DPP Inhibition Enhances the Efficacy of PD-1 Blockade by Remodeling the Tumor Microenvironment in Lewis Lung Carcinoma Model.

The remarkable efficacy of cancer immunotherapy has been established in several tumor types. Of the various immunotherapies, PD-1/PD-L1 inhibitors are most extensively used in the treatment of many cancers in clinics. These inhibitors restore the suppressed antitumor immune response and inhibit tumor progression by blocking the PD-1/PD-L1 signaling. However, the low response rate is a major limitation in the clinical application of PD-1/PD-L1 inhibitors. Therefore, combination strategies that enhance the response rate are the need of the hour. In this investigation, PT-100 (also referred to as Talabostat, Val-boroPro, and BXCL701), an orally administered and nonselective dipeptidyl peptidase inhibitor, not only augmented the effectiveness of anti-PD-1 therapy but also significantly improved T immune cell infiltration and reversed the immunosuppressive tumor microenvironment. The combination of PT-100 and anti-PD-1 antibody increased the number of CD4+ and CD8+ T cells. Moreover, the mRNA expression of T cell-associated molecules was elevated in the tumor microenvironment. The results further suggested that PT-100 dramatically reduced the ratio of tumor-associated macrophages. These findings provide a promising combination strategy for immunotherapy in lung cancer.

Fucoidan MF4 from Fucus vesiculosus inhibits Lewis lung cancer via STING-TBK1-IRF3 pathway.

Fucoidan, a sulfated polysaccharide of marine origin found in brown algae and sea cucumbers, has been identified as a neuroprotective compound. In this study, a novel fucoidan MF4 was extracted from Fucus vesiculosus and isolated using Q-Sepharose fast-flow ion-exchange chromatography. The physicochemical properties of MF4 were characterized. MF4 is primarily composed of fucose, xylose, galactose, glucose, and mannose in a molar ratio of 12.3: 4.9: 1.1: 1.0: 1.1, with an average molecular weight of 67.7 kDa. Notably, MF4 demonstrated suppression of LLC tumor growth in vivo. RNA-sequencing analysis revealed that MF4 enhanced the expression of type I interferon-associated downstream genes in macrophages. Furthermore, MF4 increased the levels of phosphorylated TBK1 and IRF3 proteins in vitro. By activating the STING-TBK1-IRF3 signaling pathway, MF4 may enhance the antitumor activity of macrophages. Taken together, MF4 has promising potential as an antitumor and immunomodulatory agent.

Effects of reprogrammed splenic CD8+ T-cells in vitro and in mice with spontaneous metastatic Lewis lung carcinoma.

BACKGROUND: Metastatic disease is a major and difficult-to-treat complication of lung cancer. Considering insufficient effectiveness of existing therapies and taking into account the current problem of lung cancer chemoresistance, it is necessary to continue the development of new treatments. METHODS: Previously, we have demonstrated the antitumor effects of reprogrammed CD8+ T-cells (rCD8+ T-cells) from the spleen in mice with orthotopic lung carcinoma. Reprogramming was conducted by inhibiting the MAPK/ERK signalling pathway through MEKi and the immune checkpoint PD-1/PD-L1. Concurrently, CD8+ T-cells were trained in Lewis lung carcinoma (LLC) cells. We suggested that rCD8+ T-cells isolated from the spleen might impede the development of metastatic disease. RESULTS: The present study has indicated that the reprogramming procedure enhances the survival and cytotoxicity of splenic CD8+ T-cells in LLC culture. In an LLC model of spontaneous metastasis, splenic rCD8 + T-cell therapy augmented the numbers of CD8+ T-cells and CD4+ T-cells in the lungs of mice. These changes can account for the partial reduction of tumors in the lungs and the mitigation of metastatic activity. CONCLUSIONS: Our proposed reprogramming method enhances the antitumor activity of CD8+ T-cells isolated from the spleen and could be valuable in formulating an approach to treating metastatic disease in patients with lung cancer.

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.

Grafted Sertoli Cells Exert Immunomodulatory Non-Immunosuppressive Effects in Preclinical Models of Infection and Cancer.

The Sertoli cells (SeCs) of the seminiferous tubules secrete a multitude of immunoregulatory and trophic factors to provide immune protection and assist in the orderly development of germ cells. Grafts of naked or encapsulated SeCs have been proved to represent an interesting therapeutic option in a plethora of experimental models of diseases. However, whether SeCs have immunosuppressive or immunomodulatory effects, which is imperative for their clinical translatability, has not been demonstrated. We directly assessed the immunopotential of intraperitoneally grafted microencapsulated porcine SeCs (MC-SeCs) in murine models of fungal infection (Aspergillus fumigatus or Candida albicans) or cancer (Lewis lung carcinoma/LLC or B16 melanoma cells). We found that MC-SeCs (i) provide antifungal resistance with minimum inflammatory pathology through the activation of the tolerogenic aryl hydrocarbon receptor/indoleamine 2,3-dioxygenase pathway; (ii) do not affect tumor growth in vivo; and (iii) reduce the LLC cell metastatic cancer spread associated with restricted Vegfr2 expression in primary tumors. Our results point to the fine immunoregulation of SeCs in the relative absence of overt immunosuppression in both infection and cancer conditions, providing additional support for the potential therapeutic use of SeC grafts in human patients.

Bifunctional molecular probe targeting tumor PD-L1 enhances anti-tumor efficacy by promoting ferroptosis in lung cancer mouse model.

PURPOSE: Immune checkpoint inhibitors (ICIs) targeting tumor-specific PD-1/PD-L1 significantly improve the overall survival rate of patients with advanced cancer by reactivating the immune system to attack cancer cells. To explore their tumor killing effect, we used the radionuclide iodine-131 (131I) to label the anti-PD-L1 antibody Atezolizumab (131I-PD-L1 mAb). METHOD: We prepared the radioimmunoassay molecular probe 131I-PD-L1 mAb by the chloramine-T method and evaluated its affinity using Lewis lung cancer (LLC) cells. The uptake of 131I-PD-L1 mAb by transplanted tumors was examined through SPECT and its in vivo distribution. We then compared the in vitro and in vivo anti-tumor efficacy of groups treated with control, PD-L1 mAb, 131I-PD-L1 mAb, and 131I-PD-L1 mAb + PD-L1 mAb combined treatment. We performed H&E staining to examine the changes in tumor, as well as the damage in major tissues and organs caused by potential side effects. The anti-tumor mechanism of 131I-PD-L1 mAb was analyzed by Western blot, RT-qPCR and immunohistochemistry (IHC). RESULT: 131I-PD-L1 mAb was highly stable and specific, and easily penetrated into tumor. 131I-PD-L1 mAb suppressed cancer cell proliferation in vitro, and inhibited tumor growth in vivo by inducing ferroptosis, thus prolonging the survival of experimental animals while demonstrating biological safety. CONCLUSION: Therefore, our study suggested that 131I-PD-L1 mAb affected the expression of tumor-related factors through ß-rays and thus promoted ferroptosis in tumor. Combined treatment showed better anti-tumor effect compared to single ICI treatment.

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.

Predicting the mechanism of action of YQYYJD prescription in the treatment of non-small cell lung cancer using transcriptomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The efficacy of the herbal formula Yiqi Yangyin Jiedu (YQYYJD) in the treatment of advanced lung cancer has been reported in clinical trials. However, the key anti-lung cancer herbs and molecular mechanisms underlying its inhibition of lung cancer are not well-understood. AIM OF THE STUDY: To identify the key anti-lung cancer herbs in the YQYYJD formula and investigate their therapeutic effect and potential mechanism of action in non-small cell lung cancer (NSCLC) using transcriptomics and bioinformatics techniques. MATERIALS AND METHODS: A mouse Lewis lung carcinoma (LLC) subcutaneous inhibitory tumor model was established with 6 mice in each group. Mice were treated with the YQYYJD split formula: Yiqi Formula (YQ), Yangyin Formula (YY), and Ruanjian Jiedu Formula (RJJD) for 14 days. The tumor volume and mouse weight were recorded, and the status of tumor occurrence was further observed by taking photos. The tumor was stained with hematoxylin-eosin to observe its histopathological changes. Immunohistochemistry was used to detect the expression of the proliferation marker Ki67 and the apoptotic marker Caspase-3 in tumor tissues. Flow cytometry was used to detect the number of CD4+ and CD8+ T cells and cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in the spleen and tumor tissues. The differential genes of key drugs against tumors were obtained by transcriptome sequencing of tumors. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed on differential genes to obtain pathways and biological processes where targets were aggregated. TIMER2.0 and TISIDB databases were used to evaluate the impact of drugs on immune cell infiltration and immune-related genes. The binding activity of the key targets and compounds was verified by molecular docking. RESULTS: YQ, YY, and RJJD inhibited the growth of subcutaneous transplanted tumors in LLC mice to varying degrees and achieved antitumor effects by inhibiting the expression of tumor cell proliferation, apoptosis, and metastasis-related proteins. Among the three disassembled prescriptions, YQ better inhibited the growth of subcutaneous transplanted tumors in LLC mice, significantly promoted tumor necrosis, significantly increased the expression of Caspase-3 protein in tumor tissue, and significantly decreased the expression of Ki-67 (P < 0.05), thereby increasing the infiltration of CD8+ T cells. YQ significantly increased the expression of CD4+ and CD8+ T cells in tumor and splenic tissues of tumor-bearing mice and up-regulated the expression of IL-2 and IFN-γ. Transcriptome sequencing and bioinformatics results showed that after YQ intervention, differentially expressed genes were enriched in more than one tumor-related pathway and multiple immune regulation-related biological functions. There were 12 key immune-related target genes. CONCLUSION: YQ was the key disassembled prescription of YQYYJD, exerting significant antitumor effects and immune regulation effects on NSCLC. It may have relieved T cell exhaustion and regulated the immune microenvironment to exert antitumor effects by changing lung cancer-related targets, pathways, and biological processes.

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.

Ag85B-ENO146-82 therapeutic vaccines enhance anti-tumor immunity by inducing CD8+ T cells and remodeling tumor microenvironment.

Lung cancer is the leading cause of cancer-related morbidity and mortality in China. However, the effect of traditional cancer treatment is limited. Herein, we designed a therapeutic cancer vaccine based on the tumor-associated antigen mENO1, which can prevent lung cancer growth in vivo, and explored the underlying mechanism of Ag85B-ENO146-82 therapy. Lewis lung carcinoma (LLC) tumor-bearing immunocompetent C57BL/6 mice that received Ag85B-ENO146-82 treatment showed antitumor effect. Further, we detected CD8+ T, CD4+ T in LLC-bearing C57BL/6 mice to understand the impact of Ag85B-ENO146-82 therapy on antitumor capacity. The Ag85B-ENO146-82 therapy induced intensive infiltration of CD4+ and CD8+ T cells in tumors, increased tumor-specific IFN-γ and TNF-α secretion by CD8+ T cells and promoted macrophage polarization toward M1 phenotype. Flow cytometric analysis revealed that CD8+ T effector memory (TEM) cells and central memory (TCM) cells were upregulated. qPCR and ELISA analysis showed that the expression of IFN-γ and TNF-α were upregulated, whereas of IL1ß, IL6 and IL10 were downregulated. This study demonstrated that Ag85B-ENO146-82 vaccine augmented antitumor efficacy, which was CD8+ T cells dependent. Our findings paved the way for therapeutic tumor-associated antigen peptide vaccines to enhance anti-tumor immunotherapy for treatment of cancer.

IL-37d enhances COP1-mediated C/EBPß degradation to suppress spontaneous neutrophil migration and tumor progression.

The spontaneous migration of bone marrow neutrophils (BMNs) is typically induced by distant tumor cells during the early stage of the tumor and critically controls tumor progression and metastases. Therefore, identifying the key molecule that prevents this process is extremely important for suppressing tumors. Interleukin-37 (IL-37) can suppress pro-inflammatory cytokine generation via an IL-1R8- or Smad3-mediated pathway. Here, we demonstrate that human neutrophil IL-37 is responsively reduced by tumor cells and the recombinant IL-37 isoform d (IL-37d) significantly inhibits spontaneous BMN migration and tumor lesion formation in the lung by negatively modulating CCAAT/enhancer binding protein beta (C/EBPß) in a Lewis lung carcinoma (LLC)-inducing lung cancer mouse model. Mechanistically, IL-37d promotes C/EBPß ubiquitination degradation by facilitating ubiquitin ligase COP1 recruitment and disrupts C/EBPß DNA binding abilities, thereby reducing neutrophil ATP generation and migration. Our work reveals an anti-tumor mechanism for IL-37 via destabilization of C/EBPß to prevent spontaneous BMN migration and tumor progression.

Diet-induced obesity does not exacerbate cachexia in male mice bearing Lewis-lung carcinoma tumors.

Cachexia is a muscle-wasting syndrome commonly observed in patients with cancer, which can significantly worsen clinical outcomes. Because of a global rise in obesity, the coexistence of cachexia in obese individuals poses unique challenges, with the impact of excessive adiposity on cachexia severity and underlying pathophysiology not well defined. Understanding the interplay between cachexia and obesity is crucial for improving diagnosis and treatment strategies for these patients; therefore, the present study examined differences in cachexia between lean and obese mice bearing Lewis lung carcinoma (LLC) tumors. Nine-week-old, male C57Bl6J mice were placed on either a chow or a high-fat diet (HFD) for 9 wk. After the diet intervention, mice were inoculated with LLC or vehicle. Markers of cachexia, such as body and muscle loss, were noted in both chow and HFD groups with tumors. Tumor weight of HFD animals was greater than that of chow. LLC tumors reduced gastrocnemius, plantaris, and soleus mass, regardless of diet. The tibialis anterior and plantaris mass and cross-sectional area of type IIb/x fibers in the gastrocnemius were not different between HFD-chow, HFD-tumor, and chow-tumor. Using RNA sequencing (RNA-seq) of the plantaris muscle from chow-tumor and HFD-tumor groups, we identified ∼400 differentially expressed genes. Bioinformatic analysis identified changes in lipid metabolism, mitochondria, bioenergetics, and proteasome degradation. Atrophy was not greater despite larger tumor burden in animals fed an HFD, and RNA-seq data suggests that partial protection is mediated through differences in mitochondrial function and protein degradation, which may serve as future mechanistic targets.NEW & NOTEWORTHY This study provides timely information on the interaction between obesity and cancer cachexia. Lean and obese animals show signs of cachexia with reduced body weight, adipose tissue, and gastrocnemius muscle mass. There was not significant wasting in the tibialis anterior, plantaris, or fast twitch fibers in the gastrocnemius muscle of obese animals with tumors. RNA-seq analysis reveals that obese tumor bearing animals had differential expression of mitochondria- and degradation-related genes, which may direct future studies in mechanistic research.

Identification of key genes in chronic intermittent hypoxia-induced lung cancer progression based on transcriptome sequencing.

BACKGROUND: Obstructive sleep apnea (OSA) is associated with increased risk of lung cancer mortality. Nevertheless, little is known about the underlying molecular mechanisms. This research aimed to investigate differentially expressed genes (DEGs) and explore their function in Lewis lung carcinoma (LLC)-bearing mice exposed to chronic intermittent hypoxia (CIH) by transcriptome sequencing. METHODS: Lung cancer tissues in LLC-bearing mice exposed to CIH or normoxia were subjected for transcriptome sequencing to examine DEGs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were employed to explore the function of DEGs. To evaluate the prognostic value of DEGs, the Kaplan-Meier survival analysis in combination with Cox proportional hazard model were applied based on The Cancer Genome Atlas. RESULTS: A total of 388 genes with 207 up-regulated and 181 down-regulated genes were differentially expressed between the CIH and normoxia control groups. Bioinformatics analysis revealed that the DEGs were related to various signaling pathways such as chemokine signaling pathway, IL-17 signaling pathway, TGF-ß signaling pathway, transcriptional misregulation in cancer, natural killer cell mediated cytotoxicity, PPAR signaling pathway. In addition, the DEGs including APOL1, ETFB, KLK8, PPP1R3G, PRL, SPTA1, PLA2G3, PCP4L1, NINJ2, MIR186, and KLRG1 were proven to be significantly correlated with poorer overall survival in lung adenocarcinoma. CONCLUSIONS: CIH caused a significant change of gene expression profiling in LLC-bearing mice. The DEGs were found to be involved in various physiological and pathological processes and correlated with poorer prognosis in lung cancer.

Whole­exome sequencing reveals Lewis lung carcinoma is a hypermutated Kras/Nras-mutant cancer with extensive regional mutation clusters in its genome.

Lewis lung carcinoma (LLC), as a widely used preclinical cancer model, has still not been genetically and genomically characterized. Here, we performed a whole-exome sequencing analysis on the LLC cell line to elucidate its molecular characteristics and etiologies. Our data showed that LLC originated from a male mouse belonging to C57BL/6L (a transitional strain between C57BL/6J and C57BL/6N) and contains substantial somatic SNV and InDel mutations (> 20,000). Extensive regional mutation clusters are present in its genome, which were caused mainly by the mutational processes underlying the SBS1, SBS5, SBS15, SBS17a, and SBS21 signatures during frequent structural rearrangements. Thirty three deleterious mutations are present in 30 cancer genes including Kras, Nras, Trp53, Dcc, and Cacna1d. Cdkn2a and Cdkn2b are biallelically deleted from the genome. Five pathways (RTK/RAS, p53, cell cycle, TGFB, and Hippo) are oncogenically deregulated or affected. The major mutational processes in LLC include chromosomal instability, exposure to metabolic mutagens, spontaneous 5-methylcytosine deamination, defective DNA mismatch repair, and reactive oxygen species. Our data also suggest that LLC is a lung cancer similar to human lung adenocarcinoma. This study lays a molecular basis for the more targeted application of LLC in preclinical research.

C-C chemokine receptor 4 (CCR4)-positive regulatory T cells interact with tumor-associated macrophages to facilitate metastatic potential after radiation.

PURPOSE: Our previous study revealed that elevated C-C motif chemokine ligand 2 (CCL2) secretion by irradiated cancer cells recruited C-C motif chemokine receptor 2 (CCR2)-positive myeloid cells and polarized M2-type tumor-associated macrophages (TAMs), promoting lung metastasis in an established mouse model. This study investigated the impact of CCL2 and TAMs on adaptive immunity. METHODS: We assessed the influence of CCL2 and TAMs on adaptive immunity through two ectopic allograft mouse models constructed with MB49 bladder cancer cells and Lewis lung carcinoma cells. Both models exhibited delayed primary tumor growth following radiation therapy (RT), but RT promoted the development of pulmonary metastases in C57BL/6 mice. Additionally, we employed a direct coculture system to investigate the interaction between macrophages and target cells in the context of adaptive immunity. RESULTS: C-C motif chemokine receptor 4 (CCR4)-positive regulatory T cells (Tregs) were recruited to the postirradiated tumor microenvironment (TME). Utilizing a CCR4 antagonist to inhibit CCL2-CCR4 activation reversed the infiltration of CCR4 + Tregs and reduced the incidence of pulmonary metastases. In addition, a positive feedback loop between M2-type TAMs and Tregs was observed. The combined blockade of the CCL2-CCR4 and CCL2-CCR2 signaling pathways further decreased the risk of RT-promoted lung metastasis. CONCLUSION: The recruitment of CCR4 + Tregs to the postirradiated TME increases the metastatic potential of tumor cells through increased interactions with M2-type TAMs. A significant reduction in post-RT lung metastases in ectopic mouse models was achieved by disrupting the recruitment of both CCR4 + Tregs and CCR2 + myeloid cells, which are TAM precursors.

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.

Human mesenchymal stem cells increase LLC metastasis and stimulate or decelerate tumor development depending on injection method and cell amount.

Mesenchymal stem cells (MSCs) being injected into the body can stimulate or decelerate carcinogenesis. Here, the direction of influence of human placenta-derived MSCs (P-MSCs) on the Lewis lung carcinoma (LLC) tumor development and metastatic potential is investigated in C57BL/6 mice depending on the injection method. After intramuscular co-inoculation of LLC and P-MSCs (LLC + P-MSCs), the growth of primary tumor and angiogenesis are slowed down compared to the control LLC on the 15th day. This is explained by the fact of a decrease in the secretion of proangiogenic factors during in vitro co-cultivation of an equal amount of LLC and P-MSCs. When P-MSCs are intravenously (i.v.) injected in the mice with developing LLC (LLC + P-MSCs(i.v.)), the tumor growth and angiogenesis are stimulated on the 15th day. A highly activated secretion of proangiogenic factors by P-MSCs in a similar in vitro model can explain this. In both the models compared to the control on the 23rd day, there is no significant difference in the tumor growth, while angiogenesis remains correspondingly decelerated or stimulated. However, in both the models, the total volume and number of lung metastases constantly increase compared to the control: it is mainly due to small-size metastases for LLC + P-MSCs(i.v.) and larger ones for LLC + P-MSCs. The increase in the rate of LLC cell dissemination after the injection of P-MSCs is explained by the disordered polyploidy and chromosomal instability, leading to an increase in migration and invasion of cancer cells. After LLC + P-MSCs co-inoculation, the tumor cell karyotype has the most complex and heterogeneous chromosomal structure. These findings indicate a bidirectional effect of P-MSCs on the growth of LLC in the early periods after injection, depending on the injection method, and, correspondingly, the number of contacting cells. However, regardless of the injection method, P-MSCs are shown to increase LLC aggressiveness related to cancer-associated angiogenesis and metastasis activation in the long term.

Co-encapsulation of fisetin and cisplatin into liposomes: Stability considerations and in vivo efficacy on lung cancer animal model.

Lung cancer is a highly vascularized tumor for which a combination between an antitumor agent, cisplatin, and an antiangiogenic molecule, fisetin, appears a promising therapeutic approach. In order to deliver both chemotherapies within the tumor, to enhance fisetin solubility and decrease cisplatin toxicity, an encapsulation of both drugs into liposomes was developed. Purification and freeze-drying protocols were optimized to improve both the encapsulation and liposome storage. The cytotoxicity of the encapsulated chemotherapies was evaluated on Lewis lung carcinoma (3LL) cell lines. The antitumor effect of the combination was evaluated in vivo on an ectopic mouse model of Lewis Lung carcinoma. The results showed that fisetin and cisplatin co-loaded liposomes were successfully prepared. Freeze-drying allowed a 30 days storage limiting the release of both drugs. The combination index between liposomal fisetin and liposomal cisplatin on 3LL cell line after 24 h of exposure showed a clear synergism: CI = 0.7 for the co loaded liposomes and CI = 0.9 for the mixture of cisplatin loaded and fisetin loaded liposomes. The co-encapsulating formulation showed in vivo efficacy against an ectopic murine model of Lewis Lung carcinoma with a probable reduction in the toxicity of cisplatin through co-encapsulation with fisetin.

Opaganib (ABC294640) Induces Immunogenic Tumor Cell Death and Enhances Checkpoint Antibody Therapy.

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies.