Author Correction: A gammaherpesvirus provides protection against allergic asthma by inducing the replacement of resident alveolar macrophages with regulatory monocytes.

In the version of this article initially published, the accession code for the RNA-seq data set deposited in the NCBI public repository Sequence Read Archive was missing from the 'Data availability' subsection of the Methods section. The accession code is SRP125477.

A gammaherpesvirus provides protection against allergic asthma by inducing the replacement of resident alveolar macrophages with regulatory monocytes.

The hygiene hypothesis postulates that the recent increase in allergic diseases such as asthma and hay fever observed in Western countries is linked to reduced exposure to childhood infections. Here we investigated how infection with a gammaherpesvirus affected the subsequent development of allergic asthma. We found that murid herpesvirus 4 (MuHV-4) inhibited the development of house dust mite (HDM)-induced experimental asthma by modulating lung innate immune cells. Specifically, infection with MuHV-4 caused the replacement of resident alveolar macrophages (AMs) by monocytes with regulatory functions. Monocyte-derived AMs blocked the ability of dendritic cells to trigger a HDM-specific response by the TH2 subset of helper T cells. Our results indicate that replacement of embryonic AMs by regulatory monocytes is a major mechanism underlying the long-term training of lung immunity after infection.

Recruitment of hepatic macrophages from monocytes is independent of IL-4Rα but is associated with ablation of resident macrophages in schistosomiasis.

Alternatively activated Mφs (AAMφ) accumulate in hepatic granulomas during schistosomiasis and have been suggested to originate in the bone marrow. What is less understood is how these Mφ responses are regulated after S. mansoni infection. Here, we investigated the role of IL-4 receptor α-chain (IL-4Rα)-signalling in the dynamics of liver Mφ responses. We observed that IL-4Rα signalling was dispensable for the recruitment of Ly6Chi monocytes and for their conversion into F4/80hi CD64hi CD11bhi Mφ. Moreover, while IL-4Rα provided an AAMφ phenotype to liver F4/80hi CD64hi CD11bhi Mφ that was associated with regulation of granuloma formation, it was dispensable for host survival. Resident F4/80hi CD64hi CD11blo Mφ did not upregulate the AAMφ signature gene Ym1. Rather, resident Mφ nearly disappeared by week 8 after infection and artificial ablation of resident Mφ in CD169DTR mice did not affect the response to S. mansoni infection. Interestingly, ablation of CD169+ cells in naive mice resulted in the accumulation of F4/80hi CD64hi CD11bhi Mφ, which was amplified when ablation occurred during schistosomiasis. Altogether, our results suggest the ablation of resident KCs after S. mansoni infection to be associated with the recruitment and accumulation of F4/80hi CD64hi CD11bhi Mφ with lyz2-dependent IL-4Rα contributing to the regulation of granuloma inflammation but being dispensable for host survival.

FDG PET/CT radiomics for predicting the outcome of locally advanced rectal cancer.

PURPOSE: The aim of this study was to investigate the prognostic value of baseline 18F-FDG PET/CT textural analysis in locally-advanced rectal cancer (LARC). METHODS: Eighty-six patients with LARC underwent 18F-FDG PET/CT before treatment. Maximum and mean standard uptake values (SUVmax and SUVmean), metabolic tumoral volume (MTV), total lesion glycolysis (TLG), histogram-intensity features, as well as 11 local and regional textural features, were evaluated. The relationships of clinical, pathological and PET-derived metabolic parameters with disease-specific survival (DSS), disease-free survival (DFS) and overall survival (OS) were assessed by Cox regression analysis. Logistic regression was used to predict the pathological response by the Dworak tumor regression grade (TRG) in the 66 patients treated with neoadjuvant chemoradiotherapy (nCRT). RESULTS: The median follow-up of patients was 41 months. Seventeen patients (19.7%) had recurrent disease and 18 (20.9 %) died, either due to cancer progression (n = 10) or from another cause while in complete remission (n = 8). DSS was 95% at 1 year, 93% at 2 years and 87% at 4 years. Weight loss, surgery and the texture parameter coarseness were significantly associated with DSS in multivariate analyses. DFS was 94 % at 1 year, 86 % at 2 years and 79 % at 4 years. From a multivariate standpoint, tumoral differentiation and the texture parameters homogeneity and coarseness were significantly associated with DFS. OS was 93% at 1 year, 87% at 2 years and 79% after 4 years. cT, surgery, SUVmean, dissimilarity and contrast from the neighborhood intensity-difference matrix (contrastNGTDM) were significantly and independently associated with OS. Finally, RAS-mutational status (KRAS and NRAS mutations) and TLG were significant predictors of pathological response to nCRT (TRG 3-4). CONCLUSION: Textural analysis of baseline 18F-FDG PET/CT provides strong independent predictors of survival in patients with LARC, with better predictive power than intensity- and volume-based parameters. The utility of such features, especially coarseness, should be confirmed by larger clinical studies before considering their potential integration into decisional algorithms aimed at personalized medicine.

[(18)F]FPRGD2 PET/CT imaging of integrin αvß3 levels in patients with locally advanced rectal carcinoma.

PURPOSE: Our primary objective was to determine if [(18)F]FPRGD2 PET/CT performed at baseline and/or after chemoradiotherapy (CRT) could predict tumour regression grade (TRG) in locally advanced rectal cancer (LARC). Secondary objectives were to compare baseline [(18)F]FPRGD2 and [(18)F]FDG uptake, to evaluate the correlation between posttreatment [(18)F]FPRGD2 uptake and tumour microvessel density (MVD) and to determine if [(18)F]FPRGD2 and FDG PET/CT could predict disease-free survival. METHODS: Baseline [(18)F]FPRGD2 and FDG PET/CT were performed in 32 consecutive patients (23 men, 9 women; mean age 63 ± 8 years) with LARC before starting any therapy. A posttreatment [(18)F]FPRGD2 PET/CT scan was performed in 24 patients after the end of CRT (median interval 7 weeks, range 3 - 15 weeks) and before surgery (median interval 4 days, range 1 - 15 days). RESULTS: All LARC showed uptake of both [(18)F]FPRGD2 (SUVmax 5.4 ± 1.5, range 2.7 - 9) and FDG (SUVmax 16.5 ± 8, range 7.1 - 36.5). There was a moderate positive correlation between [(18)F]FPRGD2 and FDG SUVmax (Pearson's r = 0.49, p = 0.0026). There was a moderate negative correlation between baseline [(18)F]FPRGD2 SUVmax and the TRG (Spearman's r = -0.37, p = 0.037), and a [(18)F]FPRGD2 SUVmax of >5.6 identified all patients with a complete response (TRG 0; AUC 0.84, 95 % CI 0.68 - 1, p = 0.029). In the 24 patients who underwent a posttreatment [(18)F]FPRGD2 PET/CT scan the response index, calculated as [(SUVmax1 - SUVmax2)/SUVmax1] × 100 %, was not associated with TRG. Post-treatment [(18)F]FPRGD2 uptake was not correlated with tumour MVD. Neither [(18)F]FPRGD2 nor FDG uptake predicted disease-free survival. CONCLUSION: Baseline [(18)F]FPRGD2 uptake was correlated with the pathological response in patients with LARC treated with CRT. However, the specificity was too low to consider its clinical routine use.

LET-dependent radiosensitization effects of gold nanoparticles for proton irradiation.

The development of new modalities and protocols is of major interest to improve the outcome of cancer treatment. Given the appealing physical properties of protons and the emerging evidence of biological relevance of the use of gold nanoparticles (GNPs), the radiosensitization effects of GNPs (5 or 10 nm) have been investigated in vitro in combination with a proton beam of different linear energy transfer (LET). After the incubation with GNPs for 24 h, nanoparticles were observed in the cytoplasm of A431 cells exposed to 10 nm GNPs, and in the cytoplasm as well as the nucleus of cells exposed to 5 nm GNPs. Cell uptake of 0.05 mg ml-1 of GNPs led to 0.78 pg Au/cell and 0.30 pg Au/cell after 24 h incubation for 10 and 5 nm GNPs respectively. A marked radiosensitization effect of GNPs was observed with 25 keV µm-1 protons, but not with 10 keV µm-1 protons. This effect was more pronounced for 10 nm GNPs than for 5 nm GNPs. By using a radical scavenger, a major role of reactive oxygen species in the amplification of the death of irradiated cell was identified. All together, these results open up novel perspectives for using high-Z metallic NPs in protontherapy.

FDG PET/CT for rectal carcinoma radiotherapy treatment planning: comparison of functional volume delineation algorithms and clinical challenges.

PET/CT imaging could improve delineation of rectal carcinoma gross tumor volume (GTV) and reduce interobserver variability. The objective of this work was to compare various functional volume delineation algorithms. We enrolled 31 consecutive patients with locally advanced rectal carcinoma. The FDG PET/CT and the high dose CT (CTRT) were performed in the radiation treatment position. For each patient, the anatomical GTVRT was delineated based on the CTRT and compared to six different functional/metabolic GTVPET derived from two automatic segmentation approaches (FLAB and a gradient-based method); a relative threshold (45% of the SUVmax) and an absolute threshold (SUV > 2.5), using two different commercially available software (Philips EBW4 and Segami OASIS). The spatial sizes and shapes of all volumes were compared using the conformity index (CI). All the delineated metabolic tumor volumes (MTVs) were significantly different. The MTVs were as follows (mean ± SD): GTVRT (40.6 ± 31.28ml); FLAB (21.36± 16.34 ml); the gradient-based method (18.97± 16.83ml); OASIS 45% (15.89 ± 12.68 ml); Philips 45% (14.52 ± 10.91 ml); OASIS 2.5 (41.6 2 ± 33.26 ml); Philips 2.5 (40 ± 31.27 ml). CI between these various volumes ranged from 0.40 to 0.90. The mean CI between the different MTVs and the GTVCT was < 0.4. Finally, the DICOM transfer of MTVs led to additional volume variations. In conclusion, we observed large and statistically significant variations in tumor volume delineation according to the segmentation algorithms and the software products. The manipulation of PET/CT images and MTVs, such as the DICOM transfer to the Radiation Oncology Department, induced additional volume variations.

PD-L1 Expression in Paired Samples of Rectal Cancer.

Immune checkpoint inhibitors and immune-related biomarkers are increasingly investigated in rectal cancer (RC). We retrospectively analysed PD-L1 expression in diagnostic biopsy and resection samples from RC patients treated at our centre between 2000 and 2020. PD-L1 immunostaining (22C3 clone) was evaluated according to tumour proportion (TPS), immune cell (ICS), and the combined positive score (CPS). Eighty-three patients were included. At diagnosis, PD-L1 expression ≥1%/≥5% was observed in 15.4%/0%, 80.7%/37.4%, and 69.2%/25.6% of patients based on TPS, ICS, and CPS, respectively. At surgery, the respective figures were 4.6%/1.5%, 60.2%/32.5%, and 50.7%/26.2%. Using the 1% cut-off and regardless of the scoring system, PD-L1 was less expressed in surgery than biopsy samples (p ≤ 0.04). In paired specimens, PD-L1-ICS reduction was especially observed following neoadjuvant long-course (chemo)radiotherapy (p = 0.03). PD-L1-ICS of ≥5% in surgical samples (HR: 0.17; p = 0.02), and a biopsy-to-surgery increase in PD-L1-ICS (HR: 0.19; p = 0.04) was predictive for longer disease-free survival, while the PD-L1-ICS of either ≥1% (HR 0.28; p = 0.04) or ≥5% (HR 0.19; p = 0.03) in surgical samples and the biopsy-to-surgery increase in PD-L1-ICS (HR: 0.20; p = 0.04) were associated with better overall survival. Our study suggests that PD-L1 expression in RC is largely reflective of immune cell infiltration, and its presence/increase in surgical samples predicts better outcomes.

The current understanding of the immune landscape relative to radiotherapy across tumor types.

Radiotherapy is part of the standard of care treatment for a great majority of cancer patients. As a result of radiation, both tumor cells and the environment around them are affected directly by radiation, which mainly primes but also might limit the immune response. Multiple immune factors play a role in cancer progression and response to radiotherapy, including the immune tumor microenvironment and systemic immunity referred to as the immune landscape. A heterogeneous tumor microenvironment and the varying patient characteristics complicate the dynamic relationship between radiotherapy and this immune landscape. In this review, we will present the current overview of the immunological landscape in relation to radiotherapy in order to provide insight and encourage research to further improve cancer treatment. An investigation into the impact of radiation therapy on the immune landscape showed in several cancers a common pattern of immunological responses after radiation. Radiation leads to an upsurge in infiltrating T lymphocytes and the expression of programmed death ligand 1 (PD-L1) which can hint at a benefit for the patient when combined with immunotherapy. In spite of this, lymphopenia in the tumor microenvironment of 'cold' tumors or caused by radiation is considered to be an important obstacle to the patient's survival. In several cancers, a rise in the immunosuppressive populations is seen after radiation, mainly pro-tumoral M2 macrophages and myeloid-derived suppressor cells (MDSCs). As a final point, we will highlight how the radiation parameters themselves can influence the immune system and, therefore, be exploited to the advantage of the patient.

Differential Biological Effects of Dietary Lipids and Irradiation on the Aorta, Aortic Valve, and the Mitral Valve.

Aims: Dietary cholesterol and palmitic acid are risk factors for cardiovascular diseases (CVDs) affecting the arteries and the heart valves. The ionizing radiation that is frequently used as an anticancer treatment promotes CVD. The specific pathophysiology of these distinct disease manifestations is poorly understood. We, therefore, studied the biological effects of these dietary lipids and their cardiac irradiation on the arteries and the heart valves in the rabbit models of CVD. Methods and Results: Cholesterol-enriched diet led to the thickening of the aortic wall and the aortic valve leaflets, immune cell infiltration in the aorta, mitral and aortic valves, as well as aortic valve calcification. Numerous cells expressing α-smooth muscle actin were detected in both the mitral and aortic valves. Lard-enriched diet induced massive aorta and aortic valve calcification, with no detectable immune cell infiltration. The addition of cardiac irradiation to the cholesterol diet yielded more calcification and more immune cell infiltrates in the atheroma and the aortic valve than cholesterol alone. RNA sequencing (RNAseq) analyses of aorta and heart valves revealed that a cholesterol-enriched diet mainly triggered inflammation-related biological processes in the aorta, aortic and mitral valves, which was further enhanced by cardiac irradiation. Lard-enriched diet rather affected calcification- and muscle-related processes in the aorta and aortic valve, respectively. Neutrophil count and systemic levels of platelet factor 4 and ent-8-iso-15(S)-PGF2α were identified as early biomarkers of cholesterol-induced tissue alterations, while cardiac irradiation resulted in elevated levels of circulating nucleosomes. Conclusion: Dietary cholesterol, palmitic acid, and cardiac irradiation combined with a cholesterol-rich diet led to the development of distinct vascular and valvular lesions and changes in the circulating biomarkers. Hence, our study highlights unprecedented specificities related to common risk factors that underlie CVD.

Humanized Mice as a Valuable Pre-Clinical Model for Cancer Immunotherapy Research.

Immunotherapy with checkpoint inhibitors opened new horizons in cancer treatment. Clinical trials for novel immunotherapies or unexplored combination regimens either need years of development or are simply impossible to perform like is the case in cancer patients with limited life expectancy. Thus, the need for preclinical models that rapidly and safely allow for a better understanding of underlying mechanisms, drug kinetics and toxicity leading to the selection of the best regimen to be translated into the clinic, is of high importance. Humanized mice that can bear both human immune system and human tumors, are increasingly used in recent preclinical immunotherapy studies and represent a remarkably unprecedented tool in this field. In this review, we describe, summarize, and discuss the recent advances of humanized mouse models used for cancer immunotherapy research and the challenges faced during their establishment. We also highlight the lack of preclinical studies using this model for radiotherapy-based research and argue that it can be a great asset to understand and answer many open questions around radiation therapy such as its presumed associated "abscopal effect".

Tumor Microenvironment Modifications Recorded With IVIM Perfusion Analysis and DCE-MRI After Neoadjuvant Radiotherapy: A Preclinical Study.

PURPOSE: Neoadjuvant radiotherapy (NeoRT) improves tumor local control and facilitates tumor resection in many cancers. Some clinical studies demonstrated that both timing of surgery and RT schedule influence tumor dissemination, and subsequently patient overall survival. Previously, we developed a pre-clinical model demonstrating the impact of NeoRT schedule and timing of surgery on metastatic spreading. We report on the impact of NeoRT on tumor microenvironment by MRI. METHODS: According to our NeoRT model, MDA-MB 231 cells were implanted in the flank of SCID mice. Tumors were locally irradiated (PXI X-Rad SmART) with 2x5Gy and then surgically removed at different time points after RT. Diffusion-weighted (DW) and Dynamic contrast enhancement (DCE) MRI images were acquired before RT and every 2 days between RT and surgery. IntraVoxel Incoherent Motion (IVIM) analysis was used to obtain information on intravascular diffusion, related to perfusion (F: perfusion factor) and subsequently tumor vessels perfusion. For DCE-MRI, we performed semi-quantitative analyses. RESULTS: With this experimental model, a significant and transient increase of the perfusion factor F [50% of the basal value (n=16, p<0.005)] was observed on day 6 after irradiation as well as a significant increase of the WashinSlope with DCE-MRI at day 6 (n=13, p<0.05). Using immunohistochemistry, a significant increase of perfused vessels was highlighted, corresponding to the increase of perfusion in MRI at this same time point. Moreover, Tumor surgical resection during this peak of vascularization results in an increase of metastasis burden (n=10, p<0.05). CONCLUSION: Significant differences in perfusion-related parameters (F and WashinSlope) were observed on day 6 in a neoadjuvant radiotherapy model using SCID mice. These modifications are correlated with an increase of perfused vessels in histological analysis and also with an increase of metastasis spreading after the surgical procedure. This experimental observation could potentially result in a way to personalize treatment, by modulating the time of surgery guided on MRI functional data, especially tumor perfusion.

Antibody-functionalized gold nanoparticles as tumor-targeting radiosensitizers for proton therapy.

AIM: This study aimed at developing antibody-functionalized gold nanoparticles (AuNPs) to selectively target cancer cells and probing their potential radiosensitizing effects under proton irradiation. MATERIALS & METHODS: AuNPs were conjugated with cetuximab (Ctxb-AuNPs). Ctxb-AuNP uptake was evaluated by transmission electron microscopy and atomic absorption spectroscopy. Radioenhancing effect was assessed using conventional clonogenic assay. RESULTS & CONCLUSION: Ctxb-AuNPs specifically bound to and accumulated in EGFR-overexpressing A431 cells, compared with EGFR-negative MDA-MB-453 cells. Ctxb-AuNPs enhanced the effect of proton irradiation in A431 cells but not in MDA-MB-453 cells. These data indicate, for the first time, that combining enhanced uptake by specific targeting and radioenhancing effect, using conjugated AuNPs, is a promising strategy to increase cell killing by protontherapy.

Caveolin plays a central role in endothelial progenitor cell mobilization and homing in SDF-1-driven postischemic vasculogenesis.

When neovascularization is triggered in ischemic tissues, angiogenesis but also (postnatal) vasculogenesis is induced, the latter requiring the mobilization of endothelial progenitor cells (EPC) from the bone marrow. Caveolin, the structural protein of caveolae, was recently reported to directly influence the angiogenic process through the regulation of the vascular endothelial growth factor (VEGF)/nitric oxide pathway. In this study, using caveolin-1 null mice (Cav(-/-)), we examined whether caveolin was also involved in the EPC recruitment in a model of ischemic hindlimb. Intravenous infusion of Sca-1(+) Lin(-) progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization in Cav(-/-) mice, suggesting a defect in progenitor mobilization. The adhesion of Cav(-/-) EPC to bone marrow stromal cells indeed appeared to be resistant to the otherwise mobilizing SDF-1 (Stromal cell-Derived Factor-1) exposure because of a defect in the internalization of the SDF-1 cognate receptor CXCR4. Symmetrically, the attachment of Cav(-/-) EPC to SDF-1-presenting endothelial cells was significantly increased. Finally, EPC transduction with caveolin small interfering RNA reproduced this advantage in vitro and, importantly, led to a more extensive rescue of the ischemic hindlimb after intravenous infusion (versus sham-transfected EPC). These results underline the critical role of caveolin in ensuring the caveolae-mediated endocytosis of CXCR4, regulating both the SDF-1-mediated mobilization and peripheral homing of progenitor cells in response to ischemia. In particular, a transient reduction in caveolin expression was shown to therapeutically increase the engraftment of progenitor cells.

Glucocorticoids modulate tumor radiation response through a decrease in tumor oxygen consumption.

PURPOSE: We hypothesized that glucocorticoids may enhance tumor radiosensitivity by increasing tumor oxygenation (pO(2)) through inhibition of mitochondrial respiration. EXPERIMENTAL DESIGN: The effect of three glucocorticoids (hydrocortisone, dexamethasone, and prednisolone) on pO(2) was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO(2) (t(max), 30 min after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO(2) measurements were done using electron paramagnetic resonance. The oxygen consumption rate of tumor cells after in vivo glucocorticoid administration was measured using high-frequency electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging. RESULTS: All glucocorticoids tested caused a rapid increase in pO(2). At t(max), tumor perfusion decreased, indicating that the increase in pO(2) was not caused by an increase in oxygen supply. Also at t(max), global oxygen consumption decreased. When irradiation (25 Gy) was applied at t(max), the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7). CONCLUSION: These results show the potential usefulness of the administration of glucocorticoids before irradiation.

Preconditioning of the tumor vasculature and tumor cells by intermittent hypoxia: implications for anticancer therapies.

Hypoxia is a common feature in tumors associated with an increased resistance of tumor cells to therapies. In addition to O(2) diffusion-limited hypoxia, another form of tumor hypoxia characterized by fluctuating changes in pO(2) within the disorganized tumor vascular network is described. Here, we postulated that this form of intermittent hypoxia promotes endothelial cell survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We found that endothelial cell exposure to cycles of hypoxia reoxygenation not only rendered them resistant to proapoptotic stresses, including serum deprivation and radiotherapy, but also increased their capacity to migrate and organize in tubes. By contrast, prolonged hypoxia failed to exert protective effects and even seemed deleterious when combined with radiotherapy. The use of hypoxia-inducible factor-1alpha (HIF-1alpha)-targeting small interfering RNA led us to document that the accumulation of HIF-1alpha during intermittent hypoxia accounted for the higher resistance of endothelial cells. We also used an in vivo approach to enforce intermittent hypoxia in tumor-bearing mice and found that it was associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (versus normoxia or prolonged hypoxia). Radioresistance was further ascertained by an increased rate of tumor regrowth in irradiated mice preexposed to intermittent hypoxia and confirmed in vitro using distinctly radiosensitive tumor cell lines. In conclusion, we have documented that intermittent hypoxia may condition endothelial cells and tumor cells in such a way that they are more resistant to apoptosis and more prone to participate in tumor progression. Our observations also underscore the potential of drugs targeting HIF-1alpha to resensitize the tumor vasculature to anticancer treatments.

Landmark detection in 2D bioimages for geometric morphometrics: a multi-resolution tree-based approach.

The detection of anatomical landmarks in bioimages is a necessary but tedious step for geometric morphometrics studies in many research domains. We propose variants of a multi-resolution tree-based approach to speed-up the detection of landmarks in bioimages. We extensively evaluate our method variants on three different datasets (cephalometric, zebrafish, and drosophila images). We identify the key method parameters (notably the multi-resolution) and report results with respect to human ground truths and existing methods. Our method achieves recognition performances competitive with current existing approaches while being generic and fast. The algorithms are integrated in the open-source Cytomine software and we provide parameter configuration guidelines so that they can be easily exploited by end-users. Finally, datasets are readily available through a Cytomine server to foster future research.

Botulinum toxin potentiates cancer radiotherapy and chemotherapy.

PURPOSE: Structural and functional abnormalities in the tumor vascular network are considered factors of resistance of solid tumors to cytotoxic treatments. To increase the efficacy of anticancer treatments, efforts must be made to find new strategies for transiently opening the tumor vascular bed to alleviate tumor hypoxia (source of resistance to radiotherapy) and improve the delivery of chemotherapeutic agents. We hypothesized that Botulinum neurotoxin type A (BoNT-A) could interfere with neurotransmitter release at the perivascular sympathetic varicosities, leading to inhibition of the neurogenic contractions of tumor vessels and therefore improving tumor perfusion and oxygenation. EXPERIMENTAL DESIGN: To test this hypothesis, BoNT-A was injected locally into mouse tumors (fibrosarcoma FSaII, hepatocarcinoma transplantable liver tumor), and electron paramagnetic resonance oximetry was used to monitor pO(2) in vivo repeatedly for 4 days. Additionally, contrast-enhanced magnetic resonance imaging was used to measure tumor perfusion in vivo. Finally, isolated arteries were mounted in wire myograph to monitor specifically the neurogenic tone developed by arterioles that were co-opted by the surrounding growing tumor cells. RESULTS: Using these tumor models, we showed that local administration of BoNT-A (two sites; dose, 29 units/kg) substantially increases tumor oxygenation and perfusion, leading to a substantial improvement in the tumor response to radiotherapy (20 Gy of 250-kV radiation) and chemotherapy (cyclophosphamide, 50 mg/kg). This observed therapeutic gain results from an opening of the tumor vascular bed by BoNT-A because we showed that BoNT-A could inhibit neurogenic tone in the tumor vasculature. CONCLUSIONS: The opening of the vascular bed induced by BoNT-A offers a way to significantly increase the response of tumors to radiotherapy and chemotherapy.

Reversal of temporal and spatial heterogeneities in tumor perfusion identifies the tumor vascular tone as a tunable variable to improve drug delivery.

Maturation of tumor vasculature involves the recruitment of pericytes that protect the endothelial tubes from a variety of stresses, including antiangiogenic drugs. Mural cells also provide mature tumor blood vessels with the ability to either relax or contract in response to substances present in the tumor microenvironment. The observed cyclic alterations in tumor blood flow and the associated deficit in chemotherapeutic drug delivery could in part arise from this vasomodulatory influence. To test this hypothesis, we focused on endothelin-1 (ET-1), which, besides its autocrine effects on tumor cell growth, is a powerful vasoconstrictor. We first document that an ET(A) receptor antagonist induced relaxation of microdissected tumor arterioles and selectively and quantitatively increased tumor blood flow in experimental tumor models. We then combined dye staining of functional vessels, fluorescent microsphere-based mapping, and magnetic resonance imaging to identify heterogeneities in tumor blood flow and to examine the reversibility of such phenomena. Data from all these techniques concurred to show that administration of an ET(A) receptor antagonist could reduce the extent of underperfused tumor areas, proving the key role of vessel tone variations in tumor blood flow heterogeneity. We also provide evidence that ET(A) antagonist administration could, despite an increase in tumor interstitial fluid pressure, improve access of cyclophosphamide to the tumor compartment and significantly influence tumor growth. In conclusion, tumor endogenous ET-1 production participates largely in the temporal and spatial variations in tumor blood flow. ET(A) antagonist administration may wipe out such heterogeneities, thus representing an adjuvant strategy that could improve the delivery of conventional chemotherapy to tumors.

Metallic nanoparticles irradiated by low-energy protons for radiation therapy: Are there significant physical effects to enhance the dose delivery?

PURPOSE: To identify which physical properties of nanoparticles are correlated with the survival fraction of cells exposed in vitro to low-energy protons in combination with nanoparticles. METHODS: The Geant4 simulation toolkit (version 10.3) was used to model nanoparticles of different sizes (5-50 nm) and materials (Ti, Zr, Hf, Ta, Au, Pt), with or without an organic capping ensuring biocompatibility and to irradiate them with 1.3 or 4 MeV protons and 5.3 MeV alpha particles. The spectra of secondary electrons inside and at the nanoparticle surface were computed, as well as electron yields, Auger and organic capping contribution, trapping in metal bulk and linear energy transfer profiles as a function of distance from the nanoparticle center. In a next step, an in silico cell model was designed and loaded with gold nanoparticles, according to experimental uptake values. Dose to the cell was evaluated macroscopically and microscopically in 100 × 100 × 100 nm³ voxels for different radiation qualities. RESULTS: The cell geometry showed that radiation enhancement is negligible for the gold concentration used and for any radiation quality. However, when the single nanoparticle geometry is considered, we observed a local LET in its vicinity considerably higher than for the water equivalent case (up to 5 keV/µm at the titanium nanoparticle surface compared to 2.5 keV/µm in the water case). The yield of secondary electrons per primary interaction with 1.3 MeV protons was found to be most favorable for titanium (1.54), platinum (1.44), and gold (1.32), although results for higher Z metals are probably underestimated due to the incomplete simulation of de-excitation cascade in outer shells. It was also found that the organic capping contributed mostly to the production of low-energy electrons, adding a spike of dose near the nanoparticle surface. Indeed, the yield for the coated gold nanoparticle increased to 1.53 when exposed to 1.3 MeV protons. Although most electrons are retained inside larger nanoparticles (50 nm), it was shown that their yield is comparable to smaller sizes and that the linear energy transfer profile is better. From a combination of ballistic and nanoparticle size factors, it was concluded that 10-nm gold nanoparticles were better inducers of additional cell killing than 5-nm gold nanoparticles, matching our previous in vitro study. CONCLUSIONS: Although effects from a physical standpoint are limited, the high linear energy transfer profile at the nanoparticle surface generates detrimental events in the cell, in particular ROS-induced damage and local heating.