Immune Cell Markers Research Articles

Regulation of glucose uptake and inflammation markers by FOXO1 and FOXO3 in skeletal muscle

ObjectiveForkhead box class O (FOXO) transcription factors regulate whole body energy metabolism, skeletal muscle mass, and substrate switching. FOXO1 and FOXO3 are highly abundant transcription factors, but their precise role in skeletal muscle metabolism has not been fully elucidated. MethodsTo elucidate the role of FOXO in skeletal muscle, dominant negative (dn) constructs for FOXO1 (FOXO1dn) or FOXO3 (FOXO3dn) were transfected by electroporation into mouse tibialis anterior muscle and glucose uptake, signal transduction, and gene expression profiles were assessed after an oral glucose tolerance test. Results were compared against contralateral control transfected muscle. ResultsFOXO1dn and FOXO3dn attenuated glucose uptake (35%, p < 0.01 and 20%, p < 0.05), GLUT4 protein (40%, p < 0.05 and 10%, p < 0.05), and subunits of the oxidative phosphorylation cascade. Intramuscular glycogen content was decreased (20%, p < 0.05) by FOXO3dn, but not FOXO1dn. Transcriptomic analysis revealed major pathways affected by FOXO1dn or FOXO3dn revolve around metabolism and inflammation. FOXO1dn increased Akt protein (140%, p < 0.001), p-AktSer473 (720%, p < 0.05) and p-AktThr308 (570%, p < 0.01), whereas FOXO3dn was without effect. FOXO1dn and FOXO3dn increased mTOR protein content (170% and 190%, p < 0.05), and p-p70S6KThr389 (420%, p < 0.01 and 300%, p < 0.01), while p-mTORSer2448 (500%, p < 0.01), was only increased by FOXO1dn. Chemokines and immune cell markers were robustly upregulated in skeletal muscle following the FOXOdn transfections, but not after control transfection. ConclusionsFOXO1 and FOXO3 regulate glucose metabolism and markers of inflammation in skeletal muscle, implicating transcriptional control governing “immunometabolic” dynamics.

Radiogenomics Profiling for Glioblastoma-related Immune Cells Reveals CD49d Expression Correlation with MRI parameters and Prognosis

Although there have been a plethora of radiogenomics studies related to glioblastoma (GBM), most of them only used genomic information from tumor cells. In this study, we used radiogenomics profiling to identify MRI-associated immune cell markers in GBM, which was also correlated with prognosis. Expression levels of immune cell markers were correlated with quantitative MRI parameters in a total of 60 GBM patients. Fourteen immune cell markers (i.e., CD11b, CD68, CSF1R, CD163, CD33, CD123, CD83, CD63, CD49d and CD117 for myeloid cells, and CD4, CD3e, CD25 and CD8 for lymphoid cells) were selected for RNA-level analysis using quantitative RT-PCR. For MRI analysis, quantitative MRI parameters from FLAIR, contrast-enhanced (CE) T1WI, dynamic susceptibility contrast perfusion MRI and diffusion-weighted images were used. In addition, PFS associated with interesting mRNA data was performed by Kaplan-Meier survival analysis. CD163, which marks tumor associated microglia/macrophages (TAMs), showed the highest expression level in GBM patients. CD68 (TAMs), CSF1R (TAMs), CD33 (myeloid-derived suppressor cell) and CD4 (helper T cell, regulatory T cell) levels were highly positively correlated with nCBV values, while CD3e (helper T cell, cytotoxic T cell) and CD49d showed a significantly negative correlation with apparent diffusion coefficient (ADC) values. Moreover, regardless of any other molecular characteristics, CD49d was revealed as one independent factor for PFS of GBM patients by Cox proportional-hazards regression analysis (P = 0.0002). CD49d expression level CD49d correlated with ADC can be considered as a candidate biomarker to predict progression of GBM patients.

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry.

Homograft (syngeneic) tumors are the workhorse of today's immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models. Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7-9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300-1,000 mm3 by 17 days. Only tumors of 400-600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

P3.04-14 TMB and Immune Checkpoint Inhibitor Gene Expression are Unrelated in NSCLC Patients

Tumor Mutation Burden (TMB) is an emerging biomarker for response to immune checkpoint inhibitor therapy (ICT). PDL1 is still the only companion diagnostic test for ICT. We sought to examine the relationship between TMB based on whole exome sequencing (WES) data and ICT gene expression. Retrospective analysis of a database from a commercial DNA tumor:normal and RNAseq platform was carried out. 112 samples were identified with tumor and normal DNA sequencing (WGS or WES) and RNA sequencing. A “high” vs “low” mutation burden was defined with a threshold of 200 exonic non-synonymous mutations. ICT gene expression of 12 checkpoint markers (CTLA4,IDO,FOXP3,LAG3,TIGIT,VEGFA,VEGFC,VEGFB,PDL2,PDL1,OX40, and TIM3) were analyzed for tissue-specific enrichment and within the TMB high and low categories. Immune-cell infiltration was estimated using RNA deconvolution based on known immune cell marker genes (Bindea et al 2013). 52 of the 112 patients had high TMB based on the defined threshold. ICT gene expression was not significantly different in the TMB high and low groups and none of the 12 ICT markers showed a significant correlation with TMB. Age and gender did not affect TMB. Quantitative TMB may be a marker of ICT efficacy independent of ICT gene expression.

P3.04-15 IDO and Tim3 Gene Expression is Correlated in NSCLC Patients with Low PDL1 Gene Expression

Tumor cells evade immunosurveillance by expression of immune checkpoint proteins PD-L1, IDO, and TIM3, even when tumor-infiltrating lymphocytes (TILs) are present. Combination of checkpoint inhibitors Pembrolizumab(PD-1i) and Epacadostat (IDOi) failed to show synergy in melanoma. Yet there are several candidate targets for checkpoint inhibition and rational selection of which combinations to explore is lacking. In particular, which checkpoints to target when PD-L1 is not expressed is largely unexplored. The purpose of this study was to assess the expression of 12 immune checkpoint genes in lung cancer patients with high and low expression of PD-L1. Retrospective review of deep whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) on 1467 unselected clinical cases from NantHealth. 112 NSCLC cases were categorized as PD-L1-low and PD-L1-high by median splitting. Expression and co-expression of 12 checkpoint markers (CTLA4,IDO,FOXP3,LAG3,TIGIT,VEGFA,VEGFC,VEGFB,PDL2,PDL1,OX40, and TIM3) were analyzed for tissue-specific enrichment and within PD-L1-defined categories. Immune-cell infiltration was estimated using RNA deconvolution based on known immune cell marker genes (Bindea et al 2013). CTLA, TIGIT and FOXP3 showed high correlations across all 112 samples as well within the PD-L1 low and high expression groups and differential expression across the two groups, with higher expression of the markers among the PD-L1 high expression group versus the low group. Within the PD-L1 high expression group, the expression of markers LAG3, TIM3, OX40, FOXP3, CTLA4 and TIGIT was both higher and more highly correlated than in the PD-L1 low group. Within the PD-L1-low category, IDO1 and TIM3 have relatively high expression and are highly correlated with each other (R=0.72, p=3.85x10ˆ-10). PD-L1 is a primary driver of immune suppression, however when PD-L1 is low there may be some differential role for IDO or TIM3. Combination IDOi and Tim3i should be considered in PD-L1 low patients.

The effect of anesthetic technique on \xb5-opioid receptor expression and immune cell infiltration in breast cancer

BackgroundClinical histological studies demonstrate that the distribution of natural killer (NK) cells, other immune cells and μ-opioid receptors (MOR) within cancer tissue can predict cancer prognosis. No clinical study has evaluated whether anesthetic technique influences immune cell and MOR expression within human breast cancer.MethodsExcised preoperative biopsies and intraoperative breast cancer specimens from 20 patients randomly chosen from patients previously enrolled in an ongoing, prospective, randomized trial (NCT00418457) investigating the effect of anesthetic technique on long-term breast cancer outcome were immunohistochemically stained and microscopically examined by two independent investigators, masked to randomization, to quantify MOR and immune cell infiltration: CD56, CD57 (NK cells), CD4 (T helper cells), CD8 (cytotoxic T cells) and CD68 (macrophages). Patients had been randomized to receive either a propofol–paravertebral anesthetic with continuing analgesia (PPA, n = 10) or balanced general anesthetic with opioid analgesia (GA, n = 10).ResultsThere were no differences between the groups in staining intensity in preoperative biopsy specimens. Expression intensity values (median 25–75%) for MOR in intraoperative resected biopsy were higher in GA 8.5 (3–17) versus PPA 1 (0–10), p = 0.04. The numbers of MOR-positive cells were also higher in GA patients. Expression and absolute numbers of CD56, CD57, CD4 and CD68 were similar in resected tumor in both groups.ConclusionGeneral anesthesia with opioid analgesia increased resected tumor MOR expression compared with propofol–paravertebral anesthetic technique, but the anesthetic technique did not significantly influence the expression of immune cell markers.

Increased macrophages and\xa0changed brain endothelial cell gene expression in the frontal cortex of people with schizophrenia displaying inflammation

Elevated pro-inflammatory cytokines exist in both blood and brain of people with schizophrenia but how this affects molecular indices of the blood–brain barrier (BBB) is unclear. Eight mRNAs relating to BBB function, a microglia and three immune cell markers were measured by qPCR in the prefrontal cortex from 37 people with schizophrenia/schizoaffective disorder and 37 matched controls. This cohort was previously grouped into “high inflammation” and “low inflammation” subgroups based on cortical inflammatory-related transcripts. Soluble intercellular adhesion molecule-1 (sICAM1) was measured in the plasma of 78 patients with schizophrenia/schizoaffective disorder and 73 healthy controls. We found that sICAM1 was significantly elevated in schizophrenia. An efflux transporter, ABCG2, was lower, while mRNAs encoding VE-cadherin and ICAM1 were higher in schizophrenia brain. The “high inflammation” schizophrenia subgroup had lower ABCG2 and higher ICAM1, VE-cadherin, occludin and interferon-induced transmembrane protein mRNAs compared to both “low inflammation” schizophrenia and “low inflammation” control subgroups. ICAM1 immunohistochemistry showed enrichment in brain endothelium regardless of diagnosis and was localised to astrocytes in some brains. Microglia mRNA was not altered in schizophrenia nor did it correlate with ICAM1 expression. Immune cell mRNAs were elevated in “high inflammation” schizophrenia compared to both “low inflammation” schizophrenia and controls. CD163+ perivascular macrophages were identified by immunohistochemistry in brain parenchyma in over 40% of “high inflammation” schizophrenia brains. People with high levels of cytokine expression and schizophrenia display changes consistent with greater immune cell transmigration into brain via increased ICAM1, which could contribute to other neuropathological changes found in this subgroup of people.

Activation of the innate immune system is evident throughout epileptogenesis and is associated with blood‐brain barrier dysfunction and seizure progression

Because brain inflammation may contribute to the pathophysiology of temporal lobe epilepsy (TLE), we investigated the expression of various inflammatory markers of the innate and adaptive immune system in the epileptogenic human and rat hippocampus in relation to seizure activity and blood-brain barrier (BBB) dysfunction. Immunohistochemistry was performed using various immune cell markers (for microglia, monocytes, macrophages, T lymphocytes, and dendritic cells) on hippocampal sections of drug-resistant TLE patients and patients who died after status epilepticus. The expression of these markers was also studied in the electrical post-status epilepticus rat model for TLE, during the acute, latent, and chronic epileptic phase. BBB dysfunction was assessed using albumin immunohistochemistry and the BBB tracer fluorescein. Monocyte infiltration, microglia, and perivascular macrophage activation were persistently increased in both epileptogenic human and rat hippocampus, whereas T lymphocytes and dendritic cells were not or were scarcely detected. In addition to this, increased expression of C-C motif ligand 2 (CCL2) and osteopontin was observed. In humans, the expression of CD68 and CCL2 was related to the duration of epilepsy and type of pathology. In rats, the expression of CD68, CCL2, and the perivascular macrophage marker CD163 was related to the duration of the initial insult and to the number of spontaneous seizures. Interestingly, the number of CD163-positive perivascular macrophages was also positively correlated to BBB dysfunction in chronic epileptic rats. These data suggest a proepileptogenic role for monocytes/macrophages and other cells of the innate immune response, possibly via increased BBB leakage, and indicate that T cells and dendritic cells, which are closely associated with the adaptive immune response, are only sparsely infiltrated during epileptogenesis in the electrical post-status epilepticus rat model. Future studies should reveal the relative importance of these immune cells and whether specific manipulation can modify or prevent epileptogenesis.

Role of Host GPR120 in Mediating Dietary Omega-3 Fatty Acid Inhibition of Prostate Cancer.

GPR120, a G protein-coupled receptor for long-chain polyunsaturated fatty acids (FAs), mediates the anti-inflammatory effects of omega-3 (ω-3) FAs. We investigated whether host or tumor GPR120 plays a role in the anti-prostate cancer effects of ω-3 FAs. MycCap prostate cancer allografts were grown in immunocompetent wild-type (WT) and GPR120 knockout (KO) mice fed ω-3 (fish oil) or ω-6 (corn oil) diets. Immune cell infiltration was quantified by flow cytometry, and gene expression of immune cell markers in isolated tumor-associated macrophages (TAMs) was quantified by quantitative real-time polymerase chain reaction. Archived tissue from a fish oil intervention trial was used to correlate gene expression of GPR120 with cell cycle progression (CCP) genes and Ki67 index (n = 11-15 per group). All statistical tests were two-sided. In WT mice (n = 7 per group), dietary ω-3 FAs decreased MycCap allograft tumor growth (mean [SD] final tumor volume ω-6 = 491 [437] mm3 vs ω-3 = 127 [77] mm3, P = .04), whereas in global GPR120KO mice (n = 7 per group) ω-3 FAs had no anticancer effects. Dietary ω-3 FAs inhibited GPR120KO-MycCaP allografts grown in WT mice (n = 8 per group; mean [SD] final tumor volume ω-6 = 776 [767] mm3 vs ω-3 = 36 [34] mm3, P = .02). Omega-3 FA treatment decreased the number of M2-like TAMs in tumor tissue and gene expression of M2 markers in isolated TAMs compared with ω-6 controls in WT (n = 7 per group) but not in GPR120KO mice (n = 7 per group). In human tissue, higher expression of stromal GPR120 correlated with greater reduction in expression of CCP genes in men with prostate cancer on a high-ω-3 diet (r = -.57, P = .04). Host GPR120 plays a central role in the anti-prostate cancer effects of dietary ω-3 FAs. Future studies are required to determine if the anticancer effects of ω-3 FAs are mediated through inhibition of M2-like macrophages and if host GPR120 status predicts anticancer effects of dietary ω-3 FAs in men with prostate cancer.

IL-8/CXCR1/2 signalling promotes tumor cell proliferation, invasion and vascular mimicry in glioblastoma

BackgroundGlioblastoma multiforme (GBM) is one of the lethal malignant tumors of the central nervous system. Despite advances made in understanding this complex disease, little has been achieved in improving clinical efficacy towards it. Factors such as chemokines play important role in shaping the tumor microenvironment which in turn plays a significant role in deciding course of tumor progression. In this study, we investigated the role of chemokine IL-8 in glioblastoma progression with particular emphasis on immunomodulation, cellular proliferation, invasion and vascular mimicry.MethodsRole of IL-8 in GBM immunology was determined by correlating the expression of IL-8 by immunohistochemistry with other immune cell markers such as CD3 and CD68. Effect of high IL-8 expression on overall survival, the difference in expression level between different GBM subgroups and anatomic structures were analyzed using other databases. Two GBM cell lines –U-87MG and LN-18 were used to study the impact of targeting IL-8-CXCR1/2 signalling using neutralizing antibodies and pharmacological antagonist. Reverse transcriptase–polymerase chain reaction and immunocytochemistry were used to determine the expression of these axes. Impact on cell viability and proliferation was assessed by MTT, proliferation marker-ki-67 and clonogenic survival assays. Multicellular tumor spheroids generated from GBM cell lines were used to study invasion in matrigel.ResultsWeak Positive correlation was observed between IL-8 and CD3 as well as between IL-8 and CD68. High IL-8 expression in GBM patients was found to be associated with dismal survival. No significant difference in IL-8 expression between different molecular subgroups of GBM was observed. In vitro targeting of IL-8-CXCR1/2 signalling displayed a significant reduction in cell viability and proliferation, and spheroid invasion. Furthermore, the presence of CD34-/CXCR1+ vessels in GBM tissues showed the involvement of IL-8/CXCR1 in vascular mimicry structure formation.ConclusionThese results suggest a direct involvement of IL-8-CXCR1/2 axes in GBM progression by promoting both cell proliferation and invasion and indirectly by promoting neovascularization in the form of vascular mimicry.

Immune Cell and Stromal Signature Associated With Progression-Free Survival of Patients With Resected Pancreatic Ductal Adenocarcinoma

Changes to the microenvironment ofpancreatic ductal adenocarcinomas (PDACs) have been associated with poor outcomes of patients. We studied the associations between composition of the pancreatic stroma (fibrogenic, inert, dormant, or fibrolytic stroma) and infiltration by inflammatory cells and times of progression-free survival (PFS) of patients with PDACs after resection. We obtained 1824 tissue microarray specimens from 385 patients included in the European Study Group for Pancreatic Cancer trial 1 and 3 and performed immunohistochemistry to detect alpha smooth muscle actin, type 1 collagen, CD3, CD4, CD8, CD68, CD206, and neutrophils. Tumors that expressed high andlow levels of these markers were compared with patient outcomes using Kaplan-Meier curves and multivariable recursive partitioning for discrete-time survival tree analysis. Prognostic index was delineated by a multivariable Cox proportional hazards model of immune cell and stromal markers and PFS. Findings were validated using 279 tissue microarray specimens from 93 patients in a separate cohort. Levels of CD3, CD4, CD8, CD68, and CD206 were independently associated with tumor recurrence. Recursive partitioning for discrete-time survival tree analysis identified a high level of CD3 as the strongest independent predictor for longer PFS. Tumors with levels of CD3 and high levels of CD206 associated with a median PFS time of 16.6 months and a median prognostic index of -0.32 (95% confidence interval [CI] -0.35 to -0.31), whereas tumors with low level of CD3 cell and low level of CD8 and high level of CD68 associated with a median PFS time of 7.9 months and a prognostic index of 0.32 (95% CI 0.050-0.32); we called these patterns histologic signatures. Stroma composition, when unassociated with inflammatory cellmarkers, did not associate significantly with PFS. In thevalidation cohort, the histologic signature resulted in an error matrix accuracy of predicted response of 0.75 (95% CI 0.64-0.83; accuracy P < .001). In an analysis of PDAC tissue microarray specimens, we identified and validated a histologic signature, based on leukocyte and stromal factors, that associates with PFS times of patients with resected PDACs. Immune cells might affect the composition of the pancreatic stroma to affect progression of PDAC. These findings provide new insights into the immune response to PDAC.

Abstract 336: Human Bone Contains Primitive Cells With Angiogenic and Immunomodulatory Properties

Previously, we have shown that treatments of cortical bone derived stem cells (CBSCs) reduce ventricular remodeling and improve cardiac functions in both mouse and pig myocardial infarction (MI) models. The goals of the present study were to isolate and characterize human CBSCs (hCBSCs). Using bone fragments obtained at the time surgeries, we isolated three morphologically distinctive hCBSC phenotypes; 1) sphere forming (SF); 2) epithelial like monolayer (ELM); and 3) larger and spindle shaped (LSS) phenotype. The hCBSC phenotypes grew without senescence in culture. Mean doubling time (hour) for SF, ELM, and LSS at the passage number 20 (P20) were 19.54, 21.21, and 20.3 respectively and at the P40 were 18.01, 17.13, and 20.56 respectively. All of the phenotypes were found to have surface expression of CD44; however, other markers detected (CD105, CD90, CD73, CD106, CD271, and CD133) on mouse CBSCs (mCBSCs) were not detected along with the markers not detected on the surface of mCBSCs (CD45, CD11b, CD31, CD34, CD117, and CD325). Erythrocytes (CD235a) and immune cell markers (CD2, CD3, CD14, CD16, CD19, and CD56) were also not detected. The hCBSCs were found without histocompatibility antigen Ia (MHC-Ia: HLA-A, HLA-B, and HLA-C) and MHC-II (HLA-DR); instead, they expressed soluble forms of MHC-Ib (HLA-G5, HLA-G6, and HLA-G7), potent inhibitor of natural killer cells and other immune cells. These features are commonly found in immune privileged cells. Further, the hCBSCs produced the immunomodulatory and anti-inflammatory factors (IL-4, IL-1RA, TGF-b and IL-10). Additionally, the hCBSCs secreted angiogenic factors that organize endothelial cells into tube like structures. From these results, the hCBSCs we isolated have totally different surface marker characteristics compared to any other known cells including mCBSCs. In addition to their angiogenic effects, the hCBSCs have immunomodulatory properties that could make them suitable for cell therapy targeting inflammatory diseases such post myocardial infarction remodeling.

Elevation of the TP53 isoform Δ133p53β in glioblastomas: an alternative to mutant p53 in promoting tumor development.

As tumor protein 53 (p53) isoforms have tumor‐promoting, migration, and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full‐length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT‐qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumor‐associated macrophage content, and various immune cell markers. Elevated levels of Δ133p53β mRNA characterised glioblastomas with increased CD163‐positive macrophages and wild‐type TP53. In situ‐based analyses found Δ133p53β expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C‐C motif chemokine ligand 2 (CCL2) expressed. Tumors with increased Δ133p53β had increased numbers of cells positive for macrophage colony‐stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine ‘mimic’ of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest that elevated Δ133p53β is an alternative pathway to TP53 mutation in glioblastoma that aids tumor progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53β to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 1563: Characterization of two new recombinant rabbit anti-PDL1 IHC staining in bladder cancer, NSCLC, and melanoma with immune cell markers

Abstract Published studies show tumor cells overexpress PD-L1 to escape the host immune defense by binding to PD-1 on surveilling T-cells causing the T-cells to shut down. Immunotherapies disrupting PD-1/PD-L1 signaling interactions have great success in treating PD-L1 positive tumors. Immunohistochemistry (IHC) is an important diagnostic tool currently used to determine the expression level of PD-L1 in tumor and immune cells. The FDA approved PD-L1 clones (SP142 and 28-8) for IHC present different staining patterns when evaluated on the same tumor tissue. In this study, we assessed both the immune cells and tumor cells IHC positive stain of PD-L1. To do this we evaluated multiple immune cell markers with multiple PD-L1 antibodies. IHC screens were done with 5 PD-L1 antibodies, 2 recombinant rabbit monoclonal antibodies (clone OR-5E3 and OR-5H8), the mouse monoclonal antibody (clone UMAB229), and the FDA approved clones (SP142 and 28-8). The immune cell markers used were CD3, CD8A, CD20, CD68, and FOXP3. NSCLC, Bladder Cancer, and Melanoma immune cells, as indicated by the CD3, CD8A, CD20, CD68 and FOXP3 staining, generated different distribution pattern in the three tumor types. The five PD-L1 antibodies did show variation in detection of both the immune and tumor cells. For example, PD-L1 clones OR-5E3 and OR-5H8 stained tumor cells stronger and picked up weak expression of PD-L1 better than clone SP142, suggesting the two antibodies have higher affinity. The mouse monoclonal anti-PDL1 clone UMAB229 picked up strong and weak staining similar or better than clones OR-5H8 and picked up the immune cells similar to SP-142. The five PD-L1 antibodies were made from different antigens which may contribute to their sensitivity and specificity to detect PD-L1 in tumor cells. This study suggests the new generation of PD-L1 antibodies may make a better tool for diagnostic screens. Citation Format: Rachel M. Gonzalez, Wei Fu, Evelin Logis, Emma Ding, Casey Chen, Xiaojie Li, Sonia Merritt, Mingjuan Liu, Amy Zhang, Yiran Wang, Guangli Wang, Donghui Ma. Characterization of two new recombinant rabbit anti-PDL1 IHC staining in bladder cancer, NSCLC, and melanoma with immune cell markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1563.

Abstract 635: Characterizing PD-L1 expression in tumor-associated cells and CTC using IncellDx and Celsee molecular diagnostic technologies

Abstract Detection of PD-L1 expression in NSCLC cancer patients is increasingly becoming a widely used molecular diagnostic tool. Evidence also suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Current methods analyze PD-L1 expression in either solid tumor biopsies or circulating tumor cells (CTC), independent of each other. In order to design a comprehensive PD-L1 treatment regime, it is imperative to look at the PD-L1 expression in cancer cells of the tumor as well as in CTCs. In this study we present a comprehensive profiling of PD-L1 expression in solid tumor biopsy and CTC of 10 lung cancer patients. We further extend the study by evaluating the co-relation of the PD-L1 expression with other immune cell infiltration markers in the lung biopsy samples. There is no single technology that can provide a comprehensive profiling of PD-L1 expression in lung biopsies as well as the CTCs isolated from the peripheral blood. Using two very different and unique molecular diagnostic technologies, IncellDx OncoTect iO Single Cell Quantitative PD-L1 assay and automated CTC analysis Celsee C-PREP GENESIS, we present the first report discussing the PD-L1 expression in tumor cells of the solid and liquid biopsies and its correlation with DNA content and infiltrating immune cell markers. Nine out of the ten patient samples evaluated showed strong correlation between DNA content and PD-L1 expression in tumor-associated cells and CTC. One aneuploid patient sample showed very low expression of PD-L1 in tumor-associated cancer cells; however, it still showed a positive staining for PD-L1 in CTC. Obtaining a comprehensive PD-L1 expression and associated immune cell marker profile in solid tumor biopsy and circulating tumor cells may be needed to design a more effective and lasting immunotherapy to treat NSCLC lung cancer patients. The results discussed in this study will strongly impact the current understanding and implementation of the molecular diagnostics tools to evaluate cancer patients in order to define a robust therapeutic regimen. Citation Format: Fariba Fazeli, Vishal Premdev Sharma, William Chiro Chow, Amanda Chargin, Priya Gogoi, Kalyan Handique, Bruce Patterson. Characterizing PD-L1 expression in tumor-associated cells and CTC using IncellDx and Celsee molecular diagnostic technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 635.

Abstract 3604: Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients

Abstract Background: Genomic instability and evolution of cancers is linked to chemotherapy sensitivity and resistance in patients with metastatic cancer. Taxane-based chemotherapies operate by promoting chromosomal instability (CIN) by increasing multipolar mitoses. Breast tumors with CIN are more sensitive to paclitaxel compared to tumors without CIN. There is a critical need for new biomarkers based on biological evaluation of mechanisms of response and resistance in metastatic cancer. Although CIN is difficult to detect directly, centrosome amplification (CA) is a common mechanism of CIN. In this study, we develop an assay to detect CA in circulating tumor cells (CTCs) as a minimally invasive assay to evaluate sensitivity to taxane-based chemotherapies. Methods: Blood samples from patients with metastatic breast and prostate cancer were collected and processed for nucleated cells using a Ficoll gradient and magnetic depletion of CD45-positive cells. EpCAM-positive CTCs were captured using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. CTCs were defined as cells positive for Hoechst, cytokeratin and negative for multiple immune and endothelial cell markers (CD45/CD11b/CD14 and CD34). EpCAM-captured CTCs were also stained with pericentrin and centrin to identify CA and evaluated for pericentrin (whole centrosome marker) and centrin (centriole marker) protein-staining intensity, number and size. Results: CA was detected in a subset of CTCs from patients with metastatic breast and prostate cancer patients. As a control, centrosomes were also analyzed in matched peripheral blood mononuclear cells from the same patients. CA was heterogeneous with 100% of patients demonstrating some degree of CA (&amp;gt;4 centrioles), ranging from 15-48% of CTCs. Conclusion: In conclusion, we report the presence of CA in EpCAM-captured CTCs from metastatic cancer patients. Evaluation of CA in CTCs using pericentrin and centrin staining may serve as a predictive biomarker of taxane based chemotherapy response and resistance. We are recruiting taxane-naïve metastatic cancer patients to test the hypothesis that CA and/or CIN predict taxane sensitivity. Citation Format: Ashok Singh, Ryan A. Denu, Jamie M. Sperger, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3604.

PO-260 Deep phenotyping of colorectal cancers by high-dimensional mass cytometry reveals tumor-specific immune landscapes

IntroductionImmune checkpoint blockade has revolutionised cancer treatment. However, clinical outcomes are highly variable as only a proportion of cancer patients benefit from this treatment. To understand the mechanisms that determine responses to current immunotherapies and for the design of alternative approaches, it is crucial to characterise the immune landscape of the tumour microenvironment. An in-depth understanding of anti-tumour immunity requires a comprehensive analysis of the immune cell populations that participate in the process of tumorigenesis. The aim of this study is to unravel local and systemic profiles of lymphoid and myeloid immune subsets in colorectal cancer (CRC) using high-dimensional immunophenotyping by mass cytometry.Material and methodsThe expression of 36 immune cell markers was simultaneously assessed at the single-cell level by mass cytometry in tumour tissues (n=19), tumor-associated lymph nodes (n=17), adjacent normal mucosa (n=4), and peripheral blood samples (n=9) derived from 18 CRC patients. In total, 9 million cells were included in the analysis. Cytosplore and HSNE (Hierarchical Stochastic Neighbour Embedding) analysis tools were used to identify and visualise the composition of the lymphoid and myeloid cell populations of the innate and adaptive immune compartments in the tissues.Results and discussionsWe identified 220 immune cell subsets, including tumor-resident CD8+CD45RO+CD103+PD-1+, CD4+CD45RO+CD103+PD-1+ and CD4+CD45RO+ICOS+CD27- T cell populations that were not found in tumor-associated lymph nodes, adjacent normal mucosa, and peripheral blood samples of CRC patients. In addition, we identified an overrepresentation of CD3-CD7+CD56+CD45RO+CD127- intermediate innate lymphoid cells in mismatch-repair deficient cancers and an enrichment of CD14- myeloid populations in mismatch-repair proficient cancers. Unsupervised clustering of the samples based on the composite immune profile separated the aforementioned groups.ConclusionHigh-dimensional immunophenotyping of colorectal cancers reveals tumor-specific immune signatures and charts the complexity of innate and adaptive immune cell populations in the tumour microenvironment. Furthermore, previously unappreciated immune cell subsets further differentiate the two main pathways of tumorigenesis in this cancer type. This work may support the delineation of alternative and tailored immunotherapeutic approaches to improve treatment in colorectal cancer.

PO-364 Effect of exercise and immunotherapy on tumour immunogenicity and growth

IntroductionSeveral preclinical studies have shown that exercise can reduce the tumour growth across a range of tumour models. We recently demonstrated that this exercise-dependent suppression of tumour growth was due to an epinephrine-dependent mobilisation and IL-6-driven activation of cytotoxic cells. In line with this, exercise markedly increased intratumoral immune cell infiltration, thus promoting an immune-stimulatory intratumoral environment. Based on these original findings, we hypothesised that exercise may enhance the effect of immune checkpoint blockade treatment given the exercise-dependent increased intratumoral immune cell infiltration.Material and methodsWe used C57Black/C mice randomised to cages with or without running wheels (EX). After 4 weeks of running (with a running distance ranging from 1.8 km to 5.4 km per mice per day), the mice were inoculated with B16 melanoma cancer cells. In addition, a subset of mice was treated with αPD-L1 antibodies (100 µg per mouse three times per week starting on day 4 after tumour cell injection).Results and discussionsFirst, we observed that voluntary wheel running increased intratumoral expression of the immune checkpoint molecules, PD-L1, B7.1 and B7.2. In parallel, voluntary wheel running increased intratumoral infiltration of cytotoxic NK and T cells, and thus intratumoral expression of the receptors, PD-1 and CD28. Next, we tested the combination therapy of exercise and αPD-L1 antibody treatment. In this setup, wheel running had an overall suppressive effect (p=0.01, 2-way ANOVA). Post hoc analysis showed that αPD-L1 +EX reduced tumour growth by 82.6%, while EX alone reduced tumour growth by 72% with no significant difference between the two interventions. qPCR analysis of these tumours revealed that combination therapy further increased the expression of immune cell markers and immune check point molecules.ConclusionIn conclusion, our results show that exercise increases the expression of immune checkpoint markers and therefore, could be a good combination partner for immune checkpoint blockade treatment. However, more studies are needed to show an effect of this combination therapy on tumour growth.

Abstract 5656: Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry

Abstract Imaging mass cytometry (IMC) is a novel technology with the potential of multiplex proteomic profiling of rare circulating and disseminated tumor cells (CTCs and DTCs) and immune cells from liquid biopsies. The high-definition single-cell analysis (HD-SCA) identifies CTCs and DTCs by the virtue of morphometric and fluorescent staining with exact locations of all the cells, which could be further characterized using downstream genomics and proteomics platform. This study aims to develop and employ metal-labeled antibody panels for multiplex proteomic characterization of CTCs, DTCs and immune cells obtained from longitudinal liquid biopsies from cancer patients on the basis of their epithelial-to-mesenchymal transition (EMT) status, cancer stem cell (CSC), dormancy, proliferation and drug resistance markers. For every antibody panel, 6-10 biomarkers were chosen, and metal-labeled antibodies were processed in a three-stage validation program using known positive and negative control cell lines. In the first stage, basal level marker expression was evaluated in positive control cells seeded on a glass slide. In the second stage, cell lines were spiked in blood and slides were prepared as per the HD-SCA protocol, stored at -800 C, and expression of biomarkers was evaluated in IMC staining. In the third stage, cell line-spiked slides were first stained in three colors immunofluorescent staining in HD-SCA, mimicking the clinical setup followed by IMC staining with metal-labeled antibodies. Antibodies showing expressions in all three stages were included to build a panel targeting EMT, CSC, vasculature mimicry, dormancy, proliferation and drug resistance biomarkers. Out of 69 metal-labeled antibodies, 24 antibodies targeting cancer and immune cell-specific biomarkers have been validated for multiplex proteomic profiling in HD-SCA platform. This includes epithelial markers EpCAM and E-cadherin; mesenchymal markers vimentin and fibronectin; CSC markers CD44, CD24, ALDH and beta-catenin; prostate-specific markers PSA, PSMA, AR-C terminal, AR-N terminal; and immune cell markers CD45, CD66a, CD3, HLA-DR, CD45RA, CD8a, CD4, CD38, CD14 and CD31. Building and validating metal-labeled antibody panels for EMT, CSC, dormancy and drug resistance biomarkers could achieve multiplex proteomic characterization of the rare cell IMC. It would be further used in monitoring treatment efficacy, mutational switchovers, and immune surveillance from longitudinal liquid biopsies collected from cancer patients to better understand the biology of a disease. Citation Format: Milind Pore, Erik Gerdtsson, Christopher Boldt, Sophia Wix, Preeya Katti, Mohan Singh, Akil Merchant, James Hicks, Peter Kuhn. Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5656.

Combined VLA-4-Targeted Radionuclide Therapy and Immunotherapy in a Mouse Model of Melanoma.

Very late antigen-4 (VLA-4; also known as integrin α4β1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT). 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of 177Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor-bearing mice. Methods: Tumor cells (1 × 106) were implanted subcutaneously in C57BL/6 mice. After 8-10 d, the mice were randomized into 8 groups. 177Lu-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 + anti-CTLA-4 or anti-PD-L1 + anti-CTLA-4, whereas TRT + ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT + ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/CD49d-positive) and about 23% immune cells (CD45-positive/CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.