Keating, Patricia

Over 90% of cancer-related deaths are due to metastasis. Tumor-cell derived extracellular vesicles or exosomes are thought to contribute to metastasis. However, there is no standardized method to isolate exosomes. We hypothesize that polymer-based kits, in particular ExoQuick-TC (EQTC), may be better for isolating exosomes when compared to ultracentrifugation. In this study, we used three different methods to isolate exosomes from 4T1 murine mammary tumor cells. Samples isolated through ultracentrifugation (UC), EQTC, and Total Exosome Isolation Reagent (TEIR) were analyzed to assess quantity and quality of exosomes by nanoparticle tracking analysis (NTA) and flow cytometry (FC). Using NTA, we found that each method yielded samples with varying average concentration and particle size. FC analysis revealed UC to be the most effective method in yielding a high number of verified exosomes. Standardizing the isolation method and assessment will help in determining the role of exosomes in cancer metastasis.

It is estimated that one in eight women will be diagnosed with breast cancer. Developing an
understanding of the tumor microenvironment is critical for developing treatments for breast cancer
patients. It has been well established that hypoxia, or a lack of oxygen, is fundamental in creating a
microenvironment that enables metastasis via eliciting angiogenic processes. Poorly differentiated blood
vessels can fashion an oxygen-deprived microenvironment that triggers the expression of transcription
factor Hypoxia Inducible Factor alpha HIF-1alpha that in turn can up regulate genes mediating a protumor
effect. Our laboratory has discovered that mammary tumors express Semaphorin7a SEMA7A, a
HIF-1alpha inducible protein. This study’s objective is to delineate the mechanism for hypoxia induction
in mammary cells. Cobalt Chloride CoCl2 was used to mimic hypoxia in mammary tumor cell cultures.
Flow cytometry was used to determine HIF-1alpha activity in the mammary cells. Benign EpH4 mammary
cells expressing low SEMA7A greatly increased its expression after response to hypoxic stimuli as
determined by qPCR and SEMA7A-specific ELISA. Pretreatment of EpH4 cells with a HIF-1alpha
inhibitor Chemotin blocked induction of SEMA7A. Paradoxically, CoCl2 did not raise expression in the
highly metastatic 4T1 cells which experience high levels of SEMA7A. Treatment of 4T1 cells with
Chemotin under normoxic conditions inhibit HIF-1alpha activity and decrease SEMA7A levels.
Determining the role of SEMA7A in the hypoxia axis could further elucidate novel pathways in breast
cancer, suggesting that malignant tumor cells can utilize HIF-1alpha in a hypoxic independent manner.

Systemic lupus erythrematosus (SLE) is an autoimmune disease characterized
by the production of anti-DNA antibodies. The primary goal of this study was to
activate T cells with specificity toward lupus B cells presenting anti-DNA antibody
idiotopes on their surface. Monocyte derived dendritic cells were obtained from
peripheral blood of healthy donors and lupus patients. Affinity purified anti-DNA
antibodies were obtained from lupus patients' plasmas. The efficacy of different
carrier proteins, conjugated to lgG, was evaluated, and KLH found to be the most
efficient for antigen uptake. The cognate dendritic cells were evaluated for their
capacity to activate autologous T cells, and generate a Th1 mediated response which was evaluated by proliferation assays and interferony secretion. During the
vaccine study a patient presented with panniculitis, CREST syndrome and a
calcinotic exudate. A secondary goal of my study was to analyze this exudate.
Calcifying nanoparticles were isolated in lymphocyte culture medium. They were
characterized by Von Kassa staining for hydroxyapatite, solubilization by the
calcium chelating agent EDT A, and light and scanning electron microscopy. A
novel method was developed, using a specific monoclonal antibody, to analyze
the calcifying nanoparticles. This method allowed for an approximate
quantification of the particles. These particles increased in numbers when
incubated for different time periods, and their numbers were decreased when
incubated in the presence of minocyclin. Concomitantly, the panniculi in the
patient underwent remission with long term antibiotic therapy. CNPs were also
obtained from fetal bovine serum and human plasma samples from both lupus
patients and healthy donors. Peripheral blood mononuclear cells from healthy
donors and lupus patients were analyzed in vitro for their reactivity when
incubated in the presence of a biofilm, generated by the calcifying nanoparticles.
Viability, proliferation, and co-stimulatory marker up-regulation were determined
in the presence or absence of the particles. Osteopontin was found highly
expressed in the supernatants of the cells grown with CNPs. Microarray of the
mononuclear cells of a healthy donor and a lupus patient incubated in the
presence or absence of CNPs was performed, and the results coincided with
those determined in vitro.