Positive Cell Types Research Articles

Phase I dose escalation trial of the first-in-class TNFR2 agonist monoclonal antibody, HFB200301, in monotherapy and in combination with tislelizumab, an anti-PD-1 monoclonal antibody, in adult patients with advanced solid tumors.

2526 Background: Tumor necrosis factor receptor-2 (TNFR2) is expressed on effector CD8+, CD4+, and regulatory T (Treg) cells, natural killer (NK) cells, and myeloid cells. Activation of TNFR2 is anticipated to yield effective anti-tumor immunity by stimulating T-cell and NK-cell activation and proliferation in the tumor microenvironment. HFB200301 is a first-in-class anti-TNFR2 agonistic monoclonal antibody that triggers both innate and adaptive immune stimulation by binding to a specific epitope on TNFR2. We report initial results of an ongoing Phase I dose escalation, multicenter study (NCT05238883) of HFB200301 in monotherapy and in combination with tislelizumab (TIS) in patients (pts) with advanced refractory solid tumors. Methods: A Phase I trial designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and initial efficacy of HFB200301 in monotherapy and in combination with TIS. Eligible pts, ≥18 years with an ECOG PS 0-1 and advanced solid tumors, will be enrolled in dose escalation at 5 different dose cohorts in monotherapy (Q4W), 3 different dose cohorts in combination with TIS (Q4W), or in dose expansion following determination of safety. Radiographic response is assessed every 8 weeks. Results: A total of 39 pts have been enrolled, 27 pts in monotherapy and 12 pts in combination. All pts received prior systemic therapy (median 2, range 1-4); 23 pts (59%) had prior checkpoint inhibitor treatment. No dose limiting toxicities were observed in either the monotherapy or combination arms. Treatment-related adverse events (TRAEs) occurred in 44% of monotherapy and in 50% of combination pts, with pruritis (11%), tremor (7%), fatigue (7%), asthenia (7%) and nausea (7%) being the most common TRAEs. No ≥ Grade 3 TRAEs were reported, and there were no drug discontinuations due to TRAEs. Preliminary HFB200301 PK analysis demonstrated non-linear clearance of HFB200301, with dose-proportional maximum concentration [Cmax], achieving sufficient exposures for peripheral target engagement in all tested doses. Emerging PD data demonstrates evidence of immune activation and expansion of CD8+ T cells, but not Tregs, in the tumor microenvironment and peripheral blood. Single cell RNAseq analysis of peripheral blood mononuclear cells demonstrated PD modulation in TNFR2 positive immune cell types post-HFB200301 treatment. Early response evaluation reveals ongoing durable clinical responses (> 6 months) in a PD-L1+ pleural mesothelioma pt and an EBV+ gastric cancer pt from combination cohorts. Conclusions: HFB200301 demonstrates a favorable safety profile, dose-dependent PK, with PD and preliminary clinical activity in monotherapy and in combination with TIS in pts with heavily pre-treated refractory solid tumors. Clinical trial information: NCT05238883 .

DMP1 POSITIVE ASTROCYTES MAINTAIN BLOOD-BRAIN BARRIER VIA TRANSFERRING MITOCHONDRIA TO VASCULAR ENDOTHELIAL CELLS

Though dentin matrix protein 1 (Dmp1) is known to play critical role in mediating bone mineralization, it has also been validated to be expressed in brain and helps maintain blood brain barrier (BBB). Our study aims to clarify the expression pattern of Dmp1 in mouse brain and explore whether intercellular mitochondrial transfer occurs between Dmp1 positive astrocytes (DPAs) and endothelial cells, and thus acting as a mechanism in maintaining BBB during aging.Single cell RNA sequencing (scRNAseq) of 1 month, 6 month, and 20 month old mice brain (n=1, respectively) was employed to identify Dmp1 positive cell types. Dmp1Cre-mGmT and Dmp1Cre-COX8a fluorescent mice were generated to visualize DPAs and investigate their mitochondrial activities. A 3D noncontact coculture system and mitochondrial transplantation were applied to study the role of mitochondrial transfer between astrocytes and bEnd.3 endothelial cells. Dmp1Cre-Mfn2f/f mice were generated by depleting the ER-mitochondria tethering protein Mfn2 in DPAs.Dmp1 was mainly expressed in astrocytes at different ages. GO analysis revealed that cell projection and adhesion of DPAs were upregulated. Confocal imaging on Dmp1Cre-mGmT mice indicated that DPAs are a cluster of astrocytes that closely adhere to blood vessels (n=3). Bioinformatics analysis revealed that mitochondrial activity of DPAs were compromised during aging. Enriched scRNAseq of fluorescent cells from Dmp1Cre-COX8a mice (n=2) and immunofluorescent imaging (n=3) validated the acquisition of extrinsic mitochondria in endothelial cells. 3D coculture of astrocytes and bEnd.3 and direct mitochondrial transplantation revealed the rescue effect of mitochondrial transfer on damaged bEnd.3. BBB was impaired after depleting Mfn2 in DPAs, expressing a similar phenotype with aging brain.Astrocytes that express Dmp1 play a significant role in maintaining BBB via transferring mitochondria to vascular endothelial cells. Compromised mitochondrial transfer between DPAs and endothelial cells might be the potential mechanism of impaired BBB during aging.

Abstract 6648: Prediction of anti-PD-1 efficacy based on immune marker densities and their spatial distribution in colorectal cancer

Abstract Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of patients with deficient DNA mismatch repair (d-MMR) metastatic colorectal cancer (CRC), yet less than half of patients receive clinical benefit. We hypothesized that immune contexture including spatial distribution of cells expressing immune molecules in the tumor microenvironment may predict immunotherapy outcome. Methods: Primary CRC tissues from consecutive patients with d-MMR metastatic CRC (N=33) were treated with anti-PD-1 antibodies at Mayo Clinic Comprehensive Cancer Center (2015-2018). Tumors were stained for PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFβR2, MHC-I, CD14, B2M, DAPI and pan-cytokeratin in 5 compartments (overall tumor, tumor epithelia, tumor stroma, peritumor inside vs outside) by multiplex immunofluorescence with digital image analysis. Features computed within each image analysis region included positive cell density, fraction of positive cell types, intensity of positive cells, and spatial distribution between distinct cell types (distances measured using image analysis software). Prior to model fitting, feature selection was performed using regularized Cox regression with LASSO. Regularization parameter was chosen based on 5-fold cross validation. A Cox proportional hazards model was fitted to predict patient progression-free survival (PFS). Results: Among patients with d-MMR CRCs, 16/33 (48.4%) were female, 10 received first-line ICI therapy, and 23 had received > 1 prior chemotherapy regimen. Median age was 61.6 years (IQR of 49.4, 73.7). Eight patients (24.2%) harbored BRAFV600E, 9 (27.2%) had mutant KRAS, and median PFS was 22.2 months (95% CI: 11.8, NR) with 20 events. PD-L1 was expressed in tumor cells, CD68+ macrophages, and CD3+ T lymphocytes. PD-1 expression on CD8+ T lymphocytes was also observed. By univariate analysis, only cell-cell distance readouts achieved statistical significance. Multivariable feature selection identified the mean number of PD-1+ cells within 10 microns of a PD-L1+ cell in the overall tumor as the strongest predictor of anti-PD-1 efficacy, and was significantly associated with PFS (HR=0.87, 95% CI: 0.79-0.95, p< 0.001). In contrast, the ratio of PD-1+/PD-L1+ cells was not predictive of treatment efficacy (p= 0.47), thereby underscoring the importance of spatial distribution for PFS prediction. Conclusion: The mean number of PD-1+ cells in proximity to PD-L1+ cells in tumors was a predictive biomarker of anti-PD-1 efficacy. Confirmatory studies are warranted. Citation Format: Bahar Saberzadeh-Ardestani, Rondell P. Graham, Qian Shi, Eze Ahanonu, Sara McMahon, Crystal Williams, Antony Hubbard, Wenjun Zhang, Andrea Muranyi, Dongyao Yan, Kandavel Shanmugam, Frank A. Sinicrope. Prediction of anti-PD-1 efficacy based on immune marker densities and their spatial distribution in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6648.

ASP210 - a Potent Oligonucleotide-Based Inhibitor for Combating Both BCR-ABL1 Dependent and Independent TKI-Resistant CML Cells

Background: Tyrosine kinase inhibitors (TKIs) have remarkably changed the management of Ph+ chronic myeloid leukemia (CML) and have dramatically contributed to substantial prolongation of survival rates and superior quality of life. However, long-term clinical experience with TKIs shows that despite the apparent therapeutic benefit, ~35% of CML patients exhibit primary resistance or intolerance to TKIs, and ~25% of those treated is forced to switch TKI at least once during therapy due to acquired resistance. Mutations in the kinase domain of BCR-ABL1 are the most comprehensively described mechanism of TKI-resistance but fail to explain ~40% of resistant cases. Nevertheless, due to the availability of several TKIs, the issue of TKI-resistance has de facto reduced to the mutation T315I and BCR-ABL1 independent resistance. TKI-resistance thus represents a persistent clinical problem to be solved and ultimately calls for alternative treatment solutions for CML patients with leukemic clones escaping TKI therapy. Oligonucleotide therapeutics have generated high expectation stemming from their potency and the field has seen great advancements leading to an apparent progress in clinical trials. Given the vastly different molecular target compared to protein inhibitors (RNA versus protein) as well as principally different mechanism of action, oligonucleotide therapeutics might represent rational and potent tools to combat TKI-resistant CML cells. Progress beyond the state-of-the art: The design of standard oligonucleotides is relatively straightforward and is sequence driven. However, the promiscuity of oligonucleotides places severe limitations on this approach since sequence-dependent off-target effects deteriorate their toxicological profile. Unconventionally, we considered additional parameters during the design of an oligonucleotide-based inhibitor, including steric and thermodynamic aspects of its binding to the target RNA, which significantly improved its safety. Here we report on a potent, safe, and highly specific oligonucleotide-based modality against BCR-ABL1 mRNA, ASP210, which effectively induces apoptosis in both BCR-ABL1 dependent and independent TKI-resistant CML cells. Methods:BCR-ABL1 positive cell lines MOLM-7 a CML-T1, both sensitive and resistant to TKIs imatinib and dasatinib were used to investigate the efficacy of ASP210, a biotinylated 34mer phosphorothioate DNA with an internal poly(ethylene glycol) linker. Briefly, MOLM-7 and CML-T1 cells and their resistant sub-lines were incubated with defined concentrations of ASP210 ranging between 0.25-5.0 µM for 10 days (applied once daily). Cell viability was monitored by trypan blue staining every day. The results were normalized to control, i.e., to untreated cells on the respective day. Of note, MOLM-7 TKI-resistant cells did not possess any mutation in the kinase domain, while both resistant sub-lines derived from CML-T1 harbored the clinically relevant mutation T315I. BCR-ABL1 negative cell line HL-60 was used as target negative control in the same experimental setup. Results:In vitro functional tests of ASP210 revealed its ability to induce cell death of all BCR-ABL1 positive cell types studied. A continuous decrease in the viability of ASP210-exposed cells has been observed already after 24 hours in all cell types including both TKI-sensitive CML cells and the derived TKI-resistant sub-lines irrespective of their T315I status. The efficacy of ASP210 was confirmed to be dose-dependent, with ASP210 causing a ~90% reduction of CML cells by day 5. Importantly, no effect on the viability of BCR-ABL1 negative HL-60 cells was observed (viability remained ≥95%) upon exposure to ASP210. Conclusions: The present study demonstrates the cytoreductive potential of ASP210 in terms of its ability to induce apoptosis in both BCR-ABL1 dependent and independent TKI-resistant CML cells, with no effect on BCR-ABL1 negative cells.

Moniezia benedeni Infection Restrain IgA+, IgG+, and IgM+ Cells Residence in Sheep (Ovis aries) Small Intestine

Secreted immunoglobulin A (SIgA), IgG, and IgM play a crucial role in forming the intestinal mucosal immune barrier, and parasites could disturb the host's immune response by releasing various immunomodulatory molecules. Moniezia benedeni is an important pathogen parasitizing in the sheep small intestine. It is aimed to explore the residence characteristics of IgA+, IgG+, and IgM+ cells in the sheep small intestine, and the influence of Moniezia benedeni infection on them. Control group (n = 6) and infected group (n = 6) were selected, respectively, and the three subtype cells residing in the small intestine were systematically observed and analyzed. The results showed that in the Control group, the three types of positive cells were all distributed diffusely, and the total densities in jejunum, duodenum and ileum was gradually declined in turn. Notably, the change trend of IgA+ and IgG+ cells densities were both congruent with the total densities, and the differences among them were significant, respectively (P < 0.05); the IgM+ cells density was the highest in duodenum, followed by jejunum and ileum, there was no significant difference between duodenum and jejunum (P > 0.05), but both significantly higher than in ileum (P < 0.05). In the Infected group, their total densities in duodenum, jejunum and ileum were gradually declined in turn. Notably, the IgA+ and IgM+ cells densities change trend was the same as the total densities, and the differences among them were significant, respectively (P < 0.05). The IgG+ cells density in duodenum was the highest, followed by ileum and jejunum and there was significantly difference among them (P < 0.05). The comparison results between Control and Infected groups showed that from the duodenum, jejunum to ileum, IgA+, IgG+, and IgM+ cells were all reduced significantly, respectively. The results suggest that the three types of positive cells were resided heterogeneously in the small intestinal mucosa, that is, significant region-specificity; Moniezia benedeni infection could not change their diffuse distribution characteristics, but strikingly, reduce their resident densities, and the forming mucosal immune barrier were significantly inhibited. It provided powerful evidence for studying on the molecular mechanism of Moniezia benedeni evasion from immune surveillance by strongly inhibiting the host's mucosal immune barrier.

Quantum dots based in-vitro co-culture cancer model for identification of rare cancer cell heterogeneity

Cancer cell heterogeneity (CCH) is crucial in understanding cancer progression and metastasis. The CCH is one of the stumbling blocks in modern medicine's therapeutics and diagnostics . An in-vitro model of co-culture systems of MCF-7, HeLa, HEK-293, with THP-1 cells showed the occurrence of EpCAM positive (EpCAM+) and EpCAM negative (EpCAM−) heterogenetic cancer cell types labeled with the Quantum Dot antibody conjugates (QDAb). This in-vitro model study could provide insights into the role of rare cancer cells manifestation and their heterogeneity in metastatic progression and risk for severe infections in these patients. We successfully report the presence of CCH based on the fluorescence ratios of the co-cultured cancer cells when treated with the QDAb. These short-term mimic co-cultures give a compelling and quite associated model for assessing early treatment responses in various cancers.

Activation of C-C motif chemokine receptor 2 modulates testicular macrophages number, steroidogenesis, and spermatogenesis progression.

The monocyte chemoattractant protein 1 (MCP-1) belongs to the CC chemokine family and acts in the recruitment of C-C motif chemokine receptor 2 (CCR2)-positive immune cell types to inflammation sites. In testis, the MCP-1/CCR2 axis has been associated with the macrophage population's functional regulation, which presents significant functions supporting germ cell development. In this context, herein, we aimed to investigate the role of the chemokine receptor CCR2 in mice testicular environment and its impact on male sperm production. Using adult transgenic mice strain that had the CCR2 gene replaced by a red fluorescent protein gene, we showed a stage-dependent expression of CCR2 in type B spermatogonia and early primary spermatocytes. Several parameters related to sperm production were reduced in the absence of CCR2 protein, such as Sertoli cell efficiency, meiotic index, and overall yield of spermatogenesis. Daily sperm production decreased by almost 40%, and several damages in the seminiferous tubules were observed. Significant reduction in the expression of important genes related to the Sertoli cell function (Cnx43, Vim, Ocln, Spna2) and meiosis initiation (Stra8, Pcna, Prdm9, Msh5) occurred in comparison to controls. Also, the number of macrophages significantly decreased in the absence of CCR2 protein, along with a disturbance in Leydig cell steroidogenic activity. In summary, our results show that the non-activation of the MCP-1/CCR2 axis disturbs the testicular homeostasis, interfering in macrophage population, meiosis initiation, blood-testis barrier function, and androgen synthesis, leading to the malfunction of seminiferous tubules, decreased testosterone levels, defective sperm production, and lower fertility index.

Dio2-CreErt2 Knockin Model to Identify T3-Generating Cells That Express Type 2 Deiodinase in Tissues

Background: Thyroid hormone promotes many developmental and homeostatic functions. Apart from adequate circulating levels, the concentration of the active hormone T3 within tissues may be amplified by type 2 deiodinase (Dio2) by conversion from the precursor T4. Dio2 is critical in auditory development, bone maturation, brain function and control of the hypothalamic-pituitary-thyroid axis. Despite its crucial role, an obstacle to studying Dio2 is that the protein has a short half-life, is at low levels and is often transiently expressed, making it difficult to identify Dio2 in tissues at cellular resolution. Methods: We derived a Dio2-CreERt2 knockin mouse that expresses tamoxifen-dependent Cre recombinase from the endogenous Dio2 gene. When crossed onto Ai6 reporter mice, following tamoxifen treatment, Dio2-CreERt2 expression is detected as fluorescent signal in specific cells in brain regions, pituitary, and other tissues. We showed previously that Dio2 is essential for hearing with rising expression levels in the cochlea prior to onset of hearing. The Dio2-CreERt2 model identified positive cell types in the cochlear spiral ligament, septal divisions and modiolus around the sensory epithelium. Dio2-positive fibrocytes were adjacent to and extended projections around blood capillary networks, the source of T4 supply. Transcriptome analysis of isolated positive cells revealed bone lineage-related origins for many of these cells. Conclusion: The Dio2-CreERt2 model detects Dio2 expression sensitively at cellular resolution. In the cochlea, Dio2-positive cell types reside in vascularized support tissues, suggesting combined endocrine and paracrine-like control of the T3 supply. Analysis of cell origins suggests novel interactions between endocrine and skeletal systems in promoting T3 action required for hearing.

Tryptophan 2,3-Dioxygenase Expression Identified in Murine Decidual Stromal Cells Is Not Essential for Feto-Maternal Tolerance

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) catalyze the rate-limiting step of tryptophan catabolism along the kynurenine pathway, which has important immuno suppressive properties, particularly in tumor cells and dendritic cells. The prominent expression of IDO1 in the placenta also suggested a role in preventing immune rejection of fetal tissues, and pharmacological inhibition of IDO1 induced abortion of allogeneic fetuses in mice. However, this was later challenged by the lack of rejection of allogeneic fetuses in IDO1-KO mice, suggesting that other mechanisms may compensate for IDO1 deficiency. Here we investigated whether TDO could contribute to feto-maternal tolerance and compensate for IDO1 deficiency in IDO1-KO mice. Expression of TDO mRNA was previously detected in placental tissues. We developed a new chimeric rabbit anti-TDO antibody to confirm TDO expression at the protein level and identify the positive cell type by immunohistochemistry in murine placenta. We observed massive TDO expression in decidual stromal cells, starting at day E3.5, peaking at day E6.5 then declining rapidly while remaining detectable until gestation end. IDO1 was also induced in decidual stromal cells, but only at a later stage of gestation when TDO expression declined. To determine whether TDO contributed to feto-maternal tolerance, we mated TDO-KO and double IDO1-TDO-KO females with allogeneic males. However, we did not observe reduced fertility. These results suggest that, despite its expression in decidual stromal cells, TDO is not a dominant mechanism of feto-maternal tolerance able to compensate for the absence of IDO1. Redundant additional mechanisms of immunosuppression likely take over in these KO mice. The massive expression of TDO during decidualization might suggest a role of TDO in angiogenesis or vessel tonicity, as previously described for IDO1.

An ancestral apical brain region contributes to the central complex under the control of foxQ2 in the beetle Tribolium.

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.

Mst1/2 kinases restrain transformation in a novel transgenic model of Ras driven non-small cell lung cancer.

Non-small cell lung cancer remains a highly lethal malignancy. Using the tamoxifen inducible Hnf1b:CreERT2 (H) transgenic mouse crossed to the LsL-KrasG12D (K) transgenic mouse, we recently discovered that an Hnf1b positive cell type in the lung is sensitive to adenoma formation when expressing a mutant KrasG12D allele. In these mice, we observe adenoma formation over a time frame of three to six months. To study specificity of the inducible Hnf1b:CreERT2 in the lung, we employed lineage tracing using an mTmG (G) reporter allele. This technique revealed recombined, GFP+ cells were predominantly SPC+. We further employed this technique in HKG mice to determine Hnf1b+ cells give rise to adenomas that express SPC and TTF1. Review of murine lung tissue confirmed a diagnosis of adenoma and early adenocarcinoma, a pathologic subtype of non-small cell lung cancer. Our expanded mouse model revealed loss of Mst1/2 promotes aggressive lung adenocarcinoma and large-scale proteomic analysis revealed upregulation of PKM2 in the lungs of mice with genetic deletion of Mst1/2. PKM2 is a known metabolic regulator in proliferating cells and cancer. Using a human lung adenocarcinoma cell line, we show pharmacologic inhibition of Mst1/2 increases the abundance of PKM2, indicating genetic loss or pharmacologic inhibition of Mst1/2 directly modulates the abundance of PKM2. In conclusion, here we report a novel model of non-small cell lung cancer driven by a mutation in Kras and deletion of Mst1/2 kinases. Tumor development is restricted to a subset of alveolar type II cells expressing Hnf1b. Our data show loss of Mst1/2 regulates levels of a potent metabolic regulator, PKM2.

Glutaminase Activity of L-Asparaginase Contributes to Durable Preclinical Activity against Acute Lymphoblastic Leukemia.

We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNaseWT) and its glutaminase-deficient Q59L mutant (ASNaseQ59L) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo, even against ASNS-negative cancer types.

Genomic analysis of benign prostatic hyperplasia implicates cellular re-landscaping in disease pathogenesis.

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.

Abstract 346: A Standard Flow Cytometry Protocol for Assessing Human Pluripotent Stem Cell-derived Cardiomyocyte Identity by Troponin Positivity

Directed differentiation of human pluripotent stem cells (hPSC) into cardiomyocytes (hPSC-CM) offers an inexhaustible supply of cells for basic science research and translational applications. However, despite significant advancements in defining the factors most critical for differentiation, the resulting cultures remain a heterogeneous mixture of cells. Ultimately, as heterogeneity can pose challenges to interpreting functional data, the ability to accurately and precisely assess cell identity in differentiation cultures is paramount to well-defined and reproducible studies. To date, a standardized flow cytometry protocol that is broadly accepted among laboratories has not been established for assessing cell type heterogeneity within hPSC-CM cultures, posing challenges to evaluating outcomes generated among laboratories and studies. A survey of studies published over the past seven years (1/2010-10/2017) reveals the wide range of antibodies and experimental conditions reported for flow cytometry-based assessment of hPSC-CM. Although our literature survey revealed that a preponderance of studies relied on TNNT2 as a marker of cardiomyocyte identity, TNNI3 is more specific to cardiomyocytes than TNNT2 throughout development. We applied targeted mass spectrometry to confirm the presence of TNNI3 in our hPSC-CM. We then investigated five commercially available anti-TNNI3 and three sample preparation techniques for use in the assessment of heterogeneity of hPSC-CM cultures by flow cytometry. Results demonstrate two of the five anti-TNNI3 clones appear suitable for marking hPSC-CM and reveal differential susceptibility of antibody clones to sample preparation conditions. To further test the rigor and utility of our flow cytometry protocol, it was shared with two collaborating laboratories and applied to heterogeneous mixtures of positive and negative cell types. Results from these two laboratories demonstrate the protocol successfully distinguishes positive and negative cell types similarly, despite using different cell lines and differentiation protocols. We propose a workflow for establishing the fit-for-purpose use of antibodies and a standard protocol for assessing hPSC-CM identity by troponin positivity.

Analysis of Pro-inflammatory Cytokine and Type II Interferon Induction by Nanoparticles.

Cytokines, chemokines, and interferons are released by the immune cells in response to cellular stress, damage and/or pathogens, and are widely used as biomarkers of inflammation. Certain levels of cytokines are needed to stimulate an immune response in applications such as vaccines or immunotherapy where immune stimulation is desired. However, undesirable elevation of cytokine levels, as may occur in response to a drug or a device, may lead to severe side effects such as systemic inflammatory response syndrome or cytokine storm. Therefore, preclinical evaluation of a test material's propensity to cause cytokine secretion by healthy immune cells is an important parameter for establishing its safety profile. Herein, we describe in vitro methods for analysis of cytokines, chemokines, and type II interferon in whole blood cultures derived from healthy donor volunteers. First, whole blood is incubated with controls and tested nanomaterials for 24h. Then, culture supernatants are analyzed by ELISA to detect IL-1β, TNFα, IL-8, and IFNγ. The culture supernatants can also be analyzed for the presence of other biomarkers secreted by the immune cells. Such testing would require additional assays not covered in this chapter and/or optimization of the test procedure to include relevant positive controls and/or cell types.

Epstein-Barr virus-encoded small RNAs in idiopathic orbital inflammatory pseudotumor tissues: a comparative case series.

To investigate the positive rate and types of cells that express Epstein-Barr virus-encoded small RNAs (EBERs) and to determine the distribution of EBER-expressing cells in idiopathic orbital inflammatory pseudotumor (IOIP) tissues. We retrospectively examined 40 archived paraffin specimens from two teaching hospitals in Southern China between January 2007 and January 2015 that were pathologically determined to exhibit IOIP. Eleven concurrent paraffin specimens of thyroid-associated ophthalmopathy (TAO) composed the control group. In situ hybridization was performed to detect EBERs. Immunohistochemistry was employed to detect CD3, CD20, Vimentin, and smooth muscle actin (SMA), and the positive rate, types of positive cells, and distribution and location of EBERs were evaluated. The positive expression rate of EBERs was 47.5% (19/40) in the IOIP group, which was significantly higher than that in the TAO group [0 (0/11), P=0.011]. In the IOIP group, the lymphocyte infiltrative subtype, fibrotic subtype, and mixed subtype exhibited EBER-positive rates of 57.1% (12/21), 12.5% (1/8), and 54.5% (6/11), respectively, and no significant differences were found between these subtypes (P=0.085). Positive signals of EBERs were mainly present in medium-small lymphocytes between or around follicles and in the nuclei of activated immunoblasts (14/19). The positive rate, types, and distribution of EBER-expressing cells in IOIP have been documented. These findings are conducive for a better understanding of the underlying mechanisms of Epstein-Barr virus infection in IOIP pathogenesis.

Abstract 2937: Utilizing multiplex chromogenic IHC and digital image analysis to evaluate immune cell content and spatial distribution within NSCLC tumor tissue

Abstract Chromogenic-based immunohistochemistry (IHC) is an established technique utilized within oncology R&amp;D, allowing cells to be visualized within the context of their tumor microenvironment. However, when quantifying chromogenic stained sections via digital image analysis, the number of targets is usually limited due to constraints in deconvolving more than 3 chromogens present on the same tissue section image. The purpose of this study was to apply a customized algorithm, including a 5-colour deconvolution process to triplex chromogen IHC plus hematoxylin stained Non-Small Cell Lung Carcinoma (NSCLC) tumor tissues in order to quantify immune cell content and spatial distribution within both tumor and stroma regions of interest (ROI). A multiplex IHC assay was developed and applied to FFPE sections of NSCLC tumor tissue to stain epithelial tumor cells (Pan CK, yellow), CD8+ cells (purple) and FoxP3+ nuclei (brown), counterstained with hematoxylin (blue). Immunostained whole slide images were generated for image analysis. A customized algorithm, which included a 5-chromogen color deconvolution process, was utilized within the Indica Labs HALO platform to separate the four IHC chromogens (including counterstain) and a fifth color (black) representing carbon deposit artefacts. Nuclear objects were formed by applying weighted optical density values for the brown, purple and blue colors, which were positive for either CD8 or FoxP3 and counterstain. A classifier was developed to automatically segment tumor from stroma. Each positive cell type was identified, using defined size and shape parameters, and quantified within each ROI. IHC multiplex staining highlighted CD8+ cells and FoxP3+ nuclei to be present in both tumor and stroma compartments. Analysis data demonstrated the number of tumor and stroma CD8+ cells was 120 and 554 cells per mm2, while the number of tumor and stroma FoxP3+ nuclei was 20 and 19 nuclei per mm2. This gave a CD8+:FoxP3+ ratio of 6 within the tumor and 29 within the stroma. The average distance of CD8+ cells in the tumor or stroma to their nearest FoxP3+ nuclei was 92µm and 174µm respectively. The average distance of CD8+ cells to their nearest tumor cell was 39µm while the average distance of FoxP3+ nuclei to their nearest tumor cell was 29µm. This study demonstrated multiplex chromogenic IHC as a valuable approach for quantifying multiple cell types within the context of their tumor microenvironment. Furthermore, up to five chromogen colors can be separated utilizing a custom 5-plex analysis algorithm, leading to a more in-depth evaluation of immune cell types present and their spatial distribution. This propounds a greater potential for interpretation of therapeutic mechanistic responses within the tumor microenvironment. Citation Format: Lorcan Sherry, Mark Anderson, Marianne Cowan, Lee Dawson, Richard Bystry, Sarah-Jane Green. Utilizing multiplex chromogenic IHC and digital image analysis to evaluate immune cell content and spatial distribution within NSCLC tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2017-2937

IL4 Primes the Dynamics of Breast Cancer Progression via DUSP4 Inhibition.

The tumor microenvironment supplies proinflammatory cytokines favoring a permissive milieu for cancer cell growth and invasive behavior. Here we show how breast cancer progression is facilitated by IL4 secreted by adipose tissue and estrogen receptor-positive and triple-negative breast cancer cell types. Blocking autocrine and paracrine IL4 signaling with the IL4Rα antagonist IL4DM compromised breast cancer cell proliferation, invasion, and tumor growth by downregulating MAPK pathway activity. IL4DM reduced numbers of CD44+/CD24- cancer stem-like cells and elevated expression of the dual specificity phosphatase DUSP4 by inhibiting NF-κB. Enforced expression of DUSP4 drove conversion of metastatic cells to nonmetastatic cells. Mechanistically, RNAi-mediated attenuation of DUSP4 activated the ERK and p38 MAPK pathways, increased stem-like properties, and spawned metastatic capacity. Targeting IL4 signaling sensitized breast cancer cells to anticancer therapy and strengthened immune responses by enhancing the number of IFNγ-positive CTLs. Our results showed the role of IL4 in promoting breast cancer aggressiveness and how its targeting may improve the efficacy of current therapies. Cancer Res; 77(12); 3268-79. ©2017 AACR.

HVEM-BTLA interactions stimulate lung innate immunity following S. pneumoniae infection.

Abstract Streptococcus pneumoniae (S. pneumoniae) is the leading cause of bacterial pneumonia in children and the elderly and it kills close to half a million children under 5 years old worldwide every year. Recent reports indicate that epithelial cells act not only as a physical barrier to pathogen invasion, but also as an extension of the immune system to prevent infection or eliminate pathogen. The herpes virus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor super family. HVEM polymorphisms are a risk factor for several inflammatory and infectious diseases. Although lymphocytes and myeloid cells express HVEM, our previous data suggested that HVEM expression by epithelial cells was critical for innate immunity in the lung. However, the mechanism of HVEM-mediated host defense against S. pneumoniae is still unknown. To address the issue, we have analyzed mice with a lung epithelial cell-specific deletion of HVEM (Cc10-cre x Hvemfl/fl). We found that Cc10-cre x Hvemfl/fl mice were highly susceptible to S. pneumoniae, with an increased bacterial load by 24h and reduced survival. Mice treated with an antibody that blocks HVEM had a similar outcome, confirming that HVEM expression is required during the infection. HVEM has three ligands: the TNF super family member LIGHT and the Ig super family members BTLA and CD160. We determined that BTLA-deficient mice showed increased bacterial load in the lung and reduced survival, similar to HVEM deficiency. Moreover, CD11c-cre x BTLA fl/fl mice were highly susceptible to S. pneumoniae. Our data show that interaction between BTLA expressed by a CD11c positive myeloid cell type and HVEM expressed by lung epithelial cells leads to a protective innate response in the lung after infection.

Dystrophin Distribution and Expression in Human and Experimental Temporal Lobe Epilepsy.

Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE).Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats.Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls.Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated in human TLE, but not in AK rats, possibly indicating a compensatory mechanism in the chronic epileptic human brain.