The enumeration of EpCAM-positive circulating tumor cells (CTCs) has allowed estimation of overall metastatic burden in breast cancer patients. However, a thorough understanding of CTCs associated with breast cancer brain metastasis (BCBM) is necessary for early identification and evaluation of treatment response to BCBM. Here we report that BCBM CTCs is enriched in a distinct sub-population of cells identifiable by their biomarker expression and mutational content. Deriving from a comprehensive analysis of CTC transcriptomes, we discovered a unique “circulating tumor cell gene signature” that is distinct from primary breast cancer tissues. Further dissection of the circulating tumor cell gene signature identified signaling pathways associated with BCBM CTCs that may have roles in potentiating BCBM. This study proposes CTC biomarkers and signaling pathways implicated in BCBM that may be used either as a screening tool for brain micro-metastasis detection or for making rational treatment decisions and monitoring therapeutic response in patients with BCBM.
Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multi-parametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice co-injected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients’ blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro co-culturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs; and induced their pro-tumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bi-directional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immuno-suppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease.
Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. To overcome these hurdles, we developed a new experimental model of clinical dormancy employing patient-isolated Circulating Tumor Cells (de novo CTCs) and their injection in xenografts with subsequent tumor monitoring and CTC characterization (ex vivo CTCs). We hypothesized that significant distinctions exist between signaling pathways of bone marrow-homing vs metastasis-competent CTCs upon transplantation in xenografts. Comparative transcriptomic analyses of ex vivo vs de novo CTCs identified increased mTOR signaling—a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy—with contrasting actions by its two complementary arms (mTORC2/mTORC1). Heightened mTORC2 downstream targets augmented quiescent CTCs (Ki67−/RBL2+ cells) in paired breast cancer tissues, along with high mTORC2 activity in solitary BMRCs and tissue-resident CTCs. Further, shRNA mediated the knockdown of RICTOR, an essential component of mTORC2, and augmented Ki67/PCNA biomarker expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling regulates CTC-associated mitotic and/or dormancy characteristics.
Recurrence is the major cause of melanoma death due to cell dissemination from primary/metastatic tumor (CTCs). During asymptomatic periods, these cells reside in bone marrow (bone-marrow resident tumor [BMRT cells]) and remain quiescent. We hypothesized that disseminated tumor cells survive in bone marrow (BM) during these periods and evolve to metastatic potency during disease progression. First, we isolated CTC-enriched, Lin-neg population from clinically advanced melanoma patients and performed biomarker expression and mutational profiling to confirm presence of putative CTCs. Lin-neg CTC population contained unique transcriptomics signature with elevated melanoma markers expression (BAGE, MAGEA1, B4GALNT1, S100A3). Further, downstream IPA analysis demonstrated that an upregulation of transcripts for genes involved in cell survival and pro-development functions with concomitant decrease in cell proliferative and inflammation properties. Second, we implanted Lin-neg population in murine xenograft models and isolated HLA+/ Melan-A+ cells population from blood and BM at endpoint. Expression of human (HLA-ABC) and melanoma markers (Melan-A, S100, NG2, CD146) on BMRTCs and CTCs was confirmed by employing: (a) immunofluorescence staining, (b) single-cell DEPArrayTM and CellSearchTM capture, (c) genomic profiling, and (d) organ-site colonization of CTCs with concomitant to BMRTCs in BM. Third, we discovered distinct transcriptomic signature of BMRTCs vs CTCs by differential gene expression profiling. Subsequent pathway analysis showed top five altered canonical pathways in BMRTCs: protein ubiquitination pathway, EIF2, actin cytoskeleton, systemic lupus erythematosus and hypoxia signaling. Of note, a strikingly elevated expression of PTEN in BMRTCs was detected. Fourth, because PTEN binds to USP7, a key component of protein ubiquitination pathway, we evaluated USP7/PTEN axis in CTC-driven BMRTCs modulated metastatic competency. The use of two clinically approved USP7 inhibitors to study their effects on the proliferative capacity of BMRTCs led to a significant reduction of CTCs at metastatic sites. This study provides critical insights to identify biomarkers of melanoma recurrence during the asymptomatic periods of the disease, fostering application of USP7 inhibitors for innovative melanoma therapies for patients with metastasis undetectable disease and/or yet to develop metastasis. Citation Format: Monika Vishnoi, Debasish Boral, Haowen N. Liu, Marc L. Sprouse, Wei Yin, Michael A. Davies, Isabella C. Glitza Oliva, Dario Marchetti. Targeting USP7/PTEN axis regulates the metastatic competency of bone marrow-resident melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3601.
Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. Further, there is no model of clinical dormancy that can comprehensively emulate the entire spectrum of heterogeneous clones of Circulating Tumor Cells (CTCs) present in cancer patients. To overcome these hurdles, we developed a new experimental model of clinical dormancy employing patient-isolated de novo CTCs and their injection in xenografts with subsequent tumor monitoring and CTC characterization (ex vivo CTCs). We hypothesized that significant distinctions exist between signaling pathways of bone marrow-homing vs metastasis-competent CTCs upon transplantation in xenografts. Comparative transcriptomic analyses of ex vivo vs de novo CTCs identified increased mTOR signaling – a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy – with contrasting actions by its two complementary arms (mTORC2/mTORC1). Heightened mTORC2 downstream targets augmented quiescent CTCs (Ki67-/RBL2+ cells) in paired breast cancer tissues, along with high mTORC2 activity in solitary BMRCs and tissue-resident CTCs. Further, shRNA mediated knockdown of RICTOR, an essential component of mTORC2, augmented Ki67/PCNA biomarker expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling regulates CTC-associated mitotic and/or dormancy characteristics. Further elucidation of mTOR-mediated CTC dormancy will provide novel strategies for therapeutic interventions in patients having early-event metastatic seeding but yet to develop clinically-detectable overt metastasis. Citation Format: Debasish Boral, Haowen N. Liu, S. Ray Kenney, Dario Marchetti. Molecular interplay between dormant bone marrow-resident tumor cells (BMRCs) and CTCs in breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-113.
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