The Anti-Proliferative Effect of PI3K/mTOR and ERK Inhibition in Monolayer and Three-Dimensional Ovarian Cancer Cell Models

Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors.

Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

Background: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. Objective: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. Methods: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. Results: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. Conclusion: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

Increased Platelet-CD4+ T Cell Aggregates Are Correlated With HIV-1 Permissiveness and CD4+ T Cell Loss

Chronic HIV-1 infection is associated with persistent inflammation, which contributes to disease progression. Platelet-T cell aggregates play a critical role in maintaining inflammation. However, the phenotypic characteristics and clinical significance of platelet-CD4+ T cell aggregates remain unclear in different HIV-infected populations. In this study, we quantified and characterized platelet-CD4+ T cell aggregates in the peripheral blood of treatment-naïve HIV-1-infected individuals (TNs), immunological responders to antiretroviral therapy (IRs), immunological non-responders to antiretroviral therapy (INRs), and healthy controls (HCs). Flow cytometry analysis and immunofluorescence microscopy showed increased platelet-CD4+ T cell aggregate formation in TNs compared to HCs during HIV-1 infection. However, the frequencies of platelet-CD4+ T cell aggregates decreased in IRs compared to TNs, but not in INRs, which have shown severe immunological dysfunction. Platelet-CD4+ T cell aggregate frequencies were positively correlated with HIV-1 viral load but negatively correlated with CD4+ T cell counts and CD4/CD8 ratios. Furthermore, we observed a higher expression of CD45RO, HIV co-receptors, HIV activation/exhaustion markers in platelet-CD4+ T cell aggregates, which was associated with HIV-1 permissiveness. High levels of caspase-1 and caspase-3, and low levels of Bcl-2 in platelet-CD4+ T cell aggregates imply the potential role in CD4+ T cell loss during HIV-1 infection. Furthermore, platelet-CD4+ T cell aggregates contained more HIV-1 gag viral protein and HIV-1 DNA than their platelet-free CD4+ T cell counterparts. The platelet-CD4+ T cell aggregate levels were positively correlated with plasma sCD163 and sCD14 levels. Our findings demonstrate that platelet-CD4+ T cell aggregate formation has typical characteristics of HIV-1 permissiveness and is related to immune activation during HIV-1 infection.

Stem Cells from Human Exfoliated Deciduous Teeth Differentiation and Characterization in to Islet like Pancreatic Cell Lineages – An Ex-vivo and In-vitro Study

Background and Objectives: Stem cells from human exfoliated deciduous teeth (SHEDs) have been demonstrated as a novel population of adult stem cells capable of multi-differentiation potential. Methods: Study samples comprise of 30 extracted exfoliating primary teeth collected from children aged 6 to 14 years. After attaining the required cell passage, flowcytometric analysis and trilineage differentiation was done to characterize SHEDs. Further SHEDs were differentiated into Islet like cell aggregates (ICAs) using Serum free media A,B&C. Differentiated ICAs were characterized by RT-PCR, immunocytochemistry, DTZ stain and insulin assay. Results: Flowcytometric analysis of SHEDs showed expression of positive markers CD73, CD90 while no expression of negative markers CD34, CD45 and HLA-DR. Isolated SHEDs had the potential to differentiate into tri-lineages and ICAs. RT-PCR analysis of derived ICAs showed up-regulated expression of GAPDH, insulin, Glut2, PDX1 and PAX6. Immunofluorescence analysis gave expression of Ngn3, Isl-1, C-peptide, Glut2 and PDX1. DTZ stained positive on derived ICAs.Insulin secretion of SHED derived ICAs were measured 26 ± 6 MIU/L at basal glucose level, 128±3 MIU/L and 240 ±9 MIU/L at stimulated glucose level which gave a statistically significant difference in mean value of insulin secreted in different concentration of glucose (p<0.001). The net insulin secretion value of SHED derived ICAs at different glucose concentration was less when compared with Min 6 cells used as positive control. Interpretation and Conclusion: Stem cells from Human Exfoliated Deciduous teeth are mesenchymal stem cells which has the unique potential to differentiate into islet like cell aggregates and serve as a promising source of insulin which under standardized protocols and experimentations can be used for stem cell based therapy for insulin dependent diabetes mellitus.

The ECM: To Scaffold, or Not to Scaffold, That Is the Question

The extracellular matrix (ECM) has pleiotropic effects, ranging from cell adhesion to cell survival. In tissue engineering, the use of ECM and ECM-like scaffolds has separated the field into two distinct areas—scaffold-based and scaffold-free. Scaffold-free techniques are used in creating reproducible cell aggregates which have massive potential for high-throughput, reproducible drug screening and disease modeling. Though, the lack of ECM prevents certain cells from surviving and proliferating. Thus, tissue engineers use scaffolds to mimic the native ECM and produce organotypic models which show more reliability in disease modeling. However, scaffold-based techniques come at a trade-off of reproducibility and throughput. To bridge the tissue engineering dichotomy, we posit that finding novel ways to incorporate the ECM in scaffold-free cultures can synergize these two disparate techniques.

Bone Marrow Biopsy T Cell Aggregates and Cytokine Transcriptional Signatures Predict Sustained Response to CAR T-Cell Therapy in B-ALL

Background CAR T-cell therapies directed against CD19 (CAR19) are used to treat relapsed/refractory (R/R) B-acute lymphoblastic leukemia (B-ALL) and show high complete remission (CR) rates compared to salvage chemotherapy. However, subsets of patients do not respond or relapse after initial CR. While previous work has demonstrated the importance of CAR T cell immunophenotype and vector integration site in long term CAR19 responders, there are currently no clinically applicable predictors of response. Given the importance of the B-ALL tumor microenvironment in mediating chemoresistance, inflammatory signaling and recruitment of immune effectors to the leukemic bone marrow, we hypothesized that the pre-CAR19 infusion bone marrow biopsy immune microenvironment may predict CAR19 responses. Methods Patients treated with CAR19 for R/R B-ALL from 2012-2017 were retrospectively identified. Marrow core biopsies were collected immediately prior to CAR19 infusion (pre-CAR) and at one month post-infusion (post-CAR). Bone marrow biopsy slides immunohistochemically stained for CD3 were digitally scanned, analyzed algorithmically for T-cell infiltration and assessed qualitatively for interstitial T cell aggregates of &gt;5 cells (Leica Aperio ImageScope). B-ALL involvement was determined by bone marrow aspirate counts and minimal residual disease (MRD) flow cytometry. Targeted RNA sequencing-based gene expression profiling (EdgeSeq Immuno-Oncology Panel, HTG Diagnostics) was performed on pre- and post-treatment biopsies, with differential expression assessed by DESeq2 within HTG Reveal software, and further analyses conducted using Metascape. Results We identified 143 bone marrow core biopsies (84 pre-treatment, 59 post-treatment) from 84 R/R B-ALL patients treated with CAR19. Pre-CAR B-ALL marrow involvement ranged from 0-95% (median: 67.5%), and the dataset included patients with relapses occurring between 1-33 months following CAR therapy (median: 6 months). Pre-CAR CD3+ T cell aggregates were significantly increased in CAR19 Sustained Responders (SR) compared to Non-Responders (NR) (92.3% versus 50%, p=0.007) and eventual relapses (CD19+: 40%, p= 0.002 and CD19-: 61.5%, p=0.019) (Figure 1A-B). A lack of T cell aggregates in pre-CAR marrow biopsies showed a sensitivity of 45.8% and a specificity of 92.3% in distinguishing NRs and eventual relapses from SRs. In one month post-CAR marrow biopsies, T cell aggregates continued to show higher frequency in SRs compared to eventual CD19+ relapses (100% versus 42.9%, p=0.007) and NRs (50%, p=0.04). Pre-CAR marrow T cell infiltration was also significantly greater in SRs compared to NRs (2146 CD3+ T cells per biopsy versus 850, p=0.038) and eventual CD19- relapses (1086, p=0.022), and trended toward a significant increase compared to eventual CD19+ relapses (1056, p=0.063) (Figure 1C). Pre-CAR marrow T cells of &lt; 920 per biopsy showed a sensitivity of 45.8% and a specificity of 92.3% in distinguishing NRs and relapses from SRs (Figure 1D). RNAseq differential gene expression analysis of pre-CAR marrow biopsies revealed a chemokine axis of CXCL9, CXCL13, CXCR5, CXCL5, CXCL2 and CCL1 in SR that was deficient in patients with eventual relapse (Figure 1E). By contrast, NRs and eventual relapses showed a pre-CAR transcriptional signature reflecting Th2 (IL5RA, IL13) and Th17 (IL17A, IL23R) skewing, in addition to higher IL2, IFNG and IL6 transcripts. Conclusion These immunohistochemical and transcriptional findings support a pivotal role for the peri-infusion bone marrow microenvironment in long-term responses to CAR19 therapy in B-ALL. A deficiency of marrow T cell aggregates and marrow-infiltrating T cells pre-CAR shows high specificity in distinguishing NRs and eventual relapses from SRs to CAR19, and marrow T cell aggregates are a persistent feature of SRs at one month post-CAR infusion. SR to CAR19 is also associated with elevated marrow expression of a suite of immune chemoattractants which may enhance the ability of infused CAR T cells and other effectors to infiltrate the B-ALL microenvironment. Combined assessment of bone marrow T cell infiltrates and transcriptional profiling could be used to improve the sensitivity and specificity of predictive algorithms for responses to CAR19 therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation

Epithelial networks are commonly generated by processes where multicellular aggregates elongate and branch. Here we focus on understanding cellular mechanisms for elongation, using an organotypic culture system as a model of mammary epithelial anlage. Isotropic cell aggregates broke symmetry and slowly elongated when transplanted into collagen 1 gels. The elongating regions of aggregates displayed enhanced cell proliferation that was necessary for elongation to occur. Strikingly, this loco-regional increase in cell proliferation occurred where collagen 1 fibrils reorganized into bundles which were polarized with the elongating aggregates. Applying external stretch as a cell-independent way to reorganize the ECM, we found that collagen polarization stimulated regional cell proliferation to precipitate symmetry-breaking and elongation. This required b1-integrin and ERK signaling. We propose that collagen polarization supports epithelial anlagen elongation by stimulating loco-regional cell proliferation. This could provide a long-lasting structural memory of the initial axis that is generated when anlage break symmetry.