M. Rita I. Young

Phase 1B study to improve immune responses in head and neck cancer patients using escalating doses of 25-hydroxyvitamin D3

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by immune inhibitory CD34+ progenitor cells, whose numbers are increased in the peripheral blood of HNSCC patients. Immune inhibitory CD34+ cells are also present within HNSCC tumors. A phase IB clinical trial was conducted with HNSCC patients to determine if treatment with the differentiation-inducer 25-hydroxyvitamin D3 could diminish CD34+ cell levels and improve a panel of immune parameters. Here we present the results of treatment with orally administered escalating doses (20, 40, 60xa0μg) of 25-hydroxyvitamin D3, with an emphasis on the six patients who received the maximum dosage of 60xa0μg per day. Peripheral blood was collected at 0, 1, 2, 4, and 6xa0weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of this pilot study demonstrated that treatment of HNSCC patients with 25-hydroxyvitamin D3 reduces the number of immune suppressive CD34+ cells, increases HLA-DR expression, increases plasma IL-12 and IFN-γ levels, and improves T-cell blastogenesis. In contrast, 25-hydroxyvitamin D3 treatment did not modulate plasma IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, or TGF-β levels.

Secretion of vascular endothelial growth factor by oral squamous cell carcinoma cells skews endothelial cells to suppress T-cell functions

Patients with oral squamous cell carcinoma (OSCC) have severe defects in antitumor immune function. Endothelial cells are potential regulators of immune cell function and have therefore been examined to determine their role in tumor-induced immune suppression. The present studies demonstrated that supernatants from endothelial cells exposed to OSCC-conditioned media (endo(OSCC-sup)) exhibited elevated levels of the immune suppressive products prostaglandin E(2) (PGE(2)) and vascular endothelial growth factor (VEGF) compared with supernatants from endothelial cells treated with medium alone (endo(medium)) or with keratinocyte-conditioned medium (endo(ker-sup)). Antibody neutralization of OSCC-derived VEGF prevented tumor-conditioned media from inducing endothelial cells to increase production of PGE(2)and VEGF. Furthermore, treatment of T-cells with supernatants from endo(OSCC-sup) resulted in diminished T-cell proliferation and decreased interferon-gamma (IFN-gamma) production compared with T-cells treated with medium or supernatants from endo(medium) or endo(ker-sup) controls. T-cell levels of granzyme B and perforin were reduced after treatment with supernatant from endo(OSCC-sup) compared with control treatments. The addition of VEGF neutralizing antibody to the OSCC-conditioned medium prevented endothelial cells from being skewed to downregulate T-cell proliferation and production of IFN-gamma, perforin, and granzyme B. Taken together, these studies provide support for the use of VEGF-targeting therapies as an immunotherapeutic agent to block induction of immune suppressive endothelial cells in patients with OSCC.

Use of α,25-Dihydroxyvitamin D3 treatment to stimulate immune infiltration into head and neck squamous cell carcinoma

Prior studies have shown that treatment of head and neck squamous cell carcinoma (HNSCC) patients with 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] reduced intratumoral levels of immune inhibitory CD34(+) progenitor cells while increasing levels of mature progeny dendritic cells. This finding was extended to a pilot study to determine whether 1,25(OH)(2)D(3) treatment concurrently increases levels of intratumoral CD4(+) and CD8(+) T cells, increases intratumoral levels of immune cells expressing the early activation marker CD69, and prolongs time to HNSCC recurrence. The clinical trial comprised 16 patients with newly diagnosed HNSCC being untreated and 16 patients being treated with 1,25(OH)(2)D(3) during the 3-week interval between cancer diagnosis and surgical treatment. Immunologic effects of treatment were monitored by immunohistochemical analyses of surgically removed HNSCC. Clinical effectiveness of 1,25(OH)(2)D(3) treatment in this study was measured by the time to HNSCC recurrence. HNSCC tissues of patients who received treatment with 1,25(OH)(2)D(3) contained increased levels of CD4(+) cells and, more significantly, CD8(+) T cells. Also prominent was an increase in cells expressing the lymphoid activation marker CD69. Results of this pilot study suggest that patients treated with 1,25(OH)(2)D(3) had a lengthier time to tumor recurrence compared with patients who were not treated before surgery.

Characterization of the evolution of immune phenotype during the development and progression of squamous cell carcinoma of the head and neck

While studies have indicated that squamous cell carcinoma of the head and neck (HNSCC) is associated with immune suppression, these studies did not analyze the immune response at the dysplastic stage. The present study utilized a mouse model of 4-nitroquinoline 1-oxide-induced oral carcinogenesis to examine the alterations in immune phenotype at the premalignant and malignant stages of HNSCC. Cervical lymph nodes of HNSCC-bearing mice were found to contain a greater number of cells, including a greater number of conventional (Tconv) and regulatory (Treg) T cells, compared to cervical lymph nodes of control and premalignant lesion-bearing mice, though the Tconv cells appear to be less proliferative and the Treg cells appear to be less suppressive at the HNSCC stage. Premalignant lesion-bearing mouse lymph nodes consist of a greater percentage of Tconv cells expressing markers for activation, memory, and exhaustion compared to both control and HNSCC-bearing mice. Also, lymph nodes’ cells from both premalignant lesion-bearing and HNSCC-bearing mice include increased levels of Th1, Tc1, and Th17 cells, with no differences in levels of Th2 cells, compared to control mice. The data show that while there is the expected increase in immunosuppressive Tregs in lymph nodes when HNSCC is present, there is also an unexpected increase in immune populations usually associated with a beneficial antitumor response, including Tconv cells and Th1 and Tc1 cells. In addition, the results demonstrate that the premalignant stage of HNSCC development is associated with a robust immune response involving an increase in inflammatory Th1, Tc1, and Th17 cells.

Tumor Secretion of VEGF Induces Endothelial Cells to Suppress T cell Functions Through the Production of PGE2

Endothelial cells are potent regulators of immune cell functions and have therefore been examined to determine their role in tumor-induced immune suppression. Previous studies by our laboratory showed that exposure to Lewis lung carcinoma (LLC)-secreted products induced endothelial cells to suppress T-cell functions in vitro. The current studies examined in vitro and in vivo the mechanism by which tumors induce the formation of suppressor endothelial cells and the means by which suppressor endothelial cells disrupt T-cell functions. In vitro studies demonstrated that inhibition of tumor-derived VEGF with neutralizing antibodies or treatment of endothelial cells with the VEGF receptor tyrosine kinase inhibitor, SU5416, prevented endothelial cells from being induced to suppress T-cell functions. Treatment of tumor-bearing mice with SU5416 blocked the development of endothelial cells that are suppressive to CD4+ and CD8+ T-cell functions. We next examined the role of suppressor endothelial cell-derived PGE2 in the inhibition of T-cell functions. Abrogation of endothelial cell PGE2 production in vitro with indomethacin prevented tumor-conditioned media from stimulating endothelial cell production of immune inhibitory activity toward T-cell functions. Similar treatment of endothelial cells from lungs of tumor-bearing mice blocked their capacity to produce T-cell-inhibitory mediators. These studies demonstrate that tumor-derived VEGF induces endothelial cells to upregulate production of PGE2 which, in turn, leads to suppression of T-cell functions.

Tumors induce the formation of suppressor endothelial cells in vivo.

Patients with solid tumors have defects in immune effector cells, which have been associated with a poorer prognosis. Previous studies by our laboratory have shown that exposure to Lewis lung carcinoma (LLC)-secreted products induces the formation of suppressor endothelial cells in vitro. The current studies examined if tumors could induce the formation of suppressor endothelial cells in vivo. Endothelial cells were immunomagnetically isolated from the lungs of tumor-bearing mice or normal controls and examined for their ability to modulate NK cell, T-cell and macrophage functions. Compared to normal controls, supernatants from endothelial cells isolated from tumor-bearing lungs had elevated secretion of PGE2, IL-6, IL-10 and VEGF. Conditioned media from endothelial cells isolated from normal lungs increased CD8+ T-cell IFN-γ and CD4+ T-cell IL-2 production in response to anti-CD3 stimulation, while media conditioned by endothelial cells from tumor-bearing lungs had a diminished stimulatory capacity. Examination of NK cell functions showed that supernatants from endothelial cells isolated from normal lungs were potent activators of NK cells, as indicated by their secretion of TNF-α and IFN-γ. Endothelial cells isolated from tumor-bearing lungs had a significantly diminished capacity to activate NK cells. Finally, supernatants from endothelial cells of tumor-bearing lungs diminished macrophage phagocytosis compared to either treatment with supernatants of normal endothelial cells or treatment with media alone. The results of these studies demonstrate that tumors induce the formation of suppressor endothelial cells in vivo and provide support for the role of endothelial cells in tumor-induced immune suppression.

1α,25-Dihydroxyvitamin D3 to skew intratumoral levels of immune inhibitory CD34+ progenitor cells into dendritic cells.

Objectives: Prior studies showed that immune inhibitory CD34+ progenitor cells, whose numbers are increased in head and neck squamous cell carcinoma (HNSCC) patients, can be differentiated into immune stimulatory dendritic cells by culture with 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3). This was extended to a pilot study to diminish intratumoral levels of CD34+ progenitor cells by inducing their maturation into dendritic cells with 1,25(OH)2D3. Study Design: Newly diagnosed HNSCC patients were untreated for 3 weeks or received 3 weeks of 1,25(OH)2D3 treatment befoer surgical treatment. Subjects and Methods: HNSCC tissue was collected by biopsy from six patients who had no prior 1,25(OH)2D3 treatment and at the time of surgical treatment from six untreated patients and 11 patients who completed 1,25(OH)2D3 treatment. Tissues were analyzed by immunohistochemistry for levels of CD34+ cells and dendritic cells. Results: After 1,25(OH)2D3 treatment, intratumoral levels of CD34+ cells and levels of immature dendritic cells declined. However, levels of intratumoral mature dendritic cells increased. Clinical effects of 1,25(OH)2D3 treatment are premature to analyze. Conclusions: Treatment of HNSCC patients with 1,25(OH)2D3 reduced levels of immune inhibitory CD34+ cells while increasing maturation of dendritic cells. This supports added studies to determine the effect of 1,25(OH)2D3 on intratumoral immune competence.

Prostaglandin E2-protein kinase a signaling and protein phosphatases-1 and -2A regulate human head and neck squamous cell carcinoma motility, adherence, and cytoskeletal organization

Human head and neck squamous cell carcinoma (HNSCC) cultures were established from cancers of two patients. These cells were used to study if phosphorylation reactions by protein kinase A (PKA) and dephosphorylation reactions by protein phosphatases-1 and -2A (PP-1/2A) regulate tumor motility and adhesion to extracellular matrix components, and if this might be associated with cytoskeletal reorganization. Both cultures were motile and adherent to collagen I, fibronectin, vitronectin and laminin. Motility and adhesiveness was dependent on production of prostaglandin E2 PGE2 and on PKA activation. Blocking PP-1/2A activity with okadaic acid resulted in a PKA-dependent increase in m otility and, in some instances, adhesiveness by the HNSCC cells. The okadaic acid-induced increase in motility and adhesiveness coincided with a reduction in filamentous actin. These data suggest PKA and PP-1/2A have opposing effects in regulating the motility, adherence, and actin polymerization.

Trials and tribulations of immunotherapy as a treatment option for patients with squamous cell carcinoma of the head and neck

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy that is the sixth most common neoplasm in the world. Despite numerous advances in treatments involving surgery, radiation, and chemotherapy, the 5-year survival has remained at less than 50% for the last 30xa0years primarily due to local recurrences [66]. Consequently, the possibility of developing immunotherapeutic approaches as a treatment for HNSCC has gained interest. The present review has 3 objectives pertaining to immunotherapeutic means to treat HNSCC patients: (1) to summarize the feasibility of such approaches, (2) to provide an overview of the obstacles to attaining protective immune reactivity, and (3) to consider how these obstacles can be overcome to stimulate immune reactivity to HNSCC. These objectives will also be considered in the context of what lessons have been learned from immunotherapeutic trials for other solid malignancies and the applicability of this information to HNSCC.

Tumor skewing of CD34+ cell differentiation from a dendritic cell pathway into endothelial cells

Patients and animals bearing tumors have increased levels of CD34+ progenitor cells, which are capable of developing into dendritic cells. However, addition of medium conditioned by murine Lewis lung carcinoma cells increases the cellularity of the CD34+ cell cultures and redirects their differentiation into endothelial cells. The resulting cells resemble endothelial cells phenotypically as well as functionally by their capacity to reorganize into cord structures. Mechanisms by which tumors induced the increased cellularity and skewing toward endothelial cells were examined. Tumor-derived VEGF contributed to the increase in cellularity, but not to the redirection of differentiation. Differentiation into endothelial cells was blocked with sTie-2, suggesting tumor-derived angiopoietins in skewing differentiation. These studies show the capacity of tumors to skew progenitor cell development toward endothelial cells and define the mediators that contribute to endothelial cell development.