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Purpose: The epidermal growth factor receptor (ERBB1) and related family member HER-2/neu (ERBB2) are often overexpressed in aggressive breast cancers and their overexpression is correlated with poor prognosis. Clinical studies using ERBB inhibitors have focused on tumor growth effects, but ERBBs can contribute to malignancy independent of their effects on tumor growth. Our studies were designed to evaluate the effect of ERBB inhibition on tumor cell motility and intravasation in vivo using clinically relevant small-molecule inhibitors.Experimental Design: Using in vivo mouse models of breast cancer, we test the effects of ERBB1 and ERBB2 inhibitors AC480 and lapatinib, ERBB1 inhibitor gefitinib, and ERBB2 inhibitor AG825 on in vivo tumor cell invasive properties in mammary fat pad tumors.Results: ERBB1 and ERBB2 inhibition rapidly (within 3 h) inhibits both tumor cell motility and intravasation. Using gefitinib, ERBB1 inhibition rapidly inhibits tumor cell motility and invasion but not intravasation, whereas ERBB2 inhibition by AG825 rapidly blocks intravasation.Conclusions: ERBB1 and ERBB2 inhibition can rapidly block tumor cell invasive properties. In addition, we differentiate for the first time the contributions of ERBB1 and ERBB2 to the key metastatic properties of in vivo tumor cell invasion and intravasation. These experiments temporally and molecularly separate two key stages in tumor cell entry into blood vessels: invasion and intravasation. These results indicate that ERBB inhibition should be considered for blocking other tumor cell malignant properties besides growth.

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Supplementary Movie S1 from ERBB1 and ERBB2 Have Distinct Functions in Tumor Cell Invasion and Intravasation

Supplementary Figures S1-S2 from ERBB1 and ERBB2 Have Distinct Functions in Tumor Cell Invasion and Intravasation

Glioblastoma Multiforme (GBM) is the most aggressive form of adult brain tumor with a median survival time of twelve months. GBM is highly resistant to conventional therapy which includes surgical resection of the tumor, radiation treatment and chemotherapy. GBM cells are highly motile and invasive resulting in infiltrative tumors with poorly defined borders. GBM tumors are heavily infiltrated with microglia cells which are known to stimulate GBM cell invasion. Our laboratory has previously demonstrated that microglia strongly stimulates GBM invasion both in-vitro and in orthotopic animal models. This interaction was found to be dependent on CSF-1R which is expressed on all tumor infiltrating macrophages/microglia. Blockade of the CSF-1R using compounds such as pexidartinib (PLX3397) can inhibit microglia/macrophage-stimulated GBM invasion in-vitro and in vivo. A variety of chemokines are upregulated in the GBM tumor microenvironment and facilitate “cross-talk” between microglia and GBM cells eliciting a chemotactic response. We have demonstrated that the chemotactic ligand, CCL3, is similarly upregulated in GBM tumors. We postulated that inhibition of CCL3 associated receptors such as C-C receptor 1 (CCR1) might also inhibit GBM invasion, thus, a CCR1 antagonist could prove efficacious for blockade of microglia-induced glioblastoma invasion in vitro. Many potent CCR1 antagonists have been described in the literature. We chose four of these compounds with two distinct structural cores, all with reported IC50’s of less than 200 nM for inhibition of CCR1 binding versus CCL3. We examined the ability of these antagonists to block microglia-stimulated glioblastoma invasion using an in-vitro coculture invasion assay. Using quantitative PCR arrays, we also show that expression of chemokines and chemokine receptor genes is greatly altered in GBM conditioned media-treated microglia. Understanding the pattern of tumor-associated macrophage/microglia chemokine secretion in GBM may present additional targets for chemotherapeutic intervention and enhance immunotherapy. Citation Format: Salvatore J. Coniglio, Poornema Ramasundaram, Neshama Fournier, Danielle S. Hamilton, Gregory Marshall, Keia Smith, Diana Habib, James R. Merritt. The chemokine receptor CCR1 is involved in microglia stimulated glioblastoma invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4548.

The ability of glioma tumor cells to infiltrate the surrounding brain parenchyma makes complete removal of these tumors nearly impossible. We investigated how microglia, the resident macrophages of the brain, stimulate glioma cell invasion. Coculture with microglia stimulates the invasion of GL261 glioma cells in-vitro. Pharmacological inhibition of EGFR using Iressa strongly inhibits microglia-stimulated invasion. Furthermore, we demonstrate using novel CSF-1R (c-fms) inhibitors that CSF-1R signaling is also required for microglial enhancement of glioma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which are unaffected by EGF stimulation, EGFR inhibition or coculture with microglia. GL261 tumors orthotopically injected into the brains of C57 mice fed with the CSF-1R inhibitor PLX3397 showed a dramatic reduction in microglia/macrophages infiltration compared with tumors from animals fed control chow. Tumors from the PLX3397-fed animals also showed a 50% reduction in invasion into the parenchyma. We discovered that GL261 cell secretion of CSF-1 is involved in upregulating EGFR ligands in microglia including AREG, EREG and TGFA. Microglia isolated from GL261 tumors express AREG mRNA whereas naïve microglia from control animals do not. Depletion of AREG from microglia using siRNA interfered with their ability to promote GL261 invasion in vitro. These data indicate that during invasion, glioma and microglia cells signal to each other using EGFR Ligands and CSF-1 similar to what has been observed in breast cancer models. The results of this study indicate the possibility of inhibiting glioblastoma invasion by targeting functions of tumor-associated microglia in addition to glioma cells. Citation Format: Salvatore J. Coniglio, Jeffrey E. Segall. Mechanisms of microglia-stimulated glioma invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4928. doi:10.1158/1538-7445.AM2013-4928

Glioblastoma multiforme (GBM) is the most malignant and lethal form of astrocytoma. GBM patients have a median survival time of approximately one year and therefore it is essential to identify novel targets and effective therapies. There is mounting evidence that microglia (specialized brain-resident macrophages) play a significant role in the development and progression of GBM tumors. We have established a microglia-glioma cell co-culture system consisting of normal microglia from C57Bl/6J mice and syngeneic GL261 glioma cells. We have demonstrated that microglia strongly stimulate the invasive behavior of GL261 cells in this system. Our preliminary data also indicate that the microglia significantly stimulate GL261 cell proliferation and resistance to ionizing radiation (IR) in these conditions. In a search for small molecule inhibitors that block the stimulatory effects of microglia on glioma cells, we have examined CNI-1493, a drug that is known to selectively interfere with macrophage function. We found that CNI-1493 potently inhibits microglia-stimulated GL261 invasion. We also examined the effect of intracranially administered CNI-1493 on orthotopic GL261 tumors in the presence or absence of fractionated whole brain irradiation (10 Gy total). We found that although CNI-1493 only has a marginal benefit in the absence of IR, it strongly stimulates animal survival in combination with IR: whereas IR treatment increases survival by 10 days, IR in combination with CNI-1493 treatment extends survival beyond 40 days. In conclusion, our observations support the notion that pharmacological targeting of microglia may be beneficial as adjuvant therapy in conjunction with ionizing radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-503. doi:1538-7445.AM2012-LB-503

The ability of glioma tumor cells to infiltrate the surrounding brain parenchyma makes complete removal of these tumors nearly impossible. We investigated how microglia, the resident macrophages of the brain, stimulate glioma cell invasion. Coculture with microglia stimulates the invasion of GL261 glioma cells in-vitro. Pharmacological inhibition of EGFR using Iressa completely inhibits microglia-stimulated invasion and sequestration of EGF using an EGF-specific function-blocking antibody also fully inhibits microglia-stimulated glioma invasion. Furthermore, we demonstrate using novel CSF-1R (c-fms) inhibitors that CSF-1R signaling is also required for microglial enhancement of glioma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which are unaffected by EGF stimulation, EGFR inhibition or coculture with microglia. In addition, GL261 tumors orthotopically injected into the brains of C57 mice fed with PLX3397 were compromised in their ability to recruit microglia/macrophages. Interestingly, tumors from these animals also showed a 50% reduction in invasion into the parenchyma. These data indicate that during invasion, glioma and microglia cells signal to each other using EGF and CSF-1 similar to what has been observed in breast cancer models. The results of this study indicate the possibility of inhibiting glioblastoma invasion by targeting functions of tumor-associated microglia in addition to glioma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 46. doi:1538-7445.AM2012-46