Cyclooxygenase-2 (COX-2) is a critically important mediator of inflammation that significantly influences tumor angiogenesis, invasion, and metastasis. We investigated the role of COX-2 expressed by triple negative breast cancer cells in altering the structure and function of the extracellular matrix (ECM). COX-2 downregulation effects on ECM structure and function were investigated using magnetic resonance imaging (MRI) and second harmonic generation (SHG) microscopy of tumors derived from triple negative MDA-MB-231 breast cancer cells, and a derived clone stably expressing a short hairpin (shRNA) molecule downregulating COX-2. MRI of albumin-GdDTPA was used to characterize macromolecular fluid transport in vivo and SHG microscopy was used to quantify collagen 1 (Col1) fiber morphology. COX-2 downregulation decreased Col1 fiber density and altered macromolecular fluid transport. Immunohistochemistry identified significantly fewer activated cancer associated fibroblasts (CAFs) in low COX-2 expressing tumors. Metastatic lung nodules established by COX-2 downregulated cells were infrequent, smaller, and contained fewer Col1 fibers.COX-2 overexpression studies were performed with tumors derived from triple negative SUM-149 breast cancer cells lentivirally transduced to overexpress COX-2. SHG microscopy identified significantly higher Col1 fiber density in COX-2 overexpressing tumors with an increase of CAFs. These data expand upon the roles of COX-2 in shaping the structure and function of the ECM in primary and metastatic tumors, and identify the potential role of COX-2 in modifying the number of CAFs in tumors that may have contributed to the altered ECM.

Cancer cells adapt to hypoxia by the stabilization of hypoxia inducible factor (HIF)-α isoforms that increase the transcription of several genes. Among the genes regulated by HIF are enzymes that play a role in invasion, metastasis and metabolism. We engineered triple (estrogen receptor/progesterone receptor/HER2/neu) negative, invasive MDA-MB-231 and SUM149 human breast cancer cells to silence the expression of HIF-1α, HIF-2α or both isoforms of HIF-α. We determined the metabolic consequences of HIF silencing and the ability of HIF-α silenced cells to invade and degrade the extracellular matrix (ECM) under carefully controlled normoxic and hypoxic conditions. We found that silencing HIF-1α alone was not sufficient to attenuate invasiveness in both MDA-MB-231 and SUM149 cell lines. Significantly reduced metastatic burden was observed in single (HIF-1α or HIF-2α) and double α-isoform silenced cells, with the reduction most evident when both HIF-1α and HIF-2α were silenced in MDA-MB-231 cells. HIF-2α played a major role in altering cell metabolism. Lipids and lipid droplets were significantly reduced in HIF-2α and double silenced MDA-MB-231 and SUM149 cells, implicating HIF in their regulation. In addition, lactate production and glucose consumption were reduced. These results suggest that in vivo, cells in or near hypoxic regions are likely to be more invasive. The data indicate that targeting HIF-1α alone is not sufficient to attenuate invasiveness, and that both HIF-1α and HIF-2α play a role in the metastatic cascade in these two cell lines.

High levels of total choline and phosphocholine (PC) are consistently observed in aggressive cancers. Choline kinase (Chk) catalyzes choline phosphorylation to produce PC in phosphatidylcholine (PtdCho) biosynthesis. PtdCho is the most abundant phospholipid in eukaryotic cell membranes and plays a dual role as the structural component of membranes and as a substrate to produce lipid second messengers such as phosphatidic acid and diacylglycerol. Chk-α, but not Chk-β, is overexpressed in various cancers, and is closely associated with tumor progression and invasiveness. We have previously shown that downregulation of mRNA using small interfering RNA (siRNA) against Chk-α (siRNA-Chk) or Chk short hairpin RNA, and the resultant decrease of Chk-α protein levels, significantly reduced proliferation in breast cancer cells and tumors. A novel potent and selective small-molecule Chk-α inhibitor, V-11-0711, that inhibits the catalytic activity of Chk has recently been developed. Here, we used triple negative MDA-MB-231 and SUM149 breast cancer cells to further investigate the role of Chk-α in cancer, by examining Chk-α protein levels, cell viability/proliferation, choline phospholipid and lipid metabolism, lipid droplet formation, and apoptosis, following treatment with V-11-0711. Under the conditions used in this study, treatment with V-11-0711 significantly decreased PC levels but did not reduce cell viability as long as Chk-α protein and PtdCho levels were not reduced, suggesting that Chk-α protein and PtdCho, but not PC, may be crucial for breast cancer cell survival. These data also support the approach of antitumor strategies that destabilize Chk-α protein or downregulate PtdCho in breast cancer treatment.

We report the development of a two-color Fourier domain Pump-Probe Optical Coherence Tomography (PPOCT) system. Tissue phantom experiments to characterize the system performance demonstrated imaging depths in excess of 725 μm, nearly comparable to the base Optical Coherence Tomography system. PPOCT A-line rates were also demonstrated in excess of 1 kHz. The physical origin of the PPOCT signal was investigated with a series of experiments which revealed that the signal is a mixture of short and long lifetime component signals. The short lifetime component was attributed to transient absorption while the long lifetime component may be due to a mixture of transient absorption and thermal effects. Ex vivo images of porcine iris demonstrated the potential for imaging melanin in the eye, where cancer of the melanocytes is the most common form of eye cancer in adults.

Collagen 1 (Col1) fibers play an important role in tumor interstitial macromolecular transport and cancer cell dissemination. Our goal was to understand the influence of Col1 fibers on water diffusion, and to examine the potential of using noninvasive diffusion tensor imaging (DTI) to indirectly detect Col1 fibers in breast lesions. We previously observed, in human MDA-MB-231 breast cancer xenografts engineered to fluoresce under hypoxia, relatively low amounts of Col1 fibers in fluorescent hypoxic regions. These xenograft tumors together with human breast cancer samples were used here to investigate the relationship between Col1 fibers, water diffusion and anisotropy, and hypoxia. Hypoxic low Col1 fiber containing regions showed decreased apparent diffusion coefficient (ADC) and fractional anisotropy (FA) compared to normoxic high Col1 fiber containing regions. Necrotic high Col1 fiber containing regions showed increased ADC with decreased FA values compared to normoxic viable high Col1 fiber regions that had increased ADC with increased FA values. A good agreement of ADC and FA patterns was observed between in vivo and ex vivo images. In human breast cancer specimens, ADC and FA decreased in low Col1 containing regions. Our data suggest that a decrease in ADC and FA values observed within a lesion could predict hypoxia, and a pattern of high ADC with low FA values could predict necrosis. Collectively the data identify the role of Col1 fibers in directed water movement and support expanding the evaluation of DTI parameters as surrogates for Col1 fiber patterns associated with specific tumor microenvironments as companion diagnostics and for staging.