miR-1246 in tumor extracellular vesicles promotes metastasis via increased tumor cell adhesion and endothelial cell barrier destruction.

Background: Tumor blood vessels play a key role in tumor metastasis. We have previously reported that tumor endothelial cells (TECs) exhibit abnormalities compared to normal endothelial cells. However, it is unclear how TECs acquire these abnormalities. Tumor cells secrete extracellular vesicles (EVs) to create a suitable environment for themselves. We have previously identified miR-1246 to be more abundant in high metastatic melanoma EVs than in low metastatic melanoma EVs. In the current study, we focused on miR-1246 as primarily responsible for acquiring abnormalities in TECs and examined whether the alteration of endothelial cell (EC) character by miR-1246 promotes cancer metastasis. Methods: We analyzed the effect of miR-1246 in metastatic melanoma, A375SM-EVs, in vivo metastasis. The role of tumor EV-miR-1246 in the adhesion between ECs and tumor cells and the EC barrier was addressed. Changes in the expression of adhesion molecule and endothelial permeability were examined. Results: Intravenous administration of A375SM-EVs induced tumor cell colonization in the lung resulting in lung metastasis. In contrast, miR-1246 knockdown in A375SM decreased lung metastasis in vivo. miR-1246 transfection in ECs increased the expression of adhesion molecule ICAM-1 via activation of STAT3, followed by increased tumor cell adhesion to ECs. Furthermore, the expression of VE-Cadherin was downregulated in miR-1246 overexpressed EC. A375SM-EV treatment enhanced endothelial permeability. VE-Cadherin was validated as the potential target gene of miR-1246 via the target gene prediction database and 3' UTR assay. Conclusion: miR-1246 in high metastatic tumor EVs promotes lung metastasis by inducing the adhesion of tumor cells to ECs and destroying the EC barrier.

Angiogenic inhibitor pre-administration improves the therapeutic effects of immunotherapy.

In lung cancer, immune checkpoint inhibitors (ICIs) are often inadequate for tumor growth inhibition. Angiogenic inhibitors (AIs) are required to normalize tumor vasculature for improved immune cell infiltration. However, in clinical practice, ICIs and cytotoxic antineoplastic agents are simultaneously administered with an AI when tumor vessels are abnormal. Therefore, we examined the effects of pre-administering an AI for lung cancer immunotherapy in a mouse lung cancer model. Using DC101, an anti-vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody, a murine subcutaneous Lewis lung cancer (LLC) model was used to determine the timing of vascular normalization. Microvessel density (MVD), pericyte coverage, tissue hypoxia, and CD8-positive cell infiltration were analyzed. The effects of an ICI and paclitaxel after DC101 pre-administration were investigated. On Day 3, increased pericyte coverage and alleviated tumor hypoxia represented the highest vascular normalization. CD8+ T-cell infiltration was also highest on Day 3. When combined with an ICI, DC101 pre-administration significantly reduced PD-L1 expression. When combined with an ICI and paclitaxel, only DC101 pre-administration significantly inhibited tumor growth, but simultaneous administration did not. AI pre-administration, and not simultaneous administration, may increase the therapeutic effects of ICIs due to improved immune cell infiltration.

Novel antiangiogenic therapy targeting biglycan using tumor endothelial cell-specific liposomal siRNA delivery system.

Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine-rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC-derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope-type nanodevice (MEND), which contains a unique pH-sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) into MEND because αV ß3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD-MEND-encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS-RC-2 tumor-derived TECs and induced in vitro RNAi-mediated gene silencing. MEND was then injected intravenously into OS-RC-2 tumor-bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT-PCR indicated that biglycan was knocked down by biglycan siRNA-containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA-containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment.

ALLD: Acute Lymphoblastic Leukemia Detector.

Acute Lymphoblastic Leukemia (ALL) is a life-threatening type of cancer wherein mortality rate is unquestionably high. Early detection of ALL can reduce both the rate of fatality as well as improve the diagnosis plan for patients. In this study, we developed the ALL Detector (ALLD), which is a deep learning-based network to distinguish ALL patients from healthy individuals based on blast cell microscopic images. We evaluated multiple DL-based models and the ResNet-based model performed the best with 98% accuracy in the classification task. We also compared the performance of ALLD against state-of-the-art tools utilized for the same purpose, and ALLD outperformed them all. We believe that ALLD will support pathologists to explicitly diagnose ALL in the early stages and reduce the burden on clinical practice overall.

miRNA-1246 in extracellular vesicles secreted from metastatic tumor induces drug resistance in tumor endothelial cells.

Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs.

Enhanced oncolytic activity of E4orf6-deficient adenovirus by facilitating nuclear export of HuR.

An AU-rich element (ARE) is RNA element that enhances the rapid decay of mRNA. The RNA binding protein HuR stabilizes ARE-mRNA by exporting it to the cytoplasm. In most of cancer cells, HuR is exported to the cytoplasm and ARE-mRNA is stabilized. In addition, the viral gene product E4orf6 exports HuR to stabilize ARE-mRNA in adenovirus-infected cells and the stabilization is required for full virus replication. Previously we showed the oncolytic activity of E4orf6-deleted adenovirus dl355, which can replicate in cancer cells where ARE-mRNA is stabilized. In this study, we examined whether the further enhancement of HuR export can stimulate the replication and the oncolytic activity of dl355. We found that ethanol treatment promoted the cytoplasmic relocalization of HuR in cancer cells. In addition, the replication efficiency of dl355 increased in ethanol-treated cells, and in response, the cytolytic activity of the virus also increased in vitro and in vivo. Upregulation of a cleaved-PARP level in infected cells mediated by ethanol is suggesting that ethanol activated the apoptosis induced by dl355. IVa2 mRNA, the only ARE-mRNA among transcripts of adenovirus was augmented by ethanol treatment. These data indicate that the enhancement of ARE-mRNA stabilization as a result of ethanol treatment upregulates the oncolytic activity of dl355 and suggests that the combined use of an oncolytic adenovirus and ethanol treatment may be a good strategy for cancer therapy.

Chemotherapy-Induced IL8 Upregulates MDR1/ABCB1 in Tumor Blood Vessels and Results in Unfavorable Outcome.

Tumor endothelial cells (TEC) lining tumor blood vessels actively contribute to tumor progression and metastasis. In addition to tumor cells, TEC may develop drug resistance during cancer treatment, allowing the tumor cells to survive chemotherapy and metastasize. We previously reported that TECs resist paclitaxel treatment via upregulation of ABCB1. However, whether TEC phenotypes are altered by anticancer drugs remains to be clarified. Here, we show that ABCB1 expression increases after chemotherapy in urothelial carcinoma cases. The ratio of ABCB1-positive TEC before and after first-line chemotherapy in urothelial carcinoma tissues (n = 66) was analyzed by ABCB1 and CD31 immunostaining. In 42 cases (64%), this ratio increased after first-line chemotherapy. Chemotherapy elevated ABCB1 expression in endothelial cells by increasing tumor IL8 secretion. In clinical cases, ABCB1 expression in TEC correlated with IL8 expression in tumor cells after first-line chemotherapy, leading to poor prognosis. In vivo, the ABCB1 inhibitor combined with paclitaxel reduced tumor growth and metastasis compared with paclitaxel alone. Chemotherapy is suggested to cause inflammatory changes in tumors, inducing ABCB1 expression in TEC and conferring drug resistance. Overall, these findings indicate that TEC can survive during chemotherapy and provide a gateway for cancer metastasis. Targeting ABCB1 in TEC represents a novel strategy to overcome cancer drug resistance. SIGNIFICANCE: These findings show that inhibition of ABCB1 in tumor endothelial cells may improve clinical outcome, where ABCB1 expression contributes to drug resistance and metastasis following first-line chemotherapy.

Advantages of Using Paclitaxel in Combination with Oncolytic Adenovirus Utilizing RNA Destabilization Mechanism.

Oncolytic virotherapy is a novel approach to cancer therapy. Ad-fosARE is a conditionally replicative adenovirus engineered by inserting AU-rich elements (ARE) in the 3'-untranslated region of the E1A gene. In this study, we examined the oncolytic activity of Ad-fosARE and used it in a synergistic combination with the chemotherapeutic agent paclitaxel (PTX) for treating cancer cells. The expression of E1A was high in cancer cells due to stabilized E1A-ARE mRNA. As a result, the efficiency of its replication and cytolytic activity in cancer cells was higher than in normal cells. PTX treatment increased the cytoplasmic HuR relocalization in cancer cells, enhanced viral replication through elevated E1A expression, and upregulated CAR (Coxsackie-adenovirus receptor) required for viral uptake. Furthermore, PTX altered the instability of microtubules by acetylation and detyrosination, which is essential for viral internalization and trafficking to the nucleus. These results indicate that PTX can provide multiple advantages to the efficacy of Ad-fosARE both in vitro and in vivo, and provides a basis for designing novel clinical trials. Thus, this virus has a lot of benefits that are not found in other oncolytic viruses. The virus also has the potential for treating PXT-resistant cancers.

Carbonic anhydrase 2 (CAII) supports tumor blood endothelial cell survival under lactic acidosis in the tumor microenvironment.

BACKGROUND: Tumor endothelial cells (TECs) perform tumor angiogenesis, which is essential for tumor growth and metastasis. Tumor cells produce large amounts of lactic acid from glycolysis; however, the mechanism underlying the survival of TECs to enable tumor angiogenesis under high lactic acid conditions in tumors remains poorly understood. METHODOLOGY: The metabolomes of TECs and normal endothelial cells (NECs) were analyzed by capillary electrophoresis time-of-flight mass spectrometry. The expressions of pH regulators in TECs and NECs were determined by quantitative reverse transcription-PCR. Cell proliferation was measured by the MTS assay. Western blotting and ELISA were used to validate monocarboxylate transporter 1 and carbonic anhydrase 2 (CAII) protein expression within the cells, respectively. Human tumor xenograft models were used to access the effect of CA inhibition on tumor angiogenesis. Immunohistochemical staining was used to observe CAII expression, quantify tumor microvasculature, microvessel pericyte coverage, and hypoxia. RESULTS: The present study shows that, unlike NECs, TECs proliferate in lactic acidic. TECs showed an upregulated CAII expression both in vitro and in vivo. CAII knockdown decreased TEC survival under lactic acidosis and nutrient-replete conditions. Vascular endothelial growth factor A and vascular endothelial growth factor receptor signaling induced CAII expression in NECs. CAII inhibition with acetazolamide minimally reduced tumor angiogenesis in vivo. However, matured blood vessel number increased after acetazolamide treatment, similar to bevacizumab treatment. Additionally, acetazolamide-treated mice showed decreased lung metastasis. CONCLUSION: These findings suggest that due to their effect on blood vessel maturity, pH regulators like CAII are promising targets of antiangiogenic therapy. Video Abstract.

Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan.

Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-κB and extracellular signal-regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.

Inhibition of multidrug transporter in tumor endothelial cells enhances antiangiogenic effects of low-dose metronomic paclitaxel.

Tumor angiogenesis plays an important role in tumor progression and metastasis. Tumor endothelial cells (TECs) are a therapeutic target of antiangiogenic chemotherapy that was recently developed and is currently being investigated in the clinic with promising results. Low-dose chemotherapy, which is the long-term administration of relatively low doses of chemotherapeutic agents, has been proposed for targeting tumor angiogenesis in various types of cancers. Although the efficacy of low-dose chemotherapy has been confirmed in several clinical models, some studies show insufficient therapeutic effect for malignant cancers. As a possible mechanism of the treatment failure, it has been considered that tumor cells may acquire resistance to this therapy. However, drug resistance by TECs may also be due to another mechanism for resistance of tumor cells to low-dose chemotherapy. We reported elsewhere that TECs were resistant to the anticancer drug paclitaxel, which is a mitotic inhibitor, concomitant with P-glycoprotein up-regulation. Verapamil, a P-glycoprotein inhibitor, abrogated TEC resistance in vitro. Herein, we demonstrated that verapamil coadministration enhanced the effects of low-dose paclitaxel concomitant with inhibiting tumor angiogenesis in a preclinical in vivo mouse melanoma xenograft model. Furthermore, verapamil coadministration reduced lung metastasis. These results suggest that inhibiting P-glycoprotein in TECs may be a novel strategy for low-dose chemotherapy targeting TECs.