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The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. To leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDXs were grown in humanized NBSGW (huNBSGW) mice engrafted with human CD34+ cord blood–derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and identification of infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for patients with ovarian cancer. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment, which may recommend these models for preclinical therapeutic trials.Significance:huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

Levels of human M-CSF detected in patient ascites fluid

Human cytokines with significantly higher levels in PDX ascites samples compared to plasma samples

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. To leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDXs were grown in humanized NBSGW (huNBSGW) mice engrafted with human CD34+ cord blood–derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and identification of infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for patients with ovarian cancer. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment, which may recommend these models for preclinical therapeutic trials.Significance:huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

Cell characterization of primary ovarian cancer cells by immunofluorescence.

Patient information

Legends for the supplementary figures

Method of immunofluorescence for the ovarian cancer cell identification

Biochemical properties of Cdc42 and Rac1 nucleotide binding

Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans. Mol Cancer Ther; 14(10); 2215–27. ©2015 AACR.

Native Americans are the least represented population in science fields. In recent years, undergraduate and graduate level summer research programs that aimed to increase the number of Native Americans in science have made some progress. As new programs are designed, key characteristics that address science self-efficacy and science identity and provide supports for Native American students’ commitment to a scientific career should be considered. In this study, we used sequential mixed methods to investigate the potential of culturally tailored internship programs on Native American persistence in science. We analyzed surveys (n = 47) and interviews (n = 4) with Native American students to understand their perceptions of themselves in relation to science research and how summer research experiences might develop science identities. Based on regression modeling, science identity, but not science self-efficacy, predicted intent to persist in science. In turn, science self-efficacy and Native American identity predicted science identity, and this suggests cultural identity is central to Native American persistence in science. In interviews, students’ comments reinforced these findings and shed light on students’ reasoning about the kinds of science experiences they sought; specifically, they chose to participate in culturally tailored internships because these programs provided a sense of belonging to the scientific community that did not conflict with their cultural identities. Based on our analysis, we propose an Indigenous science internship model and recommend that agencies target funding for culturally tailored programs from high school through early-investigator levels as well as provide inclusive programmatic and mentoring guidelines.

Supplemental Figure S3. Racemic distribution of clinical drug.

Purpose: We previously identified the R-enantiomer of ketorolac as an inhibitor of the Rho-family GTPases Rac1 and Cdc42. Rac1 and Cdc42 regulate cancer-relevant functions, including cytoskeleton remodeling necessary for tumor cell adhesion and migration. This study investigated whether administration of racemic (R,S) ketorolac after ovarian cancer surgery leads to peritoneal distribution of R-ketorolac, target GTPase inhibition in cells retrieved from the peritoneal cavity, and measureable impact on patient outcomes.Experimental Design: Eligible patients had suspected advanced-stage ovarian, fallopian tube or primary peritoneal cancer. Secondary eligibility was met when ovarian cancer was confirmed and optimally debulked, an intraperitoneal port was placed, and there were no contraindications for ketorolac administration. R- and S-ketorolac were measured in serum and peritoneal fluid, and GTPase activity was measured in peritoneal cells. A retrospective study correlated perioperative ketorolac and ovarian cancer–specific survival in ovarian cancer cases.Results: Elevated expression and activity of Rac1 and Cdc42 was detected in ovarian cancer patient tissues, confirming target relevance. Ketorolac in peritoneal fluids was enriched in the R-enantiomer and peritoneal cell GTPase activity was inhibited after ketorolac administration when R-ketorolac was at peak levels. After adjusting for age, AJCC stage, completion of chemotherapy, and neoadjuvant therapy, women given perioperative ketorolac had a lower hazard of death (HR, 0.30; 95% confidence interval, 0.11–0.88).Conclusions: Ketorolac has a novel pharmacologic activity conferred by the R-enantiomer and R-ketorolac achieves sufficient levels in the peritoneal cavity to inhibit Rac1 and Cdc42, potentially contributing to the observed survival benefit in women who received ketorolac. Clin Cancer Res; 21(22); 5064–72. ©2015 AACR.

Supplemental methods, Supplemental tables S1-S3, Supplemental Figure S1-S8 Legends. Supplemental methods for IHC of patient TMA, qPCR of patient cDNA, HPLC protocol, and GTPase activity of patient cells. Figure legends for supplemental figures S1-S8 are included. Supplemental table S1 - Patient Characteristics for IHC Microarrays OV1005 and OV8010. Supplemental table S2 - Patient Characteristics for cDNA Microarray. Supplemental table S3 - Ketorolac enantiomer concentration in serum or peritoneal fluids

Supplemental Figures S4-S7. Supplemental Figure S4 - Survival estimates based on Cox-regression for Stage I (AJCC) with completion of chemotherapy. Supplemental Figure S5 - Survival estimates based on Cox-regression for Stage II (AJCC) with completion of chemotherapy. Supplemental Figure S6 - Survival estimates based on Cox-regression for Stage III (AJCC) with completion of chemotherapy. Supplemental Figure S7 - Survival estimates based on Cox-regression for Stage IV (AJCC) with completion of chemotherapy.

Experimental and computational studies pinpoint rate-limiting step(s) in metastasis governed by Rac1. Using ovarian cancer cell and animal models, Rac1 expression was manipulated, and quantitative measurements of cell-cell and cell-substrate adhesion, cell invasion, mesothelial clearance, and peritoneal tumor growth discriminated the tumor behaviors most highly influenced by Rac1. The experimental data were used to parameterize an agent-based computational model simulating peritoneal niche colonization, intravasation, and hematogenous metastasis to distant organs. Increased ovarian cancer cell survival afforded by the more rapid adhesion and intravasation upon Rac1 overexpression is predicted to increase the numbers of and the rates at which tumor cells are disseminated to distant sites. Surprisingly, crowding of cancer cells along the blood vessel was found to decrease the numbers of cells reaching a distant niche irrespective of Rac1 overexpression or knockdown, suggesting that sites for tumor cell intravasation are rate limiting and become accessible if cells intravasate rapidly or are displaced due to diminished viability. Modeling predictions were confirmed through animal studies of Rac1-dependent metastasis to the lung. Collectively, the experimental and modeling approaches identify cell adhesion, rapid intravasation, and survival in the blood as parameters in the ovarian metastatic cascade that are most critically dependent on Rac1.

Despite substantial progress in identifying and understanding causative mutations in autosomal dominant polycystic kidney disease (ADPKD), little is known about subsequent cellular events leading to cyst formation. In prior studies we reported that Cadherin 8, a type II Cadherin, expression is sufficient to induce cyst emergence from HK-2 cells grown as tubule arrays in collagen matrix (1). However, emergent cysts did not exhibit the luminal enlargement observed in ADPKD. In this communication, we reconstitute cyst emergence with consequent cyst lumen expansion in 3D culture by stable co-expression of Cadherin 8 in combination with a constitutively active mutant of YAP1, the key effector of the HIPPO pathway. Specifically, immortalized cells derived from ADPKD cyst epithelia formed cysts with substantially larger lumen sizes when transduced with YAP1-5SA. Conversely, expression of the YAP1 inhibitor, AMOTL1, in these cells resulted in their forming cysts with smaller lumens than control cells. Our data show that cyst formation results from a sequential two-step process consisting of cyst initiation and subsequent cyst expansion. Taken together, cyst initiation induced by Cadherin 8 expression is proposed to result from decreased cell-cell adhesion while cyst expansion is driven by increased Yap1 activity.

American Indians (AIs) in New Mexico have lower cancer screening rates compared to other populations and are more likely to be diagnosed with cancer at an advanced stage of the disease as reported by Li et al. (Archives of Internal Medicine 163(1):49–56, 2003). AIs also have the lowest 5-year cancer survival rates compared to any ethnic/racial group in the USA as reported by Clegg et al. (Arch Intern Med 162:1985–1993, 2002) and Edwards et al. (Cancer 97:1407–1427, 2005). Numerous barriers such as cultural beliefs, fear, fatalism, mistrust, stigma, and lack of culturally appropriate interventions could contribute to low cancer screening rates as reported by Daley et al. (J Health Dispar Res Pract 5(2), 2012); Filippi et al. (J Prim Care Community Health 4(3):160–166, 2013); James et al. (Prev Chronic Dis 10:E170, 2013); and Schumacher et al. (Cancer Causes Control 19(7):725–737, 2008). Trained Community Health Representatives (CHRs) from the Zuni Pueblo and native Zuni undergraduate students led six 1-h focus group sessions using a structured focus group guide with probes. The focus groups were conducted among 51 participants from different age groups (20–29 years, n = 19; 30–49 years, n = 17; and 50 years and older, n = 15) stratified by sex. Focus groups were conducted in both English and Shiwi (Zuni) languages. Sessions were audio recorded, and team members took notes. CHRs transcribed the notes and audio recordings, and created a codebook for qualitative data analysis. In the focus groups, participants provided Zuni-specific cultural context, opinion, and experience regarding (1) general knowledge about cancer, (2) cancer risk, (3) cancer risk reduction, (4) personal experiences with cancer, and (5) culturally competent delivery of cancer information and resources. Understanding the perceptions of cancer within the Zuni Pueblo is an essential component in the development of interventional/preventative measures and improvement of current care. Ultimately, this information will provide a basis for the next steps in culturally sensitive cancer care for the Zuni Pueblo.

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells. Section 3.5 'Applications of G-Trap Assay' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells. Section 3.4 'Assay Runtime' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells. Section 3.3 'Molecular Assembly of GST Effector Proteins on Glutathione Beads' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells. Section 3.2 'Production of a Cleared Cell Lysate' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells. Section 3.1 'Synthesis of Glutathione Beads' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac–treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected in estrogen receptor, progesterone receptor, β-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

Aberrant Rac1 signaling contributes to cancer growth and metastasis, therefore, we investigated the effect of altering Rac1 expression and activity to pinpoint which metastatic processes and tumor microenvironment immune factors are influenced by Rac1 signaling. We have identified that the R- enantiomer of ketorolac, a drug given for pain management, is a Rac1 and Cdc42 selective inhibitor. In a retrospective study, we found that treatment with racemic ketorolac significantly increased the survival probability of ovarian cancer patients. RNA seq analyses of ascites tumor cell samples from ovarian cancer patients in a prospective study receiving racemic ketorolac for clinically indicated use in pain relief, showed significant downregulation of genes involved in endocytosis, regulation of actin cytoskeleton, ER protein processing, TNF and NOD signaling. To further distinguish the activities most directly regulated by Rac1 and important in ovarian cancer, we tested the impact of manipulating the expression of Rac1 in ovarian cancer cells on adhesive and invasive cell behaviors, as well as the impacts of gene expression relative to ketorolac treatment. Using electric cell-substrate impedance sensing (ECIS), Rac1 overexpression resulted in increased cell-cell barrier formation and cell adhesion. Whereas, CRISPR-CAS9 mediated knockdown of Rac1 resulted in decreased cell adhesion. Using Matrigel invasion chambers and mesothelial clearance assays, Rac1 overexpression led to increased ovarian cancer cell invasion when compared to parental and Rac1 knockdown cell lines. Furthermore, intraperitoneal growth of ovarian cancer cells overexpression Rac1 relative to parental control lines increased tumor burden in vivo. Conversely, with knockdown of Rac1, tumor burden and omental adhesion was decreased, as quantified using IVIS bioluminescence imaging. RNA seq analyses of tumors manipulated for Rac1 as compared to R-ketorolac will be presented. The composite data indicate that targeting Rac1 in ovarian cancer with R-ketorolac or otherwise has therapeutic benefit in decreasing tumor cell dissemination and metastasis. Citation Format: Melanie Rivera, Martha M. Grimes, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 as a driver and therapeutic target in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2898.

Cytoreductive surgery and chemotherapy are cornerstones of ovarian cancer treatment, yet disease recurrence remains a significant clinical issue. Surgery can release cancer cells into the circulation, suppress anti-tumor immunity, and induce inflammatory responses that support the growth of residual disease. Intervention within the peri-operative window is an under-explored opportunity to mitigate these consequences of surgery and influence the course of metastatic disease to improve patient outcomes. One drug associated with improved survival in cancer patients is ketorolac. Ketorolac is a chiral molecule administered as a 1:1 racemic mixture of the S- and R-enantiomers. The S-enantiomer is considered the active component for its FDA indication in pain management with selective activity against cyclooxygenase (COX) enzymes. The R-enantiomer has a previously unrecognized activity as an inhibitor of Rac1 (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42) GTPases. Therefore, ketorolac differs from other non-steroidal anti-inflammatory drugs (NSAIDs) by functioning as two distinct pharmacologic entities due to the independent actions of each enantiomer. In this review, we summarize evidence supporting the benefits of ketorolac administration for ovarian cancer patients. We also discuss how simultaneous inhibition of these two distinct classes of targets, COX enzymes and Rac1/Cdc42, by S-ketorolac and R-ketorolac respectively, could each contribute to anti-cancer activity.

The small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) is commonly overexpressed in cancer and high Rac1 levels are associated with poor patient outcomes in ovarian cancer. Rac1 drives multiple hallmarks of cancer such as proliferation, quiescence, epithelial-to-mesenchymal transition (EMT), and transcriptional programs relevant to tumor development, progression, and chemoresistance. EMT promotes metastatic dissemination, chemoresistance, and cancer stem cell properties. We find that modest overexpression of Rac1 in ovarian tumor cells causes morphologic changes consistent with EMT. Elevated Rac1 (4- fold) expression decreases colony number and increases colony area when compared to vector control cells. In addition, cell invasion through an artificial basement membrane is increased by 10-fold in cells overexpressing Rac1. An increase in mesenchymal markers (e.g., N-cadherin) and decrease in epithelial markers (e.g., E-cadherin) was observed as a consequence of Rac1 expression. Rac1 overexpression was associated with selective upregulation of Slug/Snai2 (8-fold) and Zeb2 (100-fold) mRNA levels with little impact on Snai1, Twist, or Zeb1. Slug/Snai2 expression is reportedly induced by oxidative stress and treatment of six ovarian tumor cell lines with tert-butyl hydroperoxide (TBHP) to generate oxidative stress increased the expression Slug/Snai2, Snai1, and heme oxygenase-1, an indicator of oxidative stress response. This finding confirms that elevated oxidative stress regulates certain EMT transcription factors in various ovarian cancer cells. Rac1 is an essential factor for activation of NADPH oxidases 1, 2, and 3 and generation of reactive oxygen species (ROS). Basal level of ROS was elevated in cells overexpressing Rac1 compared to vector control cells as detected by the fluorescent probe Dihydroethidium (DHE). Similarly, elevated Rac1 increased expression of the oxidative stress response genes heme oxygenase-1 (17-fold) and superoxide dismutase-1 (12-fold), further supporting that Rac1 stimulated ROS signaling in these cells. We further tested the impact of Rac1 inhibition in vivo using R-ketorolac. Analysis of tumor tissue from mice showed decreased expression of heme-oxygenase-1 and Slug/Snai2 in mice treated with R-ketorolac compared to placebo controls. R-ketorolac also decreased invasive implantation and expansion of ovarian cancer xenografts. Taken together, these findings illustrate the functional impact of Rac1 overexpression in ovarian cancer cells and suggest that Rac1 may be a viable target for further drug development to attenuate EMT in ovarian tumors. Citation Format: Martha M. Grimes, Dayna R. Dominquez, Michaela L. Granados, Alejandra Rosales, Melanie Rivera, Angela Wandinger-Ness, Laurie G. Hudson. Rac1 overexpression promotes epithelial-to-mesenchymal transition in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B59.

Rac1 is a high-value therapeutic target for cancer based on its tumor-promoting activities, yet clinical applications targeting Rac1 are in their infancy. High expression and hyperactivation of Rac1 in ovarian cancer, along with our identification of R-ketorolac as a novel Rac1 and Cdc42 selective inhibitor with translational potential, prompt us to test the hypothesis that targeting Rac1 has therapeutic utility for ovarian cancer. Ascites tumor cell samples from ovarian cancer patients in a prospective study receiving racemic ketorolac for clinically indicated use in pain relief were previously reported to show time-dependent reduction of Rac1 and Cdc42 activities post-treatment. New RNA seq data of these patient samples reveals significant downregulation of genes involved in endocytosis, regulation of actin cytoskeleton, ER protein processing, TNF and NOD signaling. Conversely, the identified downregulated genes were overexpressed and associated with worse survival in ovarian cancer patients analyzed through The Cancer Genome Atlas (TCGA). Among the downregulated genes in the NOD pathway are chemokines and proinflammatory cytokines. Follow-up cytokine panels from patients confirm that racemic ketorolac treatment reduces the levels of immunosuppressive cytokines IL-6, IL-10, and RANTES in ascites fluids. Together, these data indicate there may be a benefit to the anti-inflammatory activity of the S- enantiomer, as well as the GTPase inhibitory activity of the R-enantiomer of ketorolac for ovarian cancer treatment. Citation Format: Melanie Rivera, Martha M. Grimes, Dayna Dominguez, S. Ray Kenney, Yuna Guo, Elsa Romero, Kathryn J. Brayer, Yan Guo, Scott A. Ness, Sarah F. Adams, Carolyn Muller, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 as a therapeutic target in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B70.

New Mexico is one of the most geographically and culturally diverse states and faces serious cancer health disparities among its poor, rural, and ethnically diverse populations of American Indians (AIs). They have lower cancer screening rates compared to other populations and are more likely to be diagnosed with a later stage of cancer but less likely to receive treatment (Li et al, 2003). In turn, AIs have the lowest five-year cancer survival rates compared to any ethnic/racial group in the US (Clegg, 2002; Edwards, 2005). Numerous barriers such as cultural beliefs in health and health care, fear, fatalism, mistrust, stigma and lack of culturally appropriate interventions contribute to low cancer screening rates and low cancer survivorship (Daley et al, 2012; Filippi et al, 2013; James et al, 2013). Zuni Health Initiative conducted a community-based participatory research project in collaborations with community stakeholders from the Zuni Pueblo to assess knowledge and perceptions about common cancers among Zuni adults. We used trained Community Health Representatives (CHRs) from Zuni along with Zuni undergraduate students who led six one-hour focus group sessions using structured questionnaire with probes designed to illicit information on knowledge and perceptions about cancer among Zuni adults. The focus groups were conducted among 51 participants from different age groups (20-29 yrs, n=19; 30-49 yrs, n=17; and 50 yrs and older, n=15) stratified by gender. Focus groups were conducted in English and Shiwi language and were audio recorded, with the Zuni students maintaining extensive notes on the focus group discussions. The Zuni CHRs transcribed the audio recordings, and developed a codebook based on the transcriptions and notes for the qualitative data analysis. Overall, the responses given by participants fell into three distinct categories including, (1) a general lack of knowledge, (2) the perception that the disease is uncontrollable in nature, and (3) general negative connotations. Although some participants acknowledged that cancer could come in many forms, there were many participants who were uncertain about the different factors that contribute to the disease, as well as specific outcomes associated with the disease. Many participants mentioned cancer being uncontrollable in nature, that cancer always comes back, and expressed that death is the inevitable outcome. Finally, several participants displayed general uncertainty and discomfort when discussing cancer. There was negative connotations when participants heard the word “cancer”, as well as construed negative implications about discussing personal experiences with cancer. Findings from this formative study provides evidence that can guide the development and testing of interventions aimed at enhancing knowledge about cancer and cancer-specific screening practices. Citation Format: Safia Safi, Donica Ghahate, Jeanette Bobelu, Angela Wandinger-Ness, Thomas Faber, Shiraz Mishra, Cheryl Willman, Vallabh (Raj) Shah. Assessing knowledge and perceptions about cancer among American Indians of Zuni Pueblo, New Mexico [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B044.

Ras and Ras-related small GTPases are key regulators of diverse cellular functions that impact cell growth, survival, motility, morphogenesis, and differentiation. They are important targets for studies of disease mechanisms as well as drug discovery. Here, we report the characterization of small molecule agonists of one or more of six Rho, Rab, and Ras family GTPases that were first identified through flow cytometry-based, multiplexed high-throughput screening of 200000 compounds. The activators were categorized into three distinct chemical families that are represented by three lead compounds having the highest activity. Virtual screening predicted additional compounds with potential GTPase activating properties. Secondary dose-response assays performed on compounds identified through these screens confirmed agonist activity of 43 compounds. While the lead and second most active small molecules acted as pan activators of multiple GTPase subfamilies, others showed partial selectivity for Ras and Rab proteins. The compounds did not stimulate nucleotide exchange by guanine nucleotide exchange factors and did not protect against GAP-stimulated GTP hydrolysis. The activating properties were caused by a reversible stabilization of the GTP-bound state and prolonged effector protein interactions. Notably, these compounds were active both in vitro and in cell-based assays, and small molecule-mediated changes in Rho GTPase activities were directly coupled to measurable changes in cytoskeletal rearrangements that dictate cell morphology.

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

A FASEB Summer Research Conference entitled ‘Arf and Rab family G proteins’ was held in July 2013 at Snowmass Village, Snowmass, Colorado. Arfs and Rabs are two families of GTPases that control membrane trafficking in eukaryotic cells, and increasing evidence indicates that their functions are tightly coordinated. Because many workers in this field have focused on only one family, this meeting was designed to integrate our understanding of the two families. The conference was organized by Elizabeth Sztul (University of Alabama, Birmingham, USA) and Jim Casanova (University of Virginia, Charlottesville, USA), and provided an opportunity for ∼90 scientists to communicate their work and discuss future directions for the field. The talks highlighted the structural, functional and regulatory properties of Arf and Rab GTPases and the need to develop coordinated approaches to investigate them. Here, we present the major themes that emerged from the meeting.

Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases. Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays. Conclusion: The identified compound is a highly Cdc42-selective inhibitor. Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research., Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.