Sequential CAR T cell and targeted alpha immunotherapy in disseminated multiple myeloma.

Multiple myeloma (MM) is still an incurable disorder despite improved antibody and cellular therapies against different MM antigens. Single targeted antigens have so far been ineffective against MM with most patients relapsing after initial response. Hence, sequential immunotherapies directed at different targets are expected to perform better than monotherapy alone. Here, we optimized and established in preclinical studies the therapeutic rationale of using targeted alpha therapy (TAT) directed against CD38 antigen (225Ac-DOTA-daratumumab) with CAR T cell therapy directed at CS1 antigen in a systemic MM model. The sequential therapies compared CAR T therapy followed by TAT to TAT followed by CAR T therapy. CAR T cell monotherapy increased median survival from 49 days (d) in untreated controls to 71d with a modest improvement to 89d for 3.7 kBq of TAT given 14d later. When CAR T was followed by 7.4 kBq of TAT 29d later, sequential therapy increased median survival from 47d in untreated controls to 106d, compared to 68d for CAR T monotherapy. When CAR T therapy was followed by untargeted alpha immunotherapy using 7.4 kBq of 225Ac-DOTA-trastuzumab (anti-HER2) antibody 29d later, there was only a slight improvement in response over CAR T monotherapy demonstrating the role of tumor targeting. TAT (7.4 kBq) followed by CAR T therapy was also effective when CAR T therapy was delayed for 21d vs 14d or 28d post TAT, highlighting the importance of timing sequential therapies. Sequential targeted therapies using CS1 CAR T or 225Ac-DOTA-CD38 TAT in either order shows promise over monotherapies alone.

PEGylated Fluorescent Anti-carcinoembryonic Antigen Antibody Labels Colorectal Cancer Tumors in Orthotopic Mouse Models.

INTRODUCTION: Colorectal cancer (CRC) patients often develop liver metastasis. However, curative resection of liver metastasis is not always possible due to poor visualization of tumor margins. The present study reports the characterization of a humanized anti-carcinoembryonic antigen monoclonal antibody conjugated to a PEGylated near-infrared dye, that targets and brightly labels human CRC tumors in metastatic orthotopic mouse models. METHODS: The hT84.66-M5A (M5A) monoclonal antibody was conjugated with a polyethylene glycol (PEG) chain that incorporated a near infrared (NIR) IR800 dye to establish M5A-IR800 Sidewinder (M5A-IR800-SW). Nude mice with CRC orthotopic primary tumors and liver metastasis both developed from a human CRC cell line, were injected with M5A-IR800-SW and imaged with the Pearl Trilogy Imaging System. RESULTS: M5A-IR800-SW targeted and brightly labeled CRC tumors, both in primary-tumor and liver-metastasis models. M5A-IR800-SW at 75 µg exhibited highly-specific tumor labeling in a primary-tumor orthotopic model with a median tumor-to-background ratio of 9.77 and in a liver-metastasis orthotopic model with a median tumor-to-background ratio of 7.23 at 96 h. The precise labeling of the liver metastasis was due to lack of hepatic accumulation of M5A-IR800-SW in the liver. CONCLUSIONS: M5A-IR800-SW provided bright and targeted NIR images of human CRC in orthotopic primary-tumor and liver-metastasis mouse models. The results of the present study suggest the clinical potential of M5A-IR800-SW for fluorescence-guided surgery including metastasectomies for CRC. The lack of hepatic NIR signal is of critical importance to allow for precise labeling of liver tumors.

Phosphorylation of human CEACAM1-LF by PKA and GSK3ß promotes its interaction with ß-catenin.

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for ß-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bß but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3ß binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3ß able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of ß-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3ß may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with ß-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.

Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in the liver and secreted as biliary glycoprotein 1 (BGP1) via bile canaliculi (BCs). CEACAM1-LF is a 72 amino acid cytoplasmic domain mRNA splice isoform with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ceacam1-/- or Ser503Ala transgenic mice have been shown to develop insulin resistance and nonalcoholic fatty liver disease; however, the role of the human equivalent residue, Ser508, in lipid dysregulation is unknown. Human HepG2 hepatocytes that express CEACAM1 and form BC in vitro were compared with CEACAM1-/- cells and CEACAM1-/- cells expressing Ser508Ala null or Ser508Asp phosphorylation mimic mutations or to phosphorylation null mutations in the tyrosine ITIMs known to be phosphorylated by the tyrosine kinase Src. CEACAM1-/- cells and the Ser508Asp and Tyr520Phe mutants strongly retained lipids, while Ser508Ala and Tyr493Phe mutants had low lipid levels compared with wild-type cells, indicating that the ITIM mutants phenocopied the Ser508 mutants. We found that the fatty acid transporter CD36 was upregulated in the S508A mutant, coexpressed in BCs with CEACAM1, co-IPed with CEACAM1 and Src, and when downregulated via RNAi, an increase in lipid droplet content was observed. Nuclear translocation of CD36 associated kinase LKB1 was increased sevenfold in the S508A mutant versus CEACAM1-/- cells and correlated with increased activation of CD36-associated kinase AMPK in CEACAM1-/- cells. Thus, while CEACAM1-/- HepG2 cells upregulate lipid storage similar to Ceacam1-/- in murine liver, the null mutation Ser508Ala led to decreased lipid storage, emphasizing evolutionary changes between the CEACAM1 genes in mouse and humans.

The short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction.

Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues, which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with the generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens.

Antibody Targeted PET Imaging of 64Cu-DOTA-Anti-CEA PEGylated Lipid Nanodiscs in CEA Positive Tumors.

Lipid nanodiscs (LNDs), comprising a phospholipid bilayer encircled by two molecules of a recombinant membrane scaffold protein, can be targeted to tumors with covalently attached antibodies (Abs) or their fragments. Antibody attachment to click chemistry based PEGylated lipids on LNDs including DOTA allowed PET imaging with the positron emitter 64Cu. Carcinoembryonic antigen (CEA) positive tumors in CEA transgenic mice were chosen as a tumor target. Fab' fragments, that otherwise are rapidly cleared by the kidney due to their small size, were retained in circulation when conjugated to LNDs. Untargeted PET imaging of 64Cu-DOTA-LNDs revealed low tumor uptake (4-5% ID/g) in the range expected for the enhanced permeability retention (EPR) effect with high liver uptake (17-21% ID/g) indicating gut clearance. Fab' targeted LNDs showed little improvement over untargeted LNDs, but intact IgG targeted LNDs gave high tumor uptake (40% ID/g) with low liver (8% ID/g), demonstrating that tumor targeting with antibody conjugated LNDs is feasible.

Copper 64-labeled daratumumab as a PET/CT imaging tracer for multiple myeloma.

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (18F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.

Synthesis and evaluation of 18F-PTTCO-Cys40-Exendin-4 for PET imaging of ectopic insulinomas in rodents.

A major limitation in the development of radiolabeled Exendin-4 analogues (short half-life isotopes) is an inability to efficiently and rapidly separate final products from precursors. This is important as lack of purity in the final product decreases probe efficiency. The purpose of this study was to develop a method to prepare the high-purity imaging reagent [18F] PTTCO-Cys40-Exendin-4. To accomplish this, magnetic TCO-beads were incubated with the crude product to remove unlabeled Exendin-4. In rodents pre-treatment with purified [18F] PTTCO-Cys40-Exendin-4 (~1.85 MBq) allowed precise microPET imaging of ectopic insulinomas. Moreover, analogue uptake was successfully blocked by administering non-labelled "cold" Exendin-4. Biodistribution data revealed that [18F] PTTCO-Cys40-Exendin-4 accumulated specifically in GLP-1R-enriched insulinomas in mice, confirming results obtained using miroPET. Investigation of [18F] PTTCO-Cys40-Exendin-4 as a tracer to image portal vein-transplanted pancreatic islets is proceeding in animals.

Interferon regulatory factor 1 and a variant of heterogeneous nuclear ribonucleoprotein L coordinately silence the gene for adhesion protein CEACAM1.

The adhesion protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is widely expressed in epithelial cells as a short cytoplasmic isoform (S-iso) and in leukocytes as a long cytoplasmic isoform (L-iso) and is frequently silenced in cancer by unknown mechanisms. Previously, we reported that interferon response factor 1 (IRF1) biases alternative splicing (AS) to include the variable exon 7 (E7) in CEACAM1, generating long cytoplasmic isoforms. We now show that IRF1 and a variant of heterogeneous nuclear ribonucleoprotein L (Lv1) coordinately silence the CEACAM1 gene. RNAi-mediated Lv1 depletion in IRF1-treated HeLa and melanoma cells induced significant CEACAM1 protein expression, reversed by ectopic Lv1 expression. The Lv1-mediated CEACAM1 repression resided in residues Gly71-Gly89 and Ala38-Gly89 in Lv1's N-terminal extension. ChIP analysis of IRF1- and FLAG-tagged Lv1-treated HeLa cells and global treatment with the global epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A indicated that IRF1 and Lv1 together induce chromatin remodeling, restricting IRF1 access to the CEACAM1 promoter. In interferon γ-treated HeLa cells, the transcription factor SP1 did not associate with the CEACAM1 promoter, but binding by upstream transcription factor 1 (USF1), a known CEACAM1 regulator, was greatly enhanced. ChIP-sequencing revealed that Lv1 overexpression in IRF1-treated cells induces transcriptional silencing across many genes, including DCC (deleted in colorectal carcinoma), associated with CEACAM5 in colon cancer. Notably, IRF1, but not IRF3 and IRF7, affected CEACAM1 expression via translational repression. We conclude that IRF1 and Lv1 coordinately regulate CEACAM1 transcription, alternative splicing, and translation and may significantly contribute to CEACAM1 silencing in cancer.

CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes.

BACKGROUND: Systemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1ß. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1ß in murine neutrophils through its ITIM receptor. RESULTS: We now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1-/- mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G-) express IL-6 in the early response (< 2 h) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment perhaps due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and ß-actin in response to LPS. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and ß-actin from RP105. CONCLUSION: This novel pathway and regulation of IL-6 signaling by CEACAM1 defines a novel role for monocytes in the fever response of mice to LPS.

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

BACKGROUND: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. METHODS: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. RESULTS: The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. CONCLUSION: dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Carcinoembryonic antigen-related cell adhesion molecule-1 regulates granulopoiesis by inhibition of granulocyte colony-stimulating factor receptor.

Although carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is an activation marker for neutrophils and delays neutrophil apoptosis, the role of CEACAM1 in granulopoiesis and neutrophil-dependent host immune responses has not been investigated. CEACAM1 expression correlated with granulocytic differentiation, and Ceacam1(-/-) mice developed neutrophilia because of loss of the Src-homology-phosphatase-1 (SHP-1)-dependent inhibition of granulocyte colony-stimulating factor receptor (G-CSFR) signal transducer and activator of transcription (Stat3) pathway provided by CEACAM1. Moreover, Ceacam1(-/-) mice were hypersensitive to Listeria Monocytogenes (LM) infection with an accelerated mortality. Reintroduction of CEACAM1 into Ceacam1(-/-) bone marrow restored normal granulopoiesis and host sensitivity to LM infection, while mutation of its immunoreceptor tyrosine-based inhibitory motifs (ITIMs) abrogated this restoration. shRNA-mediated reduction of Stat3 amounts rescued normal granulopoiesis, attenuating host sensitivity to LM infection in Ceacam1(-/-) mice. Thus, CEACAM1 acted as a coinhibitory receptor for G-CSFR regulating granulopoiesis and host innate immune response to bacterial infections.

Serum-derived carcinoembryonic antigen (CEA) activates fibroblasts to induce a local re-modeling of the extracellular matrix that favors the engraftment of CEA-expressing tumor cells.

Elevated levels of the carcinoembryonic antigen (CEA; CEACAM5) in the serum of colorectal cancer (CRC) patients represent a clinical biomarker that correlates with disease recurrence. However, a mechanistic role for soluble CEA (sCEA) in tumor progression and metastasis remains to be established. In our study, we report that sCEA acts as a paracrine factor, activating human fibroblasts by signaling through both the STAT3 and AKT1-mTORC1 pathways, promoting their transition to a cancer-associated fibroblast (CaF) phenotype. sCEA-activated fibroblasts express and secrete higher levels of fibronectin, including cellular EDA+ -fibronectin (Fn-EDA) that selectively promote the implantation and adherence of CEA-expressing cancer cells. Immunohistochemical analyses of liver tissues derived from CRC patients with elevated levels of sCEA reveal that the expression of cellular Fn-EDA co-registers with CEA-expressing liver metastases. Taken together, these findings indicate a direct role for sCEA as a human fibroblast activation factor, in priming target tissues for the engraftment of CEA-expressing cancer cells, through the differentiation of tissue-resident fibroblasts, resulting in a local change in composition of the extracellular matrix.

ETS transcription factor ELF5 induces lumen formation in a 3D model of mammary morphogenesis and its expression is inhibited by Jak2 inhibitor TG101348.

The loss of expression of a single gene can revert normal tissue to a malignant phenotype. For example, while normal breast has high lumenal expression of CEACAM1, the majority of breast cancers exhibit the early loss of this gene with the concurrent loss of their lumenal phenotype. MCF7 cells that lack CEACAM1 expression and fail to form lumena in 3D culture, regain the normal phenotype when transfected with CEACAM1. In order to probe the mechanism of this gain of function, we treated these cells with the clinically relevant Jak2 inhibitor TG101348 (TG), expecting that disruption of the prolactin receptor signaling pathway would interfere with the positive effects of transfection of MCF7 cells with CEACAM1. Indeed, lumen formation was inhibited, resulting in the down regulation of a set of genes, likely involved in the complex process of lumen formation. As expected, inhibition of the expression of many of these genes also inhibited lumen formation, confirming their involvement in a single pathway. Among the genes identified by the inhibition assay, ETS transcription factor ELF5 stood out, since it has been identified as a master regulator of mammary morphogenesis, and is associated with prolactin receptor signaling. When ELF5 was transfected into the parental MCF7 cells that lack CEACAM1, lumen formation was restored, indicating that ELF5 can replace CEACAM1 in this model system of lumenogenesis. We conclude that the event(s) that led to the loss of expression of CEACAM1 is epistatic in that multiple genes associated with a critical pathway were affected, but that restoration of the normal phenotype can be achieved with reactivation of certain genes at various nodal points in tissue morphogenesis.

The adaptor SASH1 acts through NOTCH1 and its inhibitor DLK1 in a 3D model of lumenogenesis involving CEACAM1.

CEACAM1 transfection into breast cancer cells restores lumen formation in a 3D culture model. Among the top up-regulated genes that were associated with restoration of lumen formation, the adaptor protein SASH1 was identified. Furthermore, SASH1 was shown to be critical for lumen formation by RNAi inhibition. Upon analyzing the gene array from CEACAM1/MCF7 cells treated with SASH1 RNAi, DLK1, an inhibitor of NOTCH1 signaling, was found to be down-regulated to the same extent as SASH1. Subsequent treatment of CEACAM1/MCF7 cells with RNAi to DLK1 also inhibited lumen formation, supporting its association with SASH1. In agreement with the role of DLK1 as a NOTCH1 inhibitor, NOTCH1, as well as its regulated genes HES1 and HEY1, were down-regulated in CEACAM1/MCF7 cells by the action of DLK1 RNAi, and up-regulated by SASH1 RNAi. When CEACAM1/MCF7 cells were treated with a γ-secretase inhibitor known to inhibit NOTCH signaling, lumen formation was inhibited. We conclude that restoration of lumen formation by CEACAM1 regulates the NOTCH1 signaling pathway via the adaptor protein SASH1 and the NOTCH1 inhibitor DLK1. These data suggest that the putative involvement of NOTCH1 as a tumor-promoting gene in breast cancer may depend on its lack of regulation in cancer, whereas its involvement in normal lumen formation requires activation of its expression, and subsequently, inhibition of its signaling.

Induction of Lumen Formation in a Three-dimensional Model of Mammary Morphogenesis by Transcriptional Regulator ID4: ROLE OF CaMK2D IN THE EPIGENETIC REGULATION OF ID4 GENE EXPRESSION.

Concomitant loss of lumen formation and cell adhesion protein CEACAM1 is a hallmark feature of breast cancer. In a three-dimensional culture model, transfection of CEACAM1 into MCF7 breast cells can restore lumen formation by an unknown mechanism. ID4, a transcriptional regulator lacking a DNA binding domain, is highly up-regulated in CEACAM1-transfected MCF7 cells, and when down-regulated with RNAi, abrogates lumen formation. Conversely, when MCF7 cells, which fail to form lumena in a three-dimensional culture, are transfected with ID4, lumen formation is restored, demonstrating that ID4 may substitute for CEACAM1. After showing the ID4 promoter is hypermethylated in MCF7 cells but hypomethylated in MCF/CEACAM1 cells, ID4 expression was induced in MCF7 cells by agents affecting chromatin remodeling and methylation. Mechanistically, CaMK2D was up-regulated in CEACAM1-transfected cells, effecting phosphorylation of HDAC4 and its sequestration in the cytoplasm by the adaptor protein 14-3-3. CaMK2D also phosphorylates CEACAM1 on its cytoplasmic domain and mutation of these phosphorylation sites abrogates lumen formation. Thus, CEACAM1 is able to maintain the active transcription of ID4 by an epigenetic mechanism involving HDAC4 and CaMK2D, and the same kinase enables lumen formation by CEACAM1. Because ID4 can replace CEACAM1 in parental MCF7 cells, it must act downstream from CEACAM1 by inhibiting the activity of other transcription factors that would otherwise prevent lumen formation. This overall mechanism may be operative in other cancers, such as colon and prostate, where the down-regulation of CEACAM1 is observed.

miRNA-342 Regulates CEACAM1-induced Lumen Formation in a Three-dimensional Model of Mammary Gland Morphogenesis.

Lumen formation of breast epithelium is rapidly lost during tumorigenesis along with expression of cell adhesion molecule CEACAM1. CEACAM1 induces lumena in a three-dimensional culture of MCF7/CEACAM1 cells that otherwise fail to form lumena. We hypothesized miRNAs may be involved because >400 genes were up- or down-regulated in MCF7/CEACAM1 cells and miRNAs may modify global expression patterns. Comparative analysis of miRNA expression in MCF7 versus MCF7/CEACAM1 cells revealed two miRNAs significantly down-regulated (hsa-miR-30a-3p by 6.73-fold and hsa-miR-342-5p by 5.68-fold). Location of miR-342 within an intron of the EVL gene, hypermethylated and involved in tumorigenesis, suggested that miR-342 overexpression may block lumen formation. In fact, overexpression of miR-342 in MCF7/CEACAM1 cells significantly blocked lumen formation (p < 0.001). ID4, a dominant-negative inhibitor of basic helix-loop-helix transcription factors, up-regulated in MCF7/CEACAM1 cells, down-regulated in breast cancer, and containing a miR-342 binding site, was tested as a potential target of miR-342. The ratio of ID4 to miR-342 increased from 1:2 in MCF7 cells to 30:1 in MCF7/CEACAM1 cells and a miR-342 inhibitor was able to induce 3'-UTR ID4 reporter activity in MCF7 cells. Because 5-methylcytosine methyltransferase DNMT1 is also a potential target of miR-342, we inhibited miR-342 in MCF7 cells and found DNMT1 was up-regulated with no change in EVL expression, suggesting that miR-342 regulates DNMT1 expression but DNMT1 does not affect the EVL expression in these cells. We conclude that the regulation of lumen formation by miR-342 involves at least two of its known targets, namely ID4 and DNMT1.

Characterization of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy(polyethylene glycerol)-2000] and Its Complex with Doxorubicin Using Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics.

Polyethylene glycol (PEG) lipid nanoparticles (LNPs) spontaneously assemble in water, forming uniformly sized nanoparticles incorporating drugs with prolonged blood clearance compared to drugs alone. Previously, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycerol)-2000] (DSPE-PEG2000) and several drug adducts, including doxorubicin, were analyzed by a combination of physical and molecular dynamic (MD) studies. In this study, a complete chemical shift assignment of DSPE-PEG2000 plus or minus doxorubicin was achieved using nuclear magnetic resonance (NMR), one-dimensional selective nuclear Overhauser spectroscopy (1D-selNOESY), NOESY, correlation spectroscopy (COSY), total correlated spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and HSQC-TOCSY. Chemical shift perturbation, titration, relaxation enhancement, and NOESY analysis combined with MD reveal detailed structural information at the atomic level, including the location of doxorubicin in the micelle, its binding constant, the hydrophilic shell organization, and the mobility of the PEG2000 tail, demonstrating that NMR spectroscopy can characterize drug-DSPE-PEG2000 micelles with molecular weights above 180 kDa. The MD study revealed that an initial spherical organization led to a more-disorganized oblate structure in an aqueous environment and agreed with the NMR study in the details of the fine structure, in which methyl group(s) of the stearic acid in the hydrophobic core of the micelle are in contact with the phosphate headgroup of the lipid. Although the molecular size of the LNP drug complex is about 180 kDa, atomic resolution can be achieved by NMR-based methods that reveal distinct features of the drug-lipid interactions. Because many drugs have unfavorable blood clearance that may benefit from incorporation into LNPs, a thorough knowledge of their physical and chemical properties is essential to moving them into a clinical setting. This study provides an advanced basic approach that can be used to study a wide range of drug-LNP interactions.

CEACAM1 is associated with recurrence after hepatectomy for colorectal liver metastasis.

BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is re-expressed at the invasion front of colorectal cancer. CEACAM1 expression at metastatic sites remains to be investigated. The current study aims to clarify the association between CEACAM1 expression and recurrence after hepatectomy of colorectal liver metastasis and to address whether CEACAM1 induces tumor-initiating properties needed for growth at metastatic sites. METHODS: Immunohistochemical analyses for CEACAM1 were performed in 67 patients with liver metastasis of colorectal cancer who had undergone curative hepatectomy. The risk factors for postoperative recurrence were calculated based on a CEACAM1 cytoplasmic domain isoform at the primary tumor invasion front. To investigate the effects of CEACAM1 cytoplasmic isoforms on HT29 and HCT116 colorectal cancer cells, Western blotting for CD44 and CD133, flow cytometry for ALDH1 activity, and soft-agar colony formation assay were performed. RESULTS: CEACAM1 long (CEACAM1-L) and short (CEACAM1-S) cytoplasmic domain isoforms are strongly expressed on cancer cells in the liver metastases. Enhanced CEACAM1-S expression in the state of CEACAM1-L dominance at the primary tumor invasion front was an independent factor for colorectal cancer recurrence after curative hepatectomy. CEACAM1-4S-transfected HT29 and HCT116 cells had significantly higher CD44 expression and ALDH1 activity and increased the growth in anchorage-independent condition. CONCLUSIONS: High expression of CEACAM1-S at the primary lesion invasion front is associated with recurrence and prognosis of patients with colorectal liver metastasis after curative hepatectomy. The expression of CEACAM1-4S enhances the tumor-initiating property of colorectal cancer cells.

Induction of antigen-specific TH 9 immunity accompanied by mast cell activation blocks tumor cell engraftment.

The engraftment of circulating cancer cells at distal sites represents a key step in the metastatic cascade, yet remains an unexplored target for therapeutic intervention. In this study, we establish that a vaccination strategy yielding an antigen-specific TH 9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV-like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA-specific TH 9 response, wherein IL-9 secreting TH cells act in concert with CEA N domain-specific antibodies as well as activated mast cells in preventing tumor cell engraftment. The development of this immune response was dependent on TLR3, since interference with the TLR3-dsRNA complex formation led to a reduction in vaccine-imparted protection and a shift in the resulting immune response toward a TH 2 response. These findings point to the existence of an alternate tumor targeting immune mechanism that can be exploited for the purpose of developing vaccine therapies targeting tumor dissemination and engraftment.