Yvonne Lozano

Mechanisms of immune suppression in patients with head and neck cancer : Influence on the immune infiltrate of the cancer

Freshly excised human head and neck cancers (219 primary cancers; 64 metastatic lymph node cancers) were analyzed for the immune inhibitory mediators released from the cancer tissues and the immune infiltrate within the tumor. Significant levels of the immune inhibitory mediators transforming growth factor‐β (TGF‐β), prostaglandin E2 (PGE2) and interleukin‐10 (IL‐10) were released from these cancers. Also released was granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), whose secretion was associated with an intratumoral presence of CD34+ cells. We have previously shown that CD34+ cells within human head and neck cancers are immune inhibitory granulocyte‐macrophage progenitor cells. The presence of TGF‐β, PGE2 and IL‐10 was associated with a reduced content of CD8+ T‐cells within the cancers. The CD4+ cell content appeared to be less affected by these immune inhibitory mediators. Instead, parameters indicative of CD4+ cell function (p55 IL‐2 receptor expression, release of IL‐2 and IFN‐γ) were diminished in cancers that released higher levels of TGF‐β, IL‐10 and GM‐CSF and had a higher CD34+ cell content. Furthermore, metastatic cancers released higher levels of the soluble immune inhibitory mediators and lower levels of IFN‐γ and IL‐2 than did primary cancers, although CD34+ cells were similarly present in both primary and metastatic cancers. Our results show that human head and neck cancers have a multiplicity of non‐mutually exclusive mechanisms of immune suppression that are most prominently associated with reduced CD8+ cell influx and reduced influx and altered function of intratumoral CD4+ cells.

Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) have been shown to contain CD34+ natural suppressor cells that inhibit the activity of intratumoral T‐cells. The present study evaluated whether GM‐CSF production and the presence of CD34+ cells within primary HNSCC would translate into increased recurrence, metastasis or cancer‐related death during the 2 years following surgical excision. Freshly excised primary HNSCC of 20 patients that subsequently developed disease, and of 17 patients that remained with no evidence of disease were analyzed for production of GM‐CSF and for CD34+ cell content. The cancers of patients that subsequently developed recurrences or metastatic disease produced almost 4‐fold the levels of GM‐CSF and had approximately 2.5‐fold the number of CD34+ cells as did cancers of patients that remained disease‐free. In a second method of analysis, the prognostic significance of high vs. low GM‐CSF and CD34+ cell values was evaluated. These analyses showed that patients whose cancers produced high GM‐CSF levels or had a high CD34+ cell content had a disproportionately high incidence of recurrence or metastatic disease (94% and 100%, respectively), while the majority of patients whose primary cancers produced low levels of GM‐CSF or had a low CD34+ cell content remained disease‐free (16% and 19%, respectively). Our results indicate that the presence of CD34+ cells in GM‐CSF‐producing HNSCC is associated with a poorer prognosis for the cancer patients and suggest the utility of these parameters as prognostic indicators of outcome. Mechanistically, our results suggest that the presence of immune suppressive CD34+ cells in GM‐CSF‐producing HNSCC leads to increased tumor recurrence or metastasis.Int. J. Cancer 74:69–74.

Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression, and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1α,25-dihydroxyvitamin D3 reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D3 reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D3 treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D3 after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D3 treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.

Vitamin D3 treatment of tumor bearers can stimulate immune competence and reduce tumor growth when treatment coincides with a heightened presence of natural suppressor cells

By secreting granulocyte-macrophage colony-stimulating factor (GM-CSF), Lewis lung carcinoma tumors induce immune suppressive granulocyte-macrophage progenitor cells. Treating mice having established tumors and high levels of suppressor activity with vitamin D3 eliminated suppressor activity, increased anti-tumor immunity, induced an immune stimulatory cell population, and reduced tumor growth. When instead, the vitamin D3 treatment was initiated earlier, when implanted tumors first became detectable and when natural suppressor activity was less prominent, the treatment had no effect. Thus, vitamin D3 treatment can stimulate the immune competence of tumor bearers when treatment is targeted to coincide with a heightened presence of GM-CSF-induced suppressor cells.

Protein phosphatase-2A association with microtubules and its role in restricting the invasiveness of human head and neck squamous cell carcinoma cells

The role of protein phosphatase-2A (PP-2A) in regulating the motility and adhesion of human head and neck squamous cell carcinomas (HNSCC) was investigated. Immunofluorescent staining of these HNSCC cells showed PP-2A can co-localize with microtubules. That the PP-2A influences motility was shown by the increase in HNSCC cell migration through laminin and vitronectin when PP-2A was selectively inhibited with low dose okadaic acid, and by the reduction in invasion through these same matrix components by elevators of PP-2A activity. Motility of HNSCC cells through collagen I or fibronectin was not modulated by PP-2A. The reduction in HNSCC migration through vitronectin or laminin that resulted from treatment with PP-2A elevators was associated with an increase in cellular adhesiveness to these same ECM components. These studies show the association of PP-2A with the cellular cytoskeleton and its role in restricting the invasiveness of tumor cells through select extracellular matrix components.

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.

Protein kinase A regulates Lewis lung carcinoma adherence to extracellular matrix components and spontaneous metastasis

Tumor cell adhesion to and migration through the extracellular matrix (ECM) can influence their capacity to disseminate. Since prior studies with Lewis lung carcinoma (LLC) tumors had shown metastatic clones to have more protein kinase A (PKA) activity than nonmetastatic clones, the present study assessed if PKA regulates the interaction between tumor and the ECM, and how this may be associated with the metastatic capacity of the tumor cells. This was accomplished with the use of metastatic (LLC-LN7) and nonmetastatic (LLC-C8) variants that had been stably transfected to overexpress the PKA Ca subunit or to have blocked PKA activity. Cells with increased PKA activity were less adherent to vitronectin, laminin, and collagen I, and could more readily migrate through these ECM components than could transfectants with reduced PKA activity. PKA did not regulate adhesion to or migration through fibronectin, and did not appear to be associated with changes in expression of surface integrins. In addition to modulating tumor adhesion and migration in vitro, PKA activation caused an increased formation of metastases from s.c. tumors, but did not regulate formation of experimental metastases by i.v. injected tumor cells. These results suggest that PKA signaling is important for modulating the tumor-ECM interaction and can facilitate tumor transit from the primary tumor site.

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells.

Abstract Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D3 eliminated natural suppressor activity. In mice that were first treated with vitamin D3 and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D3/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D3/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D3 to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor.

Immune parameters of mice bearing human head and neck cancer.

A xenogeneic human head and neck squamous cell carcinoma (HNSCC) model in immunocompetent mice was evaluated for its requirement of cyclosporine for progressive tumor growth. Tumor growth and T cell functions were assessed in mice receiving cyclosporine treatment for various lengths of time. Tumor cells were injected s.c. on day 1 and cyclosporine was injected i.p. daily on days 1, 1–7, 1–14, 1–21, or for the entire 28 days of tumor growth. All mice developed tumors. These tumors were confirmed to be squamous carcinomas of human origin histologically and by positive staining for human MHC class I antigen expression. Tumors were largest in mice that received cyclosporine for days 1–21 or days 1–28. Increased tumor size was associated with increased serum levels of tumor-reactive antibodies, an increased intratumoral frequency of CD4+ and CD8+ cells, but a diminished production of interleukin-2 (IL-2) by the tumor infiltrate. Also correlating with increasing tumor size was splenomegaly, a decline in the frequency, but not the absolute levels, of splenic CD4+ and CD8+ cells, and a diminished capacity to proliferate in response to concanavalin A and to be stimulated to secrete IL-2. The HNSCC tumors contributed to the immune decline since T cell functions were more depressed in the tumor bearers than in control mice receiving only cyclosporine treatment. These results demonstrate that human HNSCC tumor xenografts can grow in mice even with limited cyclosporine treatment, and that the survival of these xenografts may, in part, be due to a tumor-induced decline in select T cell functions.

Bi-directional stimulation of adherence to extracellular matrix components by human head and neck squamous carcinoma cells and endothelial cells

Head and neck squamous cell carcinoma (HNSCC) cultures were established from two patients to determine if cancer and endothelial cells bi-directionally regulate their adherence to extracellular matrix components (ECM), an important process for tumor vascularization and metastasis. Soluble products from endothelial cells transiently enhanced adherence by HNSCC to ECM and increased surface levels of beta 1 and beta 4 integrins, although not beta 3. HNSCC products enhanced endothelial cell adherence to fibronectin and laminin, and beta 1 and beta 4 expression. These data show bi-directional enhancement of adherence to ECM and integrin expression among endothelial and tumor cells, which may facilitate metastasis and neovascularization.