Activation of neuroimmune pathways increases therapeutic effects of radiotherapy on poorly differentiated breast carcinoma.

Recent studies document the importance of neuronal dysfunction in cancer development and metastasis. We reported previously that both depletion of neuropeptides in capsaicin-sensitive sensory nerve endings and vagotomy increases metastasis of triple negative breast carcinoma. Of the sensory neuropeptides, Substance P (SP) is distributed widely for regulation of immune functions. We therefore examined the affects of continuous exposure to low doses of SP on brain metastatic cells of the mouse breast carcinoma (4TBM) in the presence of radiotherapy (RT) thought to increase antigenicity of cancer cells. 4TBM cells have a cancer stem cell phenotype and induce extensive visceral metastasis after orthotopic inoculation into the mammary pad. Results demonstrated that SP treatment decreases the number of tumor-infiltrating myeloid-derived suppressor cells as well as the TNF-α response to LPS challenge. SP also increased CD4+Cd25(bright) cells in draining lymph nodes of tumor-bearing animals and IFN-γ secretion from leukocyte culture prepared from lymph nodes and spleens of tumor-bearing animals. SP also prevented tumor-induced degeneration of sensory nerve endings and altered release of angiogenic factors from cancer-associated fibroblasts (CAF) and tumor explants. In accordance with these observed immunological effects, combination treatment of continuous SP with a single dose of RT induced complete tumor regression and significantly reduced or prevented metastasis in 50% of the animals while suppressing primary tumor growth and metastasis in the remaining mice. These original findings demonstrate that SP through neuroimmune modulation can prevent formation of immune suppression in the tumor microenvironment, enhance cytotoxic immunity in the presence of RT and prevent metastatic growth.

Differential characteristics of heart, liver, and brain metastatic subsets of murine breast carcinoma.

Breast carcinoma is comprised of heterogeneous groups of cells with different metastatic potential. To develop effective therapeutic strategies targeting metastatic disease, it is crucial to understand the characteristics of breast cancer cells that enable metastasis to distant organs. 4THM breast carcinoma cells are the cells of 4T1 primary tumors that metastasized to the heart. Cells of 4THM tumors which metastasized to liver (4TLM) were previously isolated. Recently macroscopic brain metastasis in 4THM injected animals, were isolated to obtain a brain metastatic cell line (4TBM). Using an orthotopic mouse model differential characteristic of cells metastasized to heart (4THM), liver (4TLM), and brain (4TBM) were compared for ability to metastasize and expression of stem cell markers. We found that 4TLM cells produced significantly more lung and liver metastasis compared to 4TBM and 4THM cells. In vitro, proliferation as well as migration rate of 4TLM cells was also significantly higher than the other cell lines. Remarkably primary tumors formed by 4TLM cells expressed significant amounts of CD34, a marker for mesenchymal malignancies. Markers of epithelial-mesenchymal transition were expressed in all metastatic cells, but the degree of expression differed. Majorities of 4TLM, 4THM, and 4TBM cells were CD44+ CD24- whereas, 12 % of 4TLM cells also expressed membranous CD24. Conditioned mediums of non-metastatic 67NR breast tumors and cancer-associated fibroblasts inhibited growth of highly metastatic 4TLM cells. Malignant cells metastasized to brain were distinguished by membranous E-cadherin expression that was markedly higher in 4TBM cells grown as spheroids suggesting E-cadherin is required for brain metastasis. Differential features of heart, brain, and liver metastatic cells in a syngenic model was shown in this study for the first time. These findings not only provide a model to explore new treatment modalities, but also demonstrate differential features of cancer cells that originally homed to a certain organ, such as liver or brain.

HSP90 inhibitor PU-H71 increases radiosensitivity of breast cancer cells metastasized to visceral organs and alters the levels of inflammatory mediators.

Heat shock protein 90 (HSP90) inhibitors are considered as new radiosensitizing agents. PU-H71, a novel HSP90 inhibitor, is under evaluation for the treatment of advanced cancer. It is however not known whether PU-H71 alters radiosensitivity of metastatic breast cancer. Hence, we here evaluated mechanisms of possible anti-tumoral and radiosensitizing effects of PU-H71 on breast carcinoma cells metastasized to vital organs such as the liver and brain. The effect of PU-H71 on proliferation of breast carcinoma cells was determined using 4T1 cells and its brain (4TBM), liver (4TLM), and heart (4THM) metastatic subsets as well as non-metastatic 67NR cells. Changes in radiation sensitivity were determined by clonogenic assays. Changes in client proteins and levels of angiogenic and inflammatory mediators from these cancer cell cultures and ex vivo cultures were detected. PU-H71 alone inhibited ERK1/2, p38, and Akt activation and reduced N-cadherin and HER2 which further documented the anti-tumoral effects of PU-H71. The combination of PU-H71 and radiotherapy induced cytotoxic effect than PU-H71 alone, and PU-H71 showed a radiosensitizing effect in vitro. On the other hand, PU-H71 and radiation co-treatment increased p38 phosphorylation which is one of the hallmarks of inflammatory response. Accordingly, IL-6 secretion was increased following PU-H71 and radiotherapy co-treatment ex vivo. Levels of angiogenic and inflammatory factors such as MIP-2, SDF-1, and VEGF were increased under in vitro conditions but not under ex vivo conditions. These results demonstrated for the first time that PU-H71 enhances therapeutic effects of radiotherapy especially in highly metastatic breast carcinoma but a possible increase in inflammatory response should also be considered.

Metastatic breast carcinoma induces vascular endothelial dysfunction in Balb-c mice: Role of the tumor necrosis factor-α and NADPH oxidase.

Although the oxidative stress and inflammation are closely related with breast cancer, there is no study directly examining the possible changes in vascular functions in the presence of breast carcinoma. The goal of the present study was to evaluate changes in vascular reactivity in tumor-bearing mice. In this study, highly metastatic breast carcinoma cells which were derived from liver or brain metastasis of 4T1 murine breast carcinoma (4TLM and 4TBM, respectively), and 67NR cells which were tumorigenic but non-metastatic cells were used. Female Balb-c mice 8-10weeks old were divided into following groups: (1) control, (2) injected with 67NR, (3) injected with 4TLM, and (4) injected with 4TBM orthotopically. Thoracic aorta was removed 23-25days after injection of tumor cells. Isometric tension studies were performed in response to potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh, an endothelium-dependent vasodilator), and sodium nitroprusside (SNP, an endothelium-independent vasodilator). Endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser 1177) (p-eNOS), gp91(phox), and tumor necrosis factor-α (TNF-α) expressions in aortic tissues were demonstrated by immunohistochemistry. The level of TNF-α in vascular tissue was measured by ELISA. The presence of tumor was resulted in significant inhibition of response to ACh in both 4TLM and 4TBM injected mice, but not 67NR injected mice. Furthermore, both KCl and Phe-induced contraction of thoracic aorta was not changed significantly in tumor-bearing animals. eNOS and p-eNOS expressions decreased while gp91(phox) and TNF-α expressions increased in endothelium of 4TLM and 4TBM mice compared to 67NR injected and control mice. Moreover, TNF-α levels of thoracic aorta in mice with metastatic breast carcinoma were significantly higher than that of 67NR mice. Tumor-induced endothelial dysfunction determined by ACh-induced relaxation improved by superoxide dismutase (SOD), apocynin (a NADPH oxidase inhibitor), and infliximab (a TNF-α monoclonal antibody). The findings of this study suggest that the presence of metastatic breast carcinoma may cause a significant reduction in endothelium-dependent relaxation of thoracic aorta via NADPH oxidase-mediated oxidative stress and TNF-α production.