A ventricular fibrillation cardiac arrest model with extracorporeal cardiopulmonary resuscitation in rats: 8 minutes arrest time leads to increased myocardial damage but does not increase neuronal damage compared to 6 minutes

IntroductionExtracorporeal cardiopulmonary resuscitation (ECPR) is an emerging strategy in highly selected patients with refractory cardiac arrest (CA). Animal models can help to identify new therapeutic strategies to improve neurological outcome and cardiac function after global ischemia in CA. Aim of the study was to establish a reproducible ECPR rat model of ventricular fibrillation CA (VFCA) that leads to consistent neuronal damage with acceptable long-term survival rates, which can be used for future research.Materials and methodsMale Sprague Dawley rats were resuscitated with ECPR from 6 min (n = 15) and 8 min (n = 16) VFCA. Animals surviving for 14 days after return of spontaneous resuscitation (ROSC) were compared with sham operated animals (n = 10); neurological outcome was assessed daily until day 14. In the hippocampal cornu ammonis 1 region viable neurons were counted. Microglia and astrocyte reaction was assessed by Iba1 and GFAP immunohistochemistry, and collagen fibers in the myocardium were detected in Azan staining. QuPath was applied for quantification.ResultsOf the 15 rats included in the 6 min CA group, all achieved ROSC (100%) and 10 (67%) survived to 14 days; in the 8 min CA group, 15 (94%) achieved ROSC and 5 (31%) reached the endpoint. All sham animals (n = 10) survived 2 weeks. The quantity of viable neurons was significantly decreased, while the area displaying Iba1 and GFAP positive pixels was significantly increased in the hippocampus across both groups that experienced CA. Interestingly, there was no difference between the two CA groups regarding these changes. The myocardium in the 8 min CA group exhibited significantly more collagen fibers compared to the sham animals, without differences between 6- and 8-min CA groups. However, this significant increase was not observed in the 6 min CA group.ConclusionOur findings indicate a uniform occurrence of neuronal damage in the hippocampus across both CA groups. However, there was a decrease in survival following an 8-min CA. Consequently, a 6-min duration of CA resulted in predictable neurological damage without significant cardiac damage and ensured adequate survival rates up to 14 days. This appears to offer a reliable model for investigating neuroprotective therapies

The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status

Abstract: Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status

Development and <i>In Vivo</i> Evaluation of Small-Molecule Ligands for Positron Emission Tomography of Immune Checkpoint Modulation Targeting Programmed Cell Death 1 Ligand 1

A substantial portion of patients do not benefit from programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) checkpoint inhibition therapies, necessitating a deeper understanding of predictive biomarkers. Immunohistochemistry (IHC) has played a pivotal role in assessing PD-L1 expression, but small-molecule positron emission tomography (PET) tracers could offer a promising avenue to address IHC-associated limitations, i.e., invasiveness and PD-L1 expression heterogeneity. PET tracers would allow for improved quantification of PD-L1 through noninvasive whole-body imaging, thereby enhancing patient stratification. Here, a large series of PD-L1 targeting small molecules were synthesized, leveraging advantageous substructures to achieve exceptionally low nanomolar affinities. Compound 5c emerged as a promising candidate (IC50 = 10.2 nM) and underwent successful carbon-11 radiolabeling. However, a lack of in vivo tracer uptake in xenografts and notable accumulation in excretory organs was observed, underscoring the challenges encountered in small-molecule PD-L1 PET tracer development. The findings, including structure–activity relationships and in vivo biodistribution data, stand to illuminate the path forward for refining small-molecule PD-L1 PET tracers

Development and <i>In Vivo</i> Evaluation of Small-Molecule Ligands for Positron Emission Tomography of Immune Checkpoint Modulation Targeting Programmed Cell Death 1 Ligand 1

A substantial portion of patients do not benefit from programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) checkpoint inhibition therapies, necessitating a deeper understanding of predictive biomarkers. Immunohistochemistry (IHC) has played a pivotal role in assessing PD-L1 expression, but small-molecule positron emission tomography (PET) tracers could offer a promising avenue to address IHC-associated limitations, i.e., invasiveness and PD-L1 expression heterogeneity. PET tracers would allow for improved quantification of PD-L1 through noninvasive whole-body imaging, thereby enhancing patient stratification. Here, a large series of PD-L1 targeting small molecules were synthesized, leveraging advantageous substructures to achieve exceptionally low nanomolar affinities. Compound 5c emerged as a promising candidate (IC50 = 10.2 nM) and underwent successful carbon-11 radiolabeling. However, a lack of in vivo tracer uptake in xenografts and notable accumulation in excretory organs was observed, underscoring the challenges encountered in small-molecule PD-L1 PET tracer development. The findings, including structure–activity relationships and in vivo biodistribution data, stand to illuminate the path forward for refining small-molecule PD-L1 PET tracers

Active immunization with a Her-2/neu-targeting Multi-peptide B cell vaccine prevents lung metastases formation from Her-2/neu breast cancer in a mouse model

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4+ and CD8+ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL

Interferon γ–Induced Human Guanylate Binding Protein 1 Inhibits Mammary Tumor Growth in Mice

Interferon γ (IFN-γ) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-γ are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-γ–mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1–expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1–expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-γ–mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-γ–induced antitumoral defense system