Urszula M. Domanska

A review on CXCR4/CXCL12 axis in oncology : No place to hide

Classical chemotherapeutic anti-cancer treatments induce cell death through DNA damage by taking advantage of the proliferative behaviour of cancer cells. The more recent approach of targeted therapy (usually protein-targeted) has led to many treatments that are currently available or are under development, all of which are designed to strike at the critical driving forces of cancer cells. The interaction of the cancer cells with their microenvironment is one of these fundamental features of neoplasms that could be targeted in such cancer treatments. Haematological and solid tumour cells interact with their microenvironment through membrane chemokine receptors and their corresponding ligands, which are expressed in the tumour microenvironment. Important representatives of this system are the chemokine ligand CXCL12 and its receptor chemokine receptor 4 (CXCR4). This interaction can be disrupted by CXCR4 antagonists, and this concept is being used clinically to harvest haematopoietic stem/progenitor cells from bone marrow. CXCR4 and CXCL12 also have roles in tumour growth and metastasis, and more recently their roles in cancer cell-tumour microenvironment interaction and angiogenesis have been studied. Our review focuses on these roles and summarises strategies for treating cancer by disrupting this interaction with special emphasis on the CXCR4/CXCL12 axis. Finally, we discuss ongoing clinical trials with several classes of CXCR4 inhibitors, and their potential additive value for patients with a (therapy resistant) malignancy by sensitising cancer cells to conventional therapy.

The chemokine network, a newly discovered target in high grade gliomas

Chemokines are small cytokines, characterised by their ability to induce directional migration of cells by binding to chemokine receptors. They are known to play a role in tumour development, angiogenesis and metastasis. Interestingly, the chemokine network also contributes to the progression of gliomas, mainly by intensifying their characteristic invasive character. The main hurdle in treatment of these tumours is their infiltration of surrounding tissues, hampering complete surgical tumour removal. Standard postsurgical treatment with radio- and chemotherapy is of limited effect. Therefore drugs that target the chemokine system in high grade gliomas might fill the gap existing in the current approach. This review presents the current knowledge of the role of chemokine network in the development of the central nervous system, in brain physiology and the involvement in brain tumour progression. Finally, current studies exploring new compounds targeting the chemokine network in cancer patient are discussed.

[99mTc]O2-AMD3100 as a SPECT tracer for CXCR4 receptor imaging

PURPOSE CXCR4 plays an important role in HIV infection, tumor progression, neurogenesis, and inflammation. In-vivo imaging of CXCR4 could provide more insight in the role of this receptor in health and disease. The aim of this study was to investigate [(99m)Tc]O₂-AMD3100 as a potential SPECT tracer for imaging of CXCR4. METHOD AMD3100 was labelled with [(99m)Tc]pertechnetate. A cysteine challenge assay was performed to test the tracer stability. Heterologous and homologous receptor binding assay and internalization assay were performed in CXCR4 expressing Jurkat-T cells. Ex vivo biodistribution was studied in healthy mice at 30, 60, and 120 min after tracer injection. Tumor uptake of the tracer was determined by microSPECT imaging in nude mice xenografted with human PC-3 prostate tumor. Specificity of tracer uptake was determined by blocking studies using an excess of unlabelled AMD3100. RESULTS AMD3100 was labelled with technetium-99m with a radiochemical yield of >98%. The tracer was stable in PBS and mouse plasma for at least 6h at 37 °C. Heterologous and homologous binding assays with AMD3100 showed IC50 values of 240 ± 10 μM, and 92 ± 5 μM for [(125)I]SDF-1α and [(99m)Tc]O₂-AMD3100 respectively, with negligible receptor internalisation. The tracer showed high uptake in liver, lungs, spleen, thymus, intestine and bone. Blocking dose of AMD3100.8HCl (20mg/kg) decreased the uptake in these organs (p<0.05). [(99m)Tc]O2-AMD3100 showed specific tumor accumulation in mice bearing PC-3 xenografts model. Time activity curves (TAC) in AMD3100 pre-treated animals tracer showed 1.7 times less tumor uptake as compared to control animals (p<0.05). CONCLUSION [(99m)Tc]O2-AMD3100 is readily labelled, is stable in plasma and displays a favourable binding affinity for the CXCR4 receptors. [(99m)Tc O₂-AMD3100 shows specific binding in organs with high CXCR4 expression and in CXCR4 positive tumors. These results justify further evaluation of this radiopharmaceutical as a potential biomarker for the non-invasive imaging of CXCR4 receptors.

Studying cancer metastasis: Existing models, challenges and future perspectives

Cancer metastasis causes most cancer-related deaths. Several model systems to study the complex and multi step process of metastasis exist, including in vitro systems, ex-vivo organ slices, Drosophila Melanogaster and zebrafish models and the use of the chorio allantoic membrane (CAM) of fertilized chicken eggs. These models are relatively easy and cheap but often lack the opportunity to study the complete metastasis cascade. More complex but also more expensive is the use of animal models including the more recently developed patient derived tumor xenografts (PDTX). In this review, we give an overview of the existing metastatic models, discuss the challenges of improving current models to enhance translation from the preclinical to the clinical setting and consider future perspectives.

Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus

Background:High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug.Methods and results:FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I–IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019).Conclusion:Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

Abstract 1511: CXCR4 inhibition sensitizes prostate cancer cells to radiotherapy in an in vitro co-culture model

Background: Tumor microenvironment is an important factor influencing radiosensitivity of cancer cells. Amongst multiple soluble factors released by tumor-associated stromal cells, chemokine CXCL12 was shown to confer pro-survival effects on cancer cells expressing chemokine receptor 4 (CXCR4). It has been recently shown that stromal microenvironment protects prostate cancer cells from chemotherapy-induced cell death via CXCR4 signaling, which was reversed by AMD3100, a specific CXCR4 inhibitor. In this study, AMD3100 was tested for its radiosensitizing capacity in prostate cancer. Methods: Both short-term (72 h) and long-term survival (17 days) of PC3 prostate cancer cells in the absence or presence of murine stromal cells (MS5) after increasing doses of gamma-irradiation (0-15 Gy) was assessed. To analyze the short-term effect of stromal cells on PC3 viability after radiation, fluorescently labeled PC3 cells were cultured with or without MS5 cells and exposed to gamma-irradiation (0-15 Gy) in the presence or absence of 25 µg/ml AMD3100. DAPI nuclear staining was used to assess the viability of PC3 after 72 hours, Long-term survival of PC3 cells was evaluated by clonogenic assay of PC3 cells seeded alone or on feeder layer of MS5-GFP stromal cells and irradiated with 0-15 Gy. Results: The short-term viability assay showed that PC3 in mono-culture show dose-dependent loss of viability after increasing doses of irradiation, showing only 18±6% (mean ± SD) viability when exposed to 15 Gy as compared to non-irradiated control cells. However, in the presence of stromal cells, viability of PC3 cells exposed to the same radiation dose remained high, reaching levels of 69±2% viability (P=0.004 vs 15 Gy in monoculture). This effect was potently reversed by AMD3100, which lowered viability of cells to 46±3% (P=0.001 vs 15 Gy in co-culture without AMD3100). Importantly, addition of AMD3100 to PC3 cultured in the absence of stromal cells did not change their viability after radiation. The long-term survival measured in clonogenic assay showed that in presence of MS5-GFP murine stromal cell line, 8% PC3-luc cells survived 6 Gy gamma-radiation dose, as compared to only 3.6% of PC3 cells that were cultured without the stromal cells (P=0.03). Conclusion: Murine stromal cells protect PC3 prostate cancer cells against irradiation-induced cell death in an in vitro co-culture system. Treatment with AMD3100 potently inhibits the protective effect of stromal cells on the prostate cancer cells exposed to gamma-irradiation. These data underscore the relevance of stromal microenvironment in tumor response to irradiation and warrant further research on the role of CXCR4 inhibitors as a therapeutic option to potentiate radiotherapy. 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 1511. doi:1538-7445.AM2012-1511

Inhibitory effects of chemotaxis inhibitory protein of S. aureus (CHIPS) in U87 glioma mouse xenografts

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Glioblastoma is the most common malignant neoplasm of the central nervous system. FPR1 is a seven-transmembrane G protein coupled receptor, present on a highly malignant human astrocytoma cell line U87, where it promotes cell motility, growth, and angiogenesis. Chemotaxis Inhibitory Protein of S. aureus (CHIPS) is a potent FPR1 inhibitor. We therefore explored the in vivo activity of CHIPS in U87 tumor bearing mice. To determine the relevance of targeting FPR1 in gliomas, we analyzed FPR1 expression in human glioma specimens. Methods: Male, nonobese diabetic (NOD)/severe combined immune deficient (SCID) mice (total 16) were pre-irradiated with 2 Gy and subcutaneously implanted in the dorsal flank with 5x106 U87 cells in 100 μL PBS. Tumor size was measured 3 times a week and volumes were calculated with the formula Lw2/2. The survival of mice was evaluated with a Log-Rank (Mantel Cox) test. When reaching the humane endpoint (tumor size > 2 cm3) tumors were excised, formalin-fixed and paraffin-embedded. Tumor sections were subjected to H&E staining and immunohistochemical analysis of expression of anti-apoptotic markers BCLxL, survivin and MCL-1, apoptotic marker cleaved caspase-3 and proliferation marker Ki67. All xenograft sections were first stained with GLUT-1 to distinguish hypoxic from normoxic areas. In addition, the expression of FPR1 on different grades of human astrocytoma tissues was evaluated by immunohistochemistry. Results: Tumor volumes of CHIPS-treated and placebo-treated mice did not differ. However, CHIPS-treated animals did survive longer as compared to control animals (41 versus 47 days, P = 0.0019). No differences in BCLxL, survivin, MCL-1 and Ki67 expression were detected between treated and untreated tumors in both hypoxic or normoxic areas. However, in normoxic areas we found an increased expression of cleaved caspase-3 in the treated group (1.4 ± 1.1 % positive cells) compared to untreated group (0.7 ± 0.4 % positive cells, P = 0.026). Furthermore, in specimens of grade I and II astrocytomas, FPR1 expression was detected in over 70% of tumor cells, with a homogeneous cytoplasmic staining pattern and moderate to high staining intensity. In grade III astrocytomas, 70% of tumor cells were FPR1 positive, but the expression pattern was less homogeneous although with high intensity. In grade IV astrocytomas, 40% of tumor cells were FPR1 positive. Conclusions: Targeting FPR1 with CHIPS prolongs survival of U87 tumor-bearing mice. FPR1 is a relevant target in glioma patients expressed in all grades of human astrocytomas. Supported by grant RUG 4622 of the Dutch Cancer Society Citation Format: Jennifer Carla Boer, Urszula Domanska, Carla de Haas, Jos van Strijp, Evelien Jongeneel, Elisabeth de Vries, Wilfred Den Dunnen, Annemiek Walenkamp. Inhibitory effects of chemotaxis inhibitory protein of S. aureus (CHIPS) in U87 glioma mouse xenografts. [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 5537. doi:10.1158/1538-7445.AM2013-5537

Abstract 1672: Mitochondrial and bacterial peptides act on the formyl peptide receptor (FPR) to promote migration and proliferation in high grade glioblastoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Recently several aberrant molecular pathways and underlying genetic defects are elucidated in astrocytomas, among which the role of the formylpeptide receptor (FPR) malignant behavior. In a highly malignant human astrocytoma cell line FPR promotes cell motility, growth, and angiogenesis by interacting with host-derived agonists originating from necrotic cell material. Until now the only known ligands of FPR are N-formyl peptides, which are cleavage products of bacterial proteins like N-formyl-methionyl-leucyl-phenylalanine (fMLF). In humans the only conserved N-formyl peptides are the cleavage products of mitochondrial proteins. This study aims to identify these peptides as responsible ligands for FPR expressing high grade glioma cells. Methods: We used FITC-fMLF and flow cytometry to confirm binding to FPR-expressing high grade human gliobastoma cell line U87. In addition, U937 cells (human promonocytic cell line) transfected with the FPR (U937FPR) and empty expression vectors were generated. The hallmark of chemokine receptor activation is a rapid and transient increase in the free intracellular Ca2+ level upon ligand binding. Formylated mitochondrial peptides fMLKLIV/fMLALV and FPRs natural peptide ligand fMLF were used to induce Ca2+ mobilization, chemotaxis as determined by the Neuro Probe® system and proliferation in U87 cells, analyzed by MTT assay. Results: FITC-fMLF bound to FPR expressing U87 and U937FPR cells. Increasing concentrations of fMLF (10−8 M-10−5 M) caused a dose dependent calcium mobilization in FPR expressing U87 cells. Increasing concentrations of fMLF/fMLKLIV/fMLALV (10−8 M- 10−5M) were tested in U937FPR cells, again showing dose dependent calcium mobilization. In the chemotaxis assay, respectively 22, 20 and 24% of U937FPR cells migrated towards fMLF/fMLKLIV/fMLALV as compared to 4% towards 0.05% FCS. A 2.5 fold increase in migration of U87 cells towards 10−7 M fMLF was found as compared to 0.05% FCS. Respectively 20, 21 and 15% increase in U87 cell proliferation was found when cells were stimulated with 10−6 M fMLF/fMLKLIV/fMLALV. Conclusions: These results indicate that mitochondrial peptides present in necrotic tumor material serve as ligands for FPR, expressed on high grade human gliobastoma cell line U87. Activation of FPR via N-formyl peptides causes increased cell growth and motility and is a potential target for anti-cancer therapy. Supported by grant RUG 4622 of the Dutch Cancer Society Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1672. doi:10.1158/1538-7445.AM2011-1672