Influence of EE on NK population in the peripheral blood and spleen of tumor-bearing mice.

Supplementary figures 1-8. Supplementary Figure 1. The amino acid sequence of scFv GC33 in the construct of anti-GPC3 CAR. Supplementary Figure 2. The expression of CD64 and CD86 in the artificial antigen-presenting cell aK562-64/86. Supplementary Figure 3. Tumor outgrowth of Huh-7 cells was completely abolished by GPC3-targted CAR T cells in vivo. Supplementary Figure 4. Growth suppression on established s.c. HCC xenografts by GPC3-targeted CAR T cells. Supplementary Figure 5. Mice bearing orthotopic Huh-7 tumors. Supplementary Figure 6. The up-regulation of Bcl-XL protein in αGPC3-28BBZ CAR T cells was stimulated by GPC3-specific antigen. Supplementary Figure 7. The GPC3 expression in the normal kidney and gastric glands. Supplementary Figure 8. The homogeneity of GPC3 expression was analyzed in the human primary HCC tissues (n=75).

Exposure to EE reduces anxiety-like behavior, decreases body weight, and increases food intake in mice.

EE prevents experimental lung metastases of murine melanoma cells.

Primers used in quantitative real-time PCR analysis.

EE increases the expression levels of INF-gamma and granzyme B in NK cells.

EE decreases serum leptin levels in wild-type and Beige mice.

Supplementary table. Primer sequences used for construction of the different CARs using the splicing PCR by overlap extension technique.

To explore the mechanism of co-evolution and potential driver of which in pancreatic ductal adenocarcinoma (PDAC) metastasis to liver, we studied key molecules involved in this progress and their translational values. Pre-metastatic niche (PMN) and macro metastatic niche (MMN) formation in mouse model were recognized via CT combined 3D organ reconstruction bioluminescence imaging. We next confirmed the expressions and distributions of SLIT2 and ROBO1 in 35 cases of human matched liver metastasis and primary PDAC samples, 14 case human PDAC liver metastasis transcriptional analysis, intrasplenic mouse models and Kras G12D / Trp53 R172H / Pdx1 -Cre (KPC) mouse models. Translational value was assessed on Slit2 fl/fl / Alb 1 -Cre ( Slit2 CKO) mice, KPC mouse model and Ex vivo tests via administration of neutralizing antibody targeting ROBO1. We also analyzed prognosis of 266 cases human PDAC tissue with or without SLIT2-ROBO1 fostered co-evolution and demonstrated the dependence receptor (DR) characteristics of ROBO1 in the following-up mechanism study. Experiments on Slit2 CKO, Slit2 CKO-RE and KPC mouse models demonstrated that disturbing SLIT2-ROBO1 mediated co-evolution in liver microenvironment via preventing their interaction could significantly attenuate liver metastasis of PDAC. We have demonstrated that co-evolution took advantage of DR characteristics in PMN and MMN. Targeting SLIT2-ROBO1 axis could be a therapeutic strategy towards metastatic PDAC.

EE does not appear to influence the expression levels of the ligands of NKG2D and CCR5 in tumors.

PDF file - 734K, Figure S1. EGFRvA is widely expressed in various cancer cell lines and tissues. Figure S2. The upregulation of EGFRvA in glioma tissues compared with paired adjacent non-neoplastic brain tissues and a poor prognosis in patients with high-grade gliomas. Figure S3. EGFRvA promotes cell migration and invasion in vitro and in vivo. Figure S4. EGFRvA caused the activation of STAT3 and increased expression of HB-EGF. Figure S5. The positive feedback regulation between HB-EGF and p-STAT3 in EGFRvA-expressing cells. Figure S6. Cell viability and cell adhesion of U87MG EGFRvA cells treated with STAT3 inhibitor AG490, and siRNA-mediated knockdown of STAT3 in U87MG transfectants. Table S1. Clinical characteristics of patients with low or high expression of EGFR or EGFRvA in 52 glioma patients. Table S2. Higher expressed genes in U87MG EGFRvA cells versus U87MG EGFR cells. Table S3. Lower expressed genes in U87MG EGFRvA cells versus U87MG EGFR cells. Table S4. Primers used in this study.

Supplementary Materials and Methods.

The development of resistance to anticancer drugs is believed to cause chemotherapy failure in pancreatic cancer (PC). The efflux of anticancer drugs mediated by ATP-binding cassette (ABC) transporters is a widely accepted mechanism for chemoresistance, but for ABCA subfamily members, which are characterized by their ability to transport lipids and cholesterol, its role in chemoresistance remains unknown. Here we found that the expression of ABCA8, a member of ABCA subfamily transporters, was significantly increased in human PC cells after gemcitabine (GEM) treatment, as well as in established GEM-resistant (Gem-R) PC cells. Importantly, ABCA8 knockdown reversed the chemoresistance phenotype of Gem-R cells, whereas ABCA8 overexpression significantly decreased the sensitivity of human PC cells to GEM, both in vitro and in vivo, demonstrating an important role of ABCA8 in regulating chemosensitivity. Moreover, our results showed that treatment with taurocholic acid (TCA), an endogenous substrate of ABCA8, also induced GEM insensitivity in PC cells. We further demonstrated that ABCA8 mediates the efflux of TCA out of PC cells, and that extracellular TCA activates extracellular signal-regulated kinase (ERK) signaling via the sphingosine 1-phosphate receptor 2 (S1PR2), which is responsible for ABCA8-induced GEM ineffectiveness. Together, these findings reveal a novel TCA-related mechanism of ABCA subfamily transporter-mediated chemoresistance that goes beyond the role of a drug pump and suggest ABCA8 or the TCA-S1RP2-ERK pathway as potential targets for improving the effectiveness of and overcoming the resistance to chemotherapy in PC.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a malignant disease with a poor prognosis. One prominent aspect of PDAC that contributes to its aggressive behavior is its altered cellular metabolism. The aim of this study was to characterize the oncogenic effects of ubiquinol-cytochrome c reductase core protein I (UQCRC1), a key component of mitochondrial complex III, in PDAC development and to assess its potential as a therapeutic target for PDAC. Experimental Design: The expression of UQCRC1 in human PDAC tissues and p48-Cre/p53Flox/WT/LSL-KrasG12D (KPC) mouse pancreatic intraepithelial neoplasias (PanINs) was determined by immunohistochemistry. The role of UQCRC1 in promoting PDAC growth was evaluated in vitro in PANC-1 and CFPAC-1 cells and in vivo in transplanted mouse models of PDAC. Extracellular flux and RNA-Seq analyses were applied to investigate the mechanism of UQCRC1 in the regulation of mitochondrial metabolism and PDAC cell growth. The therapeutic potential of UQCRC1 in PDAC was assessed by knockdown of UQCRC1 using an RNA interference approach. Results: UQCRC1 expression showed a gradual increase during the progression from PanIN stages to PDAC in KPC mice. Elevated expression of UQCRC1 was observed in 72.3% of PDAC cases and was correlated with poor prognosis of the disease. UQCRC1 promoted PDAC cell growth in both in vitro experiments and in vivo subcutaneous and orthotopic mouse models. UQCRC1 overexpression resulted in increased mitochondrial oxidative phosphorylation (OXPHOS) and ATP production. The overproduced ATP was released into the extracellular space via the pannexin 1 channel and then functioned as an autocrine or paracrine agent to promote cell proliferation through the ATP/P2Y2-RTK/AKT axis. UQCRC1 knockdown or ATP release blockage could effectively inhibit PDAC growth. Conclusion: UQCRC1 has a protumor function and may serve as a potential prognostic marker and therapeutic target for PDAC.

The overarching view of current tumor therapies simplifies cancer to a cell-biology problem in which neoplasms are caused solely by malignant cells and the exploration of carcinogenesis and tumor progression largely focuses on somatic mutations and other genetic abnormalities of cancer cells. The limited therapeutic response indicates that cancer is driven not only by endogenous oncogenic factors and reciprocal interactions within the tumor microenvironment, but also by complex systemic processes. Homeostasis is the fundamental premise of health, and is maintained by systemic regulation of neuro-endocrine-immune axis. Cancer is also a systemic disease that manifested by dysfunction of the nervous, endocrine, and immune systems. Multiple axes of regulation exist in cancer, including central-, organ-, and microenvironment-level manipulation. At each specific regulatory level, the tridirectional communication among the nervous, endocrine, and immune factors transmit flexible signaling to induce proliferation, invasion, reprogrammed metabolism, therapeutic resistance, and other malignant phenotypes of cancer cells, resulting in the extremely poor prognosis of this lethal disease. Understanding this coordinated signaling network will enable the development of new approaches for cancer treatment via behavioral and pharmacological interventions.

The overarching view of current tumor therapies simplifies cancer to a cell-biology problem in which neoplasms are caused solely by malignant cells and the exploration of carcinogenesis and tumor progression largely focuses on somatic mutations and other genetic abnormalities of cancer cells. The limited therapeutic response indicates that cancer is driven not only by endogenous oncogenic factors and reciprocal interactions within the tumor microenvironment, but also by complex systemic processes. Homeostasis is the fundamental premise of health, and is maintained by systemic regulation of neuro-endocrine-immune axis. Cancer is also a systemic disease that manifested by dysfunction of the nervous, endocrine, and immune systems. Multiple axes of regulation exist in cancer, including central-, organ-, and microenvironment-level manipulation. At each specific regulatory level, the tridirectional communication among the nervous, endocrine, and immune factors transmit flexible signaling to induce proliferation, invasion, reprogrammed metabolism, therapeutic resistance, and other malignant phenotypes of cancer cells, resulting in the extremely poor prognosis of this lethal disease. Understanding this coordinated signaling network will enable the development of new approaches for cancer treatment via behavioral and pharmacological interventions.

Background: Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood. Methods: In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression. Results: COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor. Conclusion: COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC.

Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells in vitro and MPC1 silencing enhanced liver metastases in vivo . Furthermore, we uncovered that decreased MPC1 activated the Wnt/ β -catenin pathway by promoting nuclear translocation of β -catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.