A dual approach for positive T–S fuzzy controller design and its application to cancer treatment under immunotherapy and chemotherapy

This study proposes an effective positive control design strategy for cancer treatment by resorting to the combination of immunotherapy and chemotherapy. The treatment objective is to transfer the initial number of tumor cells and immune–competent cells from the malignant region into the region of benign growth where the immune system can inhibit tumor growth. In order to achieve this goal, a new modeling strategy is used that is based on Takagi–Sugeno. A Takagi-Sugeno fuzzy model is derived based on the Stepanova nonlinear model that enables a systematic design of the controller. Then, a positive Parallel Distributed Compensation controller is proposed based on a linear co-positive Lyapunov Function so that the tumor volume and administration of the chemotherapeutic and immunotherapeutic drugs is reduced, while the density of the immune-competent cells is reached to an acceptable level. Thanks to the proposed strategy, the entire control design is formulated as a Linear Programming problem. Finally, the simulation results show the effectiveness of the proposed control approach for the cancer treatment

Doctor of Philosophy

dissertationFocused ultrasound (FUS) is a promising noninvasive and radiation-free cancer therapy that selectively delivers high-intensity acoustic energy to a small target volume. This dissertation presents original research that improves the speed, safety, and efficacy of FUS therapies under magnetic resonance imaging (MRI) guidance. First, a new adaptive model-predictive controller is presented that leverages the ability of MRI to measure temperature inside the patient at near real-time speeds. The controller uses MR temperature feedback to dynamically derive and update a patient-specific thermal model, and optimizes the treatment based on the model's predictions. Treatment safety is a key element of the controller's design, and it can actively protect healthy tissue from unwanted damage. In vivo and simulation studies indicate the controller can safeguard healthy tissue and accelerate treatments by as much as 50%. Significant tradeoffs exist between treatment speed, and safety, which makes a real-time controller absolutely necessary for carrying out efficient, effective, and safe treatments while also highlighting the importance of continued research into optimal treatment planning. Next, two new methods for performing 3D MR acoustic radiation force imaging (MR-ARFI) are presented. Both techniques measure the tissue displacement induced by short bursts of focused ultrasound, and provide a safe way to visualize the ultrasound beam's location. In some scenarios, ARFI is a necessity for proper targeting since traditional MR thermometry cannot measure temperature in fat. The first technique for performing 3D ARFI introduces a novel unbalanced bipolar motion encoding gradient. The results demonstrate that this technique is safe, and that 3D displacement maps can be attained time-efficiently even in organs that contain fat, such as breast. The second technique measures 3D ARFI simultaneously with temperature monitoring. This method uses a multi-contrast gradient recalled echo sequence which makes multiple readings of the data without increasing scan time. This improves the signal to noise ratio and makes it possible to separate the effects of tissue heating vs displacement. Both of the 3D MR-ARFI techniques complement the presented controllersince proper positioning of the focal spot is critical to achieving fast and safe treatments

Intratumoral B and T cell receptors: reconstruction and analysis

When cells divide, mistakes happen. However, an intricate surveillance system has evolved to detect and eliminate anomalous cells before they become detrimental to the host organism. In cancer, abnormal cells manage to escape the immune system and grow uncontrollably. In this sense, cancer can be considered as an oversight of the immune system, as immune escape is a defining feature of clinically detectable cancers. The role of the immune system in fighting cancer is becoming increasingly indisputable as our understanding of its underlying mechanisms expand owing to technological advances in genomics, cancer biology, and computational sciences. In particular, significant research effort is undertaken in the field of cancer immunotherapy, where the immune system is stimulated to recognize and attack cancerous cells. In this thesis, I investigate certain aspects of the immune system in the context of cancer by computationally reconstructing and analyzing intratumoral B and T cell receptors. Applying a novel immune cell receptor profiling protocol to original single-cell RNA sequencing (scRNA-seq) data obtained from melanoma patients, I present a complete computational reconstruction of intratumoral immune receptors in this cancer type. The scRNA-seq results are consistent with the presence of an ongoing intratumoral immune response, likely involving tertiary lymphoid structures and the cooperation between B and T cells. Additionally, using a dataset of paired tumor biopsies collected pre- and post-treatment, I show that B cell infiltration increases after immunotherapy in pancreatic and colorectal cancer. This thesis includes the sequences of the most clonally expanded intratumoral antibodies expressed in these biopsies which I computationally reconstructed from bulk RNA sequencing reads. Furthermore, by combining scRNA-seq and immune cell receptor profiling of samples collected from a novel mouse model, I present a comprehensive statistical analysis of gene expression in clonal tumor-reactive T cells. I also show the distribution of tumor-reactive clones across the tumor and spleen. This study forms a first proof-of-principle effort for the in-depth assessment of tumor-reactive T and B cell clones, in-vivo, and paves the way for further, more extensive experiments

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Investigating the role of HIF2a and the type I interferon signalling in cancer hypoxia

293 p.La hipoxia es una de las principales características de los tumores sólidos. A medida que el tumor crece, se generan regiones a las que no llega el suministro de oxígeno y nutrientes debido a la distancia que se encuentran los vasos sanguíneos. Bajo esta circunstancia, las células activan una serie de respuestas para poder sobrevivir, la mayoría de ellas mediadas por los factores de transcripción HIF (del inglés, hypoxia induced factors). HIF está formado por una subunidad ¿ sensible a los niveles de oxígeno, y una subunidad ß. En situaciones normales, la subunidad ¿ es hidroxilada, lo cual permite la unión del complejo von Hippel Lindau (VHL) que estimulará su degradación proteosomal. Sin embargo, en condiciones de hipoxia, HIF¿ no es hidroxilada y puede translocarse al núcleo para dimerizar con la subunidad ß y promover la transcripción de genes para hacer frente a la escasez de oxígeno y promover el desarrollo tumoral.En este sentido, el carcinoma renal de células claras (ccRCC, del inglés clear cell Renal Cell Carcinoma) es un modelo de estudio importante de los factores de transcripción HIF, no solo porque el 80% de los casos presentan el gen VHL inactivo, sino también porque HIF1¿ y HIF2¿ tienen efectos opuestos en laprogresión tumoral. En esta tesis se ha podido observar que HIF2¿ es importante en el crecimiento tumoral, sobre todo cuando la densidad celular es baja, como ocurre al inicio de la enfermedad o de las metástasis. Asimismo, HIF2¿ suprimió la capacidad de migración celular in vitro, pero estimuló la capacidad de invasión tanto in vitro como in vivo, demostrando que HIF2¿ también ejerce un papel fundamental en el desarrollo de las metástasis. Por otro lado, a diferencia de la oxidación fosforilativa que las células llevan a cabo en condiciones normales para obtener la energía, las células tumorales redirigen el metabolismo a la glicolisis aerobia, incluso en presencia de oxígeno, efecto principalmente mediado por HIF1¿. De este modo, las células de ccRCC presentaron menor capacidad de respiración mitocondrial debido a los elevados niveles de HIF2¿.Por otro lado, la hipoxia genera un ambiente inmunosupresor dentro del tumor, impidiendo, por un lado, el establecimiento de células inmune efectoras y, por otro lado, promoviendo la presencia de células inmunosupresoras. La vía del interferón tipo I, importante en la respuesta inmune contra agentes infecciosos cuando detecta material genético foráneo y también relevante en la progresión tumoral, se vio disminuida en condiciones de hipoxia, promoviendo el efecto inmunosupresor. Esta supresión se detectó tanto en células tumorales como en células normales y en condiciones basales o tras haber estimulado la vía con un agente externo que simula ARN de doble cadena (dsRNA), mostrando que el efecto inmunosupresor de la hipoxia es general. Las mitocondrias se han descrito como orgánulos altamente inmunogénicos, debido a su origen bacteriano, y recientemente se ha demostrado que el ARN de origen mitocondrial (mtRNA) es un potente estimulador de la vía del interferón tipo I si es liberado al citoplasma. En esta tesis se demuestra que la inhibición de la vía del interferón tipo I se debe a los bajos niveles de mtRNA en condiciones de hipoxia, probablemente debido a una disminución en la transcripción del ADN mitocondrial, ya que no se detectó un aumento en los niveles de enzimas implicadas en la degradación del mtRNA ni tampoco se vio un aumento en la mitofagia, proceso por el cual la célula elimina mitocondrias no funcionales o dañadas en condiciones de hipoxia. Dado que muchos tratamientos oncológicos requieren la total funcionalidad de la vía del interferón tipo I para ser exitosos, y dado el efecto inmunosupresor ejercido por hipoxia, los resultados presentados en esta tesis sugieren que además de contar con el efecto inmunosupresor ya conocido de hipoxia, se debería tener en cuenta también las causas de esta inmunosupresión, como el reciente descubrimiento de la menor transcripción del ADN mitocondrial