RESUMEN
Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.
RESUMEN
There remains a large need for a greater understanding of the metastatic process within the prostate cancer field. Our research aims to understand the adaptive - ergo potentially metastatic - responses of cancer to changing microenvironments. Emerging evidence has implicated a role of the Polyaneuploid Cancer Cell (PACC) state in metastasis, positing the PACC state as capable of conferring metastatic competency. Mounting in vitro evidence supports increased metastatic potential of cells in the PACC state. Additionally, our recent retrospective study of prostate cancer patients revealed that PACC presence in the prostate at the time of radical prostatectomy was predictive of future metastatic progression. To test for a causative relationship between PACC state biology and metastasis, we leveraged a novel method designed for flow-cytometric detection of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in subcutaneous, caudal artery, and intracardiac mouse models of metastasis. This approach provides both quantitative and qualitative information about the number and PACC-status of recovered CTCs and DTCs. Collating data from all models, we found that 74% of recovered CTCs and DTCs were in the PACC state. In vivo colonization assays proved PACC populations can regain proliferative capacity at metastatic sites following dormancy. Additional direct and indirect mechanistic in vitro analyses revealed a PACC-specific partial Epithelial-to-Mesenchymal-Transition phenotype and a pro-metastatic secretory profile, together providing preliminary evidence that PACCs are mechanistically linked to metastasis.
RESUMEN
The prostate cancer tumor microenvironment (TME) is comprised of many cell types that can contribute to and influence tumor progression. Some of the most abundant prostate cancer TME cells are macrophages, which can be modeled on a continuous spectrum of M1-like (anti-tumor macrophages) to M2-like (pro-tumor macrophages). A function of M2-like macrophages is efferocytosis, the phagocytosis of apoptotic cells. Based on literature from other models and contexts, efferocytosis further supports the M2-like macrophage phenotype. MerTK is a receptor tyrosine kinase that mediates efferocytosis by binding phosphatidylserine on apoptotic cells. We hypothesize efferocytosis in the prostate cancer TME is a tumor-promoting function of macrophages and that targeting MerTK-mediated efferocytosis will slow prostate cancer growth and promote an anti-tumor immune infiltrate. The aims of this study are to measure efferocytosis of prostate cancer cells by in vitro human M1/M2 macrophage models and assess changes in the M2-like, pro-tumor macrophage phenotype following prostate cancer efferocytosis. Additionally, this study aims to demonstrate that targeting MerTK decreases prostate cancer efferocytosis and promotes an anti-tumor immune infiltrate. We have developed methodology using flow cytometry to quantify efferocytosis of human prostate cancer cells using the LNCaP cell line. We observed that M2 macrophages efferocytose the LNCaP cell line more than M1 macrophages. Following efferocytosis of LNCaP cells by M2 human monocyte-derived macrophages (HMDMs), we observed an increase in the M2-like, pro-tumor phenotype by flow cytometry cell surface marker analysis. By qRT-PCR, flow cytometry, and Western blot, we detected greater MerTK expression in M2 than M1 macrophages. Targeting MerTK with antibody Mer590 decreased LNCaP efferocytosis by M2 HMDMs, establishing the role of MerTK in prostate cancer efferocytosis. In the prostate cancer mouse model hi-myc, Mertk KO increased anti-tumor immune infiltrate including CD8 T cells. These findings support targeting MerTK-mediated efferocytosis as a novel therapy for prostate cancer.
Asunto(s)
Neoplasias de la Próstata , Animales , Ratones , Masculino , Humanos , Tirosina Quinasa c-Mer , Fagocitosis , Macrófagos , Próstata , Microambiente TumoralRESUMEN
Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (~ 1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.
Asunto(s)
Neoplasias , Línea Celular TumoralRESUMEN
Metastatic cancer is lethal and patients who suffer bone metastases fare especially poorly. Bone-specific metastatic progression in prostate and breast cancers is a highly observed example of organ-specific metastasis, or organotropism. Though research has delineated the sequential steps of the metastatic cascade, the determinants of bone-specific metastasis have remained elusive for decades. Applying fundamental ecological principles to cancer biology models of metastasis provides novel insights into metastatic organotropism. We use critical concepts from foraging theory and movement ecology to propose that observed bone-specific metastasis is the result of habitat selection by foraging cancer cells. Furthermore, we posit that cancer cells can only perform habitat selection if and when they employ a reversible motile foraging strategy. Only a very small percentage of cells in a primary tumor harbor this ability. Therefore, our habitat selection model emphasizes the importance of identifying the rare subset of cancer cells that might exhibit habitat selection, ergo achieve bone-specific metastatic colonization.