Probes for INS

Aberrant Ca-CaM signaling has been implicated in various congenital and acquired cardiac pathologies, including arrhythmia, hypertrophy, and HF. We examined the impact of HF induced by trans-aortic constriction (TAC) on the distribution of the three CaM mRNAs (Calm 1,2 and 3) and their key protein target mRNAs (Ryr2, Scn5a, Camk2d, NOS1 and Cacna1c) in cardiomyocytes, using fluorescence in situ hybridization (RNAScope™). HF resulted in specific changes in the pattern of localization of Calms, manifested in redistribution of Calm3 from the cell periphery towards the perinuclear area and enhanced Calm2 attraction to the perinuclear area compared to sham myocytes. Additionally, HF resulted in redistribution of mRNAs for certain CaM target mRNAs. Particularly, NOS1 localization shifted from the cell periphery towards the perinuclear area, Cacna1c, Camk2d and Scn5a abundance increased at the perinuclear area, and Ryr2 attracted even closer to the cell periphery in HF myocytes compared to sham myocytes. The strength of non-random attraction/repulsion was measured as the maximal deviation between the observed distribution of nearest neighbor distances from the distribution predicted under complete spatial randomness. Consistent with the observed alterations in abundance and distribution of CaM and CaM target mRNAs, HF resulted in increased attraction between Calm1 and Scn5a, Ryr2 and Camk2d, between Calm2 and Ryr2 and Camk2d; and between Calm3 and NOS1 and Scn5a. In contrast, the attraction between Calm3 and Ryr2 decreased in HF myocytes compared to sham. Collectively, these results suggest distribution of Calms and their association with key target protein mRNAs undergo substantial alterations in heart failure. These results have new important implications for organization of Ca signaling in normal and diseased heart.

The development and subsequent adaptation of mass cytometry for the histological analysis of tissue sections has allowed the simultaneous spatial characterization of multiple components. This is useful to find the correlation between the genotypic and phenotypic profile of tumor cells and their environment in clinical-translational studies. In this revision, we provide an overview of the most relevant hallmarks in the development, implementation and application of multiplexed imaging in the study of cancer and other conditions. A special focus is placed on studies based on imaging mass cytometry (IMC) and multiplexed ion beam imaging (MIBI). The purpose of this review is to help our readers become familiar with the verification techniques employed on this tool and outline the multiple applications reported in the literature. This review will also provide guidance on the use of IMC or MIBI in any field of biomedical research.

Atrial fibrillation (AF) is commonly observed in patients with hypertension and is associated with pathologically elevated cardiomyocyte stretch. AF triggers have been linked to subcellular Ca2+ abnormalities, while their association with stretch remains elusive. Caveolae are mechanosensitive membrane structures, that play a role in both Ca2+ and cyclic adenosine monophosphate (cAMP) signaling. Therefore, caveolae could provide a mechanistic connection between cardiomyocyte stretch, Ca2+ mishandling, and AF. In isolated mouse atrial myocytes, cell stretch was mimicked by hypotonic swelling, which increased cell width (by ∼30%, p

As the most common type of HPV-independent (HPVI) endocervical adenocarcinomas (ECAs), gastric-type endocervical adenocarcinomas (GEAs) account for approximately 10% of all ECAs Although anti-HER2 therapy has been proven effective in many cancers, it has not been utilized in ECAs including GEAs, which is at least partly due to the lack of a well-defined guideline. Limited available data regarding HER2 in GEAs and ECAs have considerable variations likely caused by variations in tumor types selection, testing methods, and scoring criteria. Here, we selected 58 GEA cases to examine the HER2 status using IHC and FISH and to investigate the prognostic value and their association with other known or potential prognostic factors. When strong complete or lateral/basolateral membranous reactivity in ≥10% tumor cells was used to define HER2 positivity, relatively high prevalence of HER2 overexpression (17.2%, 10/58) and amplification (15.5%, 9/58), as well as high IHC-FISH concordance rate (90%, 9/10) was found in GEAs. A lateral/basolateral staining pattern ('U-shaped') was observed, at least focally, in the majority of HER2-positive (3+) and equivocal (2+) tumors. Notably, considerable heterogeneity of HER2 expression was observed in HER2 positive and equivocal cases (80.0% and 83.3%, respectively). HER2 overexpression and amplification were associated with worse progression-free survival (PFS) (p=0.047 and p=0.032, respectively). PD-L1 expression was associated with worse PFS (p=0.032), while mutant type p53 demonstrated no prognostic significance. Our work laid a solid foundation for the eventual development of a future standard HER testing guideline for GEAs.

Stochastic spiking is a prominent feature of Ca2+ signaling. The main noise source at the cellular level are puffs from inositol-trisphosphate receptor (IP3R) channel clusters in the membrane of the endoplasmic reticulum (ER). While the random cluster activity has been known for decades, a stringent method to derive the puff noise term acting on the cytosolic Ca2+ concentration is still lacking. We adopt a popular description of neural spike generation from neuroscience, the stochastic integrate-and-fire (IF) model, to describe Ca2+ spiking. Our model consists of two components describing i) activity of IP3R clusters and ii) dynamics of the global Ca2+ concentrations in the cytosol and in the ER. Cluster activity is modeled by a Markov chain, capturing the puff. The global Ca2+ concentrations are described by a two-variable IF model driven by the puff current. For the Markov chain we derive expressions for the statistics of interpuff interval, single-puff strength, and puff current assuming constant cytosolic Ca2+, an assumption often well met because the Ca2+ concentrations vary much slower than the cluster activity does. The latter assumption also allows to approximate the driving Ca2+ dependent puff current by a white Gaussian noise. This approximation results in an IF model with nonlinear drift and multiplicative noise. We consider this reduced model in a renewal version and in a version with cumulative refractoriness. Neglecting ER depletion, the stochastic IF model has only one variable and generates a renewal spike train, a point process with statistically independent interspike intervals (ISI). We derive analytical expressions for the mean and coefficient of variation of the ISI and suggest approximations for the ISI density and spike-train power spectrum. Taking into account ER depletion, the two-variable IF model displays cumulative refractoriness as seen in experimental data.

Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but not in those with solid tumors. Macrophages are actively recruited into, and abundantly present in the solid tumor microenvironment (sTME). Tumor- associated macrophages typically evince immunosuppressive behavior, but when engineered to be proinflammatory, may be an ideal vector to administer adoptive cellular therapy in solid tumors. Furthermore, insertion of a CAR on the macrophages confers the ability to selectively recognize and phagocytose antigen overexpressing cancer cells. Additionally, CAR macrophages reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression promotes tumorigenesis and is seen in many cancers, including but not limited to breast and gastroesophageal cancers (Table 1). CT-0508 is a cell product comprised of autologous monocyte-derived pro-inflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreasing tumor burden and prolonging survival in relevant models. CT-0508 cells were safe and effective in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. This is a FIH Phase 1 study to evaluate safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary evidence of efficacy of investigational product CT-0508 in approximately 18 subjects with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2, who have failed available therapies including anti-HER2 therapies where indicated.Filgrastim is being used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. The CT-0508 CAR macrophage product is manufactured, prepared and cryopreserved from mobilized peripheral blood monocytes. The study is enrolling Group 1 subjects, who receive CT-0508 infusion split over D1, 3 and 5. Subjects will be continually assessed for acute and cumulative toxicity. Dose limiting toxicities will be observed and addressed by a Safety Review Committee. Group 2 subjects will follow, and will receive the full CT-0508 infusion on D1. Pre and post treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (PCR, RNA scope), CT-0508 persistence in blood and in the tumor, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others. Clinical trial registry number: NCT04660929 Table 1.HER2 Positivity Frequencies Across Tumor TypesTumor typeHER2 positivity (%)ReferenceBladder cancer8-70Gandour-Edwards et al, 2002;Caner et al, 2008;Laé et al, 2010; Fleischmann et al, 2011;Charfi et al, 2013;Yan et al, 2015Breast cancer11.0-25.0Varga et al, 2013;Stenehjem et al, 2014Cervical cancer2.8-3.9Chavez-Blanco et al, 2004;Yan et al, 2015Colorectal cancer1.6-5.0Schuell et al, 2006;Ingold Heppner et al, 2014;Seo et al, 2014Esophageal cancer12.0-14.0König et al, 2013;Yoon et al, 2013;Wang et al, 2014Extrahepatic Cholangiocarcinoma6.3-9.0Yoshikawa et al, 2008;Yan et al, 2015Gallbladder cancer9.8-12.8Roa et al, 2014;Yan et al, 2015Gastric adenocarcinoma7.0-34.0Rüschoff et al, 2012;Hofmann et al, 2008Ovarian cancer26Slamon et al, 1989Salivary mucoepidermoid carcinomas17.6Glisson et al, 2004Salivary duct carcinoma30-40Skálová et al, 2003; Cornolti et al, 2007; Nardi et al, 2013Testicular cancer2.4Yan et al, 2015Uterine cancer3.0Yan et al, 2015 Citation Format: Yara George Abdou, Debora Barton, Amy Ronczka, Daniel Cushing, Michael Klichinsky, Kim Reiss Binder. A phase 1, first in human (FIH) study of adenovirally transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor (CAR) in subjects with HER2 overexpressing solid tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-03-01.

Background: Adoptive T cell therapies have led to remarkable advances among patients with hematologic malignancies, but not in those with solid tumors. Macrophages are actively recruited into, and abundantly present in the solid tumor microenvironment (sTME). Tumor- associated macrophages typically evince immunosuppressive behavior, but when engineered to be proinflammatory, may be an ideal vector to administer adoptive cellular therapy in solid tumors. Furthermore, insertion of a CAR confers on the macrophages the ability to selectively recognize and phagocytose antigen overexpressing cancer cells. Additionally, CAR macrophages reprogram the sTME and present neoantigens to T cells, leading to epitope spreading and immune memory. Human Epidermal Growth Factor Receptor 2 (HER2) is overexpressed in many cancers, including but not limited to breast and gastroesophageal cancers. CT-0508 is a cell product comprised of autologous monocyte-derived pro-inflammatory macrophages expressing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 induced targeted cancer cell phagocytosis while sparing normal cells, decreased tumor burden and prolonged survival in relevant models. CT-0508 cells were safe in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. Methods: This is a FIH Phase 1 study to evaluate safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary evidence of efficacy of investigational product CT-0508 in approximately 18 subjects with locally advanced (unresectable) or metastatic solid tumors overexpressing HER2 who have failed available therapies including anti-HER2 therapies when indicated. Filgrastim will be used to mobilize autologous hematopoietic progenitor cells for monocyte collection by apheresis. The CT-0508 CAR macrophage product will be manufactured, prepared and cryopreserved from mobilized peripheral blood monocytes. Group 1 subjects will receive CT-0508 infusion split over D1, 3 and 5. Subjects will be continually assessed for acute and cumulative toxicity. Dose limiting toxicities will be observed and addressed by a Safety Review Committee. Group 2 subjects will receive the full CT-0508 infusion on D1. Pre and post treatment biopsies and blood samples will be collected to investigate correlates of safety (immunogenicity), trafficking (PCR, RNA scope), persistence, target antigen engagement, TME modulation (single cell RNA sequencing), immune response (TCR sequencing) and others.

The mitochondrial calcium uniporter is a multi-subunit calcium channel that imports Ca2+ into mitochondria. Its MICU subunits (MICU1, MICU2, and the neuron-specific MICU3) gate the channel by blocking the pore in low Ca2+. Upon local Ca2+ elevation, Ca2+ binds to MICUs to cause MICU unblock, thus opening the pore so Ca2+ can permeate. Previous work using cell lines suggests that the uniporter in mammalian cells is exclusively regulated by a MICU1-MICU2 heterodimer. However, we show here that multiple types of electrically excitable cells, including skeletal muscle and cardiac tissues, can also possess a MICU1-MICU1 homodimer or virtually no MICUs. Kinetic analyses demonstrate that MICU1 has a higher Ca2+ affinity than MICU2, and that without MICUs the uniporter is constitutively open. As a result, uniporters with the MICU1-1 homodimer or no MICUs exhibit higher transport activities, leading to mitochondria accumulating much higher levels of matrix Ca2+. Using a Seahorse assay, we show that cells with MICU1-1 or no MICUs have impaired basal oxidative phosphorylation, likely due to increased ROS and damaged respiratory-complex proteins, including NDUFS3 and COX2. These cells, moreover, are highly susceptible to apoptosis. The disadvantage of employing MICU1-1 or omitting MICUs, however, accompanies strong physiological benefits. We show that in response to intracellular Ca2+ signals, these mitochondria import more Ca2+ and consequently produce more ATP, thus better supplying the energy required for the cellular processes initiated by the Ca2+ signals. In conclusion, this work reveals that tissues can manipulate their mitochondrial calcium uptake properties to suit their unique physiological needs by customizing their MICU regulation of the mitochondrial calcium uniporter.