Phase 2 Trial of VGT-309: Cathepsin-Based Near-Infrared Probe for Intraoperative Molecular Imaging.

BACKGROUND: Advances in intraoperative molecular imaging (IMI) may improve surgical outcomes when resecting tumors in the lung. A single-center trial was conducted using VGT-309, a cathepsin-targeted near-infrared (NIR) imaging agent that causes lung nodules to fluoresce during surgery. The endpoint of this Phase 2 study was to evaluate the frequency that IMI with VGT-309 resulted in a clinically significant event (CSE): localization of pulmonary nodules, discovery of unsuspected additional cancers, or identification of positive margins. METHODS: Patients undergoing surgical resection for known or suspected cancer in the lung received VGT-309 (0.32 mg/kg) preoperatively. During surgery, localization and resection of the nodules were performed using standard surgical techniques. NIR imaging was then used to localize nodules, seek occult lesions, and assess resection margins. Efficacy was measured by the frequency of CSEs. RESULTS: Of the 40 patients who underwent pulmonary resection with VGT-309, 17 (42.5%) had at least 1 CSE. NIR imaging identified lesions not found by standard surgical methods in 16 participants, additional cancers not found by pre-operative imaging in 1 patient, and margins within 5 mm of the closest staple line in 2 individuals. VGT-309 performance was tested across a broad range of tumor types and commercial NIR imaging systems. VGT-309 appeared safe, well-tolerated, with no infusion reactions, and no drug-related serious adverse events. CONCLUSIONS: This Phase 2 study demonstrated the utility of IMI with VGT-309 in localizing pulmonary nodules, recognizing synchronous lesions, and identifying positive margins. A multi-institutional study will further evaluate the efficacy of VGT-309.

Association of Risk Factors and Comorbidities With Chronic Pain in the Elderly Population.

INTRODUCTION/OBJECTIVE: Chronic pain disorders affect about 20% of adults in the United States, and it disproportionately affects individuals living in the neighborhoods of extreme socioeconomic disadvantage. In many instances, chronic pain has been noted to arise from an aggregation of multiple risk factors and events. Therefore, it is of importance to recognize the modifiable risk factors. The aim of this study was to investigate the comorbid medical conditions and risk factors associated with chronic pain disorders in patients aged 65 years and older. METHODS: Our team retrospectively reviewed medical records of elderly patients (65 years and older) who were evaluated in our outpatient medicine office between July 1, 2020 and June 30, 2021 for acute problems, management of chronic medical problems, or well visits. We divided our patients into a group who suffered from chronic pain disorder, and another group who did not have chronic pain disorder. The association of variables were compared between those groups. RESULTS: Of the 2431 patients, 493 (20.3%) had a chronic pain disorder. A higher frequency of females in the group with chronic pain disorder was found compared to the group without a chronic pain disorder (60.6% vs 55.2%; P = .033). The mean ages between the two groups were similar in the group with a chronic pain disorder compared to the group without (76.35 ± 7.5 year vs 76.81 ± 7.59 year; P = .228). There were significant associations of certain comorbidities in the group with a chronic pain disorder compared to the group without a chronic pain disorder, such as depression (21.9% vs 15.2%; P < .001), anxiety (27.0% vs 17.1%; P < .001), chronic obstructive pulmonary disease (8.7% vs 6.1%; P = .036), obstructive sleep apnea (16.8% vs 11.6%; P = .002), gastroesophageal reflux disease (40.8% vs 29.0%; P < .001), osteoarthritis (49.3% vs 26.1%; P < .001), other rheumatologic diseases (24.9% vs 19.4%; P = .006), and peripheral neuropathy (14.4% vs 5.3%; P < .001). CONCLUSION: Female sex, depression, anxiety, chronic obstructive pulmonary disease, obstructive sleep apnea, gastroesophageal reflux disease, osteoarthritis, other rheumatologic diseases, and peripheral neuropathy were significantly associated with chronic pain disorder in elderly patients, while BMI was not associated with chronic pain disorder.

Preoperative predictors of successful tumour localization by intraoperative molecular imaging with pafolacianine in lung cancer to create predictive nomogram.

OBJECTIVES: Intraoperative molecular imaging (IMI) uses cancer-targeted fluorescent probe to locate nodules. Pafolacianine is a Food and Drug Administration-approved fluorescent probe for lung cancer. However, it has a 8-12% false negative rate for localization. Our goal is to define preoperative predictors of tumour localization by IMI. METHODS: We performed a retrospective review of patients who underwent IMI using pafolacianine for lung lesions from June 2015 to August 2019. Candidate predictors including sex, age, body mass index, smoking history, tumour size, distance of tumour from surface, use of neoadjuvant therapy and positron emission tomography avidity were included. The outcome was fluorescence in vivo and comprehensively included those who were true or false positives negatives. Multiple imputation was used to handle the missing data. The final model was evaluated using the area under the receiver operating characteristic curve. RESULTS: Three hundred nine patients were included in our study. The mean age was 64 (standard deviation 13) and 68% had a smoking history. The mean distance of the tumours from the pleural surface was 0.4 cm (standard deviation 0.6). Smoking in pack-years and distance from pleura had an odds ratio of 0.99 [95% confidence interval: 0.98-0.99; P = 0.03] and 0.46 [95% confidence interval: 0.27-0.78; P = 0.004], respectively. The final model had an area under the receiver operating characteristic curve of 0.68 and was used to create a nomogram that gives a probability of fluorescence in vivo. CONCLUSIONS: Primary tumours that are deeper from the pleural surface, especially in patients with a higher pack-years, are associated with a decreased likelihood of intraoperative localization. We identified a nomogram to predict the likelihood of tumour localization with IMI with pafolacianine.

Thyroid hormones mediate the impact of early-life stress on ventral tegmental area gene expression and behavior.

Proper thyroid function is essential to the developing brain, including dopamine neuron differentiation, growth, and maintenance. Stress across the lifespan impacts thyroid hormone signaling and anxiety disorders and depression have been associated with thyroid dysfunction (both hypo- and hyper-active). However, less is known about how stress during postnatal development impacts thyroid function and related brain development. Our previous work in mice demonstrated that early-life stress (ELS) transiently impinged on expression of a transcription factor in dopamine neurons, Otx2, shown to be regulated by thyroid hormones. We hypothesized that thyroid hormone signaling may link experience of ELS with transcriptional dysregulation within the dopaminergic midbrain, and ultimately behavior. Here, we find that ELS transiently increases thyroid-stimulating hormone levels (inversely related to thyroid signaling) in both male and female mice at P21, an effect which recovers by adolescence. We next tested whether transient treatment of ELS mice with synthetic thyroid hormone (levothyroxine, LT4) could ameliorate the impact of ELS on sensitivity to future stress, and on expression of genes related to dopamine neuron development and maintenance, thyroid signaling, and plasticity within the ventral tegmental area. Among male mice, but not females, juvenile LT4 treatment prevented hypersensitivity to adult stress. We also found that rescuing developmental deficits in thyroid hormone signaling after ELS restored levels of some genes altered directly by ELS, and prevented alterations in expression of other genes sensitive to the second hit of adult stress. These findings suggest that thyroid signaling mediates the deleterious impact of ELS on VTA development, and that temporary treatment of hypothyroidism after ELS may be sufficient to prevent future stress hypersensitivity.

Fluorescence-guided surgery with indocyanine green to identify an idiopathic chyle leak-case report.

Background: Chylothorax is the leakage of chyle into the pleural space and is associated with up to 50% morbidity. Although, the identification of traumatic chylothoraces is well described, non-traumatic chylothoraxes, mostly idiopathic, present therapeutic challenges as they are difficult to localize. We describe an attempt at localizing and treating an idiopathic chylothorax refractory to conservative and minimally invasive techniques. This was done using indocyanine green (ICG) and was a joint case between a thoracic surgeon and an interventional radiologist. Case Description: A 50-year-old female with a recent history of coronavirus disease (COVID)-19 presented with shortness of breath. She was found to have a right pleural effusion and was admitted to the hospital, where a chest tube was inserted and pleural fluid analysis confirmed the diagnosis of a chylothorax. Conservative management was attempted but with little success. Initial magnetic resonance lymphangiogram (MRL) revealed abnormal enhancing lymphatic masses in the right paraspinal thoracic space as well as lympho-venous junction obstruction with large neck collaterals. She then underwent percutaneous lympho-venous junction angioplasty followed by multiple rounds of glue embolization without clinical improvement. The decision was then made to proceed with a thoracotomy, identification of the site of thoracic duct (TD) leakage, and a mechanical pleurodesis assisted by intraoperative imaging. Ten mg of ICG was injected into the inguinal lymph nodes. Using a camera capable of detection of near-infrared (NIR) light, we were able to visualize the site from which the ICG was extravasating in the chest. Glue was then injected in that area to further help in reducing the leak. After keeping her nil per os (NPO) and requiring one more ligation, a repeat MRL showed a markedly decreased leak into the right pleural space. Two weeks later, she was seen in clinic and reported significant improvement in her symptoms. Conclusions: This is the case of a 50-year-old female who was found to have a significant right chylothorax. She underwent conservative management, followed by tube thoracostomy, and TD ligation but was refractory to treatment. Fluorescence-guided surgery was pivotal to localize the leakage site and help seal it intraoperatively.

A pH-Activatable Nanoprobe Labels Diverse Histologic Subtypes of Human Lung Cancer During Resection.

BACKGROUND: Intraoperative molecular imaging (IMI) uses tumor-targeted optical contrast agents to improve identification and clearance of cancer during surgery. Recently, pH-activatable contrast agents have been developed but none has been tested in lung cancer. Here, we report the successful clinical translation of pegsitacianine (ONM-100), a pH-activatable nanoprobe, for fluorescence-guided lung cancer resection. METHODS: We first characterized the pH setpoint for pegsitacianine fluorescence activation in vitro. We then optimized the specificity, dosing, and timing of pegsitacianine in murine flank xenograft models of lung adenocarcinoma and squamous cell carcinoma. Finally, we tested pegsitacianine in humans undergoing lung cancer surgery as part of an ongoing phase 2 trial. RESULTS: We found that the fluorescence activation of pegsitacianine occurred below physiologic pH in vitro. Using preclinical models of lung cancer, we found that the probe selectively labeled both adenocarcinoma and squamous cell carcinoma xenografts (mean tumor-to-background ratio [TBR] > 2.0 for all cell lines). In the human pilot study, we report cases in which pegsitacianine localized pulmonary adenocarcinoma and pulmonary squamous cell carcinoma (TBRs= 2.7 and 2.4) in real time to illustrate its successful clinical translation and potential to improve surgical management. CONCLUSIONS: This translational study demonstrates the feasibility of pegsitacianine as an IMI probe to label the two most common histologic subtypes of human lung cancer.

Thyroid hormones mediate the impact of early-life stress on ventral tegmental area gene expression and behavior.

Proper thyroid function is essential to the developing brain, including dopamine neuron differentiation, growth, and maintenance. Stress across the lifespan impacts thyroid hormone signaling and anxiety disorders and depression have been associated with thyroid dysfunction (both hypo- and hyper-active). However, less is known about how stress during postnatal development impacts thyroid function and related brain development. Our previous work in mice demonstrated that early-life stress (ELS) transiently impinged on expression of a transcription factor in dopamine neurons shown to be regulated by thyroid hormones. We hypothesized that thyroid hormone signaling may link experience of ELS with transcriptional dysregulation within the dopaminergic midbrain, and ultimately behavior. Here, we find that ELS transiently increases thyroid-stimulating hormone levels (inversely related to thyroid signaling) in both male and female mice at P21, an effect which recovers by adolescence. We next tested whether transient treatment of ELS mice with synthetic thyroid hormone (levothyroxine, LT4) could ameliorate the impact of ELS on sensitivity to future stress, and on expression of genes related to dopamine neuron development and maintenance, thyroid signaling, and plasticity within the ventral tegmental area. Among male mice, but not females, juvenile LT4 treatment prevented hypersensitivity to adult stress. We also found that rescuing developmental deficits in thyroid hormone signaling after ELS restored levels of some genes altered directly by ELS, and prevented alterations in expression of other genes sensitive to the second hit of adult stress. These findings suggest that thyroid signaling mediates the deleterious impact of ELS on VTA development, and that temporary treatment of hypothyroidism after ELS may be sufficient to prevent future stress hypersensitivity.

Strategies to reduce morbidity following pleurectomy and decortication for malignant pleural mesothelioma.

BACKGROUND: Pleurectomy and decortication (PD) in malignant pleural mesothelioma has a high morbidity mostly associated with aspiration pneumonia (PNA), deep vein thrombosis (DVT), and foreign catheter sepsis. We instituted four strategies to reduce these complications and report our experience. METHODS: This was a retrospective review of patients who underwent PD at the University of Pennsylvania between 2015 and 2022. Our patients underwent standard of care PD in addition to tracheostomy and gastrostomy/jejunostomy tube with therapeutic anticoagulation (AC) leading up to surgery. Measured outcomes were postoperative PNA, DVT, and sepsis. The predicted risk of those same outcomes had patients not undergone the interventions was calculated based on the American College of Surgeons (ACS) surgical risk calculator (SRC). A McNemar's test was used to determine whether the risk of having PNA, DVT and sepsis differed between the two subgroups. RESULTS: Fifty-five patients were included in the study. The mean age was 70 years (SD 6.2) with a mean of 21 (SD 19) pack-years of smoking. PNA, DVT, and catheter-related sepsis occurred in 12, four, and seven patients, respectively. Upon using the ACS SRC prediction model of the nonintervention group, PNA, DVT and catheter related sepsis was predicted to occur in 24 (paired data OR 5, 95% CI: 1.4-17.2; McNemar's test p = 0.008), 14 (paired data OR 3.5, 95% CI: 1.15-10.6; McNemar's test p = 0.03), and 17 (paired OR 3, 95% CI: 1.09-8.3; McNemar's test p = 0.04) patients, respectively. DISCUSSION: Patients undergoing tracheostomy creation, therapeutic AC at the time of diagnosis, and gastrostomy tube placement had a reduced risk of aspiration PNA, DVT, and catheter sepsis.

Preclinical Lymph Node Model for Intraoperative Molecular Imaging of Cancer.

Purpose: Lymph node(LN) dissection is part of most oncologic resections. Intraoperatively identifying a positive LN(+ LN), that harbors malignant cells, can be challenging. We hypothesized that intraoperative molecular imaging(IMI) using a cancer-targeted fluorescent prober can identify + LNs. This study aimed to develop a preclinical model of a + LN and test it using an activatable cathepsin-based enzymatic probe, VGT-309. Procedures: In the first model, we used peripheral blood mononuclear cells (PBMC), representing the lymphocytic composition of the LN, mixed with different concentrations of human lung adenocarcinoma cell line A549. Then, they were embedded in a Matrigel® matrix. A black dye was added to mimic LN anthracosis. Model two was created using a murine spleen, the largest lymphoid organ, injected with various concentrations of A549. To test these models, we co-cultured A549 cells with VGT-309. Mean fluorescence intensity(MFI) was. An independent sample t-test was used to compare the average MFI of each A549:negative control ratio. Results: A significant difference in MFI from our PBMC control was noted when A549 cells were 25% of the LN (p = 0.046) in both 3D cell aggregate models-where the LNs native parenchyma is replaced and the one where the tumor grows over the native parenchyma. For the anthracitic equivalents of these models, the first significant MFI compared to the control was when A549 cells were 9% of the LN (p = 0.002) in the former model, and 16.7% of the LN (p = 0.033) in the latter. In our spleen model, we first noted significance in MFI when A549 cells were 16.67% of the cellular composition.(p = 0.02). Conclusions: A + LN model allows for a granular evaluation of different cellular burdens in + LN that can be assessed using IMI. This first exvivo + LN model can be used in preclinical testing of several existing dyes and in creating more sensitive cameras for IMI-guided LN detection.

Bioinspired color-near infrared endoscopic imaging system for molecular guided cancer surgery.

Significance: Fluorescently guided minimally invasive surgery is improving patient outcomes and disease-free survival, but biomarker variability hinders complete tumor resection with single molecular probes. To overcome this, we developed a bioinspired endoscopic system that images multiple tumor-targeted probes, quantifies volumetric ratios in cancer models, and detects tumors in ex vivo samples. Aim: We present a new rigid endoscopic imaging system (EIS) that can capture color images while simultaneously resolving two near-infrared (NIR) probes. Approach: Our optimized EIS integrates a hexa-chromatic image sensor, a rigid endoscope optimized for NIR-color imaging, and a custom illumination fiber bundle. Results: Our optimized EIS achieves a 60% improvement in NIR spatial resolution when compared to a leading FDA-approved endoscope. Ratio-metric imaging of two tumor-targeted probes is demonstrated in vials and animal models of breast cancer. Clinical data gathered from fluorescently tagged lung cancer samples on the operating room's back table demonstrate a high tumor-to-background ratio and consistency with the vial experiments. Conclusions: We investigate key engineering breakthroughs for the single-chip endoscopic system, which can capture and distinguish numerous tumor-targeting fluorophores. As the molecular imaging field shifts toward a multi-tumor targeted probe methodology, our imaging instrument can aid in assessing these concepts during surgical procedures.

Intraoperative molecular imaging: 3rd biennial clinical trials update.

Significance: This third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery. Aim: National and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed. Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's third clinical trials update on IMI were selected to discuss their clinical trials and endpoints. Results: Dyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed. Conclusions: IMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.

Comorbidities and Risk Factors Associated With Insomnia in the Elderly Population.

INTRODUCTION/OBJECTIVES: Sleep disorders affect around 50 to 70 million Americans, with chronic insomnia being the most common, especially in the elderly population. With an 11-fold increase in the US office visits due to insomnia, from 0.8 to 9.4 million, between 1993 and 2015, it is imperative to identify the modifiable risk factors. The aim of our study was to examine the association of risk factors and comorbid medical conditions with insomnia in patients 65 years, and older. METHODS: We performed a retrospective electronic medical record review of the patients aged 65 years and older, who visited our suburban internal medicine office between July 1, 2020 and June 30, 2021. Patients were divided into insomnia group, and the group without insomnia. The associated variables were compared. RESULTS: Among 2431 patients, 247 patients (10.2%) had insomnia. Mean ages of the patients in the insomnia group and the group without insomnia were comparable (77 ± 8.1 year vs 76 ± 7.5 year; P = .211). There was a significantly greater frequency of women in the insomnia group compared to the group without insomnia (63.2% vs 55.5%; P = .022). In the insomnia group, there were significantly higher frequencies of association of certain comorbidities compared to the group without insomnia, such as dementia (6.5% vs 3.4%; P = .015), depression (30.8% vs 14.9%; P < 0.001), anxiety disorder (34.4% vs 17.4%; P < .001), atrial fibrillation (19.4% vs 13.4%; P = .01), and chronic pain disorders (32.8% vs 18.9%; P < .001). Logistic regression analysis showed significantly greater odds of insomnia in patients who had depression (OR = 1.860, 95% CI 1.342-2.576; P < .001), anxiety (OR = 1.845, 95% CI 1.342-2.537; P < .001), and chronic pain disorders (OR = 1.901, 95% CI 1.417-2.549; P < .001). CONCLUSIONS: Female sex, dementia, depression, anxiety, chronic pain disorders, and atrial fibrillation are associated with insomnia in the elderly patients. Presence of depression, anxiety, and chronic pain disorders are associated with greater odds of having insomnia in the elderly patients.

Epidemiological, therapeutic, and survival trends in malignant pleural mesothelioma: A review of the National Cancer Database.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer of the cells lining the pleural cavity with a low overall incidence. The National Cancer Database (NCDB) released in August 2022 updated data that reflect the newest trends in MPM. METHODS: The NCDB was queried for patients diagnosed with MPM between 2004 and 2020. Variables collected included demographics, tumor characteristics, and treatment. Student's t-test and independent-samples proportions test were used for means analysis. Survival was assessed by the Kaplan-Meier method using SPSS version 28. RESULTS: A total of 41,074 patients were diagnosed with mesothelioma, with a steady incidence (0.25%) between 2004 and 2017. The mean age of diagnosis was 70 (SD 13). 73.2% of the patients were males, 69% had no comorbidities, and 93.3% were white. More patients were diagnosed at Stage 1 after 2008 (p < 0.001). Since 2010, there has been a significant increase in patients offered treatment with 73.9% receiving some therapy (p < 0.01): 50.5% received chemotherapy, 27.6% surgery, 8.6% radiation, and 5.4% immunotherapy. The median overall survival was 10.3 months from diagnosis [95% CI: 10.2-10.5]. Risk factors associated with 30-day mortality from surgical intervention included age (OR = 1.02, p < 0.001), male gender (OR = 1.3, p = 0.03), poorly differentiated grade (OR = 2.1, p < 0.001), Stage 4 (OR = 1.4, p = 0014), and epithelioid histology (OR = 0.51, p = 0.03). CONCLUSION: The current management of MPM is based on stage and histologic subtype. Due to the small numbers of patients at most academic centers, the NCDB provides a robust dataset to draw upon broad data points in treatment discussions with patients.

Prospective validation of tumor folate receptor expression density with the association of pafolacianine fluorescence during intraoperative molecular imaging-guided lung cancer resections.

PURPOSE: Pafolacianine, a folate receptor alpha-targeted NIR tracer, has demonstrated clear efficacy in intraoperative molecular imaging-guided (IMI) lung cancer surgery. However, the selection of patients who would benefit from IMI remains challenging given the variability of fluorescence with patient-associated and histopathologic factors. Our goal in this study was to prospectively evaluate whether preoperative FRα/FRß staining can predict pafolacianine-based fluorescence during real-time lung cancer resections. METHODS: This was a prospective study conducted between 2018 and 2022 that reviewed core biopsy and intraoperative data from patients with suspected lung cancer. A total of 196 patients were deemed eligible, of whom core biopsies were taken from 38 patients and assessed for FRα and FRß expression by immunohistochemistry (IHC). All patients underwent infusion of pafolacianine 24 h prior to surgery. Intraoperative fluorescence images were captured with the VisionSense bandpass filter-enabled camera. All histopathologic assessments were performed by a board-certified thoracic pathologist. RESULTS: Of the 38 patients, 5 (13.1%) were found to have benign lesions (necrotizing granulomatous inflammation, lymphoid aggregates) and 1 had metastatic non-lung nodule. Thirty (81.5%) had malignant lesions, with the vast majority (23, 77.4%) being lung adenocarcinoma (7 (22.5%) SCC). None of the benign tumors (0/5, 0%) exhibited in vivo fluorescence (mean TBR of 1.72), while 95% of the malignant tumors fluoresced (mean TBR of 3.11 ± 0.31) compared to squamous cell carcinoma (1.89 ± 0.29) of the lung and sarcomatous lung metastasis (2.32 ± 0.09) (p < 0.01). The TBR was significantly higher in the malignant tumors (p = 0.009). The median FRα and FRß staining intensities were both 1.5 for benign tumors, while the FRα and FRß staining intensities were 3 and 2 for malignant tumors, respectively. Increased FRα expression was significantly associated with the presence of fluorescence (p = 0.01), CONCLUSION: This prospective study sought to determine whether preoperative FRα and FRß expression on core biopsy IHC correlates with intraoperative fluorescence during pafolacianine-guided surgery. These results, although of small sample size, including limited non-adenocarcinoma cohort, suggest that performing FRα IHC on preoperative core biopsies of adenocarcinomas as compared to squamous cell carcinomas could provide low-cost, clinically useful information for optimal patient selection which should be further explored in advanced clinical trials.

Sodium Multivitamin Transporter-Targeted Fluorochrome Facilitates Enhanced Metabolic Evaluation of Tumors Through Coenzyme-R Dependent Intracellular Signaling Pathways.

BACKGROUND: Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT). METHODS: Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293 T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456. RESULTS: CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways. CONCLUSION: These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors.

Cathepsin detection to identify malignant cells during robotic pulmonary resection.

Background: Intraoperative molecular imaging (IMI) uses a fluorescent probe to identify occult cancers. VGT-309 is a quenched activity-based probe that is activated in the presence of cathepsins, enzymes overexpressed in cancer cells, and detected by near-infrared (NIR) light. This study aims to evaluate the sensitivity and the positive predictive value (PPV) of robotic-assisted thoracic surgery (RATS) with intraoperative molecular imaging (RIMI) using VGT-309 to localize tumors using NIR light to detect areas with increased cathepsin activity. Our secondary outcome was to compare RIMI to video-assisted thoracic surgery (VATS) with intraoperative molecular imaging (VIMI). Methods: In a phase 2 clinical trial at the University of Pennsylvania, patients (n=10) with suspicious pulmonary lesions underwent RATS. First, white light was used followed by RIMI to identify tissues with increased cathepsin activity. Then, VIMI was performed to compare the sensitivity and PPV in identifying the cathepsin activity. The resected specimens were then evaluated for fluorescence and underwent histopathological analysis for cathepsin expression. Image analysis was performed using ImageJ software. Statistical analysis was conducted using IBM SPSS Statistics software. A P value of 0.05 or less was considered significant. Results: RATS with white light identified 6 out of the 10 pulmonary nodules, whereas adding RIMI identified an additional 4 more pulmonary nodules. RIMI and VIMI were able to detect the same 8/10 (80%) nodules. The addition of VIMI did not identify any lesions that RIMI may have missed. The mean fluorescence intensity of tumors visualized by RIMI was 115.81 A.U. [standard deviation (SD) =58.57] compared to 95.6 A.U. (SD =14.81) by VIMI (P=0.41). The mean tumor-to-background ratios (TBR) of tumors visualized by RIMI was 9.20 (SD =9.12) compared to 2.29 A.U. (SD =1.11) using VIMI (P=0.1). The sensitivity of RIMI and VIMI was 88.9% which was superior to that of RATS (55.6%). The PPV of RATS was 83.3% compared to 100% in RIMI and VIMI. Conclusions: RIMI is a valuable option for visualization of occult disease using VGT-309-guided IMI through identifying areas of increased cathepsin activity. In this small series, RIMI and VIMI showed clinical equivalence in sensitivity and PPV of detecting cathepsin activity.

Tissue-Engineered Vascular Graft with Co-Culture of Smooth Muscle Cells and Human Endothelial Vein Cells on an Electrospun Poly(lactic-co-glycolic acid) Microtube Array Membrane.

Coronary artery disease is one of the major diseases that plagues today's modern society. Conventional treatments utilize synthetic vascular grafts such as Dacron® and Teflon® in bypass graft surgery. Despite the wide adaptation, these synthetic grafts are often plagued with weaknesses such as low hemocompatibility, thrombosis, intimal hyperplasia, and risks of graft infection. More importantly, these synthetic grafts are not available at diameters of less than 6 mm. In view of these challenges, we strived to develop and adapt the electrospun Poly Lactic-co-Glycolic Acid (PLGA) Microtube Array Membrane (MTAM) vascular graft for applications smaller than 6 mm in diameter. Homogenously porous PLGA MTAMs were successfully electrospun at 5.5-8.5 kV under ambient conditions. Mechanically, the PLGA MTAMs registered a maximum tensile strength of 5.57 ± 0.85 MPa and Young's modulus value of 1.134 ± 0.01 MPa; while MTT assay revealed that seven-day Smooth Muscle Cells (SMCs) and Human Umbilical Vein Endothelial Cells (HUVECs) registered a 6 times and 2.4 times higher cell viability when cultured in a co-culture setting in medium containing α-1 haptaglobulin. When rolled into a vascular graft, the PLGA MTAMs registered an overall degradation of 82% after 60 days of cell co-culture. After eight weeks of culturing, immunohistochemistry staining revealed the formation of a monolayer of HUVECs with tight junctions on the surface of the PLGA MTAM, and as for the SMCs housed within the lumens of the PLGA MTAMs, a monolayer with high degree of orientation was observed. The PLGA MTAM registered a burst pressure of 1092.2 ± 175.3 mmHg, which was sufficient for applications such as small diameter blood vessels. Potentially, the PLGA MTAM could be used as a suitable substrate for vascular engineering.

Early life stress alters transcriptomic patterning across reward circuitry in male and female mice.

Abuse, neglect, and other forms of early life stress (ELS) significantly increase risk for psychiatric disorders including depression. In this study, we show that ELS in a postnatal sensitive period increases sensitivity to adult stress in female mice, consistent with our earlier findings in male mice. We used RNA-sequencing in the ventral tegmental area, nucleus accumbens, and prefrontal cortex of male and female mice to show that adult stress is distinctly represented in the brain's transcriptome depending on ELS history. We identify: 1) biological pathways disrupted after ELS and associated with increased behavioral stress sensitivity, 2) putative transcriptional regulators of the effect of ELS on adult stress response, and 3) subsets of primed genes specifically associated with latent behavioral changes. We also provide transcriptomic evidence that ELS increases sensitivity to future stress through enhancement of known programs of cortical plasticity.

mTORC2 modulates the amplitude and duration of GFAT1 Ser-243 phosphorylation to maintain flux through the hexosamine pathway during starvation.

The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.