The tumour-associated antigen L6 (L6-Ag) is recruited to the tetraspanin-enriched microdomains: implication for tumour cell motility

Tumour-associated antigen L6 (L6-Ag, also known as TM4SF1) regulates tumour cell motility and invasiveness. We found that L6-Ag is abundant on the plasma membrane and on intracellular vesicles, on which it is co-localised with the markers for late endosomal/lysosomal compartments, including Lamp1/Lamp2 proteins and LBPA. Antibody internalisation and live-imaging experiments suggested that L6-Ag is targeted to late endocytic organelles (LEO) predominantly via a biosynthetic pathway. Mapping experiments showed that the presence of transmembrane regions is sufficient for directing L6-Ag to LEO. On the plasma membrane, L6-Ag is associated with tetraspanin-enriched microdomains (TERM). All three predicted cytoplasmic regions of L6-Ag are crucial for the effective recruitment of the protein to TERM. Recruitment to TERM correlated with the pro-migratory activity of L6-Ag. Depletion of L6-Ag with siRNA has a selective effect on the surface expression of tetraspanins CD63 and CD82. By contrast, the expression levels of other tetraspanins and β1 integrins was not affected. We found that L6-Ag is ubiquitylated and that ubiquitylation is essential for its function in cell migration. These data suggest that L6-Ag influences cell motility via TERM by regulating the surface presentation and endocytosis of some of their components

A species-specific double isotope dilution strategy for the accurate quantification of platinum–GG adducts in lung cells exposed to carboplatin

Platinum–DNA adducts, and in particular Pt–GG, have been identified as the major cytotoxic species during chemotherapy treatment with platinum containing drugs. This paper reports for the first time a strategy based on the use of double species-specific isotope dilution analysis (IDA) for the quantification of carboplatin–GG adducts formed by exposing human lung carcinoma cells to carboplatin. The main challenge posed by the use of carboplatin in this pre-clinical application, in comparison with most previously reported studies using cisplatin, includes the relatively low reactivity of this drug, thus demanding for improved limits of detection to be achieved in order to perform accurate quantification of the adducts at low ng Pt per mg DNA levels with relatively small uncertainty in micro-volumes of the biological sample. This was alleviated by developing micro-flow HPLC reversed phased methodology coupled to sector field ICP-MS (R = 300), showing a limit of detection of 0.2 ng Pt per mg DNA. To perform IDA, carboplatin–GG calibrants and spikes (194Pt-enriched GG adducts) were synthesized in house and characterised for Pt mass fraction, Pt distribution and structural composition. In order to assess the accuracy of the developed procedure, in the absence of certified reference materials, a reference sample prepared by incubation of calf thymus DNA with carboplatin and characterised in house (e.g. for its total P and Pt contents and Pt–GG concentration) was analysed in parallel. The use of this sample as a quality control of the cleavage of DNA and recovery of Pt adducts from real samples makes the described strategy particularly novel. Moreover, spike recovery experiments on the cell samples with the reference carboplatin–DNA sample were undertaken. The validated methodology was applied to cultured human lung carcinoma cells exposed to carboplatin; Pt–GG adducts were found to be at a level of 5.54 ng Pt per mg DNA with a relatively expanded combined uncertainty (k = 2) of approximately 20%. The major contributing factors to the overall measurement uncertainty were the mass fraction of Pt in the natural carboplatin–GG standard, the measured isotope ratio precision of sample and calibration blends and the blend to blend variation. The SI traceable methodology presented here will be invaluable for the provision of reference values to clinical measurements and cancer clinical trials

Comparability of scalable, automated hMSC culture using manual and automated process steps

Automation will likely to play a key role in the development of scalable manufacturing processes for cell-based therapies. In this study, we have compared the effects of manual centrifugation and automated non-centrifugation cell culture process steps, performed using TAP biosystems’ CompacT SelecT automated cell culture platform, upon hMSC morphology, number, viability, surface marker expression, Short tandem repeat (STR) profile, and paracrine function. Furthermore, the comparability between flow cytometry analyses of hMSCs, performed at multiple sites, was investigated. No significant difference in hMSC growth and characteristics was observed between cells cultured using either the manual centrifugation process step or the automated non-centrifugation process step, in which residual dissociation agent is carried over. However, some variability in paracrine activity was observed between hMSCs cultured using alternative process steps. It is also apparent that differences in analytical methods can influence the inter-laboratory reproducibility of hMSC flow cytometry analysis, although differences in culture may also contribute to the variability observed in the expression of 2 of the 8 surface markers examined. This novel investigation into the effects of these two key process steps will help to improve the understanding of the influence of automated cell culture upon various cell culture parameters, as well as upon process comparability

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging DARPin (designed ankyrin repeat protein) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells co-expressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mouse models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity towards LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057)

Comparison of Volumetric and Bead‐Based Counting of CD34 Cells by Single‐Platform Flow Cytometry

Background: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. Method: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. Results: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method. Conclusions: Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society

Comparability of scalable, automated hMSC culture using manual and automated process steps

This is an open access article published by Elsevier under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Automation will likely to play a key role in the development of scalable manufacturing processes for cell-based therapies. In this study, we have compared the effects of manual centrifugation and automated non-centrifugation cell culture process steps, performed using TAP biosystems’ CompacT SelecT automated cell culture platform, upon hMSC morphology, number, viability, surface marker expression, Short tandem repeat (STR) profile, and paracrine function. Furthermore, the comparability between flow cytometry analyses of hMSCs, performed at multiple sites, was investigated. No significant difference in hMSC growth and characteristics was observed between cells cultured using either the manual centrifugation process step or the automated non-centrifugation process step, in which residual dissociation agent is carried over. However, some variability in paracrine activity was observed between hMSCs cultured using alternative process steps. It is also apparent that differences in analytical methods can influence the inter-laboratory reproducibility of hMSC flow cytometry analysis, although differences in culture may also contribute to the variability observed in the expression of 2 of the 8 surface markers examined. This novel investigation into the effects of these two key process steps will help to improve the understanding of the influence of automated cell culture upon various cell culture parameters, as well as upon process comparability