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Purpose: Tumor vessels influence the growth and response of tumors to therapy. Imaging vascular changes in vivo using dynamic contrast-enhanced MRI (DCE-MRI) has shown potential to guide clinical decision making for treatment. However, quantitative MR imaging biomarkers of vascular function have not been widely adopted, partly because their relationship to structural changes in vessels remains unclear. We aimed to elucidate the relationships between vessel function and morphology in vivo.Experimental Design: Untreated preclinical tumors with different levels of vascularization were imaged sequentially using DCE-MRI and CT. Relationships between functional parameters from MR (iAUC, Ktrans, and BATfrac) and structural parameters from CT (vessel volume, radius, and tortuosity) were assessed using linear models. Tumors treated with anti-VEGFR2 antibody were then imaged to determine whether antiangiogenic therapy altered these relationships. Finally, functional–structural relationships were measured in 10 patients with liver metastases from colorectal cancer.Results: Functional parameters iAUC and Ktrans primarily reflected vessel volume in untreated preclinical tumors. The relationships varied spatially and with tumor vascularity, and were altered by antiangiogenic treatment. In human liver metastases, all three structural parameters were linearly correlated with iAUC and Ktrans. For iAUC, structural parameters also modified each other's effect.Conclusions: Our findings suggest that MR imaging biomarkers of vascular function are linked to structural changes in tumor vessels and that antiangiogenic therapy can affect this link. Our work also demonstrates the feasibility of three-dimensional functional–structural validation of MR biomarkers in vivo to improve their biological interpretation and clinical utility. Clin Cancer Res; 24(19); 4694–704. ©2018 AACR.

Supplementary methods and data. Supplementary Table 1. Equations of chosen models fit using ordinary least squares regression with the best Bayesian Information Criterion for iAUC90, Ktrans, and BATfrac in untreated preclinical tumors (cohort 1). Goodness of fit for each model was assessed by two measures, residuals vs fitted and Q-Q plots, which are shown in Supplementary Figure 4. Supplementary Table 2. Evaluation of model fits for iAUC90, Ktrans, and BATfrac generated from cohort 1 applied to preclinical data from cohort 2 treated with control IgG or with anti-VEGFR2 antibody DC101. Goodness of fit for each model was assessed by two measures, residuals vs fitted and Q-Q plots, which are shown in Supplementary Figures 6-7. Supplementary Table 3. Equations of chosen linear models with best Bayesian Information Criterion for iAUC90 and Ktrans in clinical tumors. Supplementary Figure 1. Signal-to-noise ratio on DCE-MRI is affected > 5 mm away from surface coil. Supplementary Figure 2. Longitudinal, repeated imaging with CT curbs tumor growth. Supplementary Figure 3. Vascular parameters (vessel length (a), tortuosity (b), and radius (c)) correlate poorly with volume of MC38 and FaDu tumors. Supplementary Figure 4. For each MR parameter of untreated preclinical tumors of cohort 1, the chosen linear model has normal residuals, as assessed by residual vs fitted plots and Q-Q plots, demonstrating goodness of fit. Supplementary Figure 5. For each MR parameter of untreated preclinical tumors of cohort 2, the tested linear model has normal residuals, as assessed by residual vs fitted plots and Q-Q plots, demonstrating goodness of fit. Supplementary Figure 6. Goodness-of-fit, as assessed by residual vs fitted plots and by Q-Q plots, for linear models applied to DC101-treated preclinical tumors of cohort 2. The chosen linear models for iAUC and BAT have residuals that exhibit departures from normality, indicating that the treatment altered these parameters. Supplementary Figure 7. For each MR parameter of clinical tumors, the chosen linear models demonstrate normal residuals, indicating goodness-of-fit.

Standardisation of animal handling procedures for a wide range of preclinical imaging scanners will improve imaging performance and reproducibility of scientific data. Whilst there has been significant effort in defining how well scanners should operate and how in vivo experimentation should be practised, there is little detail on how to achieve optimal scanner performance with best practices in animal welfare. Here, we describe a system-agnostic, adaptable and extensible animal support cradle system for cardio-respiratory-synchronised, and other, multi-modal imaging of small animals. The animal support cradle can be adapted on a per application basis and features integrated tubing for anaesthetic and tracer delivery, an electrically driven rectal temperature maintenance system and respiratory and cardiac monitoring. Through a combination of careful material and device selection, we have described an approach that allows animals to be transferred whilst under general anaesthesia between any of the tomographic scanners we currently or have previously operated. The set-up is minimally invasive, cheap and easy to implement and for multi-modal, multi-vendor imaging of small animals.

Commercial mouse chow is designed to provide a complete, nutrient-rich diet, and it can contain upwards of 100 mg/kg manganese, an essential mineral. Manganese acts as a relaxation time-shortening contrast agent for both T1and T2, and where standard chow is hydrated in the gastrointestinal tract, bright signals are produced when using T1-weighted imaging (T1WI). As a result of peristalsis, gastrointestinal hyperintensities result in temporally unstable signals, leading to image ghosting and decreased resolution from that prescribed. To avoid the problem, various methods of gastrointestinal tract modulation, including the use of intestinal cleansing with laxatives and dietary modulation, have been reported. Here, dietary modulation has been extended to the use of a biologically innocuous, long-term change of diet. In this study, we report on the use of a commercially available manganese-free chow to improve the image quality of the gastrointestinal tract. This manganese-free chow, apart from the omitted manganese which is available in tap water, is a complete diet and readily available. We investigated the time-dependent, diet-related gastrointestinal intensities on short-TR T1WI magnetic resonance imaging; monitored body mass, food and water consumption and standard blood biochemistry analysis following diet change; and determined manganese concentration in blood plasma following a five-day change to manganese-free chow. We show that the manganese-free chow presents a refinement to other gastrointestinal tract modulation, as it avoids the need for invasive procedures for gut voiding and can be provided ad libitum so that animals can be maintained with no need for prescribed diet change before imaging.

Treatment options for patients with pancreatic cancer are limited and survival prospects have barely changed over the past 4 decades. Chemoradiation treatment (CRT) has been used as neoadjuvant therapy in patients with borderline resectable disease to reduce tumour burden and increase the proportion of patients eligible for surgery. Antimetabolite drugs such as gemcitabine and 5-fluorouracil are known to sensitise pancreatic tumours to radiation treatment. Likewise, photodynamic therapy (PDT) has also been shown to enhance the effect of radiation therapy. However, PDT is limited to treating superficial lesions due to the attenuation of light by tissue. The ability of the related technique, sonodynamic therapy (SDT), to enhance CRT was investigated in two murine models of pancreatic cancer (PSN-1 and BxPC-3) in this study. SDT uses low intensity ultrasound to activate an otherwise non-toxic sensitiser, generating toxic levels of reactive oxygen species (ROS) locally. It is applicable to greater target depths than PDT due to the ability of ultrasound to propagate further than light in tissue. Both CRT and the combination of CRT plus SDT delayed tumour growth in the two tumour models. In the PSN-1 model, but not the BxPC-3 model, the combination treatment caused an increase in survival relative to CRT alone (p = 0.038). The improvement in survival conferred by the addition of SDT in this model may be related to differences in tumour architecture between the two models. MRI and US images showed that PSN-1 tumours were less well perfused and vascularised than BxPC-3 tumours. This poor vascularisation may explain why PSN-1 tumours were more susceptible to the effects of vascular damage exerted by SDT treatment.

Background Colorectal cancer (CRC) liver metastasis is highly unfavorable for patient outcome and is a leading cause of cancer-related death. Pre-clinical research of CRC liver metastasis predominately utilizes CRC cell lines grown in tissue culture. Here, we demonstrate that CRC liver metastases organoids derived from human specimens recapitulate some aspects of human disease. Methods Human CRC liver metastases pathological specimens were obtained following patient consent. Tumor disaggregates were plated and organoids were allowed to expand. CRC markers were identified by immunofluorescence. Stem cell genes were analysed by QPCR and flow cytometry. Response to drug therapy was quantified using time-lapse imaging and MATLAB analysis. Results Organoids showed global expression of the epithelial marker, EpCAM and the adenocarcinoma marker, CEA CAM1. Flow cytometry analysis demonstrated that organoids express the stem cell surface markers CD24 and CD44. Finally, we demonstrated that CRC liver metastases organoids acquire chemotherapy resistance and can be utilized as surrogates for drug testing. Conclusion These data demonstrate that CRC liver metastases organoids recapitulate some aspects of human disease and may provide an invaluable resource for investigating novel drug therapies, chemotherapy resistance and mechanism of metastasis., Highlights • Human colorectal cancer liver metastases can be cultured as organoids using rapid dissociation. • Organoids retain characteristics of human disease. • Organoids acquire chemotherapy resistance.

Purpose To develop an MR-compatible resistive heater for temperature maintenance of anaesthetized animals. Materials and Methods An MR-compatible resistive electrical heater was formed from a tightly-wound twisted pair wire, interfaced to a homeothermic maintenance controller. Fat-suppressed images and localized spectra were acquired with the twisted pair heater and a near-identical single strand heater during operation at maximum power. Data were also acquired in the absence of heating to demonstrate the insensitivity of MR to distortions arising from the passage of current through the heater elements. The efficacy of temperature maintenance was examined by measuring rectal temperature immediately following induction of general anesthesia and throughout and after the acquisition of a heater artifact-prone image series. Results Images and spectra acquired in the presence and absence of DC current through the twisted pair heater were identical whereas the passage of current through the single strand wire created field shifts and lineshape distortions. Temperature that is lost during anesthesia induction was recovered within approximately 10–20 minutes of induction, and a stable temperature is reached as the animal's temperature approaches the set target. Conclusion The twisted pair wire heater does not interfere with MR image quality and maintains adequate thermal input to the animal to maintain body temperature.

Commercial mouse chow is designed to provide a complete, nutrient-rich diet, and it can contain upwards of 100 mg/kg manganese, an essential mineral. Manganese acts as a relaxation time-shortening contrast agent for both T1 and T2, and where standard chow is hydrated in the gastrointestinal tract, bright signals are produced when using T1-weighted imaging (T1WI). As a result of peristalsis, gastrointestinal hyperintensities result in temporally unstable signals, leading to image ghosting and decreased resolution from that prescribed. To avoid the problem, various methods of gastrointestinal tract modulation, including the use of intestinal cleansing with laxatives and dietary modulation, have been reported. Here, dietary modulation has been extended to the use of a biologically innocuous, long-term change of diet. In this study, we report on the use of a commercially available manganese-free chow to improve the image quality of the gastrointestinal tract. This manganese-free chow, apart from the omitted manganese which is available in tap water, is a complete diet and readily available. We investigated the time-dependent, diet-related gastrointestinal intensities on short-TR T1WI magnetic resonance imaging; monitored body mass, food and water consumption and standard blood biochemistry analysis following diet change; and determined manganese concentration in blood plasma following a five-day change to manganese-free chow. We show that the manganese-free chow presents a refinement to other gastrointestinal tract modulation, as it avoids the need for invasive procedures for gut voiding and can be provided ad libitum so that animals can be maintained with no need for prescribed diet change before imaging.

A magnetic resonance (MR)-, computed tomography (CT)-, single-photon emission computed tomography (SPECT)-, and positron emission tomography (PET)-compatible carbon-fiber sheet resistor for temperature maintenance in small animals where space limitations prevent the use of circulating fluids was developed. A 250 Ω carbon-fiber sheet resistor was mounted to the underside of an imaging cradle. Alternating current, operating at 99 kHz, and with a power of 1-2 W, was applied to the resistor providing a cradle base temperature of ∼37°C. Temperature control was implemented with a proportional–integral–derivative controller, and temperature maintenance was demonstrated in 4 mice positioned in both MR and PET/SPECT/CT scanners. MR and CT compatibility were also shown, and multimodal MR-CT-PET-SPECT imaging of the mouse abdomen was performed in vivo. Core temperature was maintained at 35.5°C ± 0.2°C. No line-shape, frequency, or image distortions attributable to the current flow through the heater were observed on MR. Upon CT imaging, no heater-related artifacts were observed when carbon-fiber was used. Multimodal imaging was performed and images could be easily coregistered, displayed, analyzed, and presented. Carbon fiber sheet resistors powered with high-frequency alternating current allow homeothermic maintenance that is compatible with multimodal imaging. The heater is small, and it is easy to produce and integrate into multimodal imaging cradles.

A magnetic resonance (MR)-, computed tomography (CT)-, single-photon emission computed tomography (SPECT)-, and positron emission tomography (PET)-compatible carbon-fiber sheet resistor for temperature maintenance in small animals where space limitations prevent the use of circulating fluids was developed. A 250 Ω carbon-fiber sheet resistor was mounted to the underside of an imaging cradle. Alternating current, operating at 99 kHz, and with a power of 1-2 W, was applied to the resistor providing a cradle base temperature of ∼37 °C. Temperature control was implemented with a proportional–integral–derivative controller, and temperature maintenance was demonstrated in 4 mice positioned in both MR and PET/SPECT/CT scanners. MR and CT compatibility were also shown, and multimodal MR-CT-PET-SPECT imaging of the mouse abdomen was performed in vivo. Core temperature was maintained at 35.5 °C ± 0.2 °C. No line-shape, frequency, or image distortions attributable to the current flow through the heater were observed on MR. Upon CT imaging, no heater-related artifacts were observed when carbon-fiber was used. Multimodal imaging was performed and images could be easily coregistered, displayed, analyzed, and presented. Carbon fiber sheet resistors powered with high-frequency alternating current allow homeothermic maintenance that is compatible with multimodal imaging. The heater is small, and it is easy to produce and integrate into multimodal imaging cradles.

The extent of tumor oxygenation is an important factor contributing to the efficacy of radiation therapy (RTx). Interestingly, several preclinical studies have shown benefit of combining RTx with drugs that inhibit tumor blood vessel growth, i.e. angiostatic therapy. Recent findings show that proper scheduling of both treatment modalities allows dose reduction of angiostatic drugs without affecting therapeutic efficacy. We found that whilst low dose sunitinib (20 mg/kg/day) did not affect the growth of xenograft HT29 colon carcinoma tumors in nude mice, the combination with either single dose RTx (1x 5Gy) or fractionated RTx (5x 2Gy/week, up to 3 weeks) substantially hampered tumor growth compared to either RTx treatment alone. To better understand the interaction between RTx and low dose angiostatic therapy, we explored the effects of RTx on tumor angiogenesis and tissue perfusion. DCE-MRI analyses revealed that fractionated RTx resulted in enhanced perfusion after two weeks of treatment. This mainly occurred in the center of the tumor and was accompanied by increased tissue viability and decreased hypoxia. These effects were accompanied by increased expression of the pro-angiogenic growth factors VEGF and PlGF. DCE-MRI and contrast enhanced ultrasonography showed that the increase in perfusion and tissue viability was counteracted by low-dose sunitinib. Overall, these data give insight in the dynamics of tumor perfusion during conventional 2 Gy fractionated RTx and provide a rationale to combine low dose angiostatic drugs with RTx both in the palliative as well as in the curative setting.

Purpose Multi-slice scanning in the abdomen and thorax of small animals is compromised by the effects of respiration unless imaging and respiration are synchronised. To avoid the signal modulations that result from respiration motion and a variable TR, blocks of fully relaxed slices are typically acquired during inter-breath periods, at the cost of scan efficiency. This paper reports a conceptually simple yet effective prospective gating acquisition mode for multi-slice scanning in free breathing small animals at any fixed TR of choice with reduced sensitivity to respiratory motion. Methods Multi-slice scan modes have been implemented in which each slice has its own specific projection or phase encode loop index counter. When a breath is registered RF pulses continue to be applied but data are not acquired, and the corresponding counters remain fixed so that the data are acquired one TR later, providing it coincides with an inter-breath period. The approach is refined to reacquire the slice data that are acquired immediately before each breath is detected. Only the data with reduced motion artefact are used in image reconstruction. The efficacy of the method is demonstrated in the RARE scan mode which is well known to be particularly useful for tumour visualization. Results Validation in mice with RARE demonstrates improved stability with respect to ungated scanning where signal averaging is often used to reduce artefacts. SNR enhancement maps demonstrate the improved efficiency of the proposed method that is equivalent to at least a 2.5 fold reduction in scan time with respect to ungated signal averaging. A steady-state magnetisation transfer contrast prepared gradient echo implementation is observed to highlight tumour structure. Supplementary simulations demonstrate that only small variations in respiration rate are required to enable efficient sampling with the proposed method. Conclusions The proposed prospective gating acquisition scheme enables efficient multi-slice scanning in small animals at the optimum TR with reduced sensitivity to respiratory motion. The method is compatible with a wide range of complementary methods including non-Cartesian scan modes, partially parallel imaging, and compressed sensing. In particular, the proposed scheme reduces the need for continual close monitoring to effect operator intervention in response to respiratory rate changes, which is both difficult to maintain and precludes high throughput.

Purpose: Tumor vessels influence the growth and response of tumors to therapy. Imaging vascular changes in vivo using dynamic contrast-enhanced MRI (DCE-MRI) has shown potential to guide clinical decision making for treatment. However, quantitative MR imaging biomarkers of vascular function have not been widely adopted, partly because their relationship to structural changes in vessels remains unclear. We aimed to elucidate the relationships between vessel function and morphology in vivo. Experimental Design: Untreated preclinical tumors with different levels of vascularization were imaged sequentially using DCE-MRI and CT. Relationships between functional parameters from MR (iAUC, Ktrans, and BATfrac) and structural parameters from CT (vessel volume, radius, and tortuosity) were assessed using linear models. Tumors treated with anti-VEGFR2 antibody were then imaged to determine whether antiangiogenic therapy altered these relationships. Finally, functional–structural relationships were measured in 10 patients with liver metastases from colorectal cancer. Results: Functional parameters iAUC and Ktrans primarily reflected vessel volume in untreated preclinical tumors. The relationships varied spatially and with tumor vascularity, and were altered by antiangiogenic treatment. In human liver metastases, all three structural parameters were linearly correlated with iAUC and Ktrans. For iAUC, structural parameters also modified each other's effect. Conclusions: Our findings suggest that MR imaging biomarkers of vascular function are linked to structural changes in tumor vessels and that antiangiogenic therapy can affect this link. Our work also demonstrates the feasibility of three-dimensional functional–structural validation of MR biomarkers in vivo to improve their biological interpretation and clinical utility. Clin Cancer Res; 24(19); 4694–704. ©2018 AACR.

Because metastasis is associated with the majority of cancer-related deaths, its prevention is a clinical aspiration. Prostanoids are a large family of bioactive lipids derived from the activity of cyclooxygenase-1 (COX-1) and COX-2. Aspirin impairs the biosynthesis of all prostanoids through the irreversible inhibition of both COX isoforms. Long-term administration of aspirin leads to reduced distant metastases in murine models and clinical trials, but the COX isoform, downstream prostanoid, and cell compartment responsible for this effect are yet to be determined. Here, we have shown that aspirin dramatically reduced lung metastasis through inhibition of COX-1 while the cancer cells remained intravascular and that inhibition of platelet COX-1 alone was sufficient to impair metastasis. Thromboxane A2 (TXA2) was the prostanoid product of COX-1 responsible for this antimetastatic effect. Inhibition of the COX-1/TXA2 pathway in platelets decreased aggregation of platelets on tumor cells, endothelial activation, tumor cell adhesion to the endothelium, and recruitment of metastasis-promoting monocytes/macrophages, and diminished the formation of a premetastatic niche. Thus, platelet-derived TXA2 orchestrates the generation of a favorable intravascular metastatic niche that promotes tumor cell seeding and identifies COX-1/TXA2 signaling as a target for the prevention of metastasis.

Purpose Cardiac and respiratory motion derived image artefacts are reduced when data are acquired with cardiac and respiratory synchronisation. Where steady state imaging techniques are required in small animals, synchronisation is most commonly performed using retrospective gating techniques but these invoke an inherent time penalty. This paper reports the development of prospective gating techniques for cardiac and respiratory motion desensitised MRI with significantly reduced minimum scan time compared to retrospective gating. Methods Prospective gating incorporating the automatic reacquisition of data corrupted by motion at the entry to each breath was implemented in short TR 3D spoiled gradient echo imaging. Motion sensitivity was examined over the whole mouse body for scans performed without gating, with respiratory gating, and with cardio-respiratory gating. The gating methods were performed with and without automatic reacquisition of motion corrupted data immediately after completion of the same breath. Prospective cardio-respiratory gating, with acquisition of 64 k-space lines per cardiac R-wave, was used to enable whole body DCE-MRI in the mouse. Results Prospective cardio-respiratory gating enabled high fidelity steady state imaging of physiologically mobile organs such as the heart and lung. The automatic reacquisition of data corrupted by motion at the entry to each breath minimised respiratory motion artefact and enabled a highly efficient data capture that was adaptive to changes in the inter-breath interval. Prospective cardio-respiratory gating control enabled DCE-MRI to be performed over the whole mouse body with the acquisition of successive image volumes every 12–15 s at 422 μm isotropic resolution. Conclusions Highly efficient cardio-respiratory motion desensitised steady state MRI can be performed in small animals with prospective synchronisation, centre-out phase-encode ordering, and the automatic reacquisition of data corrupted by motion at the entry to each breath. The method presented is robust against spontaneous changes in the breathing rate. Steady state imaging with prospective cardio-respiratory gating is much more efficient than with retrospective gating, and enables the examination of rapidly changing systems such as those found when using DCE-MRI.

Evidence from experimental studies and clinical trials has shown that aspirin reduces the incidence of distant metastases. 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 employing mouse models of experimental and spontaneous metastasis, we have shown that aspirin and a selective COX-1 inhibitor dramatically reduced metastatic lung nodules, an effect that was not replicated by selective inhibition of COX-2. Metastasis was impaired in COX-1 deficient mice as well, suggesting a pivotal role of microenvironmental COX-1 in the metastatic process. In particular, COX-1 activity was essential during the intravascular phase of pulmonary metastasis. Using a model of platelet depletion and re-infusion we identified platelet-derived thromboxane A2 (TXA2) as the main product of COX-1 responsible for its permissive effect on metastasis. The inhibition of the COX-1-TXA2 pathway in platelets decreased the aggregation of platelets on tumor cells and was associated with a reduction in endothelial activation, in tumor cell adhesion to the endothelium, in recruitment of metastasis-promoting monocytes/macrophages and in transendothelial migration. Thus, platelet-derived TXA2 orchestrates the generation of a favorable intravascular metastatic niche that promotes tumor cell seeding and metastasis. Taken together, our data suggest that COX-1 inhibition in platelets by aspirin is sufficient to exert an anti-metastatic effect and shed a new light on COX-1-TXA2 signalling in the context of tumor cell dissemination. From this perspective, TXA2 might present a more selective therapeutic target for the prevention of metastasis. Citation Format: Serena Lucotti, Camilla Cerutti, Magali Soyer, Ana M. Gil-Bernabé, Ana L. Gomes, Philip D. Allen, Sean Smart, Bostjan Markelc, Karla Watson, Paul C. Armstrong, Jane A. Mitchell, Timothy D. Warner, Anne J. Ridley, Ruth J. Muschel. Aspirin inhibits metastasis in the intravascular phase through the blockade of COX-1-TXA2 pathway in platelets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4196.

A prominent feature of many human cancers is oncogene-driven activation of the DNA damage response (DDR) during early tumorigenesis. It has been shown previously that noninvasive imaging of the phosphorylated histone H2A variant H2AX, γH2AX, a DNA damage signaling protein, is possible using (111)In-labeled anti-γH2AX antibody conjugated to the cell-penetrating peptide transactivator of transcription (TAT). The purpose of this study was to investigate whether (111)In-anti-γH2AX-TAT detects the DDR during mammary oncogenesis in BALB-neuT mice.Mammary fat pads from BALB-neuT and wild-type mice (age, 40-106 d) were immunostained for γH2AX. (111)In-anti-γH2AX-TAT or a control probe was administered intravenously to BALB-neuT mice. SPECT was performed weekly and compared with tumor detection using palpation and dynamic contrast-enhanced MR imaging.γH2AX expression was elevated in hyperplastic lesions in the mammary fat pads of BALB-neuT mice aged 76-106 d, compared with normal fat pads from younger mice and carcinomas from older mice (13.5 ± 1.2 γH2AX foci/cell vs. 5.2 ± 1.5 [P < 0.05] and 3.4 ± 1.1 [P < 0.001], respectively). Serial SPECT imaging revealed a 2.5-fold increase in (111)In-anti-γH2AX-TAT accumulation in the mammary fat pads of mice aged 76-106 d, compared with control probe (P = 0.01). The median time to detection of neoplastic lesions by (111)In-anti-γH2AX-TAT (defined as >5% injected dose per gram of tissue) was 96 d, compared with 120 and 131 d for dynamic contrast-enhanced MR imaging and palpation, respectively (P < 0.001).DDR imaging using (111)In-anti-γH2AX-TAT identified mammary tumors significantly earlier than MR imaging. Imaging the DDR holds promise for the detection of preneoplasia and as a technique for screening cancer-prone individuals.

Pulmonary metastasis is a frequent cause of poor outcome in cancer patients. The formation of pulmonary metastasis is greatly facilitated by recruitment of myeloid cells, which are crucial for tumor cell survival and extravasation. During inflammation, homing of myeloid cells is mediated by endothelial activation, raising the question of a potential role for endothelial activation in myeloid cell recruitment during pulmonary metastasis. Here, we show that metastatic tumor cell attachment causes the induction of the endothelial activation markers vascular cell adhesion molecule-1 (VCAM-1) and vascular adhesion protein-1 (VAP-1). Induction of VCAM-1 is dependent on tumor cell-clot formation, decreasing upon induction of tissue factor pathway inhibitor or treatment with hirudin. Furthermore, inhibition of endothelial activation with a VCAM-1 blocking antibody or a VAP-1 small molecule inhibitor leads to reduced myeloid cell recruitment and diminished tumor cell survival and metastasis without affecting tumor cell adhesion. Simultaneous inhibition of VCAM-1 and VAP-1 does not result in further reduction in myeloid cell recruitment and tumor cell survival, suggesting that both act through closely related mechanisms. These results establish VCAM-1 and VAP-1 as mediators of myeloid cell recruitment in metastasis and identify VAP-1 as a potential target for therapeutic intervention to combat early metastasis.

Tissue factor (TF) expression by tumor cells correlates with metastasis clinically and supports metastasis in experimental settings. However, the precise pathways coupling TF to malignancy remain incompletely defined. Here, we show that clot formation by TF indirectly enhances tumor cell survival after arrest in the lung, during experimental lung metastasis, by recruiting macrophages characterized by CD11b, CD68, F4/80, and CX3CR1 (but not CD11c) expression. Genetic or pharmacologic inhibition of coagulation, by either induction of TF pathway inhibitor ex-pression or by treatment with hirudin, respectively, abrogated macrophage recruitment and tumor cell survival. Furthermore, impairment of macrophage function, in either Mac1-deficient mice or in CD11b-diphtheria toxin receptor mice in which CD11b-positive cells were ablated, decreased tumor cell survival without altering clot formation, demonstrating that the recruitment of functional macrophages was essential for tumor cell survival. This effect was independent of NK cells. Moreover, a similar population of macrophages was also recruited to the lung during the formation of a premetastatic niche. Anticoagulation inhibited their accumulation and prevented the enhanced metastasis associated with the formation of the niche. Our study, for the first time, links TF induced coagulation to macrophage recruitment in the metastatic process.

PURPOSE: To describe a combination of techniques using the excellent volumetric capacities of magnetic resonance imaging (MRI) while avoiding anesthesia and maintaining high-throughput capability for tumor volume measurement in the awake mouse. This approach presents an alternative to calipers which, although cheap, fast, and easy to use, introduce many biases for tumor volume estimation. MATERIALS AND METHODS: The murine CaNT subcutaneous xenograft model was used. A quiet and modestly T2-weighted spin-echo scan was acquired at 4.7T (TE = 15 msec, TR = 1100 msec, 0.5 mm isotropic resolution) while the awake mouse was held by hand in the magnet. This method was compared to standard MR in the anesthetized mouse and caliper measurements. RESULTS: The combination of techniques used allows rapid, accurate, and reproducible measurement of subcutaneous tumor volumes in awake mice. It is less sensitive to both intra- and interoperator-derived biases and avoids confounds from the compliance of the fat and skin around the tumor, as well as from the tumor itself. Moreover, the data remain available for retrieval and scrutiny and reanalysis. CONCLUSION: Rapid, accurate, and precise tumor volumetry can be performed in the awake mouse by handheld positioned MR.

Background Preclinical imaging requires anaesthesia to reduce motion-related artefacts. For direct translational relevance, anaesthesia must not significantly alter experimental outcome. This study reports on the effects of both anaesthetic and carrier gas upon the uptake of [64Cu]-CuATSM, [99mTc]-HL91 and [18F]-FMISO in a preclinical model of tumor hypoxia. Methodology/Principal Findings The effect of carrier gas and anaesthetic was studied in 6 groups of CaNT-bearing CBA mice using [64Cu]-CuATSM, [99mTc]-HL91 or [18F]-FMISO. Mice were anaesthetised with isoflurane in air, isoflurane in pure oxygen, with ketamine/xylazine or hypnorm/hypnovel whilst breathing air, or in the awake state whilst breathing air or pure oxygen. PET or SPECT imaging was performed after which the mice were killed for organ/tumor tracer quantitation. Tumor hypoxia was confirmed. Arterial blood gas analysis was performed for the different anaesthetic regimes. The results demonstrate marked influences on tumor uptake of both carrier gas and anaesthetic, and show differences between [99mTc]-HL91, [18F]-FMISO and [64Cu]-CuATSM. [99mTc]-HL91 tumor uptake was only altered significantly by administration of 100% oxygen. The latter was not the case for [18F]-FMISO and [64Cu]-CuATSM. Tumor-to-muscle ratio (TMR) for both compounds was reduced significantly when either oxygen or anaesthetics (isoflurane in air, ketamine/xylazine or hypnorm/hypnovel) were introduced. For [18F]-FMISO no further decrease was measured when both isoflurane and oxygen were administered, [64Cu]-CuATSM did show an additional significant decrease in TMR. When using the same anaesthetic regimes, the extent of TMR reduction was less pronounced for [64Cu]-CuATSM than for [18F]-FMISO (40–60% versus 70% reduction as compared to awake animals breathing air). Conclusions/Significance The use of anaesthesia can have profound effects on the experimental outcome. More importantly, all tested anaesthetics reduced tumor-hypoxia uptake. Anaesthesia cannot be avoided in preclinical studies but great care has to be taken in preclinical models of hypoxia as anaesthesia effects cannot be generalised across applications, nor disease states.

UNLABELLED: CT is widely used for anatomic referencing of PET and SPECT images of small animals but requires sufficiently high radiation doses capable of causing significant DNA damage. Therefore, we described the relationship between radiation dose, biologic damage, and image quality to determine whether CT can be used without significantly compromising radiotherapy and tumor development studies. METHODS: The CT dose index generated by the nanoSPECT/CT system was compared with measurements using EBT2 gafchromic film. The effects of micro-CT were evaluated in 2 mouse strains that differ in sensitivity to radiation. γH2AX foci analysis to determine leukocyte, liver, and jejunum DNA damage and hematoxylin and eosin staining to investigate macroscopic jejunum damage were performed. Signal-to-noise ratio, contrast-to-noise ratio, and scanner linearity were determined to assess image quality. RESULTS: For the standard settings, that is, as set by the manufacturers, EBT2 gafchromic film dosimetry showed that the nanoSPECT/CT system underestimated the absorbed dose. Moreover, significant doses were obtained, resulting in a significant increase in γH2AX formation in leukocytes, liver, and jejunum 40 min after CT, using preset parameters when compared with nonimaged controls. The jejenum response was more pronounced for the more radiosensitive strain. In contrast to leukocytes, the liver and jejunum still showed evidence of DNA damage 3 d after CT. Contrast-to-noise ratio, signal-to-noise ratio, and scanner linearity were sufficient to allow for anatomic referencing for both imaging protocols tested. CONCLUSION: Anatomic reference images can be produced with no observable DNA damage or compromising image quality using low radiographic voltage, flux, and duration.

Two hundred ninety-three children suffering from acute appendicitis have been analyzed. Five deaths occurred in this group, two of which were nonoperative. This is a marked reduction in death rate as compared to previous reports from the same service. Among the outstanding reasons assigned for the lowered mortality rate are: (1) earlier hospitalization; (2) fewer cathartics; (3) more preoperative care aimed at correcting fluid balance, especially in toxic dehydrated patients with evidence of spreading peritonitis; (4) better anesthesia and (5) the use of the McBurney incision. This incision is likewise believed to have been the chief factor in reducing hospital stay from twenty-one days in the previous report to fifteen and six-tenth days in this series. Further followup observations in cases in which drainage was employed and in which only the peritoneum was sutured is reported, with a decreased incidence of hernia in this group.

Periarteritis nodosa was first accurately described by Kussmaul and Maier1in 1866. Since that time, many detailed case reports have appeared in journals devoted to pathology or to internal medicine. The frequency with which this disease simulates acute conditions requiring surgical treatment justifies its emphasis in the surgical literature. Periarteritis nodosa or, better, polyarteritis nodosa, is primarily a disease process involving the walls of blood vessels. The lesions start with edema and subsequent necrosis in the media of the vessel wall. The process ultimately extends to the intima, with reactive cellular proliferation which frequently produces endothelial injury and thrombus formation. There is also an adventitial infiltration of polymorphonuclear leukocytes, often accompanied by eosinophils. The healing process with the laying down of connective tissue results in the formation of a nodule involving the vessel wall; lack of repair, mural degeneration and necrosis with destruction of elastic fibers may terminate in

Previous to the last decade, torsion of the spermatic cord was considered rare. In 1930 Donovan 1 collected 163 cases from the literature, in 13 of which the patients were less than 1 year of age. He reported 3 additional cases in which the anomaly was observed in infants. Since that time over 200 cases have been added to the literature, and relatively the same percentage of infantile involvement has prevailed. Three recent cases of involvement before 1 year of age occurring in the Children's Surgical Service of Bellevue Hospital are reported in this paper. The Children's Surgical Service was organized in 1917 and consists of approximately 80 beds. Children from infancy to the age of 13 are admitted. As a search of the records of this service reveals only the 3 aforementioned cases, it is apparent that torsion of the spermatic cord is still relatively uncommon in infants. In