Lewis Lung Carcinoma Tumor-bearing Research Articles

The LEAP2 Response to Cancer-Related Anorexia-Cachexia Syndrome in Male Mice and Patients.

The hormone ghrelin serves a protective role in cancer-related anorexia-cachexia syndrome (CACS)-a condition in which plasma levels of ghrelin rise, its administration lessens CACS severity, and experimentally reduced signaling by its receptor (GHSR) worsens fat loss and anorexia and accelerates death. Yet, actions for the related hormone liver-expressed antimicrobial peptide-2 (LEAP2), which is an endogenous GHSR antagonist, are unexplored in CACS. Here, we found that plasma LEAP2 and LEAP2/ghrelin ratio were lower in Lewis lung carcinoma (LLC) and RM-9 prostate cancer CACS mouse models. Ghrelin deletion exaggerated losses of tumor-free body weight and fat mass, reduced food intake, reduced soleus muscle weight, and/or lowered grip strength in LLC or RM-9 tumor-bearing mice. LEAP2 deletion lessened reductions in tumor-free body weight and fat mass and increased food intake in LLC or RM-9 tumor-bearing mice. In a 55-subject cohort of patients with CACS or weight-stable cancer, the plasma LEAP2/total ghrelin ratio was negatively correlated with 6-month weight change preceding blood collection. These data demonstrate that ghrelin deletion exacerbates CACS in the LLC and RM-9 tumor-bearing mouse models while contrastingly, LEAP2 deletion reduces measures of CACS in these tumor-bearing mouse models. Further, they suggest that lower plasma LEAP2/ghrelin ratio protects against worsened CACS.

Multifunctional elastin-like polypeptide nanocarriers for efficient miRNA delivery in cancer therapy.

The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation. Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice. Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects.

Aerobic exercise training mitigates tumor growth and cancer-induced splenomegaly through modulation of non-platelet platelet factor 4 expression

Exercise training reduces the incidence of several cancers, but the mechanisms underlying these effects are not fully understood. Exercise training can affect the spleen function, which controls the hematopoiesis and immune response. Analyzing different cancer models, we identified that 4T1, LLC, and CT26 tumor-bearing mice displayed enlarged spleen (splenomegaly), and exercise training reduced spleen mass toward control levels in two of these models (LLC and CT26). Exercise training also slowed tumor growth in melanoma B16F10, colon tumor 26 (CT26), and Lewis lung carcinoma (LLC) tumor-bearing mice, with minor effects in mammary carcinoma 4T1, MDA-MB-231, and MMTV-PyMT mice. In silico analyses using transcriptome profiles derived from these models revealed that platelet factor 4 (Pf4) is one of the main upregulated genes associated with splenomegaly during cancer progression. To understand whether exercise training would modulate the expression of these genes in the tumor and spleen, we investigated particularly the CT26 model, which displayed splenomegaly and had a clear response to the exercise training effects. RT-qPCR analysis confirmed that trained CT26 tumor-bearing mice had decreased Pf4 mRNA levels in both the tumor and spleen when compared to untrained CT26 tumor-bearing mice. Furthermore, exercise training specifically decreased Pf4 mRNA levels in the CT26 tumor cells. Aspirin treatment did not change tumor growth, splenomegaly, and tumor Pf4 mRNA levels, confirming that exercise decreased non-platelet Pf4 mRNA levels. Finally, tumor Pf4 mRNA levels are deregulated in The Cancer Genome Atlas Program (TCGA) samples and predict survival in multiple cancer types. This highlights the potential therapeutic value of exercise as a complementary approach to cancer treatment and underscores the importance of understanding the exercise-induced transcriptional changes in the spleen for the development of novel cancer therapies.

The time-course of cancer cachexia onset reveals biphasic transcriptional disruptions in female skeletal muscle distinct from males

BackgroundCancer-cachexia (CC) is a debilitating condition affecting up to 80% of cancer patients and contributing to 40% of cancer-related deaths. While evidence suggests biological sex differences in the development of CC, assessments of the female transcriptome in CC are lacking, and direct comparisons between sexes are scarce. This study aimed to define the time course of Lewis lung carcinoma (LLC)-induced CC in females using transcriptomics, while directly comparing biological sex differences.ResultsWe found the global gene expression of the gastrocnemius muscle of female mice revealed biphasic transcriptomic alterations, with one at 1 week following tumor allograft and another during the later stages of cachexia development. The early phase was associated with the upregulation of extracellular-matrix pathways, while the later phase was characterized by the downregulation of oxidative phosphorylation, electron transport chain, and TCA cycle. When DEGs were compared to a known list of mitochondrial genes (MitoCarta), ~ 47% of these genes were differently expressed in females exhibiting global cachexia, suggesting transcriptional changes to mitochondrial gene expression happens concomitantly to functional impairments previously published. In contrast, the JAK-STAT pathway was upregulated in both the early and late stages of CC. Additionally, we observed a consistent downregulation of Type-II Interferon signaling genes in females, which was associated with protection in skeletal muscle atrophy despite systemic cachexia. Upregulation of Interferon signaling was noted in the gastrocnemius muscle of cachectic and atrophic male mice. Comparison of female tumor-bearing mice with males revealed ~ 70% of DEGs were distinct between sexes in cachectic animals, demonstrating dimorphic mechanisms of CC.ConclusionOur findings suggest biphasic disruptions in the transcriptome of female LLC tumor-bearing mice: an early phase associated with ECM remodeling and a late phase, accompanied by the onset of systemic cachexia, affecting overall muscle energy metabolism. Notably, ~ 2/3 of DEGs in CC are biologically sex-specific, providing evidence of dimorphic mechanisms of cachexia between sexes. Downregulation of Type-II Interferon signaling genes appears specific to CC development in females, suggesting a new biological sex-specific marker of CC not reliant on the loss of muscle mass, that might represent a protective mechanism against muscle loss in CC in female mice.

Paraoxonase 2 (PON2) plays a limited role in murine lung tumorigenesis

Paraoxonase 2 (PON2) is a multifunctional intracellular enzyme that has received growing attention for its ability to modulate various aspects of normal and malignant cellular physiology. Recent research has revealed that PON2 is upregulated in tissues from patients with various types of solid tumors and hematologic cancers, likely due to its ability to suppress oxidative stress and evade apoptosis. However, the effects of PON2 on pulmonary oncogenesis are unknown. Here, we conducted studies to investigate how PON2 influences lung cancer cell proliferation in vitro and lung tumorigenesis in vivo using a variety of cellular and animal models. It was found that PON2 expression deficiency hampered the proliferation of cultured lung cancer cells with concomitant cell cycle arrest at the G1 phase. In addition, the loss of endogenous PON2 expression impaired key aspects of oxidative metabolism in lung adenocarcinoma cells. Moreover, we investigated how the interplay between PON2 expression in lung tumors and host mice influences lung tumor initiation and progression. PON2 status in both transplanted tumor cells and mice failed to influence the development of subcutaneously grafted Lewis lung carcinoma (LLC) tumors, orthotopically implanted LLC tumors, and oncogenic Kras-driven primary lung adenocarcinoma tumors. Importantly, the frequencies of tumor-infiltrating myeloid subsets that include myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages were not impacted by PON2 expression in LLC tumor-bearing mice. Overall, our studies indicate that PON2 plays a limited role in murine lung tumorigenesis.

Targeting complement C5a to improve radiotherapy sensitivity in non-small cell lung cancer.

Tumor local and distant relapse recurrence after radiotherapy (RT) is one of the critical factors leading to poor prognosis. The effective antitumor effects of RT are dependent upon the participation of innate and adaptive components of the immune system. C5a/C5aR1 signaling can regulate antitumor immune effect in the tumor microenvironment (TME). Thus, exploring the changes and mechanism in the TME induced by RT-mediated complement activation may provide a novel perspective for reversing radioresistance. First, fractionated radiation of 8 Gy ×3 fractions were targeted at Lewis lung carcinoma (LLC) tumor-bearing female mice to measure the infiltration of CD8+ T cell and analyze the RNA sequencing (RNA-seq) in RT-recruited CD8+ T cells. Second, tumor growth was measured in LLC tumor-bearing mice treated with RT either with or without C5aR1 inhibitor to clarify the antitumor effect of RT combined with C5aR1 inhibitor. Third, we detected the expression of C5a/C5aR1 and their signaling pathways on radiated tumor tissues. Furthermore, we investigated the expression of C5a in tumor cells at different time points after different doses of RT. In our system, RT induced the increased infiltration of CD8+ T cells and local activation of complement C5a/C5aR. Concurrent administration of RT and blocking of C5aR improved radiosensitivity and tumor-specific immune response, which was reflected by high C5aR expression in CD8+ T cells. The AKT/NF-κB pathway was found to be an important signaling pathway in C5a/C5aR axis mediation by RT. RT promotes the release of C5a from tumor cells and leads to up-regulation of C5aR1 expression via the AKT/NF-κB pathway. Inhibition of the combination of complement C5a and C5aR could improve RT sensitivity. Our work provides evidence that the combination of RT and C5aR blockade opens a new window of opportunity to promote anti-tumor therapeutic effects in lung cancer.

Ursolic Acid Alleviates Cancer Cachexia and Prevents Muscle Wasting via Activating SIRT1

Skeletal muscle wasting is the most remarkable phenotypic feature of cancer cachexia that increases the risk of morbidity and mortality. However, there are currently no effective drugs against cancer cachexia. Ursolic acid (UA) is a lipophilic pentacyclic triterpene that has been reported to alleviate muscle atrophy and reduce muscle decomposition in some disease models. This study aimed to explore the role and mechanisms of UA treatment in cancer cachexia. We found that UA attenuated Lewis lung carcinoma (LLC)-conditioned medium-induced C2C12 myotube atrophy and muscle wasting of LLC tumor-bearing mice. Moreover, UA dose-dependently activated SIRT1 and downregulated MuRF1 and Atrogin-1. Molecular docking results revealed a good binding effect on UA and SIRT1 protein. UA rescued vital features wasting without impacting tumor growth, suppressed the elevated spleen weight, and downregulated serum concentrations of inflammatory cytokines in vivo. The above phenomena can be attenuated by Ex-527, an inhibitor of SIRT1. Furthermore, UA remained protective against cancer cachexia in the advanced stage of tumor growth. The results revealed that UA exerts an anti-cachexia effect via activating SIRT1, thereby downregulating the phosphorylation levels of NF-κB and STAT3. UA might be a potential drug against cancer cachexia.

Evodiamine suppresses the progression of non-small cell lung carcinoma via endoplasmic reticulum stress-mediated apoptosis pathway in vivo and in vitro.

BackgroundEvodiamine (EVO) is one of the major components isolated from Evodia rutaecarpa (Juss.). Recent studies have shown that EVO has an anti-cancer effect. However, the pharmacological mechanism by which EVO impacts cancer is still poorly understood.ObjectivesThis study focused on asking the anti-cancer effect of EVO in human non-small cell lung carcinoma (NSCLC), and in particular to investigate whether EVO acts via modulating the endoplasmic reticulum stress (ERS)-mediated apoptosis pathway.Materials and MethodsA Lewis lung carcinoma (LLC) tumor-bearing mouse model was treated with low-dose EVO (5 mg/kg) and high-dose EVO (10 mg/kg) intraperitoneally for 14 d. The effects of EVO on tumor growth, apoptosis, and ERS were assessed. In addition, NSCLC A549 and LLC cells were treated with EVO in vitro. The effects of EVO on cell proliferation, apoptosis, and ERS were investigated. Finally, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was used to validate whether EVO induced apoptosis of NSCLC cells by modulating ERS.ResultsEVO treatment significantly inhibited tumor growth in LLC tumor-bearing mice. H&E staining indicated that EVO treatment reduced the number of tumor cells and the nucleo-plasmic ratio. Immunostaining showed that EVO treatment significantly decreased the expression of Ki-67. TUNEL staining revealed that EVO induced apoptosis in the tumor. Likewise, EVO treatment up-regulated the expression of apoptosis-related genes and proteins and increased activation of the ERS pathway in the tumor. Additionally, EVO inhibited cell proliferation and increased cell apoptotic rates in A549 and LLC cells. EVO also increased the expression levels of genes and proteins associated with ERS-mediated apoptosis pathway in vitro. The effects of EVO on apoptosis were abolished by 4-PBA treatment.ConclusionsOur study demonstrated that EVO suppresses the progression of NSCLC by modulating the ERS-mediated apoptosis pathway.

IL-17A contributes to skeletal muscle atrophy in lung cancer-induced cachexia via JAK2/STAT3 pathway.

Cachexia is a complex metabolic syndrome that occurs in approximately 50% of patients with cancer. Skeletal muscle atrophy is the primary clinical feature. Interleukin (IL)-17A, a proinflammatory factor, plays an important role in many chronic inflammatory diseases. Here, we describe a novel signaling pathway through which IL-17A induced muscle atrophy. We conducted a retrospective clinical study to investigate the relationship between IL-17A and the skeletal muscle index in patients with lung adenocarcinoma. We also investigated the involvement of JAK2/STAT3 signaling pathway regarding the main features of cachexia by injecting Lewis lung carcinoma (LLC) cells into C57BL/6 mice as a model to replicate cancer-induced cachexia. In vitro, C2C12 myotubes were treated with recombinant IL-17A, anti-IL-17A monoclonal antibody, STAT3 inhibitor AG490, and LLC-conditioned medium. Cell viability and aging were also evaluated. We found that in cancer conditions, increased serum levels of IL-17A were related to muscle wasting. JAK2/STAT3 phosphorylation was observed in the muscle of LLC tumor-bearing mice, accompanied by decreased MHC/Myog levels and increased MuRF1/Atrogin-1 levels. Administration of anti-IL-17A monoclonal antibody and AG490 slowed muscle atrophy development. Consistent with the in vivo findings, C2C12 myotubes treated with IL-17A and LLC-conditioned medium demonstrated phosphorylated JAK2/STAT3 signaling, resulting in MHC loss and myotube atrophy. IL-17A also inhibited C2C12 cell proliferation, cell cycle breaking, and cellular senescence. Our results identify that phosphorylation of IL-17A/JAK2/STAT3 signaling pathway appears to be an important component in the pathogenesis of LLC tumor-induced cachexia. Targeted therapy of IL-17A may be a promising approach to reduce skeletal muscle loss in patients with cancer.

Cancer causes dysfunctional insulin signaling and glucose transport in a muscle-type-specific manner.

Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation are not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5nM). Glucose transport was measured using 2-[3 H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389, and ribosomal-S6ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent on muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, the skeletal muscle could be a target for managing metabolic dysfunction in cancer.

Downregulation of miR-29c promotes muscle wasting by modulating the activity of leukemia inhibitory factor in lung cancer cachexia

BackgroundCancer cachexia is a wasting disorder characterized by significant weight loss, and is attributed to skeletal muscle weakness. In the process of cancer development, microRNAs act as oncogenes or tumor suppressors. Moreover, they are implicated in muscle development and wasting. This study sought to explore the mechanisms and correlation between miR-29c and muscle wasting in lung cancer cachexia.MethodsData for expression analysis were retrieved from the Cancer Genome Atlas (TCGA) database. qRT-PCR analyses were performed to explore the expression levels of miR-29c and Leukemia Inhibitory Factor (LIF). Lewis lung carcinoma (LLC) cell line was used to establish a cachexia model to explore the functions of miR-29c and LIF in lung cancer cachexia. Furthermore, in vitro (in C2C12 myotubes) and in vivo (in LLC tumor-bearing mice) experiments were performed to explore the mechanisms of miR-29c and LIF in lung cachexia.ResultsAnalysis of the lung cancer cachexia model showed that miR-29c was down-regulated, and its expression was negatively correlated with muscle catabolic activity. Overexpression of miR-29c mitigated the cachectic phenotype. Mechanistic studies showed that LIF was a direct target gene of miR-29c, and LIF was upregulated in vitro and in vivo. Analysis showed that LIF promoted muscle wasting through the JAK/STAT and MAP-kinase pathways.ConclusionsThe findings indicated that miR-29c was negatively correlated with the cachectic phenotype, and the miR-29c-LIF axis is a potential therapeutic target for cancer cachexia.

Predicting the herbal medicine triggering innate anti-tumor immunity from a system pharmacology perspective

Although the main focus of immuno-oncology has been manipulating the adaptive immune system, tumor associated macrophages (TAMs) are the main infiltrating component in the tumor microenvironment (TME) and play a critical role in cancer progression. TAMs are mainly divided into two different subtypes: macrophages with antitumor or killing activity are called M1 while tumor-promoting or healing macrophages are named M2. Therefore, controlling the polarization of TAMs is an important strategy for cancer treatment, but there is no particularly effective means to regulate the polarization process. Here, combined systems pharmacology targets and pathways analysis strategy, we uncovered Scutellariae Radix (SR) has the potential to regulate TAMs polarization to inhibit the growth of non-small cell lung cancer (NSCLC). Firstly, systems pharmacology approach was used to reveal the active components of SR targeting macrophages in TME through compound target prediction and target-microenvironment phenotypic association analysis. Secondly, in vitro experiment verified that WBB (wogonin, baicalein and baicalin), major active ingredients of SR are significantly related to macrophages and survival, initiated macrophages programming to M1-like macrophages to promoted the apoptosis of tumor cells. Finally, we evidenced that WBB effectively inhibited tumor growth in LLC (Lewis lung carcinoma) tumor-bearing mice and increased the infiltration of M1-type macrophages in TME. Overall, the systems pharmacology strategy offers a paradigm to understand the mechanism of polypharmacology of natural products targeting TME.

Diversity of draining lymph node-tumor overlapping T cell receptor repertoire reflects the anti-tumor effect of cancer immunotherapies

Abstract CD8+ tumor-infiltrating lymphocytes, the main effector cells of anti-tumor immune responses, are composed of various T cell clones. Numerous studies have demonstrated the presence of tumor-reactive T cell clones with high frequency in tumors. However, it remains unclear as to which of the two mechanisms, the expansion of limited tumor-specific clones or the induction of diverse tumor-reactive ones, is more beneficial for the action of immune checkpoint inhibitors (ICIs). To address this question, we performed a T cell receptor repertoire analysis on B16F10 and on Lewis lung carcinoma tumor-bearing mice treated with immunotherapy including anti-PD-L1 monoclonal antibody (mAb) and anti-CD4 depletion mAb, which enhance the expansion of tumor-reactive CD8+ T cells. We focused on the T cell clones that overlapped between the tumor and draining lymph node (dLN) to analyze the T cell clones that were mobilized into the anti-tumor response after treatment. In anti-PD-L1 and anti-CD4 treated mice, the total frequency of CD8+ dLN-tumor overlapping clones in the tumor was increased. Surprisingly, although the expansion of the top10 overlapping clones did not differ, ICI treatment increased the total frequency of the 11th or lower overlapping clones, indicating the broadening of the tumor-reactive T cell repertoire. Moreover, the administration of FTY720 to tumor-bearing mice, which inhibits egress of T cells from the LNs to circulation, reduced the total frequency of the 11th or lower overlapping clones in the tumor and reduced the long-term anti-tumor effect of anti-PD-L1 treatment. These results suggest that mobilization of diverse tumor-reactive clones is essential for enhancing and persistent anti-tumor effect of ICIs.

Radiotherapy programs neutrophils to an antitumor phenotype by inducing mesenchymal-epithelial transition.

BackgroundNeutrophils can play a pro-tumor or anti-tumor role depending on the tumor microenvironment. The effects of concurrent treatment with granulocyte colony-stimulating factor (G-CSF) and radiotherapy (RT) on neutrophils have not yet to be described.MethodsHypofractionated radiation of 8 Gy ×3 fractions was administered with or without recombinant G-CSF to Lewis lung carcinoma tumor-bearing C57BL/6 model mice. The activation status of cytotoxic T cells in the mice was measured, along with the levels of tumor-associated neutrophils, cytotoxic T cells, and Treg cells. Tumor growth, survival, cytokine expression, and signaling pathways underlying anti-tumor effects of tumor-associated neutrophils after treatment were also studied. To ascertain the effects of concurrent RT and G-CSF on tumor-associated neutrophils, neutrophil depletion was performed.ResultsRT affected early neutrophil infiltration, which is the first-line immune response. Subsequently, enhanced accumulation of lymphocytes, particularly CD8 cytotoxic T cells, was observed. Notably, lymphocytic infiltration was inhibited by neutrophil depletion but enhanced by G-CSF treatment. RT generated persistent DNA damage, as evidenced by an accumulation of phosphorylation of histone H2AX (γH2AX), and subsequently triggered inflammatory chemokine secretion. The chemokines CXCL1, CXCL2, and CCL5 were upregulated in both radiation-treated cells and the corresponding supernatants. Neutrophils that were newly recruited after RT improved radiosensitivity by inhibiting epithelial–mesenchymal transition via the reactive oxygen species-mediated PI3K/Akt/Snail signaling pathway, and G-CSF treatment enhanced this effect.ConclusionsThe results of this study suggest that RT activates neutrophil recruitment and polarizes newly recruited neutrophils toward an antitumor phenotype, which is enhanced by the concurrent administration of G-CSF. Mesenchymal-epithelial transition induced by reactive oxygen species accumulation plays a major role in this process. Thus, the polarization of tumor-associated neutrophils might play a role in future cancer immunotherapies.

Toll-like receptor 4 mediates the development of fatigue in the murine Lewis Lung Carcinoma model independently of activation of macrophages and microglia

Cancer-related fatigue at the time of tumor diagnosis is commonly attributed to inflammation associated with the disease process. However, we have previously demonstrated that running wheel deficits occur well before increased expression of proinflammatory cytokines in the liver and brain in a murine model of human papilloma virus-related head and neck cancer (mEER). Further, we have demonstrated that genetic deletion of type I interleukin-1 receptor and MyD88 has no effect. In the current investigation we sought to test the generality of this finding by assessing whether there is a role for toll-like receptor (TLR) 4-dependent inflammation in the fatigue-like behavior observed in mice with Lewis Lung Carcinoma (LLC) or mEER tumors. Genetic deletion of TLR4 attenuated tumor-induced elevations in liver pro-inflammatory cytokine expression in both models. However, it only abrogated wheel running deficits in LLC tumor bearing mice. To determine whether TLR4 signaling in the LLC model involves innate immune cells, mice were treated with the colony stimulating factor (CSF)-1 receptor antagonist PLX-5622 before and throughout tumor development to deplete microglia and peripheral macrophages. Administration of PLX-5622 had no protective effect on wheel running deficits in either mEER or LLC tumor models despite effective depletion of microglia and a down regulation of peripheral proinflammatory cytokine expression. These results indicate that the TLR4 signaling that mediates fatigue-like behavior in LLC mice is not dependent upon microglial or peripheral macrophage activation. Based on the literature and our data demonstrating attenuation of ubiquitin proteasome pathway activation in the gastrocnemius muscle of Tlr4−/− mice implanted with LLC cells, we interpret our current findings as indication that skeletal muscle TLR4 signaling may be involved. These results are important in that they add to the evidence that tumor-induced fatigue develops independently from classical neuroinflammation.

Abstract 5522: Evaluation of eribulin-induced alterations of the intact immune cell landscape in spleens and tumors from tumor-bearing immunocompetent mice

Abstract Eribulin, a synthetic analog of the marine sponge natural product halichondrin B, is approved in the US for certain patients with advanced breast cancer or liposarcoma. Its mechanisms of action include both cytotoxic, antimitotic effects as well as non-cytotoxic effects on tumor vasculature, phenotype and microenvironment. Known aspects of eribulin's effects on tumor phenotype, including reversal of epithelial-mesenchymal transition (EMT) and induction of cellular differentiation in various preclinical models, led us to speculate that eribulin might also alter immune homeostasis in the in vivo setting, both in tumors themselves as well as supporting immune organs such as the spleen. Here, we report on eribulin's effects on both lymphoid and myeloid lineage cellular components in both spleen and tumors from immunocompetent mice bearing subcutaneous Lewis lung carcinoma (LLC) isografts. Surprisingly, spleens of vehicle treated, LLC tumor-bearing mice are approximately twice as large spleens from non-tumor-bearing mice, with an excellent correlation (r2 = 0.92) between individual tumor size and spleen weight arguing for a direct cause and effect relationship between the presence of tumor and splenomegaly. Tumor-induced splenomegaly is associated with increased proliferation of splenic non-lymphoid (CD4-/CD8-/CD19-) cells, including both CD11b+ mature myeloid lineage cells as well as CD11b- cells. Due to high levels of P-glycoprotein (PgP) characteristic of most murine tumors, LLC tumors respond poorly to eribulin, a known PgP substrate. Thus, 3 doses of MTD eribulin (1.6 mg/kg; Q4Dx3) led to only a 30% reduction in tumor volume relative to vehicle-treated mice. Unexpectedly, however, despite this reduced tumor volume, spleens from eribulin-treated mice were twice again larger than the already-enlarged spleens from vehicle-treated tumor-bearing mice, and the clear relationship between tumor size and spleen weight was abolished. This additional eribulin-induced splenomegaly was associated with enhanced proliferation of the same CD4-/CD8-/CD19- non-lymphoid cells, CD11b+ myeloid lineage cells and CD11b- cells as in vehicle-treated, tumor-bearing mice. Evaluation of immune cell components in the associated fixed tumors from eribulin- and vehicle-treated mice is currently ongoing to assess what effect, if any, such changes in the spleen will have on the tumor immune microenvironment; these results will be presented at the meeting. In conclusion, our results point to significant and unexpected effects of eribulin treatment on the immune cell landscape in spleens and tumors from tumor-bearing immunocompetent mice. Citation Format: Haochen Wu, Keyi Zhu, Xiaolong Tu, Diana Albu, Mary Woodall-Jappe, Bruce A. Littlefield. Evaluation of eribulin-induced alterations of the intact immune cell landscape in spleens and tumors from tumor-bearing immunocompetent mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5522.

Abstract 5375: Large, polymorphonuclear circulating cancer-associated cell with dual epithelial and macrophage/myeloid phenotype is prognostic in non-small cell lung cancer and has an impact on anti-tumor immune responses

Abstract Purpose: In addition to the FDA-approved definition of a circulating tumor cell (CTC), various phenotypes of circulating cancer-associated cells have been described. A distinct large (>30µm), polymorphonuclear, circulating, cancer-associated cell (CCAC) with a dual epithelial and macrophage/myeloid phenotype has been identified in solid cancer patients. Aims of the study were to identify and characterize these hybrid CCACs in non-small cell lung cancer (NSCLC) patients and to determine their impact on cancer-associated immune responses in vitro and in vivo in a murine model of lung cancer. Experimental Design: In a prospective trial, 7.5ml of peripheral blood was collected from 167 subjects. CTCs and CCACs were enriched by size-based microfilter and immunostaining was performed (CK 8/18/19, EpCAM, CD14/45, DAPI). To mimic circulating hybrid cells, polyethylene glycol was used to induce fusion of murine RAW 264.7 macrophage cells and Lewis Lung Carcinoma (LLC) cells; fused cells were validated by flow cytometry, electron microscopy and fluorescence microscopy. Immunomodulatory effects of fused cells were determined in LLC-tumor bearing mice. Results: 78 NSCLC patients and 89 control subjects (69 chronic smokers with and without lung nodules on screening LDCT, 20 healthy never-smokers) were enrolled. Distinct, large, polymorphonuclear CK+/EpCAM+/CD14/45+ CCACs were detected at lower rates than traditional CK+/EpCAM+/CD45- CTCs, but significantly higher in subjects with NSCLC in comparison to controls (p<0.05). CCACs and traditional CTCs were absent in 20 healthy controls. ≥2 CCACs correlated with significantly poorer survival and higher recurrence rates by uni- and multivariate analysis (p<0.05). To support these findings, PEG-induced fusion cells suppressed immune responses decreased T-cell activation and increased Tregs in vitro and in LLC tumor-bearing mice. Conclusions and future perspectives: These investigations show that CCAC with a dual epithelial and macrophage/myeloid phenotype is associated with aggressive clinical behavior in NSCLC. Pro-tumorigenic mechanisms are suggested by macrophage-tumor cell fusion leading to suppressed T cell-mediated anti-tumor immune responses. In future, the impact of CCACs on tumor progression and immune responses will need to be further studied in NSCLC patients. CCACs presentCCACs mean (±SEM); median (range)CTCs presentCTCs mean (±SEM); median (range)p valueSubjects analyzed (total)167NSCLC7861 (78.2%)3.12 (±0.37); 2 (0-17)78 (100%)21.95 (±1.24); 19 (8-80)<0.05† (CCACs vs. CTCs)Median Age (range)69 (41-81)Gender• Females (%)41 (52.6%)• Males (%)37 (47.4%)Stage (AJCC 8th ed.)<0.05* (CCACs vs. CTCs)• I39 (50%)26 (66.7%)1.26 (±0.21); 1 (0-5)39 (100%)14.59 (±0.62); 14 (8-28)• II15 (19.2%)11 (73.3%)2.67 (±0.64); 2 (0-7)15 (100%)22.67 (±1.12); 23 (18-33)• III12 (15.4%)12 (100%)6.17 (±0.81); 6 (2-11)12 (100%)29.42 (±1.58); 29 (23-42)• IV12 (15.4%)12 (100%)6.67 (±1.21); 5 (3-17)12 (100%)37.5 (±4.23); 36 (25-80)(p<0.05*)(p<0.05*)Control long-term smoker subjects69Median Age (range)63 (54-74)Gender• Females (%)32 (46.4%)• Males (%)37 (53.6%)No lung nodules14007 (50%)1.36 (±0.45); 0 (0-4)Benign lung nodules5511 (20%)0.33 (±0.09); 0 (0-2)41 (74.5%)3.26 (±0.39); 3 (0-11)Healthy, never-smoker controls200 (0%)00 (0%)0 Citation Format: Yariswamy Manjunath, Kanve N. Suvilesh, Diego M. Avella, Eric T. Kimchi, Kevin F. Staveley-O'Carroll, Chelsea B. Deroche, Guangfu Li, Jussuf T. Kaifi. Large, polymorphonuclear circulating cancer-associated cell with dual epithelial and macrophage/myeloid phenotype is prognostic in non-small cell lung cancer and has an impact on anti-tumor immune responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5375.

A UPLC-Q-TOF/MS-based plasma metabolomics approach reveals the mechanism of Compound Kushen Injection-based intervention against non-small cell lung cancer in Lewis tumor-bearing mice

Compound Kushen Injection (CKI), a well-known Chinese Medicine preparation, has been used to treat non-small cell lung cancer (NSCLC) for more than 15 years, and its clinical curative effect is considered to be beneficial. This study was designed to evaluate the effects and underlying mechanisms of CKI against NSCLC using an ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based plasma metabolomics approach. 4',6-diamidino-2-phenylindole (DAPI) staining and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay were employed to assess apoptosis and the viability of A549 cells with and without CKI treatment. The weight/volume of Lewis lung carcinoma (LLC) sarcomas and histopathological examinations were used to evaluate the anti-tumor effects of CKI against NSCLC. A UPLC-Q-TOF/MS method combined with multivariate data analysis was developed to characterize metabolomic fingerprinting and to screen functional biomarkers that are linked to the CKI treatment of LLC mice, and then metabolic pathway analysis was used to investigate the therapeutic mechanism of CKI. DAPI staining and MTT dye reduction assays indicated that CKI-induced apoptosis and inhibited the proliferation of A549 cells, respectively, in a concentration-dependent manner. The sarcoma volumes and weights in LLC tumor-bearing mice in CKI-dosed groups were significantly lower than those in a model group, which was treated with physiological saline. Histopathological analysis of sections of sarcomas and left pulmonary lobes indicated that CKI exerts an ameliorative effect against LLC. Fourteen functional biomarkers that are related to the therapeutic effects of CKI on LLC were screened and identified using a metabolomics study. Analysis of metabolic pathways revealed that the therapeutic effects of CKI on LLC mainly involved glycerophospholipid metabolism, amino acid metabolism and sphingolipid metabolism. As glycerophospholipid metabolism is a crucial feature of cancer-specific metabolism, the enzymes that are involved in 1-acyl-sn-glycero-3-phosphoinositol biosynthesis were further evaluated. Western blotting results indicated that CKI modulated the abnormal biosynthesis pathway of 1-acyl-sn-glycero-3-phosphoinositol by activation of cytidine diphosphate-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and cytosolic phospholipase A2 (cPLA2), and by inhibition of lysophosphatidic acid acyltransferase gamma (AGPAT3). This study demonstrated that CKI has a favorable anti-tumor effect and that a UPLC-Q-TOF/MS-based metabolomics method in conjunction with further verifications at the biochemical level is a promising approach for investigating its underlying mechanisms.

Potential Anticancer Activities of a Combination of Curcumin, Ginger Oleoresin, and Rutin Solid Lipid Nanoparticles (Vietlife-Antican) in LLC Tumor-Bearing Mice

Natural products play an important role in medicine and human healthcare and, as a result, drugs that are derived from natural resources are increasingly being used for the treatment of various diseases, including cancer. Vietlife-Antican (VLA) is a novel combination of curcumin solid lipid nanoparticles, ginger oleoresin solid lipid nanoparticles, and rutin solid lipid nanoparticles, which all possess low toxicity and enhanced bioavailabilities. To clarify the antitumor effects of this product, cytotoxic tests of VLA were conducted in various cancer cell lines in vitro and in an in vivo tumorized mouse model induced by Lewis lung carcinoma (LLC) cells. VLA expressed some cytotoxicity against breast cancer cells (MCF7), hepatocarcinoma cells (HepG2), lung cancer cells (SK-LU-1), colorectal adenocarcinoma cells (SW480), and human leukemia cells (HL-60). Additionally, LLC tumor-bearing mice treated with VLA (600 mg/kg body weight [bw]) exhibited significant reductions in tumor growth ( P < 0.05), a longer mean survival time (34.05 days), and improved liver function. Furthermore, a combination of VLA (200 mg/kg bw) and cisplatin strongly suppressed tumor development and increased mean survival time ( P < 0.05). Interestingly, cotreatment with VLA and cisplatin also induced the serum production of interleukin (IL)-2 and IL-10, which are important antitumor and anti-inflammatory cytokines, respectively, in tumorized mice. Taken together, the present results suggest that VLA may be a very promising and efficient phytochemical product that can support cancer treatment or therapy.

P300 Mediates Muscle Wasting in Lewis Lung Carcinoma.

C/EBPβ is a key mediator of cancer-induced skeletal muscle wasting. However, the signaling mechanisms that activate C/EBPβ in the cancer milieu are poorly defined. Here, we report cancer-induced muscle wasting requires the transcriptional cofactor p300, which is critical for the activation of C/EBPβ. Conditioned media from diverse types of tumor cells as well as recombinant HSP70 and HSP90 provoked rapid acetylation of C/EBPβ in myotubes, particularly at its Lys39 residue. Overexpression of C/EBPβ with mutated Lys39 impaired Lewis lung carcinoma (LLC)-induced activation of the C/EBPβ-dependent catabolic response, which included upregulation of E3 ligases UBR2 and atrogin1/MAFbx, increased LC3-II, and loss of muscle proteins both in myotubes and mouse muscle. Silencing p300 in myotubes or overexpressing a dominant negative p300 mutant lacking acetyltransferase activity in mouse muscle attenuated LLC tumor-induced muscle catabolism. Administration of pharmacologic p300 inhibitor C646, but not PCAF/GCN5 inhibitor CPTH6, spared LLC tumor-bearing mice from muscle wasting. Furthermore, mice with muscle-specific p300 knockout were resistant to LLC tumor-induced muscle wasting. These data suggest that p300 is a key mediator of LLC tumor-induced muscle wasting whose acetyltransferase activity may be targeted for therapeutic benefit in this disease. SIGNIFICANCE: These findings demonstrate that tumor-induced muscle wasting in mice is abrogated by knockout, mutation of Lys39 or Asp1399, and pharmacologic inhibition of p300.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1331/F1.large.jpg.