Mice Bearing Lewis Lung Carcinoma Research Articles

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

BackgroundTumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated.MethodsSubcutaneous 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.ResultsTranscriptome 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.ConclusionsHypofractionated 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.

In vitro and in vivo anti-tumor effect of Trichobakin fused with urokinase-type plasminogen activator ATF-TBK.

Trichobakin (TBK), a member of type I ribosome-inactivating proteins (RIPs), was first successfully cloned from Trichosanthes sp Bac Kan 8-98 in Vietnam. Previous study has shown that TBK acts as a potential protein synthesis inhibitor; however, the inhibition efficiency and specificity of TBK on cancer cells remain to be fully elucidated. In this work, we employed TBK and TBK conjugated with a part of the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), which contains the Ω-loop that primarily interacts with urokinase-type plasminogen activator receptor, and can be a powerful carrier in the drug delivery to cancer cells. Four different human tumor cell lines and BALB/c mice bearing Lewis lung carcinoma cells (LLC) were used to evaluate the role of TBK and ATF-TBK in the inhibition of tumor growth. Here we showed that the obtained ligand fused RIP (ATF-TBK) reduced the growth of four human cancer cell lines in vitro in the uPA receptor level-dependent manner, including the breast adenocarcinoma MDA-MB 231 cells and MCF7 cells, the prostate carcinoma LNCaP cells and the hepatocellular carcinoma HepG2 cells. Furthermore, the conjugate showed anti-tumor activity and prolonged the survival time of tumor-bearing mice. The ATF-TBK also did not cause the death of mice with doses up to 48mg/kg, and they were not significantly distinct on parameters of hematology and serum biochemistry between the control and experiment groups. In conclusion, ATF-TBK reduced the growth of four different human tumor cell lines and inhibited lung tumor growth in a mouse model with little side effects. Hence, the ATF-TBK may be a target to consider as an anti-cancer agent for clinical trials.

EFFECT OF B. SUBTILIS ІМV B-7724 LECTIN ON THE ACTIVITY OF EFFECTORS OF CELLULAR ANTITUMOR IMMUNITY OF MICE WITH LEWIS LUNG CARCINOMA.

To evaluate the effect of B. subtilis IMV B-7724 lectin on the functional activity of macrophages (Mph), natural killer (NK) cells and cytotoxic lymphocytes (CTL) of mice bearing Lewis lung carcinoma (LLC). The studies were performed on C57Bl/6J mice; LLC was used as an experimental transplantable tumor. The lectin from B. subtilis IMV B-7724 was administered to LLC-bearing mice subcutaneously at a dose of 1 mg/kg of body weight for 10 days. The immunological testing was performed on days 14, 21, and 28 after tumor grafting. The cytotoxic activity of Mph, NK, and CTL was estimated in MTT-assay; the content of the stable metabolites of nitric oxide (NO) was measured by a standard Griess reaction; the arginase activity (Arg) was determined based on the measurement of urea. The administration of the B. subtilis IMV B-7724 lectin to LLC-bearing mice exerted its antitumor and antimetastatic effects partially via a significant (p < 0.05) increase of Mph and NK activities after the completion of the treatment. In the group of animals injected with lectin, the NO/Arg ratio increased significantly, indicating the prevalence of Mph with proinflammatory and antitumor properties. The cytotoxic activity of Mph exceeded the indices of untreated mice and intact control by 1.8 times and 5.3 times respectively; of NK - by 2.8 and 1.3 times respectively. The effect of treatment on the CTL activity was less pronounced. Antitumor and antimetastatic activity of the lectin from B. subtilis IMV B-7724 ensured the preservation of the cytotoxic activity of the main effectors of antitumor immunity (Mph, NK, and CTL) throughout LLC growth.

Crude polysaccharide from Danggui Buxue decoction enhanced the anti-tumor effect of gemcitabine by remodeling tumor-associated macrophages

Tumor-associated macrophages (TAMs) with an M2-phenotype mediate gemcitabine resistance to cancer by influencing the metabolic enzymes of gemcitabine and releasing competitive deoxycytidine (dC). Our previous studies showed that Danggui Buxue Decoction (DBD), a traditional Chinese medicinal recipe, enhances the anti-tumor activity of gemcitabine in vivo and alleviates gemcitabine-induced myelosuppression. However, the material basis and exact mechanism underlying its enhanced effects remain unclear. In this study, a bioactive polysaccharide consisting of arabinose, mannose, ribose, and glucose was isolated from DBD. In vivo results demonstrated that DBD crude polysaccharide (DBDP) ameliorated gemcitabine-induced immune system disorders. Moreover, DBDP improved the sensitivity of Lewis lung carcinoma-bearing mice to gemcitabine by reshaping the tumor-promoting M2-like macrophages into tumor-inhibiting M1-phenotypes. Furthermore, in vitro results further revealed that DBDP blocked the protective effects of TAMs and M2-macrophages against gemcitabine by inhibiting the excessive secretion of dC and decreasing the high expression of cytidine deaminase. In conclusion, our results demonstrated that DBDP, as the pharmacodynamic material basis of DBD, enhanced the anti-tumor activity of gemcitabine against lung cancer in vitro and in vivo, which was associated with remodeling of the M2-phenotype.

Microfluidics-Enabled Nanovesicle Delivers CD47/PD-L1 Antibodies to Enhance Antitumor Immunity and Reduce Immunotoxicity in Lung Adenocarcinoma.

The CD47/PD-L1 antibodies combination exhibits durable antitumor immunity but also elicits excessive immune-related adverse events (IRAEs) caused by the on-target off-tumor immunotoxicity, hindering their clinical benefits greatly. Here, a microfluidics-enabled nanovesicle using ultra-pH-sensitive polymer mannose-poly(carboxybetaine methacrylate)-poly(hydroxyethyl piperidine methacrylate) (Man-PCB-PHEP) is developed to deliver CD47/PD-L1 antibodies (NCPA) for tumor-acidity-activated immunotherapy. The NCPA can specifically release antibodies in acidic environment, thereby stimulating the phagocytosis of bone marrow-derived macrophages. In mice bearing Lewis lung carcinoma, NCPA shows significantly improved intratumoral CD47/PD-L1 antibodies accumulation, promoted tumor-associated macrophages remodeling to antitumoral status, and increased infiltration of dendritic cells and cytotoxic T lymphocytes, resulting in more favorable treatment effect compared to those of free antibodies. Additionally, NCPA also shows less IRAEs, including anemia, pneumonia, hepatitis, and small intestinal inflammation in vivo. Altogether, a potent dual checkpoint blockade immunotherapy utilizing NCPA with enhanced antitumor immunity and reduced IRAEs is demonstrated.

Targeting CD70 in combination with chemotherapy to enhance the anti-tumor immune effects in non-small cell lung cancer

ABSTRACT Despite the recent emergence of immune checkpoint inhibitors, clinical outcomes of metastatic NSCLC patients remain poor, pointing out the unmet need to develop novel therapies to enhance the anti-tumor immune response in NSCLC. In this regard, aberrant expression of the immune checkpoint molecule CD70 has been reported on many cancer types, including NSCLC. In this study, the cytotoxic and immune stimulatory potential of an antibody-based anti-CD70 (aCD70) therapy was explored as single agent and in combination with docetaxel and cisplatin in NSCLC in vitro and in vivo. Anti-CD70 therapy resulted in NK-mediated killing of NSCLC cells and increased production of pro-inflammatory cytokines by NK cells in vitro. The combination of chemotherapy and anti-CD70 therapy further enhanced NSCLC cell killing. Moreover, in vivo findings showed that the sequential treatment of chemo-immunotherapy resulted in a significant improved survival and delayed tumor growth compared to single agents in Lewis Lung carcinoma-bearing mice. The immunogenic potential of the chemotherapeutic regimen was further highlighted by increased numbers of dendritic cells in the tumor-draining lymph nodes in these tumor-bearing mice after treatment. The sequential combination therapy resulted in enhanced intratumoral infiltration of both T and NK cells, as well as an increase in the ratio of CD8+ T cells over Tregs. The superior effect of the sequential combination therapy on survival was further confirmed in a NCI-H1975-bearing humanized IL15-NSG-CD34+ mouse model. These novel preclinical data demonstrate the potential of combining chemotherapy and aCD70 therapy to enhance anti-tumor immune responses in NSCLC patients.

Ionic Liquid-Based Transcutaneous Peptide Antitumor Vaccine: Therapeutic Effect in a Mouse Tumor Model.

Traditional vaccinations need to be injected with needles, and since some people have a strong aversion to needles, a needle-free alternative delivery system is important. In this study, we employed ionic liquids (ILs) for transcutaneous delivery of cancer antigen-derived peptides to obtain anticancer therapeutic effects in a needle-free manner. ILs successfully increased the in vitro skin permeability of a peptide from Wilms tumor 1 (WT1), one of the more promising cancer antigens, plus or minus an adjuvant, resiquimod (R848), a toll-like receptor 7 agonist. In vivo studies demonstrated that concomitant transcutaneous delivery of WT1 peptide and R848 by ILs induced WT1-specific cytotoxic T lymphocyte (CTL) in mice, resulting in tumor growth inhibition in Lewis lung carcinoma-bearing mice. Interestingly, administrating R848 in ILs before WT1 peptides in ILs increased tumor growth inhibition effects compared to co-administration of both. We found that the prior application of R848 increased the infiltration of leukocytes in the skin and that subsequent delivery of WT1 peptides was more likely to induce WT1-specific CTL. Furthermore, sequential immunization with IL-based formulations was applicable to different types of peptides and cancer models without induction of skin irritation. IL-based transcutaneous delivery of cancer antigen-derived peptides and adjuvants, either alone or together, could be a novel approach to needle-free cancer therapeutic vaccines.

CHANGES IN THE FUNCTIONAL ACTIVITY OF MACROPHAGES UNDER THE INFLUENCE OF BACTERIAL LECTIN APPLAIED IN DIFFERENT SCHEMES

Aim: to investigate the antitumor effect and the influence of B. subtilis IMV B-7724 lectin applied as a single therapy or in combination with cisplatin on various manifestations of macrophages functional activity during the growth of a metastasizing experimental tumor. Materials and methods: the study was performed on C57Bl/6J mice bearing Lewis lung carcinoma (LLC). The effect of the lectin applied as a single therapy or in combination with cisplatin on tumor growth and the functional activity of peritoneal macrophages were evaluated. The functional activity of peritoneal macrophages was studied by the level of NO production, arginase and cytotoxic activity. Results: there was demonstrated an antimetastatic efficacy of B. subtilis IMV B-7724 lectin applied in Lewis lung carcinoma model either as a single therapy or in combination with cisplatin. In all probability, this effect was grounded by the changes in macrophages functional activity. As it is evidenced by a significant (p &lt; 0.05) suppression of macrophages’ cytotoxic activity and characteristic changes in arginase metabolism, M2 macrophages predominated in the control (untreated) tumor-bearing mice. The features of L-arginine metabolism and cytotoxic activity in peritoneal macrophages indicate the preservation of their antitumor activity (polarization toward M1 type) at the terminal stage of experimental tumor growth. Conclusions: in the animals bearing experimental tumor, the most pronounced antitumor effect was observed when the bacterial lectin was applied in combination with cisplatin. The use of B. subtilis IMV B-7724 lectin as a therapeutic agent (either as a single therapy or in combination with cisplatin) preserved the antitumor activity of macrophages and promoted their polarization toward M1 direction at the terminal stage of tumor growth.

Exosomal IL-8 derived from Lung Cancer and Colon Cancer cells induced adipocyte atrophy via NF-κB signaling pathway

BackgroundCytokines secreted in the tumor microenvironment function in cancer cachexia (CC), a common clinicopathological syndrome associated with adipocyte wasting and skeletal muscle atrophy. Extracellular vesicles (EVs) secreted by cancer cells actively engage in inter-tissue communication; EVs and enclosed cytokines are largely undefined in CC adipocytes wasting.MethodsEVs derived from Lewis lung carcinoma (LLC) and colorectal cancer C26 cells were extracted and characterized. Conditioned medium and EVs from cancer cells were applied to 3 T3-L1 adipocytes. Recombinant IL-8, IL-8 neutralizing antibody, CXCR2 and NF-κB inhibitor were examined in functional assays. Lipolysis of adipocytes was monitored by Western blots, Oil red O staining and glycerol assays. Furthermore, LLC and C26 cell lines were established as cachexia model to explore the relevance of IL-8 and NF-κB signaling in CC adipose wasting. Adipose tissues were collected for histology analyses.ResultsLLC and C26 cell-derived EVs induced lipolysis of 3 T3-L1 adipocytes. Specially, Dil-labeled EVs were effectively taken up by 3 T3-L1 adipocytes, which were motivated by the delivered IL-8 to elicit the NF-κB pathway. In comparison, special IL-8 neutralizing antibody relieved that lipolysis of 3 T3-L1 adipocytes induced by EVs together with conditioned medium of LLC and C26 cells, respectively. Consistently, both CXCR2 and NF-κB inhibitors would lessen the phenotype of lipolysis in 3 T3-L1 adipocytes. In the in vivo settings, both LLC and C26-tumor bearing mice had higher serum IL-8 levels as compared to the control groups. Two typical lipolysis markers, PGC1α and UCP1, were also up-regulated in the adipose tissues of LLC and C26-tumor mice groups, respectively.ConclusionsEVs secreted by LLC and C26 tumor cells would induce adipocyte wasting via extracellular IL-8-mediated NF-κB signaling. Our study pointed out the physiological and therapeutic values of exosomal IL-8 in CC lipolysis.

Caloric Restriction Ketogenic Diets (KR) Enhance Radiotherapy Responses in Lung Cancer Xenografts

<h3>Purpose/Objective(s)</h3> Dietary interventions can change metabolite levels in the tumor microenvironment, which might then affect cancer cell metabolism to alter tumor growth. Caloric restriction (CR) and ketogenic diet (KD) are often thought to limit tumor progression. <h3>Materials/Methods</h3> Mice bearing Lewis Lung Carcinoma xenografts were randomly divided into ad libitum diet group (AL), radiation group (RT), KR diet group (KR), or KR + RT group (KT). Mice in KR and KT group were fed a KR (KetoCal 4:1 fats: proteins+carbohydrates; 70% of total energy). Mice in RT and KT group were treated with radiation (4Gy*3f). Mice weights, tumor size, blood ketone levels and survival were monitored. <h3>Results</h3> Blood ketone levels were significantly higher for the mice consuming a KR diet compared to standard diet (3.2 mmol/L vs 0.8 mmol/L at week 3, P < .001). Some mice with KR diet lost a significant amount of weight (10%ཞ15% of starting weight) by the end of the treatment. The KR diets combined with radiation resulted in slower tumor growth in Lewis Lung Carcinoma xenografts and longer survival time (P < 0.05), relative to radiation alone. The KR diet also slowed tumor growth and prolonged survival (P < 0.05), relative to control. Hematological and pathological examinations indicated that the KR diet was safe and well tolerated, and no significant cardiac, hepatic and renal toxicity was observed. <h3>Conclusion</h3> Blood ketone levels were significantly higher for the mice consuming a KR diet compared to standard diet (3.2 mmol/L vs 0.8 mmol/L at week 3, P < .001). Some mice with KR diet lost a significant amount of weight (10%ཞ15% of starting weight) by the end of the treatment. The KR diets combined with radiation resulted in slower tumor growth in Lewis Lung Carcinoma xenografts and longer survival time (P < 0.05), relative to radiation alone. The KR diet also slowed tumor growth and prolonged survival (P < 0.05), relative to control. Hematological and pathological examinations indicated that the KR diet was safe and well tolerated, and no significant cardiac, hepatic and renal toxicity was observed.

Caloric restriction ketogenic diets (KR) in relation to radiotherapy responses in lung cancer xenografts

e20528 Background: Dietary interventions can change metabolite levels in the tumour microenvironment, which might then affect cancer cell metabolism to alter tumour growth. Caloric restriction (CR) and ketogenic diet (KD) are often thought to limit tumour progression. The current study tests the hypothesis that caloric restriction ketogenic diets (KR) enhance radiotherapy responses in lung cancer xenografts. Methods: Mice bearing Lewis Lung Carcinoma xenografts were randomly divided into ad libitum diet group (AL), radiation group (RT), KR diet group (KR), or KR + RT group (KT). Mice in KR and KT group were fed a KR (KetoCal 4:1 fats: proteins+carbohydrates;70% of total energy). Mice in RT and KT group were treated with radiation (4Gy*3f). Mice weights, tumor size, blood ketone levels and survival were monitored. Results: Blood ketone levels were significantly higher for the mice consuming a KR diet compared to standard diet (3.2 mmol/L vs 0.8 mmol/L at week 3, P &lt; .001). Some mice with KR diet lost a significant amount of weight (10%～15% of starting weight) by the end of the treatment.The KR diets combined with radiation resulted in slower tumor growth in Lewis Lung Carcinoma xenografts and longer survival time (P &lt; 0.05), relative to radiation alone. The KR diet also slowed tumor growth and prolonged survival (P &lt; 0.05), relative to control. Hematological and pathological examinations indicated that the KR diet was safe and well tolerated, and no significant cardiac, hepatic and renal toxicity was observed. Conclusions: These results showed that a KR diet enhanced radiotherapy responses in lung cancer xenografts. Further studies are needed to address the mechanism of this diet intervention.

An Integrative Metabolomic and Network Pharmacology Study Revealing the Regulating Properties of Xihuang Pill That Improves Anlotinib Effects in Lung Cancer

Lung cancer ranks as a leading cause of death. Although targeted therapies usually trigger profound initial patient responses, these effects are transient due to drug resistance and severe side effects. Xihuang Pill (XHW) is a popular Chinese medicine formula that might benefit cancer patients when used as a complementary therapy. However, its underlying mechanism when combined with anticancer drugs is not clearly understood. Here, we used an integrated strategy to reveal the regulatory properties of XHW in increasing the antitumor activity of anlotinib in lung cancer. We evaluated the anti-lung cancer effect of XHW combined with anlotinib in mice bearing Lewis lung carcinoma (LLC). We applied untargeted metabolomics to identify the differences metabolism and found that XHW improved the effects of anlotinib on lung cancer. The components and targets related to the effects of XHW treatment on lung cancer were obtained through network pharmacology. Then, by integrating the biologically active components of XHW and anlotinib as well as the treatment-responsive metabolites and their related targets, an interaction network was constructed to evaluate the combination therapy. Finally, important protein candidates for this response were verified by immunohistochemistry of tumor tissues. The results showed that XHW significantly improved the inhibitory effect of anlotinib on tumor growth in LLC-bearing mice. Additionally, 12 differentially-abundant metabolites were identified by untargeted metabolomics in the XHW/anlotinib group compared with the XHW or anlotinib groups, and they were mainly enriched in fatty acid metabolism, lipid metabolism and amino acid metabolism pathways. Anlotinib, 23 components in Shexiang, 2 components in Niuhuang, 30 components in Ruxiang and 60 components in Moyao work together to act on 30 targets to regulate hexadecanoic acid (also named palmitic acid), linoleic acid, lactosylceramide, adrenaline, arachidonic acid and lysoPC(18:1(9Z)). The results of immunohistochemistry showed that XHW combined with anlotinib reduced the expression of PDGFRA in tumors. Overall, the key metabolites of XHW that enhances the efficacy of anlotinib were regulated by a multicomponent and multitarget interaction network. Our results suggested that anlotinib combined with XHW may be a promising strategy for the treatment of lung cancer.

Synchrotron Microbeam Radiation Therapy for the Treatment of Lung Carcinoma: A Preclinical Study

In the past 3 decades, synchrotron microbeam radiation therapy (S-MRT) has been shown to achieve both good tumor control and normal tissue sparing in a range of preclinical animal models. However, the use of S-MRT for the treatment of lung tumors has not yet been investigated. This study is the first to evaluate the therapeutic efficacy of S-MRT for the treatment of lung carcinoma, using a new syngeneic and orthotopic mouse model. Lewis Lung carcinoma-bearing mice were irradiated with 2 cross-fired arrays of S-MRT or synchrotron broad-beam (S-BB) radiation therapy. S-MRT consisted of 17 microbeams with a width of 50 µm and center-to-center spacing of 400 µm. Each microbeam delivered a peak entrance dose of 400 Gy whereas S-BB delivered a homogeneous entrance dose of 5.16 Gy (corresponding to the S-MRT valley dose). Both treatments prolonged the survival of mice relative to the untreated controls. However, mice in the S-MRT group developed severe pulmonary edema around the irradiated carcinomas and did not have improved survival relative to the S-BB group. Subsequent postmortem examination of tumor size revealed that the mice in the S-MRT group had notably smaller tumor volume compared with the S-BB group, despite the presence of edema. Mice that were sham-implanted did not display any decline in health after S-MRT, experiencing only mild and transient edema between 4 days and 3 months postirradiation which disappeared after 4 months. Finally, a parallel study investigating the lungs of healthy mice showed the complete absence of radiation-induced pulmonary fibrosis 6 months after S-MRT. S-MRT is a promising tool for the treatment of lung carcinoma, reducing tumor size compared with mice treated with S-BB and sparing healthy lungs from pulmonary fibrosis. Future experiments should focus on optimizing S-MRT parameters to minimize pulmonary edema and maximize the therapeutic ratio.

Cancer Cell-Derived Extracellular Vesicles Are Prothrombotic in an in-Vitro Vessel Model

Introduction:The risk of thrombosis is markedly increased in patients with cancer, and cancer-associated thrombosis (CAT) is associated with significant morbidity and mortality. It is likely that several mechanisms contribute to the development of CAT, although these may vary among different types of cancers and under different conditions. While genetically-engineered mice may be valuable models for studying thrombosis, an in-vitro model of human vasculature would enable study of CAT mechanisms more rapidly and with less expense; however, at this point such models are not well-developed. Extracellular vesicles (EV) are cell-derived particles, most of which range from <100 nM to 500 nM in diameter. These may arise from cancer cells, stromal cells, or vascular cells, including platelets, endothelial cells and leukocytes, and circulate at elevated concentrations in patients with cancer. EV may induce thrombosis through a variety of mechanisms, including expression of tissue factor, contact activation, and potentially other mechanisms including transfer of RNA or proteins to vascular cells, enhancing their prothrombotic activity.Methods:To develop a system for evaluating the prothrombotic potential of EV on a microscale, we used the Cellix microfludic system, with which we can assess cellular adhesion to the wall of a glass capillary tube through which fluid is driven by a peristaltic pump. In these experiments human umbilical vein endothelial cells (HUVEC) were cultured in fibronectin-coated glass capillary tubes, forming an intact monolayer (Figure, top left panel). After washing non-adherent endothelial cells from the vessel, the endothelial cell-lined vessels were exposed to EV derived from cancer cell lines (U87 glioblastoma or HT29 colorectal carcinoma), or primary human fibroblasts, for four hours prior to flowing of normal human whole blood, to which calcein-AM had been added to label platelets, through the vessel at a shear rate of 67 dynes/cm2. TNFα was used as a positive control to induce an adhesive endothelial cell phenotype. In some experiments, EV were added directly to whole blood, or both preincubated with endothelial cells and added to whole blood before perfusion through the system.Results:As expected (see Figure), we observed that preincubation of TNFα with HUVEC-lined vessels significantly increased platelet adhesion to the endothelial cell monolayer (~100 arbitrary fluorescence units, AFU). In addition, either preincubation of endothelial cells with U87 or HT29-derived exosomes (Figure), or addition of cancer cell-derived exosomes to whole blood also led to markedly-increased rates of platelet adhesion (50-80 AFU) to the endothelial monolayer, and in some cases caused complete occlusion of the vessel and cessation of flow (e.g. in vitro thrombus formation). Normal fibroblast-derived EV also stimulated platelet adhesion but to a much lower extent than cancer cell-derived EV (~20 AFU). Addition of cancer cell-derived EV to both the microfluidics vessel and whole blood almost universally caused vessel occlusion. In further studies, we also found that whole blood from Lewis lung carcinoma-bearing mice was more prothrombotic in this system than that from non-tumor-bearing littermates (~60% higher), and that the absence of high molecular weight kininogen (studied using blood from tumor bearing and non-tumor-bearing kininogen deficient mice) partially protected again vascular occlusion in this model.Conclusion:These studies suggest that cancer cell-derived EV are more prothrombotic than EV derived from non-transformed cells in this model vessel lined by human endothelial cells. Additional work is needed to further define the underlying mechanism by which this occurs, but involvement of the contact activation system is likely, particularly based on preliminary results using blood from tumor-bearing kininogen deficient mice. This microfluidics system will be of significant value in future translational and mechanistic studies focused on mechanisms of CAT. [Display omitted] DisclosuresKhorana:Janssen Scientific Affairs, LLC: Consultancy, Honoraria, Research Funding; Leo: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Honoraria.

Combination of Bempegaldesleukin and Anti-CTLA-4 Prevents Metastatic Dissemination After Primary Resection or Radiotherapy in a Preclinical Model of Non-Small Cell Lung Cancer.

Surgical resection or hypo-fractionated radiation therapy (RT) in early-stage non-small cell lung cancer (NSCLC) achieves local tumor control, but metastatic relapse remains a challenge. We hypothesized that immunotherapy with anti-CTLA-4 and bempegaldesleukin (BEMPEG; NKTR-214), a CD122-preferential IL2 pathway agonist, after primary tumor RT or resection would reduce metastases in a syngeneic murine NSCLC model. Mice bearing Lewis Lung Carcinoma (LLC) tumors were treated with combinations of BEMPEG, anti-CTLA-4, and primary tumor treatment (surgical resection or RT). Primary tumor size, mouse survival, and metastatic disease at the time of death were assessed. Flow cytometry, qRT-PCR, and cytokine analyses were performed on tumor specimens. All mice treated with RT or surgical resection of primary tumor alone succumbed to metastatic disease, and all mice treated with BEMPEG and/or anti-CTLA-4 succumbed to primary tumor local progression. The combination of primary tumor RT or resection and BEMPEG and anti-CTLA-4 reduced spontaneous metastasis and improved survival without any noted toxicity. Flow cytometric immunoprofiling of primary tumors revealed increased CD8 T and NK cells and decreased T-regulatory cells with the combination of BEMPEG, anti-CTLA-4, and RT compared to RT alone. Increased expression of genes associated with tumor cell immune susceptibility, immune cell recruitment, and cytotoxic T lymphocyte activation were observed in tumors of mice treated with BEMPEG, anti-CTLA-4, and RT. The combination of BEMPEG and anti-CTLA-4 with primary tumor RT or resection enabled effective control of local and metastatic disease in a preclinical murine NSCLC model. This therapeutic combination has important translational potential for patients with early-stage NSCLC and other cancers.

Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.

Polysaccharide Isolated From Tetrastigma hemsleyanum Activates TLR4 in Macrophage Cell Lines and Enhances Immune Responses in OVA-Immunized and LLC-Bearing Mouse Models.

Tetrastigma hemsleyanum Diels et Gilg is a valuable Chinese medicinal herb with a long history of clinical application. Our previous study isolated and characterized a purified polysaccharide from the aerial part of Tetrastigma hemsleyanum (SYQP) and found it having antipyretic and antitumor effects in mice. A preliminary mechanistic study suggests these effects may be related to the binding of toll-like receptor (TLR4). The objective of this study is to further explore the detailed stimulating characteristics of SYQP on TLR4 signaling pathway and its in vivo immune regulating effect. We use HEK-BLUE hTLR4, mouse and human macrophage cell lines, as research tools. In vitro results show SYQP activated HEK-BLUE hTLR4 instead of HEK-BLUE Null cells. The secretion and the mRNA expression of cytokines related to TLR4 signaling significantly increased after SYQP treatment in both PMA-induced THP-1 and RAW264.7 macrophage cell lines. The TLR4 antagonist TAK-242 can almost completely abolish this activation. Furthermore, molecules such as IRAK1, NF-κB, MAPKs, and IRF3 in both the MyD88 and TRIF branches were all activated without pathway selection. In vivo results show SYQP enhanced antigen-specific spleen lymphocyte proliferation and serum IgG levels in OVA-immunized C57BL/6 mice. Orally administered 200 mg/kg SYQP induced obvious tumor regression, spleen weight increase, and the upregulation of the mRNA expression of TLR4-related cytokines in Lewis lung carcinoma–bearing mice. These results indicate SYQP can act as both a human and mouse TLR4 agonist and enhance immune responses in mice (p < 0.05). This study provides a basis for the development and utilization of SYQP as a new type of TLR4 agonist in the future.

Intratumoral Injection of Anlotinib Hydrogel Combined With Radiotherapy Reduces Hypoxia in Lewis Lung Carcinoma Xenografts: Assessment by Micro Fluorine-18-fluoromisonidazole Positron Emission Tomography/Computed Tomography Hypoxia Imaging.

Hypoxia is a common feature of solid tumors that increases tumor invasiveness and resistance to radiotherapy (RT) and chemotherapy. Local application of anlotinib (AL) might increase the regulation of new blood vessel growth and improve tumor hypoxia in RT. Therefore, it is essential to fully understand the drug delivery system of AL. Herein, we applied hypoxia imaging using micro fluorine-18-fluoromisonidazole positron emission tomography/computed tomography (micro 18F-FMISO PET/CT) to assess responses to intratumoral injections of an AL hydrogel (AL-HA-Tyr) combined with RT in mice bearing Lewis lung carcinoma (LLC). We formed AL-HA-Tyr by encapsulating AL with hyaluronic acid-tyramine (HA-Tyr) conjugates via the oxidative coupling of tyramine moieties catalyzed by H2O2 and horseradish peroxidase. AL-HA-Tyr restrained the proliferation of human umbilical endothelial cells (HUVECs) in colony formation assays in vitro (p < 0.001). We established a subcutaneous LLC xenograft model using C57BL/6J mice that were randomly assigned to six groups that were treated with AL, HA-Tyr, AL-HA-Tyr, RT, and RT+AL-HA-Tyr, or untreated (controls). Tumor volume and weight were dynamically measured. Post treatment changes in hypoxia were assessed in some mice using micro 18F-FMISO PET/CT, and survival was assessed in others. We histopathologically examined toxicity in visceral tissues and Ki-67, VEGF-A, γ-H2AX, and HIF-1α expression using immunohistochemistry. Direct intratumoral injections of AL-HA-Tyr exerted anti-tumor effects and improved hypoxia like orally administered AL (p > 0.05), but reduced visceral toxicity and prolonged survival. The uptake of 18F-FMISO did not significantly differ among the AL, AL-HA-Tyr, and RT+AL-HA-Tyr treated groups. Compared with the other agents, RT+AL-HA-Tyr decreased HIF-1α, Ki67, and VEGF-A expression, and increased γ-H2AX levels in tumor cells. Overall, compared with AL and AL-HA-Tyr, RT+AL-HA-Tyr improved tumor hypoxia, enhanced anti-tumor effects, and prolonged the survival of mice bearing LLC.

A novel co-culture assay to assess anti-tumor CD8+ T cell cytotoxicity via luminescence and multicolor flow cytometry

T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8+ T cells evidenced by in vitro CD8+ T cell depletion. Flow cytometry demonstrated increased expression of CD107a and production of granzyme B, IFNγ, and TNFα by CD8+ T cells. Our co-culture assay is therefore suitable as proof of principle for in vivo therapeutic studies testing immunotherapies, and specifically to assess the involvement of cytotoxic CD8+ T cells in treatment response in LLC and CT26 tumor models. We also propose this assay as an ex vivo platform for high-throughput screening of immunomodulating agents to be tested in these two murine tumor models. This assay can be adapted to other tumor models after optimizations.

Abstract 4480: Circulating and tumor associated neutrophil subtypes correlate with hyperprogressive disease (HPD) upon immunecheckpoint inhibitors (ICI) in advanced non-small cell lung cancer (aNSCLC) patients (pts) and in vivo models

Abstract Background: HPD was described in ≃10-25% of NSCLC pts upon single-agent ICI and correlated with poor prognosis. High circulating neutrophil count and neutrophils/lymphocytes ratio were associated with shorter survival and HPD in aNSCLC pts. In mouse lung cancer models, interleukin-17 (IL-17) promoted tumor growth upon ICI through increased intratumoral neutrophils. The role of specific circulating and/or tumor-associated neutrophil populations in driving HPD is unknown. Methods: aNSCLC pts treated with ICI and enrolled in a prospective monocentric study were assessed for HPD and circulating neutrophils' phenotype. HPD required 3 tumor measurements (2 before ICI, 1 upon ICI) and was defined as delta tumor growth rate (TGR) TGR (TGR upon ICI - TGR before ICI) &amp;gt;50% and/or TGR ratio (TGR upon ICI/ TGR before ICI) ≥2. Survival curves were estimated by Kaplan Meier and compared by log-rank, correlations with categorical/continuous variables were performed by Fisher/Mann-Whitney tests. The phenotype of circulating low density neutrophils (LDNs) was assessed by flow cytometry (FC) on peripheral blood mononuclear cells from fresh blood samples. Mature and immature LDNs were defined according to the expression of CD10 on CD66b+CD15+ cells, being immature the CD10− neutrophil population. The capacity of immune competent mice to undergo HPD upon anti-PD-1 treatment was further tested in C57BL/6 mice bearing Lewis Lung Carcinoma and treated with anti-murine PD-1. Tumor associated neutrophils' phenotype was assessed by FC analysis. Results: Of 46 aNSCLC, 85% were &amp;gt;65 years, 72% had adenocarcinoma histology, 89% stage IV, 22% had PD-L1 on tumor cells ≥50%, 72% received 1st line ICI. 19 (41%) pts had progression (PD) by RECIST, 4 (9%) pts were HPD by TGR ratio and/or delta-TGR. HPD [4.1 (95% CI 3.1-5) months] correlated with poorer median overall survival compared to non-HPD [6.9 (95% CI 6.1-7.8) months, p=0.03] and to PD pts [4.7 (95% CI 3.2-6.1) months, p=0.8]. Before ICI initiation, the median percentage of circulating immature CD10− CD15+ CD66b+ LDNs was significantly higher in HPD [36.6 (min 29.2; max 64.5)] compared to both non-HPD [8.9 (min 0; max 70.4); p=0.01] and PD non-HPD pts [10.1 (min 0; max 70.4), p=0.04]. HPD defined by TGR ratio and/or delta-TGR occurred in 3 (12.5%) out of 24 immune competent mice treated with anti-murine PD-1. The mean of Gr1highLy6Clow IL-17+ tumor associated neutrophils was significantly higher in HPD compared to non-HPD mice [0.25 (min 0.14; max 0.63) vs 0.06 (min 0.02; max 0.32), p=0.03]. Conclusions: Circulating immature CD10− LDNs and tumor associated IL-17+ neutrophils correlate with HPD upon ICI in aNSCLC pts and in vivo models, respectively. Characterization of neutrophils' functional properties in HPD pts and in vivo models is currently ongoing. Citation Format: Roberto Ferrara, Claudia Proto, Giuseppe Lo Russo, Diego Signorelli, Arsela Prelaj, Giulia Galli, Nicoletta Zilembo, Alessandro De Toma, Giuseppe Viscardi, Riccardo Lobefaro, Marta Brambilla, Monica Ganzinelli, Giuliano Molino, Giovanna Talarico, Marina Chiara Garassino, Mario Colombo, Sabina Sangaletti. Circulating and tumor associated neutrophil subtypes correlate with hyperprogressive disease (HPD) upon immunecheckpoint inhibitors (ICI) in advanced non-small cell lung cancer (aNSCLC) patients (pts) and in vivo models [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 4480.