Siddarth Chandrasekaran

Phenotypic switch in blood: effects of pro-inflammatory cytokines on breast cancer cell aggregation and adhesion.

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLex expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of ‘triple-negative’ breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments.

Effect of homotypic and heterotypic interaction in 3D on the E-selectin mediated adhesive properties of breast cancer cell lines.

Hematogenous metastasis involves a glycoprotein mediated adhesion cascade of tumor cells with E-selectin on the endothelial layer of the blood vessels. Cell-cell interactions play a major role in cancer metastasis and invasiveness. Intercellular communication between two cancer cells or between a cancer cell with a stromal cell in the microenvironment such as fibroblasts or inflammatory cells play an important role in metastatic invasion. Culturing tumor cells as 3D spheroids can recapitulate these physiologically relevant cell-cell interactions. The heterogeneity in primary tumors is attributed to cell subpopulations with varying degree of invasiveness. Co-culturing cancer cells with different phenotypes as 3D spheroids can mimic this heterogeneity. Here we report the effect of homotypic and heterotypic interactions in breast cancer cells cultured as 3D spheroids on polydimethylsiloxane (PDMS) on the adhesion phenotype to E-selectin. We show that breast cancer cell lines (BT20 and MCF7) propagating as 3D spheroids on PDMS exhibit a stronger interaction with human recombinant E-selectin when compared to their respective monolayer grown counterparts on tissue culture plate (TCP). Matrigel invasion assay also indicated that BT20 and MCF7 spheroids were more invasive than BT20 and MCF7 cells grown as monolayers. To mimic tumor heterogeneity in vitro, a co-culture model included tumorigenic cell lines BT20, MCF7 and a non-tumorigenic mammary epithelial cell line MCF10A. These cell lines were cultured together in equal seeding ratio on PDMS to generate co-culture spheroids. The heterotypic interactions in the co-culture model resulted in enhancement of the adhesion of the most invasive BT20 cell line to E-selectin. BT20 cells in co-culture bound to the greatest degree to soluble E-selectin compared to MCF7 and MCF10A cells in co-culture. Co-invasion assay with co-culture spheroids indicated that BT20 cells in co-culture were more invasive than MCF7 and MCF10A cells. The results presented here indicate that homotypic and heterotypic interaction of cancer cells favor adhesion to E-selectin thus representing a complexity beyond planar cell culture. Also, when cells of different phenotypes are mixed, the heterogeneity enhances the adhesive phenotype and invasiveness of the most invasive cell population. The results challenge the classic use of planar cell culture for evaluating the adhesion of cancer cells to E-selectin and establish our co-culture technique as a model that can help investigative studies in metastasis and invasiveness of breast and other types of cancers.

Microenvironment induced spheroid to sheeting transition of immortalized human keratinocytes (HaCaT) cultured in microbubbles formed in polydimethylsiloxane

The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, disease development and progression. In this study, we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100-300 μm in diameter formed in hydrophobic polydimethylsiloxane (PDMS) with ∼60-100 μm circular openings and aspect ratio ∼3.0. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15-20 cells/MB) and cultured under standard conditions, adopt a compact 3D spheroidal morphology. Within 2-3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not undergo a transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96-well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the growth media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca(2+) concentration and changes in E-cadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy.

Enriching and characterizing cancer stem cell sub-populations in the WM115 melanoma cell line

Cutaneous melanoma is an increasingly common and potentially lethal malignancy of melanocytes, the melanin producing cells normally located in the basal layer of the skin epidermis. Despite major advances in cancer chemotherapeutics and immunotherapy, the success in treating metastatic melanoma remains poor. The notion that cancer stem cells (CSCs) play a key role in melanoma progression is well received. Therefore, isolating and characterizing CSCs is of critical importance for designing new therapeutic strategies that target this unique tumor initiating cell sub-population. In this work, we present a simple in vitro method, employing cell culture on polydimethylsiloxane (PDMS) and transfer back onto standard tissue culture plate, to enrich a non-adherent spheroid (NA/S) forming and an adherent monolayer (AM) cell sub-populations from the tumorigenic WM115 melanoma cell line. The phenotypes of the morphologically distinct NA/S and AM sub-populations were further characterized by quantifying the expression of stem cell markers, CD20 and CD271. Flow cytometric analysis found 2.32% of the cells in the NA/S sub-population were CD20+ CD271+ whereas only 0.27% of the cells in the AM sub-population were CD20+ CD271+. When the NA/S sub-population was cultured back onto PDMS it resulted in the further enrichment of CD20+ CD271+ cells to 14.7%. We used microbubble arrays to quantify the in vitro clonogenic potential of the NA/S and AM cell sub-populations. Microbubbles are spherical cavities, ~160 μm in diameter with 60 μm circular openings, formed in PDMS using the gas expansion molding (GEM) process. Cells from each sub-population were seeded, under limiting dilution conditions, onto separate arrays containing 1215 microbubble wells. After five days in culture, wells seeded with 1, 2, 3 and >3 cells per microbubble well were inspected for cell proliferation. The Extreme Limiting Dilutions Analysis (ELDA) determined a ~58% clonal survival (1 in every 1.72 cells) for the NA/S sub-population and ~25% clonal survival (1 in every 3.93 cells) for the AM sub-population (= 176, p = 4.41e(-40)). These findings taken together add to the existing evidence that melanoma cells propagating as non-adherent/spheroids represent a more aggressive phenotype due to the greater presence of tumor initiating cells.

A physical sciences network characterization of circulating tumor cell aggregate transport

Circulating tumor cells (CTC) have been implicated in the hematogenous spread of cancer. To investigate the fluid phase of cancer from a physical sciences perspective, the multi-institutional Physical Sciences-Oncology Center (PS-OC) Network performed multidisciplinary biophysical studies of single CTC and CTC aggregates from a patient with breast cancer. CTCs, ranging from single cells to aggregates comprised of 2-5 cells, were isolated using the high-definition CTC assay and biophysically profiled using quantitative phase microscopy. Single CTCs and aggregates were then modeled in an in vitro system comprised of multiple breast cancer cell lines and microfluidic devices used to model E-selectin mediated rolling in the vasculature. Using a numerical model coupling elastic collisions between red blood cells and CTCs, the dependence of CTC vascular margination on single CTCs and CTC aggregate morphology and stiffness was interrogated. These results provide a multifaceted characterization of single CTC and CTC aggregate dynamics in the vasculature and illustrate a framework to integrate clinical, biophysical, and mathematical approaches to enhance our understanding of the fluid phase of cancer.

TRAIL-coated leukocytes that prevent the bloodborne metastasis of prostate cancer.

Prostate cancer, once it has progressed from its local to metastatic form, is a disease with poor prognosis and limited treatment options. Here we demonstrate an approach using nanoscale liposomes conjugated with E-selectin adhesion protein and Apo2L/TRAIL (TNF-related apoptosis-inducing ligand) apoptosis ligand that attach to the surface of leukocytes and rapidly clear viable cancer cells from circulating blood in the living mouse. For the first time, it is shown that such an approach can be used to prevent the spontaneous formation and growth of metastatic tumors in an orthotopic xenograft model of prostate cancer, by greatly reducing the number of circulating tumor cells. We conclude that the use of circulating leukocytes as a carrier for the anti-cancer protein TRAIL could be an effective tool to directly target circulating tumor cells for the prevention of prostate cancer metastasis, and potentially other cancers that spread through the bloodstream.

TRAIL-mediated apoptosis in breast cancer cells cultured as 3D spheroids.

TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44hiCD24loALDH1hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway. We show that inhibition of the COX-2/PGE2 pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44hiCD24lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.

Super natural killer cells that target metastases in the tumor draining lymph nodes.

Tumor draining lymph nodes are the first site of metastasis in most types of cancer. The extent of metastasis in the lymph nodes is often used in staging cancer progression. We previously showed that nanoscale TRAIL liposomes conjugated to human natural killer cells enhance their endogenous therapeutic potential in killing cancer cells cultured in engineered lymph node microenvironments. In this work, it is shown that liposomes decorated with apoptosis-inducing ligand TRAIL and an antibody against a mouse natural killer cell marker are carried to the tumor draining inguinal lymph nodes and prevent the lymphatic spread of a subcutaneous tumor in mice. It is shown that targeting natural killer cells with TRAIL liposomes enhances their retention time within the tumor draining lymph nodes to induce apoptosis in cancer cells. It is concluded that this approach can be used to kill cancer cells within the tumor draining lymph nodes to prevent the lymphatic spread of cancer.

In vitro assays for determining the metastatic potential of melanoma cell lines with characterized in vivo invasiveness.

The metastatic potential of cancer cells is an elusive property that is indicative of the later stages of cancer progression. The ability to distinguish between poorly and highly metastatic cells is invaluable for understanding the basic biology of cancer and to develop more treatments. In this paper, we exploit a A375 melanoma cell line series (A375P, A375MA1, A375MA2) that vary in metastatic potential, to demonstrate an in vitro screening assay using polydimethylsiloxane (PDMS) microbubble well arrays that can distinguish these cell lines by their growth characteristics in including morphology, migratory potential, and clonogenic potential. These cell lines cannot be distinguished by their growth characteristics when cultured on standard tissue culture plastic or planar PDMS. Results show that the more metastatic cell lines (A375MA1, A375MA2) have a higher proliferative potential and a distinctive radial spreading growth pattern out of the microbubble well. The A375MA2 cell line also has a higher tendency to form multicellular spheroids. The ability to successfully correlate the metastatic potential of cancer cells with their growth characteristics is essential first step toward developing a high-throughput screening assay to identify aggressive tumor cells in primary samples. The capability to culture and recover aggressive cells from microbubble wells will enable identification of candidate metastatic biomarkers which has immense clinical significance.

Focus: Two-dimensional electron-electron double resonance and molecular motions: The challenge of higher frequencies

The development, applications, and current challenges of the pulsed ESR technique of two-dimensional Electron-Electron Double Resonance (2D ELDOR) are described. This is a three-pulse technique akin to 2D Exchange Nuclear Magnetic Resonance, but involving electron spins, usually in the form of spin-probes or spin-labels. As a result, it required the extension to much higher frequencies, i.e., microwaves, and much faster time scales, with π/2 pulses in the 2-3 ns range. It has proven very useful for studying molecular dynamics in complex fluids, and spectral results can be explained by fitting theoretical models (also described) that provide a detailed analysis of the molecular dynamics and structure. We discuss concepts that also appear in other forms of 2D spectroscopy but emphasize the unique advantages and difficulties that are intrinsic to ESR. Advantages include the ability to tune the resonance frequency, in order to probe different motional ranges, while challenges include the high ratio of the detection dead time vs. the relaxation times. We review several important 2D ELDOR studies of molecular dynamics. (1) The results from a spin probe dissolved in a liquid crystal are followed throughout the isotropic → nematic → liquid-like smectic → solid-like smectic → crystalline phases as the temperature is reduced and are interpreted in terms of the slowly relaxing local structure model. Here, the labeled molecule is undergoing overall motion in the macroscopically aligned sample, as well as responding to local site fluctuations. (2) Several examples involving model phospholipid membranes are provided, including the dynamic structural characterization of the boundary lipid that coats a transmembrane peptide dimer. Additionally, subtle differences can be elicited for the phospholipid membrane phases: liquid disordered, liquid ordered, and gel, and the subtle effects upon the membrane, of antigen cross-linking of receptors on the surface of plasma membrane, vesicles can be observed. These 2D ELDOR experiments are performed as a function of mixing time, Tm, i.e., the time between the second and third π/2 pulses, which provides a third dimension. In fact, a fourth dimension may be added by varying the ESR frequency/magnetic field combination. Therefore, (3) it is shown how continuous-wave multifrequency ESR studies enable the decomposition of complex dynamics of, e.g., proteins by virtue of their respective time scales. These studies motivate our current efforts that are directed to extend 2D ELDOR to higher frequencies, 95 GHz in particular (from 9 and 17 GHz), in order to enable multi-frequency 2D ELDOR. This required the development of quasi-optical methods for performing the mm-wave experiments, which are summarized. We demonstrate state-of-the-art 95 GHz 2D ELDOR spectroscopy through its ability to resolve the two signals from a spin probe dissolved in both the lipid phase and the coexisting aqueous phase. As current 95 GHz experiments are restricted by limited spectral coverage of the π/2 pulse, as well as the very short T2 relaxation times of the electron spins, we discuss how these limitations are being addressed.