Peter Ferguson

Simple synthesis and functionalization of iron nanoparticles for magnetic resonance imaging.

Magnetic nanoparticles (NPs) are increasingly important in many biomedical applications, such as drug delivery, hyperthermia, and magnetic resonance imaging (MRI) contrast enhancement. For MRI, iron oxide NPs are the only commercial T2 or negative contrast agents, due to their biocompatibility and ease of synthesis and research in the area is highly active. The efficacy of these contrast agents depends mainly on the surface chemistry and magnetic properties of the NPs. Materials with larger magnetization could induce more efficient transverse (T2) relaxation of protons and result in greater contrast enhancement. As iron has the highest saturation magnetization at room temperature among all elements, and is biocompatible, it is an ideal candidate for MRI contrast enhancement. Nevertheless, the development of using iron NPs for magnetic applications has been challenging and limited compared to those of its oxides, due to the difficulty in preparing stable iron NPs with simple synthesis methods and precursors. 6] Under ambient conditions, iron NPs of 8 nm or smaller oxidize completely upon exposure to air. For larger NPs, an oxide shell of 3–4 nm forms instantly on the surface, forming iron/iron oxide core/shell NPs. Groundbreaking studies for the synthesis of iron NPs of larger than 8 nm has largely been achieved by decomposition of iron pentacarbonyl, [Fe(CO)5]. [6,8] Additional reports include the use of other precursors in forming iron nanocubes. However, all of these processes are limited in terms of ease of synthesis and scalability; [Fe(CO)5] is volatile and highly toxic, [5] and other processes involve precursors that are expensive and airsensitive, or require high decomposition temperatures. Here, we chose an easy to handle iron organometallic sandwich compound as the precursor and prepared singlecrystal iron NPs using a simple, low-temperature synthesis method. The iron nanocrystals oxidized naturally to form highly crystalline iron/iron oxide core/shell NPs. The ease of this synthesis facilitates the larger-scale application of stabilized iron NPs. To enable the use of these NPs in biological applications, the NP surface was modified to make the NPs water soluble. The strongly magnetic core/shell NPs are shown to be more effective T2 contrast agents for in vivo MRI and small tumor detection, compared to pure iron oxides. The successful detection of small tumors in vivo demonstrated here holds a great promise for accurate detection of early metastases in human lymph nodes, which has a large impact on the treatment and prognosis of a range of cancers. The iron/iron oxide core/shell NPs were prepared by first synthesizing iron nanocrystals by decomposition of the iron precursor [Fe(C5H5)(C6H7)], in the presence of oleylamine (OLA) stabilizing molecules. The non-carbonyl, sandwich compound was chosen for its simple preparation and ease of decomposition compared to other more stable sandwich compounds such as ferrocene. The synthesis was carried out in a closed reaction vessel under a mild hydrogen atmosphere, at 130 8C. The temperature required was lower than the usual temperature range (150–300 8C) needed for decomposition of other iron precursors in previous studies. Once [*] Dr. K. W. Feindel, Prof. P. T. Callaghan, Prof. R. D. Tilley School of Chemical and Physical Sciences and The MacDiarmid Institute for Advanced Materials and Nanotechnology, Victoria University of Wellington, Wellington 6012 (New Zealand) Fax: (+ 64)4-463-5237 E-mail: richard.tilley@vuw.ac.nz Dr. S. Cheong, Dr. B. Ingham Industrial Research Limited and The MacDiarmid Institute for Advanced Materials and Nanotechnology P. O. Box 31-310, Lower Hutt 5040 (New Zealand) Dr. P. Ferguson, Dr. I. F. Hermans Malaghan Institute of Medical Research P. O. Box 7060, Wellington 6012 (New Zealand)

ILC2s and T cells cooperate to ensure maintenance of M2 macrophages for lung immunity against hookworms

Defining the immune mechanisms underlying protective immunity to helminth infection remains an important challenge. Here we report that lung CD4(+) T cells and Group 2 innate lymphoid cells (ILC2s) work in concert to block Nippostrongylus brasiliensis (Nb) development in the parenchyma within 48 h in mice. Immune-damaged larvae have a striking morphological defect that is dependent on the expansion of IL-13-producing ILC2 and CD4(+) T cells, and the activation of M2 macrophages. This T-cell requirement can be bypassed by administration of IL-2 or IL-33, resulting in expansion of IL-13-producing ILC2s and larval killing. Depletion of ILC2s inhibits larval killing in IL-2-treated mice. Our results broaden understanding of ILC2s role in immunity to helminths by demonstrating that they not only act as alarmin sensors, but can also be sustained by CD4(+) T cells, ensuring both the prompt activation and the maintenance of IL-13-dependent M2 macrophage immunity in the lung.

Vaccination with irradiated tumor cells pulsed with an adjuvant that stimulates NKT cells is an effective treatment for glioma.

Purpose: The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor despite recent treatment advances. There is an urgent need to develop novel therapies for this disease. Experimental Design: We used the implantable GL261 murine glioma model to investigate the therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed with the immuno-adjuvant α-galactosylceramide (α-GalCer). Results: Vaccine treatment alone was highly effective in a prophylactic setting. In a more stringent therapeutic setting, administration of one dose of vaccine combined with depletion of regulatory T cells (Treg) resulted in 43% long-term survival and the disappearance of mass lesions detected by MRI. Mechanistically, the α-GalCer component was shown to act by stimulating “invariant” natural killer–like T cells (iNKT cells) in a CD1d-restricted manner, which in turn supported the development of a CD4+ T-cell–mediated adaptive immune response. Pulsing α-GalCer onto tumor cells avoided the profound iNKT cell anergy induced by free α-GalCer. To investigate the potential for clinical application of this vaccine, the number and function of iNKT cells was assessed in patients with GBM and shown to be similar to age-matched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with α-GalCer were able to stimulate iNKT cells and augment a T-cell response in vitro. Conclusions: Injection of irradiated tumor cells loaded with α-GalCer is a simple procedure that could provide effective immunotherapy for patients with high-grade glioma. Clin Cancer Res; 18(23); 6446–59. ©2012 AACR.

A self-adjuvanting vaccine induces cytotoxic T lymphocytes that suppress allergy

Epitope-based peptide vaccines encompass minimal immunogenic regions of protein antigens to allow stimulation of precisely targeted adaptive immune responses. However, because efficacy is largely determined by the functional status of antigen-presenting cells (APCs) that acquire and present peptides to cells of the adaptive immune system, adjuvant compounds are needed to enhance immunogenicity. We present here a vaccine consisting of an allergen-derived peptide conjugated to a prodrug of the natural killer-like T (NKT) cell agonist α-galactosylceramide, which is highly effective in reducing inflammation in a mouse model of allergic airway inflammation. Unlike other peptide-adjuvant conjugates that directly activate APCs through pattern recognition pathways, this vaccine encourages third-party interactions with NKT cells to enhance APC function. Therapeutic efficacy was correlated with marked increases in the number and functional activity of allergen-specific cytotoxic T lymphocytes (CTLs), leading to suppression of immune infiltration into the lungs after allergen challenge in sensitized hosts.

Total submission of pelvic lymphadenectomy tissues removed during radical prostatectomy for prostate cancer increases lymph node yield and detection of micrometastases

The detection of lymph node metastases has prognostic and therapeutic implications for patients undergoing radical prostatectomy for prostate cancer. Macroscopic identification of pelvic lymph nodes in surgical lymphadenectomy specimens can be difficult, with a potential for incomplete submission of lymph nodes for microscopic examination. This study was undertaken to determine whether complete sampling of lymphadenectomy specimens would improve the detection of metastatic disease in patients undergoing radical prostatectomy.

Strongly Magnetic Iron Nanoparticles Improve the Diagnosis of Small Tumours in the Reticuloendothelial System by Magnetic Resonance Imaging

Despite advances in non-invasive medical imaging, accurate nodal staging of malignancy continues to rely on surgery. Superparamagnetic iron oxide nanoparticles (IONP) with lymphotropic qualities have shown some promise as contrast agents for MRI of the lymph nodes, but recent large-scale studies failed to show consistent detection of tumours below 5 mm. Herein we compare imaging of splenic and lymph node tissue using iron/iron oxide core/shell nanoparticles (Fe NP) that have superior magnetic qualities to IONP, to determine whether improved negative contrast in T2-weighted MRI can enhance the diagnosis of small tumours in the reticuloendothelial system. To provide an in vivo pre-clinical model of human lymph node micrometastases, breast cancer cells were injected into the spleens of mice, providing localised areas of tumour growth. MR images of groups of tumour-bearing and sham-treated animals were generated using a 1.5 T imaging system and analysed by two independent, blinded radiologists. Fe NP improved the sensitivity and specificity of MRI when compared to IONP, enabling accurate detection of tumours as small as 1–3 mm. The use of Fe NP as contrast agents have the potential to improve the diagnostic accuracy of MRI in cancer patients, leading to more rapid and effective treatment.

Using magnetic resonance imaging to evaluate dendritic cell-based vaccination.

Cancer immunotherapy with antigen-loaded dendritic cell-based vaccines can induce clinical responses in some patients, but further optimization is required to unlock the full potential of this strategy in the clinic. Optimization is dependent on being able to monitor the cellular events that take place once the dendritic cells have been injected in vivo, and to establish whether antigen-specific immune responses to the tumour have been induced. Here we describe the use of magnetic resonance imaging (MRI) as a simple, non-invasive approach to evaluate vaccine success. By loading the dendritic cells with highly magnetic iron nanoparticles it is possible to assess whether the injected cells drain to the lymph nodes. It is also possible to establish whether an antigen-specific response is initiated by assessing migration of successive rounds of antigen-loaded dendritic cells; in the face of a successfully primed cytotoxic response, the bulk of antigen-loaded cells are eradicated on-route to the node, whereas cells without antigen can reach the node unchecked. It is also possible to verify the induction of a vaccine-induced response by simply monitoring increases in draining lymph node size as a consequence of vaccine-induced lymphocyte trapping, which is an antigen-specific response that becomes more pronounced with repeated vaccination. Overall, these MRI techniques can provide useful early feedback on vaccination strategies, and could also be used in decision making to select responders from non-responders early in therapy.

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non‐genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient‐ and in vivo‐derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase‐dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro‐apoptotic BIM or NOXA together with reduced expression of anti‐apoptotic proteins. Independent of these changes, panobinostat reduced c‐Myc and pre‐treatment of cells with siRNA against c‐Myc reduced BRAFi/HDACi drug‐induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK‐independent pathways.

Pleomorphic giant cell carcinoma of the urinary bladder: an extreme form of tumour de-differentiation.

Vesical pleomorphic giant cell carcinoma (PGCC) is a variant of urothelial carcinoma (UC) characterized by highly pleomorphic tumour with giant cells. Fewer than 10 cases have been reported, and our aim was to determine the clinical and pathological features of a series of tumours from a specialized uropathology laboratory.

Effective maybe, but is it cost‐effective? A reply

Sir: The letter from Varma and Griffiths in response to our study that investigated lymph node yield following processing of all tissues submitted from lymph node dissections in association with radical prostatectomy for prostate adenocarcinoma suggests that this should not be undertaken, on the basis of costeffectiveness. In the letter, the authors query how many additional blocks were required for the complete processing of lymphadenectomy tissues. No evidence-based guidelines for the sampling of these tissues currently exist. In our study, we identified palpable lymph nodes and then embedded the tissue completely. This necessitated the preparation of an additional three to five blocks per case. With a mean of 7.4 blocks per case, total embedding of lymphadenectomy tissue resulted in the detection of an additional 190% of lymph nodes. Varma and Griffiths make the point that a section taken through a lymph node may miss micrometastases. In support of this, they cite their unpublished study from 2007, which evaluated the utility of frozen sections of pelvic lymph nodes obtained during radical prostatectomy. They showed that frozen sections revealed metastatic tumour in 2% of cases, with a false-negative result being obtained in 3% of cases, following submission of the entire node for paraffin processing. Similar results were reported more than a decade earlier, when it was shown that frozen section assessment of lymph nodes for prostate cancer had a sensitivity of only 63.2%. These observations are not relevant to our study, as the majority of impalpable nodes are of small size, and sectioning therefore samples a substantial proportion of the node. There are no firm recommendations suggesting an optimum nodal harvest for patients undergoing radical prostatectomy for prostate cancer. In a postmortem study, it was shown that the mean number of lymph nodes removed during bilateral lymph node dissection was 22.7. This was similar to the mean number of 20.5 lymph nodes removed from patients undergoing radical prostatectomy. The authors noted that ~20 pelvic lymph nodes may serve as a guideline for sufficient standard pelvic lymph node dissection. It is of interest that, in support of these findings, total processing of our specimens resulted in the detection of a mean number of 10.8 nodes per side. This indicates that total submission of pelvic lymphadenectomy tissue provides evidence of the adequacy of lymphadenectomy. In their letter, the authors query our ability to accurately identify and count the numbers of lymph nodes present. In reality, the technique that we employ makes this straightforward. First, as noted above, the impalpable nodes discovered following complete sampling are small, and are therefore well circumscribed and easily identified as discrete nodes. Second, we embed the lymphadenectomy specimen in serial blocks, and this makes it possible to match a partially sampled node in one block with the other portion of it in the adjacent block. The presence of metastases in lymph nodes is of prognostic significance for prostate cancer. In particular, it has been shown that both the maximum diameter of nodal metastases and the grade of nodal tumour are independent predictors of outcome on multivariate analysis. This underscores the importance of total submission of lymphadenectomy specimens. In our study, we identified additional metastatic prostatic cancer in 7.3% of cases, thus permitting more accurate assignment of tumour grade to lymph node metastases. In their concluding comments, Varma and Griffiths muse that it might be possible for additional lymph nodes to be be detected in other malignancies following submission of all tissues removed as part of a curative resection for cancer in a variety of organs. In this context, they particularly mention axillary lymph node dissections and colectomies. This latter argument is inappropriate, as the authors are confusing practicality and feasibility with cost-effectiveness. There are protocols in place to ensure optimum sampling of relevant nodes for patients with breast and rectal cancer, and these have been well validated. Such protocols remain to be established for prostate cancer; hence the purpose of our study. Varma and Griffiths conclude that processing of lymphadenectomy tissue is not cost-effective, and here their argument is on shaky ground. Although the data that we reported were from a formal study based on the material submitted to a solitary, specialized, private uropathology practice, in the three pathology units (one hospital and two private pathology practices) in which the authors work, all lymphadenectomy tissue are always routinely embedded, as this provides optimal prognostic information for the surgeon and also accurately informs subsequent treatment. The additional sections that are examined as a result of this are small and the costs