Low Expressing Cell Lines Research Articles

Long non-coding RNA HOTTIP promotes renal cell carcinoma progression through the regulation of the miR-506 pathway.

HOXA transcript at the distal tip (HOTTIP), a lncRNA, induces cell proliferation and cancer progression. However, the expression and function of HOTTIP in renal cell carcinoma (RCC) were rarely reported. The role of the HOTTIP in RCC was explored in this study. HOTTIP expresses higher in RCC tissues than in normal tissues and indicates poor prognosis based on the TCGA database. The over- and low-expression HOTTIP cell line was established in this research to assess the oncogenic function of HOTTIP in RCC progression. Mechanistic analyses revealed that HOTTIP functioned as a competing endogenous RNA (ceRNA) for miR-506. RIP experiment and luciferase assay were performed to explore the mechanisms of the sponge between HOTTIP and miR-506. HOTTIP down-regulation attenuated cell proliferation, migration, and invasion, which could be rescued by miR-506 down-regulation. On the whole, this study revealed that the HOTTIP/miR-506 axis has a dominant impact on RCC progression and potentially provides a novel strategy for RCC diagnosis and therapy.

DNA Hypermethylation Inhibits the CD82 Metastasis Suppressor Gene in Gastric Cancer.

Gastric cancer, with its high global incidence and mortality rates, poses a significant challenge due to the rapid decline in patient survival upon metastasis. Understanding and combating metastasis are crucial in improving outcomes. The metastasis suppressor gene CD82 has demonstrated efficacy in inhibiting metastasis across various carcinomas but is frequently down-regulated. However, its role and regulatory mechanisms in gastric cancer remain elusive. Utilizing public data, we assessed patient survival in relation to CD82 expression. CD82 expression in gastric cancer cell lines was evaluated via western blotting, and its impact on cell mobility was assessed through wound healing and Transwell assays. The demethylation of CD82 was induced using 5-aza-deoxycytidine, while methylation levels were detected via methylation-specific PCR. Low CD82 expression correlated with poor prognosis in patients, and down-regulation and over-expression of CD82 significantly affected cell mobility. Treatment with 5-aza-deoxycytidine restored CD82 expression in low-expressing cell lines, highlighting its methylation-dependent regulation. CD82 serves as a pivotal regulator of cell mobility in gastric cancer by suppressing metastasis. Its expression is attenuated in gastric cancer cells through promoter hypermethylation.

Construction and Identification of a Human Colorectal Adenoma Epithelial Cell Line by SV40 Large T-Antigen Transfection.

Colorectal adenoma represents the critical step in the development of colorectal cancer. The establishment of an immortalized epithelial cell line of colorectal adenoma of human origin would provide a tool for studying the mechanism of precancerous lesions, screening the efficacy of novel drugs, and constructing in vivo disease models. Currently, there is no commercially available stable supply of epithelial cells from precancerous lesions. This study aimed to establish a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection. Simian vacuolating virus 40(SV40), SV40-LT overexpressed lentivirus vector, was transfected into primary human colorectal adenomatous polyp epithelial cells. The transfected cells were screened, and the screened cells were amplified to obtain the epithelial cell line: IHCRA- CELL. The cells were identified by morphological observation, cell proliferation, Quantitative real-time PCR (qPCR), and Short Tandem Repeats (STR) experiments. Morphologically, the cells showed epithelial-like characteristics, such as polygon shape, desmosomes mitochondria, and strong positive keratin staining. There was no significant difference between the transfected cells and the primary cells. Through the STR identification experiment, no matching cell lines were found in the cell lines retrieval. We successfully established a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection, which has been patented and is now preserved in the Chinese Typical Culture Preservation Center. It was verified that the transformed cells maintained the phenotype and biological characteristics of epithelial cells. This cell line can be used to study the mechanism of precancerous lesions, screen the efficacy of novel drugs, and construct in vivo disease models.

Abstract 5595: Multifaceted role Cornulin in head and neck squamous cell carcinoma: From tumor suppressor pathophysiology to prognosticating biomarker

Abstract Background: In our previous study, based on LC-MS/MS analysis, the salivary Cornulin, a relatively less explored protein, was found to be ~10-fold downregulated in Head and Neck Squamous Cell Carcinoma (HNSCC) patients. However, the exact role of Cornulin in the pathophysiology of HNSCC is not reported. Here, we investigated its role in the pathophysiology of HNSCC and elucidated its potential as a prognostic marker as well as its antitumour effect. Methods: Sandwich ELISA and immunohistochemistry were performed to estimate the salivary and tissue levels of Cornulin, respectively, in HNSCC patients, along with an analysis of the effects of Cornulin levels on disease outcome. To investigate the antitumor effects ,its levels were upregulated through lentiviral transduction in low expressing Cal27, FaDu cell lines. Subsequently, various assays were conducted to examine the antitumor effects of Cornulin upregulation. The molecular signalling pathway was elucidated by analysing transcriptome. Additionally, the effects of increased Cornulin expression were also evaluated in an in vivo nude mice model. Results: The pre-treatment salivary Cornulin levels were significantly low (p=0.0001) in HNSCC patients (n=128,146.4±5.589pg/mL) and in oral premalignant disease patients (n=25, 174.6 ± 9.924 pg/mL) with respect to healthy controls (n=84,185.2±7.170pg/mL). Also, the tumor tissue expression of Cornulin (n=113,H-score=12.70±2.396) was significantly downregulated (p<0.0001) compared to the tumor-free margin (n=72,H-score=139.6±10.34). The patients showing complete clinical response regained normal salivary levels within 6 months of completion of treatment (p<0.0001). More importantly, low salivary Cornulin level at diagnosis was associated with poor overall survival (p=0.0282), indicating it to be a potential prognostic marker. On upregulation of Cornulin in low-expressing HNSCC cell lines (Cal27 and FaDu), the cellular viability, migration, and invasion showed a significant decrease. These indicate its potential as a tumor suppressor. RNAseq data of Cornulin overexpressed cell lines and further Gene set enrichment analysis, the G2/M checkpoint pathway was found to be positively enriched. Cornulin overexpression also arrests the G1/S phase of the cell cycle. GSEA and in vitro experiments revealed Cornulin to be involved in the PI3K/Akt/mTOR signalling pathway. In the nude mice xenograft model, decreased tumor progression, and tumor volume, was documented after Cornulin overexpression. There was an increase in differentiation in the xenograft tumors formed by Cornulin overexpressed cells. Conclusion: Here, we report for the first time that intrinsic Cornulin has a role as a potent antitumor and has potential as a prognostic marker. However the exact molecular mechanism needs to be further evaluated. Citation Format: Rajandeep Kaur, Anshika Chauhan, Sushmita Ghoshal, Jaimanti Bakshi, Radhika Srinivasan, Arnab Pal. Multifaceted role Cornulin in head and neck squamous cell carcinoma: From tumor suppressor pathophysiology to prognosticating biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5595.

Co-culture platform for tuning of cancer receptor density allows for evaluation of bispecific immune cell engagers

Cancer immunotherapy, where a patient’s immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.

CNSC-07. USING CRISPR-CAS9 SCREENING TO IDENTIFY BIOMARKERS FOR MMF SENSITIVITY IN GLIOBLASTOMA MULTIFORME

Abstract Glioblastoma (GBM) the most common malignant brain tumor with a median survival of 21 months. Recurrent GBM remains resistant to available therapies. Our phase I clinical trial investigates combining mycophenolate mofetil (MMF) and temozolomide (TMZ) to target de novo purine biosynthesis in GBM. Previously, our lab revealed that GBM cells upregulate purine biosynthesis and inhibit purine salvage through the ARL13B-IMPDH2 axis during TMZ therapy. Targeting IMPDH2 with MMF/TMZ extended survival in GBM patient-derived xenograft models. In preparation for Phase II of our clinical trial, we aim to identify a predictive biomarker for MMF+ TMZ therapy. In a CRISPR-Cas9-mediated knockout screen, we identified ten genes, including SLC1A5, showing enrichment (differential β score mean values > 60), indicating sensitivity to MMF/TMZ. SLC1A5 displays a wide distribution of expression in Glioviz patient datasets and encodes the glutamine transporter ASCT2, which is crucial for de novo purine biosynthesis. RNA-seq and protein expression analysis in six glioma cell lines confirmed that high SLC1A5 expression appears to correlate with sensitivity to MMF/TMZ (IC50 0.58-21.30 μM), while low expression led to resistance (IC50 73.43-1148 μM). Knockdown of SLC1A5 in a high-expression cell line demonstrated reduced sensitivity to MMF/TMZ (IC50 2.099 vs IC50 1007 μM). To further validate our findings, we plan to perform SLC1A5 overexpression in low-expression glioma cell lines. We hypothesize that GBM patients with higher SLC1A5 expression may respond favorably to MMF/TMZ therapy due to the reliance of their tumors on de novo purine biosynthesis. We aim to combine this data with analysis of our Phase I clinical samples to retrospectively establish SLC1A5 as a biomarker for MMF/TMZ sensitivity and investigate its role in tumorigenic purine metabolism. Identifying predictive markers will enhance personalized treatment strategies for GBM, offering hope for improved outcomes.

Thyroid-stimulating hormone receptor (TSHR) as a target for imaging differentiated thyroid cancer

BackgroundOf the half a million cases of thyroid cancer diagnosed annually, 95% are differentiated thyroid cancers. Although clinical guidelines recommend surgical resection followed by radioactive iodine ablation, loss of sodium–iodine symporter expression causes up to 20% of differentiated thyroid cancers to become radioactive iodine refractory. For patients with radioactive iodine refractory disease, there is an urgent need for new diagnostic and therapeutic approaches. We evaluated the thyroid-stimulating hormone receptor as a potential target for imaging of differentiated thyroid cancer. MethodsWe immunostained tissue microarrays containing 52 Hurthle cell carcinomas to confirm thyroid-stimulating hormone receptor expression. We radiolabeled chelator deferoxamine conjugated to recombinant human thyroid-stimulating hormone analog superagonist TR1402 with 89Zr (t1/2 = 78.4 h, β+ =22.7%) to produce [89Zr]Zr-TR1402. We performed in vitro uptake assays in high–thyroid-stimulating hormone receptor and low–thyroid-stimulating hormone receptor–expressing THJ529T and FTC133 thyroid cancer cell lines. We performed in vivo positron emission tomography/computed tomography and biodistribution studies in male athymic nude mice bearing thyroid-stimulating hormone receptor–positive THJ529T tumors. ResultsImmunohistochemical analysis revealed 62% of patients (27 primary and 5 recurrent) were thyroid-stimulating hormone receptor membranous immunostain positive. In vitro uptake of 1nM [89Zr]Zr-TR1402 was 38 ± 17% bound/mg in thyroid-stimulating hormone receptor–positive THJ529T thyroid cancer cell lines compared to 3.2 ± 0.5 in the low-expressing cell line (P < .01), with a similar difference seen in FTC133 cell lines (P < .0001). In vivo and biodistribution studies showed uptake of [89Zr]Zr-TR1402 in thyroid-stimulating hormone receptor–expressing tumors, with a mean percentage of injected dose/g of 1.9 ± 0.4 at 3 days post-injection. ConclusionOur observation of thyroid-stimulating hormone receptor expression in tissue microarrays and [89Zr]Zr-TR1402 accumulation in thyroid-stimulating hormone receptor–positive thyroid cancer cells and tumors suggests thyroid-stimulating hormone receptor is a promising target for imaging of differentiated thyroid cancer.

Toll-interacting protein may affect doxorubicin resistance in hepatocellular carcinoma cell lines.

Liver cancer is the third leading cause of cancer-related deaths worldwide, and hepatocellular carcinoma (HCC) is the most common type of liver cancer. Transarterial interventions are among the chemotherapeutic approaches used in hardly operable regions prior to transplantation, and in electrochemotherapy, where doxorubicin is used. However, the efficacy of treatment is affected by resistance mechanisms. Previously, we showed that overexpression of the CUE5 gene results in doxorubicin resistance in Saccharomyces cerevisiae (S. cerevisiae). In this study, the effect of Toll-interacting protein (TOLLIP), the human ortholog of CUE5, on doxorubicin resistance was evaluated in HCC cells to identify its possible role in increasing the efficacy of transarterial interventions. The NIH Gene Expression Omnibus (GEO) and Oncomine datasets were analyzed for HCC cell lines with relatively low and high TOLLIP expression, and SNU449 and Hep3B cell lines were chosen, respectively. TOLLIP expression was increased by plasmid transfection and decreased by TOLLIP-siRNA in both cell lines and evaluated by RT-PCR and ELISA. Cell proliferation and viability were examined using xCELLigence and MTT assays after doxorubicin treatment, and growth inhibitory 50 (GI 50) concentrations were evaluated. Doxorubicin GI 50 concentrations decreased approximately 2-folds in both cell lines upon silencing TOLLIP after 48h of drug treatment. Our results showed for the first time that silencing TOLLIP in hepatocellular carcinoma cells may help sensitize these cells to doxorubicin and increase the efficacy of chemotherapeutic regimens where doxorubicin is used.

Kinetic insights into agonist-dependent signalling bias at the pro-inflammatory G-protein coupled receptor GPR84

GPR84 is an orphan G-protein coupled receptor (GPCR) linked to inflammation. Strategies targeting GPR84 to prevent excessive inflammation in disease are hampered by a lack of understanding of its precise functional role. We have developed heterologous cell lines with low GPR84 expression levels that phenocopy the response of primary cells in a label-free cell electrical impedance (CEI) sensing system that measures cell morphology and adhesion. We then investigated the signalling profile and membrane localisation of GPR84 upon treatment with 6-OAU and DL-175, two agonists known to differentially influence immune cell function. When compared to 6-OAU, DL-175 was found to exhibit a delayed impedance response, a delayed and suppressed activation of Akt, which together correlated with an impaired ability to internalise GPR84 from the plasma membrane. The signalling differences were transient and occurred only at early time points in the low expressing cell lines, highlighting the importance of receptor number and kinetic readouts when evaluating signalling bias. Our findings open new ways to understand GPR84 signalling and evaluate the effect of newly developed agonists.

Abstract 3586: In vivo targeted detection and imaging of ovarian cancer by SPMR and MRI using anti-folate receptor functionalized iron oxide nanoparticles

Abstract Ovarian cancer is the second most common and most deadly gynecological cancer in women. Early and accurate detection is crucial in improving survival rate and quality of life of patients. Folate receptor alpha (FRα) is a folate transporter that has been reported to be overexpressed in approximately 90% of ovarian cancer, which makes it a suitable molecular target to use in developing tumor imaging methods. Iron oxide nanoparticles (NPs) have been used in a variety of cancer detection/imaging applications. Here we show that anti-FRα targeted superparamagnetic iron oxide nanoparticles (SPION) can be used for in vivo detection by two different imaging methods. Superparamagnetic Relaxometry (SPMR) is a highly sensitive in vivo detection technology that is able to differentiate the magnetic signature of nanoparticles bound to tumor cells from unbound nanoparticles. Same targeted SPIONs accumulated in tumor are able to provide molecular magnetic resonance imaging (MRI) contrast as well. Our results have shown that the anti-FRα NPs can distinguish high and low FRα expression cell lines, such as KB and A549, respectively in an in vitro cell based assay by SPMR, demonstrating good sensitivity, specificity and selectivity. In vivo study using xenograph model with KB and A549 cells implanted subcutaneously on the flank region of female athymic nude mice demonstrated higher level tumor accumulation of anti-FRα NPs in KB tumor compared to A549 tumor, measurable by ex vivo tumor detection using SPMR. Using a PEG NPs (without the target ligand) as control for the mice study have shown that minimum nanoparticle tumor accumulated were observed, demonstrating the specificity of the nanoparticles in vivo. Organ distribution study indicated that anti-FRα NPs mostly accumulated in liver and spleen as these nanoparticles were cleared through these two organs. Other organs such as brain, heart, lung, kidney didn’t show appreciable accumulation. The repeat of same type of xenograph mice model used in the SPMR study is currently under the way using MRI as an alternative in vivo detection method with images taken pre- and post- dosing of the nanoparticles. Based on the specificity and sufficient accumulation of nanoparticles demonstrated in the SPMR study, it is expected that MRI will show similar performance with additional anatomical information.Our anti-FRα NPs provided targeted and specific delivery to cancerous tissue and generated measurable signal by SPMR. Furthermore, demonstration of these anti-FRα NPs generating tumor specific contrast in MRI is currently under the way. These studies lay the groundwork for use of bio-safe magnetic particles as a detection and contrast agent for early ovarian cancer detection with improved accuracy to minimize the needs of invasive biopsies/surgery as well as potential utility for monitoring therapy response or recurrence. Citation Format: Marie Zhang, Kathirvel Kandasamy, Yamitha Perera, Dan Inglese, Eric Smith-Nguyen. In vivo targeted detection and imaging of ovarian cancer by SPMR and MRI using anti-folate receptor functionalized iron oxide nanoparticles. [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 3586.

Abstract 2983: Tumor-specific CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors

Abstract T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve optimal activation. Solid tumor cells lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling at the T cell/tumor cell interface could enhance anti-tumor activity. We developed a CD28 bispecific platform that allows for the rapid generation of tumor-associated antigen (TAA) x CD28 bispecific antibodies that conditionally provide CD28 costimulation only in the presence of TAA and TCR engagement. Here, we present results for a set of bispecific antibodies with broad applicability across a range of solid tumors, including CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28. We developed optimized CD28 scFv and Fabs that cover a range of affinities and are only agonistic in the context of TCR engagement. A matrix of bispecifics pairing these CD28 binders with CEACAM5, Trop-2, STEAP1, and mesothelin antibodies of varying affinities and epitopes were generated using Xencor’s XmAb® bispecific antibody platform. Activities of these bispecifics were assessed in vitro by measuring T cell proliferation, cytokine production, and cytotoxicity in co-cultures of human cancer cell lines mixed with primary human T cells. CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28 bispecifics enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity only in concert with TCR engagement. IHC was used to compare TAA expression on cancer cell lines versus that found on cancer tissues, allowing categorization of cell lines as high density (tumor surrogate) or low density (normal tissue surrogate). CD28 bispecifics were identified with selective potency on high versus low expressing cell lines, suggesting a favorable therapeutic index. CD28 bispecific antibodies co-targeting CEACAM5, Trop-2, STEAP1, and mesothelin show promising activity and warrant further development across a range of solid tumors. Citation Format: Matthew A. Dragovich, Viralkumar Davra, Matthew S. Faber, Yoon Kyung Kim, Alex Nisthal, Veronica G. Zeng, Jonathan Jacinto, Juan E. Diaz, Thuy Truong, Jing Qi, Kendra N. Avery, Rumana Rashid, Sung-Hyung Lee, Seung Y. Chu, Christine Bonzon, Ruschelle Love, Matthew J. Bernett, James A. Ernst, Rena Bahjat, Norman J. Barlow, John R. Desjarlais, Michael Hedvat, Gregory L. Moore. Tumor-specific CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors [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 2983.

Abstract 2145: High SLFN11 expression correlates with sensitivity to lurbinectedin in small cell lung cancer (SCLC) models

Abstract Background: Lurbinectedin monotherapy received accelerated FDA approval for adults with metastatic SCLC with disease progression on or after platinum-based chemotherapy. Lurbinectedin binds to GC-rich areas of gene promoters, blocks RNA-polymerase-II and induces degradation via ubiquitin/proteasome machinery. DNA binding of lurbinectedin results in double stranded DNA breaks and DNA damage that leads to cellular apoptosis. Schlafen 11 (SLFN11), a putative DNA/RNA helicase irreversibly binds to DNA replication forks resulting in replication block and is a predictive biomarker of response to therapeutics that elicit DNA damage including cisplatin, topoisomerase I/II inhibitors and PARP inhibitors. Objective: Evaluate the role of SLFN11 expression in predicting response to lurbinectedin, a DNA damaging agent, in human SCLC cell lines and in vivo models. Methods: Cytotoxicity assays: SCLC cell lines DMS 53, DMS 114, NCI-H69, NCI-H82, NCI-H196, NCI-H209, NCI-H211, NCI-H446, NCI-H526, NCI-H841, NCI-H889, NCI-H1048, and SHP-77 were tested with lurbinectedin dose range from 100 nM to 0.01 nM. Cell viability was determined by measuring intracellular ATP to determine percent cellular viability and IC50 of lurbinectedin. SLFN11 expression of 13 SCLC cell lines were retrieved as RNAseq values from the DepMap database. RNAseq value &amp;gt;2 was assigned as SLFN11 high, values &amp;lt;2 assigned as low. Western blot analysis was used to confirm protein expression. The difference in 10log IC50 between SLFN11 high and low lines is determined using Mann-Whitney test. In vivo efficacy studies: High SLFN11 (NCI-H1048) and low SLFN11 (NCI-H889 and NCI-H69) SCLC cell lines were used for xenograft studies in BALB/c nude mice. Mice were randomly allocated to 4 groups (n=8) as tumors reached a volume of 100 - 150 mm3. Groups received either vehicle or lurbinectedin at doses of 0.06, 0.12 or 0.18 mg/kg IV, QWx 3 weeks. Immunohistochemistry was used to assess SLFN11 protein expression on xenograft tissue. Results and Conclusions: Cell viability assays in 13 SCLC models confirm high SLFN11 expressing cell lines are 4-fold more sensitive to lurbinectedin compared to low SLFN11 expressing cell lines (ΔpIC50 = 0.64; p = 0.0451). The in vivo efficacy data confirms that the SLFN11 high NCI-H1048 model is more responsive with 90% TGI compared to 35% observed in SLFN11 low models (NCI-H889 and NCI-H69) at the highest lurbinectedin dose (p-value = 0.006). Efficacy data confirms correlation to SLFN11 protein expression, consistent with the RNA level association. Both in vitro cytotoxicity and in vivo efficacy studies in SCLC models confirm that high SLFN11 expression predicts response to lurbinectedin. Cell viability studies demonstrate that a limited set of SLFN11 low expressing cell lines are sensitive to lurbinectedin, suggesting a role for other genes. Proteomics and genomics work is underway to define additional response biomarkers Citation Format: Aparna Gupta, Kedar S. Vaidya, Janneke J.T.M. Melis, Robert Hauptschein, Graham Brock, Robin Humphreys. High SLFN11 expression correlates with sensitivity to lurbinectedin in small cell lung cancer (SCLC) models [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 2145.

Abstract 6008: Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors

Abstract Immune checkpoint inhibitors (ICIs) have generated durable clinical responses in multiple tumor types, but their therapeutic efficacy is limited by development of primary or acquired resistance. The tumor microenvironment (TME) contains many factors influencing ICI responses. The ratio of effector T-cells (Teffs) and regulatory T-cells (Tregs) in tumors has been shown to be associated with the efficacy of ICIs in murine models, indicating that increasing Teffs or decreasing Tregs in tumor may prevent development of resistance to immunotherapy. There is a high and restricted expression of CC motif chemokine receptor 8 (CCR8) on tumor-infiltrating Tregs, whereas they are negligibly expressed on Teffs. Deletion of tumor-infiltrating Tregs via murine anti-CCR8 surrogates has shown effective anti-tumor activity in tumor models.LM-108 is a novel humanized anti-CCR8 monoclonal antibody with engineered IgG1 that enhances antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of CCR8 and Foxp3 on tumor-infiltrating lymphocytes (TILs) was analyzed using published single-cell sequencing data. The binding affinity of LM-108 was assessed by flow cytometry using CCR8 high- and low-expressing cell lines. In vitro measurements of ADCC and antibody-dependent cellular phagocytosis (ADCP) mediated by LM-108 were conducted in CCR8 overexpressing HEK293, CHO-K1, and Jurkat cells. Furthermore, in vivo anti-tumor activity of LM-108 as monotherapy and in combination with anti-PD-1 therapy was evaluated in syngeneic models and PD-1-resistant tumor models with human CCR8 knock-in mice. Analysis of published single-cell sequencing data showed that in comparison with CCR4, CCR8 gene expression was specifically overlapped with Foxp3 in TILs of breast cancer. Fluorescence-activated cell sorting (FACS) assay revealed that LM-108 specifically binds with high affinity to human CCR8 in a dose-dependent manner. ADCC and ADCP effects induced by LM-108 were observed in HEK293 and CHO-K1 cells with different levels of CCR8 expression. In vivo data of CT26 syngeneic model showed that LM-108 murine surrogate antibody alone or in combination with anti-PD-1 antibody significantly inhibited tumor growth. Treatment with LM-108 effectively depleted tumor-infiltrating Tregs accompanied by increase in CD8/Treg ratio in tumors, leading to significant anti-tumor activity in MC38 model. Finally, LM-108 showed a synergistic therapeutic efficacy in PD-1 resistant models in combination with anti-PD-1 antibody. To conclude, LM-108 is a novel Fc-optimized CCR8 antibody that selectively depletes tumor infiltrating Tregs thereby improving anti-tumor immune response either as monotherapy or combination therapy. Thus, LM-108 can be a promising therapeutic approach to overcome ICI resistance in cancer patients. Citation Format: Jie Luo, Wentao Huang, Junwei Yang, Jin Li, Yifan Li, Da Fei, Xia Qin, Runsheng Li. Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6008.

Abstract 621: ZL-1211 exhibits robust anti-tumor activity by enhancing ADCC and activating innate and adaptive immunity in CLDN18.2-high and -low expressing gastric cancer models

Abstract CLDN18.2 (Claudin18.2)-targeting therapeutic antibodies have shown promising clinical efficacy in ~30% of gastric cancers expressing high levels of CLDN18.2 and less pronounced activity in the majority of low expressing malignancies. ZL-1211 is a monoclonal antibody targeting CLDN18.2 engineered to promote enhanced ADCC with the goal of achieving more potent activity in a wider spectrum of high- and low-CLDN18.2 expressing tumors than the leading clinical benchmark. To further assess the potency of ZL-1211, we utilized a panel of gastric tumor cell lines that endogenously express CLDN18.2 and more accurately reflect target levels in human gastric tumors than engineered overexpressing models. ZL-1211 demonstrated more robust in vitro ADCC activity than clinical benchmark not only in CLDN18.2-high but also -low expressing gastric cell lines. Greater anti-tumor efficacy was also observed in mouse xenograft models with CLDN18.2-high, -medium, and low-expressing gastric cell lines in response to treatment with ZL-1211 compared to the clinical benchmark. NK cell depletion abrogated ZL-1211-mediated ADCC activity in vitro. ZL-1211 efficacy in vivo was also dependent on the presence of an NK compartment, as CLDN18.2-expressing gastric tumors grown in Balb/c nude with an intact NK function were sensitive to ZL-1211 treatment, while NOD-SCID with partially competent NK activity exhibited minimal response, and NCG without NK cells did not display any evidence of in vivo efficacy. Strikingly, NK cells strongly induced an inflammatory response in response to ZL-1211 treatment, including increased IFNγ, TNFα, and IL-6 production. Interestingly, ZL-1211 treatment led to NK cell and monocyte stimulation and enhanced CD8+ T cell activation, suggesting a mechanism of action that not only involves innate but also adaptive immunity. Taken together, our data suggest that ZL-1211 more effectively targets CLDN18.2-high gastric cancers as well as -low expressing malignancies that are not eligible for treatment with the leading clinical benchmark by inducing a robust ADCC response and activating innate and adaptive immunity to enhance anti-tumor efficacy. Clinical activity of ZL-1211 is currently under evaluation in a Phase I clinical trial (NCT05065710). Citation Format: Hiroyasu Konno, Tracey Lin, Bee-Chun Sun, Renyi Wu, Christopher Szeto, David Bellovin, Karl Hsu, Omar Kabbarah. ZL-1211 exhibits robust anti-tumor activity by enhancing ADCC and activating innate and adaptive immunity in CLDN18.2-high and -low expressing gastric cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 621.

Abstract 4263: A novel pegylated biparatopic antibody-drug conjugate (pb-adc) targeting cancers with low HER2+ expression

Abstract ADCs have demonstrated much improved target selectivity and efficacy comparing to the nonspecific cytotoxic small molecule drug. The goal of ADC technology is to resolve the toxicity issue inherent in potent small molecule drugs. Yet, the main problem facing current ADC technology is still a toxicity issue, evidenced by 9 out of 12 FDA approved ADCs carrying black box warning labels and many failed clinical programs due to their toxicities. Many factors contribute to the ADC’s toxicity including but not limited to random conjugation of payloads to the antibody, premature release of payload, Fc related toxicity, on-target/off-tumor toxicity etc. In this study, we developed a novel pegylated BsAb-ADC formed by site-specific conjugating a fusion protein of two scFvs that target at two different epitopes of Her2, with PEG-MMAE, a pegylated cytotoxic payload MMAE. As expected, the compound is homogeneous without any Fc related toxicity. Although the compound showed lower affinity to Her2 positive cells, it demonstrated higher in vitro cytotoxicity than T-DM1, especially for Her2 low expressing cell lines when its DAR is 4. Furthermore, the compound was comparable to or better than T-DM1 or DS-8201 in inhibiting xenograft tumor models (including Her2+ low expressing models) even when DAR is only 2. The compound also demonstrated efficient internalization without detectable efflux, suggesting improved endosome and lysosome trafficking, most probably due to the lack of Fc promoted efflux mechanism and formation of complex between biparatopic Her2 antibody and Her2. In addition, unlike T-DM1 and DS-8201, the compound did not generate cytotoxicity to megakaryocyte cell line Dami, and is less toxic to the non-cancerous HaCaT cells than T-DM1 or DS-8201. The compound is stable with no detectable release of the payload when incubating with human Plasma for a week at 37°C. The circulation T1/2 of the compound in rat is about 30 hours. In conclusion, our pegylated BsAb-ADC compound demonstrated improved efficacy with better safety profile than T-DM1 and DS-8201. Citation Format: Shumin Liu, Shuqiang Yin, Weidong Lyu, Yang Lei, Qiudong Zhuo, Zibin Wu, Shuangyu Tan, Liling Zheng, Yu (Yvonne) Wen, David Wu. A novel pegylated biparatopic antibody-drug conjugate (pb-adc) targeting cancers with low HER2+ expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4263.

Forkhead Box q1 promotes invasion and metastasis in colorectal cancer by activating the epidermal growth factor receptor pathway

BACKGROUNDColorectal cancer (CRC) is an extremely malignant tumor with a high mortality rate. Little is known about the mechanism by which forkhead Box q1 (FOXQ1) causes CRC invasion and metastasis through the epidermal growth factor receptor (EGFR) pathway.AIMTo illuminate the mechanism by which FOXQ1 promotes the invasion and metastasis of CRC by activating the heparin binding epidermal growth factor (HB-EGF)/EGFR pathway.METHODSWe investigated the differential expression and prognosis of FOXQ1 and HB-EGF in CRC using the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/index.html). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of FOXQ1 and HB-EGF in cell lines and tissues, and we constructed a stable low-expressing FOXQ1 cell line and verified it with the above method. The expression changes of membrane-bound HB-EGF (proHB-EGF) and soluble HB-EGF (sHB-EGF) in the low-expressing FOXQ1 cell line were detected by flow cytometry and ELISA. Western blotting was used to detect changes in the expression levels of HB-EGF and EGFR pathway-related downstream genes when exogenous recombinant human HB-EGF was added to FOXQ1 knockdown cells. Proliferation experiments, transwell migration experiments, and scratch experiments were carried out to determine the mechanism by which FOXQ1 activates the EGFR signaling pathway through HB-EGF, and then to evaluate the clinical relevance of FOXQ1 and HB-EGF.RESULTSGEPIA showed that the expression of FOXQ1 in CRC tissues was relatively high and was related to a lower overall survival rate. PCR array results showed that FOXQ1 is related to the HB-EGF and EGFR pathways. Knockdown of FOXQ1 suppressed the expression of HB-EGF, and led to a decrease in EGFR and its downstream genes AKT, RAF, KRAS expression levels. After knockdown of FOXQ1 in CRC cell lines, cell proliferation, migration and invasion were attenuated. Adding HB-EGF restored the migration and invasion ability of CRC, but not the cell proliferation ability. Kaplan–Meier survival analysis results showed that the combination of FOXQ1 and HB-EGF may serve to predict CRC survival.CONCLUSIONBased on these collective data, we propose that FOXQ1 promotes the invasion and metastasis of CRC via the HB-EGF/EGFR pathway.

Accurate Isolation of Circulating Tumor Cells via a Heterovalent DNA Framework Recognition Element-Functionalized Microfluidic Chip.

Detection of circulating tumor cells (CTCs) has provided a noninvasive and efficient approach for early diagnosis, treatment, and prognosis of cancer. However, efficient capture of CTCs in the clinical environment is very challenging because of the extremely rare and heterogeneous expression of CTCs. Herein, we fabricated a multimarker microfluidic chip for the enrichment of heterogeneous CTCs from peripheral blood samples of breast cancer patients. The multimarker aptamer cocktail DNA nanostructures (TP-multimarker) were modified on a deterministic lateral displacement (DLD)-patterned microfluidic chip to enhance the capture efficiency through the size selection effect of DLD arrays and the synergistic effect of multivalent aptamers. As compared to a monovalent aptamer-modified chip, the multimarker chip exhibits enhanced capture efficiency toward both high and low epithelial cell adhesion molecule expression cell lines, and the DNA nanostructure-functionalized chip enables the accurate capture of different phenotypes of CTCs. In addition, the DNA nanoscaffold makes nucleases more accessible to the aptamers to release cells with molecular integrity and outstanding cell viability.

Study on Forsythin promoting apoptosis of laryngeal carcinoma cells by regulating miRNA-1469

Objective The paper aimed to explore the mechanism of Forsythin regulating miRNA expression in laryngeal carcinoma cells, and to clarify the molecular biological mechanism of Forsythin regulating miRNA to promote the apoptosis of laryngeal carcinoma cells, providing theoretical and experimental basis for clinical application of Forsythin as an anti-laryngeal cancer treatment drug. Methods: A miR-1469 low-expression laryngeal carcinoma cell line was established. Western blot and flow cytometry were applied to detect the effect of Forsythin on cell apoptosis. Western blot was employed to detect the effects of Forsythin on P53 protein, P53 low expression, and P53 overexpression in laryngeal carcinoma cells, as well as the effects on overexpression of miRNA-1469, and on double low expression of P53 and Mcl1. Real-time PCR method was used to detect the effect of miR-1469 on p53 low expression in laryngeal carcinoma cells. Results: Flow cytometry detection of cell apoptosis showed that, after the cells with low miR-1469 expression were treated with Forsythin, the apoptosis rate was significantly reduced. Western blot detection showed that, compared with the Control group, the expression level of miR-1469 was significantly reduced after Forsythin administration in Hep2 cells with low expression of P53. Compared with the idle Control group, the apoptosis level of laryngeal carcinoma cells in Hep2 cells with low expression of P53 was significantly reduced. In Hep2 cells transfected with P53 overexpression plasmids, apoptosis level of laryngeal carcinoma cells increased. Compared with the idle Control group, the apoptosis level of laryngeal carcinoma cells in the single-transformed P53 shRNA group decreased, while the apoptosis level of the double-transformed miR-1469 mimic+P53 shRNA group increased again. After drug treatment, the apoptosis level of the single-transformed P53 shRNA group decreased, while the apoptosis level of the double-transformed Mcl1 shRNA+P53 shRNA group increased again. Conclusion: Forsythin can promote the apoptosis of laryngeal carcinoma cells by up-regulating the expression of miR-1469 and then down-regulating the expression of Mcl1. The drug can up-regulate the expression of miR-1469 by elevating the expression of P53. miR-1469 can promote the apoptosis of laryngeal carcinoma cells by inhibiting the expression of its downstream target gene Mcl1.

High-purity Isolation for Genotyping Rare Cancer Cells from Blood Using a Microfluidic Chip Cell Sorter

A multistep sorting method for enrichment of rare cells, such as circulating tumor cells, in the blood without cumbersome pretreatments required by most flow cytometry-based methods, which lead to high cost and decreased detection efficiency, was developed. After only hemolysis and cell staining, cancer cells are enriched by repetitive sorting (3×) based on nuclear-positive, cytokeratin-positive, and CD45-negative expression. Experiments using spikes of PC-9 cells showed a mean recovery of 65% and mean purity of 83%, which was retained up to 72 hours after blood draw using preservative tubes. Significant differences in expression level of programmed death-ligand 1 or vimentin were observed between high- and low-expressing cell lines, concurrently with enrichment. Next-generation sequencing analysis of recovered PC-9, A549, and MDA-MB231 cells successfully detected all known mutations. This novel isolation method applicable for preserved samples with sufficient recovery and purity may be substantially beneficial for recovering cells for subsequent molecular analysis.

Targeting DDX3X Helicase Activity with BA103 Shows Promising Therapeutic Effects in Preclinical Glioblastoma Models.

Simple SummaryIn the last ten years, the human helicase protein DDX3X turned out to be an extremely interesting target for the development of potential anticancer drugs. Herein, we discovered BA103, a novel specific inhibitor of the helicase binding site of DDX3X, which is characterized by broad-spectrum anticancer activity. BA103 revealed promising tolerability in fibroblasts and good pharmacokinetic properties. Furthermore, BA103 was able to decrease the expression of β-catenin and to reduce tumor migration. Its capability to pass the blood–brain barrier led us to investigate its potential against glioblastoma, which is a high refractory disease with poor prognosis. High efficacy was proven in both xenograft and orthotopic animal models.DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.