CD44 Protein Expression Research Articles

Immune vulnerability of ovarian cancer stem-like cells due to low CD47 expression is protected by surrounding bulk tumor cells

ABSTRACT Recurrence of advanced epithelial ovarian cancer is common despite optimal surgical debulking and initial favorable responses to chemotherapy. Evidences suggest that cancer stem cells (CSCs) have inherent resistance to conventional therapies such as chemotherapy and play a decisive role in cancer recurrence. Cancer stem cells are also believed to be able to evade immunological attack. However, this study showed a different scenario in which cancer stem-like cells are more vulnerable to immunosurveillance. Our study demonstrated that isolated murine cancer stem-like cells, stem cell antigen (SCA)-1+ ID8 and CD133+ HM-1 cells, were susceptible to phagocytosis by macrophages and consequent CD8+ T cell immunity. The increased phagocytosis of these stem cell-like cells is attributed to low CD47 protein expression. SCA-1+ ID8 cells were able to grow in syngeneic mice but were soon rejected. Restoring CD47 expression delayed this immune-mediated rejection. SCA-1+ ID8 cells showed rapid growth by mixing with bulk ID8 cells. These results suggest that stem-like cells could be protected by surrounding non-stem cancer cells from immune attack. Similarly, both isolated human CD24−/low SKOV3 stem-like cells and spheroid OVCAR3 cells expressed lower CD47 levels. Our study provided novel insights into the immune characteristics of CSCs within a tumor microenvironment. The results might lead to the design of more effective treatment strategies for ovarian cancer.

3D proteome-wide scale screening and activity evaluation of a new ALKBH5 inhibitor in U87 glioblastoma cell line

The imidazobenzoxazin-5-thione MV1035, synthesized as a new sodium channel blocker, has been tested on tumoral cells that differ for origin and for expressed NaV pool (U87-MG, H460 and A549). In this paper we focus on the effect of MV1035 in reducing U87 glioblastoma cell line migration and invasiveness. Since the effect of this compound on U87-MG cells seemed not dependent on its sodium channel blocking capability, alternative off-target interaction for MV1035 have been identified using SPILLO-PBSS software. This software performs a structure-based in silico screening on a proteome-wide scale, that allows to identify off-target interactions. Among the top-ranked off-targets of MV1035, we focused on the RNA demethylase ALKBH5 enzyme, known for playing a key role in cancer. In order to prove the effect of MV1035 on ALKBH5 in vitro coincubation of MV1035 and ALKBH5 has been performed demonstrating a consequent increase of N6-methyladenosine (m6A) RNA. To further validate the pathway involving ALKBH5 inhibition by MV1035 in U87-MG reduced migration and invasiveness, we evaluated CD73 as possible downstream protein. CD73 is an extrinsic protein involved in the generation of adenosine and is overexpressed in several tumors including glioblastoma. We have demonstrated that treating U87-MG with MV1035, CD73 protein expression was reduced without altering CD73 transcription. Our results show that MV1035 is able to significantly reduce U87 cell line migration and invasiveness inhibiting ALKBH5, an RNA demethylase that can be considered an interesting target in fighting glioblastoma aggressiveness. Our data encourage to further investigate the MV1035 inhibitory effect on glioblastoma.

232 Protecting pigs from viral infectivity with gene editing

Abstract Pig models were created that were fully resistant to Porcine Reproductive and Respiratory Syndrome (PRRS) and the alphacoronaviruses, transmissible gastroenteritis virus (TGEV) by DNA editing of the cluster of differentiation (CD163) gene and aminopeptidase N (ANPEP) gene via CRISPR/Cas, respectively. Litters of pigs were bred with a gene edit in exon 7 of CD163 that resulted no CD163 protein expression (CD163-/-). The pigs were either challenged with multiple PRRSV isolates at 3 weeks of age or bred at maturity for a challenge with pregnant sows. The challenges demonstrated that the pigs were completely resistant to infectivity to both Type 1 and 2 isolates as measured by clinical signs, viremia, antibody response and lung histopathology. In a follow-up study, pregnant CD163 edited pigs were challenged with PRRSV to determine if absence of CD163 in the dam should be sufficient to protect the fetuses that have functional CD163 protein. The CD163-/- sows carrying both the CD163-/- and CD163+/- fetuses were all negative for PRRSV nucleic acid and showed no sign of fetal or placental failure clearly demonstrating that the absence of CD163 in the sow is sufficient to protect a PRRSV-susceptible CD163+/- fetus. Gene editing of CD163 in pigs, via CRISPR/Cas9, successfully blocked PRRSV infectivity in young growing pigs and pregnant sows and their fetuses. Similar methods were used to determine if TGEV infectivity could be prevented by editing the ANPEP gene. Exon 2 of ANPEP was edited and resulted an ANPEP -/- genotype. ANPEP -/- pigs and age matched wild type pigs were challenged with TGEV. The presence of virus nucleic acid was determined by PCR in fecal samples. ANPEP -/- pigs were fully resistant to TGEV infectivity. These studies clearly show that precise gene editing can be successfully used in pigs to prevent viral infectivity of both PRRSV and TGEV.

Spectomycin B1 induces VEGFR2 de-SUMO modification to inhibit angiogenesis in nasopharyngeal carcinoma

Objective:To explore the new mechanism of spectomycin B1 in inhibiting angiogenesis of nasopharyngeal carcinoma and to provide a theoretical basis for targeted gene therapy of nasopharyngeal carcinoma. Method:Human nasopharyngeal carcinoma CNE1 cells were divided into two groups, the control group and spectomycin B1 group. Western blot was used to detect the expression levels of small ubiquitin-related modified protein（SUMO） 1 and vascular endothelial growth factor receptor 2（VEGFR2）. The angiogenesis assay was used to detect the angiogenic ability of CNE1 cells, and the apoptosis was detected by flow cytometry. The model of nasopharyngeal carcinoma-bearing mice was established, spectomycin B1 was administered, tumor volume and weight were measured, and protein expression of CD31 was detected by immunohistochemistry and microvessel density was compared. Result:Spectomycin B1 could reduce deSUMOylation of VEGFR2 protein by 4.05 times, significantly reduce the angiogenic ability of CNE1 cells, and increase the apoptosis rate by 20.68%. In the tumor-bearing mouse model, spectomycin B1 treatment could inhibit subcutaneous tumor growth rate and weight, and the blood vessel density decreased by 40.04%. Conclusion:Spectomycin B1 can inhibit neovascularization of nasopharyngeal carcinoma by inducing deSUMOylation of VEGFR2 protein.

Abstract B095: Novel anti-CD147 antibodies inducing activation of SMAD signaling and tumor shrinkage in intractable cancer models

Abstract CD147, a type I membrane protein, is highly expressed in various cancers and is involved in the growth, metastasis, and activation of inflammatory signals in cancer cells via interaction with various molecules, such as integrins, CD44, and monocarboxylate transporters. Additionally, the expression of CD147 in tumors positively correlates with poor prognosis in various cancers. Therefore, CD147 is an attractive target for cancer therapy. However, there have been no successful drugs targeting CD147 so far, possibly because it is a multifunctional protein interacting with multiple molecules, making it challenging to discover a relevant CD147-targeting drug candidate by general in vitro screening methodologies. In this study, we developed novel anti-CD147 antibodies, which showed significant antitumor efficacy in immune-deficient mice bearing human pancreatic cancer cell lines, PANC-1 or MIA PaCa-2. Six anti-CD147 antibodies showing in vivo anti-tumor efficacy were obtained; one mouse IgG3, two rat IgG2b, two rat IgG1, and one rat IgG2a antibodies. Analysis of antibody dependent cellular toxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity of chimeric and humanized antibodies with human IgG2 or IgG4P derived from the obtained 6 anti-CD147 antibodies, #147A/B/C/D/E/F, suggested no significant immune-effector function for these antibodies. Despite lacking effector function, these antibodies exhibited significant in vivo antitumor activity, suggesting that the mechanism underlying their anti-tumor efficacy did not involve their effector function. We also found that the anti-tumor efficacy of antibodies #147A and #147B positively correlated with SMAD4 protein expression rather than CD147 protein expression in 10 pancreatic cancer xenograft models (correlation coefficient of #147B: anti-tumor efficacy vs. CD147, 0.245; vs. SMAD4, 0.9328). Moreover, the administration of anti-CD147 antibodies upregulated rhoB expression in an anti-CD147 antibody-sensitive SMAD4-positive MIA PaCa-2 graft mouse model. The stable expression of SMAD4 increased the sensitivity to the anti-CD147 antibody, ch4#147A (chimeric IgG4P derived from #147A) in xenograft mice inoculated with the BxPC-3 pancreatic cancer cell line, which is originally negative for SMAD4 and has low sensitivity to anti-CD147 antibodies. These data suggest that the expression of SMAD4 and activation of SMAD signaling are functionally important for susceptibility to anti-CD147 antibody treatment. The humanized anti-CD147 IgG4P antibody, h4#147D, derived from #147D with cross reactivity to monkey CD147, showed superior anti-tumor efficacy with complete tumor reduction, compared to standard anticancer drugs, including gemcitabine, imatinib, and sorafenib, in xenograft mice bearing corresponding cancer cell lines, MIA PaCa-2, KU812 chronic myeloid leukemia, and HepG2 liver cancer cell lines, respectively. No toxicity of h4#147D was observed in cynomolgus monkeys at a single dose of 100 mg/kg. These data suggest that h4#147D might be a novel anti-CD147 antibody, which could induce SMAD activation and tumor shrinkage in multiple xenograft models. Therefore, it has potential as a promising antitumor therapeutic antibody with superior anti-tumor efficacy to existing therapy. Citation Format: Keisuke Fukuchi, Kayoko Nanai, Shoji Yamamoto, Jun Tsukada, Yusuke Totoki, Koichiro Inaki, Masato Ishigami, Naoyuki Makita, Yoko Nakano, Chigusa Yoshimura, Kozo Yoneda, Masato Amano, Kensuke Nakamura, Yoshiyuki Kanari, Yoko Oda, Haruyuki Nishigohri, Rika Nakano, Atsuko Nishida, Kenji Murakami, Yumi Matsui, Naomi Kasanuki, Shoji Midori, Satoko Funo, Sayako Takahashi, Hironobu Komori, Toshiaki Ohtsuka, Toshinori Agatsuma. Novel anti-CD147 antibodies inducing activation of SMAD signaling and tumor shrinkage in intractable cancer models [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 B095. doi:10.1158/1535-7163.TARG-19-B095

Prognostic impact of CD34 and SMA in cancer-associated fibroblasts in stage I-III NSCLC.

BackgroundEpithelial‐to‐mesenchymal transition (EMT) is a crucial step in lung cancer pathogenesis. Among others, cancer‐associated fibroblasts (CAFs) are reported to regulate this process.ObjectivesTo investigate the prognostic and clinical impact, we analyzed CD34+ and SMA+ CAFs in non‐small cell lung cancer (NSCLC).MethodsRetrospectively, immunohistochemistry was performed to study stromal protein expression of both CD34 and SMA in 304 NSCLC patients with pTNM stage I‐III disease. All tissue samples were embedded on tissue microarrays (TMAs).ResultsOur analysis revealed an association for CD34+ CAFs with G1/2 tumors and adenocarcinoma histology. Moreover CD34+ CAFs were identified as an independent prognostic factor (both for progression free survival [PFS] and overall survival [OS] in stage I‐III NSCLC). Besides, SMA+ expression correlated with higher pTNM‐tumor stages and lymphatic spread (pN stage). In turn, SMA‐negativity was associated with improved PFS, but no prognostic impact was found on OS. Of interest, neither CD34+ CAFs nor SMA+ CAFs were associated with the primary tumor size, localization and depth of infiltration (pT stage).ConclusionsCD34 was identified as an independent prognostic marker in pTNM stage I‐III NSCLC. Moreover, loss of CD34+ CAFs might influence the dedifferentiation of the NSCLC tumor from its cell origin. Finally, SMA+ CAFs are more prevalent in NSCLC tumors of higher stages and lymphonodal positive NSCLC.Key points Expression of CD34 on cancer associated fibroblasts (CAFs) is an independent prognostic factor in stage I‐III NSCLC.SMA+ cancer associated fibroblasts are associated with higher tumor stages in NSCLC and might contribute to tumor progression in NSCLC.

Biological Characteristics and Carrier Functions of Pegylated Manganese Zinc Ferrite Nanoparticles

This study was performed to investigate the biocompatibility (BC), magnetothermal effect, and DNA binding biological characteristics of manganese zinc ferrite nanoparticles (Mn0.6Zn0.4Fe2O4-NPs (MZF-NPs)) coated with pegylated manganese (PEG-MZF-NPs). Their functions as gene transfer carrier for gene therapy and magnetic medium for tumor hyperthermia were also explored. The manganese zinc ferrite nanoparticles were synthesized through high temperature cracking, and their characterizations were discovered. Hemolysis test and MTT assay were performed to evaluate biocompatibility, and their self-heating effects in the alternating magnetic field were investigated. PEG-MZF-NPs with different concentrations were measured by using 7.0 T Micro-MR scanner (MRI) to calculate the T2 value and r2 relaxation rate of each sample. The CD44-shRNA plasmids were constructed, and their ability to bind PEG-MZF-NPs were examined. The DNA release from PEG-MZF-NP/DNA complex and protection of DNA from nuclease digestion were also detected. After CD44-shRNA-EGFP were transfected into the ovarian cancer SK-OV-3 cells by using PEG-MZF-NPs as carriers, the transfection efficiency was detected by a flow cytometer and expression of CD44 mRNA and protein in cells was detected using RT-PCR and Western blot, respectively. We successfully prepared PEG-MZF-NPs with favorable dispersity, magnetic responsiveness, and BC. Typically, the excellent magnetothermal effect can be used for a tumor magnetothermal therapeutic study. In vitro MRI showed the application potential for being magnetic resonance T2 relaxation contrast agents and the possibility to achieve goal of integration of targeting diagnosis and treatment. The CD44-shRNA plasmids have been successfully constructed and concluded that PEG-MZF-NPs may serve as gene transfer carriers for gene therapy.

Expression of CD147, MMP-9 and HER-2 in patients with gastric cancer and their correlation with clinicopathology and prognosis

Objective To investigate the correlation between the expression of extracellular matrix metalloproteinase-inducible factor (CD147), matrix metalloproteinase-9 (MMP-9) and human epidermal growth factor receptor-2 (HER-2) in gastric cancer tissues and clinical pathology and prognosis. Methods From June 2016 to June 2018, 80 gastric cancer specimens from the First People's Hospital of Taizhou were collected as observation group, and another 60 normal specimens of gastric mucosa adjacent to cancer were selected as control group.The expressions of CD147, MMP-9 and HER-2 were detected by SP immunohistochemical method.The positive rates of CD147, MMP-9 and HER-2 protein expression, the positive rates of CD147, MMP-9 and HER-2 protein expression in different pathological characteristics, and the positive rates of CD147, MMP-9 and HER-2 protein expression in different prognosis were compared between the two groups. Results The positive rates of CD147 (73.75%), MMP-9 (76.25%) and HER-2 (42.50%) in the observation group were higher than those in the control group (21.67%, 25.00%, 5.00%), the differences were statistically significant (χ2=37.233, 36.288, 24.797, all P 0.05). The positive rates of CD147 (93.88%), MMP-9 (95.92%) and HER-2 (61.22%) with lymph node metastasis were higher than those without lymph node metastasis (41.94%, 45.16%, 12.90%), the differences were statistically significant (χ2=26.462, 27.012, 18.142, all P<0.05). The positive rates of CD147 (96.15%), MMP-9 (92.31%) and HER-2 (69.23%) in the death group were higher than those in the survival group (62.96%, 68.52%, 29.63%), the differences were statistically significant(χ2=9.987, 5.484, 11.263, all P<0.05). Conclusion High expression of CD147, MMP-9 and HER-2 proteins in gastric cancer tissues is closely related to invasion and metastasis, which can become a new prognostic marker and therapeutic target of gastric cancer. Key words: Gastric cancer; Paracancerous tissue of gastric mucosa; Extracellular matrix metalloproteinase-inducing factor; Matrix metalloproteinase-9; Human epidermal growth factor receptor-2; Tumor diameter; Differentiation degree; Lymph node metastasis; Prognosis

Expression of CD47 and Calr in Myeloproliferative Neoplasms and Myelodysplastic Syndrome: Potential New Therapeutical Targets

Myelodysplastic neoplasms (MPN) and myelodysplastic syndrome (MDS) are myeloid malignancies characterized by either overproduction of mature blood cells or hyperplastic bone marrow and peripheral cytopenia, but both with a tendency to evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti-phagocytic CD47, reflecting an apoptosis vs survival mechanism. Increases of both CALR and CD47 on the cell membrane have been observed in response to chemotherapy, however their role in myeloid malignancies is poorly understood. Aims: Investigate the expression and cellular localisation of CALR and CD47 in untreated and treated patients with essential thrombocythemia (ET), polycythemia vera (PV) myelofibrosis (MF), and in MDS patients in comparison with healthy controls. Methods: Mononuclear cells were collected by Ficoll separation, from peripheral blood of 27 MPN (8 PV, 16 ET, 3 MF); 14 MPN patients received cyto-reductive therapies (Hydroxyurea, Anagrelide or Ruxolitinib); 10 MDS patients and 4 controls. Cells were fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol, with CALR and CD47 protein expression analysed by western blotting. Results: Total CALR and CD47 protein expression increased in MPN samples compared with controls (CALR- 7.9 vs 5.1; CD47- 2.7 vs 2.2 fold, respectively). CD47 showed higher expression of its overall protein on MPN cell membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR expression in all MPN subtypes when patients were treated with cyto-reductive agents (ET- untreated 43.3% vs treated 2%, PV- 3.6% vs 2.2%, ET- 21% vs 11%). Interestingly we have observed a significant increase in CD47 cell membrane expression after treatment in MF and PV (CD47 in MF- untreated 11.8% vs treated 34.3%, PV-11.4% vs 35.9%), suggesting an anti-phagocytic effect being induced by cytotoxic drugs in MF and PV cells. In ET cell membranes however, CD47 expression is reduced after cyto-reductive treatment (22% vs 16.6%), suggesting instead a prophagocytic effect. Similarly, in MDS cells CD47 is overexpressed compared with controls (CD47- 11.31 vs 2.2 fold, respectively) and it mainly located to the membrane. Interestingly the degree of CD47 expression correlated to patients IPSS-R, increasing from low risk to high risk (low - 15.7%, intermediate 1 - 41.3%, intermediate 2 - 53.9% and high - 67.6%). CALR expression is also reduced in MDS cells comparing with controls when split by IPSS-R risk score (low - 9%, intermediate 1 - 16.9%, intermediate 2 - 17%, high - 17.9% Vs control - 29.1%). Summary/Conclusion: CD47, but not CALR, is overexpressed on the membrane of patients with MPN and MDS. In MDS, we observed a progressive increase in CD47 expression as the MDS evolve in accordance to the IPSS-R risk score. This opposes previous studies in solid tumours, which showed significant increases of both CALR and CD47 as disease progresses, suggesting a role for CD47 as a strong antiphagocytic signal responsible for immune survival in MPN and MDS. All MPN patients showed decreased CALR expression during cyto-reduction therapy, but we observed a significant difference in CD47 expression across different MPN subtypes. In fact, treating MF and PV patients with cyto-reductive agents, increases CD47 expression leading to a stronger anti-phagocytic signal, while in ET we observed a reduction, suggesting a more pro-phagocytic reaction to cytotoxic drugs in ET. A larger cohort of patients is required to confirm our findings and allow a further stratification based of type of cyto-toxic drugs. The use of anti-CD47 antibodies could represent a new strategy to enhance the pro-phagocytic signal via increasing the CALR expression, and in combination with standard cyto-reduction therapy, might represent a new therapeutical strategy in both MPN and MDS. Disclosures No relevant conflicts of interest to declare.

Pharmacodynamic Responses to CC-90009, a Novel Cereblon E3 Ligase Modulator, in a Phase I Dose-Escalation Study in Relapsed or Refractory Acute Myeloid Leukemia (R/R AML)

Pharmacodynamic Responses to CC-90009, a Novel Cereblon E3 Ligase Modulator, in a Phase I Dose-Escalation Study in Relapsed or Refractory Acute Myeloid Leukemia (R/R AML)

Isolation and Characterization of a CD34+ Sub-Clone in B-Cell Lymphoma

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in the US. Many types remain incurable despite response to initial therapy and achievement of complete remission (CR). Advanced laboratory techniques like multicolor flow cytometry (MCF) and polymerase chain reaction (PCR) have demonstrated persistence of rare malignant cell population post therapy referred to as minimal residual disease (MRD). However, the functional and biological characteristics of this population have not been fully elucidated. Established B-lymphoma cell lines (B-NHL) and patient-derived samples (PDS) were analyzed using 8-color FCM of leukemia and lymphoma antibody panels (28 antibodies). The CD34+ sub-population was enriched using in vitro exposure to 2-chlorodeoxyadenosine (2-CdA), and a CD34-coated magnetic beads isolation procedure (Miltenyi Biotech). Genetic analysis of CD34+ and CD34-/parent cell fractions was done by karyotyping, and by chromosomal microarray (CMA) using the oligonucleotide-single nucleotide polymorphism (Oligo-SNP), whole genome Agilent 180K GGXChip+SNP (Agilent Technologies, Inc). Sensitivity to chemotherapy was assayed by short-term in vitro exposure to drugs. Clonogenicity was determined by soft agar colony formation assay, and proliferation was determined using DNA staining with propidium iodide and flow cytometry. The side population was determined using the fluorescent vital dye Hoechst 33342 and flow cytometry. Analysis of three B-NHL cell lines revealed the presence of a minute sub-clone (&lt;1%) of monotypic B-cells that expresses CD34. This sub-population enriched several folds in response to exposure of the cells to 2-CdA. Enrichment was highest in the follicular lymphoma cell line, WSU-FSCCL reaching more than 50 folds compared with control (13.2% vs 0.26%), followed by WSU-WM, 10-fold (2.36% vs 0.23%), and least in WSU-DLCL2 (1.25% vs 0.71%). Using a CD34 microbead positive selection technique, we were able to enrich the CD34+ population up to 80% purity. Western blots confirmed higher expression of CD34 protein in the enriched population compared with parent cells. Using StemPro media (Gibco by Life Technologies), CD34+ cell fraction of WSU-WM showed more sustained growth and viability in culture over 9-day period compared with parent cells. This technique allowed the generation of large number of CD34+ cells for further characterization. Except for CD34 expression, this cell fraction expressed identical phenotype, karyotype, SNP and MCA profile to parent cells. However, the CD34+ fraction was more proliferative with an increase in S-phase cells (~60% vs 20% in control cells) and Hoechst 33342-positive cells (~40%). These cells were clonogenic even in presence of the cytotoxic agents Doxorubicin and 2-CdA that completely inhibited colony formation in parent cells. Resistance to chemotherapy was also noticed in liquid culture. A CD34+ population within clonal B-cells was also detected in 8 of 8 PDS. The lowest number of CD34+ cells was detected in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and the highest (5.35%) was seen in Richter's transformation of CLL. The isolated CD34+ cells in this study show many features of what is known as Lymphoma Stem Cells implicated in resistance to therapy and disease recurrence. Its relatedness to MRD cells is uncertain, however, since CD34 is not routinely included in MRD antibody panel. The observation that CD34+ cells are genetically identical to parent cells suggests an epigenetic modification. This conclusion is supported by our finding that Ehd3 expression is dramatically reduced in the CD34+ cell population of WSU-DLCL2 compared with parent cells. Ehd3 was recently identified as an epigenetically-silenced gene in solid tumors and in hematological malignancies including leukemia and lymphoma. The Ehd3-encoded protein belongs to the family of C-terminal Eps15 homology domain-containing (EHD) proteins, implicated in intracellular trafficking of tubular recycling endosomes (TRE), apoptosis, and cell cycle arrest. Further studies are underway to determine the contribution of Ehd3 to the biological and functional characteristics of the CD34+ cells in B-NHL. Utilizing the CD34+ monotypic B-cells in B-NHL as a model for screening new therapeutic agents and to identify potential new therapeutic strategies may lead to a more effective treatment of B-NHL. Disclosures No relevant conflicts of interest to declare.

Evaluation of the Prognostic Significance and Effect of NT5E-Adenosine Axis, a Novel Immune Checkpoint, in Diffuse Large B-Cell Lymphoma

Background: The NT5E-adenosine axis constitutes one of the most promising immunosuppressive pathways in immune-oncology. NT5E is also named as CD73, which catalyzes the conversion of AMP to adenosine, and then the extracellular adenosine within tumor microenvironment bindings to A2a adenosine receptor (A2aR) to further dampen T cell-mediated immune responses and promote tumor immune escape. Several anti-CD73 or anti-A2aR antibodies are being evaluated in different types of malignancies in clinical trials. However, the prognostic significance and effect of NT5E-adenosine axis in diffuse large B-cell lymphoma (DLBCL) remain unclear. Methods: Multiplexed immunofluorescence staining, and a professionally and automatically assessed computer-assisted platform were applied to localize and quantify the markers from the DLBCL tumor and microenvironment cells. Gene expression status was analyzed according to the microarray data. The associations among marker expression patterns or their correlations with clinicopathological characteristics were estimated with the χ2 test and two-tailed Spearman analyses. Co-culture system of CD73 positive DLBCL cells and primary CD8 positive T cells was applied to evaluate the effect of NT5E-adenosine axis in DLBCL. Results: We found that CD73 was widely expressed on tumor and immune cells in DLBCL tissue. The CD73 positive rate on tumor cells was 52.31%, and CD73 expression on tumor cells was significantly associated with several clinicopathologic parameters, including IPI score (p = 0.020), LDH levels (p = 0.016) and Ki67 expression (p = 0.014). The A2aR positive rate on tumor-infiltrating lymphocytes (TILs) was 43.08%, and A2aR expression on TILs was significantly correlated with several clinicopathologic parameters, including IPI score (p = 0.036), clinical stage (p = 0.011), B symptom (p = 0.024) and Ki67 expression (p = 0.036). No significant difference in OS was observed in 233 DLBCL patients stratified by their CD73 gene expression status according to the microarray data (p = 0.267). There was also no significant effect of total CD73 protein expression on OS for the patients. However, a significant difference in OS was found when the patients were stratified by the CD73 expression on tumor cells, and the median survival times of the patients with CD73+/Pax-5+ and those with CD73-/Pax-5+ were 57.8 months (95% CI: 46.4-69.3) and 73.5 months (95% CI: 65.9-81.2), respectively. Patients with CD73+/Pax-5+ experienced significantly poorer outcomes than those with CD73-/Pax-5+ (p = 0.027). Furthermore, the median survival times of the patients with A2aR+ TILs and those with A2aR- TILs were respectively 53.3 months (95% CI: 40.6-66.0) and 74.5 months (95% CI: 67.5-81.5), and patients with A2aR+ TILs also had a significantly shorter survival time than those with A2aR- TILs (p = 0.003). Spearman's analysis revealed that CD73+/Pax-5+ tumor cells were positively correlated with A2aR+ TILs (R=0.395, p = 0.001). In a cohort, we found that 24 exhibited CD73-/Pax-5+ as well as A2aR- TILs, and 21 exhibited CD73+/Pax-5+ as well as A2aR+ TILs. Patients with CD73+/Pax-5+ and A2aR+TILs had poorer survival than those with CD73-/Pax-5+ and A2aR- TILs (p = 0.001). Residual patients were further classified into 2 groups, in which 7 exhibited CD73-/Pax-5+ as well as A2aR+ TILs, and 13 displayed CD73+/Pax-5+ as well as A2aR- TILs. There was a significant difference in survival among these four groups, and patients with CD73+/Pax-5+ and A2aR+ TILs had the worst outcome, with a 5-year OS rate of 57.1% (p = 0.017). We also found that CD73 positivity on tumor cells weakened the favorable prognosis for patients, which was correlated with the presence of CD8+ TILs. Patients with CD73+/Pax-5+ and CD8+ TILslow had the worst clinical outcome, with a 5-year OS rate of 50%. In contrast, patients with CD73-/Pax-5+ and CD8+ TILshigh had the best clinical outcome, with a 5-year OS rate of 100% (p = 0.002). Co-culture system displayed that CD73 being expressed on the DLBCL cells suppressed the growth of CD8 positive T cells through A2aR, but not A2aR negative T cells. Conclusions: Our findings uncovered that DLBCL patients with CD73+ on tumor cells as well as A2aR+ on TILs exhibited inferior survival and NT5E-adenosine axis inhibited the growth of CD8 positive T cells, supporting the potential combination strategies using CD73/A2aR immunosuppressive blockades as treatment options for DLBCL patients. Disclosures No relevant conflicts of interest to declare.

CD20 Is an Unstable Target in Primary-Refractory High-Grade Lymphomas

Introduction: A third of patients with diffuse large B cell lymphoma (DLBCL) are not cured with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (RCHOP). Approximately 20% of DLBCLs will lose CD20-protein expression (CD20-neg) at the time of relapse. The incidence of CD20-neg relapses in other aggressive B cell lymphomas is unknown. CD20 is not only targeted by rituximab but other antibody-based therapies that are being investigated in salvage regimens. We hypothesized that prolonged exposure to anti-CD20 containing regimens may lead a CD20-neg relapse. Our goal was to determine the clinical and genetic factors associated with loss of CD20 expression in high-grade lymphomas treated with curative intent. Method: We consented 374 patients with B cell high-grade lymphomas that were treated with RCHOP or more intensive regimens at the time of diagnosis or had a histological transformation from a prior indolent lymphoma (TLy). We recorded the baseline clinical characteristics, histological diagnosis, date of first relapse and the number of treatment regimens prior to their second biopsy. CD20 expression and cell of origin (COO) were determined by immunohistochemistry and, in the latter, using Hans criteria. We performed targeted sequencing of 63 lymphoma-related genes, including MS4A1, the gene that encodes CD20. Sequencing was performed using circulating tumor DNA in the plasma or DNA from the tumor biopsy, both obtained at the time of relapse. Results: Relapse occurred in 170/374 (45%) of patients: 102/253 DLBCL, 55/96 TLy, 6/16 primary mediastinal B cell lymphoma (PMBCL) and 7/9 high grade B cell lymphomas (HGBL) with or without translocations in MYC and BCL2 or BCL6. The median age at diagnosis was 62 years old, 54% were male and 85% had an elevated international prognostic index of ≥ 2. Of these, 104 had a biopsy taken at first (47%) or subsequent relapse (53%) to confirm the diagnosis or performed in the context of a clinical trial. CD20 could be assessed in 100 cases, of which 26 had CD20-neg lymphoma cells: 15/56 (27%) of DLBCL, 7/38 (18%) of TLy, 2/3 (66%) PMBCL and 2/3 (66%) HGBL. Relapsed PMBCL and HGBL combined, appeared to have an increased risk of CD20 negativity at relapse compared to DLBCL and TLy (p=0.043). A GCB phenotype in DLBCL was present in 35% of cases and was not associated with CD20 status. The mean number of therapies given before the second biopsy was similar in both groups (CD20+ = 2.2 and CD20-neg = 2.7, p=0.2). In fact, 11/26 (44%) of CD20-neg cases had biopsies taken after RCHOP alone. Additional chemo-immunotherapy (range 2 to 8) did not increase the risk of having a CD20-neg relapse (p=0.5), suggesting that unlike follicular lymphoma, the emergence of CD20-neg cells occurs early after rituximab exposure in high-grade lymphomas. Supporting this hypothesis, patients with a CD20-neg relapse had a shorter progressive free survival (PFS) after RCHOP (1,7 vs 3,2 years, p=0.025). Patients with primary treatment failure, defined here as a PFS of &amp;lt; 1 year, had a significantly increased risk of having a CD20-neg relapse (hazard ratio 7.9, p= 0.005). Sequencing was performed in 75/100 patients. MS4A1 mutations were present in 16% of CD20-neg cases, while none were present in the CD20+ cases (p=0.003). There was no significant difference in the mutation rate of TP53 (47%) or histone-modifying genes based on CD20 expression status. Conclusion: Decreased CD20 expression occurs early in high-grade lymphomas under the selective pressure of RCHOP, 16% of which are a consequence of MS4A1 mutations, suggesting that other mechanisms also modulate CD20 expression. In patients with a PFS of &amp;lt; 1 year, a repeat biopsy would be recommended if primary anti-CD20-targeted therapy is considered. Disclosures Assouline: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Johnson:Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; BMS: Consultancy, Honoraria; Merck: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding.

PDTM-18. COMBINED SUPPRESSION OF THE mTOR AND MAPK PATHWAYS INHIBITS GROWTH, DECREASES VASCULARITY AND INDUCES APOPTOSIS OR SENESCENCE IN PEDIATRIC LOW GRADE GLIOMA

Abstract Pediatric low-grade glioma (pLGG) is the most common brain tumor in children. We and others have identified constitutive activation of the mTOR and MEK-pathway in pLGG. This led us to investigate TAK228, a mTORC1/2-inhibitor, and the FDA approved MEK-inhibitor, trametinib, in mono- and combination therapy in pLGG. We examined antitumor activity in four patient-derived pLGG cell models treated with TAK228, trametinib, and combination: JHH_NF1_PA1(NF1mut), BT40(BRAFV600E), Res186(PTEN-/-) and Res259(CDKN2A-/-). Treatment with TAK228 or trametinib reduced cell proliferation in a dose and time dependent manner (MTS-assay). The combination treatment exerted a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells (Chou-Talay method). The combination of TAK228 and trametinib increased apoptosis by up to 127% (p&lt; 0.001 by 1-way ANOVA) in Res186 and Res259 cells as measured by cleaved caspase 3 immunocytochemistry. Trametinib and combination treatment induces senescence in JHH_NF1_PA1 cell line verified through increase in p27 protein expression (Western Blot) and positive b-galactosidase staining. Mono-treatment with TAK228 or vehicle showed no signs of senescence. We tested trametinib and TAK228 against the mutant BT40BRAFV600E patient-derived cell line in immunodeficient mice. The combination treatment showed greater antitumor activity than that of either mono-treatment in vivo. BT40 tumor growth was significantly decreased in combination compared to vehicle or either agent alone (p&lt; 0.01). Combination treatment strikingly reduced the vascularization in the tumor tissue after combination treatment analyzed by CD31 and CD34 protein expression (Western Blot), compared to vehicle and mono-treatment. Our study provides evidence that pLGG-derived cell lines in vitro and in vivo are sensitive to mTORC1/2 kinase inhibition and MEK inhibition. Combination treatment with TAK228 and trametinib had a significant anti-tumor activity in vivo shown in survival rate, decreased tumor size, and reduced vascularity. These results provide the first strong rationale for combination therapy with TAK228 and trametinib for clinical consideration in pLGG.

MicroRNA-328-3p inhibits malignant progression of hepatocellular carcinoma by regulating MMP-9 level.

This study aims to explore the influence of microRNA-328-3p on proliferative and apoptotic abilities of hepatocellular carcinoma (HCC) cells, and the potential regulatory mechanisms. Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression of microRNA-328-3p in 52 tumor tissue samples and paracancerous ones of HCC patients. The potential interaction between microRNA-328-3p level and clinical indicators of HCC patients was analyzed. Subsequently, the microRNA-328-3p overexpression model was established. In addition, the influence of microRNA-328-3p on the biological functions of the HCC cells was analyzed by the cell counting kit-8 (CCK-8), colony formation assay, and flow cytometry. Finally, the potential downstream gene of microRNA-328-3p was explored by bioinformatics analysis. Recovery experiments were performed to explore the regulation mechanism. QRT-PCR results revealed that microRNA-328-3p level in tumor tissue specimens of HCC patients was remarkably lower than that in adjacent ones, and the difference was statistically significant. Compared with patients with high expression of microRNA-328-3p, those with low expression of miR-328-3p had more advanced pathological staging and lower overall survival. The overexpression of microRNA-328-3p decreased the proliferative capacity and increased apoptotic rate in HCC cells. Subsequently, MMP-9 expression was found to be highly expressed in HCC tissues and cells, and negatively correlated with microRNA-328-3p level. In addition, microRNA-328-3p overexpression significantly down-regulated the protein expressions of CD31, Ki-67, c-Myc, MMP-2, and MMP-9. In the cell reverse experiment, the overexpression of MMP-9 could counteract the influence of the overexpressed microRNA-328-3p on proliferation and apoptosis in HCC cells, so as to regulate the malignant progression of HCC. MicroRNA-328-3p could inhibit the malignant progression of HCC. Its level is remarkably associated with the pathological staging and prognosis of HCC patients. In addition, it is found that microRNA-328-3p might suppress the proliferative ability and promote apoptosis of HCC cells via modulating MMP-9.

CD147 as a key mediator of the spleen inflammatory response in mice after focal cerebral ischemia

BackgroundThe splenic inflammatory response after cerebral ischemia has been implicated in secondary brain injury. We have recently reported that CD147 plays an important role in driving brain inflammation after ischemic stroke. In this study, we hypothesized that CD147 may play a role in the splenic inflammatory response after cerebral ischemia.MethodsTransient (60 min) middle cerebral artery occlusion was induced in wild-type mice treated with an anti-CD147 antibody (αCD147) 1 h before ischemia onset. The splenic inflammatory response was evaluated at 4 and 24 h, representing the peak and early stage of splenic inflammatory activation in this model. Changes in mRNA and protein expression of CD147 and inflammatory markers were measured using RT-qPCR and western blot, respectively. Immune cells in the spleen and brain were measured using flow cytometry.ResultsCD147 expression was rapidly upregulated in the spleen at 4 and 24 h after ischemia onset. The splenic inflammatory response induced by cerebral ischemia was inhibited by αCD147 treatment as demonstrated by the reduced expression of cytokines (TNFα, IL-6, IL-1β) and monocyte chemoattractant protein-1 (MCP-1) in the spleen at 4 and 24 h after ischemia onset. Furthermore, reduced expression of Ly-6C and CCR2 coincided with a decrease in the number of Ly-6Chigh MMs subset in the spleen at 4 h after ischemia onset. This suggests αCD147 treatment abrogates cerebral ischemia-induced inflammatory activation of splenic monocytes/macrophages (MMs). In addition, the experiment in splenectomized mice showed the spleen as the major source of infiltrated Ly-6Chigh MMs subset in the ischemic brain and that brain infiltration of Ly-6Chigh MMs was reduced by αCD147 treatment. These results reveal CD147 as a key mediator of the spleen’s inflammatory activation in response to cerebral ischemia.

CD36 promotes the epithelial\u2013mesenchymal transition and metastasis in cervical cancer by interacting with TGF-\u03b2

BackgroundAccumulating evidence indicates that CD36 initiates metastasis and correlates with an unfavorable prognosis in cancers. However, there are few reports regarding the roles of CD36 in initiation and metastasis of cervical cancer.MethodsUsing immunohistochemistry, we analyzed 133 cervical cancer samples for CD36 protein expression levels, and then investigated the correlation between changes in its expression and clinicopathologic parameters. The effect of CD36 expression on the epithelial–mesenchymal transition (EMT) in cervical cancer cells was evaluated by Western immunoblotting analysis. In vitro invasion and in vivo metastasis assays were also used to evaluate the role of CD36 in cervical cancer metastasis.ResultsIn the present study, we confirmed that CD36 was highly expressed in cervical cancer samples relative to normal cervical tissues. Moreover, overexpression of CD36 promoted invasiveness and metastasis of cervical cancer cells in vitro and in vivo, while CD36 knockdown suppressed proliferation, migration, and invasiveness. We demonstrated that TGF-β treatment attenuated E-cadherin expression and enhanced the expression levels of CD36, vimentin, slug, snail, and twist in si-SiHa, si-HeLa, and C33a–CD36 cells, suggesting that TGF-β synergized with CD36 on EMT via active CD36 expression. We also observed that the expression levels of TGF-β in si-SiHa cells and si-HeLa cells were down-regulated, whereas the expression levels of TGF-β were up-regulated in C33a–CD36 cells. These results imply that CD36 and TGF-β interact with each other to promote the EMT in cervical cancer.ConclusionsOur findings suggest that CD36 is likely to be an effective target for guiding individualized clinical therapy of cervical cancer.

CD147 self-regulates matrix metalloproteinase-2 release in gingival fibroblasts after coculturing with U937 monocytic cells.

Cluster of differentiation 147 (CD147) is a multifunctional glycoprotein that functions as an inducer of matrix metalloproteinase (MMP) expression in fibroblasts. Synergistically enhanced MMP-2 expression was recently observed in the coculture of human gingival fibroblasts (HGFs) and U937 human monocytic cells; however, the responsible mechanisms have not yet been fully established. The aim of this study was to evaluate the release of soluble CD147 in HGFs after coculturing with U937 cells and its functional effect on the enhancement of MMP-2 expression in HGFs. Enzyme-linked immunosorbent assay was used to determine the amount of CD147 protein in media, whereas real-time polymerase chain reaction was performed to evaluate the mRNA levels of CD147 and MMP-2 in HGFs and U937 cells. The enzyme activities of MMP-2 released from cells were examined by zymography. Transwell coculturing and conditioned media treatments were selected to rule out the effect of direct contact of HGFs and U937 cells. The protein and mRNA expression of CD147 in HGFs were enhanced after transwell coculturing with U937 cells and exposure to U937-conditioned medium. MMP-2 enzyme activities in HGFs were also significantly increased by the coculturing methods. Administration of exogenous CD147 enhanced MMP-2 expression in HGFs, whereas treatment with cyclosporine-A, which inhibited CD147 expression, reduced U937-enhanced MMP-2 expression in HGFs. CD147 can interact with fibroblasts to stimulate the expression of MMPs associated with periodontal extracellular matrix degradation. This study has demonstrated that CD147 released from fibroblasts might play a role in monocyte-enhanced MMP-2 expression in HGFs.

Expression of CD47 and CALR in myeloproliferative neoplasms: Potential new therapeutic targets.

48 Background: In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. Increases of both CALR and CD47 on the cell membrane have been observed in response to chemotherapy, however their role in myeloid malignancies is poorly understood. We investigated the expression of CALR and CD47 in patients with essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF). Methods: Mononuclear cells were collected from peripheral blood of 27 MPN patients (8 PV, 16 ET, 3 MF), and from 4 controls. In total 14 patients received cyto-reductive therapies (Hydroxyurea, Anagrelide and Ruxolitinib). Cells were fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol, with CALR and CD47 protein expression analysed by western blotting. Results: Total CALR and CD47 protein expression increased in MPN samples comparing with controls (CALR- 7.9 vs 5.1; CD47- 2.7 vs 2.2 fold). CD47 showed higher expression on MPN cells membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR when patients are treated with cyto-reductive agents (ET- untreated 43.3% vs treated 2%, PV- 3.6% vs 2.2%, ET- 21% vs 11%). Interestingly we have observed a significant increase in CD47 after treatment in MF and PV (CD47 in MF- untreated 11.8% vs treated 34.3%, PV-11.4% vs 35.9%), suggesting an anti-phagocytic effect induced by cytotoxic drugs. In ET however, CD47 expression is reduced after treatment (22% vs 16.6%), suggesting instead a pro-phagocytic effect. Conclusions: CD47, is overexpressed on the membrane of patients with MPN suggesting a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. MPN patients decreased CALR expression during therapy, but we observed a significant difference in CD47 expression in different MPN types. Anti-CD47 antibodies could represent a new strategy to enhance the pro-phagocytic signal via increasing the CALR expression, and in combination with standard cyto-reduction therapy, might represent a new therapeutical strategy in MPN.

TGF-β2/Smad3 Signaling Pathway Activation Through Enhancing VEGF and CD34 Ameliorates Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats.

Evidence has shown the therapeutic potential of isoflurane (ISO) in cerebral stroke. The present study investigated the mechanism of ISO on vascular endothelial growth factor (VEGF) and CD34 expression in a rat model of stroke. Transient focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) for 1h followed by reperfusion for 24h in rats. ISO was administered for 1.5h when the reperfusion was initiated. Neurologic deficit scores, infarct volumes, HE staining, Nissl staining, and TUNEL staining were evaluated at 24h after reperfusion. The levels of transforming growth factor (TGF)-β2, Smad3, p-Smad3, VEGF, and CD34 proteins were detected by immunofluorescence (IF) staining and Western blot assay. Administration of ISO significantly reduced the neurologic deficit scores, infarct volumes, and damaged and apoptotic cells after cerebral ischemia/reperfusion (I/R) injury (P < 0.05). Meanwhile, ISO post-conditioning significantly increased the expression levels of TGF-β2, p-Smad3, VEGF, and CD34 (P < 0.05), whereas the expression of Smad3 showed no difference (P > 0.05). However, Pirfenidone, a TGF-β2 inhibitor, decreased the expression levels of TGF-β2, p-Smad3, VEGF, and CD34 (P < 0.05). Moreover, the protective effects of ISO post-conditioning were negated by the inhibitor. The present study indicated that ISO attenuates brain damage by activating the TGF-β2/Smad3 signaling pathway and increasing the protein expression of VEGF and CD34 in the rat MCAO model.