Platelets are considered intravascular effectors of hemostasis, yet growing evidence indicates that they can migrate across the endothelium. Whether platelet exit from the vasculature is a regulated process analogous to leukocyte trafficking and how migration is coordinated with effector functions has remained unclear. Using genetic, pharmacologic, and imaging approaches, we define the molecular program governing platelet transendothelial migration into tumors in vivo. CXCL12-CXCR4 signaling contributed to platelet extravasation in vivo, with stromal rather than tumor-derived CXCL12 acting as the dominant cue; CXCR4 disruption reduced platelet infiltration and tumor growth. Efficient vascular exit required platelet focal adhesion kinase and platelet endothelial cell adhesion molecule 1, implicating cytoskeletal remodeling and junctional adhesion. Platelet trafficking was uncoupled from effector activity: Munc13-4-dependent dense granule secretion was dispensable for extravasation but required for growth promotion, whereas Munc18-2-regulated α-granule release preserved vascular integrity and restricted passage. Disruption of the CLEC-2/podoplanin axis destabilized vessels and increased leakage. Together, these findings establish regulated platelet extravasation.

BACKGROUND:Platelets can recognize and destroy microbial pathogens through the release of antimicrobial proteins, suggesting that they may contribute to innate immune defense against infection. OBJECTIVES:This study aimed to determine whether platelets contribute to pulmonary innate immunity during bacterial pneumonia and to distinguish their antimicrobial functions from their hemostatic role. METHODS:Mice were exposed to aerosolized Gram-negative Pseudomonas aeruginosa or Gram-positive Streptococcus pneumoniae. Platelet number or function was reduced by antibody-mediated platelet depletion, thrombopoietin gene deletion, or aspirin treatment. Clinical outcomes, mortality, pulmonary pathogen burden, and lung histopathology were assessed. RESULTS:Thrombocytopenic and aspirin-treated mice had increased mortality following bacterial pneumonia. Lung congestion and pathogen burden increased in proportion to the severity of thrombocytopenia. In contrast, pulmonary hemorrhage was observed only in severely thrombocytopenic mice, indicating that impaired antimicrobial defense occurred at platelet levels sufficient to maintain hemostasis. CONCLUSION:Platelets actively protect against both Gram-negative and Gram-positive bacterial pneumonia. This protective effect is distinct from their hemostatic function: platelets contribute directly to antimicrobial host defense, whereas their hemostatic activity maintains a physical barrier that limits local pathogen spread.

BACKGROUND:Atherothrombosis, which underlies most acute coronary syndromes and is driven by intraplaque thrombosis, preferentially occurs in regions of disturbed blood flow (d-flow). Although LATS1/2 (large tumor suppressor kinases 1 and 2) are known regulators of endothelial mechanotransduction, the mechanisms by which d-flow connects endothelial senescence, proliferation, and intraplaque thrombosis remain poorly understood. METHODS:We investigated endothelial cell (EC)-specific roles of Lats1/2 using inducible EC-specific knockout mice in a partial carotid ligation model. Spatial multiomics of human and mouse plaques was performed using imaging mass cytometry, COMET sequential immunofluorescence, and spatial metabolomics. RESULTS:Tamoxifen-induced deletion of both Lats1 and Lats2 (homozygous) in ECs caused fatal edema and increased vascular permeability. In contrast, Lats1het(±)/Lats2 homo(-/-)-EC-specific knockout mice survived and developed spontaneous atherothrombotic plaques with neovascularization. Spatial proteomics revealed that LATS1/2 loss induced a senescence-associated stemness phenotype driven by CD38 upregulation. Spatial metabolomics showed sulfite and taurine accumulation, indicating SUOX (sulfite oxidase) deficiency. CD38 suppressed SUOX, demonstrated a switch into the reverse mode of mitochondrial complex V, increased succinate dehydrogenase activity, and promoted ATP consumption. Despite ATP depletion, glutamate metabolism and the citric acid cycle flux increased, sustaining EC proliferation under energetic stress. This senescence-associated stemness state promoted both proliferation and senescence, leading to fragile, leaky neovessels and intraplaque thrombotic lesions. Pharmacological CD38 inhibition attenuated these phenotypes. Similar EC states were observed in human plaques. CONCLUSIONS:Loss of Lats1/2 in ECs induces a CD38-associated senescence-associated stemness-like phenotype that promotes intraplaque thrombosis through mitochondrial metabolic reprogramming, including changes consistent with reverse-mode operation of mitochondrial complex V. These findings define a mechanistic link between disturbed flow, endothelial metabolic reprogramming, and intraplaque thrombosis and hemorrhage.

B cells constitute ∼15% to 20% of tumor-infiltrating lymphocytes in melanoma. Their presence in the tumor microenvironment correlates with improved survival and enhanced response to immune checkpoint blockade therapy. Yet, the functional contribution of B cells to melanoma immunity remains unclear. In this study, we showed that both genetic and antibody-mediated B cell depletion significantly promoted melanoma progression in mice. Immune profiling revealed that, although B cell percentages were reduced, IL-10-producing B regulatory cells (Bregs) persisted after depletion. However, the persistence of Bregs alone cannot explain the impact of B cell depletion on enhancing melanoma growth, as codepletion of B cells and CD4+ T cells, despite similar Breg levels, did not promote melanoma progression. B cell depletion also resulted in the accumulation of PD-1+ B cells, CD4+ T cells, and monocytic myeloid-derived suppressor cells into the tumor microenvironment, alongside a reduction in IFN-γ+CD8+ T cells, CXCL13+CD8+ T cells, and M1-like macrophages. Notably, plasma cell deficiency did not affect tumor growth, indicating that B cell-mediated antitumor activity is independent of antibody production. The tumor-promoting effect of B cell loss was at least partially CD4+ T cell dependent, as codepletion of B cells and CD4+ T cells reversed this phenotype and B cell depletion did not enhance tumor growth in Nu/Nu mice lacking mature T cells. Taken together, our findings reveal an antitumor role of B cells in melanoma and demonstrate that their loss promotes tumor progression through reprogramming of the tumor immune microenvironment.

Immune checkpoint regulators, such as the V-domain Ig suppressor of T cell activation (VISTA), play a critical role in shaping the tumor microenvironment (TME) and facilitating immune evasion. In ovarian cancer, VISTA exhibits more abundant and consistent expression than other immune checkpoints, including Programmed Death-Ligand 1 (PD-L1). This study examined the role of platelets in the regulation of VISTA in ovarian cancer using both in vitro and in vivo models. Our findings demonstrate that platelets upregulate VISTA expression in both myeloid and tumor cells, thereby promoting an immunosuppressive TME. Elevated VISTA levels were associated with higher platelet counts and poorer clinical outcomes. These results highlight that platelet-mediated VISTA upregulation is a potential therapeutic target for improving antitumor immune responses in ovarian cancer.

Spatial statistics are crucial for analyzing clustering patterns in various spaces, such as the distribution of trees in a forest or stars in the sky. Advances in spatial biology, such as single-cell spatial transcriptomics, enable researchers to map gene expression patterns within tissues, offering unprecedented insights into cellular functions and disease pathology. Common methods for deriving spatial relationships include density-based methods (quadrat analysis, kernel density estimators) and distance-based methods (nearest-neighbor distance [NND], Ripley's K function). While density-based methods are effective for visualization, they struggle with quantification due to sensitivity to parameters and complex significance tests. In contrast, distance-based methods offer robust frameworks for hypothesis testing, quantifying spatial clustering or dispersion, and facilitating comparisons with models such as uniform random distributions or Poisson processes [1, 2]. Ripley's K function provides a detailed measure of spatial clustering or dispersion across multiple scales by considering all pairs of points within specified distances. This is in contrast to NND, which may overlook structures that vary across scales. Ripley's K function can detect complex spatial patterns over a range of distances, making it suitable for datasets with non-uniform arrangements that exhibit different behaviors at different scales. However, its broader adoption has been hindered by computational complexity and challenges in interpretation, especially for three-dimensional data, which are common in spatial biomedical research [3-6]. To address these limitations, we introduce MDSpacer (Multi-Dimensional Spatial Pattern Analysis with Comparable and Extendable Ripley's K), a modeling tool that implements Ripley's K function for both 2D and 3D data, facilitating detailed analyses within and between groups (Figure 1A, B, Supplementary Figure S1). MDSpacer uses a novel normalization scheme (described in Supplementary Materials and Methods) that dramatically reduces computational overhead while delivering results in an easily interpretable and comparable format (Figure 1C–F). We validated this tool in two cancer research studies: one on metastatic bone cancer and another on ovarian cancer. In the metastatic bone cancer study, we used the Vessel3D analysis toolkit to extract spatial point information from 3D confocal images of murine femurs with early-stage spontaneous metastasis (Figure 1G–K, Supplementary Figures S2, S3, Supplementary Videos S1, S2). MDSpacer identified both expected clustering at short distances and unexpected dispersion patterns at larger scales between early-stage disseminated tumor cells (DTCs) and neural/glial antigen 2-positive (NG2+) mesenchymal cells in relation to other microenvironmental factors [7-9]. Interestingly, no spatial relationships were observed between DTCs and vessel bifurcations, which have been reported in other studies [8]. In the ovarian cancer study, we applied MDSpacer along with a deep learning model developed to pinpoint platelet locations in whole-slide confocal images and identified stromal cell–derived factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR4)-driven platelet clustering at the primary ovarian tumor site (Supplementary Figure S4). Our findings confirmed expected interactions and revealed new significant relationships with additional microenvironmental factors, deepening our understanding of tumor-microenvironment interactions and demonstrate the effectiveness of the MDSpacer spatial statistics tool. In the metastatic bone cancer study, we developed a murine model of spontaneous metastasis (Supplementary Figure S5A) and verified the presence of DTCs within the bone through fluorescent confocal imaging (Supplementary Figure S5B, C). Figure 1A and Supplementary Figure S6 show how point sets were isolated from the 3D confocal channels. The locations of every DTC seed cell in the femur were manually recorded, totaling 824 cancer cells across all samples. The locations of NG2+ perivascular mesenchymal stem cells (MSCs) were determined through segmentation of the fluorescent marker layer (Supplementary Figure S7). This method converts complex visual structures into simplified point sets, allowing for analysis of inherent spatial relationships using Ripley's K function (Figure 1B, Supplementary Video S3). In univariate analyses, which examine clustering within a single point event type independently of others, tumor cells and NG2+ cells consistently exhibited significant clustering across samples, particularly at specific scales (Supplementary Figure S8A, B). The branch points displayed extremely significant clustering across all scales (Supplementary Figure S8C). However, tortuous vessels did not substantially deviate from a random distribution until distances of about 80 µm (Supplementary Figure S8D). The consistent trajectories of K values across different radii underscore the uniformity of spatial distributions between samples, demonstrating that MDSpacer offers quantitative insights into biologically relevant features while maintaining internal consistency (Supplementary Figure S9). In bivariate analysis, which examine relationships between two distinct point event types, Monte Carlo simulation provides percentile intervals by randomizing point labels and repeating the process 100 times for each image (Supplementary Figure S10). Bivariate K function plots between DTCs and NG2+ cells (Supplementary Figure S11A) show that most samples exhibit significant NG2+ cell clustering near DTCs at distances under 20 µm. However, no clustering is observed at larger distances; instead, significant dispersion between NG2+ cells and DTCs is noted at larger radii. No clustering is observed at any scale between tumor cells and vessel branch points or between tumor cells and the most tortuous vessel segments (Supplementary Figure S11B, C). The K function plots examine relationships between NG2+ cells and vascular features, such as vessel branch points and the most tortuous vessel segments (Supplementary Figure S12). Results show no spatial relationship between NG2+ cells and vessel branch points across all scales. In contrast, NG2+ cells consistently cluster near the most tortuous blood vessel segments at distances shorter than 20 µm across all samples, suggesting potential biological interactions. Additional details regarding data processing and MDSpacer procedures can be found in the Supplementary Materials and Methods. In the ovarian cancer study, we hypothesized that SDF-1 secreted by ovarian cancer cells interacts with CXCR4 receptors on platelets, functioning as a chemotactic factor [10]. We developed a deep learning model for localizing platelets, achieving high accuracy (area under receiver operating characteristic [AUROC]: 0.99) in validation tests (Supplementary Figures S13, S14, Supplementary Table S1). Using Plerixafor, a CXCR4 inhibitor, we observed a significant reduction in tumor weight in Plerixafor-treated mice compared to controls (P = 0.008, Supplementary Figure S15A). Blocking SDF-1 using clustered regularly interspaced short palindromic repeats (CRISPR) targeting the SDF-1 genes led to a significant reduction in tumor weight compared to control (P < 0.001, Supplementary Figure S15B). Blocking CXCR4 reduced the number of platelets extravasated into the tumor parenchyma. Supplementary Figure S15C shows a significant reduction (P = 0.047) in platelet density in Plerixafor-treated tumor tissues compared to controls. We validated the effect using univariate MDSpacer. Platelets exhibited significant clustering within ovarian cancer tissues in both control and Plerixafor-treated mice (Supplementary Figure S16A). However, Plerixafor-treated samples showed significantly reduced platelet clustering compared to control regions across radii from 7 to 249 µm (P < 0.05), with a more pronounced difference observed across radii from 12 to 57 µm (P < 0.01). Supplementary Figure S16B, C depict the K functions for control and Plerixafor-treated mice, respectively. We overlaid vessel segmentation using anti-CD31 for endothelial cells onto platelet segmentation, enabling measurement of each platelet's proximity to its nearest vessel. We observed that within 1 µm of the vessel, the number of platelets was significantly higher in control tumor tissues compared to Plerixafor-treated tissues (Supplementary Figure S17). Additional details regarding the ovarian cancer experiments and data analysis can be found in the Supplementary Materials and Methods. In conclusion, we present a versatile point-pattern analysis platform designed for characterizing point locations and spatial relationships within large tissue samples. By extending Ripley's K function to the biomedical domain and optimizing it for multi-dimensional data, our platform enables researchers to detect spatial relationships across a range of distances. The novel MDSpacer normalization approach significantly reduces computational cost while facilitating meaningful comparisons between samples. By making Ripley's K function both user-friendly and accessible through a comprehensive software toolkit, MDSpacer offers significant potential for application across a wide array of research domains. Conceptualization: Daniel Shafiee Kermany, Ju Young Ahn, Jianting Sheng, and Stephen Tin Chi Wong. Methodology: Daniel Shafiee Kermany, Ju Young Ahn, Stephen Tin Chi Wong, Jianting Shen, Raksha Raghunathan, Matthew Vasquez, Kai Liu, Zhan Xu, Xiaoxin Hao, Min Soon Cho, Wendolyn Carlos-Alcalde, Hani Lee, Vahid Afshar-Kharghan, Hong Zhao, Weijie Zhang, and Xiang Hong-Fei Zhang. Investigation, formal analysis, and validation: Daniel Shafiee Kermany, Ju Young Ahn, Stephen Tin Chi Wong, Raksha Raghunathan, Jianting Sheng, Weijie Zhang, Lin Wang, Matthew Vasquez, Kai Liu, Zhan Xu, Min Soon Cho, Vahid Afshar-Kharghan, Xiaoxin Hao, and Xiang Hong-Fei Zhang. Resources: Hong Zhao, Stephen Tin Chi Wong, Weijie Zhang, Vahid Afshar-Kharghan, and Xiang Hong-Fei Zhang. Software, data curation, and visualization: Daniel Shafiee Kermany, Ju Young Ahn, Lin Wang, Stephen Tin Chi Wong, and Weijie Zhang. Writing - original draft: Daniel Shafiee Kermany and Stephen Tin Chi Wong. Writing - review & editing: Daniel Shafiee Kermany, Ju Young Ahn, Stephen Tin Chi Wong, Jianting Sheng, Weijie Zhang, Lin Wang, and Xiang Hong-Fei Zhang. Supervision: Stephen Tin Chi Wong, Xiang Hong-Fei Zhang, and Vahid Afshar-Kharghan. Project administration: Daniel Shafiee Kermany, Raksha Raghunathan, and Stephen Tin Chi Wong. Funding acquisition: Stephen Tin Chi Wong, Xiang Hong-Fei Zhang, and Vahid Afshar-Kharghan. We extend our gratitude to Jiasong Li from the Translational Biophotonics Laboratory at the Houston Methodist for his invaluable guidance on image acquisition, to Andrea Olvera from Kervera, LLC for her assistance with editing of the manuscript text, and to Gefei Song for her assistance with image collection and processing. Our thanks also go to the Advanced Cellular and Tissue Microscopy Core of the Houston Methodist Neal Cancer Center and Houston Methodist Research Institute for their innovative approaches in acquiring comprehensive thigh bone confocal imaging. We appreciate the support of the GPU supercomputer facility at the Laboratory for Artificial Intelligence in Medicine and Innovation, located at the Systems Medicine and Bioengineering Department, Houston Methodist. Additionally, we acknowledge the contributions of the Pathology Core at the Lester and Sue Smith Breast Center, Baylor College of Medicine, for their support in our research endeavors. The authors declare no competing interests. Daniel Shafiee Kermany, Ju Young Ahn, Matthew Vasquez, Lin Wang, Kai Liu, Raksha Raghunathan, Jianting Sheng, Hong Zhao, and Stephen Tin Chi Wong are supported by NCI U01CA252553, NCI R01CA238727, NCI R01CA177909, NCI R01CA244413, John S. Dunn Research Foundation, and Ting Tsung and Wei Fong Chao Foundation. Xiang Hong-Fei Zhang, Zhan Xu, Xiaoxin Hao, Weijie Zhang are supported by US Department of Defense DAMD W81XWH-16-1-0073 (Era of Hope Scholarship), NCI R01CA183878, NCI R01CA251950, NCI U01CA252553, DAMD W81XWH-20-1-0375, Breast Cancer Research Foundation, and McNair Medical Institute. Vahid Afshar-Kharghan, Min Soon Cho, Wendolyn Carlos-Alcalde, and Hani Lee are supported by NCI R01CA177909, NCI R01CA016672, NCI R01CA275762, and NCI P50CA217685. All animal procedures were conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine, protocol number [AN-5734]. The datasets generated and/or analyzed during the current study are available from the corresponding author by request. The code used for the analysis is publicly available at https://github.com/Translational-Biophotonics-Laboratory/mdspacer. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Lung cancer is the leading cause of cancer-related death worldwide. The most common type of lung cancer is KRAS mutant lung adenocarcinoma (KM-LUAD), which is associated with smoking and lung inflammatory conditions. Despite various approaches and recent discoveries, effective treatments for this aggressive disease remain elusive. Our previous studies along with others demonstrated that the expression of Muc5ac, a main secretory airway mucin, is high in KM-LUAD patients and correlates with poor prognosis. Using a mouse model of KM-LUAD (CC-LR mice), we previously showed that targeting Muc5ac leads to decreased lung tumor burden. Here, we further investigated the immunomodulatory aspects of Muc5ac inhibition and how it impacts tumor progression. To achieve our goal, we used CC-LR mice with and without Muc5ac gene knockout (KO) and compared their tumor microenvironment (TME) at the age of 14 weeks using flow cytometry analysis of the whole lung. We found that the total T cell percentage was significantly elevated in the lung of CC-LR/Muc5ac KO compared to CC-LR mice This increase was accompanied by a marked rise in CD8+ T cell percentage in CC-LR/Muc5ac KO compared to the control group. Additionally, the percentage of IFN-γ+ CD8+ T cells was significantly increased in CC-LR/Muc5ac KO compared to the controls, indicating an enhanced cytotoxic anti-tumor response. Interestingly the percentage of CD4+ T cells was drastically decreased in CC-LR/Muc5ac KO compared to the control group. However, the majority of remaining CD4+ T cells expressed IFN-γ, while the percentage of FOXP3+ CD4+ T cells was decreased, indicating a shift from immunosuppressive T regulatory response to an anti-tumor T helper 1 CD4 T cell response. Although the total NK cell percentage was decreased, the remaining NK cells exhibited higher IFN-γ further suggesting the induction of a cytotoxic TME. In addition, the total percentage of dendritic cells (DCs) was increased with a notable rise in monocytic DCs in the CC-LR/Muc5ac KO group. These findings suggest that the MUC5AC inhibition exerts an anti-tumor and immunostimulatory effect via enhancing the DC response which reprograms the TME from a pro-tumor immunosuppressive phenotype to an anti-tumor cytotoxic phenotype. This further emphasizes the role of mucin in KM-LUAD pathogenesis and that the targeted elimination of MUC5AC could serve as a potential therapeutic target for KM-LUAD patients. Funding: This study was supported in part by a University Cancer Foundation via the IRG program and the retention fund both awarded to SJM. Nastaran Karimi, Michael J. Clowers, Shanshan Deng, Katherine E. Larsen, Selin Oncul, Hani Lee, Avantika Krishna, Yasmina H. Rezai, Farbod Khalaj, Maria T. Grimaldo, Seyed Javad Moghaddam. Immunomodulatory role of MUC5AC in lung adenocarcinoma: A potential therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB385.

Introduction: A reliable and sustainable supply of platelets remains a critical challenge in managing thrombocytopenic patients, particularly those undergoing chemotherapy, hematopoietic stem cell transplantation, or surgical procedures. Over 2 million platelet units are transfused annually in the United States alone. However, platelet transfusion is often complicated by platelet transfusion refractoriness (PTR), characterized by an inadequate post-transfusion platelet count increment. PTR occurs in up to 30% of chronically transfused patients and is frequently caused by alloimmunization to HLA class I antigens. Alloimmune PTR presents a major clinical hurdle, as HLA-matched donor platelets are logistically difficult to source and limit timely access to effective transfusion therapy. The reliance on donor-derived apheresis platelets is further constrained by the short shelf life of platelets and limited donor availability. To address the shortage, we developed a novel strategy for the large-scale ex vivo generation of HLA class I-deficient GMP-grade megakaryocytes (MKs) and platelets from CD34⁺ cord blood (CB) cells. Methods: We have developed a GMP-compliant method to produce unedited, expanded MKs and platelets in vitro, using a multi-phase co-culture system of CB-derived CD34⁺ cells and CB-MSCs. The culture system is supplemented with dynamically titrated cytokines to simulate the bone marrow microenvironment across distinct developmental stages. Additional components include a caspase-3 inhibitor, a histone deacetylase inhibitor, and a ROCK inhibitor to enhance MK viability and platelet production. CD34⁺ cells, sourced from the MDACC Cord Blood Bank, and CB-MSCs, from the MSC Bank, are expanded and differentiated over five sequential culture phases. Platelet release is induced via shear force in a closed-loop bioreactor, and the final MK-platelet product is harvested on day 24. The product is phenotyped, assessed for in vitro aggregation, and infused into irradiated NSG mice for functional evaluation.. To overcome platelet transfusion refractoriness (PTR), we then targeted HLA class I expression on MKs. On day 3 of culture, CRISPR/Cas9 gene editing is performed using a single-guide RNA targeting exon 1 of B2 microglobulin M (B2M), delivered via the Lonza 4D-Nucleofector system. Editing conditions were optimized to minimize off-target effects while achieving robust B2M disruption. Results: After 24 days, the unedited cultures yielded ~50 × 10⁶ mature polyploid MKs and ~0.5 × 10¹⁰ platelets. Upon infusion of 4.2 × 10⁶ MKs and 0.5 × 10⁸ platelets into sublethally irradiated thrombocytopenic NSG mice, human CD41⁺/CD61⁺ platelets were detected in circulation within 1 hour. In vivo–generated platelets appeared by day 4 and peaked at day 11, with persistence in circulation through day 21, indicating sustained thrombopoietic activity. B2M knockout was then successfully integrated into the culture protocol. Flow cytometry confirmed effective downregulation of HLA class I, with only ~15% of cells expressing residual HLA-I. In vitro functional assays showed thrombin-induced aggregation of platelets, confirming functional competency. Importantly, in vitro cytotoxicity assays showed that the B2M knockout cells were resistant to allogeneic T cell–mediated killing, supporting the immune evasion potential of this product in alloimmunized patients. Conclusions: We have established a scalable, GMP-compliant platform for generating MKs and platelets from cord blood. The combined infusion of MKs and platelets enables both immediate and sustained platelet reconstitution. To address platelet transfusion refractoriness (PTR), we incorporated CRISPR/Cas9-mediated knockout of HLA class I, generating universal, donor-independent platelets with immune-evasive properties. This platform offers a renewable and potentially off-the-shelf source of platelets. Clinical trials are planned to evaluate the safety and efficacy of this approach.

Introduction: A reliable and sustainable supply of platelets remains a critical challenge in managing thrombocytopenic patients, with over 2 million platelet units transfused annually in the United States alone. This demand is common in patients undergoing treatments such as chemotherapy or bone marrow transplantation, who frequently require platelet transfusions due to the short life span of platelets. It can be further exacerbated by immune refractoriness to platelet transfusions. Currently, hospitals rely heavily on adult donor apheresis to meet this need; however, the short storage life of platelets and donor availability limit this supply. We thus developed a method of large-scale ex vivo production of good manufacturing practice (GMP) grade platelet and platelet progenitor cells from CD34+ cord blood (CB) cells co-cultured with mesenchymal stem cells (MSCs) derived from CB using hollowfiber flow-based bioreactors. Our approach aims to establish a continuous and renewable source of platelets from the large global cord blood bank inventories. Methods: We developed a dynamic protocol for the large-scale production of expanded megakaryocyte (MKs) and platelets in vitro, employing a coculture system with CB MSCs, the addition of dynamic concentrations of MK differentiation factors, and chemical inhibition of Rho-associated protein kinase (ROCK). Cord blood and CB MSCs were obtained from the MDACC Cord Blood and MSC Banks, respectively. Our manufacturing process consists of three phases: the first phase involves the co-culturing fresh CB-derived CD34+ cells (enriched to >90% using MACS enrichment columns, Milteny Biotec) with monolayers of CB MSCs cells for 3 days, using GMP media supplemented with MK early differentiation cytokines. During the second phase, the expanded CD34+ cells and MK progenitors were cultured using GMP media supplemented with late differentiation factors including high concentrations of thrombopoietin (TPO) for 7 days. At day 10, the cells were transferred to the hollowfiber bioreactor, where they were co-cultured for 9 days with MSCs in GMP media supplemented with MK late differentiation factors and a lower concentration of TPO. In the final phase, the MKs were treated with a ROCK inhibitor for 4 days, to promote further MK maturation and release of platelets. On Day 23, the bioreactor was harvested, and the final product consisting of platelets and expanded MKs were washed, analyzed by flow cytometry and infused into xenogeic NSG mice. Results: After a 23 day culture, our final product contains 113 x 106 ±29.2 megakaryocytes (CD41+CD61+) and 0.4-0.9 x 1011 platelets. The cultured MK were polyploid, and the platelets demonstrated functional equivalence to control human platelets, as confirmed by in vitro aggregation studies. To evaluate the in vivo efficacy and functionality of the product, we transfused a combination of 106 megakaryocytes and 20 x 106 platelets into sublethally irradiated NSG mice. Post-transfusion assessments revealed a significant reduction in the tail vein bleeding time 24 hours later. Moreover, human platelet production was detected in the peripheral blood of the mice up to 2 weeks following the transfusion, suggesting that the administered MKs were actively producing platelets in vivo. Importantly, the product was well-tolerated with no observed toxicity in the mice. Our results highlight the clinical feasibility of this approach. In addition to MSC co-culture and ROCK inhibition, the novelty of this approach includes the transfusion of a product containing both megakaryocytes and platelets, offering a promising strategy for addressing platelet shortages and improving patient outcomes. Conclusions: We standardized a GMP-compliant protocol for the manufacture of megakaryocytes and platelets from CD34+ derived from Cord Blood. Large-scale expansion of functional MKs and platelets can be generated efficiently in the bioreactor system for clinical use. Clinical trials to evaluate this new product are planned.

Abstract Resistance to radiation therapy significantly affects the prognosis of solid tumors. Histone deacetylase 6 (HDAC6) is a stress-responsive lysine deacetylase that has emerged as a promising target for cancer therapy, with numerous clinical trials investigating interventions to modulate its activity. While HDAC6 appears to be linked to responses to DNA-damaging therapeutics, the reported responses and underlying mechanisms remain intriguing and varied. In this study, we elucidate a novel mechanism of radioresistance mediated by HDAC6 in non-small cell lung cancer (NSCLC). We classified nine NSCLC cell lines into three groups based on their radioresistance using survival assays and correlated this resistance with the induced expression of six deacetylases. We conducted gain-of-function experiments (GOF) using plasmid transfection and loss-of-function experiments (LOF) employing inhibitors or RNAi. Furthermore, we quantified the repair of damaged dsDNA through FACS-based GFP assays and assessed the mechanism of homologous recombination repair (HRR) end-resection via single-strand quantitative PCR. Co-immunoprecipitation assays, cancer stem cell enrichment, DNA damage-induced senescence assays, reverse-phase protein assays (RPPA), and RNA sequencing were also performed to investigate gene expression changes resulting from the loss of HDAC6. Our findings revealed that HDAC6 induction by radiation is strongly correlated with NSCLC resistance to radiation leading to CSC survival and escape from radiation led senescence LOF of HDAC6 through RNAi or inhibitors suppressed resistance, while GOF achieved through wild-type or deacetylase activity-deficient mutant transfection, induced resistance. Importantly, this resistance mechanism was independent of deacetylation activity. RNAi-mediated loss of HDAC6 reduced both HRR and non-homologous end-joining, whereas pharmacological inhibition of HDAC6 activity did not. Notably, DNA end-resection, a critical step in HRR, was decreased by RNA interference but not affected by the inhibitors. RPPA assays demonstrated that Histone demethylase 1A (LSD1/KDM1A) levels decreased significantly upon HDAC6 RNA interference but remained unaffected by pharmacological intervention. Loss of LSD1 alone resulted in decreased HRR and end-resection, likely mediated through HDAC6-induced ubiquitination. Moreover, overexpression of LSD1 rescued the HDAC6 loss-induced sensitization of NSCLCs. In summary, our study unveils a novel mechanism by which HDAC6 mediates HRR after radiation through the stabilization of LSD1. This mechanism operates independently of deacetylase activity. Therefore, our findings suggest that interventions targeting both deacetylation and non-deacetylation roles of HDAC6 should be considered to overcome HDAC6-derived radioresistance in NSCLCs and enhances the radiation therapy efficacy. Citation Format: Sojung Ha, Hyejin Kim, Hani Lee, Seokgyeong Choi, Ho-young Lee, Woo-Young Kim. HDAC6 mediates the radioresistance of NSCLC through LSD1, independent of its own deacetylation activity [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 2883.

BACKGROUND:Many patients with glioblastoma multiforme (GBM) develop deep venous thrombosis or pulmonary emboli. Cell-free circulating mitochondria increase after brain injury and are associated with coagulopathy. OBJECTIVES:This study evaluated whether mitochondria play a role in the GBM-induced hypercoagulable state. METHODS:We examined the correlation between cell-free circulating mitochondria and venous thrombosis in patients with GBM and the impact of mitochondria on venous thrombosis in mice with inferior vena cava stenosis. RESULTS:Using plasma samples of 82 patients with GBM, we found that patients with GBM had a higher number of mitochondria in their plasma (GBM with venous thromboembolism [VTE],: 2.8 × 107 mitochondria/mL; GBM without VTE, 1.9 × 107 mitochondria/mL) than that in healthy control subjects (n = 17) (0.3 × 107 mitochondria/mL). Interestingly, patients with GBM and VTE (n = 41) had a higher mitochondria concentration than patients with GBM without VTE (n = 41). In a murine model of inferior vena cava stenosis, intravenous delivery of mitochondria resulted in an increased rate of venous thrombosis compared with that in controls (70% and 28%, respectively). Mitochondria-induced venous thrombi were neutrophil-rich and contained more platelets than those in control thrombi. Furthermore, as mitochondria are the only source of cardiolipin in circulation, we compared the concentration of anticardiolipin immunoglobulin G in plasma samples of patients with GBM and found a higher concentration in patients with VTE (optical density, 0.69 ± 0.04) than in those without VTE (optical density, 0.51 ± 0.04). CONCLUSION:We concluded that mitochondria might play a role in the GBM-induced hypercoagulable state. We propose that quantifying circulating mitochondria or anticardiolipin antibody concentrations in patients with GBM might identify patients at increased risk of VTE.

Previously, we found that an important homologous recombination repair protein ‘Tonsoku Like, DNA Repair Protein’ (TONSL) might be required for glioblastoma cancer stem cell (CSC). In this study, we further investigated the role of TONSL in CSC from several cancer cell lines from different tissues. We also analyzed the clinical data available from the public databases to find the clinical impact of TONSL. The higher expression of TONSL is associated with the worse prognosis of lung and gastric cancer. Interestingly, in breast cancer, while its higher expression is also the negative prognostic marker of the luminal A and HER2 positive cancers, that is rather the positive prognostic marker for luminal B and basal cancers. TONSL expression is higher in all 4 subtypes of breast cancer, and ovarian cancer than the corresponding normal tissues. The knockdown of TONSL in an ovarian cancer cell line, OVCAR8, results in the increase of G2/M cells and apoptotic cells in the bulk cultured population while increase also the senescence cells only in the CSC population leading to the severe loss of CSC population. TONSL and the close binding partner in homologous recombination repair (HRR), MMS22L, both were essential for the CSC growth but the bulk cultured cells could survive without those two in a colon cancer cell line, HCT15 suggesting the importance of HRR in colon CSC. Indeed, gemcitabine treatment induced DNA breaks showed a synergistic cytotoxicity with those two HRR factors knockdown in the CSC only. These results suggest that the TONSL and MMS2L mediated HRR is essential for the CSC and while most of bulk cultured cancer cells circumvent the HRR deficit. Collectively, these data showed that the CSCs ultimately depend on the HRR process which requires TONSL and, therefore, CSC population may be more vulnerable to HRR targeting therapeutic approaches. Citation Format: Hani Lee, SeokGyeong Choi, Sojung Ha, Woo-Young Kim. Prognostic and therapeutic impact of a homologous recombination repair protein TONSL in cancer stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6104.

Melanoma is an aggressive skin cancer that develops from the malignant transformation of pigment-producing skin cells, melanocytes. The incidence of cutaneous melanoma is remarkably high, with an estimated number of new cases in the United States in 2022 close to 100,000 patients. Several previous reports pointed out the effect of the complement system in the progression of melanoma, although the precise mechanism is largely unknown. The complement system is a crucial component of innate immunity, and it also has a significant role in adaptive immunity regulating the function of immune cells. The complement receptors C3aR1 and C5aR1 are present on the surface of various immune cells. Anaphylatoxins (C3a and C5a) generated by complement activation bind to their respective receptors, C3aR1 and C5aR1, suppress the antitumor function of immune cells and promote migration and activity of immunosuppressive cells in the tumor microenvironment. Hence, C3aR1 and C5aR1 act as immune checkpoint receptors. To identify the precise role of the complement receptors in melanoma, we challenged C3 −/−, C3aR1 −/−, and C5aR1 −/−mice with the B16F10 murine melanoma cells. We showed that the deficiency of these molecules substantially delayed tumor growth and promoted an antitumorigenic immune response. The results of this study indicate the distinct role of complement receptor signaling on melanoma growth and suggest a novel immunotherapeutic approach. Developmental Research Program Award from the MD Anderson Melanoma SPORE (P50CA221703-04)

We investigated the role of TONSL, a mediator of homologous recombination repair (HRR), in stalled replication fork double-strand breaks (DSBs) in cancer. Publicly available clinical data (tumors from the ovary, breast, stomach and lung) were analyzed through KM Plotter, cBioPortal and Qomics. Cancer stem cell (CSC)-enriched cultures and bulk/general mixed cell cultures (BCCs) with RNAi were employed to determine the effect of TONSL loss in cancer cell lines from the ovary, breast, stomach, lung, colon and brain. Limited dilution assays and ALDH assays were used to quantify the loss of CSCs. Western blotting and cell-based homologous recombination assays were used to identify DNA damage derived from TONSL loss. TONSL was expressed at higher levels in cancer tissues than in normal tissues, and higher expression was an unfavorable prognostic marker for lung, stomach, breast and ovarian cancers. Higher expression of TONSL is partly associated with the coamplification of TONSL and MYC, suggesting its oncogenic role. The suppression of TONSL using RNAi revealed that it is required in the survival of CSCs in cancer cells, while BCCs could frequently survive without TONSL. TONSL dependency occurs through accumulated DNA damage-induced senescence and apoptosis in TONSL-suppressed CSCs. The expression of several other major mediators of HRR was also associated with worse prognosis, whereas the expression of error-prone nonhomologous end joining molecules was associated with better survival in lung adenocarcinoma. Collectively, these results suggest that TONSL-mediated HRR at the replication fork is critical for CSC survival; targeting TONSL may lead to the effective eradication of CSCs.

ARL2 is a small GTP binding protein mediates many cytosolic functions including localization of Ras, dynamics of microtubule and fission of mitochondria but its nuclear function is not well elucidated yet. Here we present the novel role of ARL2 in human cancer nucleus and cancer stem cells (CSC). The cancer tissue gene expression data in The Human Protein Atlas and The Cancer Genome Atlas showed possible pro-cancer and nuclear role of ARL2 in colon cancers. The single cell RNA seq analysis suggests its function in colon stem cells. The activation mutant of K-Ras harboring colon cancer cells expressed less ARL2 than the wild type cells but increased ARL2 expression in CSC spheres. The deprivation of ARL2 in colon cancer cell lines using RNAi preferentially eliminated the CSC spheres while it resulted in minimal impact on bulk cultured cell (BCC) numbers. Suppression of ARL2 halted the BCC at M phase as expected but did not change cell cycle in CSC spheres. However, it led to accumulation of a DNA double strand break marker and apoptosis only in CSC spheres which suggest that ARL2’s requirement for double strand DNA break repair in CSC. Out of 6 somatic RAD51 family genes which all mediate the critical early events of homologous recombination repair (HRR), 5 showed significant and positive association with ARL2 in gene expression in human colon cancer tissue suggesting functional link between ARL2 and HRR. Indeed, ARL2 was required for HRR when double strand DNA breaks were induced. These data collectively showed that ARL2 may contribute to the HRR in the nucleus and the ARL2 mediated HRR is more critical for colon CSC survival. Citation Format: Seok Gyeong Choi, Hani Lee, Sojung Ha, Sukjoon Yoon, Woo-Young Kim. ARL2 is required for homologous recombination repair and colon cancer stem cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3171.

The interactions between platelets and cancer cells activate platelets and enhance tumor growth. Platelets increase proliferation and epithelial–mesenchymal transition in cancer cells, inhibit anoikis, enhance the extravasation of cancer cells, and protect circulating tumor cells against natural killer cells. Here, we have identified another mechanism by which platelets dampen the immune attack on cancer cells. We found that platelets can blunt the antitumor immune response by increasing the expression of inhibitory immune checkpoint (PD-L1) on ovarian cancer cells in vitro and in vivo. Platelets increased PD-L1 in cancer cells via contact-dependent (through NF-κB signaling) and contact-independent (through TFGβR1/Smad signaling) pathways. Inhibition of NF-κB or TGFβR1 signaling in ovarian cancer cells abrogated platelet-induced PD-L1 expression. Reducing platelet counts or inhibiting platelet functions reduced the expression of PD-L1 in ovarian cancer. On the other hand, an increase in platelet counts increased the expression of PD-L1 in tumor-bearing mice.

ARL2 regulates the dynamics of cytological components and is highly expressed in colon cancer tissues. Here, we report novel roles of ARL2 in the cell nucleus and colon cancer stem cells (CSCs). ARL2 is expressed at relatively low levels in K‐RAS active colon cancer cells, but its expression is induced in CSCs. Depletion of ARL2 results in M phase arrest exclusively in non‐CSC cultured cells; in addition, DNA break stress accumulates in CSCs leading to apoptosis. ARL2 expression is positively associated with the expression of all six RAD51 family genes, which are essential for homologous recombination repair (HRR). Furthermore, ARL2 is required for HRR and detected within chromatin compartments. These results demonstrate the requirement of ARL2 in colon CSC maintenance, which possibly occurs through mediating double‐strand break DNA repair in the nucleus.

We and other investigators have shown that platelets promote metastasis and the growth of tumors. Our rationale for conducting this study is that platelets' prometastatic and progrowth effects depend on a close encounter between platelets and cancer cells. This interaction occurs inside blood vessels with circulating tumor cells and outside blood vessels with cancer cells residing in the tumor parenchyma. Our hypothesis was that platelet extravasation is required for the effect of platelets on tumor growth. Platelets respond to environmental stimuli by activation of G protein–coupled receptors on their surface. We investigated the impact of various platelet G proteins on the growth of ovarian cancer tumors and platelet extravasation. We used mice with platelet-specific deficiency of Gαi2 (Gi), Gα13 (G13), or Gαq (Gq) in a syngeneic ovarian cancer model. We measured the total weight of tumor nodules resected from tumor-bearing mice. We developed methods for automated whole-slide image acquisition and unbiased computerized image analysis to quantify extravasated platelets. We compared the number of platelets inside tumor nodules of platelet G protein–deficient tumor-bearing mice. We found that deficiency of Gi and G13, but not Gq, in platelets resulted in smaller tumors compared with those in corresponding littermates. Deficiency of Gi and G13 in platelets reduced the number of extravasated platelets by >90%, but deficiency of Gq did not reduce the number of extravasated platelets significantly. The lack of Gi or G13 in platelets reduced platelet extravasation into the tumor and tumor growth.