A critical first step in many cell biology applications – including viral vector generation for cell and gene therapies and biotherapeutic antibody production - is transfection. Optimal transfections are vital to the success of your experiment, whereas poor transfections lead to inconclusive results and additional time spent troubleshooting. We will highlight critical protocol considerations, along with our innovative products, to improve your transfection-mediated workflows and accelerate your research.
Whether studying molecular mechanisms underlying an oncogenic process, identifying treatable targets, or testing out a novel gene therapy, having the right research tools is critical. The goal to utilize vectors for gene delivery allowing for expression of wild-type or mutant genes, or to modulate the expression of a gene, is complicated by the range of possibilities to consider in vector design. With options ranging from vector type (non-viral, viral, transposon, etc.), expression pattern (transient, stable, inducible, tissue-specific, etc.), to the ability to co-express multiple GOIs or markers, complexity bends to your specific needs. To conduct large-scale screening of cancer pathways, having a high-quality library, be it whole genome or targeted and shRNA or CRISPR mediated loss- or gain-of-function, may efficiently reveal novel targets. This webinar will highlight what we have learned about these topics and demonstrate why you should consider outsourcing these activities to VectorBuilder so you can focus your time and wisdom on the downstream experiments for biological implications and theory generation.
With accelerating development and approval of cell therapies and biologic drugs comes a need for more robust and reliable cell-based assays and validated analysis systems. In this seminar a variety of techniques for immune cell isolation, expansion, and characterization will be demonstrated in cell-based immunotherapy models using powerful cytometric instrumentation. Special focus will be given to immune effector function assays to characterize the potency and specificity of CAR-T and NK cells. Additionally, scalable screening methods based on recent innovations in high-throughput flow cytometry, partnered with regulatory-compliant acquisition and analysis software, will provide valuable additions to your portfolio of assays for immunotherapy research.
Immunotherapy has proven clinical efficacy and tremendous potential in multiple cancers. However, clinical benefits vary between patients. The research community is therefore intensively investigating how the composition of tumor-infiltrating leukocytes (TILs) contributes to patient stratification and which effects therapeutic interventions have on cell populations within the tumor microenvironment. Our goal is to support every step of your TIL analysis by providing tools and workflows to maximize the amount and quality of data obtained from limited tumor material. We will present solutions for storage of fresh tissue, automated dissociation of tumor tissue, isolation of different pure populations of viable TILs from dissociated tumor, and background-free flow cytometry analysis of targeted TIL populations.
The study of viruses is of vital importance, both to increase our understanding of animal and vegetal life, and to learn how to counteract the dangerous effects of viral infections with vaccines and anti-viral treatments. Anti-viral drug and vaccine development begin with high throughput screening phases. Virus research workflows therefore need to be designed to be able to process high numbers of samples at the same time while maintaining virus integrity and limiting bias.
Homogenization is the first step of most viral extraction workflows. To obtain reproducible results, a proper homogenization of virus-infected samples is crucial. Mechanical lysis using beads (bead beating) is the gold standard for standardized approaches to homogenization. The Precellys tissue homogenizers are the ideal instruments to evaluate viral RNA and viral titer from various tissues thanks to their 3-D bead beating technology. In this webinar, we present optimized protocols allowing for high throughput and reproducible virus analysis.
Tumor stem cells (TSCs) contribute to cancer mortality via therapeutic resistance, tumor recurrence, and metastatic mechanisms. However, the origins of the stem capacity to TSCs remains in question, but all TSCs descend from the original tumor cell-of-origin where the first oncogenic event occurred. Tumors arising from different cells-of-origin are histologically identical, but it is unknown whether TSCs that arose from different origins are molecularly and functionally distinct. Using mouse models driving identical Apc mutations from Lrig1-expressing and Mist1-expressing cells, we characterized TSCs of tumors driven from stem and non-stem cells-of-origin using single cell RNA sequencing (scRNA-seq), organoids, and multiplexed imaging. We revealed reduced stem capacity but increased class II antigen presentation ability for non-stem cell (Mist1) driven TSCs compared with stem cell (Lrig1) driven TSCs, which resulted in a favorable immune microenvironment skewed towards active cytotoxic response in Mist1-driven tumors. These results suggest that the cell-of-origin of tumorigenesis provides a specific context by which TSCs are generated, dictating their interactions with the tumor microenvironment.
Cherie’ Scurrah is a PhD Candidate in the department of Cell and Developmental Biology at Vanderbilt University where she is interested in understanding tumor heterogeneity through single cell technologies. Specifically, her thesis investigates whether the tumor cell-of-origin can dictate tumor phenotypes. Cherie’ graduated from the University of California, Irvine with a Bachelor’s of Science in Biological Sciences where she explored the role of serotonin and dopamine neurological pathways in addiction. During this time, she also investigated a rare, but deadly pediatric brain tumor, Diffuse Intrinsic Pontine Glioma, at Stanford University. She is currently finishing up her thesis work and actively looking to start an industry career as a Medical Science Liaison (MSL).
In allogeneic transplantation, stem cells may be obtained from the bone marrow, the peripheral blood, and umbilical cord blood (UCB). Previous studies have demonstrated that the use of peripheral blood in the allogeneic setting is associated with a decreased relapse rate in hematologic malignancies and improvement in overall and disease-free survival, as compared to bone marrow transplantation. Much less is known about partially HLA-matched unrelated umbilical cord blood units as an alternative graft source. In adult patients, the success has been increased by transplantation of two cord blood units, as only one of the cord blood units usually survives beyond 100 days. Delayed immune reconstitution is a major obstacle for the successful use of umbilical cord blood cell transplantation, because it increases the risk of death from serious infections. Peripheral blood specimens of transplant patients (15 matched sibling donors (MSD, control), and 15 double umbilical cord blood recipients), were investigated at days 100, and one year after transplantation. INFG and GranzymeB ELISpot assays were used to quantitate the frequency of NK and T cells that secrete cytotoxic molecules in response to 10 peptides from 5 most commonly occurring viral reactivation types of infections. We observed delays in quantitative and functional recovery of viral-specific T-cell responses, but faster recovery and functional overactivation of B- NK- in UCB group compared to MDS patients at day 100 and one year post-transplant. In summary, alterations in the balanced recovery of innate and adaptive immunity represent immune restoration disorders that are required post-transplant individualized immunomodulatory treatments.
Dr. St. Louis is appointed as an Assistant Professor at the Department of Medicine, University of Minnesota, USA. She has established translational research program in immune restoration disorders (IRD). Dr. St. Louis collaborates with the Division Hematology Oncology and Transplantation, on virus-specific immune cell reconstitution after various hematopoietic cell transplantation regimens.
Her primary research focuses on identification of biomarkers of immune reconstitution inflammatory syndrome (IRIS): in leukemia patients who received hematopoietic cell transplantation, and in AIDS patients after initiation of antiretroviral therapy. Dr. St. Louis collaborates on several clinical trials, working on stratification of patients for optimal immunomodulatory treatments.
Neha Jaiswal1,2, Harold Saavedra3 and Srikumar Chellappan1+
1Dept. of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612.
2Rosewell Park Cancer Center, Buffalo, NY.
3Ponce Health Sciences University, Ponce, PR.
Cell cycle checkpoints involve complex regulatory mechanisms that ensure proper genomic maintenance and fidelity of cell division. Especially, error-free progression through mitosis is critical for proper cell division and accurate distribution of the genetic material. The APC/C ubiquitin ligase plays a central role in exit of cells from metaphase and its activation is controlled by the cofactors Cdc20 and Cdh1. Additionally, genome stability is maintained by Spindle Assembly Checkpoint (SAC), which monitors proper attachment of chromosomes to spindle microtubules through kinetochores prior to cell division. SAC functions through mitotic checkpoint complex (MCC) composed of Cdc20, Mad2, BubR1/Mad3 and Bub3. MCC associates with APC/C rendering it inactive, halting cell cycle progression.
Tank binding kinase1 (TBK1) phosphorylates and activates various proteins involved in cell cycle progression and innate immunity. An oncogenic role for TBK1 has been proposed for certain cancer types and our studies showed a role for TBK1 in microtubule dynamics and mitosis. Similarly, the SAC kinase TTK is a major regulator of mitosis. Here we elucidate the role of TBK1 and TTK in regulating mitosis by modulating SAC.
TBK1 was depleted using CRISPR/Cas9 or inhibited using BX795, MRT67307 or amlexanox. Techniques used include immunofluorescence and proximity ligation assays, in vitro kinase assays, mass-spectrometry and micronucleation experiments.
TBK1 interacts with and phosphorylates Cdc20 and Cdh1 and depletion of TBK1 results in overexpression of SAC components. TBK1 inhibition increases the association of Cdc20 with APC/C and BubR1 indicating inactivation of APC/C. Similarly, interaction of Cdh1 with APC/C is also enhanced. These results show a TBK1-mediated regulation of SAC and APC/C. We also find that TBK1 and TTK inhibition result in reduced cell viability and mitotic aberrations indicated by centrosome amplification and increased micronucleation.
Our results indicate that TBK1 affects mitotic checkpoints and alterations in TBK1 will affect SAC, impeding mitotic progression. It is likely that combining TBK1 inhibitors with other regulators of mitosis might induce mitotic catastrophe and eliminate cancer cells.
Dr. Meenu Maan was awarded PhD from the Jawaharlal Nehru University, New Delhi, India in 2018. During her PhD she explored the role of Ubiquitin E3 ligase CHIP in autophagy mediated protein degradation. Currently, she is a Postdoctoral fellow at the Moffitt Cancer Center, Tampa, Florida. She is investigating the role of TBK1, a non-canonical Ikk kinase, in regulating cell division fidelity, EMT and cellular metabolism. Her boarder research interests include cell biology and cancer metabolism.
Cellular transformation is associated with dramatic changes in gene expression, but it is difficult to determine which regulated genes are oncogenically relevant. Here, we describe Pheno-RNA, a general approach to identify candidate genes associated with a specific phenotype. Specifically, we generate a “phenotypic series” by treating a non-transformed breast cell line with a wide variety of molecules that induce cellular transformation to various extents. By performing transcriptional profiling across this phenotypic series, the expression profile of every gene can be correlated with the transformed phenotype. We identify ~200 genes whose expression profiles are very highly correlated with the transformation phenotype, strongly suggesting their importance in transformation. Within biological categories linked to cancer, some genes show high correlations with the transformed phenotype, but others do not. Many genes whose expression profiles are highly correlated with transformation have never been linked to cancer, suggesting the involvement of heretofore unknown genes in cancer.
Rabih Darwiche obtained his B.S. in animal biology at the Lebanese University in Beirut, the capital of Lebanon. He then obtained an M.S. in integrative and molecular biology at the University of Versailles Saint Quentin En Yvelines, and an M.S. in functional biology and biophotonics at the University of Jean Monnet in France. After his Master’s degrees, he has worked in various research and scientific positions at Ghent University in Belgium, and later completed a Ph.D. in Biochemistry from the University of Fribourg in Switzerland in the laboratory of Prof. Roger Schneiter in Switzerland, with a thesis on Lipid homeostasis in yeast from 2012-2016. After earning his doctoral degree, he continued a one-year post-doctoral fellowship in his Ph.D. laboratory. After completing his Post-doc in Switzerland, he received the Swiss National Sciences Foundation fellowship and joined the group of Prof. Kevin Struhl in Boston, the U.S. at the department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School in 2018, specializing in the Cancer Cell Biology in the field of identifying genes and pathways important for cancer.
Abhinav Dey and David Sharp; Affiliation: MicroCures Inc., Bronx NY
Adoptive cell therapy with genetically modified T-cells ex vivo holds the promise of improving outcomes for patients with solid tumors and has the potential to reduce treatment complications for all cancer patients. Although T-cells that express chimeric antigen receptors (CARs) specific for CD19 have had remarkable success for B-cell– derived malignancies, which has led to their approval by the U.S. FDA, CAR T-cells have been less effective for solid tumors.
Lack of CAR T-cell efficacy in solid tumors is multifactorial. Major roadblocks include:
While there is a major focus by Pharma/Biotech/Academic institutions to address the roadblocks 1 and 2 by expansion of the repertoire of targetable antigens or engineering CAR-T cells to resist the immunosuppressive environment, very limited evidence is available on the efforts of enhancing migration of CAR T-cells to tumor sites and within tumors. The current need, thus, remains where every genetically modified CAR T-cell variety can be made to effectively migrate and penetrate the solid tumors. Successful accumulation of T cells in tumors is dependent on expression of several cellular adhesion pathways, including integrins.
At MicroCures, we are developing RNAi therapeutics that can modify T-cell motility to efficiently migrate and penetrate solid tumors. We will present data from our in-house developed assays that show that modifying microtubule assembly of T-cells by targeting Fidgetin-like 2 (FL2) can lead to enhanced tissue-homing and cellular adhesion properties of T-cells with cancer cells. Our technology can potentially be harnessed in adoptive cell therapies where an important goal is to improve the targeting efficiency of engineered CAR-T cells in solid tumors.
Abhi is a Lead Scientist at MicroCures Inc. where he is directing the R&D program for oncology and immuno-oncology. He graduated from the PhD program of Indian Institute of Science (Bangalore, India), specializing in Molecular Biophysics. As a staff scientist at the Aflac Cancer and Blood Disorders Center, he headed the preclinical assay development team for their Precision Medicine program. His team's efforts resulted in the initiation of a Phase 1 Clinical trial for pediatric cancer patients at Emory University. Previously, as a postdoctoral fellow at Emory University (Atlanta, GA), he was the recipient of two Young Investigator Awards in Cancer Research and multiple grants from various foundations.
Checkpoint blockade immunotherapy (CBI) affords systemic and durable anti-tumor immunity by targeting T cell regulatory pathway, yet fails on immunosuppressive tumors with inadequate T cell infiltration. Radiotherapy as local immunomodulatory effects kills tumor cells in an immunogenic mode and alters tumor microenvironment to synergize with CBI. To maximize anti-tumor efficacy as well as immunoadjuvant effect of radiotherapy, intratumorally enriched high-Z radiosensitizers are clinically investigated to enlarge therapeutic index. We developed nanoscale metal-organic frameworks (nMOFs) as next-generation radiosensitizers. Compared to nanoparticle-based radiosensitizer such as HfO2, Hf-based nMOF effectively amplifies energy deposition with ordered structure and facilitates diffusion of reactive species with pores. By crystal engineering, we systemically compared nMOFs with different Hf-oxo clusters to find Hf12-oxo a better X-ray absorber. By molecular engineering, we employed photosensitizing porphyrin-based linker to elicit a new therapeutic modality, radiotherapy-radiodynamic therapy (RT-RDT). Upon low-dose X-ray, Hf12-oxo clusters not only absorb incident energy to generate hydroxyl radical via radiolysis but also scatter secondary energy to proximate linker to produce singlet oxygen. Combination of nMOF-mediated RT or RT-RDT with CBI not only eradicated primary tumor, but also extended local therapeutic efficacy to distant tumor via abscopal effect, suggesting a strong anti-tumor immunity induced by nMOF technology.
Kaiyuan Ni graduated from Xiamen University in 2015 with honor and received his PhD degree at the University of Chicago. His research interests include developing novel radiosensitizer and nanomedicine for cancer treatment as well as imaging probe for cancer diagnosis.
Aayush Gupta (1), Jianling Yuan (1,2), Christopher Wilke (1,2,3), Damien C Mathew(1,2,3)*
Protein phosphatases play an integral role in cellular senescence and have been demonstrated to facilitate malignant transformation of normal healthy cells. The protein phosphatase PP2A is a known tumor suppressor and is composed of three protein chains (A, B and C). Missense mutations at residues 179, 183 and 256 on chain A have been identified in uterine cancer. Here, we utilize dynamic network analysis to computationally investigate interactions within PP2A protein chain A and chain B at the atomic level. Our results demonstrate the need for these residues to be intact in order to stabilize its binding with chain B.
An all-atom model of PP2A was created using previously published x-ray coordinates and molecular dynamics simulations were performed. Communities were formed from the trajectory of the molecular dynamics simulations using the Girvan–Newman algorithm. In this process, a sphere of fixed radius around a given C-alpha atom was created while non-bonded neighbors of the same atom type (C-alpha) were analyzed to determine the likelihood of being inside of the sphere throughout the course of the trajectory. If an atom remains inside of the sphere longer than a threshold amount of time, the atom joins a community with the first atom. The process was repeated across all C-alpha atoms to create a set of communities comprising the entire biomolecule. Nodes are links through which dynamic correlation occurs between atoms. Critical nodes are nodes connecting two communities where there are no other nodes connecting the same communities. Critical nodes from our analysis were compared against known mutations associated with uterine cancer.
Our computation model identified critical nodes to occur at residues 180, 217, and 257, highly correlating with known missense mutation sites of 179, 183, 256. We additionally recreated each reported missense mutation and reran the simulation. Under these simulation conditions, the critical nodes disappeared, implying a loss of the dynamic link between chains A and B and potentially a loss of the protein’s function. Based upon these promising preliminary results, we hypothesize that dynamic network analysis is capable of identifying critical locations in other proteins which exert their tumor suppressing function via protein-protein interaction. Such analysis may facilitate the design of targeted agents for potential therapeutic intervention.
The roles of the Major Histocompatibility Complexes (MHC) in anti-cancer immunity are well established. Cytotoxic immune cells can recognize MHC presentation of unique peptides from altered proteins on the surface of tumor cells (tumor neoantigens) or the downregulation of MHC itself and kill the affected cells. One of the hallmarks of cancer progression is the evasion of anti-tumor immunity by various mechanisms, including the upregulation of checkpoint proteins. Checkpoint inhibitors, which have recently seen dramatic success in the clinic in some cancers, act by inducing potent immune responses to tumor neoantigens. In addition, the promise of personalized medicine lies in the theoretical ability to teach the immune system through vaccination to recognize and kill tumor cells bearing MHC-presented neoantigens. Aside from MHC on cell surfaces, the presence of soluble MHC (sMHC) has been shown in a variety of bodily fluids, including blood. Soluble MHC may be an important component of the tumor arsenal for inhibiting anti-tumor immune responses. The precise function of sMHC may be disease-dependent, but it has been postulated to play an inhibitory role, presumably through signaling cognate TCRs in the absence of a cellular signal (signal 2). Presence of sMHC carrying cancer-associated neoantigens could predict success of vaccination therapy or checkpoint therapy. Furthermore, sMHC-associated peptides could help to identify neoantigens that have already induced T cell anergy and would hence not be good targets. Here, data are presented describing the plasma immunopeptidome of advanced non small-cell lung cancer subjects under a variety of treatment paradigms.
Fang Wang 1,# , Wei Hou 1,#, Lennox Chitsike 1 , Yingchen Xu 2, Carlee Bettler 1, Aldeb Perera 1,
Thomas Bank 1, Scott J. Cotler 3, Asha Dhanarajan 4, Mitchell F. Denning 4, Xianzhong Ding 4,
Peter Breslin 5,6, Wenan Qiang 7, Jun Li 8, Anthony J. Koleske 9 and Wei Qiu 1*
Departments of 1 Surgery and Cancer Biology, 3 Medicine, 4 Pathology, 5 Molecular/Cellular
Physiology and Oncology Institute, 6 Biology, Loyola University Chicago Stritch School of
Medicine, 2160 South 1st Avenue., Maywood, IL 60153, USA
2 Department of General Surgery, Beijing Tongren Hospital, Capital Medical University
7 Department of Obstetrics and Gynecology and Pathology, Northwestern University
8 Department of Applied and Computational Mathematics and Statistics, University of Notre Dame
9 Department of Molecular Biophysics and Biochemistry, Yale University
# these authors contributed equally to this work.
*Correspondence: Wei Qiu, Ph.D., Departments of Surgery and Cancer Biology, Loyola University Chicago, 2160 South 1st Avenue, Bldg. 112 Rm. 338, Maywood, IL
Email: firstname.lastname@example.org; telephone: 708-327-8191; fax: 708-327-3342
Background & Aims: We investigated whether ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1) is involved in development of hepatocellular carcinoma (HCC).
Methods: We analyzed clinical and gene expression data from The Cancer Genome Atlas. Albumin-Cre (HepWT) mice and mice with hepatocyte-specific disruption of Abl1 (HepAbl–/– mice) were given hydrodynamic injections of plasmids encoding the sleeping beauty transposase and transposons with the MET gene and a catenin beta 1 gene with an N-terminal truncation, which induces development of liver tumors. Some mice were then gavaged with the ABL1 inhibitor nilotinib or vehicle (control), daily for 4 weeks. We knocked down ABL1 with short hairpin RNAs in Hep3B and Huh7 HCC cells and analyzed their proliferation and growth as xenograft tumors in mice. We performed RNA sequencing and gene set enrichment analysis of tumors. We knocked down or overexpressed NOTCH1 and MYC in HCC cells and analyzed proliferation. We measured levels of phosphorylated ABL1, MYC, and NOTCH1 by immunohistochemical analysis of an HCC tissue microarray.
Results: HCC tissues had higher levels of ABL1 than non-tumor liver tissues, which correlated with shorter survival times of patients. HepWT mice with the MET and catenin beta 1 transposons developed liver tumors and survived a median 64 days; HepAbl–/– mice with these transposons developed tumors that were 50% smaller and survived a median 81 days. Knockdown of ABL1 in human HCC cells reduced proliferation, growth as xenograft tumors in mice, and expression of MYC, which reduced expression of NOTCH1. Knockdown of NOTCH1 or MYC in HCC cells significantly reduced cell growth. NOTCH1 or MYC overexpression in human HCC cells promoted proliferation and rescued the phenotype caused by ABL1 knockdown. The level of phosphorylated (activated) ABL1 correlated with levels of MYC and NOTCH1 in human HCC specimens. Nilotinib decreased the expression of MYC and NOTCH1 in HCC cell lines, reduced their growth of xenograft tumors in mice, and slowed growth of liver tumors in mice with MET and catenin beta 1 transposons, reducing tumor levels of MYC and NOTCH1.
Conclusions: HCC samples have increased levels of ABL1 compared with non-tumor liver tissues, and increased levels of ABL1 correlate with shorter survival times of patients. Loss or inhibition of ABL1 reduces proliferation of HCC cells and slows growth of liver tumors in mice. Inhibitors of ABL1 might be used for treatment of HCC.
KEY WORDS: hepatocarcinogenesis, mouse model, signal transduction, oncogene
An in-depth characterization of tumor-infiltrating leukocytes (TILs) is crucial to further improve cancer immunotherapies. Since TILs generally constitute only a small subpopulation in solid tumors, they can be lost in the background noise of downstream analyses such as flow cytometry or single-cell sequencing. Therefore, improved methods for pre-enrichment of TILs are necessary to increase the sensitivity of such studies and save resources spent on the analysis of contaminating cell populations. Current enrichment strategies using magnetic labeling of the target cells allow for highly efficient isolation. However, in some instances removal of residual cell surface labeling after isolation is of great importance. Therefore, we have combined the benefits of positive selection by MACS® Technology – the proven state-of-the-art method for the isolation of functional, viable cells – with a novel technology enabling the removal of labeling. The REAlease® Technology provides an easy and fast solution for the highly specific isolation of unlabeled TILs, which can be used to isolate further subpopulations of interest.
There are racial and age-related structural differences in the skin. As a promising imaging modality, optical coherence tomography (OCT) has been used to evaluate the health status of different skin lesions. Visual examination of OCT images has allowed experienced clinicians to diagnose many skin diseases. In addition, the OCT images also contain information that can be extracted using some post-image processing algorithms to aide in diagnosis. Tissue attenuation coefficient is one of the extractable information and may potentially be affected by the amount of pigmentation in different races, as well as the changes in collagen fibers in the aged.
Attenuation coefficient is an optical property that correlates with how easily light penetrates a tissue after considering scattering and absorption events. Therefore, it can provide understanding of the composition of the skin. We studied that how attenuation coefficient is influenced by different skin type (due to different amounts of melanin in the tissue) and age (due to the integrity and density of collagen and elastin fibers).
In this study, 100 participants were recruited. They were separated in 3 different age groups, and 3 different Fitzpatrick skin types. The OCT images were taken from their forehead, and their attenuation coefficient was calculated. By statistically analyzing the attenuation coefficient, we develop a baseline of this property in different skin types and at different ages which may assist in correct utility of analyzing this property in the diagnosis of skin cancer, as well as photodynamic therapy.
Detecting circulating tumor DNA for liquid biopsy applications is challenging because the molecules bearing the target of interest are only a small fraction of the total circulating cell free DNA collected in the sample. Liquid biopsy assays must be able to accurately quantify rare, single nucleotide polymorphisms, among high levels of wild-type background DNA with outstanding sensitivity and precision.
Here we demonstrate the performance of five cancer-relevant mutations in hot-spot genes down to 0.1% mutation allele frequency (MAF) using a novel dPCR platform with a 1-step walkaway workflow which can be completed in under 90 minutes. The platform utilizes a dPCR plate designed with microfluidic array partitioning (MAP) technology, which highly consistent partition yield and enables 90% of the total sample input to be analyzed. These features are critical for rare target detection for precision oncology liquid biopsy applications.
Cancer mutations in KRAS, EGFR, and PIK3CA genes were selected, and for each a series of DNA mixtures with varying mutation allele fractions were prepared by combining normal genomic DNA with mutation-containing plasmid DNA. For each assay, the total number of mutation molecules and observed mutation allele fraction (MAF) were calculated for each point of the titration series (10%, 1%, 0.1% and 0%). We identified a strong correlation between expectations and observations for both the absolute quantities and calculated MAF for all five assays (R = 1.0, p<0.001).
Additionally, MAP technology is not reliant on emulsion-based reagent partitioning, and consistently generates over 20,000 of uniformly filled micro-reactions with no required user interaction. Across 5 plates of dPCR the average number of accepted partitions per unit was 20,457 (±98) - above the typical target of 20,000 for other dPCR platforms. Mutation screening is becoming a standard for evaluating treatment options of patients diagnosed with cancer. With unparalleled precision and sensitivity, digital PCR is ideally suited for liquid biopsy applications in which low amounts of relevant mutations exist in the sample.
CRISPR/Cas9 is an incredibly powerful tool to study biology and can be leveraged for cancer research. Like any laboratory technique, it requires set up and optimization to ensure success. Synthego has specialized in genome engineering, having conducted over 100,000 edits over a 3-year period. We have established workflows to optimize and transfect hundreds of cell lines to enable cancer research. Here we some of our results as well as studies uncovering the mechanisms of cancer and drug discovery using Synthego cell lines by our customers.
Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of the intercellular networks, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and nucleic acids derived from their producing cells, and have potential as biomarkers specific to cell types and cellular states. Flow cytometry is a technique that is generally used for cell marker analysis and sorting; however, analysis of small-sized EVs (50 nm to 150 nm) can be extremely challenging and frustrating.
Here, we have developed a flow cytometric analysis method for Exosome markers using Tim4 immobilized magnetic beads, utilizing the property of PS binding to Exosome through phosphatidylserine (PS) (PS affinity*). In this method, first, Exosome is isolated from the sample using Tim4 immobilized magnetic beads. Next, a fluorescence-labeled Exosome marker antibody is bound to the Exosome bound to the magnetic beads via Tim4. Finally, the complex of magnetic beads, Exosome, and fluorescent-labeled antibody is analyzed by flow cytometry to detect the Exosome marker. The feature of this method is that the whole process from isolation to the detection of Exosome takes only about 2.5 hours, and highly reproducible results can be obtained by a simple experimental operation.
Furthermore, it can be mentioned that the Exosome marker can be detected with high sensitivity as compared with the case of using magnetic beads on which the Exosome marker antibody is immobilized.
Hematopoetic stem cell transplantation (HSCT) has become an indispensable strategy in many malignant and non malignant hematologic conditions. It has made a lot of progress over the years and proved to be an important tool in treating certain type of cancers like multiple myeloma, acute myeloid leukemia (AML), Lymphoma etc. Apart from this, it has also shown potential in rescuing the radiation induced injury to bone marrow caused after a nuclear accident. But there are many limitations to it like timely matching of HLA, Graft vs host disease, rejection, poor homing, engraftment and so on. Although the use of umbilical cord blood stem cells have overcome certain obstacles (HLA matching is not very stringent) but the limited number of HSCs per unit cord blood is still a major drawback. Here in this study, we have a proposed a possible cost effective strategy to enhance the Hematopoietic stem and Progenitor Cell (HSPC) migration, homing, proliferation and their repopulation potential in order to achieve successful and efficient transplantation even when the HSPCs are limited in number. We have reported that pulse exposure of Bone marrow cells to a small molecule increases the CXCR4 expression on HSPCs, thereby enhancing their migration and homing to the bone marrow microenvironment.
Glioblastoma Multiforme (GBM) is the most prevalent and aggressive form of malignant glioma in the United States with a five-year survival rate of only 6.8%.1 Glioma stem cells (GSCs), a subpopulation of undifferentiated, self-renewable, stem-like cells in GBM tumors, have been recently implicated in promoting GBM chemoradioresistance, metastasis, and tumorigenesis.2 Interestingly, 70-80% of low-grade glioma (LGG) and secondary GBM patients carry the IDH1R132H mutation, which has been shown to inhibit GSC aggression by impairing migration and invasion along with promoting GSC differentiation.3 However, the effects of alpha-thalassemia/mental retardation syndrome X-linked gene inactivating mutations (ATRXLoss), which often co-present with IDH1R132H in younger LGG patients, on GSC behavior after chemoradiotherapy is not well understood. Proton radiation therapy has been recognized as an effective treatment modality in LGG and GBM due to its superior ability to target tumors and induce more complex DNA damage than conventional X-ray therapy (XRT).4 Based on previous studies that associate ATRXLoss with XRT sensitivity,5 we hypothesize that ATRXLoss promotes diminished survival compared to ATRXWT in GSCs treated with proton radiation.
To test this hypothesis, we treated TS543-wtATRX and puromycin activated TS543-shATRX isogenic GSCs with 1-4 Gy proton radiation. GSC self-renewal was quantified using extreme limiting dilution analysis (ELDA).6 Our results demonstrate that ATRXLoss significantly impairs GSC self-renewal compared to ATRXWT in response to proton radiation based on neurosphere formation frequency. In the future, we will elucidate changes in the DNA damage repair of ATRXLoss isogenic GSCs to further the development of optimal treatment regiments for IDH1R132H/ATRXLoss glioma patients.
Leisha Kopp is an Applications Scientist at Mirus Bio LLC, a biotech company providing innovative transfection products to cell culture researchers worldwide. Leisha has over 15 years of molecular biology and mammalian cell culture experience in industrial labs, and her combined bench and business knowledge enables support of scientists in all stages of the drug discovery process - from R&D to commercial manufacturing. Leisha is a graduate of the University of Wisconsin-Madison, with key interests in biotherapeutic antibody discover and gene therapy.
Joan Marcano is the Northwest Territory Manager at VectorBuilder, where he focuses his efforts on establishing relationships with customers to help them achieve their research goals. Prior to joining VectorBuilder, Joan was a Postdoctoral Researcher at National Bioenergy Center of the National Renewable Energy Laboratory, Golden CO. In the laboratory, Joan focused on RNA structural biology and the application of RNA regulatory systems for synthetic biology applications.
Joan earned his B.S. in Industrial Biotechnology from the University of Puerto Rico-Mayaguez and his Ph.D. in Biochemistry from the University of Colorado at Boulder.
As a Staff Scientist at Thermo Fisher Scientific, Chris leads a cross-functional team developing fluorescent probes, labeling technologies, and cell-based assays focused on immunotherapy research. Over the last thirteen years, he has developed more than seventy novel reagents for characterizing disease on a cellular level in areas such as innate and adaptive immunity, cell growth and proliferation, apoptosis and cytotoxicity, and endocytosis. Currently he is focused on improving the development of cell therapies and therapeutic antibodies.
Carina Emery earned a BS in biochemistry from the University of Florida and an MS in life sciences from Northwestern University. At Northwestern she used a variety of mouse models to study the molecular basis of genetic disorders affecting the nervous system. The San Diego resident then spent time in the Core Genomics facility at the University of Illinois at Chicago, where she developed a passion for utilizing the latest genomic technologies to advance research. Carina moved onto becoming a Field Applications Scientist for Bio-Rad Laboratories, supporting their digital PCR and single-cell sequencing systems. Providing technical assistance to a wide variety of researchers afforded her the firsthand knowledge that no matter how advanced your molecular analysis technology, it doesn't necessarily compensate for poor quality starting material. These days, as Product Manager for Sample Preparation at Miltenyi Biotec, the dedicated biotech professional focuses on finding solutions to ensure researchers begin their experiments armed with the best possible materials.
Dr. Floriane Cohen is a Field Application Scientist at Bertin Technologies, Rockville, MA. She graduated with a PhD in Biophysics from Sorbonne Université in Paris in 2018. After working at Bertin Technologies as a Field Application Scientist in Montigny-Le-Bretonneux (France), she transferred to Bertin’s American branch, Bertin Corp, to fill the position of Field Application Scientist for the US territory.
Dr. Mathew earned his Ph.D. in Biophysics and Computational Biology at the University of Illinois - Urbana Champaign in 2010. He completed his medical physics residency at the University of Minnesota and is currently an assistant professor at the University of Minnesota Department of Radiation Oncology. Dr. Mathew is also a principal investigator at the Minnesota Supercomputing Institute and has a faculty position at the Masonic Cancer Center at the University of Minnesota. His research interests involve molecular dynamics simulations, network theory related to cancer progression and oncogenesis.
Dr. Julie Rumble studied Immunology at the University of Michigan, focusing on endogenous inhibitors of apoptosis. She went on to a postdoctoral fellowship in Neuroimmunology at the University of Michigan, studying several factors in autoimmune neuroinflammation. Dr. Rumble started at Cayman Chemical in 2015, working in the Assay Development group, and transitioned to the Immunology Group in Contract Services. Dr. Rumble has worked to expand the service offerings in the immunopeptidome profiling area as well as pursue some novel research projects to challenge the field.
Dr. Wang received her Ph.D. from Guangzhou Institutes of Biomedicine and Health (GIBH), University of Chinese Academy of Sciences, Guangzhou, China in 2015. Later, she has joined Loyola University Chicago as a Research Associate until 01/2019. To further study the epigenetic regulators involved in cancers, Dr. Wang joined Northwestern University in Chicago as a Postdoctoral Fellow in 08/2019. Her current research interests are to investigate the epigenetic regulators involved in prostate cancer and to establish new metastasis prostate cancer models.
Qiuyun Xu is a PhD student in Biomedical Engineering at Wayne State University, Detroit, MI. She earned her B.Sc. in Biotechnology at Inner Mongolia Agricultural University, and M.S. in Biomedical Engineering at Wayne State University, Detroit, MI. She has been working on researches related to molecular imaging, noninvasive skin cancer diagnosis, and tissue mimicking phantom design. She is interested in the application of radiomic feature extraction in cancer differential diagnosis.
Christina Bouwens is a Senior Application Scientist at Combinati. She has a background in digital PCR assay development and next generation sequencing technologies with specific focus on applications to cancer biology and precision medicine.
Ania Wronski, Ph.D., is the Senior Product Manager for Engineered Cells. Her primary focus is to enable researchers to improve their science through CRISPR/Cas9 technology. Ania has over a decade of laboratory experience, with a focus on cancer modeling. She has a Ph.D. from the University of Queensland, Australia and completed her postdoctoral training in the lab of Charlotte Kuperwasser at Tufts University, Boston, USA. Her scientific experience includes creating 2D and 3D in vitro and in vivo models, interrogation of gene function using molecular tools and CRISPR-based functional screening. She has also supported researchers working on drug discovery, stem cell biology, protein expression systems and the broad utilization of CRISPR as a Field Application Scientist.
Naomi Tsurutani received her Ph.D. in Virology/Immunology from Tokyo Medical and Dental University in Tokyo, Japan. Before joining FUJIFILM Wako, she worked at Emory University, UMass Medical School, and UConn Health as an immunology researcher.
At FUJIFILM Wako, she helps researchers to better perform and communicate their research by using FUJIFILM Wako products.
We are working in the field of Radiation Biology, especially focussed on the development of radiation counter measures and management of Hematopoetic form of Acute Radiation Syndrome. Apart from this, we are inclined towards finding novel and cost-effective strategies for enhancing the Hematopoietic stem cell transplantation efficiency. Furthermore, we have reported here, a small molecule capable of enhancing the homing of the transplanted Bone marrow cells.
Husam Al-hraishawi is a Ph.D. Student in the Physiology and Integrative Biology program at Rutgers, The State University of New Jersey. Husam received his Bachelor of Science in Veterinary Medicine and Surgery from the University of Baghdad - College of Veterinary Medicine (Baghdad, Iraq) in 1999. He continued his studies at the University of Baghdad, earning his Higher Diploma in Internal and Preventive Veterinary Medicine in 2004 and his Master of Science in Veterinary Medicine and Physiology in 2007. He then joined the faculty of Veterinary School of Basrah University where he stayed for two years. In 2009, he moved to a position at the Medical School of Misan University (Misan, Iraq), where he taught physiology to medical students, conducted independent research and pursued several research projects.
As opportunities for biological research are limited in Iraq, Husam applied for and was accepted into the highly competitive HCED (Higher Committee Education for Development) Program sponsored by the Iraqi Government, and in cooperation with Rutgers University, which led to his current enrollment in the Graduate Program of Molecular Biosciences. After joining Rutgers University, he completed the course requirements and joined the laboratory of Dr. Shridar Ganesan at the Rutgers Cancer Institute of New Jersey where he is currently working on the biology of oncogenic fusion proteins, with a focus on novel therapeutic approaches. After receiving his Ph.D., Husam will establish his own laboratory and continue his lifelong goal of doing research and teaching medical and graduate students. His highest hope is to be able to contribute to cancer research both locally, and to the global community.
Angel Adrian Garces is currently pursuing an M.S. in Biomedical Sciences with a concentration in Therapeutics and Pharmacology under the mentorship of Dr. David Grosshans at the University Of Texas MD Anderson Cancer Center UTHealth Graduate School Of Biomedical Sciences in Houston, TX, USA. He graduated from Rice University in 2018 with a B.S. in Inorganic Chemistry and distinction in chemical research. His current research is focused on elucidating the roles of IDH and ATRX mutations on the efficacy of proton radiation therapy in eliminating glioma stem cells (GSCs), a subpopulation of undifferentiated, self-renewable, stem-like cells present in the tumors of patients presenting with Glioblastoma Multiforme. Angel’s future goal is to translate the results of scientific investigations on the bench into the clinic as a physician-scientist.
Miguel Tam received is PhD in Clinical Immunology from the University of Gothenburg, where he studied Dendritic Cells and the regulation of the Immune System in response to bacterial infections. He continued his postgraduate studies at the University of California San Diego (UCSD) where he continued to characterize the immune response and immune regulation against infections. At UCSD Miguel discovered a known stimulatory receptor that had an opposite function in a subset of dendritic cells. After finishing his postdoctoral research, Miguel joined BioLegend where he has had several roles. Currently, Miguel is leading the Strategic and Product Marketing group for BioLegend.
Throughout the latter part of the 20th century, technologies based on the Coulter principle have helped cell biologists characterize cells and their physiological states. Very few other tools have helped to advance the understanding of cell biology more than flow cytometry. Flow cytometry allows for the detection of fluorophore-conjugated antibodies bound to their targets on individual cells. While flow cytometry has revolutionized cell biology and our understating of biological processes, it has some limitations. An area where further development is needed, is the number of parameters that can be used to characterize cells. Even with the recent advances in instrumentation and number of fluorophores available, the number of parameters or colors that can be used at the same time is in the number of dozens. A recent technology using barcoded, oligonucleotide-conjugated antibodies, has expanded the number of proteins that can be detected at once in a given cell to the hundreds, instead of dozens. This is achieved by using Next Generations Sequencing (NGS) as a readout, instead of detecting fluorescent signals. Furthermore, this revolutionary method combines single cell RNA sequencing analysis with protein detection, providing an unprecedented characterization of individual cells. The technology is powered by our Totalseq™ reagents. In this talk, I discuss multiomic data from the characterization of antigen specific T and B cells using our TotalSeq™ portfolio, and Flex-T™ oligo-conjugated MHC monomers. As our understanding of the cell and its interactions with the environment develops, more powerful and innovative tools are needed to advance the frontiers of science. Next generation tools such as TotalSeq™ are contributing to make this possible.
The PD-1/PD-L1 immune checkpoint pathway is a clinically validated drug target in the suppression of tumor immunity. Despite the clinical success of antibodies in patients with advanced cancers, there are currently no FDA-approved small molecule immunomodulators for this immune checkpoint pathway. Small molecules offer an appropriate pharmacokinetic profile for oral administration, increased tumor penetration, and shorter half-life for management of intractable immune-related side effects. Here, we present our design, synthesis, and a preliminary structure-activity relationship study of a series of C2-symmetric inhibitors. Based on structural studies, the C2-symmetric inhibitor LH1307 (2b) directly bound to PD-L1 and induced the formation of a near symmetrically arranged PD-L1 homodimer. LH1307 demonstrated an IC50 of 3 nM in a homogeneous time resolved fluorescence assay. In a cell-based PD-1/PD-L1 blockade assay, LH1306 (2a) and LH1307 were found to be about 8.2- and 2.8-fold more potent in inhibiting the PPI as compared to LH1301 (1a) and LH1305 (1b), respectively. The new C2-symmetric inhibitors could serve as new leads for further optimization into potential immunomodulators for the treatment of cancer.
Subhadwip Basu is a graduate student pursuing his Ph.D. in Medicinal Chemistry at Rutgers University. He is currently working on the design, synthesis, and biological evaluation of novel small molecule immunomodulators targeting the PD-1/PD-L1 protein-protein interaction. He has a strong passion for the discovery of effective treatments for solid and hematologic malignancies and hopes to make a significant impact in human health.
The RNAscope™ technology allows high sensitivity and spatial resolution providing pivotal single-cell information to gain better insights into tumor immunology, the complex and heterogeneous tumor microenvironment and its dynamics in cancer initiation, progression and metastasis. Using the RNAscope technology, it is possible to simultaneously visualize and assess the gene expression of cytokines, chemokines, immune cell markers, tumor cell markers and checkpoint markers within the tumor microenvironment (TME) in order to:
Anushka Dikshit, Ph.D. is an Application Scientist with Advanced Cell Diagnostics with background in cancer research and immuno-oncology. She is focused on expanding the applications of RNAscope technology for oncology research. Prior to working for ACD, Anushka was involved in identifying novel therapeutic targets for melanoma as a postdoctoral fellow at Duke University. She obtained her doctoral degree from Southern Illinois University, School of Medicine in Molecular Physiology.
Activation of T-cells is used in many different research areas such as cancer, inflammation and autoimmune diseases. Dynabeads coated with antibodies targeting CD3 and CD28 is the most published technology for T cell activation– a technology optimized for consistent activation and of T cells ready for any downstream analysis. Here we describe the activation process of T cells pre-isolated using either manual isolation methods, or the KingFisher Flex automated instrument for higher throughput, reproducibility and ease of use. Here, we present the Toolbox for T-cell activation workflow with the focus on the nature of the target cells being activated in terms of their phenotype and cytokine secretion profile.
Berit has been a Thermo Fisher Scientific Employee for more than 20 years, and is an expert in Dynabeads Magnetic Bead use for purification of cells, proteins, and exosomes.
TANK binding kinase 1 (TBK1) is a non-canonical IkB kinase, that regulates the innate immune response and is highly expressed in lung, breast, pancreatic and colon cancers. It phosphorylates and activates downstream targets such as IRF3 and c-Rel and mediates NF-kB activation and expression of pro-inflammatory genes and interferon’s indicating that enhanced TBK-1 function is associated with autoimmune diseases and cancer modulating anti-tumor immune response. In addition to its role in regulating innate immunity, TBK1 also promotes oncogenesis by phosphorylating Akt and regulating cell growth, proliferation and enhancing autophagy and mitophagy. Therefore TBK1 inhibitors are considered a promising therapy for inflammation and cancer.
Recent studies from our lab revealed a novel role for TBK1 in regulating mammalian cell division. Specifically, levels of active phospho TBK1 increases during mitosis and localize to the centrosomes, mitotic spindles and midbody and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and selective disruption of the TBK1-CEP170 complex augments microtubule stability and triggers defects in mitosis (Smitha et al, 2015). Based on these findings we proposed to see if inhibition of TBK1 would likely have a cytotoxic effect on immune mediated T cell responses.
Pilot experiment performed using JURKAT cells treated with potent TBK1 inhibitor BX795 showed higher percent of cells stained positive for granzyme-b and perforin as compared to control cells by immunofluorescence staining, indicating that microtubule stability of immune cells by TBK1 inhibition may have retained these proteins within the granules of T cells and inhibit its release to mediate apoptosis of target cells. Further we carried out in vitro cytotoxicity assay using normal human peripheral blood mononuclear cells (PBMC’s) treated with BX795 (2.5uM for 24 hrs), activated with concanavalin A (1.25ug/ml) and co-cultured with KRAS mutant A549 lung cancer cells for 72 hrs. Our results showed that BX795 treatment of PBMC’s significantly inhibited the tumor A549 cell lysis as indicated by Annexin-V / PI +ve signal.
These preliminary findings suggest that inhibiting TBK1 may be causing dysfunction of immune mediated T cell functions and enhance anti-tumor immunity due to microtubule stability. Further experiments are ongoing to study its effect on different T cell populations and cytokines.