Immune Cell Composition in Human Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the world. Immunological analysis of the tumor microenvironment (immunoscore) shows great promise for improved prognosis and prediction of response to immunotherapy. However, the exact immune cell composition in NSCLC remains unclear. Here, we used flow cytometry to characterize the immune infiltrate in NSCLC tumors, non-cancerous lung tissue, regional lymph node, and blood. The cellular identity of >95% of all CD45+ immune cells was determined. Thirteen distinct immune cell types were identified in NSCLC tumors. T cells dominated the lung cancer landscape (on average 47% of all CD45+ immune cells). CD4+ T cells were the most abundant T cell population (26%), closely followed by CD8+ T cells (22%). Double negative CD4-CD8- T cells represented a small fraction (1.4%). CD19+ B cells were the second most common immune cell type in NSCLC tumors (16%), and four different B cell sub-populations were identified. Macrophages and natural killer (NK) cells composed 4.7 and 4.5% of the immune cell infiltrate, respectively. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c+ DCs, and CD141+ DCs) which together represented 2.1% of all immune cells. Among granulocytes, neutrophils were frequent (8.6%) with a high patient-to-patient variability, while mast cells (1.4%), basophils (0.4%), and eosinophils (0.3%) were less common. Across the cohort of patients, only B cells showed a significantly higher representation in NSCLC tumors compared to the distal lung. In contrast, the percentages of macrophages and NK cells were lower in tumors than in non-cancerous lung tissue. Furthermore, the fraction of macrophages with high HLA-DR expression levels was higher in NSCLC tumors relative to distal lung tissue. To make the method readily accessible, antibody panels and flow cytometry gating strategy used to identify the various immune cells are described in detail. This work should represent a useful resource for the immunomonitoring of patients with NSCLC.

Toll-Like Receptor Ligands and Interferon-γ Synergize for Induction of Antitumor M1 Macrophages.

Tumor-associated macrophages may either promote or suppress tumor growth depending on their activation status. Interferon-γ (IFN-γ) has been identified as a key factor for inducing tumoricidal M1 phenotype in macrophages. However, it remains unclear whether IFN-γ is sufficient or if additional stimuli are required. Here, we tested IFN-γ and a panel of toll-like receptor (TLR) agonists for the ability to activate murine macrophages toward a tumoricidal M1 phenotype. The following TLR ligands were used: TLR1/TLR2 agonist Pam3CSK4, TLR2/TLR6 agonist lipotechoic acid, TLR3 agonist poly(I:C), TLR4 agonist lipopolysaccharide (LPS), TLR5 agonist flagellin, TLR7 agonist CL264, and TLR9 agonist CpG. We used an in vitro growth inhibition assay to measure both cytotoxic and cytostatic activity of mouse macrophages against Lewis lung carcinoma (LLC) and MOPC315 plasmacytoma tumor cells. Production of nitric oxide (NO) and cytokines by activated macrophages was quantified. We found that IFN-γ alone was not able to render macrophages tumoricidal. Similarly, macrophage activation with single TLR agonists was inefficient. In sharp contrast, IFN-γ was shown to synergize with TLR agonists for induction of macrophage tumoricidal activity and production of both NO and pro-inflammatory cytokines (TNF-α, IL-12p40, and IL-12p70). Furthermore, IFN-γ was shown to suppress macrophage IL-10 secretion induced by TLR agonists. NO production was necessary for macrophage tumoricidal activity. We conclude that two signals from the microenvironment are required for optimal induction of antitumor M1 macrophage phenotype. Combination treatment with IFN-γ and TLR agonists may offer new avenues for macrophage-based cancer immunotherapy.

Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time.

PURPOSE: To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system. METHODS: We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells. RESULTS: The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas. CONCLUSIONS: The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.

Regeneration of the epithelium in organ-cultured donor corneas with extended post-mortem time.

PURPOSE: The maximum post-mortem time limit for obtaining donor corneas varies between eye banks. It is not known for how long a time the epithelial cells survive post-mortem, nor is it known if donor corneas with extended post-mortem time are able to regenerate the epithelium. Therefore, we wanted to examine the epithelium in donor corneas for regenerative ability during storage in an eye bank organ culture system. METHODS: Twenty-four paired donor corneas with post-mortem time from 28 to 163 hr were obtained. One cornea of a pair was fixed immediately to serve as a control, and the second was cultured in eye bank medium at 32 degrees C for 3 days. Examination of the specimens was performed with light and scanning electron microscopy. Immunohistochemical staining methods with antibodies against K 3, K 19, vimentin and p63 were used to further characterize the cells. RESULTS: The control corneas showed decreasing amounts of epithelial cells with increasing post-mortem time. All the cultured corneas demonstrated rapid regeneration of the epithelium. After 3 days in organ culture, 10 of 12 donor corneas were covered with epithelium. CONCLUSION: Even up to 7 days post-mortem, viable cells reside in the corneal epithelium. The study demonstrates the hardiness and enormous regenerative potential of peripheral corneal cells. Donor corneas processed in an eye bank organ culture storage system will obtain an intact epithelial layer within a few days.

EGFR gene copy number heterogeneity in fine-needle aspiration cytology from breast carcinomas determined by chromogenic in situ hybridization.

Most studies have shown epidermal growth factor receptor (EGFR) overexpression to be associated with poor prognostic factors in breast carcinomas. The relationship to EGFR gene copy number is unclear. The aim of our study was to investigate the heterogeneity of the EGFR gene copy number in breast carcinomas. The material consisted of air-dried smears from 29 breast carcinomas and 3 breast cancer cell lines (MCF-7, SKBR3, and T47D). Chromogenic in situ hybridization (CISH) was done using chromogenic detection. The mean signal numbers for EGFR gene and chromosome 7 as well as the EGFR gene/chromosome 7 centromere probe (CEP7) ratio were recorded. Immunohistochemical (IHC) staining was done on the corresponding paraffin sections. The copy number of the EGFR gene in each tumor/cell line ranged from 1.2 to 5.6. The EGFR gene/CEP7 ratio showed a biological continuum ranging from 0.59 to 1.94 with a mean of 1.04. EGFR gene copy loss was found in 16.6% of cases whereas copy gain was demonstrated in 19.4%. There was no relationship between IHC protein expression of EGFR and EGFR gene copy number or EGFR gene/CEP7 ratio.In conclusion, most breast carcinomas had a balanced EGFR gene/CEP7 copy number with a mean ratio of 1.04. Almost equal subpopulations revealed limited copy gain and copy loss. EGFR high dosage amplification, like in HER-2, was not demonstrated. Demonstration of EGFR gene copy loss might have a potential as a surrogate marker for EGFR gene mutation and/or deletion.