Danny Allen

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)

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical.

UNLABELLED (111)In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, (111)In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether (111)In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6MBq/μg) can amplify treatment-related DNA damage for therapeutic gain. METHODS MDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with (111)In-DTPA-anti-γH2AX-Tat (3MBq, 6MBq/μg) or a control radioimmunoconjugate, (111)In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. (111)In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies. RESULTS The number of γH2AX foci was greater after exposure of cells to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat compared to IR alone (20.6±2.5 versus 10.4±2.3 foci/cell; P<.001).(111)In-DTPA-anti-γH2AX-Tat resulted in a reduced surviving fraction in cells co-treated with IR (4Gy) versus IR alone (5.2%±0.9% versus 47.8%±2.8%; P<.001). Similarly, bleomycin (25-200μg/mL) plus (111)In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either (111)In-DTPA-anti-γH2AX-Tat or IR alone (-0.002±0.004 versus 0.036±0.011 and 0.031±0.014mm(3)/day, respectively, P<.001). CONCLUSION (111)In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo.

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.

Acute vascular response to cediranib treatment in human non-small-cell lung cancer xenografts with different tumour stromal architecture

Highlights • We studied cediranib, a VEGFR tyrosine kinase inhibitor in lung cancer xenografts.• Gadolinium-enhanced DCE-MRI was used to study acute vascular responses.• Acute vascular response was associated with tumour stromal architecture.• Tumour growth inhibition by cediranib was linked to acute vascular response.• Acute vascular changes are a potential predictive marker of response to cediranib.

Anti-inflammatory Microglia/Macrophages As a Potential Therapeutic Target in Brain Metastasis

Brain metastasis is a common complication of cancer patients and is associated with poor survival. Histological data from patients with brain metastases suggest that microglia are the major immune population activated around the metastatic foci. Microglia and macrophages have the ability to polarize to different phenotypes and to exert both tumorigenic and cytotoxic effects. However, the role of microglia/macrophages during the early stages of metastatic growth in the brain has not yet been determined. The aim of this study was to profile microglial/macrophage activation in a mouse model of breast cancer brain metastasis during the early stages of tumor growth, and to assess the role of the anti-inflammatory microglial/macrophage population, specifically, during this phase. Following intracerebral injection of 5 × 103 4T1-GFP mammary carcinoma cells into female BALB/c mice, robust microglial/macrophage activation around the 4T1 metastatic foci was evident throughout the time-course studied (28 days) and correlated positively with tumor volume (R2 = 0.67). Populations of classically (proinflammatory) and alternatively (anti-inflammatory) activated microglia/macrophages were identified immunohistochemically by expression of either induced nitric oxide synthase/cyclooxygenase 2 or mannose receptor 1/arginase 1, respectively. Temporally, levels of both pro- and anti-inflammatory cells were broadly stable across the time-course. Subsequently, selective depletion of the anti-inflammatory microglia/macrophage population by intracerebral injection of mannosylated clodronate liposomes significantly reduced metastatic tumor burden (p < 0.01). Moreover, increased levels of apoptosis were associated with tumors in clodronate liposome treated animals compared to controls (p < 0.05). These findings suggest that microglia/macrophages are important effectors of the inflammatory response in the early stages of brain metastasis, and that targeting the anti-inflammatory microglial/macrophage population may offer an effective new therapeutic avenue for patients with brain metastases.

Improved outcome of 131I-mIBG treatment through combination with external beam radiotherapy in the SK-N-SH mouse model of neuroblastoma

Purpose To assess the efficacy of different schedules for combining external beam radiotherapy (EBRT) with molecular radiotherapy (MRT) using 131I-mIBG in the management of neuroblastoma. Materials and methods BALB/c nu/nu mice bearing SK-N-SH neuroblastoma xenografts were assigned to five treatment groups: 131I-mIBG 24 h after EBRT, EBRT 6 days after 131I-mIBG, EBRT alone, 131I-mIBG alone and control (untreated). A total of 56 mice were assigned to 3 studies. Study 1: Vessel permeability was evaluated using dynamic contrast-enhanced (DCE)-MRI (n = 3). Study 2: Tumour uptake of 131I-mIBG in excised lesions was evaluated by γ-counting and autoradiography (n = 28). Study 3: Tumour volume was assessed by longitudinal MR imaging and survival was analysed (n = 25). Tumour dosimetry was performed using Monte Carlo simulations of absorbed fractions with the radiation transport code PENELOPE. Results Given alone, both 131I-mIBG and EBRT resulted in a seven-day delay in tumour regrowth. Following EBRT, vessel permeability was evaluated by DCE-MRI and showed an increase at 24 h post irradiation that correlated with an increase in 131I-mIBG tumour uptake, absorbed dose and overall survival in the case of combined treatment. Similarly, EBRT administered seven days after MRT to coincide with tumour regrowth, significantly decreased the tumour volume and increased overall survival. Conclusions This study demonstrates that combining EBRT and MRT has an enhanced therapeutic effect and emphasizes the importance of treatment scheduling according to pathophysiological criteria such as tumour vessel permeability and tumour growth kinetics.

Monitoring response to anti-angiogenic mTOR inhibitor therapy in vivo using 111In-bevacizumab

BackgroundThe ability to image vascular endothelial growth factor (VEGF) could enable prospective, non-invasive monitoring of patients receiving anti-angiogenic therapy. This study investigates the specificity and pharmacokinetics of 111In-bevacizumab binding to VEGF and its use for assessing response to anti-angiogenic therapy with rapamycin.Specificity of 111In-bevacizumab binding to VEGF was tested in vitro with unmodified radiolabelled bevacizumab in competitive inhibition assays. Uptake of 111In-bevacizumab in BALB/c nude mice bearing tumours with different amounts of VEGF expression was compared to that of isotype-matched control antibody (111In-IgG1κ) with an excess of unlabelled bevacizumab. Intratumoural VEGF was evaluated using ELISA and Western blot analysis. The effect of anti-angiogenesis therapy was tested by measuring tumour uptake of 111In-bevacizumab in comparison to 111In-IgG1κ following administration of rapamycin to mice bearing FaDu xenografts. Uptake was measured using gamma counting of ex vivo tumours and effect on vasculature by using anti-CD31 microscopy.ResultsSpecific uptake of 111In-bevacizumab in VEGF-expressing tumours was observed. Rapamycin led to tumour growth delay associated with increased relative vessel size (8.5 to 10.3, P = 0.045) and decreased mean relative vessel density (0.27 to 0.22, P = 0.0015). Rapamycin treatment increased tumour uptake of 111In-bevacizumab (68%) but not 111In-IgGκ and corresponded with increased intratumoural VEGF165.Conclusions111In-bevacizumab accumulates specifically in VEGF-expressing tumours, and changes after rapamycin therapy reflect changes in VEGF expression. Antagonism of mTOR may increase VEGF in vivo, and this new finding provides the basis to consider combination studies blocking both pathways and a way to monitor effects.