Harnessing microRNA-enriched extracellular vesicles for liquid biopsy.

Extracellular microRNAs (miRNAs) can be detected in body fluids and hold great potential as cancer biomarkers. Extracellular miRNAs are protected from degradation by binding various proteins and through their packaging into extracellular vesicles (EVs). There is evidence that the diagnostic performance of cancer-associated extracellular miRNAs can be improved by assaying EV-miRNA instead of total cell-free miRNA, but several challenges have hampered the advancement of EV-miRNA in liquid biopsy. Because almost all types of cells release EVs, cancer cell-derived EVs might constitute only a minor fraction of EVs in body fluids of cancer patients with low volume disease. Furthermore, a given cell type can release several subpopulations of EVs that vary in their cargo, and there is evidence that the majority of EVs contain low copy numbers of miRNAs. In this mini-review, we discuss the potential of several candidate EV membrane proteins such as CD147 to define cancer cell-derived EVs, and approaches by which subpopulations of miRNA-rich EVs in body fluids might be identified. By integrating these insights, we discuss strategies by which EVs that are both cancer cell-derived and miRNA-rich could be isolated to enhance the diagnostic performance of extracellular miRNAs.

Revisiting the Use of Normal Saline for Peritoneal Washing in Ovarian Cancer.

The omentum is the predominant site of ovarian cancer metastasis, but it is difficult to remove the omentum in its entirety. There is a critical need for effective approaches that minimize the risk of colonization of preserved omental tissues by occult cancer cells. Normal saline (0.9% sodium chloride) is commonly used to wash the peritoneal cavity during ovarian cancer surgery. The omentum has a prodigious ability to absorb fluid in the peritoneal cavity, but the impact of normal saline on the omentum is poorly understood. In this review article, we discuss why normal saline is not a biocompatible solution, drawing insights from clinical investigations of normal saline in fluid resuscitation and from the cytopathologic evaluation of peritoneal washings. We integrate these insights with the unique biology of the omentum and omental metastasis, highlighting the importance of considering the absorptive ability of the omentum when administering agents into the peritoneal cavity. Furthermore, we describe insights from preclinical studies regarding the mechanisms by which normal saline might render the omentum conducive for colonization by cancer cells. Importantly, we discuss the possibility that the risk of colonization of preserved omental tissues might be minimized by using balanced crystalloid solutions for peritoneal washing.

Normal saline remodels the omentum and stimulates its receptivity for transcoelomic metastasis.

The omentum contains immune cell structures called milky spots that are niches for transcoelomic metastasis. It is difficult to remove the omentum completely, and there are no effective strategies to minimize the risk of colonization of preserved omental tissues by cancer cells that circulate in the peritoneal fluid. Normal saline is commonly administered into the peritoneal cavity for diagnostic and intraoperative lavage. Here we show that normal saline, when administered into the peritoneal cavity of mice, is prominently absorbed by the omentum, exfoliates its mesothelium, and induces expression of CX3CL1, the ligand for CX3CR1, within and surrounding the omental vasculature. Studies using CX3CR1-competent and CX3CR1-deficient mice showed that the predominant response in the omentum following saline administration is an accumulation of CX3CR1+ monocytes/macrophages that expand milky spots and promote neoangiogenesis within these niches. Moreover, saline administration promoted the implantation of cancer cells of ovarian and colorectal origin onto the omentum. By contrast, these deleterious effects were not observed following i.p. administration of lactated Ringer's solution. Our findings suggest that normal saline stimulates the receptivity of the omentum for cancer cells and that the risk of colonization can be minimized by using a biocompatible crystalloid for lavage procedures.

The glycoprotein CD147 defines miRNA-enriched extracellular vesicles that derive from cancer cells.

Extracellular vesicles (EVs) are ideal for liquid biopsy, but distinguishing cancer cell-derived EVs and subpopulations of biomarker-containing EVs in body fluids has been challenging. Here, we identified that the glycoproteins CD147 and CD98 define subpopulations of EVs that are distinct from classical tetraspanin+ EVs in their biogenesis. Notably, we identified that CD147+ EVs have substantially higher microRNA (miRNA) content than tetraspanin+ EVs and are selectively enriched in miRNA through the interaction of CD147 with heterogeneous nuclear ribonucleoprotein A2/B1. Studies using mouse xenograft models showed that CD147+ EVs predominantly derive from cancer cells, whereas the majority of tetraspanin+ EVs are not of cancer cell origin. Circulating CD147+ EVs, but not tetraspanin+ EVs, were significantly increased in prevalence in patients with ovarian and renal cancers as compared to healthy individuals and patients with benign conditions. Furthermore, we found that isolating miRNAs from body fluids by CD147 immunocapture increases the sensitivity of detecting cancer cell-specific miRNAs, and that circulating miRNAs isolated by CD147 immunocapture more closely reflect the tumor miRNA signature than circulating miRNAs isolated by conventional methods. Collectively, our findings reveal that CD147 defines miRNA-enriched, cancer cell-derived EVs, and that CD147 immunocapture could be an effective approach to isolate cancer-derived miRNAs for liquid biopsy.

Extracellular Vesicle Membrane-Associated Proteins: Emerging Roles in Tumor Angiogenesis and Anti-Angiogenesis Therapy Resistance.

The tumor vasculature is essential for tumor growth and metastasis, and is a prime target of several anti-cancer agents. Increasing evidence indicates that tumor angiogenesis is stimulated by extracellular vesicles (EVs) that are secreted or shed by cancer cells. These EVs encapsulate a variety of biomolecules with angiogenic properties, and have been largely thought to stimulate vessel formation by transferring this luminal cargo into endothelial cells. However, recent studies have revealed that EVs can also signal to recipient cells via proteins on the vesicular surface. This review discusses and integrates emerging insights into the diverse mechanisms by which proteins associate with the EV membrane, the biological functions of EV membrane-associated proteins in tumor angiogenesis, and the clinical significance of these proteins in anti-angiogenic therapy.

Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake.

Cancer-derived small extracellular vesicles (sEVs) induce stromal cells to become permissive for tumor growth. However, it is unclear whether this induction solely occurs through transfer of vesicular cargo into recipient cells. Here we show that cancer-derived sEVs can stimulate endothelial cell migration and tube formation independently of uptake. These responses were mediated by the 189 amino acid isoform of vascular endothelial growth factor (VEGF) on the surface of sEVs. Unlike other common VEGF isoforms, VEGF189 preferentially localized to sEVs through its high affinity for heparin. Interaction of VEGF189 with the surface of sEVs profoundly increased ligand half-life and reduced its recognition by the therapeutic VEGF antibody bevacizumab. sEV-associated VEGF (sEV-VEGF) stimulated tumor xenograft growth but was not neutralized by bevacizumab. Furthermore, high levels of sEV-VEGF were associated with disease progression in bevacizumab-treated cancer patients, raising the possibility that resistance to bevacizumab might stem in part from elevated levels of sEV-VEGF.

Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum.

Ovarian cancer preferentially metastasizes to the omentum, a fatty tissue characterized by immune structures called milky spots, but the cellular dynamics that direct this tropism are unknown. Here, we identified that neutrophil influx into the omentum is a prerequisite premetastatic step in orthotopic ovarian cancer models. Ovarian tumor-derived inflammatory factors stimulated neutrophils to mobilize and extrude chromatin webs called neutrophil extracellular traps (NETs). NETs were detected in the omentum of ovarian tumor-bearing mice before metastasis and of women with early-stage ovarian cancer. NETs, in turn, bound ovarian cancer cells and promoted metastasis. Omental metastasis was decreased in mice with neutrophil-specific deficiency of peptidylarginine deiminase 4 (PAD4), an enzyme that is essential for NET formation. Blockade of NET formation using a PAD4 pharmacologic inhibitor also decreased omental colonization. Our findings implicate NET formation in rendering the premetastatic omental niche conducive for implantation of ovarian cancer cells and raise the possibility that blockade of NET formation prevents omental metastasis.

The homeobox gene DLX4 regulates erythro-megakaryocytic differentiation by stimulating IL-1ß and NF-κB signaling.

Megakaryocyte and erythroid development are tightly controlled by a repertoire of cytokines, but it is not clear how cytokine-activated signaling pathways are controlled during development of these two lineages. Here, we identify that expression of DLX4, a transcription factor encoded by a homeobox gene, increases during megakaryopoiesis but decreases during erythropoiesis. Enforced expression of DLX4 in CD34(+) stem and progenitor cells and in bipotent K562 cells induced lineage markers and morphologic features of megakaryocytes and repressed erythroid marker expression and hemoglobin levels. Converse results were obtained when DLX4 was knocked down. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression revealed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. DLX4 also induced gene signatures that are associated with nuclear factor κB (NF-κB) signaling. The ability of DLX4 to promote megakaryocyte development at the expense of erythroid generation was diminished by blocking NF-κB activity or by repressing IL1B, a transcriptional target of DLX4. Collectively, our findings indicate that DLX4 exerts opposing effects on the megakaryocytic and erythroid lineages in part by inducing IL-1ß and NF-κB signaling.

The homeoprotein DLX4 stimulates NF-κB activation and CD44-mediated tumor-mesothelial cell interactions in ovarian cancer.

Ovarian cancers often highly express inflammatory cytokines and form implants throughout the peritoneal cavity. However, the mechanisms that drive inflammatory signaling and peritoneal metastasis of ovarian cancer are poorly understood. We previously identified that high expression of DLX4, a transcription factor encoded by a homeobox gene, is associated with reduced survival of ovarian cancer patients. In this study, we identified that DLX4 stimulates attachment of ovarian tumor cells to peritoneal mesothelial cells in vitro and increases the numbers of peritoneal implants in xenograft models. DLX4 induced expression of the cell surface molecule CD44 in ovarian tumor cells, and inhibition of CD44 abrogated the ability of DLX4 to stimulate tumor-mesothelial cell interactions. The induction of CD44 by DLX4 was attributed to increased activity of NF-κB that was stimulated by the inflammatory cytokine IL-1ß, a transcriptional target of DLX4. The stimulatory effects of DLX4 on CD44 levels and tumor-mesothelial cell interactions were abrogated when IL-1ß or NF-κB was inhibited in tumor cells. Furthermore, DLX4 expression levels strongly correlated with NF-κB activation and disease stage in clinical specimens of ovarian cancer. Collectively, these findings indicate that DLX4 induces CD44 by stimulating IL-1ß-mediated NF-κB activity, thereby promoting peritoneal metastasis of ovarian cancer.

The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer.

BACKGROUND: Homeobox genes encode transcription factors that control patterning of virtually all organ systems including the vasculature. Tumor angiogenesis is stimulated by several homeobox genes that are overexpressed in tumor cells, but the mechanisms of these genes are poorly understood. In this study, we investigated the mechanisms by which DLX4, a homeobox gene that is associated with increased tumor microvessel density, stimulates ovarian tumor angiogenesis. METHODS: Expression of DLX4 and nitric oxide synthases was analyzed in publicly available transcriptional profiles of ovarian cancer clinical specimens. Levels of inducible nitric oxide synthase (iNOS) were evaluated by quantitative RT-PCR, flow cytometry and nitric oxide assays using ovarian cancer cell lines in which DLX4 was overexpressed or knocked down. Signal Transducer and Activator of Transcription 1 (STAT1) expression and activity were evaluated by luciferase reporter assays, immunofluorescence staining, Western blot and immunoprecipitation. Endothelial cell growth and tumor angiogenesis were evaluated in in vitro assays and xenograft models. RESULTS: We identified that DLX4 induces expression of iNOS, an enzyme that stimulates angiogenesis by generating nitric oxide. Analysis of datasets of two independent patient cohorts revealed that high DLX4 expression in ovarian cancer is strongly associated with elevated expression of iNOS but not of other nitric oxide synthases. Studies using STAT1-expressing and STAT1-deficient cells revealed that DLX4 interacts with STAT1 and induces iNOS expression in part by stimulating STAT1 activity. Expression of DLX4 in ovarian cancer cells stimulated endothelial cell growth in vitro and increased microvessel density in xenograft models, and these stimulatory effects of DLX4 were abrogated when its induction of iNOS was inhibited. CONCLUSION: These findings indicate that DLX4 promotes ovarian tumor angiogenesis in part by stimulating iNOS expression.

HOXA9 promotes homotypic and heterotypic cell interactions that facilitate ovarian cancer dissemination via its induction of P-cadherin.

BACKGROUND: Epithelial ovarian cancer (EOC) is a lethal disease that frequently involves the peritoneal cavity. Dissemination of EOC is a multi-step process in which exfoliated tumor cells survive in the peritoneal fluid as multi-cellular aggregates and then form invasive implants on peritoneal surfaces. The mechanisms that control this process are poorly understood. We previously identified that high expression of the developmental patterning gene HOXA9 is associated with poor survival in EOC patients. In this study, we investigated the significance and mechanisms of HOXA9 in controlling aggregation and implantation of floating EOC cells. METHODS: HOXA9 was inhibited by shRNAs or expressed in EOC cells that were propagated in suspension cultures and in the peritoneal cavity of mice. Cell death was assayed by flow cytometry and ELISA. Cell aggregation, attachment and migration were evaluated by microscopy, transwell chamber assays and histopathologic analysis. DNA-binding of HOXA9 and its effect on expression of the cell adhesion molecule P-cadherin were assayed by chromatin immunoprecipitation, quantitative RT-PCR and Western blot. HOXA9 and P-cadherin expression was evaluated in publicly available datasets of EOC clinical specimens. RESULTS: We identified that HOXA9 promotes aggregation and inhibits anoikis in floating EOC cells in vitro and in xenograft models. HOXA9 also stimulated the ability of EOC cells to attach to peritoneal cells and to migrate. HOXA9 bound the promoter of the CDH3 gene that encodes P-cadherin, induced CDH3 expression in EOC cells, and was associated with increased CDH3 expression in clinical specimens of EOC. Inhibiting P-cadherin in EOC cells that expressed HOXA9 abrogated the stimulatory effects of HOXA9 on cell aggregation, implantation and migration. Conversely, these stimulatory effects of HOXA9 were restored when P-cadherin was reconstituted in EOC cells in which HOXA9 was inhibited. CONCLUSION: These findings indicate that HOXA9 contributes to poor outcomes in EOC in part by promoting intraperitoneal dissemination via its induction of P-cadherin.

Therapeutic strategies for targeting the ovarian tumor stroma.

Epithelial ovarian cancer is the most lethal type of gynecologic malignancy. Sixty percent of women who are diagnosed with ovarian cancer present with advanced-stage disease that involves the peritoneal cavity and these patients have a 5-year survival rate of less than 30%. For more than two decades, tumor-debulking surgery followed by platinum-taxane combination chemotherapy has remained the conventional first-line treatment of ovarian cancer. Although the initial response rate is 70%-80%, most patients with advanced-stage ovarian cancer eventually relapse and succumb to recurrent chemoresistant disease. A number of molecular aberrations that drive tumor progression have been identified in ovarian cancer cells and intensive efforts have focused on developing therapeutic agents that target these aberrations. However, increasing evidence indicates that reciprocal interactions between tumor cells and various types of stromal cells also play important roles in driving ovarian tumor progression and that these stromal cells represent attractive therapeutic targets. Unlike tumor cells, stromal cells within the tumor microenvironment are in general genetically stable and are therefore less likely to become resistant to therapy. This concise review discusses the biological significance of the cross-talk between ovarian cancer cells and three major types of stromal cells (endothelial cells, fibroblasts, macrophages) and the development of new-generation therapies that target the ovarian tumor microenvironment.

Heterotypic cellular interactions in the ovarian tumor microenvironment: biological significance and therapeutic implications.

The majority of women who are diagnosed with epithelial ovarian cancer present with extensive peritoneal carcinomatosis and are rarely cured by conventional chemotherapy. Ovarian cancer cells typically disseminate by shedding into the peritoneal fluid and implant on the mesothelium-lined peritoneal surfaces that overlie connective and white adipose tissues. Emerging evidence indicates that ovarian tumor progression is orchestrated by dynamic interplay between tumor cells and a variety of stromal cells such as adipocytes, endothelial cells, fibroblasts, mesenchymal stem cells, macrophages, and other immune cells. This mini-review discusses the biological significance of the heterotypic cellular interactions in the ovarian tumor microenvironment and the therapeutic implications of targeting these interactions.

P-cadherin promotes ovarian cancer dissemination through tumor cell aggregation and tumor-peritoneum interactions.

UNLABELLED: More than 60% of patients who are diagnosed with epithelial ovarian cancer (EOC) present with extensive peritoneal carcinomatosis. EOC cells typically disseminate by shedding into the peritoneal fluid in which they survive as multicellular aggregates and then implant onto peritoneal surfaces. However, the mechanism that facilitates aggregation and implantation of EOC cells is poorly understood. The cell adhesion molecule P-cadherin has been reported to be induced during early progression of EOC and to promote tumor cell migration. In this study, P-cadherin not only promoted migration of EOC cells, but also facilitated the assembly of floating EOC cells into multicellular aggregates and inhibited anoikis in vitro. Furthermore, inhibiting P-cadherin by short hairpin RNAs (shRNA) or a neutralizing antibody prevented EOC cells from attaching to peritoneal mesothelial cells in vitro. In mouse intraperitoneal xenograft models of EOC, inhibition of P-cadherin decreased the aggregation and survival of floating tumor cells in ascites and reduced the number of tumor implants on peritoneal surfaces. These findings indicate that P-cadherin promotes intraperitoneal dissemination of EOC by facilitating tumor cell aggregation and tumor-peritoneum interactions in addition to promoting tumor cell migration. IMPLICATIONS: Inhibiting P-cadherin blocks multiple key steps of EOC progression and has therapeutic potential.

Adaptation of ovarian cancer cells to the peritoneal environment: Multiple mechanisms of the developmental patterning gene HOXA9.

The lethality of ovarian cancer stems from its propensity to involve the peritoneal cavity. However, the mechanisms that enable ovarian cancer cells to readily adapt to the peritoneal environment are not well understood. Here, we describe our recent studies in which we identified the mechanisms by which the transcription factor encoded by the patterning gene HOXA9 promotes the aggressive behavior of ovarian cancer. Firstly, we identified that HOXA9 promotes ovarian tumor growth and angiogenesis by activating the gene encoding transforming growth factor-ß2 (TGF-ß2), which in turn stimulates peritoneal fibroblasts and mesenchymal stem cells to acquire features of cancer-associated fibroblasts. Secondly, by inducing TGF-ß2 and chemokine (C-C motif) ligand 2, HOXA9 stimulates peritoneal macrophages to acquire an immunosuppressive phenotype. Thirdly, HOXA9 stimulates attachment of ovarian cancer cells to peritoneal mesothelial cells by inducing expression of P-cadherin. By inducing P-cadherin, HOXA9 also enables floating cancer cells in the peritoneal cavity to form aggregates and escape anoikis. Together, our studies demonstrate that HOXA9 enables ovarian cancer cells to adapt to the peritoneal environment and 'educates' different types of stromal cells to become permissive for tumor growth. Our studies provide new insights into the regulation of tumor-stroma interactions in ovarian cancer and implicate several key effector molecules as candidate therapeutic targets.

Expression of the homeobox gene HOXA9 in ovarian cancer induces peritoneal macrophages to acquire an M2 tumor-promoting phenotype.

Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-ß2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8(+) tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages.

Homeobox Gene Deregulation: Impact on the Hallmarks of Cancer.

Homeobox genes comprise a super-family of evolutionarily conserved genes that play essential roles in controlling body plan specification and cell fate determination. Substantial evidence indicates that leukemogenesis is driven by abnormal expression of homeobox genes that control hematopoiesis. In solid tumors, aberrant expression of homeobox genes has been increasingly found to modulate diverse processes such as cell proliferation, cell death, metastasis, angiogenesis and DNA repair. This review discusses how homeobox genes are deregulated in solid tumors and the functional significance of this deregulation in the hallmarks of cancer.

Dual functions of the homeoprotein DLX4 in modulating responsiveness of tumor cells to topoisomerase II-targeting drugs.

Topoisomerase II (TOP2)-targeting poisons such as anthracyclines and etoposide are commonly used for cancer chemotherapy and kill tumor cells by causing accumulation of DNA double-strand breaks (DSB). Several lines of evidence indicate that overexpression of TOP2A, the gene encoding topoisomerase IIα, increases sensitivity of tumor cells to TOP2 poisons, but it is not clear why some TOP2A-overexpressing (TOP2A-High) tumors respond poorly to these drugs. In this study, we identified that TOP2A expression is induced by DLX4, a homeoprotein that is overexpressed in breast and ovarian cancers. Analysis of breast cancer datasets revealed that TOP2A-high cases that also highly expressed DLX4 responded more poorly to anthracycline-based chemotherapy than TOP2A-high cases that expressed DLX4 at low levels. Overexpression of TOP2A alone in tumor cells increased the level of DSBs induced by TOP2 poisons. In contrast, DLX4 reduced the level of TOP2 poison-induced DSBs irrespective of its induction of TOP2A. DLX4 did not stimulate homologous recombination-mediated repair of DSBs. However, DLX4 interacted with Ku proteins, stimulated DNA-dependent protein kinase activity, and increased erroneous end-joining repair of DSBs. Whereas DLX4 did not reduce levels of TOP2 poison-induced DSBs in Ku-deficient cells, DLX4 stimulated DSB repair and reduced the level of TOP2 poison-induced DSBs when Ku was reconstituted in these cells. Our findings indicate that DLX4 induces TOP2A expression but reduces sensitivity of tumor cells to TOP2 poisons by stimulating Ku-dependent repair of DSBs. These opposing activities of DLX4 could explain why some TOP2A-overexpressing tumors are not highly sensitive to TOP2 poisons.

HOXA9 promotes ovarian cancer growth by stimulating cancer-associated fibroblasts.

Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-ß2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.