Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery.

Glioblastoma multiforme (GBM) is one of the most intractable of human cancers, principally because of the highly infiltrative nature of these neoplasms. Tracking and eradicating infiltrating GBM cells and tumor microsatellites is of utmost importance for the treatment of this devastating disease, yet effective strategies remain elusive. Here we report polymeric nanoparticle-engineered human adipose-derived stem cells (hADSCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as drug-delivery vehicles for targeting and eradicating GBM cells in vivo. Our results showed that polymeric nanoparticle-mediated transfection led to robust up-regulation of TRAIL in hADSCs, and that TRAIL-expressing hADSCs induced tumor-specific apoptosis. When transplanted in a mouse intracranial xenograft model of patient-derived glioblastoma cells, hADSCs exhibited long-range directional migration and infiltration toward GBM tumor. Importantly, TRAIL-overexpressing hADSCs inhibited GBM growth, extended survival, and reduced the occurrence of microsatellites. Repetitive injection of TRAIL-overexpressing hADSCs significantly prolonged animal survival compared with single injection of these cells. Taken together, our data suggest that nanoparticle-engineered TRAIL-expressing hADSCs exhibit the therapeutically relevant behavior of "seek-and-destroy" tumortropic migration and could be a promising therapeutic approach to improve the treatment outcomes of patients with malignant brain tumors.

Matrix Stiffness Modulates Patient-Derived Glioblastoma Cell Fates in Three-Dimensional Hydrogels.

Cancer progression is known to be accompanied by changes in tissue stiffness. Previous studies have primarily employed immortalized cell lines and 2D hydrogel substrates, which do not recapitulate the 3D tumor niche. How matrix stiffness affects patient-derived cancer cell fate in 3D remains unclear. In this study, we report a matrix metalloproteinase-degradable poly(ethylene-glycol)-based hydrogel platform with brain-mimicking biochemical cues and tunable stiffness (40-26,600 Pa) for 3D culture of patient-derived glioblastoma xenograft (PDTX GBM) cells. Our results demonstrate that decreasing hydrogel stiffness enhanced PDTX GBM cell proliferation, and hydrogels with stiffness 240 Pa and below supported robust PDTX GBM cell spreading in 3D. PDTX GBM cells encapsulated in hydrogels demonstrated higher drug resistance than 2D control, and increasing hydrogel stiffness further enhanced drug resistance. Such 3D hydrogel platforms may provide a valuable tool for mechanistic studies of the role of niche cues in modulating cancer progression for different cancer types.

GTP cyclohydrolase I activity from Rickettsia monacensis strain Humboldt, a rickettsial endosymbiont of Ixodes pacificus.

The complete folate biosynthesis pathway exists in the genome of a rickettsial endosymbiont of Ixodes pacificus, Rickettsia monacensis strain Humboldt (formerly known as Rickettsia species phylotype G021). Recently, our lab demonstrated that the folA gene of strain Humboldt, the final gene in the folate biosynthesis pathway, encodes a functional dihydrofolate reductase enzyme. In this study, we report R. monacensis strain Humboldt has a functional GTP cyclohydrolase I (GCH1), an enzyme required for the hydrolysis of GTP to form 7,8-dihydroneopterin triphosphate in the folate biosynthesis pathway. The GCH1 gene of R. monacensis, folE, share homology with the folE gene of R. monacensis strain IrR/Munich, with a nucleotide sequence identity of 99%. Amino acid alignment and comparative protein structure modeling have shown that the FolE protein of R. monacensis has a conserved core subunit of GCH1 from the T-fold structural superfamily. All amino acid residues, including conserved GTP binding sites and zinc binding sites, are preserved in the FolE protein of R. monacensis. A recombinant GST-FolE protein from R. monacensis was overexpressed in Escherichia coli, purified by affinity chromatography, and assayed for enzyme activity in vitro. The in vitro enzymatic assay described in this study accorded the recombinant GCH1 enzyme of R. monacensis with a specific activity of 0.81 U/mg. Our data suggest folate genes of R. monacensis strain Humboldt have the potential to produce biochemically active enzymes for de novo folate synthesis, addressing the physioecological underpinnings behind tick-Rickettsia symbioses.

A comparative study of brain tumor cells from different age and anatomical locations using 3D biomimetic hydrogels.

Brain tumors exhibit vast genotypic and phenotypic diversity depending on patient age and anatomical location. Hydrogels hold great promise as 3D in vitro models for studying brain tumor biology and drug screening, yet previous studies were limited to adult glioblastoma cells, and most studies used immortalized cell lines. Here we report a hydrogel platform that supports the proliferation and invasion of patient-derived brain tumor cell cultures (PDCs) isolated from different patient age groups and anatomical locations. Hydrogel stiffness was tuned by varying poly(ethylene-glycol) concentration. Cell adhesive peptide (CGRDS), hyaluronic acid, and MMP-cleavable crosslinkers were incorporated to facilitate cell adhesion and cell-mediated degradation. Three PDC lines were compared including adult glioblastoma cells (aGBM), pediatric glioblastoma cells (pGBM), and diffuse pontine intrinsic glioma (DIPG). A commonly used immortalized adult glioblastoma cell line U87 was included as a control. PDCs displayed stiffness-dependent behavior, with 40 Pa hydrogel promoting faster tumor proliferation and invasion. Adult GBM cells exhibited faster proliferation than pediatric GBM, and DIPG showed slowest proliferation. These results suggest both patient age and tumor location affects brain tumor behaviors. Adult GBM PDCs also exhibited very different cell proliferation and morphology from U87. The hydrogel reported here can provide a useful tool for future studies to better understand how age and anatomical locations impacts brain tumor progression using 3D in vitro models.

The folA gene from the Rickettsia endosymbiont of Ixodes pacificus encodes a functional dihydrofolate reductase enzyme.

Although nonpathogenic bacterial endosymbionts have been shown to contribute to their arthropod host's fitness by supplying them with essential vitamins and amino acids, little is known about the nutritional basis for the symbiotic relationship of endosymbionts in ticks. Our lab has previously reported that Rickettsia species phylotype G021 in Ixodes pacificus carries all five genes for de novo folate synthesis, and that these genes are monophyletic with homologs from other Rickettsia species. In this study, the rickettsial folate synthesis folA gene, coding for dihydrofolate reductase, was PCR amplified, cloned into an expression vector, and overexpressed in E. coli. Bioinformatic analysis identified that the FolA protein of phylotype G021 has the conserved DHFR domain, NADP binding sites, and substrate binding sites of bacterial dihydrofolate reductase. SDS-PAGE results showed that recombinant rickettsial FolA protein was overexpressed in BL21(DE3) E. coli in its soluble form. Affinity chromatography was used to purify the protein, and in vitro enzyme assays were performed to assess the biochemical activity of dihydrofolate reductase. The specific activity of recombinant FolA from phylotype G021 was determined to be 16.1 U/mg. This study has revealed that Rickettsia species phylotype G021 of I. pacificus is capable of producing a functional enzyme of the folate biosynthesis pathway, addressing the nutritional interactions behind the symbiosis between Rickettsia species phylotype G021 and its host.

Targeting Tumor Hypoxia Using Nanoparticle-engineered CXCR4-overexpressing Adipose-derived Stem Cells.

Hypoxia, a hallmark of malignant tumors, often correlates with increasing tumor aggressiveness and poor treatment outcomes. Due to a lack of vasculature, effective drug delivery to hypoxic tumor regions remains challenging. Signaling through the chemokine SDF-1α and its receptor CXCR4 plays a critical role in the homing of stem cells to ischemia for potential use as drug-delivery vehicles. To harness this mechanism for targeting tumor hypoxia, we developed polymeric nanoparticle-induced CXCR4-overexpressing human adipose-derived stem cells (hADSCs). Using glioblastoma multiforme (GBM) as a model tumor, we evaluated the ability of CXCR4-overexpressing hADSCs to target tumor hypoxia in vitro using a 2D migration assay and a 3D collagen hydrogel model. Compared to untransfected hADSCs, CXCR4-overexpressing hADSCs showed enhanced migration in response to hypoxia and penetrated the hypoxic core within tumor spheres. When injected in the contralateral brain in a mouse intracranial GBM xenograft, CXCR4-overexpressing hADSCs exhibited long-range migration toward GBM and preferentially penetrated the hypoxic tumor core. Intravenous injection also led to effective targeting of tumor hypoxia in a subcutaneous tumor model. Together, these results validate polymeric nanoparticle-induced CXCR4-overexpressing hADSCs as a potent cellular vehicle for targeting tumor hypoxia, which may be broadly useful for enhancing drug delivery to various cancer types.