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

In the paper

“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].”

What the sources say

• Partly supported

  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)

  cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
• ?Not checked — no full text of the sourcecites [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

In the paper

“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.”

What the sources say

• Supported

  The Results section reports reduced macrophage recruitment in mammary tumors of α4Y991A mice and suppressed myeloid-cell infiltration in other α4Y991A tumor models. The citing sentence attributes the α4Y991A entry defect to this paper; it presents the Rac2−/− comparison as the citing authors’ own finding. (LLM, conf=0.92)

  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

In the paper

“Other investigators have shown that α4β1 is involved in tumor progression involving the TLR4 signaling pathway and p110γ axis.”

What the sources say

• Partly supported

  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)

  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 sourceExperimental — accuracy not yet measured

In the paper

“Rac2 is only expressed in hematopoietic and endothelial cells whereas Rac1 and Rac3 are ubiquitously expressed in mammalian systems [3], [4], [5].”

What the sources say

• ?Not checked — no full text of the sourcecites [3] J Didsbury, 1989, rac, a novel ras-related family of proteins that are botulinum toxin substrates, J Biol Chem source
• Partly supported

  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)

  cites [4] L Haataja, 1997, Characterization of RAC3, a novel member of the Rho family, J Biol Chem sourceExperimental — accuracy not yet measured
• ?Could not verify

  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)

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

In the paper

“The α4β1/αvβ3 integrins are expressed in hematopoietic and endothelial cells [14] and play a key role in inflammation [15], [29], [46], [47].”

What the sources say

• ?Not checked — no full text of the sourcecites [14] CJ Avraamides, 2008, Integrins in angiogenesis and lymphangiogenesis, Nat Rev Cancer source
• ?Not checked — no full text of the sourcecites [15] H Jin, 2006, Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors, Cancer Res source
• Partly supported

  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)

  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 sourceExperimental — accuracy not yet measured

In the paper

“we took advantage of the reports that M2 macrophages can be generated under conditions of MCSF stimulation in vitro [39], [40].”

What the sources say

• ?Not checked — no full text of the sourcecites [39] AJ Fleetwood, 2009, GM-CSF- and M-CSF-dependent macrophage phenotypes display differential dependence on type I interferon signaling, J Leukoc Biol source
• Supported

  The introduction reports that anti-inflammatory type-2 human macrophages, termed M-2, have been generated in the presence of M-CSF. The Phenotyping section also describes this paper's in vitro culture of murine bone-marrow cells with M-CSF, and the Discussion describes the resulting cells as M2-like. (LLM, conf=0.92)

  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

In the paper

“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].”

What the sources say

• Supported

  The Results section reports that RAC2 deficiency markedly reduces α4β1- and αv-integrin-directed migration in bone marrow-derived endothelial cells, and it notes prior work showing RAC2-dependent integrin-directed macrophage migration. The Discussion reports that RAC2 is required for postnatal neovascularization in vivo. Page numbers were not provided in our copy. (LLM, conf=0.90)

  cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res source
• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source

In the paper

“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].”

What the sources say

• ?Could not verify

  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)

  cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
• ?Not checked — no full text of the sourcecites [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

In the paper

“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].”

What the sources say

• ?Could not verify

  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)

  cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
• ?Not checked — no full text of the sourcecites [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

In the paper

“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].”

What the sources say

• ?Not checked — no full text of the sourcecites [48] Y Imai, 2002, Intracellular signaling in M-CSF-induced microglia activation: role of Iba1, Glia source
• Partly supported

  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)

  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 sourceExperimental — accuracy not yet measured

In the paper

“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].”

What the sources say

• Partly supported

  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)

  cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
• ?Not checked — no full text of the sourcecites [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

In the paper

“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].”

What the sources say

• ?Could not verify

  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)

  cites [1] P De, 2009, Expression of RAC2 in endothelial cells is required for the postnatal neovascular response, Exp Cell Res sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source
• ?Not checked — no full text of the sourcecites [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

In the paper

“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).”

In the paper

“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).”

In the paper

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

In the paper

“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).”

In the paper

“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).”

In the paper

“we and other laboratories have confirmed that these Rac2-/- mice do not exhibit any defects in organ development.”

In the paper

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

In the paper

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

In the paper

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

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

“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.”

In the paper

“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].”

What the sources say

• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source

In the paper

“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.”

In the paper

“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].”

What the sources say

• ?Not checked — no full text of the sourcecites [19] A Mantovani, 2005, Macrophage polarization comes of age, Immunity source
• ?Not checked — no full text of the sourcecites [20] S Gordon, 2005, Monocyte and macrophage heterogeneity, Nat Rev Immunol source

In the paper

“Previous work from our laboratory demonstrated that specific α4β1 and αvβ3/αvβ5 integrin directed migration in macrophages requires Syk and Rac2 [2].”

What the sources say

• ?Not checked — no full text of the sourcecites [2] D Pradip, 2003, Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase, J Biol Chem source

In the paper

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

In the paper

“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].”

What the sources say

• ?Not checked — no full text of the sourcecites [44] GH Fong, 1995, Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium, Nature source
• ?Not checked — no full text of the sourcecites [45] F Shalaby, 1995, Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice, Nature source

In the paper

“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].”

What the sources say

• ?Not checked — no full text of the sourcecites [19] A Mantovani, 2005, Macrophage polarization comes of age, Immunity source

In the paper

“Lawrence et al has suggested that MCSF receptor activates IRF4 transcription factor to promote M2 differentiation of macrophages [37].”

What the sources say

• ?Not checked — no full text of the sourcecites [37] T Lawrence, 2011, Transcriptional regulation of macrophage polarization: enabling diversity with identity, Nat Rev Immunol source

In the paper

“The ComBat method [57] was then used to remove the batch effect.”

What the sources say

• ?Not checked — no full text of the sourcecites [57] WE Johnson, 2007, Adjusting batch effects in microarray expression data using empirical Bayes methods, Biostatistics source

In the paper

“which are considered as M2 markers [33].”

What the sources say

• ?Not checked — no full text of the sourcecites [33] A Mantovani, 2004, The chemokine system in diverse forms of macrophage activation and polarization, Trends Immunol source

In the paper

“All cells were cultured in DMEM media containing 10% FBS and tested for mycoplasma before implanting in animals.”

In the paper

“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.”

In the paper

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

In the paper

“Importantly, malignant tumors have found a way to co-op this mechanism for M2 macrophage transition as an important component of tumor progression.”

In the paper

“In the present study, we provide a novel role of macrophage Rac2 in controlling tumor growth and M2 macrophage differentiation.”

In the paper

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

In the paper

“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.”

In the paper

“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].”

What the sources say

• ?Not checked — no full text of the sourcecites [36] CL Elsegood, 2006, M-CSF induces the stable interaction of cFms with alphaVbeta3 integrin in osteoclasts, Int J Biochem Cell Biol source