SUMMARY The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a “normal” human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor micro-tubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

The transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC-cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. IntroductionCellular uptake of cobalamin (Cbl, vitamin B 12 ) in mammalian cells is mediated by receptors expressed on the cell surface. 1 Transcobalamin (TC, TCII), a plasma protein secreted by the endothelial cells, binds the Cbl absorbed in the distal ileum and carries between 10% and 30% of the total circulating Cbl. 2,3 TC saturated with Cbl specifically binds to the receptor (TCblR) and is internalized by endocytosis. The TC is degraded in the lysosome, and the free Cbl released is converted into Cbl cofactors. 4,5 Methylcobalamin is a cofactor for the cytosolic enzyme methionine synthase in the conversion of homocysteine to methionine using N 5 -methyltetrahydrofolate 6 ; therefore, homocysteine is elevated in both Cbl and folate deficiency. 7 The mitochondrial enzyme methylmalonyl CoA mutase converts methylmalonyl CoA to succinyl CoA and requires 5Јdeoxyadenosylcobalamin as a cofactor. The elevated methylmalonic acid in Cbl deficiency is a direct consequence of a block in this pathway. 8 The definitive purification of TC 9 followed by the identification of vascular endothelium as the source of TC in blood 10 ultimately led to the cloning of the cDNA and the gene encoding this protein. 11,12 Attempts to purify the receptor have yielded ambiguous results 13,14 ; however, the functional properties of TCblR have been well characterized in cell culture models. 15,16 We have previously described the functional and structural properties of TCblR based on binding of TC-Cbl to TCblR from human placenta and by crosslinking studies. 17,18 Our data on the properties and structure of this receptor differ from 2 other reports describing the purification of this protein. 13,14 The report by Bose et al 14 described a receptor with different structural constituents. Since their first report, numerous publications by this group have described the structural and functional characterization of a putative receptor from human placenta. [19][20][21][22][23][24][25] However, they did not establish the functional specificity of their receptor for TC-Cbl and have not identified the primary structure and the gene encoding the receptor. This report describes the purification and definitive identification of the primary structure and the gene encoding a receptor for the cellular uptake of TC-Cbl. This unique receptor has the specificity and affinity required for the cellular uptake of holo TC and differs from the 72/144 kDa monomer/dimer protein reported to be the receptor for TC-Cbl. 14 MethodsActigel ALD agarose (Sterogene Bioseparations, Carlsbad, CA), Ultralink matri...

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Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function.

The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01-0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected.

The membrane receptor TCblR/CD320 binds transcobalamin (TC) saturated with vitamin B12 [cobalamin (Cbl)] and mediates cellular uptake of the vitamin. The specificity of TC for Cbl and of the receptor for TC-Cbl ensures efficient uptake of Cbl into cells. The high-affinity interaction of TCblR with TC-Cbl (Ka=10 nM(-1)) was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extracellular region (aa 32-229) is needed for TC-Cbl binding, but the N-glycosylation sites (N126, N195, and N213) are of no importance for this function. Deleting the cysteine-rich region (aa 95-141) that separates the two low-density lipoprotein receptor type A (LDLR-A) domains does not affect TC-Cbl binding (Ka = 19-24 nM(-1)). The two LDLR-A domains (aa 54-89 and 132-167) with the negatively charged acidic residues involved in Ca(2+) binding are critical determinants of ligand binding. The cytoplasmic tail is apparently crucial for internalization of the ligand. Within this region, the RPLGLL motif and the PDZ binding motifs (QERL/KESL) appear to be involved in initiating and completing the process of ligand internalization. Mutations and deletions of these regions involved in binding and internalization of TC-Cbl are likely to produce the biochemical and clinical phenotype of Cbl deficiency.

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Cellular uptake of cobalamin (Cbl) occurs by endocytosis of transcobalamin saturated with Cbl by the transcobalamin receptor (TCblR/CD320). The cell cycle-associated overexpression of this receptor in many cancer cells provides a suitable target for delivering chemotherapeutic drugs and cytotoxic molecules to these cells while minimizing the effect on the normal cell population. We have used monoclonal antibodies to the extracellular domain of TCblR to deliver saporin-conjugated secondary antibody to various cell lines propagating in culture. A molar ratio of 2.5:10 nmol/L of primary:secondary antibody concentration was identified as the lowest concentration needed to produce the optimum cytotoxic effect. The effect was more pronounced when cells were seeded at lower density, suggesting lack of cell division in a fraction of the cells at higher density as the likely explanation. Cells in suspension culture, such as K562 and U266 cells, were more severely affected than adherent cultures, such as SW48 and KB cells. This differential effect of the anti-TCblR-saporin antibody conjugate and the ability of an anti-TCblR antibody to target proliferating cells were further evident by the virtual lack of any effect on primary skin fibroblasts and minimal effect on bone marrow cells. These results indicate that preferential targeting of some cancer cells could be accomplished through the TCblR.Mol Cancer Ther; 9(11); 3033-40. ©2010 AACR.