Cancer dormancy refers to the prolonged clinical disease-free time between removal of the primary tumor and recurrence, which is common in prostate cancer (PCa), breast cancer, esophageal cancer, and other cancers. PCa disseminated tumor cells (DTC) are detected in both patients with no evidence of disease (NED) and advanced disease (ADV). However, the molecular and cellular nature of DTC is unknown. We performed a first-in-field study of single DTC transcriptomic analyses in cancer patients to identify a molecular signature associated with cancer dormancy. We profiled eighty-five individual EpCAM+/CD45− cells from the bone marrow of PCa patients with NED or ADV. We analyzed 44 DTC with high prostate-epithelial signatures, and eliminated 41 cells with high erythroid signatures and low prostate epithelial signatures. DTC were clustered into 3 groups: NED, ADV_1, and ADV_2, in which the ADV_1 group presented a distinct gene expression pattern associated with the p38 stress activated kinase pathway. Additionally, DTC from the NED group were enriched for a tumor dormancy signature associated with head and neck squamous carcinoma and breast cancer. This study provides the first clinical evidence of the p38 pathway as a potential biomarker for early recurrence and an attractive target for therapeutic intervention.
Prostate cancer; dormancy; metastasis; p38; gene expression
As therapies for systemic cancer improve and patients survive longer, the risk for brain metastases increases. We evaluated whether immune mechanisms are involved in the development of brain metastasis.
We conducted our studies using BALB/c mice bearing syngeneic 4T1 mammary adenocarcinoma cells in the mammary gland.
The brains of mice bearing 4T1 tumors at day 14 had no detectable metastatic tumor cells but presented with marked accumulation of bone marrow–derived CD11b+Gr1+ myeloid cells, which express high levels of inflammatory chemokines S100A8 and S100A9. In vitro, S100A9 attracts 4T1 cells through Toll-like receptor 4 and CD11b+Gr1+ myeloid cells through Toll-like receptor 4 and the receptor for advanced glycation end-products. Systemic treatment of 4T1-bearing mice with anti-Gr1 (RB6-8C5) monoclonal antibody reduces accumulation of CD11b+Gr1+ myeloid cells in the day-14 premetastatic brain as well as subsequent brain metastasis of 4T1 cells detected on day 30. Furthermore, treatment of 4T1 tumor-bearing mice with the cyclooxygenase-2 inhibitor celecoxib or genetic disruption of cyclooxygenase-2 in 4T1 cells inhibits the inflammatory chemokines and infiltration of CD11b+Gr1+ myeloid cells in the premetastatic brain and subsequent formation of brain metastasis.
Our results suggest that the primary tumor induces accumulation of CD11b+Gr1+ myeloid cells in the brain to form “premetastatic soil” and inflammation mediators, such as S100A9, that attract additional myeloid cells as well as metastatic tumor cells. Celecoxib and anti-Gr1 treatment may be useful for blockade of these processes, thereby preventing brain metastasis in patients with breast cancer.
brain metastasis; breast cancer; CCL2; myeloid-derived suppressor cells; prostaglandin-E2; S100A8/A9
Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.
To evaluate the efficacy and oral activity of two promising indoles, (2-(1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound II] and (2-(1H-indol-5-ylamino)-thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound IAT], in paclitaxel- and docetaxel-resistant tumor models in vitro and in vivo.
The in vitro drug-like properties, including potency, solubility, metabolic stability, and drug-drug interactions were examined for our two active compounds. An in vivo pharmacokinetic study and antitumor efficacy study were also completed to compare their efficacy with docetaxel.
Both compounds bound to the colchicine-binding site on tubulin, and inhibited tubulin polymerization, resulting in highly potent cytotoxic activity in vitro. While the potency of paclitaxel and docetaxel was compromised in a multidrug-resistant cell line that overexpresses P-glycoprotein, the potency of compounds II and IATwas maintained. Both compounds had favorable drug-like properties, and acceptable oral bioavailability (21–50%) in mice, rats, and dogs. Tumor growth inhibition of greater than 100% was achieved when immunodeficient mice with rapidly growing paclitaxel-resistant prostate cancer cells were treated orally at doses of 3–30 mg/kg of II or IAT.
These studies highlight the potent and broad anticancer activity of two orally bioavailable compounds, offering significant pharmacologic advantage over existing drugs of this class for multidrug resistant or taxane-refractory cancers.
paclitaxel resistant cancer; P-glycoprotein; pharmacokinetics; tubulin; xenograft
Tracheal intubation in the Intensive Care Unit (ICU) can be challenging as patients often have anatomic and physiologic characteristics that make intubation particularly difficult. Video laryngoscopy (VL) has been shown to improve first attempt success compared to direct laryngoscopy (DL) in many clinical settings and may be an option for ICU intubations.
All intubations performed in this academic medical ICU during a 13-month period were entered into a prospectively collected quality control database. After each intubation, the operator completed a standardized form evaluating multiple aspects of the intubation including: patient demographics, difficult airway characteristics (DACs), method and device(s) used, medications used, outcomes and complications of each attempt. Primary outcome was first attempt success. Secondary outcomes were grade of laryngoscopic view, ultimate success, esophageal intubations, and desaturation. Multivariate logistic regression was performed for first attempt and ultimate success.
Over the 13-month study period (January 2012-February 2013), a total of 234 patients were intubated using VL and 56 patients were intubated with DL. First attempt success for VL was 184/234 (78.6%; 95% CI 72.8 to 83.7) while DL was 34/56 patients (60.7%; 95% CI 46.8 to 73.5). Ultimate success for VL was 230/234 (98.3%; 95% CI 95.1 to 99.3) while DL was 52/56 patients (91.2%; 95% CI 81.3 to 97.2). In the multivariate regression model, VL was predictive of first attempt success with an odds ratio of 7.67 (95% CI 3.18 to 18.45). VL was predictive of ultimate success with an odds ratio of 15.77 (95% CI 1.92 to 129). Cormack-Lehane I or II view occurred 199/234 times (85.8%; 95% CI 79.5 to 89.1) and a median POGO (Percentage of Glottic Opening) of 82% (IQR 60 to 100) with VL, while Cormack-Lehane I or II view occurred 34/56 times (61.8%; 95% CI 45.7 to 71.9) and a median POGO of 45% (IQR 0 to 78%) with DL. VL reduced the esophageal intubation rate from 12.5% with DL to 1.3% (P = 0.001) but there was no difference in desaturation rates.
In the medical ICU, video laryngoscopy resulted in higher first attempt and ultimate intubation success rates and improved grade of laryngoscopic view while reducing the esophageal intubation rate compared to direct laryngoscopy.
Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE.
The chemokine CCL2, which is best known for its chemotactic functions, is expressed not only by immune cells, but also by several types of malignant and stromal cells. CCL2 has been shown to exert both pro- and anti-tumor effects. However, recent results demonstrate a main role for CCL2 in tumor progression and metastasis, suggesting that this chemokine may constitute a therapeutic target for anticancer drugs. Mammary carcinoma models, including models of implantable, transgenic, and chemically-induced tumors, were employed in the setting of Ccl2 or Ccr2 knockout mice or CCL2 neutralization with a monoclonal antibody to further investigate the role of the CCL2/CCR2 signaling axis in tumor progression and metastatic spread. In our implantable tumor models, an anti-CCL2 monoclonal antibody inhibited the growth of primary malignant lesions in a biphasic manner and reduced the number of metastases. However, in Ccl2−/− or Ccr2−/− mice developing implanted or transgenic tumors, the number of pulmonary metastases was increased despite a reduction in the growth rate of primary neoplasms. Transgenic Mtag.Ccl2−/− or Mtag.Ccr2−/− mice also exhibited a significantly earlier of disease onset. In a chemical carcinogenesis model, anti-CCL2 monoclonal antibody inhibited the growth of established lesions but was ineffective in the tumor induction phase. In contrast to previous studies indicating a role for CCL2 in the establishment of metastases, we have demonstrated that the absence of CCL2/CCR2-signaling results in increased metastatic disease. Thus, the CCL2/CCR2 signaling axis appears to play a dual role in mediating early tumor immunosurveillance and sustaining the growth and progression of established neoplasms. Our findings support the use of anti-CCL2 therapies for the treatment of established breast carcinoma, although the complete abrogation of the CCL2 signaling cascade may also limit immunosurveillance and support metastatic spread.
breast cancer; CCL2; CCR2; chemokine; immunosurveillance; metastases; monoclonal antibody therapy; tumor progression
A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide prior to intra-cardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)-2, interleukin (IL)-6, and vascular endothelial growth factor (VEGF)-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the pre-metastatic murine host significantly reduced the pro-metastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.
Cyclophosphamide; prostate cancer; bone metastasis; myeloid cells; CCL2
The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.
prostate cancer; bone metastases; chemokine; CCL2; docetaxel; bone metastases
The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein–1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non–small-cell lung cancer (NSCLC). Anti-murine–CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8+ T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.
tumor immunology; CCL2; lung cancer; mesothelioma; tumor-associated macrophages
Macrophages abundantly found in the tumor microenvironment enhance malignancy1. At metastatic sites a distinct population of metastasis associated macrophages (MAMs) promote tumor cell extravasation, seeding and persistent growth2. Our study has defined the origin of these macrophages by showing Gr1+ inflammatory monocytes (IMs) are preferentially recruited to pulmonary metastases but not primary mammary tumors, a process also found for human IMs in pulmonary metastases of human breast cancer cells. The recruitment of these CCR2 (receptor for chemokine CCL2) expressing IMs and subsequently MAMs and their interaction with metastasizing tumor cells is dependent on tumor and stromal synthesized CCL2 (FigS1). Inhibition of CCL2/CCR2 signaling using anti-CCL2 antibodies blocks IM recruitment and inhibits metastasis in vivo and prolongs the survival of tumor-bearing mice. Depletion of tumor cell-derived CCL2 also inhibits metastatic seeding. IMs promote tumor cell extravasation in a process that requires monocyte-derived VEGF. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer (Fig S2)3-6. Our data provides the mechanistic link between these two clinical associations and indicates new therapeutic targets for treating metastatic breast disease.
Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immune responses and facilitate tumor growth. Precursors for these immune cell populations migrate to the tumor site in response to tumor secretion of chemokines, such as monocyte chemoattractant protein-1 (MCP-1/CCL2), which was originally purified and identified from human gliomas. In syngeneic mouse GL261 glioma and human U87 glioma xenograft models, we evaluated the efficacy of systemic CCL2 blockade by monoclonal antibodies (mAb) targeting mouse and/or human CCL2. Intraperitoneal (i.p.) administration of anti-mouse CCL2 mAb as monotherapy (2 mg/kg/dose, twice a week) significantly, albeit modestly, prolonged the survival of C57BL/6 mice bearing intracranial GL261 glioma (p=0.0033), which was concomitant with a decrease in TAMs and MDSCs in the tumor microenvironment. Similarly, survival was modestly prolonged in severe combined immunodeficiency (SCID) mice bearing intracranial human U87 glioma xenografts treated with both anti-human CCL2 mAb and anti-mouse CCL2 antibodies (2 mg/kg/dose for each, twice a week) compared to mice treated with control IgG (p=0.0159). Furthermore, i.p. administration of anti-mouse CCL2 antibody in combination with temozolomide (TMZ) significantly prolonged the survival of C57BL/6 mice bearing GL261 glioma with 8 of 10 treated mice surviving longer than 70 days, while only 3 of 10 mice treated with TMZ and isotype IgG survived longer than 70 days (p=0.0359). These observations provide support for development of mAb-based CCL2 blockade strategies in combination with the current standard TMZ-based chemotherapy for treatment of malignant gliomas.
glioma; chemokine; CCL2; monoclonal antibody; chemotherapy; temozolomide
Since an immuno-inhibitory environment exists within tumors, successful vaccines will likely require additional approaches to alter the tumor microenvironment. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immuno-inhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine-CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the HPV-E7 antigen expressed by a non-small cell lung cancer line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus expressing Interferon-α to treat a non-immunogenic, non-small cell lung cancer line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intra-tumoral CD8+ T-cells that were more activated and more anti-tumor antigen specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T-regulatory (T-reg) cells. CCL2 appears to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.
CCL2; Cancer immunotherapy; Lung Cancer; Mesothelioma; T-lymphocytes
Extracellular matrix metalloprotease inducer (EMMPRIN) is a tumor surface protein that promotes growth and is overexpressed in head and neck cancer. These features make it a potential therapeutic target for monoclonal antibody (mAb) – based therapy. Because molecular therapy is considered more effective when delivered with conventional cytotoxic agents, anti-EMMPRIN therapy was assessed alone and in combination with external beam radiation.
Using a murine flank model, loss of EMMPRIN function was achieved by transfection with a small interfering RNA against EMMPRIN or treatment with a chimeric anti-EMMPRIN blocking mAb. Cytokine expression was assessed for xenografts, tumor cells, fibroblasts, and endothelial cells.
Animals treated with anti-EMMPRIN mAb had delayed tumor growth compared with untreated controls, whereas treatment with combination radiation and anti-EMMPRIN mAb showed the greatest reduction in tumor growth (P = 0.001). Radiation-treated EMMPRIN knockdown xenografts showed a reduction in tumor growth compared with untreated knockdown controls (P = 0.01), whereas radiation-treated EMMPRIN – expressing xenografts did not show a delay in tumor growth. Immunohistochemical evaluation for Ki67 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) resulted in a reduction in proliferation (P = 0.007) and increased apoptosis in anti-EMMPRIN mAb – treated xenografts compared with untreated controls (P = 0.087). In addition, we provide evidence that EMMPRIN suppression results in decreased interleukin 1β (IL-1β), IL-6, and IL-8 cytokine production, in vitro and in vivo.
These data suggest that anti-EMMPRIN antibody inhibits tumor cell proliferation in vivo and may represent a novel targeted treatment option in head and neck squamous cell carcinoma.
Monocyte chemoattractant protein 1 (MCP-1; CCL2) is a recently identified prominent regulator of prostate cancer growth and metastasis. The purpose of this study was to investigate the mechanistic role of CCL2 in prostate cancer growth in bone. The present study found that CCL2 was upregulated in osteoblasts (3-fold by PC3 and 2-fold by VCaP conditioned media) and endothelial cell (2-fold by PC3 and VCaP conditioned media). PTHrP treatment of osteoblastic cells upregulated CCL2 and was blocked by a PTHrP antagonist suggesting prostate cancer-derived PTHrP plays an important role in elevation of osteoblast derived CCL2. CCL2 indirectly increased blood vessel formation in endothelial cells through VEGF-A, which was up-regulated 2-fold with administration of CCL2 in prostate cancer cells. In vivo, anti-CCL2 treatment suppressed tumor growth in bone. The decreased tumor burden was associated with decreased bone resorption (serum TRAP5b levels were decreased by 50~60% in anti-CCL2 treated animals from VCaP or PC-3 cell osseous lesions) and micro-vessel density was decreased by 70% in anti-CCL2 treated animals with bone lesions from VCaP cells. These data suggest that a destructive cascade is driven by tumor cell-derived PTHrP mediated induction of CCL2, which facilitates tumor growth via enhanced osteoclastic and endothelial cell activity in bone marrow. Taken together, CCL2 mediates the interaction between tumor-derived factors and host-derived chemokines acting in cooperation to promote skeletal metastasis.
CCL2; prostate cancer; parathyroid hormone related protein
The ability of CCL2 to influence prostate cancer tumorigenesis and metastasis may occur through two distinct mechanisms: 1) a direct effect on tumor cell growth and function, and 2) an indirect effect on the tumor microenvironment by the regulation of macrophage mobilization and infiltration into the tumor bed. We have previously demonstrated that CCL2 exerts a direct effect on prostate cancer epithelial cells by the regulation of their growth, invasion, and migration, resulting in enhanced tumorigenesis and metastasis. Here we describe an indirect effect of CCL2 on prostate cancer growth and metastasis by regulating monocyte/macrophage infiltration into the tumor microenvironment and by stimulating a phenotypic change within these immune cells to promote tumor growth (tumor-associated macrophages). VCaP prostate cancer cells were subcutaneously injected in male SCID mice and monitored for tumor volume, CD68+ macrophage infiltration, and microvascular density. Systemic administration of anti-CCL2 neutralizing antibodies (CNTO888 and C1142) significantly retarded tumor growth and attenuated CD68+ macrophage infiltration, which was accompanied by a significant decrease in microvascular density. These data suggest that CCL2 contributes to prostate cancer growth through the regulation of macrophage infiltration and enhanced angiogenesis within the tumor.
Monocyte chemoattractant protein 1; prostate cancer; chemokine; tumor-associated macrophage; angiogenesis