Cord Blood CD34 Research Articles

Impact of fetal umbilical cord blood CD34+ cells on breast cancer cell lines: a mechanism of fetal microchimerism?

Introduction Fetal microchimerism could be involved in the regulation of breast cancer oncogenesis. CD34+ cells could be of a particular interest as up to 12% of the CD34+ population in maternal blood are of fetal origin. The aim of this research was to analyze the impact of umbilical cord blood (UCB) CD34+ on MCF-7 and MDA-MB-231 breast cancer cell lines, in order to uncover novel biological mechanisms and suggest novel treatment options for breast cancer. Methods UCB CD34+ cells were obtained from healthy women at full-term delivery. Direct cultures were grown with MCF-7 and MDA-MB-231 cells. Proliferation, migration, invasion, and transcriptomic analysis of breast cancer cells were compared between cultures exposed and non-exposed to UCB CD34+ cells. Interactions between UCB CD34+ and breast cancer cells were analyzed under fluorescent microscopy. Functional analyses were generated with QIAGEN's Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Results Direct contact between UCB CD34+ and breast cancer cell lines induced a reduction in the proliferative capacities of MCF-7 and MDA-MB-231 and diminished the migration abilities of MDA-MB-231 cells. In 3D co-culture, UCB CD34+ cells were attracted by tumor spheroids and incorporated into tumor cells. These cell-to-cell interactions were responsible for transcriptome modifications coherent with observed functional modifications. Among the cytokines secreted by UCB CD34+, IFN was identified as a potential upstream regulator responsible for the molecular modifications observed in transcriptomic analysis of MCF-7 breast cancer cells exposed to UCB CD34+ cells, as was IL-17A in MDA-MB-231 cells. Conclusion Direct cell-to-cell contact induced functional modifications in breast cancer cells. Interactions between UCB CD34+ and breast cancer cells could induce cell fusion and signal transmission via cytokines. Further analysis of direct cell-to-cell interactions should be performed at a molecular level to further understand the potential role of fetal CD34+ cells in breast cancer.

Development of a quantitative prediction algorithm for human cord blood-derived CD34+ hematopoietic stem-progenitor cells using parametric and non-parametric machine learning models

The transplantation of CD34+ hematopoietic stem-progenitor cells (HSPCs) derived from cord blood serves as the standard treatment for selected hematological, oncological, metabolic, and immunodeficiency disorders, of which the dose is pivotal to the clinical outcome. Based on numerous maternal and neonatal parameters, we evaluated the predictive power of mathematical pipelines to the proportion of CD34+ cells in the final cryopreserved cord blood product adopting both parametric and non-parametric algorithms. Twenty-four predictor variables associated with the cord blood processing of 802 processed cord blood units randomly sampled in 2020–2022 were retrieved and analyzed. Prediction models were developed by adopting the parametric (multivariate linear regression) and non-parametric (random forest and back propagation neural network) statistical models to investigate the data patterns for determining the single outcome (i.e., the proportion of CD34+ cells). The multivariate linear regression model produced the lowest root-mean-square deviation (0.0982). However, the model created by the back propagation neural network produced the highest median absolute deviation (0.0689) and predictive power (56.99%) in comparison to the random forest and multivariate linear regression. The predictive model depending on a combination of continuous and discrete maternal with neonatal parameters associated with cord blood processing can predict the CD34+ dose in the final product for clinical utilization. The back propagation neural network algorithm produces a model with the highest predictive power which can be widely applied to assisting cell banks for optimal cord blood unit selection to ensure the highest chance of transplantation success.

A millifluidic bioreactor allows the long term culture of primary lymphocytes or CD34+ hematopoietic cells while allowing the detection of tumorigenic expansion.

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore, genetic engineering of the above-mentioned primary human cells has several safety needs, including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work, we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB, we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4+ T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34+ cells. A fraction of these cells is viable for up to 3months. Next, we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4+ or CD34+ primary cells, and their growth was followed. By this approach, we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment, whereas tumor cells, after an adaptation phase, expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells.

Heterozygous missense CSF1R variants hamper in vitro CD34+-derived dendritic cell generation but not in vivo dendritic cell development

Colony stimulating factor 1 receptor (CSF1R) is an essential receptor for both colony stimulating factor 1 (CSF1) and interleukin (IL) 34 signaling expressed on monocyte precursors and myeloid cells, including monocytes, dendritic cells (DC), and microglia. In humans, dominant heterozygous pathogenic variants in CSF1R cause a neurological condition known as CSF1R-related disorder (CSF1R-RD), typically with late onset, previously referred to as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). CSF1R-RD is characterized by microglia reduction and altered monocyte function; however, the impact of pathogenic CSF1R variants on the human DC lineage remains largely unknown.We previously reported that cord blood CD34+ stem cell-derived DCs generated in vitro originate specifically from CSF1R expressing precursors. In this study, we examined the DC lineage of four unrelated patients with late-onset CSF1R-RD who carried heterozygous missense CSF1R variants (c.2330G>A, c.2375C>A, c.2329C>T, and c.2381T>C) affecting different amino acids in the protein tyrosine kinase domain of CSF1R. CD34+ stem cells and CD14+ monocytes were isolated from peripheral blood and subjected to an in vitro culture protocol to differentiate towards conventional DCs and monocyte-derived DCs, respectively. Flow cytometric analysis revealed that monocytes from patients with late-onset CSF1R-RD were still able to differentiate into monocyte-derived DCs in vitro, whereas the ability of CD34+ stem cells to differentiate into conventional DCs was impaired. Strikingly, the peripheral blood of patients contained all naturally occurring DC subsets. We conclude that the in vitro abrogation of DC-development in patients with heterozygous pathogenic missense CSF1R variants does not translate to an impairment in DC development in vivo and speculate that CSF1R signalling in vivo is compensated, which needs further study.

Abstract 30: In-Depth Characterization of B-Lineage Populations Arising During Cord Blood CD34+ HSPC Differentiation for ETV6::RUNX1 Progenitor Identification

Abstract 30: In-Depth Characterization of B-Lineage Populations Arising During Cord Blood CD34+ HSPC Differentiation for ETV6::RUNX1 Progenitor Identification

More balance toward activating receptors and cytotoxic activity of NK cells ex vivo differentiated from human umbilical cord blood-derived CD34+ stem cells in comparison with peripheral blood NK cells

Adoptive immunotherapies that use functional NK cells depend on the availability of sufficient numbers of these cells. We expanded umbilical cord blood (UCB)-CD34+ HSCs for 2 weeks and then differentiated them into NK cells and compared their function to peripheral blood (PB) NK cells. We assessed NKG2D, NKG2A, NKp30, NKp44, NKp46, and the expression of CD107a, CD57, CD69, FasL, PD-1, and IFN-γ level in two groups after co-culture with K562 cell line. We found that UCB-CD34+-derived NK cells express significantly more NKG2D, NKp44, and NKp46 receptors than PB NK cells. PB NK cells expressed significantly higher NKG2A and CD57 than UCB-CD34+-derived NK cells. In addition, UCB-CD34+-derived NK cells significantly expressed CD107a more than PB NK cells. Based on our findings, UCB-CD34+ cells can be a potentially advantageous source with strong cytotoxic function to produce allogeneic NK cells for adoptive cancer immunotherapy.

Targeting the redox vulnerability of acute myeloid leukaemia cells with a combination of auranofin and vitamin C.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by complex molecular and cytogenetic abnormalities. Pro-oxidant cellular redox status is a common hallmark of AML cells, providing a rationale for redox-based anticancer strategy. We previously discovered that auranofin (AUF), initially used for the treatment of rheumatoid arthritis and repositioned for its anticancer activity, can synergize with a pharmacological concentration of vitamin C (VC) against breast cancer cell line models. In this study, we observed that this drug combination synergistically and efficiently killed cells of leukaemic cell lines established from different myeloid subtypes. In addition to an induced elevation of reactive oxygen species and ATP depletion, a rapid dephosphorylation of 4E-BP1 and p70S6K, together with a strong inhibition of protein synthesis were early events in response to AUF/VC treatment, suggesting their implication in AUF/VC-induced cytotoxicity. Importantly, a study on 22 primary AML specimens from various AML subtypes showed that AUF/VC combinations at pharmacologically achievable concentrations were effective to eradicate primary leukaemic CD34+ cells from the majority of these samples, while being less toxic to normal cord blood CD34+ cells. Our findings indicate that targeting the redox vulnerability of AML with AUF/VC combinations could present a potential anti-AML therapeutic approach.

Identification and validation of ferroptosis related markers in erythrocyte differentiation of umbilical cord blood-derived CD34+ cell by bioinformatic analysis.

Ferroptosis has been observed to play an important role during erythrocyte differentiation (ED). However, the biological gene markers and ferroptosis mechanisms in ED remain unknown. We downloaded the datasets of ED in human umbilical cord blood-derived CD34+ cells from the Gene Expression Omnibus database. Using median differentiation time, the sample was categorized into long and short groups. The differentially expressed ferroptosis-related genes (DE-FRGs) were screened using differential expression analysis. The enrichment analyses and a protein-protein interaction (PPI) network were conducted. To predict the ED stage, a logistic regression model was constructed using the least absolute shrinkage and selection operator (LASSO). Overall, 22 DE-FRGs were identified. Ferroptosis-related pathways were enriched using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. Gene Set Enrichment Analysis and Gene Set Variation Analysis revealed the primary involvement of DE-FRGs in JAK-STAT, MAPK, PI3K-AKT-mTORC1, WNT, and NOTCH signaling pathways. Ten-hub DE-FRGs were obtained using PPI analysis. Furthermore, we constructed mRNA-microRNA (miRNA) and mRNA-transcription factor networks. Immune cell infiltration levels differed significantly during ED. LASSO regression analysis established a signature using six DE-FRGs (ATF3, CDH2, CHAC1, DDR2, DPP4, and GDF15) related to the ED stage. Bioinformatic analyses identified ferroptosis-associated genes during ED, which were further validated. Overall, we identified ferroptosis-related genes to predict their correlations in ED. Exploring the underlying mechanisms of ferroptosis may help us better understand pathophysiological changes in ED and provide new evidence for clinical transformation.

Molecular and biochemical evaluation of oxidative effects of cord blood CD34+ MPs on hematopoietic cells

A graft source for allogeneic hematopoietic stem cell transplantation is umbilical cord blood, which contains umbilical cord blood mononuclear cells (MNCs and mesenchymal stem cells, both an excellent source of extracellular microparticles (MPs). MPs act as cell communication mediators, which are implicated in reactive oxygen species formation or detoxification depending on their origin. Oxidative stress plays a crucial role in both the development of cancer and its treatment by triggering apoptotic mechanisms, in which CD34+ cells are implicated. The aim of this work is to investigate the oxidative stress status and the apoptosis of HL-60 and mononuclear cells isolated from umbilical cord blood (UCB) following a 24- and 48-hour exposure to CD34 + microparticles (CD34 + MPs). The activity of superoxide dismutase, glutathione reductase, and glutathione S-transferase, as well as lipid peroxidation in the cells, were employed as oxidative stress markers. A 24- and 48-hour exposure of leukemic and mononuclear cells to CD34 + -MPs resulted in a statistically significant increase in the antioxidant activity and lipid peroxidation in both cells types. Moreover, CD34 + MPs affect the expression of BCL2 and FAS and related proteins and downregulate the hematopoietic differentiation program in both HL-60 and mononuclear cells. Our results indicate that MPs through activation of antioxidant enzymes in both homozygous and nonhomozygous cells might serve as a means for graft optimization and enhancement.

A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer’s patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-β, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

Abstract LB120: Stem cell-derived ACUA CAR-NKT allogeneic cell therapies harness orthogonal mechanisms of action for the treatment of solid tumors

Abstract Despite the remarkable success of chimeric antigen receptor T cell (CAR-T) therapies in treating hematological malignancies, their use in solid tumor indications has been met with distinct challenges. The immunosuppressive tumor microenvironment (TME) and tumor antigen heterogeneity are two significant factors that may adversely impact the efficacy of these ground-breaking therapies. Engineered natural killer T (NKT) cells harbor biological features distinct from CAR-T cell or CAR-NK cell therapies and thus represent a novel therapeutic approach to deploy “off the shelf” to patients. Appia Bio’s ACUA platform is capable of generating large quantities of highly pure CAR-NKT cells from cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) for clinical use. We have demonstrated that multiple distinct mechanisms of anti-tumor activity are operative in CAR-NKT cells, including CAR-mediated killing, NK receptor-mediated killing, adjuvant effects on antigen-specific T cells and immature dendritic cells, as well as the ability to deplete of pro-tumorigenic macrophages.When transduced with a CAR, ACUA-derived CAR-NKT cells show potent CAR-mediated tumor cell killing, similar to CAR-T cells. In contrast to conventional T cells, CAR-NKT cells also demonstrate potent CAR antigen-independent tumor cell killing, mediated partially through NKG2D and DNAM-1 activating NK receptors. CAR-NKT cells, but not conventional CAR-T cells, enhanced the function of NY-ESO specific T cells in controlling multiple rounds of tumor rechallenge, through the secretion of soluble components. In regard to other adjuvant effects which may provide additional benefit in vivo, ACUA-derived NKT cells enhanced the activation of peripheral blood monocyte-derived dendritic cells, which can regulate beneficial Th1 type immune responses. To explore how ACUA-derived NKT cells could reshape the TME, we generated immunosuppressive M2-polarized macrophages and observed that ACUA-derived NKT cells can either kill or reprogram these cells to a pro-inflammatory phenotype following co-culture. Notably, these immune cell adjuvant mechanisms are specific to ACUA-derived NKT cells and are not observed with conventional CAR-T cells. In summary, these mechanistic studies underscore the advantages of ACUA-derived NKT cells over conventional CAR-T cells and highlight their potential as a promising differentiated allogeneic therapy for the treatment of solid tumors. Citation Format: Xi Wang, Andrew J. Hou, Michael A. Christopher, Yannick Bulliard, Jason S. Damiano. Stem cell-derived ACUA CAR-NKT allogeneic cell therapies harness orthogonal mechanisms of action for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB120.

Abstract LB074: Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies

Abstract Autologous chimeric antigen receptor (CAR)-T therapies have elicited high rates of durable complete responses in B cell lymphomas. Unfortunately, disease progression, manufacturing failures, and prohibitive costs highlight the need for an alternative source of cells that would provide a consistent and readily available therapy for patients. The “Appia Cells Utilized for Allogeneic” (ACUA) platform is an innovative process that differentiates cord blood-derived CD34+ hematopoietic stem and progenitor cells into large numbers of natural killer T (NKT) cells to support off-the-shelf treatment. NKT cells express an invariant T cell receptor (iTCR) and are naturally non-alloreactive, making them an ideal cell type for an allogeneic product. The ACUA platform circumvents the challenge of expanding NKT cells from the periphery where the cells comprise less than 1% of T cells. API-192 is an ACUA-derived, NKT cell product engineered to express dual CARs targeting CD19 and CD20 to prevent tumor antigen escape. At research scale, API-192 is a highly pure product (>90% iTCR+ 19CAR+ 20CAR+) that secretes soluble interleukin-15 for improved expansion and persistence. The transgenic iTCR is not expressed above physiologic levels and upon engagement, API-192 proliferates in response to and is cytotoxic against CD1d+ tumor cells in a ligand-dependent manner. In response to Raji and Nalm6 tumor cells, API-192 secretes pro-inflammatory cytokines, proliferates, and is cytotoxic in a dose- and CAR-dependent manner. API-192 can also control multiple rounds of tumor cell rechallenge in vitro. Using human tumor xenograft mouse models, we show that API-192 expands, controls tumor growth, and prolongs survival. Furthermore, API-192 persists in the periphery, enabling control of a tumor rechallenge at 70 days after initial dosing of effector cells. Indicative of a favorable immunogenic profile, API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less interferon (IFN)-γ release by NK and T cells in a mixed lymphocyte reaction (MLR) assay. To address a theoretical concern that endogenous TCRαβ rearrangement could occur during the ACUA process, we performed genomic DNA based TCR sequencing to quantify the level of TCRα or TCRβ rearrangement and preliminary data suggested negligible levels (<0.2%). Using a MLR assay, we demonstrate that compared to conventional CAR-T cells, API-192 secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with a low graft vs host risk. In an autonomous growth assay, API-192 expands at a low level and subsequently contracts, suggesting a lack of dysregulated growth. Collectively, these results demonstrate the favorable manufacturability, potency, and safety of API-192 and its potential as a novel allogeneic therapy. Citation Format: Lanny Gov, Jiaji Yu, Maha Qubain, Saige L. Pompura, Sean D. Allen, Michael A. Christopher, Jeff Wiezorek. Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB074.

VISTA drives macrophages towards a pro-tumoral phenotype that promotes cancer cell phagocytosis yet down-regulates T cell responses

BackgroundVISTA is a well-known immune checkpoint in T cell biology, but its role in innate immunity is less established. Here, we investigated the role of VISTA on anticancer macrophage immunity, with a focus on phagocytosis, macrophage polarization and concomitant T cell activation.MethodsMacrophages, differentiated from VISTA overexpressed THP-1 cells and cord blood CD34+ cell-derived monocytes, were used in phagocytosis assay using B lymphoma target cells opsonized with Rituximab. PBMC-derived macrophages were used to assess the correlation between phagocytosis and VISTA expression. qRT-PCR, flow cytometry, and enzyme-linked immunosorbent assay were performed to analyze the impact of VISTA on other checkpoints and M1/M2-like macrophage biology. Additionally, flow cytometry was used to assess the frequency of CD14+ monocytes expressing VISTA in PBMCs from 65 lymphoma patients and 37 healthy donors.ResultsEctopic expression of VISTA in the monocytic model cell line THP-1 or in primary monocytes triggered differentiation towards the macrophage lineage, with a marked increase in M2-like macrophage-related gene expression and decrease in M1-like macrophage-related gene expression. VISTA expression in THP-1 and monocyte-derived macrophages strongly downregulated expression of SIRPα, a prominent ‘don’t eat me’ signal, and augmented phagocytic activity of macrophages against cancer cells. Intriguingly, expression of VISTA’s extracellular domain alone sufficed to trigger phagocytosis in ∼ 50% of cell lines, with those cell lines also directly binding to recombinant human VISTA, indicating ligand-dependent and -independent mechanisms. Endogenous VISTA expression was predominantly higher in M2-like macrophages compared to M0- or M1-like macrophages, with a positive correlation observed between VISTA expression in M2c macrophages and their phagocytic activity. VISTA-expressing macrophages demonstrated a unique cytokine profile, characterized by reduced IL-1β and elevated IL-10 secretion. Furthermore, VISTA interacted with MHC-I and downregulated its surface expression, leading to diminished T cell activation. Notably, VISTA surface expression was identified in monocytes from all lymphoma patients but was less prevalent in healthy donors.ConclusionsCollectively, VISTA expression associates with and drives M2-like activation of macrophages with a high phagocytic capacity yet a decrease in antigen presentation capability to T cells. Therefore, VISTA is a negative immune checkpoint regulator in macrophage-mediated immune suppression.

SET domain containing 2 promotes megakaryocyte polyploidization and platelet generation through methylation of α-tubulin

BackgroundMegakaryocytes (MKs) are polyploid cells responsible for producing ∼1011 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the promise for the production of clinical-grade platelets from stem cells, overcoming significant current limitations in platelet transfusion medicine. Previous work identified that loss of the epigenetic regulator SET domain containing 2 (SETD2) was associated with an increased platelet count in mice. However, the role of SETD2 in megakaryopoiesis remains unknown. ObjectivesHere, we examined how SETD2 regulated MK development and platelet production using complementary murine and human systems. MethodsWe manipulated the expression of SETD2 in multiple in vitro and ex vivo models to assess the ploidy of MKs and the function of platelets. ResultsThe genetic ablation of Setd2 increased the number of high-ploidy bone marrow MKs. Peripheral platelet counts in Setd2 knockout mice were significantly increased ∼2-fold, and platelets exhibited normal size, morphology, and function. By knocking down and overexpressing SETD2 in ex vivo human cell systems, we demonstrated that SETD2 negatively regulated MK polyploidization by controlling methylation of α-tubulin, microtubule polymerization, and MK nuclear division. Small-molecule inactivation of SETD2 significantly increased the production of high-ploidy MKs and platelets from human-induced pluripotent stem cells and cord blood CD34+ cells. ConclusionThese findings identify a previously unrecognized role for SETD2 in regulating megakaryopoiesis and highlight the potential of targeting SETD2 to increase platelet production from human cells for transfusion practices.

Abstract 1420: Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC

Abstract NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 induces cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy in aPD1 resistant KRAS/STK11mt NSCLC in a humanized mouse model. Humanized mice were generated by transplanting human cord blood-derived CD34 stem cells into NSG mice. Mice harboring > 25% human CD45 cells were considered humanized. Lung metastases were developed in humanized mice by injecting KRAS/STK11mt/aPD1R A549 cells, which were treated with i.v. injection of NPRL2 nanoparticles (DOTAP-NPRL2) with or without pembrolizumab (aPD1). NPRL2 treatment reduced lung metastases, whereas pembrolizumab was ineffective. The antitumor effect was greater in humanized than non-humanized mice suggesting the role of immune cells. The antitumor effect was associated with increased infiltration of human cytotoxic immune cells and decreased numbers of Treg in tumors. NPRL2+ pembrolizumab was not synergistic in this resistant model but was synergistic in KRASwt/aPD1S H1299 tumors. Cytotoxic immune cells in tumors were associated with the antitumor effect. Consistent with A549, NPRL2 showed a significantly strong antitumor effect on other KRASmt/aPD1R syngeneic LLC2 tumors whereas aPD1 was not effective. The antitumor effect of NPRL2 was correlated with HLA-DR+ DC, CD11c DC, TILs and NK cells in TME. The antitumor effect of NPRL2 was abolished upon in-vivo depletion of CD4, CD8 T, and MΦ in the LLC2 tumor model. However, no effect was found on in-vivo depletion of NK cells. Nanostring analysis on lung metastasis resulted in a distinct pattern of human gene expression by NPRL2. T cell functional genes, including IFNγ, CD8b, CD7, TNFSF18, ITGA1, GATA3 and TBX21 were significantly increased. Conversely, the negative regulatory genes, including FOXP3, TGFB1, TGFB2, and IL-10RA were inhibited. NPRL2 also downregulated T cell co-inhibitory molecules, including CTLA4, ICOS, LAG3, PDCD1, CD274, IDO1, PDCD1LG2, CD47, and KLRB1. Stably expressing NPRL2 clones were established, and tumors in humanized mice with these clones exhibited significantly slower growth compared to controls. TME analysis showed an upregulation of human CD45, CD3, CD8 T, HLA-DR+ DC and a downregulation of Tregs, CD3+PD1+T, MDSC, and CD163+ TAM in tumors expressing NPRL2. The stable cells showed a substantial increase in both colony formation inhibition and apoptosis. Stable clones showed heightened sensitivity to carboplatin in colony formation, apoptosis, and PARP cleavage assays. Stable expression of NPRL2 resulted in the downregulation of both AKT-mTOR and MAPK pathways by inhibition of pAKT, pmTOR, pPRAS40, p4E-BP1, pS6, and pERK1/2. Taken together, NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation Citation Format: Ismail M. Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Jack A. Roth. Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC [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 1420.

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [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 1530.

Mesenchymal stem/stromal cells from human pluripotent stem cell-derived brain organoid enhance the ex vivo expansion and maintenance of hematopoietic stem/progenitor cells

BackgroundMesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported.MethodsFlow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34+ (UCB-CD34+) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs.ResultsThe stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34+ HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34+ HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34+ HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34+ HSPCs by antagonizing Wnt activation.ConclusionsA novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell–cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.

In vivo and in vitro effects of cord blood hematopoietic stem and progenitor cell (HSPC) expansion using valproic acid and/or nicotinamide

BackgroundHigh self-renewal capacity and most permissive nature of umbilical cord blood (CB) results with successful transplant outcomes but low hematopoietic stem and progenitor cell (HSPC) counts limits wider use. In order to overcome this problem ex vivo expansion with small molecules such as Valproic acid (VPA) or Nicotinamide (NAM) have been shown to be effective. To the best of our knowledge, the combinatory effects of VPA and NAM on HSPC expansion has not been studied earlier. The aim of this study was to analyze ex vivo and in vivo efficacy of VPA and NAM either alone or in combination in terms of expansion and engraftment. MethodsA total of 44 CB units were included in this study. To determine the ex vivo and in vivo efficacy, human CB CD34+ cells were expanded with VPA and/or NAM and colony forming unit (CFU) assay was performed on expanded HSPC. Xenotransplantation was performed simultaneously by intravenous injection of expanded HSPC to NOD-SCID gamma (NSG) mice (n = 22). Significance of the difference between the expansion groups or xenotransplantation models was analyzed using t-test, Mann-Whitney, ANOVA or Kruskal-Wallis tests as appropriate considering the normality of distributions and the number of groups analyzed. ResultsIn vitro CD34+ HSPC expansion fold relative to cytokines-only was significantly higher with VPA compared to NAM [2.23 (1.07–5.59) vs 1.48 (1.00–4.40); p < 0.05]. Synergistic effect of VPA+NAM has achieved a maximum relative expansion fold at 21 days (D21) of incubation [2.95 (1.00–11.94)]. There was no significant difference between VPA and VPA+NAM D21 (p = 0.44). Fold number of colony-forming unit granulocyte-macrophage (CFU-GM) colonies relative to the cytokine-only group was in favor of NAM compared to VPA [1.87 (1.00–3.59) vs 1.00 (1.00–1.81); p < 0.01]. VPA+NAM D21 [1.62 (1.00–2.77)] was also superior against VPA (p < 0.05). There was no significant difference between NAM and VPA+NAM D21. Following human CB34+ CB transplantation (CBT) in the mouse model, fastest in vivo leukocyte recovery was observed with VPA+NAM expanded cells (6 ± 2 days) and the highest levels of human CD45 chimerism was detectable with VPA-expanded CBT (VPA: 5.42 % at day 28; NAM: 2.45 % at day 31; VPA+NAM 1.8 % at day 31). ConclusionOur study results suggest using VPA alone, rather than in combination with NAM or NAM alone, to achieve better and faster expansion and engraftment of CB HSPC.

Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

RasGRP3 Role in Umbilical Cord Blood CD34+ Cell Homing and Engraftment

RasGRP3 Role in Umbilical Cord Blood CD34+ Cell Homing and Engraftment