Abstract Metastatic breast cancer (MBC) remains one of the leading causes of cancer-related mortality among women in the United States. Cancer stem cells (CSCs), which are characterized by immune evasion, self-renewal, and regenerative potential, are thought to be critical drivers of metastasis and recurrence.Here, we performed single-cell RNA sequencing on eight MBC samples from accessible secondary tumor sites with diverse hormone receptor and HER2 status, including three pleural effusions, one ascites, and five breast to bone metastases. To investigate the relationship of these metastatic cells to their microenvironment, we utilized the package CellChat which uses expression of receptor-ligand pairs to infer communication between populations.Across all samples, we identified a rare population of non-immune (CD45- CD24-) cells that consistently formed a distinct transcriptional cluster and expressed canonical stemness and cancer markers, including CD44, CD47, ALDH1A3, MET, HER3, THY1, and PROCR. Notably, they exhibited elevated expression of the RNA- and DNA-editing enzymes ADAR1 and APOBEC3C which are deaminases previously implicated in mutagenesis, splicing dysregulation, and cancer progression. This population also showed consistent downregulation of retrotransposable elements, a proposed mechanism of immune evasion in leukemia stem cells and consistent with upregulation of base deaminases, which are known repressors of retroelement activation. We uncovered a high degree of autocrine and paracrine signaling from this CSC population, particularly via the IL-6 pathway which is a known inducer of ADAR1 and APOBEC3C, and we performed downstream in vitro and in vivo analysis in patient-derived xenograft mice and nanobioreactor tumor organoid models to investigate the inflammatory and base deaminase activation through cytokine arrays and flow cytometry.Our findings highlight a conserved metastatic niche remodeling CSC-like population across MBC patients that may drive metastasis through base editing, inflammatory signaling, and retroelement repression, and targeting these pathways could offer new avenues for diagnostics and intervention. Citation Format: Claire Engstrom, Jessica Pham, Wenxue Ma, Emma Klacking, Kendale Wirtjes, Patrick Chang, Inge van der Werf, Catriona H. M. Jamieson. Inflammatory niche remodeling by metastatic breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6112.

Abstract Cancer stemness properties, including enhanced survival, malignant regeneration, telomere deregulation, genomic and epitranscriptomic instability, fuel metastases, and have been linked to stress, retrotransposon and inflammatory cytokine activation, which can occur in low earth orbit (LEO). In NASA Axiom 1, 2 and 3 missions to the ISS, confocal imaging, WGS, RNA-seq and scRNA-seq of lentiviral FUCCI2BL cell cycle and ADAR1-GFP reporter transduced erythroleukemia (TF-1a), colorectal (Caco-2) and metastatic breast cancer (MBC; MDA-MB-231 and patient samples) revealed proliferation, significant genomic instability, HERV and LINE-1 retrotransposon deregulation, and APOBEC3C and ADAR1 activation. Moreover, in Axiom 2 and 3 missions with ADAR1-reporter expressing MBC organoids and in humanized MBC mouse models, an ADAR1p150 splicing modulator, rebecsinib (IND 153126), prevented tumor propagation. Thus, cancer studies in LEO may accelerate the development of innovative cancer therapeutics and countermeasures for long-term spaceflight. Citation Format: Jessica Pham, Wenxue Ma, Claire Engstrom, Patrick Chang, Shuvro P. Nandi, Inge van der Werf, Emma Klacking, Teresa Sposito, Kendale Wirtjes, Thomas Frias, Antonio Ruiz, Jane Isquith, Luisa Ladel, Christina N. Wu, Jana Stoudemire, Pinar Mesci, Kay T. Yeung, Rebecca A. Shatsky, Anna A. Khachatrian, James J. La Clair, Michael D. Burkart, Peggy Wentworth, Curtis L. Scribner, Sheldon R. Morris, Thomas Whisenant, Karla Mack, Ludmil B. Alexandrov, Catriona H. Jamieson. Predicting and preventing cancer stemness in low Earth orbit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7631.

Human hematopoietic stem and progenitor cell (HSPC) fitness declines following exposure to stressors that reduce survival, dormancy, telomere maintenance, and self-renewal, thereby accelerating aging. While previous National Aeronautics and Space Administration (NASA) research revealed immune dysfunction in low-earth orbit (LEO), the impact of spaceflight on human HSPC aging had not been studied. To study HSPC aging, our NASA-supported Integrated Space Stem Cell Orbital Research (ISSCOR) team developed bone marrow niche nanobioreactors with lentiviral bicistronic fluorescent, ubiquitination-based cell-cycle indicator (FUCCI2BL) reporter for real-time HSPC tracking in artificial intelligence (AI)-driven CubeLabs. In monthlong International Space Station (ISS) missions (SpX-24, SpX-25, SpX-26, and SpX-27) compared with ground controls, FUCCI2BL reporter, whole-genome and transcriptome sequencing, and cytokine arrays demonstrated cell-cycle, inflammatory cytokine, mitochondrial gene, human repetitive element, and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) deregulation together with clonal hematopoietic mutations. Furthermore, HSPC functionally organized multi-omics aging (HSPC-FOMA) analyses revealed reduced telomere maintenance, adenosine deaminase acting on RNA1 (ADAR1) p150 self-renewal gene expression, and replating capacity indicative of space-associated HSPC aging that may limit long-duration spaceflight.

Recent advancements in transcriptomics and proteomics have opened the possibility for spatially resolved molecular characterization of tissue architecture with the promise of enabling a deeper understanding of tissue biology in either homeostasis or disease. The wealth of data generated by these technologies has recently driven the development of a wide range of computational methods. These methods have the requirement of advanced coding fluency to be applied and integrated across the full spatial omics analysis process, thus presenting a hurdle for widespread adoption by the biology research community. To address this, we introduce SPEX (Spatial Expression Explorer), a web-based analysis platform that employs modular analysis pipeline design, accessible through a user-friendly interface. SPEX's infrastructure allows for streamlined access to open-source image data management systems, analysis modules, and fully integrated data visualization solutions. Analysis modules include essential steps covering image processing, single-cell analysis, and spatial analysis. We demonstrate SPEX's ability to facilitate the discovery of biological insights in spatially resolved omics datasets from healthy tissue to tumor samples.

Previous reports revealed immune dysfunction, chromosomal abnormalities, cytokine deregulation, and telomere alterations after prolonged spaceflight. However, the stress of space on hematopoietic stem and progenitor cells (HSPCs) and the resilience properties maintaining lifelong hematopoiesis and immunity were not studied. We performed HSPC functionally organized multi-omics aging and resilience (HSPC-FOMA-R) analyses in 9 astronauts before, during, and after three short-duration International Space Station (ISS) missions. Whole-genome sequencing (with telomere length analysis and mitochondrial and clonal mutational profiling), whole-transcriptome sequencing (with RNA editing and retrotransposon analyses), single-cell RNA sequencing, cytokine arrays, and fluorescence-activated cell sorting (FACS) analyses assessed HSPC and immune subpopulation survival dynamics. We show that spaceflight is associated with partially reversible changes in HSPC survival and self-renewal, adenosine deaminase associated with RNA1 (ADAR1), telomere maintenance, mobilization, cell cycle, and “fight or flight” gene expression. Combined with clonal hematopoietic mutations, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3C) activation, and retrotransposon deregulation, HSPC-FOMA-R analyses are needed before extended missions.

Genetically modified, induced pluripotent stem cells (iPSCs) offer a promising allogeneic source for the generation of functionally enhanced, chimeric antigen receptor (CAR) T cells. However, the signaling of CARs during early T cell development and the removal of the endogenous T cell receptor required to prevent alloreactivity pose significant challenges to the production of mature conventional CAR T cells from iPSCs. Here, we show that TCR-null, CD8αβ CAR T cells can be efficiently generated from iPSCs by engineering stage-specific onset of CAR expression and signaling to both permit conventional T cell development and to induce efficient positive selection. CAR T cells produced using this approach displayed a uniform, naïve T cell phenotype and demonstrated superior antigen-specific cytotoxicity compared to iPSC-derived effector memory CAR T cells. Multimodal sequencing revealed CAR-mediated positive selection induced the persistent upregulation of key transcription factors involved in naïve T cell development. Achieving precise control of CAR expression and signaling in developmentally sensitive T precursors will be critical to realizing the full potential for "off-the-shelf", iPSC-derived cellular therapies.

Previous reports revealed immune dysfunction, chromosomal abnormalities, cytokine deregulation, and telomere length dynamics following prolonged spaceflight. However, the stress of space on hematopoietic stem and progenitor cells (HSPCs) that maintain lifelong hematopoiesis and immune responses was not studied. We performed HSPC functionally organized multi-omics aging (HSPC-FOMA) analyses in 7 astronauts before, during, and after short-duration ISS missions. Specifically, whole genome sequencing with telomere length, mitochondrial and clonal mutational profiling; whole transcriptome sequencing with RNA editing and retrotransposon analyses; single-cell RNA sequencing; cytokine arrays; and FACS-analyses were performed to assess HSPC and immune subpopulation survival dynamics. Overall, the observed space-associated stem cell hallmarks of aging, including spaceflight-dependent alterations in HSPC survival and self-renewal, adenosine deaminase associated with RNA1 (ADAR1), telomere maintenance, mobilization and cell cycle gene expression combined with space-associated clonal hematopoietic mutations, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3C) activation and retrotransposon deregulation warrant countermeasure development to enable long-duration spaceflight.

Abstract Purpose: The purpose of this study is to determine how T cell engagers (TCEs) impact the localization and phenotype of key T-cell subsets in preclinical solid tumor models. Background: TCEs are a class of immunotherapy that have demonstrated substantial benefit to patients with hematologic cancers and have shown evidence of clinical activity in solid tumor malignancies. Mechanisms of response to TCEs, particularly in solid tumors, are not well understood. Identification of diagnostic strategies to stratify patients that are more likely to respond is expected to enhance the clinical benefit of TCEs. Friedrich et al. (Cancer Cell 2023) found that effector CD8 T cell subsets significantly expanded in TCE responsive but not refractory patients in multiple myeloma using single cell RNA sequencing (scRNAseq). Furthermore, the increased abundance of exhausted T cells at baseline was associated with a worse prognosis. We hypothesize that analogous populations exist in preclinical solid tumor models, which have distinct localization and cellular interactions within the TCE treated tumor microenvironment. Methods: Either hCD3 transgenic C57BL/6J or wildtype FVB mice were implanted with either ID8-LyPD1 or Fo5-HER2 tumors. Mice were grouped at a tumor volume of ∼ 300mm^3 and treated with either 10mpk of LyPD1 TCE, 5mpk of HER2 TCE, or vehicle. scRNAseq data was produced at day 1 and 4 post-treatment according to the 10X gene expression kit protocol and was processed in R using Seurat. Flow cytometry data was generated using commercially available antibodies, acquired on a BD FACSymphony and analyzed using FlowJo. Xenium data was generated using a custom probe panel according to the 10X Xenium protocol and was processed in either Python or R using the Squidpy or Seurat packages. Results: In the ID8-LyPD1 TCE model, we identified proliferative effector (CD8.pe) and terminally differentiated (CD8.Tex) CD8 populations by scRNAseq. The abundance of CD8.pe increased 3-fold and CD8.Tex expanded 2-fold upon TCE treatment. We then validated the expansion of these CD8 subsets by flow cytometry. Notably, increases in the number of CD8.pe per gram of tumor significantly correlated with reductions in tumor volume. Due to the impact of TCE treatment on these CD8 subsets and the importance of CD8.pe in tumor outcomes, we profiled the localization and cellular interactions of CD8s in preclinical solid tumors. Cell-cell communication inference analysis suggested that CD8s were more frequently targets of cellular interactions in TCE treated tumors. Utilizing Xenium spatial transcriptomics we verified that CD8.pe and CD8.Tex were more abundant in TCE treated samples and that their interactions with other key cell subsets were enhanced by TCE treatment. Conclusions: In summary we found that key CD8 subsets were expanded by TCE treatment in preclinical solid tumor models, that the abundance of CD8.pe correlates with reduced tumor burden, and that TCE treatment promotes cellular interactions among CD8s and other key cell subsets in the tumor microenvironment. Citation Format: Billy Tomaszewski, Matthew Curtis, Conrad Foo, Gu Zhang, Patrick Chang, Robyn Clark, Thao Nguyen, Joshua Webster, Sandra Rost, Raj Jesudason, Robert Piskol, Klara Totpal, Lisa McGinnis, Kevin Walsh, Weilan Ye. T cell engager therapy affects the spatial distribution and phenotype of T cells in the tumor microenvironment [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR002.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

Atezolizumab (anti-PD-L1), combined with carboplatin and etoposide (CE), is now a standard of care for extensive-stage small-cell lung cancer (ES-SCLC). A clearer understanding of therapeutically relevant SCLC subsets could identify rational combination strategies and improve outcomes. We conduct transcriptomic analyses and non-negative matrix factorization on 271 pre-treatment patient tumor samples from IMpower133 and identify four subsets with general concordance to previously reported SCLC subtypes (SCLC-A, -N, -P, and -I). Deeper investigation into the immune heterogeneity uncovers two subsets with differing neuroendocrine (NE) versus non-neuroendocrine (non-NE) phenotypes, demonstrating immune cell infiltration hallmarks. The NE tumors with low tumor-associated macrophage (TAM) but high T-effector signals demonstrate longer overall survival with PD-L1 blockade and CE versus CE alone than non-NE tumors with high TAM and high T-effector signal. Our study offers a clinically relevant approach to discriminate SCLC patients likely benefitting most from immunotherapies and highlights the complex mechanisms underlying immunotherapy responses.

Introduction The International Space Station (ISS) is a uniquely accelerating environment to study micro- and macro-environmental stressors in the context of modeling human diseases by simulating inflammation, aging, immune dysfunction, and accumulation of mutations. Studies done in low earth orbit (LEO) may offer accelerated insights into human health, and specifically, human hematopoietic stem cell (HSC) and immune cell health and function. Stem cells are resilient to environmental stressors: they can self-renew without differentiating, can differentiate into tissue-specific progenitors, and maintain longevity while retaining dormancy. As a follow-on study to ex-vivo nanobioreactor cultures sent to the ISS in four iterative 30-45 day missions, we hope to study the effects of spaceflight on the astronauts conducting these experiments in low earth orbit (LEO). Methods Under a NASA IRB-approved protocol, we collected peripheral blood from seven astronauts, who flew in Axiom Mission 2 and Axiom Mission 3 (Ax-2, Ax-3). CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated to identify functional, genomic, and single-cell transcriptomic changes across ten different timepoints by clonogenic assay, 90X whole genome sequencing (WGS), and single-cell RNA sequencing (scRNA-seq) respectively. To date, timepoints include two pre-flight, one during mission, and 2 (Ax-3) to 3 (Ax-2) post-flight. Parallel studies were also conducted to profile the immune subsets by fluorescence-activated cell sorting and scRNA-seq analysis. Results To investigate the effects of spaceflight on HSC function, we performed clonogenic survival and self-renewal assays. The percent of multilineage colonies from Ax-2 and Ax-3 was significantly reduced inflight compared to pre- and post-mission. HSPCs had significantly increased self-renewal capacity upon return (R + 1 day) in Ax-2. The self-renewal capacity of HSPCs from Ax-3 were significantly decreased inflight when compared to other timepoints. Interestingly, Ax-3 HSPCs begin to lose their self-renewal capacity in anticipation of launch at launch - 2 days (L - 2 days). Upon return, HSPCs appear to regain their cloning capacity after 30-days (R + 30 days). To corroborate these findings, scRNA-seq analyses revealed that while spaceflight induces significant changes in self-renewal genes, results indicate that a fight-or-flight response may be occurring immediately pre-mission. This suggests that changes seen inflight may be compounded by changes leading up to flight. Analysis is ongoing to identify mutational burden and mutational signatures associated with each timepoint, as well as targeted FACS analysis to profile the immune subsets. Conclusion Preliminary results from Ax-2 and Ax-3 show dynamic changes in stem cell function associated with pre-, during, and post-flight. Some changes to HSCs are individual-specific, indicating variable responses to environmental stressors. Functional and scRNA-seq analyses also suggest that duration spent in LEO is a contributing factor to HSC recovery. This longitudinal study has the potential to provide valuable insight into human health on Earth and inform on the effects of longer duration missions and future missions beyond the ISS.

Introduction Maintenance of hematopoietic stem cell (HSC) fitness is predicated on tightly regulated cell cycle transit, self-renewal and multi-lineage differentiation potential in supportive niches but can be altered detrimentally by inflammatory microenvironments and difficult to quantify. Here, we report on the development of a NASA-funded 3D biosensing nanobioreactor system (Pham...Jamieson. BioRxiv 2024) that enables real-time confocal fluorescent microscopic tracking of HSPC cell cycle kinetics with our dual fluorescence lentiviral FUCCI2BL cell cycle indicator (Pineda...Jamieson. Scientific Reports 2016), quantification of ADAR1 self-renewal gene activity with a lentiviral ADAR1 GFP reporter and cell fate decisions in response to niche cues over a 4 to 6 week period. Methods Niche nanobioreactors were constructed from 2-port, fluoroethyl polymer (FEP) film bags that are gas permeable, chemically inert, and transparent for imaging. A porcine gelatin sponge (J&J MedTech) matrix was cut to size to function as a physical scaffold before resealing with a heat sealer under sterile conditions. Subsequently, these nanobioreactors were utilized in experiments involving aged bone marrow samples collected from routine knee and hip replacement surgeries under an IRB-approved protocol. Upon collection, mononuclear cells were isolated by Ficoll-Paque density centrifugation and subjected to CD34+ immunomagnetic bead selection. The CD34- bone marrow stromal cells were either seeded directly into the nanobioreactor following magnetic bead selection, or irradiated at 80 cGy prior to seeding into the nanobioreactor. The CD34+ cells were lentivirally transduced with the FUCCI2BL or our ADAR1-NanoLuc-GFP reporter (Crews...Jamieson. Cell Stem Cell 2023) and cultured for 48 hours or directly seeded into the same nanobioreactor as the autologous CD34- cell fraction in a 1:4 ratio. At baseline, cells were stained for immune cell lineage markers CD3, CD19, CD14, and CD56, as well as a panel of additional surface markers and evaluated by FACS analysis to assess the frequency of progenitor cell subsets. The nanobioreactor cultures were analyzed weekly for up to 6 weeks. Cultures were also analyzed at baseline and weekly by whole transcriptome RNA sequencing (RNA-seq) and single cell RNA sequencing (sc-RNA-seq). Results Both RNA-seq and weekly FACS analysis demonstrated that a population of ADAR1-expressing hematopoietic stem cells (HSCs, CD34+CD38-Lin-) can be consistently detected in the nanobioreactor for four to six weeks as can FUCCI-red dormant HSCs. Upon four weeks of culture, we also observed the maintenance of progenitor populations such as common myeloid progenitors (CMP), megakaryocyte-erythroid progenitors (MEP), granulocyte-macrophage progenitors (GMP), multipotent progenitors (MPP), and lymphoid-primed multipotent progenitors (LMPP). When compared to traditional 2D cell culture, the 3D nanobioreactor system performs significantly better in maintaining populations of stem and progenitor cells. Furthermore, unirradiated CD34- cell populations, including immune cells, are also maintained in the nanobioreactor. Over a four week period, a stable population of T cells is detected, while B cells persist at low frequencies. Additionally, myeloid cells are maintained and, in some cases, expanded in the nanobioreactor. Conclusions Our 3D biosensing nanobioreactor culture system enables the prolonged maintenance of primary human HSPCs and effectively supports the culture of immune cells. This system can serve as an in vitro model system to study both HSPC and niche dynamics. Further investigation into additional immune cell subsets and their functionality following culture in the nanobioreactor is currently underway.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient’s HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.