Targeting Regulatory T Cells: A Novel Approach to Therapy in Dogs with Cancer
Regulatory T Cells Foster Tumor Growth and Are Associated with a Poor Prognosis in Cancer Patients
Regulatory T cells (Treg) are a distinct subset of the CD4+ T cell population that actively suppress the proliferation of harmful self-reactive T cells and are therefore critical to the maintenance of tolerance to self tissues and prevention of autoimmune disease (1,2). In cancer patients, however, Treg potently suppress cellular immune responses against tumors and inhibit the response to therapy in a wide range of human malignancies (1-6). Recent studies have shown that tumors actively suppress the development of effective antitumor immunity in part through recruitment and activation of Treg using several mechanisms. For example, tumor-induced production of the chemokine CCL22 has been shown to drive recruitment of Treg to tumor tissue in people with ovarian and prostatic carcinoma, and high Treg levels are typically associated with more advanced disease (4,7-11). Alternatively, tumor production of immunosuppressive cytokines, such as TGF-B and IL-10, facilitates conversion of CD4+ non-regulatory T cells (i.e., CD4+ T-helper cells) into Treg that then accumulate within tumor tissues and tumor-draining lymph nodes and suppress the function of tumor-specific cytotoxic T cells (12,13). Independent of the mechanism for their recruitment, high numbers of intratumoral or systemic Treg is a poor prognostic indicator for many cancers.
Targeting Treg with Metronomic Chemotherapy
Metronomic dosing of chemotherapy is defined as the administration of low, repeated doses of chemotherapy drugs without prolonged interruptions in drug administration. Unlike traditional chemotherapy which targets rapidly dividing tumor cells, key targets of metronomic therapy appear to be tumor-associated endothelial cells and circulating endothelial cell precursors, both of which play an important role in tumor angiogenesis and tumor cell viability. More recently, Treg have also been shown to be targeted by metronomic chemotherapy, although the mechanism for this effect is not as well understood.
The selective toxicity of metronomic chemotherapy on Treg has been best studied for the alkylating agent cyclophosphamide (CYC). In mice and in humans, administration of low doses of CYC leads to a transient but profound decrease in the number of circulating Treg and directly inhibits Treg function (14-18). In rodent tumor models administration of even a single, low dose of CYC has been shown to dramatically reduce Treg accumulation in tumors and restore the efficacy of antitumor immunotherapy (18). In a study of people with end stage cancer, administration of daily low-dose oral CYC therapy led to significantly lower Treg numbers within 4 weeks; Treg depletion was also associated with marked restoration of CD4+ and CD8+ -T cells and natural killer (NK) cell functions (14). Based on these findings, numerous Phase II clinical trials are underway to evaluate the combination of metronomic CYC with various forms of tumor immunotherapy (19-21).
The therapeutic benefit of continuously administered low-dose CYC therapy in veterinary cancer patients has not been systematically studied. Evaluation of a metronomic chemotherapy protocol has been recently reported in one small clinical trial (22). In this study, dogs with splenic hemangiosarcoma received a combination of 3 drugs (cyclophosphamide, etoposide and piroxicam). The investigators found that the median survival time for dogs treated with the metronomic regimen compared favorably with those treated with conventional single-agent doxorubicin. However, the study's design did not permit determination of single drug efficacy or optimal drug dosing and biological endpoints, such as Treg levels and tumor angiogenesis, were not assessed. More recently, an abstract reported a significant treatment effect from low-dose CYC and piroxicam in dogs with incompletely-resected soft tissue sarcoma (23). In this study the disease-free interval was significantly longer in dogs receiving metronomic chemotherapy than for a control population of dogs treated with surgery alone.
Evaluation of Treg Depletion in Dogs with Cancer
The Impact of Surgery and Chemotherapy on Treg Levels in Dogs with Osteosarcoma
As preliminary step in testing the therapeutic benefit of Treg depletion in canine cancer patients, we are currently conducting a study to correlate Treg numbers with treatment outcome, disease free interval and overall survival in dogs with appendicular osteosarcoma (OSA). Our laboratory has recently reported the use of a flow cytometry-based assay for the identification and quantification of canine Treg (24). In the OSA study, Treg numbers are measured in the blood and lymph nodes immediately prior to limb amputation, one day postoperatively, and again 7 days after the first chemotherapy treatment with either doxorubicin or carboplatin. Although we are still in the process of patient accrual, review of the preliminary data shows that the number of Treg in blood and lymph nodes of dogs with OSA are significantly lower 24-hours after limb amputation than in the pre-operative period (7.1% vs. 4.8%, p < .05 for blood and 18.1% vs. 11.1%, p < .01 for lymph nodes). This data is presented in Figure 1 (below) and also shows that Treg numbers in the blood (A) and lymph nodes (B) of dogs with OSA following limb amputation drops to the level found in healthy, age-matched control dogs. These findings suggest that resection of the primary tumor may have a systemic inhibitory effect on Treg levels in canine OSA patients and therefore may provide a window of opportunity for introduction of novel therapies such as tumor vaccines. The duration of Treg depletion and correlation with prognosis as well as the impact of chemotherapy on Treg numbers are still under investigation.
%Treg in healthy dogs and in dogs with OSA, before and 24-hours after surgery.
Using Metronomic Cyclophosphamide Therapy to Decrease Treg in Dogs with Soft Tissue Sarcoma
In a separate study, we are evaluating the ability of metronomic dosing of single-agent, oral CYC to decrease Treg numbers, both locally within tumor tissue or tumor-draining lymph nodes, and in the peripheral circulation of dogs with soft tissue sarcoma (STS). Using the flow cytometry-based assay, we are enumerating Treg in the blood and tumor-draining lymph nodes (sampled via fine-needle aspiration) before, and at 2 and 4 weeks after starting CYC. The extent of Treg infiltration into tumors was recently reported to be a strong predictor of biologic behavior for some tumor types (25,26). Therefore tumor biopsies are also obtained from each dog prior to treatment and at the same post-treatment time points and evaluated for Treg infiltration. An additional objective is to determine if metronomic CYC decreases tumor angiogenesis; this objective will be based on tumor microvessel density assessment and enumeration of circulating endothelial precursor cells. Although the primary goals of this study are to determine whether low dose cyclophosphamide therapy depletes Treg and/or exhibits antiangiogenic activity, tumor growth inhibition is an important secondary endpoint. It is anticipated that this study will guide the design of larger clinical trials and provide a foundation for more logical application of metronomic chemotherapy to veterinary cancer patients.
Conclusions and Future Directions
A primary mechanism of immune evasion for many tumors is the recruitment and activation of Treg which, in turn, suppress the development of effective antitumor immunity. In addition to their negative association with prognosis, high levels of Treg are likely to contribute to the failure of many forms of anticancer therapy, especially tumor immunotherapy. Treg depletion is therefore an attractive strategy in improving treatment outcomes for a wide range of malignancies.
Metronomic chemotherapy represents one of a number of strategies in selectively targeting Treg and is an especially attractive treatment approach because of the minimal cost and toxicity associated with administration of low doses of chemotherapy drugs such as CYC. We are currently conducting several studies designed to determine the impact of conventional and metronomic chemotherapy as well as surgery on Treg levels in dogs with common malignancies. We expect that the selective depletion of Treg will be an important step in improving the efficacy of anticancer therapy in human as well as in veterinary patients.
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