Breast Carcinoma Metastatic in the Brain

The TWEAK receptor, Fn14, is a promising candidate for active targeting of cancer nanotherapeutics to many solid tumor types, including metastatic breast and primary brain cancers. Targeting of therapeutic nanoparticles (NPs) has been accomplished using a range of targeting moieties including monoclonal antibodies and related fragments, peptides, and small molecules. Here, we investigated a full-length Fn14-specific monoclonal antibody, ITEM4, or an ITEM4-Fab fragment as a targeting moiety to guide the development of a clinical formulation. We formulated NPs with varying densities of the targeting moieties while maintaining the decreased nonspecific adhesivity with receptor targeting (DART) characteristics. To model the conditions that NPs experience following intravenous infusion, we investigated the impact of serum exposure in relation to the targeting moiety type and surface density. To further evaluate performance at the cancer cell level, we performed experiments to assess differences in cellular uptake and trafficking in several cancer cell lines using confocal microscopy, imaging flow cytometry, and total internal reflection fluorescence microscopy. We observed that Fn14-targeted NPs exhibit enhanced cellular uptake in Fn14-high compared to Fn14-low cancer cells and that in both cell lines uptake levels were greater than observed with control, nontargeted NPs. We found that serum exposure increased Fn14-targeted NP specificity while simultaneously reducing the total NP uptake. Importantly, serum exposure caused a larger reduction in cancer cell uptake over time when the targeting moiety was an antibody fragment (Fab region of the monoclonal antibody) compared with the full-length monoclonal antibody targeting moiety. Lastly, we uncovered that full monoclonal antibody-targeted NPs enter cancer cells via clathrin-mediated endocytosis and traffic through the endolysosomal pathway. Taken together, these results support a pathway for developing a clinical formulation using a full-length Fn14 monoclonal antibody as the targeting moiety for a DART cancer nanotherapeutic agent.

Inactivation of sensory neurons expressing transient receptor potential vanilloid 1 (TRPV1) enhances breast cancer metastasis. Sensory neurons have profound effects on immune response to a wide range of diseases including cancer. Hence, activation of sensory nerves using feasible approaches such as specific TRPV1 agonists may inhibit breast cancer metastasis through neuroimmune pathways. TRPV1 agonists are considered for the treatment of pain and inflammatory diseases.

We here first determined the effects of four different TRPV1 agonists on proliferation of three different metastatic breast carcinoma cells since TRPV1 is also expressed in cancer cells. Based on the results obtained under in-vitro conditions, brain metastatic breast carcinoma cells (4TBM) implanted orthotopically into the mammary-pad of Balb-c mice followed by olvanil treatment (i.p.). Changes in tumor growth, metastasis and immune response to cancer cells were determined.

Olvanil dose-dependently activated sensory nerve fibers and markedly suppressed lung and liver metastasis without altering the growth of primary tumors. Olvanil (5 mg/kg) systemically increased T cell count, enhanced intra-tumoral recruitment of CD8+ T cells and increased IFN-γ response to irradiated cancer cells and Con-A. Anti-inflammatory changes such as increased IL-10 and decrease IL-6 as well as S100A8+ cells were observed following olvanil treatment.

Our results show that anti-metastatic effects of olvanil is mainly due to activation of neuro-immune pathways since olvanil dose used here is not high enough to directly activate immune cells. Furthermore, olvanil effectively depletes sensory neuropeptides; hence, olvanil is a good non-pungent alternative to capsaicin.

Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant nonmalignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. Here we demonstrate that targeting TAMs at distinct stages of the metastatic cascade using an inhibitor of colony-stimulating factor 1 receptor (CSF1R), BLZ945, in murine breast-to-brain metastasis models leads to antitumor responses in prevention and intervention preclinical trials. However, in established brain metastases, compensatory CSF2Rb-STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuroinflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, blockade of CSF1R combined with inhibition of STAT5 signaling via AC4-130 led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage.