BACKGROUND: T cells drive acute cellular rejection (ACR) and its progression to chronic lung allograft dysfunction (CLAD) following lung transplantation. International Society for Heart and Lung Transplantation grade A1 ACR without associated allograft dysfunction is often untreated, yet some patients develop progressive graft dysfunction. T-cell composition of A1 ACR lesions may have prognostic value; therefore, protein-level and epigenetic techniques were applied to transbronchial biopsy tissue to determine whether differential T-cell infiltration in recipients experiencing a first episode of stable grade A1 ACR (StA1R) is associated with early CLAD. METHODS: Sixty-two patients experiencing a first episode of StA1R were divided into those experiencing CLAD within 2 years (n = 13) and those remaining CLAD-free for 5 or more years (n = 49). Imaging mass cytometry (IMC) was used to profile the spectrum and distribution of intragraft T cell phenotypes on a subcohort (n = 16; 8 early-CLAD and 8 no early-CLAD). Immunofluorescence was used to quantify CD4(+), CD8(+), and FOXP3+ cells. Separately, CD3+ cells were fluorescently labeled, micro-dissected, and the degree of Treg-specific demethylated region methylation was determined. RESULTS: PhenoGraph unsupervised clustering on IMC revealed 50 unique immune cell subpopulations. Methylation and immunofluorescence analyses demonstrated no significant differences in Tregs between early-CLAD and no early-CLAD groups. Immunofluorescence revealed that patients who developed CLAD within 2 years of lung transplantation showed greater CD8(+) T cell infiltration compared to those who remained CLAD-free for 5 or more years. CONCLUSIONS: In asymptomatic patients with a first episode of A1 rejection, greater CD8+ T cell content may be indicative of worse long-term outlook. (c) 2024 The Authors. Published by Elsevier Inc. on behalf of International Society for Heart and Lung Transplantation.

BackgroundT cells drive acute cellular rejection (ACR) and its progression to chronic lung allograft dysfunction (CLAD) following lung transplantation. ISHLT grade A1 ACR without associated allograft dysfunction is often untreated, yet some patients develop progressive graft dysfunction. T cell composition of A1 ACR lesions may have prognostic value, therefore protein-level and epigenetic techniques were applied to transbronchial biopsy (TBB) tissue to determine whether differential infiltration of T cells in recipients experiencing a first episode of stable grade A1 ACR (StA1R) is associated with early CLAD.MethodsSixty-two patients experiencing a first episode of StA1R were divided into those experiencing CLAD within 2 years (n=13) and those remaining CLAD-free for 5≤ years (n=49). Imaging mass cytometry (IMC) was used to profile the spectrum and distribution of intragraft T cell phenotypes on a subcohort (n=16; 8 early-CLAD and 8 no-early-CLAD). Immunofluorescence was used to quantify CD4+, CD8+ and FOXP3+ cells. Separately, CD3+ cells were fluorescently labelled, micro-dissected and, using bisulfite conversion and pyrosequencing, the degree of Treg-specific demethylated region methylation was determined.ResultsPhenoGraph unsupervised clustering on IMC revealed 50 unique immune cell subpopulations. Methylation and immunofluorescence analyses demonstrated no significant differences in Tregs between early-CLAD and no-early-CLAD groups. Immunofluorescence revealed that patients who developed CLAD within 2 years of LTx showed greater CD8+ T cell infiltration compared to those who remained CLAD-free for 5 or more years.ConclusionsIn asymptomatic patients with a first episode of A1 rejection, greater CD8+ T cell content may be indicative of worse long-term outlook.

Abstract Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

Supplementary Figure Legends 1-8 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

Supplementary Figure 4 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

Supplementary Figure 2 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

Supplementary Information from Targeting Focal Adhesion Kinase with Dominant-Negative FRNK or Hsp90 Inhibitor 17-DMAG Suppresses Tumor Growth and Metastasis of SiHa Cervical Xenografts

PDF file - 111K, Effects of the combination of AZD6244 with gemcitabine plus cisplatin

Supplementary Figure 3 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

PDF file - 66K, Effect of AZD6244 on cell cycle phase distribution and DNA synthesis of human biliary cancer cells

PDF file - 161K, Time course of recovery of cell cycle phase distribution and proliferation following AZD6244 treatment in vitro

Supplementary Figure 8 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

PDF file - 93K, Effect of single drug treatment of AZD6244 or gemcitabine on OCIP55 mice model

Supplementary Figures 1-9 from Targeting Focal Adhesion Kinase with Dominant-Negative FRNK or Hsp90 Inhibitor 17-DMAG Suppresses Tumor Growth and Metastasis of SiHa Cervical Xenografts

Supplementary Figure 5 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

Supplementary Methods, Tables and Figures Supplementary Table 1: Summary of whole genome sequencing results including performance metrics and time to reporting (N=62). Supplementary Table 2: Summary of GATA6 RNA in situ results and its correlation with RNA subtype and histologic grade. Supplementary Figure 1: WGS Performance Characteristics. Supplementary Figure 2: Both COMP-0037 and COMP-0057 had germline BRCA2 p.S1982fs mutations but only COMP-0037 had a second hit (somatic LOH of wild type allele) and showed hallmarks of double strand break repair deficiency. Supplementary Figure 3: BRCA2 mutation status of COMP-0057. Supplementary Figure 4: Subtypes of advanced PDAC by Moffitt tumour classification Supplementary Figure 5: Correlation between Moffitt tumor RNA subtypes and GATA6 gene expression. Supplementary Figure 6: Correlation between GATA6 expression measured by RNASeq and RNA in situ hybridization. Supplementary Figure 7: Differentially expressed pathways between Moffitt's basal like and classical RNA subtypes Supplementary Figure 8: CDK4 amplification identified in COMP-0008 (A) and CDK6 amplification identified in COMP-0010 (B) validated by fluorescence in-situ hybridization.

Supplementary Figure 1 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

PDF file - 99K, Characterization of biliary cancer xenografts

Supplementary Figure 7 from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer

Supplementary Information: Primer Sequence from Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer