Dose-response of EBT3 radiochromic films to proton and carbon ion clinical beams

We investigated the dose-response of the external beam therapy 3 (EBT3) films for proton and carbon ion clinical beams, in comparison with conventional radiotherapy beams; we also measured the film response along the energy deposition-curve in water. We performed measurements at three hadrontherapy centres by delivering monoenergetic pencil beams (protons: 63-230 MeV; carbon ions: 115-400 MeV/u), at 0.4-20 Gy dose to water, in the plateau of the depth-dose curve. We also irradiated the films to clinical MV-photon and electron beams. We placed the EBT3 films in water along the whole depth-dose curve for 148.8 MeV protons and 398.9 MeV/u carbon ions, in comparison with measurements provided by a plane-parallel ionization chamber. For protons, the response of EBT3 in the plateau of the depth-dose curve is not different from that of photons, within experimental uncertainties. For carbon ions, we observed an energy dependent under-response of EBT3 film, from 16% to 29% with respect to photon beams. Moreover, we observed an under-response in the Bragg peak region of about 10% for 148.8 MeV protons and of about 42% for 398.9 MeV/u carbon ions. For proton and carbon ion clinical beams, an under-response occurs at the Bragg peak. For carbon ions, we also observed an under-response of the EBT3 in the plateau of the depth-dose curve. This effect is the highest at the lowest initial energy of the clinical beams, a phenomenon related to the corresponding higher LET in the film sensitive layer. This behavior should be properly modeled when using EBT3 films for accurate 3D dosimetry

Molecular Mechanisms Directing Migration and Retention of Natural Killer Cells in Human Tissues

A large body of data shows that Natural Killer (NK) cells are immune effectors exerting a potent cytolytic activity against tumors and virus infected cells. The discovery and characterization of several inhibitory and activating receptors unveiled most of the mechanisms allowing NK cells to spare healthy cells while selectively attacking abnormal tissues. Nevertheless, the mechanisms ruling NK cell subset recirculation among the different compartments of human body have only lately started to be investigated. This is particularly true for pathological settings such as tumors or infected tissues but also for para-physiological condition like pregnant human uterine mucosa. It is becoming evident that the microenvironment associated to a particular clinical condition can deeply influence the migratory capabilities of NK cells. In this review we describe the main mechanisms and stimuli known to regulate the expression of chemokine receptors and other molecules involved in NK cell homing to either normal or pathological/inflamed tissues, including tumors or organs such as lung and liver. We will also discuss the role played by the chemokine/chemokine receptor axes in the orchestration of physiological events such as NK cell differentiation, lymphoid organ retention/egress and recruitment to decidua during pregnancy

Overview of the Soluble and Membrane-bound Tumor Factors Limiting NKmediated Immune Surveillance

Many evidences suggest that NK cells are effective in patrolling for and eliminating tumors in their onset phase, but hardly limit the progression of large established solid tumors. Beside the transition of tumor cells towards a more aggressive phenotype, the NK cell efficacy might be limited by a complex immunosuppressive milieu present in the tumor microenvironment. Indeed, different mechanisms damping NK cell function have been shown in these last years. These include a plethora of tumor-derived immunomodulatory soluble factors (TGF-\u3b2, MIF, adenosine, LKynurenin, PGE2) as well as soluble ligands (MICA, ULBP-2, PVR, B7-H6) that compete with membrane-bound tumor ligands for binding to activating NK receptors. During NK-tumor cell contact the NK cell function can also be inhibited by the engagement on NK cells of different inhibitory receptors. The specific ligands might be either constitutively expressed at the tumor cell surface (HLA-I, B7-H3, PVR) or de novo induced/up-regulated (PD-Ls) by immunostimulatory factors (IFN-\u3b3, TNF-\u3b1). These are largely released during the active phases of the immune responses and exert an unwanted side effect called \u201ctumor adaptive immune resistance\u201d. This review aims to summarize the best-known molecular mechanisms that, at various times and in different ways, can limit the efficacy of the NK-mediated immune surveillance of tumors

Novel Immunoregulatory Functions of IL-18, an Accomplice of TGF-\u3b21

TGF-\u3b21 is a pleiotropic factor exerting a strong regulatory role in several cell types, including immune cells. In NK cells it profoundly alters the surface expression of crucial activating and chemokine receptors. To understand which soluble signals might better contrast these effects, we cultured human NK cells in the presence of TGF-\u3b21 and different innate and adaptive cytokines, generally referred as "immunostimulatory". These included IL-2, IL-15, IL-21, IL-27, and IL-18. Unexpectedly, IL-18 strengthened rather than contrasting important TGF-\u3b21-mediated functions. In particular, IL-18 further reduced the expression of CX\u2083CR1 and NKp30, leading to the virtual abrogation of the triggering capability of this activating receptor. Moreover, IL-18 further increased the expression of CXCR4. The IL-18-mediated additive effect on NKp30 and CXCR4 expression involved transcriptional regulation and activation of MEK/ERK and/or p38MAPK. A proteomic approach quantified both surface and intracellular proteins significantly modified in cytokine-treated NK cells, thus giving global information on the biological processes involving TGF-\u3b21 and IL-18. Our data support the concept that IL-18 may have a different behavior depending on the type of soluble factors characterizing the microenvironment. In a TGF-\u3b21 rich milieu such as tumors, it may contribute to the impairment of both NK cells recruitment and killing capability

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

Identification of PVR (CD155) and Nectin-2 (CD112) as Cell Surface Ligands for the Human DNAM-1 (CD226) Activating Molecule

Human natural killer (NK) cells express a series of activating receptors and coreceptors that are involved in recognition and killing of target cells. In this study, in an attempt to identify the cellular ligands for such triggering surface molecules, mice were immunized with NK-susceptible target cells. On the basis of a functional screening, four mAbs were selected that induced a partial down-regulation of the NK-mediated cytotoxicity against the immunizing target cells. As revealed by biochemical analysis, three of such mAbs recognized molecules of ∼70 kD. The other mAb reacted with two distinct molecules of ∼65 and 60 kD, respectively. Protein purification followed by tryptic digestion and mass spectra analysis, allowed the identification of the 70 kD and the 65/60 kD molecules as PVR (CD155) and Nectin-2 δ/α (CD112), respectively. PVR-Fc and Nectin-2-Fc soluble hybrid molecules brightly stained COS-7 cells transfected with the DNAM-1 (CD226) construct, thus providing direct evidence that both PVR and Nectin-2 represent specific ligands for the DNAM-1 triggering receptor. Finally, the surface expression of PVR or Nectin-2 in cell transfectants resulted in DNAM-1–dependent enhancement of NK-mediated lysis of these target cells. This lysis was inhibited or even virtually abrogated upon mAb-mediated masking of DNAM-1 (on NK cells) or PVR or Nectin-2 ligands (on cell transfectants)

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&#8211;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.This work has been supported by Fondazione Regionale per la Ricerca Biomedica, project nr. 110 - JTC PerPlanRT ERA PerMed, GA 779282.Publicad

NKp44, a Novel Triggering Surface Molecule Specifically Expressed by Activated Natural Killer Cells, Is Involved in Non–Major Histocompatibility Complex–restricted Tumor Cell Lysis

After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non–major histocompatibility complex (MHC)–restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)–mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcγR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3ζ or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation