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EPR oximetry is established as a viable method for measuring the tissue oxygen level (partial pressure of oxygen, pO2) in animal models; however, it has not yet been established for measurements in humans. EPR oximetry requires an oxygen-sensing paramagnetic probe (molecular or particulate) to be placed at the site/organ of measurement, which may pose logistical and safety concerns, including invasiveness of the probe-placement procedure as well as lack of temporal stability and sensitivity for long-term (repeated) measurements, and possible toxicity in the short- and long-term. In the past, we have developed an implantable oxygen-sensing probe, called OxyChip, which we have successfully established for oximetry in pre-clinical animal models (Hou et al. Biomed. Microdevices 20, 29, 2018). Currently, OxyChip is being evaluated in a limited clinical trial in cancer patients. A major limitation of OxyChip is that it is a large (1.4 mm3) implant and hence not suitable for measuring oxygen heterogeneity that may be present in solid tumors, chronic wounds, etc. In this report, we describe the development of a substantially smaller version of OxyChip (0.07 mm3 or 70 cubic micron), called mChip, that can be placed in the tissue of interest using a 23G syringe-needle with minimal invasiveness. Using in vitro and in vivo models, we have shown that the microchip provides adequate EPR sensitivity, stability, and biocompatibility and thus enables robust, repeated, and simultaneous measurement from multiple implants providing mean and median pO2 values in the implanted region. The mChips will be particularly useful for those applications that require repeated measurements of mean/median pO2 in superficial tissues and malignancies.

Oxygen-sensing implants, composed of paramagnetic microcrystals embedded in a biocompatible polymer, are increasingly being used for electron paramagnetic resonance (EPR) oximetry in animal models and human subjects. The implants are stable and designed to stay in the tissues for indefinite periods. However, it is not known whether the crystals that may be exposed on the surface of the implants or leached out from the implants will induce cytotoxicity thereby compromising their biocompatibility over the long term. The goal of the current study was to evaluate the cytotoxicity of the implants and crystalline particulates under in vitro conditions. Apoptosis and cell viability studies were performed using L6, a rat muscle cell line and AsPC-1, a cancer cell line derived from human pancreatic adenocarcinoma. The results indicated that neither the intact implants nor their components elicit cytotoxicity, thus establishing their biocompatibility for use in human subjects.

PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin- and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.

Endothelial diaphragms are subcellular structures critical for mammalian survival with poorly understood biogenesis. Plasmalemma vesicle associated protein (PLVAP) is the only known diaphragm component and is necessary for diaphragm formation. Very little is known about PLVAP regulation. Phorbol esters (PMA) are known to induce de novo PLVAP expression and diaphragm formation. We show that this induction relies on the de novo production of soluble factors that will act in an autocrine manner to induce PLVAP transcription and protein expression. We identified vascular endothelial growth factor‐A (VEGF‐A) signalling through VEGFR2 as a necessary but not sufficient downstream event as VEGF‐A inhibition with antibodies and siRNA or pharmacological inhibition of VEGFR2 only partially inhibit PLVAP upregulation. In terms of downstream pathways, inhibition of MEK1/Erk1/2 MAP kinase blocked PLVAP upregulation, whereas inhibition of p38 and JNK MAP kinases or PI3K and Akt had no effect on PMA‐induced PLVAP expression. In conclusion, we show that VEGF‐A along with other secreted proteins act synergistically to up‐regulate PLVAP in MEK1/Erk1/2 dependent manner, bringing us one step further into understanding the genesis of the essential structures that are endothelial diaphragms.

Endothelial diaphragms are subcellular structures critical for mammalian survival with poorly understood biogenesis. Plasmalemma vesicle associated protein ( PLVAP ) is the only known diaphragm component and is necessary for diaphragm formation. Very little is known about PLVAP regulation. Phorbol esters ( PMA ) are known to induce de novo PLVAP expression and diaphragm formation. We show that this induction relies on the de novo production of soluble factors that will act in an autocrine manner to induce PLVAP transcription and protein expression. We identified vascular endothelial growth factor‐A ( VEGF ‐A) signalling through VEGFR 2 as a necessary but not sufficient downstream event as VEGF ‐A inhibition with antibodies and si RNA or pharmacological inhibition of VEGFR 2 only partially inhibit PLVAP upregulation. In terms of downstream pathways, inhibition of MEK 1/Erk1/2 MAP kinase blocked PLVAP upregulation, whereas inhibition of p38 and JNK MAP kinases or PI 3K and Akt had no effect on PMA ‐induced PLVAP expression. In conclusion, we show that VEGF ‐A along with other secreted proteins act synergistically to up‐regulate PLVAP in MEK 1/Erk1/2 dependent manner, bringing us one step further into understanding the genesis of the essential structures that are endothelial diaphragms.

Electron paramagnetic resonance (EPR) spectroscopy using oxygen-sensing implants can provide reliable and repeated measurements of the partial pressure of oxygen (pO2) over a period of months or longer; however, it does not provide accurate information about the distribution of tissue oxygenation. While EPR imaging has the capability to provide spatially resolved oxygen data, it is time-consuming and not optimized for discrete number of implants. Previous reports suggest multi-site algorithms, which would require either the implants to be aligned in a certain way so as to deconvolve them using a linear magnetic field gradient or sparse imaging of the implants from a small number of 3D projections. In this paper, we present a simpler and much faster method to estimate the pO2 histogram from a composite, single-scan EPR spectrum measured without applying field gradients to separate the EPR signals from multiple randomly placed oxygen-sensing implants. The method is optimized for a discrete number of implants, validated using simulations, experimental phantoms and in animal models. The results established the composite spectral fitting algorithm as a reliable and robust tool for multi-site oximetry.

Pancreatic cancer has a 5-year survival rate below 10% and the treatment options are limited. Signal transducer and activator of transcription (STAT3) is a constitutively expressed protein in human pancreatic cancers and is associated with their poor prognosis. Targeting of STAT3 signaling using novel therapeutic agents is a potential strategy for pancreatic cancer treatment. Diarylidenylpiperidone (DAP) compounds, such as H-4073 and HO-3867, have been shown to be STAT3 inhibitors in several human ovarian cancers. Particularly, HO-3867 is an N-hydroxypyrroline derivative of DAP that has targeted cytotoxicity toward cancer cells without affecting healthy cells. In the present study, we evaluated the anticancer efficacy of H-4073 and HO-3867 in a human pancreatic cell line (AsPC-1). We found that both the compounds exhibited potential cytotoxicity to AsPC-1 cells by inducing G2/M cell-cycle arrest, apoptosis, and cell death, by mitochondrial damage and inhibition of STAT3 phosphorylation. In summary, H-4073 and HO-3867 are cytotoxic to AsPC-1 cells and seem to act through similar mechanisms, including STAT3 inhibition, cell-cycle arrest, and apoptosis.

(1) Summarize revisions made to the implantable resonator (IR) design and results of testing to characterize biocompatibility; (2) Demonstrate safety of implantation and feasibility of deep tissue oxygenation measurement using electron paramagnetic resonance (EPR) oximetry. In vitro testing of the revised IR and in vivo implantation in rabbit brain and leg tissues. Revised IRs were fabricated with 1–4 OxyChips with a thin wire encapsulated with two biocompatible coatings. Biocompatibility and chemical characterization tests were performed. Rabbits were implanted with either an IR with 2 oxygen sensors or a biocompatible-control sample in both the brain and hind leg. The rabbits were implanted with IRs using a catheter-based, minimally invasive surgical procedure. EPR oximetry was performed for rabbits with IRs. Cohorts of rabbits were euthanized and tissues were obtained at 1 week, 3 months, and 9 months after implantation and examined for tissue reaction. Biocompatibility and toxicity testing of the revised IRs demonstrated no abnormal reactions. EPR oximetry from brain and leg tissues were successfully executed. Blood work and histopathological evaluations showed no significant difference between the IR and control groups. IRs were functional for up to 9 months after implantation and provided deep tissue oxygen measurements using EPR oximetry. Tissues surrounding the IRs showed no more tissue reaction than tissues surrounding the control samples. This pre-clinical study demonstrates that the IRs can be safely implanted in brain and leg tissues and that repeated, non-invasive, deep-tissue oxygen measurements can be obtained using in vivo EPR oximetry.

Pancreatic adenocarcinoma is an aggressive cancer with poor clinical prognosis and limited therapeutic options. There is a significant lack of effective, safe, and targeted therapies for successful treatment of pancreatic cancer. In this report, we describe the anticancer efficacy of two novel compounds, N-methylpiperazinyl diarylidenylpiperidone (L-2663) and its pro-nitroxide conjugate (HO-4589) evaluated on human pancreatic adenocarcinoma (AsPC-1) cell line and xenograft tumor in mice. Using flow cytometry, we determined the effect of the L-2663 and HO-4589 drugs in inducing mitochondrial toxicity, triggering cell-cycle arrest, and apoptosis. EPR spectroscopy was used to quantify cellular uptake, metabolic conversion and stability of HO-4589 in cells and in vivo monitoring of tumor oxygenation as a function of growth. The results established different antiproliferative efficacy of the L-2663 and HO-4589 compounds, with a targeted action on cancer cells while being less toxic to noncancerous cells. The study may have important implications in the future designs of safe and effective chemotherapeutic agents for the treatment of pancreatic cancer.

PV1 is an endothelial‐specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour‐bearing mice by single‐dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down‐regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC‐1 and BxPC‐3). The effect observed is because of down‐regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down‐regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.