Correlation of CXCL12 Expression and FoxP3+ Cell Infiltration with Human Papillomavirus Infection and Clinicopathological Progression of Cervical Cancer

Human cervical cancer is an immunogenic tumor with a defined pattern of histopathological and clinical progression. Tumor-infiltrating T cells contribute to immune control of this tumor; however, cervical cancer dysregulates this immune response both through its association with human papillomavirus (HPV) infection and by producing cytokines and chemokines. Animal tumor models have revealed associations between overproduction of the chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) and dysregulation of tumor-specific immunity. We therefore proposed that CXCL12 expression by cervical precancerous and cancerous lesions correlates with histopathological progression, loss of immune control of the tumor, and HPV infection. We found a significant association between cancer stage and CXCL12 expression for squamous and glandular lesions as well as with the HPV16+ (high-risk) status of the neoplastic lesions. Cancer progression was correlated with increasing levels of FoxP3 T-cell infiltration in the tumor. FoxP3 and CXCL12 expression significantly correlated for squamous and glandular neoplastic lesions. These observations were supported by enzyme-linked immunosorbent assay and Western blotting. In addition, we demonstrated CXCL12 expression by dyskaryotic cells in ThinPrep cervical smears. This study robustly links increased CXCL12 expression and FoxP3⁺-cell infiltration to HPV infection and progression of cervical cancer. It supports the detection of CXCL12 in cervical smears and biopsies as an additional biomarker for this disease.Human cervical cancer is an immunogenic tumor, and several tumor-specific antigens have been identified in recent years.^1,2,3 Persistent infection with high-risk human papillomavirus (HPV) (types 16 and 18) has been shown to be an important risk factor for malignant transformation of the cervical epithelium.^4,5 Tumor immunity in this context results from the positive recognition of tumor antigens by immature dendritic cells, which subsequently migrate to draining lymph nodes and interact with and activate both helper CD4⁺ T cells and effector antigen-specific CD8⁺ T cells.¹ Activated and targeted antigen-specific cytotoxic CD8⁺ T cells subsequently circulate through the peripheral blood to the tumor and then selectively transmigrate into the cancer itself and kill tumor cells. Immune control of cervical cancer is therefore critically dependent on the activation and migration of tumor-specific cytotoxic T cells into the tumor and the subsequent killing of neoplastic cells. The success of the immune response in destroying cervical cancer is thought to depend on the relationship between the kinetics of tumor growth and the efficacy of tumor-specific cytotoxic T cells (in balance with regulatory T cells) in infiltrating the tumor and killing neoplastic cells over time. It is well established that the magnitude of T-cell infiltration within certain forms of cancer correlates with a favorable prognosis.⁶Tumors, including cervical cancer, evade immune recognition using a number of different mechanisms, including the down-regulation of major histocompatibility class I antibodies, the immunological ignorance to tumor antigens, the lack of costimulatory molecules and antigen loss, or the expression of inhibitory molecules.^7,8 Several studies have also shown that tumors, including cervical cancer, overexpress specific chemokines, such as stromal cell-derived factor 1 (SDF-1 or CXCL12), that are thought to dysregulate the immune response to the tumor.^9,10,11 Chemokines are a superfamily of 8- to 11-kDa proteins found in humans that have been shown to be critical in causing directional movement of immune cells in vitro and in determining the localization of leukocytes in models of inflammatory, immune-mediated diseases, and cancer.^11,12,13 Chemokines have been shown to act as chemoattractants for leukocyte subpopulations including T cells and dendritic cells. Chemokines signal via G_αi protein-coupled receptors on the cell surface and subsequently induce directed cell movement in response to a gradient of the chemokines.¹⁴The chemokine CXCL12 is a known T-cell chemoattractant that selectively binds the receptor CXCR4.^9,10,11 Until recently CXCL12 was thought to have chemoattractant activity only for T cells. We recently demonstrated that the chemokine CXCL12 could serve as a bidirectional cue, attracting T cells at low concentrations and repelling at high concentrations in vitro and in vivo.¹⁵ Certain murine models of melanoma and ovarian cancer indicate that the effect of CXCL12 on the tumor immune response is dose dependent.¹⁶ Low levels of CXCL12 expression result in infiltration of the tumor by T cells, a delay in tumor growth, and the development of a long-lived tumor-specific T cell-mediated immune response. In contrast, high levels of CXCL12 expression result in reduced infiltration of the tumor by tumor antigen-specific T cells¹⁷ and the suppression of antitumor immune responses via various mechanisms including the intratumoral accumulation of FoxP3⁺ suppressor T cells.^18,19We hypothesized that secretion of CXCL12 by cervical tumors is correlated with progression of the tumor and loss of immune control. To explore this hypothesis we first examined whether clinicopathological progression of cervical cancer is associated with an increase in CXCL12 expression and a concomitant reduction in immune cell infiltration into the tumor as the tumor progresses from a preinvasive to an invasive state. CXCL12 expression and T-cell infiltration were studied in tissue sections of normal cervix, preinvasive, microinvasive, and invasive squamous cervical cancer, and preinvasive and invasive adenocarcinoma using immunohistochemical (IHC) staining methods in concert with quantitative digital image analysis, Western blotting, and enzyme-linked immunosorbent assay (ELISA). Because HPV is associated with cervical cancer, we also examined the relationship between HPV status and CXCL12 expression in normal, preinvasive, and invasive cervical cancer. In this way we demonstrated that CXCL12 is a robust biomarker for clinical progression that is correlated both with HPV infection and the accumulation of FoxP3⁺ T cells in the tumor.