Pre-metastatic Phase Research Articles

Abstract 1502: Deciphering the spatial and temporal dynamics of premetastatic niche in lung mediated by Treg and myeloid cell interactions

Abstract Introduction: The lung is a frequent site for metastases, where pre-metastatic niches (PMN) form in anticipation of tumor cell arrival. A deeper understanding of the molecular and immune features underlying the PMN formation may facilitate developing innovative strategies to prevent and treat metastasis more effectively in its early stages. Methods: In this study, we developed mouse models by subcutaneously implanting the 344SQ cell line, derived from the metastasis of a genetically engineered mouse model (GEMM) harboring KrasG12D/+; p53R172H/+ mutations, which reliably metastasizes to the lungs. We collected paired samples of the subcutaneous tumors, peripheral blood mononuclear cells (PBMCs), and lung tissue at pre-metastatic, early-metastatic, and late-metastatic phases. These samples were subjected to single-cell RNA sequencing (scRNA-seq), single-cell TCR/BCR sequencing (scTCR/BCR-seq), and spatial gene expression profiling with 10X Genomics Visium technology. Results: ScRNA analysis revealed large-scale comprehensive longitudinal changes in cell components throughout the invasion and metastasis cascade, which were validated by spatial gene expression. Furthermore, analysis of Visium data deduces the spatial information of these dynamic changes.We identified a subset of tumor-associated Tregs (TA_Tregs, enriched in the subcutaneous tumor tissues) that existed in small amounts in the lung tissue in the pre-metastatic phase when there are no tumor cells detectable in the lung. These cells were characterized by high expression of Treg-associated suppressive genes (Tnfrsf9, Tnfrsf4, Tnfrsf18, Traf1, Nfkbia), inhibitory molecules (Ikzf2, Il2ra, Tight, Pdcd1), cytokines and chemokines (Ccr5, Ccr8, Ccl5, Cxcr6) and other Treg effector molecules (Id2, Hif1a, Rora, Il1r2, Itgav, Stat1, Icos, Areg). The scTCR-seq results and trajectory analysis revealed the TA_Tregs might derive from minor clones of lung resident Tregs (LR_Tregs) and play an active role in remodeling an immunosuppressive microenvironment by closely interacting with other cell components in the TME. We also observed a significant alteration in the ratio of macrophage subpopulations between Macro_Spic and Macro_Vcan. This ratio exhibited a continuous decrease in lung tissues, which could be an early indication of the initiation of pre-metastatic niche (PMN) conditioning. Conclusion: By simultaneously characterizing cellular gene expression, immune repertoires, and their corresponding spatial information, we revealed the dynamic changes of the lung tissue microenvironment (LTME) during from a normal state to pro-metastasis state at single-cell resolution. The LTME of metastatic organs may be primed before the establishment of metastasis. Citation Format: Yanhua Tian, Jian-Rong Li, Bo Zhu, Jared Fradette, Hong Chen, Chenyang Li, Leticia Rodriguez, Daniel Kraushaar, Luisa Solis, Alexandre Reuben, Ignacio Wistuba, Don L. Gibbons, Chao Cheng, Jianjun Zhang. Deciphering the spatial and temporal dynamics of premetastatic niche in lung mediated by Treg and myeloid cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1502.

Abstract 1300: Single-cell and spatial transcriptomic profiling reveal the cascade of immune events during progression and metastasis of non-small cell lung cancer

Abstract Introduction: Lung cancer is the leading cause of cancer-related deaths worldwide and metastasis accounts for 90% of cancer-related mortality. A better understanding of the cascade of molecular and immune events underlying evolution during tumor progression and metastasis may help develop new strategies to prevent and treat metastasis more effectively. Methods: In this study, we utilized two mouse models of metastatic lung cancer: a genetically engineered mouse model (GEMM) with mutant Kras and Trp53 (KP, KrasLSL-G12D/+; Trp53fl/fl), which forms lung adenocarcinoma with high metastatic potential, and a syngeneic model using cell lines derived from the metastatic KP_GEMMs that faithfully develop lung metastases. Paired samples of the primary tumor, peripheral blood mononuclear cells, lung and subcutaneous tumors were serially collected at different time points (pre-metastatic, early-metastatic, and late-metastatic phases) and subjected to single-cell RNA sequencing (scRNA-seq), scTCR/BCR-seq, and spatial gene expression by using 10X Genomics (Visium). Results: ScRNA analysis revealed large-scale comprehensive longitudinal changes in cell components throughout the invasion and metastasis cascade, which was validated by spatial gene expression. Furthermore, analysis of Visium data deduces the spatial information of these dynamic changes. We found that NK and B cells from both the lung tissues and PBMC mainly participated in the early-metastatic stage and vanished in the late-metastatic phase. We also identified a subset of tumor-associated Tregs (TA_Tregs) that existed in small amounts in the lung tissue in the pre-metastatic phase and greatly expanded in the late-metastatic phase. These cells were characterized by high expression of Treg-associated suppressive genes (Tnfrsf9, Tnfrsf4, Tnfrsf18, Traf1, Nfkbia), inhibitory molecules (Ikzf2, Il2ra, Tight, Pdcd1), cytokines and chemokines (Ccr5, Ccr8, Ccl5, Cxcr6) and other Treg effector molecules (Id2, Hif1a, Rora, Il1r2, Itgav, Stat1, Icos, Areg). The scTCR-seq results and trajectory analysis revealed the TA_Tregs might derive from minor clones of lung resident Tregs (LR_Tregs) and play an active role in remodeling an immunosuppressive microenvironment by closely interacting with other cell components in the TME. Conclusion: By simultaneously characterizing cellular gene expression, immune repertoires, and their corresponding spatial information, we revealed the dynamic changes of the tumor immune microenvironment (TIME) during tumor progression and metastasis at single-cell resolution. The TIME of metastatic organs may be primed before the establishment of metastasis. Treatment targeting these molecular and immune events during pre- and early metastatic phases may provide a novel avenue to prevent metastasis of lung cancer. Citation Format: Yanhua Tian, Jian-Rong Li, Bo Zhu, Jared Fradette, Hong Chen, Chenyang Li, Sijia Cui, Daniel Kraushaar, Junya Fujimoto, Leticia Rodriguez, Luisa Maren Solis Soto, Alexandre Reuben, Ignacio Wistuba, Don Gibbons, Cheng Chao, Jianjun Zhang. Single-cell and spatial transcriptomic profiling reveal the cascade of immune events during progression and metastasis of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1300.

Suppression of CEBPδ recovers exhaustion in anti-metastatic immune cells

The pre-metastatic microenvironment consists of pro-metastatic and anti-metastatic immune cells in the early stages of cancer, when the primary tumor begins to proliferate. Redundantly, pro-inflammatory immune cells predominated during tumor growth. Although it is well known that pre-metastatic innate immune cells and immune cells fighting primary tumor cells become exhausted, the mechanism by which this occurs is unknown. We discovered that anti-metastatic NK cells were mobilized from the liver to the lung during primary tumor progression and that the transcription factor CEBPδ, which was upregulated in a tumor-stimulated liver environment, inhibited NK cell attachment to the fibrinogen-rich bed in pulmonary vessels and sensitization to the environmental mRNA activator. CEBPδ-siRNA treated anti-metastatic NK cells regenerated the binding proteins that support sitting in fibrinogen-rich soil, such as vitronectin and thrombospondin, increasing fibrinogen attachment. Furthermore, CEBPδ knockdown restored an RNA-binding protein, ZC3H12D, which captured extracellular mRNA to increase tumoricidal activity. Refreshed NK cells using CEBPδ-siRNA with anti-metastatic abilities would work at metastatic risk areas in the pre-metastatic phase, resulting in a reduction in lung metastasis. Furthermore, tissue-specific siRNA-based therapy in lymphocyte exhaustion may be beneficial in the treatment of early metastases.

Abstract B022: pH-low insertion peptide detects lactic acidosis contributing metastatic niche formation in lungs

Abstract Background: Pre-metastatic niche refers to the environment prepared for efficient colonization of disseminated cancer cells in specific organs. Primary tumor secretome facilitates pre-metastatic niche through a series of molecular and cellular changes. This study focuses on interstitial pH in organs with pre-metastatic niche because dysregulated pH homeostasis plays a well-established role in tumor metastasis. In primary tumor, extracellular acidification directly promotes invasion/migration of cancer cells for extravasation. Meanwhile, little is known regarding interstitial pH in organs that are distant from primary tumor. Methods: BALB/c mice were subcutaneously inoculated with 4T1.2 cells or 66cl4 cells (mouse breast cancer cell lines) with resistance against 6-thioguanine. Absence of metastatic cells in their lungs was confirmed by long-termed culture of dissociated pulmonary cells in presence of 6-thioguanine; it defines Day 21 from inoculation as a pre-metastatic phase. Alternatively, 4T1.2 cell-conditioned media (4T1.2-CM) or control media was intraperitoneally administered every day for 21 consecutive days. pH-low insertion peptide (pHLIP® peptide) stably labels cells exposed to acidic conditions, which is feasible to evaluate interstitial acidity in each organ. Alexa Fluor® 750-labelled pHLIP® peptide was injected into mice, and fluorescent signals in harvested organs were detected by IVIS Lumina XRMS Series III. For a metastasis model, mice first received 4T1.2-CM or in combination with sodium oxamate (a lactate dehydrogenase inhibitor) followed by an intravenous injection of 66cl4 cells expressing luciferase. Results: At the pre-metastatic phase, 4T1.2 tumor-bearing mice showed great accumulation of pHLIP® peptide and high amounts of lactate in their lungs. By contrast, there was no significant fluorescence from pHLIP® peptide in lungs of low-metastatic 66cl4 cell-inoculated mice. Notable accumulation of pHLIP® peptide was also observed in the lungs of 4T1.2-CM-given mice. Intravenously injected 66cl4 cells colonized more aggressively in the acidic lungs than in control ones. Suppression of the lung acidification by sodium oxamate attenuated the metastatic burden, which significantly improved survival of the mice. In the acidic lungs, pHLIP® peptide was localized in alveolar type II (AT2) cells expressing surfactant protein C. Sorted AT2 cells from the acidic lungs strongly expressed glycolysis-related proteins. Conclusions: This study using pHLIP® peptide indicates a highly glycolytic activity with increased lactic acid in the lungs of 4T1.2 tumor-bearing mice at the pre-metastatic phase. Mechanistically, soluble factors derived from 4T1.2 cells could transform AT2 cells into acidic cells, which might provide metastasis-promoting function. This study will shed light on understanding the whole picture of pre-metastatic niche and give scientific insights to develop novel diagnosis and therapy for tumor metastasis. Citation Format: Toma Matsui, Yuki Toda, Anna Mosnikova, Oleg A. Andreev, Shigekuni Hosogi, Yana K. Reshetnyak, Eishi Ashihara. pH-low insertion peptide detects lactic acidosis contributing metastatic niche formation in lungs [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B022.

Fourier Transform Infrared Polarization Contrast Imaging Recognizes Proteins Degradation in Lungs upon Metastasis from Breast Cancer.

Simple SummarySeveral lung extracellular matrix (ECM) proteins are involved in the formation of a metastatic niche in pulmonary metastasis and they accompany the cancer progression. Its gradual remodeling does not induce compositional changes of its components, but it is related to the re-distribution of individual proteins, their cross-linking and spatial arrangement within the tissue. The combination of FTIR and FTIR polarization contrast (PCI) imaging, as rapid, non-destructive, and label-free techniques, allows for the determination of protein alternations occurring in lungs that are affected by breast cancer metastasis. Both have the potential to characterize biochemical changes of the metastatic target, can determine phenotypes of tissue structures, and deliver a novel spectroscopic marker panel for the recognition of metastasis environment.The current understanding of mechanisms underlying the formation of metastatic tumors has required multi-parametric methods. The tissue micro-environment in secondary organs is not easily evaluated due to complex interpretation with existing tools. Here, we demonstrate the detection of structural modifications in proteins using emerging Fourier Transform Infrared (FTIR) imaging combined with light polarization. We investigated lungs affected by breast cancer metastasis in the orthotopic murine model from the pre-metastatic phase, through early micro-metastasis, up to an advanced phase, in which solid tumors are developed in lung parenchyma. The two IR-light polarization techniques revealed, for the first time, the orientational ordering of proteins upon the progression of pulmonary metastasis of breast cancer. Their distribution was complemented by detailed histological examination. Polarized contrast imaging recognised tissue structures of lungs and showed deformations in protein scaffolds induced by inflammatory infiltration, fibrosis, and tumor growth. This effect was recognised by not only changes in absorbance of the spectral bands but also by the band shifts and the appearance of new signals. Therefore, we proposed this approach as a useful tool for evaluation of progressive and irreversible molecular changes that occur sequentially in the metastatic process.

Effects of Platycodin D on S100A8/A9-induced inflammatory response in murine mammary carcinoma 4T1 cells

Activation of the inflammatory signaling pathway is the most vital part of the pre-metastatic events of breast cancer. Platycodin D (PlaD) shows favorable pharmacological activities in anti-inflammatory and anti-tumor effect. The main purpose of this study was to survey the effects of PlaD on S100A8/A9-induced inflammation in mouse mammary carcinoma 4T1 cells. S100A8/A9 immunolocalization and expression in pre-metastatic lung tissue were assessed by immunofluorescence staining and ELISA. 4T1 cells were treated with 2.5 μg/mL recombinant S100A8/A9 heterodimer and 7.5, 10, or 12.5 μM of PlaD. After 24 h of incubation, cell viability, migration, and invasion were evaluated by CCK-8, wound-healing, and transwell assay, respectively. Nuclear translocation of NF-κB p65 was determined by immunostaining and western blot. The levels of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α were detected by ELISA. The results showed that S100A8/A9 was actively increased and released into the extracellular space during the pre-metastatic phase of breast cancer. PlaD treatment attenuated S100A8/A9-induced growth, migration, and invasion of 4T1 cells. Furthermore, PlaD decreased the levels of IL-1β, IL-6, and TNF-α by inhibiting nuclear translocation of NF-κB p65. In conclusion, this study demonstrated that PlaD inhibited S100A8/A9-induced inflammatory response in 4T1 cells by suppressing the expression of IL-6, IL-1β, and TNF-α via inhibition of NF-κB signaling pathways.

152 - Pulmonary and systemic nitric oxide deficiency in the early phase of lung metastasis in 4T1 metastatic breast cancer murine model

The role of nitric oxide (•NO) in metastatic cancer progression has not been fully explained. In the present work we characterized the development of •NO deficiency in the lung and examined changes in •NO and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in Balb/c mice. Furthermore, orthotopic and intravenous models of 4T1 metastatic cancer in mice were used to study the differential effects of L-NAME-induced •NO deficiency on the primary tumor and the metastatic burden. •NO was assessed in isolated aorta and lungs using EPR spin-trapping and chemiluminescence, while NOx in blood were measured using either EPR, chemiluminescence or Griess reaction. Early pulmonary metastasis was correlated with lung inflammation. Pulmonary endothelial function and systemic •NO availability in 4T1 breast cancer-bearing mice were compromised prior to the development of pulmonary metastasis,preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT). Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway. L-NAME-induced •NO-deficiency showed that •NO plays a major role in the primary tumor development, but it is not the key mediator responsible for the regulation of cancer cell extravasation to the lungs in our model, despite clear-cut •NO-deficiency in the lung, reflected by a fall in systemic •NO bioavailability, both occurring early in the pre-metastatic phase.

FEATURES OF EXPRESSION OF KI-67 AND NFE2L2 IN TARGET ORGANS OF MELANOMA METASTASIS IN PREMETASTATIC PHASE

Tumor microenvironment is known to be an active participant of tumor development. Tumor microenvironment is functional cell community interacting with cancer cells and promoting proliferation, invasion and metastasis of tumor cells. In the article features of Ki-67 and NFE2L2 expression levels in target organs of melanoma metastasis in premetastatic phase are investigated. Increased Ki-67expression levels in lung tissue, liver and kidneys and decreased NFE2L2 levels in liverandkidneys can be the markers of structural and functional reorganization of melanoma metastasis target organs in premetastatic phase of melanoma development.

Chronic psychological stress promotes lung metastatic colonization of circulating breast cancer cells by decorating a pre-metastatic niche through activating β-adrenergic signaling.

Numerous studies have indicated that primary tumors induce the formation of a pre-metastatic niche in distant organs by secreting tumor-derived factors. The present study shows that pre-exposure to chronic stress enhanced lung colonization efficiency by circulating tumor cells, suggesting that chronic stress critically influences pre-metastatic lungs before the arrival of disseminated tumor cells. Ablation of the sympathetic nerve function by 6-OHDA or blockage of the β-adrenergic signaling by propranolol remarkably suppressed stress-induced lung metastasis. Depletion of circulating monocytes or lung macrophages strongly abolished stress-induced lung seeding by tumor cells, whereas treatment of mice with the β-adrenergic agonist isoproterenol (ISO) during the pre-metastatic phase promoted the infiltration of macrophages to the lung. Meanwhile, the numbers of monocytes in peripheral blood, spleen, and bone marrow were remarkably increased in response to ISO stimulation. These data indicate that the β-adrenergic signaling promotes lung metastatic colonization by tumor cells through increased output of monocytes in the pre-metastatic phase and infiltration of macrophages into the pre-metastatic lung. Mechanistic studies revealed that ISO stimulation upregulated the expression of CCL2 in pulmonary stromal cells and CCR2 in monocytes/macrophages, leading to the recruitment and infiltration of macrophages into the pre-metastatic lung. By inducing a response of monocytes/macrophages driven by the CCL2/CCR2 axis, stress-related catecholamine may act as a crucial factor in regulating the pre-metastatic niche for and lung colonization by tumor cells. Our data demonstrate that disturbance of host macro-environmental homeostasis has an influence on future metastatic organs. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SURVIVIN as a marker for quiescent-breast cancer stem cells-An intermediate, adherent, pre-requisite phase of breast cancer metastasis.

Cancer stem cells drive the metastatic cascade by undergoing epithelial to mesenchymal transition (EMT) and again mesenchymal to epithelial transition (MET). Using multiple breast cancer cell lines including cigarette smoke induced breast cancer cells and tumor derived primary cells from patient sample; we developed a breast cancer metastasis model and reported the existence of an adherent, distinct pre-metastatic phase, quiescent-breast cancer stem cells (Q-BCSCs) prior to attaining an EMT. SURVIVIN was found to be expressed in Q-BCSCs. Time dependant biphasic expression of SURVIVIN in Q-BCSCs reveals that Q-BCSCs is a pre-metastatic phase distinct from both epithelial and mesenchymal counterparts. SURVIVIN favours metastasis and up-regulates WNT/β-CATENIN pathway in a PI3 K/AKT-dependant manner for self-renewal. Knockdown of SURVIVIN in Q-BCSCs lost the metastatic property of cells by inhibiting invasion, EMT-MET, PI3 K/AKT/WNT cascade, and induced apoptosis. Thus, our data suggest the existence of a novel pre-metastatic phase (Q-BCSCs) before EMT and SURVIVIN acts as a marker for Quiescent-BCSCs.

Vascular endothelial growth factor secreted by breast cancer cells plays a critical role in the formation of pre-metastatic niche in the mouse lung

To explore the formation of pre-metastatic niche in the mouse lung and to study the underlying molecular mechanisms whereby primary breast carcinoma-derived factors mediate recruitment of bone marrow-derived cells (BMDCs) and affect the formation of pre-metastatic lung environment before the arrival of tumor cells. Mammary carcinoma 4T1 cells were inoculated into the mammary gland to construct mouse model of breast cancer. Confocal microscopy was used to detect the recruitment of BMDCs in the pre-metastatic lungs. The expression of factors in the mouse sera and 4T1 cell culture media was assayed using RayBio Custom mouse cytokine antibody array kit. The mice were injected daily with recombinant VEGF for 7 consecutive days to observe the effect of VEGF on BMDCs recruitment in the mouse lung. No BMDCs were observed in the lungs of control and 4T1-tumor-bearing mice on day 0. On day 7 and 14, clusters of BMDCs observed in the lungs of 4T1-tumor-bearing mice were 8.7±2.2/objective field and 48.8±3.2/objective field, respectively, significantly higher than those in the control mice (1.1±0.8/objective field and 3.1±1.7/objective field) (P<0.05 for both). Confocal microscopic observation found that metastatic breast cancer cells preferentially facilitate BMDCs recruitment sites in the pre-metastatic mouse lungs. The levels of VEGF, GM-CSF, and IL-6 in the serum of 4T1-tumor-bearing mice were significantly increased compared with those in the control group (P<0.05 for all). However, VEGF was detected only in the culture media of 4T1 cells. The amount of BMDCs in the mouse lung tissue was (22.8±3.6)/objective field in the VEGF group and (3.1±0.4)/objective field in the control group (P<0.05). There were 36.8±5.4 metastatic foci in the lung tissue of VEGF group and 12.6±2.2 in the control group (P<0.05). The results of this study demonstrate that primary breast cancer cells can alter the lung microenvironment during the pre-metastatic phase and induce the formation of pre-metastatic niche. Primary tumor cell-derived VEGF may be a crucial factor responsible for the formation of pre-metastatic niche.

TGFβ modulates inflammatory cytokines and growth factors to create premetastatic microenvironment and stimulate lung metastasis.

The formation of tumor-promoting premetastatic microenvironment plays a pivotal role on metastatic progression. Understanding how the primary tumor can promote the formation of premetastatic microenvironment in the lung will aid discovery of a final cure for metastatic breast cancer. The murine 4T1 mammary carcinoma cells were injected into the mammary fat pads of the BALB/c mice. Days 0-14 were considered the premetastatic phase. Lung tissues were examined using hematoxylin-eosin staining and transmission electron microscopy. After intravenous injection of TGFβ1 pretreated 4T1 cells, the relative pulmonary vascular permeability was quantified, the extravasation, survival, and proliferation of tumor cells in premetastatic lungs were evaluated, and the levels of S100A8, S100A9, VEGF, and Angpt2 were detected in tumor-bearing mice. The results showed that during the premetastatic phase, an inflammatory response and inflammation-induced vascular hyperpermeability were established, leading to an abnormal pulmonary microenvironment, which facilitated extravasation of circulating tumor cells, and subsequent survival and proliferation of metastatic tumor cells in a TGFβ-dependent manner. Moreover, the expressions of S100A8, S100A9, VEGF, and Angpt2 were increased, and an induction of these genes by TGFβ was further observed in premetastatic lungs. Thus, this study demonstrated that TGFβ promoted the creation of premetastatic microenvironment by modulating certain crucial inflammatory cytokines and growth factors, and finally enhanced the ability of circulating cells to seed the lung.

Primary breast cancer induces pulmonary vascular hyperpermeability and promotes metastasis via the VEGF-PKC pathway.

The lung is one of the most frequent target organs for breast cancer metastasis. When breast cancer cells from a primary tumor do not colonize the lung, which we named the premetastatic phase, the microenvironment of the lung has already been influenced by the primary tumor. However, little is known about the exact premetastatic alteration and regulatory mechanisms of the lung. Here, we used 4T1 cells (a mouse breast cancer cell line which can specifically metastasize to the lung) to build a mouse breast cancer model. We found that primary breast tumor induced increased pulmonary vascular permeability in the premetastatic phase, which facilitated the leakage of rhodamine-dextran and the extravasation of intravenous therapy injected cancer cells. Furthermore, tight junctions (TJs) were disrupted, and the expression of zonula occludens-1(ZO-1), one of the most important components of tight junctions, was decreased in the premetastatic lung. In addition, elevated serum vascular endothelial growth factor (VEGF) was involved in the destabilization of tight junctions and the VEGF antagonist bevacizumab reversed the primary tumor-induced vascular hyperpermeability. Moreover, activation of the protein kinase C (PKC) pathway disrupted the integrity of TJs and accordingly, the disruption could be alleviated by blocking VEGF. Taken together, these data demonstrate that primary breast cancer may induce tight junction disruptions in the premetastatic lung via the VEGF-PKC pathway and promote pulmonary vascular hyperpermeability before metastasis. © 2015 Wiley Periodicals, Inc.

The miR-30 Family Inhibits Pulmonary Vascular Hyperpermeability in the Premetastatic Phase by Direct Targeting of Skp2.

Before metastasis, primary tumor can create a premetastatic niche in distant organ to facilitate the dissemination of tumor cells. In the premetastatic phase, the permeability of pulmonary vasculatures is increased to accelerate the extravasation of circulating tumor cells. However, it is not clear whether local miRNAs contribute to the vascular hyperpermeability of the premetastatic niche. The expression of total miRNAs was determined using microarray in series of premetastatic lungs from tumor-bearing mice. Significantly differentially expressed miRNAs were identified and validated with qRT-PCR. Vascular permeability assays, vascular mimic systems, and orthotopic tumor models were used to investigate roles of selected miRNAs and target genes in premetastatic hyperpermeability. We identified a miRNA signature in premetastatic lungs. Among these miRNAs, miR-30a, b, c, d, and e were significantly attenuated. Subsequent investigations elucidated that lung fibroblast-derived miR-30s stabilized pulmonary vessels. Overexpression of miR-30s in lungs postponed metastasis and extended overall survival of B16 tumor-bearing mice. Following studies uncovered that Skp2 was directly targeted by miR-30s. Overexpression of Skp2 could disrupt pulmonary vessels, promote lung metastasis, and decrease overall survival of B16 tumor-bearing mice. These findings illuminate a novel mechanism for the modulation of premetastatic niches by miR-30s, which suggest that miR-30s represent not only promising targets for antimetastasis therapy but also indicators for metastasis.

Abstract B03: Hypoxia-induced carbonic anhydrase IX promotes MDSC recruitment and establishment of the breast cancer premetastatic niche by stimulating G-CSF production

Abstract The recruitment of bone marrow-derived cells (BMDCs) to distant tissue sites prior to the arrival of disseminated tumor cells has been shown to facilitate metastasis in a preclinical setting through the establishment of the pre-metastatic niche. Primary tumor hypoxia has been shown to play a pivotal role in the production of chemokines and cytokines responsible for the mobilization of these BMDCs, especially in breast cancer. Carbonic anhydrase IX (CAIX) expression is highly upregulated by breast cancer cells in hypoxia where its activity contributes to cell survival through the buffering of intracellular pH. Preclinically, CAIX has been demonstrated to be required for breast tumor growth and metastasis, whereas clinically, CAIX is an established indicator of poor prognosis and distant metastasis in breast cancer patients. However, the mechanism by which CAIX exerts its pro-metastatic function is incompletely understood. We investigated whether CAIX function in the hypoxic niche of the primary tumor was involved in establishment of the pre-metastatic niche through the production of soluble mediators of this process. Using shRNA-mediated depletion of CAIX in the metastatic 4T1 murine breast cancer cells and orthotopic tumors, we demonstrate that CAIX is required for the mobilization and recruitment of myeloid-derived suppressor cells (MDSCs) to pre-metastatic niches through the stimulation of granulocyte colony stimulating factor (G-CSF) production, a potent stimulator of MDSC mobilization and recruitment to pre-metastatic niches. We further demonstrate that CAIX expression is required for the activation of NF-κB by hypoxic breast cancer cells and that constitutive activation of the NF-κB pathway upon reducing CAIX expression by shRNA restores G-CSF secretion by hypoxic breast cancer cells. This is the first demonstration of a role for CAIX in the pre-metastatic phase of breast cancer metastasis and indicates that CAIX regulates the expression of G-CSF and recruitment of granulocytic MDSCs to pre-metastatic niches. Citation Format: Shawn C. Chafe, Yuanmei Lou, Jaclyn Sceneay, Marylou Vallejo, Paul C. McDonald, Andreas Möller, Shoukat Dedhar. Hypoxia-induced carbonic anhydrase IX promotes MDSC recruitment and establishment of the breast cancer premetastatic niche by stimulating G-CSF production. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B03. doi:10.1158/1538-7445.CHTME14-B03

Vascular endothelial growth factor plays a critical role in the formation of the pre-metastatic niche via prostaglandin E2.

Factors secreted by primary tumors can alter the microenvironment at distant organ sites, generating pre-metastatic niches for subsequent metastatic cancer cell colonization. Breast cancer cells have a propensity to home preferentially to the lung, but the underlying molecular mechanisms whereby primary breast carcinoma-derived factors affect the pre-metastatic lung environment before the arrival of the tumor cells are poorly understood. In this study, 4T1 mammary carcinoma cells were subcutaneously injected into the mammary glands of mice, resulting in the induction of inflammation, increased total vessel density and recruitment of bone marrow-derived cells (BMDCs) in pre-metastatic lungs. Subsequent examination revealed that the sites of inflammatory cell clusters in the lungs were tumor metastasis sites. Moreover, vascular endothelial growth factor (VEGF) induced prostaglandin E2(PGE2) production in mouse pulmonary microvascular endothelial cells (MPVECs) and enhanced the adhesion of 4T1 cells. Treatment with the cyclooxygenase-2 inhibitor celecoxib significantly reduced 4T1 cell adhesion to MPVECs, and also reduced cancer metastasis and the inflammatory response. These results suggest that VEGF may be an underlying carcinoma-derived factor responsible for formation of the pre-metastatic niche in the lung of 4T1 cell-bearing mice. This study, therefore, demonstrated that primary tumors can alter the lung microenvironment during the pre-metastatic phase by triggering an inflammatory response and PGE2 production. Primary tumor-derived VEGF might thus be a crucial factor responsible for the formation of the pre-metastatic niche by inducing PGE2 production.

Ephrin-A1 expression induced by S100A8 is mediated by the toll-like receptor 4

The deregulation of Eph/ephrin protein expression has been shown to lead to tumor development and progression. Both mRNA and protein expression analyses using clinical samples have demonstrated that ephrin-A1 is over-expressed in various cancers and positively correlates with a poor prognosis for cancer patients. The prognosis of cancer patients depends on metastasis to distant organs. We previously demonstrated that ADAM12 metalloproteinase cleaved ephrin-A1 and ADAM12-cleaved ephrin-A1 enhanced vascular permeability by degrading VE-cadherin and the EphA2 receptor at the plasma membrane. An increase of soluble ephrin-A1 levels in the serum facilitated tumor cell recruitment to the lungs, which resulted in lung metastasis. We also found that ephrin-A1 was overexpressed in 3LL tumors, a highly metastatic tumor, in mice and TNFα, an authentic positive regulator of ephrin-A1, was not elevated in the tumors, whereas S100A8 was. Moreover, S100A8 induced ephrin-A1 expression mediated by the toll-like receptor 4 (TLR4). S100A8 is known to be an endogenous ligand for TLR4 and its expression was shown to be increased in the lungs at the premetastatic phase. Thus, S100A8 and ephrin-A1 contribute to lung metastasis. Therefore, elucidating the regulation mechanism of ephrin-A1 overexpression is of importance and may lead to the development of therapeutic drugs against tumor growth and metastasis.

Primary tumor regulates the pulmonary microenvironment in melanoma carcinoma model and facilitates lung metastasis

Lung is a common site for the occurrence of melanoma metastasis, the mechanism by which primary melanoma affects the lungs before tumor cells arrival is poorly understood. The aim of this study was to explore lung microenvironment response to primary melanoma in the premetastatic phase. Melanoma cells (B16) were implanted into the Balb/c mice back, pulmonary inflammation response was analyzed by wet/dry ratios and H&E staining, the relationship between inflammation cells and metastatic foci was analyzed by bone marrow transplant mouse model, pulmonary vasculature was further analyzed by whole mount staining, and the circulating levels of proinflammatory cytokines in sera were evaluated by mouse cytokine array. In the premetastatic stage, significant inflammation response in lungs was induced by a distant primary melanoma, inflammation cells colonize in premetastatic sites before tumor cells arrived, and the sites of inflammation cells clusters are tumor metastasis sites. VEGF, M-CSF and TNF-α may be the underlying factors responsible for the increased metastasis in the B16-bearing mice. Treatment with celecoxib had effects on inflammation response and reduced cancer metastasis. In the premetastatic phase, the melanoma induces pulmonary inflammation response, changes the lung environment and then facilitates lung metastasis. Thus, inhibition of lung inflammation may provide potential targets for the prevention of metastasis.

Combining Carbon Ion Radiotherapy and Local Injection of α-Galactosylceramide–Pulsed Dendritic Cells Inhibits Lung Metastases in an In Vivo Murine Model

Our previous report indicated that carbon ion beam irradiation upregulated membrane-associated immunogenic molecules, underlining the potential clinical application of radioimmunotherapy. The antimetastatic efficacy of local combination therapy of carbon ion radiotherapy and immunotherapy was examined by use of an in vivo murine model. Tumors of mouse squamous cell carcinoma (NR-S1) cells inoculated in the legs of C3H/HeSlc mice were locally irradiated with a single 6-Gy dose of carbon ions (290 MeV/nucleon, 6-cm spread-out Bragg peak). Thirty-six hours after irradiation, α-galactosylceramide-pulsed dendritic cells (DCs) were injected into the leg tumor. We investigated the effects on distant lung metastases by counting the numbers of lung tumor colonies, making pathologic observations, and assessing immunohistochemistry. The mice with no treatment (control) presented with 168 ± 53.8 metastatic nodules in the lungs, whereas the mice that received the combination therapy of carbon ion irradiation and DCs presented with 2.6 ± 1.9 (P = 0.009) at 2 weeks after irradiation. Immunohistochemistry showed that intracellular adhesion molecule 1, which activates DCs, increased from 6 h to 36 h after irradiation in the local tumors of the carbon ion-irradiated group. The expression of S100A8 in lung tissue, a marker of the lung pre-metastatic phase, was decreased only in the group with a combination of carbon ions and DCs. The combination of carbon ion radiotherapy with the injection of α-galactosylceramide-pulsed DCs into the primary tumor effectively inhibited distant lung metastases.

Pulmonary Vascular Destabilization in the Premetastatic Phase Facilitates Lung Metastasis

Before metastasis, certain organs have already been influenced by primary tumors. However, the exact alterations and regulatory mechanisms of the premetastatic organs remain poorly understood. Here, we report that, in the premetastatic stage, angiopoietin 2 (Angpt2), matrix metalloproteinase (MMP) 3, and MMP10 are up-regulated in the lung by primary B16/F10 tumor, which leads to the increased permeability of pulmonary vasculatures and extravasation of circulating tumor cells. Subsequent studies show that Angpt2, MMP3, and MMP10 have a synergistic effect on disrupting vascular integrity in both in vitro and in vivo models. Lentivirus-based in vivo RNA interference of Angpt2, MMP3, and MMP10 attenuates the pulmonary vascular permeability and suppresses the infiltration of myeloid cells in the premetastatic lung. Moreover, knocking down these factors significantly inhibits the spontaneous lung metastasis in the model by orthotopic implantation of MDA-MB-231-Luc-D3H1 cells in nude mice. Further investigations reveal that the malignancy of tumor cells is positively correlated with their capabilities to induce the expression of Angpt2, MMP3, and MMP10. Luciferase reporter assay and chromatin immunoprecipitation assay also suggest that transforming growth factor-beta1 and tumor necrosis factor-alpha signaling are involved in the regulation of these premetastatic factors. Our study shows that pulmonary vascular destabilization in the premetastatic phase promotes the extravasation of tumor cells and facilitates lung metastasis, which may provide potential targets for clinical prevention of metastasis.