Differential in vivo biodistribution of 131I-labeled exosomes from diverse cellular origins and its implication for theranostic application.

Exosomes are critical mediators of intercellular crosstalk and are regulator of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosomes research has been thwarted by our limited knowledge of the most efficient isolation method and their in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 µm syringe filter and 100 k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes. We also demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.

Inhibition of Epithelial-Mesenchymal Transition and Metastasis by Combined TGFbeta Knockdown and Metformin Treatment in a Canine Mammary Cancer Xenograft Model.

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-ß) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-ß, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-ß1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-ß1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mice's lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-ß1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-ß1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-ß1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-ß1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-ß1 silencing and their effect on EMT.

Arachidonic Acid Metabolite as a Novel Therapeutic Target in Breast Cancer Metastasis.

Metastatic breast cancer (BC) (also referred to as stage IV) spreads beyond the breast to the bones, lungs, liver, or brain and is a major contributor to the deaths of cancer patients. Interestingly, metastasis is a result of stroma-coordinated hallmarks such as invasion and migration of the tumor cells from the primary niche, regrowth of the invading tumor cells in the distant organs, proliferation, vascularization, and immune suppression. Targeted therapies, when used as monotherapies or combination therapies, have shown limited success in decreasing the established metastatic growth and improving survival. Thus, novel therapeutic targets are warranted to improve the metastasis outcomes. We have been actively investigating the cytochrome P450 4 (CYP4) family of enzymes that can biosynthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an important signaling eicosanoid involved in the regulation of vascular tone and angiogenesis. We have shown that 20-HETE can activate several intracellular protein kinases, pro-inflammatory mediators, and chemokines in cancer. This review article is focused on understanding the role of the arachidonic acid metabolic pathway in BC metastasis with an emphasis on 20-HETE as a novel therapeutic target to decrease BC metastasis. We have discussed all the significant investigational mechanisms and put forward studies showing how 20-HETE can promote angiogenesis and metastasis, and how its inhibition could affect the metastatic niches. Potential adjuvant therapies targeting the tumor microenvironment showing anti-tumor properties against BC and its lung metastasis are discussed at the end. This review will highlight the importance of exploring tumor-inherent and stromal-inherent metabolic pathways in the development of novel therapeutics for treating BC metastasis.

Major Challenges and Potential Microenvironment-Targeted Therapies in Glioblastoma.

Glioblastoma (GBM) is considered one of the most malignant, genetically heterogeneous, and therapy-resistant solid tumor. Therapeutic options are limited in GBM and involve surgical resection followed by chemotherapy and/or radiotherapy. Adjuvant therapies, including antiangiogenic treatments (AATs) targeting the VEGF-VEGFR pathway, have witnessed enhanced infiltration of bone marrow-derived myeloid cells, causing therapy resistance and tumor relapse in clinics and in preclinical models of GBM. This review article is focused on gathering previous clinical and preclinical reports featuring major challenges and lessons in GBM. Potential combination therapies targeting the tumor microenvironment (TME) to overcome the myeloid cell-mediated resistance problem in GBM are discussed. Future directions are focused on the use of TME-directed therapies in combination with standard therapy in clinical trials, and the exploration of novel therapies and GBM models for preclinical studies. We believe this review will guide the future of GBM research and therapy.

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression.

The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of 'hot' spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

MicroRNA as a promising molecular biomarker in the diagnosis of breast cancer.

Introduction: Breast cancer represents the most prevalent malignancy among women. Recent advancements in translational research have focused on the identification of novel biomarkers capable of providing valuable insights into patient outcomes. Furthermore, comprehensive investigations aimed at discovering novel miRNAs, unraveling their biological functions, and deciphering their target genes have significantly contributed to our understanding of the roles miRNAs play in tumorigenesis. Consequently, these investigations have facilitated the way for the development of miRNA-based approaches for breast cancer prognosis, diagnosis, and treatment. However, conducting a more extensive array of studies, particularly among diverse ethnic groups, is imperative to expand the scope of research and validate the significance of miRNAs. This study aimed to assess the expression patterns of circulating miRNAs in plasma as a prospective biomarker for breast cancer patients within a population primarily consisting of individuals from Black, Indigenous, and People of Color (BIPOC) communities. Methods: We evaluated 49 patients with breast cancer compared to 44 healthy women. Results and discussion: All miRNAs analyzed in the plasma of patients with breast cancer were downregulated. ROC curve analysis of miR-21 (AUC = 0.798, 95% CI: 0.682-0.914, p <0.0001), miR-1 (AUC = 0.742, 95% CI: 0.576-0.909, p = 0.004), miR-16 (AUC = 0.721, 95% CI: 0.581-0.861, p = 0.002) and miR-195 (AUC = 0.672, 95% CI: 0.553-0.792, p = 0.004) showed better diagnostic accuracy in discrimination of breast cancer patients in comparison with healthy women. miR-210, miR-21 showed the highest specificities values (97.3%, 94.1%, respectively). Following, miR-10b and miR-195 showed the highest sensitivity values (89.3%, and 77.8%, respectively). The panel with a combination of four miRNAs (miR-195 + miR-210 + miR-21 + miR-16) had an AUC of 0.898 (0.765-0.970), a sensitivity of 71.4%, and a specificity of 100.0%. Collectively, our results highlight the miRNA combination in panels drastically improves the results and showed high accuracy for the diagnosis of breast cancer displaying good sensitivity and specificity.

Differential biodistribution of intravenously administered endothelial progenitor and cytotoxic T-cells in rat bearing orthotopic human glioma.

BACKGROUND: A major challenge in the development of cell based therapies for glioma is to deliver optimal number of cells (therapeutic dose) to the tumor. Imaging tools such as magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) has been used in cell tracking and/or biodistribution studies. In this study, we evaluate the dynamic biodistribution of systemic injected labeled cells [human cord blood derived endothelial progenitor cells (EPCs) and cytotoxic T-cells (CTLs)] in rat glioma model with in vivo SPECT imaging. METHODS: Human cord blood EPCs, T-cells and CD14⁺ cells (monocytes/dendritic cells) were isolated using the MidiMACS system. CD14⁺ cells were converted to dendritic cells (DC) and also primed with U251 tumor cell line lysate. T-cells were co-cultured with irradiated primed DCs at 10:1 ratio to make CTLs. Both EPCs and CTLs were labeled with In-111-oxine at 37°C in serum free DMEM media. Glioma bearing animals were randomly assigned into three groups. In-111 labeled cells or In-111 oxine alone were injected through tail vein and SPECT imaging was performed on day 0, 1, and 3. In-111 oxine activity in various organs and tumor area was determined. Histochemical analysis was performed to further confirm the migration and homing of injected cells at the tumor site. RESULTS: EPCs and CTLs showed an In-111 labeling efficiency of 87.06 ± 7.75% and 70.8 ± 12.9% respectively. Initially cell migration was observed in lung following inravenous administration of In-111 labeled cells and decreased on day 1 and 3, which indicate re-distribution of labeled cells from lung to other organs. Relatively higher In-111 oxine activity was observed in tumor areas at 24 hours in animals received In-111 labeled cells (EPCs or CTLs). Histiological analysis revealed iron positive cells in and around the tumor area in animals that received labeled cells (CTLs and EPCs). CONCLUSION: We observed differential biodistribution of In-111-oxine labeled EPCs and CTLs in different organs and intracranial glioma. This study indicates In-111 oxine based SPECT imaging is an effective tool to study the biodistribution of therapeutically important cells.

The Role of miRNAs in the Prognosis of Triple-Negative Breast Cancer: A Systematic Review and Meta-Analysis.

Breast cancer is one of the most common malignancies among women around the world. The basal or triple-negative subtype (TNBC) is a heterogeneous group of tumors, characterized by its aggressive and metastatic nature, with low survival and worse prognosis. Research on genetic biomarkers, such as microRNAs (miRs) in TNBC, demonstrate their relevance in the prognosis of the disease. Therefore, the objective of this research was to verify the role of miRs in the prognosis of TNBC. A search was carried out in the PubMed (MEDLINE), Web of Science, and Scopus databases, with articles in the English language from 2010 to 2022. Only articles that analyzed the role of miRNAs in the prognosis of TNBC and that met the criteria of the MOOSE method were included. For the preparation and planning of this systematic review, a PRISMA checklist and the MOOSE method were used. The Newcastle-Ottawa Scale was used to analyze the quality of the included studies. The excluded criteria considered were: (1) studies that presented duplication in the databases; (2) reviews of the literature, clinical case reports, meta-analyses, conference abstracts, letters to the editor, theses, dissertations, and book chapters; (3) studies that stratified only women diagnosed with other subtypes of breast cancer subtypes; (4) experiments without a control or comparison group. After the bibliographic survey of the 2.274 articles found, 43 articles met the inclusion criteria, totaling 5421 patients with TNBC analyzed for this review. Six miRs (miR-155, miR-21, miR-27a/b/, miR-374a/b, miR-30a/c/e, and miR-301a) were included in the meta-analysis. A low expression of miR-155 was associated with reduced overall survival (OS) (HR: 0.68, 95% CI: 0.58-0.81). A high expression of miR-21 was a predictor of OS reduction (HR: 2.56; 95% CI: 1.49-4.40). In addition, high levels of miR-27a/b and miR-301a/b were associated with lower OS, while the decreased expression levels of miR-30 and miR-374a/b were associated with worse relapse-free survival (RFS) and shorter disease-free survival (DFS), respectively. The present study revealed that miRs play essential roles in the development of metastases, in addition to acting as suppressors of the disease, thus improving the prognosis of TNBC. However, the clinical application of these findings has not yet been investigated.

Critical immunosuppressive effect of MDSC­derived exosomes in the tumor microenvironment.

Myeloid­derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME). Along with the role of MDSC immunosuppression and antitumor immunity, MDSCs facilitate tumor growth, differentiation, and metastasis in several ways that are yet to be explored. Like any other cell type, MDSCs also release a tremendous number of exosomes, or nanovesicles of endosomal origin, that participate in intercellular communications by dispatching biological macromolecules. There have been no investigational studies conducted to characterize the role of MDSC­derived exosomes (MDSC exo) in modulating the TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant level of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed a higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper­activating or exhausting CD8 T­cells and induce reactive oxygen species production that elicits activation­induced cell death. We confirmed the depletion of CD8 T­cells in vivo by treating mice with MDSC exo. We also observed a reduction in pro­inflammatory M1­macrophages in the spleen of those animals. Our results indicate that the immunosuppressive and tumor­promoting functions of MDSCs are also implemented by MDSC­derived exosomes which would open up a new avenue of MDSC research and MDSC­targeted therapy.

Changes in the tumor microenvironment and outcome for TME-targeting therapy in glioblastoma: A pilot study.

Glioblastoma (GBM) is a hypervascular and aggressive primary malignant tumor of the central nervous system. Recent investigations showed that traditional therapies along with antiangiogenic therapies failed due to the development of post-therapy resistance and recurrence. Previous investigations showed that there were changes in the cellular and metabolic compositions in the tumor microenvironment (TME). It can be said that tumor cell-directed therapies are ineffective and rethinking is needed how to treat GBM. It is hypothesized that the composition of TME-associated cells will be different based on the therapy and therapeutic agents, and TME-targeting therapy will be better to decrease recurrence and improve survival. Therefore, the purpose of this study is to determine the changes in the TME in respect of T-cell population, M1 and M2 macrophage polarization status, and MDSC population following different treatments in a syngeneic model of GBM. In addition to these parameters, tumor growth and survival were also studied following different treatments. The results showed that changes in the TME-associated cells were dependent on the therapeutic agents, and the TME-targeting therapy improved the survival of the GBM bearing animals. The current GBM therapies should be revisited to add agents to prevent the accumulation of bone marrow-derived cells in the TME or to prevent the effect of immune-suppressive myeloid cells in causing alternative neovascularization, the revival of glioma stem cells, and recurrence. Instead of concurrent therapy, a sequential strategy would be better to target TME-associated cells.

Generation of Novel Diagnostic and Therapeutic Exosomes to Detect and Deplete Protumorigenic M2 Macrophages.

Given their protumorigenic function and prevalence in most malignant tumors with lower survival; early detection, and intervention of CD206-positive M2 macrophages may boost the clinical outcome. To determine in vivo distribution of M2 macrophages, 111In-oxine-based radiolabeling of the targeted exosomes is adopted. When these radiolabeled targeted exosomes are injected into breast tumor-bearing mice, exosomes accumulate at the periphery of the primary tumor, metastatic foci in the lungs, spleen, and liver. Ex vivo quantification of radioactivity also shows similar distribution. Injecting DiI dye-labeled exosomes into the same mice shows adherence of exosomes to the CD206-positive M2 macrophages on ex vivo fluorescent microscopy imaging. In addition, these engineered exosomes are utilized to carry the Fc portion of lgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. It is demonstrated that M2 macrophage targeting therapeutic exosomes deplete M2 macrophages both in vitro and in vivo, and reduce tumor burden, increasing survival in a metastatic breast cancer model.

The role of melatonin on miRNAs modulation in triple-negative breast cancer cells.

Melatonin, a hormone secreted by pineal gland, exerts antimetastatic effects by reducing tumor cell proliferation, migration and invasion. MicroRNAs (miRNAs) are small, non-coding RNAs that play a crucial role in regulation of gene expression and biological processes of the cells. Herein, we search for a link between the tumor/metastatic-suppressive actions of melatonin and miRNA expression in triple-negative breast cancer cells. We demonstrated that melatonin exerts its anti-tumor actions by reducing proliferation, migration and c-Myc expression of triple negative breast cancer cells. By using Taqman-based assays, we analyzed the expression levels of a set of miRNAs following melatonin treatment of triple negative breast cancer cells and we identified 17 differentially expressed miRNAs, 6 down-regulated and 11 up-regulated. We focused on the anti-metastatic miR-148b and the oncogenic miR-210 both up-regulated by melatonin treatment and studied the effect of their modulation on melatonin-mediated impairment of tumor progression. Surprisingly, when miR-148b or miR-210 were depleted in triple-negative breast cancer cells, using a specific miR-148b sponge or anti-miR-210, melatonin effects on migration inhibition and c-myc downregulation were still visible suggesting that the increase of miR-148b and miR-210 expression observed following melatonin treatment was not required for the efficacy of melatonin action. Nevertheless, ours results suggest that melatonin exhibit a compound for metastatic trait inhibition, especially in MDA-MB-231 breast cancer cells even if a direct link between modulation of expression of certain proteins or miRNAs and melatonin effects has still to be established.

Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat.

Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while alpha-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27(Kip1) and p16(INKA) remained unchanged, whereas p21(Cip1) was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21(Cip1) is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.

Activation of Interleukin-1 Beta in Arterialized Vein Grafts and the Influence of the -511C/T IL-1ß Gene Polymorphism.

The interleukin-1 family is associated with innate immunity and inflammation. The latter has been linked to the genesis of cardiovascular diseases. We, therefore, investigated whether interleukin-1 beta (IL-1ß) is activated during arterialization of vein grafts. First, we examined the activation of IL-1ß using the rat arterialized jugular vein serially sampled for up to 90 days. IL-1ß expression increased 18 times on day 1 in the arterialized rat jugular vein and remained five times above nonarterialized vein levels for up to 90 days. Similarly, IL-1ß expression increased early (1-5 days) in human vein graft autopsy samples compared with late phases (1-4 years). Activation was also detected in ex vivo arterialized human saphenous veins. Upon stratification of the results, we uncovered a T allele promoter attenuating effect in IL-1ß activation in response to hemodynamic stress. Altogether, the results show that IL-1ß is activated during arterialization of vein grafts in rats and humans, and this response is modulated by -511C/T IL-1ß gene polymorphism. It is tempting to speculate that the activation of IL-1ß, and consequently local inflammation, modulates early vascular remodeling and that the gene polymorphism may be useful in predicting outcomes or assisting in interventions.

Therapeutic Potential of Melatonin in the Regulation of MiR-148a-3p and Angiogenic Factors in Breast Cancer.

BACKGROUND: The high mortality rate of breast cancer is related to the occurrence of metastasis, a process that is promoted by tumor angiogenesis. MicroRNAs are small molecules of noncoding mRNA that play a key role in gene regulation and are directly involved in the progression and angiogenesis of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors angiogenesis and may be associated with tumor growth and metastasis. Melatonin is an oncostatic agent with a capacity of modifying the expression of innumerable genes and miRNAs related to cancer. OBJECTIVE: The aim of this study was to evaluate the role of melatonin and the tumor suppressor miR- 148a-3p on angiogenesis of breast cancer. METHOD: MDA-MB-231 cells were treated with melatonin and modified with the overexpression of miR-148a-3p. The relative quantification in real-time of miR-148a-3p, IGF-IR and VEGF was performed by real-time PCR. The protein expression of these targets was performed by immunocytochemistry and immunohistochemistry. Survival, migration and invasion rates of tumor cells were evaluated. Finally, the xenograft model of breast cancer was performed to confirm the role of melatonin in the tumor. RESULTS: The melatonin was able to increase the gene level of miR-148a-3p and decreased the gene and protein expression of IGF-1R and VEGF, both in vitro and in vivo. In addition, it also had an inhibitory effect on the survival, migration and invasion of breast tumor cells. CONCLUSION: Our results confirm the role of melatonin in the regulation of miR-148a-3p and decrease of angiogenic factors.

Evaluation of Angiogenesis Process after Metformin and LY294002 Treatment in Mammary Tumor.

BACKGROUND: The angiogenesis process is regulated by many factors, such as Hypoxia-Inducible Factor-1 (HIF-1) and Vascular Endothelial Growth Factor (VEGF). Metformin has demonstrated its ability to inhibit cell growth and the LY294002 is the major inhibitor of PI3K/AKT/mTOR pathway that has antiangiogenic properties. METHODS: Canine mammary tumor cell lines CMT-U229 and CF41 were treated with metformin and LY294002. Cell viability, protein and gene expression of VEGF and HIF-1 were determined in vitro. For the in vivo study, CF41 cells were inoculated in female athymic nude mice treated with either metformin or LY294002. The microvessel density by immunohistochemistry for CD31 as well as the gene and protein expression of HIF-1 and VEGF were evaluated. RESULTS: The treatment with metformin and LY294002 was able to reduce the cellular viability after 24 hours. The protein and gene expression of HIF-1 and VEGF decreased after treatment with metformin and LY294002. In the in vivo study, there was a decrease in tumor size, protein and gene expression of HIF-1 and VEGFA, in addition to the decreasing of CD31 expression after all treatments. CONCLUSION: Our results demonstrate the effectiveness of metformin and LY294002 in controlling the angiogenesis process in mammary tumors by VEGF and HIF-1, the most important angiogenic markers.

CXCR2-Expressing Tumor Cells Drive Vascular Mimicry in Antiangiogenic Therapy-Resistant Glioblastoma.

BACKGROUND: Glioblastoma (GBM) was shown to relapse faster and displayed therapeutic resistance to antiangiogenic therapies (AATs) through an alternative tumor cell-driven mechanism of neovascularization called vascular mimicry (VM). We identified highly upregulated interleukin 8 (IL-8)-CXCR2 axis in tumor cells in high-grade human glioma and AAT-treated orthotopic GBM tumors. METHODS: Human GBM tissue sections and tissue array were used to ascertain the clinical relevance of CXCR2-positive tumor cells in the formation of VM. We utilized U251 and U87 human tumor cells to understand VM in an orthotopic GBM model and AAT-mediated enhancement in VM was modeled using vatalanib (anti-VEGFR2) and avastin (anti-VEGF). Later, VM was inhibited by SB225002 (CXCR2 inhibitor) in a preclinical study. RESULTS: Overexpression of IL8 and CXCR2 in human datasets and histological analysis was identified as a bonafide candidate to validate VM through in vitro and animal model studies. AAT-treated tumors displayed a higher number of CXCR2-positive GBM-stem cells with endothelial-like phenotypes. Stable knockdown of CXCR2 expression in tumor cells led to decreased tumor growth as well as incomplete VM structures in the animal models. Similar data were obtained following SB225002 treatment. CONCLUSIONS: The present study suggests that tumor cell autonomous IL-8-CXCR2 pathway is instrumental in AAT-mediated resistance and VM formation in GBM. Therefore, CXCR2 can be targeted through SB225002 and can be combined with standard therapies to improve the therapeutic outcomes in clinical trials.

Vascular Mimicry: A Novel Neovascularization Mechanism Driving Anti-Angiogenic Therapy (AAT) Resistance in Glioblastoma.

Glioblastoma (GBM) is a hypervascular neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT therapy resistance. Alternative neovascularization mechanisms were shown to be at work in these resilient tumors to counter the AAT therapy insult. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties and lay down vascular patterned networks reminiscent of host endothelial blood vessels. The VM channels served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands of the tumor in the event of the ensuing hypoxia resulting from AAT. In our previous studies, we have demonstrated that AAT accelerates VM in GBM. In this review, we will focus on the origins of VM, visualizing VM in AAT-treated tumors and the development of VM as a resistance mechanism to AAT.

HET0016 decreases lung metastasis from breast cancer in immune-competent mouse model.

Distant metastasis is the primary cause of death in the majority of the cancer types. Recently, much importance has been given to tumor microenvironment (TME) in the development of invasive malignant tumors, as well as the metastasis potential. The ability of tumor cells to modulate TME and to escape immune-mediated attack by releasing immunosuppressive cytokines has become a hallmark of breast cancer. Our study shows the effect of IV formulation of HET0016 (HPßCD-HET0016) a selective inhibitor of 20-HETE synthesis, administered intravenously in immune-competent in vivo mouse model of murine breast cancer. 4T1 luciferase positive cells were implanted to the mammary fat pad in Balb/c mice. Treatment started on day 15, and was administered for 5 days a week for 3 weeks. The development of metastasis was detected via optical imaging. Blood, spleen, lungs, bone marrow and tumor were collected for flow cytometry, to investigate changes in myeloid-derived suppressive cells (MDSCs) populations and endothelial phenotype. Tumor and lungs were collected for protein analysis. Our results show that HPßCD-HET0016: (1) decreased tumor volume and lung metastasis compared to the vehicle group; (2) reduced migration and invasion of tumor cells and levels of metalloproteinases in the lungs of animals treated with HPßCD-HET0016 via PI3K/AKT pathway; and (3) decreased expression of pro-inflammatory cytokines, growth factors and granulocytic MDSCs population in the lung microenvironment in treated animals. Thus, HPßCD-HET0016 showed potential in treating lung metastasis in a preclinical mouse model and needs further investigations on TME.

Vascular mimicry in glioblastoma following anti-angiogenic and anti-20-HETE therapies.

Glioblastoma (GBM) is one hypervascular and hypoxic tumor known among solid tumors. Antiangiogenic therapeutics (AATs) have been tested as an adjuvant to normalize blood vessels and control abnormal vasculature. Evidence of relapse exemplified in the progressive tumor growth following AAT reflects development of resistance to AATs. Here, we identified that GBM following AAT (Vatalanib) acquired an alternate mechanism to support tumor growth, called vascular mimicry (VM). We observed that Vatalanib induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer-side. The PAS+ matrix is positive for basal laminae (laminin) indicating vascular structures. Vatalanib treated GBM displayed various stages of VM such as initiation (mosaic), sustenance, and full-blown VM. Mature VM structures contain red blood cells (RBC) and bear semblance to the functional blood vessel-like structures, which provide all growth factors to favor tumor growth. Vatalanib treatment significantly increased VM especially in the core of the tumor, where HIF-1α was highly expressed in tumor cells. VM vessels correlate with hypoxia and are characterized by co-localized MHC-1+ tumor and HIF-1α expression. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at periphery of the tumors. In summary, AAT induced resistance characterized by VM is an alternative mechanism adopted by tumors to make functional vessels by transdifferentiation of tumor cells into endothelial-like cells to supply nutrients in the event of hypoxia. AAT induced VM is a potential therapeutic target of the novel formulation of HET0016. Our present study suggests that HET0016 has a potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.