Ronald J. Buckanovich

Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A.

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of β-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse β-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and β-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow–derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that β-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: β-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.

Endothelin B receptor mediates the endothelial barrier to T cell homing to tumors and disables immune therapy.

In spite of their having sufficient immunogenicity, tumor vaccines remain largely ineffective. The mechanisms underlying this lack of efficacy are still unclear. Here we report a previously undescribed mechanism by which the tumor endothelium prevents T cell homing and hinders tumor immunotherapy. Transcriptional profiling of microdissected tumor endothelial cells from human ovarian cancers revealed genes associated with the absence or presence of tumor-infiltrating lymphocytes (TILs). Overexpression of the endothelin B receptor (ETBR) was associated with the absence of TILs and short patient survival time. The ETBR inhibitor BQ-788 increased T cell adhesion to human endothelium in vitro, an effect countered by intercellular adhesion molecule-1 (ICAM-1) blockade or treatment with NO donors. In mice, ETBR neutralization by BQ-788 increased T cell homing to tumors; this homing required ICAM-1 and enabled tumor response to otherwise ineffective immunotherapy in vivo without changes in systemic antitumor immune response. These findings highlight a molecular mechanism with the potential to be pharmacologically manipulated to enhance the efficacy of tumor immunotherapy in humans.

Aldehyde Dehydrogenase in Combination with CD133 Defines Angiogenic Ovarian Cancer Stem Cells That Portend Poor Patient Survival

Markers that reliably identify cancer stem cells (CSC) in ovarian cancer could assist prognosis and improve strategies for therapy. CD133 is a reported marker of ovarian CSC. Aldehyde dehydrogenase (ALDH) activity is a reported CSC marker in several solid tumors, but it has not been studied in ovarian CSC. Here we report that dual positivity of CD133 and ALDH defines a compelling marker set in ovarian CSC. All human ovarian tumors and cell lines displayed ALDH activity. ALDH(+) cells isolated from ovarian cancer cell lines were chemoresistant and preferentially grew tumors, compared with ALDH(-) cells, validating ALDH as a marker of ovarian CSC in cell lines. Notably, as few as 1,000 ALDH(+) cells isolated directly from CD133(-) human ovarian tumors were sufficient to generate tumors in immunocompromised mice, whereas 50,000 ALDH(-) cells were unable to initiate tumors. Using ALDH in combination with CD133 to analyze ovarian cancer cell lines, we observed even greater growth in the ALDH(+)CD133(+) cells compared with ALDH(+)CD133(-) cells, suggesting a further enrichment of ovarian CSC in ALDH(+)CD133(+) cells. Strikingly, as few as 11 ALDH(+)CD133(+) cells isolated directly from human tumors were sufficient to initiate tumors in mice. Like other CSC, ovarian CSC exhibited increased angiogenic capacity compared with bulk tumor cells. Finally, the presence of ALDH(+)CD133(+) cells in debulked primary tumor specimens correlated with reduced disease-free and overall survival in ovarian cancer patients. Taken together, our findings define ALDH and CD133 as a functionally significant set of markers to identify ovarian CSCs.

Combination cediranib and olaparib versus olaparib alone for women with recurrent platinum-sensitive ovarian cancer: a randomised phase 2 study

BACKGROUND Olaparib is a poly(ADP-ribose) polymerase inhibitor and cediranib is an anti-angiogenic agent with activity against VEGF receptor (VEGFR) 1, VEGFR2, and VEGFR3. Both oral agents have antitumour activity in women with recurrent ovarian cancer, and their combination was active and had manageable toxicities in a phase 1 trial. We investigated whether this combination could improve progression-free survival (PFS) compared with olaparib monotherapy in women with recurrent platinum-sensitive ovarian cancer. METHODS In our randomised, open-label, phase 2 study, we recruited women (aged ≥18 years) who had measurable platinum-sensitive, relapsed, high-grade serous or endometrioid ovarian, fallopian tube, or primary peritoneal cancer, or those with deleterious germline BRCA1/2 mutations from nine participating US academic medical centres. We randomly allocated participants (1:1) according to permuted blocks, stratified by germline BRCA status and previous anti-angiogenic therapy, to receive olaparib capsules 400 mg twice daily or the combination at the recommended phase 2 dose of cediranib 30 mg daily and olaparib capsules 200 mg twice daily. The primary endpoint was progression-free survival analysed in the intention-to-treat population. The phase 2 trial is no longer accruing patients. An interim analysis was conducted in November, 2013, after 50% of expected events had occurred and efficacy results were unmasked. The primary analysis was performed on March 31, 2014, after 47 events (66% of those expected). The trial is registered with ClinicalTrials.gov, number NCT01116648. FINDINGS Between Oct 26, 2011, and June 3, 2013, we randomly allocated 46 women to receive olaparib alone and 44 to receive the combination of olaparib and cediranib. Median PFS was 17·7 months (95% CI 14·7-not reached) for the women treated with cediranib plus olaparib compared with 9·0 months (95% CI 5·7-16·5) for those treated with olaparib monotherapy (hazard ratio 0·42, 95% CI 0·23-0·76; p=0·005). Grade 3 and 4 adverse events were more common with combination therapy than with monotherapy, including fatigue (12 patients in the cediranib plus olaparib group vs five patients in the olaparib monotherapy group), diarrhoea (ten vs none), and hypertension (18 vs none). INTERPRETATION Cediranib plus olaparib seems to improve PFS in women with recurrent platinum-sensitive high-grade serous or endometrioid ovarian cancer, and warrants study in a phase 3 trial. The side-effect profile suggests such investigations should include assessments of quality of life and patient-reported outcomes to understand the effects of a continuing oral regimen with that of intermittent chemotherapy. FUNDING American Recovery and Reinvestment Act grant from the National Institutes of Health (NIH) (3 U01 CA062490-16S2); Intramural Program of the Center for Cancer Research; and the Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH.

Human ovarian carcinoma–associated mesenchymal stem cells regulate cancer stem cells and tumorigenesis via altered BMP production

Accumulating evidence suggests that mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment; however, controversy exists regarding their role in solid tumors. In this study, we identified and confirmed the presence of carcinoma-associated MSCs (CA-MSCs) in the majority of human ovarian tumor samples that we analyzed. These CA-MSCs had a normal morphologic appearance, a normal karyotype, and were nontumorigenic. CA-MSCs were multipotent with capacity for differentiating into adipose, cartilage, and bone. When combined with tumor cells in vivo, CA-MSCs promoted tumor growth more effectively than did control MSCs. In vitro and in vivo studies suggested that CA-MSCs promoted tumor growth by increasing the number of cancer stem cells. Although CA-MSCs expressed traditional MSCs markers, they had an expression profile distinct from that of MSCs from healthy individuals, including increased expression of BMP2, BMP4, and BMP6. Importantly, BMP2 treatment in vitro mimicked the effects of CA-MSCs on cancer stem cells, while inhibiting BMP signaling in vitro and in vivo partly abrogated MSC-promoted tumor growth. Taken together, our data suggest that MSCs in the ovarian tumor microenvironment have an expression profile that promotes tumorigenesis and that BMP inhibition may be an effective therapeutic approach for ovarian cancer.

Tumor vascular proteins as biomarkers in ovarian cancer.

PURPOSE This study aimed to identify novel ovarian cancer biomarkers and potential therapeutic targets through molecular analysis of tumor vascular cells. METHODS Immunohistochemistry-guided laser-capture microdissection and genome-wide transcriptional profiling were used to identify genes that were differentially expressed between vascular cells from human epithelial ovarian cancer and healthy ovaries. Tumor vascular markers (TVMs) were validated through quantitative real-time polymerase chain reaction (qRT-PCR) of immunopurified tumor endothelial cells, in situ hybridization, immunohistochemistry, and Western blot analysis. TVM expression in tumors and noncancerous tissues was assessed by qRT-PCR and was profiled using gene expression data. RESULTS We identified a tumor vascular cell profile of ovarian cancer that was distinct from the vascular profile of normal ovary and other tumors. We validated 12 novel ovarian TVMs. These were expressed by immunopurified tumor endothelial cells and localized to tumor vasculature. Select TVMs were found to be specifically expressed in ovarian cancer and were absent in all normal tissues tested, including female reproductive tissues with physiologic angiogenesis. Many ovarian TVMs were expressed by a variety of other solid tumors. Finally, overexpression of any one of three ovarian TVMs by vascular cells was associated with decreased disease-free interval (all P < .005). CONCLUSION We have identified for the first time the molecular profile of ovarian tumor vasculature. We demonstrate that TVMs may serve as potential biomarkers and molecular targets for ovarian cancer and a variety of other solid tumors.

Depletion of Dendritic Cells Delays Ovarian Cancer Progression by Boosting Antitumor Immunity

Dendritic cells (DC) and cytokines that expand myeloid progenitors are widely used to treat cancer. Here, we show that CD11c(+)DEC205(+) DCs coexpressing alpha-smooth muscle actin and VE-cadherin home to perivascular areas in the ovarian cancer microenvironment and are required for the maintenance of tumor vasculature. Consequently, depletion of DCs in mice bearing established ovarian cancer by targeting different specific markers significantly delays tumor growth and enhances the effect of standard chemotherapies. Tumor growth restriction was associated with vascular apoptosis after DC ablation followed by necrosis, which triggered an antitumor immunogenic boost. Our findings provide a mechanistic rationale for selectively eliminating tumor-associated leukocytes to promote antitumor immunity while impeding tumor vascularization and to develop more effective DC vaccines based on a better understanding of the tumor microenvironment.

Metformin targets ovarian cancer stem cells in vitro and in vivo.

PURPOSE Studies in non-gynecologic tumors indicate that metformin inhibits growth of cancer stem cells (CSC). Diabetic patients with ovarian cancer who are taking metformin have better outcomes than those not taking metformin. The purpose of this study was to directly address the impact of metformin on ovarian CSC. METHODS The impact of metformin on ovarian cancer cell line growth and viability was assessed with trypan blue staining. Aldehyde dehydrogenase (ALDH) expressing CSC were quantified using FACS®. Tumor sphere assays were performed to determine the impact of metformin on cell line and primary human ovarian tumor CSC growth in vitro. In vivo therapeutic efficacy and the anti-CSC effects of metformin were confirmed using both tumor cell lines and ALDH(+) CSC tumor xenografts. RESULTS Metformin significantly restricted the growth of ovarian cancer cell lines in vitro. This effect was additive with cisplatin. FACS analysis confirmed that metformin reduced ALDH(+) ovarian CSC. Consistent with this, metformin also inhibited the formation of CSC tumor spheres from both cell lines and patient tumors. In vivo, metformin significantly increased the ability of cisplatin to restrict whole tumor cell line xenografts. In addition, metformin significantly restricted the growth of ALDH(+) CSC xenografts. This was associated with a decrease in ALDH(+) CSC, cellular proliferation, and angiogenesis. CONCLUSIONS Metformin can restrict the growth and proliferation of ovarian cancer stem cells in vitro and in vivo. This was true in cell lines and in primary human CSC isolates. These results provide a rationale for using metformin to treat ovarian cancer patients.

Ovarian cancer stem cell markers: prognostic and therapeutic implications.

Cancer stem cells are rare chemotherapy resistant cells within a tumor which can serve to populate the bulk of a tumor with more differentiated daughter cells and potentially contribute to recurrent disease. Ovarian cancer is a disease for which at the time of initial treatment we can obtain complete clinical remission in the majority of patients. Unfortunately, most will relapse and succumb to their disease. This clinical course is in line with the cancer stem cell model. In the past 5 years a significant amount of work has been done to identify cells with characteristics of ovarian cancer stem cells. This review will focus specifically on the markers used to define human ovarian cancer stem cells, the prognostic implications of the expression of these cancer stem cell markers in patients primary tumors, and the potential of these cancer stem cell markers to serve as therapeutic targets.

F3-targeted cisplatin-hydrogel nanoparticles as an effective therapeutic that targets both murine and human ovarian tumor endothelial cells in vivo

Recent studies indicate that ovarian cancer may be highly responsive to antivascular therapeutics. We have developed an antivascular tumor therapeutic using the F3 peptide to target cisplatin-loaded nanoparticles (F3-Cis-Np) to tumor vessels. We show that although F3-Cis-Np bind with high specificity to both human ovarian tumor cells and tumor endothelial cells in vitro, they only show cytotoxic activity against the tumor endothelial cells. In vivo these nanoparticles bind primarily to tumor endothelial cells. Therapeutic studies in both flank and orthotopic i.p. murine ovarian tumor models, as well as human tumor xenograft models, show rapid tumor regression with treatment. Treatment was associated with significant vascular necrosis consistent with an antivascular effect. Furthermore, treatment was active in both platinum-sensitive and platinum-resistant cell lines. Importantly, we show that F3-Cis-Np bind to human tumor endothelial cells in vitro and to human tumor vessels in vivo. Therapy targeting human vasculature in vivo with F3-Cis-Np led to near complete loss of all human tumor vessels in a murine model of human tumor vasculature. Our studies indicate that F3-targeted vascular therapeutics may be an effective treatment modality in human ovarian cancer.