OE33 Cells Research Articles

Preclinical evaluation of a novel EGFR&c-Met bispecific near infrared probe for visualization of esophageal cancer and metastatic lymph nodes.

This study aimed to establish a near infrared fluorescent (NIRF) probe based on an EGFR&c-Met bispecific antibody for visualization of esophageal cancer (EC) and metastatic lymph nodes (mLNs). EGFR and c-Met expression were assessed by immunohistochemistry. EGFR&c-Met bispecific antibody EMB01 was labeled with IRDye800cw. The binding of EMB01-IR800 was assessed by enzyme linked immunosorbent assay, flow cytometry, and immunofluorescence. Subcutaneous tumors, orthotopic tumors, and patient-derived xenograft (PDX) were established for in vivo fluorescent imaging. PDX models using lymph nodes with or without metastasis were constructed to assess the performance of EMB01-IR800 in differential diagnosis of lymph nodes. The prevalence of overexpressing EGFR or c-Met was significantly higher than single marker either in EC or corresponding mLNs. The bispecific probe EMB01-IR800 was successfully synthesized, with strong binding affinity. EMB01-IR800 showed strong cellular binding to both Kyse30 (EGFR overexpressing) and OE33 (c-Met overexpressing) cells. In vivo fluorescent imaging showed prominent EMB01-IR800 uptake in either Kyse30 or OE33 subcutaneous tumors. Likewise, EMB01-IR800 exhibited superior tumor enrichment in both thoracic orthotopic esophageal squamous cell carcinoma and abdominal orthotopic esophageal adenocarcinoma models. Moreover, EMB01-IR800 produced significantly higher fluorescence in patient-derived mLNs than in benign lymph nodes. This study demonstrated the complementary overexpression of EGFR and c-Met in EC. Compared to single-target probes, the EGFR&c-Met bispecific NIRF probe can efficiently depict heterogeneous esophageal tumors and mLNs, which greatly increased the sensitivity of tumor and mLN identification.

CCT6A promotes esophageal squamous cell carcinoma cell proliferation, invasion and epithelial-mesenchymal transition by activating TGF-β/Smad/c-Myc pathway.

Chaperonin-containing TCP1 subunit 6A (CCT6A) facilitates several malignant cancer behaviors, but its regulation of esophageal squamous cell carcinoma (ESCC) has not been reported. This study aimed to investigate the effect of CCT6A on cell proliferation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) and its interaction with the TGF-β/Smad/c-Myc pathway in ESCC. CCT6A expression was detected in ESCC and normal esophageal epithelial cell lines by RT‒qPCR and western blotting. Furthermore, CCT6A siRNA, negative control (NC) siRNA, CCT6A encoding plasmid and NC encoding plasmid were transfected into OE21 and TE-1 cells. Subsequently, CCT6A siRNA- and NC siRNA-transfected cells were treated with TGF-β for rescue experiments. Cell proliferation, apoptosis, invasion, and E-cadherin/N-cadherin and p-Smad2/p-Smad3/c-Myc expression were detected. CCT6A expression was increased in KYSE-180, TE-1, TE-4 and OE21 cells compared with HET-1A cells. In both OE21 and TE-1 cells, CCT6A knockdown inhibited cell proliferation, invasion and N-cadherin expression while promoting cell apoptosis and E-cadherin expression; meanwhile, CCT6A overexpression had the opposite effects. Furthermore, in both OE21 and TE-1 cells, CCT6A knockdown decreased p-Smad2/Smad2, p-Smad3/Smad3 and c-Myc/GAPDH expression; CCT6A overexpression had the opposite effects. Next, TGF-β facilitated cell proliferation, invasion, and N-cadherin, p-Smad2/Smad2, p-Smad3/Smad2 and c-Myc/GAPDH expression while repressing cell apoptosis and E-cadherin expression in OE21 and TE-1 cells; importantly, TGF-β could compensate for the regulation of CCT6A knockdown on these activities. CCT6A facilitates ESCC malignant activities by activating the TGF-β/Smad/c-Myc pathway, which sheds light on the identification of a possible therapeutic target in the management of ESCC.

Abstract 6700: Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma

Abstract Background: While the incidence of esophageal adenocarcinoma (EAC) is rapidly increasing, the 5-year survival rate of this disease remains poor at less than 25%, with a substantial number of patients exhibiting inherent or acquired resistance to chemotherapy. Circulating tumor DNA (ctDNA) isolated from liquid biopsies of EAC patients holds clinical biomarker potential in this disease, but our understanding of how chemotherapy affects ctDNA release remains limited. Methods: In order to study the impact of cancer treatment on ctDNA emission in vitro, we first sought to establish a chemo-resistant model of EAC. As such, the EAC cell line OE19 was exposed to discontinuous cisplatin treatments for three months in order to establish drug resistance. Following this, established chemo-resistant OE19 cells and parental (chemo-sensitive) OE19 cells were exposed to five daily cisplatin treatments, and cell culture supernatant was collected every day throughout treatment. ctDNA was subsequently isolated from cell culture supernatant and quantified by Qubit fluorometer dsDNA HS assay. ctDNA quantities were additionally assessed by droplet digital PCR (ddPCR) assay using primers and probes targeting the TP53 N310K mutation in the OE19 cell line. Finally, ctDNA fragment lengths were analyzed using the Agilent Bioanalyzer 2100. Results: A chemo-resistant model of the OE19 cell line was established, as exemplified by marked morphological changes in the cells and significantly increased cell viability determined by CCK8 assay after 48-hour treatments of various doses of cisplatin (p<0.05). ctDNA levels, as measured by both Qubit fluorometer and mutant copies detected by ddPCR assay, increased after chemotherapy treatment for both chemo-sensitive and resistant cells. Notably, ctDNA emission was higher with larger amounts cell death, with chemo-sensitive cells emitting significantly higher levels of ctDNA compared to chemo-resistant cells throughout treatment (p<0.05). Fragmentomics analysis revealed ctDNA peaks corresponding to apoptosis (~167 bp) and necrosis (~10,000 bp). Moreover, chemotherapy treatment caused a shift in average fragment size, with larger fragments being observed during chemotherapy treatment in both chemo-sensitive and resistant cells. Conclusion: This study reveals that in vitro cancer models can be used to successfully study ctDNA emission during pre-clinical drug analyses. ctDNA release was found to be correlated to cytotoxicity and cell death, providing us with valuable insight into how clinical liquid biopsy data should be interpreted. Finally, fragment size analysis of ctDNA reveals a potential novel biomarker for treatment response in cancer patients. Citation Format: Alexandra Bartolomucci, Sarah Tadhg Ferrier, Thupten Tsering, Xin Su, Jonathan Cools-Lartigue, Julia V. Burnier. Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma [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 6700.

Abstract 1745: Targeting therapeutic resistance of esophageal adenocarcinoma through modulating the epithelial-to-mesenchymal transition via NFκB-mediated signaling

Abstract Esophageal adenocarcinoma (EAC) is the major subtype of esophageal cancer in Westernized countries, characterized by rising incidence and poor prognosis with a five-year survival rate less than 20%. Only 15% of EAC patients have a complete histopathological response to standard chemotherapy, indicating the urgent need to identify mechanisms associated with therapeutic resistance for improved therapeutic efficacy and patient outcomes. Herein, we performed transcriptional profiling utilizing tissues collected from EAC patients prior to treatment initiation. First, EAC patients (n=67) were stratified into 5 therapeutic response groups based on the changes in TNM pre- to post-treatment; second, bioinformatic approaches were utilized to identify molecular changes associated with response to treatment and patient survival. Third, we employed OE19 and OE33 human EAC cell lines to investigate the efficacy of cranberry proanthocyanidins (C-PAC), a promising anti-inflammatory and anti-cancer agent which shows synergistic effects with carboplatin in other cancer types, at mitigating molecular pathways which drive therapeutic resistance. Our therapeutic response data set revealed epithelial-mesenchymal transition (EMT) as a top pathway associated with differential response to therapy and patient survival. Subsequent gene-set enrichment analysis identified that EMT is significantly associated with NFκB and STAT3 signaling pathways, two nodes with documented proinflammatory roles in EAC progression. Deconvolution analysis was conducted further supporting EMT enrichment creates an immune-suppressive tumor microenvironment in EAC patients. Next, EAC cell viability, migration and interrogation of EMT signaling proteins were conducted following treatment with paclitaxel and carboplatin (chemo drugs) alone or in combination with C-PAC. Combination treatment of C-PAC and chemo drugs significantly inhibited EAC cell viability and migration capability, compared to cells treated with chemo drugs alone, with synergistic effects observed in OE33 cells. Moreover, combination treatment also significantly decreased NFκB1 expression and expressions of EMT markers and transcription factors (Snail, MMP-2, MMP-9, and Vimentin) whereas cells treated only with chemo drugs showed minimal modulation. Knockdown experiments of NFκB1 using RNA interference are underway to investigate the relationship between canonical NFκB1 signaling and EMT. Primary cell lines and organoids developed from isolated EAC patient tissues will also be used in the future to validate experimental findings. The results suggest C-PAC may be used as a safe and novel therapeutic strategy to improve chemotherapy efficacy targeting EAC. Citation Format: Yun Zhang, Katherine M. Weh, Connor L. Howard, Jean-Jack Riethoven, Jennifer L. Clarke, Michelle P. Lee, Kiran H. Lagisetty, Jules Lin, Rishindra M. Reddy, Andrew C. Chang, Amy B. Howell, Laura A. Kresty. Targeting therapeutic resistance of esophageal adenocarcinoma through modulating the epithelial-to-mesenchymal transition via NFκB-mediated signaling [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 1745.

Abstract 6357: Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile

Abstract Background/Purpose: CD47 has been validated to be expressed on various tumor types including acute myeloid leukemia, myelodysplastic syndrome and other hematologic or solid tumors. As primary ‘don’t eat me’ signal, high expression of CD47 on tumor cells interacts with SIRPα negatively regulating the phagocytosis level. However, poor safety profile as well as insufficient combinatory effect remain to be the issues limiting the clinical outcomes of CD47-targeting molecules. Herein, we used rational design strategies to generate SCR9168 with potent affinity improvement, and maintained favorable safety profiles in different animal species, including cynomolgus monkeys. Procedures: Mutagenesis was introduced to the residues critical for CD47/SIRPα binding interface. Affinity was measured by SPR analysis. In vitro activities were determined by biochemical- or cell-based binding, blocking and antibody-dependent cellular phagocytosis (ADCP) assays. The efficacy in vivo was assessed with the OE19 xenograft model. Pharmacokinetics (PK) and safety were monitored in both mice and cynomolgus monkeys. Results: The affinity of SCR9168 achieves 178 pM for human CD47 which is nearly 50-fold increase in comparison with wild-type SIRPα. Enhanced affinity has also been confirmed in binding to OE19 or DLD1 cell lines, as well as in blocking human SIRPα binding to Raji cells. SCR9168 remains strong binding potency to monkey, mouse or rat CD47 which allows the PK and safety assessment using these animal species. The combined effects of SCR9168 and multiple therapeutic antibodies targeting tumor-associated antigens (TAAs) have been evaluated with ADCP assays using human monocyte-derived macrophages. SCR9168 markedly increases the ratio of phagocytic cells combining with antibodies, such as cetuximab. However, due to the inert function of Fc fragment, no phagocytosis of red blood cells or platelets was caused after the incubation in vitro. In addition, SCR9168 treatment in combination with trastuzumab leads to significantly improved suppression of tumor growth in a dose-dependent manner. Moreover, administration of two doses at day 1 and day 11 in cynomolgus monkeys results in no toxicity events related to SCR9168 based on the data from hematological analysis. Conclusion: SCR9168 demonstrates the best-in-class potential among SIRPα mutein molecules. It elicits improved and dose-dependent efficacy in phagocytosis or tumor suppression combining with therapeutic antibodies, such as trastuzumab or cetuximab. Favorable safety profile with no phagocytosis of RBC or platelets in vitro as well as no hematological toxicity observation in cynomolgus monkeys allows broader dose range exploration in early clinical phase. SCR9168 is currently in development stage and IND enabling will be expected in the end of 2023. Citation Format: Yingying Hu, Yayuan Fu, Lei Liu, Wenjing Li, Liting Xue, Zhiyong Yu, Yun Zhang, Meijuan Gao, Yixin Tan, Fudong Wang, Yadan Wu, Jie Li, Zhenzhen Li, Feng Zhou, Wenqing Yang, Zhuoxiao Cao, Renhong Tang. Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile [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 6357.

Abstract 1284: The effect of Semaphorin 7A on breast cancer metastases and chemoresistance in young women

Abstract Our overall goal is to understand drivers of chemoresistance in triple-negative breast cancer (TNBC) and the mechanisms that contribute to metastatic spread in young women. TNBC is more frequently diagnosed in women aged <40 and confers worse overall and distant-metastatic-free survival. Our lab identified a ß1 integrin-ligand, Semaphorin 7A (SEMA7A), that is increased in TNBC and young women's breast cancer (BC). Our central hypothesis is that SEMA7A is increased in TNBC and signals via ß1-integrin to activate epithelial-to-mesenchymal plasticity (EMP) and chemoresistance. We have incorporated genetically manipulated TNBC cell lines and ex vivo tumor samples to define how SEMA7A expression impacts metastasis and chemoresistance. We have also incorporated our Young Women’s BC Cohort to analyze SEMA7A protein expression in human tumors and mammary epithelial tissue. In this cohort, we found that the normal mammary epithelium and tumors exhibit higher SEMA7A expression in women with postpartum BC, defined as BC diagnosed within 10 years postpartum, who are between 30&39-years-old. This sub-population also has increased tumor-associated lymphatic vessel density, as well as increased lymphovascular invasion and LN involvement. Using a 9-month-old mouse model, which is equivalent to a 35-year-old woman, we have shown that mammary tumor growth and metastasis are increased compared to young 8-week-old mice, which can be mimicked by SEMA7A OE tumors in the young mice; these results suggest that the aged microenvironment can replicate the tumor promotional effects of SEMA7A. Similarly, in orthotopic models, SEMA7A OE cells metastasize more frequently to lymph nodes (LNs) and lungs compared to controls. Additionally, via a tail vein injection, we observe that SEMA7A OE cells survive in circulation, seed the lung, and outgrow faster compared to controls. In vitro, SEMA7A promotes migration and invasion via ß1-integrin signaling. Finally, ex vivo metastatic tumor cells, when compared to parental cells, exhibit evidence of EMP. Namely, prior to dissemination, SEMA7A OE cells are mostly mesenchymal-like. Ex vivo, SEMA7A OE cells from LN metastasis express both epithelial/mesenchymal markers, suggesting a hybrid phenotype and maintenance of an invasive phenotype. Whereas those from the lung are more epithelial, which can be more proliferative. EMP is also known to promote chemoresistance and we have found that SEMA7A OE cells are resistant to caspase-mediated cell death in the presence of paclitaxel, a known chemotherapeutic utilized for TNBC, and have increased activation of pAkt, which can mediate pro-survival phenotypes. Overall, our results suggest that the aged mammary gland and SEMA7A expression can promote metastatic spread and chemoresistance and that these patient populations should be considered for direct and indirect targeting of SEMA7A by novel therapeutics. Citation Format: Kelsey Kines, Sarah Tarullo, Virginia Borges, Traci Lyons. The effect of Semaphorin 7A on breast cancer metastases and chemoresistance in young women [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 1284.

Mir-421 and mir-550a-1 are potential prognostic markers in esophageal adenocarcinoma

ObjectiveTo identify the prognostic indicators of esophageal adenocarcinoma (EAC) for future EAC diagnosis and treatment.MethodsThe EAC dataset from The Cancer Genome Atlas was screened for differentially expressed microRNAs (miRNAs) and mRNAs associated with EAC. Weighted gene coexpression network analysis was performed to cluster miRNAs or mRNA with similar expression patterns to identify the miRNAs or mRNA that are highly associated with EAC. Prognostic miRNAs for overall survival (OS) were identified using Cox proportional-hazards regression analysis and least absolute shrinkage and selection operator based on survival duration and status. Two types of miRNAs were selected to develop a prognostic signature model for EAC using multiple Cox regression analysis. Furthermore, the signature was validated using internal validation sets 1 and 2. The receiver operating characteristic curve and concordance index were used to evaluate the accuracy of the signature and validation sets. The expression of miR-421, miR-550a-3p, and miR-550a-5p was assessed using quantitative polymerase chain reaction (qPCR). The proliferation, invasion, and migration of EAC cells were assessed using CCK8 and transwell assays. The OS of target mRNAs was assessed using Kaplan–Meier analysis. Functional enrichment analysis of the target mRNAs was performed using Metascape.ResultsThe prognostic signature and validation sets comprising mir-421 and mir-550a-1 had favorable predictive power in OS. Compared with the patients with EAC in the high-expression group, those assigned to the low-expression group displayed increased OS according to survival analysis. Differential and qPCR analysis showed that miR-421, miR-550a-3p, and miR-550a-5p were highly expressed in the EAC tissues and cell lines. Moreover, the downregulation of miR-421 and miR-550a-3p with inhibitor markedly suppressed the proliferation, invasion, and migration in OE33 cells compared with the negative control. A total of 20 target mRNAs of three miRNAs were predicted, among which seven target mRNAs—ASAP3, BCL2L2, LMF1, PPM1L, PTPN21, SLC18A2, and NR3C2—had prognostic value; PRKACB, PDCD4, RPS6KA5, and BCL2L2 were enriched in the miRNA cancer pathway.ConclusionPrognostic indicators of EAC may be useful in future EAC diagnosis and treatment.

Baicalein Inhibits the Growth of Transplanted Esophageal Cancer in Mice and the Effect on the Expression of PAK4

We studied the mechanism underlying the effect of baicalein on the growth of transplanted esophageal cancer in NOG mice and its effect on the expression of PAK4. For that purpose, we developed a new model of transplanted esophageal cancer (human esophageal cancer OE19 cells (107 cells/ml) were inoculated to NOG mice). Three experimental groups with transplanted esophageal cancer cells received baicalein in different doses (1, 1.5, and 2 mg/kg). In 32 days, the tumors were resected, and the expression of PAK4 and the level of activated PAK4 were assayed by reverse transcription PCR and Western blotting, respectively. The results showed a dose-depending anti-tumor effect of baicalein on the transplanted esophageal cancer in NOG mice: this effect of baicalein (determined by the size and weight of the tumor) increased with increasing the dose of the substance. Furthermore, the anti-tumor effect of baicalein was also confirmed by reduction of PAK4 expression. Thus, baicalein can inhibit tumor growth by inhibiting activation of PAK4. Therefore, our results showed that baicalein could inhibit the growth of esophageal cancer cells by inhibiting the activity of PAK4, which can be an important mechanism of its antitumor effect.

FAM83B promotes the invasion of primary lung adenocarcinoma via PI3K/AKT/NF-κB pathway

ObjectsThe family with sequence similarity 83B (FAM83B) is one of the markers for poor prognosis in several carcinomas, but the expression and the mechanism resulted in malignant phenotype in lung adenocarcinoma (LUAD) remain to be elucidated.Methods Data of RNA-seq in LUAD were downloaded from the cancer genome atlas (TCGA) database for differential expression and survival analysis, and immunohistochemistry was employed to analyze the protein expression of FAM83B in 126 cases of primary LUAD. The LUAD cell lines were collected for the detection of the effects on migration and invasion. Then, western blot was performed to measure the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and activation of PI3K/AKT/NF-κB pathway.ResultsFAM83B was overexpressed in multiple types of carcinomas; The differential expression analysis revealed that the level of FAM83B was higher in LUAD than that in para-carcinoma; The patients with overexpression of FAM83B were with shorter overall survival (OS), disease specific survival (DSS) and progress free interval (PFI); Enrichment analysis suggested it was related to the focal adhesion of LUAD. Immunohistochemistry analysis demonstrated that higher FAM83B expression was positively related to lymph node metastasis in primary. Scratch assay and Borden chamber assay showed that the overexpression of FAM83B promoted migration and invasion activity in vitro. Furthermore, high level of FAM83B accelerated the tumorigenesis in vivo. Western blot showed that TIMP-1 was upregulated in H1299/FAM83B OE cells accompanying by the activation of PI3K/AKT/NF-κB pathway.ConclusionsFAM83B was a marker for poor prognosis of LUAD and it might promote the expression of TIMP-1 by activating PI3K/AKT/NF-κB pathway and then affect the ECM balance, which resulted in the migration and invasion of LUAD.

Alantolactone inhibits oesophageal adenocarcinoma cells through nuclear factor erythroid 2-related factor 2-mediated reactive oxygen species increment.

Oesophageal adenocarcinoma (EAC) is a highly lethal cancer associated with a rapidly rising incidence and a poor prognosis. Alantolactone, a sesquiterpene lactone isolated from inula helenium, has anti-inflammatory, antimicrobial, neuroprotective activities, and anticancer properties. In the present study, the anticancer effects of alantolactone on the human EAC cells were investigated in vitro and in vivo. After treated with alantolactone, the cell viability of KYAE-1, KYAE-2, OE19, and OE33 cells reduced significantly compared with that of the control cells. Alantolactone induced apoptosis of the EAC cell lines by inhibiting the protein expression levels of nuclear factor erythroid2-related factor 2 (Nrf2). Furthermore, the apoptosis-inducing effect of alantolactone was enhanced by Nrf2 knockdown while reduced by overexpression of Nrf2. Antioxidant α-tocopherol and glutathione can protect EAC cell lines against alantolactone. A xenograft nude mice model showed that alantolactone can inhibit EAC growth in vivo. Alantolactone inhibits oesophageal adenocarcinoma cells through Nrf2-mediated reactive oxygen species (ROS) increment. Alantolactone maybe a potential therapeutical candidate for treating EAC.

Nutrient deprivation and hypoxia alter T cell immune checkpoint expression: potential impact for immunotherapy

AimUse of immune checkpoint blockade to enhance T cell-mediated immunity within the hostile tumour microenvironment (TME) is an attractive approach in oesophageal adenocarcinoma (OAC). This study explored the effects of the hostile TME, including nutrient deprivation and hypoxia, on immune checkpoint (IC) expression and T cell phenotypes, and the potential use of nivolumab to enhance T cell function under such conditions.Methods and ResultsICs were upregulated on stromal immune cells within the tumour including PD-L2, CTLA-4 and TIGIT. OAC patient-derived PBMCs co-cultured with OE33 OAC cells upregulated LAG-3 and downregulated the co-stimulatory marker CD27 on T cells, highlighting the direct immunosuppressive effects of tumour cells on T cells. Hypoxia and nutrient deprivation altered the secretome of OAC patient-derived PBMCs, which induced upregulation of PD-L1 and PD-L2 on OE33 OAC cells thus enhancing an immune-resistant phenotype. Importantly, culturing OAC patient-derived PBMCs under dual hypoxia and glucose deprivation, reflective of the conditions within the hostile TME, upregulated an array of ICs on the surface of T cells including PD-1, CTLA-4, A2aR, PD-L1 and PD-L2 and decreased expression of IFN-γ by T cells. Addition of nivolumab under these hostile conditions decreased the production of pro-tumorigenic cytokine IL-10.ConclusionCollectively, these findings highlight the immunosuppressive crosstalk between tumour cells and T cells within the OAC TME. The ability of nivolumab to suppress pro-tumorigenic T cell phenotypes within the hostile TME supports a rationale for the use of immune checkpoint blockade to promote anti-tumour immunity in OAC.Graphical abstractStudy schematic: (A) IC expression profiles were assessed on CD45+ cells in peripheral whole blood and infiltrating tumour tissue from OAC patients in the treatment-naïve setting. (B) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then co-cultured for 48 h with OE33 cells. T cell phenotypes were then assessed by flow cytometry. (C) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then further cultured under conditions of nutrient deprivation or hypoxia for 48 h and T cell phenotypes were then assessed by flow cytometry. Key findings: (A) TIGIT, CTLA-4 and PD-L2 were upregulated on CD45+ immune cells and CTLA-4 expression on CD45+ cells correlated with a subsequent decreased response to neoadjuvant regimen. (B) Following a 48 h co-culture with OE33 cells, T cells upregulated LAG-3 and decreased CD27 co-stimulatory marker. (C) Nutrient deprivation and hypoxia upregulated a range of ICs on T cells and decreased IFN-γ production by T cells. Nivolumab decreased IL-10 production by T cells under nutrient deprivation-hypoxic conditions.

PHF19 Inhibits Multiple Myeloma Cell Response to Immunotherapy Via Promoting Immunosuppressive Microenvironment

Introduction: The multiple myeloma (MM) bone marrow (BM) microenvironment promotes tumor cell growth, survival, and drug resistance, while also conferring immunosuppression. This involves crosstalk between the myeloma cells and the components of BM microenvironment which can be facilitated or hindered by epigenetic modifiers altering the gene expression of receptors. Among those, regulatory T (Tregs) cells are essential to control peripheral tolerance and response to foreign and tumor antigens. Tregs within the BM niche play a significant role in the suppression of antitumor immune responses and limiting the beneficial response of immunotherapy in MM. Here, we investigated the impact of the Polycomb-like protein PHF19/PCL3, an epigenetic gene whose high level predicts poor prognosis in patients with MM, on the immunosuppressive BM microenvironment. Methods & Results: We first investigated how PHF19 overexpression would affect the microenvironment by using an ex vivo co-culture of MM cells with PBMCs or purified CD3+ T cells. MM cells that overexpressed PHF19 by lenti-virus infection (PHF19-OE cells) induced 2-4-fold higher conversion of conventional T cells (Tcon) into induced regulatory T cells (iTregs) (n=5, day 4). In addition, PHF19-OE MM cells increased the frequency of iTreg at later time points (day 7) and upregulated cell proliferation (CCND1, CCND2), anti-apoptosis (BCL2, BCL2L1), and Treg-related genes (TGFβ1, IL-10, Foxp3) (P < .0001). Moreover, enhanced checkpoint marker (PD1, TIM3, LAG3) expression was observed on Tcon co-cultured with PHF19-OE vs control MM cells. There were elevated IL-10 and TGFβ1 levels in the culture supernatants of PHF19-OE MM cells, alone and in co-cultures, and antibodies to these cytokines partially neutralized the Treg inhibition of Tcon proliferation triggered by PHF19-OE MM cells. Thus, PHF19 seems to be important in facilitating the crosstalk between MM cells and immune cells in the BM. CD38 expression on the MM cell surface is commonly targeted in immunotherapy. However, CD38 expression was downregulated on PHF19-OE vs -KD MM cells, and PHF19 mRNA expression was negatively correlated to CD38 expression in patient MM cells (Spearman's ρ = -0.38 and -0.2 for GSE5900 and GSE31161, P < .004). As expected, PHF19-OE MM cells were less susceptible than PHF19-KD MM cells to in vitro NK-mediated lysis induced by daratumumab (dara) or isatuximab, which target CD38, as evidenced by fewer CD107a+ and IFN γ+ NK effector cells. Moreover, dara-induced cytotoxicity was diminished in xenografted NSG mice with PHF19-OE vs control cells and reconstituted with human NK cells. These resistant PHF19-OE AMO1 tumors had lower expression of CD38, STAT1, and IFN-related genes, which produced higher levels of human κ light chain, soluble BCMA, and TGFβ1 in matched serum samples. Accordingly, PHF19-OE resulted in significantly decreased mouse survival. To find the mediators of the immunosuppression induced by PHF19, we used ATAC-seq and CHIP-seq analysis in MM cell lines. We found that a number of genes essential for T cell-mediated tumor immunity was downregulated in the PHF19 OE group vs control, including key genes in the interferon-γ (IFN-γ) signaling pathway, which is a pathway that leads to cytokine-mediated cell death. Among the genes of the IFN-γ pathway, 56 genes had less chromatin accessibility in PHF19 OE vs control cells. RNA-seq analysis confirmed that PHF19 OE repressed the expression of CD38 and the IFN-γ and IFN-α signaling pathway. Furthermore, PHF19 OE altered the expression of genes in NF-κB signaling triggered by TNF-α signaling pathway, which are associated with resistance to immunotherapy. Finally, activation of the PI3K/AKT pathway was also identified in PHF19 OE cells, which directly promotes MM cell proliferation and survival, as well as enhances resistance to immunotherapy via upregulation of PD-L1 on the MM cell surface. Conclusions: Taken together, these data indicate that PHF19 promotes an immunosuppressive BM milieu by promoting iTreg frequency and the expression of immunosuppressive cytokines, as well as decreasing CD38 expression on MM cells and diminishing their sensitivity to IFN-γ-mediated killing. These results suggest the potential clinical utility of targeting PHF19 to overcome immunosuppression and thereby enhance or restore sensitivity to CD38-targeted therapy in MM.

High Expression of BCAT1 Affects DNA Damage Response By Inhibiting α-KG-Dependent Histone Demethylase

Branched-chain amino acids (BCAA) are essential for tumor growth and proliferation. Branched-chain aminotransferase 1(BCAT1) correlates with AML aggression, not only that, it has been reported that overexpression of BCAT1 in leukemia cells decreases intracellular αKG and impairs the activities of αKG-dependent dioxygenase. In this study, we firstly analyzed the prognostic significance of BCAT1 expression in Cytogenetically normal AML(CN-AML) patients. We found that patients with high levels of BCAT1 had poor prognosis, the median overall survival time is shorter in BCAT1 high group (356 days versus 570 days,p=0.004)(Figure A).Next, we performed gene knockout in THP-1 and MV4-11 cells and gene overexpressing in OCI-AML3 cells. In summary, BCAT1 suppression of BCAT1 markedly limited cell proliferation and colony-forming ability by blocking cell circle at G0/G1 phase(Figure B,C),whereas overexpression contributed to colony-forming and cell growth(Figure D,E). In vivo experiments confirmed that BCAT1 knockout can reduce the tumor burden of xenograft mice, while BCAT1 overexpression remarkably increased tumor burden (Figure F).Survival analysis showed that the BCAT1 OE group significantly shortened survival time(22.5 days vs 26.0 days), and the median survival time of the knockout group was 2.5 days longer than control(26.0 days vs 28.5 day ).(Figure G). To further clarify the mechanism by which BCAT1 affects the proliferation of AML cells, we collected specimens from 87 AML patients and performed RNA-Seq analysis. Through Gene Set Enrichment Analysis, we found that the DNA Genes down-regulated in response to ultraviolet (UV) radiation pathway was enriched in the high expression group of BCAT1(Figure H). Therefore, we further examined the effect of BCAT1 expression level on DNA damage repair in vitro.DNA damage foci studies were performed in control and BCAT1 OE cells. More γ-H2AX focis in BCAT1 overexpressed cells were observed. About 48.20% of BCAT1 OE cell were obeseved ＞5 γ-H2AX foci, which was much higher than the control group(13.6%). And significant regression of γ-H2AX levels in BCAT1 overexpressing cells after the addition of DM-αKG(21%)(Figure I).We used western blot to detect DSB repair-related proteins and found that ATM was also down-regulated in BCAT1 overexpressing cells, along with an upregulation γ-H2AX and the changes of these proteins can also be rescued by exogenous supplementation of DM-aKG(Figure J). High expression of BCAT1 reduces the level of intracellular α-KG and inhibits the activity of α-KG-dependent dioxygenases. Histone demethylase KDM2-7 are αKG-dependent dioxygenase, so we speculated that the decrease in a-KG caused by high BCAT1 expression would limit the activity of histone demethylase, thereby affecting the cellular histone methylation levels. We detected several classic histone methylation markers after BCAT1 overexpression, results showed that the levels of H3K9me3 were significantly up-regulated after BCAT1 overexpression, which could be reversed by supplementation of DM-α-KG(Figure K). Therefore, we believe that the regulation of cellular histone methylation levels by BCAT1 is affected by α-KG levels. H3K9me3 is an epigenetic suppressive marker. IDH1mut cells inhibited ATM expression by upregulating histone H3K9 methylation levels and impede cellular DNA damage repair. The elevated DNA damage level in BCAT1 overexpression condition may be therapeutically exploited using therapies that induce or augment DNA damage, such as chemotherapy and PARP inhibitor, Next, we tried to compare the responses of BCAT1 overexpression and control AML cells to the PARP inhibitor BMN673(Talazoparib). After treatment with BMN673, the survival fraction of BCAT1 overexpressing AML cells was lower than that of negative control in MTS assay. Vice versa, The IC50 of BMN673 in BCAT1 knockout AML cells is much larger than that of the control(Figure L). In summary, this study verified BCAT1 high expression is an independent prognostic predictor of CN-AML.BCAT1 reduces intracellular αKG levels, inhibits αKG-dependent histone demethylation enzymes to inhibit H3K9me3 demethylation, thereby suppressing ATM expression and inhibiting DNA damage response in AML cells. Therefore, BCAT1 high expressing AML cells have higher sensitivity to PARP inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

From phenotypical investigation to RNA-sequencing for gene expression analysis: A workflow for single and pooled rare cells.

Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

Abstract 14973: Upregulated Sphingosine-1-Phosphate Receptor 3 Disrupts Endothelial Cell Barrier and Mitochondrial Network Dynamics

Introduction: Ventilator-Induced Lung Injury (VILI) exacerbates existing Acute Lung Injury (ALI) of ICU patients via mechanical ventilation. However, the cellular and molecular mechanism behind VILI is still poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive lipid which usually enhances vascular endothelial barrier via S1P receptor 1 (S1PR1) to protect against ALI. VILI associated mechanical stress reduces S1PR1 while increases S1P receptor 3 (S1PR3) expression. S1PR3 couples to Rho, a central regulator of cytoskeleton and mitochondrial network dynamics. Therefore, the upregulation of S1P-S1PR3 pathway is highly likely to be involved in the deterioration of endothelial barrier and mitochondrial function during VILI. Methods and Results: Cyclic stretch (18%) significantly increased S1PR3 levels (1.85±0.12 fold, n=3, p&lt;0.05) in human lung endothelial cells (EC). After S1PR3 overexpression (S1PR3 OE), EC basal resistance was reduced (62.8±4.3%, n=8, p&lt;0.05) in ECIS experiments. Moreover, S1P (100 nM) increased 37.2±4.1% resistance in control cells (n=5, p&lt;0.05), while decreased 32.0±3.2% resistance in S1PR3 OE cells (n=5, p&lt;0.05). In contrast to S1P-induced junctional distribution of VE-cadherin in control cells, S1P failed to cause any VE-cadherin junctional redistribution in S1PR3 OE cells as shown by Immunofluorescence (Fig. 1). We further confirmed that S1PR3 OE suppresses VE-cadherin expression in human ECs by western blot. In addition, in S1PR3 OE cells, S1P challenge induced an increased mitochondrial ROS release as shown by MitoSOX assay (6.87±1.10 fold, n=3, p&lt;0.05) and a mitochondrial fission phenotype that was not observed in control cells. Conclusions: VILI associated mechanical stress upregulates S1PR3, leading to the reprograming of endothelial cell cytoskeleton regulatory machinery and mitochondrial network dynamics. S1PR3 might be a promising target to ameliorate the damage caused by mechanical ventilation.

Antitumor effects of lactate transport inhibition on esophageal adenocarcinoma cells

As a consequence of altered glucose metabolism, cancer cell intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs). MCT 1 and MCT4 are frequently overexpressed in tumors, and recently, MCT inhibition has been reported to exert antineoplastic effects. In the present study, MCT1 and MCT4 levels were assessed in esophageal adenocarcinoma (EAC) cells and the effects of the MCT-1 selective inhibitor AZD3965, hypoxia, and a glucose overload were evaluated in vitro. Two EAC cell lines (OE33 and OACM5.1C) were treated with AZD3965 (10–100 nM) under different conditions (normoxia/hypoxia) and also different glucose concentrations, and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi), and lactate levels were evaluated. MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small proportion of OACM5.1C cells. Glucose addition did not have any effect on apoptosis nor cell proliferation. AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia. MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress. AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4 and also increased the sodium/hydrogen exchanger 1 (NHE-1) expression on these cells. These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition.

SPARC expression in tumor microenvironment induces partial epithelial-to-mesenchymal transition of esophageal adenocarcinoma cells via cooperating with TGF-β signaling.

Secreted protein, acidic and rich in cysteine (SPARC) has been characterized as an oncoprotein in esophageal squamous cell carcinoma (ESCC), but its involvement in the pathological development of esophageal adenocarcinoma (ESAD) remains poorly understood. In this study, we aimed to explore the sources of SPARC in the tumor microenvironment (TME) and its functional role in ESAD. Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA)-esophageal cancer (ESCA) and Genotype-Tissue Expression (GTEx). ESAD tumor cell line OE33 and OE19 cells were used as in vitro cell models. Results showed that SPARC upregulation was associated with unfavorable disease-specific survival (DSS) in ESAD. ESAD tumor cells (OE33 and OE19) had no detectable SPARC protein expression. In contrast, IHC staining in ESAD tumor tissues suggested that peritumoral stromal cells (tumor-associated fibroblasts and macrophages) were the dominant SPARC source in TME. Exogenous SPARC induced partial epithelial-to-mesenchymal transitionof ESAD cells, reflected by reduced CDH1 and elevated ZEB1/VIM expression at both mRNA and protein levels. Besides, exogenous SPARC enhanced tumor cell invasion. When TGFBR2 expression was inhibited, the activation of TGF-β signaling induced by exogenous SPARC was impaired. However, the activating effects were rescued by overexpressing mutant TGFBR2 resistant to the shRNA sequence. Copresence of exogenous SPARC and TGF-β1 induced higher expression of mesenchymal markers and enhanced the invading capability of ESAD cells than TGF-β1 alone. In conclusion, this study suggests a potential cross-talk between ESAD tumor stromal cells and cancer cells via a SPARC-TGF-β1 paracrine network.

Evaluation of phenolic profiles, antioxidant activities, and cytotoxic and apoptotic potentials of Phlomis angustissima and Phlomis fruticosa, medicinal plants from Turkey

The genus Phlomis belongs to the family Lamiaceae and has over 100 species distributed across Europe, Asia, North Africa, and Turkey. Some Phlomis species are consumed as herbal teas for various diseases in traditional medicine. This study investigates the phenolic profiles, antioxidant activities, and cytotoxic properties of the methanol, ethyl acetate, and water extracts of leaves and flowers of P. angustissima, which is endemic to Turkey, and of P. fruticosa. Basic phenolics were identified by RP-HPLC-DAD. Antioxidant parameters and total- phenolics, -flavonoids, and -tannins were analysed spectrophotometrically. MTT cell proliferation was detected with a microplate reader. Apoptosis was analysed by flow cytometry. Among the identified phenolics, hesperidin, kaempferol, catechin, epicatechin, epigallocatechin, eupatorin, and chlorogenic, vanillic, syringic, p-coumaric, ferulic, and benzoic acids are reported in this study for the first time. The values of radical scavenging capacities (DPPH·, HO·, and NO·) and antioxidant activities (metal chelating and reducing power) were found to be significant. Both Phlomis species are rich in total-phenolics, -flavonoids, and -tannins. The best IC50 values of the P. angustissima and P. fruticosa extracts against five different cancer cell lines were, respectively determined as 23±1.42 and 26±1.68 ppm against ACC-201 cells, 40±2.32 and 28±2.1 ppm against OE-33 cells, and 80±3.21 ppm against HeLa cells (P. fruticosa), and the death types of the cancer cells were apoptotic. The results obtained for P. angustissima and P. fruticosa show that they are promising in terms of future applicability in food and medical fields as natural biopotential resources.

428. CHARACTERIZATION OF CD82/KAI1 IN ESOPHAGEAL ADENOCARCINOMA

Abstract The incidence of esophageal adencocarcinoma (EAC) has increased in last decades. Regardless to improved perioperative chemotherapies and surgical approaches, the prognosis for EAC in advanced stages is poor. CD82 is a tumor suppressor gene, and its downregulation correlates with invasive growth, metastases formation and advanced clinical stages. CD82 might influence the Wnt/ß-catenin signaling pathway. We used an in vitro Barrett’s esophagus cell culture model, represented by esophageal squamous cell epithelium (EPC1, EPC2), metaplasia (CP-A), dysplasia (CP-B) and esophageal adenocarcinoma (OE33, OE19, SKGT4, FLO1). Antisense LNA Oligonucleotides (GapmeRs, Qiagen) were used to achieve a transient knockdown of CD82 in OE33 and OE19 cells. mRNA and proteins were analyzed by q-RT-PCR and western blot analyses, respectively. A fluorescence antibody specific to CD82 was used for flow cytometry analysis. Proliferation assay, colony formation assay and boyden chamber assay were performed after CD82-knockdown to further analyze cell proliferation, cell growth and survival, colony forming and cell migration. CD82 was expressed in all investigated cell lines by flow cytometry with a significant overexpression in OE19 and FLO1 cells. A stable downregulation of CD82 was achieved in OE33 and OE19 cells. Proliferation assays showed a significantly higher proliferation rate at 72 hours after CD82-knockdown in OE33 cells. A significantly higher mRNA expression of vimentin was observed after CD82-knockdown in OE33. Colony formation assay showed a significantly higher colony count after CD82-knockdown in both cell lines. Cell migration was increased after CD82-downregulated in OE33 and OE19 cells. CD82 might influence the expression of mesenchymal marker proteins, cell migration abilities and the process of colony forming and thus enhance the metastatic potential of OE33 and OE19 cells. Low expression of CD82 could alter metastasis and cancer progression but further molecular characterizations are needed to elucidate the impact on carcinogenesis of EAC.