Recruitment Of Bone Marrow-derived Cells Research Articles

The Role of Osteocytes in Pre-metastatic Niche Formation.

The formation of a pre-metastatic niche (PMN), in which primary cancer cells prime the distant site to be favorable to their engraftment and survival, may help explain the strong osteotropism observed in multiple cancers, such as breast and prostate. PMN formation, which includes extracellular matrix remodeling, increased angiogenesis and vascular permeability, enhanced bone marrow-derived cell recruitment and immune suppression, has mostly been described in soft tissues. In this review, we summarize current literature of PMN formation in bone. We also present evidence of a potential role for osteocytes to be the primary mediators of PMN development. Osteocytes regulate the bone microenvironment in myriad ways beyond canonical bone tissue remodeling, including changes that contribute to PMN formation. Perilacunar tissue remodeling, which has been observed in both bone and non-bone metastatic cancers, is a potential mechanism by which osteocyte-cancer cell signaling stimulates changes to the bone microenvironment. Osteocytes also protect against endothelial permeability, including that induced by cancer cells, in a loading-mediated process. Finally, osteocytes are potent regulators of cells within the bone marrow, including progenitors and immune cells, and might be involved in this aspect of PMN formation. Osteocytes should be examined for their role in PMN formation.

The Mechanisms of Systemic Inflammatory and Immunosuppressive Acute-on-Chronic Liver Failure and Application Prospect of Single-Cell Sequencing.

Acute-on-chronic liver failure (ACLF) is a complex clinical syndrome, and patients often have high short-term mortality. It occurs with intense systemic inflammation, often accompanied by a proinflammatory event (such as infection or alcoholic hepatitis), and is closely related to single or multiple organ failure. Liver inflammation begins when innate immune cells (such as Kupffer cells (KCs)) are activated by binding of pathogen-associated molecular patterns (PAMPs) from pathogenic microorganisms or damage-associated molecular patterns (DAMPs) of host origin to their pattern recognition receptors (PRRs). Activated KCs can secrete inflammatory factors as well as chemokines and recruit bone marrow-derived cells such as neutrophils and monocytes to the liver to enhance the inflammatory process. Bacterial translocation may contribute to ACLF when there are no obvious precipitating events. Immunometabolism plays an important role in the process (including mitochondrial dysfunction, amino acid metabolism, and lipid metabolism). The late stage of ACLF is mainly characterized by immunosuppression. In this process, the dysfunction of monocyte and macrophage is reflected in the downregulation of HLA-DR and upregulation of MER tyrosine kinase (MERTK), which weakens the antigen presentation function and reduces the secretion of inflammatory cytokines. We also describe the specific function of bacterial translocation and the gut-liver axis in the process of ACLF. Finally, we also describe the transcriptomics in HBV-ACLF and the recent progress of single-cell RNA sequencing as well as its potential application in the study of ACLF in the future, in order to gain a deeper understanding of ACLF in terms of single-cell gene expression.

Antineoplastic agents in chemotherapy facilitating tumor growth and angiogenesis in the interval administrations

AimsThere is emerging evidence that antineoplastic agents and the cytotoxic effects on tumor tissues attenuate the benefits of chemotherapy due to tumor microenvironment changes. Nevertheless, the actual relationship between chemotherapy and recurrent tumors in which the genotypes differ from the original tumor after chemotherapy is unclear. Materials and methodsBone marrow transplantation, flow cytometer, immune inhibition and immunofluorescence will be utilized to investigate the effect of antineoplastic agents on bone-marrow-derived cells (BMDCs) release and recruitment, and to explore the pathways and mechanisms of antineoplastic agents in promoting tumor growth. Key findingsTumor growth and angiogenesis were significantly enhanced in the mouse model after treatment with antineoplastic agents such as cyclophosphamide, 5-fluorouracil, or cisplatin, along with large increases in proangiogenic vascular endothelial growth factor receptor-2 (VEGFR2+), β3+, CD11b+Gr-1+, and VEGFR2+β3+ BMDCs in circulating blood. BMDC recruitment and VEGFR2 and β3 mRNA transcription in tumor tissues were also enhanced by antineoplastic agents. Antineoplastic-agent-treated BMDCs markedly augmented tumor and endothelial cell proliferation, and β3 mRNA transcription in endothelial cells (ECs). SignificanceThe results suggested that antineoplastic-agent treatment augmented the tumor microenvironment by mobilizing proangiogenic BMDCs, enhancing BMDC recruitment and angiogenesis, and increasing BMDC-mediated tumor and EC functions. These results led to tumor growth and angiogenesis aggravation. It is paramount to elucidate the potential mechanism by which the cellular and molecular effects triggered by the antineoplastic agents attenuate the effects of cancer therapy, and thereafter to explore possible methods for improving tumor treatment efficacy.

Uterine administration of C-X-C motif chemokine ligand 12 increases the pregnancy rates in mice with induced endometriosis

To study the effect of intrauterine injection of C-X-C motif chemokine ligand 12 (CXCL12), also known as a stem cell chemoattractant (stromal cell-derived factor 1), on fertility and endometrial receptivity in mice with endometriosis. Laboratory study. Academic Medical Center. Fifty-six mice underwent chemotherapy and bone marrow transplantation. Thirty-six of these mice underwent either surgery to induce endometriosis (n = 20) or sham surgery (n = 16). Injection of CXCL12 as a potential therapeutic agent to improve fertility in endometriosis. Pregnancy rate, bone marrow-derived cell (BMDC) recruitment and endometrial receptivity markers. The mice with or without endometriosis received a single uterine injection of either CXCL12 or placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. Mice were euthanized after delivery, and implantation markers homeobox A11, alpha-v beta-3 integrin, and progesterone receptor were analyzed by immunohistochemistry, whereas green fluorescent protein positive BMDC recruitment was quantified by immunohistochemistry and immunofluorescence. The sham surgery groups without endometriosis had the highest cumulative pregnancy rate (100%) regardless of CXCL12 treatment. The endometriosis group treated with placebo had the lowest pregnancy rate. An increased pregnancy rate was noted in the endometriosis group after treatment with CXCL12. There was also an increase in BMDC recruitment and endometrial expression of progesterone receptor and alpha-v beta-3 integrin in the endometriosis group that received CXCL12 compared with that in the endometriosis group that received placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. These results suggest that CXCL12 has a potential role as a therapeutic agent in women with infertility related to endometriosis and potentially other endometrial receptivity defects.

New insights into antiangiogenic therapy resistance in cancer: Mechanisms and therapeutic aspects.

Angiogenesis is a hallmark of cancer and is required for tumor growth and progression. Antiangiogenic therapy has been revolutionarily developing and was approved for the treatment of various types of cancer for nearly two decades, among which bevacizumab and sorafenib continue to be the two most frequently used antiangiogenic drugs. Although antiangiogenic therapy has brought substantial survival benefits to many cancer patients, resistance to antiangiogenic drugs frequently occurs during clinical treatment, leading to poor outcomes and treatment failure. Cumulative evidence has demonstrated that the intricate interplay among tumor cells, bone marrow-derived cells, and local stromal cells critically allows for tumor escape from antiangiogenic therapy. Currently, drug resistance has become the main challenge that hinders the therapeutic efficacies of antiangiogenic therapy. In this review, we describe and summarize the cellular and molecular mechanisms conferring tumor drug resistance to antiangiogenic therapy, which was predominantly associated with redundancy in angiogenic signaling molecules (e.g., VEGFs, GM-CSF, G-CSF, and IL17), alterations in biological processes of tumor cells (e.g., tumor invasiveness and metastasis, stemness, autophagy, metabolic reprogramming, vessel co-option, and vasculogenic mimicry), increased recruitment of bone marrow-derived cells (e.g., myeloid-derived suppressive cells, tumor-associated macrophages, and tumor-associated neutrophils), and changes in the biological functions and features of local stromal cells (e.g., pericytes, cancer-associated fibroblasts, and endothelial cells). We also review potential biomarkers to predict the response to antiangiogenic therapy in cancer patients, which mainly consist of imaging biomarkers, cellular and extracellular proteins, a certain type of bone marrow-derived cells, local stromal cell content (e.g., pericyte coverage) as well as serum or plasma biomarkers (e.g., non-coding RNAs). Finally, we highlight the recent advances in combination strategies with the aim of enhancing the response to antiangiogenic therapy in cancer patients and mouse models. This review introduces a comprehensive understanding of the mechanisms and biomarkers associated with the evasion of antiangiogenic therapy in cancer, providing an outlook for developing more effective approaches to promote the therapeutic efficacy of antiangiogenic therapy.

Promotion of tumor progression induced by continuous low-dose administration of antineoplastic agent gemcitabine or gemcitabine combined with cisplatin

Major findingThis study observes a previously neglected pharmacological phenomenon and investigates its mechanism of that the continuous low-dose administration of some antineoplastic agents in certain dose ranges can promote tumorigenesis and tumor progression in vitro and in vivo, through stimulation of tumor cell functions directly as well as enhancement of tumor angiogenesis by BMDCs recruitment indirectly. The results alert to a potential risk in current empirically based continuous low-dose chemotherapy regimens such as metronomic chemotherapy. Background and objectivesThere are indications that certain antineoplastic agents at low dosages may exhibit abnormal pharmacological actions, such as promoting tumor growth. However, the phenomenon still needs to be further confirmed, and its underlying mechanisms have not yet been fully elucidated. MethodsGemcitabine (GEM) and cisplatin (CDDP) were employed as representative antineoplastic agents to observe effects of continuous low-dose chemotherapy with GEM or GEM combined with CDDP (GEM+CDDP) on tumor formation and growthin xenograft tumor models in vivo. Tumor and endothelial cell functions, apoptosis, cell cycle analysis, as well as bone marrow derived cells (BMDCs) mobilization, were evaluated with transwell, MTT or flow cytometry analysis in vitro, respectively. Histological methods were employed to assess angiogenesis in tumor tissues. ResultsThe results showed that tumor formation and growth were both significantly promoted by GEM or GEM+CDDP at as low as half of the metronomic dosages, which were accompanied by enhancements of angiogenesis in tumor tissues and the release of proangiogenic BMDCs in the circulating blood. Additionally, GEM or GEM+CDDP at low concentrations dramatically facilitated the proliferation, migration, and invasion of tumor cells in vitro. Cell-cycle arrest, activation of associated apoptotic proteins, and inhibition of apoptosis were also observed in tumor cells. ConclusionsThese findings indicate that, the continuous low-dose administration of GEM and GEM+CDDP can promote tumorigenesis and tumor progression in vivo by inhibiting apoptosis, mobilizing BMDCs, and promoting angiogenesis in certain dose ranges. These findings urge further investigations to avoid the potential risks in current empiric continuous low-dose chemotherapy regimens with antineoplastic agents.

Cancer-Associated Stromal Cells Promote the Contribution of MMP2-Positive Bone Marrow-Derived Cells to Oral Squamous Cell Carcinoma Invasion

Simple SummaryBased on its invasiveness, oral squamous cell carcinoma (OSCC) shows two different subtypes: less-invasive verrucous squamous carcinoma (VSCC) or highly invasive squamous cell carcinoma (SCC). The stromal component influences OSCC progression and invasion. On the other hand, bone marrow-derived cells (BMDCs) are recruited into tumors and involved in tumor development. We hypothesized that stromal factors might also affect the relation of BMDCs and tumor invasion. We established the OSCC models transplanted with stromal cells from VSCC and SCC, and we compared the potential stromal factors of VSCC and SCC for the involvement of BMDCs in tumor invasion. Our study showed that stromal factors IL6 and IL1B might promote the contribution of MMP-2 positive BMDCs to OSCC invasion.Tumor stromal components contribute to tumor development and invasion. However, the role of stromal cells in the contribution of bone marrow-derived cells (BMDCs) in oral squamous cell carcinoma (OSCC) invasion is unclear. In the present study, we created two different invasive OSCC patient-derived stroma xenografts (PDSXs) and analyzed and compared the effects of stromal cells on the relation of BMDCs and tumor invasion. We isolated stromal cells from two OSCC patients: less invasive verrucous OSCC (VSCC) and highly invasive conventional OSCC (SCC) and co-xenografted with the OSCC cell line (HSC-2) on green fluorescent protein (GFP)-positive bone marrow (BM) cells transplanted mice. We traced the GFP-positive BM cells by immunohistochemistry (IHC) and detected matrix metalloproteinase 2 (MMP2) expression on BM cells by double fluorescent IHC. The results indicated that the SCC-PDSX promotes MMP2-positive BMDCs recruitment to the invasive front line of the tumor. Furthermore, microarray analysis revealed that the expressions of interleukin 6; IL-6 mRNA and interleukin 1 beta; IL1B mRNA were higher in SCC stromal cells than in VSCC stromal cells. Thus, our study first reports that IL-6 and IL1B might be the potential stromal factors promoting the contribution of MMP2-positive BMDCs to OSCC invasion.

Pre-metastatic Niche Formation in Different Organs Induced by Tumor Extracellular Vesicles.

Primary tumors selectively modify the microenvironment of distant organs such as the lung, liver, brain, bone marrow, and lymph nodes to facilitate metastasis. This supportive metastatic microenvironment in distant organs was termed the pre-metastatic niche (PMN) that is characterized by increased vascular permeability, extracellular matrix remodeling, bone marrow-derived cells recruitment, angiogenesis, and immunosuppression. Extracellular vesicles (EVs) are a group of cell-derived membranous structures that carry various functional molecules. EVs play a critical role in PMN formation by delivering their cargos to recipient cells in target organs. We provide an overview of the characteristics of the PMN in different organs promoted by cancer EVs and the underlying mechanisms in this review.

Pro-Inflammatory Cytokines in the Formation of the Pre-Metastatic Niche.

Simple SummaryThe formation of the pre-metastatic niche, a favorable microenvironment in an organ distant from a primary tumor, is critical for tumor metastasis. We review the role of a key player, a class of proteins named pro-inflammatory cytokines secreted from both tumor cells and other cells in tissues, in helping to build the pre-metastatic niche. Various drugs have been developed to target pro-inflammatory cytokines, and their effects on tumor metastases are under investigation. Future clinical studies should focus on combining those drugs and applying them during cancer surgery, a critical moment for the establishment of the pre-metastatic niche.In the presence of a primary tumor, the pre-metastatic niche is established in secondary organs as a favorable microenvironment for subsequent tumor metastases. This process is orchestrated by bone marrow-derived cells, primary tumor-derived factors, and extracellular matrix. In this review, we summarize the role of pro-inflammatory cytokines including interleukin (IL)-6, IL-1β, CC-chemokine ligand 2 (CCL2), granulocyte-colony stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), stromal cell-derived factor (SDF)-1, macrophage migration inhibitory factor (MIF), and Chemokine (C–X–C motif) ligand 1 (CXCL1) in the formation of the pre-metastatic niche according to the most recent studies. Pro-inflammatory cytokines released from tumor cells or stromal cells act in both autocrine and paracrine manners to induce phenotype changes in tumor cells, recruit bone marrow-derived cells, and form an inflammatory milieu, all of which prime a secondary organ’s microenvironment for metastatic cell colonization. Considering the active involvement of pro-inflammatory cytokines in niche formation, clinical strategies targeting them offer ways to inhibit the establishment of the pre-metastatic niche and therefore attenuate metastatic progression. We review clinical trials targeting different inflammatory cytokines in patients with metastatic cancers. Due to the pleiotropy and redundancy of pro-inflammatory cytokines, combined therapies should be designed in the future.

Integrin α5β1 promotes BMCs mobilization and differentiation to exacerbate choroidal neovascularization

Choroidal neovascularization (CNV) is an acknowledged pathogenic mechanism of various ocular diseases, and in situ cells and mobilized bone marrow-derived cells (BMCs) are thought to participate in this process. We aimed to evaluate the roles of integrin α5 in BMCs and vascular endothelial cells (VECs) in the CNV process mediated by SDF-1/CXCR4 signaling. Adult wild-type mice were engrafted with whole BMCs obtained from GFP transgenic mice and then laser injured to induce CNV. BMCs and RF/6A cells were cultured to discover the mechanism of CNV in vitro. BMCs were mobilized to CNV areas, which expressed elevated SDF-1 and CXCR4. When SDF-1 was intravitreally injected, the number of BMCs was profoundly increased. In the SDF-1-treated group, the levels of integrin α5 expressed on BMCs and VECs were significantly higher than those on the cells in the control group. SDF-1 significantly increased the expression and positive ratio of integrin α5, which was involved in the recruitment and differentiation of BMCs into BMC-derived VECs, and these effects were suppressed by the CXCR4 inhibitor AMD3100. The PI3K/AKT pathway rather than the ERK pathway mediated SDF-1/CXCR4 induction of integrin α5. Integrin α5 suppression efficiently prevented the production of TGF-β and bFGF but not VEGF. Inhibiting the SDF-1/CXCR4-PI3K/AKT-integrin α5 axis reduced CNV severity. Integrin α5 participates in BMC recruitment and differentiation in SDF-1/CXCR4-induced CNV and inhibition of this pathway may be a new approach to inhibit CNV.

CXCL12 Attracts Bone Marrow-Derived Cells to Uterine Leiomyomas.

Uterine leiomyomas, also known as fibroids or myomas, are a common benign gynecologic tumor found in women of reproductive age. Though advances have been made in understanding leiomyomas, the etiology and pathogenesis of this disease are not fully characterized. Current evidence supports a role of putative human uterine stem/progenitor cells in the onset of uterine disease such as uterine myomas. In this study, we report that increased expression of CXCL12 in leiomyomas recruits bone marrow-derived cells (BMDCs) that may contribute to leiomyoma growth. Tissue was collected from leiomyomas or control myometrium from women with or without leiomyomas. qRT-PCR analysis showed increased expression of CXCL12 and decreased CXCR4 expression in the leiomyoma and myometrium of women with leiomyoma compared with normal myometrium. Increased CXCL12 protein secretion from cultured myoma cells was confirmed by ELISA. Further, we found that BMDCs migration was increased toward leiomyoma conditioned medium compared with conditioned medium from normal myometrium. CXCR4 antagonist AMD3100 completely blocked this migration. Engraftment of BMDCs significantly increased in myoma of mouse uteri treated with CXCL12 compared with placebo. We conclude that CXCL12 may play a role in leiomyomas growth by attracting bone marrow-derived cells to leiomyoma. Therefore, CXCL12 and its receptors are novel targets for leiomyoma therapy.

Cadherin-11 blockade reduces inflammation-driven fibrotic remodeling and improves outcomes after myocardial infarction.

Over one million Americans experience myocardial infarction (MI) annually, and the resulting scar and subsequent cardiac fibrosis gives rise to heart failure. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not been studied in myocardium after MI. MI was induced by ligation of the left anterior descending artery in mice with either heterozygous or homozygous knockout of CDH11, wild-type mice receiving bone marrow transplants from Cdh11-deficient animals, and wild-type mice treated with a functional blocking antibody against CDH11 (SYN0012). Flow cytometry revealed significant CDH11 expression in noncardiomyocyte cells after MI. Animals given SYN0012 had improved cardiac function, as measured by echocardiogram, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 reduced bone marrow-derived myeloid cells and increased proangiogenic cells in the heart 3 days after MI. Cardiac fibroblast and macrophage interactions increased IL-6 secretion in vitro. Our findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI and that targeting CDH11 with a blocking antibody improves outcomes by altering recruitment of bone marrow-derived cells, limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.

Adult bone marrow progenitors become decidual cells and contribute to embryo implantation and pregnancy.

Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However, whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here, we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua, where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy, we used Homeobox a11 (Hoxa11)-deficient mice, having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11−/− mice results in stromal expansion, gland formation, and marked decidualization otherwise absent in Hoxa11−/− mice. Moreover, in Hoxa11+/− mice, which have increased pregnancy losses, BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively, these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma, thereby playing important roles in decidualization and pregnancy.

Differential expression profiles of the transcriptome in bone marrow-derived cells in lung cancer revealed by next generation sequencing and bioinformatics.

A pre-metastatic niche (PMN) facilitates cancer metastasis through mobilization and recruitment of bone marrow-derived cells (BMDCs) and associated factors. In bone marrow, hematogenous cells, including osteoclasts, macrophages and lymphocytes, and mesenchymal cells, including mesenchymal stem cells, osteoblasts and adipocytes, are involved in PMN formation. Patients with lung cancer and metastasis have a poor prognosis and shortened median survival time. Bone marrow has been considered fertile ground for dormant and proliferating tumor cells, and mobilizing and recruiting BMDCs and immune cells can establish a PMN. However, the role of BMDCs in PMN formation is not yet fully understood. The present study aimed to investigate the association between BMDCs and PMN in bone marrow tissue samples. The results demonstrated that bone marrow served an important role in lung cancer progression and that eight pathways were potentially involved, including ‘T-cell receptor signaling pathway’, ‘osteoclast differentiation’, ‘MAPK signaling pathway’, ‘VEGF signaling pathway’, ‘leukocyte transendothelial migration’, ‘signaling pathways regulating the pluripotency of stem cells’, ‘oxytocin signaling pathway’ and ‘cell adhesion molecules (CAMs)’. In addition, the present study investigated the role of BMDCs in facilitating lung cancer metastasis. In conclusion, the results from the present study suggested that molecular alterations in gene expression may provide a novel signature in lung cancer, which may aid in the development of novel diagnostic and therapeutic strategies for patients with lung cancer and bone metastasis.

Estrogen enhances tumor growth and angiogenesis indirectly via mediation of bone marrow‑derived cells as well as directly through stimulation of tumor and endothelial cells.

Estradiol (E2) is a prime culprit for enhancing the progression of female hormone‑related cancers. Bone marrow‑derived cells (BMDCs) have been found to play a pivotal role in tumor growth. Estrogen receptors (ERs) are also found on certain subtypes of BMDCs, in addition to endothelial cells (ECs) and certain tumor cells. However, the role of BMDCs in E2‑induced tumor biology is still unclear. Thus, the effects of E2 on ER‑negative 4T1 breast cancer growth, the mobilization and recruitment of BMDCs, and interactions among BMDCs, ECs, and 4T1 cells were investigated. The results showed that E2 potentiated 4T1 tumor growth and angiogenesis in mice subjected to sham operation, ovariectomy (OVX), or OVX and E2 replacement treatment. E2 supplementation in mice with OVX upregulated the transcription of stromal cell‑derived factor‑1 (SDF‑1) mRNA in tumor tissues and enhanced the recruitment of BMDCs into tumor tissues invivo. E2 deficiency significantly decreased proangiogenic CXCR4+, β3+, Sca‑1+ and CXCR4+β3+ BMDCs circulating in the peripheral blood. Cell‑based system analyses showed that E2 augmented the transcription of β3 mRNA in ECs, increased the adhesion of BMDCs to ECs. In addition, E2 enhanced the BMDC‑induced EC proliferation and migration, the BMDC‑induced 4T1 proliferation and the 4T1‑stimulated EC proliferation in addition to enhancing the proliferation of tumor cells and the migration of ECs invitro. Therefore, E2 enhanced the growth of breast tumors by stimulating tumor cells and ECs directly, as well as by increasing proangiogenic BMDC mobilization and recruitment leading to augmentation of the tumor and EC functions indirectly by cell proliferation assay. These findings reveal a separate mechanism via which E2 promotes the growth of female hormone‑dependent tumors, which may be useful in explorations of new therapies for related cancers.

Abstract 4182: Impacting early events in metastatic spread: Targeted therapeutics to disrupt formation of the pre-metastatic niche and development of lung metastases

Abstract There is now growing interest in understanding the early events which lead to the successful formation of solid tumor metastatic spread because this information may lead to early interventional approaches. Bone marrow derived cells (BMDCs) of various lineages are gradually being identified as central to the ongoing growth of the primary tumor as well as the successful development of metastasis. Upon distant stimulation from the primary tumor, BMDCs migrate from the bone marrow to organ sites and self-assemble into a pre-metastatic niche that hosts migrating tumor cells from the primary site. Thus, disruption of this niche or selective targeting the migratory tumor cells may inhibit metastatic spread. Using GFP+ BMDC's from genetically engineered mice, we characterized the role that recruitment of BMDCs may play in breast cancer metastasis. We assessed the BMDC recruitment profile in lethally irradiated female nu/nu mice transplanted with GFP+ BMDCs from donor mice, followed by orthotopic placement of MDA-MB-231/luc cells or injected with MDA-MB-231/luc conditioned media for 30 days. Flow cytometry results show a gradual increase in the recruitment of CD11b+VEGFR-1+ cells in all the tissues examined from tumor-bearing mice, particularly lungs and lymph nodes. Recruitment of these cells to the liver in mice treated only with MDA-MB-231/luc conditioned media was also apparent and at higher levels than to livers of mice with the orthotopically-placed tumors. Significant recruitment of CD11b+VEGFR-2+ BMDCs was observed to liver, lung and lymph nodes. Interestingly, MDA-MB-231/luc conditioned media appeared to recruit this subset of cells more strongly to liver and lung tissues than did signaling from orthotopic tumors, while spleen and lymph node showed less recruitment of CD11b+VEGFR-2+ BMDCs. Treatment with GrB/VEGF fusion construct - a novel granzyme B-based pro-apoptotic fusion protein which specifically targets cells harboring VEGFRs - indicated that GrB/VEGF normalized recruitment of VEGFR-1+ or VEFGR-2+ cells to lungs when assessed two weeks after the final treatment. GrB/VEGF treatment also increased recruitment of F4/80+ macrophages to the lung, including those with the CD11b+Gr-1- signature, which were otherwise significantly reduced. GrB/VEGF also impacted the BMDC profile of several cell lineages in the primary tumor. Studies are ongoing to understand the impact of GrB/VEGF systemic administration on lung metastasis. Identifying key players in modulating the formation of the pre-metastatic niche and the early development of metastasis will be critical in designing targeted therapeutic approaches to inhibit the metastatic process. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. Impacting early events in metastatic spread: Targeted therapeutics to disrupt formation of the pre-metastatic niche and development of lung metastases [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 4182.

Bone Marrow-Derived Cells Trafficking to the Oviduct: Effect of Ischemia-Reperfusion Injury.

The oviduct/fallopian tube is a crucial organ in the mammalian reproductive tract; it plays a critical role in gamete transportation and early embryo development. In women, torsion of the fallopian tubes can cause ischemia and reperfusion (IR) injury. In this study, we tested the effect of this injury on recruitment of bone marrow-derived cells (BMDCs) to the oviducts of reproductive age female mice. Bone marrow-derived cells were collected from ubiquitin-green fluorescent protein-positive male mice and transplanted into wild-type female mice. Ischemia and reperfusion injury was performed in half of the mice, while controls received equivalent surgery without oviduct injury. Two weeks following injury, recruitment of BMDCs to the oviducts was analyzed in both groups. Ischemia and reperfusion injury caused a greater than 2-fold increase in BMDC recruitment to the injured oviducts compared to those without injury. Specifically, the recruitment of BMDCs was localized to the stroma of the oviduct. We demonstrate that IR injury to oviduct recruits BMDCs to this tissue and suggest that BMDCs have function in the healing process.

LXHY formula inhibits choroidal neovascularization development via inhibiting the recruitment and adhesion of BMCs to the retina

Objective: The objective of this study is to investigate the inhibitory effect of LXHY, a Chinese medicine compound formula, on choroidal neovascularization (CNV) and to find the possible working mechanism. Methods: CNV was induced in C57BL/6 mice by krypton laser and bone marrow-derived cells (BMCs) isolated from enhanced green fluorescent protein (EGFP) transgenic mice were injected through tail vein 0.5–1 h after the laser surgery. The BMC-treated mice were randomly divided into two groups gavaged with either distilled water (DW group) or LXHY formula solution from day 1 after laser surgery. On days 7, 14, and 28 after treatment, histopathologic examination, fundus fluorescein angiography, and choroidal flatmount assay were performed to measure the CNV severity and BMC recruitment. CXCR4 levels in peripheral blood were measured by enzyme-linked immunosorbent assay. Stromal cell-derived factor-1α (SDF-1α), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) were detected by immunofluorescent staining. Results: On days 7 and 14 after treatment, CNV lesions in the LXHY-treated mice showed less recruitment of BMCs and were smaller in size compared to DW-treated mice. Histological examination also confirmed less severe CNV lesions in the LXHY group. CXCR4 levels in peripheral blood in the LXHY group were less than that of DW group on days 7 and 14. Moreover, the expression levels of SDF-1α, ICAM-1, and VCAM-1 at the lesion sites in the LXHY group were lower compared with the DW group. Conclusion: This experiment indicated that LXHY formula could inhibit CNV formation and development, probably by inhibiting the recruitment and attachment of BMCs into CNV area.

Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

PurposeWe investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)–derived cells to the neurosensory retina (NSR) and RPE layer.MethodsGFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 μm) with 30 applications were placed 50 to 100 μm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed.ResultsWithin 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle–dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid.ConclusionsSRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle–dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.

Abstract 1628: Early events in metastatic spread: new approaches using targeted therapeutics to disrupt formation of the pre-metastatic niche and development of lung metastases

Abstract Solid tumors release soluble factors causing migration of a subset of normal bone marrow derived cells (BMDCs) - primarily VEGFR-1+ hematopoietic progenitor cells and VEGFR-2+ circulating endothelial progenitor cells - from the bone marrow to organ sites. These normal BMDCs self-assemble into a pre-metastatic niche- a micro-environment eventually hosting migratory tumor cells from the primary site. Disruption of this niche or selective targeting the migratory tumor cells may inhibit metastatic spread. The significance and specific characteristics of the BMDCs is still unclear. We have previously observed migration of VEGFR-1+ BMDCs to the lung and lymph nodes, and VEGFR-2+ BMDCs to liver, lung and lymph nodes. Using GFP+ BMDC’s from genetically engineered mice, we characterized the role that recruitment of BMDCs may play in breast cancer metastasis. We assessed the BMDC recruitment profile in lethally irradiated female nu/nu mice transplanted with GFP+ BMDCs from donor mice, followed by orthotopic placement of MDA-MB-231/luc cells or injected with MDA-MB-231/luc conditioned media for 30 days. Flow cytometry results show a gradual increase in the recruitment of CD11b+VEGFR-1+ cells in all the tissues examined from tumor-bearing mice. Recruitment of these cells to the liver in mice treated only with MDA-MB-231/Luc conditioned media was also apparent, but the increase was not as high as in livers of mice with the orthotopically-placed tumors. Recruitment of CD11b+VEGFR-2+ BMDCs was also observed but only in liver and lung. Interestingly, conditioned media seemed to recruit this subset of cells more strongly to these two tissues than signaling from orthotopic tumors. Spleen and lymph node showed minimal recruitment of CD11b+VEGFR-2+ BMDCs. We have previously developed the GrB/VEGF fusion construct- a novel pro-apoptotic fusion protein which specifically targets cells harboring VEGFR2 and have utilized this agent to specifically target BMDCs which are VEGFR2 +. Our preliminary data indicate that treatment with GrB/VEGF does not significantly alter the recruitment of VEGFR-1+ or VEFGR-2+ cells to lungs, when assessed two weeks after the final treatment. We observed increased recruitment of F4/80+ macrophages to the lung. On the other hand, CD11b+Gr-1- BMDCs were significantly reduced following GrB/VEGF treatment, although the F4/80+ subpopulation carrying this signature increased. Studies are ongoing to determine whether systemic administration of this agent can disrupt the formation of the niche and the eventual establishment of metastatic tumors. Understanding the role of BMDCs in metastatic spread and the formation of the pre-metastatic niche and their role in the early development of metastasis will be critical in designing targeted therapeutic approaches to inhibit the metastatic process. Research sponsored, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. Early events in metastatic spread: new approaches using targeted therapeutics to disrupt formation of the pre-metastatic niche and development of lung metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1628. doi:10.1158/1538-7445.AM2017-1628