Navigating the Blood-Brain Barrier: Challenges and Therapeutic Strategies in Breast Cancer Brain Metastases.

Breast cancer (BC) is the most common cancer in women, with metastatic BC being responsible for the highest number of deaths. A frequent site for BC metastasis is the brain. Brain metastasis derived from BC involves the cooperation of multiple genetic, epigenetic, angiogenic, and tumor-stroma interactions. Most of these interactions provide a unique opportunity for development of new therapeutic targets. Potentially targetable signaling pathways are Notch, Wnt, and the epidermal growth factor receptors signaling pathways, all of which are linked to driving BC brain metastasis (BCBM). However, a major challenge in treating brain metastasis remains the blood-brain barrier (BBB). This barrier restricts the access of unwanted molecules, cells, and targeted therapies to the brain parenchyma. Moreover, current therapies to treat brain metastases, such as stereotactic radiosurgery and whole-brain radiotherapy, have limited efficacy. Promising new drugs like phosphatase and kinase modulators, as well as BBB disruptors and immunotherapeutic strategies, have shown the potential to ease the disease in preclinical studies, but remain limited by multiple resistance mechanisms. This review summarizes some of the current understanding of the mechanisms involved in BC brain metastasis and highlights current challenges as well as opportunities in strategic designs of potentially successful future therapies.

Generation of prolactin-inducible protein (Pip) knockout mice by CRISPR/Cas9-mediated gene engineering.

Prolactin-inducible protein (PIP) is a multifunctional glycoprotein that is highly expressed and found in the secretions of apocrine glands such as salivary, lacrimal, and sweat glands including the mammary glands. PIP has been implicated in various diseases, including breast cancer, gross cystic disease of the breast, keratoconus of the eye, and the autoimmune Sjögren's syndrome. Here we have generated a Pip knockout (KO) mouse using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRSPR-associated (Cas)9 system. The Cas9 protein and two single guide RNAs targeting specific regions for both exons 1 and 2 of the Pip gene were microinjected into mouse embryos. The deletions and insertions promoted by CRISPR/Cas9 system on the Pip gene successfully disrupted Pip protein coding, as confirmed by PCR genotyping, sequencing, and ultimately Western blot analysis. This mouse model was generated in the inbred C57Bl/6J mouse, which exhibits lower genetic variation. This novel CRISPR Pip KO mouse model will not only be useful for future studies to interrogate the multifunctional role of PIP in physiological processes but will facilitate a broader understanding of the function of PIP in vivo while providing unprecedented insight into its role in a spectrum of diseases attributed to the deregulation of the PIP gene.

Comparison of Fixation Methods for the Detection of Claudin 1 and E-Cadherin in Breast Cancer Cell Lines by Immunofluorescence.

The tight junction membrane protein claudin 1 and the adherens junction protein E-cadherin play critical roles in cell-cell communication and in cell signaling. As a result, their protein levels and distribution in cancer have been a focus of cancer researchers in recent years. The loss of sensitivity to contact inhibition and the establishment of invasive properties in cancer are thought to be a result of the mislocalization of these membrane proteins to the cytoplasm. However, reports on their distribution and levels have been inconsistent. It is therefore critical that the techniques used to determine the cellular localization of these proteins be both consistent and reliable. This study was undertaken to determine the optimal fixation method, methanol or formalin, for the detection of claudin 1 and E-cadherin by immunofluorescence in five different human breast cancer cell lines. Both methods exhibited staining of the cell membrane and cytoplasm, but the strongest and most distinct signals were obtained using methanol fixation. Interestingly, cell-specific differences were also observed that appeared to be associated with levels of claudin 1 and E-cadherin as seen by Western blotting. Therefore, when evaluating cellular localization of the junction proteins claudin 1 and E-cadherin, expression level and cell type differences must be considered.

The Breast Tumor Microenvironment: A Key Player in Metastatic Spread.

The tumor microenvironment plays a pivotal role in the tumorigenesis, progression, and metastatic spread of many cancers including breast. There is now increasing evidence to support the observations that a bidirectional interplay between breast cancer cells and stromal cells exists within the tumor and the tumor microenvironment both at the primary tumor site and at the metastatic site. This interaction occurs through direct cell to cell contact, or by the release of autocrine or paracrine factors which can activate pro-tumor signaling pathways and modulate tumor behavior. In this review, we will highlight recent advances in our current knowledge about the multiple interactions between breast cancer cells and neighboring cells (fibroblasts, endothelial cells, adipocytes, innate and adaptive immune cells) in the tumor microenvironment that coordinate to regulate metastasis. We also highlight the role of exosomes and circulating tumor cells in facilitating breast cancer metastasis. We discuss some key markers associated with stromal cells in the breast tumor environment and their potential to predict patient survival and guide treatment. Finally, we will provide some brief perspectives on how current technologies may lead to the development of more effective therapies for the clinical management of breast cancer patients.

The Prolactin Inducible Protein Modulates Antitumor Immune Responses and Metastasis in a Mouse Model of Triple Negative Breast Cancer.

The prolactin inducible protein (PIP) is expressed to varying degrees in more than 90% of breast cancers (BCs). Although high levels of PIP expression in BC has been shown to correlate with better prognosis and patient response to chemotherapy, some studies suggest that PIP may also play a role in metastasis. Here, we investigated the role of PIP in BC using the well-established 4T1 and E0771 mouse BC cell lines. Stable expression of PIP in both cell lines did not significantly alter their proliferation, migration, and response to anticancer drugs in vitro compared to empty vector control. To assess the effect of PIP expression on breast tumorigenesis in vivo, the 4T1 syngeneic transplantable mouse model was utilized. In immunocompetent syngeneic BALB/c mice, PIP-expressing 4T1 primary tumors displayed delayed tumor onset and reduced tumor growth, and this was associated with higher percentages of natural killer cells and reduced percentages of type 2 T-helper cells in the tumor environment. The delayed tumor onset and growth were abrogated in immunodeficient mice, suggesting that PIP-mediated modulation of primary tumor growth involves an intact immune system. Paradoxically, we also observed that PIP expression was associated with a higher number of 4T1 colonies in the lungs in both the immunocompetent and immunodeficient mice. Gene expression analysis of PIP-expressing 4T1 cells (4T1-PIP) revealed that genes associated with tumor metastasis such as CCL7, MMP3 and MMP13, were significantly upregulated in 4T1-PIP cells when compared to the empty vector control (4T1-EV) cells. Collectively, these studies strongly suggest that PIP may possess a double-edge sword effect in BC, enhancing both antitumor immunity as well as metastasis.

Semaphorin 3E Promotes Susceptibility to Leishmania major Infection in Mice by Suppressing CD4+ Th1 Cell Response.

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ-secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05-0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Isolation and Preparation of Bone Marrow-Derived Immune Cells for Metabolic Analysis.

The isolation of immune cells from the bone marrow is important for obtaining sufficient numbers for downstream analysis. Immune cells derived from the bone marrow may be subjected to metabolic assays for analysis or used to test the effect of infectious agents on immune cells. Here, we describe a process for the isolation of macrophages, dendritic cells, and neutrophils from mice. Using the methods described herein, specific immune cells with purity above 85-90% can be obtained from the bone marrow of mice.

Leishmania Immunity: Advancing Immunotherapy and Vaccine Development.

Parasitic diseases still constitute a major global health problem affecting billions of people around the world. These diseases are capable of becoming chronic and result in high morbidity and mortality. Worldwide, millions of people die each year from parasitic diseases, with the bulk of those deaths resulting from parasitic protozoan infections. Leishmaniasis, which is a disease caused by over 20 species of the protozoan parasite belonging to the genus Leishmania, is an important neglected disease. According to the World Health Organization (WHO), an estimated 12 million people are currently infected in about 98 countries and about 2 million new cases occur yearly, resulting in about 50,000 deaths each year. Current treatment methods for leishmaniasis are not very effective and often have significant side effects. In this review, we discussed host immunity to leishmaniasis, various treatment options currently being utilized, and the progress of both immunotherapy and vaccine development strategies used so far in leishmaniasis. We concluded with insights into what the future holds toward the fight against this debilitating parasitic disease.

The prolactin inducible protein/gross cystic disease fluid protein-15 deficient mice develop anomalies in lymphoid organs.

The Prolactin Inducible Protein (PIP) is a 15 kDa protein secreted by normal apocrine glands, including salivary, lacrimal and sweat glands. PIP levels are normally low in the mammary glands of healthy individuals, but high levels have been observed in pathological conditions of the breast such as benign breast cystic disease and breast cancer. While the function of PIP is not well elucidated, accumulating evidence strongly point to a role in both innate and adaptive immunity. Using PIP deficient mice (Pip-/- mice) our laboratory demonstrated that loss of PIP function led to impaired T helper type 1 response and cell mediated immunity. In the present study we provide additional supporting evidence showing abnormal lymphocytic distribution in primary and secondary lymphoid organs of Pip-/- mice. Significant morphological changes in the Eustachian tube, an immune-protected site where PIP is normally found, were also associated with the absence of PIP. Collectively, these results further support an immuno-regulatory role for PIP and have implications for a spectrum of immune-related illnesses including otitis media and hearing loss as well as breast cancer.

Regulation of Immunity in Breast Cancer.

Breast cancer affects millions of women worldwide, leading to many deaths and significant economic burden. Although there are numerous treatment options available, the huge potentials of immunotherapy in the management of localized and metastatic breast cancer is currently being explored. However, there are significant gaps in understanding the complex interactions between the immune system and breast cancer. The immune system can be pro-tumorigenic and anti-tumorigenic depending on the cells involved and the conditions of the tumor microenvironment. In this review, we discuss current knowledge of breast cancer, including treatment options. We also give a brief overview of the immune system and comprehensively highlight the roles of different cells of the immune system in breast tumorigenesis, including recent research discoveries. Lastly, we discuss some immunotherapeutic strategies for the management of breast cancer.