Macrophages From Irradiated Tumors Express Higher Levels of iNOS, Arginase-I and COX-2, and Promote Tumor Growth

Purpose: To investigate the effects of single and fractionated doses of radiation on tumors and tumor-associated macrophages (TAMs), and to elucidate the potential of TAMs to influence tumor growth.

Methods and Materials: A murine prostate cell line, TRAMP-C1, was grown in C57Bl/6J mice to 4-mm tumor diameter and irradiated with either 25 Gy in a single dose, or 60 Gy in 15 fractions. The tumors were removed at the indicated times and assessed for a variety of markers related to TAM content, activation status, and function.

Results: In tumors receiving a single radiation dose, arginase (Arg-I), and cycloxygenase-2 (COX-2) mRNA expression increased as a small transient wave within 24 h and a larger persistent wave starting after 3 days. Inducible nitric oxide synthase (iNOS) mRNA was elevated only after 3 days and continued to increase up to 3 weeks. After fractionated irradiation, Arg-1 and COX-2 mRNA levels increased within 5 days, whereas iNOS was increased only after 10 fractions of irradiation had been given. Increased levels of Arg-I, COX-2, and, to a lesser extent, iNOS protein were found to associate with TAMs 1–2 weeks after tumor irradiation. Function of TAMs were compared by mixing them with TRAMP-C1 cells and injecting them into mice; TRAMP-C1 cells mixed with TAMs from irradiated tumors appeared earlier and grew significantly faster than those mixed with TAMs from unirradiated tumors or TRAMP-C1 alone.

Conclusions: Tumor-associated macrophages in the postirradiated tumor microenvironment express higher levels of Arg-1, COX-2, and iNOS, and promote early tumor growth in vivo.

Introduction

The microenvironment of normal tissues is altered in response to irradiation so as to be conducive to a healing response. Part of this involves cytokine production leading to recruitment and activation of immune cells (1). Phagocytes and other immune cells infiltrate the irradiated site (2, 3, 4). This response is initiated within hours of exposure, but recurs in a cyclical pattern days to months later (3, 5, 6, 7). For example, irradiation of lungs in mice leads to waves of expression of chemokines (8) and pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-1, and infiltration by polymorphonuclear leukocytes, lymphocytes, and macrophages that express markers, indicating their influx from the circulation (2, 4). Although this pattern of response has been observed in several normal tissues, much less is known about how the tumor microenvironment responds to irradiation, in particular with immune cell infiltration, despite its obvious relevance to therapeutic outcome of cancer treatment.

It has been known for many years that tumor-associated macrophages (TAMs) are a major cellular component of human (9) and murine cancers (10, 11), yet there is still no consensus as to their role in cancer growth and metastasis. This lack of consensus can be reconciled by the hypothesis that they have a double-edged role depending upon whether they have a type 1 (M1) or type 2 (M2) phenotype; the former are able to kill tumor cells, present antigen, and produce immune-stimulatory cytokines, whereas the latter promote angiogenesis, tumor growth, and metastasis, and suppress T-cell function (see review in (12, 13)). The content, phenotype, and function of TAMs are defined by the microenvironment and remain constant over most of the growth period of experimental tumors, if left unperturbed (14). Most TAMs in most tumors express a M2 phenotype, unless manipulated (15).

Reports on the effects of therapy, especially radiation therapy, on TAM number, phenotype and function are rare. Experimental studies indicate that macrophages are relatively radioresistant and that the balance of cell types after noncurative irradiation is initially disturbed, but reestablishes itself with time (16). The time after irradiation when the balance of cell types within the tumor is most perturbed may be the optimal one for therapeutic intervention aimed at boosting tumor immunity, because the tumor burden will be less, radiation may provide some “danger” signaling enhancing antigen presentation, and the balance of cytokines and the redox status may be more favorable for immune cell activation and expression of effector function.

Several pathways can influence the phenotype and function of macrophages, but one that can have a major impact is driven by L-arginine metabolism. L-arginine is metabolized by nitric oxide synthases (NOSs) to form L-citrulline and nitric oxide (NO), which have a variety of biologic effects and in general promote inflammatory and immune reactions (17, 18). However, in contrast, tumor growth can be promoted by depletion of L-arginine by arginase produced by TAMs. This can be mediated by enhanced production of L-ornithine and polyamine, negation of NO-mediated tumor cytotoxicity (19), and appearance of TAMs that suppress T-cell function (20, 21, 22). In this study, we examine the effect of in vivo irradiation on TAM function in murine prostate tumors with particular reference to the arginase pathway. COX-2 expression was also examined because it is linked to control of inflammation, is elevated in prostate cancers, and is associated with increased proliferation, invasiveness, and angiogenesis (23, 24). The hypothesis was that irradiation might affect TAM phenotype and function and this could contribute to or inhibit accelerated tumor growth and repopulation during and after radiation therapy. Our results indicate that the irradiated tumor microenvironment favors the development of a macrophage phenotype that favors tumor growth and indicates the need for further studies aimed at targeting this cell population for improved radiotherapeutic benefit in cancer.