Little is known about the complexity and plasticity of circulating tumor cell (CTC) biology in different compartments of the fluid microenvironment during tumor metastasis. Here we integrated phenomics, genomics, and targeted proteomics to characterize CTC phenotypic and genotypic heterogeneity in paired peripheral blood (PB) and bone marrow aspirate (BMA) from a metastatic prostate cancer patient following the rapid disease progression, using the High-Definition Single Cell Assay 3.0 (HDSCA3.0). Uniquely, we identified a subgroup of genetically clonal CTCs that acquired a mesenchymal-like state and its presence was significantly associated with one subclone that emerged along the clonal lineage. Higher CTC abundance and phenotypic diversity were observed in the BMA than PB and differences in genomic alterations were also identified between the two compartments demonstrating spatial heterogeneity. Single cell copy number profiling further detected clonal heterogeneity within clusters of CTCs (also known as microemboli or aggregates) as well as phenotypic variations by targeted proteomics. Overall, these results identify epithelial and mesenchymal CTCs in the clonal lineage of an aggressive prostate cancer case and also demonstrate a single cell multi-omic approach to deconvolute the heterogeneity and association of CTC phenotype and genotype in multi-medium liquid biopsies of metastatic prostate cancer.

Osteosarcoma is the most common malignant bone tumor in young patients and remains a significant clinical challenge, particularly at the metastatic stage. Studies detailing the immunosuppressive mechanisms within the metastatic osteosarcoma microenvironment are needed to elucidate the cellular communities in the metastatic microenvironment that support metastatic growth and to identify therapeutic approaches to target the cross-talk between cancer cells and their microenvironment. In this study, we performed spatial transcriptional profiling on a cohort of osteosarcoma pulmonary metastases from pediatric patients. The data revealed a conserved spatial gene expression pattern resembling a foreign body granuloma, characterized by peripheral inflammatory signaling, fibrocollagenous encapsulation, lymphocyte exclusion, and peritumoral macrophage accumulation. The intratumoral microenvironment of these lesions, however, lacked inflammatory signaling. Exploration of spatially distinct drug–gene interactions identified the CXCR4 signaling axis, which displayed spatial heterogeneity and complexity, as a potential therapeutic target that bridges both the intra- and extratumoral microenvironments. Collectively, this study reveals that metastatic osteosarcoma comprises multiple regionally distinct immunosuppressive microenvironments. Significance: Exploration of spatially resolved microenvironments in metastatic osteosarcoma tissues reveals how the tissue architecture promotes immunosuppression and identifies actionable processes to enhance immunotherapy efficacy.

Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient’s overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs.

Tumor immunosuppression affects survival and treatment efficacy. Tumor NOS2/COX2 coexpression strongly predicts poor outcome in estrogen receptor–negative (ER ^– ) breast cancer by promoting metastasis, drug resistance, cancer stemness, and immune suppression. Herein, a spatially distinct NOS2/COX2 and CD3 ⁺ CD8 ⁺ PD1 ^– T effector (T _Eff ) cell landscape correlated with poor survival in ER ^– tumors. NOS2 was primarily expressed at the tumor margin, whereas COX2 together with B7H4 was associated with immune desert regions lacking T _Eff cells, where a higher ratio of tumor NOS2 or COX2 to T _Eff cells predicted poor survival. Also, programmed cell death ligand 1/programmed cell death 1, regulatory T cells (T _Regs ), and IDO1 were primarily associated with stroma-restricted T _Eff cells. Regardless of the survival outcome, CD4 ⁺ T cells and macrophages were primarily in stromal lymphoid aggregates. Finally, in a 4T1 model, COX2 inhibition led to increased CD8 ⁺ T _Eff /CD4 ⁺ T _Reg ratio and CD8 ⁺ T _Eff infiltration while Nos2 deficiency had no significant effect, thus reinforcing our observations that COX2 is an essential component of immunosuppression through CD8 ⁺ T _Eff cell exclusion from the tumor. Our study indicates that tumor NOS2/COX2 expression plays a central role in tumor immune evasion, suggesting that strategies combining clinically available NOS2/COX2 inhibitors with immune therapy could provide effective options for the treatment of aggressive and drug-resistant ER ^– breast tumors.

Immune therapy is the new frontier of cancer treatment. Therapeutic radiation is a known inducer of immune response and can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2) that is highly expressed in aggressive triple negative breast cancer (TNBC). A clinical cohort of TNBC tumors revealed poor radiation therapeutic efficacy in tumors expressing high COX2. Herein, we show that radiation combined with adjuvant NSAID (indomethacin) treatment provides a powerful combination to reduce both primary tumor growth and lung metastasis in aggressive 4T1 TNBC tumors, which occurs in part through increased antitumor immune response. Spatial immunological changes including augmented lymphoid infiltration into the tumor epithelium and locally increased cGAS/STING1 and type I IFN gene expression were observed in radiation-indomethacin–treated 4T1 tumors. Thus, radiation and adjuvant NSAID treatment shifts “immune desert phenotypes” toward antitumor M1/TH1 immune mediators in these immunologically challenging tumors. Importantly, radiation-indomethacin combination treatment improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared with radiation treatment alone. These results show that clinically available NSAIDs can improve radiation therapeutic efficacy through increased antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs.

Estrogen receptor–negative breast cancer is an aggressive subtype with limited therapeutic options. Elevated nitric oxide synthase 2 (NOS2) and COX2 mediate immunosuppression and poor survival in these tumors. Therefore, the influence of tumor NOS2/COX2 on immune architecture was examined in 16 African American and five Caucasian estrogen receptor–negative tumors. Elevated tumor NOS2/COX2 limited CD8+ T-cell infiltration at 5-year survival. Distinct CD8+/−NOS2+/−COX2+/− phenotypes defining metastatic and cancer stem cell niches and immune desert regions were identified. These results were supported by an unbiased, unsupervised nonlinear dimensionality reduction technique, Uniform Manifold Approximation and Projection for Dimension Reduction, incorporating spatial relations between cells and validated in a separate gene expression cohort using NOS2/CD8 and COX2/CD8 ratios. Additionally, elongated tumor cells were specifically in CD8−NOS2+COX2+ regions, suggesting metastatic hot spots. This work demonstrates predictive power of spatial analyses of CD8/NOS2/COX2 architecture and supports the use of clinically available NOS2/COX2 inhibitors for improved survival in patients with these aggressive tumors. Significance: This work identifies CD8−NOS2+COX2+ and CD8−NOS2−COX2+ unique cellular neighborhoods that drive the tumor immune spatial architecture of CD8+ T cells predictive of clinical outcome and can be targeted with clinically available NOS inhibitors and NSAIDs.