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Cited by 128Immune cells in cancerCancer Cells and MetastasisMechanisms of cancer metastasis

Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

Open access
Joshi, Shweta, Singh, Alok R., Zulcic, Muamera, Bao, Lei, Messer, Karen, Ideker, Trey, Dutkowski, Janusz, Durden, Donald L.·2014·PLoS ONE
DOI 10.1371/journal.pone.0095893PMC PMC4000195
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Abstract

Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

AI snapshot

Written from the paper’s full text.

TL;DR

Rac2 is required in macrophages for tumor growth, angiogenesis, invasion, metastasis, and M1-to-M2 differentiation in mouse tumor models. The authors propose a macrophage-autonomous axis in which MCSF receptor and α4β1 integrin co-signal through Rac2 to promote M2 polarization and tumor progression.

Key findings

  • Rac2-/- mice showed significantly reduced growth of syngeneic LLC, B16 melanoma, and NB9464D tumors (p<0.001), reduced tumor angiogenesis by CD31 microvessel density (P<0.05), and less invasion into muscularis compared with WT mice.
  • Rac2-/- mice had an 80% reduction in B16F10 lung metastatic nodules (p<0.001), reduced orthotopic Panc02 pancreatic tumor growth (P<0.05), and reduced regional colonic lymph node metastasis.
  • Macrophages sorted from LLC tumors in WT mice expressed higher M2 markers (Cox2, uPA, MMP9, MMR, arginase, VEGF), while Rac2-/- tumor macrophages expressed higher M1 markers IL1 and TNFα; WT tumor macrophages also had higher arginase activity (p<0.05) and lower nitrite production (p<0.001).
  • α4Y991A knock-in mice were defective in tumor growth (p<0.001), metastasis by 90% (p<0.001), and M2 macrophage differentiation; MCSF stimulation activated Rac2 but not Rac1 about 5-fold, and injection of WT BMDMs—but not WT GBMDMs or Rac2-/- BMDMs—reversed tumor growth and metastatic defects in Rac2-/- mice.

Methods

  • Genetic mouse models: Rac2-/- and α4Y991A knock-in mice, with syngeneic/orthotopic tumor implants including LLC, B16F10, NB9464D, and Panc02; tumor growth, angiogenesis, invasion, experimental metastasis, and spontaneous lymph node metastasis were assessed.
  • Bone marrow-derived macrophages (BMDM) and GMCSF-cultured macrophages (GBMDM) were analyzed by RT-PCR, FACS, arginase/NOS assays, Rac2 GTP pull-down, haptotaxis/invasion assays, microarray (Affymetrix Mouse Genome 1.0 ST; FDR<5%), and metabolomic profiling (UHLC/MS/MS2 and GC/MS).
  • Macrophage autonomy was tested by injecting WT, Rac2-/-, or GBMDM into Rac2-/- mice; interactome analysis used literature-curated M1/M2 drivers, BioGRID protein interactions, and network propagation.

Limitations

  • The paper states that the molecular mechanisms controlling the M1-M2 transition remain incompletely understood, that M1/M2 classification is “somewhat inexact and artificial,” and that future studies are needed to validate additional cooperating signaling elements.
  • Evident from the text: all experiments are in mouse models, with no human validation; some supporting data are described as “data not shown”; and the interactome map is predictive/iterative rather than fully experimentally validated.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0095893

Cites

66 of 66 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

13 of 42 cited sources checked · 3 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Rac2-/- mice orthotopically implanted with Panc02 cells in the pancreas display a marked reduction in regional colonic lymph node metastasis.

    “Rac2-/- mice orthotopically implanted with Panc02 cells in pancreas display a marked reduction in regional colonic lymph node metastasis (Figure 2D & Figure S2)”
    Quote in the paperSubstantial claim
  • Findingown data

    Arginase activity is higher in macrophages isolated from LLC tumors injected into WT animals compared with Rac2-/- mice.

    “higher arginase activity (p<0.05) and lower nitrite production (NOS) (p<0.001) was observed in macrophages isolated from LLC tumors injected into WT animals as compared to Rac2-/- mice (Figure 4D–E).”
    Quote in the paperSubstantial claim
  • Findingown data

    Tumor-induced angiogenesis is greatly reduced in subcutaneously implanted LLC and B16 tumors in Rac2-/- mice compared with WT animals.

    “Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis.”
    • Also stated in: § Rac2 promotes tumor growth, angiogenesis and invasion · plain
      “angiogenesis was greatly reduced in subcutaneously implanted LLC and B16 tumors in the Rac2-/- mice compared with WT animals (P<0.05; Figure 1C).”
    Quote in the paperSubstantial claim
  • Findingown data

    Tumor growth of syngeneic tumors (LLC, B16 melanoma, and neuroblastoma 9464D) is significantly reduced in Rac2-/- mice compared with WT mice.

    “Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis.”
    • Also stated in: § Rac2 promotes tumor growth, angiogenesis and invasion · plain
      “tumor growth of 4 different syngeneic tumors which included: Lewis lung carcinoma (LLC), B16 melanoma and neuroblastoma (9464D) was significantly reduced in the Rac2-/- mice.”
    Quote in the paperSubstantial claim
  • Findingown data

    Nitrite production is lower in macrophages isolated from LLC tumors injected into WT animals compared with Rac2-/- mice.

    “higher arginase activity (p<0.05) and lower nitrite production (NOS) (p<0.001) was observed in macrophages isolated from LLC tumors injected into WT animals as compared to Rac2-/- mice (Figure 4D–E).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 26
  • May combine two claims. 24
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Rac2 knockout mice display a number of hematopoietic defects mostly in blood cell-specific receptor function or hematopoietic-specific effector mechanisms and in kinase pathway-activated cell survival.

    “the Rac2 knockout mice display a number of hematopoietic defects mostly in the context of blood cell-specific receptor function or hematopoietic-specific effector mechanisms and also in kinase pathway-activated cell survival [1], [2], [6], [7], [8].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res source

      The Introduction (page unavailable) reports neutrophil, macrophage, and mast-cell defects in Rac2 knockout mice. The Results discuss integrin-directed signaling and SYK-dependent RAC2 activation, but in endothelial cells; the cited paper does not establish kinase pathway-activated cell-survival defects in the knockout mice. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
    • cites [6] AW Roberts, 1999, Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense, Immunity source

  • May combine two claims.

    Findingcitation

    α4Y991A knock-in mice display a defect in macrophage entry into the tumor microenvironment, whereas Rac2-/- mice do not.

    “As expected, our data demonstrate that the phenotype of the α4Y991A knock-in differs significantly from the Rac2 -/- mice in that α4Y991A knock-in mice as reported by Schmidt et al [29] display a defect in Mθ entry into the TME whereas no such defect is noted in Rac2-/- mice.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [29] MC Schmid, 2011, Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kgamma, a single convergent point promoting tumor inflammation and progression, Cancer Cell source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    α4β1 is involved in tumor progression involving the TLR4 signaling pathway and p110γ axis.

    “Other investigators have shown that α4β1 is involved in tumor progression involving the TLR4 signaling pathway and p110γ axis.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] MC Schmid, 2011, Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kgamma, a single convergent point promoting tumor inflammation and progression, Cancer Cell source

      The Discussion states that TLR/IL1R signaling activates p110γ, which promotes α4β1 activation and tumor progression; the Results also link p110γ-mediated α4β1 activation to tumor growth. Our copy does not identify TLR4 specifically, so the claim's narrower receptor attribution is not established. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Rac2 is only expressed in hematopoietic and endothelial cells, whereas Rac1 and Rac3 are ubiquitously expressed in mammalian systems.

    “Rac2 is only expressed in hematopoietic and endothelial cells whereas Rac1 and Rac3 are ubiquitously expressed in mammalian systems [3], [4], [5].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [3] J Didsbury, 1989, rac, a novel ras-related family of proteins that are botulinum toxin substrates, J Biol Chem source
    • cites [4] L Haataja, 1997, Characterization of RAC3, a novel member of the Rho family, J Biol Chem source

      The Results (page unavailable) report RAC1 and RAC3 mRNA in all human cell lines studied and RAC2 mRNA mainly in hematopoietic cell lines. They do not establish ubiquitous RAC1 or RAC3 expression across mammalian systems or the claimed endothelial-cell expression of RAC2; RAC3 was detected in only some of the tissues listed. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [5] D Diekmann, 1995, Rac GTPase interacts with GAPs and target proteins through multiple effector sites, EMBO J source

      The Introduction discusses rac activity in phagocytic cells, and the Methods identify a human rac1 construct, but neither passage describes where the Rac isoforms are expressed. Searches for Rac2, Rac3, and tissue distribution found no passage supporting the claimed restricted or ubiquitous expression patterns. Page numbers were not available in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    α4β1/αvβ3 integrins are expressed in hematopoietic and endothelial cells and play a key role in inflammation.

    “The α4β1/αvβ3 integrins are expressed in hematopoietic and endothelial cells [14] and play a key role in inflammation [15], [29], [46], [47].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [14] CJ Avraamides, 2008, Integrins in angiogenesis and lymphangiogenesis, Nat Rev Cancer source
    • cites [15] H Jin, 2006, Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors, Cancer Res source
    • cites [29] MC Schmid, 2011, Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kgamma, a single convergent point promoting tumor inflammation and progression, Cancer Cell source

      The Introduction states that integrins play key roles in inflammation, and the Results identify α4β1 as important for myeloid-cell trafficking to tumors. The paper does not establish the same inflammatory role for αvβ3; in the Results, inhibiting αvβ3 had no effect on the myeloid-cell adhesion tested. The expression claim has a separate citation in the citing sentence. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    M2 macrophages can be generated under conditions of MCSF stimulation in vitro.

    “we took advantage of the reports that M2 macrophages can be generated under conditions of MCSF stimulation in vitro [39], [40].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [39] AJ Fleetwood, 2009, GM-CSF- and M-CSF-dependent macrophage phenotypes display differential dependence on type I interferon signaling, J Leukoc Biol source
    • cites [40] AJ Fleetwood, 2007, Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation, J Immunol source

  • May combine two claims.

    Premisecitation

    α4β1/αvβ3-induced migration requires Rac2 in macrophages and endothelial cells, and this pathway regulates the postnatal angiogenic response in vivo.

    “The current investigation began with our initial observation that the provisional integrins, α4β1/αvβ3 induced migration requires a specific isoform of Rac, Rac2 in macrophages and endothelial cells and that this pathway regulated the postnatal angiogenic response in vivo [1], [2].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res source
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the absence of Rac2, macrophages and/or neutrophils display impaired phagocytosis.

    “Previous findings clearly demonstrate that in the absence of Rac2, macrophages and/or neutrophils display suppressed reactive oxygen species (ROS) production, defective chemotaxis, impaired phagocytosis, and decreased microbial killing [1], [2], [6], [7], [8].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res source

      The Introduction reports neutrophil and macrophage defects in Rac2-knockout mice, and the Results mention impaired macrophage migration, but neither identifies impaired phagocytosis. A search for “phagocytosis” in our copy found no passage addressing that specific claim. (LLM, conf=0.73)

      Experimental — accuracy not yet measured
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
    • cites [6] AW Roberts, 1999, Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense, Immunity source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the absence of Rac2, macrophages and/or neutrophils display suppressed reactive oxygen species (ROS) production.

    “Previous findings clearly demonstrate that in the absence of Rac2, macrophages and/or neutrophils display suppressed reactive oxygen species (ROS) production, defective chemotaxis, impaired phagocytosis, and decreased microbial killing [1], [2], [6], [7], [8].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res source

      The Results discuss RAC2-dependent macrophage migration, while the paper’s experiments concern endothelial migration and neovascularization. A References entry names a study of RAC2 and neutrophil NADPH oxidase activation, but no passage in our copy reports suppressed ROS production in RAC2-deficient macrophages or neutrophils (searched: ROS, reactive oxygen species, oxidative burst, and NADPH oxidase). (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
    • cites [6] AW Roberts, 1999, Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense, Immunity source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MCSF signaling activates its receptor FMS tyrosine kinase, which sequentially activates PI-3 kinase and Rac in microglia and bone marrow derived cells.

    “existing literature provides evidence that MCSF signaling activates its receptor the FMS tyrosine kinase which sequentially activates PI-3 kinase and Rac in microglia and bone marrow derived cells [48], [49].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [48] Y Imai, 2002, Intracellular signaling in M-CSF-induced microglia activation: role of Iba1, Glia source
    • cites [49] V Vedham, 2005, Vav activation and function as a rac guanine nucleotide exchange factor in macrophage colony-stimulating factor-induced macrophage chemotaxis, Mol Cell Biol source

      The paper identifies c-fms as the M-CSF receptor and reports that M-CSF-induced Rac activation requires PI 3-kinase in bone marrow-derived macrophages (Abstract; “Macrophages lacking SHIP display elevated chemotactic responses,” page not provided). Its Animals section identifies the cells studied as bone marrow-derived macrophages; our copy does not establish the same signaling sequence in microglia. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the absence of Rac2, macrophages and/or neutrophils display defective chemotaxis.

    “Previous findings clearly demonstrate that in the absence of Rac2, macrophages and/or neutrophils display suppressed reactive oxygen species (ROS) production, defective chemotaxis, impaired phagocytosis, and decreased microbial killing [1], [2], [6], [7], [8].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res source

      The Results section states that previous work found RAC2 necessary for macrophage integrin-directed migration. That supports a related migration defect, but integrin-directed migration is not the same as chemotaxis, and this paper does not report defective chemotaxis in RAC2-deficient macrophages or neutrophils. No page number is available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
    • cites [6] AW Roberts, 1999, Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense, Immunity source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the absence of Rac2, macrophages and/or neutrophils display decreased microbial killing.

    “Previous findings clearly demonstrate that in the absence of Rac2, macrophages and/or neutrophils display suppressed reactive oxygen species (ROS) production, defective chemotaxis, impaired phagocytosis, and decreased microbial killing [1], [2], [6], [7], [8].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res source

      The Results section discusses RAC2-dependent macrophage migration, while the References list a study of neutrophil function and host defense. Neither passage states that Rac2-deficient macrophages or neutrophils have decreased microbial killing. Searches for microbial killing, killing, and infection found no passage addressing that outcome. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
    • cites [6] AW Roberts, 1999, Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense, Immunity source

  • May combine two claims.

    Findingown data

    Engagement of α5β1 with CH271 and α2β1 with collagen does not result in appreciable Rac2 activation, and there is minimal migration defect on these matrix proteins in Rac2-/- macrophages.

    “In contrast, engagement of α5β1 with CH271 and α2β1 with collagen does not result in an appreciable activation of Rac2 and there is minimal migration defect on these matrix proteins observed in Rac2-/- murine macrophages (Figure 3A, right upper panel).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Metastasis is markedly reduced in Rac2-/- mice compared with WT mice in experimental and spontaneous models.

    “Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis.”
    • Also stated in: § Rac2 promotes tumor metastasis · plain
      “marked increase in metastatic foci in the lungs of WT mice compared with an 80% reduction in the number of nodules in Rac2-/- mice injected with B16F10 melanoma (p<0.001; Figure 2A–B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BMDMs isolated from WT mice express Rac2, whereas Rac2-/- BMDMs do not.

    “The analysis confirms that BMDMs isolated from WT mice express Rac2 as compared to negative result in Rac2-/- BMDMs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In simultaneous local injections, the right hemithorax injected with WT BMDMs showed luciferase activity and B16 metastatic nodules, while the left hemithorax injected with WT GBMDMs or Rac2-/- BMDMs showed no B16 melanoma or luciferase signal and minimal metastatic nodules.

    “The right hemithorax (injected with WT BMDMs) showed luciferase activity and B16 metastatic nodules while there was no B16 melanoma or luciferase signal and minimal metastatic nodules detected within the left hemithorax (injected with WT GBMDM vs. Rac2-/- BMDM) (Figure 6E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GBMDM express higher levels of proinflammatory cytokines, while BMDM express increased levels of Mgl1 and MMR mRNA, which are considered M2 markers.

    “real time PCR analysis done on macrophages cultured in MCSF (BMDM) or GMCSF (GBMDM) demonstrated that GBMDM express higher levels of proinflammatory cytokines, while BMDM express increased levels of Mgl 1 and MMR mRNA which are considered as M2 markers (Figure S4A).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    Rac2-/- mice do not exhibit defects in organ development.

    “we and other laboratories have confirmed that these Rac2-/- mice do not exhibit any defects in organ development.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MCSF receptor cosignals through α4β1 integrin to promote tumor growth, metastasis and M2 macrophage differentiation by specifically activating Rac2.

    “we provide evidence that MCSF receptor cosignals through α4β1 integrin to promote these events by specifically activating Rac2 (Figure 6A).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The interaction network spanning the top 100 genes was identified and annotated with protein and gene expression changes between Rac2-/- and WT conditions.

    “The interaction network spanning the top 100 genes was identified and annotated with protein and gene expression changes between the Rac2-/- and WT conditions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Only MCSF, not GMCSF, stimulation activates Rac2.

    “Moreover, our results also suggest that only MCSF but not GMCSF stimulations activate Rac2 (Figure 6A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Rac2 is required to promote tumor growth, invasion, angiogenesis and metastasis potentially by promoting alternative activation of macrophages into the M2 phenotype.

    “These results further support our model that Rac2 is required to promote tumor growth, invasion, angiogenesis and metastasis potentially by promoting alternative activation of macrophages into the M2 phenotype.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo.

    “the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    The polarization of M1 into M2 macrophages promotes tumor growth, angiogenesis, invasion and metastasis.

    “A defining feature of tumor inflammation is the polarization of M1 into M2 macrophages which promotes tumor growth, angiogenesis, invasion and metastasis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Macrophage entry into the tumor is likely necessary but not sufficient to drive M1 to M2 differentiation of tumor-associated macrophages.

    “from our data in Rac2-/- mice, we conclude that macrophage entry into the tumor is likely necessary but not sufficient to drive M1 to M2 differentiation of TAMs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Rac2 is activated downstream of the α4β1 integrin and the CSF1 receptor.

    “Our current results establish that the Rac2 GTPase controls tumor growth, invasion and metastasis and that Rac2 is activated downstream of the α4β1integrin, the CSF1 receptor and that Rac2 is required for macrophage differentiation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The Rac2 GTPase controls tumor growth, invasion and metastasis.

    “Our current results establish that the Rac2 GTPase controls tumor growth, invasion and metastasis and that Rac2 is activated downstream of the α4β1integrin, the CSF1 receptor and that Rac2 is required for macrophage differentiation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Syk is necessary and sufficient to convert α4β1 integrin-dependent migration in COS7 cells to Rac2 dependency.

    “We transduced a nonmyeloid cell, COS7 which expresses Rac1 and α4β1 but not Rac2 or Syk kinase with Syk and/or Rac2 and were able to determine that Syk was necessary and sufficient to convert α4β1 integrin dependent migration in COS7 cells to Rac2 dependency [2].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source

  • May combine two claims.

    Interpretationown data

    The signaling pathway elucidated in this report extends from the cell surface receptors MCSF receptor and integrin α4β1 to activation of Rac2, which controls M2 macrophage differentiation, tumor growth and metastasis in vivo.

    “Figure 7A shows a schematic representation of the signaling pathway elucidated in this report. The pathway extends from the cell surface receptors MCSF receptor and integrin α4β1 to the activation of the Rac2 to control differentiation of M2 macrophage differentiation, tumor growth and metastasis in vivo.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    M2 macrophages are involved in polarized Th2 inflammatory reactions and characterized by expression of arginase-1 and mannose and scavenger receptors.

    “In contrast, M2 macrophages are involved in polarized Th2 inflammatory reactions and characterized by expression of arginase-1 and mannose and scavenger receptors [19], [20].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] A Mantovani, 2005, Macrophage polarization comes of age, Immunity source
    • cites [20] S Gordon, 2005, Monocyte and macrophage heterogeneity, Nat Rev Immunol source

  • Classified as probably not a substantial claim.

    Premisecitation

    Specific α4β1 and αvβ3/αvβ5 integrin directed migration in macrophages requires Syk and Rac2.

    “Previous work from our laboratory demonstrated that specific α4β1 and αvβ3/αvβ5 integrin directed migration in macrophages requires Syk and Rac2 [2].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source

  • May combine two claims.

    Interpretationown data

    WT and Rac2-/- BMDMs are M2 and M1 skewed, respectively.

    “independently support RT-PCR results generated in our knockout models that WT and Rac2-/- BMDMs are M2 and M1 skewed, respectively.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    VEGF-/- and VEGF+/- mice were embryonic lethal or associated with profound defects in vasculogenesis and developmental angiogenesis.

    “This was an important distinction since in other knockout models e.g. VEGF-/- and +/- mice were embryonic lethal or associated with profound defects in vasculogenesis and developmental angiogenesis [44], [45].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [44] GH Fong, 1995, Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium, Nature source
    • cites [45] F Shalaby, 1995, Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice, Nature source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    M1 macrophages are IL-12high, IL-23high, IL-10low; produce high levels of inducible nitric oxide synthetase; secrete inflammatory cytokines such as IL-1β, IL-6, and TNF; and are inducer and effector cells in Th1 type inflammatory responses.

    “M1 macrophages are IL-12high, IL-23high, IL-10low; produce high levels of inducible nitric oxide synthetase (iNOS); secrete inflammatory cytokines such as IL-1β, IL-6, and TNF; and are inducer and effector cells in Th1 type inflammatory responses [19].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] A Mantovani, 2005, Macrophage polarization comes of age, Immunity source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MCSF receptor activates IRF4 transcription factor to promote M2 differentiation of macrophages.

    “Lawrence et al has suggested that MCSF receptor activates IRF4 transcription factor to promote M2 differentiation of macrophages [37].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] T Lawrence, 2011, Transcriptional regulation of macrophage polarization: enabling diversity with identity, Nat Rev Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Batch effect was removed using the ComBat method.

    “The ComBat method [57] was then used to remove the batch effect.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [57] WE Johnson, 2007, Adjusting batch effects in microarray expression data using empirical Bayes methods, Biostatistics source

  • Classified as probably not a substantial claim.

    Premisecitation

    CCL2 and CCL22 are considered M2 markers.

    “which are considered as M2 markers [33].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [33] A Mantovani, 2004, The chemokine system in diverse forms of macrophage activation and polarization, Trends Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All cells were tested for mycoplasma before implanting in animals.

    “All cells were cultured in DMEM media containing 10% FBS and tested for mycoplasma before implanting in animals.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All procedures involving animals were approved by the University of California San Diego Animal Care Committee.

    “All procedures involving animals were approved by the University of California San Diego Animal Care Committee, which serves to ensure that all federal guidelines concerning animal experimentation are met.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    All samples demonstrated RNA integrity (RIN) of 7 or greater.

    “All samples demonstrated RNA integrity (RIN) of 7 or greater.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    Malignant tumors have co-opted this mechanism for M2 macrophage transition as an important component of tumor progression.

    “Importantly, malignant tumors have found a way to co-op this mechanism for M2 macrophage transition as an important component of tumor progression.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The coordinate regulation of the macrophage transcriptomic program downstream of CSF1R/α4β1 stimulation required for the complex physiologic transition from M1 to M2 remains unclear.

  • May combine two claims.

    Interpretationown data

    Macrophage Rac2 controls tumor growth and M2 macrophage differentiation.

    “In the present study, we provide a novel role of macrophage Rac2 in controlling tumor growth and M2 macrophage differentiation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The molecular entities involved in the global rearrangement of the transcriptional profile during alternative macrophage activation are still largely unknown.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All tumor experiments were performed three to four times with n = 8-10.

    “All tumor experiments were performed three to four times with n = 8-10.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The BMDMs and GBMDMs injected in the mice were 90% viable at the time of injection, as determined by trypan blue and replating.

    “The BMDMs and GBMDMs injected in the mice were 90% viable, as determined by trypan blue and replating a small aliquot of cells, at the time of injection.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    M-CSF stimulation induces association between CSF-1 receptor (FMS) and αVβ3 integrin in osteoclasts.

    “a study by Faccio et al has illustrated that M-CSF stimulation induces association between CSF-1 receptor (FMS) and αVβ3 integrin in osteoclasts [36].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] CL Elsegood, 2006, M-CSF induces the stable interaction of cFms with alphaVbeta3 integrin in osteoclasts, Int J Biochem Cell Biol source
45 of 45

  • The paper relies on this without support.

    The α4Y991A knock-in mutation specifically disrupts α4β1 integrin signaling relevant to Rac2 activation and does not have confounding effects on other pathways.

    “We next examined if α4Y991A knock-in mice bearing a point mutation in α4 integrin tail (Y991A), has any defect in activating Rac2.”
    “We observed that the α4Y991A k/in mice are defective in tumor growth (p<0.001), polarization of macrophages and metastasis (by 90%, p<0.001) (Figure 3B–D).”

  • The paper relies on this without support.

    The molecular entities involved in the global rearrangement of the transcriptional profile during alternative macrophage activation are still largely unknown.

    “Despite considerable progress, the molecular entities involved in the global rearrangement of the transcriptional profile occurring during alternative macrophage activation are still largely unknown.”

  • The paper relies on this without support.

    The coordinate regulation of the macrophage transcriptomic program downstream of CSF1R/α4β1 stimulation required for the complex physiologic transition from M1 to M2 remains unclear.

    “The coordinate regulation of the macrophage transcriptomic program downstream of CSF1R/α4β1 stimulation is required for the complex physiologic transition from M1 to M2 remains unclear.”

  • The paper relies on this without support.

    There is no literature on the interaction of CSF-1 receptor and α4β1 integrin.

    “Although there is no literature on the interaction of CSF-1 receptor and α4β1 integrin”
4 of 4

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • B16 macrophage-injection schedules disagree

    Major

    The results describe 5-6 daily macrophage injections before a B16F10 challenge on day 5. The Figure 6 caption instead describes one injection two days before B16F10 inoculation, followed by injections every third day.

  • Rac2 expression is described as hematopoietic-specific despite endothelial expression

    Minor

    One passage says Rac2 is specifically expressed in the hematopoietic compartment, while the introduction says it is also expressed in endothelial cells.

  • LLC tumors in the macrophage-injection experiment have two harvest days

    Minor

    The Figure 6 caption says the treated LLC tumors were removed on day 21, whereas the methods for the same macrophage-reconstitution experiment say they were harvested on day 25.

  • Figure 3A panel references reverse migration and Rac2-activation assays

    Minor

    The results text points to the left panel for Rac2 activation and the right upper panel for migration data, but the figure caption assigns migration to the left panel and Rac2 activation to the right upper panel.

  • Figure 6B-C is described as a B16 challenge but depicts LLC tumors

    Minor

    The results link Figure 6B-C to injections followed by a B16F10 melanoma challenge. The figure caption identifies those panels instead as the volume and mass of subcutaneously implanted LLC tumors.

5 of 5

Cites

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Related

Authors
Joshi, S., Singh, A. R., Zulcic, M., Bao, L., Messer, K., Ideker, T., Dutkowski, J., & Durden, D. L.
Affiliations
University of California San Diego (US)
Corresponding author
Durden, Donald L.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-04-25
Volume
9
Issue
4
Pages
e95893
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0095893
PMC
PMC4000195
OpenAlex
W2058100343
Cited by
128
References
66
Citations by year
2026: 5 · 2025: 11 · 2024: 16 · 2023: 12 · 2022: 6 · 2021: 7 · 2020: 12 · 2019: 14 · 2018: 13 · 2017: 11 · 2016: 9 · 2015: 8 · 2014: 4
FWCI
4.15
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, other-oa)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health (CA23100, CA94233, HL091385)
  • University of California, San Diego (P30 CA23100)
  • National Heart, Lung, and Blood Institute (R21 HL091385, HL091385)
  • National Cancer Institute (CA94233-09, R01 CA094233, P30CA023100, P30 CA23100-22, R56 CA094233)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mechanisms of cancer metastasis
Keywords
Rac2, small GTPases, tumor-associated macrophages, macrophage polarization, macrophage differentiation, metastasis, tumor growth, angiogenesis, alpha4beta1 integrin, macrophage colony-stimulating factor receptor, extracellular matrix signaling, bone marrow-derived macrophages, protein-protein interaction network, systems biology
MeSH terms
RAC2 GTP-Binding Protein, Animals, Cell Differentiation, Enzyme Activation, Lung Neoplasms, Macrophages, Melanoma, Experimental, Mice, Inbred C57BL, Neoplasm Invasiveness, Neoplasm Transplantation, Neovascularization, Pathologic, Signal Transduction, Receptor, Macrophage Colony-Stimulating Factor, Mice, Knockout, Platelet Endothelial Cell Adhesion Molecule-1, rac GTP-Binding Proteins, Integrin alpha4beta1, Cell Line, Tumor, Tumor Burden

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-19.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-19.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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