Soluble PD-L1 as a diagnostic and prognostic biomarker in resectable gastric cancer patients.

BACKGROUND: In this study, we compared programmed death-ligand 1 (PD-L1) expression in primary tissue samples and its soluble form (sPD-L1) concentration in matched preoperative plasma samples from gastric cancer patients to understand the relationship between tissue and plasma PD-L1 expression and to determine its diagnostic and prognostic value. METHODS: PD-L1 expression in tissue was assessed by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), and sPD-L1 concentration in plasma was quantified by ELISA. The levels of the CD274 gene, which encodes for PD-L1 protein, were examined as part of bulk tissue RNA-sequencing analyses. Additionally, we evaluated the association between sPD-L1 levels and various laboratory parameters, disease characteristics, and patient outcomes. RESULTS: GC patients had significantly higher levels of sPD-L1 in their plasma (71.69 pg/mL) compared to healthy controls (35.34 pg/mL) (p < 0.0001). Moreover, sPD-L1 levels were significantly correlated with tissue PD-L1 protein, CD274 mRNA expression, larger tumor size, advanced tumor stage, and lymph node metastasis. Elevated sPD-L1 levels (> 103.5 ng/mL) were associated with poor overall survival (HR = 2.16, 95%CI 1.15-4.08, p = 0.017). Furthermore, intratumoral neutrophil and dendritic cell levels were directly correlated with plasma sPD-L1 concentration in the GC patients. CONCLUSIONS: sPD-L1 was readily measurable in GC patients, and its level was associated with GC tissue PD-L1 expression, greater inflammatory cell infiltration, disease progression, and survival. Thus, sPD-L1 may be a useful minimally invasive diagnostic and prognostic biomarker in GC patients.

A tumor microenvironment-based classification of gastric cancer for more effective diagnosis and treatment.

With approximately one million diagnosed cases and over 700,000 deaths recorded annually, gastric cancer (GC) is the third most common cause of cancer-related deaths worldwide. GC is a heterogeneous tumor. Thus, optimal management requires biomarkers of prognosis, treatment selection, and treatment response. The Cancer Genome Atlas program sub-classified GC into molecular subtypes, providing a framework for treatment personalization using traditional chemotherapies or biologics. Here, we report a comprehensive study of GC vascular and immune tumor microenvironment (TME)-based on stage and molecular subtypes of the disease and their correlation with outcomes. Using tissues and blood circulating biomarkers and a molecular classification, we identified cancer cell and tumor archetypes, which show that the TME evolves with the disease stage and is a major determinant of prognosis. Moreover, our TME-based subtyping strategy allowed the identification of archetype-specific prognostic biomarkers such as CDH1-mutant GC and circulating IL-6 that provided information beyond and independent of TMN staging, MSI status, and consensus molecular subtyping. The results show that integrating molecular subtyping with TME-specific biomarkers could contribute to improved patient prognostication and may provide a basis for treatment stratification, including for contemporary anti-angiogenesis and immunotherapy approaches.

Inhibition of CXCR4 Enhances the Efficacy of Radiotherapy in Metastatic Prostate Cancer Models.

Radiotherapy (RT) is a standard treatment for patients with advanced prostate cancer (PCa). Previous preclinical studies showed that SDF1α/CXCR4 axis could mediate PCa metastasis (most often to the bones) and cancer resistance to RT. We found high levels of expression for both SDF1α and its receptor CXCR4 in primary and metastatic PCa tissue samples. In vitro analyses using PCa cells revealed an important role of CXCR4 in cell invasion but not radiotolerance. Pharmacologic inhibition of CXCR4 using AMD3100 showed no efficacy in orthotopic primary and bone metastatic PCa models. However, when combined with RT, AMD3100 potentiated the effect of local single-dose RT (12 Gy) in both models. Moreover, CXCR4 inhibition also reduced lymph node metastasis from primary PCa. Notably, CXCR4 inhibition promoted the normalization of bone metastatic PCa vasculature and reduced tissue hypoxia. In conclusion, the SDF1α/CXCR4 axis is a potential therapeutic target in metastatic PCa patients treated with RT.

Inhibition of STAT3 Promotes Effector T Cell Infiltration But also Immunosuppression in the HCC Tumor Microenvironment.

BACKGROUND/AIM: STAT3 is involved in the progression of several cancers, and has been proposed as target for therapy. Indeed, the multitargeted tyrosine kinase inhibitor drug regorafenib, which indirectly inhibits STAT3, can significantly enhance the effects of anti-programmed death receptor (PD)-1 therapy in hepatocellular carcinoma (HCC) models. Here, we studied the impact of a direct STAT3 inhibitor on the tumor microenvironment and PD-1 blockade efficacy in HCC models. MATERIALS AND METHODS: Orthotopic mouse models of HCC (RIL-175 and HCA-1 grafts in syngeneic mice) were used to test the efficacy of the selective STAT3 inhibitor STX-0119 alone or combined with anti-PD-1 antibodies. We evaluated the effects of therapy on tumor vasculature and the immune microenvironment using immunofluorescence (IF), cell viability assay and quantitative real-time (qRT)-PCR in tumor tissues. RESULTS: Combining anti-PD-1 antibodies with a STX-0119 failed to show a growth delay or survival benefit compared to each agent alone or control in any of the HCC models. Interestingly, evaluation of intratumoral CD8+ T cell infiltration by IF showed a significant increase after one-week treatment with STX-0119 (p=0.034). However, STX-0119 treatment simultaneously promoted increased immunosuppression in the tumor microenvironment by increasing the proportion of Tregs, tissue hypoxia and α-SMA activated cancer-associated fibroblasts (CAFs) measured by IF. Consistent with these findings, we found increased immature tumor vessels by IF and VEGF, Tgf-ß and Vash2 expression by qPCR. CONCLUSION: Pharmacologic STAT3 inhibition could significantly enhance CD8+ T cell infiltration in HCC but also significantly alter the immunosuppression and vascular abnormalization in the tumor microenvironment.

Placental growth factor promotes tumour desmoplasia and treatment resistance in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC)-a rare liver malignancy with limited therapeutic options-is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression. DESIGN: We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems. RESULTS: PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC. Conclusion PlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC.

Current Landscape of Sonodynamic Therapy for Treating Cancer.

Recent advancements have tangibly changed the cancer treatment landscape. However, curative therapy for this dreadful disease remains an unmet need. Sonodynamic therapy (SDT) is a minimally invasive anti-cancer therapy involving a chemical sonosensitizer and focused ultrasound. A high-intensity focused ultrasound (HIFU) beam is used to destroy or denature targeted cancer tissues. Some SDTs are based on unfocused ultrasound (US). In some SDTs, HIFU is combined with a drug, known as a chemical sonosensitizer, to amplify the drug's ability to damage cancer cells preferentially. The mechanism by which US interferes with cancer cell function is further amplified by applying acoustic sensitizers. Combining multiple chemical sonosensitizers with US creates a substantial synergistic effect that could effectively disrupt tumorigenic growth, induce cell death, and elicit an immune response. Therefore, the minimally invasive SDT treatment is currently attracting attention. It can be combined with targeted therapy (double-targeting cancer therapy) and immunotherapy in the future and is expected to be a boon for treating previously incurable cancers. In this paper, we will consider the current state of this therapy and discuss parts of our research.

Regorafenib combined with PD1 blockade increases CD8 T-cell infiltration by inducing CXCL10 expression in hepatocellular carcinoma.

BACKGROUND AND PURPOSE: Combining inhibitors of vascular endothelial growth factor and the programmed cell death protein 1 (PD1) pathway has shown efficacy in multiple cancers, but the disease-specific and agent-specific mechanisms of benefit remain unclear. We examined the efficacy and defined the mechanisms of benefit when combining regorafenib (a multikinase antivascular endothelial growth factor receptor inhibitor) with PD1 blockade in murine hepatocellular carcinoma (HCC) models. BASIC PROCEDURES: We used orthotopic models of HCC in mice with liver damage to test the effects of regorafenib-dosed orally at 5, 10 or 20 mg/kg daily-combined with anti-PD1 antibodies (10 mg/kg intraperitoneally thrice weekly). We evaluated the effects of therapy on tumor vasculature and immune microenvironment using immunofluorescence, flow cytometry, RNA-sequencing, ELISA and pharmacokinetic/pharmacodynamic studies in mice and in tissue and blood samples from patients with cancer. MAIN FINDINGS: Regorafenib/anti-PD1 combination therapy increased survival compared with regofarenib or anti-PD1 alone in a regorafenib dose-dependent manner. Combination therapy increased regorafenib uptake into the tumor tissues by normalizing the HCC vasculature and increasing CD8 T-cell infiltration and activation at an intermediate regorafenib dose. The efficacy of regorafenib/anti-PD1 therapy was compromised in mice lacking functional T cells (Rag1-deficient mice). Regorafenib treatment increased the transcription and protein expression of CXCL10-a ligand for CXCR3 expressed on tumor-infiltrating lymphocytes-in murine HCC and in blood of patients with HCC. Using Cxcr3-deficient mice, we demonstrate that CXCR3 mediated the increased intratumoral CD8 T-cell infiltration and the added survival benefit when regorafenib was combined with anti-PD1 therapy. PRINCIPAL CONCLUSIONS: Judicious regorafenib/anti-PD1 combination therapy can inhibit tumor growth and increase survival by normalizing tumor vasculature and increasing intratumoral CXCR3+CD8 T-cell infiltration through elevated CXCL10 expression in HCC cells.

Potential Circulating Biomarkers of Recurrence after Hepatic Resection or Liver Transplantation in Hepatocellular Carcinoma Patients.

Background: Improving surgical outcomes in hepatocellular carcinoma (HCC) patients would greatly benefit from biomarkers. Angiogenesis and inflammation are hallmarks of HCC progression and therapeutic targets. Methods: We retrospectively evaluated preoperative clinical variables and circulating (plasma) biomarkers of angiogenesis and inflammation in a cohort of HCC patients who underwent liver resection (LR) or transplantation (LT). Biomarker correlation with outcomes-freedom of liver recurrence (FLR), disease-free survival (DFS) and overall survival (OS)-was tested using univariate and multivariate Cox regression analyses. Results: Survival outcomes associated with sVEGFR1, VEGF and VEGF-C in LT patients and with IL-10 in LR patients. Moreover, in LT patients within Milan criteria, higher plasma VEGF and sVEGFR1 were associated with worse outcomes, while in those outside Milan criteria lower plasma VEGF-C associated with better outcomes. Multivariate analysis indicated that adding plasma VEGF or VEGF-C to a predictive model including Milan criteria and AFP improved prediction of DFS and OS (all p < 0.05). Conclusion: Survival outcomes after LR or LT differentially associated with angiogenic and inflammatory biomarkers. High plasma VEGF correlated with poorer prognosis within Milan criteria while low plasma VEGF-C associated with better prognosis outside Milan criteria. These candidate biomarkers should be further validated to improve patient stratification.

Dual Programmed Death Receptor-1 and Vascular Endothelial Growth Factor Receptor-2 Blockade Promotes Vascular Normalization and Enhances Antitumor Immune Responses in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown. APPROACH AND RESULTS: We recapitulated these clinical outcomes using orthotopic-grafted or induced-murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8-positive (CD8+ ) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2-positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4+ cells. We also found that under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody. CONCLUSIONS: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy-resistant and anti-PD-1 therapy-responsive HCC models.

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice.

Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognoses, and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma, or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in cancer patients remains to be determined.

Morphological characterization of pulmonary microvascular disease in bronchopulmonary dysplasia caused by hyperoxia in newborn mice.

PURPOSE: Pulmonary microvascular injury is associated with the pathogenesis of bronchopulmonary dysplasia (BPD). To characterize the mechanisms of pulmonary vascular disease resulting from BPD, we studied the ultrastructural changes affecting pulmonary microvasculature. METHODS: Newborn ICR mice were exposed to 85% hyperoxia or normoxia for 14 days, and then normal air replacement conditions for the following 7 days. At postnatal day (P)14 and P21, lungs were harvested for ultrastructural examination and assessment of pulmonary hypertension. RESULTS: The ultrastructure of pulmonary microvasculature in the hyperoxia-exposed lungs revealed a collapsed capillary lumen. This was due to the abnormal morphology of endothelial cells (ECs) characterized by heterogeneously thick cytoplasm. Compared to normal air controls, the specimens displayed also remarkably thick blood-air barriers (BABs), most of which were occupied by EC layer components. Structural changes were accompanied by increased pulmonary artery medial thickness and right ventricular hypertrophy (RVH). Moreover, abnormalities in ECs persisted even after exposure to 7 days of normal air replacement conditions. Results were confirmed by morphometric quantification. CONCLUSION: Our results suggest that the abnormal morphology of capillary ECs and thick BABs correlates with pulmonary artery remodeling and RVH. These ultrastructural changes might represent possible mechanisms of secondary pulmonary hypertension in BPD.

Requisite role of vasohibin-2 in spontaneous gastric cancer formation and accumulation of cancer-associated fibroblasts.

The vasohibin (VASH) family consists of two genes, VASH1 and VASH2. VASH1 is mainly expressed in vascular endothelial cells and suppresses angiogenesis in an autocrine manner, whereas VASH2 is mainly expressed in cancer cells and exhibits pro-angiogenic activity. Employing adenomatous polyposis coli gene mutant mice, we recently reported on the role of Vash2 in the spontaneous formation of intestinal tumors. In this study, we used K19-Wnt1/C2mE (Gan) mice and examined the role of Vash2 in spontaneous gastric cancer formation. Gan mice spontaneously develop gastric tumors by activation of Wnt and prostaglandin E2 signaling pathways in gastric mucosa after 30 weeks of age. Expression of Vash2 mRNA was significantly increased in gastric tumor tissues compared with normal stomach tissues. When Gan mice were crossed with the Vash2-deficient (Vash2LacZ/LacZ ) strain, gastric cancer formation was significantly suppressed in Vash2LacZ/LacZ Gan mice. Normal composition of gastric mucosa was partially maintained in Vash2LacZ/LacZ Gan mice. Knockout of Vash2 caused minimal reduction of tumor angiogenesis but a significant decrease in cancer-associated fibroblasts (CAF) in tumor stroma. DNA microarray analysis and real-time RT-PCR showed that mRNA levels of epiregulin (Ereg) and interleukin-11 (Il11) were significantly downregulated in gastric tumors of Vash2LacZ/LacZ Gan mice. Furthermore, conditioned medium of gastric cancer cells stimulated migration of and α-smooth muscle actin expression in fibroblasts, whereas conditioned medium of VASH2 knockdown cells attenuated these effects in vitro. These results suggest that VASH2 plays an important role in gastric tumor progression via the accumulation of CAF accompanying upregulation of EREG and IL-11 expression.

Overcoming sorafenib evasion in hepatocellular carcinoma using CXCR4-targeted nanoparticles to co-deliver MEK-inhibitors.

Sorafenib is a RAF inhibitor approved for several cancers, including hepatocellular carcinoma (HCC). Inhibition of RAF kinases can induce a dose-dependent "paradoxical" upregulation of the downstream mitogen-activated protein kinase (MAPK) pathway in cancer cells. It is unknown whether "paradoxical" ERK activation occurs after sorafenib therapy in HCC, and if so, if it impacts the therapeutic efficacy. Here, we demonstrate that RAF inhibition by sorafenib rapidly leads to RAF dimerization and ERK activation in HCCs, which contributes to treatment evasion. The transactivation of RAF dimers and ERK signaling promotes HCC cell survival, prevents apoptosis via downregulation of BIM and achieves immunosuppression by MAPK/NF-kB-dependent activation of PD-L1 gene expression. To overcome treatment evasion and reduce systemic effects, we developed CXCR4-targeted nanoparticles to co-deliver sorafenib with the MEK inhibitor AZD6244 in HCC. Using this approach, we preferentially and efficiently inactivated RAF/ERK, upregulated BIM and down-regulated PD-L1 expression in HCC, and facilitated intra-tumoral infiltration of cytotoxic CD8+ T cells. These effects resulted in a profound delay in tumor growth. Thus, this nano-delivery strategy to selectively target tumors and prevent the paradoxical ERK activation could increase the feasibility of dual RAF/MEK inhibition to overcome sorafenib treatment escape in HCC.

Electron microscopic study of capillary network remodeling in the extensor digitorum longus muscle of normal adult rat.

Capillary networks demonstrate structural changes during maturation, aging, vascular disease, and cancer. Their morphological structure and function have an important influence on each other. Understanding the process of morphological vascular changes in the capillary network with advancing age may help overcome fatal vascular diseases. Aging-related structural changes of the capillary segments may accompany degeneration and regeneration of muscle fibers and serve to remodel the capillary network as a means of adapting to the changing environment. However, difficulty in obtaining human samples has hampered clarification of these microstructural changes. Herein, we examined serial ultrathin sections of capillary segments in the extensor digitorum longus muscle of normal mature (12 months old) rats in an attempt to analyze their structural changes. After bifurcation, a minimum of one capillary segment was filled with erythrocytes and was found to have fenestrations and plural endothelial disruptions, or pores, at the fenestrated portions. Some of the stagnated erythrocytes demonstrated extended protrusions, and their processes appeared to penetrate the basal lamina through the pores. These findings can also show that capillary segments are involved in partial remodeling of the capillary network. A better understanding of age-related structural changes of the capillary networks will help in fine-tuning novel vascular therapy for not only several fatal vascular diseases but also malignant tumors.

Endothelial cell transplantation in tumors restores normal vasculature, reduces tumor hypoxia, and suppresses tumor outgrowth.

OBJECTIVES: Vascular normalization, or restoration of the normal structure and function of blood vessels, using molecular-targeted therapy, has emerged as a potential strategy for treating malignant cancer and other vascular disorders. We hypothesized that restoring tumor blood vessels to their normal state would alleviate hypoxic conditions and potentially enhance the delivery of anticancer drugs. Our objective was to determine if transplanting normal endothelial cells into tumor-bearing mice could trigger vascular normalization. METHODS: Tumor cells were injected into the dorsal subcutis of severe combined immunodeficiency (SCID) mice (day 0). Tumor-bearing mice were injected intraperitoneally with cisplatin at day 14 to create scaffolds for blood vessel formation in the tumors. At day 28, human microvascular endothelial cells (HMVECs) or human embryonic stem-derived endothelial cells (ESECs) were transplanted into the necrotic regions of the tumor to induce normal angiogenesis. RESULTS: Microscopic observation revealed that the transplanted HMVECs or ESECs formed anastomoses with the host mouse vasculature. In addition, blood vessels with blood flow could be detected after 14d. Blood vessels reconstituted by HMVECs or ESECs exhibited normal vasculature, and tumor growth was significantly inhibited upon treatment. CONCLUSION: Reconstruction of tumor blood vessels to their normal state alleviated hypoxic conditions and improved the efficiency of drug delivery; the present approach provides a useful model for the development of new cancer therapies.

CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice.

UNLABELLED: Sorafenib, a broad tyrosine kinase inhibitor, is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC) but provides limited survival benefits. Recently, immunotherapy has emerged as a promising treatment strategy, but its role remains unclear in HCCs, which are associated with decreased cytotoxic CD8(+) T-lymphocyte infiltration in both murine and human tumors. Moreover, in mouse models after sorafenib treatment intratumoral hypoxia is increased and may fuel evasive resistance. Using orthotopic HCC models, we now show that increased hypoxia after sorafenib treatment promotes immunosuppression, characterized by increased intratumoral expression of the immune checkpoint inhibitor programmed death ligand-1 and accumulation of T-regulatory cells and M2-type macrophages. We also show that the recruitment of immunosuppressive cells is mediated in part by hypoxia-induced up-regulation of stromal cell-derived 1 alpha. Inhibition of the stromal cell-derived 1 alpha receptor (C-X-C receptor type 4 or CXCR4) using AMD3100 prevented the polarization toward an immunosuppressive microenvironment after sorafenib treatment, inhibited tumor growth, reduced lung metastasis, and improved survival. However, the combination of AMD3100 and sorafenib did not significantly change cytotoxic CD8(+) T-lymphocyte infiltration into HCC tumors and did not modify their activation status. In separate experiments, antibody blockade of the programmed death ligand-1 receptor programmed death receptor-1 (PD-1) showed antitumor effects in treatment-naive tumors in orthotopic (grafted and genetically engineered) models of HCC. However, anti-PD-1 antibody treatment had additional antitumor activity only when combined with sorafenib and AMD3100 and not when combined with sorafenib alone. CONCLUSION: Anti-PD-1 treatment can boost antitumor immune responses in HCC models; when used in combination with sorafenib, anti-PD-1 immunotherapy shows efficacy only with concomitant targeting of the hypoxic and immunosuppressive microenvironment with agents such as CXCR4 inhibitors.

Role of the vasohibin family in the regulation of fetoplacental vascularization and syncytiotrophoblast formation.

Vasohibin-1 (VASH1) and vasohibin-2 (VASH2), the 2 members of the vasohibin family, have been identified as novel regulators of angiogenesis. VASH1 ceases angiogenesis, whereas VASH2 stimulates sprouting. Here we characterized their functional role in the placenta. Immunohistochemical analysis of human placental tissue clarified their distinctive localization; VASH1 in endothelial cells and VASH2 in trophoblasts. We then used a mouse model to explore their function. Wild-type, Vash1((-/-)), and Vash2((-/-)) mice on a C57BL6 background were used in their first pregnancy. As expected, the fetal vascular area was increased in the Vash1((-/-)) mice, whereas it was decreased in the Vash2((-/-)) mice relative to wild-type. In addition, we noticed that the Vash2((-/-)) mice at 18.5dpc displayed thinner villi of the labyrinth and larger maternal lacunae. Careful observation by an electron microscopy revealed that the syncytiotrophoblast formation was defective in the Vash2((-/-)) mice. To test the possible involvement of VASH2 in the syncytiotrophoblast formation, we examined the fusion of BeWo cells, a human trophoblastoid choriocarcinoma cell line. The forskolin treatment induced the fusion of BeWo cells, and the knockdown of VASH2 expression significantly inhibited this cell fusion. Conversely, the overexpression of VASH2 by the infection with adenovirus vector encoding human VASH2 gene significantly increased the fusion of BeWo cells. Glial cell missing-1 and endogenous retrovirus envelope glycoprotein Syncytin 1 and Syncytin 2 are known to be involved in the fusion of trophoblasts. However, VASH2 did not alter their expression in BeWo cells. These results indicate that VASH1 and VASH2 showed distinctive localization and opposing function on the fetoplacental vascularization. Moreover, our study shows for the first time that VASH2 expressed in trophoblasts is involved in the regulation of cell fusion for syncytiotrophoblast formation.

Vasohibin-2 modulates tumor onset in the gastrointestinal tract by normalizing tumor angiogenesis.

BACKGROUND: Vasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of ApcMin/+ mice and ApcMin/+/Vash2-/- mice. METHODS: ApcMin/+ mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. ApcMin/+ mice were mated with Vash2-/- mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2-/- gene by PCR. Intestinal tumors from ApcMin/+ mice and ApcMin/+/Vash2-/- mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed. RESULTS: We found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in ApcMin/+ mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2-/- mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2-/- mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine. CONCLUSION: We propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.

Role of uterine contraction in regeneration of the murine postpartum endometrium.

The endometrium undergoes continuous repair and regeneration without scarring throughout the reproductive life of women. However, the mechanisms responsible for this complete restoration remain mostly unexplored. We hypothesized that the ischemic state and local hypoxia present after parturition may create a special microenvironment for endometrial healing, and that this ischemia might be caused by reduction in organ volume via postpartum uterine contraction. Here, we developed a mouse model using a combination of cesarean section and the administration of a beta 2 adrenergic receptor agonist (ritodrine hydrochloride) in postpartum mice that had been ovariectomized to exclude the effect of ovarian hormones. Our results revealed that transient hypoxia indeed occurred in postpartum uteri. Furthermore, we found that the number of M2 macrophages, which play a central role in wound healing, peaked on Postpartum Day 3 and gradually decreased thereafter in hypoxic injury sites. Almost concurrently, significant upregulation of vascular endothelial growth factor and transforming growth factor beta (TGFbeta) was observed. In particular, the antifibrotic factor TGFbeta3 was released during the endometrial healing process. These changes were significantly suppressed by inhibition of uterine contraction. Taken together, these results suggest that uterine contraction is essential, not only for hemostasis, but also for endometrial regeneration, leading to a process that involves the activation of macrophages, increased endometrial cell proliferation, and upregulation of nonfibrotic growth factors. This study paves the way to a novel approach for investigating the process of scarless wound healing.