An analysis modality for vascular structures combining tissue-clearing technology and topological data analysis.

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.

Suppression of Tumor Growth in a Rabbit Hepatic Cancer Model by Boron Neutron Capture Therapy With Liposomal Boron Delivery Systems.

BACKGROUND/AIM: Tumor cell destruction by boron neutron capture therapy (BNCT) is attributed to the nuclear reaction between 10B and thermal neutrons. The accumulation of 10B atoms in tumor cells without affecting adjacent healthy cells is crucial for effective BNCT. We previously reported that several types of liposomal boron delivery systems (BDS) delivered effective numbers of boron atoms to cancer tissues, and showed tumor-growth suppression after thermal neutron irradiation. In the present study, we examined the effects of BNCT after intra-arterial infusion of 10B-borono-dodecaborate (10BSH) by liposomal BDS in rabbit hepatic cancer models. MATERIALS AND METHODS: We prepared 10BSH-entrapped transferrin-conjugated polyethylene glycol liposomes constructed with distearoyl-boron lipid (TF-PEG-DSBL), and performed thermal neutron irradiation at the Kyoto University Institute for Integrated Radiation and Nuclear Science after intra-arterial infusion into rabbit VX-2 hepatic tumors. RESULTS: Concentrations of 10B in VX-2 tumors on delivery with TF-PEG-DSBL liposomes reached 25 ppm on day 3 after the injection. Tumor growth was suppressed by thermal neutron irradiation after intra-arterial injection of this 10BSH-containing liposomal BDS, without damage to normal cells. CONCLUSION: The present results demonstrate the applicability of 10B-containing TF-PEG-DSBL liposomes as a novel intra-arterial boron carrier in BNCT for cancer.

Visualization of the cancer cell cycle by tissue-clearing technology using the Fucci reporter system.

Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.

Systemic administration of monovalent follistatin-like 3-Fc-fusion protein increases muscle mass in mice.

Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor ß (TGF-ß) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-ß family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.

Tumor Growth Suppression With Novel Intra-arterial Chemotherapy Using Epirubicin-entrapped Water-in-oil-in-water Emulsion In Vivo.

BACKGROUND/AIM: A mixture of anticancer agents and iodized poppy seed oil (IPSO) has been widely used for intra-arterial chemotherapy of hepatocellular carcinoma. However, the anticancer agents can easily separate from IPSO, so the therapeutic potential is limited. We developed epirubicin-entrapped water-in-oil-in-water emulsion (WOW-Epi) using a double-membrane emulsification technique. MATERIALS AND METHODS: We delivered WOW-Epi through a hepatic arterial injection to VX2 hepatic tumor rabbit model (1.2 mg/kg). RESULTS: VX2 tumor growth was selectively suppressed in the WOW-Epi-treated group compared with the control treated groups. The accumulation of WOW in nearby cancer cells was confirmed via electron-microscopy. Endocytosis seemed to be the mechanism underlying the uptake of WOW. CONCLUSION: WOW-Epi led to tumour growth suppression in vivo. WOW does not cause toxicity to arterial vessels. WOW-Epi will be hopefully used for repeated intra-arterial chemotherapy to HCC patients in the near future.

Blocking VCAM-1 inhibits pancreatic tumour progression and cancer-associated thrombosis/thromboembolism.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the deadliest cancer. Cancer-associated thrombosis/thromboembolism (CAT), frequently observed in PDAC, is known as a poor prognostic factor. Here, we investigated the underlying mechanisms between PDAC and CAT, and performed a trial of therapeutic approach for PDAC using a genetically engineered mouse model, PKF (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox ). DESIGN: Presence of CAT in PKF mice was detected by systemic autopsy. Plasma cytokines were screened by cytokine antibody array. Murine and human plasma atrial natriuretic peptide (ANP) and soluble vascular cell adhesion molecule 1 (sVCAM-1) were determined by ELISA. Distribution of VCAM-1 in PKF mice and human autopsy samples was detected by immunohistochemistry. PKF mice were treated with anti-VCAM-1 antibody and the effects on survival, distribution of CAT and the tumour histology were analysed. RESULTS: We found spontaneous CAT with cardiomegaly in 68.4% PKF mice. Increase of plasma ANP and sVCAM-1 was observed in PKF mice and PDAC patients with CAT. VCAM-1 was detected in the activated endothelium and thrombi. Administration of anti-VCAM-1 antibody to PKF mice inhibited tumour growth, neutrophil/macrophage infiltration, tumour angiogenesis and progression of CAT; moreover, it dramatically extended survival (from 61 to 253 days, p<0.01). CONCLUSION: Blocking VCAM-1/sVCAM-1 might be a potent therapeutic approach for PDAC as well as CAT, which can contribute to the prognosis. Increase of plasma ANP and sVCAM-1 might be a diagnostic approach for CAT in PDAC.

Neurotensin receptor 1 signaling promotes pancreatic cancer progression.

Pancreatic cancer is one of the cancers with the poorest prognosis, with a 5-year survival rate of approximately 5-10%. Thus, it is urgent to identify molecular targets for the treatment of pancreatic cancer. Using serial transplantations in a mouse pancreatic orthotopic inoculation model, we previously produced highly malignant pancreatic cancer sublines with increased tumor-forming abilities in vivo. Here, we used these sublines to screen molecular targets for the treatment of pancreatic cancer. Among the genes with increased expression levels in the sublines, we focused on those encoding cell surface receptors that may be involved in the interactions between cancer cells and the tumor microenvironment. Based on our previous RNA-sequence analysis, we found increased expression levels of neurotensin (NTS) receptor 1 (NTSR1) in highly malignant pancreatic cancer sublines. Furthermore, re-analysis of clinical databases revealed that the expression level of NTSR1 was increased in advanced pancreatic cancer and that high NTSR1 levels were correlated with a poor prognosis. Overexpression of NTSR1 in human pancreatic cancer cells Panc-1 and SUIT-2 accelerated their tumorigenic and metastatic abilities in vivo. In addition, RNA-sequence analysis showed that MAPK and NF-κB signaling pathways were activated upon NTS stimulation in highly malignant cancer sublines and also revealed many new target genes for NTS in pancreatic cancer cells. NTS stimulation increased the expression of MMP-9 and other pro-inflammatory cytokines and chemokines in pancreatic cancer cells. Moreover, the treatment with SR48692, a selective NTSR1 antagonist, suppressed the activation of the MAPK and NF-κB signaling pathways and induction of target genes in pancreatic cancer cells in vitro, while the administration of SR48692 attenuated the tumorigenicity of pancreatic cancer cells in vivo. These findings suggest that NTSR1 may be a prognostic marker and a molecular target for pancreatic cancer treatment.

Soluble VCAM-1 promotes gemcitabine resistance via macrophage infiltration and predicts therapeutic response in pancreatic cancer.

Pancreatic cancer is one of the malignant diseases with the worst prognosis. Resistance to chemotherapy is a major difficulty in treating the disease. We analyzed plasma samples from a genetically engineered mouse model of pancreatic cancer and found soluble vascular cell adhesion molecule-1 (sVCAM-1) increases in response to gemcitabine treatment. VCAM-1 was expressed and secreted by murine and human pancreatic cancer cells. Subcutaneous allograft tumors with overexpression or knock-down of VCAM-1, as well as VCAM-1-blocking treatment in the spontaneous mouse model of pancreatic cancer, revealed that sVCAM-1 promotes tumor growth and resistance to gemcitabine treatment in vivo but not in vitro. By analyzing allograft tumors and co-culture experiments, we found macrophages were attracted by sVCAM-1 to the tumor microenvironment and facilitated resistance to gemcitabine in tumor cells. In a clinical setting, we found that the change of sVCAM-1 in the plasma of patients with advanced pancreatic cancer was an independent prognostic factor for gemcitabine treatment. Collectively, gemcitabine treatment increases the release of sVCAM-1 from pancreatic cancer cells, which attracts macrophages into the tumor, thereby promoting the resistance to gemcitabine treatment. sVCAM-1 may be a potent clinical biomarker and a potential target for the therapy in pancreatic cancer.

Exosomes of Epstein-Barr Virus-Associated Gastric Carcinoma Suppress Dendritic Cell Maturation.

The Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is characterized by the infiltration of lymphocytes and a unique tumor microenvironment. Exosomes from cancer cells are essential for intercellular communication. The aims of this study were to investigate the secretion of EBVaGC exosomes and their physiological effect on dendritic cell maturation in vitro and to characterize dendritic cells (DCs) in EBVaGC in vivo. Western blotting analysis of CD63 and CD81 of exosomes from EBV-infected gastric cancer cell lines indicated an increase in exosome secretion. The fraction of monocyte-derived DCs positive for the maturation marker CD86 was significantly suppressed when incubated with exosomes from EBV-infected gastric cancer cell lines. Immunohistochemical analysis of GC tissues expressing DC markers (S100, Langerin, CD1a, CD83, CD86, and BDCA-2) indicated that the density of DCs was generally higher in EBVaGC than in EBV-negative GC, although the numbers of CD83- and CD86-positive DCs were decreased in the group with high numbers of CD1a-positive DCs. A low number of CD83-positive DCs was marginally correlated with worse prognosis of EBVaGC in patients. EBVaGC is a tumor with abundant DCs, including immature and mature DCs. Moreover, the maturation of DCs is suppressed by exosomes from EBV-infected epithelial cells.

Chd4 choreographs self-antigen expression for central immune tolerance.

Autoreactive T cells are eliminated in the thymus to prevent autoimmunity by promiscuous expression of tissue-restricted self-antigens in medullary thymic epithelial cells. This expression is dependent on the transcription factor Fezf2, as well as the transcriptional regulator Aire, but the entire picture of the transcriptional program has been obscure. Here, we found that the chromatin remodeler Chd4, also called Mi-2ß, plays a key role in the self-antigen expression in medullary thymic epithelial cells. To maximize the diversity of self-antigen expression, Fezf2 and Aire utilized completely distinct transcriptional mechanisms, both of which were under the control of Chd4. Chd4 organized the promoter regions of Fezf2-dependent genes, while contributing to the Aire-mediated induction of self-antigens via super-enhancers. Mice deficient in Chd4 specifically in thymic epithelial cells exhibited autoimmune phenotypes, including T cell infiltration. Thus, Chd4 plays a critical role in integrating Fezf2- and Aire-mediated gene induction to establish central immune tolerance.

Single-dose toxicity study by intra-arterial injection of 10BSH entrapped water-in-oil-in-water emulsion for boron neutron capture therapy to hepatocellular carcinoma.

We developed a mixing medical device by attaching Shirasu porous glass Millipore membrane to prepare water-in-oil-in-water (WOW) emulsion in a shorter time to be applied as 10B-entrapped WOW emulsion for hepatocellular carcinoma (HCC) treatment. Single-dose toxicity studies by intra-arterial injection of 10BSH-entrapped WOW were performed in rabbits and pig, and no side effects were observed. We hope to proceed to the preclinical and clinical studies for further evaluation of 10B compound as multidisciplinary treatments for HCC.

ASCL1 promotes tumor progression through cell-autonomous signaling and immune modulation in a subset of lung adenocarcinoma.

The master regulator of neuroendocrine differentiation, achaete-scute complex homolog 1 (ASCL1) defines a subgroup of lung adenocarcinoma. However, the mechanistic role of ASCL1 in lung tumorigenesis and its relation to the immune microenvironment is principally unknown. Here, the immune landscape of ASCL1-positive lung adenocarcinomas was characterized by immunohistochemistry. Furthermore, ASCL1 was transduced in mouse lung adenocarcinoma cell lines and comparative RNA-sequencing and secretome analyses were performed. The effects of ASCL1 on tumorigenesis were explored in an orthotopic syngeneic transplantation model. ASCL1-positive lung adenocarcinomas revealed lower infiltration of CD8+, CD4+, CD20+, and FOXP3+ lymphocytes and CD163+ macrophages indicating an immune desert phenotype. Ectopic ASCL1 upregulated cyclin transcript levels, stimulated cell proliferation, and enhanced tumor growth in mice. ASCL1 suppressed secretion of chemokines, including CCL20, CXCL2, CXCL10, and CXCL16, indicating effects on immune cell trafficking. In accordance with lower lymphocytes infiltration, ASCL1-positive lung adenocarcinomas demonstrated lower abundance of CXCR3-and CCR6-expressing cells. In conclusion, ASCL1 mediates its tumor-promoting effect not only through cell-autonomous signaling but also by modulating chemokine production and immune responses. These findings suggest that ASCL1-positive tumors represent a clinically relevant lung cancer entity.

Long-term Risk of Malignancy in Branch-Duct Intraductal Papillary Mucinous Neoplasms.

BACKGROUND & AIMS: Long-term outcomes of patients with branch-duct intraductal papillary mucinous neoplasms (IPMNs), particularly those after 5 years of surveillance, have not been fully evaluated in large studies. We analyzed incidences of IPMN-derived carcinoma and concomitant ductal adenocarcinoma (pancreatic ductal adenocarcinoma [PDAC]) over 20 years in a large population of patients. METHODS: We identified 1404 consecutive patients (52% women; mean age, 67.5 years) with a diagnosis of branch-duct IPMN, from 1994 through 2017, at the University of Tokyo in Japan. Using a competing risk analysis, we estimated cumulative incidence of pancreatic carcinoma, overall and by carcinoma type. We used competing risks proportional hazards models to estimate subdistribution hazard ratios (SHRs) for incidences of carcinomas. To differentiate IPMN-derived and concomitant carcinomas, we collected genomic DNA from available paired samples of IPMNs and carcinomas and detected mutations in GNAS and KRAS by polymerase chain reaction and pyrosequencing. RESULTS: During 9231 person-years of follow-up, we identified 68 patients with pancreatic carcinomas (38 patients with IPMN-derived carcinomas and 30 patients with concomitant PDACs); the overall incidence rates were 3.3%, 6.6%, and 15.0% at 5, 10, and 15 years, respectively. Among 804 patients followed more than 5 years, overall cumulative incidence rates of pancreatic carcinoma were 3.5% at 10 years and 12.0% at 15 years from the initial diagnosis. The size of the IPMN and the diameter of the main pancreatic duct associated with incidence of IPMN-derived carcinoma (SHR 1.85; 95% confidence interval 1.38-2.48 for a 10-mm increase in the IPMN size and SHR 1.56; 95% confidence interval 1.33-1.83 for a 1-mm increase in the main pancreatic duct diameter) but not with incidence of concomitant PDAC. CONCLUSIONS: In a large long-term study of patients with branch-duct IPMNs, we found the 5-year incidence rate of pancreatic malignancy to be 3.3%, reaching 15.0% at 15 years after IPMN diagnosis. We observed heterogeneous risk factor profiles between IPMN-derived and concomitant carcinomas.

The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension.

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of Atoh8 did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, Atoh8-deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, ATOH8 expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2α (HIF-2α) and decreased its abundance, leading to reduced induction of HIF-2α target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.

c-Ski accelerates renal cancer progression by attenuating transforming growth factor ß signaling.

Although transforming growth factor beta (TGF-ß) is known to be involved in the pathogenesis and progression of many cancers, its role in renal cancer has not been fully investigated. In the present study, we examined the role of TGF-ß in clear cell renal carcinoma (ccRCC) progression in vitro and in vivo. First, expression levels of TGF-ß signaling pathway components were examined. Microarray and immunohistochemical analyses showed that the expression of c-Ski, a transcriptional corepressor of Smad-dependent TGF-ß and bone morphogenetic protein (BMP) signaling, was higher in ccRCC tissues than in normal renal tissues. Next, a functional analysis of c-Ski effects was carried out. Bioluminescence imaging of renal orthotopic tumor models demonstrated that overexpression of c-Ski in human ccRCC cells promoted in vivo tumor formation. Enhancement of tumor formation was also reproduced by the introduction of a dominant-negative mutant TGF-ß type II receptor into ccRCC cells. In contrast, introduction of the BMP signaling inhibitor Noggin failed to accelerate tumor formation, suggesting that the tumor-promoting effect of c-Ski depends on the inhibition of TGF-ß signaling rather than of BMP signaling. Finally, the molecular mechanism of the tumor-suppressive role of TGF-ß was assessed. Although TGF-ß signaling did not affect tumor angiogenesis, apoptosis of ccRCC cells was induced by TGF-ß. Taken together, these findings suggest that c-Ski suppresses TGF-ß signaling in ccRCC cells, which, in turn, attenuates the tumor-suppressive effect of TGF-ß.

Blocking CXCLs-CXCR2 axis in tumor-stromal interactions contributes to survival in a mouse model of pancreatic ductal adenocarcinoma through reduced cell invasion/migration and a shift of immune-inflammatory microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense stromal reaction (desmoplasia). We have previously reported that mice with conditional KrasG12D mutation and knockout of TGF-ß receptor type II (Tgfbr2), PKF mice, develop PDAC with desmoplasia modulated by CXC chemokines that are produced by PDAC cells through tumor-stromal interaction. In this study, we further discovered that PDAC and cancer-associated fibroblast (CAF) accelerated each other's invasion and migration through the CXC chemokines-receptor (CXCLs-CXCR2) axis. Heterozygous knockout of Cxcr2 in PKF mice (PKF2h mice) prolonged survival and inhibited both tumor angiogenesis and PDAC microinvasion. Infiltration of neutrophils, myeloid-derived suppressor cells (MDSCs), and arginase-1+ M2-like tumor-associated macrophages (TAMs) significantly decreased in the tumors of PKF2h mice, whereas inducible nitric oxide synthase (iNOS)+ M1-like TAMs and apoptotic tumor cells markedly increased, which indicated that blockade of the CXCLs-CXCR2 axis resulted in a shift of immune-inflammatory microenvironment. These results suggest that blocking of the CXCLs-CXCR2 axis in tumor-stromal interactions could be a therapeutic approach against PDAC progression.

SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity.

Glioblastoma is one of the most aggressive forms of cancers and has a poor prognosis. Genomewide analyses have revealed that a set of core signaling pathways, the p53, RB, and RTK pathways, are commonly deregulated in glioblastomas. However, the molecular mechanisms underlying the tumorigenicity of glioblastoma are not fully understood. Here, we show that the lysine deacetylase SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cells, including glioblastoma stem cells. Furthermore, we demonstrate that SIRT2 regulates p73 transcriptional activity by deacetylation of its C-terminal lysine residues. Our results suggest that SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells and that SIRT2 may be a promising molecular target for the therapy of glioblastoma.

Pancreatic tumor microenvironment confers highly malignant properties on pancreatic cancer cells.

Tumor microenvironment plays a pivotal role in cancer progression; however, little is known regarding how differences in the microenvironment affect characteristics of cancer cells. Here, we investigated the effects of tumor microenvironment on cancer cells by using mouse tumor models. After three cycles of inoculation and extraction of human pancreatic cancer cells, including SUIT-2 and Panc-1 cells, from tumors, distinct cancer cell lines were established: 3P cells from the pancreas obtained using the orthotopic tumor model and 3sc cells from subcutaneous tissue obtained using the subcutaneous tumor model. On re-inoculation of these cells, the 3sc cells and, more prominently, the 3P cells, exhibited higher tumorigenic activity than the parental cells. The 3P cells specifically exhibited low E-cadherin expression and high invasiveness, suggesting that they were endowed with the highest malignant characteristics. RNA-sequence analysis demonstrated that distinct signaling pathways were activated in each cell line and that the 3P cells acquired a cancer stem cell-like phenotype. Among cancer stem cell-related genes, those specifically expressed in the 3P cells, including NES, may be potential new targets for cancer therapy. The mechanisms underlying the development of highly malignant cancer cell lines were investigated. Individual cell clones within the parental cells varied in tumor-forming ability, indicating the presence of cellular heterogeneity. Moreover, the tumor-forming ability and the gene expression profile of each cell clone were altered after serial orthotopic inoculations. The present study thus suggests that both selection and education processes by tumor microenvironment are involved in the development of highly malignant cancer cells.

TUFT1 interacts with RABGAP1 and regulates mTORC1 signaling.

The mammalian target of rapamycin (mTOR) pathway is commonly activated in human cancers. The activity of mTOR complex 1 (mTORC1) signaling is supported by the intracellular positioning of cellular compartments and vesicle trafficking, regulated by Rab GTPases. Here we showed that tuftelin 1 (TUFT1) was involved in the activation of mTORC1 through modulating the Rab GTPase-regulated process. TUFT1 promoted tumor growth and metastasis. Consistently, the expression of TUFT1 correlated with poor prognosis in lung, breast and gastric cancers. Mechanistically, TUFT1 physically interacted with RABGAP1, thereby modulating intracellular lysosomal positioning and vesicular trafficking, and promoted mTORC1 signaling. In addition, expression of TUFT1 predicted sensitivity to perifosine, an alkylphospholipid that alters the composition of lipid rafts. Perifosine treatment altered the positioning and trafficking of cellular compartments to inhibit mTORC1. Our observations indicate that TUFT1 is a key regulator of the mTORC1 pathway and suggest that it is a promising therapeutic target or a biomarker for tumor progression.

Dendritic cell vaccine induces antigen-specific CD8+ T cells that are metabolically distinct from those of peptide vaccine and is well-combined with PD-1 checkpoint blockade.

The success of immune checkpoint blockade has unequivocally demonstrated that anti-tumor immunity plays a pivotal role in cancer therapy. Because endogenous tumor-specific T-cell responsiveness is essential for the success of checkpoint blockade, combination therapy with cancer vaccination may facilitate tumor rejection. To select the best vaccine strategy to combine with checkpoint blockade, we compared dendritic cell-based vaccines (DC-V) with peptide vaccines for induction of anti-tumor immunity that could overcome tumor-induced immunosuppression. Using B16 melanoma and B16-specific TCR-transgenic T-cells (pmel-1), we found that DC-V efficiently primed and expanded pmel-1 cells with an active effector and central memory phenotype that were not exhausted. Vaccine-primed cells were metabolically distinct from naïve cells. DC-V-primed pmel-1 cells contained the population that shifted metabolic pathways away from glycolysis to mitochondrial oxidative phosphorylation. They displayed better effector function and proliferated more than those induced by peptide vaccination. DC-V inhibited tumor growth in prophylactic and therapeutic settings. Only DC-V but not peptide vaccine showed augmented anti-tumor activity when combined with anti-PD-1 therapy. Thus, DC-V combined with PD-1 checkpoint blockade mediates optimal anti-cancer activity in this model.