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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2018)

Cancer has long ranked as #1 in the view of doctors and health professionals due to its high death rate. Recent development of molecular engineering and synthetic biology sheds lights on cancer diagnosis and treatment. For example, CAR-T based immunotherapy has become an emerging field in cancer therapeutics. However, major challenges still remain for cancer diagnosis and therapeutics, such as time-consuming detection in cancer diagnosis and uncontrolled cytotoxicity in CAR-T therapy. Therefore, there is a critical need to engineer molecular tools for more rapid and better control of live cells.

Rapid and efficient measurement of cancer cells is a major challenge in early cancer diagnosis. Fluorescent proteins have revolutionized biology by allowing live cell visualization. Therefore, biosensors based on fluorescent proteins could potentially be developed as the point-of-care detection methods of live cancer cells. In chapter 2 we discussed the development of a FRET based EphA4 biosensor. EphA4 is involved in cellular mechanotransduction and is an engaging target for cancer research. We discovered that EphA4 has different activation at subcellular membrane locations in single live cells, which provides powerful tools to monitor the dynamic molecular activities at subcellular compartments.

Various synthetic biology approaches have been adopted for targeted cancer therapeutics. For example, optogenetics integrating optical and genetics has enabled the sensing and control of molecular events in living systems. However, there is a critical need to remotely manipulate live cells with high precision deep in the body. Mechanical manipulation, in contrast to optogenetics, could allow deep and non-invasive control of these cells. Chapter 3 goes on to discuss the combination of developing a laser based mechanical stimulation tools and molecular biosensors to activate and study mechano-sensitive cells. Together, Chapter 2 and Chapter 3 built the basis for developing and integrating engineered molecular tools with novel clinical therapeutic stimulation devices. Chapter 4, as a major work of this thesis, presents the engineering of a remote controlled mechanogenetics system which integrates ultrasound mediated mechanical stimulation and gene activation of CD19CAR for controllable application in cancer immunotherapy. Collectively, our suite of engineered molecular tools shows potential in extrapolation to advance biomedical and clinical applications.

    Cover page: Development of mechanogenetic tools for subcellular applications in cancer immunotherapy
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2015)
    Cells within the body are subject to various forces; however, the details concerning the way in which cells respond to mechanical stimuli are not well understood. We demonstrate that laser-induced shockwaves (LIS) combined with biosensors based on fluorescence resonance energy transfer (FRET) is a promising new approach to study biological processes in single live cells. As "proof-of-concept," using a FRET biosensor, we show that in response to LIS, cells release intracellular calcium. With the parameters used, cells retain their morphology and remain viable. LIS combined with FRET permits observation of the cells immediate response to a sudden shear force.
      Cover page: Laser‐induced shockwave paired with FRET: A method to study cell signaling
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2018)
      While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2021)
        Focused ultrasound can deliver energy safely and non-invasively into tissues at depths of centimetres. Here we show that the genetics and cellular functions of chimeric antigen receptor T cells (CAR-T cells) within tumours can be reversibly controlled by the heat generated by short pulses of focused ultrasound via a CAR cassette under the control of a promoter for the heat-shock protein. In mice with subcutaneous tumours, locally injected T cells with the inducible CAR and activated via focused ultrasound guided by magnetic resonance imaging mitigated on-target off-tumour activity and enhanced the suppression of tumour growth, compared with the performance of non-inducible CAR-T cells. Acoustogenetic control of the activation of engineered T cells may facilitate the design of safer cell therapies.
          Cover page: Control of the activity of CAR-T cells within tumours via focused ultrasound.
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2022)
          Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.
            Cover page: Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signalingCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2018)
            The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.
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