Probes for INS

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.

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.

Epithelial-mesenchymal signaling in the gastrointestinal system is vital in establishing regional identity during organogenesis and maintaining adult stem cell homeostasis. Although recent work has demonstrated that Wnt ligands expressed by mesenchymal cells are required during gastrointestinal development and stem cell homeostasis, epigenetic mechanisms driving spatiotemporal control of crosstalk remain unknown. Here, we demonstrate that gastrointestinal mesenchymal cells control epithelial fate and function through Polycomb Repressive Complex 2-mediated chromatin bivalency. We find that while key lineage-determining genes possess tissue-specific chromatin accessibility, Polycomb Repressive Complex 2 controls Wnt expression in mesenchymal cells without altering accessibility. We show that reduction of mesenchymal Wnt secretion rescues gastrointestinal fate and proliferation defects caused by Polycomb Repressive Complex 2 loss. We demonstrate that mesenchymal Polycomb Repressive Complex 2 also regulates niche signals to maintain stem cell function in the adult intestine. Our results highlight a broadly permissive chromatin architecture underlying regionalization in mesenchymal cells, then demonstrate further how chromatin architecture in niches can influence the fate and function of neighboring cells.

Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.

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.

RESULTS : Short-term lineage tracing data showed that _Lrig1_, _Lgr6_ and _Axin2_ label basal cells in MG ducts and acini. Long-term lineage tracing results showed that clones of labeled cells persist through multiple rounds of ductal and acinar renewal and give rise to differentiated progeny, identifying _Lrig1_+, _Lgr6_+ and _Axin2+_ ductal and acinar basal cells as self-renewing SCs. Forced expression of GLI2ΔN enhanced basal proliferation, caused expansion of _Lrig1_+ SCs, and lead to replacement of lipid-filled meibocytes by proliferative and poorly differentiated acinar cells. Transcriptional profiling of GLI2ΔN-expressing and control MGs revealed that forced GLI2ΔN expression caused greatly increased expression of _Lrig1_ and _Lgr6_ and suppressed expression of meibocyte differentiation genes.

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a "hit-and-run" adeno-associated virus-assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.