Grady E. Carlson

Simple modifications to methimazole that enhance its inhibitory effect on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression by human endothelial cells

The expression of vascular cell adhesion molecule-1 (VCAM-1) on the vascular endothelium can be increased by pro-inflammatory cytokines [e.g. tumor necrosis factor-α (TNF-α)]. VCAM-1 contributes to leukocyte adhesion to, and emigration from, the vasculature which is a key aspect of pathological inflammation. As such, a promising therapeutic approach for pathological inflammation is to inhibit the expression of VCAM-1. Methimazole [3-methyl-1, 3 imidazole-2 thione (MMI)] is routinely used for the treatment of Graves׳ disease and patients treated with MMI have decreased levels of circulating VCAM-1. In this study we used cultured human umbilical vein endothelial cells (HUVEC) to investigate the effect of MMI structural modifications on TNF-α induced VCAM-1 expression. We found that addition of a phenyl ring at the 4-nitrogen of MMI yields a compound that is significantly more potent than MMI at inhibiting 24h TNF-α-induced VCAM-1 protein expression. Addition of a para methoxy to the appended phenyl group increases the inhibition while substitution of a thiazole ring for an imidazole ring in the phenyl derivatives yields no clear difference in inhibition. Addition of the phenyl ring to MMI appears to increase toxicity as does substitution of a thiazole ring for an imidazole ring in the phenyl MMI derivatives. Each of the compounds reduced TNF-α-induced VCAM-1 mRNA expression and had a functional inhibitory effect, i.e. each inhibited monocytic cell adhesion to 24h TNF-α-activated HUVEC under fluid flow conditions. Combined, these studies provide important insights into the design of MMI-related anti-inflammatory compounds.

Dynamic biochemical tissue analysis detects functional L-selectin ligands on colon cancer tissues

A growing body of evidence suggests that L-selectin ligands presented on circulating tumor cells facilitate metastasis by binding L-selectin presented on leukocytes. Commonly used methods for detecting L-selectin ligands on tissues, e.g., immunostaining, are performed under static, no-flow conditions. However, such analysis does not assay for functional L-selectin ligands, specifically those ligands that promote adhesion under shear flow conditions. Recently our lab developed a method, termed dynamic biochemical tissue analysis (DBTA), to detect functional selectin ligands in situ by probing tissues with L-selectin-coated microspheres under hemodynamic flow conditions. In this investigation, DBTA was used to probe human colon tissues for L-selectin ligand activity. The detection of L-selectin ligands using DBTA was highly specific. Furthermore, DBTA reproducibly detected functional L-selectin ligands on diseased, e.g., cancerous or inflamed, tissues but not on noncancerous tissues. In addition, DBTA revealed a heterogeneous distribution of functional L-selectin ligands on colon cancer tissues. Most notably, detection of L-selectin ligands by immunostaining using HECA-452 antibody only partially correlated with functional L-selectin ligands detected by DBTA. In summation, the results of this study demonstrate that DBTA detects functional selectin ligands to provide a unique characterization of pathological tissue.

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Abstract 3991: Differentiating esophageal cancer cells from normal cells using ligand-conjugated microspheres

Cancer of the esophagus has a dismal overall prognosis and low 5 year survival rate due to its aggressive nature and the fact that it often presents at a late stage. Biochemical changes present on transforming tissue provide an opportunity for the early detection of cancer within the esophagus and thus the promise of a more favorable prognosis and a higher survival rate. Recently, there has been an increasing effort to detect cancer of the esophagus by introducing, during an endoscopic procedure, soluble molecules (ligands) cognate to moieties preferentially expressed on transforming tissue. The success of this approach depends on the selective binding of the ligand to transforming tissue relative to normal tissue. For soluble ligands, the factors that dictate the selective binding depend on a very small number of factors. In contrast, if the ligands are conjugated to particles, there are a large number of controllable factors that can be manipulated to “engineer” the detection scheme and thus optimize selective recognition of transforming tissue. In this study, we utilized an in vitro system to investigate the feasibility of the ligand-conjugated particle approach. First, we explored the surface chemistry of an esophageal adenocarcinoma cell line, OE19, relative to a normal esophageal cell line, HEEpiC, using flow cytometric analysis. Among other differences, we found that the OE19 cell line expresses relatively high levels of the tetrasaccharides sialyl Lewis A (sLea) and sialyl Lewis X (sLex). sLea and sLex are known cognate molecules for the selectin family of adhesion molecules, in particular E-selectin. Thus, we conjugated an E-selectin construct to 10 μm diameter microspheres. The E-selectin construct consisted of the extracellular domain of E-selectin fused to the Fc domain of IgG. Flow cytometric analysis revealed that the E-selectin construct was conjugated to the microspheres and that the E-selectin portion of the molecule was available for binding. To roughly simulate the introduction of the conjugated microspheres during an endoscopic procedure, a parallel plate flow chamber was used. A planar substrate of either OE19 or HEEpiC cells was placed in the flow chamber and a suspension of E-selectin or IgG (negative control) microspheres were perfused through the flow chamber. We observed that the E-selectin microspheres exhibited significantly greater adhesion to the OE19 cells relative to the HEEpiC cells. In contrast, IgG microspheres exhibited negligible adhesion to the OE19 and HEEpiC cells. Combined, this study provides proof of concept for an assay approach that could be engineered to detect transforming tissue present within the esophagus. Citation Format: Mahboubeh S. Noori, Sarah J. Bodle, Grady E. Carlson, David S. Drozek, Monica M. Burdick, Douglas J. Goetz. Differentiating esophageal cancer cells from normal cells using ligand-conjugated microspheres. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3991.

Abstract 1609: The epithelial to mesenchymal transition regulates the expression of E-selectin ligands on breast cancer cell lines

Breast cancer cells garner invasive and stem-like characteristics through the epithelial to mesenchymal transition (EMT). Consequently, the EMT has been implicated in cancer metastasis. While many of the effects of the EMT have been elucidated, it is currently unknown how the EMT modifies the expression of E-selectin ligands on breast cancer cells. These ligands are often sialofucosylated glycans that mediate cell adhesion with E-selectin molecules presented by endothelial cells lining the blood vessel walls at the metastatic site, which is commonly bone marrow. Although E-selectin is widely recognized for its role in promoting adhesion during tumor cell extravasation, it also maintains the quiescence of hematopoietic stem cells in the vascular niche of the bone marrow. Thus in this investigation, we studied the effects of the EMT on the E-selectin ligand activities of breast cancer cell lines and their behavior in an E-selectin rich environment. The EMT was induced in breast cancer cell lines by ectopic expression of either the Snail or Twist transcription factors. Through the EMT breast cancer cells upregulated the expression of vimentin and N-cadherin, yet reduced the expression of CD24, E-cadherin, and E-selectin ligands. Breast cancer cell lines were assayed for E-selectin ligand activity using laminar flow assays, flow cytometry, qPCR, and were cultured on E-selectin-coated tissue culture plates. In laminar flow assays breast cancer cells were perfused over E-selectin substrates using physiological flow conditions. Breast cancer cells expressing Snail or Twist adhered to E-selectin from the free fluid stream in significantly fewer numbers and demonstrated fewer E-selectin ligands in flow cytometry than the vector controls. Additionally, qPCR revealed that in comparison to vector controls breast cancer cells expressing Snail or Twist downregulated the expression of α-(1,3) fucosyltransferase and α-(1,4) fucosyltransferase, which catalyze terminal fucosylations necessary for E-selectin ligand function. Interestingly, on E-selectin coated plates breast cancer cells expressing Snail or Twist avoided prolonged exposure to E-selectin by forming mammospheres, yet vector controls did not. Collectively, these data demonstrate that the EMT regulates the expression of E-selectin ligands on breast cancer cells, and causes the cells to become sensitive to culture in an E-selectin rich environment. Citation Format: Grady Earl Carlson, Venktesh Shirure, Alexander O. Ostermann, Emily A. Blaha, Louis F. Delgadillo, David F.J. Tees, Fabian Benencia, Monica Burdick. The epithelial to mesenchymal transition regulates the expression of E-selectin ligands on breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1609.

Abstract 1920: Stem-like triple negative breast cancer cells exhibit a distinct response to selectin/selectin ligand interactions

Circulating tumor cells (CTCs) often over-express sialofucosylated glycans, which bind to E-selectin presented on vascular endothelial cells. This interaction facilitates the initial tethering and rolling adhesion events in CTC extravasation during cancer metastasis. Though selectins and their ligands are well-established cell adhesion molecules, relatively little is known about their roles as cell signaling molecules for regulating growth, migration, and apoptosis in breast cancer (BC). To investigate the effects of selectin/selectin ligand interactions on BC cell signaling and adhesion, Hs578T, and MDA-MB-231 stem-like triple negative cell lines and BT-20, MCF-7, and ZR-75-1 non-stem-like hormone-receptor-positive cell lines were grown on tissue culture plates pre-treated with recombinant E-selectin or perfused over E-selectin-presenting substrate at physiologically relevant laminar flow conditions. In culture, cell lines demonstrated a dose dependent response on substrates pre-treated with increasing concentrations (1-10 µg/ml) of recombinant E-selectin. More specifically, cell lines exhibited little change on substrate treated with low concentrations of recombinant E-selectin (1-5 µg/ml) relative to human Fc fragment and fibronectin controls, yet displayed varying degrees of membrane blebbing and, in some cases, aggregate formation and reduced proliferation on substrate treated with 10 µg/ml of recombinant E-selectin. Notably, the stem-like triple negative cell lines, specifically Hs578T cells, had the most pronounced response to culture on E-selectin substrate as defined by blebbing and formation of cell aggregates. Furthermore, when Hs578T cells were cultured on substrate with adjacent areas treated with either recombinant E-selectin (10 µg/ml) or fibronectin (molar equivalent), cells failed to attach and grow on E-selectin but successfully seeded and proliferated in culture on fibronectin. In the laminar flow assay, Hs578T and MDA-MB-231 stem-like triple negative cell lines had four fold fewer adhesions on E-selectin substrate relative to the adhesion of BT-20, MCF-7, and ZR-75-1 non-stem-like hormone-receptor-positive cell lines. Moreover, the average rolling velocity of the stem-like triple negative cell lines on E-selectin substrate was significantly greater than the average rolling velocities of the other cell lines, which illustrates that under dynamic flow conditions stem-like triple negative BC cell lines have relatively fewer and weaker interactions with E-selectin. These data show that BC cells, especially Hs578T stem-like triple negative BC cells, differ in response to selectin/selectin ligand interaction under static and dynamic conditions, and illustrate that threshold levels of selectin/selectin ligand interaction may regulate cell signaling in certain types of BC cell lines. Thus targeting these interactions may lead to novel BC therapeutics. Citation Format: Grady E. Carlson, Luis F. Delgadillo, Emily A. Blaha, Fabian Benencia, Monica M. Burdick. Stem-like triple negative breast cancer cells exhibit a distinct response to selectin/selectin ligand interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1920. doi:10.1158/1538-7445.AM2014-1920

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Abstract 203: Dynamic biochemical tissue analysis provides a unique method to detect functional in situ L-selectin ligands and unveils intercellular heterogeneity in colon cancer tissues.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC A growing body of evidence suggests that E-,P-, and L-selectin, a family of cell adhesion molecules, engage their ligands on colon cancer cells to initiate adhesion with the blood vessel wall at the secondary site during metastasis. This initial adhesion is a “rolling” adhesion in which selectin/selectin ligand bonds break and reform under forces in hemodynamic flow. Current standards for tissue analyses, such as immunostaining, do not assay for functional in situ selectin ligands, i.e., those that promote rolling. Recently, our lab developed a novel tissue analysis, termed dynamic biochemical tissue analysis (DBTA), which detects functional selectin ligands in situ by probing pathological tissues with selectin-coated microspheres. Due to the application of hydrodynamic flow in the assay, the selectin coated microspheres “roll” on tissue sections presenting functional selectin ligands. When colon cancer tissue sections of diverse histological classifications were probed with L-selectin-coated microspheres in DBTA, functional L-selectin ligands were detected (i.e., L-selectin microspheres rolled on the surface of the tissue sections). Rolling velocities were a function of hemodynamic shear stress, illustrating the unique kinetic and tensile properties of force-dependent L-selectin/L-selectin ligand bonds. More specifically, DBTA preserved the catch-slip behavior of selectin/selectin ligand bonds. That is, rolling became stabilized at physiologic shear stresses between 0.5-0.75 dyne/cm2, but outside this range microsphere rolling was increasingly erratic as bond breakage exceeded bond formation necessary to preserve adhesion. Differences in microsphere rolling velocities varied with position on the tissue surface and thereby demonstrated the heterogeneous expression of functional L-selectin ligands on colon cancer tissues. In summary, DBTA provides a unique method to detect functional in situ L-selectin ligands and unveils intercellular heterogeneity such that, with further development, DBTA may promote potential diagnostics and prognostics for cancer characterization. Citation Format: Grady E. Carlson, Venktesh S. Shirure, Vicente A. Resto, Ramiro Malgor, Douglas J. Goetz, Monica M. Burdick. Dynamic biochemical tissue analysis provides a unique method to detect functional in situ L-selectin ligands and unveils intercellular heterogeneity in colon cancer tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 203. doi:10.1158/1538-7445.AM2013-203

Abstract 441: Dynamic biochemical tissue analysis of selectin ligands present on tissue derived from solid tumors

Identification and characterization of molecular markers on cancerous tissue can lead to novel diagnostics and prognostics for cancer. Markers of interest include sialofucosylated molecules such as sialyl Lewis X and sialyl Lewis A (CA19-9), which are expressed on both lipids and proteins, and are often upregulated on certain cancerous tissues. Mounting evidence suggests that circulating cancer cells use these molecules to bind E-, P- and L- selectin (adhesion molecules present on leukocytes, platelets, and endothelial cells), to facilitate dissemination indicating that the presence of selectin ligands may correlate with metastatic potential. The hallmark of selectin-mediated binding is “rolling,” i.e. adhesion governed by the unique kinetic and tensile properties of selectin-selectin ligand bonds occurring under dynamic (flow) conditions. At present, the analysis of pathological tissue (e.g. immunohistochemistry) is performed under static conditions that only reveal high affinity interactions between the probing molecule and the tissue. Traditional biochemical tissue analysis is woefully inadequate for investigating selectin ligands on cancerous tissue, as it does not allow a detailed exploration of ligand chemistry in situ under biophysical conditions fit for selectin binding. Recently, we established a new assay for probing tissue, termed dynamic biochemical tissue analysis (DBTA), in which the probing molecule is conjugated to microspheres that are contacted with tissue samples under fluid shear. In our current investigation, E-, P- and L-selectin microspheres rolled on colon cancer tissue microarrays in DBTA. The adhesive interactions appeared to be specific, as the microspheres were released from the tissue samples and no further adhesion events were observed when EDTA was perfused through the system. Additionally, the adhesion of the microspheres with a given tissue section was dependent on the selectin. E-selectin microspheres were the most promiscuous, binding at high levels to many of the tissue samples, while the adhesion of the P- and L-selectin microspheres was more select. For a given type of selectin microsphere, the observed adhesion was not homogeneous; it was localized on discrete regions of the tissue sample. Notably, HECA-452 staining (an antibody that recognizes sialyl Lewis X and related moieties) in conjunction with selectin microsphere rolling revealed that HECA-452 reactivity only partially correlated with selectin binding. This observation strongly suggests that DBTA yields tissue characterization that is distinct compared to a high affinity immunohistochemistry assay. In conclusion, DBTA provides a controlled environment in which the shear-dependent selectin-selectin ligand interactions may be observed, providing a unique characterization of cancerous tissue applicable to developing novel diagnostic and prognostic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 441. doi:1538-7445.AM2012-441