Physician Scientist Award – Interim Report
- Role of Chemokines in Head and Neck Cancer
- Principal Investigator: Robert L. Ferris, MD, PhD
- Institution: University of Pittsburgh
Chemokines are small, secreted molecules that mediate homing and recruitment of immune cells, in response to inflammation, through a family of G-protein linked receptors. These receptors are classified based on the position of disulfide-liked cysteine residues, as well as their angiogenic effects. By recruiting antigen-specific cells to the site of an inflammatory response, this family of mediators creates a bridge between innate and adaptive immunity. Chemokines can be made and secreted by many different cell types, including tumor cells. Exploiting the role of chemokines to recruit dendritic cells and lymphocytes to the primary tumor site and to secondary lymphoid organs, chemokine-expressing tumor cells may be used to enhance anti-tumor immunity. Recently, tumor cells have also been shown to express chemokine receptors that enable their access to the lymphatic system and facilitate metastatic spread to lymph nodes, fulfilling Paget’s hypothesis over one hundred years later. Chemokine-expressing tumor cells might be able to determine the nature of mononuclear cell infiltrates at the tumor site and influence interactions of infiltrating immune cells with the tumor. By chronically stimulating CCRs on these immune cells, leading to receptor desensitization, tumors may also contribute to the immunosuppressive effect of the neoplastic cells. We propose to study the chemokine receptor (CCR) and ligand (CCL) expression by HNC cell lines and in primary explants of human head and neck tumor samples. To evaluate the role of CCR expression on lymphocytes and dendritic cells (DC) in tumor-directed migration of these cells, a murine HPV-induced oral cavity carcinoma established in my laboratory will be utilized. Initial ex vivo chemotaxis experiments and subsequent in vivo peptide/DC-based vaccinations will be performed to assess whether intratumoral CCL expression contributes to increased recruitment and better efficacy of the vaccine. Overall these studies are designed to identify the CCR and CCL relevant to progression of HNC and to provide initial data on their possible clinical utility as components of future vaccination therapies for HNC.
Briefly describe progress in completing the project:
The two-year grant is based on studying the role of chemokines and chemokine receptors in head and neck cancer, with a focus on metastasis and the harnessing of these chemo-attracting cytokines for improved tumor-specific vaccination in a murine tumor model.
Aim 1 focuses on investigating whether chemokine receptors are expressed by head and neck cancer cell lines and fresh tumor tissues, identifying which chemokine receptors are expressed, and if these are functionally active. Furthermore this first aim of the grant proposed to analyze whether chemokine receptors could be responsible for some metastatic phenotype seen in HNC.
Our progress on Aim 1 has certainly been within expectations. First, we have shown that a number of chemokine receptors are selectively expressed on metastatic but not primary tumor cells, using established, paired cell lines and fresh tumor tissues from both primary and metastatic lesions from the same patients. Using RT-PCR, we first successfully showed that the chemokine receptor CCR6 is expressed on primary tumor derived cell lines and tissues, but not on metastatic tumors from the same patient. Furthermore the chemokine receptor, CCR7, was expressed on both primary and metastatic tumors, but recent quantitative RT-PCR has shown that CCR7 is approximately tenfold higher on the metastatic tumors. These molecular studies have been confirmed functionally to show that the metastatic tumors migrate in response to CCR7 ligands, MIP3-ß and SLC, but significantly less well in the primary tumors which express lower levels of CCR7 by qRT-PCR. However, the migration to the CCR6 ligand (MIP3-a) was seen only in the primary tumors, which were the only cells in tissues to express the appropriate receptor for this ligand CCR6.
Our interpretation of these findings is that SCCHN metastasis parallels the maturation of dendritic cells, which are immune cells which sample sites of inflammation in the periphery, before migrating to secondary lymphoid organs, such as lymph nodes. These dendritic cells express CCR6 in the periphery just as our primary tumor cell lines and fresh tissues did. However, mature dendritic cells downregulate CCR6 and upregulate CCR7, so that they can migrate to the regional lymph nodes and stimulate an immune response. This is the exact pattern we have seen in four paired cell lines and six fresh primary versus metastatic tumor samples from patients in my clinical practice. Thus it appears from our work currently that metastatic SCCHN cells downregulate CCR6 and upregulate CCR7, exploiting a common immune mechanism for dendritic cells, as well as memory T lymphocytes, to migrate to the regional lymph nodes. This may explain at least one mechanism for tumor cell metastasis which is seen in HNC to occur nearly universally in the regional lymph nodes.
What work is currently underway?
The second and third aims in this project are focused on studying the role of chemokine ligands expressed at the tumor site, to recruit lymphocytes and antigen presenting cells for enhanced anti-tumor vaccine efficacy. Our preliminary work has focused on establishing the reagents, both in vitro and in vivo for the mouse experiments. Our success has been reasonable to date lending confidence that this vaccine protocol will be at least partially completed in the next twelve to 14 months.
First, we have established a tumor model as shown in our preliminary data using TC-1, an HPV-transformed cell line, in the mouse floor of mouth. Since our grant was funded we have also obtained the human HLA A2.1-transfected TC-1 from a collaborator at Johns Hopkins, enabling vaccination with human peptides for easier correlation to human t-cell derived antigens. We have constructed two adenovirus vectors through cloning the genes for two chemokine ligands, MIP-3a and MIP-3ß. These have been inserted into a shuttle vector and recombinant adeno-virus vectors constructed over the past year. We propose first to inject into the established tumors recombinant MIP-3a which recruits immature dendritic cells or recombinant MIP-3ß which attracts mature dendritic cells and memory T-lymphocytes to the tumor site. We then will follow up with the recombinant adenoviral vectors expressing these two chemokines to compare their individual effect. The HLA A2.1 transgenic mice have been obtain and are currently being bred in our facility. Some delay was encountered since my laboratory moved to a new facility which required the animals to be purchase again to avoid quarantine. However these mouse experiments can now move forward as the second round of breeding is underway, and we are optimizing our strategy for procuring and growing mouse dendritic cells, which we propose to use as our cancer vaccine.
What work has not yet been initiated?
We had planned to compare CCR expression between HPV-positive and HPV-negative HNC. Levels of soluble CCL in tumor cell supernatants and patient sera and saliva are to be measured and correlated with disease activity and progression to assess the potential utility of CCL as potential tumor markers associated with metastatic spread. This work is being planned, to be performed first on patient serum using the Luminex machine, a multiplex ELISA technique that can quantitate multiple soluble proteins in bodily fluids. These assays are currently being validated, as the machine arrived at our institution a few months ago.
Will you be able to complete this project on time?
We have described above that Aim 1 is essentially complete with interesting, novel data having been obtained. This is being prepared for publication in the next several months and will be presented at the AAOHNSF meeting in September, 2003. The latter portion of the project will likely be at least partially completed in the next 12 months. Animal studies sometimes take longer than anticipated, notwithstanding our move of the laboratory and initiating an animal facility which had air handling problems for the first 2-3 months of our relocation to the new facility, somewhat hampering our progress. However in the meantime we have established the reagents to perform these experiments and with the conclusion of the project in Aim 1, anticipate that the next 12 months will focus heavily on the mouse vaccination and chemokine-enhanced immunotherapy portion of the grant. Luminex cytokine/chemokine studies on serum will be performed over the next year also.
Clinical Applications, Either Immediate or Potential, of This Research:
Since we are showing evidence that chemokine receptors play an important role in metastasis of head and neck cancer to the lymph nodes in the neck, it is conceivable that blockers or inhibitors of these receptors might be able to decrease or eliminate the metastatic spread of such tumors. Further more this opens a new field of study for the signaling and invasive potential of metastatic versus non-metastatic head and neck cancer, which could lead to other signaling interference to prevent metastasis in the future.
We hope that in one year’s time we will have additional data on whether harnessing chemokine ligand signaling at the tumor site may improve an established vaccine protocol in our mouse tumor model. If so this would have important application for current human cancer vaccine trials, including one currently underway at the University of Pittsburgh. For instance our current head and neck cancer vaccine trial includes direct injection of a patients own dendritic cells, a potent antigen presenting cell to stimulate a cancer specific immune response, into the patient groin lymph nodes under ultrasound guided needle infusion. If our hypothesis is correct, that chemokines can attract dendritic cells and memory T cells to the tumor site or to the lymph node, this would enable an elegant way to avoid a painful and somewhat inefficient vaccine delivery process that we currently use to deliver dendritic cells to desired sites. We anticipate updating this hypothesis in the coming year.
Other Pertinent Information:
We are grateful for the continued support of the American Academy of Otolaryngology Head and Neck Surgery Foundation, and the American Head and Neck Society, who make possible this surgeon-scientist grant mechanism. We have been gratified to obtain some promising data which will likely be incorporated into an upcoming grant for external funding. We hope that the coming year’s time will prove fruitful in achieving the proposed work as described above and in our original grant proposal.