Exercise accelerates recruitment of CD8+ T cell to promotes anti-tumor immunity in lung cancer via epinephrine.

BACKGROUND AND PURPOSE: In recent years, there has been extensive research on the role of exercise as an adjunctive therapy for cancer. However, the potential mechanisms underlying the anti-tumor therapy of exercise in lung cancer remain to be fully elucidated. As such, our study aims to confirm whether exercise-induced elevation of epinephrine can accelerate CD8+ T cell recruitment through modulation of chemokines and thus ultimately inhibit tumor progression. METHOD: C57BL/6 mice were subcutaneously inoculated with Lewis lung cancer cells (LLCs) to establish a subcutaneous tumor model. The tumor mice were randomly divided into different groups to performed a moderate-intensity exercise program on a treadmill for 5 consecutive days a week, 45 min a day. The blood samples and tumor tissues were collected after exercise for IHC, RT-qPCR, ELISA and Western blot. In addition, another group of mice received daily epinephrine treatment for two weeks (0.05 mg/mL, 200 µL i.p.) (EPI, n = 8) to replicate the effects of exercise on tumors in vivo. Lewis lung cancer cells were treated with different concentrations of epinephrine (0, 5, 10, 20 µM) to detect the effect of epinephrine on chemokine levels via ELISA and RT-qPCR. RESULTS: This study reveals that both pre- and post-cancer exercise effectively impede the tumor progression. Exercise led to an increase in EPI levels and the infiltration of CD8+ T cell into the lung tumor. Exercise-induced elevation of EPI is involved in the regulation of Ccl5 and Cxcl10 levels further leading to enhanced CD8+ T cell infiltration and ultimately inhibiting tumor progression. CONCLUSION: Exercise training enhance the anti-tumor immunity of lung cancer individuals. These findings will provide valuable insights for the future application of exercise therapy in clinical practice.

Single-cell RNA sequencing reveals recruitment of the M2-like CCL8high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy.

BACKGROUND: Tumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated. METHODS: Subcutaneous Lewis lung carcinoma (LLC) murine model was treated with hypofrationated radiotherapy (8 Gy × 3F). Single-cell RNA sequencing was utilized to identify subclusters and functions of TAMs. Multiplex assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure serum chemokine levels. Bindarit was used to inhibit CCL8, CCL7, and CCL2. The infiltration of TAMs after combination treatment with hypofractionated radiotherapy and Bindarit was quantified with flow cytometry, while the influx of CD206 and CCL8 was assessed by immunostaining. RESULTS: Transcriptome analysis identified a distinct subset of M2-like macrophages characterized by elevated Ccl8 expression level following hypofractionated radiotherapy in LLC-bearing mice. Remarkbly, hypofractionated radiotherapy not only promoted CCL8high macrophages infiltration but also reprogrammed them by upregulating immunosuppressive genes, thereby fostering an immunosuppressive tumor microenvironment. Additioinally, hypofractionated radiotherapy enhanced the CCL signaling pathway, augmenting the pro-tumorigenic functions of CCL8high macrophages and boosting TAMs recruitment. The adjunctive treatment combining hypofractionated radiotherapy with Bindarit effectively reduced M2 macrophages infiltration and prolonged the duration of local tumor control. CONCLUSIONS: Hypofractionated radiotherapy enhances the infiltration of CCL8high macrophages and amplifies their roles in macrophage recruitment through the CCL signaling pathway, leading to an immunosuppressive tumor microenvironment. These findings highlight the potential of targeting TAMs and introduces a novel combination to improve the efficacy of hypofractionated radiotherapy.

Age-Related Features of the Response of Cancer Stem Cells and T Cells in Experimental Lung Cancer.

The responses of tumor stem cells and various populations of CD4 and CD8 T cells of young and aged C57BL/6 mice were studied in a lung cancer model. Using Lewis lung carcinoma cell line, an orthotopic model of lung cancer was modeled. Cancer stem cells, circulating tumor cells, and various populations of CD4 and CD8 T cells in the blood and lung tissue were studied by cytometry. We revealed age-related differences in the content of various populations of CD4 and CD8 T cells in the blood and lungs of intact young and aged mice. Age-related features of the reaction of various populations of cancer stem cells and CD4 and CD8 T cells in the blood and lungs of animals in the Lewis lung carcinoma were shown.

MHY1485 potentiates immunogenic cell death induction and anti-cancer immunity following irradiation.

Recent in vitro experiments showed that combined treatment with MHY1485, a low-molecular-weight compound, and X-ray irradiation significantly increased apoptosis and senescence in tumor cells, which was associated with oxidative stress, endoplasmic reticulum (ER) stress and p21 stabilization, compared to radiation treatment alone. However, evidence for MHY1485 treatment-mediated suppression of tumor growth in animals is still lacking. Furthermore, it has been shown that ER stress enhances immunogenic cell death (ICD) in tumor cells, as it can exert a favorable influence on the anti-cancer immune system. In the present study, we examined whether co-treatment of MHY1485 and X-ray irradiation induces ICD and in vivo tumor growth suppression using the CT26 and Lewis lung carcinoma murine tumor cell lines. We found that MHY1485 + X-ray treatment promotes ICD more effectively than X-ray treatment alone. MHY1485 suppresses tumor growth in vivo under co-treatment with X-rays and increases INF-γ, tumor necrosis factor, interleukin-2 and interleukin-12 levels in the spleen as well as the presence of CD8+ cells in the tumor. The results suggest that MHY1485 treatment leads to the conversion of irradiated tumors into effective vaccines. Thus, MHY1485 is a promising lead compound for use in combination with radiotherapy.

Obesity worsens mitochondrial quality control and does not protect against skeletal muscle wasting in murine cancer cachexia.

BACKGROUND: More than 650 million people are obese (BMI > 30) worldwide, which increases their risk for several metabolic diseases and cancer. While cachexia and obesity are at opposite ends of the weight spectrum, leading many to suggest a protective effect of obesity against cachexia, mechanistic support for obesity's benefit is lacking. Given that obesity and cachexia are both accompanied by metabolic dysregulation, we sought to investigate the impact of obesity on skeletal muscle mass loss and mitochondrial dysfunction in murine cancer cachexia. METHODS: Male C57BL/6 mice were given a purified high fat or standard diet for 16 weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25 days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). RESULTS: Obese LLC mice experienced significant tumour-free body weight loss similar to lean (-12.8% vs. -11.8%, P = 0.0001) but had reduced survival (33.3% vs. 6.67%, χ2  = 10.04, P = 0.0182). Obese LLC mice had reduced muscle weights (-24%, P < 0.0354) and mCSA (-16%, P = 0.0004) with similar activation of muscle p65 (P = 0.0337), and p38 (P = 0.0008). ADP-dependent coupled respiration was reduced in both Obese and Obese LLC muscle (-30%, P = 0.0072) consistent with reductions in volitional cage activity (-39%, P < 0.0001) and grip strength (-41%, P < 0.0001). TEM revealed stepwise reductions in intermyofibrillar and subsarcolemmal mitochondrial size with Obese (IMF: -37%, P = 0.0009; SS: -21%, P = 0.0101) and LLC (IMF: -40%, P = 0.0019; SS: -27%, P = 0.0383) mice. Obese LLC mice had increased pAMPK (T172; P = 0.0103) and reduced FIS1 (P = 0.0029) and DRP1 (P < 0.0001) mitochondrial fission proteins, which were each unchanged in Lean LLC. Further, mitochondrial TEM analysis revealed that Obese LLC mice had an accumulation of damaged and dysfunctional mitochondria (IMF: 357%, P = 0.0395; SS: 138%, P = 0.0174) in concert with an accumulation of p62 (P = 0.0328) suggesting impaired autophagy and clearance of damaged mitochondria. Moreover, we observed increases in electron lucent vacuoles only in Obese LLC muscle (IMF: 421%, P = 0.0260; SS: 392%, P = 0.0192), further supporting an accumulation of damaged materials that cannot be properly cleared in the obese cachectic muscle. CONCLUSIONS: Taken together, these results demonstrate that obesity is not protective against cachexia and suggest exacerbated impairments to mitochondrial function and quality control with a particular disruption in the removal of damaged mitochondria. Our findings highlight the need for consideration of the severity of obesity and pre-existing metabolic conditions when determining the impact of weight status on cancer-induced cachexia and functional mitochondrial deficits.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology.

Photocontrolled, biologically active compounds are an emerging class of "smart" drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient's body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

EFFECT OF LACTATE DEHYDROGENASE INHIBITION BY OXAMATE ON LEWIS LUNG CARCINOMA CELLS WITH DIFFERENT METASTATIC POTENTIAL.

BACKGROUND: Today, the ability for metabolic reprogramming is considered one of the distinguishing features of metastatically active tumor cells, a classic example of which is aerobic glycolysis. Despite a large number of studies in this direction, the question of the relationship between the intensity of aerobic glycolysis and the metastatic potential of tumor cells remains almost completely open. The work aimed to investigate the effect of the lactate dehydrogenase (LDH) inhibitor on the viability and several characteristics of Lewis lung carcinoma cells with different metastatic potential. MATERIALS AND METHODS: High-metastatic (LLC) and low-metastatic (LLC/R9) variants of Lewis lung carcinoma cells were used. After 24 h of tumor cells incubation with or without 40 mM sodium oxamate, cell viability, the concentration of glucose and lactate in the incubation medium, distribution of cells by the cell cycle phases, and intracellular ROS production were estimated. RESULTS: It was revealed that regardless of the metastatic potential, LLC cells are heterogeneous in terms of both the involvement of aerobic glycolysis in their growth and survival processes and the sensitivity to the cytotoxic/cytostatic action of an LDH inhibitor. 35% of cells of either LLC variant form an oxamate-resistant subpopulation while 65% are oxamate-sensitive. The rate of glucose consumption of LLC/R9 cells in the absence of oxamate is almost twice higher compared to LLC and, as a result, the sensitivity of these cells to the cytotoxic/cytostatic effect of oxamate also is significantly higher (the IC50 for LLC/R9 cells is by 35.8% lower than that for LLC cells, p < 0.05). Approximately one-third of the cells of both LLC and LLC/R9 variants can survive and proliferate when aerobic glycolysis is completely inhibited by oxamate. This indicates metabolic reprogramming (either pre-existing or dynamically arising in response to inhibition of glycolysis) of this subpopulation of cells, within which not only the survival of cells but also their proliferative activity is most likely based on glutamine metabolism. CONCLUSIONS: Such metabolic heterogeneity of metastatically active cells indicates that inhibition of glycolysis as monotherapy is insufficient for effective antimetastatic therapy. Presumably, more effective would be to involve various inhibitors of metabolic processes that ensure the metabolic plasticity of metastatic cells.

Exosomal EIF5A derived from Lewis lung carcinoma induced adipocyte wasting in cancer cachexia.

Cancer cachexia is a systemic inflammation-driven syndrome, characterized by muscle atrophy and adipose tissue wasting, with progressive weight loss leading to serious impairment of physiological function. Extracellular vesicles (EVs) derived from cancer cells play a significant role in adipocyte lipolysis, yet the mechanism remain uneclucidated. In this study, EVs derived from Lewis lung carcinoma (LLC) cells were extracted and characterized. 3T3-L1 and HIB1B adipocytes were cultured with conditioned medium or EVs from LLC, and LLC cells were used to establish a cancer cachexia mouse model. EVs derived from LLC cells were taken up by 3T3-L1 and HIB1B adipocytes, and derived exosomal EIF5A protein-induced lipolysis of adipocytes. High level of EIF5A was expressed in EVs from LLC cells, exosomal EIF5A is linked to lipid metabolism. Elevated expression of EIF5A is associated with shorter overall survival in lung cancer patients. Western blots, glycerol release and Oil red O staining assays were used to evaluate lipolysis of adipocytes. The reduction of lipolysis in 3T3-L1 and HIB1B adipocytes is achieved through silencing EIF5A or treating with pharmacologic inhibitor GC7 in vitro, and suppressing the expression of EIF5A in LLC cells by infected with shRNA or GC7 treatment partly alleviated white and brown adipose tissue lipolysis in vivo. Mechanistically, EIF5A directly binds with G protein-coupled bile acid receptor 1 (GPBAR1) mRNA to promote its translation and then activates cAMP response element binding protein (CREB) signaling pathway to induce lipolysis. This study demonstrates that exosomal EIF5A from LLC cells, with hypusinated EIF5A, has a lipolytic effect on adipocyte and adipose tissues in cancer cachexia model. Exosomal EIF5A could be involved in lipolysis and these findings indicate that a novel regulator and potential target for cachexia treatment.

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

BACKGROUND: Cancer-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. RESULTS: We 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. CONCLUSION: Our 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.

Effects of S-pindolol in mouse pancreatic and lung cancer cachexia models.

BACKGROUND: It is known that S-pindolol attenuates muscle loss in animal models of cancer cachexia and sarcopenia. In cancer cachexia, it also significantly reduced mortality and improved cardiac function, which is strongly compromised in cachectic animals. METHODS: Here, we tested 3 mg/kg/day of S-pindolol in two murine cancer cachexia models: pancreatic cancer cachexia (KPC) and Lewis lung carcinoma (LLC). RESULTS: Treatment of mice with 3 mg/kg/day of S-pindolol in KPC or LLC cancer cachexia models significantly attenuated the loss of body weight, including lean mass and muscle weights, leading to improved grip strength compared with placebo-treated mice. In the KPC model, treated mice lost less than half of the total weight lost by placebo (-0.9 ± 1.0 vs. -2.2 ± 1.4 g for S-pindolol and placebo, respectively, P < 0.05) and around a third of the lean mass lost by tumour-bearing controls (-0.4 ± 1.0 vs. -1.5 ± 1.5 g for S-pindolol and placebo, respectively, P < 0.05), whereas loss of fat mass was similar. In the LLC model, the gastrocnemius weight was higher in sham (108 ± 16 mg) and S-pindolol tumour-bearing (94 ± 15 mg) mice than that in placebo (83 ± 12 mg), whereas the soleus weight was only significantly higher in the S-pindolol-treated group (7.9 ± 1.7 mg) than that in placebo (6.5 ± 0.9). Grip strength was significantly improved by S-pindolol treatment (110.8 ± 16.2 vs. 93.9 ± 17.1 g for S-pindolol and placebo, respectively). A higher grip strength was observed in all groups; whereas S-pindolol-treated mice improved by 32.7 ± 18.5 g, tumour-bearing mice only show minimal improvements (7.3 ± 19.4 g, P < 0.01). CONCLUSIONS: S-pindolol is an important candidate for clinical development in the treatment of cancer cachexia that strongly attenuates loss of body weight and lean body mass. This was also seen in the weight of individual muscles and resulted in higher grip strength.

Development of skeletal muscle fibrosis in a rodent model of cancer cachexia.

Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under-investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Female and male C57BL6/J mice were injected with phosphate-buffered saline or Lewis Lung Carcinoma (LLC) at 8-week of age, and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on cocultured C2C12 and 3T3 cells exposed to LLC conditioned media. Immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis were used to investigate fibrosis and fibrosis-related signaling in skeletal muscle. Collagen deposition in skeletal muscle was increased in the 1wk, LT, and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general, female mice displayed earlier alterations in extracellular matrix (ECM)-related genes beginning at 1wk post-LLC injection. Whereas this was not seen in males. While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early-stage alterations in ECM-related genes contrary to what was noted in female mice.

Proteins Secreted by Lung Cancer Cells Induce the Onset of Proteinuria via Focal Adhesion Kinase Signaling in Mice.

Paraneoplastic nephrotic syndrome (PNS) is a complication seen in cancer patients. Ultrastructural examination shows the accumulation of proteins and the presence of foot process (FP) effacement in the glomeruli of PNS patients. Previously, we reported that orthotopic xenografts of Lewis lung carcinoma 1 in C57BL/6 mice caused them to develop lung cancer with albuminuria. This implies that these mice can be used as a model of human disease and suggests that Lewis lung carcinoma 1 cell-secreted proteins (LCSePs) contain nephrotoxic molecules and cause inflammation in renal cells. As podocyte effacement was present in glomeruli in this model, such podocyte injury may be attributable to either soluble LCSeP or LCSeP deposits triggering pathological progression. LCSePs in conditioned media was concentrated for nephrotoxicity testing. Integrin-focal adhesion kinase (FAK) signaling and inflammatory responses were evaluated in podocytes either exposed to soluble LCSePs or seeded onto substrates with immobilized LCSePs. FAK phosphorylation and interleukin-6 expression were higher in podocytes attached to LCSePs substrates than in those exposed to soluble LCSePs. Notably, LCSeP-based haptotaxis gave rise to altered signaling in podocytes. When podocytes were stimulated by immobilized LCSePs, FAK accumulated at focal adhesions, synaptopodin dissociated from F-actin, and disrupting the interactions between synaptopodin and α-actinin was observed. When FAK was inhibited by PF-573228 in immobilized LCSePs, the association between synaptopodin and α-actinin was observed in the podocytes. The association of synaptopodin and α-actinin with F-actin allowed FP stretching, establishing a functional glomerular filtration barrier. Therefore, in this mouse model of lung cancer, FAK signaling prompts podocyte FP effacement and proteinuria, indicative of PNS.

EXT418, a novel long-acting ghrelin, mitigates Lewis lung carcinoma induced cachexia in mice.

BACKGROUND: Ghrelin is a potential therapy for cachexia due to its orexigenic properties and anabolic effects on muscle and fat. However, its clinical use is limited by the short half-life of active (acylated) ghrelin (~11 min in humans). EXT418 is a novel long-acting, constitutively active ghrelin analog created by covalently linking it to a vitamin D derivative. Here, we evaluated the effects and mechanisms of action of EXT418 on Lewis lung carcinoma (LLC)-induced cachexia in mice. METHODS: Male C57BL/6J mice (5- to 7-month-old) were implanted with 1 × 106 heat-killed (HK) or live LLC cells. When the tumour was palpable, mice were injected with vehicle (T + V) or EXT418 daily (T + 418 Daily, 0.25 mg/kg/day) or every other day (T + 418 EOD, 0.5 mg/kg/EOD) for up to 14 days, whereas HK-treated mice were given vehicle (HK + V). Subsets of T + 418 Daily or EOD-treated mice were pair-fed to the T + V group. Body composition and grip strength were evaluated before tumour implantation and at the end of the experiment. Molecular markers were probed in muscles upon termination. RESULTS: In tumour-bearing mice, administration of EXT418 daily or EOD partially prevented weight loss (T + V vs. T + 418 Daily, P = 0.030; and vs. T + 418 EOD, P = 0.020). Similar effects were observed in whole body fat and lean body mass. Grip strength in tumour-bearing mice was improved by EXT418 daily (P = 0.010) or EOD (P = 0.008) administration compared with vehicle-treated mice. These effects of EXT418 on weight and grip strength were partially independent of food intake. EXT418 daily administration also improved type IIA (P = 0.015), IIB (P = 0.037) and IIX (P = 0.050) fibre cross-sectional area (CSA) in tibialis anterior (TA) and EXT418 EOD improved CSA of IIB fibres in red gastrocnemius (GAS; P = 0.005). In skeletal muscles, tumour-induced increases in atrogenes Fbxo32 and Trim63 were ameliorated by EXT418 treatments (TA and GAS/plantaris, PL), which were independent of food intake. EXT418 administration decreased expression of the mitophagy marker Bnip3 (GAS/PL; P ≤ 0.010). Similar effects of EXT418 EOD were observed in p62 (GAS/PL; P = 0.039). In addition, EXT418 treatments ameliorated the tumour-induced elevation in muscle Il6 transcript levels (TA and GAS/PL), independently of food intake. Il-6 transcript levels in adipose tissue and circulating IL-10 were elevated in response to the tumour but these increases were not significant with EXT418 administration. Tumour mass was not altered by EXT418. CONCLUSIONS: EXT418 mitigates LLC-induced cachexia by attenuating skeletal muscle inflammation, proteolysis, and mitophagy, without affecting tumour mass and partially independent of food intake.

Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer.

Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non-small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells-such as M2 tumor-associated macrophages (TAM)-thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti-PD-1-resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFß levels. The addition of anti-PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

Cell Therapy with Human Reprogrammed CD8+ T-Cells Has Antimetastatic Effects on Lewis Lung Carcinoma in C57BL/6 Mice.

Using a model of Lewis lung carcinoma (LLC) in vitro and in vivo, we previously demonstrated increased antitumor activity in CD8+ T-cells reprogrammed with an MEK inhibitor and PD-1 blocker. In this follow-up study, we carried out the reprogramming of human CD8+ T-cells (hrT-cell) using the MEK inhibitor and PD-1 blocker and targeted LLC cells. The effects of hrT-cell therapy were studied in a mouse model of spontaneous metastasis of a solid LLC tumor. We found antimetastatic activity of hrT-cells, a decrease in the number of cancer cells and cancer stem cells in the lungs, and an increase in the number of T-cells in the blood (including effector T-cells). Thus, reprogramming of human CD8+ T-cells with an MEK inhibitor and PD-1 blocker with targeted training by tumor target cells is a potential platform for developing a new approach to targeted lung cancer therapy.

LP07 and LLC preclinical models of lung cancer induce divergent anabolic deficits and expression of pro-inflammatory effectors of muscle wasting.

Preclinical models have been instrumental to elucidate the mechanisms underlying muscle wasting in lung cancer (LC). We investigated anabolic deficits and the expression of proinflammatory effectors of muscle wasting in the LP07 and Lewis lung carcinoma (LLC) tumor models. Tumor growth resulted in significant weakness in LP07 but not in LLC mice despite similar reductions in gastrocnemius muscle mass in both models. The LP07 tumors caused a reduction in ribosomal (r)RNA and a decrease in rRNA gene (rDNA) transcription elongation, whereas no changes in ribosomal capacity were evident in LLC tumor-bearing mice. Expression of RNA Polymerase I (Pol I) elongation-associated subunits Polr2f, PAF53, and Znrd1 mRNAs was significantly elevated in the LP07 model, whereas Pol I elongation-related factors FACT and Spt4/5 mRNAs were elevated in the LLC mice. Reductions in RPS6 and 4E-BP1 phosphorylation were similar in both models but were independent of mTOR phosphorylation in LP07 mice. Muscle inflammation was also tumor-specific, IL-6 and TNF-α mRNA increased with LLC tumors, and upregulation of NLRP3 mRNA was independent of tumor type. In summary, although both models caused muscle wasting, only the LP07 model displayed muscle weakness with reductions in ribosomal capacity. Intracellular signaling diverged at the mTOR level with similar reductions in RPS6 and 4E-BP1 phosphorylation regardless of tumor type. The increase in proinflammatory factors was more pronounced in the LLC model. Our results demonstrate novel divergent anabolic deficits and expression of proinflammatory effectors of muscle wasting in the LP07 and LLC preclinical models of lung cancer.NEW & NOTEWORTHY We provide novel data demonstrating significant divergence in anabolic deficits and the expression of proinflammatory effectors of muscle wasting consequent to different lung-derived tumors.

Modified Bu-Fei decoction inhibits lung metastasis via suppressing angiopoietin-like 4.

BACKGROUND: Modified Bu-Fei decoction (MBFD), a formula of traditional Chinese medicine, is used for treating lung cancer in clinic. The actions and mechanisms of MBFD on modulating lung microenvironment is not clear. PURPOSE: Lung microenvironment is rich in vascular endothelial cells (ECs). This study is aimed to examine the actions of MBFD on tumor biology, and to uncover the underlying mechanisms by focusing on pulmonary ECs. METHODS: The Lewis lung carcinoma (LLC) xenograft model and the metastatic cancer model were used to determine the efficacy of MBFD on inhibiting tumor growth and metastasis. Flow cytometry and trans-well analysis were used to determine the role of ECs in anti-metastatic actions of MBFD. The in silico analysis and function assays were used to identify the mechanisms of MBFD in retarding lung metastasis. Plasma from lung cancer patients were used to verify the effects of MBFD on angiogenin-like protein 4 (ANGPTL4) in clinical conditions. RESULTS: MBFD significantly suppressed spontaneous lung metastasis of LLC tumors, but not tumor growth, at clinically relevant concentrations. The anti-metastatic effects of MBFD were verified in metastatic cancer models created by intravenous injection of LLC or 4T1 cells. MBFD inhibited lung infiltration of circulating tumor cells, without reducing tumor cell proliferations in lung. In vitro, MBFD dose-dependently inhibited trans-endothelial migrations of tumor cells. RNA-seq assay and verification experiments confirmed that MBFD potently depressed endothelial ANGPTL4 which is able to broke endothelial barrier and protect tumor cells from anoikis. Database analysis revealed that high ANGPTL4 levels is negatively correlated with overall survival of cancer patients. Importantly, MBFD therapy reduced plasma levels of ANGPTL4 in lung cancer patients. Finally, MBFD was revealed to inhibit ANGPTL4 expressions in a hypoxia inducible factor-1α (HIF-1α)-dependent manner, based on results from specific signaling inhibitors and network pharmacology analysis. CONCLUSION: MBFD, at clinically relevant concentrations, inhibits cancer lung metastasis via suppressing endothelial ANGPTL4. These results revealed novel effects and mechanisms of MBFD in treating cancer, and have a significant clinical implication of MBFD therapy in combating metastasis.

АNTIMETASTATIC EFFECT OF B. SUBTILIS IMV B-7724 LECTIN OBSERVED IN LEWIS LUNG CARCINOMA MODEL.

AIM: To study the antitumor and antimetastatic effects of B. subtilis IMV B-7724 lectin used in neoadjuvant and adjuvant settings in vivo. MATERIALS AND METHODS: Studies were performed on C57Bl/6J mice; Lewis lung carcinoma (LLC) was used as an experimental tumor. В. subtilis ІМV В-7724 lectin was administered to tumor-bearing mice or to mice which underwent surgical resection of the primary tumor. The lectin was injected subcutaneously, 10 times, at a single dose of 5 or 1 mg/kg of body weight. The standard indicators of tumor growth and metastasis were evaluated. RESULTS: Independently of the application settings, the lectin at a dose of 1 mg/kg of b.w. caused more pronounced effect than at a dose of 5 mg/kg of b.w. The administration of B. subtilis IMV B-7724 lectin to the mice with LLC in neoadjuvant setting did not cause notable antitumor effect but led to a significant decrease in the number and volume of lung metastases. The lectin administration in adjuvant setting significantly inhibited metastasis: the metastasis inhibition index reached 63.0% and 100% in the mice treated with the lectin at a dose of 5 mg/kg and 1 mg/kg respectively. The mean survival time of the treated animals significantly increased. CONCLUSION: A pronounced antimetastatic effect of B. subtilis IMV B-7724 lectin administered in an adjuvant setting was demonstrated.

Metabolomes of Lewis lung carcinoma metastases and normal lung tissue from mice fed different diets.

Metastasis is a devastating aspect of cancer. This study tested the hypothesis that metabolome of metastases differs from that of host organs by using the spontaneous metastasis model of Lewis lung carcinoma (LLC). In a 2 × 2 design, male C57BL/6 mice with or without a subcutaneous LLC inoculation were fed the standard AIN93G diet or a high-fat diet (HFD) for 12 weeks. Lung metastases from injected mice and the lungs from non-injected mice were harvested at the end of study for untargeted metabolomics of primary metabolism by using gas chromatography time-of-flight mass spectrometry. We identified 91 metabolites for metabolomic analysis. The analysis demonstrated that amino acid and energy metabolism were altered the most in LLC metastases compared to the lungs. A 60% decrease in glutamine and a 25-fold elevation in sorbitol were observed in metastases. Cholesterol and its metabolite dihydrocholesterol were 50% lower in metastases than in the lungs. The HFD elevated arachidonic acid and its precursor linoleic acid in the lungs from noncancer-bearing mice, reflecting the dietary fatty acid composition of the HFD. This elevation did not occur in metastases from HFD-fed LLC-bearing mice, suggesting alterations in lipid metabolism during LLC metastatic progression. Understanding the differences in metabolome between pulmonary LLC metastases and the normal healthy lungs can be useful in designing targeted studies for prevention and treatment of cancer spread using this LLC spontaneous metastasis model.

Hypothalamic-pituitary-adrenal axis activation and glucocorticoid-responsive gene expression in skeletal muscle and liver of Apc mice.

BACKGROUND: Cancer patients at advanced stages experience a severe depletion of skeletal muscle compartment together with a decrease in muscle function, known as cancer cachexia. Cachexia contributes to reducing quality of life, treatment efficiency, and lifespan of cancer patients. However, the systemic nature of the syndrome is poorly documented. Here, we hypothesize that glucocorticoids would be important systemic mediators of cancer cachexia. METHODS: To explore the role of glucocorticoids during cancer cachexia, biomolecular analyses were performed on several tissues (adrenal glands, blood, hypothalamus, liver, and skeletal muscle) collected from ApcMin/+ male mice, a mouse model of intestine and colon cancer, aged of 13 and 23 weeks, and compared with wild type age-matched C57BL/6J littermates. RESULTS: Twenty-three-week-old Apc mice recapitulated important features of cancer cachexia including body weight loss (-16%, P < 0.0001), muscle atrophy (gastrocnemius muscle: -53%, P < 0.0001), and weakness (-50% in tibialis anterior muscle force, P < 0.0001), increased expression of atrogens (7-fold increase in MuRF1 transcript level, P < 0.0001) and down-regulation of Akt-mTOR pathway (3.3-fold increase in 4EBP1 protein content, P < 0.0001), together with a marked transcriptional rewiring of hepatic metabolism toward an increased expression of gluconeogenic genes (Pcx: +90%, Pck1: +85%), and decreased expression of glycolytic (Slc2a2: -40%, Gk: -30%, Pklr: -60%), ketogenic (Hmgcs2: -55%, Bdh1: -80%), lipolytic/fatty oxidation (Lipe: -50%, Mgll: -60%, Cpt2: -60%, Hadh: -30%), and lipogenic (Acly: -30%, Acacb: -70%, Fasn: -45%) genes. The hypothalamic pituitary-adrenal axis was activated, as evidenced by the increase in the transcript levels of genes encoding corticotropin-releasing hormone in the hypothalamus (2-fold increase, P < 0.01), adrenocorticotropic hormone receptor (3.4-fold increase, P < 0.001), and steroid biosynthesis enzymes (Cyp21a1, P < 0.0001, and Cyp11b1, P < 0.01) in the adrenal glands, as well as by the increase in corticosterone level in the serum (+73%, P < 0.05), skeletal muscle (+17%, P < 0.001), and liver (+24%, P < 0.05) of cachectic 23-week-old Apc mice. A comparative transcriptional analysis with dexamethasone-treated C57BL/6J mice indicated that the activation of the hypothalamic-pituitary-adrenal axis in 23-week-old ApcMin/+ mice was significantly associated with the transcription of glucocorticoid-responsive genes in skeletal muscle (P < 0.05) and liver (P < 0.001). The transcriptional regulation of glucocorticoid-responsive genes was also observed in the gastrocnemius muscle of Lewis lung carcinoma tumour-bearing mice and in KPC mice (tibialis anterior muscle and liver). CONCLUSIONS: These findings highlight the role of the hypothalamic-pituitary-adrenal-glucocorticoid pathway in the transcriptional regulation of skeletal muscle catabolism and hepatic metabolism during cancer cachexia. They also provide the paradigm for the design of new therapeutic strategies.