In glioblastoma, the benefit from temozolomide chemotherapy is largely limited to a subgroup of patients (30-35%) with tumors exhibiting methylation of the promoter region of the O 6-methylguanine-DNA methyltransferase (MGMT) gene. In order to allow more patients to benefit from this treatment, we explored magnetic resonance image-guided microbubble-enhanced low-intensity pulsed focused ultrasound (LIFU) to transiently open the blood-brain barrier and deliver a first-in-class liposome-loaded small molecule MGMT inactivator in mice bearing temozolomide-resistant gliomas. We demonstrate that a liposomal O 6-(4-bromothenyl)guanine (O 6 BTG) derivative can efficiently target MGMT, thereby sensitizing murine and human glioma cells to temozolomide in vitro. Furthermore, we report that image-guided LIFU mediates the delivery of the stable liposomal MGMT inactivator in the tumor region resulting in potent MGMT depletion in vivo. Treatment with this new liposomal MGMT inactivator facilitated by LIFU-mediated blood-brain barrier opening reduced tumor growth and significantly prolonged survival of glioma-bearing mice, when combined with temozolomide chemotherapy. Exploring this novel combined approach in the clinic to treat glioblastoma patients with MGMT promoter-unmethylated tumors is warranted.

The α6β1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6β1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRβ expression and AKT–mTOR signalling. Taken together, we show that pericyte α6β1-integrin regulates tumour blood vessels by both controlling PDGFRβ and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis.

Transforming growth factor (TGF)-β contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-β and TGF-β isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-β in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-β in glioblastoma models. mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than or in most cell lines. Inhibition of mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting or mRNA expression or protein levels. Moreover, inhibition of expression reduces invasiveness Interestingly, depletion of also attenuates signaling evoked by TGF-β or TGF-β In orthotopic syngeneic (SMA-560) and xenograft (LN-308) glioma models, expression of as well as of the downstream target, , was reduced, while and levels were unaffected following systemic treatment with -specific antisense oligonucleotides. We conclude that TGF-β might function as a gatekeeper controlling downstream signaling despite high expression of TGF-β and TGF-β isoforms. Targeting TGF-β may represent a promising strategy interfering with aberrant TGF-β signaling in glioblastoma. .

Purpose: Transforming growth factor (TGF)-b is expressed at high levels by glioma cells and contributes to the malignant phenotype of glioblastoma. However, its therapeutic targeting remains challenging. Here, we examined an alternative therapeutic approach of TGFb inhibition using two novel phosphorothioate-locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, ISTH1047 and ISTH0047, which specifically target TGFb 1 and TGFb 2 .Experimental Design: We characterized the effects of ISTH1047 and ISTH0047 on TGFb 1/2 expression, downstream signaling and growth of human LN-308, LN-229, and ZH-161 cells as well as murine SMA-560 glioma cells in vitro. Furthermore, we assessed their target inhibition and effects on survival in orthotopic xenogeneic and syngeneic rodent glioma models in vivo.Results: Both antisense oligonucleotides specifically silenced their corresponding target and abrogated SMAD2 phosphorylation in several glioma cell lines. Moreover, inhibition of TGFb 1 or TGFb 2 expression by ISTH1047 or ISTH0047 reduced the migration and invasiveness of LN-308 and SMA-560 glioma cells. Systemic antisense oligonucleotide administration to glioma-bearing mice suppressed TGFb 1 or TGFb 2 mRNA expression as well as the expression of the downstream target PAI-1 in orthotopic gliomas. Gliomabearing mice had significantly prolonged survival upon systemic treatment with ISTH1047 or ISTH0047, which was associated with a reduction of intratumoral SMAD2 phosphorylation and, in a fully immunocompetent model, with increased immune cell infiltration.Conclusions: Targeting TGFb expression with the novel LNA antisense oligonucleotides ISTH1047 or ISTH0047 results in strong antiglioma activity in vitro and in vivo, which may represent a promising approach to be examined in human patients with glioma.

Liposomal delivery is a well-established approach to increase the therapeutic index of drugs, mainly in the field of cancer chemotherapy. Here, we report the preparation and characterization of a new liposomal formulation of a derivative of lomeguatrib, a potent O-methylguanine-DNA methyltransferase (MGMT) inactivator. The drug had been tested in clinical trials to revert chemoresistance, but was associated with a low therapeutic index. A series of lomeguatrib conjugates with distinct alkyl chain lengths - i.e. C12, C14, C16, and C18 - was synthesized, and the MGMT depleting activity as well as cytotoxicity were determined on relevant mouse and human glioma cell lines. Drug-containing liposomes were prepared and characterized in terms of loading and in vitro release kinetics. The lipophilic lomeguatrib conjugates did not exert cytotoxic effects at 5 μM in the mouse glioma cell line and exhibited a similar MGMT depleting activity pattern as lomeguatrib. Overall, drug loading could be improved by up to 50-fold with the lipophilic conjugates, and the slowest leakage was achieved with the C18 derivative. The present data show the applicability of lipophilic lomeguatrib derivatization for incorporation into liposomes, and identify the C18 derivative as the lead compound for in vivo studies.

Focused ultrasound (FUS) exposure in the presence of microbubbles (MBs) has been successfully used in the delivery of various sizes of therapeutic molecules across the blood-brain barrier (BBB). While acoustic pressure is correlated with the BBB opening size, real-time control of BBB opening to avoid vascular and neural damage is still a challenge. This arises mainly from the variability of FUS-MB interactions due to the variations of animal-specific metabolic environment and specific experimental setup. In this study, we demonstrate a closed-loop cavitation control framework to induce BBB opening for delivering large therapeutic molecules without causing macro tissue damages. To this end, we performed in mice long-term (5 min) cavitation monitoring facilitated by using long-circulating MBs. Monitoring the long-term temporal kinetics of the MBs under varying level of FUS pressure allowed to identify in-situ, animal specific activity regimes forming a pressure-dependent activity bands. This enables to determine the boundaries of each activity band (i.e. steady oscillation, transition, inertial cavitation) independent from the physical and physiological dynamics of the experiment. However, such a calibration approach is time consuming and to speed up characterization of the in-situ, animal specific FUS-MB dynamics, we tested a novel method called "pre-calibration" that closely reproduces the results of long-term monitoring but with a much shorter duration. Once the activity bands are determined from the pre-calibration method, an operation band can be selected around the desired cavitation dose. To drive cavitation in the selected operation band, we developed an adaptive, closed-loop controller that updates the acoustic pressure between each sonication based on measured cavitation dose. Finally, we quantitatively assessed the safety of different activity bands and validated the proposed methods and controller framework. The proposed framework serves to optimize the FUS pressure instantly to maintain the targeted

BackgroundProstate cancer is the second leading cause of cancer-related death in men in the USA; death occurs when patients progress to metastatic castration-resistant prostate cancer (CRPC). Although immunotherapy with the Food and Drug Administration‐approved vaccine sipuleucel‐T, which targets prostatic acid phosphatase (PAP), extends survival for 2–4 months, the identification of new immunogenic tumor-associated antigens (TAAs) continues to be an unmet need.MethodsWe evaluated the differential expression profile of castration-resistant prostate epithelial cells that give rise to CRPC from mice following an androgen deprivation/repletion cycle. The expression levels of a set of androgen-responsive genes were further evaluated in prostate, brain, colon, liver, lung, skin, kidney, and salivary gland from murine and human databases. The expression of a novel prostate-restricted TAA was then validated by immunostaining of mouse tissues and analyzed in primary tumors across all human cancer types in The Cancer Genome Atlas. Finally, the immunogenicity of this TAA was evaluated in vitro and in vivo using autologous coculture assays with cells from healthy donors as well as by measuring antigen-specific antibodies in sera from patients with prostate cancer (PCa) from a neoadjuvant clinical trial.ResultsWe identified a set of androgen-responsive genes that could serve as potential TAAs for PCa. In particular, we found transglutaminase 4 (Tgm4) to be highly expressed in prostate tumors that originate from luminal epithelial cells and only expressed at low levels in most extraprostatic tissues evaluated. Furthermore, elevated levels of TGM4 expression in primary PCa tumors correlated with unfavorable prognosis in patients. In vitro and in vivo assays confirmed the immunogenicity of TGM4. We found that activated proinflammatory effector memory CD8 and CD4 T cells were expanded by monocyte-derived dendritic cell (moDCs) pulsed with TGM4 to a greater extent than moDCs pulsed with either PAP or prostate-specific antigen (PSA), and T cells primed with TGM4-pulsed moDCs produce functional cytokines following a prime/boost regiment or in vitro stimulation. An IgG antibody response to TGM4 was detected in 30% of vaccinated patients, while fewer than 8% of vaccinated patients developed antibody responses to PSA or prostate-specific membrane antigen (PSMA).ConclusionsThese results suggest that TGM4 is an immunogenic, prostate-restricted antigen with the potential for further development as an immunotherapy target.

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