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- All Subjects: Cancer
- Creators: Lake, Douglas
- Resource Type: Text
Description
Programmed cell death ligand-1 (PD-L1) is an overexpressed protein on many tumor cell types. PD-L1 is involved in normal immune regulation, playing an important role in self-tolerance and controlling autoimmunity. However, ligation of PD-L1 to PD-1 on activated T cells leads to tumor-mediated T cell suppression. Inhibiting the PD-1/PD-L1 pathway has emerged as an effective target for anti-tumor immunotherapies. Monoclonal antibodies (mAbs) targeting tumor-associated antigens such as PD-L1 have proven to be effective checkpoint blockades, improving therapeutic outcomes for cancer patients and receiving FDA approval as first line therapies for some cancers. A single chain variable fragment (scFv) is composed of the variable heavy and light chain regions of a mAb, connected by a flexible linker. We hypothesized that scFv proteins based on the published anti-PD-L1 monoclonal antibody sequences of atezolizumab and avelumab would bind to cell surface PD-L1. Four single chain variable fragments (scFvs) were constructed based on the sequences of these mAbs. PCR was used to assemble, construct, and amplify DNA fragments encoding the scFvs which were subsequently ligated into a eukaryotic expression vector. Mammalian cells were transfected with the scFv and scFv-IgG plasmids. The scFvs were tested for binding to PD-L1 on tumor cell lysates by western blot and to whole tumor cells by staining and flow cytometry analysis. DNA sequence analysis demonstrated that the scFv constructs were successfully amplified and cloned into the expression vectors and recombinant scFvs were produced. The binding capabilities of the scFvs constucts to PD-L1 protein were confirmed by western blot and flow cytometry analysis. This lead to the idea of constructing a CAR T cell engineered to target PD-L1, providing a possible adoptive T cell immunotherapy.
ContributorsPfeffer, Kirsten M. (Author) / Lake, Douglas (Thesis director) / Ho, Thai (Committee member) / Hastings, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Through a standpoint feminist perspective (Harding 2009) I conducted a situational analysis (Clarke, 2015) that examined academic literature and cancer support discussion boards (DBs) to identify how Western biomedicine, specifically oncology, can integrate complementary and alternative medicine (CAM) to improve cancer treatment in children. The aims of this project were: 1) to identify the CAM treatments that are being used to alleviate the side effects from oncological treatments and/or treat pediatric cancers; 2) to compare the subjective experience of CAM to Western biomedicine of cancer patients who leave comments on Group Loop, Cancer Compass and Cancer Forums, which are online support groups (N=20). I used grounded theory and situational mapping to analyze discussion threads. The participants identified using the following CAM treatments: herbs, imagery, prayer, stinging nettle, meditation, mind-body therapies and supplements. The participants turned to CAM treatments when their cancer was late-stage or terminal, often as an integrative and not exclusively to treat their cancer. CAM was more "effective" than biomedical oncology treatment at improving their overall quality of life and functionality. We found that youth on discussion boards did not discuss CAM treatments like the adult participants, but all participants visited these sites for support and verification of their cancer treatments. My main integration recommendation is to combine mind-body CAM therapies with biomedical treatment. This project fills the gap in literature that ignores the ideas of vulnerable populations by providing the experiences of adult and pediatric cancer patients, and that of their families. It is applicable to areas of the social studies of medicine, patient care, and families suffering from cancer. KEYWORDS: Cancer; Complementary and Alternative Medicine; Situational Analysis; Standpoint Feminism
ContributorsEsposito, Sydney Maria (Author) / Martinez, AirÃÂn (Thesis director) / Hruschka, Daniel (Committee member) / School of Human Evolution and Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Exosomes have been known to secrete an increased amount of miRNA and noncoding genes that are abnormally expressed in various cancer subtypes. Thus, they may be an early marker for pediatric cancer types that are more difficult to diagnosis without invasive techniques, and may also help identify progression of the disease. In the project, six types of pediatric cancer cell lines, along with their extracted exosomes, were analyzed and tested for different monoclonal antibodies through western blot analysis. The genes EWS-FLI1 and FGFR4 were also identified in some cancer cell lines through Reverse-Transcriptase Polymerase Chain Reaction analysis (RT-PCR). The results were indicative of similar protein markers being found in both the originating cells and their corresponding exosomes.
ContributorsKaur Bhinder, Harsimran (Author) / Lake, Douglas (Thesis director) / Azorsa, David (Committee member) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Cancer is one of the leading causes of death in the world and represents a tremendous burden on patients, families and societies. S. Typhimurium strains are specifically attracted to compounds produced by cancer cells and could overcome the traditional therapeutic barrier. However, a major problem with using live attenuated Salmonella as anti-cancer agents is their toxicity at the dose required for therapeutic efficacy, but reducing the dose results in diminished efficacy. In this project, we explored novel means to reduce the toxicity of the recombinant attenuated Salmonella by genetically engineering those virulence factors to facilitate maximal colonization of tumor tissues and reduced fitness in normal tissues. We have constructed two sets of Salmonella strains. In the first set, each targeted gene was knocked out by deletion of the gene. In the second set, the predicted promoter region of each gene was replaced with a rhamnose-regulated promoter, which will cease the synthesis of these genes in vivo, a rhamnose-free environment.
ContributorsBenson, Lee Samuel (Author) / Kong, Wei (Thesis director) / Martin, Thomas (Committee member) / Lake, Douglas (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Center for Infectious Diseases and Vaccinology (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of the SFK member Lyn as determined by multiplex Luminex assay and verified by immunoprecipitation. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells.
ContributorsJameson, Nathan Meade (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Tran, Nhan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in vivo tumor growth study utilizing the pancreatic tumor cell line MIAPaCa-2 containing a QSOX1-silencing shRNA construct revealed that QSOX1 expression supports a proliferative phenotype. These preliminary studies suggest that suppressing the enzymatic activity of QSOX1 could represent a novel therapeutic strategy to inhibit proliferation and invasion of malignant neoplasms.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
ContributorsHanavan, Paul D (Author) / Lake, Douglas (Thesis advisor) / LaBaer, Joshua (Committee member) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
Quiescin Sulfhydryl Oxidase 1 (QSOX1) generates disulfide bonds in its client substrates via oxidation of free thiols. Localized to the Golgi and secreted, QSOX1 helps to fold proteins into their active form. Early work with QSOX1 in cancer began with the identification of a peptide from the long form of QSOX1 in plasma from patients with pancreatic ductal adenocarcinoma. Subsequent work confirmed the overexpression of QSOX1 in numerous cancers in addition to pancreatic, including those originating in the breast, lung, brain, and kidney. For my work, I decided to answer the question, “How does inhibition of QSOX1 effect the cancer phenotype?” To answer this I sought to fulfill the following goals A) determine the overexpression parameters of QSOX1 in cancer, B) identify QSOX1 small molecule inhibitors and their effect on the cancer phenotype, and C) determine potential biological effects of QSOX1 in cancer. Antibodies raised against rQSOX1 or a peptide from QSOX1-L were used to probe cancer cells of various origins for QSOX1 expression. High-throughput screening was utilized to identify 3-methoxy-n-[4(1pyrrolidinyl)phenyl]benzamide (SBI-183) as a lead inhibitor of QSOX1 enzymatic activity. Characterization of SBI-183 activity on various tumor cell lines revealed inhibition of viability and invasion in vitro, and inhibition of growth, invasion, and metastasis in vivo, a phenotype that was consistent with QSOX1 shKnockdown cells. Subsequent work identified 3,4,5-trimethoxy-N-[4-(1-pyrrolidinyl)phenyl]benzamide (SPX-009) as an SBI-183 analog with stronger inhibition of QSOX1 enzymatic activity, resulting in a more potent reduction in tumor invasion in vitro. Additional work with QSOX1 shKnockdown and Knockout (KO) cell lines confirmed current literature that QSOX1 is biologically active in modulation of the ECM. These results provide evidence for the master regulatory role of QSOX1 in cancer, making it an attractive chemotherapeutic target. Additionally, the small molecules identified here may prove to be useful probes in further elucidation of QSOX1 tumor biology and biomarker discovery.
ContributorsFifield, Amber (Author) / Lake, Douglas (Thesis advisor) / Ho, Thai (Committee member) / Rawls, Jeffery (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2020
Description
Cancer researchers have traditionally used a handful of markers to understand the origin of tumors and to predict therapeutic response. Additionally, performing machine learning activities on disparate data sources of varying quality is fraught with inherent bias. The Caris Life Sciences Molecular Database (CMD) is an immense resource for discovery as it contains over 215,000 molecular profiles of tumors with consistently gathered clinical grade molecular data along with immense amounts of clinical outcomes data. This resource was leveraged to generate two artificial intelligence algorithms aiding in diagnosis and one for therapy selection.
The Molecular Disease Classifier (MDC) was trained on 34,352 cases and tested on 15,473 unambiguously diagnosed cases. The MDC predicted the correct tumor type out of thirteen possibilities in the labeled data set with sensitivity, specificity, PPV, and NPV of 90.5%, 99.2%, 90.5% and 99.2% respectively when considering up to 5 predictions for a case.
The availability of whole transcriptome data in the CMD prompted its inclusion into a new platform called MI GPSai (MI Genomic Prevalence Score). The algorithm trained on genomic data from 34,352 cases and genomic and transcriptomic data from 23,137 cases and was validated on 19,555 cases. MI GPSai can predict the correct tumor type out of 21 possibilities on 93% of cases with 94% accuracy. When considering the top two predictions for a case, the accuracy increases to 97%.
Finally, a 67 gene molecular signature predictive of efficacy of oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer was developed - FOLFOXai. The signature was predictive of survival in an independent real-world evidence (RWE) dataset of 412 patients who had received FOLFOX/BV in 1st line and inversely predictive of survival in RWE data from 55 patients who had received 1st line FOLFIRI. Blinded analysis of TRIBE2 samples confirmed that FOLFOXai was predictive of OS in both oxaliplatin-containing arms (FOLFOX HR=0.629, p=0.04 and FOLFOXIRI HR=0.483, p=0.02).
The Molecular Disease Classifier (MDC) was trained on 34,352 cases and tested on 15,473 unambiguously diagnosed cases. The MDC predicted the correct tumor type out of thirteen possibilities in the labeled data set with sensitivity, specificity, PPV, and NPV of 90.5%, 99.2%, 90.5% and 99.2% respectively when considering up to 5 predictions for a case.
The availability of whole transcriptome data in the CMD prompted its inclusion into a new platform called MI GPSai (MI Genomic Prevalence Score). The algorithm trained on genomic data from 34,352 cases and genomic and transcriptomic data from 23,137 cases and was validated on 19,555 cases. MI GPSai can predict the correct tumor type out of 21 possibilities on 93% of cases with 94% accuracy. When considering the top two predictions for a case, the accuracy increases to 97%.
Finally, a 67 gene molecular signature predictive of efficacy of oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer was developed - FOLFOXai. The signature was predictive of survival in an independent real-world evidence (RWE) dataset of 412 patients who had received FOLFOX/BV in 1st line and inversely predictive of survival in RWE data from 55 patients who had received 1st line FOLFIRI. Blinded analysis of TRIBE2 samples confirmed that FOLFOXai was predictive of OS in both oxaliplatin-containing arms (FOLFOX HR=0.629, p=0.04 and FOLFOXIRI HR=0.483, p=0.02).
ContributorsAbraham, Jim (Author) / Spetzler, David (Thesis advisor) / Frasch, Wayne (Thesis advisor) / Lake, Douglas (Committee member) / Compton, Carolyn (Committee member) / Arizona State University (Publisher)
Created2020
Description
Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis advisor) / Mason, Hugh (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2020
Description
Human Papillomavirus (HPV) is the most commonly transmitted STI and isresponsible for an estimated 5% of cancer cases worldwide. HPV infection is implicated
in 70% of cervical cancer incidence and is also responsible for a variety of oropharyngeal
and anogenital cancers. While vaccination has greatly reduced the cervical cancer
burden in developed countries, HPV infection remains high in developing countries due
to high cost and poor access to healthcare. Several studies have highlighted the
presence of anti-HPV antibodies following infection and their potential use as
biomarkers for developing novel screening methods. Progression from initial infection to
cancer is slow, thus presenting an opportunity for effective screening programs.
Biomarker screening is an important area of cancer detection and Lateral Flow Assays
(LFA) are a low cost, easy to use alternative to other screening methods that require
extensive training and laboratory space. Therefore, this project proposes as a hypothesis
that the development of an LFA screening for HPV specific IgG can provide clinically
relevant data for the early detection of cervical dysplasia. This project adapts an LFA in a
multiplexed format for fluorescence-based serologic detection of HPV specific IgG in
patient plasma.
ContributorsJohns, William (Author) / Anderson, Karen (Thesis advisor) / Lake, Douglas (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2023