Higher Chondrogenic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells Compared to Chondrocytes-EVs In Vitro

The inability of cartilage to self-repair necessitates an effective therapeutic approach to restore damaged tissues. Extracellular vesicles (EVs) are attractive options because of their roles in cellular communication and tissue repair where they regulate the cellular processes of proliferation, differentiation, and recruitment. However, it is a challenge to determine the relevant cell sources for isolation of EVs with high chondrogenic potential. The current study aims to evaluate the chondrogenic potential of EVs derived from chondrocytes (Cho-EV) and mesenchymal stem cells (MSC-EV). The EVs were separately isolated from conditioned media of both rabbit bone marrow MSCs and chondrocyte cultures. The isolated vesicles were assessed in terms of size, morphology, and surface marker expression. The chondrogenic potential of MSCs in the presence of different concentrations of EVs (50, 100, and 150 μg/ml) was evaluated during 21 days, and chondrogenic surface marker expressions were checked by qRT-PCR and histologic assays. The extracted vesicles had a spherical morphology and a size of 44.25 ± 8.89 nm for Cho-EVs and 112.1 ± 10.10 nm for MSC-EVs. Both groups expressed the EV-specific surface markers CD9 and CD81. Higher expression of chondrogenic specified markers, especially collagen type II (COL II), and secretion of glycosaminoglycans (GAGs) and proteoglycans were observed in MSCs treated with 50 and 100 μg/ml MSC-EVs compared to the Cho-EVs. The results from the use of EVs, particularly MSC-EVs, with high chondrogenic ability will provide a basis for developing therapeutic agents for cartilage repair.

Progenitor Cells in Healthy and Osteoarthritic Human Cartilage Have Extensive Culture Expansion Capacity while Retaining Chondrogenic Properties

Objective Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. Design ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Results Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. Conclusions This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.

Monocyte Maturation Mediators Upregulate CD83, ICAM-1 and MHC Class 1 Expression on Ewing’s Sarcoma, Enhancing T Cell Cytotoxicity

Ewing’s sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1319-specific TCR-transgenic T cells are able to control EwS growth in a preclinical mouse model as well as in a patient with metastatic disease. However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1β and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.

The Therapeutic Modalities of Sickle Cell Disease Reprogram the Platelet Functional-Bioenergetic Profile

BACKGROUND: Sickle cell disease (SCD) is a group of inherited hemoglobinopathies that continues to be highly morbid and lethal. SCD-associated platelet hyperreactivity is a well-recognized contributor to the pathophysiology of the disease via complex interactions with the immune system and endothelium. Aberrant platelet bioenergetics have been implicated as a biological mechanism for SCD-associated platelet hyperreactivity, however, little is known about the impact current medical interventions (e.g., hydroxyurea [HU] and red blood cell [RBC] exchange transfusions) have on the platelet functional-bioenergetic profile. In this study we investigate the effects of hydroxyurea and RBC exchange transfusions on reprograming the platelet functional-bioenergetic profile and provide insight into biological pathways that may be amenable to intervention. METHODS: Platelets from sex-, race-, and aged-matched adult healthy control subjects and adult patients with homozygous SCD (HbSS), actively being treated with hydroxyurea (HU group) or RBC exchange transfusions (RBC exchange transfusion group), were isolated and washed following standard protocols. Platelet activation by flow cytometry was determined at baseline and following activation with thrombin (0.075U/ml) and ADP (1.25uM). Platelet-activated fibrinogen binding site (αIIbβIII), P-selectin, and phosphatidylserine (PS) surface marker expression (as measured by mean fluorescence intensity [MFI]) was determined with PAC-1, P-selectin, and lactadherin antibodies, respectively. The bioenergetic profile of washed platelets was determined by the 24-well format Seahorse extracellular flux analyzer. Statistical analyses were performed using the one-way ANOVA. Correlations were performed by 2-tailed nonparametric Spearman correlations and linear regression analysis with 95% confidence interval (GraphPad Software v9.1.2). Data expressed as mean plus or minus standard error of the mean (SEM). Differences were considered significant at p < 0.05. RESULTS: Platelets from patients in the HU group exhibited increased surface marker expression of αIIbβIII (p = 0.004), P-selectin (p = 0.003), and PS (p = 0.003) at resting conditions when compared to the RBC exchange transfusion group and healthy controls. Additionally, an increase in PS expression was seen in the HU group upon activation with ADP (p = 0.0003). No significant differences were seen in the platelet functional profile after activation with thrombin. The platelet bioenergetic profile in the HU group demonstrated an elevated proton leak (p = 0.03) when compared to the RBC exchange transfusion group. Elevated proton leak in SCD was found to have positive correlation with P-selectin and PS expression (Figure 1). CONCLUSION: While therapeutic interventions have improved overall outcomes in patients with SCD, adverse events continue to be a deterrent to many patients prompting the need for safer, more tolerable, and cost-effective alternatives. We have identified that while HU has little impact on the hyperreactive and procoagulant platelet phenotype in SCD, RBC exchange transfusions appear to mitigate the phenotype and reprogram the bioenergetic profile. Amongst treatment groups, a strong correlation was found between platelet activation markers (i.e., P-selectin and PS) and proton leak, suggesting an interplay between alterations in platelet bioenergetics and SCD-associated platelet hyperreactivity. Further studies are needed to elucidate the metabolic pathways that are responsible for the aberrant platelet functional-bioenergetic profile seen in SCD. These observations are important as targeting the platelet bioenergetic profile via less invasive and toxic therapeutic modalities may be equally efficacious as current interventions. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Characterization of T-Cell Exhaustion in Rapid Progressing Multiple Myeloma Using Cross Center Scrna-Seq Study

Introduction: Multiple myeloma (MM) is a complex hematological malignancy with the heterogenous immune bone marrow (BM) environment contributing to tumor growth, drug resistance, and immune escape. T-Cells play a critical role in the clearance of malignant plasma cells from the tumor environment. However, T-Cells in multiple myeloma demonstrate impaired cytotoxicity, proliferation, and cytokine production due to the activation of immune inhibitory receptors from ligands produced by the myeloma cells. In this study, we investigate the behavior of T-Cells in MM patients by using single-cell RNA-Seq (scRNA-Seq) to compare the transcriptomic profiles of BM T-Cells of patients with rapid progressing (FP; PFS < 18mo) and non-progressing (NP; PFS > 4yrs) disease. Methods: Newly diagnosed MM patients (n=18) from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study (NCT01454297) were identified as either rapid progressors or non-progressors based on their progression free survival since diagnosis. To capture transcriptomic data, scRNA-Seq was performed on 48 aliquots of frozen CD138-negative BM cells at three medical centers/universities (Beth Israel Deaconess Medical Center, Boston, Washington University in St. Louis, and Mount Sinai School of Medicine, NYC). Samples were collected at diagnosis prior to treatment. Surface marker expression for 29 proteins was captured for at least one sample per patient using CITE-Seq. After integration and batch correction, clustering was performed to identify cells of T or NK lineage. Uniform Manifold Approximation and Projection (UMAP) and differential expression were used to identify T-Lymphoid subtypes, and differences in NP and FP samples. Results: In this study, single cell transcriptomic profiles were identified for ~102,207 cells from 48 samples of 18 MM patients. 40,328 T (CD3+) and NK (CD3-, NKG7+) cells were isolated, and subclustered for further analysis (Fig 1A). Using differentially expressed markers for each cluster, the T-Lymphoid subset was refined into seven subtypes, consisting of various CD4+ T-Cells, CD8+ T-Cells, and NK cells (Fig 1B). The CD8+ cells were divided into three distinct phenotypes, namely a GZMK-, GZMB- CD8+ T-Cell cluster, a GZMK+ CD8+ Exhausted T-Cell cluster enriched in TIGIT and multiple chemokines (CCL3, CCL4, XCL2), and a GZMB+ NkT cluster enriched in cytolytic markers (PRF1, GNLY, NKG7) (Fig 1C). Differential expression between NP and FP samples in this CD8+ subset showed enrichment of the NkT cytotoxic markers in NP samples, while FP samples were enriched in the CD8+ Exhausted chemokine markers (Fig 1D). Furthermore, the proportion of CD8+ Exhausted T-Cells was enriched in FP samples (p.val < 0.05) (Fig 1E). Exhaustion markers were measured through both RNA and surface marker levels. In RNA, TIGIT was uniquely associated with the FP-enriched CD8+ Exhausted T-Cell cluster, and CD160 was uniquely expressed in FP samples (Fig 1F). CITE-Seq surface marker expression confirms enrichment of both TIGIT and PD1 in the CD8+ Exhausted T-Cell cluster, and along with more exhaustion in FP samples (p.val < 0.01). Conclusion: In this study, we have identified significant differences in T-Cell activity in patients with non-progressing and rapid-progressing multiple myeloma. T-Cells in rapid progressing patients appear to be in a suppressed state, with low cytolytic activity and enriched exhaustion markers. This GZMK+ T-Cell population shows strong similarities with an aging-associated subtype of effector memory T-Cells found to be enriched in older populations (Mogilenko et al, Immunity 54, 2021). These findings will be further validated in an expanded study, consisting both of a larger number of samples, and multiple samples at different timepoints from the same patient. Figure 1 Figure 1. Disclosures Jayasinghe: MMRF: Consultancy; WUGEN: Consultancy. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Kumar: Carsgen: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Roche-Genentech: Consultancy, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

Novel in Vitro and In Vivo Drug Screening Platform for ALL.

Acute Lymphoblastic leukemia (ALL) is a malignancy of bone marrow. Accumulation of mutations in lymphoid progenitor cells give rise to either B-ALL or T-ALL. Treatment for ALL has improved in recent years, yet relapse of the disease and development of resistance are observed in patients. Lack of suitable and robust in vitro and in vivo drug testing platforms for primary ALL cells along with the lack of rapid development of novel therapeutics drugs encompassing the heterogeneity of the disease contribute to the delay in approved patient treatments. We have developed a short-term culture system that supports the survival of primary B-ALL and T-ALL cells. Our ALL bank includes patient-derived specimens with complete cytogenetics and surface marker expression information. Different culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL and T-ALL specimens. Cell growth/viability was assessed using the Cell Titer-Glo ® assay. Primary B-ALL cells survived in the optimized media for 3 days and a heterogenous dose dependent response was observed across the models to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. BCR-ABl - B-ALL patient samples were found to be resistant to imatinib in contrast to BCR-ABL + samples which were sensitive to imatinib. Similarly, culture conditions optimized for T-ALL primary cells supported the survival until day 6 and displayed a diverse response to standard of care drugs like venetoclax, imatinib, vincristine, cytarabine and methotrexate, reflecting the heterogeneity of the patient derived specimens. Immunophenotypic characterization of ALL cells grown in culture displayed retention of the B and T cells surface marker expression. Further, a patient derived pre-clinical xenograft model was developed in NCG mice to study in vivo ALL drug efficacy. 100% engraftment was observed for B-ALL primary cells, with latency of engraftment (>3%) in peripheral blood varying from 15 days to 3.5 months. 30-90% of the bone marrow cells were occupied by human CD45 cells. Infiltration of human B-ALL cells were observed in the spleen causing splenomegaly. 8 out of the 14 models having high penetrance were passaged until P3. Flow analysis at each passage demonstrated surface marker expression displaying low divergence from the primary samples. Additionally, evaluation of ex vivo drug response from B-ALL PDX splenocytes was largely concordant with the primary specimen ex vivo data in three of the models evaluated. In an in vivo drug efficacy study administration of venetoclax, CHOP and R-CHOP inhibited the proliferation of B-ALL cells. Significant reduction of B-ALL cells was observed while on treatment with Venetoclax. At termination of the study, up to 80% reduction of human B-ALL cells was observed in whole blood, bone marrow, and spleen after treatment with CHOP and R-CHOP in comparison to the vehicle cohort. Similarly, patient derived T-ALL pre-clinical xenograft model development is in progress. Thus, we have developed a robust in vitro drug testing platform for B-ALL and T-ALL to evaluate drug efficacy. We also demonstrate that NCG mice support the growth and proliferation of primary B-ALL cells and have successfully developed an in vivo platform that will facilitate the testing of clinically relevant chemotherapeutic drugs for ALL. Disclosures No relevant conflicts of interest to declare.

Characterizing the Metabolic Determinants of Thromboinflammation in Myeloproliferative Neoplasms

BACKGROUND: Myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic disorders characterized by an overproduction of terminally differentiated myeloid elements. Venous and arterial thrombotic events represent the leading causes of morbidity and mortality in patients with JAK2 positive MPNs (essential thrombocythemia [ET] and polycythemia vera [PV]) in chronic phase. Platelet hyperreactivity is a hallmark finding of MPNs but the biological mechanisms remain to be elucidated. We have previously identified that platelets from patients with PV and ET exhibit dysmorphic mitochondria, however, the detailed bioenergetic and metabolomic implications of such alterations in mitochondrial mass and morphology remain to be investigated. Here in, we aim to identify the metabolic determinants that underpin thromboinflammation in patients with JAK2 positive MPNs in chronic phase. METHODS: Platelets from sex and age-matched healthy control subjects and individuals with either JAK2 positive PV or ET were isolated and washed following standard protocols. Platelet activation by flow cytometry was determined at baseline conditions. Platelet activated fibrinogen binding site (αIIbβIII), P-selectin, and phosphatidylserine (PS) surface marker expression (as measured by mean fluorescence intensity [MFI]) was determined with PAC-1 and P-selectin antibodies and lactadherin, respectively. The bioenergetic profile of washed platelets was determined by the 24-well format Seahorse extracellular flux analyzer. Metabolomic profile was developed with the Vanquish UHPLC system coupled online to a Q Exactive mass spectrometer. Statistical analyses were performed using the unpaired 2-tailed Student t test (GraphPad Software v9.1.2). Data expressed as mean plus or minus standard error of the mean (SEM). Significance was determined at p < 0.05. RESULTS: Platelets from patients with MPNs had higher surface marker expression of PS under resting conditions. A trend of higher surface marker expression of αIIbβIII and P-selectin was seen (Figure 1). The platelet bioenergetic profile in individuals with MPN demonstrated decreased basal respiration (p = 0.0008), ATP-linked respiration (p = 0.001), and basal extracellular acidification rate (ECAR) (p = 0.09) (Figure 2). Liquid chromatography-mass spectrometry-based metabolomics revealed elevated pentose phosphate pathway metabolites (pentose phosphates and 6-Phospho-D-gluconate), and diminished adenosine diphosphate (ADP) and adenosine triphosphate (ATP) pools (Figure 3). CONCLUSION: The findings from this study suggest that platelets from patients with JAK2 positive PV and ET have a procoagulant phenotype given the increased expression of PS, an anionic phospholipid known to facilitate the generation of thrombin. The decreased basal respiration, ATP-linked respiration, basal ECAR (a surrogate marker of glycolysis), and energy pools (ADP and ATP) in platelets from MPN patients suggest a hypometabolic phenotype. Additionally, it appears there is preferential shunting of glucose into the pentose phosphate pathway. Such metabolic switch in conjunction with the hypometabolic state of platelets in MPN, could potentially represent a compensatory mechanism of platelets against oxidative stress. The bioenergetic dysregulation of platelets in MPNs may precipitate a cascade of events that leads to the loss of plasma membrane integrity (increased PS exposure) therefore increasing its procoagulant potential. Higher amounts of platelet PS could underly the elevated incidence of thrombosis observed in patients with JAK2 positive ET and PV. Further studies are underway to identify the inciting factor(s) that lead to the platelet bioenergetic failure and elevated PS exposure seen in patients with MPNs. These investigations will provide significant mechanistic insight for identifying therapies aimed at preventing thrombotic events in patients with MPNs. Figure 1 Figure 1. Disclosures Nemkov: Omix Thecnologies: Other: Co-founder. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

BRAF and MEK Inhibitors Affect Dendritic-Cell Maturation and T-Cell Stimulation

BRAF and MEK inhibitor (BRAFi/MEKi) combinations are currently the standard treatment for patients with BRAFV600 mutant metastatic melanoma. Since the RAS/RAF/MEK/ERK-pathway is crucial for the function of different immune cells, we postulated an effect on their function and thus interference with anti-tumor immunity. Therefore, we examined the influence of BRAFi/MEKi, either as single agent or in combination, on the maturation of monocyte-derived dendritic cells (moDCs) and their interaction with T cells. DCs matured in the presence of vemurafenib or vemurafenib/cobimetinib altered their cytokine secretion and surface marker expression profile. Upon the antigen-specific stimulation of CD8+ and CD4+ T cells with these DCs or with T2.A1 cells in the presence of BRAFi/MEKi, we detected a lower expression of activation markers on and a lower cytokine secretion by these T cells. However, treatment with any of the inhibitors alone or in combination did not change the avidity of CD8+ T cells in peptide titration assays with T2.A1 cells. T-helper cell/DC interaction is a bi-directional process that normally results in DC activation. Vemurafenib and vemurafenib/cobimetinib completely abolished the helper T-cell-mediated upregulation of CD70, CD80, and CD86 but not CD25 on the DCs. The combination of dabrafenib/trametinib affected DC maturation and activation as well as T-cell activation less than combined vemurafenib/cobimetinib did. Hence, for a potential combination with immunotherapy, our data indicate the superiority of dabrafenib/trametinib treatment.

Surface Marker Expression in Small and Medium/Large Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Naive or Apoptotic Condition Using Orthogonal Techniques

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a promising resource for regenerative medicine. Small MSC-EVs represent the active EV fraction. A bulk analysis was applied to characterise MSC-EVs’ identity and purity, with the assessment of single EV morphology, size and integrity using electron microscopy. We applied different methods to quantitatively analyse the size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC-EVs obtained using sequential ultracentrifugation. Bone marrow, adipose tissue and umbilical cord MSC-EVs were compared in naive and apoptotic conditions. As detected by electron microscopy, the 100k EV size < 100 nm was confirmed by super-resolution microscopy and ExoView. Single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanins. ExoView allowed a comparative screening of single MSC-EV tetraspanin and mesenchymal markers. A semiquantitative bead-based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC-EVs. Apoptotic MSC-EVs were released in higher numbers, without significant differences in the naive fractions in surface marker expression. These results show a consistent profile of MSC-EV fractions among the different sources and a safer profile of the 100k MSC-EV population for clinical application. Our study identified suitable applications for EV analytical techniques.

Tumor-Derived Factors Differentially Affect the Recruitment and Plasticity of Neutrophils

Neutrophils play a key role in cancer biology. In contrast to circulating normal-density neutrophils (NDN), the amount of low-density neutrophils (LDN) significantly increases with tumor progression. The correlation between these neutrophil subpopulations and intratumoral neutrophils (TANs) is still under debate. Using 4T1 (breast) and AB12 (mesothelioma) tumor models, we aimed to elucidate the source of TANs and to assess the mechanisms driving neutrophils’ plasticity in cancer. Both NDN and LDN were found to migrate in response to CXCL1 and CXCL2 exposure, and co-infiltrate the tumor site ex vivo and in vivo, although LDN migration into the tumor was higher than NDN. Tumor-derived factors and chemokines, particularly CXCL1, were found to drive neutrophil phenotypical plasticity, inducing NDN to transition towards a low-density state (LD-NDN). LD-NDN appeared to differ from NDN by displaying a phenotypical profile similar to LDN in terms of nuclear morphology, surface receptor markers, decreased phagocytic abilities, and increased ROS production. Interestingly, all three subpopulations displayed comparable cytotoxic abilities towards tumor cells. Our data suggest that TANs originate from both LDN and NDN, and that a portion of LDN derives from NDN undergoing phenotypical changes. NDN plasticity resulted in a change in surface marker expression and functional activity, gaining characteristics of LDN.