Prescription coverage in indigent patients affects the use of long-acting opioids in the management of cancer pain.

PURPOSE: We tested the hypothesis that prescription coverage affects the prescribing of long-acting opiates to indigent inner city minority patients with cancer pain. MATERIALS AND METHODS: We conducted a chart review of 360 patients treated in the Oncology Practice at University of Medicine and Dentistry of New Jersey University Hospital, who were prescribed opiate pain medications. Half the patients were charity care or self-pay (CC/SP), without the benefit of prescription coverage, and half had Medicaid, with unlimited prescription coverage. We evaluated patients discharged from a hospitalization, who had three subsequent outpatient follow-up visits. We compared demographics, pain intensity, the type and dose of opiates, adherence to prescribed pain regimen, unscheduled emergency department visits, and unscheduled hospitalizations. RESULTS: There was a significantly greater use of long-acting opiates in the Medicaid group than in the CC/SP group. The Medicaid group had significantly more African American patients and a greater rate of smoking and substance use, and the CC/SP group disproportionately more Hispanic and Asian patients and less smoking and substance use. Hispanic and Asian patients were less likely to have long-acting opiates prescribed to them. Pain levels and adherence were equivalent in both groups and were not affected by any of these variables except stage of disease, which was equally distributed in the two groups. CONCLUSION: Appropriate use of long-acting opiates for equivalent levels of cancer pain was influenced only by the availability of prescription coverage. The group without prescription coverage and receiving fewer long-acting opiates had disproportionately more Hispanic and Asian patients.

Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta.

Mesenchymal stem cells (MSCs) have been shown to support breast cancer growth. Because MSCs also increase the frequency of regulatory T cells (T(regs)), this study tested the hypothesis that human MSCs, via Tregs, protect breast cancer cells (BCCs) from immune clearance MSCs suppressed the proliferation of PBMCs when the latter were exposed to gamma-irradiated BCCs. Similarly, MSCs showed significant inhibition of PBMC migration toward BCCs and a corresponding decrease in CXCL12. MSCs also inhibited NK cell and CTL functions, which correlated with reduced numbers of CD8(+) and CD56(+) cells compared with parallel cultures without MSCs. The reduced NK and CTL activities correlated with a decrease in intracellular and secreted granzyme B. To explain these immunosuppressive findings, we compared T(reg) levels after coculture with MSCs and found an approximately 2-fold increase in T(regs), with associated decreases in antitumor Th1 cytokines and increases in Th2 cytokines. MSC-derived TGF-beta1 was largely responsible for the increase in T(regs) based on knockdown studies. In the presence of T(reg) depletion, PBMC proliferation and effector functions were partially restored. Together, these studies show an MSC-mediated increase in T(regs) in cocultures of PBMCs and BCCs. The results could be explained, in part, by the increase in Th2-type cytokines and MSC-generated TGF-beta1. These findings demonstrate immune protection by MSCs to BCCs. The reduction in immune cell proliferation and recruitment mediated by MSCs has implications for treatment of breast cancer with chemotherapy.

A pilot phase II trial of all-trans retinoic acid (Vesanoid) and paclitaxel (Taxol) in patients with recurrent or metastatic breast cancer.

PURPOSE: We investigated a combination therapy with weekly paclitaxel and all trans-retinoic acid (ATRA) for tolerability, response to treatment, time to progression and survival in previously treated patients with metastatic or recurrent breast cancer. Our rationale was based on preclinical studies demonstrating potentiation of the cytotoxic effects of taxanes and induction of differentiation by ATRA. PATIENTS AND METHODS: Seventeen patients with previously treated metastatic or recurrent breast cancer were enrolled to a regimen of all-trans retinoic acid (Vesanoid, tretinoin, Hoffman-La Roche, Inc.) 45 mg/m(2) PO daily for 4 days starting 2 days before a 1 h treatment with paclitaxel (Taxol, Bristol-Myers Squibb, Plainsboro, NJ) 80 mg/m(2) IV administered weekly for 3 weeks, repeated in 28 day cycles until disease progression or until no longer tolerated. Patients were evaluated for toxicity, response, time to progression and survival. Patients were primarily African American and Latino, representative of the population served by our Cancer Center. RESULTS: The regimen was relatively well tolerated. There were nine grade 3 and one grade 4 toxic events. We administered 162 treatment cycles with a mean of 7.5 per patient (range 1-22, median 5). Three patients had a partial response (17.6%) and ten patients had stable disease (58.8%), with an overall clinical benefit of 76.4%. Median time to progression was 6.0 months (range 1-21, mean 7.7 months). Fourteen evaluable patients had a median survival of 16 months (range 1-68 months, mean 25.2 months). CONCLUSIONS: The data suggest this is a well tolerated regimen with modest response rates but with time to progression and survival rates similar to those reported for paclitaxel alone and relatively high rates of stable disease in this sample of patients.

Specific N-cadherin-dependent pathways drive human breast cancer dormancy in bone marrow.

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region.

In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-ß-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention.

Non-canonical effects of anthrax toxins on haematopoiesis: implications for vaccine development.

Anthrax receptor (ATR) shares similarities with molecules relevant to haematopoiesis. This suggests that anthrax proteins might bind to these mimicking molecules and exert non-specific haematopoietic effects. The haematopoietic system is the site of immune cell development in the adult. As such, ATR ligand, protective antigen (PA) and the other anthrax proteins, lethal factor, edema factor, could be significant to haematopoietic responses against Bacillus anthracis infection. Because haematopoiesis is the process of immune cell development, effects by anthrax proteins could be relevant to vaccine development. Here, we report on effects of anthrax proteins and toxins on early and late haematopoiesis. Flow cytometry shows binding of PA to haematopoietic cells. This binding might be partly specific because flow cytometry and Western blots demonstrate the presence of ATR1 on haematopoietic cell subsets and the supporting stromal cells. Functional studies with long-term initiating cell and clonogenic assays determined haematopoietic suppression by anthrax toxins and stimulation by monomeric proteins. The suppressive effects were not attributed to cell death, but partly through the induction of haematopoietic suppressors, interleukin (IL)-10 and CCL3 (MIP-1alpha). In summary, anthrax proteins affect immune cell development by effects on haematopoiesis. The type of effect, stimulation or suppression, depend on whether the stimulator is a toxin or monomeric protein. The studies show effects of anthrax proteins beginning at the early stage of haematopoiesis, and also show secondary mediators such as IL-10 and CCL3. The roles of other cytokines and additional ATR are yet to be investigated.

Exosomes from differentially activated macrophages influence dormancy or resurgence of breast cancer cells within bone marrow stroma.

Breast cancer (BC) cells (BCCs) can retain cellular quiescence for decades, a phenomenon referred to as dormancy. BCCs show preference for the bone marrow (BM) where they can remain dormant for decades. Targeting BCCs within the BM is a challenge since the dormant BCCs reside within BM stroma, also residence for hematopoietic stem cells (HSCs). Dormant BCCs could behave as cancer stem cells (CSCs). The CSCs and HSCs are similar by function and also, by commonly expressed genes. The method by which dormant BCCs transition into clinically metastatic cells remains unclear. This study tested the hypothesis that macrophages (MΦs) within BM stroma, facilitates dormancy or reverse this state into metastatic cells. MΦs exhibiting an M2 phenotype constitute ~10% of cultured BM stroma. The M2 MΦs form gap junctional intercellular communication (GJIC) with CSCs, resulting in cycling quiescence, reduced proliferation and carboplatin resistance. In contrast, MΦs expressing the M1 phenotype reversed BC dormancy. Activation of M2a MΦs via the toll-like receptor 4 (TLR4) switched to M1 phenotype. The switch can occur by direct activation of M2a MΦs, or indirectly through activation of mesenchymal stem cells. M1 MΦ-derived exosomes activated NFкB to reverse quiescent BCCs to cycling cells. Using an in vivo model of BC dormancy, injected Mi MOs sensitized BCCs to carboplatin and increased host survival. In summary, we have shown how BM stromal MΦs, through exosomes, regulate the behavior of BCCs, by either inducing or reversing dormancy.

Phase I trial of pegylated liposomal doxorubicin with hyperthermic intraperitoneal chemotherapy in patients undergoing cytoreduction for advanced intra-abdominal malignancy.

BACKGROUND: Cytoreduction coupled with hyperthermic intraperitoneal chemotherapy (HIPEC) is an attractive treatment option for a select group of patients with abdominal-only malignancy. The present phase I study examined the safety and pharmacokinetics of intraperitoneal pegylated liposomal doxorubicin (PLD) used in the context of HIPEC in patients with advanced abdominal-only malignancies. METHODS: Patients with advanced abdominal malignancies underwent maximal cytoreduction and HIPEC with escalating doses of PLD (15-100 mg/m(2)). Perfusate, serum, and tissue doxorubicin levels were measured in five patients undergoing HIPEC at the maximum tolerated dose. RESULTS: Twenty-one patients were enrolled in this trial. The maximum dose evaluated in this trial was 100 mg/m(2) and was well tolerated. The most common grade 3/4 complications were superficial wound infection and prolonged ileus. One patient developed an anastomotic leak in the postoperative period, requiring re-exploration. The median postoperative length of stay was 7 days (range, 4-29 days), three patients required readmissions within 30 days, and there were no operative mortalities The median follow-up time for was 13.7 months (range, 3-38 months). The median overall survival was 30.6 months with a median disease-free survival of 25 months. CONCLUSIONS: We report that HIPEC with PLD following maximal cytoreduction in patients with advanced abdominal-only gastrointestinal or gynecologic malignancies is well tolerated. Encouraging survival after cytoreduction and HIPEC with PLD suggest that a phase II trial to verify activity is indicated.

Influence of prescription benefits on reported pain in cancer patients.

OBJECTIVE: To identify associations between prescription coverage and cancer pain and its sequelae in indigent patients. DESIGN AND SETTING: A retrospective chart review at UMDNJ-University Hospital. PATIENTS AND OUTCOME MEASURES: Charts from 20 patients with Medicaid and 20 patients categorized as Self-pay/Charity Care were analyzed for the influence of insurance coverage on reported pain at the time of a hospital discharge and at three subsequent clinic visits. Patient and disease characteristics, pain regimens, doses, reported pain and its impact were determined. RESULTS: The groups were statistically indistinguishable except for age and ethnicity. The Medicaid group was younger and had a majority of African Americans while the Self-pay/Charity Care patients had a majority of Hispanics. Lower doses of transdermal fentanyl were prescribed to Self-pay/Charity Care patients. Self-pay/Charity Care patients tended to report higher pain levels, but this was statistically significant only at the second clinic visit. The clinical significance of differences in pain intensity was reflected in differences in unscheduled visits and admissions. Adherence to pain regimens improved in the Medicaid group and diminished in the Self-pay/Charity Care group, but the differences did not achieve statistical significance. Lack of funds as the reason for non-adherence was only given by Self-pay/Charity Care patients. CONCLUSION: Indigent patients without prescription coverage trended toward reporting more cancer pain, received lower doses of transdermal fentanyl, and trended to lower adherence to pain regimens due to financial reasons. The trends observed in this pilot study will guide the design of a hypothesis-driven regression analysis.

Evaluation of a developmental hierarchy for breast cancer cells to assess risk-based patient selection for targeted treatment.

This study proposes that a novel developmental hierarchy of breast cancer (BC) cells (BCCs) could predict treatment response and outcome. The continued challenge to treat BC requires stratification of BCCs into distinct subsets. This would provide insights on how BCCs evade treatment and adapt dormancy for decades. We selected three subsets, based on the relative expression of octamer-binding transcription factor 4 A (Oct4A) and then analysed each with Affymetrix gene chip. Oct4A is a stem cell gene and would separate subsets based on maturation. Data analyses and gene validation identified three membrane proteins, TMEM98, GPR64 and FAT4. BCCs from cell lines and blood from BC patients were analysed for these three membrane proteins by flow cytometry, along with known markers of cancer stem cells (CSCs), CD44, CD24 and Oct4, aldehyde dehydrogenase 1 (ALDH1) activity and telomere length. A novel working hierarchy of BCCs was established with the most immature subset as CSCs. This group was further subdivided into long- and short-term CSCs. Analyses of 20 post-treatment blood indicated that circulating CSCs and early BC progenitors may be associated with recurrence or early death. These results suggest that the novel hierarchy may predict treatment response and prognosis.

Mesenchymal Stem Cell-Derived Exosomes Stimulate Cycling Quiescence and Early Breast Cancer Dormancy in Bone Marrow.

Dormant breast cancers resurge as metastatic disease after a long dormancy period in the bone marrow, where cancer cells interact with mesenchymal stem cells (MSC). However, the nature of early interactions between breast cancer cells and MSCs in the bone marrow microenvironment that facilitate adaptation to a quiescent state remains poorly understood. Here, we report that breast cancer cells prime MSC to release exosomes containing distinct miRNA contents, such as miR-222/223, which in turn promotes quiescence in a subset of cancer cells and confers drug resistance. Building on these results, we developed a novel, nontoxic therapeutic strategy to target dormant breast cancer cells based on systemic administration of MSC loaded with antagomiR-222/223. In an immunodeficient mouse model of dormant breast cancer, this therapy sensitized breast cancer cells to carboplatin-based therapy and increased host survival. Overall, our findings illuminate the nature of the regulatory interactions between breast cancer cells and MSCs in the evolution of tumor dormancy and resurgence in the micrometastatic microenvironment of the bone marrow. Cancer Res; 76(19); 5832-44. ©2016 AACR.

A Perspective of Immunotherapy for Breast Cancer: Lessons Learned and Forward Directions for All Cancers.

Immunotherapy for cancer has been a focus 50 years ago. At the time, this treatment was developed prior to cloning of the cytokines, no knowledge of regulatory T-cells, and very little information that mesenchymal stem cells (MSCs) (originally colony forming unit-fibroblasts [CFU-F]) could be licensed by the inflammatory microenvironment to suppress an immune response. Given the information available at that time, mononuclear cells from the peripheral blood were activated ex vivo and then replaced in the patients with tumor. The intent was to harness these activated immune cells to target the cancer cells. These studies did not lead to long-term responses because the activated cells when reinfused into the patients were an advantage to the resident MSCs, which can home the tumor and then become suppressive in the presence of the immune cells. The immune suppression caused by MSCs would also expand regulatory T-cells, resulting instead in tumor protection. As time progressed, these different fields converged into a new approach to use immunotherapy for cancer. This article discusses these approaches and also reviews chimeric antigen receptor in the context of future treatments for solid tumors, including breast cancer.

Phase I/II trial of accutane as a potentiator of carboplatin and paclitaxel in squamous cell carcinomas.

This study investigated the toxicity and efficacy of a 13-cis retinoic acid, carboplatin, and paclitaxel (Taxol) regimen in 18 patients with recurrent or metastatic squamous cell carcinomas (12 head and neck, 4 cervix, 1 esophagus, and 1 anus). Three patients were treated at each dose level with fenretamide (Accutane) 1 mg/kg/d orally for 14 days, carboplatin AUC of 5 mg/ml.min intravenously (IV) and paclitaxel at a dose of 135, 155, 175, 195, 205, or 225 mg/m(2) IV on day 8 every 4 weeks for 6 cycles. Fifteen evaluable patients had a total of 72 treatment cycles. There were 21 grade III or IV toxicities distributed among all the dose levels, including neutropenia, anemia, thrombocytopenia, elevated prothrombin time/partial thromboplastin time, elevated alkaline phosphatase, weight loss, alopecia, and three deaths from aspiration pneumonia and septic shock. The maximum tolerated dosage included 205 mg/m(2) paclitaxel. There was one complete response, three partial responses, and 2 stable diseases. The three partial responses were in the four patients with cervical cancer. Responses did not correlate with expression of retinoic acid receptor subtypes. Toxicity profiles and overall response rates were comparable to prior studies with similar chemotherapy regimens alone. The data support further study in a phase II trial.

Treg/Th17 polarization by distinct subsets of breast cancer cells is dictated by the interaction with mesenchymal stem cells.

Breast cancer (BC) cells (BCCs) exist within a hierarchy beginning with cancer stem cells (CSCs). Unsorted BCCs interact with mesenchymal stem cells (MSCs) to induce regulatory T cells (Tregs). This study investigated how distinct BCC subsets interacted with MSCs to polarize T-cell response, Tregs versus T helper 17 (Th17). This study tested BC initiating cells (CSCs) and the relatively more mature early and late BC progenitors. CSCs interacted with the highest avidity to MSCs. This interaction required CXCR4 and connexin 43 (Cx43)-dependant gap junctional intercellular communication (GJIC). This interaction induced Treg whereas interactions between MSCs and the progenitors induced Th17 response. The increases in Treg and Th17 depended on MSCs but not CTLA-4, which was increased in the presence of MSCs. Studies with BM stroma (fibroblasts) and MSCs from the same donors, indicated specific effects of MSCs. In total, MSC-CSC interaction required CXCR4 for GJIC. This led to increased Tregs and TGFß, and decreased Th17. In contrast, late and early BCCs showed reduced formation of GJIC, decreased Treg and increased Th17 and IL-17. These findings have significance to the methods by which CSCs evade the immune response. The findings could provide methods of intervention to reverse immune-mediated protection and support of BC.

Developmental regulation of TAC1 in peptidergic-induced human mesenchymal stem cells: implication for spinal cord injury in zebrafish.

Human mesenchymal stem cells (MSCs) are easy to expand, are relatively safe, and can be transplanted in allogeneic recipients as off-the-shelf cells. MSCs can be induced to form functional peptidergic neurons and express the neurotransmitter gene, TAC1. Expression of TAC1 requires that the repressor gene, RE-1 silencing transcription factor (REST), is decreased. This study investigated the molecular pathway in TAC1 induction as MSCs differentiated into neurons and then applied the findings in a model of spinal cord injury (SCI) in zebrafish. We studied the developmental roles of the 2 cAMP response element (CRE) sites: CRE1 and CRE2. Activator protein-1 (AP-1) binding site overlaps with CRE2 (CRE2/AP-1). Reporter gene studies with the 5' regulatory region of TAC1 containing wild-type or mutant CRE sites and, parallel studies with ectopically expressed inhibitor of cAMP proteins (inducible cAMP early repressor) indicated that CRE1 and CRE2/AP-1 are activated at days 6 and 12, respectively. Studies with protein kinase-A (PKA) and Jun N-terminal kinase (JNK) inhibitors in the reporter gene studies, chromatin immunoprecipation assay, and ectopic expression of REST indicated the following pathways: Decrease of REST activated upstream c-Jun N-terminal kinase (JNK). In turn, JNK activated ATF-2 and AP-1 for interaction with CRE1 and CRE2/AP-1, respectively. To apply the finding to SCI, we transplanted 6-day-induced MSCs in transgenic HB9-GFP zebrafish larvae with SCI, in the presence or absence of JNK inhibitors. Imaging and functional studies showed significant improvement in the fish. The repair mechanism involved the activation of JNK. The findings have long-term implications for SCI repair with MSCs.

Delineation of breast cancer cell hierarchy identifies the subset responsible for dormancy.

The bone marrow (BM) is a major organ of breast cancer (BC) dormancy and a common source of BC resurgence. Gap junctional intercellular communication (GJIC) between BC cells (BCCs) and BM stroma facilitates dormancy. This study reports on a hierarchy of BCCs with the most immature subset (Oct4(hi)/CD44(hi/med)/CD24(-/+)) demonstrating chemoresistance, dormancy, and stem cell properties: self-renewal, serial passaging ability, cycling quiescence, long doubling time, asymmetric division, high metastatic and invasive capability. In vitro and in vivo studies indicated that this subset was responsible for GJIC with BM stroma. Similar BCCs were detected in the blood of patients despite aggressive treatment and in a patient with a relatively large tumor but no lymph node involvement. In brief, these findings identified a novel BCC subset with stem cell properties, with preference for dormancy and in the circulation of patients. The findings establish a working cellular hierarchy of BCCs based on phenotype and functions.

AMD3100-mediated production of interleukin-1 from mesenchymal stem cells is key to chemosensitivity of breast cancer cells.

Breast cancer cells (BCCs) can remain quiescent for a long period, before detection and during remission. Mesenchymal stem cells (MSCs) exert both protective and growth support of BCCs. Intercellular interactions between MSCs and BCCs partly occur through membrane-bound CXCL12 (SDF-1α) and its receptor, CXCR4. MSCs can protect BCCs by suppressing immune cytotoxicity and concomitant induction of regulatory T-cells. This study investigated how the cellular interactions between MSCs and BCCs can be targeted to sensitize the BCCs to chemotherapy. Knockdown of CXCR4 and CXCL12 indicated that these molecules are involved in reduced proliferation of MDA-MB-231 and T47D BCCs. We therefore treated co-cultures of MSCs and BCCs with the CXCR4 antagonist, AMD3100, and showed that this treatment led to cycling of BCCs with increased sensitivity to carboplatin, although the effectiveness of carboplatin required the presence of AMD3100. Cytokine array analyses and transwell cultures indicated that AMD3100 caused an increase in BCC proliferation by inducing the production of IL-1α and IL-1ß in MSCs after uncoupling from BCCs. The findings with cell lines were validated with primary BCCs from the blood of patients, and in nude BALB/ c mice. MDA-MB-231 was injected in the dorsal flank of mice. The tumors were treated with IL-1 receptor antagonist, AMD3100 and/ or carboplatin. The results verified a critical role for IL-1 in transitioning MSCs from protective to supportive with respect to BCC growth. The clinical significance of these studies was further highlighted in preliminary studies that detected circulating MSCs in obese, but not non-obese patients. Since obese breast cancer patients show poor outcome, these findings underscore that importance of MSCs in consideration for future development of efficient therapy.

Gap junction-mediated import of microRNA from bone marrow stromal cells can elicit cell cycle quiescence in breast cancer cells.

Bone marrow (BM) metastasis of breast cancer (BC) can recur even decades after initial diagnosis and treatment, implying the long-term survival of disseminated cancer cells in a dormant state. Here we investigated the role of microRNAs (miRNA) transmitted from BM stroma to BC cells via gap junctions and exosomes in tumor cell quiescence. MDA-MB-231 and T47D BC cells arrest in G(0) phase of the cell cycle when cocultured with BM stroma. Analyses of miRNA expression profiles identified numerous miRNAs implicated in cell proliferation including miR-127, -197, -222, and -223 targeting CXCL12. Subsequently, we showed that these CXCL12-specific miRNAs are transported from BM stroma to BC cells via gap junctions, leading to reduced CXCL12 levels and decreased proliferation. Stroma-derived exosomes containing miRNAs also contributed to BC cell quiescence, although to a lesser degree than miRNAs transmitted via gap junctions. This study shows that the transfer of miRNAs from BM stroma to BC cells might play a role in the dormancy of BM metastases.

Mesenchymal stem cells in early entry of breast cancer into bone marrow.

BACKGROUND: An understanding of BC cell (BCC) entry into bone marrow (BM) at low tumor burden is limited when compared to highly metastatic events during heavy tumor burden. BCCs can achieve quiescence, without interfering with hematopoiesis. This occurs partly through the generation of gap junctions with BM stroma, located close to the endosteum. These events are partly mediated by the evolutionary conserved gene, Tac1. METHODOLOGY/PRINCIPAL FINDINGS: This study focuses on the role of mesenchymal stem cells (MSCs), Tac1, SDF-1 and CXCR4 in BCC entry into BM. The model is established in studies with low numbers of tumor cells, and focuses on cancer cells with low metastatic and invasion potential. This allowed us to recapitulate early event, and to study cancer cells with low invasive potential, even when they are part of larger numbers of highly metastatic cells. A novel migration assay showed a facilitating role of MSCs in BCC migration across BM endothelial cells. siRNA and ectopic expression studies showed a central role for Tac1 and secondary roles for SDF-1alpha and CXCR4. We also observed differences in the mechanisms between low invasive and highly metastatic cells. The in vitro studies were verified in xenogeneic mouse models that showed a preference for low invasive BCCs to BM, but comparable movement to lung and BM by highly metastatic BCCs. The expressions of Tac1 and production of SDF-1alpha were verified in primary BCCs from paired samples of BM aspirates and peripheral blood. CONCLUSIONS/SIGNIFICANCE: MSC facilitate BCC entry into BM, partly through Tac1-mediated regulation of SDF-1alpha and CXCR4. We propose a particular population of BCC with preference for BM could be isolated for characterization. This population might be the subset that enter BM at an early time period, and could be responsible for cancer resurgence and resistance to current therapies.