Tumor Cell Types Research Articles

MBD2 facilitates tumor metastasis by mitigating DDB2 expression

Despite past extensive studies, the pathoetiologies underlying tumor metastasis remain poorly understood, which renders its treatment largely unsuccessful. The methyl-CpG-binding domain 2 (MBD2), a “reader” to interpret DNA methylome-encoded information, has been noted to be involved in the development of certain types of tumors, while its exact impact on tumor metastasis remains elusive. Herein we demonstrated that patients with LUAD metastasis were highly correlated with enhanced MBD2 expression. Therefore, knockdown of MBD2 significantly attenuated the migration and invasion of LUAD cells (A549 and H1975 cell lines) coupled with attenuated epithelial–mesenchymal transition (EMT). Moreover, similar results were observed in other types of tumor cells (B16F10). Mechanistically, MBD2 selectively bound to the methylated CpG DNA within the DDB2 promoter, by which MBD2 repressed DDB2 expression to promote tumor metastasis. As a result, administration of MBD2 siRNA-loaded liposomes remarkably suppressed EMT along with attenuated tumor metastasis in the B16F10 tumor-bearing mice. Collectively, our study indicates that MBD2 could be a promising prognostic marker for tumor metastasis, while administration of MBD2 siRNA-loaded liposomes could be a viable therapeutic approach against tumor metastasis in clinical settings.

Immune Infiltration in Tumor and Adjacent Non-Neoplastic Regions Codetermines Patient Clinical Outcomes in Early-Stage Lung Cancer

IntroductionIn recent years, the proportion of patients with NSCLC diagnosed at an early stage has increased continuously. MethodsIn this study, we analyzed samples and data collected from 119 samples from 67 early stage patients with NSCLC, including 52 pairs of tumor and adjacent non-neoplastic samples, and performed RNA-sequencing analysis with high sequencing depth. ResultsWe found that immune-related genes were highly enriched among the differentially expressed genes and observed significantly higher inferred immune infiltration levels in adjacent non-neoplastic samples than in tumor samples. In survival analysis, the infiltration of certain immune cell types in tumor, but not adjacent non-neoplastic, samples were associated with overall patient survival, and excitingly, the differential infiltration between paired samples (tumor minus non-neoplastic) was more prognostic than expression in either non-neoplastic or tumor tissues. We also performed B cell receptor (BCR) and T cell receptor (TCR) repertoire analysis and observed more BCR/TCR clonotypes and increased BCR clonality in tumor than in non-neoplastic samples. Finally, we carefully quantified the fraction of the five histologic subtypes in our adenocarcinoma samples and found that higher histologic pattern complexity was associated with higher immune infiltration and low TCR clonality in the tumor-proximal regions. ConclusionsOur results indicated significantly differential immune characteristics between tumor and adjacent non-neoplastic samples and suggested that the two regions provided complementary prognostic values in early-stage NSCLCs.

Analyzing the immunological variability in T cell/tumor interactions to identify a subpopulation of CD8+ T cells (Spark T cells) that drives succesful cancer immunotherapy

Abstract Thousands of patients have benefited from the growing use of cancer immunotherapies. However, the success of these therapies can be highly variable. Strikingly, some murine tumor models show large variability in the outcome of cancer immunotherapies, even when the mice, tumor cells and anti-tumor immune cells injected into mice are all genetically identical. Here, we sought to analyze this variability in adoptive cell therapies, in order to identify the immune population driving this variability. To search for the occurrence of large variability ex vivo, we extracted mouse TCR-transgenic CD8+ T cells, and co-cultured them with antigen-expressing tumor cells. By using multiplexed in vitro assays and single-cell analysis of thousands of samples, we identified conditions (e.g. cell numbers, antigen quality, tumor cell types) where large variations in the immune activation against cancer cells is observed even between technical replicates. We then developed a quantitative framework that uses statistical modeling and machine learning to extract useful information from this immunotherapeutic variability. Our framework allowed the prediction and identification of a rare population of naïve CD8+ T cells (“Spark T cells”) that is necessary and sufficient to spark massive anti-tumor immune reactions. We are currently performing experiments to test the efficacy and functional significance of the identified immune population in vivo. We are also applying this framework in human TCR-engineered T cell blasts to identify the equivalent of the Spark T cells in humans. We envision this framework being applied to identify other relevant immune cell types that act as catalysts for successful cancer immunotherapies. Supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute

Abstract A020: CETSA profiling unveils novel targets engaged by anti-tumor drug rigosertib to inhibit RAS-MAPK signaling and trigger NLRP3 inflammasome activation

Abstract Background: The importance of styryl benzyl sulfones that induce G2/M cell cycle arrest and apoptosis in tumor cells has raised interest in cancer therapeutics. Rigosertib (ON 01910.Na) has been characterized as a disruptor of multiple signaling pathways including RAS-MAPK signaling through multiple mechanisms. The aim of our study was to further elucidate the mechanism of action and to identify potential novel targets engaged by rigosertib. Methods: Thermal shift, NanoBiT, in vitro tubulin polymerization and a variety of biochemical, molecular and cell biological assays were performed to characterize the binding of rigosertib to previously proposed targets and its effect on RAS-MAPK signaling and tumor cell survival. A mass spectrometry-based Cellular Thermal Shift Assay (CETSA-MS) that serves as a key tool to identify the targets in the entire proteome engaged by chemical ligands or small molecules of interest at physiological levels in cells, was conducted in multiple cell types to identify potential new protein targets of rigosertib. Results: Our data suggest that rigosertib exerts its inhibitory effect on RAS-MAPK signaling through ROS induced activation of JNK signaling. While we confirm that rigosertib might affect microtubules at higher concentrations, the main cellular target(s) responsible for the induction of stress and JNK-mediated inhibition of RAS-RAF-MEK signaling needs to be further characterized. By performing comparative CETSA profiling of rigosertib and colchicine, a classical anti mitotic drug that binds to soluble tubulin, we are able to identify unique targets of rigosertib in multiple tumor cell types. In particular, we identify two ROS-related proteins, ERO1A and NQO2 that potentially contribute to the induction of ROS-dependent JNK activation. NEK7, a relevant protein for microtubule organization and essential for NLRP3 inflammasome activation, is another potential target for rigosertib. Indeed, rigosertib activates NLRP3-dependent inflammatory responses via NEK7 and Caspase-1 to trigger IL-1β secretion. Conclusion: These results suggest that rigosertib may represent an effective compound for inhibition of RAS-MAPK signaling through ROS-mediated JNK activation. Our findings reveal a rigosertib-dependent mechanism for NLRP3 activation that might be the cause of rigosertib synergistic effect with immune checkpoint blockers to induce cell death in advanced KRAS-mutated non-small cell lung cancer patients. This MOA suggests rigosertib can modulate the mutated RAS pathway no matter the mutation present, now confirmed with clinical data. Further clinical trials in a number of RAS mutated cancers are continuing. Citation Format: Petros Kechagioglou, Camille Dupont, Hajime Yurugi, Alexey Chernobrovkin, Kristina Riegel, Stephen Cosenza, Steven M. Fruchtman, Krishnaraj Rajalingam. CETSA profiling unveils novel targets engaged by anti-tumor drug rigosertib to inhibit RAS-MAPK signaling and trigger NLRP3 inflammasome activation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A020.

T-ALL Cells as Tool Cells for CAR T Therapy.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy derived from T cells. Numerous CAR T therapies have been successfully applied to treat hematologic malignancies in the clinic. Nevertheless, there remain several challenges to the extensive application of CAR T cell therapy in T cell malignancies, especially in T-ALL. The main reason for CAR T therapy limitations is that T-ALL cells and normal T cells share antigens, which improves the difficulty of sorting pure T cells, resulting in product contamination, and would lead to CAR T cell fratricide. Thus, we considered creating a CAR on T-ALL tumor cells (CAR T-ALL) to prevent fratricide and eliminate tumor cells. We found that T-ALL cells transduced with CAR would actually commit fratricide. However, CAR T-ALL could kill only tumor cells on T-ALL cell lines, and other types of tumor cells had no killing function after being transferred with CAR. Furthermore, we created CD99 CAR with expression controlled by the Tet-On system on Jurkat cells, which could avoid the fratricide of CAR T-ALL during proliferation, ensuring the controllability of the killing time and effect. Jurkat transduced with a CAR-targeting antigen, which was expressed on other cancer cells, could kill other cancer cell lines, demonstrating that T-ALL cells could be used as tool cells for cancer therapy. Our study supplied a new feasible treatment regimen for cancer treatment in the clinic.

Abstract 6615: Novel digital pathology tools for exploring role of myeloid cells in modulating next generation immunotherapies

Abstract Purpose: Deeper understanding of the immune landscape in tumor microenvironments is critical for creation of next generation immunotherapies (I-O). While significant investments were made to understand the role of T-cells, the contribution of myeloid cells in limiting the effectiveness of I-O therapies is not well understood primarily due to limitations posed by conventional immunohistochemistry (IHC) analysis that typically provides information on a single target. We developed a clinical grade multiplex fluorescence IHC (mFIHC) assay to simultaneously evaluate different subsets of myeloid cells (e.g., M1, M2 and MDSC) as well as PD-L1 receptors associated with these subsets as the latter is known to have profound positive impact on patients treated with PD-1 blockers (Liu et al., CCR, 2019). In addition, we developed an algorithm to identify the microenvironment using imaging datasets from AQUA® (Automated Quantitative Analysis) and HALO to understand the spatial relationship between tumor cells and myeloid cell subsets as well as immune checkpoint (IC) expression. mFIHC combined with hypothesis driven spatial profiling algorithms (e.g., AQUA Technology) were found to provide superior predictors of I-O therapies in a systematic meta-analysis of over 8000 patients treated with PD1/L1 pathway blockers (Lu et al., JAMA Oncol 2019). Furthermore, spatial profiling by AQUA has proven to robustly identify powerful biomarker predictors (PD1/PDL1) in melanoma (Johnson et al., CCR, 2018). Implementation of mFIHC coupled with robust image analysis may provide great insight into immune surveillance, mechanisms of resistance and patient stratification. Study Design: We designed a novel mFIHC assay (CD68, CD163, HLA-DR, IDO-1, PD-L1, PanMel-CK) and evaluated various myeloid and tumor cell types on tissue sections (e.g., non-small cell lung cancer, gastric carcinoma, breast carcinoma, and renal cell carcinoma). Following the initial evaluation, the mFIHC assay was validated for clinical trial application using automated staining (Leica Bond RX) and imaging (PhenoImager HT). We conducted image analysis using AQUA and HALO technologies. Results: Analysis of samples by AQUA and HALO revealed unique myeloid microenvironments in the examined tissue types. Microenvironment data from AQUA and HALO were assessed by analyzing enrichment of macrophage phenotypes in areas dense with checkpoint markers. Our analysis revealed unique myeloid cell clusters in the assessed tumor microenvironments. Conclusion: We anticipate this hypothesis driven clinical trial grade mFIHC assay design combined with novel analyses algorithms will aid in the better understanding of the immunomodulatory effects of myeloid cells in several cancer types and the development of next generation I-O therapies. Citation Format: Richard Van Krieken, Lisa Adams, Beiru Chen, Emmanuel Pacia, Naveen Dakappagari, Brian Bourke-Martin, Jennifer Bordeaux. Novel digital pathology tools for exploring role of myeloid cells in modulating next generation immunotherapies [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 6615.

Abstract 3678: Rewiring of amino acid metabolism in neurofibromatosis type 1-related tumors

Abstract Neurofibromatosis type 1 (NF1) is a tumor-predisposing genetic disorder caused by heterozygous mutations in the NF1 gene encoding for the tumor suppressor Neurofibromin. Biallelic NF1 inactivation in Schwann cells leads to the onset of neurofibromas, benign neoplasms that can cause severe medical complications and give rise to malignant peripheral nerve sheath tumors (MPNSTs), for which no treatment exists. Neurofibroma cells undergo bioenergetic changes required to sustain their malignant transformation. Our group has shown that inhibition of the mitochondrial chaperone TRAP1, a master metabolic regulator, hampers MPNST cell tumorigenicity. A growing number of evidence indicates that urea cycle (UC) dysregulation can redirect metabolic substrates to specific biosynthetic routes in many tumor cell types. We have found that a complete UC is absent in MPNST cell models, as they lack ornithine transcarbamylase (OTC), a key UC enzyme that converts ornithine into citrulline. In the absence of TRAP1, MPNST cells strongly reduce arginine levels and accumulate ornithine through the activity of the mitochondrial enzymes arginase 2 (ARG2) and ornithine aminotransferase (OAT). These changes can reverberate on glutamine and proline levels ultimately affecting extracellular matrix deposition, motility and ROS homeostasis, as well as on ornithine-dependent polyamine biosynthesis that can regulate cell growth and proliferation. Surprisingly, in the absence of TRAP1 MPNST cells also accumulate citrulline, even though they do not express either OTC or any nitric oxide synthase (NOS) isoform that converts arginine into citrulline. We ascribe citrulline production to the activity of dimethylarginine dimethylaminohydrolase (DDAH) enzymes, involved in the catabolism of asymmetric dimethylarginines. Induction of DDAHs indicates an elevated protein catabolism that could contribute to tune MPNST cell tumorigenicity. A thorough comprehension of the amino acid metabolism rewiring in NF1-related tumors may unveil new targetable liabilities of cancer cells to be exploited for the design of new effective therapeutic approaches. Citation Format: Martina La Spina. Rewiring of amino acid metabolism in neurofibromatosis type 1-related 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 3678.

Abstract 464: AAC-11 survival pathways as therapeutic target in cancer: AAC-11 leucine-zipper domain derived peptides exert potent antitumor effects and exhibit favorable stability, pharmacokinetic and toxicology profiles

Abstract Background: Due to their basal stress phenotype associated to transformation, cancer cells are addicted to non-mutated, non-oncogenic proteins that do not bear such vital functions in normal cells, a phenomenon referred as Non-Oncogenic Addiction (NOA). Targeting these NOAs could therefore induce selective killing of cancer cells, opening several therapeutic opportunities. Recent data suggest that the stress-related scaffold protein AAC-11 (anti-apoptosis clone 11, also known as Api5) is critically involved in cancer cells resistance to chemotherapies, metastatic potential and escape from the immune system. Methods: We have developed inactivating peptides based on the fusion of a cell penetrating sequence and portions of the leucine-zipper domain of AAC-11, which functions as a protein-protein interaction module. These peptides induce cancer cells death, through the inhibition of protein-protein interactions between AAC-11 and its partners, while sparing normal cells. We now describe further characterization of our lead peptide, JRT39, which contains residues 377 to 379 of AAC-11 linked to the cell-penetrating peptide “penetratin”. Results: In vitro, JRT39 causes cell death in a wide spectrum of cancer cell lines with IC50 ranging from 5 µM to 30 µM depending on tumor cell type. In particular, JRT39 showed selective efficacy towards primary cells from Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL) or Sézary syndrome (SS) patients, while sparing normal hematopoietic cells, with an IC50 of 5-15 µM. Mechanistically, JRT39 induces membranolysis of cancer cells through binding to p21-activated kinase 1 (PAK1) in AML or SS cells plasma membrane, where PAK1 is overexpressed. In addition, JRT39 exerted potent anti-tumor activity in vivo in disseminated or subcutaneous AML, APL (Acute Promyelocytic Leukemia) and SS-patient derived preclinical murine models. Preliminary pharmacokinetic studies revealed that JRT39 is stable in human serum and has a plasma half-life of ~1.5-2.5 hours after intravenous (IV) administration to dogs or cynomolgus monkeys, with concentration-time data fitting 2-compartment model. Finally, JRT39 was well tolerated at 5 and 10 mg/kg after single or repeated (daily) IV injections. Conclusions: Combined, our preclinical data confirm that interfering with AAC-11-related survival pathways is a promising novel anticancer strategy and support the development of JRT39 for the treatment of cancer. Citation Format: Louise Jeammet, Emile Adicéam, Justine Habault, Anna Kaci, Jeannig Berrou, Mélanie Dupont, Nicolas Thonnart, Ewa Pasquereau-Kotula, Anne Marie-Cardine, Armand Bensussan, Marika Pla, Hervé Dombret, Claude Gardin, Martine Bagot, Jean-Christophe Rain, Hélène Sicard, Jérôme Tiollier, Thorsten Braun, Jean-Luc Poyet. AAC-11 survival pathways as therapeutic target in cancer: AAC-11 leucine-zipper domain derived peptides exert potent antitumor effects and exhibit favorable stability, pharmacokinetic and toxicology profiles [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 464.

Abstract 2229: Cell state transitions shape the intratumoral composition of small cell lung carcinoma

Abstract Introduction: Small cell lung carcinoma (SCLC) is characterized by rapid growth, early metastases, and initial response followed by almost invariable resistance to therapy. Studies to date have not determined the extent that diverse transcriptional programs drive SCLC and contribute to its lethality. We sought to characterize the intra-tumoral transcriptional heterogeneity of SCLC. We identify multivalent, distinct, and commutable transcriptional states that confer discrete functions in individual SCLC tumors. Methods: We developed a biorepository of patient-derived xenografts (PDX) (n = 64) and matched PDX-derived ex vivo lines. We used multi-omic profiling (RNAseq, scRNAseq, and ATAC seq), single-cell fluorescence tracking of fate-defining transcription factor (TF)-driven states, and mathematical and statistical models (Markov chain) to study the topology of the SCLC transcriptional landscape and its plasticity. Human tumor material and associated clinical data were obtained after informed written consent on an IRB-approved prospective registry. Results: We show that individual SCLC tumors are more heterogenous than previously appreciated, displaying distinctive equilibria in the proportion of cells within well-delimited cellular states (ASCL1, NEUROD1 and YAP1). We also show that transcriptional states undergo transitions, which we identified as a mechanism for maintaining cell state diversity. We measured the kinetics of state transitions using single-cell fluorescence tracking of ex vivo cultures and found that these measure were associated significantly with transition estimates using stochastic transition theory (i.e. Markov chains). ATAC-seq profiling indicated a role for the epigenome in the state diversity of SCLC. Namely, there was preferential promoter accessibility to Ascl1, NeuroD1, and Yap1 in a manner consistent with gene and protein expression in the respective subpopulations. Our results indicate that the transition rates between cell types in individual tumors were largely governed by tendencies to reach an equilibrium state that are critical for configuring intratumoral cell state proportions. Conclusion: In conclusion, we demonstrate that TF driven cell states can transition to maintain an equilibrium in cell state proportions. Our work advances a model of cellular states and program diversity in SCLC and nominates new therapeutic strategies designed to limit the plasticity of this lethal cancer. Citation Format: Priyanka Gopal, Aaron Petty, Kevin Rogacki, Titas Bera, Rohan Bareja, Craig Peacock, Mohamed Abazeed. Cell state transitions shape the intratumoral composition of small cell lung carcinoma [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 2229.

Abstract 5162: Discovery of INCB098377: a potent inhibitor of phosphoinositide 3-kinase gamma (PI3Kγ)

Abstract Immune checkpoint blockade has shown impressive efficacy in patients with inflamed tumors, although minimal activity has been observed in tumors lacking T cells. Myeloid cells are one of the most abundant cell types in both inflamed and non-inflamed tumors, and may contribute to immune checkpoint blockade resistance. The plasticity of macrophages enables them to directly and indirectly modulate T cell responses, and directly kill tumor cells via phagocytosis. This suggests that targeting myeloid cells could be an effective therapeutic approach. Class I PI3Ks are a family of dual specificity lipid and protein kinases. Unlike other class I PI3Ks, PI3Kγ is predominantly expressed in myeloid cells. PI3Kγ has been shown to be a key mediator that drives the immunosuppressive macrophage program by stimulating AKT/mTOR signaling and promote C/EBPβ expression while inhibiting NF-кB activity (Keneda MM. Nature. 2016;17:437-442). Here, we present the discovery and characterization of INCB098377, a potent and selective PI3Kγ inhibitor. Specific inhibition of PI3Kγ with INCB098377 may induce anti-tumor activity by reshaping the tumor immune microenvironment. In cell-based assays, INCB098377 has an IC50 of 1.4 nM and is greater than 100-fold selective over other PI3K isoforms. It also shows a favorable PK profile in several animal species. Treatment of M2 polarized macrophages with INCB098377 resulted in changes towards a more pro-inflammatory phenotype. CD163 and CD206 were decreased, whereas HLA-DR and co-stimulatory CD80/86 molecules were increased. MHC-I expression was unchanged, suggesting a role for these macrophages in MHC-II-mediated antigen presentation. Furthermore, INCB098377 treatment reduced macrophage-mediated immunosuppression and restored T cell proliferation in M2 polarized macrophages co-cultured with allogeneic human T cells. In vivo, significant tumor growth inhibition was observed with once-daily dosing of 10 mg/kg INCB098377 in both syngeneic and humanized mouse tumor models without toxicity. Moreover, efficacy was observed in inflamed and non-inflamed tumor models. Consistent with the proposed mechanism of action, INCB098377 inhibited phospho-AKT levels in vivo and in human PBMCs. Treatment with INCB098377 induced pro-inflammatory responses without macrophage depletion suggests that robust tumor microenvironment changes are responsible for observed anti-tumor efficacy. In addition, INCB098377 inhibited neutrophil migration in the Carrageenan-induced paw inflammation model. INCB098377, a potent and selective inhibitor of PI3Kγ, shows effective anti-tumor activity in a variety of mouse and humanized cancer models through the inhibition of immunosuppressive cells trafficking into the tumor, modulation of myeloid cell function, and enhancement of T cell proliferation. Acknowledgments: Diana Alvarez Arias and Stephen Douglass contributed equally to this study. Citation Format: Diana A. Arias, Stephen Douglass, Lisa Truong, Qian Wang, Kathy H. Wang, Gengjie Yang, Michael Hansbury, Sybil O’Connor, Kevin Bowman, Robert Collins, Matthew Stubbs, Leslie Hall, Christina Stevens, Christopher Maddage, Brent Douty, Maryanne Covington, Lynn Leffet, Eddy Yue, Andrew Combs, Sunkyu Kim, Niu Shin, Holly Koblish, Rodrigo Hess. Discovery of INCB098377: a potent inhibitor of phosphoinositide 3-kinase gamma (PI3Kγ). [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 5162.

Abstract 4527: CD8 T cell-melanoma cell interactions in response and resistance to ipilimumab plus nivolumab: Biopsy analysis of SWOG S1616

Abstract Background: In the randomized S1616 trial (NCT03033576), patients with metastatic melanoma refractory to anti-PD-1-based therapy had improved progression free survival (PFS, HR = 0.63, p = 0.037) and objective response to ipilimumab plus nivolumab (ipi/nivo, RR 28%) compared to ipilimumab alone (ipi, RR = 9%). We hypothesized that reversal of resistance to PD-1 blockade with CTLA-4 blockade would result in increased CD8 T-cell infiltration in patient biopsies. Methods: Multiplex ion beam imaging (MIBI) for 32 protein markers, spanning tumor, immune, and stromal cell types, was used to evaluate the tumor microenvironment in melanoma tumor biopsies collected at baseline (N=21 samples) and one month on-treatment (N=22; N=16 paired timepoints). Tumor-immune cell dynamics were determined by comparing baseline and on-treatment biopsies within clinical groups (ipi/nivo response, N=10; ipi/nivo no-response, N=9; ipi no-response, N=8). Results: Multiplex analysis in ipi/nivo responsive patients demonstrated increased proportions of CD8 (median 1.6X increase), CD4 (2.1X), regulatory T cells (1.6X), and monocytes (1.4X), with a paralleled 8.1X decrease in melanoma cells over the course of treatment. This was confirmed by histopathologic evidence of tumor regression, necrosis, and immune infiltrate in on-treatment, responding tumor biopsies, as determined by dermatopathologists. Melanoma cells had direct, cell-membrane interactions with CD8 T cells in ipi/nivo responsive patients, both at baseline (median 26%) and on-treatment (median 86%). The shared interface between melanoma and CD8 T-cells had polarized expression of CD3, CD8, and CD45RO. There was no change in T-cell infiltration nor tumor cell content over the course of treatment in ipi/nivo non-responders nor ipi non-responders; similar proportions of melanoma cells were interacting with CD8 T-cells at baseline (median 24%) and on-treatment (27%). Conclusion: Reversal of resistance to PD-1 blockade is associated with increased frequency of CD8 T-melanoma cell interactions and pathological response, reflective of different stages of antitumor immune responses. Response to ipi/nivo in the anti-PD-1 refractory setting was further associated with increased tumor-polarized and activated CD8 T cells interacting with melanoma cells, demonstrated by the colocalized expression of immune synapse proteins at tumor-CD8-T-cell membrane interfaces. Citation Format: Katie M. Campbell, Lawrence Kuklinski, Zaid Bustami, Jessica Maxey, Egmidio Medina, Sandra Santulli-Marotto, Cynthia R. Gonzalez, Nataly Naser Aldeen, Kari Kendra, Sapna Patel, Siwen Hu-Lieskovan, James Moon, Shay Bellasea, Christine N. Spencer, Marshall A. Thompson, Michael Wu, Ari Vanderwalde, Philip O. Scumpia, Antoni Ribas. CD8 T cell-melanoma cell interactions in response and resistance to ipilimumab plus nivolumab: Biopsy analysis of SWOG S1616. [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 4527.

Abstract 5418: AI-driven image analysis enables simplified, label-free cytotoxicity screening

Abstract The increasing use of precious, patient-derived cells has driven the need for non-perturbing and label-free cell measurements, particularly in the oncology field. To address this we developed the Incucyte® AI Cell Health Analysis Software Module, which uses two pre-trained deep neural networks to perform automated, unbiased analysis of Phase contrast images to segment individual cells and perform label-free Live/Dead cell classification. The neural networks which perform cell instance segmentation and infer cell viability were trained on a wide diversity of cell types with varied morphologies, ensuring that the analysis is applicable across a variety of adherent and suspension tumor cell types. Here, we demonstrate the application of this analysis across diverse and commonly used biological models of breast cancer, glioblastoma, and B-cell lymphoma. In each case, cells were treated with chemotherapeutic compounds or biosimilar antibodies and Phase contrast images were acquired at regular intervals over 3 - 4 days using the Incucyte® Live-Cell Analysis System. Using the Incucyte® AI Cell Health Analysis cells were accurately segmented and the percentage of dead cells were quantified over time without the requirement for a fluorescent reporter or other exogenous label, and with limited user input. Four breast cancer cell lines were treated with a panel of chemotherapeutics designed to target specific expression patterns. AI Cell Health analysis showed that Estrogen receptor (ER) inhibitor Tamoxifen selectively induced >60% cell death only in ER positive cell lines BT474 and MCF7; dual epidermal growth factor receptor (EGFR/HER2) inhibitor Lapatinib induced cell death in AU565, BT474 and MCF7 which express these surface markers. In contrast, Lapatinib and Tamoxifen induced morphological change - but minimal cell death - in triple negative MDA-MB-231 cells. Three glioblastoma cell lines A172, U87 and T98G were treated with a larger panel of chemotherapeutic compounds and for four of the active compounds, efficacy was also determined. Cisplatin, doxorubicin, vinblastine and taxol induced concentration-dependent cell death in A172 and T98G cells; U87 cells displayed resistance to each of these compounds with a maximal 46.5% cell death induced by doxorubicin. Ramos B-cell lymphoma cells were exposed to increasing concentrations of monoclonal antibody Rituximab and the biosimilar Truxima®. The antibodies induced specific cell death via the surface marker CD20 in a time and concentration-dependent manner with similar efficacy (IC50 Rituximab 94.7 ng/mL; Truxima® 110.3 ng/mL), while antibody control IgG1 remained non-perturbing to cells. These results demonstrate that the Incucyte® AI Cell Health Analysis is applicable to a broad range of cancer types cultured in 2D monolayer. This unbiased method enables accurate, label-free quantification of cytotoxic effects induced by clinically relevant therapeutics. Citation Format: Gillian Lovell, Daniel A. Porto, Jasmine Trigg, Nevine Holtz, Nicola Bevan, Timothy Dale, Daniel Appledorn. AI-driven image analysis enables simplified, label-free cytotoxicity screening. [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 5418.

Distinct cellular composition between normal surgical margins and tumor tissues in oral squamous cell carcinoma

Adequate resection of normal surgical margins is important. However, the clear distinction between the normal surgical margins and tumor tissues is still difficult. Here, this study analyzed the variety of cell types in tumors and the normal surgical margins using a computational approach. The composition of cell types was compared between the two tissues by statistical and machine learning approaches. The results showed the distinct cellular composition between tumor-adjacent and tumor tissues. In particular, endothelial cells were highly represented and macrophages were underrepresented at the normal surgical margin. Moreover, the normal surgical margin and tumor tissues could be discriminated using a machine learning algorithm. The results will help to understand cellular differences between normal surgical margins and tumor tissues and to provide potentials for tumor detection and treatment.

Diagnostic value and biological significance of CD71 in lymphoid disorders

Abstract Background The CD71 expression is tightly regulated by intracellular iron level, cell proliferation, or erythropoiesis at levels of receptor recycling, as well as transcriptional or posttranscriptional control. In the previous studies, CD71 was expressed by most of acute leukemia and chronic granulocytic leukemia in blast crisis with a variable density of expression. Only few studies have observed CD71 as a diagnostic and prognostic marker in cases of acute lymphocytic leukemia (ALL) as a type of acute leukemia. Therefore, our study aimed to evaluate the diagnostic and biological significance of CD71 in lymphoid disorders. Aim and objectives This study aimed to evaluate the diagnostic value and biological significance of CD71 in lymphoid disorders. Patients and methods The present research was carried out on 60 patients diagnosed with lymphoid disorders (benign lymphocytosis, ALL, CLL, lymphoma, and multiple myeloma) who presented to Assiut University Hospital from July 2020 till July 2021. Results CD71 expression was significantly higher in acute disorders (immature cell-type tumors of malignant lymphoid disorders) than in benign lymphocytosis and chronic disorders (immature cell-type tumors of malignant lymphoid disorders), with strong relation with the positivity of CD34 and TdT. Conclusion CD71 helps to differentiate between lymphoid disorders and further helps in discriminating the differentiation status of ALL. High expression of CD71 might reflect a much more aggressive and immature cell type. However, further prospective investigations are needed to document the possible relation of CD71 with response to treatment and relapse rate. Moreover, high expression of CD71 with clonal evolution in leukemia may predict the possibility of being an important molecular target in future therapy of acute leukemia.

High-Performance Detection of Exosomes Based on Synergistic Amplification of Amino-Functionalized Fe3O4 Nanoparticles and Two-Dimensional MXene Nanosheets

Exosomes derived from cancer cells have been recognized as a promising biomarker for minimally invasive liquid biopsy. Herein, a novel sandwich-type biosensor was fabricated for highly sensitive detection of exosomes. Amino-functionalized Fe3O4 nanoparticles were synthesized as a sensing interface with a large surface area and rapid enrichment capacity, while two-dimensional MXene nanosheets were used as signal amplifiers with excellent electrical properties. Specifically, CD63 aptamer attached Fe3O4 nanoprobes capture the target exosomes. MXene nanosheets modified with epithelial cell adhesion molecule (EpCAM) aptamer were tethered on the electrode surface to enhance the quantification of exosomes captured with the detection of remaining protein sites. With such a design, the proposed biosensor showed a wide linear range from 102 particles μL-1 to 107 particles μL-1 for sensing 4T1 exosomes, with a low detection limit of 43 particles μL-1. In addition, this sensing platform can determine four different tumor cell types (4T1, Hela, HepG2, and A549) using surface proteins corresponding to aptamers 1 and 2 (CD63 and EpCAM) and showcases good specificity in serum samples. These preliminary results demonstrate the feasibility of establishing a sensitive, accurate, and inexpensive electrochemical sensor for detecting exosome concentrations and species. Moreover, they provide a significant reference for exosome applications in clinical settings, such as liquid biopsy and early cancer diagnosis.

FAK/IL-8 axis promotes the proliferation and migration of gastric cancer cells.

Gastric cancer (GC) is one of the most common malignancies in China and is associated with high mortality. The occurrence and development of gastric cancer are related to genetic and environmental factors. Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor protein tyrosine kinase that is activated by the extracellular matrix and growth factors. FAK is highly expressed in cancer and promotes its development by regulating cancer cell proliferation, migration, and angiogenesis. The expression of IL-8 is increased in many types of malignant tumor cells and is linked to their proliferation, migration, invasion, angiogenesis, and EMT. In this study, we found FAK to be essential for the proliferation, migration, and peritoneal metastasis of gastric cancer cells. To examine the molecular regulatory mechanisms of FAK in the peritoneal dissemination of gastric cancer, we performed RNA-seq analysis of MKN-45-FAK-/- and MKN45 cells and demonstrated that IL-8 was downregulated in FAK-deficient cells. Conversely, we confirmed that IL-8 activates FAK activity. We established that IL-8 promotes the proliferation, colony formation, andmigration of gastric cancer cells that are partially mediated by FAK. Thus, we propose that an IL-8-FAK-IL-8 positive feedback loop effects the proliferation and migration of gastric cancer cells.

A Comparative Study of Cancer Cells Susceptibility to Silver Nanoparticles Produced by Electron Beam.

Silver nanoparticles (AgNPs) have a wide range of bioactivity, which is highly dependent on particle size, shape, stabilizer, and production method. Here, we present the results of studies of AgNPs cytotoxic properties obtained by irradiation treatment of silver nitrate solution and various stabilizers by accelerating electron beam in a liquid medium. The results of studies of morphological characteristics of silver nanoparticles were obtained by transmission electron microscopy, UV-vis spectroscopy, and dynamic light scattering measurements. MTT test, alamar blue test, flow cytometry, and fluorescence microscopy were used to study the anti-cancer properties. As biological objects for standard tests, adhesive and suspension cell cultures of normal and tumor origin, including prostate cancer, ovarian cancer, breast cancer, colon cancer, neuroblastoma, and leukemia, were studied. The results showed that the silver nanoparticles obtained by irradiation with polyvinylpyrrolidone and collagen hydrolysate are stable in solutions. Samples with different stabilizers were characterized by a wide average size distribution from 2 to 50 nm and low zeta potential from -7.3 to +12.4 mV. All AgNPs formulations showed a dose-dependent cytotoxic effect on tumor cells. It has been established that the particles obtained with the combination of polyvinylpyrrolidone/collagen hydrolysate have a relatively more pronounced cytotoxic effect in comparison to samples stabilized with only collagen or only polyvinylpyrrolidone. The minimum inhibitory concentrations for nanoparticles were less than 1 μg/mL for various types of tumor cells. It was found that neuroblastoma (SH-SY5Y) is the most susceptible, and ovarian cancer (SKOV-3) is the most resistant to the action of silver nanoparticles. The activity of the AgNPs formulation prepared with a mixture of PVP and PH studied in this work was higher that activity of other AgNPs formulations reported in the literature by about 50 times. The results indicate that the AgNPs formulations synthesized with an electron beam and stabilized with polyvinylpyrrolidone and protein hydrolysate deserve deep study for their further use in selective cancer treatment without harming healthy cells in the patient organism.

Single-cell profiling of the copy-number heterogeneity in colorectal cancer.

With functionally heterogeneous cells, tumors comprise a complex ecosystem to promote tumor adaptability and evolution under strong selective pressure from the given microenvironment. Diversifying tumor cells or intra-tumor heterogeneity is essential for tumor growth, invasion, and immune evasion. However, no reliable method to classify tumor cell subtypes is yet available. In this study, we introduced the single-cell sequencing combined with copy number characteristics to identify the types of tumor cells in microsatellite stable (MSS) colorectal cancer (CRC). To characterize the somatic copy number alteration (SCNA) of MSS CRC in a single cell profile, we analyzed 26 tissue samples from 19 Korean patients (GSE132465, the Samsung Medical Center [SMC] dataset) and then verified our findings with 15 tissue samples from five Belgian patients (GSE144735, the Katholieke Universiteit Leuven 3 [KUL3] dataset). The Cancer Genome Atlas (TCGA) cohort, GSE39582 cohort, and National Cancer Center (NCC) cohort (24 MSS CRC patients were enrolled in this study between March 2017 and October 2017) were used to validate the clinical features of prognostic signatures. We employed single cell RNA-sequencing data to identify three types of tumor cells in MSS CRC by their SCNA characteristics. Among these three types of tumor cells, C1 and C3 had a higher SCNA burden; C1 had significant chromosome 13 and 20 amplification, whereas C3 was the polar opposite of C1, which exhibited deletion in chromosome 13 and 20. The three types of tumor cells exhibited various functions in the tumor microenvironment and harbored different mutations. C1 and C2 were linked to the immune response and hypoxia, respectively, while C3 was critical for cell adhesion activity and tumor angiogenesis. Additionally, one gene (OLFM4) is identified as epithelium-specific biomarker of better prognosis of CRC (TCGA cohort: P = 0.0110; GSE39582 cohort: P = 0.0098; NCC cohort: P = 0.0360). On the basis of copy number characteristics, we illustrated tumor heterogeneity in MSS CRC and identified three types of tumor cells with distinct roles in tumor microenvironment. By understanding heterogeneity in the intricate tumor microenvironment, we gained an insight into the mechanisms of tumor evolution, which may support the development of therapeutic strategies.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) as an undetermined tool in tumor cells.

In the tumor microenvironment, the function of T cells is a fate-changer for tumor progression. In the meantime, CD28 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are vital role players in the controlling activity of T cells as an activator and deactivator, respectively. In T cells in comparison to CD28, the molecular mechanism of CTLA-4 is unclear. In addition, despite the fact that most tumor cell types express CTLA-4, its role in tumor cells is not well understood and only few studies focused on the role of CTLA-4 signaling in tumor cells. It is illustrated that CTLA-4 signaling causes PD-L1 expression in tumor cells. However, numerous characteristics of CTLA-4 signaling in tumor cells are ambiguous and require to be described. In this article, we proposed that the CTLA-4 signaling during immunotherapy with anti-CTLA-4 antibodies may cause poor responses by patients. In addition, we attract attention to several fundamental questions regarding CTLA-4 signaling in tumor cells. Overall, the CTLA-4 signaling function and the related gaps about its role in tumor cells in the present review are challenged.

Spatial analysis of the metastatic brain tumor immune and extracellular matrix microenvironment

Metastatic cancer is responsible for the overwhelming majority of cancer-related deaths, with metastatic tumors being the most common neoplasms affecting the central nervous system. One of the major factors regulating tumor biology is the tumor microenvironment. However, little is known about the cellular and non-cellular composition of metastatic brain tumors and how tumor cell ontogeny influences the metastatic brain tumor microenvironment. By integrating multiplex immunohistochemistry and histopathological analysis to investigate composition and the spatial relationship between neoplastic cells, infiltrating and brain resident immune cells and the extracellular matrix, we demonstrate that metastatic brain tumors exhibit differences in extracellular matrix deposition, compared with the most common primary brain tumor type, glioblastoma, and that the dominant immune cell types in metastatic brain tumors are immunosuppressive macrophages, which preferentially localize to extracellular matrix-rich stromal regions.