Anemia of inflammation (AI) is associated with the immune response that occurs when inflammatory diseases interfere with the body's ability to absorb and use iron. Cancer and other chronic illnesses produce inflammatory cytokines, some of which can diminish the production of red blood cells by affecting erythropoietin levels. Cancer cells might also modify system iron homeostasis for their survival and proliferation. To date only a few studies have addressed the role of cancer in AI. For this reason we selected the tumor cell line B16−F10, which expresses the transferrin receptor−1 (TfR1), and whose growth can be modulated by iron−chelating agents. Cancer cell line B16−F10 also triggers a profound anemia as the tumor progresses. However, the rapid expansion of the tumor poorly represent human models and limits the analyses that can be done to evaluate the role of iron metabolism and inflammation in the etiology of the anemia. For this reason we modified the tumor cell line B16−F10 melanoma with an inducible suicide gene to prevent tumors from expanding too quickly. Using this model in comparison with controls, effects on inflammation and iron metabolism were assessed in tumorigenic mice at different time points.
B16−F10 cells were modified with an oncoretroviral vector that encodes an engineered fusion molecule that activates a proapoptotic molecule when used with a homodimerizer molecule (AP). B16−F10 cells with an inducible suicide gene were injected into C57BL/6 mice. After the appropriate tumor size was obtained the mice were injected with the inducer to control the growth of the tumor. Bone marrow and spleen cells from mice were analyzed by flow cytometry (FACS) to assess erythropoiesis. Serum was analyzed for iron content and transferrin saturation by spectroscopy and for markers of inflammation by cytokine microarray. Several organs were also collected for analysis of inflammation and iron− related gene expression.
Mice analyzed 20 to 28 days after injection of B16−F10 cells and without treatment exhibited large tumors, had lower hemoglobin (7.1±1.5 vs. 12.9±1.6 g/dL) and higher reticulocytes (15.28±3.9 vs. 2.13±0.26 × 105 cells/mL) compared to control mice (p<0.05). Analysis of inflammatory cytokines and FACS carried out using CD44, Ter119 and CD71 markers suggested that anemia was associated with inflammation and ineffective erythropoiesis both in the spleen and bone marrow (BM). Serum iron and Tf saturation levels were not significantly altered. Mice treated with 5 mg/kg of AP eliminated or shrunk the tumors to undetectable sizes and had similar hematological parameters as control mice. Tumorigenic mice treated with less than 3 mg/kg of AP reduced tumor growth but did not eradicate the tumor. These animals still exhibited anemia, abnormal erythropoiesis but had lower reticulocyte counts than untreated mice with similar tumor sizes analyzed at earlier time points.
As hemoglobin decreased in mice, apoptosis and ROS increased in erythroid populations in the BM and spleen. Inspection of BM and spleen indicated absence of metastasis. Altogether, these analyses suggested that tumor formation was indirectly responsible for the cell death of the erythroid cells, most likely by an inflammatory mechanism. Compared to normal mice, RT-qPCR analysis indicated that hepcidin, ferroportin, divalent metal transporter-1, and TfR1 were reduced in the liver of mice at day 14 and even more at day 28 during tumor development. In contrast, inflammatory markers including interleukin-6 (IL6) and IL6R were increased significantly in the serum or liver respectively, during the early phases of tumor development, but not at day 28 post-tumor injection. Altogether, these analyses suggest that an inflammatory mechanism affects erythropoiesis, but that the anemia has a dominant effect on iron metabolism, repressing hepcidin expression.
In conclusion, the ineffective erythropoiesis is likely responsible for the anemia observed during tumor progression mediated by B16−F10 melanoma cells. Both apoptosis and ROS of progenitor erythroid cells appear to contribute to the development of anemia, with IL6 and other inflammatory markers as potential players in this process. This study also successfully showed tumor growth can be controlled with an inducible suicide gene, which is useful for analyzing inflammation and iron metabolism effects in mice with more slowly progressing melanoma tumors.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.