Tumor-derived Extracellular Vesicles Research Articles

Exploring the Immunomodulatory Potential of Pancreatic Cancer-Derived Extracellular Vesicles through Proteomic and Functional Analyses

Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network (p = 1 × 10−16) and were involved in the “Immune System” (FDR: 1.10 × 10−24 and 3.69 × 10−19, respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor.

Abstract PO3-06-05: Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer

Abstract Background: Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). Recently, the complementary prognostic value of tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) has been reported. We have previously confirmed the strong prognostic significance of CTCs and tdEVs in inflammatory breast cancer (IBC). While previous studies have reported the association of CTCs with circulating tumor DNA (ctDNA) alterations in MBC, no evidence is available for tdEVs. This study aimed to analyze the association of tdEVs with ctDNA alterations, to investigate the molecular pathways underlying their presence. Moreover, we explored potential differences of this association in patients (pts) with IBC to provide a comprehensive liquid biopsy-based portrait of this aggressive subtype of BC. Methods: Blood samples were collected from 355 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). For CTCs and tdEVs analysis, 7.5 mL of blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Positivity cutoff was ≥5 for CTCs, while tdEV cutoff points were < 20 (low), 20-79 (intermediate), and ≥80 (high). For ctDNA analysis, matched plasma samples (± 1 month) were analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs) and copy number variations (CNVs), which were classified into oncogenic pathways based on defined profiles generated on the Cancer Genome Atlas database (p53, PI3K, ER, RTK, RAS, RAF, WNT, MYC, cell cycle, notch). Associations between ctDNA pathways alterations and tdEVs were tested in the overall population by multinomial logistic regression and corrected for significant clinical characteristics. Differences in liquid biopsy features between IBC and non-IBC subgroups were analyzed through Fisher's exact test. Results: Of the 355 MBC pts, 210 (62%) had HR+ BC, 61 (17%) had HER2+ BC, and 68 (20%) had triple-negative BC. Eighty-three (23%) had a diagnosis of IBC and had a numerically lower tdEV count. Also, lower tdEVs were detected in HER2+ BC as compared with HR+. Significantly higher tdEVs were observed among pts with lobular histology, liver, and bone metastases. CTC count was significantly associated with tdEV number. A matched plasma sample for ctDNA analysis was available for 175 pts (62 IBC and 113 non-IBC). In the overall population, SNVs in ER pathway were associated with intermediated/high levels of tdEVs (p=0.004 and 0.008). A similar association was observed for CNVs in the cell cycle pathway (p=0.008 and 0.007). Moreover, associations with ≥80 tdEVs were observed for PI3K SNVs and CNVs (p=0.039 and 0.004, respectively), RTK CNVs (p=0.023), and MYC CNVs (p=0.001). In multivariable analysis, clinical characteristics associated with higher tdEVs were lobular histology (p=0.014 for 20-79 and < 0.001 for ≥80) and bone metastases (p=0.019 for 20-79 and 0.002 for ≥80). When considering clinical variables of interest, only ER SNVs and MYC CNVs were significantly associated with intermediate (p=0.031) and high (p=0.022) tdEV counts, respectively. While the association with ER SNVs and cell cycle CNVs was significant both in the IBC and non-IBC subgroups, others were specific for a certain subgroup: higher tdEVs were significantly associated with PI3K SNVs only in non-IBC pts whereas the significant association with CNVs in PI3K and MYC pathways was observed only in pts with IBC. Conclusion: Detection of tdEVs was associated with particular genomic profiles. These alterations seem to be different in IBC further underlying a different biology of this BC subtype. Additional studies are needed to explore how to integrate different liquid-biopsy based biomarkers in the management of pts with MBC. Citation Format: Eleonora Nicolò, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz Arcos, Mara Serena Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda Kaylan Strickland, Youbin Zhang, Paolo D’Amico, Andrew Davis, Jeannine Donahue, Huiping liu, William Gradishar, Ami Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-05.

Advanced Nanoencapsulation-Enabled Ultrasensitive Analysis: Unraveling Tumor Extracellular Vesicle Subpopulations for Differential Diagnosis of Hepatocellular Carcinoma via DNA Cascade Reactions.

Tumor-derived extracellular vesicles (tEVs) hold immense promise as potential biomarkers for the precise diagnosis of hepatocellular carcinoma (HCC). However, their clinical translation is hampered by their inherent characteristics, such as small size and high heterogeneity and complex environment, including non-EV particles and normal cell-derived EVs, which prolong separation procedures and compromise detection accuracy. In this study, we devised a DNA cascade reaction-triggered individual EV nanoencapsulation (DCR-IEVN) strategy to achieve the ultrasensitive and specific detection of tEV subpopulations via routine flow cytometry in a one-pot, one-step fashion. DCR-IEVN enables the direct and selective packaging of multiple tEV subpopulations in clinical serum samples into flower-like particles exceeding 600 nm. This approach bypasses the need for EV isolation, effectively reducing interference from non-EV particles and nontumor EVs. Compared with conventional analytical technologies, DCR-IEVN exhibits superior efficacy in diagnosing HCC owing to its high selectivity for tEVs. Integration of machine learning algorithms with DCR-IEVN resulted in differential diagnosis accuracy of 96.7% for the training cohort (n = 120) and 93.3% for the validation cohort (n = 30), effectively distinguishing HCC, cirrhosis, and healthy donors. This strategy offers a streamlined workflow and rapid assay completion and requires only small-volume serum samples and routine clinical devices, facilitating the clinical translation of tEV-based tumor diagnosis.

Codetection of Proteins and RNAs on Extracellular Vesicles for Pancreatic Cancer Early Diagnosis.

Tumor-derived extracellular vesicles (EVs) carry tumor-specific proteins and RNAs, thus becoming prevalent targets for early cancer diagnosis. However, low expression of EV cargos and insufficient diagnostic power of individual biomarkers hindered EVs application in clinical practice. Herein, we propose a multiplex Codetection platform of proteins and RNAs (Co-PAR) for EVs. Co-PAR adopted a pair of antibody-DNA probes to recognize the same target protein, which in turn formed a double-stranded DNA. Thus, the target protein could be quantified by detecting the double-stranded DNA via qPCR. Meanwhile, qRT-PCR simultaneously quantified the target RNAs. Thus, with a regular qPCR instrument, Co-PAR enabled the codetection of multiplex proteins and RNAs, with the sensitivity of 102 EVs/μL (targeting CD63) and 1 EV/μL (targeting snRNA U6). We analyzed the coexpressions of three protein markers (CD63, GPC-1, HER2) and three RNA markers (snRNA U6, GPC-1 mRNA, miR-10b) on EVs from three pancreatic cell lines and 30 human plasma samples using Co-PAR. The diagnostic accuracy of the 6-biomarker combination reached 92.9%, which was at least 6.2% higher than that of 3-biomarker combinations and at least 13.5% higher than that of 6 single biomarkers. Co-PAR, as a multiparameter detection platform for EVs, has great potential in early disease diagnosis.

Tumor-derived extracellular vesicles regulate macrophage polarization: role and therapeutic perspectives.

Extracellular vesicles (EVs) are important cell-to-cell communication mediators. This paper focuses on the regulatory role of tumor-derived EVs on macrophages. It aims to investigate the causes of tumor progression and therapeutic directions. Tumor-derived EVs can cause macrophages to shift to M1 or M2 phenotypes. This indicates they can alter the M1/M2 cell ratio and have pro-tumor and anti-inflammatory effects. This paper discusses several key points: first, the factors that stimulate macrophage polarization and the cytokines released as a result; second, an overview of EVs and the methods used to isolate them; third, how EVs from various cancer cell sources, such as hepatocellular carcinoma, colorectal carcinoma, lung carcinoma, breast carcinoma, and glioblastoma cell sources carcinoma, promote tumor development by inducing M2 polarization in macrophages; and fourth, how EVs from breast carcinoma, pancreatic carcinoma, lungs carcinoma, and glioblastoma cell sources carcinoma also contribute to tumor development by promoting M2 polarization in macrophages. Modified or sourced EVs from breast, pancreatic, and colorectal cancer can repolarize M2 to M1 macrophages. This exhibits anti-tumor activities and offers novel approaches for tumor treatment. Therefore, we discovered that macrophage polarization to either M1 or M2 phenotypes can regulate tumor development. This is based on the description of altering macrophage phenotypes by vesicle contents.

Abstract 3758: Evaluation of breast cancer-derived extracellular vesicles as diagnostic indicator for HER2 status

Abstract HER2-low is a new distinct type of breast cancer that is characterized by low levels of the HER2 protein. As treatment paradigms for HER2-low breast cancer are rapidly evolving with the approval of antibody-drug conjugates, identification of HER2-low breast cancer with more sensitive and reproducible HER2 assays is needed. We aimed to investigate the utility of liquid biopsy in isolating tumor-derived extracellular vesicles (TDEs) for diagnosis of HER2 status. We adopted an affinity-based approach to isolate TDEs and performed CNV analysis of the ERBB2/neu genes using the tumor DNA present in TDEs (etDNA). HER2 status of etDNA had been determined by droplet digital PCR using HER2/EIF2C ratio, a highly accurate method to quantify DNA copy number. In our study, we utilized a variety of breast cancer cell lines categorized as HER2-low (MDA-MB-231, MCF7, ZR-75-1) and HER2-high (MDA-MB-453, BT-474, SK-BR-3) to confirm HER2 expression in extracellular vesicles (EVs) by flow cytometry. This analysis successfully demonstrated that EVs effectively reflect the HER2 status of the tumor cells, evidenced by a strong Pearson correlation coefficient (r=0.987, p<0.001). Further clinical validation was conducted with plasma samples from 22 breast cancer patients. In the clinical study, we observed a significant correlation between the CNV of tumor DNA present in TDEs and the HER2 status of primary tumors (p<0.01). Additionally, the Receiver Operating Characteristic (ROC) analysis yielded an impressive area under the curve (AUC) value of 0.838 (p<0.01), demonstrating a high level of accuracy in differentiating between high and low HER2 groups, with a sensitivity of 55.6% and a specificity of 100%. In summary, we show potential diagnostic tools for real-time, less invasive determination of HER2 status in primary tumors, providing continuous monitoring methods for HER2 status. Although further validation in larger cohorts and prospective studies is needed, our study indicates that TDE analysis could contribute to the assays for identification of the HER2 status in breast cancer patients. Citation Format: Sol Moon, Min Woo Kim, Young Kim, Suji Lee, Hyojung Lee, Joon Ye Kim, Seung Il Kim, Jee Ye Kim. Evaluation of breast cancer-derived extracellular vesicles as diagnostic indicator for HER2 status [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 3758.

Abstract 7500: Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC)

Abstract Background: Invasive lobular carcinoma (ILC) has distinctive clinical and molecular features compared with invasive ductal carcinoma (IDC). CTCs and tdEVs are independent prognostic factors in MBC, however, liquid biopsy studies focusing on ILC are to date scarce. Higher CTC levels have been observed in ILC than IDC. One study including 28 ILC pts suggested the use of higher CTC cutoff for prognosis stratification. The clinical significance of tdEVs in ILC is unexplored. This study aimed to assess differences in the distribution and prognostic value of CTCs and tdEVs between ILC and IDC. Methods: Blood samples were collected from 304 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). Blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Association of CTCs and tdEVs with overall survival (OS) was tested in ILC and IDC pts. A count of CTCs ≥ 5 was considered high. The cutoff levels for tdEVs were < 20 (low), 20-79 (intermediate), and ≥ 80 (high), as previously reported. Additional exploratory cutoffs for CTCs (≥ 20, ≥ 50, ≥ 80, and ≥ 100) and tdEVs (based on quartiles of tdEV distribution in the overall population and ILC) were also evaluated. Results: Of the 304 pts, 47 had ILC while 257 had IDC. 56% of pts received first-line therapy. Median CTC count was 8 [interquartile range (IQR) 2-35] in ILC and 1 (IQR 0-8) in IDC pts (P <.001). A significantly higher median tdEVs count was observed in ILC (36; IQR 18-115) than in IDC (11; IQR 2-86) pts (P =.002). High levels of CTCs (≥5) were not associated with OS in ILC (P =.88), even when higher cutoffs (≥ 20, ≥ 50, and ≥ 80) were used. Only the detection of ≥ 100 CTCs was significantly associated with OS in ILC pts (median OS 36 vs 10 months; HR 3.4; P =.028). In contrast, the prognostic effect of CTCs ≥ 5 was strong in IDC and maintained for all cutoffs. Similarly, while the prognostic value for tdEVs was confirmed for IDC using both 20-80 and exploratory cutoffs (3-105 and 18-155), no association between tdEV levels and OS was observed in ILC regardless of the cutoffs used. Conclusion: ILC is associated with higher CTCs and tdEVs compared to IDC, probably due to the impaired cell-cell adhesion that characterizes ILC. Unlike IDC, the prognostic effect for the established cutoff of ≥ 5 CTC was not observed in ILC. A higher CTC cutoff could be considered for this subtype of BC. For tdEV no association with OS was observed, regardless of the cutoff used. These findings emphasize that ILC is a distinct entity also in the liquid biopsy features. Because of the low prevalence of ILC, research efforts should be directed at combining data from ILC pts to better characterize this subtype of BC and understand the reason behind the different associations with OS as compared to IDC. Citation Format: Eleonora Nicolo, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz-Arcos, Mara S. Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda K. Strickland, Caterina Gianni, Youbin Zhang, Paolo D'Amico, Andrew A. Davis, Jeannine Donahue, Huiping Liu, William J. Gradishar, Ami N. Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC) [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 7500.

Abstract 5118: Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner

Abstract Glypican 2 (GPC2) chimeric antigen receptor (CAR) T cells are safe and efficacious in neuroblastoma and are currently being tested in a first-in-human Phase 1 clinical trial at the Children’s Hospital of Philadelphia. Tumor-derived extracellular vesicles (TEVs) are nanosized vesicles secreted by cancer cells, often enriched with glypicans. However, the presence of GPC2 on neuroblastoma TEVs and any interaction with GPC2 CAR T cells has not been explored. High-levels of GPC2 were found on TEVs isolated from neuroblastoma preclinical models [7 cell lines, circulating EVs from 6 patient-derived xenograft (PDX)-bearing mice] and from the peripheral blood of 16 neuroblastoma patients, but not on circulating EVs from 7 non-tumor bearing mice or 12 healthy human donors. The level of circulating GPC2+ EVs isolated from the peripheral blood of PDX-bearing mice positively correlated with PDX size (R=0.96; P<0.0001) and parent PDX GPC2 expression. To evaluate how GPC2 on EVs modulates CAR T cell functionality, we generated EVs with varied levels of GPC2 (Hi/Lo/Neg) and co-incubated each EV subset with GPC2 CAR T cells. Here using immunofluorescence and flow assays we found that EVs bind GPC2 CAR T cells proportional to the amount of GPC2 on their surface. Further, GPC2Hi EV-CAR T cell synapses induced potent T cell activation shown by CD69/Granzyme B expression and release of IL-2/IFN-γ. GPC2Hi EV pre-incubated CAR T cells also displayed enhanced target neuroblastoma cell cytotoxicity, inducing 4-fold more specific cytotoxicity than CAR T cells incubated with control GPC2Neg EVs. In vivo we observed that injection of GPC2Hi EVs into GPC2Lo neuroblastoma SK-N-AS xenografts genetically altered to have significantly decreased endogenous EV production (via RAB27A knock-out) greatly enhanced the efficacy of co-infused GPC2 CAR T cells (p<0.05). Similarly, in an isogenic SK-N-AS-GPC2 xenograft murine model treated with a limiting number of GPC2 CAR T cells concurrently with the EV-inhibiting drug GW4869, GPC2 CAR efficacy was dependent on the presence of GPC2Hi EVs. Here, pharmacologic EV inhibition in mice decreased GPC2 CAR efficacy which could be rescued by intratumoral injection of GPC2Hi EVs. In both in vivo models, the presence of intratumoral GPC2Hi EVs resulted in a significantly enhanced tumor infiltration of activated (CD69+/CD25+) GPC2 CAR T cells (p<0.05). Taken together, GPC2Hi EVs are selectively secreted from neuroblastomas and bind and activate GPC2 CAR T cells enhancing their anti-tumor cytotoxicity. To further capitalize on these findings, we engineered GPC2Hi EVs to have an extended circulation half-life through albumin binding or tumor targeting via GD2 binding that are currently being tested in vivo and results will be reported. EVs offer a versatile platform for CAR antigen presentation and should be further validated as a strategy to enhance CAR T cell efficacy for solid tumors. Citation Format: Anna M. Giudice, Stephanie Matlaga, Guillem Pascual-Pasto, Patrick M. Schuerch, Geoffrey Rouin, Brendan McIntyre, Vincent P. Zecchino, Kristopher R. Bosse. Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner [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 5118.

Abstract 1843: Integrating machine learning with microfluidic technologies for proteomic profiling of extracellular vesicles in triple-negative breast cancer

Abstract Accurate diagnosis of breast cancer using circulating biomarkers present in plasma remains an important challenge. In particular, protein changes in tumor-derived extracellular vesicles (tdEVs) have emerged as potential biomarkers for breast cancer diagnosis because they accurately reflect dynamic changes in tumors. In this study, we compared the proteomes of extracellular vesicle (EV) isolated from the plasma of 100 breast cancer patients and 30 healthy individuals who visited Severance Hospital between Mar 2010 and Dec 2015. Microfluidic chip-based protocol that facilitates the removal of contaminants such as albumin and immunoglobulins was used for the extraction of enriched tdEVs with small amounts of plasma. Comparative analysis of the proteomes identified 26 significant biomarkers that could indicate differences between breast cancer patients and healthy individuals. Using the LsBoost-CNN-SVM hybrid machine learning algorithm, we especially identified key EV protein biomarkers for detecting triple-negative breast cancer (TNBC), a breast cancer subtype with a poor prognosis and a high recurrence rate, as well as lacking therapeutic and diagnostic targets. Remarkably, a signature consisting of three EV proteins, specifically extracellular matrix protein 1 (ECM1), mannose-binding lectin 2 (MBL2), and biotinidase (BTD), effectively distinguished TNBC patients from healthy individuals. In our proteomic analysis set (n=73), this signature exhibited impressive performance, achieving a sensitivity of 93.3% and specificity of 93% in the accurate discrimination of TNBC from the control group. The validation set (n=40) confirmed these findings, with 100% sensitivity and 80% specificity. This signature not only served as a diagnostic tool but also provided valuable insights into the risk of recurrence and patient prognosis. We found a novel diagnostic approach that holds the potential to revolutionize breast cancer diagnostics by enhancing the reliability of tumor-related information obtained from blood samples. Citation Format: Min Woo Kim, Jee Ye Kim, Young Kim, Suji Lee, Sol Moon, Ju-yong Hyon, Kyung-A Hyun, Yeji Yang, Seongmin Ha, Sunyoung Park, Hogyeong Gawk, Haeji Lee, Eun Hee Han, Jin Young Kim, Hyo-Il Jung, Young-Ho Chung, Seung Il Kim. Integrating machine learning with microfluidic technologies for proteomic profiling of extracellular vesicles in triple-negative breast cancer [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 1843.

Abstract 3841: Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies

Abstract Despite the remarkable efficacy of chimeric antigen receptor T -cell therapy (CART) for the treatment of B-cell malignancies, durable remission remains limited often due to tumor relapse. We have previously demonstrated that tumor-derived extracellular vesicles (EVs) induced CART cell failure in preclinical models. To characterize the impact of tumor EV induced CART cell dysfunction, we assessed microRNA (miRNA) profiles contained within tumor EVs from two independent patient cohorts and studied how they contributed to CART dysfunction. Our first cohort included patients with chronic lymphocytic leukemia (n=50). We previously showed that CLL patient derived EVs induce a state of CART cell dysfunction. To understand this dysfunction, we interrogated the miRNA cargo significantly enriched in CLL EVs and how their coculture impacts CART signaling by RNAseq. Here, we found a significant enrichment in miR-125a within CLL EVs as compared to healthy donor controls (p=.0001). CART cocultured with CLL EVs showed alterations in T cell activation pathways, where miR-125a targets TNFAIP3 and NFkB pathways were activated. These findings implicate miR-125a from CLL EVs in CART dysfunction. Next, we aimed to validate the impact of miRNA on CART cell outcomes in the clinic. We interrogated miRNA profiles from patients with diffuse large B cell lymphoma (DLBCL) treated with CART cell therapy (CART19-28ζ, axicabtagene ciloleucel). EV miRNA cargo was compared between responders (complete remission at 6 months, n=10) and non-responders (no response 1 month post CART19, n=10) to identify differentially enriched miRNAs across groups. miR-125a was again significantly upregulated in EVs of non-responders as compared to responders to CART19 treatment (fold change=3.28, padj=.018). To study the direct effect of miR-125a on CART cell functions, we generated and studied the effector functions of CART19-28z following transfection with miR-125a or negative miR control. CART19 cells transfected with miR-125a showed a dose dependent decrease in killing of lymphoma cells as compared to miR-control (p=.0001). miR-125a also significantly increased the rate of apoptosis in CART19 cells (fold change=1.95, p=.024) and decreased the level of CART proliferation (fold change=.71, p=.013) as compared to controls. Notably, miR-125a did not impact tumor cell growth, further verifying a direct CART effect. In summary, our studies demonstrate the impact of tumor derived miRNAs on CART cell outcomes. They identify that miR-125a is 1) associated with CLL EV induced CART dysfunction, 2) significantly elevated in non-responders to CART therapy in the clinic, and 3) able to decrease CART19 efficacy through reduced CART killing/proliferation and increased apoptosis. These results highlight a novel mechanism of CART19 failure induced through miRNA cargo delivery by tumor-derived EVs and the role of miR-125a in inducing CART19 dysfunction. Citation Format: Olivia Sirpilla, R. Leo Sakemura, Ismail Can, Truc N. Huynh, James H. GIrsch, Claudia Manriquez Roman, Carli M. Stewart, Kun Yun, Ekene J. Ogbodo, Long Mai, Omar Gutierrez Ruiz, Makena Rodriguez, Brooke Kimball, Hong Xia, Jose C. Villasboas, Stephen Ansell, Nora Bennani, Patrick B. Johnston, Jonas Paludo, Januario Castro, Mohamed Kharfandabaja, Sameer Parikh, Neil Kay, Yi Lin, Elizabeth Siegler, Saad Kenderian. Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies [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 3841.

Abstract 3633: Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy

Abstract Introduction: Aqueous humor (AH), fluid in the anterior chamber of the eye, is a liquid biopsy source of tumor information in uveal melanoma (UM). Extracellular vesicles (EVs) have recently been characterized in the AH of retinoblastoma eyes, however, have not been investigated in UM. EVs are small membrane-enclosed particles secreted by virtually every cell type with a role in intercellular communications. We hypothesize that tumor-derived EVs are detectable in the AH of UM eyes and that understanding the type of vesicles present may help us better understand their function, particularly for cancer. Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis, leading to the discovery of clinically-relevant biomarkers for multiple cancer types. As the diversity and role of sEVs in the AH of UM patients has never previously been reported, this project is a first step towards investigating new biomarkers of clinical utility. Our aim is to use single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically tetraspanin co-expression patterns. Methods: sEVs were analyzed from paired pre- and post-brachytherapy AH samples collected from 19 UM patients. AH samples from 5 glaucoma patients served as the control cohort. Unprocessed AH underwent Nanoparticle Tracking Analysis for size quantification and Single Particle-Interferometric Reflectance Imaging Sensor analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequently converted to percentages for analysis. Statistics were performed using Mann-Whitney U, ANOVA, and Tukey’s tests. Results: AH derived from UM eyes (pre-brachytherapy) displayed a broader sEV size distribution than glaucoma eyes, with more diverse sEV subpopulations. Both tumor and nontumor AH samples demonstrated a dominant CD63+ sEV subpopulation. A significantly higher percentage of CD63/81+ sEVs (P= 0.0217) and CD9/63/81+ sEVs (P= 0.0146) were found in post-brachytherapy samples compared to pre-brachytherapy. Conclusions: sEV subpopulations in UM eyes demonstrate increased heterogeneity in comparison to nontumor eyes. Our results are consistent with prior studies reporting that the CD63+ sEV subtype is an AH-specific biomarker enriched across multiple ocular diseases. The increase in CD63/81+ sEVs and CD9/63/81+ sEVs after brachytherapy is presumably a result of radiation-induced release of these vesicles, suggesting a tumor-derived origin of these subpopulations. CD63/81+ sEV subpopulation was previously reported to also be a tumor-derived subpopulation in AH of retinoblastoma eyes. Further study of the cargo carried by these sEV subpopulations may uncover molecular mechanisms pivotal to UM and other ocular cancers. Citation Format: Shreya Sirivolu, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy [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 3633.

Abstract 6965: Characterizing the proteome landscape of extracellular vesicles from prostate cancer cell lines by mass spectrometry

Abstract Multiple subtypes of prostate cancer can emerge in advanced stages of disease, such as tumors that maintain androgen receptor features (AR+), those without AR but with neuroendocrine features (AR-/NE+), and those without AR or NE features (AR-/NE-). These latter subtypes are particularly aggressive and incredibly lethal. Extracellular vesicles (EVs) are small particles that are mediators of cell-cell communication and have been nominated as potential biomarkers of disease state. We hypothesized that the proteome of EVs contains information about their originating cell that can distinguish between prostate cancer subtypes. To address this, we isolated EVs from cell lines of differing subtypes – AR+ (LNCaP, LNCaP95, C4-2, 22RV1), AR-/NE+(NCI-H660, EF1, LASCPC-01), and AR-/NE- (PC3, DU145) – and prepared the EV proteome for quantitative, label-free mass spectrometry analysis. This identified 2,000+ proteins in the EVs for each cell line where EVs from similar subtypes clustered together. Interestingly, we also found that EVs contained enough protein information to utilize developed gene signatures for AR or NE activity. EVs isolated from AR-/NE+ cells contained known NE signature proteins such as SYP, CHGA, and UCHL1. EVs from the AR+ cell lines displayed >50% AR activity driven by PSA and STEAP1 proteins in addition to other significant distinguishing protein markers. This supports the potential for using the proteome of tumor-derived EVs to identify novel signature molecular markers besides those already known to the field for developing liquid-based biomarker assays. In addition, our work characterizes the EV surfaceome which is critical for enriching signals from tumor-derived EVs feasible for clinical diagnostics assay development. While PSMA has been a reliable marker for cells reliant on AR signaling, less is known about protein markers in the AR- subset. Our work highlights TROP2 as especially enriched in the EVs from AR-/NE- cell lines, supporting its relevance as a target in patients without treatment options. To evaluate disease outcomes, further clinical studies are needed to develop specific and sensitive tests based on the EV proteome derived from advanced prostate cancer patients. Citation Format: Megan L. Ludwig, Abderrahman Day, Hannah E. Bergom, Zoi Sychev, Alec Horrmann, Justin Hwang, Justin M. Drake. Characterizing the proteome landscape of extracellular vesicles from prostate cancer cell lines by mass spectrometry [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 6965.

Abstract 5569: Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas

Abstract Tissue fibrosis and extracellular matrix (ECM) stiffening have been shown to promote tumor cell proliferation, metastasis, and chemoresistance in a range of cancers. Stiffness of the ECM can lead to changes in cancer cell behavior, including enhanced proliferation, activation of mechano-sensitive signaling pathways, and potential genomic damage, with implications for cancer progression and immune responses. Recent research suggests stiff matrix may also induce the release of tumor-derived small extracellular vesicles (sEVs) that promote tumor growth. Tumor-derived sEVs carry bioactive cargo, such as micro-RNAs (miRNAs) and DNA fragments, and function as a unique form of intracellular communication promoting cell survival, growth, and metastasis. Understanding these mechano-sensing mechanisms is essential for identifying potential targets in precision medicine for solid tumor treatment. However, the influence of tumor stiffness on secretory cues, and their subsequent role in immunomodulation and/or chemo-resistance has not been extensively studied. This project aims to explore how tumor stiffness can alter cell responses to genotoxic stress and how these responses can perpetuate feedback to the immune system through the release of secretory cues such as sEVs. Here, we outline the isolation and characterization of tumor-derived sEVs obtained from culture media of oral squamous cell carcinoma (OSCC) spheroids, cultured at varying stiffness (0.2kPa-1.5kPa). In this project, we use OSCC lines that are either sensitive or resistant to cisplatin (HSC-3 and HN-5, respectively). To explore the immunomodulatory effects of these tumor-derived sEVs, we first established a consistent supply of CD206+ve macrophages differentiated from human induced pluripotent stem cells (iMacs), following the established protocol of Douthwaite et al (2021). iMacs were then treated with sEVs with their resulting immunophenotype, and activation assessed. These preliminary experiments provide a step towards understanding how the biophysical environment influences tumor cell resistance to therapy and how sEVs can modulate the relationship between cancer cells and macrophages. Citation Format: Bethany R. Bareham, Matthew Dibble, Leila Mouhib, Aitor Bermejo Arteagabeitia, Subhankar Mukhopadhyay, Maddy Parsons. Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas [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 5569.

Abstract 4283: NAMPT-enriched small extracellular vesicle promotes liver cancer via activation of SLC27A4-mediated glycolysis

Abstract Introduction: Tumor-derived small extracellular vesicles (sEV) act as a major mediator of the tumor microenvironment (TME) and are reported to regulate various metabolic pathways. Methods: Small extracellular vesicles (sEV) were collected from normal human hepatocyte and metastatic HCC cell lines using differential ultracentrifugation. Mass Spectrometry proteomic analysis was employed to identify NAMPT to be enriched in metastatic HCC-sEV. The glycolytic effect of NAMPT-sEV was analyzed by seahorse glycolytic assay. Result: Mass spectrometry protein analysis revealed nicotinamide phosphoribosyl transferase (NAMPT) to be upregulated in metastatic HCC-sEV. NAMPT is a rate-limiting enzyme essential for maintaining cellular level of nicotinamide dinucleotide (NAD+). However, the underlying pathway of sEV-NAMPT-induced metabolic consequence in HCC remains largely unknown. This study elucidated sEV-NAMPT’s carcinogenic role by establishing NAMPT-knockdown (NAMPT-KD) stable clone in metastatic HCC cell lines, which significantly hampered the promoting effect of HCC-sEV in glycolysis. sEV-mediated in vitro and in vivo tumor growth was also inhibited. Through sEV treatment, we found that sEV-NAMPT significantly elevate SLC27A4 in both transcription and protein level of recipient cells. Mechanistically, it is demonstrated that SLC27A4 expression was enhanced by NF-κB transcription factor, which is activated upon binding of sEV-NAMPT to toll-like receptor 4 (TLR4). SLC27A4 mainly function as a long-chain fatty acid transporter or acyl-CoA synthetase. Lipidomic and metabolomic analysis unveiled SLC27A4 to be positively correlated with intracellular triacylglycerol (TG) and dihydroxyacetone phosphate (DHAP) level. Elevation of TG aggravates lipolysis by hepatic lipase and promotes conversion of glycerol-3-P to DHAP, which is an intermediate metabolite that transit between lipid metabolism and glycolysis. This study uncovers a regulatory axis of sEV-NAMPT-mediated SLC27A4 in glycolysis, apart from conventional fatty acid-related metabolisms. Clinically, we explored the therapeutic potential of targeting sEV-NAMPT by inhibitor FK866. Treatment of NAMPT-inhibitor significantly hampered tumor growth in various HCC in vivo models, which suggested an alternative therapeutic strategy for HCC. Conclusion: NAMPT is enriched in metastatic HCC sEV. sEV-NAMPT regulates the promoting effect of HCC-sEV in glycolysis and oncogenesis. SLC27A4 function as the downstream target that promotes HCC progression via glycolysis. NAMPT inhibitor FK866 are proven to reduce in vitro and in vivo tumorigenic ability of metastatic HCC-sEV, which may serve as potential HCC treatment option. Citation Format: Lot Sum Cherlie Yeung, Tu Him ng, Judy Wai Ping Yam. NAMPT-enriched small extracellular vesicle promotes liver cancer via activation of SLC27A4-mediated glycolysis [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 4283.

Abstract 5358: The influence of extracellular vesicles on immune cells in PDAC

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is bad. The tumor microenvironment consists of an abundance of immunosuppressive immune cells and anti-inflammatory cytokines that contribute to local and systemic immunosuppression in PDAC. Tumor cells can influence the tumor microenvironment and, with that, the present immune cells through the secretion of cytokines or chemokines or the secretion of extracellular vesicles (EVs). EVs are a media of communication among the tumor cells themselves, but also with other cells of the body, with the goal to promote tumor expansion and metastasis and decrease any anti-tumor-directed forces of the immune system. Our lab has shown that those EVs play a significant role in tumor growth and metastasis in PDAC. We have found MUC1 to be an important marker for tumor-derived EVs (tdEVs), associated with their activity and influence on cell proliferation and tumor growth in vitro and in vivo. Determining which immune cell populations are most targeted and impacted by tdEVs is essential to improving our understanding of the role of EVs in PDAC. Methods: TdEVs were isolated from murine pancreatic cancer cell lines through differential ultracentrifugation. EVs were then labeled with a membrane dye, which allows tracking of the EVs in the mouse. We designed an in vivo study to investigate the effects that tdEVs have on different immune cell populations, as well as tumor growth and metastasis. This study has two distinct phases: a 3-week “Pre-Education Phase”, with injection of either MUC1+ or MUC1KO EVs, is followed by orthotopic implantation of tumor cells in the pancreas. Data: We hypothesized that tdEVs impact effector immune cells in PDAC, leading to phenotypic, transcriptional, and functional changes that result in a decreased anti-tumor immune response. The highest uptake of EVs was found in macrophages and dendritic cells (DCs). Furthermore, most immune cells preferably take up MUC1+ as compared to MUC1KO EVs. After three weeks of EV education, RNA sequencing of the liver showed differential gene expression in a variety of genes related to processes associated with B cells. Treatment with MUC1+ EVs downregulated B cell-related processes in the liver, B cell differentiation, proliferation and activity are reduced. Several genes coding for immunoglobulin parts were affected as well. Immune cell profiling of the blood and spleen showed that MUC1+ EVs reduce NK and macrophage numbers in the spleen as compared to MUC1KO EV treatment after three weeks. Additionally, EV-treatment increased CD8+ T cell numbers in the blood but decreased CD8+ T cells in the spleen. Conclusion: We have shown that tdEVs induce phenotypic and transcriptional changes in immune cell populations and alter immune cell frequencies in vivo. Ongoing research in our lab involves analysis of phenotypic and transcriptional changes after tumor implantation as well as functional differences in EV-treated immune cells in vitro and in vivo. Citation Format: Clara S. Mundry, Ying Huang, Paul M. Grandgenett, Michael A. Hollingsworth. The influence of extracellular vesicles on immune cells in PDAC [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 5358.

Abstract 3637: Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and is the second leading cause of cancer death in the United States. Carcinoembryonic antigen (CEA) is a biomarker commonly used in the management of CRC. It is typically present at low levels in the blood of healthy adults but is elevated in CRC samples and some other cancers. CEA is most commonly used to monitor treatment efficacy and as a critical part of post-treatment surveillance for residual disease, recurrence, and/or progression/metastasis. The CRC recurrence rate averages >25%, and liver metastasis occurs in 25-30% of cases. Improved CEA assays could enable earlier determination of treatment failure, detection of lower levels of minimal residual disease (MRD) after treatment, or earlier detection of recurrence. CEA is a GPI-linked membrane protein secreted from CRC cells mainly by enzymatic cleavage of its GPI anchor. However, we have identified alternative secretion of CEA in association with extracellular vesicles (EVs). We hypothesized that the soluble CEA produced by canonical shedding and the alternatively secreted CEA may provide distinct information about the tumor or its microenvironment and serve as independent but complementary biomarkers of CRC. We developed ultrasensitive, multi-marker electrochemiluminescence (ECL) immunoassays for measuring intact EVs presenting CEA, CD73, or both in human plasma. We observed that plasma levels of CEA+EVs and CD73+EVs were elevated in some late-stage CRC samples compared to controls. CEA and CD73 double-positive EV (CEA+CD73+EVs) levels were also elevated in the same samples, exhibiting a 3-fold improved separation of these samples with CRC from controls, compared to detection of CEA+EVs. Additionally, approximately 25% of stage IV CRC samples had elevated CEA+EV and CEA+CD73+EV levels while exhibiting low soluble CEA. We confirmed this result in an independent sample set that included samples across all CRC stages, and also identified a few earlier-stage samples that exhibited high CEA+EVs and CEA+CD73+EVs without elevated soluble CEA. Since CEA+CD73+EVs and soluble CEA assays identified different subsets of samples, we combined the two assays into a single classifier for late stage CRC and controls, yielding improved receiver operator characteristic (ROC) with area under the curve (AUC) of 0.975 versus 0.927 for the standard soluble CEA assay or 0.788 for CEA+CD73+EVs alone. These results suggest that assaying EVs presenting multiple surface markers can increase specificity for the detection of tumor-derived EVs relative to a single marker and that assaying both soluble and EV-associated forms of CEA in plasma may improve the value of CEA measurement in CRC. Citation Format: Lucie Hebert, Misk Al-Ameen, Talis Spriggs, Collin Nelson, Evan A. Gizzie, Jeffrey L. Franklin, Robert J. Coffey, David A. Routenberg, Jacob N. Wohlstadter. Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA [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 3637.

Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

Extracellular vesicle-mediated communication between CD8+ cytotoxic T cells and tumor cells.

Tumors pose a significant global public health challenge, resulting in numerous fatalities annually. CD8+ T cells play a crucial role in combating tumors; however, their effectiveness is compromised by the tumor itself and the tumor microenvironment (TME), resulting in reduced efficacy of immunotherapy. In this dynamic interplay, extracellular vesicles (EVs) have emerged as pivotal mediators, facilitating direct and indirect communication between tumors and CD8+ T cells. In this article, we provide an overview of how tumor-derived EVs directly regulate CD8+ T cell function by carrying bioactive molecules they carry internally and on their surface. Simultaneously, these EVs modulate the TME, indirectly influencing the efficiency of CD8+ T cell responses. Furthermore, EVs derived from CD8+ T cells exhibit a dual role: they promote tumor immune evasion while also enhancing antitumor activity. Finally, we briefly discuss current prevailing approaches that utilize functionalized EVs based on tumor-targeted therapy and tumor immunotherapy. These approaches aim to present novel perspectives for EV-based tumor treatment strategies, demonstrating potential for advancements in the field.

Tumor-derived extracellular vesicles as a biomarker for breast cancer diagnosis and metastasis monitoring

It is imperative to explore biomarkers that are both precise and readily accessible in the comprehensive management of breast cancer.A multicenter cohort, including 512 breast cancer patients and 198 nonneoplastic individuals, was recruited to detect the level of tumor-derived extracellular vesicles using our method based on dual DNA tetrahedral nanostructures. The level of tumor-derived extracellular vesicles was significantly higher in newly diagnosed breast cancer patients than in nonneoplastic individuals at a cutoff value of 3.58 U/μL. For postoperative metastasis monitoring, the level of tumor-derived extracellular vesicles was significantly higher in breast cancer patients with metastasis than in those without metastasis at a cutoff value of 3.91 U/μL. Its efficacy of diagnosis and metastasis monitoring was superior to traditional tumor markers. Elevated level of tumor-derived extracellular vesicles served as a predictive biomarker for diagnosis and metastasis monitoring in breast cancer patients.

Tumor-derived apoptotic extracellular vesicle-mediated intercellular communication promotes metastasis and stemness of lung adenocarcinoma

Apoptosis has long been recognized as a significant mechanism for inhibiting tumor formation, and a plethora of stimuli can induce apoptosis during the progression and treatment of tumors. Moreover, tumor-derived apoptotic extracellular vesicles (apoEVs) are inevitably phagocytosed by live tumor cells, promoting tumor heterogeneity. Understanding the mechanism by which apoEVs regulate tumor cells is imperative for enhancing our knowledge of tumor metastasis and recurrence. Herein, we conducted a series of in vivo and in vitro experiments, and we report that tumor-derived apoEVs promoted lung adenocarcinoma (LUAD) metastasis, self-renewal and chemoresistance. Mechanistically, we demonstrated that apoEVs facilitated tumor metastasis and stemness by initiating the epithelial-mesenchymal transition program and upregulating the transcription of the stem cell factor SOX2. In addition, we found that ALDH1A1, which was transported by apoEVs, activated the NF-κB signaling pathway by increasing aldehyde dehydrogenase enzyme activity in recipient tumor cells. Furthermore, targeting apoEVs-ALDH1A1 significantly abrogated these effects. Collectively, our findings elucidate a novel mechanism of apoEV-dependent intercellular communication between apoptotic tumor cells and live tumor cells that promotes the formation of cancer stem cell-like populations, and these findings reveal that apoEVs-ALDH1A1 may be a potential therapeutic target and biomarker for LUAD metastasis and recurrence.