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-beta (TGF-beta), 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-beta, 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-gamma) were diminished in cancers that released higher levels of TGF-beta, 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-gamma 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.

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte-macrophage colonystimulating factor (GM-CSF) have been shown to contain CD34 1 natural suppressor cells that inhibit the activity of intratumoral T-cells. The present study evaluated whether GM-CSF production and the presence of CD34 1 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 1 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 1 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 1 cell values was evaluated. These analyses showed that patients whose cancers produced high GM-CSF levels or had a high CD34 1 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 1 cell content remained disease-free (16% and 19%, respectively). Our results indicate that the presence of CD34 1 cells in GM-CSFproducing 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 1 cells in GM-CSF-producing HNSCC leads to increased tumor recurrence or metastasis. Int. J. Cancer 74:69-74.r 1997 Wiley-Liss, Inc.Head and neck cancers, most of which are squamous cell carcinomas (HNSCC), are vulnerable to immune effector cells such as macrophages, natural killer cells and cytotoxic T-lymphocytes (Chikamatsu et al., 1995;Rabinowich et al., 1992). However, patients with HNSCC cancers are particularly prone to have defects in their immune defenses (Gooding et al., 1995;Hadden et al., 1994). Part of this immune suppression has been shown to be directly induced by soluble immune suppressive factors produced by the cancer (O'Mahony et al., 1993). However, cancers can also mediate immune suppression indirectly by stimulating immune suppressor cell activity. The immune suppressor cells that may be induced by human HNSCC have not been extensively studied or described, but have been well documented in many other cancer types of both humans and animals. Some human cancers induce suppressive T-cells that are inhibitory to immune functions, including anti-tumor T-cell reactivities (Chakraborty et al., 1991). Suppression of T-cell function in patients with HNSCC as well as with other cancers can also be due to monocytes or macrophages secreting the immune suppressive mediator prostaglandin E 2...

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Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte-macrophage colonystimulating factor (GM-CSF) have been shown to contain CD34 1 natural suppressor cells that inhibit the activity of intratumoral T-cells. The present study evaluated whether GM-CSF production and the presence of CD34 1 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 1 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 1 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 1 cell values was evaluated. These analyses showed that patients whose cancers produced high GM-CSF levels or had a high CD34 1 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 1 cell content remained disease-free (16% and 19%, respectively). Our results indicate that the presence of CD34 1 cells in GM-CSFproducing 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 1 cells in GM-CSF-producing HNSCC leads to increased tumor recurrence or metastasis. Int. J. Cancer 74:69-74.r 1997 Wiley-Liss, Inc.Head and neck cancers, most of which are squamous cell carcinomas (HNSCC), are vulnerable to immune effector cells such as macrophages, natural killer cells and cytotoxic T-lymphocytes (Chikamatsu et al., 1995;Rabinowich et al., 1992). However, patients with HNSCC cancers are particularly prone to have defects in their immune defenses (Gooding et al., 1995;Hadden et al., 1994). Part of this immune suppression has been shown to be directly induced by soluble immune suppressive factors produced by the cancer (O'Mahony et al., 1993). However, cancers can also mediate immune suppression indirectly by stimulating immune suppressor cell activity. The immune suppressor cells that may be induced by human HNSCC have not been extensively studied or described, but have been well documented in many other cancer types of both humans and animals. Some human cancers induce suppressive T-cells that are inhibitory to immune functions, including anti-tumor T-cell reactivities (Chakraborty et al., 1991). Suppression of T-cell function in patients with HNSCC as well as with other cancers can also be due to monocytes or macrophages secreting the immune suppressive mediator prostaglandin E 2...

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colony-stimulating 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 1 alpha,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.

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Elevators of cAMP, such as prostaglandin E2 (PGE2), activate protein kinase A (PKA) and induce PKA-stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC-LN7). Non-metastatic LLC (LLC-C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP-1/2A) within non-metastatic LLC-C8, the effects of the PP-1/2A inhibitor okadaic acid on the migration and invasion by non-metastatic LLC-C8 cells was measured. Okadaic acid stimulated motility of non-metastatic LLC-C8 cells to a level that was comparable to that of metastatic LLC-LN7 cells. PGE2 further increased the motility of the non-metastatic LLC-C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose-response studies with concentrations of okadaic acid that selectively inhibited PP-2A or both PP-2A and PP-1 showed a progressive increase in migration of non-metastatic LLC-C8 cells, suggesting that both PP-1 and PP-2A limit their motility. By contrast, metastatic LLC-LN7 cells were more motile than were non-metastatic LLC-C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP-1/2A enzyme activities in the LLC variants showed more activity in non-metastatic LLC-C8 than in metastatic LLC-LN7 cells. The identity of the PP whose activity was increased in the non-metastatic LLC-C8 was assessed by using okadaic acid, which selectively inhibits PP-2A activity at low concentrations and PP-1 and PP-2A at high concentrations, and calyculin A, which inhibits PP-2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP-1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP-1 and of PP-2A in non-metastatic LLC-C8 cells, but the presence of PP-1 and a reduction in PP-2A in metastatic LLC-LN7 cells. The sum of these data suggests that PKA-stimulated motility is restricted both by PP-1 and by PP-2A in non-metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.

Metastatic Lewis lung carcinoma (LLC-LN7) cells have increased protein kinase A (PKA) activity and are more invasive in vitro than are non-metastatic (LLC-C8) cells. To determine whether PKA mediates the in vitro invasiveness and in vivo metastatic capabilities of these tumor cells, the LLC variants were stably transfected to over-express the C alpha subunit of PKA, and thus to have increased PKA activity, or to express a mutant cAMP-resistant PKA R1 alpha subunit which blocks PKA activation. Wild-type LLC-LN7 tumor cells were invasive in vitro and in vivo, recurred after tumor excision and metastasized to the lungs. However, they lost these properties after transfection to express the mutant R1 alpha that blocks PKA activation. The non-invasive, non-recurring and non-metastatic LLC-C8 cells gained the capacity to invade, to recur following tumor excision and to metastasize when transfected to express the PKA C alpha subunit.