Chloé B. Steen

• Zhang, Wubing; Brown, Erin L.; Usmani, Abul; Earland, Noah; Kang, Minji & Olelewe, Chibuzor [Show all 29 contributors for this article] (2026). Non-invasive profiling of the tumour microenvironment with spatial ecotypes. Nature. ISSN 0028-0836. 654(8120), p. 1076–1087. doi: 10.1038/s41586-026-10452-4. Full text in Research Archive Show summary

  Multicellular programs in the tumour microenvironment (TME) drive cancer pathogenesis and response to therapy but remain challenging to identify and profile clinically1,2,3. Here, we present a machine-learning framework for multi-analyte profiling of spatially dependent cell states and multicellular ecosystems, termed spatial ecotypes (SEs). By integrating over 10 million single-cell and spot-level spatial transcriptomes from diverse human carcinomas and melanomas, we identified nine SEs with broad conservation, each of which has unique biology, geospatial features and clinical outcome associations, including several linked to immunotherapy response. Notably, SEs were distinguishable by DNA methylation profiling and were recoverable from plasma cell-free DNA (cfDNA) using deep learning. In cfDNA from nearly 100 patients with melanoma, SE levels exhibited striking associations with immunotherapy response. Our data reveal fundamental units of TME organization and demonstrate a multimodal platform for profiling solid and liquid TMEs, with implications for improved risk stratification and therapy personalization.

• Kang, Minji; Gulati, Gunsagar S.; Brown, Erin L.; Qi, Zhen; Avagyan, Susanna & Armenteros, Jose Juan Almagro [Show all 14 contributors for this article] (2025). Improved reconstruction of single-cell developmental potential with CytoTRACE 2. Nature Methods. ISSN 1548-7091. 22(11), p. 2258–2263. doi: 10.1038/s41592-025-02857-2. Full text in Research Archive Show summary

  While single-cell RNA sequencing has advanced our understanding of cell fate, identifying molecular hallmarks of potency—a cell’s ability to differentiate into other cell types—remains a challenge. Here we introduce CytoTRACE 2, an interpretable deep learning framework for predicting absolute developmental potential from single-cell RNA sequencing data. Across diverse platforms and tissues, CytoTRACE 2 outperformed previous methods in predicting developmental hierarchies, enabling detailed mapping of single-cell differentiation landscapes and expanding insights into cell potency.

• Bai, Baoyan; Wise, Jillian Frances; Vodak, Daniel; Nakken, Sigve; Sharma, Ankush & Blaker, Yngvild Nuvin [Show all 23 contributors for this article] (2024). Multi-omics profiling of longitudinal samples reveals early genomic changes in follicular lymphoma. Blood Cancer Journal. 14:147(1), p. 1–12. doi: 10.1038/s41408-024-01124-5. Full text in Research Archive Show summary

  Follicular lymphoma (FL) is the most common indolent type of B-cell non-Hodgkin lymphoma. Advances in treatment have improved overall survival, but early relapse or transformation to aggressive disease is associated with inferior outcome. To identify early genetic events and track tumor clonal evolution, we performed multi-omics analysis of 94 longitudinal biopsies from 44 FL patients; 22 with transformation (tFL) and 22 with relapse without transformation (nFL). Deep whole-exome sequencing confirmed recurrent mutations in genes encoding epigenetic regulators (CREBBP, KMT2D, EZH2, EP300), with similar mutational landscape in nFL and tFL patients. Calculation of genomic distances between longitudinal samples revealed complex evolutionary patterns in both subgroups. CREBBP and KMT2D mutations were identified as genetic events that occur early in the disease course, and cases with CREBBP KAT domain mutations had low risk of transformation. Gains in chromosomes 12 and 18 (TCF4), and loss in 6q were identified as early and stable copy number alterations. Identification of such early and stable genetic events may provide opportunities for early disease detection and disease monitoring. Integrative analysis revealed that tumors with EZH2 mutations exhibited reduced gene expression of numerous histone genes, including histone linker genes. This might contribute to the epigenetic dysregulation in FL.

• Vahid, Milad R.; Brown, Erin L.; Steen, Chloe Beate; Zhang, Wubing; Jeon, Hyun Soo & Kang, Minji [Show all 8 contributors for this article] (2023). High-resolution alignment of single-cell and spatial transcriptomes with CytoSPACE. Nature Biotechnology. ISSN 1087-0156. 41, p. 1543–1548. doi: 10.1038/s41587-023-01697-9. Full text in Research Archive
• Spasevska, Ivana; Sharma, Ankush; Steen, Chloe Beate; Josefsson, Sarah Elisabet; Blaker, Yngvild Nuvin & Kolstad, Arne [Show all 26 contributors for this article] (2023). Diversity of intratumoral regulatory T cells in B-cell non-Hodgkin lymphoma. Blood Advances. ISSN 2473-9529. 7(23), p. 7216–7230. doi: 10.1182/bloodadvances.2023010158. Full text in Research Archive Show summary

  Abstract Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3− Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.

• Wise, Jillian; Nakken, Sigve; Steen, Chloe Beate; Vodak, Daniel; Trøen, Gunhild & Johannessen, Bjarne [Show all 24 contributors for this article] (2020). Mutational dynamics and immune evasion in diffuse large B-cell lymphoma explored in a relapse-enriched patient series. Blood Advances. ISSN 2473-9529. 4(9), p. 1859–1866. doi: 10.1182/bloodadvances.2019001325. Full text in Research Archive Show summary

  • Diagnostic and relapse diffuse large B-cell lymphoma (DLBCL) biopsies reveal increased mutational burden/loss of heterozygosity in HLA-A. • Serially sampled tumor biopsies provide insight into therapeutic targets and evolutionary divergence in relapsed/refractory DLBCL.

• Steen, Chloe Beate; Leich, Ellen; Myklebust, June Helen; Lockmer, Sandra; Wise, Jillian & Wahlin, Björn Engelbrekt [Show all 14 contributors for this article] (2019). A clinico-molecular predictor identifies follicular lymphoma patients at risk of early transformation after first-line immunotherapy. Haematologica. ISSN 0390-6078. 104(10), p. 460–464. doi: 10.3324/haematol.2018.209080. Full text in Research Archive
• Newman, Aaron M; Steen, Chloe Beate; Liu, Chih Long; Gentles, Andrew J.; Chaudhuri, Aadel A & Scherer, Florian [Show all 12 contributors for this article] (2019). Determining cell type abundance and expression from bulk tissues with digital cytometry. Nature Biotechnology. ISSN 1087-0156. 37, p. 773–782. doi: 10.1038/s41587-019-0114-2. Full text in Research Archive
• Våtsveen, Thea Kristin; Myhre, Marit Renee; Steen, Chloe Beate; Wälchli, Sébastien; Lingjærde, Ole Christian & Bai, Baoyan [Show all 14 contributors for this article] (2018). Artesunate shows potent anti-tumor activity in B-cell lymphoma. Journal of Hematology & Oncology. 11:23, p. 1–12. doi: 10.1186/s13045-018-0561-0. Full text in Research Archive Show summary

  Background Although chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma. Methods We included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action. Results Drug screening identified artesunate as a highly potent anti-lymphoma drug.Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis. Conclusions Artesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for treatment of B-cell lymphoma.

• Lockmer, Sandra; Østenstad, Bjørn; Hagberg, Hans; Holte, Harald; Johansson, Ann-Sofie & Wahlin, Björn Engelbrekt [Show all 13 contributors for this article] (2018). Chemotherapy-free initial treatment of advanced indolent lymphoma has durable effect with low toxicity: Results from two Nordic lymphoma group trials with more than 10 years of follow-up. Journal of Clinical Oncology. ISSN 0732-183X. 36(33), p. 3315–3323. doi: 10.1200/JCO.18.00262. Full text in Research Archive
• Josefsson, Sarah Elisabet Göthberg; Huse, Kanutte; Kolstad, Arne; Beiske, Klaus; Pende, Daniela & Steen, Chloe Beate [Show all 13 contributors for this article] (2017). T cells expressing checkpoint receptor TIGIT are enriched in follicular lymphoma tumors and characterized by reversible suppression of T-cell receptor signaling. Clinical Cancer Research. ISSN 1078-0432. 24(4), p. 870–881. doi: 10.1158/1078-0432.CCR-17-2337. Full text in Research Archive Show summary

  Purpose: T cells infiltrating follicular lymphoma (FL) tumors are considered dysfunctional, yet the optimal target for immune checkpoint blockade is unknown. Characterizing coinhibitory receptor expression patterns and signaling responses in FL T-cell subsets might reveal new therapeutic targets. Experimental Design: Surface expression of 9 coinhibitory receptors governing T-cell function was characterized in T-cell subsets from FL lymph node tumors and from healthy donor tonsils and peripheral blood samples, using high-dimensional flow cytometry. The results were integrated with T-cell receptor (TCR)-induced signaling and cytokine production. Expression of T-cell immunoglobulin and ITIM domain (TIGIT) ligands was detected by immunohistochemistry. Results: TIGIT was a frequently expressed coinhibitory receptor in FL, expressed by the majority of CD8 T effector memory cells, which commonly coexpressed exhaustion markers such as PD-1 and CD244. CD8 FL T cells demonstrated highly reduced TCR-induced phosphorylation (p) of ERK and reduced production of IFNγ, while TCR proximal signaling (p-CD3ζ, p-SLP76) was not affected. The TIGIT ligands CD112 and CD155 were expressed by follicular dendritic cells in the tumor microenvironment. Dysfunctional TCR signaling correlated with TIGIT expression in FL CD8 T cells and could be fully restored upon in vitro culture. The costimulatory receptor CD226 was downregulated in TIGIT+ compared with TIGIT− CD8 FL T cells, further skewing the balance toward immunosuppression. Conclusions: TIGIT blockade is a relevant strategy for improved immunotherapy in FL. A deeper understanding of the interplay between coinhibitory receptors and key T-cell signaling events can further assist in engineering immunotherapeutic regimens to improve clinical outcomes of cancer patients.

• Knudsen, Markus Dines; Lange, Thomas de; Botteri, Edoardo; Nguyen, Dung-Hong; Evensen, Helge & Steen, Chloe Beate [Show all 10 contributors for this article] (2016). Favorable lifestyle before diagnosis associated with lower risk of screen-detected advanced colorectal neoplasia. World Journal of Gastroenterology (WJG). ISSN 1007-9327. 22(27), p. 6276–6286. doi: 10.3748/wjg.v22.i27.6276. Full text in Research Archive
• Eikenes, Åsmund Husabø; Malerød, Lene; Christensen, Anette Lie; Steen, Chloe Beate; Mathieu, Juliette & Nezis, Ioannis P [Show all 10 contributors for this article] (2015). ALIX and ESCRT-III Coordinately Control Cytokinetic Abscission during Germline Stem Cell Division In Vivo. PLoS Genetics. ISSN 1553-7390. 11(1). doi: 10.1371/journal.pgen.1004904. Full text in Research Archive
• Randen, Ulla; Trøen, Gunhild; Tierens, Anne; Steen, Chloe Beate; Warsam, Abdirashid Ali & Beiske, Klaus [Show all 9 contributors for this article] (2014). Primary cold agglutinin associated lymphoproliferative disease: a B-cell lymphoma of the bone marrow distinct from lymphoplasmacytic lymphoma. Haematologica. ISSN 0390-6078. 99(3), p. 497–504. doi: 10.3324/haematol.2013.091702. Full text in Research Archive

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• Zhang, Wubing; Brown, Erin L.; Usmani, Abul; Earland, Noah; Jeon, Hyun Soo & Alahi, Irfan [Show all 22 contributors for this article] (2025). Abstract 153: High-resolution profiling of the tumor microenvironment with spatial ecotypes. Cancer Research. ISSN 0008-5472. 85(8_Supplement_1), p. 153–153. doi: 10.1158/1538-7445.am2025-153. Full text in Research Archive Show summary

  Background: Multicellular programs in the tumor microenvironment (TME) drive cancer pathogenesis and response to therapy but remain challenging to identify and profile clinically. Here, we present Spatial EcoTyper, a multimodal machine learning framework for large-scale profiling of spatially dependent cell states and multicellular ecosystems, termed spatial ecotypes (SEs). Methods and Findings: By integrating five million single-cell and spot-level spatial transcriptomes from 10 diverse human neoplasms, including carcinomas and melanomas, we identified a rich tapestry of conserved regional plasticity across nine TME cell types. We then applied Spatial EcoTyper to deeply dissect these data, revealing nine novel SEs with broad conservation - each with unique biology, TME cell states, geospatial features, and clinical outcome associations. SEs were linked to specific biogeographic domains, including the tumor core, margin, and adjacent stroma, and were remarkably well-validated in 235 held-out tumor samples profiled by either single-cell RNA sequencing, sequencing-based spatial transcriptomics (10x Visium), or high-resolution imaging-based spatial transcriptomics (Vizgen MERSCOPE and NanoString CosMx). By deconvolving SEs from over 1,200 bulk tumor RNA-seq profiles, we discovered striking associations between SE levels and response to immune checkpoint inhibitors (ICIs), outperforming nearly 40 potential correlates of ICI response, including previously described signatures of tertiary lymphoid structures and tumor tissue PD-L1 expression. Notably, baseline levels of a proinflammatory SE - localized to the tumor core and defined by a hub of unique T cell, macrophage, fibroblast, and endothelial cell states - strongly forecasted ICI benefit, while another SE - localized to the tumor margin and defined by myofibroblast and hypoxia-associated endothelial cell states - portended ICI resistance. Conclusion: Our data reveal fundamental units of TME organization and demonstrate a platform for large-scale profiling of spatial cellular ecosystems in any tissue, with implications for improved forecasting of immunotherapy response and the development of novel therapeutic strategies. Citation Format: Wubing Zhang,Erin L. Brown,Abul Usmani,Noah Earland,Hyun Soo Jeon,Irfan Alahi,Nicholas P. Semenkovich,Janella C. Schwab,Chloé B. Steen,Chloe M. Sachs,Bilge Gungoren,Faridi Qaium,Peter K. Harris,Jacqueline Mudd,Qingyuan Cai,Antonella Bacchiocchi,Ryan C. Fields,Mario Sznol,Ruth Halaban,David Y. Chen,Aadel A. Chaudhuri,Aaron M. Newman. High-resolution profiling of the tumor microenvironment with spatial ecotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 153.

• Gleyzer, Rachel; Khameneh, Farnaz; Qi, Zhen; Zhang, Wubing; Steen, Chloe Beate & Kang, Minji [Show all 14 contributors for this article] (2025). Abstract 113: Decoding developmental programs driving tumorigenesis and immunotherapy resistance in triple-negative breast cancer. doi: 10.1158/1538-7445.am2025-113. Full text in Research Archive Show summary

  Abstract Background: Triple-negative breast cancer (TNBC), defined by a lack of hormone receptors and lack of HER2 amplification, is the deadliest subtype of breast cancer. TNBC kills 1 in 3 patients and while promising new drugs based on PARP inhibition and anti-PD1 immunotherapy can extend survival in selected patients, 30-40% of patients relapse or fail therapy. Increasing evidence suggests that TNBC tumors harbor stem-like tumorigenic cancer cells with high plasticity, capable of replenishing cancer cell populations and linked to adverse outcomes. Unfortunately, the phenotypic diversity, microenvironmental contexture, and functional significance of these cells remain poorly understood and available marker genes are not specific, precluding rational drug development. Methods: We created a multimodal single-cell RNA-seq atlas of 80 TNBC patients, including clinical covariates such as BRCA1/2 mutations, race/ethnicity, and prior treatment status. We then defined differentiation-associated malignant cell states using a novel interpretable deep learning approach for determining single-cell potency. We subsequently interrogated geospatial features, including localized microenvironments of less differentiated malignant cells, and linked them to immune checkpoint inhibitor (ICI) response. Results: Our quantitative analysis shows that TNBC is enriched in cancer cells with high developmental potency as compared to other breast cancer subtypes, consistent with its aggressive phenotype. Critically, we observed a strong association between enrichment of high potency cells and resistance to ICI treatment across human and mouse bulk expression data, both within TNBC and across other cancer types, a finding that we experimentally validated in a murine model of TNBC. In scRNA-seq and spatial transcriptomics data, we defined striking relationships between less differentiated cancer cells and context-dependent T cell states indicative of strong immunosuppressive capability. In line with this, we found that knockout of genes associated with immature cancer cells promotes TNBC cell killing by T cells in a co-culture CRISPR screen. Ongoing experiments include perturbations of novel therapeutic targets emerging from this work, with the goal of improving immunotherapy responses and ultimately extending patient survival. Conclusion: We anticipate that our approach will expand our understanding of developmental landscapes in TNBC and lead to new opportunities for developing targeted drugs in this devastating disease. Citation Format: Rachel Gleyzer,Farnaz Khameneh,Zhen Qi,Wubing Zhang,Chloé B. Steen,Minji Kang,Maya Maalouf,Melanis Ghadimi,Makenna Lindsey,Sally Bobo,Thomas J. Lomis,Frederick M. Dirbas,Michael F. Clarke,Aaron M. Newman. Decoding developmental programs driving tumorigenesis and immunotherapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 113.

• Sharma, Ankush; Kongsrud, Karoline; Spasevska, Ivana; Lamont, Colin Harry; Steen, Chloe Beate & Lorenz, Susanne [Show all 10 contributors for this article] (2025). Deciphering Tumor Microenvironment Complexity in Lymphoma Using Spatial Transcriptomics. Full text in Research Archive Show summary

  The tumor microenvironment (TME) in lymphomas, such as Diffuse Large B-cell Lymphoma (DLBCL) and Follicular Lymphoma (FL), is highly heterogeneous and plays a critical role in modulating therapeutic outcomes, including responses to CD19 CAR T-cell therapy. To unravel the spatial complexity of the TME, we employed an integrative approach combining spatial transcriptomics (Visium, 10x Genomics) and spatial proteomics (imaging mass cytometry, IMC) on FFPE tissue sections from FL and DLBCL patients. Using tools such as CytoSPACE and CARD, we mapped single-cell RNA-seq data to spatial coordinates and validated cellular compositions with IMC data. We identified spatially distinct immune and stromal cell populations, including GC B cells, Tregs, macrophages, dendritic cells, and endothelial cells, across follicular and T-cell rich regions. CytoSPACE-based mapping revealed consistent enrichment of key markers (e.g., CD79B, FOXP3, GRZA) with corresponding protein expression profiles. We also developed R based ShinySpatial framework to visualize spatial data enhancing the accessibility and interpretability of complex spatial datasets. This pilot study lays the groundwork for identifying spatial biomarkers that predict therapeutic responses and resistance in lymphoma, with ongoing integration of data from patients enrolled in the JULIET, BELINDA, and ELARA trials.

• Steen, Chloe Beate (2024). Pan-cancer Cartography of Human Tumors with CytoSPACE. Full text in Research Archive
• Knudsen, Markus Dines; Botteri, Edoardo; Steen, Chloe Beate; Hoff, Geir; Lange, Thomas de & Bernklev, Tomm [Show all 8 contributors for this article] (2015). Baseline lifestyle characteristics in participants in bowel cancer screening in Norway and in controls. Full text in Research Archive
• Randel, Kristin Ranheim; Schult, Anna Lisa; Botteri, Edoardo; Steen, Chloe Beate; Børmer, Ole P. & Hoff, Geir [Show all 7 contributors for this article] (2015). Bowel Cancer Screening in Norway (BCSN)-A Randomized Pilot Study Comparing Flexible Sigmoidoscopy (FS) to Fecal Immunochemical Test (FIT). Full text in Research Archive
• Lange, Thomas de; Jørgensen, Anita; Børmer, Ole P.; Sandvei, Per; Steen, Chloe Beate & Ursin, Giske [Show all 7 contributors for this article] (2014). COLORECTAL CANCER SCREENING PILOT IN NORWAY - COMPARATIVE EFFECTIVENESS RESEARCH OF FLEXIBLE SIGMOIDOSCOPY (FS) AND FECAL IMMUNOCHEMICAL TEST(FIT). Full text in Research Archive

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