjuvant settings. Combination studies with paclitaxel, doxorubicin, cisplatin, gemcitabine, and topotecan were done on the LLC model, using opportune regimens. In a neoadjuvant setting, sunitinib inhibited Renca-luc tumor growth, prolonging survival despite an increase in lung metastasis; treatment after primary tumor surgery (adjuvant setting) or on established metastasis prolonged survival and decreased metastasis. Sunitinib increased lung metastasis from mice bearing early-stage LLC, but did not affect established metastases (no acceleration) from advanced tumors. Combinations with doxorubicin, topotecan, gemcitabine, but not cisplatin and paclitaxel, counteracted the increase in metastasis from LLC, partly reflecting their antitumor activity. Histology analysis after sunitinib confirmed tumor vascular changes and increased hypoxia. Topotecan at suboptimal daily doses reduced sunitinib-related metastasis, reducing tumor hypoxia. Tyrosine kinase inhibitors, as sunitinib, can have adverse malignant effects mainly in the neoadjuvant setting. The addition of chemotherapy might influence metastasis, depending on each drug mechanism of action and its regimen of administration. Mol Cancer Ther; 12(10); 2237–47. ©2013 AACR.

Angiogenesis inhibitors have shown antitumor efficacy when combined with chemotherapy. It has been proposed that this potentiation is related to better drug delivery to the tumor due to improved vessel functionality. This work combined classical pharmacokinetics and imaging analysis to study the delivery and distribution of paclitaxel after bevacizumab in ovarian cancer. The study was conducted on ovarian cancer models (A2780-1A9 and IGROV-1) xenografted orthotopically under the bursa of the nude mouse ovary. Mice bearing tumors in the ovary, treated with bevacizumab (150μg/mouse) and 24 hours later with paclitaxel (60mg/kg), were sacrificed and tumor excised for analysis, or monitored for anti-tumor activity. Paclitaxel spatial distribution after bevacizumab was analyzed by MALDI-Mass Spectrometry Imaging (MALDI-MSI) on frozen tumor slices, and compared with adjacent histochemical images stained for CD31 (vessel analysis) and Ki-67 (proliferation/necrosis); total paclitaxel in tumor homogenates, liver and plasma was evaluated by high-performance liquid chromatography (HPLC). Tumor growth of A2780-1A9 and IGROV-1, carrying the firefly luciferase gene luc2, was monitored by Bioluminescence imaging (BLI) and survival recorded. Bevacizumab decreased tumor vessel number/size compared to vehicle treated mice, but potentiated the antitumor activity of paclitaxel in both models. MALDI-MSI showed that after bevacizumab, paclitaxel was more homogenously distributed and mainly in actively proliferating areas of the tumor where vessels were uniformly diffused, despite paclitaxel concentration did not increase after antiangiogenic drug. No changes in paclitaxel systemic exposure was found in normal tissue. These findings suggest that a more uniform distribution of paclitaxel in the tumor of the mouse ovary is responsible for the better antitumor activity of the combination. A.D. and I.F.N. are the recipients of a fellowship from the Italian Foundation for Cancer Research (FIRC). Citation Format: Marta Cesca, Lavinia Morosi, Alexander Berndt, Ilaria Fuso Nerini, Alessandra Decio, Massimo Zucchetti, Raffaella Giavazzi. Bevacizumab-improved distribution of paclitaxel in ovarian cancer xenografts potentiates antitumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3377.

Background: Bevacizumab is being incorporated as first-line therapy with standard-of-care chemotherapy on epithelial ovarian carcinoma (EOC). We investigated bevacizumab combined with chemotherapy on tumour progression and mouse survival in EOC xenograft models. Methods: Bevacizumab was administered concomitantly with cisplatin plus paclitaxel (DDP+PTX), continued after induction (maintenance) or started after chemotherapy. The effect on tumour progression was monitored by bioluminescence imaging (BLI) (1A9-luc xenograft). Tumour dissemination into the peritoneal organs and ascites formation (HOC22 xenograft) was evaluated by histological analysis at the end of treatment (interim) and at euthanasia (survival). The effects on overall survival (OS) were investigated in both EOC models. Results: Bevacizumab with PTX+DDP delayed tumour progression in mice bearing EOC xenografts. OS was significantly extended, with complete responses, by bevacizumab continued after stopping chemotherapy in the HOC22 xenograft. Bevacizumab alone inhibited ascites formation, with only limited effect on tumour burden, but combined with PTX+DDP reduced ascites and metastases. Bevacizumab started after induction with PTX+DDP and maintained was equally effective on tumour progression and survival on 1A9-luc xenograft. Conclusion: Bevacizumab combined with chemotherapy not only affected tumour progression, but when administered as maintenance regimen significantly prolonged survival, reducing ascites, and tumour dissemination. We believe our findings are consistent with the clinical results and shed light on the potential effects of this kind of treatment on tumour progression.