John Beech

Recruitment of a myeloid cell subset (CD11b/Gr1mid) via CCL2/CCR2 promotes the development of colorectal cancer liver metastasis

Liver metastasis from colorectal cancer is a leading cause of cancer mortality. Myeloid cells play pivotal roles in the metastatic process, but their prometastatic functions in liver metastasis remain incompletely understood. To investigate their role, we simulated liver metastasis in C57BL/6 mice through intrasplenic inoculation of MC38 colon carcinoma cells. Among the heterogeneous myeloid infiltrate, we identified a distinct population of CD11b/Gr1mid cells different from other myeloid populations previously associated with liver metastasis. These cells increased in number dramatically during establishment of liver metastases and were recruited from bone marrow by tumor‐derived CCL2. Liver metastasis of Lewis lung carcinoma cells followed this pattern but this mechanism is not universal as liver colonization by B16F1 melanoma cells did not recruit similar subsets. Inhibition of CCL2 signaling and absence of its cognate receptor CCR2 reduced CD11b/Gr1mid recruitment and decreased tumor burden. Depletion of the CD11b/Gr1mid subset in a transgenic CD11b‐diphtheria toxin receptor mouse model markedly reduced tumor cell proliferation. There was no evidence for involvement of an adaptive immune response in the prometastatic effects of CD11b/Gr1mid cells. Additionally, an analogous myeloid subset was found in liver metastases of some colorectal cancer patients. Conclusion: Collectively, our findings highlight the importance of myeloid cells—in this case a selective CD11b/Gr1mid subset—in sustaining development of colorectal cancer liver metastasis and identify a potential target for antimetastatic therapy. (HEPATOLOGY 2013)

Delta-Like Ligand 4–Notch Blockade and Tumor Radiation Response

BACKGROUND The microenvironment plays an important role in regulating tumor response to radiotherapy. Ionizing radiation can disrupt tumor vasculature, and Notch pathway inhibition can interfere with functional angiogenesis. We explored the potential cooperativity between Notch inhibition and ionizing radiation in delaying tumor growth. METHODS Human colorectal carcinoma LS174T cells, which express the Notch ligand delta-like ligand 4 (DLL4), and human head and neck cancer FaDu cells, which do not, were grown as subcutaneous xenografts in nude mice. The mice were treated with dibenzazepine (DBZ), a γ-secretase inhibitor that blocks all Notch signaling, or a DLL4-specific blocking monoclonal antibody, alone or in combination with ionizing radiation (n = 5-10 mice per group), and response was assessed by tumor growth delay. Microbubble contrast Doppler ultrasound was used to measure tumor blood flow. Tumor Notch activity was monitored by in vivo bioluminescence from a Notch luciferase reporter. Vessel density was assessed using Chalkley vessel counting. All statistical tests were two-sided. RESULTS In LS174T xenografts, the average time for tumor volumes to reach four times the starting volume was longer for mice treated with the DLL4 monoclonal antibody than for mice treated with DBZ (16.4 vs 9.5 days, difference = 6.9 days, 95% confidence interval [CI] = 3.7 to 10.1 days, P < .001). Both Notch inhibitors suppressed tumor Notch activity within 24 hours of administration compared with vehicle (change in luciferase activity, vehicle vs DBZ: 103% vs 28%, difference = 75%, 95% CI = 39% to 109%, P = .002; vehicle vs DLL4 antibody: 172% vs 26%, difference = 146%, 95% CI = 86% to 205%, P < .001). Administration of the DLL4 antibody or DBZ after ionizing radiation resulted in a supra-additive growth delay compared with vehicle (vehicle vs DLL4 antibody + ionizing radiation: 6.8 vs 44.3 days, difference = 37.5 days, 95% CI = 32 to 43 days, P < .001; vehicle vs DBZ + ionizing radiation: 7.1 vs 24.4 days, difference = 17.3 days, 95% CI = 15.9 to 18.6 days, P < .001). Treatment of mice with the DLL4 antibody alone or in combination with ionizing radiation increased tumor vessel density but reduced tumor blood flow. Combination therapy with DLL4 antibody and ionizing radiation resulted in extensive tumor necrosis in LS174T xenografts and enhanced tumor growth delay in FaDu xenografts. CONCLUSION The combination of specific DLL4-Notch blockade and ionizing radiation impairs tumor growth by promoting nonfunctional tumor angiogenesis and extensive tumor necrosis, independent of tumor DLL4 expression.

Cd11b+ myeloid cells support hepatic metastasis through down‐regulation of angiopoietin‐like 7 in cancer cells

Myeloid cells are known to mediate metastatic progression. Here, we attempted to elucidate the mechanisms underlying these effects by identifying gene expression alterations in cancer cells forming hepatic metastases after myeloid cell depletion. Hepatic metastases are heavily infiltrated by CD11b+ myeloid cells. We established hepatic metastases in transgenic CD11b‐diphtheria toxin receptor mice by intrasplenic injection of MC38 colon and Lewis lung carcinoma cells before depleting myeloid cells with diphtheria toxin. Myeloid cell depletion inhibited metastatic growth with a marked diminishment of tumor vasculature. Expression of ANGPTL7 (angiopoietin‐like 7), a protein not previously linked to metastasis, was highly up‐regulated in cancer cells after myeloid cell depletion. This effect was duplicated in tissue culture, where coculture of cancer cells with tumor‐conditioned myeloid cells from liver metastases or myeloid cell conditioned media down‐regulated ANGPTL7 expression. Analogous to myeloid cell depletion, overexpression of ANGPTL7 in cancer cells significantly reduced hepatic metastasis formation and angiogenesis. We found that ANGPTL7 itself has strong antiangiogenic effects in vitro. Furthermore, analysis of The Cancer Genome Atlas colorectal and breast cancer data sets revealed striking ANGPTL7 underexpression in cancerous compared to normal tissues. Also, ANGPTL7 was down‐regulated in metastatic liver colonies of colorectal cancer patients compared to their adjacent liver tissue. Conclusion: Myeloid cells promote liver metastasis by down‐regulating ANGPTL7 expression in cancer cells; our findings implicate ANGPTL7 as a mediator of metastatic progression and a potential target for interference with liver metastases. (Hepatology 2015;62:521–533

The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions

Significance Acidic pH may distinguish aggressive from more indolent cancers. The limitation on testing this hypothesis to date has been the difficulty of measuring acidic pH in cancers. Here we show that retention of the pH low insertion peptide (pHLIP) Variant 3 (Var3) reflects acidic pH. Using pHLIP Var3, we show its ability to detect cancer with a low false-positive rate in a genetically engineered model of murine breast cancer, paving the way for testing this probe in clinical situations. Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using 31P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

Recruitment of myeloid cells to the tumor microenvironment supports liver metastasis.

Tumor-infiltrating immune cells play important roles in metastasis. We have recently revealed the recruitment of a specific myeloid cell subset (CD11b/Gr1mid) to hepatic metastases. Such a recruitment relies on CCL2/CCR2 signaling and acts to sustain metastatic growth. A similar cell subset was identified in patients bearing hepatic metastases of colorectal cancer, highlighting the potential therapeutic relevance of our findings.

MRI-guided radiotherapy of the SK-N-SH neuroblastoma xenograft model using a small animal radiation research platform

Objective: Neuroblastoma has one of the lowest survival rates of all childhood cancers, despite the use of intensive treatment regimens. Preclinical models of neuroblastoma are essential for testing new multimodality protocols, including those that involve radiotherapy (RT). The aim of this study was to develop a robust method for RT planning and tumour response monitoring based on combined MRI and cone-beam CT (CBCT) imaging and to apply it to a widely studied mouse xenograft model of neuroblastoma, SK-N-SH. Methods: As part of a tumour growth inhibition study, SK-N-SH xenografts were generated in BALB/c nu/nu mice. Mice (n = 8) were placed in a printed MR- and CT-compatible plastic cradle, imaged using a 4.7-T MRI scanner and then transferred to a small animal radiation research platform (SARRP) irradiator with on-board CBCT. MRI/CBCT co-registration was performed to enable RT planning using the soft-tissue contrast afforded by MRI prior to delivery of RT (5 Gy). Tumour response was assessed by serial MRI and calliper measurements. Results: SK-N-SH xenografts formed soft, deformable tumours that could not be differentiated from surrounding normal tissues using CBCT. MR images, which allowed clear delineation of tumours, were successfully co-registered with CBCT images, allowing conformal RT to be delivered. MRI measurements of tumour volume 4 days after RT correlated strongly with length of survival time. Conclusion: MRI allowed precision RT of SK-N-SH tumours and provided an accurate means of measuring tumour response. Advances in knowledge: MRI-based RT planning of murine tumours is feasible using an SARRP irradiator.

Subcutaneous tumor volume measurement in the awake, manually restrained mouse using MRI.

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.

A resistive heating system for homeothermic maintenance in small animals

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 animals 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.

Ultrasonography-Guided Intracardiac Injection : An Improvement for Quantitative Brain Colonization Assays

Brain metastasis is a frequent occurrence in patients with cancer, with devastating consequences. The current animal models for brain metastasis are highly variable, leading to a need for improved in vivo models that recapitulate the clinical disease. Herein, we describe an experimental brain metastasis model that uses ultrasonographic guidance to perform intracardiac injections. This method is easy to perform, giving consistent and quantitative results. Demonstrating the utility of this method, we have assessed a variety of metastatic cell lines for their ability to develop into brain metastases. Those cell lines that were competent at brain colonization could be detected in the brain vasculature 4 hours after intracardiac injection, and a few adherent cells persisted until colonization occurred. In contrast, those cell lines that were deficient in brain colonization were infrequently found 4 hours after introduction into the arterial circulation and were not detected at later time points. All of these cells were capable of brain colonization after intraparenchymal injection. We propose that adherence to the brain vasculature may be the key limiting step that determines the ability of a cancer cell to form brain metastases successfully. Identifying brain endothelium-specific adhesion molecules may enable development of screening modalities to detect brain-colonizing cancer cells and therapies to prevent these metastatic cells from seeding the brain.

High-Resolution Magnetic Resonance Imaging of the Regenerating Adult Zebrafish Heart.

The adult zebrafish is a well-established model for studying heart regeneration, but due to its tissue opaqueness, repair has been primarily assessed using destructive histology, precluding repeated investigations of the same animal. We present a high-resolution, non-invasive in vivo magnetic resonance imaging (MRI) method incorporating a miniature respiratory and anaesthetic perfusion set-up for live adult zebrafish, allowing for visualization of scar formation and heart regeneration in the same animal over time at an isotropic 31 µm voxel resolution. To test the method, we compared well and poorly healing cardiac ventricles using a transgenic fish model that exhibits heat-shock (HS) inducible impaired heart regeneration. HS-treated groups revealed persistent scar tissue for 10 weeks, while control groups were healed after 4 weeks. Application of the advanced MRI technique allowed clear discrimination of levels of repair following cryo- and resection injury for several months. It further provides a novel tool for in vivo time-lapse imaging of adult fish for non-cardiac studies, as the method can be readily applied to image wound healing in other injured or diseased tissues, or to monitor tissue changes over time, thus expanding the range of questions that can be addressed in adult zebrafish and other small aquatic species.