The tumour microenvironment links complement system dysregulation and hypoxic signalling.

The complement system is an innate immune pathway typically thought of as part of the first line of defence against "non-self" species. In the context of cancer, complement has been described to have an active role in facilitating cancer-associated processes such as increased proliferation, angiogenesis and migration. Several cellular members of the tumour microenvironment express and/or produce complement proteins locally, including tumour cells. Dysregulation of the complement system has been reported in numerous tumours and increased expression of complement activation fragments in cancer patient specimens correlates with poor patient prognosis. Importantly, genetic or pharmacological targeting of complement has been shown to reduce tumour growth in several cancer preclinical models, suggesting that complement could be an attractive therapeutic target. Hypoxia (low oxygen) is frequently found in solid tumours and has a profound biological impact on cellular and non-cellular components of the tumour microenvironment. In this review, we focus on hypoxia since this is a prevailing feature of the tumour microenvironment that, like increased complement, is typically associated with poor prognosis. Furthermore, interesting links between hypoxia and complement have been recently proposed but never collectively reviewed. Here, we explore how hypoxia alters regulation of complement proteins in different cellular components of the tumour microenvironment, as well as the downstream biological consequences of this regulation

Theranostic nanoparticles enhance the response of glioblastomas to radiation

YesDespite considerable progress with our understanding of glioblastoma multiforme (GBM) and the precise delivery of radiotherapy, the prognosis for GBM patients is still unfavorable with tumor recurrence due to radioresistance being a major concern. We recently developed a cross-linked iron oxide nanoparticle conjugated to azademethylcolchicine (CLIO-ICT) to target and eradicate a subpopulation of quiescent cells, glioblastoma initiating cells (GICs), which could be a reason for radioresistance and tumor relapse. The purpose of our study was to investigate if CLIO-ICT has an additive therapeutic effect to enhance the response of GBMs to ionizing radiation. Methods: NSG™ mice bearing human GBMs and C57BL/6J mice bearing murine GBMs received CLIO-ICT, radiation, or combination treatment. The mice underwent pre- and post-treatment magnetic resonance imaging (MRI) scans, bioluminescence imaging (BLI), and histological analysis. Tumor nanoparticle enhancement, tumor flux, microvessel density, GIC, and apoptosis markers were compared between different groups using a one-way ANOVA and two-tailed Mann-Whitney test. Additional NSG™ mice underwent survival analyses with Kaplan–Meier curves and a log rank (Mantel–Cox) test. Results: At 2 weeks post-treatment, BLI and MRI scans revealed significant reduction in tumor size for CLIO-ICT plus radiation treated tumors compared to monotherapy or vehicle-treated tumors. Combining CLIO-ICT with radiation therapy significantly decreased microvessel density, decreased GICs, increased caspase-3 expression, and prolonged the survival of GBM-bearing mice. CLIO-ICT delivery to GBM could be monitored with MRI. and was not significantly different before and after radiation. There was no significant caspase-3 expression in normal brain at therapeutic doses of CLIO-ICT administered. Conclusion: Our data shows additive anti-tumor effects of CLIO-ICT nanoparticles in combination with radiotherapy. The combination therapy proposed here could potentially be a clinically translatable strategy for treating GBMs

Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism.

Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers

Evaluating the Reproducibility of Mouse Anatomy under Rotation in a Custom Immobilization Device for Conformal FLASH Radiotherapy

The observation of an enhanced therapeutic index for FLASH radiotherapy in mice has created interest in practical laboratory-based FLASH irradiators. To date, systems capable of 3D conformal FLASH irradiation in mice have been lacking. We are developing such a system, incorporating a high-current linear accelerator to produce a collimated X-ray beam in a stationary beamline design, rotating the mouse about a longitudinal axis to achieve conformal irradiation from multiple beam directions. The purpose of this work was to evaluate the reproducibility of mouse anatomy under rotation at speeds compatible with conformal FLASH delivery. Three short-hair mice and two hairless mice were immobilized under anesthesia in body weight-specific contoured plastic molds, and subjected to three rotational (up to 3 revolutions/s) and two non-rotational movement interventions. MicroCT images were acquired before and after each intervention. The displacements of 11 anatomic landmarks were measured on the image pairs. The displacement of the anatomical landmarks with any of the interventions was 0.5 mm or less for 92.4% of measurements, with a single measurement out of 275 (11 landmarks × 5 interventions × 5 mice) reaching 1 mm. There was no significant difference in the displacements associated with rotation compared to those associated with moving the immobilized mouse in and out of a scanner or with leaving the mouse in place for 5 min with no motion. There were no significant differences in displacements between mice with or without hair, although the analysis is limited by small numbers, or between different anatomic landmarks. These results show that anatomic reproducibility under rotation speed corresponding to FLASH irradiation times appears to be compatible with conformal/stereotactic irradiation in mice

FLASH Irradiation Results in Reduced Severe Skin Toxicity Compared to Conventional-Dose-Rate Irradiation

Radiation therapy, along with surgery and chemotherapy, is one of the main treatments for cancer. While radiotherapy is highly effective in the treatment of localized tumors, its main limitation is its toxicity to normal tissue. Previous preclinical studies have reported that ultra-high dose-rate (FLASH) irradiation results in reduced toxicity to normal tissues while controlling tumor growth to a similar extent relative to conventional-dose-rate (CONV) irradiation. To our knowledge this is the first report of a dose-response study in mice comparing the effect of FLASH irradiation vs. CONV irradiation on skin toxicity. We found that FLASH irradiation results in both a lower incidence and lower severity of skin ulceration than CONV irradiation 8 weeks after single-fraction hemithoracic irradiation at high doses (30 and 40 Gy). Survival was also higher after FLASH hemithoracic irradiation (median survival >180 days at doses of 30 and 40 Gy) compared to CONV irradiation (median survival 100 and 52 days at 30 and 40 Gy, respectively). No ulceration was observed at doses 20 Gy or below in either FLASH or CONV. These results suggest a shifting of the dose-response curve for radiation-induced skin ulceration to the right for FLASH, compared to CONV irradiation, suggesting the potential for an enhanced therapeutic index for radiation therapy of cancer

Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice

Radiation therapy is the most effective cytotoxic therapy for localized tumors. However, normal tissue toxicity limits the radiation dose and the curative potential of radiation therapy when treating larger target volumes. In particular, the highly radiosensitive intestine limits the use of radiation for patients with intra-abdominal tumors. In metastatic ovarian cancer, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of abdominal metastases. However, TAI fell out of favor due to high toxicity of the intestine. Here we utilized an innovative preclinical irradiation platform to compare the safety and efficacy of TAI ultra-high dose rate FLASH irradiation to conventional dose rate (CONV) irradiation in mice. We demonstrate that single high dose TAI-FLASH produced less mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and spared cell death in crypt base columnar cells compared to TAI-CONV irradiation. Importantly, TAI-FLASH and TAI-CONV irradiation had similar efficacy in reducing tumor burden while improving intestinal function in a preclinical model of ovarian cancer metastasis. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of abdominal radiotherapy, with potential application to metastatic ovarian cancer

Development of molecular targeted imaging methods for detection of lung metastasis and angiogenesis

The 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 &lt; 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&amp;ast;-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.</p

Development of molecular targeted imaging methods for detection of lung metastasis and angiogenesis

The 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 &lt; 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 &alpha;v&beta;3-expressing endothelium in vivo, using a conjugate of cyclic penta-peptides c(RGDyK) and 2.8 &mu;m MPIO [c(RGDyK)-MPIO]. To this end, I demonstrate in Chapter 5 that c(RGDyK)-MPIO specifically binds to &alpha;v&beta;3-expressing endothelium in subcutaneous tumours and yields quantifiable contrast effects on T2&ast;-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.This thesis is not currently available via ORA

Multicellular spheroids as in vitro models of oxygen depletion during FLASH irradiation

Purpose The differential response of normal and tumor tissues to ultra-high dose rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study is to investigate these effects within hypoxic multicellular tumor spheroids, through simulations and experiments. Materials and Methods Physicobiological equations were derived to model (i) the diffusion and metabolism of oxygen within spheroids; (ii) its depletion through reactions involving radiation-induced radicals; and (iii) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored post-radiation. Clonogenic survival of two other cell lines was also investigated. Results The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroid compared to conventional irradiation, but no significant difference was observed for well-oxygenated 2D-cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy. Conclusion Tumor spheroids can be used as a model to study FLASH irradiation in vitro . The improved survival of tumor spheroids receiving FLASH radiation confirms that ultra-fast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect

Molecular magnetic resonance imaging of angiogenesis in vivo using polyvalent cyclic RGD-iron oxide microparticle conjugates.

Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule α(v)β(3) integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for α(v)β(3), to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 μm MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P &lt; 0.001) and flow (44-fold increase; P &lt; 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P &lt; 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T(2)* relaxation times than the control agent in both tumour models (melanoma P &lt; 0.001; colorectal P &lt; 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P &lt; 0.05) and colorectal (P &lt; 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with α(v)β(3) expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to α(v)β(3) expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging