BRAF(V600E) mutation together with loss of Trp53 or pTEN drives the origination of hairy cell leukemia from B-lymphocytes.

Hairy cell leukemia (HCL) is a B-lymphoma induced by BRAF(V600E) mutation. However, introducing BRAF(V600E) in B-lymphocytes fails to induce hematological malignancy, suggesting that BRAF(V600E) needs concurrent mutations to drive HCL ontogeny. To resolve this issue, here we surveyed human HCL genomic sequencing data. Together with previous reports, we speculated that the tumor suppressor TP53, P27, or PTEN restrict the oncogenicity of BRAF(V600E) in B-lymphocytes, and therefore that their loss-of-function facilitates BRAF(V600E)-driven HCL ontogeny. Using genetically modified mouse models, we demonstrate that indeed BRAF(V600E)KI together with Trp53KO or pTENKO in B-lymphocytes induces chronic lymphoma with pathological features of human HCL. To further understand the cellular programs essential for HCL ontogeny, we profiled the gene expression of leukemic cells isolated from BRAF(V600E)KI and Trp53KO or pTENKO mice, and found that they had similar but different gene expression signatures that resemble that of M2 or M1 macrophages. In addition, we examined the expression signature of transcription factors/regulators required for germinal center reaction and memory B cell versus plasma cell differentiation in these leukemic cells and found that most transcription factors/regulators essential for these programs were severely inhibited, illustrating why hairy cells are arrested at a transitional stage between activated B cells and memory B cells. Together, our study has uncovered concurrent mutations required for HCL ontogeny, revealed the B cell origin of hairy cells and investigated the molecular basis underlying the unique pathological features of the disease, with important implications for HCL research and treatment.

Application of phage display for T-cell receptor discovery.

The immune system is tasked to keep our body unharmed and healthy. In the immune system, B- and T-lymphocytes are the two main components working together to stop and eliminate invading threats like virus particles, bacteria, fungi and parasite from attacking our healthy cells. The function of antibodies is relatively more direct in target recognition as compared to T-cell receptors (TCR) which recognizes antigenic peptides being presented on the major histocompatibility complex (MHC). Although phage display has been widely applied for antibody presentation, this is the opposite in the case of TCR. The cell surface TCR is a relatively large and complex molecule, making presentation on phage surfaces challenging. Even so, recombinant versions and modifications have been introduced to allow the growing development of TCR in phage display. In addition, the increasing application of TCR for immunotherapy has made it an important binding motif to be developed by phage display. This review will emphasize on the application of phage display for TCR discovery as well as the engineering aspect of TCR for improved characteristics.

The stability of R-spine defines RAF inhibitor resistance: A comprehensive analysis of oncogenic BRAF mutants with in-frame insertion of αC-ß4 loop.

Although targeting BRAF mutants with RAF inhibitors has achieved promising outcomes in cancer therapy, drug resistance remains a remarkable challenge, and underlying molecular mechanisms are not fully understood. Here, we characterized a previously unknown group of oncogenic BRAF mutants with in-frame insertions (LLRins506 or VLRins506) of αC-ß4 loop. Using structure modeling and molecular dynamics simulation, we found that these insertions formed a large hydrophobic network that stabilizes R-spine and thus triggers the catalytic activity of BRAF. Furthermore, these insertions disrupted BRAF dimer interface and impaired dimerization. Unlike BRAF(V600E), these BRAF mutants with low dimer affinity were strongly resistant to all RAF inhibitors in clinic or clinical trials, which arises from their stabilized R-spines. As predicted by molecular docking, the stabilized R-spines in other BRAF mutants also conferred drug resistance. Together, our data indicated that the stability of R-spine but not dimer affinity determines the RAF inhibitor resistance of oncogenic BRAF mutants.

IL13RA2 Is Differentially Regulated in Papillary Thyroid Carcinoma vs Follicular Thyroid Carcinoma.

CONTEXT: The interleukin-13 receptor alpha2 (IL13RA2), which is known to be overexpressed in glioblastoma multiforme, plays a role in various cellular processes such as cell migration that may contribute to tumor progression. Studies have attributed IL13RA2 to invasion and metastasis in cancers of the ovary, breast, and pancreas, but the pathological role of IL13RA2 in thyroid cancer is still unclear. OBJECTIVE: This study aims to evaluate IL13RA2 expression in thyroid carcinomas and to examine the role of IL13RA2 in the progression of papillary thyroid carcinoma (PTC). METHODS: IL13RA2 immunochemical staining was performed on tissue microarrays of 137 thyroid carcinomas from patients, and the differential profile of IL13RA2 was validated in thyroid cancer cell lines. In PTC cell lines, we functionally assessed the effects of IL13RA2 underexpression and overexpression on cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT) by using CCK-8, transwell migration assay, quantitative RT-PCR, and Western blot analysis. RESULTS: IL13RA2 expression was significantly correlated with advanced tumor T stage (pT3 or pT4; P = 0.001) and regional lymph node metastasis (pN1; P < 0.001). The staining scores of IL13RA2 were significantly higher in PTC compared with follicular subtypes (P < 0.001) and correlated with advanced tumor stage among PTC samples (pT3 or pT4; P = 0.028). Knockdown of IL13RA2 in B-CPAP cells significantly reduced cell viability, cell migration, and EMT markers including N-cadherin, Vimentin, and Snail. Exogenous overexpression of IL13RA2 in K1 cells increased cell migration and EMT, although cell proliferation was not affected. CONCLUSION: IL13RA2 is differentially regulated in PTC and is involved in cell migration by enhancing EMT.

Ultrafast Single-Cell Level Enzymatic Tumor Profiling.

In the context of tumor analysis, the implementation of precision medicine requires on-time clinical measurements, which requires rapid large-scale single-cell screening that obtains cell population distributions and functions in tumors to determine disease progression for therapeutics. In this study, a high-throughput screening (HTS) platform integrating optical fluorescence detectors and a computational method was developed as a droplet-based microfluidic flow cytometer (Droplet-µFC) to comprehensively analyze multiple proteolytic activities of a patient-derived tumor (with ∼0.5-2 M cells) at single-cell resolution within 2 h. The data-driven analytical method identified distinct cell types and status through protease profiling with high precision. Multiple protease activities of single cells harvested from a tumor were thus determined with a throughput of ∼100 cells per second. This platform was used to screen protease activities of a wide range of cell types, forming a library. With the development of advanced computational clustering and cell mapping, rapid quantitative tumor profiling with a comprehensive description of cell population distributions and functions could be obtained for clinical treatments.

Intra-patient and inter-patient comparisons of DNA damage response biomarkers in Nasopharynx Cancer (NPC): analysis of NCC0901 randomised controlled trial of induction chemotherapy in locally advanced NPC.

BACKGROUND: Inter-patient heterogeneity in radiation-induced DNA damage responses is proposed to reflect intrinsic variations in tumour and normal tissue radiation sensitivity, but the prediction of phenotype by a molecular biomarker is influenced by clinical confounders and assay reproducibility. Here, we characterised the intrapatient and inter-patient heterogeneity in biomarkers of DNA damage and repair and radiation-induced apoptosis. METHODS: We enrolled 85 of 172 patients with locally advanced nasopharynx cancer from a randomised controlled phase II/III trial of induction chemotherapy added to chemo-radiotherapy. G0 blood lymphocytes were harvested from these patients, and irradiated with 1, 4, and 8 Gy ex vivo. DNA damage induction (1 Gy 0.5 h) and repair (4 Gy 24 h) were assessed by duplicate γH2AX foci assays in 50-100 cells. Duplicate FLICA assays performed at 48 h post-8 Gy were employed as surrogate of radiation-induced apoptosis; %FLICA-positive cells were quantified by flow cytometry. RESULTS: We observed limited intrapatient variation in γH2AX foci and %FLICA readouts; median difference of duplicate foci scores was - 0.37 (IQR = - 1.256-0.800) for 1 Gy 0.5 h and 0.09 (IQR = - 0.685-0.792) for 4 Gy 24 h; ICC of ≥0.80 was observed for duplicate %FLICA0Gy and %FLICA8Gy assays of CD4+ and CD8+ T lymphocytes. As expected, we observed wide inter-patient heterogeneity in both assays that was independent of intrapatient variation and clinical covariates, with the exception of age, which was inversely correlated with %FLICAbackground-corrected (Spearman R = - 0.406, P < 0.001 [CD4+]; R = - 0.220, P = 0.04 [CD8+]). Lastly, an exploratory case-control analysis indicates increased levels of γH2AX foci at 4 Gy 24 h in patients with severe late radiotherapy-induced xerostomia (P = 0.05). CONCLUSION: Here, we confirmed the technical reproducibility of DNA damage response assays for clinical implementation as biomarkers of clinical radiosensitivity in nasopharynx cancer patients.

The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants.

Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the ß3-αC loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.

Matrix metalloproteinase-1 facilitates MSC migration via cleavage of IGF-2/IGFBP2 complex.

The specific mechanism underlying the tumor tropism of human mesenchymal stem cells (MSCs) for cancer is not well defined. We previously showed that the migration potential of MSCs correlated with the expression and protease activity of matrix metalloproteinase (MMP)-1. Furthermore, highly tumor-tropic MSCs expressed higher levels of MMP-1 and insulin-like growth factor (IGF)-2 than poorly migrating MSCs. In this study, we examined the functional roles of IGF-2 and MMP-1 in mediating the tumor tropism of MSCs. Exogenous addition of either recombinant IGF-2 or MMP-1 could stimulate MSC migration. The correlation between IGF-2, MMP-1 expression, and MSC migration suggests that MMP-1 may play a role in regulating MSC migration via the IGF-2 signaling cascade. High concentrations of IGF binding proteins (IGFBPs) can inhibit IGF-stimulated functions by blocking its binding to its receptors and proteolysis of IGFBP is an important mechanism for the regulation of IGF signaling. We thus hypothesized that MMP-1 acts as an IGFBP2 proteinase, resulting in the cleavage of IGF-2/IGFBP2 complex and extracellular release of free IGF-2. Indeed, our results showed that conditioned media from highly migrating MSCs, which expressed high levels of MMP-1, cleaved the IGF-2/IGFBP2 complex. Taken together, these results showed that the MMP-1 secreted by highly tumor-tropic MSCs cleaved IGF-2/IGFBP2 complex. Free IGF-2 released from the complex may facilitate MSC migration toward tumor.

Interleukin-13 receptor alpha 2 cooperates with EGFRvIII signaling to promote glioblastoma multiforme.

The interleukin-13 receptor alpha2 (IL-13Rα2) is a cancer-associated receptor overexpressed in human glioblastoma multiforme (GBM). This receptor is undetectable in normal brain which makes it a highly suitable target for diagnostic and therapeutic purposes. However, the pathological role of this receptor in GBM remains to be established. Here we report that IL-13Rα2 alone induces invasiveness of human GBM cells without affecting their proliferation. In contrast, in the presence of the mutant EGFR (EGFRvIII), IL-13Rα2 promotes GBM cell proliferation in vitro and in vivo. Mechanistically, the cytoplasmic domain of IL-13Rα2 specifically binds to EGFRvIII, and this binding upregulates the tyrosine kinase activity of EGFRvIII and activates the RAS/RAF/MEK/ERK and STAT3 pathways. Our findings support the "To Go or To Grow" hypothesis whereby IL-13Rα2 serves as a molecular switch from invasion to proliferation, and suggest that targeting both receptors with STAT3 signaling inhibitor might be a therapeutic approach for the treatment of GBM.

Exploiting molecular genomics in precision radiation oncology: a marriage of biological and physical precision.

Achieving local tumour control is paramount for cure in head and neck and prostate cancers. With the transition to precision radiotherapy (RT) techniques, survival rates have improved in the majority of these cancers, but a substantial proportion of 30-40% still relapse following primary treatment. Recent large-scale molecular profiling studies have revealed unique biological events that could explain for tumour aggression and resistance to therapies, redefining the molecular taxonomy of head and neck and prostate cancers. Here, we reviewed the key findings from these studies, highlighting those relevant for clinical stratification. We also proposed novel combinatorial clinicomolecular models to identify subsets of patients with aggressive localised tumours and limited metastases, and to inform on the optimal management of these patients using molecular targeted agents, immunotherapy, and RT.

Human mesenchymal stem cells preferentially migrate toward highly oncogenic human hepatocellular carcinoma cells with activated EpCAM signaling.

The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein that is regarded as one of the markers for tumor initiating cells (TIC) in human hepatocellular carcinoma (HCC). Much work has been directed towards targeting these TICs as a mean of placing these master regulators of cell proliferation and drug resistance under control. Human bone marrow-derived mesenchymal stem cells are known to exhibit an innate property of tumor tropism. However, the possible relationship between MSC and TIC is not well understood. In this study, we show that MSC migration to HCC can be effectively inhibited by TACE and γ-secretase inhibitors that stop the activation of EpCAM signaling event. Silencing of EpCAM expression through siRNA and antibody approaches also resulted in impaired MSC migration. By contrast, increase levels of EpICD proteins in HCC cells and HCC mouse xenografts resulted in enhanced MSC migration. Taken together, these findings show that MSC is drawn to the more oncogenic population of HCC, and could potentially serve as a cell-based carrier of therapeutic genes to target EpICD-enriched hepatic tumor cells.

Recent discoveries concerning the tumor - mesenchymal stem cell interactions.

Tumor microenvironment plays a crucial role in coordination with cancer cells in the establishment, growth and dissemination of the tumor. Among cells of the microenvironment, mesenchymal stem cells (MSCs) and their ability to evolve into cancer associated fibroblasts (CAFs) have recently generated a major interest in the field. Numerous studies have described the potential pro- or anti-tumorigenic action of MSCs. The goal of this review is to synthesize recent and emerging discoveries concerning the mechanisms by which MSCs can be attracted to tumor sites, how they can generate CAFs and by which way MSCs are able to modulate the growth, response to treatments, angiogenesis, invasion and metastasis of tumors. The understanding of the role of MSCs in tumor development has potential and clinical applications in terms of cancer management.

Soluble Factors from Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells: Preparation of Conditioned Medium and Its Effect on Tumor Cells.

Mesenchymal stem cells (MSCs) possess some unique features (inherent tumor tropism, anti-inflammatory and immunosuppressive properties) that are not commonly found in conventional anti-cancer agents. These cells are known to secrete a vast array of proteins including growth factors, cytokines, chemokines, extracellular matrix metalloproteinases, and their corresponding inhibitors which exhibit profound effects on the microenvironment. However, the lack of a uniform method for culturing MSCs and their paracrine factors has hindered our understanding of MSC biology. In this chapter, we describe methods for the isolation, in vivo expansion, and phenotypic characterization of MSCs. In addition, methods for the collection and concentration of conditioned medium from these MSCs are described. Using tumor cells that constitutively express fluorescence reporter proteins, the effect of conditioned medium on tumor cell viability can be easily tested in vitro.

Isolating dividing neural and brain tumour cells for gene expression profiling.

BACKGROUND: The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints. NEW METHOD: We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis. RESULTS: 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression. COMPARISON WITH EXISTING METHOD: BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA. CONCLUSIONS: The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation.

Transcriptional profiling of dividing tumor cells detects intratumor heterogeneity linked to cell proliferation in a brain tumor model.

Intratumor heterogeneity is a primary feature of high-grade gliomas, complicating their therapy. As accumulating evidence suggests that intratumor heterogeneity is a consequence of cellular subsets with different cycling frequencies, we developed a method for transcriptional profiling of gliomas, using a novel technique to dissect the tumors into two fundamental cellular subsets, namely, the proliferating and non-proliferating cell fractions. The tumor fractions were sorted whilst maintaining their molecular integrity, by incorporating the thymidine analog 5-ethynyl-2'-deoxyuridine into actively dividing cells. We sorted the actively dividing versus non-dividing cells from cultured glioma cells, and parental and clonally derived orthotopic tumors, and analyzed them for a number of transcripts. While there was no significant difference in the transcriptional profiles between the two cellular subsets in cultured glioma cells, we demonstrate ∼2-6 fold increase in transcripts of cancer and neuronal stem cell and tumor cell migration/invasion markers, and ∼2-fold decrease in transcripts of markers of hypoxia and their target genes, in the dividing tumor cells of the orthotopic glioma when compared to their non-proliferative counterparts. This suggests the influence of the brain microenvironment in transcriptional regulation and, thereby, the physiology of glioma cells in vivo. When clonal glioma cells were derived from a parental glioma and the resultant orthotopic tumors were compared, their transcriptional profiles were closely correlated to tumor aggression and consequently, survival of the experimental animals. This study demonstrates the resolution of intratumor heterogeneity for profiling studies based on cell proliferation, a defining feature of cancers, with implications for treatment design.

Redox-Active Mn Porphyrin-based Potent SOD Mimic, MnTnBuOE-2-PyP(5+), Enhances Carbenoxolone-Mediated TRAIL-Induced Apoptosis in Glioblastoma Multiforme.

Glioblastoma multiforme is the most malignant tumor of the brain and is challenging to treat due to its highly invasive nature and heterogeneity. Malignant brain tumor displays high metabolic activity which perturbs its redox environment and in turn translates to high oxidative stress. Thus, pushing the oxidative stress level to achieve the maximum tolerable threshold that induces cell death is a potential strategy for cancer therapy. Previously, we have shown that gap junction inhibitor, carbenoxolone (CBX), is capable of enhancing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis in glioma cells. Since CBX is known to induce oxidative stress, we hypothesized that the addition of another potent mediator of oxidative stress, powerful SOD mimic MnTnBuOE-2-PyP(5+) (MnBuOE), could further enhance TRAIL-driven therapeutic efficacy in glioma cells. Our results showed that combining TRAIL + CBX with MnBuOE significantly enhances cell death of glioma cell lines and this enhancement could be further potentiated by CBX pretreatment. MnBuOE-driven cytotoxicity is due to its ability to take advantage of oxidative stress imposed by CBX + TRAIL system, and enhance it in the presence of endogenous reductants, ascorbate and thiol, thereby producing cytotoxic H2O2, and in turn inducing death of glioma cells but not normal astrocytes. Most importantly, combination treatment significantly reduces viability of TRAIL-resistant Asian patient-derived glioma cells, thus demonstrating the potential clinical use of our therapeutic system. It was reported that H2O2 is involved in membrane depolarization-based sensitization of cancer cells toward TRAIL. MnBuOE is entering Clinical Trials as a normal brain radioprotector in glioma patients at Duke University increasing Clinical relevance of our studies.

Combined treatment of Nimotuzumab and rapamycin is effective against temozolomide-resistant human gliomas regardless of the EGFR mutation status.

BACKGROUND: The treatment of glioblastoma multiforme (GBM) is an unmet clinical need. The 5-year survival rate of patients with GBM is less than 3%. Temozolomide (TMZ) remains the standard first-line treatment regimen for gliomas despite the fact that more than 90% of recurrent gliomas do not respond to TMZ after repeated exposure. We have also independently shown that many of the Asian-derived glioma cell lines and primary cells derived from Singaporean high-grade glioma patients are indeed resistant to TMZ. This issue highlights the need to develop new effective anti-cancer treatment strategies. In a recent study, wild-type epidermal growth factor receptor (wtEGFR) has been shown to phosphorylate a truncated EGFR (known as EGFRvIII), leading to the phosphorylation of STAT proteins and progression in gliomagenesis. Despite the fact that combination of EGFR targeting drugs and rapamycin has been used before, the effect of mono-treatment of Nimotuzumab, rapamycin and combination therapy in human glioma expressing different types of EGFR is not well-studied. Herein, we evaluated the efficacy of dual blockage using monoclonal antibody against EGFR (Nimotuzumab) and an mTOR inhibitor (rapamycin) in Caucasian patient-derived human glioma cell lines, Asian patient-derived human glioma cell lines, primary glioma cells derived from the Mayo GBM xenografts, and primary short-term glioma culture derived from high-grade glioma patients. METHODS: The combination effect of Nimotuzumab and rapamycin was examined in a series of primary human glioma cell lines and glioma cell lines. The cell viability was compared to TMZ treatment alone. Endogenous expressions of EGFR in various GBM cells were determined by western blotting. RESULTS: The results showed that combination of Nimotuzumab with rapamycin significantly enhanced the therapeutic efficacy of human glioma cells compared to single treatment. More importantly, many of the Asian patient-derived glioma cell lines and primary cells derived from Singaporean high-grade gliomas, which showed resistance to TMZ, were susceptible to the combined treatments. CONCLUSIONS: In conclusion, our results strongly suggest that combination usage of Nimotuzumab and rapamycin exert higher cytotoxic activities than TMZ. Our data suggest that this combination may provide an alternative treatment for TMZ-resistant gliomas regardless of the EGFR status.

Paracrine factors of human fetal MSCs inhibit liver cancer growth through reduced activation of IGF-1R/PI3K/Akt signaling.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world. The multikinase inhibitor sorafenib only demonstrated marginal improvement in overall survival for advanced disease prompted the search for alternative treatment options. Human mesenchymal stem cells (MSCs) have the ability to home to tumor cells. However, its functional roles on the tumor microenvironment remain controversial. Herein, we showed that conditioned media derived from human fetal MSC (CM-hfMSCs) expressed high level of the insulin growth factor binding proteins IGFBPs and can sequester free insulin-like growth factors (IGFs) to inhibit HCC cell proliferation. The inhibitory effect of IGFBPs on IGF signaling was further evident from the reduction of activated IGF-1R and PI3K/Akt, leading eventually to the induction of cell cycle arrest. We also demonstrated that CM-hfMSCs could enhance the therapeutic efficacy of sorafenib and sunitinib. To the best of our knowledge, this is the first report to show that CM-hfMSCs has a tumor-specific, antiproliferative effect that is not observed with normal human hepatocyte cells and patient-derived matched normal tissues. Our results thus suggest that CM-hfMSCs can provide a useful tool to design alternative/adjuvant treatment strategies for HCC, especially in related function to potentiate the effects of chemotherapeutic drugs.

Matrix metalloproteinase-1-mediated mesenchymal stem cell tumor tropism is dependent on crosstalk with stromal derived growth factor 1/C-X-C chemokine receptor 4 axis.

Human bone marrow-derived mesenchymal stem cells (MSCs) have the unique ability to home toward injuries or tumor sites. We have previously shown that the tumor-tropic property is dependent on the intrinsic expression and activity of the matrix remodeling gene, matrix metalloproteinase 1 (MMP-1). Herein, crosstalk between MMP-1/protease activated receptor 1 (PAR-1) and the G-protein coupled receptor stromal-derived growth factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR-4) in facilitating cell migration was investigated. Gain-of-function and RNA interference (RNAi) technology were used to evaluate the interplay between the key players. The downstream effect on the tumor-tropic migration of MSCs was investigated using modified Boyden chamber assay. Neutralizing PAR-1 activation using monoclonal antibody and targeted knockdown of MMP-1 using RNAi resulted in decreased expression of SDF-1, which was not observed in control-RNAi-transfected cells. Overexpression of CXCR-4 failed to promote MSC migration; the percentage of migrated cells toward tumor cell conditioned medium was similar to the vector-transduced and the CXCR-4-transduced MSCs. Furthermore, inhibition of SDF-1/CXCR-4 signaling using AMD3100 reduced MSC migration through the deregulation of MMP-1 promoter activities, protein expression, and metalloproteinase activity. Collectively, our results showed that MMP-1-mediated MSC tumor tropism is dependent on crosstalk with the SDF-1/CXCR-4 axis.

Signaling molecules and pathways involved in MSC tumor tropism.

Human bone marrow is a reservoir containing cells with different self-renewal capabilities, such as mesenchymal stem cells (MSC) and hematopoeitic stem cells (HSC). MSC in particular have been increasingly used in preclinical and clinical treatment of tissue regenerative disorder. Understanding the molecular mechanisms underlying MSC homing and mobilization is critical to the design of rational cell therapy approaches. In this review, we will discuss the key molecular mechanisms that govern the homing of MSC to bone marrow, the mobilization of MSC to tumors and injured sites via circulation, and strategies that enhance MSC migration.