Quantitative Immunofluorescence Analysis Research Articles

Keratinocyte-Expressed Podoplanin is Dispensable for Multi-Step Skin Carcinogenesis.

Podoplanin is a small transmembrane mucin-like glycoprotein that plays a crucial role in the development of the lung, heart and lymphatic vascular system. Its expression is upregulated in several types of human carcinomas and podoplanin levels in squamous cell carcinomas (SCCs) of the oral cavity and the lung correlate with cancer invasiveness, lymph node metastasis and shorter survival time of patients, indicating that podoplanin promotes tumor progression. However, its role during the early stages of carcinogenesis remain unclear. We generated mice with a specific deletion of podoplanin in epidermal keratinocytes (K5-Cre;Pdpnflox/flox mice) and subjected them to a multistep chemical skin carcinogenesis regimen. The rate of tumor initiation; the number, size and differentiation of tumors; and the malignant transformation rate were comparable in K5-Cre;Pdpnflox/flox mice and Pdpnflox/flox control mice. However, tumor cell invasion was reduced in K5-Cre;Pdpnflox/flox mice, in particular single cell invasion. Quantitative immunofluorescence analyses revealed that peritumoral lymphangiogenesis was reduced in K5-Cre;Pdpnflox/flox mice, whereas there were no major changes of tumor-associated immune cell subpopulations. Thus, keratinocyte-expressed podoplanin is dispensable for the early steps of skin carcinogenesis but contributes to the progression of established tumors.

SO038A TRANSLATIONAL KIDNEY ORGANOID SYSTEM BOLSTERS HUMAN RELEVANCE OF CLINICAL DEVELOPMENT CANDIDATE

Abstract Background and Aims A major challenge in drug discovery is gaining confidence in the human relevance of pre-clinical animal studies. While human iPSC-derived organoids offer exciting opportunities to address this, concerns about applicability and scalability remain. Here, we report a high-throughput human organoid platform for assessment of kidney disease targeting compounds. Method In vitro & in vivo transplanted under athymic rat kidney capsule, differentiated organoids were characterized using single cell RNA sequencing (scRNA-Seq), NanoString, & immunofluorescence techniques. Immunofluorescence quantitative analysis of aggregated actin per in vitro organoid in a protamine sulfate (PS) podocyte injury model was used to evaluate efficacy of GFB-887, a sub-type selective, small molecule transient receptor potential canonical 5 (TRPC5) inhibitor. In pharmacokinetic studies, GFB-887 was orally administered into rats, then the presentation of GFB-887 was measured in rat plasma, rat kidney, & in vivo transplanted human organoids. In pharmacodynamic studies of transplanted human organoids, rats were co-perfused with GFB-887 & PS or orally dosed with GFB-887 prior to PS, then immunofluorescence quantitative analysis of mean synaptopodin intensity in podocytes of the in vivo transplanted organoids was used to evaluate efficacy of GFB-887. Results We confirmed platform reproducibility by scRNA-Seq and derived a NanoString panel for efficient quality control. Organoid transplantation in rats for 2 to 4 weeks promoted organoid maturation and vascularization. In functional studies, cyclosporine A (CsA), a calcineurin inhibitor clinically utilized for the treatment of nephrotic syndrome, and GFB-887, a novel sub-type selective TRPC5 inhibitor currently in clinical development, protected in vitro kidney organoids from injury. Pharmacodynamic studies with GFB-887 delivered orally to rats were also successfully performed in human transplanted organoids. Conclusion These data show how human organoids can deliver confidence in taking development candidate compounds to the clinic, fulfilling their promise to revolutionize drug discovery.

Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice

Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1KO-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO-KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6β4 into sEVs-a process that required CD82. Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice.

Complement component C1q plays a critical role in VLRA/VLRC-mediated immune response

In jawless vertebrates, the lamprey complement component C1q (LC1q) acts as a lectin and activates lamprey complement component C3 (LC3) in association with mannose-binding lectin (MBL)-associated serine protease (MASP) via the lectin pathway. Furthermore, LC1q may interact with variable lymphocyte receptor B (VLRB) in a complex with antigens and mediate the activation of LC3, leading to cytolysis. In the present study, we found, for the first time, that LC1q plays a critical role in VLRA/VLRC-mediated immune response. Escherichia coli, Shigella flexneri, Aeromonas hydrophila, Pseudomonas plecoglossicida, Aeromonas allosaccharophila, P. luteola, Brevundimonas diminuta, and Bacillus cereus were isolated from infected Lampetra morii in our laboratory and identified using the 16s rRNA method. A. hydrophila was confirmed as a rapidly spreading lethal pathogen in the larvae of L. morii and was used in subsequent immune stimulation experiments. The results of real-time quantitative polymerase chain reaction (Q-PCR) and immunofluorescence analyses indicated that the RNA and protein expression levels of LC1q were upregulated following exposure to 107 cfu/mL of A. hydrophila, compared to the levels of the naïve group. We obtained LC1q morphants (LC1q MO) of lamprey larvae by morpholino-mediated knockdowns. We found that LC1q played key roles in the embryonic development of lamprey. The median lethal time (LT50) of LC1q MO larvae was 2 d after being exposed to the pathogens, whereas the LT50 of control MO was 5 d. The drastic decrease in LT50 values after LC1q knockdown implies that LC1q plays a critical role in lamprey immune response. Gene expression profiles of LC1q-deficient A. hydrophila, control MO A. hydrophila, wild type A. hydrophila, and naive 1-month-old ammocoetes larvae were compared by examining the expression levels of a selected panel of orthologous genes. It is worth mentioning that LC1q MO affected the VLRA+/VLRC + population genes but did not affect the VLRB + populations. Immunohistochemical data indicated that LC1q deficiency also affected VLRA and VLRC but not VLRB. Thus, LC1q plays a critical role in VLRA/VLRC-mediated immune response in lamprey.

A novel immunocompetent model of metastatic prostate cancer-induced bone pain.

Over 70% to 85% of men with advanced prostate cancer (PCa) develop bone metastases characterized by severe bone pain and increased likelihood of bone fracture. These clinical features result in decreased quality of life and act as a predictor of higher mortality. Mechanistically, the skeletal pathologies such as osteolytic lesions and abnormal osteoblastic activity drive these symptoms. The role of immune cells in bone cancer pain remains understudied, here we sought to recapitulate this symptomology in a murine model. The prostate cancer bone metastasis-induced pain model (CIBP) was established by transplanting a mouse prostate cancer cell line into the femur of immunocompetent mice. Pain development, gait dynamics, and the changes in emotional activities like depression and anxiety were evaluated. Animal tissues including femurs, dorsal root ganglion (DRG), and spinal cord were collected at killing and microcomputed tomography (μCT), histology/immunohistochemistry, and quantitative immunofluorescent analysis were performed. Mice receiving prostate cancer cells showed a significantly lower threshold for paw withdrawal responses induced by mechanical stimulation compared with their control counterparts. Zero maze and DigiGait analyses indicated reduced and aberrant movement associated emotional activity compared with sham control at 8-weeks postinjection. The μCT analysis showed osteolytic and osteoblastic changes and a 50% reduction of the trabecular volumes within the prostate cancer group. Neurologically we demonstrated, increased calcitonin gene-related peptide (CGRP) and neuronal p75NTR immune-reactivities in both the projected terminals of the superficial dorsal horn and partial afferent neurons in DRG at L2 to L4 level in tumor-bearing mice. Furthermore, our data show elevated nerve growth factor (NGF) and TrkA immunoreactivities in the same segment of the superficial dorsal horn that were, however, not colocalized with CGRP and p75NTR . This study describes a novel immunocompetent model of CIBP and demonstrates the contribution of NGF and p75NTR to chronic pain in bone metastasis.

Dual role of Ca2+-activated Cl\u2212 channel transmembrane member 16A in lipopolysaccharide-induced intestinal epithelial barrier dysfunction in vitro

Dysfunction of intestinal epithelial Cl− currents and channels have previously been reported in inflammatory intestinal diseases. However, the expression and function of the newly identified Ca2+-activated Cl− channel transmembrane member 16A (TMEM16A) in the intestinal epithelium is unclear. In this study, we investigated the effects of TMEM16A on intestinal epithelial barrier function in vitro. Intestinal epithelial barrier dysfunction was modeled by lipopolysaccharide (LPS)-induced cell damage in intestinal epithelial IEC-6 cells and the effects of TMEM16A knockdown and overexpression on cell apoptosis and tight junctions were studied. Corresponding mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence analysis, respectively. TMEM16A expression was significantly increased by LPS, possibly via a process involving the transcription factor nuclear factor-κB and both Th1 and Th2 cytokines. Low- and high-dose LPS dysregulated tight junctions (high-myosin light-chain kinase expression) and cell apoptosis-dependent cell barrier dysfunction, respectively. TMEM16A aggravated cell barrier dysfunction in IEC-6 cells pretreated with low-dose LPS by activating ERK1/MLCK signaling pathways, but protected against cell barrier dysfunction by activating ERK/Bcl-2/Bax signaling pathways in IEC-6 cells pretreated with high-dose LPS. We concluded that TMEM16A played a dual role in LPS-induced epithelial dysfunction in vitro. The present results indicated the complex regulatory mechanisms and targeting of TMEM16A may provide potential treatment strategies for intestinal epithelial barrier damage, as well as forming the basis for future studies of the expression and function of TMEM16A in normal and inflammatory intestinal diseases in vivo.

Loss of HAT1 expression confers BRAFV600E inhibitor resistance to melanoma cells by activating MAPK signaling via IGF1R

BRAF inhibitors (BRAFi) have been approved for the clinical treatment of BRAF-mutant metastatic melanoma. Although initial responses to BRAFi are generally favorable, acquired BRAFi resistance emerges rapidly, resulting in treatment failure. Only some of the underlying mechanisms responsible for BRAFi resistance are currently understood. Here, we showed that the genetic inhibition of histone acetyltransferase 1 (HAT1) in BRAF-mutant melanoma cells resulted in BRAFi resistance. Using quantitative immunofluorescence analysis of patient sample pairs, consisting of pre-treatment along with matched progressed BRAFi + MEKi-treated melanoma samples, HAT1 downregulation was observed in 7/11 progressed samples (~63%) in comparison with pre-treated samples. Employing NanoString-based nCounter PanCancer Pathway Panel-based gene expression analysis, we identified increased MAPK, Ras, transforming growth factor (TGF)-β, and Wnt pathway activation in HAT1 expression inhibited cells. We further found that MAPK pathway activation following the loss of HAT1 expression was partially driven by increased insulin growth factor 1 receptor (IGF1R) signaling. We showed that both MAPK and IGF1R pathway inhibition, using the ERK inhibitor SCH772984 and the IGF1R inhibitor BMS-754807, respectively, restored BRAFi sensitivity in melanoma cells lacking HAT1. Collectively, we show that the loss of HAT1 expression confers acquired BRAFi resistance by activating the MAPK signaling pathway via IGF1R.

Abstract P3-09-17: Preoperative pembrolizumab increases sTILs in treatment naïve stage I/IIA TNBC patients in a window of opportunity trial

Abstract Background: High levels of stromal tumor-infiltrating lymphocytes (sTILs) are significantly associated with chemosensitivity and improved overall survival for patients with triple negative breast cancer (TNBC). Our aim is to determine if we can significantly increase sTILs from biopsy to lumpectomy in a presurgical window of opportunity trial with pembrolizumab and intraoperaperative radiation (IORT) in treatment-naïve early stage TNBC compared to matched IORT only controls. We also evaluate the changes in the spatial distribution of intraepithelial TILs (iTILs) by utilizing a deep learning algorithm. Our secondary aim is to characterize the tumor immune microenvironment (TME) using quantitative multiplex immunofluorescence (qmIF) and transcriptomic analysis of tumor-associated and immune genes. Methods: Seven women with node negative TNBC <2.5 cm were enrolled in either our presurgical trial with pembrolizumab and IORT as an upfront boost (NCT02977468) or a parallel study with IORT alone (NCT03165487). The treatment consisted of two doses of pre-operative pembrolizumab (200mg IV) given alone with the second dose -10 to -18 days from surgery; 20Gy IORT was delivered during surgery to the lumpectomy bed. All patients completed standard of care adjuvant chemotherapy and whole breast irradiation. Initial analyses performed include assessments of sTILs and iTILs on H&E sections from the initial biopsy and lumpectomy. iTILs were identified with a deep learning algorithm in pathologist annotated regions with invasive carcinoma, excluding in situ legions. Deep learning derived percentages of iTILs were validated through comparison with manual estimates performed by a pathologist per international guidelines. Spatial clustering analysis with iTILs was performed by calculating the Ball-Hall index for each case to measure the mean dispersion through all the clusters. Results: Seven patients were enrolled between Nov 2017-Apr 2019; 3 patients received pembrolizumab+IORT; and 4 patients received IORT alone. Initial pathology assessment of sTILs demonstrated no significant change between biopsy and lumpectomy in control patients. No consistent patterns of iTILs dispersion were observed in the control patients. Out of the 3 pembrolizumab treated patients, one patient demonstrated a marked increase in sTILs (20% to 60%) with a shift from a neutrophil rich to lymphoplasmacytic stromal infiltrate where the mean dispersion of the iTILs doubled (180 to 360). In the other 2 pembrolizumab patients, one remained stable and one showed a slight increase in sTILs. The iTILs dispersion increased significantly in these patients (from 40 to 90 and 10 to 280, respectively). A corresponding spatial analysis of sTILs is ongoing. Additionally, qmIF and transcriptomic analysis of tumor-associated and immune genes is currently underway to compare biopsy and surgical specimens. Conclusion: In this preliminary analysis, we observe preoperative pembrolizumab-induced increases in sTILs with increased mean dispersion of iTILs in treatment naïve TNBC. Our initial analysis in this ongoing trial appears promising after examining changes in both the density and spatial structure of TILs. Further investigation is warranted to elucidate the impact of preoperative pembrolizumab in the TME of early stage TNBC and other potential therapeutic implications. Citation Format: Rami Vanguri, Hua Guo, Shuobo Boboila, Tahsin Kurc, Han Le, Shahira Abousamra, Le Hou, Dimitris Samaras, Rajarsi Gupta, Joel Saltz, Kevin Gardner, Hanina Hibshoosh, Kevin Kalinsky, Eileen Connolly. Preoperative pembrolizumab increases sTILs in treatment naïve stage I/IIA TNBC patients in a window of opportunity trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-17.

Spatial and biochemical interactions between bone marrow adipose tissue and hematopoietic stem and progenitor cells in rhesus macaques.

Recent developments in in situ microscopy have enabled unparalleled resolution of the architecture of the bone marrow (BM) niche for murine hematopoietic stem and progenitor cells (HSPCs). However, the extent to which these observations can be extrapolated to human BM remains unknown. In humans, adipose tissue occupies a significant portion of the BM medullary cavity, making quantitative immunofluorescent analysis difficult due to lipid-mediated light scattering. In this study, we employed optical clearing, confocal microscopy and nearest neighbor analysis to determine the spatial distribution of CD34+ HSPCs in the BM in a translationally relevant rhesus macaque model. Immunofluorescent analysis revealed that femoral BM adipocytes are associated with the branches of vascular sinusoids, with half of HSPCs localizing in close proximity of the nearest BM adipocyte. Immunofluorescent microscopy and flow cytometric analysis demonstrate that BM adipose tissue exists as a multicellular niche consisted of adipocytes, endothelial cells, granulocytes, and macrophages. Analysis of BM adipose tissue conditioned media using liquid chromatography-tandem mass spectrometry revealed the presence of multiple bioactive proteins involved in regulation of hematopoiesis, inflammation, and bone development, with many predicted to reside inside microvesicles. Pretreatment of purified HSPCs with BM adipose tissue conditioned media, comprising soluble and exosomal/microvesicle-derived factors, led to enhanced proliferation and an increase in granulocyte-monocyte differentiation potential ex vivo. Our work extends extensive studies in murine models, indicating that BM adipose tissue is a central paracrine regulator of hematopoiesis in nonhuman primates and possibly in humans.

Abstract A050: Validation of β-catenin as a tumor segmentation marker for delineating tumor from stromal tissues in quantitative multiplex immunofluorescence analysis of formalin-fixed, paraffin-embedded biopsy specimens

Abstract Background: Immunofluorescence assay (IFA) analysis of pharmacodynamic biomarkers in tumor biopsy tissue is limited by lack of a tumor histology–agnostic methodology for segmenting tumor tissue from the surrounding stroma. We have developed a tumor segmentation methodology utilizing β-catenin staining in combination with cell morphology to manually segment tumor regions from surrounding mesenchyme. Here, we show extensive validation of the use of β-catenin as a general tumor segmentation marker through the examination of various tumor tissue microarrays (TMAs), including both carcinoma and sarcoma specimens. Methods: We examined TMAs comprised of tumors from 4 different histologies: colorectal carcinoma (CRC), non-small cell lung cancer (NSCLC), breast cancer, and sarcoma. For each TMA specimen, we quantified tumor content via hematoxylin and eosin (H&E) staining and then assessed tumor expression of β-catenin and other established phenotypic markers of tumor differentiation by IFA and immunohistochemical (IHC) analysis, respectively. To assess b-catenin expression in tumor vs. stroma, we used a pathologist-guided annotation of specific tumor marker staining to segment tumor from stroma on sections adjacent to those stained with an immunofluorescence-conjugated β-catenin antibody; tissue overlay and area reconciliation enabled determination of the ratio of β-catenin expression in tumor vs. stroma in regions of interest (ROIs) within each tumor core. The tumor markers utilized for each histology were: carcinoembryonic antigen (CEA) and pan-cytokeratin (pan-CK) for CRC and NSCLC; MUC1, CEA, and pan-CK for breast; and desmin and pan-CK for sarcoma. Results: We detected β-catenin immunofluorescence in 169 of the 185 (91%) carcinoma TMA tumors (58/61 CRC, 57/62 NSCLC, 54/62 breast). The mean β-catenin fluorescence intensity threshold ratios in tumor vs. stroma were: CRC (3.6), NSCLC (1.5) and breast (1.1). Although IHC staining of pan-CK was detected in 183/185 (99%) carcinoma tumors (59/61 CRC, 61/62 NSCLC, 61/62 breast), pan-CK immunofluorescence is more diffuse and cytoplasmic in nature compared to β-catenin and therefore less robust as a marker for IFA-based automated tumor tissue segmentation. In addition, we examined β-catenin expression in sarcoma TMAs, detecting β-catenin in 45 of 98 sarcoma TMAs examined (46%), which was superior to both pan-CK (9 of 98; 9%) and the known sarcoma marker desmin (37 of 98 (38%). Conclusions: Together, these results demonstrate that our β-catenin–based tumor segmentation method is useful for the majority of carcinoma specimens and allows automated co-registration of tumor and pharmacodynamic biomarkers defined by tumor versus stromal areas. The same approach worked for certain sarcoma specimens. We have since developed a Definiens-based algorithm that allows automated β-catenin–based tumor segmentation for specimens from entire biopsy cohorts of NCI-sponsored clinical trials, including those examining DNA damage response and immuno-oncology biomarkers. Funded by NCI Contract No HHSN261200800001E. Citation Format: Tony Navas, Robert J. Kinders, Hala Makhlouf, Scott M. Lawrence, Rodrigo Chuaqui, Krisitin Fino, Angie Dull, Katherine Ferry-Galow, Deborah Wilsker, Alice P. Chen, Shivaani Kummar, Ralph E. Parchment, James H. Doroshow. Validation of β-catenin as a tumor segmentation marker for delineating tumor from stromal tissues in quantitative multiplex immunofluorescence analysis of formalin-fixed, paraffin-embedded biopsy specimens [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A050. doi:10.1158/1535-7163.TARG-19-A050

Single-cell transcriptome analysis reveals differential nutrient absorption functions in human intestine.

The intestine plays an important role in nutrient digestion and absorption, microbe defense, and hormone secretion. Although major cell types have been identified in the mouse intestinal epithelium, cell type-specific markers and functional assignments are largely unavailable for human intestine. Here, our single-cell RNA-seq analyses of 14,537 epithelial cells from human ileum, colon, and rectum reveal different nutrient absorption preferences in the small and large intestine, suggest the existence of Paneth-like cells in the large intestine, and identify potential new marker genes for human transient-amplifying cells and goblet cells. We have validated some of these insights by quantitative PCR, immunofluorescence, and functional analyses. Furthermore, we show both common and differential features of the cellular landscapes between the human and mouse ilea. Therefore, our data provide the basis for detailed characterization of human intestine cell constitution and functions, which would be helpful for a better understanding of human intestine disorders, such as inflammatory bowel disease and intestinal tumorigenesis.

Vascular Endothelial Growth Factor A Signaling Promotes Spinal Central Sensitization and Pain-related Behaviors in Female Rats with Bone Cancer.

Cancer pain is a pervasive clinical symptom impairing life quality. Vascular endothelial growth factor A has been well studied in tumor angiogenesis and is recognized as a therapeutic target for anti-cancer treatment. This study tested the hypothesis that vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 contribute to bone cancer pain regulation associated with spinal central sensitization. This study was performed on female rats using a metastatic breast cancer bone pain model. Nociceptive behaviors were evaluated by mechanical allodynia, thermal hyperalgesia, spontaneous pain, and CatWalk gait analysis. Expression levels were measured by real-time quantitative polymerase chain reaction, western blot, and immunofluorescence analysis. Excitatory synaptic transmission was detected by whole-cell patch-clamp recordings. The primary outcome was the effect of pharmacologic intervention of spinal vascular endothelial growth factor A/vascular endothelial growth factor receptor 2-signaling on bone cancer pain behaviors. The mRNA and protein expression of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 were upregulated in tumor-bearing rats. Spinal blocking vascular endothelial growth factor A or vascular endothelial growth factor receptor 2 significantly attenuated tumor-induced mechanical allodynia (mean ± SD: vascular endothelial growth factor A, 7.6 ± 2.6 g vs. 5.3 ± 3.3 g; vascular endothelial growth factor receptor 2, 7.8 ± 3.0 g vs. 5.2 ± 3.4 g; n = 6; P < 0.0001) and thermal hyperalgesia (mean ± SD: vascular endothelial growth factor A, 9.0 ± 2.4 s vs. 7.4 ± 2.7 s; vascular endothelial growth factor receptor 2, 9.3 ± 2.5 s vs. 7.5 ± 3.1 s; n = 6; P < 0.0001), as well as spontaneous pain and abnormal gaits. Exogenous vascular endothelial growth factor A enhanced excitatory synaptic transmission in a vascular endothelial growth factor receptor 2-dependent manner, and spinal injection of exogenous vascular endothelial growth factor A was sufficient to cause pain hypersensitivity via vascular endothelial growth factor receptor 2-mediated activation of protein kinase C and Src family kinase in naïve rats. Moreover, spinal blocking vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 pathways suppressed protein kinase C-mediated N-methyl-D-aspartate receptor activation and Src family kinase-mediated proinflammatory cytokine production. Vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 contributes to central sensitization and bone cancer pain via activation of neuronal protein kinase C and microglial Src family kinase pathways in the spinal cord.

Kunitz-type protease inhibitor BmSPI51 plays an antifungal role in the silkworm cocoon

The protease inhibitors found in silkworm cocoons can be divided into several families, a majority of which contain serpin, TIL, or Kunitz domains. Previously, it has been reported that TIL-type protease inhibitors have antimicrobial activity. To date, however, it has not been determined whether the Kunitz-type protease inhibitor BmSPI51, the most abundant of cocoon protease inhibitors, plays an antimicrobial role. Thus, in this study, we sought to determine the biological role of BmSPI51 in silkworm cocoons. Our results obtained from real-time quantitative reverse transcription PCR and immunofluorescence analyses indicate that BmSPI51 is expressed exclusively in the silk glands during the larval fifth instar stage and is subsequently secreted into cocoon silk. Moreover, at a molar ratio of 1:1, BmSPI51 produced via prokaryotic expression exhibited inhibitory activity against trypsin and also proved to be highly stable over wide ranges of temperature and pH values. The expression of BmSPI51 was also found to be significantly upregulated in the larval fat body after infection with three species of fungi, namely, Candida albicans, Beauveria bassiana, and Saccharomyces cerevisiae. In vitro inhibition tests revealed that BmSPI51 significantly inhibited the sporular growth of all three of these fungal species. Further, results obtained from a binding assay showed that BmSPI51 binds to β-d-glucan and mannan on the surface of fungal cells. In this study, we, thus, revealed the antimicrobial activity of BmSPI51 and its underlying mechanism in silkworm, thereby contributing to our present understanding of defense mechanisms in silkworm cocoons.

Patient-derived organoids as a potential model to predict response to PD-1/PD-L1 checkpoint inhibitors

Selection of cancer patients for treatment with immune checkpoint inhibitors remains a challenge due to tumour heterogeneity and variable biomarker detection. PD-L1 expression in 24 surgical chordoma specimen was determined immunohistochemically with antibodies 28-8 and E1L3N. The ability of patient-derived organoids to detect treatment effects of nivolumab was explored by quantitative and qualitative immunofluorescence and FACS analysis. The more sensitive antibody, E1L3N (ROC = 0.896, p = 0.001), was associated with greater tumour diameters (p = 0.014) and detected both tumour cells and infiltrating lymphocytes in 54% of patients, but only 1–15% of their cells. Organoids generated from PD-L1-positive patients contained both tumour cells and PD-1/CD8-positive lymphocytes and responded to nivolumab treatment with marked dose-dependent diameter reductions of up to 50% and increased cell death in both PD-L1-positive and negative organoids. Patient-derived organoids may be valuable to predict individual responses to immunotherapy even in patients with low or no immunohistochemical PD-L1 expression.

Optimization of Microenvironments Inducing Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Endothelial Cell-Like Cells.

Stem cell engineering is appealing consideration for regenerating damaged endothelial cells (ECs) because stem cells can differentiate into EC-like cells. In this study, we demonstrate that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into EC-like cells under optimal physiochemical microenvironments. TMSCs were preconditioned with Dulbecco's Modified Eagle Medium (DMEM) or EC growth medium (EGM) for 4days and then replating them on Matrigel to observe the formation of a capillary-like network under light microscope. Microarray, quantitative real time polymerase chain reaction, Western blotting and immunofluorescence analyses were used to evaluate the expression of gene and protein of EC-related markers. Preconditioning TMSCs in EGM for 4days and then replating them on Matrigel induced the formation of a capillary-like network in 3h, but TMSCs preconditioned with DMEM did not form such a network. Genome analyses confirmed that EGM preconditioning significantly affected the expression of genes related to angiogenesis, blood vessel morphogenesis and development, and vascular development. Western blot analyses revealed that EGM preconditioning with gelatin coating induced the expression of endothelial nitric oxide synthase (eNOS), a mature EC-specific marker, as well as phosphorylated Akt at serine 473, a signaling molecule related to eNOS activation. Gelatin-coating during EGM preconditioning further enhanced the stability of the capillary-like network, and also resulted in the network more closely resembled to those observed in human umbilical vein endothelial cells. This study suggests that under specific conditions, i.e., EGM preconditioning with gelatin coating for 4days followed by Matrigel, TMSCs could be a source of generating endothelial cells for treating vascular dysfunction.

Melatonin protects against lipopolysaccharide-induced epididymitis in sheep epididymal epithelial cells in vitro

Melatonin has protective effects against inflammation but its role in epididymitis is unknown. We addressed this in the present study using lipopolysaccharide (LPS)-stimulated sheep epididymal epithelial cells as an in vitro inflammation model. We found that interleukin (IL)-1β, IL-6, tumor necrosis factor α, and cyclooxygenase (COX)-2 mRNA levels; COX-2 and Toll-like receptor (TLR)-4 protein levels; and nuclear factor (NF)-κB p65 phosphorylation were increased by LPS treatment. These effects were reversed in a dose-dependent manner by melatonin (10−11–10−7 M). Quantitative reverse transcription PCR and immunofluorescence analyses showed that the melatonin receptors MT1 and MT2 were expressed in sheep epididymal epithelial cells. The inhibitory effect of melatonin on inflammation was abrogated by the MT1 and MT2 receptor antagonist luzindole and the MT2 ligand 4-phenyl-2-propanamide tetraldehyde. Thus, melatonin exerted anti-inflammatory effect in epididymal epithelial cells by inhibiting TLR4/NF-κB signaling, suggesting its potential as an effective drug for the treatment of epididymitis in sheep.

Role of KRAS in regulating normal human airway basal cell differentiation

BackgroundKRAS is a GTPase that activates pathways involved in cell growth, differentiation and survival. In normal cells, KRAS-activity is tightly controlled, but with specific mutations, the KRAS protein is persistently activated, giving cells a growth advantage resulting in cancer. While a great deal of attention has been focused on the role of mutated KRAS as a common driver mutation for lung adenocarcinoma, little is known about the role of KRAS in regulating normal human airway differentiation.MethodsTo assess the role of KRAS signaling in regulating differentiation of the human airway epithelium, primary human airway basal stem/progenitor cells (BC) from nonsmokers were cultured on air-liquid interface (ALI) cultures to mimic the airway epithelium in vitro. Modulation of KRAS signaling was achieved using siRNA-mediated knockdown of KRAS or lentivirus-mediated over-expression of wild-type KRAS or the constitutively active G12 V mutant. The impact on differentiation was quantified using TaqMan quantitative PCR, immunofluorescent and immunohistochemical staining analysis for cell type specific markers. Finally, the impact of cigarette smoke exposure on KRAS and RAS protein family activity in the airway epithelium was assessed in vitro and in vivo.ResultssiRNA-mediated knockdown of KRAS decreased differentiation of BC into secretory and ciliated cells with a corresponding shift toward squamous cell differentiation. Conversely, activation of KRAS signaling via lentivirus mediated over-expression of the constitutively active G12 V KRAS mutant had the opposite effect, resulting in increased secretory and ciliated cell differentiation and decreased squamous cell differentiation. Exposure of BC to cigarette smoke extract increased KRAS and RAS protein family activation in vitro. Consistent with these observations, airway epithelium brushed from healthy smokers had elevated RAS activation compared to nonsmokers.ConclusionsTogether, these data suggest that KRAS-dependent signaling plays an important role in regulating the balance of secretory, ciliated and squamous cell differentiation of the human airway epithelium and that cigarette smoking-induced airway epithelial remodeling is mediated in part by abnormal activation of KRAS-dependent signaling mechanisms.

A multilayer micromechanical elastic modulus measuring method in ex vivo human aneurysmal abdominal aortas

Abdominal aortic aneurysms (AAA) are common and potentially life-threatening aortic dilatations, due to the effect of hemodynamic changes on the aortic wall. Previous research has shown a potential pathophysiological role for increased macroscopic aneurysmal wall stiffness; however, not investigating micromechanical stiffness. We aimed to compile a new protocol to examine micromechanical live aortic stiffness (elastic moduli), correlated to histological findings with quantitative immunofluorescence (QIF). Live AAA biopsies (n = 7) and non-dilated aortas (controls; n = 3) were sectioned. Local elastic moduli of aortic intima, media and adventitia were analysed in the direction towards the lumen and vice versa with nanoindentation. Smooth muscle cells (SMC), collagen and fibroblasts were examined using QIF. Nanoindentation of AAA vs. controls demonstrated a 4-fold decrease in elastic moduli (p = 0.022) for layers combined and a 26-fold decrease (p = 0.017) for media-to-intima direction. QIF of AAA vs. controls revealed a 4-, 3- and 6-fold decrease of SMC, collagen and fibroblasts, respectively (p = 0.036). Correlations were found between bidirectional intima and media measurements (ρ = 0.661, p = 0.038) and all QIF analyses (ρ = 0.857–0.905, p = 0.002–0.007). We present a novel protocol to analyse microscopic elastic moduli in live aortic tissues using nanoindentation. Hence, our preliminary findings of decreased elastic moduli and altered wall composition warrant further microscopic stiffness investigation to potentially clarify AAA pathophysiology and to explore potential treatment by wall strengthening. Statement of SignificanceAlthough extensive research on the pathophysiology of dilated abdominal aortas (aneurysms) has been performed, the exact underlying pathways are still largely unclear. Previously, the macroscopic stiffness of the pathologic and healthy aortic wall has been studied. This study however, for the first time, studied the microscopic stiffness changes in live tissue of dilated and non-dilated abdominal aortas. This new protocol provides a device to analyse the alterations on cellular level within their microenvironment, whereas previous studies studied the aorta as a whole. Outcomes of these measurements might help to better understand the underlying origin of the incidence and progression of aneurysms and other aortic diseases.

Abstract 2068: A highly selective dual Haspin-kinase and PIM-inhibitor overcomes RAF/MEK-inhibitor resistance in melanoma

Abstract Intrinsic or acquired resistance to targeted therapies, such as RAF/MEK inhibitors in melanoma, represents a significant clinical challenge. We performed a small molecule screen in isogenic sensitive (S) and RAF/MEK-inhibitor-resistant (RMR) melanoma cell lines and identified a nominal PIM kinase inhibitor (Comp-1) with high activity against A375-S and A375-RMR, with an EC50of ~100 nM. While the phosphorylation of some PIM targets, including 4EBP1 and MYC, were significantly inhibited in a dose-dependent fashion, phosphorylation of BAD was not reduced as one would expect. To determine the on-target activity of this compound, we generated A375 cell lines overexpressing WT PIM isoforms 1-3 or their kinase-dead (KD) forms, followed by treatment with PIMi-1. We found only modest shifts in EC50and hypothesized that the beneficial effects of this compounds were due to inhibition of an alternative target. A screen of Comp-1, along with structurally related compounds, against 468 human kinases revealed exquisite affinity for Haspin kinase (HK). We determined the IC50of Comp-1 against HK to be 14.9 nM. The only described role of HK is phosphorylation of Histone H3 at Thr 3 (H3T3) and thereby regulation of mitotic progression. Quantitative single-cell immunofluorescence (IF) analysis of Comp-1 treated A375 confirmed complete abrogation of p-H3T3, indicating on-target activity in vitro. To understand the temporal effects of HK inhibition with Comp-1, we performed live-cell imaging and single-cell analysis of A375 transduced with a mitotic phase-specific reporter. Cells experienced mitotic stress or mitotic slippage resulting in DNA-damage and an increase gamma-H2AX foci per nuclei. In line with these observation, we observed increased phosphorylation p53 and CHECK2. Furthermore, a portion of cells developed micronuclei in a dose-dependent fashion providing a substrate for the cytosolic DNA-sensing machinery, including the cGAS-STING pathway, and rationale for combining Comp-1 with immunotherapies. Ongoing experiments in tumor-bearing BALB/c mice indicate safety and potentially enhanced efficacy of Comp-1 when combined with either anti-CTLA-4 or anti-PD-1 therapy. Ex vivotreatment of patient-derived tumor-infiltrating lymphocytes (TILs) with Comp-1 revealed no toxic effects. A pan-cancer RNA-seq analysis revealed significant upregulation of HK in 34 of 37 tumor types supporting its role as a potential therapeutic target. Overall, our work identified a highly selective Haspin Kinase inhibitor with activity against RAF/MEK inhibitor resistant melanoma, insights into the underlying mechanisms, and potential in vivo benefit when combined with immune checkpoint inhibitors. Citation Format: Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Peter K. Sorger, Kai Wucherpfennig, Benjamin Izar. A highly selective dual Haspin-kinase and PIM-inhibitor overcomes RAF/MEK-inhibitor resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2068.

The Aberrant Expression of the Mesenchymal Variant of FGFR2 in the Epithelial Context Inhibits Autophagy.

Signaling of the epithelial splice variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, epithelial mesenchymal transition (EMT) and tumorigenic features. Here we analyzed in the human keratinocyte cell line, as well as in primary cultured cells, the possible impact of FGFR2c forced expression on the autophagic process. Biochemical and quantitative immunofluorescence analysis, coupled to the use of autophagic flux sensors, specific substrate inhibitors or silencing approaches, showed that ectopic expression and the activation of FGFR2c inhibit the autophagosome formation and that AKT/MTOR is the downstream signaling mainly involved. Interestingly, the selective inhibition of AKT or MTOR substrates caused a reversion of the effects of FGFR2c on autophagy, which could also arise from the imbalance of the interplay between AKT/MTOR pathway and JNK1 signaling in favor of JNK1 activation, BCL-2 phosphorylation and possibly phagophore nucleation. Finally, silencing experiments of depletion of ESRP1, responsible for FGFR2 splicing and consequent FGFR2b expression, indicated that the switching from FGFR2b to FGFR2c isoform could represent the key event underlying the inhibition of the autophagic process in the epithelial context. Our results provide the first evidence of a negative impact of the out-of-context expression of FGFR2c on autophagy, suggesting a possible role of this receptor in the modulation of the recently proposed negative loop between autophagy and EMT during carcinogenesis.