Bone marrow derived cells in tumor angiogenesis and growth: are they the good,the bad or the evil?

Increasing evidence implicates an important role for a variety of bone marrow derived cells (BMDCs) in tumor angiogenesis and metastatic tumor growth. These cells are derived either from the hematopoietic or mesenchymal cell lineage, and they are distinguished, in part, by the expression of the panhematopoietic marker ‐ CD45. Some of these cell populations can colonize tumors perivascularily, and appear to promote angiogenesis and tumor cell proliferation by paracraine mechanisms, whereas others can contribute “directly” to the growth of tumor vessel capillaries or metastases. In this review we focus in particular on the role of hemangiocytes or recruited bone marrow derived circulating cells (RBCCs) in neovascularization, the contribution of VEGFR1+ hematopoietic stem cells and endothelial precursor cells in metastasis, and the involvement of myeloid derived suppressor CD11b+/Gr‐1+ cells in the resistance of tumors to certain antiangiogenic drugs, e.g., VEGF blocking antibodies.     

T Cells?Induce Pre-Metastatic Osteolytic?Disease and Help Bone Metastases Establishment in a Mouse Model of Metastatic Breast Cancer

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. Here we show that tumor-specific T cells induce osteolytic bone disease before bone colonization. T cells pro-metastatic activity correlate with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocks bone loss and metastasis. Our results unveil an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.     

The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase

A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-kappaB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3-TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.     

Prostate cancer-derived exosomes promote osteoblast differentiation and activity through phospholipase D2

Prostate cancer (PCa) is the most frequent cancer in men aged 65 and over. PCa mainly metastasizes in the bone, forming osteosclerotic lesions, inducing pain, fractures, and nerve compression. Cancer cell-derived exosomes participate in the metastatic spread, ranging from oncogenic reprogramming to the formation of pre-metastatic niches. Moreover, exosomes were recently involved in the dialog between PCa cells and the bone metastasis microenvironment. Phospholipase D (PLD) isoforms PLD1/2 catalyze the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), regulating tumor progression and metastasis. PLD is suspected to play a role in exosomes biogenesis. We aimed to determine whether PCa-derived exosomes, through PLD, interact with the bone microenvironment, especially osteoblasts, during the metastatic process. Here we demonstrate for the first time that PLD2 is present in exosomes of C4-2B and PC-3 cells. C4-2B-derived exosomes activate proliferation and differentiation of osteoblasts models, by stimulating ERK 1/2 phosphorylation, by increasing the tissue-nonspecific alkaline phosphatase activity and the expression of osteogenic differentiation markers. Contrariwise, when C4-2B exosomes are generated in the presence of halopemide, a PLD pan-inhibitor, they lose their ability to stimulate osteoblasts. Furthermore, the number of released exosomes diminishes significantly (−40%). When the PLD product PA is combined with halopemide, exosome secretion is fully restored. Taken together, our results indicate that PLD2 stimulates exosome secretion in PCa cell models as well as their ability to increase osteoblast activity. Thus, PLD2 could be considered as a potent player in the establishment of PCa bone metastasis acting through tumor cell derived-exosomes.     

Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts

Tumor metastasis is a hallmark of cancer. The communication between cancer-derived exosomes and stroma plays an irreplaceable role in facilitating pre-metastatic niche formation and cancer metastasis. However, the mechanisms underlying exosome-mediated pre-metastatic niche formation during colorectal cancer (CRC) liver metastasis remain incompletely understood. Here we identified HSPC111 was the leading upregulated gene in hepatic stellate cells (HSCs) incubated with CRC cell-derived exosomes. In xenograft mouse model, CRC cell-derived exosomal HSPC111 facilitated pre-metastatic niche formation and CRC liver metastases (CRLM). Consistently, CRC patients with liver metastasis had higher level of HSPC111 in serum exosomes, primary tumors and cancer-associated fibroblasts (CAFs) in liver metastasis than those without. Mechanistically, HSPC111 altered lipid metabolism of CAFs by phosphorylating ATP-citrate lyase (ACLY), which upregulated the level of acetyl-CoA. The accumulation of acetyl-CoA further promoted CXCL5 expression and secretion by increasing H3K27 acetylation in CAFs. Moreover, CXCL5-CXCR2 axis reinforced exosomal HSPC111 excretion from CRC cells and promoted liver metastasis. These results uncovered that CRC cell-derived exosomal HSPC111 promotes pre-metastatic niche formation and CRLM via reprogramming lipid metabolism in CAFs, and implicate HSPC111 may be a potential therapeutic target for preventing CRLM.Subject terms: Cancer metabolism, Metastasis, Epithelial-mesenchymal transition     

Oxidative stress regulates expression of VEGFR1 in myeloid cells: link to tumor-induced immune suppression in renal cell carcinoma

Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.     

Extracellular vesicle-mediated transport: Reprogramming a tumor microenvironment conducive with breast cancer progression and metastasis

Breast cancer metastatic progression to critical secondary sites is the second leading cause of cancer-related mortality in women. While existing therapies are highly effective in combating primary tumors, metastatic disease is generally deemed incurable with a median survival of only 2, 3 years. Extensive efforts have focused on identifying metastatic contributory targets for therapeutic antagonism and prevention to improve patient survivability. Excessive breast cancer release of extracellular vesicles (EVs), whose contents stimulate a metastatic phenotype, represents a promising target. Complex breast cancer intercellular communication networks are based on EV transport and transference of molecular information is in bulk resulting in complete reprogramming events within recipient cells. Other breast cancer cells can acquire aggressive phenotypes, endothelial cells can be induced to undergo tubule formation, and immune cells can be neutralized. Recent advancements continue to implicate the critical role EVs play in cultivating a tumor microenvironment tailored to cancer proliferation, metastasis, immune evasion, and conference of drug resistance. This literature review serves to frame the role of EV transport in breast cancer progression and metastasis. The following five sections will be addressed: (1) Intercellular communication in developing a tumor microenvironment & pre-metastatic niche. (2) Induction of the epithelial-to-mesenchymal transition (EMT). (3). Immune suppression & evasion. (4) Transmission of drug resistance mechanisms. (5) Precision medicine: clinical applications of EVs.     

LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

Gastric cancer is anatomically proximal to peritoneum. Gastric cancer peritoneal metastasis is a complex biological process which is corresponded with disharmony within dysfunctional adipose tissue and metabolism reprogramming. Laminin gamma 1 (LAMC1) is highly expressed in cancer cells of peritoneal metastatic sites, however, the mechanism of LAMC1-metiated gastric cancer metastases to adipose tissue-rich peritoneum remains unclear. In our study, immunohistochemical staining, single cell sequencing, a co-culture model, luciferase reporter, RNA immunoprecipitation (RIP), Chromatin immunoprecipitation (CHIP) and single-molecular magnetic tweezers assays were conducted, and our results showed that LAMC1 related to Perilipin-1 content was highly expressed in peritoneal metastatic sites and mainly secreted by tumor cells. Gastric cancer cells secreted LAMC1 in an autocrine manner to detached from the primary site and promoted preadipocytes mature, rupture and release of free fatty acids (FFAs) in the peritoneal microenvironment to form pre-metastatic niche by the paracrine pathway. Reversely, differentiated preadipocyte-derived conditioned medium inhibited glycolysis and enhanced fatty acid oxidation (FAO) rate to promote cell proliferation, mesenchymal-epithelial transformation which led to tumor peritoneal colonization. In terms of biological mechanisms, one of differentiated preadipocyte-derived FFAs, palmitic acid-activated STAT3 inhibited miR-193a-3p by binding to its promoter directly; Using single-molecular magnetic tweezers, this binding manner was proved to be stable, reversable and ATP-dependent. Moreover, miR-193a-3p regulated LAMC1 in a post-translational manner. Furthermore, high LAMC1 expression in serum predicted a higher risk of peritoneal metastasis. In conclusion, our results illustrated that palmitic acid/p-STAT3/miR-193a-3p/LAMC1 pathway promotes preadipocyte differentiation, pre-metastatic niche formation and gastric cancer cell colonization to peritoneum.     

FRIZZLED7 Is Required for Tumor Inititation and Metastatic Growth of Melanoma Cells

Metastases are thought to arise from cancer stem cells and their tumor initiating abilities are required for the establishment of metastases. Nevertheless, in metastatic melanoma, the nature of cancer stem cells is under debate and their contribution to metastasis formation remains unknown. Using an experimental metastasis model, we discovered that high levels of the WNT receptor, FZD7, correlated with enhanced metastatic potentials of melanoma cell lines. Knocking down of FZD7 in a panel of four melanoma cell lines led to a significant reduction in lung metastases in animal models, arguing that FZD7 plays a causal role during metastasis formation. Notably, limiting dilution analyses revealed that FZD7 is essential for the tumor initiation of melanoma cells and FZD7 knockdown impeded the early expansion of metastatic melanoma cells shortly after seeding, in accordance with the view that tumor initiating ability of cancer cells is required for metastasis formation. FZD7 activated JNK in melanoma cell lines in vitro and the expression of a dominant negative JNK suppressed metastasis formation in vivo, suggesting that FZD7 may promote metastatic growth of melanoma cells via activation of JNK. Taken together, our findings uncovered a signaling pathway that regulates the tumor initiation of melanoma cells and contributes to metastasis formation in melanoma.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.     

Blockade of Fas Signaling in Breast Cancer Cells Suppresses Tumor Growth and Metastasis via Disruption of Fas Signaling-initiated Cancer-related Inflammation

Mechanisms for cancer-related inflammation remain to be fully elucidated. Non-apoptotic functions of Fas signaling have been proposed to play an important role in promoting tumor progression. It has yet to be determined if targeting Fas signaling can control tumor progression through suppression of cancer-related inflammation. In the current study we found that breast cancer cells with constitutive Fas expression were resistant to apoptosis induction by agonistic anti-Fas antibody (Jo2) ligation or Fas ligand cross-linking. Higher expression of Fas in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. To determine whether blockade of Fas signaling in breast cancer could suppress tumor progression, we prepared an orthotopic xenograft mouse model with mammary cancer cells 4T1 and found that blockade of Fas signaling in 4T1 cancer cells markedly reduced tumor growth, inhibited tumor metastasis in vivo, and prolonged survival of tumor-bearing mice. Mechanistically, blockade of Fas signaling in cancer cells significantly decreased systemic or local recruitment of myeloid derived suppressor cells (MDSCs) in vivo. Furthermore, blockade of Fas signaling markedly reduced IL-6, prostaglandin E2 production from breast cancer cells by impairing p-p38, and activity of the NFκB pathway. In addition, administration of a COX-2 inhibitor and anti-IL-6 antibody significantly reduced MDSC accumulation in vivo. Therefore, blockade of Fas signaling can suppress breast cancer progression by inhibiting proinflammatory cytokine production and MDSC accumulation, indicating that Fas signaling-initiated cancer-related inflammation in breast cancer cells may be a potential target for treatment of breast cancer.     

Contribution of myeloid-derived suppressor cells to tumor-induced immune suppression, angiogenesis, invasion and metastasis

Growing evidence suggests that myeloid-derived suppressor cells (MDSCs), which have been named "immature myeloid cells" or "myeloid suppressor cells" (MSCs), play a critical role during the progression of cancer in tumor-bearing mice and cancer patients. As their name implies, these cells are derived from bone marrow and have a tremendous potential to suppress immune responses. Recent studies indicated that these cells also have a crucial role in tumor progression. MDSCs can directly incorporate into tumor endothelium.They secret many pro-angiogenic factors as well. In addition, they play an essential role in cancer invasion and metastasis through inducing the production of matrix metalloproteinases (MMPs), chemoattractants and creating a pre-metastatic environment. Increasing evidence supports the idea that cancer stem cells (CSCs) are responsible for tumorigenesis, resistance to therapies, invasion and metastasis.Here, we hypothesize that CSCs may "hijack" MDSCs for use as alternative niche cells, leading to the maintenance of stemness and enhanced chemo- and radio-therapy resistance. The countermeasure that directly targets to MDSCs may be useful for against angiogenesis and preventing cancer from invasion and metastasis. Therefore, the study of MDSCs is important to understand tumor progression and to enhance the therapeutic efficacy against cancer.     

Tumor-derived exosomal miR-3157-3p promotes angiogenesis,vascular permeability and metastasis by targeting TIMP/KLF2 in non-small cell lung cancer

Metastasis is the main cause of death in patients with advanced lung cancer. The exosomes released by cancer cells create tumor microenvironment, and then accelerate tumor metastasis. Cancer-derived exosomes are considered to be the main driving force for metastasis niche formation at foreign sites, but the mechanism in Non-small cell lung carcinoma (NSCLC) is unclear. In metastatic NSCLC patients, the expression level of miR-3157-3p in circulating exosomes was significantly higher than that of non-metastatic NSCLC patients. Here, we found that miR-3157-3p can be transferred from NSCLC cells to vascular endothelial cells through exosomes. Our work indicates that exosome miR-3157-3p is involved in the formation of pre-metastatic niche formation before tumor metastasis and may be used as a blood-based biomarker for NSCLC metastasis. Exosome miR-3157-3p has regulated the expression of VEGF/MMP2/MMP9 and occludin in endothelial cells by targeting TIMP/KLF2, thereby promoted angiogenesis and increased vascular permeability. In addition, exosome miR-3157-3p promoted the metastasis of NSCLC in vivo.Subject terms: Cancer microenvironment, Non-small-cell lung cancer     

Cyclin A1 Modulates the Expression of Vascular Endothelial Growth Factor and Promotes Hormone-Dependent Growth and Angiogenesis of Breast Cancer

Alterations in cellular pathways related to both endocrine and vascular endothelial growth factors (VEGF) may contribute to breast cancer progression. Inhibition of the elevated levels of these pathways is associated with clinical benefits. However, molecular mechanisms by which endocrine-related pathways and VEGF signalling cooperatively promote breast cancer progression remain poorly understood. In the present study, we show that the A-type cyclin, cyclin A1, known for its important role in the initiation of leukemia and prostate cancer metastasis, is highly expressed in primary breast cancer specimens and metastatic lesions, in contrasting to its barely detectable expression in normal human breast tissues. There is a statistically significant correlation between cyclin A1 and VEGF expression in breast cancer specimens from two patient cohorts (p<0.01). Induction of cyclin A1 overexpression in breast cancer cell line MCF-7 results in an enhanced invasiveness and a concomitant increase in VEGF expression. In addition, there is a formation of protein–protein complexes between cyclin A1 and estrogen receptor ER-α cyclin A1 overexpression increases ER-α expression in MCF-7 and T47D cells. In mouse tumor xenograft models in which mice were implanted with MCF-7 cells that overexpressed cyclin A1 or control vector, cyclin A1 overexpression results in an increase in tumor growth and angiogenesis, which is coincident with an enhanced expression of VEGF, VEGFR1 and ER-α Our findings unravel a novel role for cyclin A1 in growth and progression of breast cancer, and suggest that multiple cellular pathways, including cell cycle regulators, angiogenesis and estrogen receptor signalling, may cooperatively contribute to breast cancer progression.     

Arresting supporters: targeting neutrophils in metastasis

It is becoming increasingly clear that leukocytes dynamically regulate cancer progression and metastasis, and among leukocytes, granulocytic cells abundantly accumulate in metastatic organs; however, their function in metastasis remains controversial. In a recent report in Nature, Wculek and Malanchi clarify the role of mature neutrophils as mediators of metastatic initiation and provide a targeted approach to prevent the pro-metastatic activity of neutrophils in breast cancer models.Metastasis remains the primary cause of death in solid tumors. Metastatic tumors often respond poorly to standard therapies, underscoring the need for new and effective drugs targeting metastasis¹. Several factors are thought to determine the survival and growth of disseminated cancer cells in a distant microenvironment. These include cell-autonomous processes such as tumor-reinitiating capacity at the metastatic site and cell-extrinsic interactions with non-cancer cells in the tumor microenvironment.Research over the past decades has demonstrated that leukocytes dynamically regulate cancer progression and metastasis². Some of these paracrine interactions are tumor suppressive (NK or CD8⁺ T cells), while many others (macrophage subsets or platelets) support metastatic progression³. Among leukocytes, neutrophils represent the most abundant class of circulating immune cells. Although their role as the first responders in infection is well characterized, their function in metastasis is poorly understood and remains controversial. Some studies have demonstrated a tumor-promoting effect of neutrophils during metastatic progression^4,5,6, whereas others have found a contrasting, anti-tumor function of these cells⁷. Other close cousins of neutrophils, granulocytic myeloid-derived suppressor cells (G-MDSCs) and Ly6G⁺Ly6C⁺ granulocytes, have been shown to promote metastatic progression^8,9,10. It is currently unclear whether subpopulations of granulocytic cells with such distinct functions coexist in metastatic tissues, whether they are localized differentially within the metastatic organ or regulated temporally in metastasis. In a recent paper in Nature, Wculek and Malanchi¹¹ clarified some of these open questions and defined the role of neutrophils as mediators of metastatic initiation.In line with previous findings⁶, Wculek and Malanchi confirmed that CD11b⁺Ly6G⁺ neutrophils accumulated in the pre-metastatic lung before the arrival of tumor cells in the genetically engineered MMTV-PyMT mammary cancer mouse model, and the number of these neutrophils subsequently increased with metastatic progression¹¹. Since granulocyte-colony stimulating factor (G-CSF) regulates neutrophil production and mobilization from the bone marrow, neutrophil accumulation and lung metastasis were expectedly impaired in Gcsf-null mice. Surprisingly, G-CSF deficiency in MMTV-PyMT tumor cells did not alter either lung neutrophil accumulation or metastasis, although these tumor cells are known to secrete G-CSF¹⁰. However, lung metastasis was reduced when neutrophils were depleted in mice that were genetically engineered with an inducible, neutrophil-targeted diphtheria toxin. As a complementary strategy, the authors showed that depletion of neutrophils using anti-Ly6G blocking antibody in the pre-metastatic phase decreased spontaneous metastasis. Using labeled MMTV-PyMT⁺ tumor cells, the authors showed reduced lung colonization of these tumor cells upon neutrophil depletion and further delineated that the neutrophils support metastatic initiation. One of the strengths of this study lies in the demonstration of the role of neutrophils in metastasis using multiple experimental strategies and models.One question that arises is how neutrophils support metastatic initiation. Using the MMTV-PyMT model, the authors showed that neutrophil-derived signals promote metastatic initiation by increasing the number of metastatic initiating cancer cells (MICs). Among the neutrophil-derived factors, the authors identified high levels of leukotrienes (LTs). LTs are eicosanoids that belong to a family of molecules that are derived from arachidonic acid. LTs mediate inflammatory processes and increase vasodilation and vascular permeability¹². Interestingly, LT stimulation of mouse MMTV-PyMT⁺ breast cancer cells enhanced tumor initiation potential of these cancer cells both in vitro and in vivo. In addition, the authors showed that the LT receptors (LTRs; BLT2 and CysLT2) are enriched among the MICs in mouse and human breast cancer cell lines. LTs are products of the arachidonate 5-lipoxygenase (Alox5) enzyme. Therefore, to address the role of the neutrophil Alox5-LT pathway in metastasis, the authors generated bone marrow chimeric mice using the Alox5-null mice, implanted MMTV-PyMT mammary cancer cells and quantified lung metastasis. Alox5 inhibition decreased spontaneous lung metastasis.LTs are important mediators of inflammatory and allergic responses¹². As such, Alox5 inhibitors such as Zileuton (Zil) are in use for treating asthma. Using Zil, this study revealed a therapeutic strategy to block the pro-metastatic activity of neutrophils in multiple mouse models of breast cancer. The authors showed that lung metastasis was reduced with Zil treatment; however, the primary tumor load or lung neutrophil accumulation remained unaffected. It needs to be explored how LT inhibition selectively affects pro-metastatic neutrophils and whether there are any side effects of long-term LT inhibition in preclinical cancer models. From a translational aspect, combination therapy with standard care will further determine the efficacy of this asthma drug as an anti-metastatic agent in breast cancer.A corollary question is how LTs produced by neutrophils benefit the MICs. The authors showed that LT treatment increased MIC proliferation through ERK/MAPK activation. Interestingly, activation of the MAPK pathway in cancer cells is a common feature of several paracrine inputs from granulocytic cells (Figure 1). Granulocytic myeloid derived suppressor cells (G-MDSC) recruited by CXCL1/2 chemokines secrete pro-survival, S100A8/9 factors that activate MAPK signaling in metastatic cancer cells⁹. Metastatic tumors secrete G-CSF that mobilizes Ly6G⁺Ly6C⁺ granulocytes to the lungs¹⁰. Ly6G⁺Ly6C⁺ cells produce Bv8, a small protein belonging to the prokineticin family, which binds to cancer cell Bv8/prokineticin receptor and activates the MAPK signaling pathway, thereby promoting cancer cell migration. Therefore, different granulocyte-derived factors ranging from lipid eicosanoids to Bv8 and S100A8/9 proteins trigger MAPK pathway activation in metastatic cancer cells, which subsequently promotes their proliferation, migration and survival in metastatic organs (Figure 1).Open in a separate windowFigure 1Diverse paracrine inputs from granulocytic cells activate the MAPK signaling pathway in cancer cells, which increases their metastatic fitness. Drugs that inhibit these paracrine inputs and reduce metastasis in mouse models are also shown.Based on these encouraging experimental results, it is necessary to evaluate the clinical importance of these findings. In this regard, Wculek and Malanchi showed that LTR expression was detected in a cohort of human breast primary and lymph node metastasis. Further investigation is warranted to explore how the neutrophil paracrine LT-LTR-MAPK pathway might be linked to lung metastasis and survival in breast cancer patients. Validation of these experimental findings from mouse models in patient samples would pave the way towards clinical translation of these important findings. In summary, the study by Wculek and Malanchi addresses the functional contribution of a key player, neutrophils, in the metastatic microenvironment, and provides a targeted approach to prevent pro-metastatic activity of these cells in breast cancer models.     

Silencing of CD44 Gene Expression in Human 143-B Osteosarcoma Cells Promotes Metastasis of Intratibial Tumors in SCID Mice

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells'' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.     

Organotropic metastasis: role of tumor exosomes

A recent paper in Nature shows that tumor exosomes expressing unique integrins can determine organotropic metastasis by preparing pre-metastatic niche through their integrins-mediated fusion with and fertilization of organ-specific resident cells.Tumor metastasis is a critical step in malignant progression of tumors and has been implicated in the failure of most cancer therapeutics. One salient feature of metastasis is that some types of cancer cells preferentially colonize and metastasize to specific organs, under the control of a range of cellular and molecular programs¹. Many studies focus largely on identifying cell-intrinsic determinants of such organotropic metastasis, including genes and chemokine receptors expressed on cancer cells². The adhesion and extracellular matrixc molecules, such as integrins, tenascin and periostin, have been also shown to promote colonization of metastatic cancer cells³. In 2005, Dr Lyden and colleagues proposed a term of pre-metastatic niche to describe the phenomenon that primary tumor could promote its own metastasis by recruiting bone marrow-derived cells to the distant organ and establish supportive metastatic environments⁴. Besides, tumor-derived soluble factors such as lysyl oxidase⁵ have been also reported to induce organ-specific metastasis by formation of pre-metastatic niche in certain sites. However, the exact mechanism for metastatic organotropism is still unclear. In recent years, tumor-derived exosomes have been demonstrated to promote cancer progression⁶. Exosomes are small membrane-bound vesicles (30-100 nm) containing functional biomolecules (including proteins, RNA, DNA and lipids) that can be horizontally transferred to recipient cells⁷. For example, brain astrocyte-derived exosomes can promote the outgrowth of brain metastatic cancer cells by transferring PTEN-targeting microRNA-19a to these cancer cells⁸. Nevertheless, whether molecules present on tumor exosomes can determine organ-specific metastasis is an unresolved question.Lyden and colleagues in their recent Nature paper gave the answer that tumor exosome integrins can determine organotropic metastasis by fusing with organ-specific resident cells to establish pre-metastatic niche through activating Src phosphorylation and pro-inflammatory S100 expression⁹ (Figure 1). The authors isolated exosomes from organotropic human and mouse breast and pancreatic cancer cell lines known to primarily metastasize to the lung, liver, or both⁹. Then, they retro-orbitally injected these near infrared (NIR) or red fluorescently labeled exosomes into nude mice and quantified exosome biodistribution and uptake in distant organs after 24 h by NIR whole-lung imaging and confocal microscopy. Using this approach, they observed that exosomes from different cancer models selectively interact with the same future metastatic organs as their cell of origin. Thus, the authors proposed that exosomes could promote organ-specific metastasis. To test exosome-mediated education of target organs functionally, they injected luciferase-expressing 4175-LuT (LuT, Lung-tropic) or 1833-BoT (BoT, bone-tropic) cells into 4175-LuT or 1833-BoT exosome-educated mice, and found that education with 4175-LuT-derived exosomes increased the lung metastatic capacity of 4175-LuT tumors, and interestingly, even significantly redirected the bone-tropic 1833-BoT cells to disseminate in the lung. These observations suggested that certain type of tumor exosomes can prepare pre-metastatic niches to facilitate organ-specific metastasis and redirect metastatic distribution even for cancer cells poorly capable of metastasizing to these sites.Open in a separate windowFigure 1Tumor exosomes direct organ-specific metastasis via integrins. Tumor-derived exosomes transport proteins, nucleic acids and lipids to specific organ and fuse with resident cells, which can prepare distant organ site as pre-metastatic niche. Lyden and colleagues⁹ report that exosomes derived from different type of cancer cells can display different integrin proteins on their surface; ITGα₆β₄- and ITGα₆β₁-expressing exosomes preferentially interact with fibroblasts and epithelial cells in lung, and ITGα_vβ₅-expressing exosomes preferentially fuse with Kupffer cells in liver. Once uptaken, tumor exosomes induce cellular changes (Src activation and pro-inflammatory S100 gene expression) in the target organ, thus promoting cancer cell colonization and organ-specific metastasis.To further dissect the molecular mechanisms involved in organ-specific metastasis, through quantitative mass spectrometry of lung-, liver- and brain-tropic exosomes, they identified integrins (ITGs) — the most highly represented cell adhesion receptor proteins in exosomes — as determinants of metastatic organotropism. Subsequent analysis showed that lung-tropic exosomes expressing ITGα₆β₄ and ITGα₆β₁ could interact with S100A4-positive fibroblasts and surfactant protein C-positive epithelial cells in laminin-rich lung microenvironments, ITGα_vβ₅-expressing pancreatic exosomes co-localized with F4/80⁺ macrophages and fused with Kupffer cells in fibronectin-rich liver niches, and brain-tropic 831-BrT exosomes interacted mainly with CD31⁺ brain endothelial cells. Furthermore, inhibiting the exosomal integrins ITGβ₄ and ITGβ₅ expression via short hairpin RNAs or blocking their binding by HYD-1/RGD peptides markedly reduced exosome uptake as well as lung and liver metastasis, respectively. Thus, the authors demonstrated that certain exosomal integrins govern organ-specific metastasis by fusing with target cells in an organ-specific manner. When using RNA sequencing to analyze the gene expression in distinct cells targeted by exosomes, they found that exosomal ITGs could prominently upregulate pro-inflammatory S100 gene expression (i.e., several S100 genes (S100A4, -A6, -A10, -A11, -A13 and -A16) were upregulated in 4175-LuT exosome-educated WI-38 fibroblasts; S100A8 and S100P were upregulated in BxPC-3-LiT exosome-educated Kupffer cells). Moreover, exosomal ITGβ₄ uptake could increase Src or phosphorylated Src (pSrc) levels in resident cells. S100 proteins promote metastasis¹⁰, and ITGα₆β₄ can active Src and S100A4 expression¹¹. Taken together, the mechanisms for exosomal integrins in promoting tumor organotropic metastasis in this Nature paper⁹ may be summarized as follows: (1) promotion of adhesion by fusing with specific resident cells; (2) activation of Src signaling pathways and inflammatory responses (pro-inflammatory S100 gene expression) in these target cells, thus preparing favorable pre-metastatic niche for further metastasis (Figure 1).Finally, their clinical data showed that ITGβ₄ level is higher in exosomes from breast cancer patients with lung metastasis and increased exosomal ITGα_v in pancreatic cancer patients who developed liver metastasis than in those without metastasis. These results indicated that exosomal integrins isolated from circulating plasma may be used as organotropism biomarkers to predict organ-specific metastasis in cancer patients.The study by Lyden and colleagues⁹ expands our understanding of the organ-specific metastasis mechanisms and also highlights a crucial role of exosomes in promoting tumor metastasis. Indeed, another recent work in Nature Cell Biology shows that pancreatic cancer exosomes can increase liver metastatic burden by transferring macrophage migration inhibitory factor (MIF) to Kupffer cells and by recruiting immune cells to initiate pre-metastatic niche formation in the liver¹². Thus, strategies targeting these particular exosomal integrin molecules in this Nature paper⁹ may not be enough to prevent organ-specific metastasis. Besides, the limited integrin repertoire of bone-tropic exosomes identified by the proteome analysis calls for further investigation. This study also raises several intriguing questions: Do exosomal integrins have any effect on the recruitment of bone marrow-derived cells or immunosuppressive cells in priming pre-metastatic niche? What is the function of other exosome components such as RNA and DNA in determining organ-specific metastasis? Are these results universal to other type of cancer metastasis? Most importantly, how to translate this finding into clinical applications? Addressing these questions will help us unveil the mystery of organotropic metastasis.     

Expression of the B-Cell Receptor Component CD79a on Immature Myeloid Cells Contributes to Their Tumor Promoting Effects

The role of myeloid derived suppressor cells (MDSCs) in promoting tumorigenesis is well-established, and significant effort is being made to further characterize surface markers on MDSCs both for better diagnosis and as potential targets for therapy. Here we show that the B cell receptor adaptor molecule CD79a is unexpectedly expressed on immature bone marrow myeloid cells, and is upregulated on MDSCs generated in multiple different mouse models of metastatic but not non-metastatic cancer. CD79a on MDSCs is upregulated and activated in response to soluble factors secreted by tumor cells. Activation of CD79a on mouse MDSCs, by crosslinking with a specific antibody, maintained their immature phenotype (CD11b+Gr1+), enhanced their migration, increased their suppressive effect on T cell proliferation, and increased secretion of pro-tumorigenic cytokines such as IL-6 and CCL22. Furthermore, crosslinking CD79a on myeloid cells activated signaling through Syk, BLNK, ERK and STAT3 phosphorylation. In vivo, CD79+ myeloid cells showed enhanced ability to promote primary tumor growth and metastasis. Finally we demonstrate that CD79a is upregulated on circulating myeloid cells from lung cancer patients, and that CD79a+ myeloid cells infiltrate human breast tumors. We propose that CD79a plays a functional role in the tumor promoting effects of myeloid cells, and may represent a novel target for cancer therapy.