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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2010)

The dynamic nature of cellular attachment and detachment which regulates cell function must be characterized to understand essential biological phenomena such as embryonic development, inflammatory immune response, wound repair, cancer metastasis, etc. Although extensive efforts have been devoted toward cell motility manipulation for a better understanding cellular dynamics, these efforts have remained in their infancy. A programmable manipulation of adherent-cell motility therefore has remained a challenging task. The goal of this research is to design addressable, multi-functional, and reusable biological platforms, nicknamed biological breadboards (BBBs), to enable the spatiotemporal manipulation of cell motility at cellular and even subcellular levels, offering a new method to analyze cellular dynamics in a quantitative way. In this thesis, this method for cell motility manipulation using BBBs and their applications to quantitative characterizations of single-cellular and intracellular dynamics are summarized.

The BBBs consisting of M×N gold electrodes, identical and independently operated, on Pyrex glass substrate are designed to secure their addressability, thereby achieving a high-degree-of-freedom in programmable manipulation of adherent cell motility. This programmable manipulation of adherent cell motility is implicated by two fundamental mechanisms of cell attachment and detachment. An arginine-glycine-aspartic (RGD) acid-terminated thiol is introduced to achieve those mechanisms by tethering the RGD to the gold electrodes via thiol linker. The first mechanism is solved by a RGD which is a recognition sequence for integrin binding to many extracellular matrix proteins. The RGD-terminated thiol connects a RGD peptide into the gold electrodes using its thiol end which shows a spontaneous chemisorption of R-S-H + Au -> R-S-AU + 1/2H2 where R is a substituent, thereby enhancing cell adhesion on the BBB. The second mechanism depends on a rapid reductive desorption of the thiol-gold self-assembled monolayer under negative bias voltage, R-S-Au + H+ + e- -> R-S-H + Au. This reaction makes the RGD-terminated thiol and even a cell or a part of the cell thereon detached from the gold electrodes. The BBBs are fabricated with microfabrication processes (optical lithography, e-beam evaporation, and lift-off) and their surfaces are modified by two surface-treatments of polyethylene glycol treatment on Pyrex glass and RGD-terminated thiol treatment on gold electrodes. The first is intended to achieve a cell-resistive surface which suppresses cell spreading on them, whereas the second is planned to tether a RGD peptide into gold via thiol linker, thereby providing a cell-adhesive surface.

In the experimental studies with a mouse embryonic fibroblast cell line (NIH 3T3), cellular and subcellular detachments have been made with the fabricated BBBs both to quantitatively characterize cellular and subcellular detachment dynamics and to investigate the addressability, multi-functionality, and reusability of the BBBs, thereby evaluating the BBBs as biological platforms for programmable manipulation of adherent cell motility. In addition, the changes in viscoelastic properties of the detached and retracting thin layer of a NIH 3T3 cell have been investigated.

    Cover page: Programmable Manipulation of Adherent Cell Motility Using Biological Breadboards
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2019)
    BACKGROUND: Surgical orthotopic implantation of human colon cancer tissue to the ceca of mice has been used to mimic behavior of cancer in human patients for the development of precision cancer medicine. However, with the current method of serosal surface implantation (SSI) of pieces of human colon cancer tissue, cancer cells are exposed to the peritoneum, which can artificially increase the rate of peritoneal carcinomatosis (PC) during the disease course. The objective of the present study was to introduce a tumor-sealing method (TSM) and compare it with SSI for the ability to produce clinically-relevant metastases without artificial PC. MATERIALS AND METHODS: HCT116 colon cancer cells transfected with green fluorescence protein (GFP) were cultured and then injected into the subcutaneous layer of athymic nude mice. Subcutaneous tumors were allowed to grow sufficiently to supply adequate tumor for orthotopic implantation. For SSI, a 1 mm3-sized tumor fragment was sutured to partially torn serosa of the cecum. For TSM, the blind end of the cecum was folded over the tumor fragment and sealed with sutures. At 20 days after implantation, all mice were opened to visualize PC by intravital fluorescence imaging. At necropsy, distant metastasis was investigated using frozen section of whole blocks of organs. RESULTS: At 20 days after implantation, PC rates in the SSI group and the TSM group were 80% (12/15) and 20% (3/15), respectively (p<0.001). The liver metastasis rate was 41.7% (5/12) in the SSI group and 50% (5/10) in the TSM group (p=0.696). The lung metastasis rate was 0% (0/12) in the SSI group and 10% (1/10) in the TSM group (p=0.201). The mean survival of mice without PC on the 20th day was significantly longer than that of mice with PC on the 20th day (69.1±14.7 vs. 44.5±12.4 days, p=0.001). CONCLUSION: These results suggest that TSM might be a more patient-like and useful method as a model of metastatic colon cancer than SSI.
      Cover page: Tumor-sealing Surgical Orthotopic Implantation of Human Colon Cancer in Nude Mice Induces Clinically-relevant Metastases Without Early Peritoneal Carcinomatosis.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2023)
      Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.
        Cover page: mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching.
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2021)
        Obesity is a major public health problem related to various chronic health conditions. Lactobacillus species has been reported in obese individuals; however, its role is unknown. We compared the abundance and composition of Lactobacillus species by analyzing feces from 64 healthy control subjects and 88 obese subjects. We isolated one Lactobacillus strain from the feces of a subject with obesity and further analyzed its genetic and molecular features. We found that an increased abundance and higher prevalence of Lactobacillus sakei distinguished the fecal microbiota of the obese group from that of healthy subjects and that it was related to the increased levels of reactive oxygen species (ROS) induced by higher fat intake. The L. sakei ob4.1 strain, isolated from the feces of a subject with obesity, showed high catalase activity, which was regulated by oxidative stress at the gene transcription level. L. sakei ob4.1 maintained colon epithelial cell adhesion ability under ROS stimulation, and treatment with saturated fatty acid increased colon epithelial ROS levels in a dose-dependent manner; however, L. sakei ob4.1 did not change the level of fat-induced colon epithelial ROS. Exposing mice to a high-fat diet revealed that high-fat-diet-induced colon ROS was associated with the increased colonization of L. sakei ob4.1 through catalase activity. Four-week supplementation with this strain in mice fed a high-fat diet did not change their body weights or ROS levels. A high-fat diet induces changes in the colon environment by increasing ROS levels, which provides a colonization benefit to an L. sakei strain with high catalase activity. IMPORTANCELactobacillus provides many health benefits; its various species are widely used as probiotics. However, an increased abundance of Lactobacillus has been reported in obesity, and the role of Lactobacillus strains in obesity remains unknown. We found a high abundance of the Lactobacillus sakei species in a group of obese subjects and examined its relationship with a high-fat diet and reactive oxygen species (ROS) in the feces. To find the underlying mechanism, we analyzed and characterized an L. sakei strain isolated from a severely obese individual. We found that higher gut oxidative stress could link high-fat-diet-induced obesity and L. sakei. This translational research identifies the roles of the host gut environment in the colonization and survival of L. sakei.
          Cover page: High-Fat-Diet–Induced Oxidative Stress Linked to the Increased Colonization of Lactobacillus sakei in an Obese Population
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2008)

          Noninvasive serial monitoring of the fate of transplanted cells would be invaluable to evaluate the potential therapeutic use of human hepatocyte transplantation. Therefore, we assessed the feasibility of bioluminescent imaging using double or triple fusion lentiviral vectors in a NOD-SCID mouse model transplanted with immortalized human fetal hepatocytes. Lentiviral vectors driven by the CMV promoter were constructed carrying reporter genes: firefly luciferase and green fluorescence protein with or without herpes simplex virus type 1 thymidine kinase. Human fetal hepatocytes immortalized by telomerase reconstitution (FH-hTERT) were successfully transduced with either of these fusion vectors. Two million stably transduced cells selected by fluorescence-activated cell sorting were injected into the spleens of NOD-SCID mice pretreated with methylcholanthrene and monocrotaline. The transplanted mice were serially imaged with a bioluminescence charged-coupled device camera after D-luciferin injection. Bioluminescence signal intensity was highest on day 3 (6.10 +/- 2.02 x 10(5) p/s/cm2/sr, mean +/- SEM), but decreased to 2.26 +/- 1.54 x 10(5) and 7.47 +/- 3.09 x 10(4) p/s/cm2/sr on day 7 and 10, respectively (p = 0.001). ELISA for human albumin in mice sera showed that levels were similar to those of control mice on day 2 (3.25 +/- 0.92 vs. 2.84 +/- 0.59 ng/ml, mean +/- SEM), peaked at 18.04 +/- 3.11 ng/ml on day 7, and decreased to 8.93 +/- 1.40 and 3.54 +/- 0.87 ng/ml on day 14 and 21, respectively (p = 0.02). Real-time quantitative RT-PCR showed gene expression levels of human albumin, alpha1-antitrypsin, and transferrin in mouse liver were 60.7 +/- 6.5%, 26.0 +/- 1.4%, and 156.8 +/- 62.4% of those of primary human adult hepatocytes, respectively, and immunohistochemistry revealed cells with human albumin and alpha1-antitrypsin expression in the mouse liver. In conclusion, our study demonstrated that bioluminescent imaging appears to be a sensitive, noninvasive modality for serial monitoring of transplanted hepatic stem cells.

            Cover page: Use of bioluminescent imaging to assay the transplantation of immortalized human fetal hepatocytes into mice.
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2014)
            Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation. Glutamine is catabolyzed to α-ketoglutarate (αKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. Here we demonstrate that mTORC1 positively regulates GLS and glutamine flux through this enzyme. We show that mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, our data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN.
              Cover page: The mTORC1/S6K1 Pathway Regulates Glutamine Metabolism through the eIF4B-Dependent Control of c-Myc Translation
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2018)
              The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.
                Cover page: mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2018)
                Pancreatic cancer is a highly lethal disease in part due to incomplete tumor resection. Targeting by tumor-specific antibodies conjugated with a fluorescent label can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we describe a patient-derived orthotopic xenograft model of pancreatic cancer that recapitulated the disease on a gross and microscopic level, along with physiologic clinical manifestations. We additionally show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively highlight the pancreatic cancer and its metastases in this model with a tumor-to-background ratio of 3.5 (SEM 0.9). The present results demonstrate the clinical potential of this labeling technique for fluorescence-guided surgery of pancreatic cancer.
                  Cover page: Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2018)
                  Cancer of unknown primary (CUP) is a recalcitrant disease with poor prognosis because it lacks standard first-line therapy. CUP consists of diverse malignancy groups, making personalized precision therapy essential. The present study aimed to identify an effective therapy for a CUP patient using a patient-derived orthotopic xenograft (PDOX) model. This paper reports the usefulness of the PDOX model to precisely identify effective and ineffective chemotherapy and to compare the efficacy of S. typhimurium A1-R with first-line chemotherapy using the CUP PDOX model. The present study is the first to use a CUP PDOX model, which was able to precisely distinguish the chemotherapeutic course. We found that a carboplatinum (CAR)-based regimen was effective for this CUP patient. We also demonstrated that S. typhimurium A1-R was more effective against the CUP tumor than first-line chemotherapy. Our results indicate that S. typhimurium A1-R has clinical potential for CUP, a resistant disease that requires effective therapy.
                    Cover page: Patient-derived orthotopic xenograft models for cancer of unknown primary precisely distinguish chemotherapy, and tumor-targeting <i>S. typhimurium</i> A1-R is superior to first-line chemotherapy.
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2024)
                    The role of the serine/glycine metabolic pathway (SGP) has recently been demonstrated in tumors; however, the pathological relevance of the SGP in thyroid cancer remains unexplored. Here, we perform metabolomic profiling of 17 tumor-normal pairs; bulk transcriptomics of 263 normal thyroid, 348 papillary, and 21 undifferentiated thyroid cancer samples; and single-cell transcriptomes from 15 cases, showing the impact of mitochondrial one-carbon metabolism in thyroid tumors. High expression of serine hydroxymethyltransferase-2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is associated with low thyroid differentiation scores and poor clinical features. A subpopulation of tumor cells with high mitochondrial one-carbon pathway activity is observed in the single-cell dataset. SHMT2 inhibition significantly compromises mitochondrial respiration and decreases cell proliferation and tumor size in vitro and in vivo. Collectively, our results highlight the importance of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer and suggest that SHMT2 is a potent therapeutic target.
                      Cover page: Unraveling the role of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer by multi-omics analyses.
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