Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations.Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells.

Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.

Abstract The role of glycoprotein-NMB (GPNMB) in the immune system is varied. GPNMB expression in macrophages and dendritic cells promotes innate immune responses, whereas GPNMB-expressing myeloid-derived suppressor (MDSC) cells suppress adaptive immune responses (T-cell function). While functional roles for GPNMB expressed within cells of the immune system are emerging, the influence of tumor-derived GPNMB expression on the immune landscape within mammary tumors has not been well characterized. To investigate this further, we have generated two GPNMB-deficient breast cancer cell populations (Lung Metastatic-4T1 [LM-4T1] and E0771 cells) to determine the influence of GPNMB on the tumor immune microenvironment (TME). While loss of GPNMB significantly impaired tumor growth of both these breast cancer models in syngeneic mice, this difference was not apparent with LM-4T1 injected in athymic nude mice. These observations suggest GPNMB may promote tumor growth by modulating the T-cell function. We have used traditional immunohistochemical analyses to broadly characterize the T cells present in the TME of E0771 and LM-4T1 breast tumors, which express or lack GPNMB. We have observed a significant increase in both CD8+ and CD4+ immune cells in GPNMB-deficient LM-4T1 and E0771 tumors compared to parental cells at an experimental endpoint of matched tumor volumes. To determine the temporal response of the immune system to LM-4T1 cells, we have isolated early developing lesions and stained for CD8, CD4, Fox3p, and granzyme B positive cells. We observed a significant increase in CD4+ and a trend for elevated granzyme B+ cells at early timepoints in GPNMB-deficient tumors compared to LM-4T1 parental cells. Taken together, these results suggest GPNMB suppresses an early recruitment of CD4+ cells where, in the absence of GPNMB, this would result in elevated CD8+ and CD4+ cells in these tumors. We are currently characterizing the contribution of CD4+ cells in the progression of these tumor models. Citation Format: Matthew G. Annis, April A.N. Rose, Ryuhjin Ahn, Brian E. Hsu, Josie Ursini-Siegel, Peter M. Siegel. GPNMB expression modulates the tumor immune microenvironment in mouse models of breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B88.

Neutrophils represent the immune system’s first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

Additional file 5: Figure S5. p66ShcA does not alter the mesenchymal properties of 4T1-derived triple negative breast cancers. (A) Immunoblot analysis of whole cell lysates isolated from 4T1-537 parental, p66-CR (VC), p66-CR (WT) and p66-CR (S36A) mammary tumors (n = 18 each) using ShcA-, E-Cadherin, Vimentin and Tubulin-specific antibodies. (B-D) Densitometric quantification of mammary tumors shown in panel A for the (B) p66ShcA/Tubulin, (C) p66ShcA/p52ShcA, (D) E-Cadherin/Tubulin and (E) Vimentin/Tubulin ratios. The data is normalized to the parental 4T1-537 tumors.

Background The p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro- and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis. Methods We tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo. Results We show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site. Conclusion This study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.

Neutrophils are the first leukocytes recruited to sites of inflammation, where they execute anti-microbial functions to eliminate infectious agents. These functions include phagocytosis, release of reactive oxygen species and the formation of neutrophil extracellular traps via NETosis. Neutrophils are receiving increasing attention in the context of cancer, where these same neutrophil-associated functions are also important for modulating tumor growth and metastatic progression. Neutrophils are phenotypically heterogeneous and, depending on the context, exert anti- or pro-tumorigenic functions. Increasing evidence also suggests an important role of neutrophils and their involvement in promoting multiple steps of the metastatic cascade. The steps include: (1) local invasion and intravasation of cancer cells into circulation, (2) survival of cancer cells in the bloodstream and extravasation at a distant site, (3) early cancer cell seeding/survival, and (4) progressive growth of cancer cells to form macroscopic metastases. Although neutrophil functions designed to eliminate infectious agents can also eliminate tumor cells, their dysregulation can promote tumor growth and enable metastasis at multiple steps along the metastatic cascade. In this review, we will provide an overview of the current advances in neutrophil biology in the context of cancer. We also discuss the emerging field of immunometabolism, in which the rewiring of alternative metabolic pathways within neutrophils can impact their pro-tumorigenic/pro-metastatic functions.

Neutrophils are phenotypically heterogeneous and exert either anti- or pro-metastatic functions. We show that cancer-cell-derived G-CSF is necessary, but not sufficient, to mobilize immature low-density neutrophils (iLDNs) that promote liver metastasis. In contrast, mature high-density neutrophils inhibit the formation of liver metastases. Transcriptomic and metabolomic analyses of high- and low-density neutrophils reveal engagement of numerous metabolic pathways specifically in low-density neutrophils. iLDNs exhibit enhanced global bioenergetic capacity, through their ability to engage mitochondrial-dependent ATP production, and remain capable of executing pro-metastatic neutrophil functions, including NETosis, under nutrient-deprived conditions. We demonstrate that NETosis is an important neutrophil function that promotes breast cancer liver metastasis. iLDNs rely on the catabolism of glutamate and proline to support mitochondrial-dependent metabolism in the absence of glucose, which enables sustained NETosis. These data reveal that distinct pro-metastatic neutrophil populations exhibit a high degree of metabolic flexibility, which facilitates the formation of liver metastases.

The translation of mRNAs into proteins serves as a critical regulatory event in gene expression. In the context of cancer, deregulated translation is a hallmark of transformation, promoting the proliferation, survival, and metastatic capabilities of cancer cells. The best-studied factor involved in the translational control of cancer is the eukaryotic translation initiation factor 4E (eIF4E). We and others have shown that eIF4E availability and phosphorylation promote metastasis in mouse models of breast cancer by selectively augmenting the translation of mRNAs involved in invasion and metastasis. However, the impact of translational control in cell types within the tumor microenvironment (TME) is unknown. Here, we demonstrate that regulatory events affecting translation in cells of the TME impact cancer progression. Mice bearing a mutation in the phosphorylation site of eIF4E (S209A) in cells comprising the TME are resistant to the formation of lung metastases in a syngeneic mammary tumor model. This is associated with reduced survival of prometastatic neutrophils due to decreased expression of the antiapoptotic proteins BCL2 and MCL1. Furthermore, we demonstrate that pharmacological inhibition of eIF4E phosphorylation prevents metastatic progression in vivo, supporting the development of phosphorylation inhibitors for clinical use.

Germline mutations in STK11, which encodes the tumor suppressor liver kinase B1 (LKB1). promote Peutz-Jeghers syndrome (PJS), a cancer predisposition syndrome characterized by the development of gastrointestinal (GI) polyps. Here, we report that heterozygous deletion of Stk11 in T cells (LThet mice) is sufficient to promote GI polyposis. Polyps from LThet mice, Stk11(+/-) mice, and human PJS patients display hallmarks of chronic inflammation, marked by inflammatory immune-cell infiltration, signal transducer and activator of transcription 3 (STAT3) activation, and increased expression of inflammatory factors associated with cancer progression [interleukin 6 (IL-6), IL-11 and CXCL2]. Targeting either T cells, IL-6, or STAT3 signaling reduced polyp growth in Stk11(+/-) animals. Our results identify LKB1-mediated inflammation as a tissue-extrinsic regulator of intestinal polyposis in PJS, suggesting possible therapeutic approaches by targeting deregulated inflammation in this disease.

Abstract Translational control has emerged as a critical determinant in tumorigenesis. The mRNA cap-binding protein eIF4E is an oncoprotein that plays an important role in cancer initiation and progression. We recently demonstrated that eIF4E phosphorylation on serine 209 by the Map Kinase Integrating Kinases (MNK1/2) promotes epithelial to mesenchymal transition, invasion and metastasis. We recently discovered a critical importance of eIF4E phosphorylation in the tumor microenvironment (TME) for the metastatic process. To investigate the role of eIF4E phosphorylation in the TME, we utilized a syngeneic model in which a murine mammary tumor cell line (66cl4) was orthotopically injected into mice bearing the non-phosphorylatable eIF4E ^S209A mutation and their WT counterparts. Tumor growth was monitored by caliper measurements, and tissues were collected for histological assessment and immunohistochemistry. The onset of tumor formation was significantly delayed in eIF4E ^S209A mice. However, established tumors showed no differences in growth, proliferation, apoptosis, vascularization or immune infiltration. Strikingly, eIF4E ^S209A mice were nearly completely resistant to the formation of lung metastases from primary mammary tumors. Metastatic potential in this model was correlated with neutrophil accumulation in the spleen, blood and lungs of WT mice, which was significantly reduced in eIF4E ^S209A mice. Furthermore, treatment of tumor-bearing WT mice with an inhibitor of MNK1/2 significantly reduced lung metastasis without affecting primary tumor growth. Considering that eIF4E phosphorylation in both the TME and cancer cells promotes metastasis, our findings support the development of MNK inhibitors for metastasis prevention. Citation Format: Nathaniel Robichaud, Qianyu Guo, Sonia del Rincon, Wilson H. Miller, Peter Siegel, Brian Hsu, Nahum Sonenberg. Translational control of the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA09.

Potential conflict of interest: Nothing to report. See Article On page 1920 Neutrophils are emerging as important modulators of cancer progression and metastasis, exhibiting both antimetastatic and prometastatic functions.1 Originally considered to be a terminally differentiated and homogenous cell population that was engaged as a first line of defense against microbial infections, neutrophils are now viewed as a highly “plastic” immune cell type in the context of cancer, which can undergo polarization within the tumor microenvironment.2 Neutrophils present in the metastatic microenvironment can mediate tumor cell killing and impair the establishment of metastases.3 In other contexts, infiltrating neutrophils function to promote cancer progression and metastasis.5 With respect to liver metastases, neutrophils promote the formation and growth of hepatic metastases from multiple solid cancers.7 Neutrophils positively impact the metastatic process through a variety of functions, including their ability to enhance cancer cell entrapment/seeding within neutrophil extracellular traps,7 promote cancer cell extravasation,9 and stimulate angiogenesis.8 In the context of tumor angiogenesis, there are several mechanisms through which neutrophils play a proangiogenic role. First, neutrophils express a variety of proangiogenic factors. The best example is the observation that tumor‐associated neutrophils contain significant pools of intracellular vascular endothelial growth factor (VEGF) that can be released upon stimulation,12 and tumor‐associated neutrophils exhibit increased transcription of VEGF mRNA.13 Impairment of Bv8 (prokineticin‐2), a neutrophil‐derived angiogenic factor, with anti‐Bv8 antibodies has been shown to reduce recruitment of myeloid cells, decrease blood vessel density, and inhibit tumor growth upon inhibition.14 In addition to directly producing angiogenic factors, neutrophils can indirectly mobilize angiogenic factors stored in the local extracellular matrix (ECM) through the release of proteases. Indeed, neutrophil‐derived matrix metalloproteinase 9 (MMP‐9) catalyzes the release of VEGF bound to the ECM by proteolytic cleavage, inducing an angiogenic switch.15 Moreover, there are studies that identify a distinct subpopulation of proangiogenic neutrophils (defined as CD49d+VEGFR1high C‐X‐C chemokine receptor [CXCR]4high neutrophils) that are recruited by VEGF‐A to the sites of tissue hypoxia. These proangiogenic neutrophils release larger amounts of MMP‐9 compared to inflammatory neutrophils recruited to infectious sites.16 Fibroblast growth factor 2 (FGF2/basic FGF) can also be liberated from ECM stores through the action of neutrophil‐derived proteases.17 In the current issue, Gordon‐Weeks et al. expand our understanding of neutrophil‐dependent angiogenesis.8 In this study, the investigators demonstrate the presence of infiltrating neutrophils in liver metastases derived from human xenografts as well as in murine models of liver metastasis. Isolated neutrophils (Ly6G+/CXCR2+/CD45+) from HT29‐derived liver metastases exhibited multilobed nuclear morphology, indicating that these infiltrating cells correspond to mature, segmented neutrophils. Interestingly, no differences were observed in circulating low‐density neutrophils that have recently been described in tumor‐bearing animal models.18 To functionally interrogate the role of neutrophils in the expansion and growth of liver metastases, neutrophil depletion experiments were performed only after the cancer cells had seeded the liver. Depletion of neutrophils after 7 or 14 days post‐tumor‐cell injection reduced the metastatic burden at endpoint, whereas neutrophil depletion after 21 days did not. In addition, tumor cell proliferation and vascular density was diminished in liver metastases that formed in neutrophil‐depleted animals. These data argue that neutrophils act early in the colonization phase to enhance the growth of colorectal cancer (CRC) liver metastases by promoting angiogenesis. To gain a mechanistic understanding of how liver‐metastasis–associated neutrophils were contributing to an angiogenic response, mRNA expression profiles of metastasis‐associated neutrophils were compared to neutrophils isolated from normal livers. From these analyses, FGF2 mRNA levels were found to be elevated in neutrophils isolated from CRC liver metastases, whereas angiogenic factors such as VEGF were not significantly different. Moreover, FGF2 expression was localized to Ly6G+ cells and deposited within ECM of liver metastases in situ, and neutrophil depletion resulted in a significant reduction of FGF2 expression in hepatic lesions. Finally, the investigators demonstrate that an FGF2 neutralizing antibody, when administered 1 week post‐tumor‐cell injection, resulted in a reduction of vascular density, cancer cell proliferation, and liver‐metastatic burden. These results phenocopied the consequences of neutrophil depletion; however, mice treated with the neutralizing FGF2 antibody exhibited the same degree of neutrophil infiltration compared to control cohorts. These data argue that blocking neutrophil‐derived FGF2 resulted in impaired angiogenesis rather than a secondary effect resulting from impaired neutrophil recruitment. Indeed, the critical role of FGF2 for enhancing angiogenesis associated with the establishment of liver metastases was demonstrated by the fact that CRC cells engineered to overexpress FGF2 were capable of efficiently forming liver metastases even in the context where neutrophils were depleted. This study demonstrates that neutrophils recruited into the liver‐metastatic microenvironment produce FGF2, which drives angiogenesis and growth of nascent hepatic metastases. These results support previous work demonstrating that neutrophils can indirectly liberate FGF2 from ECM stores through release of heparanase, which was also observed in the current study to be highly elevated in neutrophils recruited to liver metastases (Fig. 1). They also make the novel observation that infiltrating neutrophils are themselves a significant source of FGF2. The precise mechanism of FGF2 release and activation is still in question. Is FGF‐2 that is produced by neutrophils deposited within the ECM and subsequently released by neutrophil‐derived heparanase to induce angiogenesis, or do cancer cells within the growing metastases secrete their own proteases that release and activate FGF2? Given the growing realization that neutrophils represent a much more heterogeneous cell population in the context of cancer, another interesting question is the relationship between the FGF2‐producing neutrophils described in the current study and previously described proangiogenic neutrophils (CD49d+VEGFR1high CXCR4high)—are they the same or distinct neutrophil subsets? Regardless, the demonstration that neutrophil‐derived FGF2 is important for a robust angiogenic response within liver metastases provides a target for a subset of patients where traditional anti‐VEGF therapies have failed.Figure 1: Neutrophil‐derived FGF2 supports the growth of liver‐metastatic cancer cells. Cancer cells first seed the liver. Metastasis‐associated neutrophils that express FGF2 are subsequently recruited to the growing lesions. FGF2 produced by neutrophils stimulates angiogenesis through direct release of active FGF2 or through the release of FGF2 bound to the ECM. Release of ECM‐bound stores of FGF2 may result from heparanase released by neutrophils or by proteases produced by cancer cells. Author names in bold designate shared co‐first authorship.

In previous studies we found that macrophages (MФs) from SH2-containing inositol-5'-phosphatase (SHIP) deficient mice are M2 polarized while their wild type (WT) counterparts are M1 polarized and that this difference in MФ phenotype can be recapitulated during in vitro derivation from bone marrow if mouse plasma (MP), but not fetal calf serum, is added to standard M-CSF-containing cultures. In the current study we investigated the mechanism by which MP skews SHIP-/- but not +/+ MФs to an M2 phenotype. Our results suggest that SHIP-/- basophils constitutively secrete higher levels of IL-4 than SHIP+/+ basophils and this higher level of IL-4 is sufficient to skew both SHIP+/+ and SHIP-/- MФs to an M2 phenotype, but only when MP is present to increase the sensitivity of the MФs to this level of IL-4. MP increases the IL-4 sensitivity of both SHIP+/+ and -/- MФs not by increasing cell surface IL-4 or CD36 receptor levels, but by triggering the activation of Erk and Akt and the production of ROS, all of which play a critical role in sensitizing MФs to IL-4-induced M2 skewing. Studies to identify the factor(s) in MP responsible for promoting IL-4-induced M2 skewing suggests that all-trans retinoic acid (ATRA), TGFβ and prostaglandin E2 (PGE2) all play a role. Taken together, these results indicate that basophil-secreted IL-4 plays an essential role in M2 skewing and that ATRA, TGFβ and PGE2 within MP collaborate to dramatically promote M2 skewing by acting directly on MФs to increase their sensitivity to IL-4.

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

INTRODUCTION:Breast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells.METHODS:Using in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1+ (Ly-6C and Ly-6G) or specifically Ly-6G+ cells was performed to functionally interrogate the role of Ly-6G+ infiltrates in promoting metastasis to these organs.RESULTS:We show that T lymphocytes (CD3+), myeloid-derived (Gr-1+) cells and neutrophils (Ly-6G+ or NE+) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1+ cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G+ cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b+/Ly-6G+ neutrophils that infiltrate and surround the liver metastases are polarized toward an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis.CONCLUSIONS:Our results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G+ cells within the liver microenvironment.

Claudin-2 enhances breast cancer liver metastasis and promotes the development of colorectal cancers. The objective of our current study is to define the regulatory mechanisms controlling Claudin-2 expression in breast cancer cells. We evaluated the effect of several Src Family Kinase (SFK) inhibitors or knockdown of individual SFK members on Claudin-2 expression in breast cancer cells. We also assessed the potential effects of pan-SFK and SFK-selective inhibitors on the formation of breast cancer liver metastases. This study reveals that pan inhibition of SFK signaling pathways significantly elevated Claudin-2 expression levels in breast cancer cells. In addition, our data demonstrate that pan-SFK inhibitors can enhance breast cancer metastasis to the liver. Knockdown of individual SFK members reveals that loss of Yes or Fyn induces Claudin-2 expression; whereas, diminished Lyn levels impairs Claudin-2 expression in breast cancer cells. The Lyn-selective kinase inhibitor, Bafetinib (INNO-406), acts to reduce Claudin-2 expression and suppress breast cancer liver metastasis. Our findings may have major clinical implications and advise against the treatment of breast cancer patients with broad-acting SFK inhibitors and support the use of Lyn-specific inhibitors.

We recently demonstrated that both murine and human carcinomas grow significantly slower in mice on low carbohydrate (CHO), high protein diets than on isocaloric Western diets and that a further reduction in tumor growth rates occur when the low CHO diets are combined with the cyclooxygenase-2 inhibitor, celecoxib. Following upon these studies, we asked herein what effect low CHO, high protein diets, with or without celecoxib, might have on tumor metastasis. In the highly metastatic 4T1 mouse mammary tumor model, a 15% CHO, high protein diet supplemented with celecoxib (1 g/kg chow) markedly reduced lung metastases. Moreover, in longer-term studies using male Transgenic Adenocarcinoma of the Mouse Prostate mice, which are predisposed to metastatic prostate cancer, the 15% CHO diet, with and without celecoxib (0.3 g/kg chow), gave the lowest incidence of metastases, but a more moderate 25% CHO diet containing celecoxib led to the best survival. Metabolic studies with 4T1 tumors suggested that the low CHO, high protein diets may be forcing tumors to become dependent on amino acid catabolism for survival/growth. Taken together, our results suggest that a combination of a low CHO, high protein diet with celecoxib substantially reduces metastasis.