In this issue of Cancer Cell, Liu et al. use mitochondrial DNA mutations to reconstruct clonal lineages of the innate immune compartment in human tumors. They show that intratumoral type 3 dendritic cells (DC3s) arise from circulating monocytes and that a monocyte's fate in the tumor microenvironment is programmed peripherally, prior to tissue entry.

Abstract Introduction Tumor neoantigens have emerged as promising candidates for personalized immunotherapies, however, confirming their presentation and immunogenicity remains challenging due to low neoantigen expression and limited detection sensitivity. Moreover, the spatial organization of neoantigen-specific T cell clones relative to their target tumor cells and other cell types within the tumor microenvironment remains incompletely defined due to technological limitations. To bridge this gap, we developed Slide-GoTags. Methods Slide-GoTags integrates single-nucleus spatial transcriptomics with targeted genotyping and TCR sequencing from a single 20 µm tissue section. Slide-tags spatial barcoding and the 10x Genomics 5’ droplet-based platform are followed by targeted RNase H—dependent PCR and long-read sequencing, enabling genotypes and TCR repertoires to be linked to gene expression profiles and spatial coordinates via shared cell barcodes. Results Slide-GoTags was applied to murine and human tumors, revealing colocalization of clonally expanded, neoantigen-specific T cells with tumor cells expressing their cognate neoantigen, validated by known neoantigen-TCR pairs. T cell functional state and clonotype avidity were linked to neoantigen proximity. Moreover, we identified IFN-γ—rich immunogenic niches consistently enriched for neoantigen-specific progenitor-exhausted T cells, marked by enhanced local T cell activation, clonal expansion, and immune-modulatory interactions with antigen-presenting cells within the TME. Spatially resolved receptor—ligand analysis revealed co-stimulatory and inhibitory interactions that shape immune engagement and regulation within these niches. Conclusion In this study, we define the spatial architecture of neoantigen directed T cell immunity shaped by proximity to neoantigen expressing tumor cells, TCR avidity, and receptor-ligand interactions within tumors, providing a framework for spatially informed TCR deorphanization and the development of personalized T cell therapy. Funding Source This work was supported in part by the NIH/NCI (CA276865), the Mark Foundation for Cancer Research Endeavor Award, the Dana-Farber/Harvard Cancer Center Kidney SPORE (P50CA101942), and the Dana-Farber/Harvard Cancer Center Support Grant (P30CA006516). A.N Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Immune aging impairs T cell-mediated tumor control as well as cancer immunotherapy outcomes. The most important drivers of T cell dysfunction in aged tumors remain unknown. We performed single-cell CRISPR screens to identify Dusp5 and Zfp219 as key regulators of CD8+ T cell persistence and effector differentiation within aged tumors. Loss of Dusp5 increased extracellular signal-regulated kinase (ERK) phosphorylation and globally enhanced T cell proliferation. Conversely, Zfp219 deletion induced epigenetic reprogramming and increased expression of cytotoxic molecules, enhancing antitumor immunity specifically in aging. Levels of the human ortholog ZNF219 were higher within intratumoral CD8+ T cells from older cancer patients, which correlates with worse survival following immunotherapy. Zfp219 ablation synergized with immune checkpoint inhibitors to expand effector-like CD8+ T cells, leading to tumor clearance in aged mice. Our findings highlight Dusp5 and Zfp219 as critical drivers of age-related T cell dysfunction that can be targeted to rejuvenate antitumor immunity in older cancer patients.

Abstract Introduction The alpha-variant wave of the COVID-19 pandemic provided a unique opportunity to study, at single-cell resolution, how near-universal exposure to the same pathogen can lead to either effective or dysfunctional immune responses in humans. Methods We analyzed 2.5 million circulating immune cells from 428 patients across time points (840 PBMC samples), encompassing three contemporaneous SARS-CoV-2 cohorts: acutely infected patients at five WHO disease severity levels and three time points, patients from the first randomized control trial to study efficacy of tocilizumab in management of COVID-19, and convalescent patients three months after infection. We used linear modeling to integrate multiple data types – single-cell RNA-seq, CITE-seq, TCR and BCR sequencing, viral load measurements, viral neutralization assays, detection of 75 autoantibodies, HLA genotype data, and serum proteomics covering 1,463 targets – to derive the most comprehensive view to-date of the biological features of COVID-19 disease severity. Results We show that myeloid-derived suppressor cells (MDSCs) act as a key immunologic pivot point in severe COVID-19. Myeloid dysfunction is marked by impaired antigen presentation and drives a non-productive adaptive immune response. Severe disease is also linked to autoantibodies targeting type I interferons, specific HLA-DQB1 allelic variants, and serum IL-6 levels. Tocilizumab treatment eliminates CLU-expressing MDSCs and ISG-positive myeloid subsets, restores antigen presentation, and reactivates productive adaptive immunity. In convalescence 3-months post-infection, we found persistently high ICOS expression in regulatory T cells. Conclusion Overall, we define distinct innate and adaptive host immune responses associated with acute, IL-6—responsive, and convalescent SARS-CoV-2 infection. Our multimodal and high-dimensional dataset with curated clinical metadata provides a foundational and clinically relevant resource for modeling host immune response biology in health and disease. Funding Source We acknowledge the following funding sources: this work was supported by several training grants, including a NIAID grant T32AR007258 (to KS), three NHLBI grants 5T32HL116275-13 (to CC), 5T32HL129970-09 (to APN), and the K08HL157725 (to PS), as well as the American Heart Association Career Development Award (to PS). PS was also supported by the Brigham and Women’s Hospital Innovation Evergreen Fund. EY was supported by funding from the Stanford Medical Scholars program. RJX acknowledges supports from NIH DK43351 and U19AI142784. RJX and AR were supported by the Manton Foundation and the Klarman Cell Observatory. PJU was supported by Third Rock Ventures; Henry Gustav Floren Trust; Stanford Department of Medicine Team Science Program; Stanford Medicine Office of the Dean; and National Institutes of Health R01 grants AI175771 and AI182319-02. RPB acknowledges funding support from the Massachusetts General Hospital Executive Committee on Research, the American Lung Association, and the Broad Institute’s Next Generation Scholar award. MBG, MRF, and NH were supported by an American Lung Association COVID-19 Action Initiative grant. MBG and MRF were supported by a grant from the Executive Committee on Research at MGH. NH acknowledges was supported by NIH/NIAID U19 AI082630, a Chair and gift from Sandra, Sarah, and Arthur Irving. ACV acknowledges funding support from the COVID-19 Clinical Trials Pilot grant from the Executive Committee on Research at MGH; a COVID-19 Chan Zuckerberg Initiative grant (2020-216954); the funds from the Manton Foundation and the Klarman Family Foundation; the Broad Institute’s Next Generation Scholar award; the MGH Howard M. Goodman Fellowship; the National Institutes of Health (DP2CA247831); work at the Broad Institute was supported by a gift from an anonymous donor. Topic Categories Computational and Systems Immunology (COMP)

Targeting cells for ablation, gene therapy or drug delivery is an ongoing challenge in development of therapeutics. With the goal of efficiently labeling cell surfaces based on target recognition, we linked the well-established engineered ultraID biotin ligase to cell-surface affinity reagents to enable catalytic installation of tags on the surface of cells. Biotin ligase conjugated to nanobodies against cell surface proteins exhibited an order of magnitude increase in cell labeling compared to nanobody alone. Biotin labeling of cells remained efficient at low nanomolar concentrations of the conjugate, and was stable for at least 4 h. By testing a swath of antibody-biotin ligase conjugates against various targets and cell types, we confirmed the feasibility and generalizability of this approach. Administration of biotin ligase-conjugated antibodies into animals efficiently biotinylated the expected target cells. Biotinylated target cells were subsequently detectable with fluorescent streptavidin injected into the same mice. Enzyme mediated cell targeting thus provides an efficient and modular platform for biotinylation and detection of cells expressing specific surface targets with potential applications for research and therapeutics.

Lupus nephritis (LN), a severe manifestation of systemic lupus erythematosus (SLE), is a heterogeneous disease driven by diverse immune and tissue cell types. We obtained 538,194 single-cell and 142,881 single-nuclear profiles from kidney biopsies of 155 patients with LN and 30 preimplantation transplant biopsy controls, along with 327,326 single-cell blood profiles. We characterized key stromal and immune cell types and cell states; moreover, we distinguished cell states that were tissue specific from those that were also present in the blood. We observed that LN pathological features were associated with particular cell states. For example, after controlling for the effects of chronic tissue damage, we observed that expansion of glomerular and scar-associated macrophage populations correlated with increasing inflammatory disease activity. Scar-associated macrophages appear to drive LN fibrosis and, in active disease, infiltrate the glomeruli more than other myeloid cells. These observations support that therapeutic targeting of myeloid populations may offer a strategy to prevent renal inflammation and ongoing kidney damage in LN.

UV radiation (UVR) drives high mutational burdens, yet precursor melanocytes accumulate these mutations without triggering immune clearance. Here, we investigated whether melanocyte-intrinsic transcriptional program(s) underlie immune tolerance to mutations resulting from UVR exposure. In primary human melanocytes, expression of PD-L1 (CD274) was dependent on microphthalmia-associated transcription factor (MITF), a crucial regulator of melanocyte development and an intermediate in the UV-tanning pathway. MITF directly activated PD-L1 transcription by binding a conserved upstream enhancer containing functional E-box elements. MITF determined both baseline melanocytic PD-L1 expression in healthy skin and its induction following UVR, independent of interferon signaling. Melanocyte-restricted Pd-l1 deletion in mice triggered CD8+ T cell infiltration and depigmentation after long-term UVB exposure, recapitulating features of human vitiligo. PD-L1-deficient human induced pluripotent stem cell (iPSC)-derived melanocytes underwent increased apoptosis and were more susceptible than PD-L1-intact melanocytes to gp100-specific CD8+ T cell killing. Thus, a melanocyte-intrinsic MITF-PD-L1 tolerance program protects melanocytes from autoimmune destruction, potentially facilitating early immune evasion during melanoma development and conversely underlying the responsiveness of melanoma to PD-1/PD-L1 blockade.

Immune aging is being increasingly recognized as a critical barrier to effective cancer immunotherapy, as the aged tumor microenvironment (TME) drives T cell dysfunction and impairs immune control of cancer. However, the key molecular drivers of this process as well as potential targets to rescue T cell dysfunction in aged tumors remain incompletely understood. Therefore, we performed in vivo single-cell CRISPR screens in CD8 + T cells within aged tumors and tumor-draining lymph nodes (tdLNs). We identified Dusp5 and Zfp219 as key regulators of T cell persistence and effector differentiation in aged hosts. Loss of Dusp5 , a negative regulator of ERK signaling, increased ERK1/2 phosphorylation and enhanced T cell proliferation in both young and aged tumors. In contrast, loss of Zfp219 , a transcriptional repressor, induced epigenetic reprogramming of cytotoxic gene programs, thereby increasing granzyme secretion and enhancing antitumor immunity. Moreover, expression of the human ortholog gene ZNF219 is increased within intratumoral CD8 + T cells in older cancer patients. High ZNF219 expression correlates with poorer survival following immune checkpoint blockade (ICB) and reduces persistence of human intratumoral T cells. Notably, Zfp219 ablation synergized with anti-PD-1 blockade in mice to expand effector-like CD8 + T cells, leading to significantly enhanced anti-tumor immunity and tumor clearance in aged hosts. Together, these findings highlight Dusp5 and Zfp219 as critical drivers of age-related T cell dysfunction and as potential therapeutic targets to rejuvenate T cell antitumor immunity in older cancer patients.

Abstract Introduction Human leukocyte antigen (HLA) class I presents intracellular peptides to the immune system on the cell surface. Since this process is crucial for the recognition of cancer cells and the initiation of anti-tumor immunity, peptides presented by HLA are valuable immunotherapy targets. More stable peptide HLA (pHLA) complexes provoke superior immune responses. However, how peptide sequence motifs contribute to pHLA stability is not well understood. Methods We developed a high-throughput assay to quantify stability of thousands of user-defined pHLA produced in E. coli. Peptide libraries and the desired HLA are produced and form pHLA complexes in E. coli. pHLA are purified and stability is evaluated by treating pHLA with a thermal gradient and recovering only the peptides which remain HLA-bound after heat treatment. Peptide depletion over the temperature range is monitored by quantitative tandem mass tag (TMT) enabled mass spectrometry. Results Our new E. coli-based method is reliable for assessing pHLA stability. Detected HLA-binding peptides have the expected binding motifs, and stability data strongly correlates with current gold-standard data. We are able to generate large peptide stability datasets (1,800+ peptides) in one scaled experiment — five times larger than currently available datasets. We show that peptide motifs and anchor residue combinations potentially drive pHLA stability. Additionally, peptides were included in user-defined libraries with public immunogenicity annotations. We observed that immunogenic peptides were significantly more stable than non-immunogenic peptides. Conclusion We generated customizable pHLA stability datasets which show how peptide sequence motifs affect pHLA stability, and may be helpful for improving our mechanistic understanding of pHLA stability. Further, since peptide stability is related to immunogenicity, these large-scale pHLA stability datasets will be useful for improving peptide immunogenicity predictions for the development of immunotherapeutics. Funding Source NIH R01CA155010, Mark Foundation for Cancer Research, Moderna Topic Categories Classical and Non-Classical Antigen Presenting Cells (APC)

Melanoma is a common and aggressive cancer, with rising incidence in most developed countries. Major discoveries in melanoma biology have been rapidly translated, allowing cures for patients in late-stage disease. Despite these advances, many tumors remain refractory, in part due to an incomplete understanding of the genes and pathways gained or lost during melanoma tumorigenesis. To address this gap and provide a broadly useful resource for the scientific community, we established melPDomiX, a multi-omics cohort of melanoma-patient-derived xenografts. By linking mutations with transcriptomic and proteomic features, melPDomiX enables systematic characterization of gain- and loss-of-function alterations in treatment-refractory melanoma. Using multi-omics integration and structural-context representation, we demonstrate how this resource distinguishes gain- from loss-of-function variants and uncovers new candidate melanoma drivers and therapeutic targets. Together, melPDomiX provides a comprehensive, deeply profiled set of tumor models that supports mechanistic discovery and facilitates the development of improved treatments for this devastating heterogeneous malignancy.

Objectives Lupus nephritis (LN) is a common, potentially fatal manifestation of systemic lupus erythematosus. We aimed to gain new insights into the immune responses underlying LN and their relation to the histologic heterogeneity observed in this disease, focusing on myeloid cells. Methods We used single-cell RNA-sequencing (scRNA-seq) data of dissociated kidney samples from 156 patients with LN and 30 healthy individuals. We applied spatial transcriptomics (ST), utilising a gene panel designed to capture all myeloid subsets identified in the scRNA-seq data, to profile kidney samples acquired from 6 patients with LN and 2 controls. Results We generated a catalogue of the myeloid subsets found in LN kidneys. Our analyses indicated that an increase in irreversible tissue damage, as measured by the National Institutes of Health chronicity index (CI), is associated with a gradual switch of the local immune response from one dominated by monocytes and macrophages to one featuring expanded CD4 T, GZMK+ CD8 T, B, and dendritic cells, with a parallel decrease in the interferon response. In proliferative/mixed LN only, the degree of active inflammation correlates with the expansion of disease-specific macrophage (DMac) subsets, which later contract as the CI increases. Trajectory analysis of the scRNA-seq data suggested that DMacs arise from both infiltrating monocytes and tissue-resident macrophages; this was supported by the ST data, as well as cell cultures. DMacs are implied to interact with parietal epithelial cells, promoting the development of glomerulosclerosis. Conclusions We suggest a detailed picture of the changes in the kidney immune mechanisms in LN as this disease progresses.

Abstract Induction of anti-tumor immunity by radiation therapy (RT) has been observed in multiple preclinical models and in selected patients and can lead to durable systemic tumor regression. Modulation of anti-tumor immunity by RT is thought to act primarily via cGAS-STING activation leading to type I interferon (IFN) response pathways, which coordinate T cell priming and recruitment and adaptive resistance such as PD-L1 induction. Individual studies have demonstrated both immune activating and immune suppressive effects of RT in various contexts, and the determinants of this wide variability are poorly understood. Discovering divergent and convergent mechanisms of type I IFN response to RT across diverse cancer cell states may thus define biomarkers and treatment strategies to enhance RT-driven immune activation. We treated 100 human cancer cell lines with 2 Gy or 8 Gy RT or the STING agonist DiABZI and performed single cell RNA sequencing. We scored cell lines using a composite of IFN response genes as well as individual IFN response genes including ISG15, MX1, CXCL10 and IFNB. We then selected four cell lines with high radiation-induced IFN response for mechanistic studies: two derived from pancreatic ductal adenocarcinoma and two from breast carcinoma. We observed distinct kinetics of ISG15 and MX1 activation between 2 Gy and 8 Gy RT and DiABZI as well as between cell lines. Notably, knockout of STING abrogated DiABZI response but paradoxically increased ISG15 induction by radiation in MDA-MB-231, but not SUIT-2. Our study highlights wide variability among cancer cell states in modulation of IFN response signatures by radiation and emphasizes the need to elucidate mechanisms of radiation-induced immune activation and suppression across diverse cancer types. Citation Format: Olivia Kaneko, Bon Ham Yip, Arnav Mehta, Nir Hacohen, Ryan J. Park. Identifying cancer cell intrinsic regulators of radiation-induced interferon signaling [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 2559.

Improved methods to identify therapeutically relevant tumor neoantigens and their cognate T cells would aid the development of precision medicines for cancer. Here, we developed Slide-GoTags, a droplet-based single-nucleus spatial transcriptomics approach that characterizes neoantigen-specific immunity by integrating targeted transcript genotyping and T cell receptor (TCR) sequencing with single-nucleus RNA sequencing from the same slice of frozen tissue. Application of Slide-GoTags to mouse and human tumors revealed colocalization of clonally expanded, neoantigen-specific T cells with tumor cells expressing their cognate neoantigen. We also identified distinct spatial immune landscapes shaped by anti-PD1 or anti-CTLA4 blockade in mouse colorectal tumors. Across human tumor types, Slide-GoTags detected TCR–neoantigen interactions through spatial proximity and identified an enrichment of interferon-driven immunogenicity niches in immunologically ‘hot’ tumors compared to ‘cold’ tumors. These niches harbored three T cell clonotypes that colocalized with genotyped neoantigens, highlighting a spatially organized antitumor immune response. Collectively, Slide-GoTags establishes a framework for in situ mapping of T cell–tumor interactions directly from individual tissue. Spatial relationships between tumor neoantigens and cognate T cells are studied.

Many cancer patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses. Circulating biomarkers have the potential to identify patients with primary resistance or early progression on therapy to alter treatment course and potentially avoid unnecessary toxicity. Unbiased multimodal proteomic profiling in blood has been underexplored due to the previously limited scalability of multiplexing technologies or cohorts lacking time-series sampling. To address this, we performed plasma proteomic profiling of >2900 proteins and high-dimensional mass cytometry of peripheral blood lymphocytes across serial time points in 250 metastatic melanoma patients on ICB treatment. We further obtained 92 patient-matched tumor samples, which were processed for single-cell and/or bulk RNA sequencing. Proteins upregulated post-ICB were associated with inflammatory pathways involving the activation of effector immune functions. Expression of genes corresponding to these proteins was higher in immune cells involved in recruitment and tumor reactivity. Expression of genes corresponding to plasma proteins more abundant in non-responders was highest in suppressive myeloid subsets and malignant cells. We further posit the involvement of these non-responder genes in immunosuppressive and pro-tumor interactions, which we confirm using publicly available spatial transcriptomic data. We also find that epithelial-specific proteins in the circulation of responders post-ICB associate with patient toxicity and likely originate from degradation of healthy tissues. Together, these data represent extensive potential peripheral biomarker characterization using paired blood and tumor samples in melanoma patients treated with ICB, and begin to elucidate the complex interplay between tumors and the systemic immune response within the host.

While cancer immunotherapies have primarily focused on activation of cytotoxic CD8 cells, CD4 T cell activity is also associated with survival and immunotherapeutic response in numerous cancers. We applied integrated single-cell RNA sequencing and multiplexed protein epitope profiling to breast cancer samples to resolve the complexity of immune cell states within the tumor microenvironment. This approach enhanced phenotypic resolution, identifying three distinct states within the CD4 T follicular helper-like (Tfh) cell cluster. A CXCR4high progenitor state gave rise to two differentiated states: an IGFL2high subset resembling conventional Tfh cells and localised to B cell-rich lymphoid aggregates, and a CD103+ subset, exhibiting features of tissue residency, exhaustion, and cytotoxicity, which co-localised with tumor foci. CD103+ Tfh-like cells were found to interact with CXCL10+ macrophages through production of CCL chemokines and CSF1. A higher CD103+ Tfh to IGFL2high Tfh ratio, together with the selective clonal expansion of the CD103+ subset, was strongly associated with improved tumour immunity and superior responses to anti-PD-1 checkpoint blockade, surpassing the predictive value of exhausted CD8 T cells. These findings integrate Tfh and CD4 with cytotoxic potential in breast cancer, offering new insight into anti-tumor immunity and response to checkpoint blockade.

Microsatellite stable (MSS) pancreatic ductal adenocarcinoma (PDAC) is refractory to immune checkpoint blockade. We conducted a single-arm phase II trial ( NCT04361162 ) combining nivolumab, ipilimumab and radiation therapy to treat patients with pre-treated metastatic MSS PDAC (n=30). We integrated longitudinal profiling of 32 pre- and on-treatment tumor biopsies from 22 patients, yielding 245,529 single-nucleus and 128,295 single-cell transcriptomes including 27,215 T-cells with paired TCR clonotypes, as well as Visium spatial transcriptomics from 13 biopsies, and peripheral blood TCR-sequencing from 25 patients. While clinical activity was limited overall, one patient achieved a durable complete response with no evidence of disease 4 years after trial enrollment. This response was marked by a therapy-associated shift in the state composition of pre-existing CD8 T cell clonotypes from GZMK+ to exhausted and predicted tumor-reactive states, durable maintenance of associated clonotypes in the blood after 1 year, interferon-polarized macrophage and fibroblast programs, and high levels of ACKR1+ venous endothelium. Across independent PDAC cohorts, high ACKR1 expression was associated with improved survival, greater intratumoral TCR richness and clonality, and increased tumor-blood TCR sharing. These findings suggest that productive immunotherapy responses in PDAC require not only tumor-reactive T cells, but also a stromal-vascular niche capable of supporting their recruitment, recirculation and persistence. This may have implications for the design of future immunotherapy and vaccine strategies for PDAC.

The addition of anti-programmed cell death protein 1 (aPD-1) to 5-fluorouracil (5-FU)/platinum in advanced gastric cancer (GC) yields variable responses. To understand chemotherapy-immunotherapy cooperativity, we previously reported a phase II trial sequentially adding pembrolizumab to 5-FU/platinum. In this study, we use single-cell RNA sequencing and T cell receptor (TCR) sequencing to analyze 66,813 T cells from primary tumor biopsies pre-treatment, post-chemotherapy, and post-immunotherapy in 33 patients. We observe greater abundance, persistence, and recruitment of T cells with transcriptionally predicted tumor-reactivity in patients with prolonged progression-free survival (slow progressors). Increased B cell abundance and predicted B cell to T cell interactions support T cell memory and co-stimulation, providing a mechanism for increased abundance and persistence of progenitor-exhausted and tumor-reactive T cells in slow progressors. T cell clones emerging in the tumor after immunotherapy are present in the blood before treatment only in slow progressors. We thus highlight mechanisms that may drive durable responses to chemoimmunotherapy in GC.

BACKGROUND:Acquired resistance to immune checkpoint inhibitors (ICIs) remains a significant challenge in the treatment of metastatic melanoma. Phenotypic plasticity, such as dedifferentiation and transdifferentiation, is an increasingly recognized mechanism of treatment resistance. METHODS:We present a case of a 70-year-old man with metastatic melanoma who experienced progression through sequential treatments including pembrolizumab in combination with the HDAC inhibitor entinostat and ipilimumab. During treatment, a histologically distinct pleomorphic rhabdomyosarcoma (RMS) emerged at metastatic sites. Longitudinally acquired tumor samples representing both phenotypes were analyzed using whole-exome sequencing (WES), RNA sequencing (RNA-seq), and high-plex tissue imaging (spatial proteomics). FINDINGS:WES revealed driver mutations (e.g., NRAS and NF1) and loss of heterozygosity (LOH) shared between phenotypes indicating a common ancestral clone. Phylogenetic analysis demonstrated an early divergence of the phenotypes, with each later acquiring unique mutations. RNA-seq showed mutually exclusive expression of lineage-specific markers as well as epithelial-mesenchymal transition and myogenic gene set enrichment in the RMS samples. High-plex imaging identified distinct tumor microenvironments, with RMS lesions enriched in CD163+ macrophages. CONCLUSIONS:This case provides molecular evidence of phenotypic plasticity occurring under the selective pressure of immune checkpoint inhibitor therapy. FUNDING:This study was supported by the National Institutes of Health (K12CA087723 and K08CA234458), the Doris Duke Charitable Foundation, and Adelson Medical Research Foundation.