Intraperitoneal Tumor Growth Research Articles

RIPK4 Promotes Cell Invasion and the Epithelial-Mesenchymal Transition in Ovarian Cancer.

To investigate the clinical role and biological function of receptor-interacting protein kinase 4 (RIPK4) in ovarian cancer (OC). We conducted a comprehensive analysis of the expression and prognostic role of RIPK4 in OC using various public databases including The Cancer Genome Atlas, Oncomine, and Kaplan-Meier plotter. In vitro studies included wound healing, cell migration and invasion, cell proliferation, and cell apoptosis assays as well as vascular mimicry experiments. In vivo studies were conducted using subcutaneous and intraperitoneal xenografts. Our findings revealed that RIPK4 was significantly overexpressed in OC tissue compared to normal ovarian tissue. Moreover, the overexpression of RIPK4 was associated with advanced-stage disease and a poor prognosis in OC patients. RIPK4 silencing resulted in significant inhibition of intraperitoneal tumor growth, invasion, and vascular mimicry in OC cells. Furthermore, downregulation of RIPK4 inhibited the epithelial-mesenchymal transition of OC cells both in vitro and in vivo by promoting the expression of E-cadherin and inhibiting the expression of N-cadherin. The results of this study suggest that RIPK4 may function as an oncogene in the development and prognosis of OC.

Ang-2 is a potential molecular marker for lymphatic metastasis and better response to bevacizumab therapy in ovarian cancer.

In ovarian cancer, there are two main routes of metastasis, namely intraperitoneal and retroperitoneal. Their biologic background is poorly understood. Identifying molecular markers involved might enable the development of tailored therapy regimens. Moreover, no reliable markers for response to anti-angiogenic treatment with bevacizumab are yet established. Angiopoietin-2 (Ang-2) is an angiogenic growth factor, involved in lymphatic activation and is associated with tumor progression. Here, we assessed the potential of Ang-2 as a molecular marker in metastasis and treatment of ovarian cancer. In our study, quantitative and qualitative protein Ang-2 expression in tumor tissue of ovarian cancer patients was analyzed by Western blot (n = 138) and immunohistochemistry (n = 58). Further, Ang-2 levels in blood samples were quantified in enzyme-linked immunosorbent assay (n = 38). Expression levels of different tumor spread patterns were evaluated, and survival analyses were made. We observed that Ang-2 expression is significantly higher in tumors with retroperitoneal dissemination (pT1a-pT3b, pN1) compared to those showing intraperitoneal tumor growth (pT3c, pN0). In addition, patients with high Ang-2 expression have significantly longer overall survival compared to patients with low Ang-2 expression. Patients with high Ang-2 expression benefit significantly from therapy with bevacizumab. All in all, Ang-2 may serve as a molecular marker for patients with tumors prone to spread to lymph nodes and for patients who might benefit from bevacizumab therapy.

Attenuation of Sialylation Augments Antitumor Immunity and Improves Response to Immunotherapy in Ovarian Cancer.

Blocking sialylation augments antitumor immunity and enhances response to immune checkpoint blockade therapy, highlighting a potential therapeutic approach for treating patients with high-grade serous ovarian cancer.

Abstract 2363: Ovarian tumor-intrinsic FAK-dependent regulation of the matrisome

Abstract High-grade serous ovarian cancer (HGSOC) is deadliest of gynecological malignancies. Ovarian tumor cells can directly spread throughout the peritoneal space as single cells that grow into tumorspheres concomitant with the generation of malignant ascites that contains growth factors and extracellular matrix (ECM) proteins. Greater than 70% of advanced stage HGSOC patients exhibit gains or amplifications for the Focal Adhesion Kinase (FAK) gene, a tyrosine kinase that is activated by ECM proteins. In other solid tumors, changes in ECM and associated proteins (termed the Matrisome) are generated in part by tumor associated fibroblasts. Herein we compared transcriptional (RNA sequencing) and proteomic (mass spectrometry) changes using two ascites generating models: MOVCAR, a SV40 T antigen under control of the Mullerian inhibitory substance type II receptor; and KMF, a spontaneously aggressive ID8 derivative containing KRas, Myc, and FAK gene amplifications. FAK inactivation was generated by crossing MOVCAR with FAK floxed mice or CRISPR to disrupt FAK expression in KMF. Comparisons were made with FAK-null or cells re-expressing FAK wildtype or a FAK kinase-inactive point mutant (K454R) by lentiviral transduction with tumor cells grown as spheroids in vitro or harvested after intraperitoneal tumor growth in mice. FAK expression and activity were required for optimal tumor growth in mice and a common set of differentially expressed genes were identified upon FAK loss and re-expression. Gene ontology analyses revealed significant differences in Matrisome, morphogenesis, and epithelial proliferation within spheroids and tumors lacking FAK. Parallel protein mass spectrometry analyses revealed that thirty-four Matrisome proteins were down regulated (> log2 fold change) including collagens -1, -3 and -5, fibulin-1 and -2, elastin, tenascin, and fibronectin. Several of these proteins were regulated by FAK expression and not necessarily intrinsic FAK activity. Bioinformatic analysis of the TCGA ovarian tumor database showed that FAK, collagen-3 and collagen-5 expression negatively correlate with patient survival. As FAK is highly tyrosine phosphorylated in tumor spheroids isolated from patients and from our mouse models, our results support the hypothesis that elevated tumor-intrinsic FAK expression results in increased ECM production that can feed-forward to drive increased FAK activity. These results may impact the clinical testing of small molecule inhibitors of FAK activity. Citation Format: Marjaana Ojalill, Joanna Zhang, Ujjwal Suwal, Pekka Rappu, Jyrki Heino, Denise C. Connolly, Dwayne G. Stupack, David D. Schlaepfer. Ovarian tumor-intrinsic FAK-dependent regulation of the matrisome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2363.

Supramolecular Loading of DNA Hydrogels with Dye-Drug Conjugates for Real-Time Photoacoustic Monitoring of Chemotherapy.

A longstanding problem with conventional cancer therapy is the nonspecific distribution of chemotherapeutics. Monitoring drug release in vivo via noninvasive bioimaging can thus have value, but it is difficult to distinguish loaded from released drug in live tissue. Here, this work describes an injectable supramolecular hydrogel that allows slow and trackable release of doxorubicin (Dox) via photoacoustic (PA) tomography. Doxis covalently linked with photoacoustic methylene blue (MB) to monitor Doxbefore, during, and after release from the hydrogel carrier. The conjugate (MB-Dox) possesses an IC50 of 161.4× 10-9 m against human ovarian carcinoma (SKOV3) cells and loads into a DNA-clad hydrogel with 91.3% loading efficiency due to MB-Dox's inherent intramolecular affinity to DNA. The hydrogel is biodegradable by nuclease digestion, which causes gradual release of MB-Dox. This release rate is tunable based on the wt% of the hydrogel. This hydrogel maintains distinct PA contrast on the order of days when injected in vivo and demonstrates activatable PA spectral shifts during hydrogel degradation. The released and loaded payload can be imaged relative to live tissue via PA and ultrasound signal being overlaid in real-time. The hydrogel slowed the rate of the murine intraperitoneal tumor growth 72.2% more than free Dox.

Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids

Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed invitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing invivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.

Altered intraperitoneal immune microenvironment in patients with peritoneal metastases from gastric cancer.

BackgroundThe peritoneal cavity contains many site-specific immune cells which constitute a unique immune microenvironment. However, it is unclear how the local immune signature is altered in patients with peritoneal metastases (PM).MethodsPeritoneal lavage fluid or ascites were obtained from 122 patients with various stages of gastric cancer (GC). Cells recovered from peritoneal fluids were immunostained with mAbs for lymphocyte-, macrophage- and tumor cell-specific antigens and the frequencies of leukocyte subsets and antigen expression levels were evaluated with multi-color flowcytometry.ResultsThe proportions of CD8(+) T cells, CD3(+)CD56(+) NKT-like cells, and CD3(-)CD56(+) NK cells to CD45(+) leukocytes were significantly reduced in patients with PM compared to those without PM. In patients with PM, the rates of CD8 (+) T cells and NKT-like cells correlated inversely with the tumor leukocyte ratio (TLR), the relative frequency of CD326(+) tumor cells to CD45(+) leukocytes. In contrast, the proportion of CD19(+) B cells was significantly increased in patients with PM, and their proportion correlated positively with the TLR and peritoneal carcinomatosis index (PCI) score. In patients with PM, CD14(+) macrophages tended to be increased with enhanced expression of CD14, CD16 and a M2-macrophage marker, CD163. In particular, macrophages in patients with high TLR contained many granules with high side scatter and CD14 expression in their flow profile compared to those without PM.ConclusionPM are accompanied by a drastic change in phenotypes of lymphocyte and macrophage in the peritoneal cavity, which might be involved in the development and progression of intraperitoneal tumor growth.

Use of Transabdominal Ultrasound for the detection of intra-peritoneal tumor engraftment and growth in mouse xenografts of epithelial ovarian cancer.

To evaluate intraperitoneal (IP) tumor engraftment, metastasis and growth in a pre-clinical murine epithelial ovarian cancer (EOC) model using both transabdominal ultrasound (TAUS) and bioluminescence in vivo imaging system (IVIS). Ten female C57Bl/6J mice at six weeks of age were included in this study. Five mice underwent IP injection of 5x106 ID8-luc cells (+ D- luciferin) and the remaining five mice underwent IP injection of ID8-VEGF cells. Monitoring of tumor growth and ascites was performed weekly starting at seven days post-injection until study endpoint. ID8-luc mice were monitored using both TAUS and IVIS, and ID8-VEGF mice underwent TAUS monitoring only. Individual tumor implant dimension and total tumor volume were calculated. Average luminescent intensity was calculated and reported per mouse abdomen. Tumor detection was confirmed by gross evaluation and histopathology. All data are presented as mean +/- standard deviation. Overall, tumors were successfully detected in all ten mice using TAUS and IVIS, and tumor detection correlated with terminal endpoint histology/ H&E staining. For TAUS, the smallest confirmed tumor measurements were at seven days post-injection with mean long axis of 2.23mm and mean tumor volume of 4.17mm3. However, IVIS imaging was able to detect tumor growth at 14 days post-injection. Ascites formation was detected in mice at 21 days post-injection. TAUS is highly discriminatory for monitoring EOC in pre-clinical murine model, allowing for detection of tumor dimension as small as 2 mm and as early as seven days post-injection compared to IVIS. In addition, TAUS provides relevant information for ascites development and detection of multiple small metastatic tumor implants. TAUS provides an accurate and reliable method to detect and monitor IP EOC growth in mouse xenografts.

Metformin Prevents Peritoneal Dissemination via Immune-suppressive Cells in the Tumor Microenvironment.

Metformin, a drug for type 2 diabetes, also exerts anticancer effects. This study addressed the immunological effects of metformin on peritoneal dissemination. We developed a mouse model of peritoneal dissemination via intraperitoneal injection of RLmale1, an X-ray-induced leukemia cell line, into BALB/c mice. Cell-surface markers, cytokine production, and myeloid-derived suppressor cells (MDSCs) were examined in cells from spleen and peritoneal lavage fluid. Metformin-treated mice exhibited suppressed intraperitoneal tumor growth and extended survival, and these effects were lost in mice with severe combined immunodeficiency. MDSCs induction was inhibited in metformin-treated mice. Although MDSC mobilization into the peritoneal cavity was correlated with suppression of interferon-γ production by tumor-infiltrating lymphocytes, the T-helper 1 ability of these lymphocytes was preserved in metformin-treated mice. Our findings demonstrate the action of metformin on both intraperitoneal tumors and immune-suppressive cells and might contribute to the development of immunotherapy against peritoneal dissemination.

Immune Effects of M51R Vesicular Stomatitis Virus Treatment of Carcinomatosis From Colon Cancer

BackgroundThe purpose of this study was to analyze the oncolytic and immunomodulatory functions of an M protein mutant of vesicular stomatitis virus (M51R VSV) in a murine model of peritoneal surface dissemination from colon cancer (PSD from CRC). MethodsLuciferase-expressing CT26 peritoneal tumors were established in Balb/c mice to evaluate the impact of M51R VSV treatment on intraperitoneal tumor growth and overall survival. The mice were treated with either intraperitoneal phosphate buffered saline (n = 10) or 5 × 106 PFU M51R VSV (n = 10) at 5 d after tumor implantation. Tumor bioluminescence was measured every 3 d during the 60-day study period. The immunomodulatory effect of M51R VSV treatment was evaluated in mice treated with either intraperitoneal phosphate buffered saline (n = 21) or M51R VSV (n = 21). Peritoneal lavages were collected at days 1, 3, and 7 after M51R VSV treatment for flow cytometry and multiplex cytokine bead analysis. ResultsA single, intraperitoneal treatment with M51R VSV inhibited the growth of PSD from CRC as evidenced by decreased bioluminescence and improved survival. This treatment approach also resulted in significantly higher frequencies of peritoneal CD4+ T (10.95 ± 1.17 versus 6.19 ± 0.44, P = 0.004) and B1b cells (5.01 ± 0.97 versus 2.20 ± 0.2, P = 0.024). On the other hand, treatment with M51R VSV resulted in fewer myeloid-derived suppressor cells relative to controls (10.66 ± 1.48 versus 14.47 ± 1.06, P = 0.035). M51R-treated peritoneal cavities also contained lower concentrations of immunosuppressive monocyte chemoattractant protein-1 and interleukin 6 cytokines relative to controls. ConclusionsOur findings suggest that M51R VSV alters the innate and adaptive immune responses in PSD from CRC. Future studies will delineate specific components of antitumor immunity that result in its therapeutic effect.

Oncolytic Maraba virus armed with tumor antigen boosts vaccine priming and reveals diverse therapeutic response patterns when combined with checkpoint blockade in ovarian cancer

BackgroundCancer immunotherapies are emerging as promising treatment strategies for ovarian cancer patients that experience disease relapse following first line therapy. As such, identifying strategies to bolster anti-tumor immunity and limit immune suppression, while recognizing diverse patterns of tumor response to immunotherapy is critical to selecting treatment combinations that lead to durable therapeutic benefit.MethodsUsing a pre-clinical mouse model, we evaluated a heterologous prime/boost vaccine in combination with checkpoint blockade to treat metastatic intraperitoneal ovarian cancer. Vaccine-elicited CD8+ T cell responses and changes in the tumor microenvironment following treatment were analyzed and compared to treatment outcome. Kinetics of intraperitoneal tumor growth were assessed using non-invasive magnetic resonance imaging (MRI).ResultsVaccine priming followed by antigen-armed oncolytic Maraba virus boosting elicited robust tumor-specific CD8+ T cell responses that improved tumor control and led to unique immunological changes in the tumor, including a signature that correlated with improved clinical outcome of ovarian cancer patients. However, this treatment was not curative and T cells in the tumor microenvironment (TME) were functionally suppressed. Combination PD-1 blockade partially overcame the adaptive resistance in the tumor observed in response to prime/boost vaccination, restoring CD8+ T cell function in the TME and enhancing the therapeutic response. Non-invasive MRI of tumors during the course of combination treatment revealed heterogeneous radiologic response patterns following treatment, including pseudo-progression, which was associated with improved tumor control prior to relapse.ConclusionsOur findings point to a key hierarchical role for PD-1 signaling and adaptive immune resistance in the ovarian TME in determining the functional fate of tumor-specific CD8+ T cells, even in the context of robust therapy mediated anti-tumor immunity, as well as the ability of multiple unique patterns of therapeutic response to result in durable tumor control.

Abstract 3801: Reversing chemoresistance in high grade serous ovarian cancer

Abstract Background: High grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype. Although it is initially responsive to platinum-containing chemotherapy, the emergence of chemo-resistant cells is inevitable. Most women with HGSOC die of platinum-resistant cancer, and the five-year overall survival for ovarian cancer has changed little in recent decades. The aim of this project is to identify and validate existing, licensed drugs that selectively target resistant cancer cells in combination with cisplatin or carboplatin. Methods: Established chemo-sensitive HGSOC cell lines Ovcar4, COV318 and OVSAHO were passaged in vitro with increasing doses of either Cisplatin or Carboplatin to achieve a 5-10 fold difference in IC50 in resistant compared to sensitive parental cells, reflecting the chemo-resistance commonly found in patient tumor samples. We screened a library of 1170 FDA-approved drugs in Ovcar4 and carboplatin-resistant Ovcar4 (Ov4Carbo) cell lines and viability was analyzed using Cell Titre Glow (CTG). Ovcar4 or Ov4Carbo cells were injected and intraperitoneal tumor growth was assessed in female CD1 nude mice as in vivo model to assess the effects of drug combinations. Results: Using a high throughput drug screen, we identified Chloroxine as one of the top hits that re-sensitized resistant Ov4Carbo cells to Carboplatin. We have further validated Chloroxine in the platinum sensitive/resistant cell pairs in our novel HGSOC cell line panel (Ovcar4/Ov4Cis; Ov4CarboRFP; Ov4Carbo single cell clone 7 and COV318/Cov318Cis). Previous literature has identified kappa-type opioid receptor, OPRK1 as a potential binding target for Chloroxine. We found that the OPRK1 agonist, U50488 reproduced the synergy with Carboplatin in resistant Ov4Carbo cells, as observed with Chloroxine. Additionally, OPRK1 antagonist, nor- Binaltorphimine (nor-BNI) reduced the combined effects of Chloroxine and Carboplatin in Ov4Carbo cells suggesting that Chloroxine acts via OPRK1 receptor. We have tested Ovcar4, Ov4Carbo and Ov4Cis in vivo and found that all formed tumors in the peritoneum of nude mice and sequential bioluminescent readings correlated with survival. We are currently testing the effects of Chloroxine or OPRK1 agonist, U50488 in combination with Carboplatin in our in-vivo models. Conclusion: We have identified Chloroxine, an FDA-approved clinical drug that combines synergistically with carboplatin or cisplatin to induce cell death in chemo-resistant cell lines. We expect that understanding the mechanism of this synergy will provide novel insights into the pathophysiology of chemotherapy resistance in high grade serous ovarian cancer. In addition, as the pharmacokinetics and toxicity profiles of these FDA-approved drugs have already been established, this ‘repurposing’ of commonly used drugs will allow for a faster bench-to-bedside translation. Citation Format: Jayeta Saxena, Francesco Nicolini, Michelle Lockley. Reversing chemoresistance in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3801.

Abstract A54: Generation and characterization of a novel panel of platinum-resistant HGSOC models

Abstract Background: High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype. Although it is initially responsive to platinum-containing chemotherapy, the emergence of chemoresistant cells is inevitable. Most women with HGSOC die of platinum-resistant cancer, and the five-year survival for ovarian cancer has changed little in recent decades. There is therefore a need to develop chemotherapy-resistant preclinical models to discover novel therapies for women with these resistant cancers. Methods: Established chemo-sensitive HGSOC cell lines Ovcar4, COV318 and OVSAHO were passaged in vitro with increasing doses of either cisplatin or carboplatin. Doses of carboplatin and cisplatin were incremented by 0.1μM when cells reached confluence. Resistance was assessed by clonogenic assay. To reflect the extent of chemoresistance commonly found in patient tumor samples, dose escalation was stopped once a 5-10 fold difference in IC50 was achieved in resistant compared to sensitive parental cells. Cells were then grown in parallel in the presence or absence of chemotherapy drug to determine whether resistance was maintained. Resistant cell lines were characterized based on molecular markers, cell growth, migration, and spheroid formation. Cells were lentivirally transfected with fluorescent proteins and Firefly luciferase. Intraperitoneal tumor growth was assessed in female CD1 nude mice. Survival was compared and tumors were sectioned and analyzed using H&E staining and IHC for p53, PAX8, and Ki67 expression. Results: All six platinum-resistant cell lines achieved a 5-10 fold increase in resistance based on IC50 values. Maintenance of resistance when drug was withdrawn varied between different cell lines. In vitro proliferation, migration, and spheroid formation varied between matched sensitive and resistant cells. The carboplatin-resistant cells, Ov4Carbo and OVSAHOCarbo, proliferated more rapidly compared to the parental cells from which they were derived (Ovcar4 and OVSAHO) and compared to their cisplatin-resistant counterparts (Ov4Cis and OVSAHOCis). Carboplatin-resistant Ovcar4 cells (Ov4Carbo) also formed spheroids more effectively and displayed greater in vitro migration than Ovcar4 and Ov4Cis. We have tested Ovcar4, Ov4Carbo, and Ov4Cis in vivo and found that all formed tumors in the peritoneum of nude mice. Sequential bioluminescent readings correlated with survival. In keeping with our in vitro findings, these three cells lines exhibited different behavior in vivo, with Ov4Carbo being the most aggressive. Median survival was 6 weeks (Ov4Carbo), 17.4 weeks (Ovcar4), and 20 weeks (Ov4Cis). At necropsy, Ov4Carbo cells were found to be locally invasive and produced large volumes of hemorrhagic ascites, while Ov4Cis metastasized exclusively to lymph nodes without production of ascites or local invasion. H&E staining and IHC confirmed HGSOC histology in all 3 cell lines. Conclusion: We have generated and characterized six new platinum-resistant HGSOC cancer cell lines and demonstrated that they display a range of phenotypes in vitro and in vivo. This novel cell panel will provide a diverse resource to investigate chemotherapy resistance and is a new platform for the development of novel therapies targeting chemoresistant HGSOC cancers. Citation Format: Joseph I. Hoare, Jayeta Saxena, Helen Hockings, Jackie McDermott, Ashley Browne, Michelle Lockley. Generation and characterization of a novel panel of platinum-resistant HGSOC models. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A54.

Crosstalk Between Apoptosis and Autophagy Is Regulated by the Arginylated BiP/Beclin-1/p62 Complex.

Emerging evidence demonstrates that autophagy and apoptosis are interconnected and their interplay greatly affects cell death. However, the key regulators in this crosstalk remain elusive. Therefore, the role of N-terminal arginylated BiP (R-BiP)/Beclin-1/p62 complex was examined in the crosstalk between apoptosis and autophagy during combination chemotherapy with mitomycin C and bortezomib using immunoblot, immunoprecipitation, and cellular imaging assays in wild-type (WT) and genetically engineered colorectal cancer cells. In addition, the tumoricidal efficacy of the combinatorial treatment in a nude mouse tumor xenograft model of colorectal cancer was assessed. Bortezomib combined with mitomycin C synergistically induced cytotoxicity and apoptosis rather than autophagy. Mechanistically, this combination inactivated Akt and subsequently induced Beclin-1 (BECN1) dephosphorylation at Ser 234/295. Dephosphorylation of Beclin-1 resulted in increased cleavage of Beclin-1 and disruption of the R-BiP/Beclin-1/p62 complex, which led to switching autophagy to the synergistic induction of apoptosis. Importantly, the combination significantly suppressed LS174T intraperitoneal xenograft tumor growth, induced Akt inactivation and Beclin-1 cleavage, and decreased autophagy in vivo Moreover, the tumoricidal efficacy of the combinatorial treatment was less effective, in vitro and in vivo, in HCT116 tumors harboring a Beclin-1 caspase 8 cleavage site mutant knock-in.Implications: This study uncovers that the R-BiP/Beclin-1/p62 complex has an important role in the crosstalk between apoptosis and autophagy. The results also propose how mono-drug resistance can be overcome using potent combinations to improve anticancer therapy. Mol Cancer Res; 16(7); 1077-91. ©2018 AACR.

Effects of nivolumab in peritoneal carcinamatosis of malign melanoma in mouse model

To evaluate the efficacy of nivolumab and comparison with dacarbazine (DTIC) on peritoneal carcinomatosis of malignant melanoma in mouse model. Mouse skin melanoma cells was injected under the capsule of the peritoneal surface in the left side of the abdomen. On postoperative day ten, mouses randomised into three groups. Group 1: Control, Group 2: HIPEC (Hyperthermic intraperitoneal chemotherapy) with DTIC and Group 3: HIPEC with Nivolumab. After the sacrification on postoperative day fifteen, peritoneum evaluated macroscopically and histopathologically by using peritoneal regression grading score (PRGS). In the 15th day exploration, all animals developed extensive intraperitoneal tumor growth in Group 1. In Group 2 and Group 3 median tumor size was 0.7±0.3cm and 0.3±0.2cm respectively (p: 0.023). Peritoneal carcinomatosis index (PCI) were significantly lower in Group 3 than other groups (p: 0.019). The lowest total tumor nodules in group 3 was 4 ± 2. The PGRS score was found significantly lower in Group 3 than other groups (p: 0.03). Lymphocytic response rate was found higher in the Group 3. It has been found that nivolumab significantly better than DTIC on peritoneal metastases of malign melanoma in mouse models. Nivolumab treatment gives promising results with pathological evidence in the treatment of metastatic disease of malignant melanoma.

MicroRNA-18a inhibits ovarian cancer growth via directly targeting TRIAP1 and IPMK.

The role of microRNA-18a (miRNA/miR-18a) as a tumor suppressor or promoter in a number of different types of cancer has been reported. However, to date, the expression and the effects of miR-18a in epithelial ovarian cancer (EOC) remain elusive. In the present study, the expression of miR-18a in patient EOC tissues and ovarian cancer cell lines was investigated using the reverse transcription-quantitative polymerase chain reaction. Luciferase assays and western blotting were performed to detect the potential direct targets of miR-18a. An A2780cp intraperitoneal mouse model, and Cell Counting Kit 8, flow cytometry and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assays, were used to investigate the effect of miR-18a on tumor growth in vivo and in vitro. The results indicated that the expression of miR-18a was reduced in EOC tissue and in the investigated ovarian cancer cell lines compared with non-malignant (normal) ovarian tissues and the human ovarian epithelium cell line, respectively. Overexpression of miR-18a in the A2780s and A2780cp cell lines significantly induced cell cycle arrest and apoptosis. It was demonstrated that miR-18a directly targets tumor protein p53-regulating inhibitor of apoptosis gene 1 and inositol phosphate multikinase, hence regulating the expression of downstream targets. The A2780cp intraperitoneal mouse model was employed and the results indicated that miR-18a may inhibit A2780cp intraperitoneal tumor growth in vivo by inhibiting proliferation and inducing apoptosis. Together, the results of the present study demonstrated that miR-18a has a role as a tumor suppressor by inhibiting proliferation and inducing apoptosis. Assessment of miR-18a expression may provide a novel method for diagnosis and be a therapeutic target for EOC.

Abstract 2301: Intraperitoneal CAR-T infusion results in durable protection against peritoneal metastases and control of systemic tumor growth

Abstract Colorectal carcinomatosis due to peritoneal metastases (PM) is a significant cause of morbidity and mortality. Present treatments fail to cure the majority of patients with PM. We are developing a chimeric antigen receptor T cell (CAR-T) regional infusion immunotherapy platform for PM. We treated mice with established CEA+ MC38 PMs with anti-CEA CAR-Ts either via regional intraperitoneal (IP) or systemic tail vein (TV) infusion. When compared to TV infusion, IP delivery resulted in significantly improved responses (37-fold reduction in tumor growth, p<0.01). To measure the durability of protection from IP tumor growth, we re-challenged mice with IP tumor following successful treatment with IP CAR-Ts. Mice who had received initial IP CAR-T treatment were protected from tumor growth following re-challenge for 20 days compared to control animals (1390-fold reduction in tumor growth, p = 0.016). Assessment of IP tumor 10 and 28 days following tumor re-challenge revealed a high proportion of intratumoral CAR-Ts (Day 10- 70.0%, Day 28- 46.6%; p = 0.10) and the proportion of CAR-Ts with an effector memory phenotype (CD44+CD62L-CCR7-, Day 10- 19.3%, Day 28- 60.0%; p<0.01). In separate experiments, we studied the effect of IP CAR-T infusions on tumor growth at distant sites. We established both IP and subcutaneous (SQ) flank tumors in mice prior to CAR-T infusions. Following treatment, IP CAR-T infusion resulted in a significant decrease in SQ tumor growth on day 8 compared to control (5.7-fold, p = 0.03), and a trend toward improvement compared to TV (4.9-fold, p = 0.224). While CAR-Ts were not detected in whole blood, SQ tumor, or draining lymph nodes following IP CAR-T infusion, serum IFNγ levels were significantly elevated on day 4 compared to control mice (p = 0.04), and returned to baseline by day 7. Our findings demonstrate that regional IP CAR-T delivery provides durable protection against IP tumor growth in association with CAR-T effector memory programming, in addition to a systemic anti-tumor effect. Citation Format: Marissa Cunetta, Gary Point, Zhi Liu, Prajna Guha, Josephine Darpolor, Matthew Lima, N J. Espat, Nader Hanna, Cherif Boutros, Richard P. Junghans, Steven Katz. Intraperitoneal CAR-T infusion results in durable protection against peritoneal metastases and control of systemic tumor growth. [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 2301.

Pharmacological focal adhesion kinase (FAK) inhibition suppresses cisplatin-resistant human ovarian carcinoma growth

Objectives: FAK is a cytoplasmic protein tyrosine kinase promoting ovarian cancer tumor growth and metastasis. Phase I and II clinical trials are testing small molecule inhibitors to FAK in different cancers. Determination of cancer cell susceptibility to FAK inhibition is an important yet undefined parameter. We test the hypothesis that elevated FAK activity contributes to the growth of aggressive cisplatin (CP)–resistant ovarian cancer. Methods: Growth of paired CP-sensitive (A2780 and OVCAR10) and CP-resistant (A2780-CP and OVCAR10-CP) human ovarian cells was evaluated in adherent, nonadherent, and methylcellulose spheroid culture in the presence or absence of 100 nM concentration of FAK inhibitor (FAKi). Quantification of cancer stemlike cells (CSCs) and cell cycle analyses was performed using flow cytometry for aldehyde dehydrogenase (ALDH) activity and propidium iodide DNA staining, respectively. Cell apoptosis was evaluated with Annexin-V and (7-aminoactinomycin D) 7-AAD staining. Changes in cellular protein expression were determined with quantitative immunoblotting. Intraperitoneal tumor growth in mice was evaluated for combined inhibitory effects of CP and FAKi administration. Results: FAK tyrosine phosphorylation and CSC protein markers were elevated in CP-resistant cells, which was reversed by FAKi addition. On immunoblotting, the levels of the transcription factor OCT4, ALDH, and the cell surface protein N-cadherin were increased in CP-resistant cells. FAKi addition selectively prevented CP-resistant cell growth as spheroids. The combination of FAKi with CP exhibited a combined effect on the CP-resistant cells, with significant decreases in spheroid and tumor growth compared with either agent alone. When comparing the percentage of CSCs with flow cytometry, FAKi addition dramatically reduced the ALDH-positive cell population in both parental and CP-resistant cells. This was associated with a G1 cell cycle growth phase arrest, decreased cyclin D protein expression, but not increased cell apoptosis. (See Fig. 1.) Conclusions: CSCs are implicated in the molecular mechanisms of cisplatin-resistance, and we find that FAK inhibition prevents CP-resistant CSC growth. Because FAKi therapy is well tolerated with limited adverse events, our results support further testing of FAKi in either primary or recurrent settings of CP-resistant ovarian cancer.

Hypoxia Promotes Synergy between Mitomycin C and Bortezomib through a Coordinated Process of Bcl-xL Phosphorylation and Mitochondrial Translocation of p53.

Colorectal peritoneal carcinomatosis (CPC) exhibits severe tumor hypoxia, leading to drug resistance and disease aggressiveness. This study demonstrates that the combination of the chemotherapeutic agent mitomycin C with the proteasome inhibitor bortezomib induced synergistic cytotoxicity and apoptosis, which was even more effective under hypoxia in colorectal cancer cells. The combination of mitomycin C and bortezomib at sublethal doses induced activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase and resulted in Bcl-xL phosphorylation at Serine 62, leading to dissociation of Bcl-xL from proapoptotic Bak. Interestingly, the intracellular level of p53 became elevated and p53 translocated to the mitochondria during the combinatorial treatment, in particular under hypoxia. The coordinated action of Bcl-xL phosphorylation and p53 translocation to the mitochondria resulted in conformational activation of Bak oligomerization, facilitating cytochrome c release and apoptosis induction. In addition, the combinatorial treatment with mitomycin C and bortezomib significantly inhibited intraperitoneal tumor growth in LS174T cells and increased apoptosis, especially under hypoxic conditions in vivo. This study provides a preclinical rationale for the use of combination therapies for CPC patients. The combination of a chemotherapy agent and proteasome inhibitor at sublethal doses induced synergistic apoptosis, in particular under hypoxia, in vitro and in vivo through coordinated action of Bcl-xL and p53 on Bak activation.

Effect of CO2 pneumoperitoneum on the proliferation of human ovarian cancer cell line SKOV-3 and the expression of NM23-H1 and MMP-2.

This study aims to investigate the impacts of CO2 pneumoperitoneum on the growth of ovarian cancer in nude mice and the expression of tumor metastasis suppressor gene (NM23-H1) and matrix metalloproteinase -2 (MMP-2) in SKOV-3 ovarian cancer cell line cancer tissue. Forty five nude mice were used to establish ovarian cancer xenograft models by intraperitoneal injection of human ovarian cancer cell line SKOV-3. Murine xenograft models were divided into four groups: control group (only anesthetized for 0.5 h), laparotomy group (laparotomy for 0.5 h), CO2 pneumoperitoneum of 0.5 h, and CO2 pneumoperitoneum of 1 h group. Mice were killed after 12 weeks to observe intraperitoneal tumor growth and detected mRNA expression of NM23-H1 and MMP-2 in tumor tissues by RT-PCR. Our data show that xenograft tumors grew faster in the CO2 pneumoperitoneum groups than that in control and laparotomy groups and even faster in the CO2 pneumoperitoneum of 1 h group. The mRNA expression of NM23-H1 in CO2 pneumoperitoneum groups was significantly lower than that in control group and laparotomy group (P < 0.01). Moreover, the longer duration of CO2 pneumoperitoneum negatively correlated with lower expression of NM23-H1 (P < 0.01). In contrast to NM23-H1, MMP-2 expression was significantly higher in CO2 pneumoperitoneum groups than that in the control group and laparotomy group (P < 0.01) and positively correlated with the duration of CO2 pneumoperitoneum (P < 0.01). In addition, there was a negative correlation between the expression of NM23-H1 and MMP-2 (r = -0.984, P < 0.05). The CO2 pneumoperitoneum could promote the proliferation and metastasis of human ovarian cancer in nude mice. This effect was positively correlated with the duration of CO2 pneumoperitoneum.