The stromal cells of the tumor microenvironment play a critical role in tumor progression, and are therefore attractive targets for cancer therapy. We previously used the stromal targeting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), to demonstrate how tumor and stromal interactions can be manipulated to favor anti-tumor activity against human melanoma xenografts in immunodeficient mice, mediated by neutrophils and macrophages. An ideal stromal targeting agent would be effective against established metastases as well as primary tumors. In order to design such therapies, and to assess their potential, a better understanding of the tumor microenvironment at the various sites is required.
The aim of this study was to characterize the tumor stroma of established B16-F10 murine melanoma tumors at subcutaneous, pulmonary, and intracranial sites, in syngeneic C57BL/6 mice. Immunofluorescence-immunohistochemistry and flow cytometry were used to identify infiltrating leukocyte populations within B16-F10 tumors at these different sites. Furthermore, the antitumor activity of DMXAA against this model was investigated.
In subcutaneous tumors, monocytic myeloid cells (CD11b+ F4/80-/dim) and macrophages (CD11b+ F4/80+) were found predominantly around the tumor periphery. Lymphocyte infiltration was variable, with B-cells (CD19+ B220+) and T-cells (CD3+ CD4+ and CD3+ CD8+) rarely detected in some tumors, but scattered diffusely or formed dense lymphoid structures throughout necrotic regions in other tumors. Lung resident alveolar macrophages (CD11c+ CD11bdim F4/80+) appeared to be the most common leukocyte associated with metastatic foci, along with interstitial macrophages (CD11b+ F4/80+). In the presence of metastases, the total leukocyte abundance in the lungs was increased by nearly 70% (p < 0.05) compared to healthy lungs, owing largely to an increase in B-cells and T-cells scattered throughout the lung tissue; however these cells were not specifically associated with metastatic foci. In contrast, lymphocytes were scarcely detected in intracranial tumors or in brain tissue, where resident microglia (CD45dim CD11b+ CD11c+) were the most abundant. Microglia were found mainly at the invasive edge of intracranial tumors, while peripheral macrophages (CD45+ CD11b+ F4/80+) localised near vessels within the central core. Although DMXAA induced haemorrhagic necrosis in subcutaneous B16-F10 tumors, antitumor activity overall was modest, and no significant survival benefit was observed compared to untreated tumor-bearing mice.
Common at all sites was the presence of myeloid cells associated with tumor tissue, including resident and peripheral macrophages. DMXAA was recently shown to be an agonist for stimulator of interferon genes (STING) protein which triggers IFN-β signalling and innate immune gene expression in macrophages. DMXAA induces innate immunity against melanoma xenografts, where a similar presence of myeloid cells is observed before treatment. However, this activity was modest in aggressive B16-F10 melanomas. The precise reasons for this are unclear, but may involve immunosuppressive mechanisms. Our center is developing novel compounds to overcome immune-related resistance, such as inhibitors of indoleamine-2,3-dioxygenase, to be used with vaccines against melanoma. B16 metastatic melanoma is one challenging model that will be used to test these.
Citation Format: Kimiora Henare, Raymond Yung, P. Rod Dunbar, Cristin G. Print, Lai-Ming Ching. Characterizing the tumor stroma of B16 melanoma at different sites. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A19. doi:10.1158/1538-7445.CHTME14-A19