Unsorted Cells Research Articles

Leveraging Xenobiotic-Responsive Cancer Stemness in Cell Line-Based Tumoroids for Evaluating Chemoresistance: A Proof-of-Concept Study on Environmental Susceptibility.

Emerging evidence suggests that cancer stemness plays a crucial role in tumor progression, metastasis, and chemoresistance. Upon exposure to internal or external stress, ribosomes stand sentinel and facilitate diverse biological processes, including oncological responses. In the present study, ribosome-inactivating stress (RIS) was evaluated for its modulation of cancer cell stemness as a pivotal factor of tumor cell reprogramming. Based on the concept of stress-responsive cancer cell stemness, we addressed human intestinal cancer cell line-based off-the-shelf spheroid cultures. Intestinal cancer cell line-based spheroids exhibited heightened levels of CD44+CD133+ cancer stemness, which was improved by chemical-induced RIS. Further evaluations revealed the potential of these stress-imprinted spheroids as a platform for chemoresistance screening. Compared to adherent cells, stemness-improved spheroid cultures displayed reduced apoptosis in response to 5-fluorouracil (5-FU), a frontline chemotherapeutic agent against colorectal cancer. Moreover, serial subcultures with repeated RIS exposure maintained and even enhanced cancer stemness and chemoresistance patterns. In particular, isolated CD44+CD133+ cancer stem cells exhibited higher chemoresistance compared to unsorted cells. To elucidate the mechanisms underlying RIS-induced stemness, RNA-seq analysis identified Wnt signaling pathways and stemness-associated signals as notable features in spheroids exposed to RIS. Loss-of-function studies targeting connective tissue growth factor (CTGF), a negative regulator of Wnt signaling, revealed that CTGF-deficient spheroids exhibited improved cancer stemness and resistance to 5-FU, with RIS further enhancing these effects. In conclusion, this proof-of-concept study demonstrates the feasibility of leveraging stress-responsive cancer stemness for the development of spheroid-based platforms for chemoresistance evaluation and elucidation of pathophysiological processes of colorectal tumorigenesis under environmental stress.

Reproducible 3D culture of multicellular tumor spheroids in supramolecular hydrogel from cancer stem cells sorted by sedimentation field-flow fractionation

Three-dimensional (3D) cancer models, such as multicellular tumor spheroids (MCTS), are biological supports used for research in oncology, drug development and nanotoxicity assays. However, due to various analytical and biological challenges, the main recurring problem faced when developing this type of 3D model is the lack of reproducibility. When using a 3D support to assess the effect of biologics, small molecules or nanoparticles, it is essential that the support remains constant over time and multiples productions. This constancy ensures that any effect observed following molecule exposure can be attributed to the molecule itself and not to the heterogeneous properties of the 3D support. In this study, we address these analytical challenges by evaluating for the first time the 3D culture of a sub-population of cancer stem cells (CSCs) from a glioblastoma cancer cell line (U87-MG), produced by a SdFFF (sedimentation field-flow fractionation) cell sorting, in a supramolecular hydrogel composed of single, well-defined molecule (bis-amide bola amphiphile 0.25% w/v) with a stiffness of 0.4 kPa. CSCs were chosen for their ability of self-renewal and multipotency that allow them to generate fully-grown tumors from a small number of cells.The results demonstrate that CSCs cultured in the hydrogel formed spheroids with a mean diameter of 336.67 ± 38.70 µm by Day 35, indicating reproducible growth kinetics. This uniformity is in contrast with spheroids derived from unsorted cells, which displayed a more heterogeneous growth pattern, with a mean diameter of 203.20 ± 102.93 µm by Day 35. Statistical analysis using an unpaired t-test with unequal variances confirmed that this difference in spheroid size is significant, with a p-value of 0.0417 (p < 0.05).These findings demonstrate that CSC-derived spheroids, when cultured in a well-defined hydrogel, exhibit highly reproducible growth patterns compared to spheroids derived from unsorted cells, making them a more reliable 3D model for biological research and drug testing applications.

Computing cell state discriminates the aberrant hematopoiesis and activated microenvironment in Myelodysplastic syndrome (MDS) through a single cell genomic study

BackgroundMyelodysplastic syndrome (MDS) is a complicated hematopoietic malignancy characterized by bone marrow (BM) dysplasia with symptoms like anemia, neutropenia, or thrombocytopenia. MDS exhibits considerable heterogeneity in prognosis, with approximately 30% of patients progressing to acute myeloid leukemia (AML). Single cell RNA-sequencing (scRNA-seq) is a new and powerful technique to profile disease landscapes. However, the current available scRNA-seq datasets for MDS are only focused on CD34+ hematopoietic progenitor cells. We argue that using entire BM cell for MDS studies probably will be more informative for understanding the pathophysiology of MDS.MethodsFive MDS patients and four healthy donors were enrolled in the study. Unsorted cells from BM aspiration were collected for scRNA-seq analysis to profile overall alteration in hematopoiesis.ResultsStandard scRNA-seq analysis of unsorted BM cells successfully profiles deficient hematopoiesis in all five MDS patients, with three classified as high-risk and two as low-risk. While no significant increase in mutation burden was observed, high-risk MDS patients exhibited T-cell activation and abnormal myelogenesis at the stages between hematopoietic stem and progenitor cells (HSPC) and granulocyte–macrophage progenitors (GMP). Transcriptional factor analysis on the aberrant myelogenesis suggests that the epigenetic regulator chromatin structural protein-encoding gene HMGA1 is highly activated in the high-risk MDS group and moderately activated in the low-risk MDS group. Perturbation of HMGA1 by CellOracle simulated deficient hematopoiesis in mouse Lineage-negative (Lin-) BM cells. Projecting MDS and AML cells on a BM cell reference by our newly developed MarcoPolo pipeline intuitively visualizes a connection for myeloid leukemia development and abnormalities of hematopoietic hierarchy, indicating that it is technically feasible to integrate all diseased bone marrow cells on a common reference map even when the size of the cohort reaches to 1,000 patients or more.ConclusionThrough scRNA-seq analysis on unsorted cells from BM aspiration samples of MDS patients, this study systematically profiled the development abnormalities in hematopoiesis, heterogeneity of risk, and T-cell microenvironment at the single cell level.

Abstract 5458: Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta

Abstract Transcriptional plasticity of cancer cells promotes tumor progression and treatment resistance via generation of phenotypically distinct cell states. We previously showed that Ewing sarcoma (EwS) cells exhibit inter- and intra-tumoral transcriptional heterogeneity and that they undergo state changes in response to cues from the tumor microenvironment (TME). More recently, we identified phenotypically distinct EwS tumor cell subpopulations in vivo that deposit pro-tumorigenic extracellular matrix (ECM) proteins, adopting similar functions to cancer-associated fibroblasts (CAFs). Here, we sought to identify the upstream regulators of these CAF-like EwS cells with the goal of defining molecular mechanisms that control EwS plasticity and creation of ECM-remodeling states. Integration of single cell proteogenomic sequencing (CITE-seq) with immunofluorescence and digital spatial profiling of cell line xenografts and patient tumors validated the cell-surface protein CD73 (NT5E) as a biomarker of ECM-secreting EwS cell subpopulations in vitro and in vivo. The transcriptomes of CD73+ CAF-like cells were compared to isogenic CD73- cells (n=9 cell lines) and highly significant enrichment of gene ontology terms related to TGF-beta signaling (p.adj = &amp;lt;4.4x10-14) was noted. EwS cells were previously reported to be non-responsive to TGF-beta due to profound transcriptional repression of the TGF-beta type 2 receptor (TGFBR2) by EWS::FLI1. In support of this, TGFBR2 expression was low in bulk measurements of EwS cells. However, TGFBR2 mRNA and surface protein expression were significantly increased in CD73+ subpopulations. Exposure of EwS cells to TGF-beta ligands (TGFB1 or TGFB2) strongly induced the CAF-like gene signature, including ECM genes BGN, COL1A1, SPARC, and TNC (p. adj &amp;lt; 8.5x10-5), and this was augmented in CD73+ cell populations. Transduction of EwS cells with dominant negative-TGFBR2 inhibited both basal and TGF-beta-induced expression of CAF-like signature genes and blocked SMAD2 phosphorylation. To test the functional significance of the TGF-beta response, unsorted EwS cells were plated in 2D or as tumor spheroids in collagen I-rich 3D cultures and exposed to TGFB1 or SB-505124, a TGF-beta inhibitor. TGFB1 treatment had no effect on cell viability or proliferation in 2D but increased invasion into collagen in 3D. Inhibition of TGF-beta reduced CAF-like gene expression in 2D cultures and diminished both viability and invasion in 3D. Finally, our studies revealed that NT5E (CD73) is a TGF-beta induced target in EwS cells and that CD73+ cells upregulate expression of TGFB2. Thus, an autocrine signaling circuit is created in which TGF-beta activity in CAF-like tumor cell subpopulations is sustained by CD73+ tumor cell-derived ligand. Together these studies demonstrate that TGF-beta is a key determinant of cell plasticity and ECM remodeling in EwS. Citation Format: Emma Wrenn, April A. Apfelbaum, Katherine Braun, Erin R. Rudzinski, Xuemei Deng, Aya Miyaki, Trisha Lipson, Nicolas M. Garcia, Virginia J. Hoglund, Elizabeth R. Lawlor. Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5458.

Abstract 5261: Immunologic signatures of peripheral blood cells in patients with advanced pancreatic cancer under lenvatinib-pembrolizumab maintenance therapy

Abstract Background: The treatment of pancreatic cancer is limited by chemotherapy toxicities which impacts quality of life. This is a phase 2 clinical trial of lenvatinib and pembrolizumab as maintenance therapy in patients with advanced pancreatic cancer. We observed preclinical activity of this combination in pancreatic mouse models. We hypothesize that this combination may address unmet needs to minimize toxicities associated with standard treatments, stimulate anti-tumor T cell immunity, and improve the progression free and overall survival. Exploratory studies aim to characterize immune-related systemic changes in peripheral blood over the course of therapy and to identify global changes in expression of tumor-related genes in needle biopsy samples of liver metastatic lesions. Methods: Multiplex gene expression analysis of unsorted peripheral blood mononuclear cells (PBMC) was performed using PanCancer Immune Profiling Panel on NanoString nCounter platform. PBMC samples from pre- through week 32 of treatment were assessed for differential expression of 730 immune profiling genes defining T cell functions and associated immune categories. Results: PBMC immune profiling from 4/5 patients show a distinctive pattern of increased activation of T cells during the first 9 weeks of treatment followed by a sharp inhibition starting at weeks 9-12 after 3-4 cycles of treatment (CD3E, CD247, LCK, TBX1, TCF7, STAT4, IL2RG). Subsequent upregulation of macrophage-derived scavenger, inflammatory, and immunosuppressive gene expression patterns continued for the remainder of treatment (CD14, MSR1, MARCO, CD68, TGFB1, IL10RA). The median PFS of first 10 patients was 21.5 (9-32) weeks. Conclusions: Immunotherapy resistance may be related to rapidly developing terminal differentiation and unresponsiveness of T cells in parallel with upregulation of macrophage-derived inflammatory and immunosuppressive processes. Studies are on-going to prevent inhibition of the T-cells. Citation Format: Vincent Chung, Ferdynand Kos, Edwin Manual, Shannon Dempsey, Don J. Diamond. Immunologic signatures of peripheral blood cells in patients with advanced pancreatic cancer under lenvatinib-pembrolizumab maintenance therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5261.

Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.

Predicting leukemic transformation in myelodysplastic syndrome using a transcriptomic signature

Background: For prediction on leukemic transformation of MDS patients, emerging model based on transcriptomic datasets, exhibited superior predictive power to traditional prognostic systems. While these models were lack of external validation by independent cohorts, and the cell origin (CD34+ sorted cells) limited their feasibility in clinical practice.Methods: Transformation associated co-expressed gene cluster was derived based on GSE58831 (‘WGCNA’ package, R software). Accordingly, the least absolute shrinkage and selection operator algorithm was implemented to establish a scoring system (i.e., MDS15 score), using training set (GSE58831 originated from CD34+ cells) and testing set (GSE15061 originated from unsorted cells).Results: A total of 68 gene co-expression modules were derived, and the ‘brown’ module was recognized to be transformation-specific (R2 = 0.23, p = 0.005, enriched in transcription regulating pathways). After 50,000-times LASSO iteration, MDS15 score was established, including the 15-gene expression signature. The predictive power (AUC and Harrison’s C index) of MDS15 model was superior to that of IPSS/WPSS in both training set (AUC/C index 0.749/0.777) and testing set (AUC/C index 0.933/0.86).Conclusion: By gene co-expression analysis, the crucial gene module was discovered, and a novel prognostic system (MDS15) was established, which was validated not only by another independent cohort, but by a different cell origin.

Ultrasensitive detection and monitoring of central nervous system tumors from plasma using personalized whole-genome ctDNA profiling.

2064 Background: Patients with the central nervous system (CNS) tumors are largely followed up by imaging. Current plasma-based liquid biopsy techniques have limited utility in neuro-oncology due to a low circulating cell-free tumor DNA (ctDNA) burden, blood-brain barrier, and low number of mutations in coding regions. Whole genome sequencing (WGS)-derived patient specific mutational signature from a matched tumor-normal WGS can provide a personalized, highly sensitive and specific approach to detect mutations in ctDNA and provide blood-based monitoring in brain tumor patients. Furthermore, it can be performed on lower amount of peripheral blood since WGS requires less sequencing depth compared to targeted ctDNA panels. Methods: We have profiled a cohort of 28 extra- and intra-axial adult and pediatric brain tumors including adult and pediatric low- and high-grade glioma (9), meningiomas (11), medulloblastomas (5), ependymomas (2), neurocytoma (1). Tumor DNA was extracted from archival pathology tissue, normal DNA from unsorted white blood cells, and ctDNA from 1-2 mL of post-surgery plasma. WGS was performed with 40x coverage for Tumor-Normal DNA and 20x for ctDNA. Using WGS of matched Tumor-Normal and plasma samples, we derived a personalized mutational pattern using SNVs, indels, and copy numbers for quantification and ultra-sensitive detection of ctDNA in plasma samples. An AI-based error suppression model was implemented to filter out the noise in the cell-free DNA (cfDNA) while the personalized mutational signature was used to detect the ctDNA in the cfDNA and to amplify the somatic signal to determine the Tumor Fraction at the time of diagnosis, during the therapy or surveillance period. The ctDNA Tumor Fraction (TF) was compared to the clinical status and MRI-based imaging. Results: All subtypes of brain tumors contained enough mutations to derive personalized mutational signatures. Most mutations were distributed in the noncoding DNA. TF correlated with clinical status and with the disease course on imaging at given time points reaching a 10-4 minimal residual disease detection sensitivity. We were able to detect ctDNA across all WHO grades ranging from WHO 1 meningioma to WHO 4 glioblastoma and medulloblastoma. Furthermore, we were able to detect tumor-specific copy number aberrations such as MYCN amplification in plasma samples and mutational signatures. Conclusions: Here we demonstrate that patient-specific WGS tumor signature in ctDNA from plasma can be used for sensitive monitoring of adults and children with primary low- and high-grade CNS tumors.

Comprehensive CRISPR-enabled functional genomics profiling to identify targetable cancer genes in primary acute myeloid leukemia (AML).

3088 Background: Acute myeloid leukemia (AML) is a life-threatening hematopoietic malignancy affecting both older and younger patient populations. Despite the approval of multiple new therapeutics during the past decade, including new targeted agents, treatment outcomes for AML remain poor, with &lt; 10% 5-year survival in patients over 60 years of age, and &lt; 50% 5-year survival in younger patients. Also, comprehensive genomic profiling (CGP) for common molecular lesions (e.g., FLT3 mutation) incompletely stratifies the patient population, indicating that a more comprehensive approach to personalized medicine could be of significant clinical benefit. To this end we developed Fx Heme, an in vitro functional genomics platform using CRISPR/Cas9 technology in primary cancer patient samples. Previously, we have shown it to stratify clinical response in AML patients treated with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g., GCLAM). Here we profile an expanded cohort to systematically characterize the AML functional genomics landscape and identify potential new targets for AML drug development. Methods: Unsorted primary tumor cells from patient samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library which was designed to inhibit expression of genes encoding the targets of FDA-approved targeted oncology drugs, as well as internal negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated for each gene to generate gene-level statistics, enabling identification of gene dependencies. Results: Comprehensive CRISPR/Cas9 functional genomics profiling was carried out retrospectively on pre-therapy samples from 59 AML patients. There were no false positives identified among negative controls. Functional dependencies on multiple genes were observed, with approximately half of patients (n=31; 52.5%) showing at least one gene dependency. We observed on average 6.7 dependencies per positive sample, with 5 of them revealing more than 10 dependencies. In addition to the targets of drugs approved to treat AML (FLT3, 10.2%; IDH1/2, 3.4%; SMO, 3.4%), a diversity of targetable genes for new therapeutic approaches to AML were identified across multiple known oncogenic signaling pathways. The prevalence of these recurrent gene dependencies ranged from 3.3% to 23.7% in our AML cohort. Conclusions: In summary, comprehensive functional genomic profiling using Fx Heme provides an effective platform to identify therapeutic opportunities in AML based on dependencies identified for targets of drugs not currently used to treat AML. These insights are now being utilized to develop additional treatment options and more effective personalization of treatment for AML.

Transcriptome and Proteome Profiling of Primary Human Gastric Interstitial Cells of Cajal Predicts Pacemaker Networks

Interstitial cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells required for normal GI motility. Dysfunctions in ICC have been reported in patients with GI motility disorders, such as gastroparesis, who exhibit debilitating symptoms and greatly reduced quality of life. While the proteins, calcium-activated chloride channel anoctamin-1 (ANO1) and the receptor tyrosine kinase (KIT), are known to be expressed by human ICC, relatively little is known about the broad molecular circuitry underpinning human ICC functions. The present study therefore investigates the transcriptome and proteome of ANO1-expressing, KITlow/CD45-/CD11B- ICC obtained from primary human gastric tissue. Excess human gastric tissue resections were obtained from sleeve gastrectomy patients. ICC were purified using fluorescence-activated cell sorting (FACSorting). Then, ICC were characterized by using immunofluorescence, real-time polymerase chain reaction, RNA-sequencing and mass spectrometry. Compared to unsorted cells, real-time polymerase chain reaction showed the KITlow/CD45-/CD11B- ICC had: a 9-fold (P < 0.05) increase in ANO1 expression; unchanged KIT expression; and reduced expression for genes associated with hematopoietic cells (CD68, > 10-fold, P < 0.001) and smooth muscle cells (DES, > 4-fold, P < 0.05). RNA-sequencing and gene ontology analyses of the KITlow/CD45-/CD11B- cells revealed a transcriptional profile consistent with ICC function. Similarly, mass spectrometry analyses of the KITlow/CD45-/CD11B- cells presented a proteomic profile consistent with ICC activities. STRING-based protein interaction analyses using the RNA-sequencing and proteomic datasets predicted protein networks consistent with ICC-associated pacemaker activity and ion transport. These new and complementary datasets provide a valuable molecular framework for further understanding how ICC pacemaker activity regulates smooth muscle contraction in both normal GI tissue and GI motility disorders.

Abstract 1062: A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme

Abstract AML is the most common acute leukemia in adults over 50 years of age. Current therapeutic approaches are focused on high or low dose chemotherapy in the front line setting, with use of molecularly targeted agents in unfit patients or the relapsed/refractory setting. However, routine genomic profiling for common molecular lesions (e.g. FLT3 mutation) incompletely stratifies the target patient population, and more personalized and comprehensive approaches could be of significant clinical benefit. To this end, we have developed an in vitro functional genomics platform for the systematic characterization of gene dependency on drug targets using CRISPR/Cas9 technology. Methods: Retrospective pre-therapy samples were obtained for a cohort of AML patients who subsequently went onto therapy with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g. GCLAM). Unsorted primary tumor cells from these samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library. This customized sgRNA library (Fx Heme) was designed to inhibit expression of genes encoding the targets of all FDA-approved oncology drugs, as well as internal positive and negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Dependency on targets of sorafenib was compared to clinical outcome (complete response (CR) without minimal residual disease (MRD), incomplete response, CR with MRD, or refractory) following treatment with sorafenib plus chemotherapy. Results: Among the 22 AML patients analyzed, 14 reached CR and 8 were classified as incomplete responders (refractory or MRD). Gene dependency on sorafenib targets including FLT3, KIT, PDGFR, and all RAF isoforms was frequently observed (12/22 cases). Among cases indicating any sorafenib target gene dependency, 5/12 (42%) demonstrated dependence on 2 or more sorafenib target genes. Overall, Fx Heme achieved 78.6% sensitivity, 87.5% specificity, and 91.7% PPV for prediction of observed clinical outcome. In contrast, stratification based on FLT3 mutation (internal tandem duplication or tyrosine kinase domain mutation) demonstrated 64.3% sensitivity, 50% specificity, 69.2% PPV. In summary, our results establish feasibility of CRISPR-based Fx Heme comprehensive functional genomic profiling for AML precision medicine, and validate this approach in a retrospective study cohort. Functional genomics may contribute to more effective personalization of AML treatment by uncovering drug target dependencies not readily identified by conventional genomic profiling, in an unbiased, comprehensive and patient-specific fashion. Citation Format: Stephane Corbel, Karl Hodel, Kathleen Tran, Maci Meyers, Jing Tong, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath. A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1062.

Stem Cell-Derived Gene Expression Scores Predict Survival and Leukemic Transformation in Myelofibrosis

Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) with survival outcomes ranging from months to years and variable risk of transformation to acute myeloid leukemia. Allogeneic bone marrow transplantation (BMT) can be curative but is associated with high treatment-related morbidity and mortality, therefore accurate risk stratification is important to guide clinical decision making in MF. Current risk prediction models use clinical and/or genomic features but do not consider the properties of the disease-driving stem cell population. Here, we use transcriptomic variation corresponding to both intra- and inter-patient heterogeneity among MF stem cells to generate novel gene expression-based scores predictive of survival and leukemic transformation in MF. To train and validate novel prognostic scores in MF, we identified 358 patients from an MPN registry at the Princess Margaret Cancer Centre (ClinicalTrials.gov Identifier: NCT02760238) from whom peripheral blood (PB) cells were collected near the date of MF diagnosis. All patients were diagnosed with either primary, post-PV, post-ET or pre-fibrotic MF with clinical follow-up of up to 12.2 years. RNA was extracted from unsorted PB mononuclear cells and RNA sequencing (RNAseq) was performed at an average depth of 50 million reads per sample. We randomly split our MF cohort into training (70%; n=250) and test sets (30%; n=108) and utilized a repeated nested cross validation approach together with statistical regression, to generate and assess the performance of models to predict survival within the training set. We tested 36,000 models derived from 36 initial MF-related genesets, ranging from stem-cell specific genesets to the whole transcriptome. The most accurate models by cross validation (median multivariable p-value = 6e-5) were produced from our retrospective identification of highly variable genes in single-cell RNAseq data derived from 82,255 Lin-CD34+ MF stem and progenitor cells across 15 patients (Psaila et al., 2020). Thus, features of intra- and inter-patient heterogeneity among MF stem and progenitor cells proved to be the most relevant for predicting survival. From these features, we derived our final model calculated as the weighted sum of gene expression across 24 genes (termed MPN24). We categorized patients with MPN24 scores above or below the training cohort median as MPN24 high or low, respectively. This model was validated in the test set, with high and low score patients experiencing 5-year survival rates of 71% [95% CI 57-88%] and 21% [95% CI 9%-52%], respectively, when censored at time of BMT (HR = 5.3 [95% CI 2.6-10.5]; p=2.1e-6) (Figure 1A). MPN24 retained independent prognostic value in multivariable analysis incorporating age, sex, DIPSS category, ECOG status, fibrosis grade, constitutional symptoms, and PB blast percentage (adjusted HR = 5.7 [95% CI 2.2-14]; p = 3e-4). Importantly, DIPSS classification remained a significant covariate, indicating that MPN24 and DIPSS capture distinct features of disease. We therefore developed a new three-tier classification scheme integrating both DIPSS and MPN24 scores (Figure 1B). Patients classified as low-, intermediate- or high-risk in this new classification scheme experienced 5-year survival rates of 88.2% [95% CI 77.9% - 99.9%], 39.3% [95% CI 19.9% - 77.7%] and 10.8% [95% CI 2.1% - 55.8%], respectively (likelihood ratio test p = 1e-8). Finally, from the MPN24 genes we derived a 13 gene subscore (MPN13) predictive of leukemic transformation in the training set. Patients in the test set scoring above the 80th percentile from the training set were classified as high risk and the remaining as low risk. Although total leukemic samples were limiting, MPN13 was significantly associated with risk of transformation (p=4.7e-03) with low and high-risk patients experiencing 3-year cumulative incidences of transformation of 5.2% [95% CI 0.2%-10.2%] and 28.6% [95% CI 3.1%-54.0%] respectively, after adjusting for death as a competing risk. Future studies will transfer our scores to the Nanostring platform for testing in independent cohorts. In summary, we used transcriptional variation among MF stem and progenitor cells to derive novel gene expression scores predictive of survival and leukemic transformation and developed a new integrated 3-tier model for predicting risk in MF patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

T Cells Expressing a Fully-Human Anti-BCMA Chimeric Antigen Receptor with a Heavy-Chain-Only Antigen-Recognition Domain Exhibit Rapid and Durable Activity Against Multiple Myeloma

Chimeric antigen receptor (CAR)-expressing T cells targeting B-cell maturation antigen (BCMA) are effective multiple myeloma therapy, but improvement in durability of responses is needed. We incorporated a fully-human heavy-chain-only anti-BCMA binding domain (FHVH33) into a CAR. Since it is only a heavy chain domain with no linker or artificial junctions, FHVH33 should be less immunogenic than a scFv. The CAR containing FHVH33 is called FHVH33-CD8BBZ. FHVH33-CD8BBZ contains CD8α hinge and transmembrane domains, a 4-1BB domain, and a CD3ζ domain; it was encoded by a γ-retroviral vector (Lam et al. Nature Communications 2020). T cells expressing FHVH33-CD8BBZ (FHVH-T) were produced from unsorted blood mononuclear cells in 7 days. Twenty-five patients received 300 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine on days -5 to -3 followed by infusion of FHVH-T on day 0. Five dose levels were assessed, and 6x106 CAR+ T cells/kg was selected for an expansion cohort based on efficacy and manufacturing factors (Table). Patients had received a median of 6 lines of therapy (range 3 to 10) prior to enrollment. Twenty-three of 25 patients (92%) obtained partial response (PR) or better. Eighteen patients (72%) attained best responses of stringent complete response (sCR) or very good partial response (VGPR). Median progression-free survival (PFS) of all treated patients is 65 weeks to date; eleven patients have ongoing responses. One striking feature of the anti-myeloma activity of FHVH-T was the rapidity of myeloma elimination. Some myeloma markers including serum free light chains, urine monoclonal light chains, and bone marrow plasma cells decrease rapidly after any type of effective therapy while intact immunoglobulin monoclonal proteins decrease slowly due to long serum half-lives, which are 20-25 days for most types of IgG. Following rapidly changing myeloma markers such as free light chains shows the actual tempo of myeloma elimination without the confounding factor of long serum half-lives of intact immunoglobulins. Among the 12 patients with rapidly changing main myeloma markers, most anti-myeloma activity occurred within the first month after FHVH-T infusion since myeloma markers underwent a 99-100% reduction in all 12 patients by 1 month after FHVH-T infusion. In 10/12 patients with rapidly changing main myeloma markers, a 99-100% decrease had occurred by 14 days after FHVH-T infusion. We assessed FHVH-33 T early after infusion during the time of most anti-myeloma activity. Blood CAR+ cell levels were assessed by quantitative PCR (qPCR). The median peak blood CAR+ cell number was 121 (range 3 to 3414) cells/μL. Peak CAR+ cell levels occurred a median of 12 (range 7 to 15) days after FHVH-T infusion. One month after infusion, the median blood CAR+ cell number was 11 (range 0 to 305) cells/μL. Median blood CAR+ cell levels one month after infusion were 20 cells/μL in patients with a PFS of 12 months or more versus 4 cells/μL in patients with PFS less than 12 months (P=0.0273). The CD4/CD8 ratio of CAR+ T cells decreased from the time of infusion until the time of peak blood CAR+ cells (P=0.0004). From the time of infusion until the time of peak blood CAR+ cells, there was an increase the percentage of T cells with an effector memory phenotype (C-C-chemokine receptor-7 (CCR7)-negative and CD45RA-negative), (P<0.001). Expression of CCR7 on infusion CD4+ FHVH-T correlated with peak CAR+ cell level (P=0.0018). Frequency of infusion CD4+ FHVH-T with a central memory phenotype (CCR7+, CD45RA-negative) correlated with peak and 1-month post-infusion CAR+ cell levels. Peak interferon (IFN)γ and interleukin (IL)-6 levels were associated with Grade 3 or 4 CRS (P=0.0034). Peak serum levels of IFNγ, IL2, IL6, IL8, IL10, and IL15 were higher (P<0.003 for all) in patients with neurologic toxicity. Single-cell RNA sequencing revealed extensive changes in FHVH-T gene expression between infusion and the time of peak blood CAR+ cell levels. Surprisingly, humoral immune responses against the fully-human CAR expressed by FHVH-T were detected in 4/12 evaluated patients after one FHVH-T infusion. In one patient who received 2 FHVH-T treatments, a humoral anti-CAR response was associated with lack of efficacy and absence of blood FHVH-T. FHVH-T therapy led to durable responses with most anti-myeloma activity occurring within 14 days after FHVH-T infusion. Blood levels of FHVH-T correlated with CD4+CCR7+ infusion cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A switchable secrete-and-capture system enables efficient selection of Pichia pastoris clones producing high yields of Fab fragments

Pichia pastoris (syn. Komagataella phaffii) represents a commonly used expression system in the biotech industry. High clonal variation of transformants, however, typically results in a broad range of specific productivities for secreted proteins. To isolate rare clones with exceedingly high product titers, an extensive number of clones need to be screened. In contrast to high-throughput screenings of P. pastoris clones in microtiter plates, secrete-and-capture methodologies have the potential to efficiently isolate high-producer clones among millions of cells through fluorescence-activated cell sorting (FACS).Here, we describe a novel approach for the non-covalent binding of fragment antigen-binding (Fab) proteins to the cell surface for the isolation of high-producing clones. Eight different single-chain variable fragment (scFv)-based capture matrices specific for the constant part of the Fabs were fused to the Saccharomyces cerevisiae alpha-agglutinin (SAG1) anchor protein for surface display in P. pastoris. By encoding the capture matrix on an episomal plasmid harboring inherently unstable autonomously replicating sequences (ARS), this secrete-and-capture system offers a switchable scFv display. Efficient plasmid clearance upon removal of selective pressure enabled the direct use of isolated clones for subsequent Fab production. Flow-sorted clones (n = 276) displaying high amounts of Fabs showed a significant increase in median Fab titers detected in the cell-free supernatant (CFS) compared to unsorted clones (n = 276) when cells were cultivated in microtiter plates (factor in the range of ∼21–49). Fab titers of clones exhibiting the highest product titer observed for each of the two approaches were increased by up to 8-fold for the sorted clone. Improved Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask cultivation of selected candidates (factor in the range of ∼2–3). Hence, the developed display-based selection method proved to be a valuable tool for efficient clone screening in the early stages of our bioprocess development.

Panning for gold: Purifying mesencephalic dopaminergic progenitors differentiated from human pluripotent stem cells.

In a recently published study, Xu et al. used two surface markers, CLSTN2 and PTPRO, to generate highly purified donor dopaminergic neurons and achieved stable and predictable therapeutic outcomes by transplantation into the brain of PD animal models (Xu et al., 2022).

Application of immune enhanced organoids in modeling personalized Merkel cell carcinoma research

Merkel cell carcinoma (MCC) is a rare neuroendocrine cutaneous cancer, with incidence of less than 1/100,000, low survival rates and variable response to chemotherapy or immunotherapy. Herein we explore the application of patient tumor organoids (PTOs) in modeling personalized research in this rare malignancy. Unsorted and non-expanded MCC tumor cells were isolated from surgical specimens and suspended in an ECM based hydrogel, along with patient matched blood and lymph node tissue to generate immune enhanced organoids (iPTOs). Organoids were treated with chemotherapy or immunotherapy agents and efficacy was determined by post-treatment viability. Nine specimens from seven patients were recruited from December 2018-January 2022. Establishment rate was 88.8% (8/9) for PTOs and 77.8% (7/9) for iPTOs. Histology on matched patient tissues and PTOs demonstrated expression of MCC markers. Chemotherapy response was exhibited in 4/6 (66.6%) specimens with cisplatin and doxorubicin as the most effective agents (4/6 PTO sets) while immunotherapy was not effective in tested iPTO sets. Four specimens from two patients demonstrated resistance to pembrolizumab, correlating with the corresponding patient’s treatment response. Routine establishment and immune enhancement of MCC PTOs is feasible directly from resected surgical specimens allowing for personalized research and exploration of treatment regimens in the preclinical setting.

Abstract 893: GD2 is a potential biomarker for glioblastoma cancer stem cells

Abstract Introduction: Treatment resistance and tumor recurrence of Glioblastoma (GBM) have been associated with cancer stem cells (CSCs), a subpopulation of cancer cells defined by capabilities of sphere-formation in 3D culture, self-renewal, migration, and survival in serum-deprived conditions. Therefore, therapies specifically targeting CSCs are urgently needed. Previous works have identified Ganglioside GD2 as a biomarker of triple negative breast cancer CSCs and suggested that GD2 is highly expressed in GBM cells. Here, we hypothesize that GD2 is also a potential biomarker of GBM CSCs and is involved with cancer stemness. Methods: GBM cells U-87 MG and U-118 MG were sorted as GD2- and GD2+ cells via FACS. Cells were cultured in 2D and 3D. 3D neurosphere size was measured and GD2 in 2D and 3D cultures was quantified via flow cytometry. Transwell migration assay was used to examine the migration capability of GD2- and GD2+ cells. Unsorted cells were cultured in either complete (10% serum) or deprived (1% serum) media and their GD2 was quantified via flow cytometry. Results: To test our hypothesis, we determined whether GD2+ cells express CSCs capabilities of sphere-formation in 3D culture, migration, and survival in serum-deprived conditions. First, we examined the sphere-forming capability by culturing GD2-, GD2+, and unsorted cells in 3D and found that they all formed neurospheres. However, GD2+ neurosphere size was significantly larger compared with unsorted and GD2- neurospheres for day 1 and 2. Interestingly, the significant difference was no longer seen on day 6. Analysis for GD2 expression in the spheres revealed that GD2- spheres contained GD2+ cells, suggesting that GD2- cells could spontaneously dedifferentiate to generate CSCs. To test this, we sorted GD2- and GD2+ cells into 2D conditions and quantified daily GD2 expression via flow cytometry. We found that GD2- seeded cells spontaneously expressed GD2 over six days, explaining the presence of GD2 in GD2- neurospheres. GD2+ cells also demonstrated the enhanced capability to migrate of CSCs with a significantly higher number of migrated cells compared with GD2- cells in the transwell migration assay. To examine the ability to survive in serum-deprived conditions, we cultured unsorted cells in either complete or deprived media. We found that these cells proliferated in the serum-deprived condition at approximately the same level as complete media. GD2 analysis by flow cytometry also showed a remarkably higher percentage of GD2+ cells in deprived media, suggesting that GD2 is involved in driving cell survival and maintaining cancer stemness in serum-deprived conditions. Conclusion: Our data suggests that GD2+ cells demonstrate characteristics of CSCs and GD2 is a potential biomarker of CSCs in GBM. Therefore, GD2 could be utilized as a differential therapeutic target for GBM. Further mechanistic studies are necessary to elucidate how GD2 is synthesized and maintains the stemness of GBM CSCs. Citation Format: Khoa Nguyen, Minh Tran, Thomas Cheng, Kurtis Willingham, Reza Izadpanah, Matthew Burow. GD2 is a potential biomarker for glioblastoma cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 893.

Serial Analysis of the T-Cell Receptor β-Chain Repertoire in People Living With HIV Reveals Incomplete Recovery After Long-Term Antiretroviral Therapy.

Long-term antiretroviral therapy (ART) in people living with HIV (PLHIV) is associated with sustained increases in CD4+ T-cell count, but its effect on the peripheral blood T-cell repertoire has not been comprehensively evaluated. In this study, we performed serial profiling of the composition and diversity of the T-cell receptor β-chain (TRB) repertoire in 30 adults with HIV infection before and after the initiation of ART to define its long-term impact on the TRB repertoire. Serially acquired blood samples from 30 adults with HIV infection collected over a mean of 6 years (range, 1-12) years, with 1-4 samples collected before and 2-8 samples collected after the initiation of ART, were available for analysis. TRB repertoires were characterized via high-throughput sequencing of the TRB variable region performed on genomic DNA extracted from unsorted peripheral blood mononuclear cells. Additional laboratory and clinical metadata including serial measurements of HIV viral load and CD4 + T-cell count were available for all individuals in the cohort. A previously published control group of 189 TRB repertoires from peripheral blood samples of adult bone marrow transplant donors was evaluated for comparison. ART initiation in PLHIV was associated with a sustained reduction in viral load and a significant increase in TRB repertoire diversity. However, repertoire diversity in PLHIV remained significantly lower than in the control group even after long-term ART. The composition of TRB repertoires of PLHIV after ART also remained perturbed compared to the control cohort, as evidenced by large persistent private clonal expansions, reduced efficiency in the generation of TRB CDR3 amino acid sequences, and a narrower range of CDR3 lengths. Network analysis revealed an antigen-experienced structure in the TRB repertoire of PLHIV both before and after ART initiation that was quite distinct from the structure of control repertoires, with a slight shift toward a more naïve structure observed after ART initiation. Though we observe significant improvement in TRB repertoire diversity with durable viral suppression in PLHIV on long-term ART, the composition and structure of these repertoires remain significantly perturbed compared to the control cohort of adult bone marrow transplant donors.

Normalization of Oxygen‐Induced Retinopathy in the Mouse Supplementation with Myo/Nog Cells

IntroductionRetinopathy of Prematurity (ROP) is the leading cause of blindness in children, affecting 50% of infants born prior to gestational week 32. ROP is the result of postpartum supplemental oxygen administered to compensate for underdeveloped lungs. However, hyperoxia inhibits proper development supplying the retina. When the infants are returned to ambient air, an imbalance is present between the oxygen concentration and metabolic demands of retinal neurons. This leads to a rapid induction of angiogenesis, leaky blood vessels, neovascular tuft formation, and cell death. Myo/Nog cells that express MyoD, Noggin, and brain‐specific angiogenesis inhibitor 1 (BAI1) exhibit neuroprotective properties in the eye and brain and are proposed to play a role in regulating angiogenesis in ROP.MethodsAn Oxygen‐induced retinopathy (OIR) protocol was implemented in neonatal C57BL/6J mice by exposing them on postnatal days 7‐12 (P7‐P12) to hyperoxia (75% oxygen) and then returning them to normal air (21% oxygen) from P12 until P21. On P15, groups receiving treatments were intravitreally injected with 1 µL of phosphate‐buffered saline (PBS) containing 2000 Myo/Nog cells isolated from the brain with the BAI1 antibody (BAI1+ group), 2000 unsorted brain cells (BAI1‐ group), or PBS alone (PBS group). Electroretinography (ERG) was conducted on P21 to assess retinal function. Eyes were extracted and either dissected for flat‐mount observation of vasculature and tufts, or processed for histology. The thickness of the retina and number of TUNEL+ apoptotic cells were quantified in tissue sections of eyes processed for histology. Adobe Photoshop (Adobe, San Jose, CA) was used to splice together 9 images that made up each whole retina from flat‐mounted eyes. The blood vessels were selected using the “Lasso tool” on photoshop, quantified by number of pixels, and then compared to the total number of pixels in the whole retina.ResultsOIR induced neovascular tuft formation. Exogenously added Myo/Nog cells normalized the retinopathy induced by the OIR model, reducing the number of tufts and improving the quality of the vasculature. The vasculature was most developed in retinas injected with brain derived Myo/Nog cells. ERG data shows a trend towards improvement of visual function with Myo/Nog cell injection.ConclusionMyo/Nog cell regulation of retinal vascularization may be related to the production of BAI1. Improved visual function seen with injection of Myo/Nog cells may be related to both a reduction of neovascular tufts and direct effects on retinal neurons. Therefore Myo/Nog cells have therapeutic potential in the prevention of ROP by normalizing the vasculature and preventing neuronal cell death.

Exploring the Neuroprotective Role of Myo/Nog Cells in a Glaucomatous Mouse

IntroductionMyo/Nog cells express MyoD, Noggin and brain‐specific angiogenesis inhibitor 1 (BAI1). They are critical for eye development and respond to injury and stress in the neonatal and adult retina. Their depletion in the embryo results in eye and retinal malformations. Myo/Nog cells increase in number and migrate to areas of injury in response to stress. Cell death in the retina increases with Myo/Nog cell depletion and decreases with their addition to the vitreous. Here we examine the effects and locations of endogenous and exogenous Myo/Nog cells in mice eyes following the induction of glaucoma.MethodsThe anterior chamber of the right eye of C57BL/6J mice was injected with magnetic microbeads and guided to the iridocorneal angle using a handheld magnet, preventing the outflow of aqueous. Intraocular pressure was measured in both eyes following microbead injection on Days 0, 4, 7, 14, D21, and 28. On D7, three of the four groups were given bilateral injections into the anterior chamber of either PBS, unsorted brain cells, or Myo/Nog cells isolated from the brain. The fourth group did not receive any follow‐up injections. Eyes were collected on D32 and labeled with an antibody to BAI1 to locate Myo/Nog cells. H&amp;E staining was used to count living cells in the retinal ganglion layer (RGC) and measure the thickness of the nerve fiber layer and inner nuclear layer (NFL:INL).ResultsEndogenous and exogenous Myo/Nog cells increased in glaucomatous eyes around areas of stress, including the cornea, trabecular meshwork, ciliary body and retina. The highest concentration of Myo/Nog cells was present around the RGC layer and cornea. Some Myo/Nog cells had phagocytized microbeads. Glaucomatous eyes treated with Myo/Nog cells displayed a lower concentration of endogenous Myo/Nog cells when compared to PBS and mixed population treatment groups. Addition of Myo/Nog cells also decreased loss of RGC cells and increased NFL:INL thickness in glaucomatous eyes compared to the control groups.ConclusionConsistent with other types of ocular injury, Myo/Nog cells accumulate in areas of stress following damage from increased intraocular pressure. Addition of Myo/Nog cells was neuroprotective in the glaucomatous retina.