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Maheswaran, Shyamala

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Maheswaran

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Shyamala

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Maheswaran, Shyamala
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    Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells
    (2014) Ting, David; Wittner, Ben; Ligorio, Matteo; Jordan, Nicole Vincent; Shah, Ajay M.; Miyamoto, David; Aceto, Nicola; Bersani, Francesca; Brannigan, Brian W.; Xega, Kristina; Ciciliano, Jordan C.; Zhu, Huili; MacKenzie, Olivia C.; Trautwein, Julie; Arora, Kshitij S.; Shahid, Mohammad; Ellis, Haley L.; Qu, Na; Bardeesy, Nabeel; Rivera, Miguel; Deshpande, Vikram; Ferrone, Cristina; Kapur, Ravi; Ramaswamy, Sridhar; Shioda, Toshi; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel
    SUMMARY Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.
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    HER2 expression identifies dynamic functional states within circulating breast cancer cells
    (2016) Jordan, Nicole Vincent; Bardia, Aditya; Wittner, Ben; Benes, Cyril; Ligorio, Matteo; Zheng, Yu; Yu, Min; Sundaresan, Tilak K.; Licausi, Joseph A.; Desai, Rushil; O’Keefe, Ryan M.; Ebright, Richard; Boukhali, Myriam; Sil, Srinjoy; Onozato, Maristela Lika; Iafrate, Anthony; Kapur, Ravi; Sgroi, Dennis; Ting, David; Toner, Mehmet; Ramaswamy, Sridhar; Haas, Wilhelm; Maheswaran, Shyamala; Haber, Daniel
    Circulating tumor cells (CTCs) in women with advanced estrogen receptor-positive/HER2-negative breast cancer acquire a HER2-positive subpopulation following multiple courses of therapy1,2. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here, we analyzed CTCs from 19 ER+/HER2− patients, 84% of whom had acquired CTCs expressing HER2. Cultured CTCs maintain discrete HER2+ and HER2− subpopulations: HER2+ CTCs are more proliferative but not addicted to HER2, consistent with activation of multiple signaling pathways. HER2− CTCs show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2− CTCs interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. While HER2+ and HER2− CTCs have comparable tumor initiating potential, differential proliferation favors the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2− phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic CTC-derived tumor models. Together, these results point to distinct yet interconverting phenotypes within patient-derived CTCs, contributing to progression of breast cancer and acquisition of drug resistance.
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    Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells
    (Nature Publishing Group UK, 2017) Fachin, Fabio; Spuhler, Philipp; Martel-Foley, Joseph M.; Edd, Jon F.; Barber, Thomas A.; Walsh, John; Karabacak, Murat; Pai, Vincent; Yu, Melissa; Smith, Kyle; Hwang, Henry; Yang, Jennifer; Shah, Sahil; Yarmush, Ruby; Sequist, Lecia; Stott, Shannon; Maheswaran, Shyamala; Haber, Daniel; Kapur, Ravi; Toner, Mehmet
    Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm–50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15–20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.
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    Ultra-fast vitrification of patient-derived circulating tumor cell lines
    (Public Library of Science, 2018) Sandlin, Rebecca; Wong, Keith H. K.; Tessier, Shannon; Swei, Anisa; Bookstaver, Lauren D.; Ahearn, Bennett E.; Maheswaran, Shyamala; Haber, Daniel; Stott, Shannon; Toner, Mehmet
    Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.
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    Bevacizumab terminates homeobox B9-induced tumor proliferation by silencing microenvironmental communication
    (BioMed Central, 2014) Hoshino, Yoshinori; Hayashida, Tetsu; Hirata, Akira; Takahashi, Hidena; Chiba, Naokazu; Ohmura, Mitsuyo; Wakui, Masatoshi; Jinno, Hiromitsu; Hasegawa, Hirotoshi; Maheswaran, Shyamala; Suematsu, Makoto; Kitagawa, Yuko
    Background: Homeobox B9 (HOXB9), a transcriptional factor, regulates developmental processes and tumor progression and has recently been recognized as one of important transcriptional factors related to angiogenesis. This study aimed to investigate the role of HOXB9 in tumorigenesis and angiogenesis. Methods: We examined the expression of HOXB9 in colorectal cancer using qPCR and in situ hybridization. We also examined the effect of HOXB9 overexpression in colorectal cancer using a proliferation assay, ELISA, a multiplex assay, and xenograft models. The clinical significance of HOXB9 was statistically evaluated in resected specimens. Results: HOXB9 was expressed in colorectal cancer specimens. HOXB9 induced angiogenesis and tumor proliferation in vitro, which resulted in high tumorigenicity in vivo and poor overall survival. Bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, remarkably suppressed tumor proliferation by inhibiting angiogenesis in HOXB9-overexpressing xenografts, and it improved overall survival and provided prolonged progression-free survival in HOXB9-overexpressing patients. A comprehensive multiplex assay of the supernatant of cancer cells co-cultured with human vascular endothelial cells and fibroblasts indicated significantly higher interleukin-6 (IL6) levels than those in the supernatant of monocultured cells. HOXB9 overexpression in clinical specimens was significantly correlated with increased IL6 expression. An IL6-neutralizing antibody inhibited VEGF secretion and tumor proliferation in the co-culture system. Conclusions: HOXB9 promotes the secretion of angiogenic factors, including VEGF, to induce tumor proliferation through microenvironmental production of cytokines including IL6 signaling. Moreover, silencing of VEGF or IL6 terminates cytokine release in tumor microenvironment. Thus, HOXB9 and IL6 may be potential biomarkers for bevacizumab treatment.
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    Dynamic Chromatin Modification Sustains Epithelial-Mesenchymal Transition following Inducible Expression of Snail-1
    (2014) Javaid, Sarah; Zhang, Jianmin; Anderssen, Endre; Black, Josh C.; Wittner, Ben; Tajima, Ken; Ting, David; Smolen, Gromoslaw A.; Zubrowski, Matthew; Desai, Rushil; Maheswaran, Shyamala; Ramaswamy, Sridhar; Whetstine, Johnathan; Haber, Daniel
    SUMMARY Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6–48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.
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    SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes
    (Nature Pub. Group, 2015) Tajima, Ken; Yae, Toshifumi; Javaid, Sarah; Tam, Oliver; Comaills, Valentine; Morris, Robert; Wittner, Ben; Liu, Mingzhu; Engstrom, Amanda; Takahashi, Fumiyuki; Black, Joshua C.; Ramaswamy, Sridhar; Shioda, Toshihiro; Hammell, Molly; Haber, Daniel; Whetstine, Johnathan; Maheswaran, Shyamala
    Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer.
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    A microfluidic device for label-free, physical capture of circulating tumor cell-clusters
    (2015) Sarioglu, A. Fatih; Aceto, Nicola; Kojic, Nikola; Donaldson, Maria C.; Zeinali, Mahnaz; Hamza, Bashar; Engstrom, Amanda; Zhu, Huili; Sundaresan, Tilak K.; Miyamoto, David; Luo, Xi; Bardia, Aditya; Wittner, Ben; Ramaswamy, Sridhar; Shioda, Toshi; Ting, David; Stott, Shannon; Kapur, Ravi; Maheswaran, Shyamala; Haber, Daniel; Toner, Mehmet
    Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identify CTC-clusters in 30–40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis.
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    The Role of Physical Stabilization in Whole Blood Preservation
    (Nature Publishing Group, 2016) Wong, Keith H. K.; Sandlin, Rebecca; Carey, Thomas R.; Miller, Kathleen L.; Shank, Aaron T.; Oklu, Rahmi; Maheswaran, Shyamala; Haber, Daniel; Irimia, Daniel; Stott, Shannon; Toner, Mehmet
    The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase—a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.
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    Deformability of Tumor Cells versus Blood Cells
    (Nature Publishing Group, 2015) Shaw Bagnall, Josephine; Byun, Sangwon; Begum, Shahinoor; Miyamoto, David; Hecht, Vivian C.; Maheswaran, Shyamala; Stott, Shannon; Toner, Mehmet; Hynes, Richard O.; Manalis, Scott R.
    The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.