Lipid-based carriers represent the leading technology for nanoparticle (NP) drug delivery. To this end, the incorporation of gold NPs into lipid coronas (LC) has long been of scientific and practical interest. However, challenges persist in confining the inorganic payload within the LC, while achieving high loading efficiency and control over the surface functionalization of Au NPs. Here, phospholipids are self-assembled on Au NPs suprastructures, generating lipid-coated nanoconstructs at an Au loading>90% in mass and maintaining the desired functionalization of the gold surface with 4-mercaptophenylboronic acid (MPBA). The resulting LC-MPBA-Au NPs exhibit the colloidal behaviour of the lipid vesicles together with the plasmonic properties of the Au suprastructures. According to molecular dynamics calculations, the phospholipids readily assemble on Au nanoparticle aggregates, with MPBA favouring the process. In addition, calculations suggest that surface corrugation of Au suprastructures promotes lipid coating. This indicates that preformed Au suprastructures characterized by surface irregularities are essential for attaining optimal loading efficiency within the LC. These nanohybrids hold significant promise for multimodal theranostics, as they combine high biocompatibility and efficient cellular uptake, can be tracked by computed tomography imaging, and possesses the physical characteristics of a bimodal radiosensitizer suitable for two complementary radiotherapy modalities based on X-ray irradiation and boron neutron capture.

Despite more than half of all oncological patients undergo X-ray radiotherapy (XRT), significant efforts are required to improve its efficacy against hypoxic tumor regions and, at the same time, to expand the therapeutic window to spare normal tissues. The use of radiosensitizers, the personalization of radiation dose planning aided by imaging with magnetic resonance imaging (MRI) and X-ray computed tomography (CT), and the implementation of boron neutron capture therapy (BNCT) are three strategies to encompass the limits of XRT. Here, these three strategies are leveraged by designing and achieving a theranostic platform based on trimetallic Au-Fe-B nanoparticles (NPs). According to density functional theory calculations, chemodegradable Au-Fe-B nanostructures are not achievable under thermodynamic equilibrium conditions. Hence, Au-Fe-B NPs were synthesized by laser ablation in liquid, because it is a nonequilibrium process, followed by a tailored cleaning protocol. The Au-Fe-B NPs were coated with biocompatible polymers and showed several useful properties for nanomedicine application, such as chemical degradation in a physiological environment, contrast ability for MRI and CT, in vitro radiosensitization efficacy for XRT and BNCT, and consistent intracellular uptake. These functionalities can enable advanced studies on tumor treatment with complementary therapeutic strategies guided by anatomic imaging, leading to more effective oncological protocols.

PURPOSE:Minibeam radiotherapy (MBRT) uses small parallel beams of radiation to create a highly modulated dose pattern. The aim of this study is to develop an optical radioluminescence imaging (RLI) approach to perform real-time dose measurement for MBRT. METHODS:MBRT was delivered using an image-guided small animal irradiator equipped with a custom collimator. Five slabs of plastic scintillators with a thicknesses of 0.5, 1, 2, 3 and 10 mm were placed on top of a mouse phantom, to localize and measure the delivered dose. A thin radioluminescence film (Gd2O2S:Tb) was used to obtain the mini beam dose profile that was compared against GafChromic (GC) films measurements. The RLI signal was detected with a CMOS camera placed at 90 deg with respect to the beam axis. Monte Carlo (MC) simulations were also performed using TOPAS for comparison with the experimental results. RESULTS:The measured peak to valley dose ratio (PVDR) obtained with RLI was 16.7 in line with GC films measurements. The differences between peak and valley dimension were less that 3% with respect to GC measurements. Using RLI performed with the scintillator slabs, it was possible to localize and measure in real-time MBRT delivery on the mouse phantom. CONCLUSIONS:We proposed a novel method for MBRT dose localization and measurement in real-time based on RLI. The results we obtained are in good agreement with GC film measurements.

Minibeam Radiation Therapy (MBRT) delivers spatially fractionated beams, generating alternating high-dose peaks and low-dose valleys, with potential benefits in tumor control and normal tissue sparing. This study presents the development of a Monte Carlo (MC) Treatment Planning System (TPS) for preclinical MBRT, preceded by an investigation of various collimator configurations and a comparison of MC simulations with experimental data. Multiple simulations were conducted to evaluate different combinations of attenuator materials (lead, tungsten) and spacer materials (PMMA, air), as well as various slit widths (0.5-1 mm). Among the tested configurations, tungsten and PMMA emerged as the most suitable materials. As expected, the peak-to-valley dose ratio (PVDR) decreased with depth, while the full-width at half-maximum (FWHM) slightly increased. Fluka and Topas simulations showed good agreement with experimental measurements from gafchromic MDV3 films. A MC-based TPS was implemented to compute dose distributions in mice using CT data and to extract key dosimetric parameters, including PVDR and FWHM. Simulations were performed for a subcutaneous glioblastoma tumor as a case study. Within the tumor volume, the TPS estimated PVDR values ranging from 27 to 16 and a depth-dependent FWHM increase of about 5%. This system provides a robust platform for preclinical MBRT research, supporting treatment planning and delivery optimization.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a short survival time after standard therapy administration including radiotherapy (RT) associated with temozolomide (TMZ). Here, we investigated the effects of radiochemotherapy in association with metformin (MET), a drug targeting cell metabolism on a syngeneic GBM mouse model using Positron Emission Tomography imaging with [18F]FLT and [18F]VC701 and single-cell RNA-sequencing analysis. The addition of drugs to RT significantly increased survival and [18F]FLT showed an early predictive response of combined therapy. We identified the presence of heterogeneous tumor populations with different treatment sensitivity and a complex immune evasive microenvironment. Tumor cells surviving to treatments showed immune response, among the main differentially modulated biological functions and a potential role of long non-coding RNAs (lncRNAs) in treatment resistance. Association with TMZ or TMZ plus MET reduced the pro-tumor phenotype of immune reaction acting more on myeloid cells the first and on lymphocytes the latter. Off note, MET add-on counteracted the immune-evasive phenotype particularly of T cells suggesting a potential role of MET also in adopted immunity.

Supplementary Figures and Tables

BackgroundIn adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.MethodsWe simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.ResultsWe show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.ConclusionThese results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

PURPOSE:The purpose of this study is to investigate the dosimetric characteristics of a collimator for minibeam radiotherapy (MBRT) with film dosimetry and Monte Carlo (MC) simulations. The outcome of MBRT with respect to conventional RT using a glioma preclinical model was also evaluated. METHODS:A multi-slit collimator was designed to be used with commercial small animal irradiator. The collimator was built by aligning 0.6 mm wide and 5 mm thick parallel lead leaves at 0.4 mm intervals. Dosimetry characteristics were evaluated by Gafchromic (CG) films and TOPAS Monte Carlo (MC) code. An in vivo experiment was performed using a glioma preclinical model by injecting two million GL261cells subcutaneously and treating with 25 Gy, single fraction, with MBRT and conventional RT. Survival curves and acute radiation damage were measured to compare both treatments. RESULTS:A satisfactory agreement between experimental results and MC simulations were obtained, the measured FWHM and distance between the peaks were respectively 0.431 and 1.098 mm. In vivo results show that MBRT can provide local tumor control for three weeks after RT treatment and a similar survival fraction of open beam radiotherapy. No severe acute effects were seen for the MBRT group. CONCLUSIONS:We developed a minibeam collimator and presented its dosimetric features. Satisfactory agreement between MC and GC films was found with differences consistent with uncertainties due to fabrication and set-up errors. The survival curves of MBRT and open field RT are similar while atoxicity is dramatically lower with MBRT, preliminarily confirming the expected effect.

Background In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. Methods We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCR ED ) and permanently disrupted LAG3 , TIM-3 or 2B4 genes (IR KO ) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCR ED -IR KO and IR competent (TCR ED -IR COMP ) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro . Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. Results We show that upon chronic stimulation, TCR ED -IR KO cells are superior to TCR ED -IR COMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo . Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. Conclusion These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

In this work the effect of combining ultrasound (US) hyperthermia (HT) with radiotherapy (RT) was investigated. The treatment was applied to a GBM xenograft nude mouse model obtained by injecting $$2 \times 10^{6}$$ U87 luc+ cells. The combined treatment group received 6 Gy and HT at $$43^\circ$$ for 8 min. The ultrasound field was generated by a closed-loop computationally controlled system, consisting of a High Intensity Focused Ultrasound (HIFU) transducer with centre frequency 3.57 MHz, a power amplifier, a function generator and a MATLAB controller. A mechanical cone adaptor has been designed to use the HIFU beam at a pre-defined post-focal distance. Two thermocouples were placed between the mechanical cone and the mice skin to measure and control the temperature during the HT treatment. Radiotherapy was carried out by using a dedicated small animal image guided radiotherapy system. Measurements of tumor volume performed with a caliper showed good tumor control for the RT-HT group with respect to the RT or control groups for up to 21 days after treatment. The mean value of the normalized (before therapy) tumor volume was almost equal to 0.5 for two weeks after treatment with an increase to 1.5 at sacrifice. The control and HT groups showed a higher value of about 1.5 during the first two weeks and 3.5 at the end of the follow-up period. We concluded that the use of HT as a radiosensitizer can improve the outcome for glioblastoma treatments.

T regulatory cells (Tregs) play a key role in modulating T cell responses. Clinical trials showed that Tregs modulate graft versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity (graft-versus-leukemia [GvL]) is largely unknown. Enforced interleukin-10 (IL-10) expression converts human CD4(+) T cells into T regulatory type 1 (Tr1)-like (CD4(IL-10)) cells that suppress effector T cells in vitro and xenoGvHD in humanized mouse models. In the present study, we show that CD4(IL-10) cells mediate anti leukemic effects in vitro and in vivo in a human leukocyte antigen (HLA) class I-dependent but antigen-independent manner. The cytotoxicity mediated by CD4(IL-10) cells is granzyme B (GzB) dependent, is specific for CD13(+) target cells, and requires CD54 and CD112 expression on primary leukemic target blasts. CD4(IL-10) cells adoptively transferred in humanized mouse models directly mediate anti-tumor and anti-leukemic effects. In addition, when co-transferred with peripheral blood mononuclear cells (PBMCs), CD4(IL-10) cells contribute to the GvL activity but suppress xenoGvHD mediated by the PBMCs. These findings provide for the first time a strong rationale for CD4(IL-10) cell immunotherapy to prevent GvHD and promote GvL in allo-HSCT for myeloid malignancies.

Bladder mechanical properties are critical for organ function and tissue homeostasis. Therefore, alterations of tissue mechanics are linked to disease onset and progression. This study aims to characterize the tissue elasticity of the murine bladder wall considering its different anatomical components, both in healthy conditions and in actinic cystitis, a state characterized by tissue fibrosis. Here, we exploit Brillouin microscopy, an emerging technique in the mechanobiology field that allows mapping tissue mechanics at the microscale, in non-contact mode and free of labeling. We show that Brillouin imaging of bladder tissues is able to recognize the different anatomical components of the bladder wall, confirmed by histopathological analysis, showing different tissue mechanical properties of the physiological bladder, as well as a significant alteration in the presence of tissue fibrosis. Our results point out the potential use of Brillouin imaging on clinically relevant samples as a complementary technique to histopathological analysis, deciphering complex mechanical alteration of each tissue layer of an organ that strongly relies on mechanical properties to perform its function.