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Microalgal biotechnology shows considerable promise as a sustainable contributor to a broad range of industrial avenues. The field is however limited by processing methods that have commonly hindered the progress of high throughput screening, and consequently development of improved microalgal strains. We tested various microplate reader and flow cytometer methods for monitoring the commercially relevant pigment fucoxanthin in the marine diatom Phaeodactylum tricornutum. Based on accuracy and flexibility, we chose one described previously to adapt to live culture samples using a microplate reader and achieved a high correlation to HPLC (R2 = 0.849), effectively removing the need for solvent extraction. This was achieved by using new absorbance spectra inputs, reducing the detectable pigment library and changing pathlength values for the spectral deconvolution method in microplate reader format. Adaptation to 384-well microplates and removal of the need to equalize cultures by density further increased the screening rate. This work is of primary interest to projects requiring detection of biological pigments, and could theoretically be extended to other organisms and pigments of interest, improving the viability of microalgae biotechnology as a contributor to sustainable industry.

We investigated two non-ionising mutagens in the form of ultraviolet radiation (UV) and ethyl methanosulfonate (EMS) and an ionising mutagen (X-ray) as methods to increase fucoxanthin content in the model diatom Phaeodactylum tricornutum. We implemented an ultra-high throughput method using fluorescence-activated cell sorting (FACS) and live culture spectral deconvolution for isolation and screening of potential pigment mutants, and assessed phenotype stability by measuring pigment content over 6 months using high-performance liquid chromatography (HPLC) to investigate the viability of long-term mutants. Both UV and EMS resulted in significantly higher fucoxanthin within the 6 month period after treatment, likely as a result of phenotype instability. A maximum fucoxanthin content of 135 ± 10% wild-type found in the EMS strain, a 35% increase. We found mutants generated using all methods underwent reversion to the wild-type phenotype within a 6 month time period. X-ray treatments produced a consistently unstable phenotype even at the maximum treatment of 1000 Grays, while a UV mutant and an EMS mutant reverted to wild-type after 4 months and 6 months, respectively, despite showing previously higher fucoxanthin than wild-type. This work provides new insights into key areas of microalgal biotechnology, by (i) demonstrating the use of an ionising mutagen (X-ray) on a biotechnologically relevant microalga, and by (ii) introducing temporal analysis of mutants which has substantial implications for strain creation and utility for industrial applications.

Fluorescence-Activated Cell Sorting (FACS) is a powerful method with many applications in microalgal research, especially for screening and selection of cells with improved phenotypes. However, the technology requires review of gene expression changes responsible for enhanced phenotypes in sorted populations. Phaeodactylum tricornutum cells were sorted using FACS with excitation/emission parameters targeted to favouring the industrially-relevant carotenoid fucoxanthin. The resulting cultures showed significantly higher growth rate (1.10 ×), biomass (1.30 ×), chlorophyll a levels (1.22 ×) and fucoxanthin content (1.28 ×) relative to the wild-type strain. RNA-seq was used to elucidate the underlying molecular-level regulatory changes associated with these traits and represents the first study do so on FACS-sorted microalgal cultures. Transcriptome analysis corroborated evidence of increased chlorophyll a and fucoxanthin, showing enrichment for the genes/pathways for tetrapyrrole biosynthesis and for suites of genes directly related to photosynthesis. Only three genes were upregulated in the MEP (non-mevalonate) pathway to carotenoid biosynthesis pathway, suggesting either a strong influence of IDI, CRTISO5 and ZEP1 on fucoxanthin biosynthesis or a post-transcriptional or post-translational mechanism for the observed increase in fucoxanthin content.

BackgroundLocally advanced oral cavity squamous cell carcinoma (OCSCC) presents a significant clinical challenge despite being partially responsive to standard treatment modalities. This study investigates the prognostic implications of programmed death-ligand 1 (PD-L1) expression in these tumors, focusing on its association with treatment outcomes and the immune microenvironment.MethodsWe assessed tumor-infiltrating lymphocytes (TILs) in 132 patients with OCSCC to evaluate their impact on survival. Multiplex immunohistochemistry staining for CD3, CD68, CD11c, PD-L1, and P40 was used to explore correlations with clinical outcomes in patients with early-stage (n=22) and locally advanced (n=36) OCSCC. These initial findings were validated through differential gene expression analysis, gene set enrichment, and immune cell deconvolution in a The Cancer Genome Atlas cohort of 163 locally advanced OCSCC tumors. Additionally, single-cell RNA sequencing (scRNA-seq) on a smaller cohort (n=10) further characterized the PD-L1hi or PD-L1lo cancer cells in these tumors.ResultsElevated PD-L1 expression was associated with poor outcomes in patients with locally advanced OCSCC undergoing standard adjuvant therapy, irrespective of “hot” or “cold” classification based on TILs assessment. PD-L1hi tumors exhibited an active immune response phenotype, enriched with M1 macrophages, CD8+ T cells and T regulatory cells in the tumor microenvironment. Notably, the negative impact of PD-L1 expression on outcomes was primarily attributed to its expression by cancer cells, rather than immune cells. Furthermore, scRNA-seq revealed that immune interactions were not essential for PD-L1 upregulation in cancer cells, instead, complex regulatory networks were involved. Additionally, PD-L1lo locally advanced tumors exhibited more complex pathway enrichment and diverse T-cell populations compared with those in the early-stage.ConclusionOur findings underscore the prognostic significance of PD-L1 expression in locally advanced OCSCC, and unveil the complex interplay between PD-L1 expression, immune responses, and molecular pathways in the tumor microenvironment. This study provides insights that may inform future therapeutic strategies, including the possibility of tailored immunotherapeutic approaches for patients with PD-L1hi locally advanced OCSCC.

We investigated two non-ionising mutagens in the form of ultraviolet radiation (UV) and ethyl methanosulfonate (EMS) and an ionising mutagen (X-ray) as methods to increase fucoxanthin content in the model diatom Phaeodactylum tricornutum . We implemented an ultra-high throughput method using fluorescence-activated cell sorting (FACS) and live culture spectral deconvolution for isolation and screening of potential pigment mutants, and assessed phenotype stability by measuring pigment content over 6 months using high-performance liquid chromatography (HPLC) to investigate the viability of long-term mutants. Both UV and EMS resulted in significantly higher fucoxanthin within the 6 month period after treatment, likely as a result of phenotype instability. A maximum fucoxanthin content of 135 ± 10% wild-type found in the EMS strain, a 35% increase. We found mutants generated using all methods underwent reversion to the wild-type phenotype within a 6 month time period. X-ray treatments produced a consistently unstable phenotype even at the maximum treatment of 1000 Grays, while a UV mutant and an EMS mutant reverted to wild-type after 4 months and 6 months, respectively, despite showing previously higher fucoxanthin than wild-type. This work provides new insights into key areas of microalgal biotechnology, by (i) demonstrating the use of an ionising mutagen (X-ray) on a biotechnologically relevant microalga, and by (ii) introducing temporal analysis of mutants which has substantial implications for strain creation and utility for industrial applications.

BackgroundChimeric antigen receptor (CAR-T) cells are a promising new therapy for patients with cancer. However, in contrast to their success in B cell malignancies, CAR-T cells targeting solid cancers have had limited success so far due to their poor proliferation and poor long-term persistence in vivo. To address this issue, we used naïve T cells to generate second-generation CAR-T cells recognizing the tumor antigen Lewis Y (LeY), termed ‘early’ CAR-T cells.MethodsPurified naïve T cells were activated by CD3/CD28 soluble tetrameric antibody complex, retrovirally transduced (LeY scFv-CD3z-CD28 CAR) and expanded in IL-7/IL-15. The early LeY CAR-T cell function was tested in vitro for cytotoxicity (Cr-release and degranulation), proliferation, and cytokine secretion by CBA, either de novo or following chronic stimulation for 1 month. Finally, early CAR-T cell persistence and anti-tumor efficacy was assessed in the OVCAR3-NSG model, in the presence or absence of anti-PD-1.ResultsThe early-CAR-T cells comprised stem cell memory-like (CD95+, CD62L+, CD45RA+) and central memory phenotype (CD95+, CD62L+, CD45RA-) T cells with increased expression of ICOS, Ki67, TCF7 and CD27 (Figure 1). The early-CAR-T cells retained potent antigen-specific cytotoxicity, and secreted significantly higher levels of cytokines (IFN-?, TNF-a and IL-2) and increased proliferation compared to conventional CAR-T cells. Importantly, early-CAR-T cells had a significantly higher proliferative capacity after long-term chronic stimulation compared to conventional CAR-T cells (figure 2), and CD4+ CAR-T cells were critical for effective early CD8+ CAR-T cell proliferation capacity in vitro (figure 3). Early CAR-T cells had significantly better in vivo tumor control compared to conventional CAR-T cells (Figure 4), this was associated with increased CAR-T cell persistence. Because chronically stimulated early-LeY-CAR-T cells expressed PD-1 (figure 2), and OVCAR-3 cells expressed PD-L1 when co-cultured with LeY-CAR-T cells (figure 5), we combined early LeY-CAR-T cells with anti-PD-1 therapy and observed complete tumour regression in these mice. Interestingly, early LeY-CAR-T cell plus anti-PD-1 treatment also enhanced the percentage of circulating stem-cell memory like CAR-T cells in vivo (figure 5).Abstract 126 Figure 1Early-CAR-T protocol, including Naïve-T cells purification and expansion in IL-7 and IL-15 promotes the maintenance of a TSCM and TCM phenotype. A) Scheme of the 7-day production protocol for Early-CAR-T cells. B) Phenotype by FACs of the conventional CAR-T cells and the Early-CAR-T cells. Pooled data in triplicate for 6 donors. C) Phenotype by Mass cytometry comparing the Conventional-CAR-T cells vs Early-CAR-T cells vs Early-CD8-CAR-T cells. Data for one donor representative of 3 different donorsAbstract 126 Figure 2Early-CAR-T cells are comparable in vitro to conventional CAR-T cells in terms of killing but have a better proliferation capacity that persists after chronic stimulation. The long-term stimulated early- CAR-T cells maintain their memory phenotype and upregulated PD-1. A) Chromium release assay against the LeY+ cell line (OVCAR3), data for one donor representative of 3 other donors. B) Cytokine secretion evaluated by CBA after coculture with the LeY+ cell line (OVCAR3) or with the LeY- cell line (MDA-MB435). C) Division index of CAR-T cells quantified with CTV. D) Evaluation of the differentiation, proliferation and cytotoxicity of the CAR-T cells after chronic stimulationAbstract 126 Figure 3Early-CD4+- CAR-T cells are critical for the proliferation capacity of the Early-CD8+-CAR-T cells. A) Scheme of the CD4-depletion protocol to compare Early-CD8-CAR-T proliferation with or without CD4-T cells. B) Division index of CD4-depleted Early-CAR-T cells, CD8-T cells from bulk Early-CAR-T cells, and from CD4+ T cells from bulk Early-CAR-T-cells quantified with CTVAbstract 126 Figure 4Early-CAR-T cells show in vivo a better persistence and a better proliferation capacity associated with a better tumoral control. A) Design of the in vivo experiment (n=7 mice per group) B) T-cell persistence in peripheral blood was measured by FACS. C) Speakman correlation (Day 13) between Tumor size and% CAR-T- cells. D) Tumor kinetic and Kaplan-Meier analysis of survival of OVCAR-bearing NSG mice treated with Conventional CAR-T cells, or Early-CAR-T cells or Low-dose of Early-CAR-T cellsAbstract 126 Figure 5Anti-PD1 treatment enhance the efficacy of the Early-CAR-T cells. A) Upregulation of PD-L1 on OVCAR3 when expanded in the supernatant from co-culture of OVCAR3 with LeY-CAR-T cells. B) Design of the in vivo experiment (n=7 mice per group). C) T-cell persistence, phenotype and anti-human IgG4 in peripheral blood were measured by FACS. D) Tumor kinetic of OVCAR-bearing NSG mice treated with Early-CAR-T cells or Early-CAR-T cells + NivolumabConclusionsOur early CAR-T cells have better cytokine secretion and proliferation than conventional CAR-T cells. Early CAR-T cells also have superior anti-tumor efficacy in vivo, they have better persistence and maintain the circulating T cell memory pool. Importantly, low dose early-LeY-CAR-T cells combined with anti-PD1-treatment leads to complete clearance of LeY+ solid tumors in vivo. The early CAR-T cell production protocol is directly translatable for improving CAR-T cell efficacy in clinical trials for patients with solid tumors.