Producing Medicinal Phytochemicals from Phyllanthus acuminatus in Plant - Cell Suspension Cultures

Pharmaceutical use is not feasible for important medicinal compounds derived from certain plant materials, including Phyllanthus acuminatus roots, due to their low natural abundance. New technologies in non-traditional biomass generation are needed to produce these remarkable natural compounds. Therefore, this article describes a methodology for establishing Phyllanthus acuminatus plant-cell suspensions from callus cultures: An evaluation on inoculum concentration and agitation speed displayed significant changes in plant cell growth kinetics. It was determined that treatment with 2 g of inoculum in 25 mL of medium and 100 rpm agitation creates the best conditions for generating thick cell suspensions. Likewise, treatment with 2 g of inoculum and 120 rpm agitation produces the best conditions for establishing fine cell suspensions. Phytochemical comparison through high-resolution mass spectrometry of P. acuminatus roots and plant cell suspension extracts confirmed presence in the plant cell culture of multiple phyllantostatins of pharmaceutical interest. Here, we demonstrate that Phyllanthus acuminatus can be cultured in plant cell suspensions to produce secondary metabolites of medical interest – technology that could be scaled up for implementation in industrial bioprocesses.

Follicular Lymphoma Tumor-Cell Transcriptional Programs Associate with Distinct Somatic Alterations and Tumor-Immune Microenvironments

Background: Follicular lymphoma (FL) exhibits significant clinical, cellular, molecular, and genetic heterogeneity. Our understanding of FL biology and molecular classifications of FL, to date, has largely been driven by pathologic classification (Grade 1-3b), genetic classification (m7-FLIPI), or gene expression profiling (IR-1/2; Huet-23), along with limited studies on small cohorts or targeted panels. In order to further understand the biological underpinnings and complexity of FL, large-scale and integrated whole exome sequencing (WES) and RNA sequencing (RNAseq) studies are needed. Using a highly-annotated cohort of 93 FL tumors with matched RNAseq, WES, and CyTOF data, we have explored the transcriptomic signature of purified FL B cells and identified unique molecular subsets that are defined by distinct pathway activation, immune content, and genomic signatures. Methods: Frozen cell suspensions from 93 untreated FL (Grade 1-3b) patients' tumor biopsies, enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE, were used for the study. DNA was isolated from whole tumor cell suspensions, and RNA was isolated from both whole tumor and B cell-enriched cell suspensions. RNAseq and WES were performed in the Mayo Clinic Genome Analysis Core. RNAseq and WES data were processed using a standard pipeline and novel driver genes were identified using Chasm+ driver analysis. Copy number variants were identified from WES data using GISTIC 2.0. NMF clustering and single sample gene set testing for B cell lineage and tumor microenvironment (TME) signatures were performed in R using the NMF and singscore packages. Results: Unsupervised clustering of RNAseq data identified three distinct expression programs which separated patient B cell samples into 3 groups: Group 1 (G1, n=20), Group 2 (G2, n=24), Group 3 (G3, n=43). While no clinical attributes were defined by any single group, G1 consisted of cases that had less aggressive characteristics (63% Stage I-II, 79% FLIPI 0-1). To identify unique transcriptional pathways driving the three expression programs, we scored gene level contributions to NMF expression programs and employed gene set enrichment analysis. This revealed significant pathway association with type-I IFN signaling (G1), DNA repair and stress response (G2), and epigenetic modulation (G3) as differentiating programs between the 3 groups (FDR q<0.001). VIPER master regulator activity inferencing revealed that these pathways were likely being controlled by differential activity in NF-kB, IRFs, STAT1, BCL6, and FOXO1. Each program significantly enriched for, but were not defined by, portions of specific germinal center programs, such as pre-memory (G1), light-zone-to-dark-zone transition (G2), and a pre-light-zone intermediate (G3). We next assessed the connection between B cell programs and the tumor microenvironment (TME) using available paired CyTOF data on 67 cases, which revealed an active TME in G1, with an abundance of CD8 T cell and NK cell populations, a wide variety of immune content in G2 that consisted mostly of Tfh and myeloid cells, and a poorly populated immune compartment in G3 compared to G1 and G2. Finally, somatic driver mutations and copy number alterations from WES were identified and associated with the three clusters. The three groups distinguished themselves by significant enrichment of copy number alterations (TNFAIP3-loss , 1q23-gain, 1q32-gain) in G2, while 10q-loss and mutations in BCL2 and chromatin modifiers (KMT2D and CREBBP) enriched in G3. G1, overall, had lower alteration burden and had weak associations with any specific alterations, suggesting an alternative mechanism for driving the G1 program. Conclusion: In this study, we have identified three unique FL tumor B cell groups, defined by specific transcriptional programs. Pathways such as inflammation, DNA damage response, and chromatin modification contribute most to the differences between B cell samples and group membership. Additionally, each program associated with specific genetic events as well as TME composition, highlighting potential drivers of these B cell states. This study improves the understanding of the mechanisms driving FL tumors and motivates further investigation into transcriptional consequences of genetic events as well as potential tumor intrinsic factors that may influence the TME. Figure 1 Figure 1. Disclosures Maurer: BMS: Research Funding; Genentech: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding. Rimsza: NanoString Technologies: Other: Fee-for-service contract. Link: MEI: Consultancy; Genentech/Roche: Consultancy, Research Funding; Novartis, Jannsen: Research Funding. Habermann: Tess Therapeutics: Other: Data Monitoring Committee; Seagen: Other: Data Monitoring Committee; Incyte: Other: Scientific Advisory Board; Morphosys: Other: Scientific Advisory Board; Loxo Oncology: Other: Scientific Advisory Board; Eli Lilly & Co.,: Other: Scientific Advisor. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. King: Celgene/BMS: Research Funding. Cerhan: Genentech: Research Funding; Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding. Novak: Celgene/BMS: Research Funding.

BIOM-10. PRECLINICAL PLATFORM FOR THE IDENTIFICATION OF DEUTERIUM MAGNETIC RESONANCE SPECTROSCOPY-BASED BIOMARKERS OF BRAIN TUMOR METABOLISM

Metabolic reprogramming is a fundamental hallmark of cancer, which can be exploited for non-invasive tumor imaging. Deuterium magnetic resonance spectroscopy (2H-MRS) recently emerged as a novel, translational method of interrogating flux from 2H-labeled substrates to metabolic products. However, to date, preclinical studies have been performed in vivo, an endeavor which suffers from low-throughput and potential wastage of animal life, especially when considering studies of treatment response. Developing in vitro assays for monitoring metabolism of 2H-labeled substrates will enhance throughput, lead to the rapid evaluation of new 2H-based probes and enable identification of treatment response biomarkers, thereby allowing the best 2H-based probes to be translated for further in vivo assessment. The goal of this study was to develop a preclinical cell-based platform for quantifying metabolism of 2H-labeled probes in brain tumor models. Since the Warburg effect, which is characterized by elevated glycolytic production of lactate, is a metabolic phenotype of cancer, including brain tumors, we examined metabolism of 2H-glucose or 2H-pyruvate in patient-derived glioblastoma (GBM6) and oligodendroglioma (BT88) cells and compared to normal human astrocytes (NHACONTROL). Following incubation in media containing [6,6’-2H]glucose or [U-2H]pyruvate, 2H-MR spectra obtained from live cell suspensions showed elevated 2H-lactate production in GBM6 and BT88 cells relative to NHACONTROL. Importantly, 2H-lactate production from [6,6’-2H]glucose or from [U-2H]pyruvate was reduced in GBM6 or BT88 cells subjected to irradiation and temozolomide, which is standard of care for glioma patients, pointing to the utility of this method for detecting response to therapy. Collectively, we have, for the first time, demonstrated the ability to quantify metabolism of 2H-MRS probes in live cell suspensions and validated the utility of our assay for differentiating tumor from normal cells and assessing response to therapy. Our studies will expedite the identification of novel 2H-MRS probes for imaging brain tumors and potentially other types of cancer.

889 DuoBody®-CD3x5T4 induces efficient T-cell activation and killing of patient-derived head and neck cancer cells in vitro and ex vivo

Background5T4, also known as trophoblast glycoprotein, is expressed in many solid cancers, including non-small cell lung cancer, triple-negative breast cancer, bladder, esophageal, prostate, uterine and head and neck squamous cell carcinomas (HNSCCs). DuoBody-CD3x5T4 is a CD3 bispecific antibody that efficiently induces T-cell mediated cytotoxicity of 5T4-positive tumor cells. Currently, DuoBody-CD3x5T4 is being evaluated in a first-in-human clinical trial (NCT04424641) in solid cancers in partnership between Genmab and AbbVie. In this study we explored the preclinical mechanism-of-action of DuoBody-CD3x5T4 in vitro and ex vivo, using HNSCC as a case study.Methods5T4 protein expression in HNSCC tumor specimens was determined by immunohistochemistry (IHC) and flow cytometry. T-cell mediated cytotoxicity and T-cell activation induced by DuoBody-CD3x5T4 were studied in co-cultures of healthy donor T cells and patient-derived HNSCC cell lines in vitro. Lastly, the capacity of DuoBody-CD3x5T4 to activate tumor-infiltrating lymphocytes (TILs) was analyzed in freshly dissociated 5T4-expressing HNSCC tumor specimens ex vivo.ResultsIHC analysis confirmed expression of 5T4 in HNSCC oral biopsies, including specimens from primary tumors, recurrent tumors and lymph node metastases. Patient-derived HNSCC cell lines (n=22) demonstrated 5T4 expression on the plasma membrane, ranging from 10,000 - 61,000 5T4 molecules per cell. Moreover, 5T4 expression was evident on EGFR+CD45- tumor cells in single-cell suspensions from freshly dissociated HNSCC biopsies, independent of the tumor site. DuoBody-CD3x5T4 demonstrated potent, target-dependent cytotoxicity in vitro in co-cultures of healthy donor T cells and patient-derived HNSCC cell lines across the range of 5T4 expression levels tested. Tumor cell kill was associated with CD4+ and CD8+ T-cell activation and granzyme B secretion. Importantly, DuoBody-CD3x5T4 induced potent activation of autologous TILs in single-cell suspensions from freshly dissociated HNSCC biopsies. Notably, T-cell activation (as assessed by expression of CD69, CD25 and CD137) was also observed in PD-1+ TILs, suggesting that DuoBody-CD3x5T4 was able to engage antigen-experienced T cells in the tumor microenvironment. In this autologous assay, preliminary data showed that 5T4-expressing HNSCC tumor cells were specifically eradicated.Conclusions5T4 was broadly expressed in HNSCC cell lines, tumor biopsies and primary tumor cell suspensions. DuoBody-CD3x5T4 activated healthy donor T cells in co-cultures with patient-derived HNSCC cell lines, resulting in secretion of granzyme B and efficient tumor cell kill. In single-cell suspensions from freshly dissociated 5T4+ HNSCC biopsies, DuoBody-CD3x5T4 activated autologous CD4+ and CD8+ TILs, including PD-1+ TILs. This dataset adds to the preclinical evidence for targeting 5T4-expressing solid cancers with DuoBody-CD3x5T4.Ethics ApprovalWritten informed consent was obtained from all patients from whom fresh tumor biopsies were used for research, as part of the HNcol protocol at the Department of Otolaryngology|Head and Neck Surgery of Amsterdam UMC (VUmc) as approved by the Institutional Review Board (2008.071|A2016.035). Archival FFPE specimens were used for scientific research in agreement with the medical ethical guidelines described in the Code of Conduct for Proper Secondary Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies (Federa) in accordance with the Declaration of Helsinki and after Biobank approval (BUP2019-74).

Feasibility of using digital confocal microscopy for cytopathological examination in clinical practice

Optical imaging modalities are emerging as digital microscopy tools for tissue examination. The investigation of these techniques for potential applications in anatomic pathology practice has focused primarily on surgical pathology and has not included cytopathological specimens. We evaluated the feasibility of using digital confocal microscopy (CM) to examine cytopathological specimens. Smears and cell suspensions collected in RPMI solution were prepared from tissue scrapes obtained from surgical resections of breast, lung, liver, and kidney. Air-dried smears and cell pellets obtained from centrifugation of the cell suspensions were stained with 0.6 mM acridine orange and imaged with a CM platform. After completion of imaging, the smears were stained with Diff-Quik (DQ), and cell pellets were routinely processed, embedded in paraffin wax, cut, and stained with hematoxylin and eosin (H&E). We evaluated the mean time to acquire digital CM images; quality of images based on the extent of tissue recognition (0%, grade 0; 1–19%, grade 1; 20–50%, grade 2; >50%, grade 3); comparison of images with DQ- and H&E-stained specimens; and ability to make specific diagnoses. We imaged 91 smears and 52 cell pellets and acquired digital CM images within 2–3 min, with 92% and 88% of images, respectively, from smears and cell pellets showing grade 3 quality. On the basis of CM images, 8 smears (9%) and 7 cell pellets (14%) were categorized as benign, and 83 (91%) and 45 (88%), respectively, as malignant. Specific diagnoses were made by using digital CM images of smears and cell pellets that matched accurately with corresponding DQ- and H&E-stained preparations. The results of our first feasibility study clearly indicated the utility of CM as a next-generation digital microscopy tool for evaluating cytology specimens. Prospective clinical studies are warranted for validating our findings for potential incorporation into cytopathological clinical practice.