Cell-like Phenotype Research Articles

Clinical and Biological Effects of Combined CD27 and CD20 Antibody Therapy in Relapsed/Refractory B-Cell Lymphoma: The Riva Trial

Clinical and Biological Effects of Combined CD27 and CD20 Antibody Therapy in Relapsed/Refractory B-Cell Lymphoma: The Riva Trial

Targeting Bruton Tyrosine Kinase for the Treatment of Vitreoretinal Lymphoma: Report of 11 Consecutive Patients

Background:Vitreoretinal lymphoma (VRL) is a rare intraocular malignancy, which is regarded as a part of the central nervous system lymphoma (CNSL) and shows a close relationship with CNS progression. Currently, no standard treatment approaches have been defined yet, although intravitreal methotrexate with or without systemic chemotherapy are generally used. Bruton tyrosine kinase (BTK) is a vital effector molecule in the progress of B-cell proliferation. VRL, as well as PCNSL, display typically an activated B cell-like (ABC) phenotype of diffuse large B-cell lymphoma (DLBCL), with frequent CD79B and MYD88 mutations, which may represent a strong biological rationale for the use of BTK inhibitors. Previous studies have demonstrated that BTK inhibitors could penetrate the blood-brain barrier, and achieve 70-90% response rate in patients with PCNSL. However, whether BTK inhibitors could penetrate the blood-eye barrier and provide benefits to patients with VRL remains unknown.Methods:In this retrospective study, 11 consecutive patients with VRL were treated with BTK inhibitors monotherapy (zanubrutinib 160mg orally twice daily, or orelabrutinib 150mg orally daily), and they were intended to be treated for 2 years, or till disease progression, or unacceptable toxicity, whichever occurs first. All enrolled patients were pathologically confirmed as vitreoretinal DLBCL using vitreous biopsy, and PET-CT scan were performed to rule out the systemic involvement of lymphoma. Patients with pretreated or concurrent CNSL were allowed in this study. Cytokine analysis (including IL-10 and IL-6) in the anterior chamber of the eye was done using Cytometric Bead Array (CBA) at one month after initiation of treatment, and every three months thereafter or as symptom indicated. Fundus photograph and optical coherence tomography (OCT) were done at each follow-up. Magnetic resonance imaging (MRI) of brain was done every three months. This study was approved by the Ethic Committee of Beijing Tongren Hospital and written informed consent was provided by all patients.Results:11 consecutive patients with VRL were treated since May 2020, among whom 5 patients were enrolled in our prospective study evaluating the efficacy and safety of zanubrutinib in the treatment of VRL (ChiCTR2000037921), and the other 6 patients were treated with off-label use of BTK inhibitors, and those 11 patients were analyzed combined in this report. The female to male ratio was 7:4, with a median of 61 years old (41-73). 3 patients had pretreated PCNSL and were in complete remission at the time of VRL relapse, and one patient (YSX2) had concurrent PCNSL and VRL. All the other 7 patients had isolated VRL, among whom 3 patients were diagnosed as de novo VRL and the other 4 patients were previously treated with repeated intravitreal methotrexate and had disease relapse at the time of BTK inhibitor treatment. The detailed efficacy information was shown in Figure 1. All but one patients achieved complete remission (CR) after one months of BTK inhibitor treatment, with symptoms resolved, disappearance of infiltration lesions in OCT, and IL-10 levels returned to normal. One patient (YSX2) suffered from disease progression in the brain and withdrew from the study after 2 months of treatment, while the eye symptoms were significantly improved. At a median follow up of 7.5 months (4-15), 4 patients were confirmed to have disease progression, with a progression-free survival (PFS) of 2, 7, 7.5, and 9 months, respectively. All the other 7 patients have durable CR. 4 patients treated with off-label use of zanubrutinib discontinued BTKi due to economic reasons, and remained in CR till the last follow-up. One patient (YSX1) stopped BTK inhibitor after 2 months, and suffered from disease relapse 5.5 months later without any other treatments. BTK inhibitors were well-tolerated, with grade 1 ecchymosis in 3/11 patients, grade 3 hypertension in 1/11 patients, grade 2 arthralgia in 1/11 patients, and grade 1 liver dysfunction in 2/11 patients. No patients stopped the drug due to adverse events, and one patient reduced the dosage due to uncontrolled hypertension. All patients were alive at the time of this report.Conclusions:In conclusion, targeting BTK in VRL is feasible and our findings can be a foundation for a paradigm shift in treatment options for this rare disease. A well-designed prospective study in a larger cohort of patients is needed to validate our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:USE OF ZANUBRUTINIB FOR THE TREATMENT OF DIFFUSE LARGE B CELL LYMPHOMA

Stemness Signature Predicts Outcomes and Identifies Candidates for Targeted Therapy in Diffuse Large B-Cell Lymphoma

Background: Mounting studies have shown that tumor cells have stem cell-like phenotypes called stemness. The stemness on prognosis and the immunological properties of diffuse large B-cell lymphoma (DLBCL) has not been systematically explored. This study aimed to explore the relationship between stemness, prognosis and immune microenvironment and further to identified potential therapeutic target related to stemness.Methods: The gene expression and clinical information of 1398 diffuse large B-cell lymphoma sample were obtained to calculated stemness based on the single-sample gene set enrichment analysis (ssGSEA). Survival analysis was performed for stemness phenotypes and the association of stemness with clinical features was assessed further. Weighted gene co-expression network analysis (WGCNA) to screen the relevant key genes systematically for stemness of DLBCL. Cell cycle assay and apoptosis assay were used to evaluate the role of cell division cycle 7 (CDC7) inhibitors in DLBCL cell lines.Results: Stemness was significantly associated with overall survival (OS) and progress free survival (PFS) of DLBCL patients. Notably, prolonged OS and PFS was observed in low stemness groups compared to high group, and the international prognostic index (IPI) with a high risk had higher stemness. Furthermore, DLBCL stemness were associated with tumor purity, immune score and stroma score. From stemness module, 16 genes in the co-expression were chosen as key genes and further identified CDC7 as candidate target gene for antitumor. CDC7 inhibitors involving in dequalinium chloride which was approved by Food and Drug Administration (FDA) for anti-microbial and simurosertib which in a phase I clinical trial for multiple solid tumors show antitumor in DLBCL cell line of SU-DHL-2 and SU-DHL-10.Conclusion: In a word, the study integrated DLBCL stemness, clinical characteristics and immunophenotyping, providing an idea of targeting for cold tumors of DLBCL, and also verified the efficacy of inhibitors of key gene of stemness CDC7 in DLBCL. It is significant to develop novel therapeutic targets for diffuse large B cell lymphoma.Figure Legends:Figure (A-B) An overview of the association between known clinical and molecular features (IPI, Hams, gender, and stage) and stemness index in DLBCL cohort. Columns represent samples sorted by stemness index from low to high (top row). Rows represent known clinical and molecular. (C-D) Overall survival and progression free survival of stemness index in DLBCL patients of GSE117556 by Kaplan-Meier analysis. (E-F) Overall survival and progression free survival of stemness index in DLBCL patients of GSE31312 by Kaplan-Meier analysis. (G-I) Boxplots of tumor purity, immune score, stromal score in individual samples stratified by stemness index. (J) WGCNA analysis of differentially expressed genes in DLBCL. Different colors correspond to related modules. (K) Correlation coefficient between the modules and clinical traits with stemness. P-values are listed in the modules. (L-N) Scatter plot analysis of different modules in the MEblue, MEbrown, and MEturquoise modules. (O) Signor analysis of 16 key genes in DisNor. (P) K-M curves of the expression of CDC7 for the DLBCL patients. (Q) Boxplots of the expression of CDC7 in individual samples stratified by Hams. (R) Apoptosis ratio of SU-DHL-10 cells were treated with dequalinium chloride and simurosertib for 48 h. Data are mean ± SD of triplicate experiments. For dequalinium chloride and simurosertib compared with control an unpairedt-test was used. (S) Cell cycle of SU-DHL-10 cells were treated with dequalinium chloride and simurosertib for 48 h. Data are mean ± SD of triplicate experiments. For dequalinium chloride and simurosertib compared with control an unpairedt-test was used. (T) Cell viability of SU-DHL-2 and SU-DHL-10 cells treated with dequalinium chloride and simurosertib at the IC50 concentrations. * p<0.05. ** p<0.01, *** p<0.001. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HDACi Targets IKZF1 Deletion High-Risk Acute Lymphoblastic Leukemia By Inducing IKZF1 Expression and Rescuing IKZF1 Function in Vitro and In Vivo

The IKZF1 gene codes the transcription factor IKAROS with key regulatory functions in lymphopoiesis. Recurrent focal IKZF1 deletions (IKZF1 del), which affects the coding regions of IKZF1, have been identified as poor outcome in 30-40% adult B-ALL. Loss of IKAROS confer stem cell-like phenotype, upregulation of self-renewal capacity and cell-adhesion molecules, and TKI resistance. However, there are not specifically therapeutic options for IKZF1 del ALL and current protocols could not abrogate the adverse effect of IKZF1 del.Considering that IKAROS acts as a key component of the nucleosome remodeling and deacetylation (NuRD) complex engaging in development and metabolism, we speculate that epigenetic drugs, such as HDACi, might play a potent role in IKZF1 del high-risk B-ALL.Firstly, B-ALL cell lines (IKZF1 del: MUTZ-5, MHH-CALL-4; IKZF1 wt: NALM6) and primary patient samples (n=10, 5 with IKZF1 del and 5 with IKZF1 wt) were treated with different HDACi, including valproic acid, vorinostat, romidepsin, RGFP966 and a novel HDAC-selective inhibitor tucidinostat. But noteworthily, only tucidinostat yielded specific and selective proliferation inhibition in IKZF1 del cell line(IC 50=1.377±0.05) and IKZF1 del patients samples (IC 50=2.318±0.07), compared with the effect on IKZF1 wt cells. Interestingly, tucidinostat induced remarked increase of mRNA and protein of IKZF1 expression in leukemia bulk and IKZF1 del single cell. Seahorse metabolic flux assay, lactate and ATP measurements was performed and revealed that tucidinostat treatment reduced glycolysis (P=0.0067), lactic acid (P＜0.0001) and ATP level (P＜0.0001) in IKZF1 del B-ALL cell lines. To verify metabolic change is depend on IKZF1 induction or not,dominant-negative Ikaros isoform 6 (DN-IK6), deletion of exons 4-7, was transfected into IKZF1 wt Nalm-6 cell line to negative regulate of IKZF1 wide-type expression. Overexpression of DN-IK6 in Nalm-6, increases sensitivity to tucidinostat, glycolytic capacity(p=0.05) and glycolytic reserve (p=0.012) also increases. While tucidinostat treating with the IK6-Nalm-6, tucidinostat would restore the transcriptional repressor function of the remaining wild-type IKZF1 allele and decrease glycolytic capacity(p=0.011) and glycolytic reserve(p=0.014). Notably, the metabolic rate-limiting enzymes HK2 and PKM2 were strongly repressed. These data indicate that tucidinostat reverses the metabolic reprogramming of glycolysis or Warburg effect in IKZF1 del B-ALL in an IKZF1-inducing dependent manner. For in vivo study, PDX model with immunodeficient NOD/SCID/IL2Rgnull mice were injected with heavily-treated refractory/relapsed IKZF1 del B-ALL patient samples (n=2) and treated with tucidinostat with different dosage of 5-12.5mg/kg/day. Administration of tucidinostat observed IKAROS expression trajectory and resulted in prolonged animal survival in IKZF1 del B-ALL PDX model（P＜0.0001). Secondary transplantation of ALL cells from tucidinostat or vehicle-treated (1 x10 6) recipients revealed significantly improved survival in tucidinostat -treated group (p= 0.0235). These results indicate that tucidinostat treatment might elimination leukemia-initiating cells.Additionally, to profile the IKZF1 del B-ALL chromatin accessibility changes after tucidinostat-treatment. We performed ATAC-seq and observed a clear increase in accessibility at TCA cycle related gene and decrease in accessibility at glycolysis related gene.Furthermore, tucidinostat, formerly known as chidamide, was added to an open-label, one-arm PDT-Ph-like-ALL trial targeting adult Ph-like ALL, which is characterized with high frequency in IKZF1 deletions (Clinicaltrials.gov. NCT03564470). Preliminary data of PDT-Ph-like-ALL indicate that tucidinostat was effective and well-tolerated, yielded promising response in IKZF1 del Ph-like ALL (ASH2018, poster 4011; EHA 2019, PF181).Collectively, our study demonstrates that the novel HDAC-selective inhibitor, tucidinostat, could specifically target IKZF1 del high-risk B-ALL, by restoring the IKZF1 expression, resulting in attenuation of proliferation, reverse the Warburg effect and improvement of the survival in PDX model and preliminary data in clinical trial. These findings provide mechanistic insights and a promising therapeutic strategy for IKZF1 haploinsufficiency alterations B-ALL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Post-Hypoxic Cells Promote Metastatic Recurrence after Chemotherapy Treatment in TNBC.

Simple SummaryIntratumoral hypoxia is a negative prognostic factor in breast cancer progression and recurrence. By implementing a hypoxia fate-mapping system, we followed cells that experience intratumoral hypoxia in vivo and determined that these cells have an increased ability to metastasize compared to cells that were never exposed to hypoxia. In this work, we investigate whether cells that experienced intratumoral hypoxia are also resistant to chemotherapy. By utilizing both in vivo and ex vivo models, we conclude that metastatic cells found in the lung and liver, that were exposed to hypoxia in the primary tumor, are less sensitive to doxorubicin and paclitaxel and drive recurrence after treatment. Our studies also suggest that chemoresistance is associated with a cancer stem cell-like phenotype that is maintained in post-hypoxic cells.Hypoxia occurs in 90% of solid tumors and is associated with treatment failure, relapse, and mortality. HIF-1α signaling promotes resistance to chemotherapy in cancer cell lines and murine models via multiple mechanisms including the enrichment of breast cancer stem cells (BCSCs). In this work, we utilize a hypoxia fate-mapping system to determine whether triple-negative breast cancer (TNBC) cells that experience hypoxia in the primary tumor are resistant to chemotherapy at sites of metastasis. Using two orthotopic mouse models of TNBC, we demonstrate that cells that experience intratumoral hypoxia and metastasize to the lung and liver have decreased sensitivity to doxorubicin and paclitaxel but not cisplatin or 5-FU. Resistance to therapy leads to metastatic recurrence caused by post-hypoxic cells. We further determined that the post-hypoxic cells that metastasize are enriched in pathways related to cancer stem cell gene expression. Overall, our results show that even when hypoxic cancer cells are reoxygenated in the bloodstream they retain a hypoxia-induced cancer stem cell-like phenotype that persists and promotes resistance and eventually recurrence.

Tumor-associated macrophages in osteosarcoma.

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. It is an aggressive tumor with a tendency to spread to the lung, which is the most common site of metastasis. Patients with advanced OS with metastases have poor prognoses despite the application of chemotherapy, thus highlighting the need for novel therapeutic targets. The tumor microenvironment (TME) of OS is confirmed to be essential for and supportive of tumor growth and dissemination. The immune component of the OS microenvironment is mainly composed of tumor-associated macrophages (TAMs). In OS, TAMs promote tumor growth and angiogenesis and upregulate the cancer stem cell-like phenotype. However, TAMs inhibit the metastasis of OS. Therefore, much attention has been paid to investigating the mechanism of TAMs in OS development and the progression of immunotherapy for OS. In this article, we aim to summarize the roles of TAMs in OS and the major findings on the application of TAMs in OS treatment.

SOX12 Promotes Stem Cell-Like Phenotypes and Osteosarcoma Tumor Growth by Upregulating JAGGED1.

SOX12 plays a role in promoting the growth of some tumors; however, its role in osteosarcoma remains unclear. From gene expression omnibus (GEO) and tumor alterations relevant for genomics-driven therapy (TARGET) databases, Kaplan–Meier analyses were conducted to establish relationships between SOX12 expression and osteosarcoma survival and recurrence in osteosarcoma patients. We also performed in vitro and in vivo assays to determine SOX12 function in osteosarcoma etiology. SOX12 expression was increased in osteosarcoma; high SOX12 expression levels were related to a poor prognosis and a high disease recurrence in patients. Moreover, SOX12 expression in osteosarcoma cell lines was increased, similar to osteosarcoma cancer stem cells. We also observed that SOX12 knockdown inhibited the spheroidization and expression of stemness markers in osteosarcoma cells and tumor formation in nude mice. In addition, SOX12 knockdown inhibited JAGGED1 and HES1 expression. Similarly, JAGGED1 knockdown also inhibited the formation of osteosarcoma cancer stem cells into pellets and reduced the expression of stemness markers and tumor formation capabilities in nude mice. Finally, during SOX12 knockdown, JAGGED1 overexpression rescued osteosarcoma cells from spheroidizing. SOX12 promotes stem cell-like phenotypes and osteosarcoma tumor growth by upregulating JAGGED1.

GOLPH3/CKAP4 promotes metastasis and tumorigenicity by enhancing the secretion of exosomal WNT3A in non-small-cell lung cancer

Cancer metastasis is the main cause of mortality associated with non-small-cell lung cancer (NSCLC), accounting for up to 70% of deaths among patients. The mechanisms underlying distal metastasis remain largely unknown. Golgi phosphoprotein 3 (GOLPH3) correlates negatively with overall survival in multiple tumors. In this study, we evaluated the function of GOLPH3 in NSCLC distal metastasis. GOLPH3 was expressed at high levels in samples from patients with NSCLC and was positively associated with clinicopathologic characteristics including clinical stage (P < 0.001), T (P = 0.001), N (P = 0.007), and M (P = 0.001) classification. Functionally, Transwell and wound-healing assays suggested that GOLPH3 overexpression enhances NSCLC cell migration and invasion abilities. Tumor-sphere formation and flow cytometry assays demonstrated that GOLPH3 overexpression enhances a stem cell-like phenotype of NSCLC cells. Metastasis models established by tail vein and intracardiac injection confirmed the pro-metastatic function of GOLPH3 in vivo. A subcutaneous tumor formation model confirmed that GOLPH3 overexpression increased the tumorigenicity of NSCLC cells. Mechanistically, gene set enrichment analysis revealed a positive association of GOLPH3 mRNA expression with WNT-activated gene signatures. Luciferase-reporter and nuclear extract assays showed that GOLPH3 overexpression enhances metastasis and tumorigenicity through activation of the WNT/β-catenin pathway. Immunoprecipitation-mass spectrometry and gene ontology analysis demonstrated that GOLPH3 interacts with cytoskeleton-associated protein 4 (CKAP4) in exosome-mediated distal metastasis. We found that GOLPH3 decreased the amount of plasma membrane-localized CKAP4 and increased the amount of exosome-localized CKAP4 to promote the formation of CKAP4-containing exosomes. Furthermore, we demonstrated that CKAP4 binds exosomal WNT3A to enhance its secretion. Therefore, the GOLPH3/CKAP4 axis plays a crucial role in promoting exosomal-WNT3A secretion to enhance and maintain the stem-like phenotype and metastasis in NSCLC, thus indicating the therapeutic potential of GOLPH3 in patients with NSCLC metastasis.

Generation of Pure Highly Functional Human Anti-Tumor Specific Cytotoxic T Lymphocytes With Stem Cell-Like Memory Features for Melanoma Immunotherapy.

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201+ MART-1+ tumor cells. Moreover, they harbored a suitable phenotype for immunotherapy, with effector memory, central memory and, most importantly, stem cell-like memory T cell features. Notably, the cells harboring stem cell-like memory phenotype features were capable of self-renewal and of differentiation into potent effector anti-tumor T cells. These “off-the-shelf” AAPCs represent a unique tool to rapidly and easily expand large numbers of long-lived highly functional pure specific CTLs with stem cell-like memory T cell properties, for the development of efficient adoptive immunotherapy strategies against cancers.

Glutamine promotes escape from therapy-induced senescence in tumor cells.

Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

Bone marrow-derived mesenchymal stem cells transplanted into a vascularized biodegradable tube containing decellularized allogenic nerve basal laminae promoted peripheral nerve regeneration; can it be an alternative of autologous nerve graft?

Previously, we showed silicone nerve conduits containing a vascular bundle and decellularized allogenic basal laminae (DABLs) seeded with bone marrow-derived mesenchymal stem cells (BMSCs) demonstrated successful nerve regeneration. Nerve conduits should be flexible and biodegradable for clinical use. In the current study, we used nerve conduits made of polyglycoric acid (PGA) fiber mesh, which is flexible, biodegradable and capillary-permeable. DABLs were created using chemical surfactants to remove almost all cell debris. In part 1, capillary infiltration capability of the PGA tube was examined. Capillary infiltration into regenerated neural tissue was compared between the PGA tube with blood vessels attached extratubularly (extratubularly vascularized tube) and that containing blood vessels intratubularly (intratubularly vascularized tube). No significant difference was found in capillary formation or nerve regeneration between these two tubes. In part 2, a 20 mm gap created in a rat sciatic nerve model was bridged using the extratubularly vascularized PGA tube containing the DABLs with implantation of isogenic cultured BMSCs (TubeC+ group), that containing the DABLs without implantation of the BMSCs (TubeC- group), and 20 mm-long fresh autologous nerve graft (Auto group). Nerve regeneration in these three groups was assessed electrophysiologically and histomorphometrically. At 24 weeks, there was no significant difference in any electrophysiological parameters between TubeC+ and Auto groups, although all histological parameters in Auto group were significantly greater than those in TubeC+ and TubeC- groups, and TubeC+ group demonstrated significant better nerve regeneration than TubeC- group. The transplanted DABLs showed no signs of immunological rejection and some transplanted BMSCs were differentiated into cells with Schwann cell-like phenotype, which might have promoted nerve regeneration within the conduit. This study indicated that the TubeC+ nerve conduit may become an alternative to nerve autograft.

Combined Treatment with Acalabrutinib and Rapamycin Inhibits Glioma Stem Cells and Promotes Vascular Normalization by Downregulating BTK/mTOR/VEGF Signaling.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with a median duration of survival of approximately 14 months after diagnosis. High resistance to chemotherapy remains a major problem. Previously, BTK has been shown to be involved in the intracellular signal transduction including Akt/mTOR signaling and be critical for tumorigenesis. Thus, we aim to evaluate the effect of BTK and mTOR inhibition in GBM. We evaluated the viability of GBM cell lines after treatment with acalabrutinib and/or rapamycin through a SRB staining assay. We then evaluated the effect of both drugs on GBM stem cell-like phenotypes through various in vitro assay. Furthermore, we incubated HUVEC cells with tumorsphere conditioned media and observed their angiogenesis potential, with or without treatment. Finally, we conducted an in vivo study to confirm our in vitro findings and analyzed the effect of this combination on xenograft mice models. Drug combination assay demonstrated a synergistic relationship between acalabrutinib and rapamycin. CSCs phenotypes, including tumorsphere and colony formation with the associated expression of markers of pluripotency are inhibited by either acalabrutinib or rapamycin singly and these effects are enhanced upon combining acalabrutinib and rapamycin. We showed that the angiogenesis capabilities of HUVEC cells are significantly reduced after treatment with acalabrutinib and/or rapamycin. Xenograft tumors treated with both drugs showed significant volume reduction with minimal toxicity. Samples taken from the combined treatment group demonstrated an increased Desmin/CD31 and col IV/vessel ratio, suggesting an increased rate of vascular normalization. Our results demonstrate that BTK-mTOR inhibition disrupts the population of GBM-CSCs and contributes to normalizing GBM vascularization and thus, may serve as a basis for developing therapeutic strategies for chemoresistant/radioresistant GBM.

Importance of lymphocyte-stromal cell interactions in autoimmune and inflammatory rheumatic diseases.

Interactions between lymphocytes and stromal cells have an important role in immune cell development and responses. During inflammation, stromal cells contribute to inflammation, from induction to chronicity or resolution, through direct cell interactions and through the secretion of pro-inflammatory and anti-inflammatory mediators. Stromal cells are imprinted with tissue-specific phenotypes and contribute to site-specific lymphocyte recruitment. During chronic inflammation, the modified pro-inflammatory microenvironment leads to changes in the stromal cells, which acquire a pathogenic phenotype. At the site of inflammation, infiltrating B cells and T cells interact with stromal cells. These interactions induce a plasma cell-like phenotype in B cells and T cells, associated with secretion of immunoglobulins and inflammatory cytokines, respectively. B cells and T cells also influence the stromal cells, inducing cell proliferation, molecular changes and cytokine production. This positive feedback loop contributes to disease chronicity. This Review describes the importance of these cell interactions in chronic inflammation, with a focus on human disease, using three selected autoimmune and inflammatory diseases: rheumatoid arthritis, psoriatic arthritis (and psoriasis) and systemic lupus erythematosus. Understanding the importance and disease specificity of these interactions could provide new therapeutic options.

NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype.

Abstract 2322: Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer

Abstract Nutrient deprivation is one of the hallmark conditions of a rapidly growing tumor. Rapid proliferation of tumor cells leads to increased consumption of metabolites such as glucose and glutamine resulting in metabolic stress. However, phenotypic and functional status of tumor cells under these conditions is not clear. We hypothesize that nutrient deprivation-induced metabolic stress results in a breast cancer stem cell-like (BCSC) phenotype in triple negative breast cancer (TNBC). We found that serum-deprived conditions result in significant upregulation of GD2+ cells compared to serum rich conditions: from 9%±2% to 26%±5% in MDA-MB-231 and 7%±2% to 29%±5% in SUM159 cells. Interestingly, this affect was compensated when the cells were supplemented with serum-rich medium. In addition, a positive correlation was observed between GD2+ cell number and tumor volume in vivo, suggesting metabolic stress in the growing tumors results in GD2+ phenotype in TNBC cells. Next, TNBC cells cultured under glucose deprivation or in the presence of glycolysis inhibitor, 2-deoxy glucose (2DG) showed a 2-fold increase in GD2+ cells compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 expression. Nutrient deprived conditions also showed a significant increase in the expression of stress markers ATF4 and SESN2 demonstrating TNBC cells under metabolic stress. In addition, the above conditions also showed a 2.5-fold upregulation of the mammosphere formation in TNBC cells compared to nutrient rich conditions. Global metabolomics profiling identified glutathione biosynthesis to be most highly upregulated in GD2+ cells. Furthermore, we found that supplementation of glutamine (precursor for glutathione biosynthesis) increases the percentage of GD2+ cells. Carbon and nitrogen tracing using 13C- and 15N-labeled glutamine by mass spectrometry showed its direct contribution to GD2 biosynthesis. Treatment with glutamine transporter (ASCT2) inhibitor V9302 reduced GD2+ cell number by 70%-80% in TNBC cell lines and dramatically inhibited mammosphere and soft agar colony formation, and migration of TNBC cell lines. Mechanistic studies revealed that V9302 inhibits glutathione pathway and induces ROS levels in TNBC cells. Additionally, V9302 treatment inhibited AKT-ERK-mTOR-4E-BP1 pathway and combination of V9302 with paclitaxel synergizes TNBC cell killing. We also identified that, V9302 treatment induced ferroptosis (iron-dependent cell death) in TNBC cells. Finally, V9302 treatment significantly inhibited tumor growth (p&amp;lt;0.0001) in TNBC-PDX model. In conclusion, metabolic stress results in GD2+ BCSC phenotype in TNBC cells and glutamine directly contributes to GD2 biosynthesis. Targeting ASCT2 could complement conventional chemotherapy in TNBC. Citation Format: Appalaraju Jaggupilli, Stanley Ly, Khoa Nguyen, Roshan Borkar, Bin Yuan, Danthasinghe Waduge Piyarathna, Yuanqing Yan, Helen Piwnica-Worms, Henry Charles Manning, Michael Andreeff, Nagireddy Putluri, Lokesh Battula. Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2322.

PCGF1 promotes epigenetic activation of stemness markers and colorectal cancer stem cell enrichment

Colorectal cancer (CRC) stem cells are resistant to cancer therapy and are therefore responsible for tumour progression after conventional therapy fails. However, the molecular mechanisms underlying the maintenance of stemness are poorly understood. In this study, we identified PCGF1 as a crucial epigenetic regulator that sustains the stem cell-like phenotype of CRC. PCGF1 expression was increased in CRC and was significantly correlated with cancer progression and poor prognosis in CRC patients. PCGF1 knockdown inhibited CRC stem cell proliferation and CRC stem cell enrichment. Importantly, PCGF1 silencing impaired tumour growth in vivo. Mechanistically, PCGF1 bound to the promoters of CRC stem cell markers and activated their transcription by increasing the H3K4 histone trimethylation (H3K4me3) marks and decreasing the H3K27 histone trimethylation (H3K27me3) marks on their promoters by increasing expression of the H3K4me3 methyltransferase KMT2A and the H3K27me3 demethylase KDM6A. Our findings suggest that PCGF1 is a potential therapeutic target for CRC treatment.

Effects of bisphenol A exposure during cardiac cell differentiation

Heart development requires a precise temporal regulation of gene expression in cardiomyoblasts. Therefore, the transcriptional changes in differentiating cells can lead to congenital heart diseases. Although the genetic mutations underlie most of these alterations, exposure to environmental contaminants, such as bisphenol A (BPA), has been recently considered as a risk factor as well. In this study we investigated the genotoxic and epigenotoxic effects of BPA throughout cardiomyocyte differentiation. H9c2 cells (rat myoblasts) were exposed to 10 and 30 μM BPA before and during the last two days of cardiac-driven differentiation. Then, we have analysed the phenotypic and molecular modifications (at transcriptional, genetic and epigenetic level). The results showed that treated myoblasts developed a skeletal muscle cell-like phenotype. The transcriptional changes induced by BPA in genes codifying proteins involved in heart differentiation and function depend on the window of exposure to BPA. The exposure before differentiation repressed the expression of heart transcription factors (Hand2 and Gata4), whereas exposure during differentiation reduced the expression of cardiac-specific genes (Tnnt2, Myom2, Sln, and Atp2a1). Additionally, significant effects were observed regarding DNA damage and histone acetylation levels after the two periods of BPA exposure: in cells exposed to the toxicant the percentage of DNA repair foci (formed by the co-localization of γH2AX and 53BP1) increased in a dose-dependent manner, whereas the treatment with the toxicant triggered a decrease in the epigenetic marks H3K9ac and H3K27ac. Our in vitro results reveal that BPA seriously interferes with the process of cardiomyocyte differentiation, which could be related to the reported in vivo effects of this toxicant on cardiogenesis.

PERK-eIF2α-ERK1/2 axis drives mesenchymal-endothelial transition of cancer-associated fibroblasts in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by remarkable desmoplasia, usually driven by cancer-associated fibroblasts (CAFs), influencing patient prognosis. CAFs are a group of plastic cells responsible for tumor growth and metastasis. Fibroblasts have been reported to directly contribute to angiogenesis by undergoing mesenchymal-endothelial transition (MEndoT) after ischemic injury in the heart, brain, and hindlimbs. However, whether CAFs can undergo similar transdifferentiation in the hostile tumor microenvironment and directly contribute to tumor angiogenesis remains unclear. Herein, we provide evidence that CAFs can adopt an endothelial cell-like phenotype and directly contribute to tumor angiogenesis in vitro and in vivo. Furthermore, this program is regulated by the PERK-eIF2α-ERK1/2 axis. Pharmacological inhibition of PERK with GSK2606414 limited the phenotypic transition of CAFs. In conclusion, our results suggest that CAFs contribute to tumor angiogenesis by undergoing the MEndoT, thus representing therapeutic targets for improving PDAC prognosis.

Scleraxis expressing scleral cells respond to inflammatory stimulation

The sclera is an ocular tissue rich of collagenous extracellular matrix, which is built up and maintained by relatively few, still poorly characterized fibroblast-like cells. The aims of this study are to add to the characterization of scleral fibroblasts and to examine the reaction of these fibroblasts to inflammatory stimulation in an ex vivo organotypic model. Scleras of scleraxis-GFP (SCX-GFP) mice were analyzed using immunohistochemistry and qRT-PCR for the expression of the tendon cell associated marker genes scleraxis (SCX), mohawk and tenomodulin. In organotypic tissue culture, explanted scleras of adult scleraxis GFP reporter mice were exposed to 10 ng/ml recombinant interleukin 1-ß (IL1-ß) and IL1-ß in combination with dexamethasone. The tissue was then analyzed by immunofluorescence staining of the inflammation- and fibrosis-associated proteins IL6, COX-2, iNOS, connective tissue growth factor, MMP2, MMP3, and MMP13 as well as for collagen fibre degradation using a Collagen Hybridizing Peptide (CHP) binding assay. The mouse sclera displayed a strong expression of scleraxis promoter-driven GFP, indicating a tendon cell-like phenotype, as well as expression of scleraxis, tenomodulin and mohawk mRNA. Upon IL1-ß stimulation, SCX-GFP+ cells significantly upregulated the expression of all proteins analysed. Moreover, IL1-ß stimulation resulted in significant collagen degradation. Adding the corticosteroid dexamethasone significantly reduced the response to IL1-ß stimulation. Collagen degradation was significantly enhanced in the IL1-ß group. Dexamethasone demonstrated a significant rescue effect. This work provides insights into the characteristics of scleral cells and establishes an ex vivo model of scleral inflammation.

Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers.

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.