Targeting Chemokines and Chemokine Receptors in Melanoma and Other Cancers

The tumor microenvironment is highly heterogeneous. It is composed of a diverse array of immune cells that are recruited continuously into lesions. They are guided into the tumor through interactions between chemokines and their receptors. A variety of chemokine receptors are expressed on the surface of both tumor and immune cells rendering them sensitive to multiple stimuli that can subsequently influence their migration and function. These features significantly impact tumor fate and are critical in melanoma control and progression. Indeed, particular chemokine receptors expressed on tumor and immune cells are strongly associated with patient prognosis. Thus, potential targeting of chemokine receptors is highly attractive as a means to quench or eliminate unconstrained tumor cell growth

Foxp3 expression in macrophages associated with RENCA tumors in mice.

The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings. We detected Foxp3 protein in macrophages infiltrating mouse renal cancer tumors injected subcutaneously or in the kidney. Expression was demonstrated using flow cytometry and Western blot with two individual monoclonal antibodies. Further analyses confirmed Foxp3 expression in macrophages by RT PCR, and studies using ribonucleic acid-sequencing (RNAseq) demonstrated a previously unknown Foxp3 messenger (m)RNA transcript in tumor-associated macrophages. In addition, depletion of Foxp3+ cells using diphtheria toxin in Foxp3DTR mice reduced the frequency of type-2 macrophages (M2) in kidney tumors. Collectively, these results indicate that tumor-associated macrophages could express Foxp3

Generation of novel chimeric antigen receptors to enhance the specificity and activity of T cells for the adoptive immunotherapy for cancer

© 2014 Dr. Connie DuongAdoptive immunotherapy is a promising treatment for cancer, with response rates of up to 70% in metastatic melanoma. To broaden this approach, T cells have been genetically modified to express chimeric antigen receptors (CARs) to endow T cells with anti-tumour activity capable of recognising a range of different cancer types. This approach has shown encouraging results in recent clinical trials for the treatment of haematological malignancies, however it has shown only moderate activity against solid cancers. To date, only a small number of molecules involved in T cell signaling have been incorporated into CARs, resulting in their suboptimal activity. Therefore improvements in CARs are needed in order to realise the full potential of adoptively transferred T cells. We proposed that using multiple or alternate signaling domains could enhance CAR-mediated T cell function. In this thesis, we describe the use of a DNA library of signaling molecules to investigate novel combinations of signaling molecules that could mediate enhanced CAR activity in the Jurkat T cell line and primary human T cells. A novel single-chain variable receptor was discovered comprising DAP10, CD3ζ and CD27 signaling domains that was able to trigger enhanced T cell activity in vitro and in an adoptive transfer mouse model. Clinical trials utilising CAR modified T cells have in some cases resulted in resulted in severe autoimmunity due to T cell recognition of tumour-associated antigens expressed on normal tissues. It is anticipated that as the application and efficacy of adoptive immunotherapy increases, toxicity against normal tissue will become increasingly common. To address this, we proposed that a T cell will respond less against normal tissue if endowed with a tumour-associated antigen-specific activating CAR co-localised with a chimeric inhibitory receptor (CIR) that is capable of turning off the T cell following engagement of antigen on normal tissue. We generated several novel chimeric inhibitory receptors and demonstrated expression of both CAR and CIR in T cells, which were then characterised for function against tumour-associated and normal tissue antigen expressing cell lines. In conclusion, the combination of these novel chimeric receptors may lead to a more efficacious but safer therapy for cancer

Passive Voltage Boosting Methods for Wireless Energy Harvesting Systems

Wireless power transfer has significantly improved the user experience in device charging and is currently an area of active research. Some major limitations of wireless charging systems include size constraints due to low frequency operation, the need for close proximity and tight alignment for efficient power transfer, and limited use of the device while charging. This work proposes several techniques to passively boost the voltage harvested and decrease the size of the power module. The rectifier and DC-DC converter explored in this dissertation extend technology proposed in existing literature that uses a resonator as the high quality factor component in the antenna matching network and energy storage element, respectively. This project proposes further development of these technologies by multiplexing the resonator between the rectifier and converter to reduce power module size and decrease generated electromagnetic interference by eliminating the need for magnetics. Preliminary testing has shown a resonator is more effective than an inductor (by a maximum of 30 percent) in boosting the rectifier output voltage and can boost the rectifier output voltage by a maximum of 8 times compared to an unmatched rectifier at 18 MHz on a commercial-grade printed circuit board. Initial DC-DC converter simulations with NMOS switches and realistic diodes show a 100 kHz resonator can store energy in a boost DC-DC converter on the order of nanojoules for a 10 V to 14 V step up conversion with 68 percent efficiency. This is similar in performance to a simulated inductor-based converter, which has a 78 percent efficiency when operating with the same step up ratio. The resonator-based DC-DC converter was built on a printed circuit board (PCB) using discrete transistors and a 185 kHz ceramic resonator. Simulations and measurements both showed the proposed discrete transistors did not effectively boost a 5 V input voltage by 1.4 times as was shown previously. Different methods to address this issue such as a floating driver and PMOS switches were proposed. An integrated design was also implemented using the open-source SkyWater 130 nm CMOS process design kit (PDK). A magnetoelectric (ME) antenna is proposed to decrease the size of the antenna by operating at acoustic resonance. Another benefit is more lenient alignment rules, which provide more device usage flexibility while charging. The maximum voltage output from the fabricated ME antenna is 0.3 V with a Qi transmitter producing 0.8 W as the source. The size of the antenna is 2 cm by 1.5 cm, which is smaller than the commercial Qi receiving antennas of 4 cm by 4 cm. When used to harvest vibrational energy, a slightly larger antenna (3 cm by 1.5 cm) could provide a maximum output power of 0.3 uW for an input vibration acceleration of approximately +/- 8 g

Targeting Chemokines and Chemokine Receptors in Melanoma and Other Cancers

The tumor microenvironment is highly heterogeneous. It is composed of a diverse array of immune cells that are recruited continuously into lesions. They are guided into the tumor through interactions between chemokines and their receptors. A variety of chemokine receptors are expressed on the surface of both tumor and immune cells rendering them sensitive to multiple stimuli that can subsequently influence their migration and function. These features significantly impact tumor fate and are critical in melanoma control and progression. Indeed, particular chemokine receptors expressed on tumor and immune cells are strongly associated with patient prognosis. Thus, potential targeting of chemokine receptors is highly attractive as a means to quench or eliminate unconstrained tumor cell growth

Characterising murine hippocampal iron homeostasis, in relation to markers of brain inflammation and metabolism, during ageing

Metal ions (Fe, Cu, and Zn) are essential to a healthy brain function, with the amount, localisation, and chemical form often tightly controlled. Evidence points towards loss of metal ion homeostasis within the ageing brain; in particular brain Fe accumulation appears to be a hallmark of ageing, which may place the brain at a greater risk of neurodegenerative disease. Unfortunately, the cause or consequence of altered brain metal ion homeostasis during ageing remains unknown, and there is a lack of data comparing brain metal ion homeostasis with other events of the ageing process (e.g. brain metabolism, brain inflammation). This study has utilised a multi-modal approach that incorporated: X-ray fluorescence microscopy for elemental mapping of metal ion homeostasis, Perl's Fe histochemistry, FTIR spectroscopic biochemical imaging of lactate and protein aggregates, and immuno-fluorescence analysis of markers of brain inflammation and Fe storage proteins (heavy-chain ferritin, light-chain ferritin, and mitochondrial ferritin). Interestingly, while age-related Fe accumulation was observed in corpus callosum white matter of murine (C56BL/6J) brain tissue (concomitant with elevated levels of markers of brain inflammation and altered metabolism), Fe content was not altered within the hippocampus (a decrease in total Zn within the mossy fibres was observed). Ultimately, the results of this study demonstrate an important association between elevated brain Fe and brain inflammation during natural ageing. This study also highlights that future research is required to image different chemical forms of Fe with respect to changes in brain metabolism and inflammation, as well as localising these changes to specific cell types

Aging and cancer: The role of macrophages and neutrophils

Impaired immune function has been implicated in the declining health and higher incidence of cancer in the elderly. However, age-related changes to immunity are not completely understood. Neutrophils and macrophages represent the first line of defence yet their ability to phagocytose pathogens decrease with aging. Cytotoxic T lymphocytes are critical in eliminating tumors, but T cell function is also compromised with aging. T cell responses can be regulated by macrophages and may depend on the functional phenotype macrophages adopt in response to microenvironmental signals. This can range from pro-inflammatory, anti-tumorigenic M1 to anti-inflammatory, pro-tumorigenic M2 macrophages. Macrophages in healthy elderly adipose and hepatic tissue exhibit a more pro-inflammatory M1 phenotype compared to young hosts whilst immunosuppressive M2 macrophages increase in elderly lymphoid tissues, lung and muscle. These M2-like macrophages demonstrate altered responses to stimuli. Recent studies suggest that neutrophils also regulate T cell function and, like macrophages, neutrophil function is modulated with aging. It is possible that age-modified tissue-specific macrophages and neutrophils contribute to chronic low-grade inflammation that is associated with dysregulated macrophage-mediated immunosuppression, which together are responsible for development of multiple pathologies, including cancer. This review discusses recent advances in macrophage and neutrophil biology in healthy aging and cancer

Loss of MXD3 induces apoptosis of Reh human precursor B acute lymphoblastic leukemia cells.

MXD3 is a transcription factor that plays an important role in proliferation of human DAOY medulloblastoma cells. Here, we demonstrate that MXD3 is highly enriched in human precursor B acute lymphoblastic leukemia (preB ALL) samples compared to mobilized peripheral blood mononuclear cells, bone marrow, or hematopoietic stem cells from healthy donors. MXD3 knock-down in the preB ALL cell line Reh resulted in decreased cell numbers with no change in G0/G1, S or G2/M populations but increased apoptosis compared to control cells. Our results suggest that MXD3 is important for survival of Reh preB ALL cells, possibly as an anti-apoptotic factor