Search

Sporadic colorectal cancer initiates with mutations in APC or its degradation target β-catenin, producing TCF-dependent nuclear transcription driving tumorigenesis. The intestinal epithelial receptor, GUCY2C, with its canonical paracrine hormone guanylin, regulates homeostatic signaling along the crypt-surface axis opposing tumorigenesis. Here, we reveal that expression of the guanylin hormone, but not the GUCY2C receptor, is lost at the earliest stages of transformation in APC-dependent tumors in humans and mice. Hormone loss, which silences GUCY2C signaling, reflects transcriptional repression mediated by mutant APC-β-catenin-TCF programs in the nucleus. These studies support a pathophysiological model of intestinal tumorigenesis in which mutant APC-β-catenin-TCF transcriptional regulation eliminates guanylin expression at tumor initiation, silencing GUCY2C signaling which, in turn, dysregulates intestinal homeostatic mechanisms contributing to tumor progression. They expand the mechanistic paradigm for colorectal cancer from a disease of irreversible mutations in APC and β-catenin to one of guanylin hormone loss whose replacement, and reconstitution of GUCY2C signaling, could prevent tumorigenesis

Gastrointestinal cancers remain a tremendous burden on society. Despite advances in therapy options, including chemotherapy and radiation, cancer mortality from recurrences and metastases occur frequently. Cancer stem cells (CSCs) drive disease recurrence and metastasis, as these cells are uniquely equipped to self-renew and evade therapy. Therefore, cancer eradication requires treatment strategies that target CSCs in addition to differentiated cancer cells. This review highlights current literature on therapies targeting CSCs in gastrointestinal cancer.

In 2017, the US Food and Drug Administration approved the first two novel cellular immunotherapies using synthetic, engineered receptors known as chimeric antigen receptors (CARs), tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta), expressed by patient-derived T cells for the treatment of hematological malignancies expressing the B-cell surface antigen CD19 in both pediatric and adult patients. This approval marked a major milestone in the use of antigen-directed "living drugs" for the treatment of relapsed or refractory blood cancers, and with these two approvals, there is increased impetus to expand not only the target antigens but also the tumor types that can be targeted. This state-of-the-art review will focus on the challenges, advances, and novel approaches being used to implement CAR T-cell immunotherapy for the treatment of solid tumors.

Background Many studies have explored molecular markers of carotid plaque development and vulnerability to rupture, usually having examined whole carotid plaques. However, there are regional differences in plaque morphology and known shear-related mechanisms in areas surrounding the lipid core. Objective To determine whether there are regional differences in protein expression along the long axis of the carotid plaque and how that might produce gaps in our understanding of the carotid plaque molecular signature. Methods Levels of 7 inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12 p70, IFN-γ, and TNF-α) and caspase-3 were analyzed in prebifurcation, bifurcation, and postbifurcation segments of internal carotid plaques surgically removed from symptomatic and asymptomatic patients. Expression profiles of miRNAs and mRNAs were determined with microarrays for the rupture-prone postbifurcation segment for comparison with published whole plaque results. Results Expression levels of all proteins examined, except IL-10, were lowest in the prebifurcation segment and significantly higher in the postbifurcation segment. Patient group differences in protein expression were observed for the prebifurcation segment; however, no significant differences were observed in the postbifurcation segment between symptomatic and asymptomatic patients. Expression profiles from postbifurcation carotid plaques identified 4 novel high priority miRNAs differentially expressed between patient groups (miR-214, miR-484, miR-942, and miR-1287) and 3 high-confidence miRNA:mRNA targets, including miR-214:APOD, miR-484:DACH1, and miR-942:GPR56. Conclusion The results demonstrate regional differences in protein expression for the first time and show that focus on the rupture-prone postbifurcation region leads to prioritization for further study of novel miRNA gene regulation mechanisms.

Background Colorectal cancer (CRC) presents a significant public health burden, responsible for the second most cancer-related deaths in the United States, with an increasing incidence in young adults observed globally.1,2 While the blockade of immune checkpoints received FDA approval as a CRC therapeutic, only patients with microsatellite instability, accounting for 15% of sporadic cases, demonstrate partial or complete responses.3 We present a third-generation chimeric antigen receptor (CAR)-T cell directed towards the extracellular domain of the mucosal antigen guanylyl cyclase C (GUCY2C), which is over-expressed in 80% of CRC cases, as a therapeutic alternative for late stage disease. Here, we demonstrate that human GUCY2C CAR-T cells can selectively kill GUCY2C-expressing colorectal cancer cells in vitro and produce inflammatory cytokines in response to antigenic stimulation. Methods Peripheral blood mononuclear (PBMCs) cells were isolated from leukoreduction filters obtained from the Thomas Jefferson University Hospital Blood Donor Center (IRB #18D.495). Magnetic Activated Cell Sorting (MACS) technology was used to negatively select pan-T cells (Miltenyi Biotec), followed by activation and expansion using anti-CD3, anti-CD28, and anti-CD2 coated microbeads (Miltenyi Biotec) and supplemented with IL-7 and IL-15 (Biological Resources Branch Preclinical Biologics Repository – NCI). T-cells were transduced with a lentiviral vector encoding the anti-GUCY2C CAR. Our CAR utilizes a single chain variable fragment of human origin directed towards the extracellular domain of GUCY2C, the CD28 hinge, transmembrane, and intracellular signaling domain (ICD), 4-1BB (CD137) ICD, and CD3ζ ICD. CAR-T cells were used for experiments between 10 to 14 days after activation in vitro using the xCELLigence real time cytotoxicity assay and intracellular cytokine staining. Results GUCY2C-directed CAR-T cells specifically lysed the GUCY2C-expressing metastatic CRC cell line T84, while the control CAR did not. GUCY2C-negative CRC cells were not killed by either. In addition to cell killing, GUCY2C-directed CAR-T cells of both the CD8+ and CD4+ co-receptor lineage produced the inflammatory cytokines IFN-γ and TNFα in response to GUCY2C antigen. Conclusions We demonstrate that human GUCY2C-directed CAR-T cells can selectively target GUCY2C-expressing cancer cells. We hypothesize that GUCY2C-directed CAR-T cells present a viable therapeutic option for metastatic CRC. In vivo animal models to examine this potential are currently on-going. Acknowledgements This work was supported by the Department of Defense Congressionally Directed Medical Research Programs (W81XWH-17-1-0299, W81XWH-191-0263, and W81XWH-19-1-0067) to AES and Targeted Diagnostic & Therapeutics to SAW. AES is also supported by a DeGregorio Family Foundation Award. SAW is supported by the National Institutes of Health (NIH) (R01 CA204881, R01 CA206026, and P30 CA56036), and the Department of Defense Congressionally Directed Medical Research Program W81XWH-17-PRCRP-TTSA. SAW and AES were also supported by a grant from The Courtney Ann Diacont Memorial Foundation. SAW is the Samuel M.V. Hamilton Professor of Thomas Jefferson University. JS, EC, and AZ were supported by an NIH institutional award T32 GM008562 for Postdoctoral Training in Clinical Pharmacology. Ethics Approval This study was approved by the Thomas Jefferson University Institutional Review Board (IRB Control #18D.495) and the Institutional Animal Care and Use Committee (Protocol #01529). References Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin2020;70: 7–30. doi:10.3322/caac.21590 Araghi M, Soerjomataram I, Bardot A, Ferlay J, Cabasag CJ, Morrison DS, et al. Changes in colorectal cancer incidence in seven high-income countries: a population-based study. Lancet Gastroenterol Hepatol 2019;4: 511–518. doi:10.1016/S2468-1253(19)30147-5 Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz H-J, Morse MA, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol 2017;18: 1182–1191. doi:10.1016/S1470-2045(17)30422-9

BackgroundAdenovirus serotype 5 (Ad5) is a commonly used viral vector for transient delivery of transgenes, primarily for vaccination against pathogen and tumor antigens. However, endemic infections with Ad5 produce virus-specific neutralizing antibodies (NAbs) that limit transgene delivery and constrain target-directed immunity following exposure to Ad5-based vaccines. Indeed, clinical trials have revealed the limitations that virus-specific NAbs impose on the efficacy of Ad5-based vaccines. In that context, the emerging focus on immunological approaches targeting cancer self-antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectors.MethodsHere, we evaluated the ability of a chimeric adenoviral vector (Ad5.F35) derived from the capsid of Ad5 and fiber of the rare adenovirus serotype 35 (Ad35) to induce immune responses to the tumor-associated antigen guanylyl cyclase C (GUCY2C).ResultsIn the absence of pre-existing immunity to Ad5, GUCY2C-specific T-cell responses and antitumor efficacy induced by Ad5.F35 were comparable to Ad5 in a mouse model of metastatic colorectal cancer. Furthermore, like Ad5, Ad5.F35 vector expressing GUCY2C was safe and produced no toxicity in tissues with, or without, GUCY2C expression. Importantly, this chimeric vector resisted neutralization in Ad5-immunized mice and by sera collected from patients with colorectal cancer naturally exposed to Ad5.ConclusionsThese data suggest that Ad5.F35-based vaccines targeting GUCY2C, or other tumor or pathogen antigens, may produce clinically relevant immune responses in more (≥90%) patients compared with Ad5-based vaccines (~50%).

Background: Intracortical electrode arrays that can record extracellular action potentials from small, targeted groups of neurons are critical for basic neuroscience research and emerging clinical applications. In general, these electrode devices suffer from reliability and variability issues, which have led to comparative studies of existing and emerging electrode designs to optimize performance. Comparisons of different chronic recording devices have been limited to single-unit (SU) activity and employed a bulk averaging approach treating brain architecture as homogeneous with respect to electrode distribution. New method: In this study, we optimize the methods and parameters to quantify evoked multi-unit (MU) and local field potential (LFP) recordings in eight mice visual cortices. Results: These findings quantify the large recording differences stemming from anatomical differences in depth and the layer dependent relative changes to SU and MU recording performance over 6-months. For example, performance metrics in Layer V and stratum pyramidale were initially higher than Layer II/III, but decrease more rapidly. On the other hand, Layer II/III maintained recording metrics longer. In addition, chronic changes at the level of layer IV are evaluated using visually evoked current source density. Comparison with existing method(s): The use of MU and LFP activity for evaluation and tracking biological depth provides a more comprehensive characterization of the electrophysiological performance landscape of microelectrodes. Conclusions: A more extensive spatial and temporal insight into the chronic electrophysiological performance over time will help uncover the biological and mechanical failure mechanisms of the neural electrodes and direct future research toward the elucidation of design optimization for specific applications.

Chronic implantation of microelectrodes into the cortex has been shown to lead to inflammatory gliosis and neuronal loss in the microenvironment immediately surrounding the probe, a hypothesized cause of neural recording failure. Caspase-1 (aka Interleukin 1β converting enzyme) is known to play a key role in both inflammation and programmed cell death, particularly in stroke and neurodegenerative diseases. Caspase-1 knockout (KO) mice are resistant to apoptosis and these mice have preserved neurologic function by reducing ischemia-induced brain injury in stroke models. Local ischemic injury can occur following neural probe insertion and thus in this study we investigated the hypothesis that caspase-1 KO mice would have less ischemic injury surrounding the neural probe. In this study, caspase-1 KO mice were implanted with chronic single shank 3 mm Michigan probes into V1m cortex. Electrophysiology recording showed significantly improved single-unit recording performance (yield and signal to noise ratio) of caspase-1 KO mice compared to wild type C57B6 (WT) mice over the course of up to 6 months for the majority of the depth. The higher yield is supported by the improved neuronal survival in the caspase-1 KO mice. Impedance fluctuates over time but appears to be steadier in the caspase-1 KO especially at longer time points, suggesting milder glia scarring. These findings show that caspase-1 is a promising target for pharmacologic interventions.

Esophageal carcinoma rarely results in intracranial metastases but when it does, the patient prognosis is grim. Because of its rarity outcomes after stereotactic radiosurgery (SRS) are not known. We sought to evaluate the outcomes of SRS in the management of esophageal cancer that has spread to the brain. This single institution retrospective analysis reviewed our experience with esophageal metastasis from 1987 to 2013. Thirty patients (36 SRS procedures) with a median age of 59 (37-86 years) underwent Gamma knife(®) SRS. The esophageal origin was adenocarcinoma in 26 patients (87%), squamous cell carcinoma in 3 patients (10%), and mixed neuroendocrine carcinoma in 1 patient (3%). Fifteen patients were treated for a single metastasis and 15 patients were treated for multiple metastases for a total of 87 tumors. The median tumor volume was 5.7 cm(3) (0.5-44 cm(3)) with a median marginal dose of 17 Gy (12-20 Gy). The median survival time from the diagnosis of brain metastasis was 8 months and the median survival from SRS was 4.2 months. This corresponded to a 6-month survival of 45% and a 12-month survival of 19% after SRS. A higher KPS at the time of procedure was associated with an increase in survival (p = 0.023). The local tumor control rate in this group was 92%. Four patients had repeat SRS for new metastatic deposits. One patient developed a new neurological deficit after SRS. SRS proved an effective means of providing local control for esophageal metastases to the brain. Concomitant systemic disease progression at the time of brain metastasis resulted in poor long-term survival.

OBJECT Non–small cell lung cancer (NSCLC) is the most frequent cancer that metastasizes to brain. Stereotactic radiosurgery (SRS) has become the management of choice for most patients with such metastatic tumors. Therefore, the authors endeavored to elucidate the survival and SRS outcomes for patients with NSCLC metastasis at their center. METHODS In this single-institution retrospective analysis, the authors reviewed their experience with NSCLC metastasis during a 10-year period from 2001 to 2010. Seven hundred twenty patients underwent Gamma Knife radiosurgery. A total of 1004 SRS procedures were performed, and 3143 tumors were treated. The NSCLC subtype was adenocarcinoma in 386 patients, squamous cell carcinoma in 111 patients, and large cell carcinoma in 34 patients. The median aggregate tumor volume was 4.5 cm3 (range 0.1–88 cm3). RESULTS The median survival time after diagnosis of brain metastasis from NSCLC was 12.6 months, and the median survival after SRS was 8.5 months. The 1-, 2-, and 5-year survival rates after SRS were 39%, 21%, and 10%, respectively. Postradiosurgery survival was decreased in patients treated with prior whole-brain radiation therapy compared with SRS alone (p = 0.003). Aggregate tumor volume was inversely related to survival after SRS (p < 0.001), and the histological subgroups demonstrated significant survival differences (p = 0.023). The overall local tumor control rate in the entire group was 92.8%. One hundred seventy-four patients (24%) underwent repeat SRS for new or resistant metastatic deposits. CONCLUSIONS Stereotactic radiosurgery is an effective means of providing local control for NSCLC metastases. Neurological function and survival benefit from serial patient monitoring and repeat SRS for new tumors.

OBJECT A visual field deficit resulting from the management of an arteriovenous malformation (AVM) significantly impacts a patient's quality of life. The present study was designed to investigate the clinical and radiological outcomes of stereotactic radiosurgery (SRS) performed for AVMs involving the postgeniculate visual pathway. METHODS In this retrospective single-institution analysis, the authors reviewed their experience with Gamma Knife surgery for postgeniculate visual pathway AVMs performed during the period between 1987 and 2009. RESULTS During the study interval, 171 patients underwent SRS for AVMs in this region. Forty-one patients (24%) had a visual deficit prior to SRS. The median target volume was 6.0 cm3 (range 0.4–22 cm3), and 19 Gy (range 14–25 Gy) was the median margin dose. Obliteration of the AVM was confirmed in 80 patients after a single SRS procedure at a median follow-up of 74 months (range 5–297 months). The actuarial rate of total obliteration was 67% at 4 years. Arteriovenous malformations with a volume < 5 cm3 had obliteration rates of 60% at 3 years and 79% at 4 years. The delivered margin dose proved significant given that 82% of patients receiving ≥ 22 Gy had complete obliteration. The AVM was completely obliterated in an additional 18 patients after they underwent repeat SRS. At a median of 25 months (range 11–107 months) after SRS, 9 patients developed new or worsened visual field deficits. One patient developed a complete homonymous hemianopia, and 8 patients developed quadrantanopias. The actuarial risk of sustaining a new visual deficit was 3% at 3 years, 5% at 5 years, and 8% at 10 years. Fifteen patients had hemorrhage during the latency period, resulting in death in 9 of the patients. The annual hemorrhage rate during the latency interval was 2%, and no hemorrhages occurred after confirmed obliteration. CONCLUSIONS Despite an overall treatment mortality of 5%, related to latency interval hemorrhage, SRS was associated with only a 5.6% risk of new visual deficit and a final obliteration rate close to 80% in patients with AVMs of the postgeniculate visual pathway.

Beta-adrenergic receptors (βARs) have long been associated with fear disorders and with learning and memory. However, the contribution of these receptors to Pavlovian fear conditioning, a leading behavioral model for studying fear learning and memory, is still poorly understood. The aim of this study was to investigate the involvement of βAR activation in the acquisition, consolidation and expression of fear conditioning. We focused on manipulations of βARs in the lateral nucleus of the amygdala (LA) because of the well-established contribution of this area to fear conditioning. Specifically, we tested the effects of intra-LA microinfusions of the βAR antagonist, propranolol, on learning and memory for auditory Pavlovian fear conditioning in rats. Pre-training propranolol infusions disrupted the initial acquisition, short-term memory (STM), and long-term memory (LTM) for fear conditioning, but infusions immediately after training had no effect. Further, infusion of propranolol prior to testing fear responses did not affect fear memory expression. These findings indicate that amygdala βARs are important for the acquisition but not the consolidation of fear conditioning.