Transplantation In Mice Research Articles

Targeting Epigenetic Resistance Mechanisms to PI3 Kinase Inhibition in Leukemic Stem Cells

Acute myeloid leukemia (AML) is a heterogeneous disease associated with poor survival and frequent relapse rates due to the inability to effectively target leukemic stem cells (LSCs) and acquired resistance to existing treatments. Development of treatments targeting LSCs and overcoming acquired resistance is crucial to improve the survival of AML patients. The PI3 kinase (PI3K)/AKT signaling pathway is activated in 80% of AML patients, across various AML subtypes, making PI3K an attractive therapeutic target for this disease. Several PI3K inhibitors, such as copanlisib, are clinically approved for other indications, with a reasonable toxicity profile. The effects of PI3K inhibitors on LSCs however, remains unknown. Several catalytic isoforms of PI3K are expressed in hematopoietic cells. To determine whether these PI3K isoforms play a role in LSC self-renewal, we performed colony assays on KMT2A-AF9 retrovirally transduced hematopoietic stem and progenitor cells (HSPCs) with either genetic deletion or pharmacologic inhibition of PI3K isoforms. We found that disruption of PI3K signaling reduces the self-renewal of LSCs and promotes myeloid differentiation in both mouse LSCs and human AML cell lines. KMT2A-AF9 induces leukemic transformation via aberrant upregulation of key self-renewal genes in hematopoietic progenitors, such as HoxA9 and Meis1 (PMID: 16862118). We found that pharmacologic inhibition of PI3K isoforms leads to a reduction in HoxA9 and Meis1 expression in LSC colonies. In the KMT2A-AF9 retroviral bone marrow transplantation model of AML, genetic deletion of the PI3K isoform Pik3ca leads to a reduction in LSC numbers in the bone marrow and prolonged survival (Fig 1A), although it is not curative. In addition, we observed significant prolongation of survival upon secondary transplantation of Pik3ca leukemic cells, suggesting a reduction in functional LSCs. Transcriptome analysis on LSCs isolated from pre-clinical bone marrow aspirates of KMT2A-AF9 primary transplant mice revealed downregulation of the target genes of HoxA9, an important driver of LSC self-renewal. We also observed enrichment of the targets of EZH2, a histone methyltransferase that catalyzes the H3K27 trimethylation repressive mark. AKT is known to phosphorylate EZH2 at Ser21, leading to inhibition of EZH2 function (PMID: 16224021). Therefore, we hypothesized that Pik3ca deletion can promote LSC differentiation by de-repressing EZH2 activation via AKT inactivation. To determine whether PI3K inactivation differentiates KMT2A-AF9 leukemic cells via dephosphorylation of EZH2, we performed Western blot analysis on bone marrow cells from Pik3ca KO and WT leukemic mice. As expected, Pik3ca deletion significantly reduced AKT phosphorylation. Unexpectedly, a reduction of total EZH2 protein levels was also observed in Pik3ca KO leukemic cells (Fig 1B), suggesting that EZH2 downregulation may be a non-genetic acquired resistance mechanism to PI3K inactivation. Histone proteomics analysis at two different timepoints after treatment with the PI3K inhibitor copanlisib revealed rapid and dynamic changes in histone modifications, including H3K27me3 and H3K4me3, consistent with epigenetic mechanisms of resistance to PI3K inhibition. EZH1, a homolog of EZH2, has been reported to compensate for EZH2 function (PMID: 26219303, PMID: 31189531). Thus, we hypothesize that PI3K inhibition may sensitize leukemic cells to EZH1/2 dual inhibition by reducing EZH2 levels. We found that dual inhibition with copanlisib and the EZH1/2 inhibitor valemetostat leads to a combinatorial effect to strongly decrease the proliferation of AML cell lines and AML patient samples. Additionally, we found that treatment of KMT2A-AF9 AML mice with these compounds induces myeloid differentiation and leads to a reduction in leukemia initiating cell capacity, as demonstrated by prolonged survival upon secondary transplantation. Overall, our data suggests that PI3K plays an important role in leukemic stem cells, and that EZH2 downregulation is a non-genetic mechanism of resistance to PI3K inhibition. Additionally, our findings indicate that combining PI3K inhibitors with EZH1/2 dual inhibitors could be a promising therapeutic approach to reduce LSCs, prevent resistance, and prolong survival in AML patients.

Combination of Selinexor with BTK Inhibitor for Central Nervous System Diffuse Large B-Cell Lymphoma, Possible Mechanisms and Therapeutic Potential Exploration

Background: Primary central nervous system lymphoma (PCNSL) is a rare aggressive extra-nodal non-Hodgkin lymphoma (NHL) of which over 90% are diffuse large B-cell lymphoma (DLBCL). High-dose methotrexate (HD-MTX)-based regimens are the dominant therapy for newly diagnosed PCNSL, but the patients' complete remission rate (CRR) is merely about 50%. Bruton tyrosine kinase (BTK), a critical kinase in the B cell receptor (BCR) signaling pathway, contributes to the degradation of FOXO3a and promotes abnormal B cell proliferation. Recent studies revealed that BTK inhibitors (BTKi) play a role in various B cell lymphomas, including DLBCL, and have also shown promising efficacy in PCNSL. However, the clinical management of PCNSL is still challenging for its limited treatment options and poor prognosis, especially when patients developed acquired resistance to BTKi. Selinexor (KPT-330) is a first-ever oral small molecule Exportin-1 (nuclear output protein XPO1) inhibitor with a molecular weight of 443.31 Da, which can efficiently cross the blood-brain barrier. Currently Selinexor was found to promote the aggregation of FOXO3a and could synergistically inhibit PI3K/AKT and NF-κB pathways with BTKi to overcome acquired BTKi resistance. To investigate the therapeutic potential of Selinexor and BTKi in PCNSL, we examined the combination both in DLBCL cells and A20 transplantation mice, and further explored the clinical efficacy in PCNSL patients. Methods and Results: ABC-DLBCL cell lines (OCI-LY3 and OCI-LY10) were treated with Selinexor for 48 hours. Western blot analysis showed that Selinexor inhibited NF-κB, P-STAT3, and NFATc1 signaling pathways in DLBCL in vitro. Combining Selinexor(1μM) with ICP-022(2μM), a BTKi, resulted in more notable inhibition of the above tumor signaling pathways. A20 murine B-cell lymphoma transplantation models were divided into a control group, Selinexor monotherapy group, ICP-022 monotherapy group, and combination group. Tumor volume was measured after 30 days (Figures 1A), and the number of CD4+ T cells and CD8+ T cells and the corresponding cellular expression of PD-1 and TIM-3 in the spleen and tumor tissues of mice were detected. The results showed that the tumor volume in the combination group was minimized, and the number of CD4+ T cells and CD8+ T cells in spleens and tumors in this group was significantly increased, while PD-1 and TIM-3 expression was downregulated (Figures 1B-C). Based on these results, a prospective, open-label, single-arm, multicenter study was conducted with MSZ regimen (MTX, BTKi, Selinexor) for the treatment of PCNSL (Trial Registration Number: ChiCTR2200062154). Newly diagnosed primary and secondary central DLBCL patients (18-80 years old) received a 6-cycle MSZ regimen (HDMTX 3.5 g/m 2 3-h infusion d1, Zanubrutinib 160 mg Bid po, Selinexor 40 mg Biw po, 3 weeks per cycle) and evaluated by MRI/PET. 5 patients (2 GCB-DLBCL, 3 ABC-DLBCL) were enrolled, with a median age of 65 years (range: 54-73 years), 100% achieved metabolic remission (Figure 2A) and maintained long-term efficacy. 4 patients achieved early deep remissions of over 75% reduction with 1 cycle of treatment, and 1 patient had a complete remission after 2 cycles of treatment (Figures 2B). 3 patients chose radiation therapy as consolidation after the end of induction chemotherapy. 2 others were on maintenance therapy with Selinexor combined with Zanubrutinib, one of them has completed the two-year maintenance treatment and maintained sustained remission till now. Only 1 patient experienced several grade 3 adverse events (AEs) including anemia, decreased white blood cell and platelet counts, and elevated alanine aminotransferase.1 patient developed mild renal insufficiency, which recovered 6 months after the end of treatment. Patients were generally well-tolerated and there were no treatment-related deaths. Conclusion: This is the first report describing that Selinexor combined with BTKi can synergistically inhibit the DLBCL tumor signaling pathway and repairing the immune microenvironment. Correspondingly, in treatment of central DLBCL patients, the novel therapeutic strategy of MSZ regimen has achieved impressive results, with a CRR of 100%. Most patients attained early and rapid deep remissions with durable efficacy. This study provides theoretical support and preliminary clinical data for the combined application of Selinexor and BTK inhibitors.

Targeting EYA1 Activity in MLL- Rearranged Leukemia

The Mixed Lineage Leukemia gene (also known as MLL or KMT2A) is involved in chromosomal translocations in a subtype of acute myeloid (AML) and lymphoblastic leukemias (ALL). MLL translocations have been identified with more than 80 partner genes, and the most common AML translocation partner is MLLT3 (AF9). MLL-rearranged (MLL-r) leukemia is associated with a sudden onset, aggressive progression, and poor prognosis in comparison to non-MLL-r (MLL-nr) leukemias, and there is a need for more effective therapies. Previous work determined that AF9 directly interacts with multiple different regulatory proteins, including the BCL6 corepressor (BCOR). An AF9 point mutation (E531R) was identified that selectively disrupts BCOR binding to AF9 and MLL-AF9. The loss of BCOR binding to MLL-AF9 abrogated its leukemogenic ability in a mouse transplant model. RNA-seq analysis determined that of the MLL-AF9 direct target genes, EYA1 was the most down-regulated in the MLL-AF9 (E531R) point mutant-transformed cells. Addback of EYA1 into MLL-AF9 (E531R) cells partially rescued its leukemogenic ability, suggesting that EYA1 has an important role in MLL-AF9 leukemogenesis. EYA1 is a transcriptional coactivator and protein tyrosine phosphatase that is expressed during embryonic development and its re-expression has been implicated in several cancers, including MLL-r leukemia. The objective of this study is to investigate the in vitroand in vivo effects of EYA1 inhibition in MLL-AF9 transformed murine bone marrow stem/progenitor cells and in human MLL-rearranged (MLL-r) and non-rearranged (MLL-nr) leukemia cell lines. Cells were treated with small molecule inhibitors at varied concentrations and time points. Effects of inhibition were assessed with viability, cell morphology, cell cycle, surface and intracellular marker expression analyses, and in vivo transplant studies. EYA1 inhibition reduced cell viability by 50% - 90% in MLL-AF9 transformed murine bone marrow (mBM) cells and multiple human AML cell lines. EYA1 inhibition did not affect some human B-ALL cell lines, which also express undetectable levels of EYA1, indicating a correlation between the response to EYA1 inhibition and EYA1 expression. In MLL-r and MLL-nr cells, cell cycle analysis indicated a reduction in cells entering S-phase, stalling in G0/G1, with EYA1 inhibition as well as an induction in cellular senescence. Inhibiting EYA1 activity induced cell morphology changes toward a more differentiated state in MLL-AF9 mBM and several MLL-r and -nr cell lines. To confirm these qualitative results, cells were stained for myeloid differentiation markers, and cell surface expression of CD11b, Gr1, and CD14 increased in a dose- and time-dependent manner in all cell types, while HSC markers CD117 (c-KIT) and CD34 were reduced in MLL-AF9 mBM and human AML cells, respectively. In assessing the mechanism of EYA1 in MLL-r leukemia, MLL-AF9 cells treated with EYA1 inhibitors showed increased RNA pol II CTD Tyr1 phosphorylation levels (known EYA1 substrate) compared to vehicle-treated cells. Thus, unregulated EYA1 phosphatase activity targeting RNA Pol II CTD may contribute to leukemia development. Mice that received MLL-AF9 mBM cells treated with an EYA1 inhibitor had a significantly decreased rate of disease progression and increased survival compared to control. Bone marrow cells isolated from these mice showed increased expression of myeloid differentiation markers CD11b and Gr1 compared to vehicle-treated mice, confirming that EYA1 activity contributes significantly to leukemogenesis. The inhibition of EYA1 induces leukemia cell differentiation, cell cycle arrest and senescence, and cell death. Ongoing experiments include additional in vivo murine leukemia, further mechanistic analyses, and combinatorial experiments to evaluate the efficacy of a multi-target approach with EYA1 inhibition and other targeted-therapies. This work may provide a promising, novel therapeutic approach to MLL-r and MLL-nr leukemias that will help improve patient outcomes.

JAK2V617F Activates IL-10R Signalling Potentially through Transactivation of JAK1

Classical myeloproliferative neoplasms (MPNs) arise from somatic mutations in haematopoietic stem cells (HSCs) that lead to unregulated proliferation of mature myeloid cells. The most common mutation associated with MPNs is JAK2 V617F, this causes constitutive JAK-STAT signalling leading to excessive production of mature myeloid cells. In humans, for MPN to arise, a JAK2 V617F HSC must have a selective advantage over WT HSC. However, in mouse MPN models Jak2 V617F HSCs do not display an overt selective advantage. This suggests that unique selective pressures may be present among those predisposed to acquire MPN that allow for the emergence of Jak2 V617F clones. We have identified defects in the IL-10R signalling pathway among MPN patients, with evidence that this defect is an intrinsic not acquired abnormality. In mice, we found that the blockade of IL-10R extends the cycling of WT but not Jak2 V617FHSC in response to inflammatory stimuli and hastens proliferation-induced WT HSC exhaustion. We hypothesized that IL-10R blockade may create an environment that affords Jak2 V617F HSC a selective advantage, indeed we found that IL-10R blocking antibody allowed Jak2 V617F cells to outcompete WT cells in mouse transplant experiments. From these mouse data, we hypothesized that Jak2 V617Fenhances IL-10R signalling. To test this hypothesis, we created Ba/F3 cell lines co-expressing IL-10Rα and Jak2 V617F. We found that Jak2 V617F enhances growth at limiting concentrations of IL-10. We also found that cytokine-independent IL-10Rα Jak2 V617F clones (but never IL-10Rα Jak2 WTor IL-10Rα cells) emerge at day 8-10 in culture after withdrawal of cytokines. We performed a limiting dilution assay to pinpoint the proportion of IL-10R Jak2 V617F cells capable of surviving IL-3 withdrawal. One in 126 IL-10Rα Jak2 V617F cells has transforming ability, whereas 1 in 1.4 MPL Jak2 V617F has transforming ability. Together, these data support our hypothesis that Jak2 V617F enhances IL-10R signalling and can activate IL-10Rα, however with weaker transformation potential compared to Jak2V617F's activation of MPL signalling. JAK2 canonically does not participate in the IL-10R pathway. It has been previously shown that JAK1 can transactivate JAK2 when JAK2 V617F cells are under the stress of persistent JAK2 inhibitor exposure, and this is the purported mechanism of JAK inhibitor resistance among MPN patients. We hypothesized that JAK2 V617F can transactivate JAK1, leading to the activation of IL-10R. The same transfected BaF3 cells aforementioned were serum starved for 4 hours and stimulated with mIL-10 for 15 minutes to induce signalling. By way of Western Blot analysis, cytokine-independent Jak2 V617F IL-10Rα Ba/F3 cells showed constitutive activation of phosphorylated JAK1 and JAK2 as well as STAT3 and STAT5, consistent with transactivation. Studies to demonstrate direct interactions between JAK2 V617F and JAK1 are currently ongoing.

Improvement of Inflammation and Abnormal Vascularization by TSP1 Treatment Combined with ADSCs Transplantation in Mice with Induced Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease with a certain degree of chronic inflammation and abnormal ovarian angiogenesis in reproductive women. Mesenchymal stem cells (MSCs) have potent immunomodulatory properties to regulate ovarian function, while thrombospondin 1 (TSP1) improves the abnormal formation of ovarian vessels. The present study investigated the efficacy of the combined use of adipose-derived mesenchymal stem cells (ADSCs) and TSP1 in PCOS mice. The PCOS model is established using dehydroepiandrosterone (DHEA) by subcutaneous injection. Ovarian apoptosis is assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Real-time quantitative PCR (RT-PCR) and western blotting are used to detect the expression of inflammatory factors and the levels of angiogenesis-related factors in ovarian tissues. Inflammatory cells count and ovarian angiogenesis are evaluated by immunofluorescence staining. This research shows that TSP1 and ADSCs treatment can significantly reduce the inflammatory state of PCOS mice, relieve the degree of ovarian cell apoptosis, optimize the ovarian histological manifestations, and restore the levels of related hormones. The proportion of CD31-positive cells in PCOS mice returned to near-normal levels. The synergistic use of ADSCs and TSP1 therapy can exert a more impressive effect by inhibiting the ovarian inflammatory response and regulating the balance of angiogenesis than the single application in PCOS mice.

Interleukin 4 improved adipose-derived stem cells engraftment via interacting with fibro/adipogenic progenitors in dystrophic mice

Adipose-derived stem cells (ADSC) therapy shows promise as an effective treatment for dystrophinopathy. Fibro-/adipogenic progenitors (FAPs) play an essential role in the myogenesis of muscle satellite cells and contribute to muscle fibrosis and adipocyte infiltration. The interleukin 4 (IL-4) pathway acts as a switch that regulates the functions of FAPs. The interaction between FAPs and engrafted cells remains unclear. In this study, we used a co-culture system to investigate possible crosstalk between the FAPs of dystrophic mice and ADSC overexpressing IL4 (IL4-ADSC) and control ADSC. Systemic transplantation of IL4-ADSC and control ADSC in dystrophic mice was conducted for 16 weeks, after which motor function and molecular improvements were evaluated. Overexpression of IL4 in ADSC significantly promoted myogenesis in vitro, increasing the expression of Pax7, Myogenin, and MyHC. Co-culture indicated that although myoblasts derived from control ADSC promoted adipogenic and fibrogenic differentiation of FAPs, FAPs did not significantly affect myogenesis of ADSC-derived myoblasts. However, overexpression of IL4 in ADSC inhibited their myotube-dependent promotion of FAPs differentiation on the one hand and promoted FAPs to enhance myogenesis on the other. Dystrophic mice administered with IL4-ADSC-derived myoblasts displayed significantly better motor ability, more engrafted cells showing dystrophin expression, and less muscle fibrosis, intramuscular adipocytes, and macrophage infiltration than mice administered control-ADSC-derived myoblasts. In conclusion, IL4 activation enhanced the therapeutic potential of ADSC transplantation in dystrophic mice, possibly by improving the myogenesis of IL4-ADSC and altering the crosstalk between engrafted stem cells and resident FAPs.

The Effects of IL-23/IL-18-Polarized Neutrophils on Renal Ischemia-Reperfusion Injury and Allogeneic-Skin-Graft Rejection in Mice.

Neutrophils display heterogeneity and plasticity with different subgroups and immune-regulatory functions under various surrounding conditions. Neutrophils induced by IL-23/IL-18 (referred to N(IL-23+IL-18) neutrophils) have a unique gene-expression profile, with highly expressing IL-17, MHC-II, and costimulatory molecules. The adoptive transfer of N(IL-23+IL-18) neutrophils significantly increased the pathogenesis in a renal ischemia-reperfusion injury mouse model. N(IL-23+IL-18) neutrophils directly and efficiently induced allogeneic T cell proliferation in vitro. N(IL-23+IL-18) neutrophils enhanced the syngeneic T cell response to allogeneic antigens in mixed-lymphocyte reaction assays. The adoptive transfer of the donor or host N(IL-23+IL-18) neutrophils significantly enhanced the antidonor antibody production in an allogeneic-skin-transplanted mouse model, accompanied by increased Tfh cells in the spleens. Therefore, the neutrophil subset induced by IL-23/IL-18 promotes tissue injury and antidonor humoral response in the allogeneic transplantation mouse model.

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF <0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

Knockdown of SLC39A14 inhibits glioma progression by promoting erastin-induced ferroptosis SLC39A14 knockdown inhibits glioma progression

BackgroundFerroptosis is a newly classified form of regulated cell death with implications in various tumor progression pathways. However, the roles and mechanisms of ferroptosis-related genes in glioma remain unclear.MethodsBioinformatics analysis was employed to identify differentially expressed ferroptosis-related genes in glioma. The expression levels of hub genes were assessed using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). To explore the role of SLC39A14 in glioma, a series of in vitro assays were conducted, including cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), flow cytometry, wound healing, and Transwell assays. Enzyme-linked immunosorbent assay (ELISA) was utilized to measure the levels of indicators associated with ferroptosis. Hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining were performed to illustrate the clinicopathological features of the mouse transplantation tumor model. Additionally, Western blot analysis was used to assess the expression of the cGMP-PKG pathway-related proteins.ResultsSeven ferroptosis-related hub genes, namely SLC39A14, WWTR1, STEAP3, NOTCH2, IREB2, HIF1A, and FANCD2, were identified, all of which were highly expressed in glioma. Knockdown of SLC39A14 inhibited glioma cell proliferation, migration, and invasion, while promoting apoptosis. Moreover, SLC39A14 knockdown also facilitated erastin-induced ferroptosis, leading to the suppression of mouse transplantation tumor growth. Mechanistically, SLC39A14 knockdown inhibited the cGMP-PKG signaling pathway activation.ConclusionSilencing SLC39A14 inhibits ferroptosis and tumor progression, potentially involving the regulation of the cGMP-PKG signaling pathway.

PDGFR-beta signaling mediates endogenous neurogenesis after postischemic neural stem/progenitor cell transplantation in mice

ABSTRACT Objective Although platelet-derived growth factor receptor (PDGFR)-β mediates the self-renewal and multipotency of neural stem/progenitor cells (NSPCs) in vitro and in vivo, its mechanisms of activating endogenous NSPCs following ischemic stroke still remain unproven. Methods The exogenous NSPCs were transplanted into the ischemic striatum of PDGFR-β conditionally neuroepithelial knockout (KO) mice at 24 h after transient middle cerebral artery occlusion (tMCAO). 5-Bromo-2’-deoxyuridine (BrdU) was intraperitoneally injected to label the newly formed endogenous NSPCs. Infarction volume was measured, and behavioral tests were performed. In the subventricular zone (SVZ), proliferation of endogenous NSPCs was tested, and synapse formation and expression of nutritional factors were measured. Results Compared with control mice, KO mice showed larger infarction volume, delayed neurological recovery, reduced numbers of BrdU positive cells, decreased expression of neurogenic factors (including neurofilament, synaptophysin, and brain-derived neurotrophic factor), and decreased synaptic regeneration in SVZ after tMCAO. Moreover, exogenous NSPC transplantation significantly alleviated neurologic dysfunction, promoted neurogenesis, increased expression of neurologic factors, and diminished synaptic deformation in SVZ of FL mice after tMCAO but had no beneficial effect in KO mice. Conclusion PDGFR-β signaling may promote activation of endogenous NSPCs after postischemic NSPC transplantation, and thus represents a novel potential regeneration-based therapeutic target.

TMIC-60. CROSS-SPECIES ANALYSIS OF HUMAN AND NEW MOUSE MODELS OF GBM SUBTYPES IDENTIFIES VISTA AS A PROMISING IMMUNOTHERAPY TARGET

Abstract Immunotherapy is a promising treatment modality for GBM; however, clinical trials thus far have failed to provide significant clinical benefit to most patients. GBM is one of the “immune cold” tumors characterized by poor infiltration of T cells and the abundance of immune-suppressive myeloid cells that block T cell infiltration and/or activation. A major bottleneck to developing more effective immunotherapies is the paucity of faithful preclinical models to discover and validate new treatments. Here, we report multi-dimensional analyses of six different transplantable mouse glioma models in the C57BL6/J background that represent the three human GBM molecular subtypes. We performed whole exome sequencing, STR fingerprinting, immune phenotyping, and single-cell RNA-sequencing of each model and confirmed that the new models exhibit highly heterogeneous and immune suppressive TME, similar to human GBM. In addition, we performed cross-species comparisons of glioma and immune cell subtypes between human and mouse GBMs at the single-cell level and demonstrate strong concordances in molecular phenotypes and cellular heterogeneity. These result in qualitative and quantitative differences in the cell: cell communication among different stromal cells and glioma cells in each model, and we propose that these interactions shape the local niche and immune suppressive neighborhoods. Notably, in both human tissues and mouse models, PD1/PDL1 expression is low or sporadic, and, unlike the GL261 model, the new mouse models are resistant to anti-PD1 and CTLA4 treatment in vivo. Finally, we discovered that VISTA is a widely expressed immune checkpoint in both human and mouse GBM and demonstrate the efficacy of anti-VISTA and anti-PD1 combination therapy in the most immune-suppressive mouse model. In summary, we demonstrate the utility of these immune-competent mouse models and nominate anti-VISTA+PD1 treatment as a promising novel combination therapy for GBM.

DUSP1 protects against ischemic acute kidney injury through stabilizing mtDNA via interaction with JNK

The mechanism underlying acute kidney injury (AKI) and AKI-to-Chronic kidney disease (CKD) transition remains unclear, but mitochondrial dysfunction may be a key driving factor. Literature reports suggest that dual-specificity phosphatase 1 (DUSP1) plays a critical role in maintaining mitochondrial function and structural integrity. In this study, ischemic Acute Kidney Injury (AKI) and post-ischemic fibrosis models were established by clamping the renal pedicle with different reperfusion times. To investigate the role of DUSP1, constitutional Dusp1 knockout mice and tubular-specific Sting knockout mice were used. Mitochondrial damage was assessed through electron microscopy observation, measurements of mitochondrial membrane potential, mtDNA release, and BAX translocation. We found that Dusp1 expression was significantly upregulated in human transplant kidney tissue and mouse AKI tissue. Dusp1 gene deletion exacerbated acute ischemic injury, post-ischemic renal fibrosis, and tubular mitochondrial dysfunction in mice. Mechanistically, DUSP1 could directly bind to JNK, and DUSP1 deficiency could lead to aberrant phosphorylation of JNK and BAX mitochondria translocation. BAX translocation promoted mitochondrial DNA (mtDNA) leakage and activated the cGAS-STING pathway. Inhibition of JNK or BAX could inhibit mtDNA leakage. Furthermore, STING knockout or JNK inhibition could significantly mitigate the adverse effects of DUSP1 deficiency in ischemic AKI model. Collectively, our findings suggest that DUSP1 is a regulator for the protective response during AKI. DUSP1 protects against AKI by preventing BAX-induced mtDNA leakage and blocking excessive activation of the cGAS-STING signaling axis through JNK dephosphorylation.

MYC overexpression and SMARCA4 loss cooperate to drive medulloblastoma formation in mice

Group 3 medulloblastoma is one of the most aggressive types of childhood brain tumors. Roughly 30% of cases carry genetic alterations in MYC, SMARCA4, or both genes combined. While overexpression of MYC has previously been shown to drive medulloblastoma formation in mice, the functional significance of SMARCA4 mutations and their suitability as a therapeutic target remain largely unclear. To address this issue, we combined overexpression of MYC with a loss of SMARCA4 in granule cell precursors. Both alterations did not increase proliferation of granule cell precursors in vitro. However, combined MYC overexpression and SMARCA4 loss successfully induced tumor formation in vivo after orthotopic transplantation in recipient mice. Resulting tumors displayed anaplastic histology and exclusively consisted of SMARCA4-negative cells although a mixture of recombined and non-recombined cells was injected. These observations provide first evidence for a tumor-promoting role of a SMARCA4 deficiency in the development of medulloblastoma. In comparing the transcriptome of tumors to the cells of origin and an established Sonic Hedgehog medulloblastoma model, we gathered first hints on deregulated gene expression that could be specifically involved in SMARCA4/MYC driven tumorigenesis. Finally, an integration of RNA sequencing and DNA methylation data of murine tumors with human samples revealed a high resemblance to human Group 3 medulloblastoma on the molecular level. Altogether, the development of SMARCA4-deficient medulloblastomas in mice paves the way to deciphering the role of frequently occurring SMARCA4 alterations in Group 3 medulloblastoma with the perspective to explore targeted therapeutic options.

Inhibition of Sphingosine Kinase 2 Enhances Immunotherapy in Mouse Model of Multiple Myeloma

Introduction: Sphingolipid metabolism, in particular the ceramide:sphigosine 1-phophate (S1P) rheostat, is increasingly recognized as a key pathway in cancer biology, inflammation and immune responses. Sphingosine kinase (SK) 1 and 2 catalyze the formation of S1P. We previously reported that SK2 but not SK1 was overexpressed in multiple myeloma (MM) cells. Treatment with opaganib, an SK2-specific inhibitor, downregulated the expression of c-Myc and Mcl-1, resulting in apoptosis of MM cells. A phase I clinical trial of opaganib in patients with relapsed/refractory MM demonstrated the safety and evidence of anti-myeloma activity of SK2 inhibition. However, the roles of SK2 in host's anti-tumor immunity are unclear. We herein determined the effects of SK2 on the functions of T cells and myeloid derived suppressor cells (MDSCs), the underlying mechanisms, and potential clinical application of SK2 inhibition in enhancing immunotherapy. Methods: For tumor models, transplantable mouse VK*Myc myeloma cells, CT-2A gliomas cells, B16F10 melanoma cells, or TRAMP C2 prostate cancer cells were implanted into SK2 -/- knockout (KO), SK1 -/- KO or WT mice. Multi-color flow cytometry was used to measure various immune cells. Anti-CD8 antibody (clone YST-169.4) and anti-Gr-1 antibody (clone RB6-8C5) were used to deplete CD8 T cells and MDSCs in vivo, respectively. Adaptive transfer of enriched CD8 T cells and bone marrow derived MDSCs was performed. Cytokines and chemokines were measured by multiplex assay. Transcriptome sequencing and omics analyses were performed on mouse primary CD8 T cells and MDSCs. CRISPR/Cas9 genetic knockout experiments were used to validate genes identified from the sequencing results. Mouse BCMA CAR T model was established using syngeneic VK*Myc myeloma cells and anti-mouse BCMA CAR T cells. Additionally, the combinatorial effects of opaganib and 4-1BB agonist were examined in VK* Myc myeloma mouse model. Results: When we injected VK*Myc myeloma cells IV into SK1 -/- KO, SK2 -/- KO or WT mice, none of SK2 -/- KO mice developed myeloma or died from myeloma whereas 85-90% of WT recipient mice and all SK1 -/- KO mice developed myeloma and died. Similarly, tumor development was significantly attenuated in SK2 -/- KO mice when CT-2A gliomas cells (intracranial injection), B16F10 melanoma cells (IV) or TRAMP C2 prostate cancer cells (IV) were implanted, demonstrating broad anti-tumor effects of SK2 deletion. We found that the homing of myeloma cells was not affected in SK2 -/- KO mice. SK2 -/- KO recipient mice showed significantly increased number of CD8 T cells and decreased number of MDSCs. Depletion of CD8 T cells using CD8 antibody rendered SK2 -/- KO mice susceptible to myeloma development and administration of anti-Gr-1 antibody increased CD8 T cells and made SK2 WT mice resistant to myeloma development. Adaptive transfer of SK2 -/- KO CD8 T cells suppressed myeloma development, indicative of a critical role of SK2 in T cell regulation. Cytokine profiling revealed significantly increased level of IFNγ and IL-12 and suppressed level of IL-6, GM-CSF, CCL2, TNFα, and IL-10 in SK2 -/- KO recipient mice. CD8 T cells isolated from SK2 -/- KO mice were more proliferative in response to CD3/CD28 antibody stimulation, had higher expression of CD69, Granzyme B and IFNγ but lower level of PD-1, LAG-3, TIGIT and TIM-3, and were more cytotoxic against myeloma cells. Differential gene expression analyses showed up-regulation of IL-36gamma and kallikrein 1-related peptidase b22 (KLK1B22) in SK2 -/- KO CD8 T cells. Knockout of IL-36gamma or KLK1B22 with CRISPR/Cas9 reversed the effects of SK2 deletion on T cell activation and exhaustion, demonstrating the important role of IL-36gamma and KLK1B22 in SK2 mediated T cell regulation. Compared to T cells from WT mice, T cells from SK2 -/- KO mice exhibited enhanced anti-myeloma activities in our VK*Myc myeloma anti-BCMA CAR T mouse model. Finally, treatment of mice with opaganib increased the number of T cells and decreased the number of MDSCs and showed synergistic anti-myeloma activities when combined with 4-1BB agonist in VK*Myc myeloma model. Conclusions: Our study demonstrated that inhibition of SK2 enhances anti-tumor immunity by promoting CD8 T cell activation likely through IL-36gamma and KLK1B22. These studies provide rationale for clinical trials investigating the combination of opaganib with CAR T therapy or other immunotherapy in cancer treatment.

Pgam1 Regulate Adaptive NK Cells in Anti-Viral and Anti-Tumor Response

Natural killer (NK) cells are the innate immune cells that play a role in the anti-viral infections and tumor cells. The adaptive NK cells are characterized with expression of the HLA-E-specific CD94/NKG2C activating receptor. The reconstitution of NKG2C+ adaptive NK cells post HSCT is much faster in NKG2C homozygous(NKG2C wt/wt) donors than NKG2C heterozygous donors (NKG2C wt/del), which resulted in lower incidence of CMV infection. In this study, we aimed to find out the mainly regulators of adaptive NK cells between NKG2C wt/wt and NKG2C wt/del donors. RNA-sequencing analysis were performed. Results shown that NK cells from NKG2C wt/wt donors constant upregulated glycolysis and biosynthesis-related genes, while downregulated in TGF-beta signaling and FOXO signaling pathway(Figure A and B). In order to determine the role of glycolysis in adaptive NK cells, upregulated glycolysis related genes were assessed by q-PCR, and PGAM1 is considered as the most important gene in the expansion of adaptive NK cells(Figure C). PGAM1 is upregulated upon NK cells stimulation via cytokine or target cells, which is related to the CD107a expression. HKB99 is an allosteric inhibitor for PGAM1, which is considered as a potential therapeutic target for many cancers nowadays. NK cells were added with HKB99 in vitro for 12h hours to evaluate the anti-K562 function. After that, Glycolysis is inhibited with the decreasing of ECAR while the OCR is not affected (FigureD). The adding of PGAM1 inhibitor resulted in decreasing of CD107a expression in NK cells, especially in the adaptive NK cells. NK cells activation signaling pathway is also inhibited, including the phosphorylation of mTORC1 protein S6, Syk, zap70, and the PLC-gamma. The PI3K/AKT is inhibited after the PGAM1 inhibition. After that,supplementation of pyruvate is efficient to rescue the inhibition caused by PGAM1 inhibitor. 5mM pyruvate adding can restore the cytokine release of NK cells and the phosphorylation of S6 and syk/zap70(Figure E). PGAM1-NCR-cre mice was constructed in which PGAM1 is knock out in NK cells specifically. In KO group, the ratio of NK cells and the maturation of NK cells are decreased. Upon cytokine stimulation, the activator receptors expression is less in KO mice. Besides, PGAM1 deficiency NK cells exhibited glucose uptake deficiency and disadvantages in proliferation both in vivo and in vitro (Figure F). The differentiation (CD11b+CD27-) and activation marker (DNAM1, Ly49H) are decreased in the competing transplantation mouse model, while the exhaustion markers such as PD-1 and NKG2A is upregulated. Further, MCMV infection models shown that PGAM1 deficiency mice with lower ratio of Ly49H+ adaptive NK cells at the early stage of infection, while Ly49G2 and Ly49C+ cells are not affected(Figure G). Further, Ly49H+ adaptive NK cells shown less expression of Ki67 in PB-NK cells, with the activating markers CD25 is decreased. Higher MCMV load induced inflammation is occurred in PGAM1 deficiency NK cells. Anti-tumor effect was also evaluated. The apoptosis of YAC-1 cells is decrease in coincidence with CD107a expression decreasing. MLL-AF9 leukemia mice model shown in day 18, the Ly49H+ adaptive NK cells in PGAM1 deficiency mice is less than WT mice, and the tumor burden is higher and accompanied with shorter survival under tumor circumstances(Figure H). RNA seq analysis shown compared to WT mice, the downregulated genes in PGAM1 deficiency NK cells are enriched in leukocyte activation and innate immune response also interfered the cytokine-mediated signaling pathway. At last, we monitored expression of PGAM1 and the function of NK cells in patients whose CMV is reactive post HSCT. PGAM1 is elevated post CMV infection, which is correlated with CD107a secretion. ROC curve suggests that PGAM1 may act as a predictor for refractory recurrence CMV infection in patients post HSCT(Figure I). In summary, our studies shown the glycolysis is effectively support the faster adaptive NK cells expansion and CMV response. PGAM1-mediated glycolysis is significant for NK cells anti-tumor and anti-viral function.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

Heterogeneous Intercellular Communication of Hematopoietic Stem Cells in the Mouse Bone Marrow

Introduction.Hematopoietic stem cells (HSCs) are responsible for maintaining the homeostasis of the blood and immune systems. This process must rapidly adapt to bleeding, infection, injury, and disease. HSCs reside in specialized microenvironments in the bone marrow, called “niche”. Several bone marrow cell types have been proposed as the key components of the niche. It is well established that HSCs rely on molecular signals from the niche cells to survive, migrate, proliferate, and differentiate. Recent studies suggest that HSCs differentiate heterogeneously. Additionally, the bone marrow is a heterogeneous environment consisting of more than thirty cell types. This study will test the hypothesis that individual HSCs are engaged in heterogeneous intercellular signaling in the bone marrow. Materials and Methods.We utilized CellChat (Jin et al., 2021), a database for ligand-receptor pairs and associated pathways, to predict cell-cell signaling at the single cell level. We used single cell RNA sequencing data from mouse hematopoietic stem and progenitor cells, blood and immune cells, and non-hematopoietic cells from the bone marrow to determine putative cell-cell interactions. The predicted signaling interactions were then adjusted based on the cell-cell spatial interaction revealed by PhenoCycler measurement (Akoya Biosciences). Mouse bone marrow transplantation experiments were performed to test the functional significance of the identified signaling pathway interactions. Results.We found that individual HSCs send and receive different levels of molecular signals across various signaling pathways. In addition to the expected interactions with the known HSC niche cells, we also found new interactions between HSCs and their progeny cells including myeloid progenitors and immune cells. These cell-cell signaling interactions are consistent with the spatial interaction frequencies of the corresponding cell types as revealed by our imaging data from the PhenoCycler analysis. Further, we found that some signaling pathways are positively or negatively correlated across individual HSCs, indicating complex intercellular signaling networks. The most significant negative correlation was found between HSCs sending MHC-I signals and sending PARs signals (Figure). Moreover, this MHC-I/PARs signaling correlation network also involves signaling from the HSC niche, including those that are known to play roles in HSC homing such as ESAM and VCAM. Therefore, we tested the engraftment efficiency of HSCs that receive PARs and those that do not using the HSC mouse transplantation model. We found that PAR1 + HSCs engraft much less efficiently. Finally, we present the HSC Interaction Data Explorer (HIDE), a database that comprehensively depicts the interactions between HSCs and other bone marrow cells including the involved signaling pathways, ligands and receptors of the pathways, and the pathway correlations. Conclusion.Our study uncovered novel interactions of HSCs with myeloid progenitor cells and immune cells, which were validated by spatial interaction data. These interactions indicate feedback signaling in regulating blood cell regeneration. Our findings further revealed signaling pathway correlation networks across individual HSCs, and we showed their functional significance using transplantation experiments. Finally, we present HIDE as a resource that informs the interactions between HSCs and various bone marrow cells, the relevant signaling pathways, and the pathway correlations. Figure Legend. HSC signaling correlation networks. Each node shows a signaling pathway and direction (e.g., HSCs sending MHC-I). The edges represent the spearman correlation score.

Fecal Microbiota Transplantation in Mice Exerts a Protective Effect Against Doxorubicin-Induced Cardiac Toxicity by Regulating Nrf2-Mediated Cardiac Mitochondrial Fission and Fusion.

Aims: The relationship between the gut microbiota and cardiovascular system has been increasingly clarified. Fecal microbiota transplantation (FMT), used to improve gut microbiota, has been applied clinically for disease treatment and has great potential in combating doxorubicin (DOX)-induced cardiotoxicity. However, the application of FMT in the cardiovascular field and its molecular mechanisms are poorly understood. Results: During DOX-induced stress, FMT alters the gut microbiota and serum metabolites, leading to a reduction in cardiac injury. Correlation analysis indicated a close association between serum metabolite indole-3-propionic acid (IPA) and cardiac function. FMT and IPA achieve this by facilitating the translocation of Nfe2l2 (Nrf2) from the cytoplasm to the nucleus, thereby activating the expression of antioxidant molecules, reducing reactive oxygen species production, and inhibiting excessive mitochondrial fission. Consequently, mitochondrial function is preserved, leading to the mitigation of cardiac injury under DOX-induced stress. Innovation: FMT has the ability to modify the composition of the gut microbiota, providing not only protection to the intestinal mucosa but also influencing the generation of serum metabolites and regulating the Nrf2 gene to modulate the balance of cardiac mitochondrial fission and fusion. This study comprehensively demonstrates the efficacy of FMT in countering DOX-induced myocardial damage and elucidates the pathways linking the microbiota and the heart. Conclusion: FMT alters the gut microbiota and serum metabolites of recipient mice, promoting nuclear translocation of Nrf2 and subsequent activation of downstream antioxidant molecule expression, while inhibiting excessive mitochondrial fission to preserve cardiac integrity. Correlation analysis highlights IPA as a key contributor among differentially regulated metabolites.

Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.

The upregulation of immune checkpoints after photodynamic therapy reducing immune effect for treating breast cancer.

The immune effect induced by photodynamic therapy (PDT) has a limited effect on breast tumor. This study hypothesized that suppressive immune checkpoints on T cells might upregulate after PDT, which may reduce the antitumor effect of PDT for treating breast tumor. This study explored the alteration of immune checkpoint for the first time. A bilateral subcutaneous transplanted breast tumor mice model was established, and right tumors imitated primary tumors, and left tumors imitated distant tumors. Primary tumors were treated with PDT mediated by hematoporphyrin derivatives (HpD-PDT). Costimulatory molecules (ICOS, OX40, and 4-1BB) and immune checkpoints (PD1, LAG-3, CTLA-4, TIM-3, TIGIT) on tumor infiltrating T cells after HpD-PDT were analyzed by flow cytometry. Antitumor and immune effects were also assessed after HpD-PDT combined with anti-PD1 and LAG-3 antibodies. Primary tumors were suppressed, but distant tumors could not be inhibited after HpD-PDT. The number of T cells was increased, but function did not enhance after HpD-PDT. Additionally, costimulatory molecules (ICOS, OX40, and 4-1BB) were not elevated, but the suppressive immune checkpoints on tumor infiltrating T cells were upregulated after HpD-PDT. Notably, PD1+ LAG-3+ CD4+ T and PD1+ LAG-3+ CD8+ T cells were significantly increased. When PD1 and LAG-3 blockade combined with HpD-PDT, both primary and distant tumors were significantly suppressed, and antitumor immune effects were significantly enhanced. HpD-PDT could upregulate the PD1+ LAG-3+ CD4+ T and PD1+ LAG-3+ CD8+ T cells. Dual blockade of PD1 and LAG-3 immune checkpoints can enhance the antitumor effect of HpD-PDT.