1,721,169 research outputs found
Identification of recruited myeloid cells important for the development of hepatic metastasis
Get PDFHepatic metastases are a frequent cause of mortality in colon cancer patients. Many patients with hepatic metastasis have large tumour burden, signaling the need for therapies capable of down-staging metastatic disease. Research evidence indicates that immune cells promote the metastasis of various primary cancers. We wish to determine whether immune cells play a role in the promotion of hepatic colon cancer metastasis. Using the well-characterised method of intrasplenic tumour cell injection, we developed hepatic metastases in both immunocompetent and immunoincompetent mice using a range of murine and human cancer cell lines. We analysed the immune cell infiltrates associated with hepatic metastases using flow cytometry and identified chemokines responsible for their recruitment using targeted protein arrays. The effect of immune cell depletion or inhibition of immune cell recruitment was determined using various in-vivo imaging techniques. Hepatic metastases developed using the murine colon cancer cell line MC38 were associated with CD11b+/Gr1mid/CCR2+ monocytes, the recruitment of which was delayed by inhibition of tumour-derived CCL2. In contrast, human HT29, HCT-116 and LoVo hepatic metastases in SCID mice were associated with infiltrates of CD45+/CXCR2+ neutrophils recruited in response to tumour-derived Macrophage Inhibitory Factor (MIF). Depletion of Gr1mid cells in CD11b-DTR transgenic mice delayed MC38 metastasis development, whilst neutrophil depletion using anti-Ly6G antibodies significantly inhibited the growth of HT29, HCT-116 and LoVo hepatic metastases. The neutrophils recruited to HT29, HCT-116 and LoVo hepatic metastases promoted angiogenesis, potentially through the expression of fibtroblast growth factor-2. This work demonstrates a role for myeloid cells in the development of hepatic metastasis from colon cancer and in doing so identifies various potential therapeutic targets
The role of macrophages in the host response to radiation
Get PDFEmerging pre-clinical data suggests a role for radiation in eliciting antitumor immunity. Effective anti-tumour responses can be hindered by highly immunosuppressive tumour microenvironments. We aimed to investigate the role of macrophages in the immune response to radiation.
We used the murine colorectal cancer cell line MC38 and the pancreatic cell line KPC for in vitro and in vivo work. To examine the immune response following irradiation, subcutaneous xenografts were analysed by flow cytometry. Further characterisation of macrophages was carried out by gene expression analyses of RNA from isolated cells, along with ex-vivo functional assays. To determine the effect of macrophage depletion, we used an antibody targeting macrophage colony stimulating factor (CSF-1).
Single dose 10Gy irradiation to murine tumors, generated from colorectal (MC38) and pancreatic (KPC) cell lines induced Colony Stimulating Factor 1 (CSF-1). Coincident with the elevation in CSF1, CD11b+ F480+ macrophages increased in the tumors, peaking five days following irradiation. These tumor-associated macrophages (TAMs) from irradiated tumors were skewed toward an immunosuppressive phenotpye (CD206hi iNOSlo) and were more effective in suppressing CD8 T cell expansion ex vivo.
Macrophage depletion via anti-CSF (aCSF) reduced macrophage numbers yet only achieved tumor growth delay when combined with radiation. The tumor growth delay from aCSF after radiation was abrogated by depletion of CD8 T cells. There was enhanced recognition of tumor cell antigens by T cells isolated from irradiated tumors, consistent with increased antigen priming. Radiation stimulated increased expression of PD-L1 on tumor cells, limiting antitumor responses. The addition of anti-PD-L1 resulted in improved tumor suppression and even regression in the highly resistant murine pancreatic cancer model.
In summary we show that adaptive immunity induced by radiation is limited by the recruitment of highly M2 polarised immunosuppressive macrophages. Macrophage depletion with aCSF partly reduced the immunosuppression after radiation, but additional treatment with anti-PD-L1 was required to achieve tumor regression.</p
Characterising the role of circulating immune cells in brain metastasis
Get PDFBrain metastasis is a frequent occurrence in cancer patients and carries a high mortality rate. The incidence of brain metastasis is on the rise, highlighting the need for improved therapeutic intervention. Immune cells have been shown to promote disseminated tumour cells to colonise the lung and liver. Therefore, we aim to determine whether immune cells also facilitate brain metastasis by describing the host immune response to tumour cells attached to the brain vasculature.We developed a model of brain metastasis by using ultrasound guidance to perform intracardiac injection of tumour cells. Using this method, we identified highly and weakly brain metastatic cell lines. To understand how cancer cells develop into brain metastases, we analysed brains harvested 4 h- 14 d after tumour injections.At 4 h after intracardiac injection, only cell lines that developed into brain metastases were found adhered to the brain vasculature in high numbers. A small number of arrested tumour cells clustered with CD45⁺ immune cells. These tumour-CD45 clusters persisted over time whilst the frequency of solitary tumour cells declined. Tumour-associated CD45⁺ immune cells were identified to be Ly6G⁺Gr-1⁺CD11c⁻ myeloid cells. Considerably more tumour-CD45⁺ immune cell clusters were found within the brain vasculature when tumour cells were injected into mice bearing a primary tumour. Increased tumour-CD45⁺ immune cells clusters correlated with an increased number of brain metastases in the same group of mice. We also found a positive association between increased tumour-immune clusters and levels of tumour and host derived G-CSF. To establish a causal relationship between tumour cell-CD45 clusters and metastases, we developed an experimental setup for transcranial imaging. Our results suggest that tumour recruited immune cells may promote tumour cell colonisation of the brain and provides a framework for further investigation
The effects of radiation on the extracellular matrix
Get PDFThe extracellular matrix (ECM) is a complex meshwork of insoluble fibrillary proteins and signalling factors interacting together to provide architectural and instructional cues to surrounding cells. Alterations in ECM organisation or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases and cancer. Due to its complexity, ECM involvement in cancer is still poorly understood, and its role in ionising radiation response is unknown.
We used subcutaneous mouse models of colorectal and pancreatic cancer to investigate the effects of ionising radiation (IR) on tumour microenvironment. In particular, we looked at the effects of radiation on Col5a1, a regulatory fibril- forming collagen that forms heterotypic fibrils with Col1a1. Immunostainings showed that Col5a1 protein expression decreased in irradiated tumours compared with the non-irradiated controls, whereas Col1a1 expression did not change. This result was confirmed by RT-PCR and Western Blot analysis. Using picrosirius red staining, we found that collagen deposition was significantly altered by irradiation. Besides, five collagen structural parameters (alignment, density, width, length and straightness) were analysed using second harmonic generation imaging, a highly specific technology for detection of collagen fibres. Our data indicated a significant decrease in collagen width after irradiation in a pancreatic cancer model, whereas all parameters were significantly changed in the colorectal cancer model. Col5a1 gene knock-down significantly decreased cell survival upon IR and cell migration as assessed using colony formation assays and 3D-spheroid imaging
To confirm our data, we generated a knockout Col5a1 KPC cell line and injected it subcutaneously in vivo in C57/BL6 mice. The lack of Col5a1 in the in vivo tumour led to a reduction in tumour growth; however, radiation seemed to activate a compensatory effect which refrained the irradiation inhibition effect on tumour growth. In vitro migration assay and cell growth assay, results showed a similar pattern.
Col5a1 knock-down and knockout significantly inhibited cell proliferation, inhibited cell migration in vitro and inhibited tumour growth in vivo. Our data, therefore, present Col5a1 as a possible novel prognostic marker and a potential therapeutic target
The effects of chemotherapy agents on hypoxia in colorectal cancer
Get PDFTumour hypoxia describes the phenomenon of reduced oxygen tension within solid
tumours, resulting in an increase in morbidity, mortality and metastasis. We aim to identify
chemotherapy agents that may reduce oxygen consumption and ultimately, improve tumour
hypoxia and radiosensitivity.
Four colorectal cancer cell lines (COLO320DM, DLD1, HCT116 and HT29) and a nontransformed
cell line (MRC5) were investigated. Clonogenic and cytotoxicity assays of a range
of agents were used to determine sub-lethal concentrations that would alter consumption
without cytotoxicity. The oxygen consumption and mitochondrial function of treated cells were
assessed with the XF96 Analyzer. Flow cytometry, high performance liquid chromatography
and western blot were performed to delineate mechanisms of action. The most responsive cell
lines were grown as spheroids for hypoxia modelling before being tested in vivo for radiation
sensitivity. In vivo tumour hypoxia analysis was achieved with the in vivo imaging system.
The oxygen consumption of all cancer cell lines was markedly reduced after treatment with
a number of the agents compared to the non-transformed cell line. DLD1 and HCT116 cell lines
showed the greatest balance of resistance to toxicity and reduction in oxygen consumption.
Hypoxia imaging of the subsequent spheroids and in vivo tumours further demonstrated a
reduction in hypoxia consistent with drug induced decrease in oxygen consumption. This also
conferred a relative enhancement in radiosensitivity in both in vitro and in vivo settings.
Despite decades of clinical use these known chemotherapeutics have not been
implicated in alterations in oxidative metabolism. These results raise the prospect of
using them in an alternative way to potentially improve clinical outcome.</p
Development of molecular targeted imaging methods for detection of lung metastasis and angiogenesis
Get PDFThe focus of this thesis is the development of two molecularly targeted imaging methods, in both cases based on contrast agents encompassing micron-sized microparticles of iron oxide (MPIO). MPIO are obligate intravascular agents and as presented in this thesis the half-life in the blood circulation is < 1min. In the first approach described in the thesis, the overall goal was to detect metastasis in mouse lungs, very early in metastatic development, by targeting vascular cell adhesion molecule 1 (VCAM-1) using conjugates of an anti-VCAM-1 antibody and 1 μm MPIO (VCAM-MPIO). In Chapter 3, I demonstrate specific retention of VCAM-MPIO in the vasculature of a lung metastasis model, and also the very short blood half-life of the contrast agent; both of which suggest the potential for in vivo detection. In Chapter 4, I show that whilst the bound VCAM-MPIO do not sufficiently dephase the signal obtained with the bright lung MRI approaches used (hyperpolarized 3He/129Xe or 19F MRI), it is possible to sensitively detect the presence of lung metastases in vivo using radiolabelled VCAM-MPIO (89Zr-DFO-VCAM-MPIO) in combination with PET imaging. The overall goal of the second approach described, was to detect and characterize tumour angiogenesis by targeting αvβ3-expressing endothelium in vivo, using a conjugate of cyclic penta-peptides c(RGDyK) and 2.8 μm MPIO [c(RGDyK)-MPIO]. To this end, I demonstrate in Chapter 5 that c(RGDyK)-MPIO specifically binds to αvβ3-expressing endothelium in subcutaneous tumours and yields quantifiable contrast effects on T2*-weighted MRI. Furthermore, I have implemented in this approach gadolinium DCE imaging, providing dynamic vascular information. To date there is no reported detection method for pulmonary metastasis at the micrometastastic stage, as presented in this thesis. Translation of this method into clinic could allow for earlier therapeutic intervention and, thus, more effective treatment. The angiogenesis characterization imaging method presented here may provide a sensitive approach for the characterization of heterogeneity in tumour angiogenesis/vascularity and monitoring of anti-angiogenic therapies
Aspirin affects early phases of metastasis through the inhibition of COX-1-thromboxane A2 axis
Get PDFMetastasis is the major cause of cancer related mortality, due to a poor understanding of the metastatic process and a subsequent lack of effective anti-metastatic therapies. Evidence from experimental studies and clinical trials has shown that aspirin reduces the incidence of distant metastases. It is well established that aspirin inhibits cyclooxygenase (COX)-1 and COX-2, triggering anti-thrombotic and anti-inflammatory effects, respectively. However, the mechanisms underlying the anti-metastatic effect of aspirin are still largely unknown.
By using an experimental model of pulmonary metastasis, we have found that the anti-metastatic effect of aspirin is associated with the inhibition of COX-1. In support of this, metastasis establishment was impaired in COX-1 deficient mice, suggesting a pivotal role of this isoform in the metastatic process. Looking in more detail into the metastatic cascade, we found that COX-1 contributes to the intravascular phase of metastasis and promotes the early persistence of tumour cells in the lung vasculature. In particular, COX-1 inhibition decreased the interaction of platelets with tumour cells and was associated with the reduction of endothelial activation, of tumour cell adhesion to the endothelium, of recruitment of metastasis-promoting monocytes/macrophages and of transendothelial migration. We have identified platelet-derived thromboxane A2 (TXA2) as the main product of COX-1 responsible for its permissive effect on metastasis. Indeed, TXA2 delivered to mice in combination with aspirin was able to abrogate the anti-metastatic effect of aspirin.
Taken together, our data suggest that the inhibition of COX-1:TXA2 axis by aspirin is sufficient to exert an anti-metastatic effect. In particular, the inhibition of platelet-derived TXA2 seems to affect multiple early steps of the haematogenous transit of tumour cells. In this perspective, TXA2 might represent a more selective therapeutic target for the prevention of metastasis.</p
Mechanisms of induction of type I/III interferons in cancer cells by ionizing radiation and the role of tumour cell-derived type I IFN and type I IFN signalling in anti-tumour immune response
Get PDFInterferons (IFNs) are a group of cytokines that have been shown to play an
important role in both radiotherapy and the anti-tumour immune response. How
ionizing radiation (IR) induces IFNs in the tumour is still not fully understood. We
found that IR directly induced dose- and time-dependent upregulation of I/III IFNs in
cancer cells. This induction occurs via cytosolic DNA sensors-STING-TBK1-IRFs
signalling axis and to less extent, the NF-κB pathway in colorectal cancer cells.
Furthermore, Type I/III acts as a feedback loop to further enhance IFNs expression
via upregulating key factors in the signalling axis.
In vivo, cancer cells not the immune cells actually have the greatest
contribution to overall intratumoural production of type I IFN in the MC38 tumours. By
using a variety of animal models, we demonstrated that tumour-derived type I IFN
has critical contribution to the anti-tumour immune response at local site. Strikingly
and unexpectedly, abrogation of type I IFN signalling via IFNAR1 KO in cancer cells
led to remarkable improvement of tumour response to IR. This phenomenon is
mainly driven by significantly enhanced susceptibility of cancer cells to CD8+ T cellmediated
killing via PD-L1/Galectin-9-independent mechanisms. Overall, these
findings add another layer of complexity to the understanding of irradiation-induced
anti-tumour immunity and cancer immune evasion, and may provide novel insights
into combinational therapy of radiotherapy and immunotherapy.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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