In vivo modeling recapitulates radiotherapy delivery and late-effect profile for childhood medulloblastoma.

Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor, with 5-year survival rates > 70%. Cranial radiotherapy (CRT) to the whole brain, with posterior fossa boost (PFB), underpins treatment for non-infants; however, radiotherapeutic insult to the normal brain has deleterious consequences to neurocognitive and physical functioning, and causes accelerated aging/frailty. Approaches to ameliorate radiotherapy-induced late-effects are lacking and a paucity of appropriate model systems hinders their development. Methods: We have developed a clinically relevant in vivo model system that recapitulates the radiotherapy dose, targeting, and developmental stage of childhood medulloblastoma. Consistent with human regimens, age-equivalent (postnatal days 35-37) male C57Bl/6J mice received computerized tomography image-guided CRT (human-equivalent 37.5 Gy EQD2, n = 12) ±â€…PFB (human-equivalent 48.7 Gy EQD2, n = 12), via the small animal radiation research platform and were longitudinally assessed for > 12 months. Results: CRT was well tolerated, independent of PFB receipt. Compared to a sham-irradiated group (n = 12), irradiated mice were significantly frailer following irradiation (frailty index; P = .0002) and had reduced physical functioning; time to fall from a rotating rod (rotarod; P = .026) and grip strength (P = .006) were significantly lower. Neurocognitive deficits were consistent with childhood MB survivors; irradiated mice displayed significantly worse working memory (Y-maze; P = .009) and exhibited spatial memory deficits (Barnes maze; P = .029). Receipt of PFB did not induce a more severe late-effect profile. Conclusions: Our in vivo model mirrored childhood MB radiotherapy and recapitulated features observed in the late-effect profile of MB survivors. Our clinically relevant model will facilitate both the elucidation of novel/target mechanisms underpinning MB late effects and the development of novel interventions for their amelioration.

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also show that brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

Evaluation of the Targeting and Therapeutic Efficiency of Anti-EGFR Functionalised Nanoparticles in Head and Neck Cancer Cells for Use in NIR-II Optical Window.

Gold nanoparticles have been indicated for use in a diagnostic and/or therapeutic role in several cancer types. The use of gold nanorods (AuNRs) with a surface plasmon resonance (SPR) in the second near-infrared II (NIR-II) optical window promises deeper anatomical penetration through increased maximum permissible exposure and lower optical attenuation. In this study, the targeting and therapeutic efficiency of anti-epidermal growth factor receptor (EGFR)-antibody-functionalised AuNRs with an SPR at 1064 nm was evaluated in vitro. Four cell lines, KYSE-30, CAL-27, Hep-G2 and MCF-7, which either over- or under-expressed EGFR, were used once confirmed by flow cytometry and immunofluorescence. Optical microscopy demonstrated a significant difference (p < 0.0001) between targeted AuNRs (tAuNRs) and untargeted AuNRs (uAuNRs) in all four cancer cell lines. This study demonstrated that anti-EGFR functionalisation significantly increased the association of tAuNRs with each EGFR-positive cancer cell. Considering this, the MTT assay showed that photothermal therapy (PTT) significantly increased cancer cell death (>97%) in head and neck cancer cell line CAL-27 using tAuNRs but not uAuNRs, apoptosis being the major mechanism of cell death. This successful targeting and therapeutic outcome highlight the future use of tAuNRs for molecular photoacoustic imaging or tumour treatment through plasmonic photothermal therapy.

Hematopoietic Stem Cell Gene Therapy for Brain Metastases Using Myeloid Cell-Specific Gene Promoters.

BACKGROUND: Brain metastases (BrM) develop in 20-40% of cancer patients and represent an unmet clinical need. Limited access of drugs into the brain because of the blood-brain barrier is at least partially responsible for therapeutic failure, necessitating improved drug delivery systems. METHODS: Green fluorescent protein (GFP)-transduced murine and nontransduced human hematopoietic stem cells (HSCs) were administered into mice (n = 10 and 3). The HSC progeny in mouse BrM and in patient-derived BrM tissue (n = 6) was characterized by flow cytometry and immunofluorescence. Promoters driving gene expression, specifically within the BrM-infiltrating HSC progeny, were identified through differential gene-expression analysis and subsequent validation of a series of promoter-green fluorescent protein-reporter constructs in mice (n = 5). One of the promoters was used to deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to BrM in mice (n = 17/21 for TRAIL vs control group). RESULTS: HSC progeny (consisting mostly of macrophages) efficiently homed to macrometastases (mean [SD] = 37.6% [7.2%] of all infiltrating cells for murine HSC progeny; 27.9% mean [SD] = 27.9% [4.9%] of infiltrating CD45+ hematopoietic cells for human HSC progeny) and micrometastases in mice (19.3-53.3% of all macrophages for murine HSCs). Macrophages were also abundant in patient-derived BrM tissue (mean [SD] = 8.8% [7.8%]). Collectively, this provided a rationale to optimize the delivery of gene therapy to BrM within myeloid cells. MMP14 promoter emerged as the strongest promoter construct capable of limiting gene expression to BrM-infiltrating myeloid cells in mice. TRAIL delivered under MMP14 promoter statistically significantly prolonged survival in mice (mean [SD] = 19.0 [3.4] vs mean [SD] = 15.0 [2.0] days for TRAIL vs control group; two-sided P = .006), demonstrating therapeutic and translational potential of our approach. CONCLUSIONS: Our study establishes HSC gene therapy using a myeloid cell-specific promoter as a new strategy to target BrM. This approach, with strong translational value, has potential to overcome the blood-brain barrier, target micrometastases, and control multifocal lesions.

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs.