High-dimensional single-cell analysis of human natural killer cell heterogeneity.

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Imatinib and Nilotinib Off-Target Effects on Human NK Cells, Monocytes, and M2 Macrophages.

Tyrosine kinase inhibitors (TKIs) are used in the clinical management of hematological neoplasms. Moreover, in solid tumors such as stage 4 neuroblastomas (NB), imatinib showed benefits that might depend on both on-target and immunological off-target effects. We investigated the effects of imatinib and nilotinib on human NK cells, monocytes, and macrophages. High numbers of monocytes died upon exposure to TKI concentrations similar to those achieved in patients. Conversely, NK cells were highly resistant to the TKI cytotoxic effect, were properly activated by immunostimulatory cytokines, and degranulated in the presence of NB cells. In NB, neither drug reduced the expression of ligands for activating NK receptors or upregulated that of HLA class I, B7-H3, PD-L1, and PD-L2, molecules that might limit NK cell function. Interestingly, TKIs modulated the chemokine receptor repertoire of immune cells. Acting at the transcriptional level, they increased the surface expression of CXCR4, an effect observed also in NK cells and monocytes of patients receiving imatinib for chronic myeloid leukemia. Moreover, TKIs reduced the expression of CXCR3 (in NK cells) and CCR1 (in monocytes). Monocytes also decreased the expression of M-CSFR, and low numbers of cells underwent differentiation toward macrophages. M0 and M2 macrophages were highly resistant to TKIs and maintained their phenotypic and functional characteristics. Importantly, also in the presence of TKIs, the M2 immunosuppressive polarization was reverted by TLR engagement, and M1-oriented macrophages fully activated autologous NK cells. Our results contribute to better interpreting the off-target efficacy of TKIs in tumors and to envisaging strategies aimed at facilitating antitumor immune responses.

Synchrotron radiation external beam rotational radiotherapy of breast cancer: proof of principle.

The principle of rotational summation of the absorbed dose for breast cancer treatment with orthovoltage X-ray beams was proposed by J. Boone in 2012. Here, use of X-ray synchrotron radiation for image guided external beam rotational radiotherapy treatment of breast cancer is proposed. Tumor irradiation occurs with the patient in the prone position hosted on a rotating bed, with her breast hanging from a hole in the bed, which rotates around a vertical axis passing through the tumor site. Horizontal collimation of the X-ray beam provides for whole breast or partial breast irradiation, while vertical translation of the bed and successive rotations allow for irradiation of the full tumor volume, with dose rates which permit also hypofractionated treatments. In this work, which follows a previous preliminary report, results are shown of a full series of measurements on polyethylene and acrylic cylindrical phantoms carried out at the Australian Synchrotron, confirmed by Geant4 Monte Carlo simulations, intended to demonstrate the proof of principle of the technique. Dose measurements were carried out with calibrated ion chambers, radiochromic films and thermoluminescence dosimeters. The photon energy investigated was 60 keV. Image guidance may occur with the transmitted beam for contrast-enhanced breast computed tomography. For a horizontal beam collimation of 1.5 cm and rotation around the central axis of a 14 cm-diameter polyethylene phantom, a periphery-to-center dose ratio of 14% was measured. The simulations showed that under the same conditions the dose ratio decreases with increasing photon energy down to 10% at 175 keV. These values are comparable with those achievable with conventional megavoltage radiotherapy of breast cancer with a medical linear accelerator. Dose painting was demonstrated with two off-center `cancer foci' with 1.3 Gy and 0.6 Gy target doses. The use of a radiosensitizing agent for dose enhancement is foreseen.

TLR activation of tumor-associated macrophages from ovarian cancer patients triggers cytolytic activity of NK cells.

We analyzed the functional outcome of the interaction between tumor-associated macrophages (TAMs) and natural killer (NK) cells. TAMs from ascites of ovarian cancer patients displayed an alternatively activated functional phenotype (M2) characterized by a remarkably high frequency and surface density of membrane-bound IL-18. Upon TLR engagement, TAMs acquired a classically activated functional phenotype (M1), released immunostimulatory cytokines (IL-12, soluble IL-18), and efficiently triggered the cytolytic activity of NK cells. TAMs also induced the release of IFN-γ from NK cells, which however was significantly lower compared with that induced by in vitro-polarized M2 cells. Most tumor-associated NK cells displayed a CD56(bright) , CD16(neg) or CD56(bright) , CD16(dim) phenotype, and very poor cytolytic activities, despite an increased expression of the activation marker CD69. They also showed downregulation of DNAM-1, 2B4, and NTB-A activating receptors, and an altered chemokine receptor repertoire. Importantly however, when appropriately stimulated, NK cells from the patients, including those cells isolated from ascites, efficiently killed autologous TAMs that expressed low, "nonprotective" levels of HLA class I molecules. Overall, our data show the existence of a complex tumor microenvironment in which poorly cytolytic/immature NK cells deal with immunosuppressive tumor-educated macrophages.

Neuroblastoma-derived TGF-ß1 modulates the chemokine receptor repertoire of human resting NK cells.

In this study, we show that neuroblastoma (NB) cell conditioning affects the chemokine receptor repertoire of human resting NK cells. In particular, NB cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX3CR1 in the CD56(dim) subset. On the contrary, the expression of CXCR1 and CCR7 remained unaltered. The phenomenon was dependent on the release by NB cells of TGF-ß1, and rTGF-ß1 induced a chemokine receptor repertoire identical to that of NB-conditioned NK cells. The immune modulatory role of TGF-ß1 appears to be dose dependent because low amounts of the cytokine were sufficient to modulate CXCR4 and CX3CR1 expression, intermediate amounts modified that of CXCR3, and high amounts were necessary to downregulate the expression of the NKp30 activating receptor. Notably, a similar receptor modulation was observed in rTGF-ß2-conditioned NK cells. Finally, the analysis of NK cells from patients with stage 4 NB suggests that NB conditioning could exert in vivo an immune modulatory effect resembling that emerged from in vitro experiments. Altogether our data propose a novel tumor escape-mechanism based on the modulation of chemokine receptors that play pivotal roles in NK cells bone marrow homing, egress, or recruitment into peripheral tissues.

Inter-institutional variability of knowledge-based plan prediction of left whole breast irradiation.

PURPOSE: Within a multi-institutional project, we aimed to assess the transferability of knowledge-based (KB) plan prediction models in the case of whole breast irradiation (WBI) for left-side breast irradiation with tangential fields (TF). METHODS: Eight institutions set KB models, following previously shared common criteria. Plan prediction performance was tested on 16 new patients (2 pts per centre) extracting dose-volume-histogram (DVH) prediction bands of heart, ipsilateral lung, contralateral lung and breast. The inter-institutional variability was quantified by the standard deviations (SDint) of predicted DVHs and mean-dose (Dmean). The transferability of models, for the heart and the ipsilateral lung, was evaluated by the range of geometric Principal Component (PC1) applicability of a model to test patients of the other 7 institutions. RESULTS: SDint of the DVH was 1.8 % and 1.6 % for the ipsilateral lung and the heart, respectively (20 %-80 % dose range); concerning Dmean, SDint was 0.9 Gy and 0.6 Gy for the ipsilateral lung and the heart, respectively (<0.2 Gy for contralateral organs). Mean predicted doses ranged between 4.3 and 5.9 Gy for the ipsilateral lung and 1.1-2.3 Gy for the heart. PC1 analysis suggested no relevant differences among models, except for one centre showing a systematic larger sparing of the heart, concomitant to a worse PTV coverage, due to high priority in sparing the left anterior descending coronary artery. CONCLUSIONS: Results showed high transferability among models and low inter-institutional variability of 2% for plan prediction. These findings encourage the building of benchmark models in the case of TF-WBI.

M-CSF induces the expression of a membrane-bound form of IL-18 in a subset of human monocytes differentiating in vitro toward macrophages.

IL-18 is a proinflammatory cytokine belonging to the "IL-1 family" that has been shown to play a prominent role in the induction of type 1 immune responses. Here, we show that M-CSF induces the expression of a membrane-bound form of IL-18 (mIL-18) in a subset of human blood monocytes differentiating toward macrophages. While monocytes, DC, and GM-CSF-treated monocytes did not express mIL-18, its expression was detected in approximately 30-40% of M-CSF-primed macrophages differentiating from both CD16(-) and CD16(+) monocytes. Treatment with the caspase-1 inhibitor significantly reduced mIL-18 expression suggesting the requirement of an assembled inflammasome for IL-18 surface expression. Polarization toward M2 did not modify mIL-18 expression. On the contrary, LPS stimulation of both M0 and M2 (mIL-18(+) ) macrophages induced shedding of mIL-18, which was likely mediated by the activation of cellular protease(s). Importantly, the soluble form IL-18 (sIL-18) induced in autologous resting NK cells both the expression of CCR7 and the production of high amounts of IFN-γ, which was virtually abrogated by Ab-mediated neutralization of sIL-18. Overall our data shed new light on the cells and mechanisms leading to the release of sIL-18, the major IFN-γ-inducing factor in both physiological and pathological immune responses.

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells.

Soluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G-mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56brigh) and CD56dim NK cells. Such sHLA-G-mediated down-modulations were reverted by adding anti-HLA-G or anti-ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G-treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G-mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G-mediated inhibition of NK-cell functions.

The interaction of human natural killer cells with either unpolarized or polarized macrophages results in different functional outcomes.

The cross-talk among cells of the innate immunity can greatly affect both innate and adaptive responses. Here we analyzed the molecular interactions between human natural killer (NK) cells and autologous macrophages. Activated NK cells killed M0 and M2, whereas M1 macrophages were more resistant to lysis because of their higher expression of HLA class I molecules. Following exposure to LPS or bacillus Calmette-Guérin, M0 and M2, but not polarized (endotoxin tolerant) M1 macrophages, induced strong activation of resting NK cells. The expression of CD69 and CD25 activation markers and the acquisition of cytotoxicity against tumor cells and immature dendritic cells required soluble factors being mostly contact independent. On the contrary, IFN-γ production was contact dependent and required the interaction of DNAM-1 and 2B4 (on NK) with their ligands on macrophages as well as IL-18. IL-18 was involved also in the acquisition of CCR7 by NK cells. Interestingly, M0 and M2 cells expressed a membrane-bound form of IL-18, which was released in small amounts after LPS treatment. Our data indicate that, upon interaction with M0 macrophages exposed to microbial products, NK cells may amplify classical type 1 immune responses. In addition, M1-polarizing stimuli can rescue M2 macrophages from their immunomodulatory state and shape their functional behavior toward NK stimulatory capability.

Monocyte and Macrophage in Neuroblastoma: Blocking Their Pro-Tumoral Functions and Strengthening Their Crosstalk with Natural Killer Cells.

Over the past decade, immunotherapy has represented an enormous step forward in the fight against cancer. Immunotherapeutic approaches have increasingly become a fundamental part of the combined therapies currently adopted in the treatment of patients with high-risk (HR) neuroblastoma (NB). An increasing number of studies focus on the understanding of the immune landscape in NB and, since this tumor expresses low or null levels of MHC class I, on the development of new strategies aimed at enhancing innate immunity, especially Natural Killer (NK) cells and macrophages. There is growing evidence that, within the NB tumor microenvironment (TME), tumor-associated macrophages (TAMs), which mainly present an M2-like phenotype, have a crucial role in mediating NB development and immune evasion, and they have been correlated to poor clinical outcomes. Importantly, TAM can also impair the antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells upon the administration of anti-GD2 monoclonal antibodies (mAbs), the current standard immunotherapy for HR-NB patients. This review deals with the main mechanisms regulating the crosstalk among NB cells and TAMs or other cellular components of the TME, which support tumor development and induce drug resistance. Furthermore, we will address the most recent strategies aimed at limiting the number of pro-tumoral macrophages within the TME, reprogramming the TAMs functional state, thus enhancing NK cell functions. We also prospectively discuss new or unexplored aspects of human macrophage heterogeneity.

B7-H3 in Pediatric Tumors: Far beyond Neuroblastoma.

B7-H3 is a 4Ig transmembrane protein that emerged as a tumor-associated antigen in neuroblastoma. It belongs to the B7 family, shows an immunoregulatory role toward NK and T cells, and, therefore, has been included in the growing family of immune checkpoints. Besides neuroblastoma, B7-H3 is expressed by many pediatric cancers including tumors of the central nervous system, sarcomas, and acute myeloid leukemia. In children, particularly those affected by solid tumors, the therapeutic protocols are aggressive and cause important life-threatening side effects. Moreover, despite the improved survival observed in the last decade, a relevant number of patients show therapy resistance and fatal relapses. Immunotherapy represents a new frontier in the cure of cancer patients and the targeting of tumor antigens or immune checkpoints blockade showed exciting results in adults. In this encouraging scenario, researchers and clinicians are exploring the possibility to use immunotherapeutics targeting B7-H3; these include mAbs and chimeric antigen receptor T-cells (CAR-T). These tools are rapidly evolving to improve the efficacy and decrease the unwanted side effects; drug-conjugated mAbs, bi-tri-specific mAbs or CAR-T, and, very recently, NK cell engagers (NKCE), tetra-specific molecules engaging a tumor-associated antigen and NK cells, have been generated. Preclinical data are promising, and clinical trials are ongoing. Hopefully, the B7-H3 targeting will provide important benefits to cancer patients.

Knowledge-based plan optimization for prostate SBRT delivered with CyberKnife according to RTOG0938 protocol.

PURPOSE: To extend the knowledge-based (KB) automatic planning approach to CyberKnife in the case of Stereotactic Body Radiation Therapy (SBRT) for prostate cancer. METHODS: Seventy-two clinical plans of patients treated according to the RTOG0938 protocol (36.25 Gy/5fr) with CyberKnife were exported from the CyberKnife system to Eclipse to train a KB-model using the Rapid Plan tool. The KB approach provided dose-volume objectives for specific OARs only and not PTV. Bladder, rectum and femoral heads were considered in the model. The KB-model was successfully trained on 51 plans and then validated on 20 new patients. A KB-based template was tuned in the Precision system for both sequential optimization (SO) and VOLO optimization algorithms. Plans of the validation group were re-optimized (KB-TP) using both algorithms without any operator intervention and compared against the original plans (TP) in terms of OARs/PTV dose-volume parameters. Paired Wilcoxon signed-rank tests were performed to assess statistically significant differences (p < 0.05). RESULTS: Regarding SO, automatic KB-TP plans were generally better than or equivalent to TP plans. PTVs V95% was slightly worse while OARs sparing for KB-TP was significantly improved. Regarding VOLO optimization, the PTVs coverage was significantly better for KB-TP while there was a limited worsening in the rectum. A significant improvement was observed in the bladder in the range of low-intermediate doses. CONCLUSIONS: An extension of the KB optimization approach to the CyberKnife system has been successfully developed and validated in the case of SBRT prostate cancer.

Selective sparing of bladder and rectum sub-regions in radiotherapy of prostate cancer combining knowledge-based automatic planning and multicriteria optimization.

Background and Purpose: The association between dose to selected bladder and rectum symptom-related sub-regions (SRS) and late toxicity after prostate cancer radiotherapy has been evidenced by voxel-wise analyses. The aim of the current study was to explore the feasibility of combining knowledge-based (KB) and multi-criteria optimization (MCO) to spare SRSs without compromising planning target volume (PTV) dose delivery, including pelvic-node irradiation. Materials and Methods: Forty-five previously treated patients (74.2 Gy/28fr) were selected and SRSs (in the bladder, associated with late dysuria/hematuria/retention; in the rectum, associated with bleeding) were generated using deformable registration. A KB model was used to obtain clinically suitable plans (KB-plan). KB-plans were further optimized using MCO, aiming to reduce dose to the SRSs while safeguarding target dose coverage, homogeneity and avoiding worsening dose volume histograms of the whole bladder, rectum and other organs at risk. The resulting MCO-generated plans were examined to identify the best-compromise plan (KB + MCO-plan). Results: The mean SRS dose decreased in almost all patients for each SRS. D1% also decreased in the large majority, less frequently for dysuria/bleeding SRS. Mean differences were statistically significant (p < 0.05) and ranged between 1.3 and 2.2 Gy with maximum reduction of mean dose up to 3-5 Gy for the four SRSs. The better sparing of SRSs was obtained without compromising PTVs coverage. Conclusions: Selectively sparing SRSs without compromising PTV coverage is feasible and has the potential to reduce toxicities in prostate cancer radiotherapy. Further investigation to better quantify the expected risk reduction of late toxicities is warranted.

Real-world validation of Artificial Intelligence-based Computed Tomography auto-contouring for prostate cancer radiotherapy planning.

Background and purpose: Artificial Intelligence (AI)-based auto-contouring for treatment planning in radiotherapy needs extensive clinical validation, including the impact of editing after automatic segmentation. The aims of this study were to assess the performance of a commercial system for Clinical Target Volumes (CTVs) (prostate/seminal vesicles) and selected Organs at Risk (OARs) (rectum/bladder/femoral heads + femurs), evaluating also inter-observer variability (manual vs automatic + editing) and the reduction of contouring time. Materials and methods: Two expert observers contoured CTVs/OARs of 20 patients in our Treatment Planning System (TPS). Computed Tomography (CT) images were sent to the automatic contouring workstation: automatic contours were generated and sent back to TPS, where observers could edit them if necessary. Inter- and intra-observer consistency was estimated using Dice Similarity Coefficients (DSC). Radiation oncologists were also asked to score the quality of automatic contours, ranging from 1 (complete re-contouring) to 5 (no editing). Contouring times (manual vs automatic + edit) were compared. Results: DSCs (manual vs automatic only) were consistent with inter-observer variability (between 0.65 for seminal vesicles and 0.94 for bladder); editing further improved performances (range: 0.76-0.94). The median clinical score was 4 (little editing) and it was <4 in 3/2 patients for the two observers respectively. Inter-observer variability of automatic + editing contours improved significantly, being lower than manual contouring (e.g.: seminal vesicles: 0.83vs0.73; prostate: 0.86vs0.83; rectum: 0.96vs0.81). Oncologist contouring time reduced from 17 to 24 min of manual contouring time to 3-7 min of editing time for the two observers (p < 0.01). Conclusion: Automatic contouring with a commercial AI-based system followed by editing can replace manual contouring, resulting in significantly reduced time for segmentation and better consistency between operators.

The Italian young medical physicist scenario: Results from the young AIFM group survey.

PURPOSE: The young working group of the Italian Association of Medical and Health Physics (AIFM) designed a survey to assess the current situation of the under 35 AIFM members. METHODS: An online survey including 65 questions was designed to gather personal information, educational issues, working and research experience, and to evaluate the AIFM activities. The survey was distributed to the under 35 members between November 2022 and February 2023, through the young AIFM mailing list and social media. RESULTS: 160 answers from 230 affiliates (70%, 31 years median age) were obtained. The results highlighted that 87% of the respondents had a fixed term/permanent employment, mainly in public hospitals (58%). Regarding Medical Physicists (MPs) training, 54% of the students left their region of origin due to the training plan (40%) and the availability of scholarships (25%) in the chosen university. Most of the respondents have no Radiation Protection Expert title, while the remaining 20%, 6%, and 3% are qualified to the first, second, and third level, respectively. Several young MPs (62.2%) were involved in research activities; however, only 28% had teaching experience, mainly within their workplace (20%, safety courses), during AIFM courses (4%), or university lectures (3%). CONCLUSIONS: This survey reported the current situation of the under 35 AIFM members, highlighting the "brain drain" phenomenon from the south to the north of Italy, mainly due to the lack of post-graduate schools, scholarships, and job opportunities. The obtained results will help the future working program of the AIFM.

miR-24-3p down-regulates the expression of the apoptotic factors FasL and BIM in human natural killer cells.

MicroRNAs are involved in the regulation of different functions in immune and non-immune cells. Here we show that miR-24-3p functionally interacts with FASLG mRNA and down-regulates its expression. This interaction occurs in human natural killer cells (NK), leading to the modulation of FasL surface expression. Moreover, miR-24-3p also modulates the mRNA and protein expression of BIM in NK cells. Thus, it likely contributes to the control of both the extrinsic and intrinsic apoptotic pathways. In line with this hypothesis, inhibition of miR-24-3p improves both initiator caspase-8 and effector caspase-3 and -7 activities, increases cell apoptosis, and reduces cell viability. Our data suggest that miR-24-3p can act as a survival factor in NK cells, affecting the FasL-mediated killing of Fas expressing cells and the BIM-dependent cell death. More generally, miR-24-3p may condition the level of cell apoptosis, which increases at the contraction phase of the immune response when the clearance of various expanded effector cells is needed.

Tumor Microenvironment and Hydrogel-Based 3D Cancer Models for In Vitro Testing Immunotherapies.

In recent years, immunotherapy has emerged as a promising novel therapeutic strategy for cancer treatment. In a relevant percentage of patients, however, clinical benefits are lower than expected, pushing researchers to deeply analyze the immune responses against tumors and find more reliable and efficient tools to predict the individual response to therapy. Novel tissue engineering strategies can be adopted to realize in vitro fully humanized matrix-based models, as a compromise between standard two-dimensional (2D) cell cultures and animal tests, which are costly and hardly usable in personalized medicine. In this review, we describe the main mechanisms allowing cancer cells to escape the immune surveillance, which may play a significant role in the failure of immunotherapies. In particular, we discuss the role of the tumor microenvironment (TME) in the establishment of a milieu that greatly favors cancer malignant progression and impact on the interactions with immune cells. Then, we present an overview of the recent in vitro engineered preclinical three-dimensional (3D) models that have been adopted to resemble the interplays between cancer and immune cells and for testing current therapies and immunotherapeutic approaches. Specifically, we focus on 3D hydrogel-based tools based on different types of polymers, discussing the suitability of each of them in reproducing the TME key features based on their intrinsic or tunable characteristics. Finally, we introduce the possibility to combine the 3D models with technological fluid dynamics platforms, reproducing the dynamic complex interactions between tumor cells and immune effectors migrated in situ via the systemic circulation, pointing out the challenges that still have to be overcome for setting more predictive preclinical assays.

Strategies for Potentiating NK-Mediated Neuroblastoma Surveillance in Autologous or HLA-Haploidentical Hematopoietic Stem Cell Transplants.

High-risk neuroblastomas (HR-NB) still have an unacceptable 5-year overall survival despite the aggressive therapy. This includes standardized immunotherapy combining autologous hemopoietic stem cell transplantation (HSCT) and the anti-GD2 mAb. The treatment did not significantly change for more than one decade, apart from the abandonment of IL-2, which demonstrated unacceptable toxicity. Of note, immunotherapy is a promising therapeutic option in cancer and could be optimized by several strategies. These include the HLA-haploidentical αßT/B-depleted HSCT, and the antibody targeting of novel NB-associated antigens such as B7-H3, and PD1. Other approaches could limit the immunoregulatory role of tumor-derived exosomes and potentiate the low antibody-dependent cell cytotoxicity of CD16 dim/neg NK cells, abundant in the early phase post-transplant. The latter effect could be obtained using multi-specific tools engaging activating NK receptors and tumor antigens, and possibly holding immunostimulatory cytokines in their construct. Finally, treatments also consider the infusion of novel engineered cytokines with scarce side effects, and cell effectors engineered with chimeric antigen receptors (CARs). Our review aims to discuss several promising strategies that could be successfully exploited to potentiate the NK-mediated surveillance of neuroblastoma, particularly in the HSCT setting. Many of these approaches are safe, feasible, and effective at pre-clinical and clinical levels.

Breast radiotherapy with kilovoltage photons and gold nanoparticles as radiosensitizer: An in vitro study.

PURPOSE: We investigated the dose enhancement and internalization of gold nanoparticles (AuNPs) used as a radiosensitizer agent for rotational radiotherapy of breast cancer using a kilovoltage (kV) X-ray beam. METHODS: Human breast cancer cells MDA-MB-231 were incubated with or without 100 µg/mL (4.87 nM) or 200 µg/mL (9.74 nM) 15 nm AuNPs and irradiated with 100 kV, 190 kV, or 6 MV X-rays. To assess the toxicity of the AuNPs, we performed a Sulforhodamine B assay. Using atomic absorption spectroscopy, scanning electron microscopy, transmission electron microscopy, and time-lapse optical microscopy (rate of 2 frames per minute), we carried out a quantitative assessment of the amount of gold internalized by MDA-MB-231 cells and a characterization of the static and dynamical aspects of this internalization process. RESULTS: No effect of AuNPs alone was shown on cell viability. Time-lapse optical microscopy showed for the first time AuNPs cellular uptake and the dynamics of AuNPs internalization. Electron microscopy demonstrated AuNPs localization in endosomal vesicles, preferentially in the perinuclear region. After irradiation at doses up to 2 Gy, cell survival fraction curves showed increased mortality with AuNPs, with respect to irradiation without AuNPs. The highest effect of radioenhancement by AuNPs (at 9.74 nM AuNPs concentration) was observed at 190 kV showing a dose enhancement factor of 1.33 ± 0.06 (1.34 ± 0.02 at 100 kV), while at 6 MV it was 1.14 ± 0.06. CONCLUSIONS: The observed radio-sensitization effect is promising for future radio-enhanced kV radiotherapy of breast cancer and quantitatively in the order of previous observations for 15 nm AuNPs. These results of a significant dose enhancement were obtained at 15 nm AuNPs concentration as low as several nanomolar units, at dose levels typical of a single dose fraction in a radiotherapy session. Dynamical behavior of the 3D spatial distribution of 15 nm AuNPs outside the nucleus of single breast cancer cell was observed, with possible implications for future models of AuNPs sensitization.

A multi-organ-on-chip to recapitulate the infiltration and the cytotoxic activity of circulating NK cells in 3D matrix-based tumor model.

The success of immunotherapeutic approaches strictly depends on the immune cells interaction with cancer cells. While conventional in vitro cell cultures under-represent the complexity and dynamic crosstalk of the tumor microenvironment, animal models do not allow deciphering the anti-tumor activity of the human immune system. Therefore, the development of reliable and predictive preclinical models has become crucial for the screening of immune-therapeutic approaches. We here present an organ-on-chip organ on chips (OOC)-based approach for recapitulating the immune cell Natural Killer (NK) migration under physiological fluid flow, infiltration within a 3D tumor matrix, and activation against neuroblastoma cancer cells in a humanized, fluid-dynamic environment. Circulating NK cells actively initiate a spontaneous "extravasation" process toward the physically separated tumor niche, retaining their ability to interact with matrix-embedded tumor cells, and to display a cytotoxic effect (tumor cell apoptosis). Since NK cells infiltration and phenotype is correlated with prognosis and response to immunotherapy, their phenotype is also investigated: most importantly, a clear decrease in CD16-positive NK cells within the migrated and infiltrated population is observed. The proposed immune-tumor OOC-based model represents a promising approach for faithfully recapitulating the human pathology and efficiently employing the immunotherapies testing, eventually in a personalized perspective. An immune-organ on chip to recapitulate the tumor-mediated infiltration of circulating immune cells within 3D tumor model.