Shoumei Bai

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.

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.

Identifying an ovarian cancer cell hierarchy regulated by bone morphogenetic protein 2

Significance Significant controversy persists regarding a hierarchical vs. stochastic model of cancer. Using a microfluidic single-cell culture device, we define for the first time, to our knowledge, the differentiation capacity of primary human ovarian cancer cells. We demonstrate that ovarian cancer follows a hierarchical model with rare stochastic events. Defining the differentiation capacity allowed us to explain apparently paradoxical actions of bone morphologenetic protein 2 (BMP2); BMP2 suppresses growth in vitro by suppressing bulk cell proliferation, but promotes growth in vivo by promoting cancer stem-like cell (CSC) expansion. This work supports BMP2 signaling as a critical therapeutic target regulating ovarian CSC growth. Whether human cancer follows a hierarchical or stochastic model of differentiation is controversial. Furthermore, the factors that regulate cancer stem-like cell (CSC) differentiation potential are largely unknown. We used a novel microfluidic single-cell culture method to directly observe the differentiation capacity of four heterogeneous ovarian cancer cell populations defined by the expression of the CSC markers aldehyde dehydrogenase (ALDH) and CD133. We evaluated 3,692 progeny from 2,833 cells. We found that only ALDH+CD133+ cells could generate all four ALDH+/−CD133+/− cell populations and identified a clear branched differentiation hierarchy. We also observed a single putative stochastic event. Within the hierarchy of cells, bone morphologenetic protein 2 (BMP2) is preferentially expressed in ALDH−CD133− cells. BMP2 promotes ALDH+CD133+ cell expansion while suppressing the proliferation of ALDH−CD133− cells. As such, BMP2 suppressed bulk cancer cell growth in vitro but increased tumor initiation rates, tumor growth, and chemotherapy resistance in vivo whereas BMP2 knockdown reduced CSC numbers, in vivo growth, and chemoresistance. These data suggest a hierarchical differentiation pattern in which BMP2 acts as a feedback mechanism promoting ovarian CSC expansion and suppressing progenitor proliferation. These results explain why BMP2 suppresses growth in vitro and promotes growth in vivo. Together, our results support BMP2 as a therapeutic target in ovarian cancer.

Endothelin receptor-A is required for the recruitment of antitumor T cells and modulates chemotherapy induction of cancer stem cells

Background: The endothelin receptor-A (ETRA) plays an important role in tumor cell migration, metastasis, and proliferation. The endothelin receptor B (ETRB) plays a critical role in angiogenesis and the inhibition of anti-tumor immune cell recruitment. Thus dual blockade of ETRA and ETRB could have significant anti-tumor effects. Results: Dual ETRA/ETRB blockade with macitentan (or the combination of the ETRA and ETRB antagonists BQ123 and BQ788) did not enhance antitumor immune cell recruitment. In vitro studies demonstrate that ETRA inhibition prevents the induction of ICAM1 necessary for immune cell recruitment. When used as a single agent against human tumor xenografts, macitentan demonstrated non-significant anti-tumor activity. However, when used in combination with chemotherapy, macitentan specifically reduced tumor growth in cell lines with CD133+ cancer stem cells. We found that ETRA is primarily expressed on CD133+ CSC in both cell lines and primary human tumor cells. ETRA inhibition of CSC prevented chemotherapy induced increases in tumor stem cells. Furthermore, ETRA inhibition in combination with chemotherapy reduced the formation of tumor spheres. Methods: We tested the dual ETRA/ETRB antagonist macitentan in conjunction with (1) an anti-tumor vaccine and (2) chemotherapy, in order to assess the impact of dual ETRA/ETRB blockade on anti-tumor immune cell infiltration and ovarian tumor growth. In vitro murine and human cell line, tumor sphere assays and tumor xenograft models were utilized to evaluate the effect of ETRA/ETRB blockade on cell proliferation, immune cell infiltration and cancer stem cell populations. Conclusions: These studies indicate a critical role for ETRA in the regulation of immune cell recruitment and in the CSC resistance to chemotherapy.

New models of hematogenous ovarian cancer metastasis demonstrate preferential spread to the ovary and a requirement for the ovary for abdominal dissemination

Emerging evidence suggest that many high-grade serous ovarian cancers (HGSOC) start in the fallopian tube. Cancer cells are then recruited to the ovary and then spread diffusely through the abdomen. The mechanism of ovarian cancer spread was thought to be largely due to direct shedding of tumor cells into the peritoneal cavity with vascular spread being of limited importance. Recent work challenges this dogma, suggesting hematogenous spread of ovarian cancer may play a larger role in ovarian cancer cell metastasis than previously thought. One reason the role of vascular spread of ovarian cancer has not been fully elucidated is the lack of easily accessible models of vascular ovarian cancer metastasis. Here, we present 3 metastatic models of ovarian cancer which confirm the ability of ovarian cancer to hematogenously spread. Strikingly, we observe a high rate of metastasis to the ovary with the development of ascites in these models. Interestingly, oophorectomy resulted in a complete loss of peritoneal metastases and ascites. Taken together, our data indicate that hematogenously disseminated HGSOC cells have a unique tropism for the ovary and that hematogenous spread in ovarian cancer may be more common than appreciated. Furthermore, our studies support a critical role for the ovary in promoting HGSOC cell metastasis to the abdomen. The models developed here represent important new tools to evaluate both the mechanism of cancer cell recruitment to the ovary and understand and target key steps in ovarian cancer metastasis.

A Novel Model for Evaluating Therapies Targeting Human Tumor Vasculature and Human Cancer Stem–like Cells

Human tumor vessels express tumor vascular markers (TVM), proteins that are not expressed in normal blood vessels. Antibodies targeting TVMs could act as potent therapeutics. Unfortunately, preclinical in vivo studies testing anti-human TVM therapies have been difficult to do due to a lack of in vivo models with confirmed expression of human TVMs. We therefore evaluated TVM expression in a human embryonic stem cell-derived teratoma (hESCT) tumor model previously shown to have human vessels. We now report that in the presence of tumor cells, hESCT tumor vessels express human TVMs. The addition of mouse embryonic fibroblasts and human tumor endothelial cells significantly increases the number of human tumor vessels. TVM induction is mostly tumor-type-specific with ovarian cancer cells inducing primarily ovarian TVMs, whereas breast cancer cells induce breast cancer specific TVMs. We show the use of this model to test an anti-human specific TVM immunotherapeutics; anti-human Thy1 TVM immunotherapy results in central tumor necrosis and a three-fold reduction in human tumor vascular density. Finally, we tested the ability of the hESCT model, with human tumor vascular niche, to enhance the engraftment rate of primary human ovarian cancer stem-like cells (CSC). ALDH(+) CSC from patients (n = 6) engrafted in hESCT within 4 to 12 weeks whereas none engrafted in the flank. ALDH(-) ovarian cancer cells showed no engraftment in the hESCT or flank (n = 3). Thus, this model represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology.

EGFL6 regulates the asymmetric division, maintenance, and metastasis of ALDH+ ovarian cancer cells

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH+ ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH+ ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH+ cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH+ cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.

Sampling from single-cell observations to predict tumor cell growth in-vitro and in-vivo

Cancer stem-like cells (CSCs) are a topic of increasing importance in cancer research, but are difficult to study due to their rarity and ability to rapidly divide to produce non-self-cells. We developed a simple model to describe transitions between aldehyde dehydrogenase (ALDH) positive CSCs and ALDH(-) bulk ovarian cancer cells. Microfluidics device-isolated single cell experiments demonstrated that ALDH+ cells were more proliferative than ALDH(-) cells. Based on our model we used ALDH+ and ALDH(-) cell division and proliferation properties to develop an empiric sampling algorithm and predict growth rate and CSC proportion for both ovarian cancer cell line and primary ovarian cancer cells, in-vitro and in-vivo. In both cell line and primary ovarian cancer cells, the algorithm predictions demonstrated a high correlation with observed ovarian cancer cell proliferation and CSC proportion. High correlation was maintained even in the presence of the EGF-like domain multiple 6 (EGFL6), a growth factor which changes ALDH+ cell asymmetric division rates and thereby tumor growth rates. Thus, based on sampling from the heterogeneity of in-vitro cell growth and division characteristics of a few hundred single cells, the simple algorithm described here provides rapid and inexpensive means to generate predictions that correlate with in-vivo tumor growth.

Abstract TMEM-019: NEW OVARIAN CANCER METASTASIS MODELS DEMONSTRATE PREFERENTIAL HEMATOGENOUS SPREAD OF OVARIAN CANCER CELLS TO THE OVARY AND A REQUIREMENT FOR THE OVARY FOR ABDOMINAL DISSEMINATION

BACKGROUND: Mounting evidence suggests that many high grade serous ‘ovarian’ cancers (HGSOC) start in the fallopian tube. Cancer cells are then recruited to the ovary and subsequently spread diffusely through the abdomen. The mechanism of ovarian cancer spread has long been thought to be largely due to direct shedding of tumor cells into the peritoneal cavity with vascular spread being of limited importance. Recent work challenges this dogma, suggesting hematogenous spread of ovarian cancer may play a larger role in ovarian cancer cell metastasis than previously thought. One reason the role of vascular spread of ovarian cancer has not been fully elucidated is the lack of easily accessible models of vascular ovarian cancer metastasis. METHODS: We developed three metastatic models of ovarian cancer which confirm the ability of ovarian cancer to hematogenously spread. A murine tail vein injection model using human ovarian cancer cell lines was used to observe the general pattern of hematogenous spread when cells are directly introduced into the vasculature. A subcutaneous murine ovarian tumor model was developed to investigate patterns of metastasis in the setting of a fully murine tumor microenvironment. Finally, a human subcutaneous xenograft model using primary patient derived ovarian cancer cells, human carcinoma-associated mesenchymal stem cells and human endothelial cells was developed to investigate patterns of metastasis in the setting of a humanized microenvironment. RESULTS: In all three models, we demonstrate the formation of distant metastasis via vascular spread. Strikingly, we observe a high rate of metastasis to the ovary (40-100%) in all three models representing a disproportionately large fraction of total metastatic burden. Mice which developed ovarian metastatic disease also developed further intra-abdominal metastatic disease and ascites. Interestingly, in the tail vein injection model, oophorectomy prior to ovarian cancer cell injection resulted in a complete loss of peritoneal metastases, ascites and decreased burden of liver metastasis. This ovary tropism appears to be unique to ovarian cancer cells as intravenous tail vein injection of breast cancer and lung cancer cell lines, while resulting in typical sites of disease in the lung and bone, did not result in ovary involvement. CONCLUSIONS: Taken together our data indicates that hematogenously disseminated high grade serous ovarian cancer cells have a unique tropism for the ovary and that hematogenous spread in ovarian cancer may be more common than previously appreciated. Furthermore our studies support a critical role for the ovary in promoting high grade serous ovarian cancer cell metastasis to the abdomen. The models developed here represent important new tools to evaluate both the mechanism of cancer cell recruitment to the ovary and to understand and target key steps in ovarian cancer metastasis. Citation Format: Lan G Coffman, Daniela Burgos-Ojeda, Rong Wu, Kathleen Cho, Shoumei Bai, and Ronald J Buckanovich. NEW OVARIAN CANCER METASTASIS MODELS DEMONSTRATE PREFERENTIAL HEMATOGENOUS SPREAD OF OVARIAN CANCER CELLS TO THE OVARY AND A REQUIREMENT FOR THE OVARY FOR ABDOMINAL DISSEMINATION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-019.

Abstract 3760: EGFL6 is a tumor vascular regulator of ovarian CSC asymmetric division and a novel therapeutic target.

Cancer stem cells (CSC) are closely associated with tumor vasculature in the CSC niche. Tumor vascular cells are known to provide critical growth, differentiation, and survival cues for CSC. Unfortunately little is known about the specific factors produced by vascular cells. EGFL6 is a factor specifically secreted by ovarian tumor vascular cells. EGFL6 is reported to regulate the differentiation of normal stem cells. We examined the impact of EGFL6 on ovarian cancer cell growth. We found that EGFL6 significantly promoted ovarian cancer cell growth in vitro. Ovarian cancer cell proliferation was associated with a decrease in the percentage of ALDH+ ovarian CSC. Using a novel single cell microfluidic culture system we found that EGFL6 acts specifically on ALDH+ CSC to promote proliferation via asymmetric division. We used a in vivo novel model of human tumor vasculature to express EGFL6 in vivo and demonstrate that EGFL6 significantly promotes ovarian tumor growth. Finally we generated an EGFL6 blocking antibody and found that anti-EGFL6 antibodies could restrict tumor growth and delay disease recurrences. Thus EGFL6 is an import vascular regulator of ovarian CSC and EGL6 blocking antibodies represent a potentially important ovarian CSC targeting therapeutic. Citation Format: Ronald J. Buckanovich, Shoumei Bai, Ning Deng, Patrick Ingram, Euisik Yoon. EGFL6 is a tumor vascular regulator of ovarian CSC asymmetric division and a novel therapeutic target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3760. doi:10.1158/1538-7445.AM2013-3760