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Towards targeting chemokine receptors CCR7 and CCRL1 to control the crossroads of tumor-host interactions

Final Report Summary - CRITICS (Towards targeting chemokine receptors CCR7 and CCRL1 to control the crossroads of tumor-host interactions.)

Chemokines are secreted proteins which induce cell movement and also affect other cell functions. Two classes of chemokine receptors exist: classical G-protein coupled receptors (GPCRs) and atypical receptors (ACRs). Whereas GPCRs mediate cell migration and other cell responses, the ACRs primarily modify chemokine availability by either degrading or transporting them. Chemokines contribute at multiple levels to the development and spread of cancer. 1. They control the homing and positioning of immune cells within lymphoid organs which will have profound effects on the ability to trigger immune responses including the anti-tumour responses. 2. Chemokines also direct leukocytes and other cells into cancer tissues and thus determine the cell contents within the cancer microenvironment, which, in turn, influence tumour homeostasis and the quality and magnitude of anti-tumour immune responses. 3. Chemokines can in addition act directly on tumour cells to affect their survival, growth and induce their spread (metastasis). All these chemokine-driven effects are mediated via GPCRs. Much less is known on how ACRs influence tumour-host interactions.

In this project we have taken an interest in crosstalk between classical and atypical chemokine receptors, in the regulation of immune responses in the context of tumour development with a focus on the chemokines CCL19 and CCL21 that binds the classical GPCR CCR7 and the ACR CCRL1. The homeostatic chemokines CCL19 and CCL21, both acting through the classical chemokine receptor CCR7, have pivotal roles in the establishment of functional microenvironments within lymphoid organs. Through their expression on high endothelial venules and afferent lymphatics they direct the homing of T-cells and dendritic cells (DCs) to the lymph nodes where the expression of these ligands by the FRC network contributes to the positioning of the cells. Defects in this system will affect the establishment of T-cell/DC cellular contacts which are necessary for the execution of effective lymph node driven immune responses.

Using retroviral techniques we have established B16 melanoma with stable expression of mouse CCR7, CCL19, CLL21 and CCRL1 together with the reporter gene luciferase for in vivo monitoring of the tumour growth. These in vitro engineered and validated tumour cell lines have then been used in wild type and mouse knockout models with deficiency in the receptors CCR7 and CCRL1. At the beginning of this project, a high profile paper, by Shields JD et al., 2010 in Science, presented data suggesting that expression of host CCR7 is necessary for the establishment and growth of B16 melanoma in the mouse host and that expression of the CCR7 ligand CCL21 creates an immunosuppressive tumour promoting environment. However, our data in part conflicts with published findings as B16 melanoma establishment, growth or progression in our hands do not depend on host CCR7. We have tested this in a range of 104 to 105 tumour cell number inocula and under all conditions CCR7ko mice display equal or higher frequency of tumours after inoculation and equal or higher progression rates compared to wild type mice. To complement the B16-melanoma model, used in mice of the C57BL6 background, we also established a 4T1 luc breast cancer model for Balb/c mice. Again 4T1 breast cancer in CCR7ko/Balbc mice established in frequencies comparable to wild type Balb/c mice and progressed in rates equal or higher compared to wild type Balb/c mice.

Our data also show that tumour establishment is independent on the presence or absence of expression of the CCR7 ligands CCL19 or CCL21 in B16 melanoma. In line with these data expression the ACR CCRL1 does not impact growth of the tumours. Moreover, we cannot detect recruitment of CCR7 positive Lti cells or regulatory T-cells into CCL19 or CCL21 expressing tumours as was reported by Shields et al. 2010. However, our data do show a change in the immunological environment dependent on tumour expression of CCL21 and CCL19, with recruitment of more naïve T-cells, which are characterized by high expression the receptor CCR7. In a therapeutic setting this may be of high relevance and this is an question we plan to address in the future. We know from our data so far that CCR7ko mice do not respond to vaccination with irradiated tumour cells whereas wild type mice display a measurable partial response. To take this further we have set up an experimental monitorable system for vaccination using transferred melanoma specific T-cells from the pmel T-cell receptor transgenic mouse line, which will be used in combination of cellular and peptide based vaccination regimens.

Analysis of tumour growth in CCRL1ko mice revealed that these mice display a defect in homing of dendritic cell to the lymph node. Preliminary data indicated a delay in tumour vaccination responses. The defect was present also in CCRL1ko mice under homeostatic condition. As a side project we have further evaluated this phenotype to understand the mechanism of this before we could take it further in the experimental tumour system. This part of the project has here led to several international collaborations and has produced data that is of fundamental importance for the understanding of chemokine gradient formation. The data from this project is now submitted for publication.

In terms of the original 4 research objectives, we have completed RO2 and RO1, RO3 and RO4 are completed to large part. The work on these will be continued with the aim to reach publication in a near future. Importantly, our work has shed important new light into the role of the chemokine axis CCL19/21 in tumourigenesis that will guide our further studies in the right direction. The data on the role of CCRL1 in the lymph node will reach the scientific community and has impact not only for the CCL19/21 chemokine axis in tumourigenesis but also for the basic understanding of chemokine gradient formation and regulation of dendritic cell homing to the lymph node. The data has already been presented at multiple International conferences and was received with great interest by the scientific community.