Metabolic reprogramming of lipid metabolism is a hallmark of cancer. Consumption of a high-fat obesogenic diet enhances spontaneous metastasis using a Lewis lung carcinoma (LLC) model. In order to gain further insights into the mechanisms by which dietary fats impact cancer progression, we conducted a lipidomic analysis of primary tumors originated from LLC from mice fed with a standard AIN93G diet or a soybean oil-based high-fat diet (HFD). Hierarchical clustering heatmap analysis of phosphatidylcholine (PC) lipids and phosphatidylethanolamine (PE) lipids demonstrated an increase in polyunsaturated fatty acids (PUFA)-containing phospholipids and a decrease in monounsaturated fatty acids (MUFA)-containing lipids in tumors from mice fed the HFD. The quantities of 51 PC and 24 PE lipids differed in primary tumors of LLC from mice fed the control diet and the HFD. Analysis of triacylglycerol (TAG) lipids identified differences in 32 TAG (by brutto structure) between the two groups; TAG analysis by neutral loss identified 46 PUFA-containing TAG species that were higher in mice fed with the HFD than in the controls. Intake of the HFD did not alter the expression of the de novo lipogenesis enzymes (fatty acid synthase, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase-1). Our results demonstrate that the dietary fatty acid composition of the HFD is reflected in the higher order lipidomic composition of primary tumors. Subsequent studies are needed to investigate how these lipidomic changes may be used for targeted dietary intervention to reduce tumor growth and malignant progression.

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.

Immune checkpoint inhibitor (ICI) therapy is first-line treatment for many advanced non-small cell lung cancer (aNSCLC) patients. Predicting response could help guide selection of intensified or alternative anti-cancer regimens. We hypothesized that radiomics and laboratory variables predictive of ICI response in a murine model would also predict response in aNSCLC patients.

The coxsackie and adenovirus receptor (CAR) is a member of the junctional adhesion molecule (JAM) family of adhesion receptors and is localised to epithelial cell tight and adherens junctions. CAR has been shown to be highly expressed in lung cancer where it is proposed to promote tumor growth and regulate epithelial mesenchymal transition (EMT), however the potential role of CAR in lung cancer metastasis remains poorly understood. To better understand the role of this receptor in tumor progression, we manipulated CAR expression in both epithelial-like and mesenchymal-like lung cancer cells. In both cases, CAR overexpression promoted tumor growth in vivo in immunocompetent mice and increased cell adhesion in the lung after intravenous injection without altering the EMT properties of each cell line. Overexpression of WTCAR resulted in increased invasion in 3D models and enhanced β1 integrin activity in both cell lines, and this was dependent on phosphorylation of the CAR cytoplasmic tail. Furthermore, phosphorylation of CAR was enhanced by substrate stiffness in vitro, and CAR expression increased at the boundary of solid tumors in vivo. Moreover, CAR formed a complex with the focal adhesion proteins Src, Focal Adhesion Kinase (FAK) and paxillin and promoted activation of the Guanine Triphosphate (GTP)-ase Ras-related Protein 1 (Rap1), which in turn mediated enhanced integrin activation. Taken together, our data demonstrate that CAR contributes to lung cancer metastasis via promotion of cell-matrix adhesion, providing new insight into co-operation between cell-cell and cell-matrix proteins that regulate different steps of tumorigenesis.

Exosomes are nanovesicles produced by a number of different cell types and regarded as important mediators of cell-to-cell communication. Although bronchoalveolar lavage fluid (BALF) has been shown to be involved in the development of tumors, its role in lung cancer (LC) remains unclear. In this article, we systemically studied BALF-derived exosomes in LC. C57BL/6 mice were injected with Lewis lung carcinoma cells and exposed to non-typeable Haemophilus influenza (NTHi) lysate. The analysis showed that the growth of lung tumors in these mice was significantly enhanced compared with the control cohort (only exposure to air). Characterization of the exosomes derived from mouse BALF demonstrated elevated levels of tumor necrosis factor alpha and interleukin-6 in mice exposed to NTHi lysates. Furthermore, abnormal BALF-derived exosomes facilitated the development of LC in vitro and in vivo. The internalization of the BALF-derived exosomes contributed to the development of LC tumors. Collectively, our data demonstrated that exosomes in BALF are a key factor involved in the growth and progression of lung cancer.

Lung cancer is one of the leading causes of cancer-related deaths in the United States. A major hurdle for improved therapies is immune suppression mediated by the tumor and its microenvironment. The lung tumor microenvironment (TME) contains large numbers of tumor-associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization of the tumor, and provide pro-tumor factors to promote tumor growth. CD11b is highly expressed on myeloid cells, including TAMs, where it forms a heterodimeric integrin receptor with CD18 (known as CD11b/CD18, Mac-1, CR3, and αMβ2), and plays an important role in recruitment and biological functions of these cells, and is a validated therapeutic target. Here, we describe our pre-clinical studies targeting CD11b in the context of lung cancer, using pharmacologic and genetic approaches that work via positive allosteric modulation of CD11b function. GB1275 is a novel small molecule modulator of CD11b that is currently in Phase 1/2 clinical development. We assess GB1275 treatment effects on tumor growth and immune infiltrates in the murine Lewis Lung Carcinoma (LLC) syngeneic tumor model. Additionally, as an orthogonal approach to determine mechanisms of action, we utilize our recently developed novel CD11b knock-in (KI) mouse that constitutively expresses CD11b containing an activating isoleucine to glycine substitution at residue 332 in the ligand binding CD11b A-domain (I332G) that acts as a positive allosteric modulator of CD11b activity. We report that pharmacologic modulation of CD11b with GB1275 significantly reduces LLC tumor growth. CD11b KI mice similarly show significant reduction in both the size and rate of LLC tumor growth, as compared to WT mice, mimicking our observed treatment effects with GB1275. Tumor profiling revealed a significant reduction in TAM infiltration in GB1275-treated and in CD11b KI mice, increase in the ratio of M1/M2-like TAMs, and concomitant increase in cytotoxic T cells. The profiling also showed a significant decrease in CCL2 levels and a concomitant reduction in Ly6Chi monocytes in circulation in both groups. These findings suggest that positive allosteric modulation of CD11b reduces TAM density and reprograms them to enhance the adaptive immune response and is a novel therapeutic strategy against lung cancer.

Lung cancer is one of the most common cancer types in the world. Despite existing treatment strategies, overall patient survival remains low and new targeted therapies are required. Acidification of the tumor microenvironment drives the growth and metastasis of many cancers. Acid sensors such as acid-sensing ion channels (ASICs) may become promising targets for lung cancer therapy. Previously, we showed that inhibition of the ASIC1 channels by a recombinant analogue of mambalgin-2 from Dendroaspis polylepis controls oncogenic processes in leukemia, glioma, and melanoma cells. Here, we studied the effects and molecular targets of mambalgin-2 in lung adenocarcinoma A549 and Lewis cells, lung transformed WI-38 fibroblasts, and lung normal HLF fibroblasts. We found that mambalgin-2 inhibits the growth and migration of A549, metastatic Lewis P29 cells, and WI-38 cells, but not of normal fibroblasts. A549, Lewis, and WI-38 cells expressed different ASIC and ENaC subunits, while normal fibroblasts did not at all. Mambalgin-2 induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma cells. In line, acidification-evoked inward currents were observed only in A549 and WI-38 cells. Gene knockdown showed that the anti-proliferative and anti-migratory activity of mambalgin-2 is dependent on the expression of ASIC1a, α-ENaC, and γ-ENaC. Using affinity extraction and immunoprecipitation, mambalgin-2 targeting of ASIC1a/α-ENaC/γ-ENaC heteromeric channels in A549 cells was shown. Electrophysiology studies in Xenopus oocytes revealed that mambalgin-2 inhibits the ASIC1a/α-ENaC/γ-ENaC channels with higher efficacy than the ASIC1a channels, pointing on the heteromeric channels as a primary target of the toxin in cancer cells. Finally, bioinformatics analysis showed that the increased expression of ASIC1 and γ-ENaC correlates with a worse survival prognosis for patients with lung adenocarcinoma. Thus, the ASIC1a/α-ENaC/γ-ENaC heterotrimer can be considered a marker of cell oncogenicity and its targeting is promising for the design of new selective cancer therapeutics.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies. Studies have shown that engulfment and cell motility 3 (ELMO3) is highly expressed in NSCLC and can be used as a novel biomarker, but its underlying mechanism remains to be explored. The aim of this study was to investigate the mechanism by which ELMO3 may be down-regulated by COX-2 inhibitors to inhibit NSCLC. NSCLC tissue and adjacent normal lung tissue from 24 patients were used to detect the mRNA and protein expression of ELMO3, COX-2, and other related proteins by Western blot, RT-PCR, and Immunohistochemical analysis. Lewis Lung carcinoma (LLC) cells were used to investigate the effects and the mechanism of siELMO3 and COX-2 inhibitor. C57BL/6 mice inoculated with LLC cells by subcutaneous (s.c.) injection were used to detect the in vivo effects of cox-2 inhibitor. The expression of ELMO3 and cyclooxygenase-2 (COX-2) in human NSCLC tissues was significantly increased compared with that in the adjacent normal tissues. ELMO3 exhibited a positive correlation with COX-2 expression. Moreover, knockdown of ELMO3 suppressed the epithelial-mesenchymal transition (EMT), adhesion, and metastasis of Lewis lung carcinoma (LLC) cells. Importantly, Parecoxib, a selective inhibitor of COX-2, significantly reduced the expression of ELMO3 and EMT in LLC cells and LLC-bearing mice. Furthermore, it could inhibit the growth, adhesion and metastasis of LLC cells in vitro. Our results demonstrate that down regulation of ELMO3 suppressed growth and metastasis of lung cancer by inhibiting EMT. Parecoxib could reduce ELMO3 expression and suppress growth and metastasis of lung cancer, which might be a useful chemotherapeutic agent for inhibiting metastasis and recurrence of NSCLC.

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.

Lung cancer is a type of cancer with higher morbidity and mortality. In spite of the impressive response rates of nab-paclitaxel (nab-PTX) or programmed cell death-1 (PD-1) and its ligand inhibitors, the effective treatment remains limited. Currently, alternative strategies aim at drug combination of nab-PTX and PD-1/PD-L1 inhibitors. Even as the clinical impact of the combined agents continues to increase, basic research studies are still limited and the mechanisms underlying this synergy are not well studied. In this study, we evaluated the antitumor efficacy and the molecular mechanisms of action of nab-PTX in combination with anti-PD-1 antibody, using Lewis lung carcinoma (LLC) cell and subcutaneously transplanted tumor models. The combination of nab-PTX and anti-PD-1 antibody displayed stronger antitumor effects, manifested at tumor volume, proliferation and apoptosis through Ki67 and TUNEL staining. In-vivo experiments showed significant increases in CD4+ T cells, CD8+ T cells, IFN-γ, TNF-α, IL-2, PF, and Gzms-B, exerting antitumor effects with reductions in MDSCs and IL-10 after the treatments. Furthermore, transcriptomic analysis indicated 20 overlapped differentially expressed genes, and Serpin peptidase inhibitor clade C Member 1 (Serpinc1) was downregulated during treatment in vivo, whose expression level was markedly related to metastasis and overall survival of lung cancer patients. Functional enrichment analysis of the target gene revealed primary GO terms related to tumor, which warrants further investigation. We also found that Serpinc1 overexpression promoted cell proliferation, migration, and invasion and inhibited cell apoptosis of LLC cells in vitro, possibly regulating the associated factors via the Pi3K/AKT pathway. In summary, our results reveal the synergistic antitumor responses of nab-PTX combined with anti-PD-1 antibody, in which Serpinc1 may play an important role, providing a target gene for combination treatment strategy.

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.

N-glycolylneuraminic acid (NeuGc) is a sialic acid molecule usually found in mammalian cells as terminal constituents of different membrane glycoconjugates such as gangliosides. The NeuGcGM3 ganglioside has been described as a tumor antigen for non-small cell lung cancer (NSCLC) in humans. Racotumomab is an anti-NeuGc-containing gangliosides anti-idiotype monoclonal antibody (mAb) (formerly known as 1E10) that has received attention as a potential active immunotherapy for advanced lung cancer in clinical trials. In this work, we have examined the antitumor activity of racotumomab in combination or not with chemotherapy, using the 3LL Lewis lung carcinoma as a preclinical model of NSCLC in C57BL/6 mice. Vaccination with biweekly doses of racotumomab at 50-200 μg/dose formulated in aluminum hydroxide (racotumomab-alum vaccine) demonstrated a significant antitumor effect against the progression of lung tumor nodules. Racotumomab-alum vaccination exerted a comparable effect on lung disease to that of pemetrexed-based chemotherapy (100 mg/kg weekly). Interestingly, chemo-immunotherapy was highly effective against lung nodules and well-tolerated, although no significant synergistic effect was observed as compared to each treatment alone in the present model. We also obtained evidence on the role of the exogenous incorporation of NeuGc in the metastatic potential of 3LL cells. Our preclinical data provide support for the combination of chemotherapy with the anti-idiotype mAb racotumomab, and also reinforce the biological significance of NeuGc in lung cancer.

Erythropoietin receptor (EPOR) is widely expressed in healthy and malignant tissues. In certain malignancies, EPOR stimulates tumor growth. In healthy tissues, EPOR controls processes other than erythropoiesis, including mitochondrial metabolism. We hypothesized that EPOR also controls the mitochondrial metabolism in cancer cells. To test this hypothesis, we generated EPOR-knockdown cancer cells to grow tumor xenografts in mice and analyzed tumor cellular respiration via high-resolution respirometry. Furthermore, we analyzed cellular respiratory control, mitochondrial content, and regulators of mitochondrial biogenesis in vivo and in vitro in different cancer cell lines. Our results show that EPOR controls tumor growth and mitochondrial biogenesis in tumors by controlling the levels of both, pAKT and inducible NO synthase (iNOS). Furthermore, we observed that the expression of EPOR is associated with the expression of the mitochondrial marker VDAC1 in tissue arrays of lung cancer patients, suggesting that EPOR indeed helps to regulate mitochondrial biogenesis in tumors of cancer patients. Thus, our data imply that EPOR not only stimulates tumor growth but also regulates tumor metabolism and is a target for direct intervention against progression.

Background: The mechanisms underlying low intensity ultrasound (LIUS) mediated suppression of inflammation and tumorigenesis remain poorly determined. Methods: We used microarray datasets from NCBI GEO Dataset databases and conducted a comprehensive data mining analyses, where we studied the gene expression of 299 cell death regulators that regulate 13 different cell death types (cell death regulatome) in cells treated with LIUS. Results: We made the following findings: (1) LIUS exerts a profound effect on the expression of cell death regulatome in cancer cells and non-cancer cells. Of note, LIUS has the tendency to downregulate the gene expression of cell death regulators in non-cancer cells. Most of the cell death regulator genes downregulated by LIUS in non-cancer cells are responsible for mediating inflammatory signaling pathways; (2) LIUS activates different cell death transcription factors in cancer and non-cancer cells. Transcription factors TP-53 and SRF- were induced by LIUS exposure in cancer cells and non-cancer cells, respectively; (3) As two well-accepted mechanisms of LIUS, mild hyperthermia and oscillatory shear stress induce changes in the expression of cell death regulators, therefore, may be responsible for inducing LIUS mediated changes in gene expression patterns of cell death regulators in cells; (4) LIUS exposure may change the redox status of the cells. LIUS may induce more of antioxidant effects in non-cancer cells compared to cancer cells; and (5) The genes modulated by LIUS in cancer cells have distinct chromatin long range interaction (CLRI) patterns to that of non-cancer cells. Conclusions: Our analysis suggests novel molecular mechanisms that may be utilized by LIUS to induce tumor suppression and inflammation inhibition. Our findings may lead to development of new treatment protocols for cancers and chronic inflammation.

Although abscopal tumor regression remains a rare phenomenon, interest in exploiting how radiation stimulates the immune system to induce systemic abscopal response is increasing. Here, we tested the hypothesis that tumor immunogenicity determined the ability of radiotherapy to induce abscopal effects. We established highly (MC-38 and E.G7-OVA) or poorly (LL/2 and B16-F10) immunogenic tumor models in this study and treated them with sham radiation, a single dose of 15 Gy, or three fractions of 5 Gy on three consecutive days. Alterations in the tumor microenvironment after radiation were examined by flow cytometry and RNA sequencing. Our results demonstrated the positive correlation between tumor immunogenicity and the abscopal effect of radiotherapy. The single dose of 15 Gy radiation was an effective regimen for inducing abscopal effects in highly immunogenic tumors. Local radiation reshaped the tumor microenvironment of irradiated and non-irradiated distant tumors by increasing CD8 T-cell infiltration and reducing suppressive immune cell accumulation. However, radiation alone was insufficient to elicit abscopal effects in poorly immunogenic tumors. No significant alterations were detected in the non-irradiated distant tumor microenvironment after radiation of poorly immunogenic tumors. In addition, tumor immunogenic subtypes were associated with the radiological response and clinical outcome of patients receiving radiotherapy. These findings indicated that tumor immunogenicity was the dominant characteristic that could predict the abscopal effect of radiotherapy. Our study provides an in-depth understanding of the immunological mechanisms involved in abscopal effects and highlights the impact of tumor heterogeneity on the therapeutic efficacy of radiotherapy and their combination with immunotherapy in clinical trials.

Although immune checkpoint and targeted therapies offer remarkable benefits for lung cancer treatment, some patients do not qualify for these regimens or do not exhibit consistent benefit. Provided that lung cancer appears to be driven by transforming growth factor beta signaling, we investigated the single drug potency of Pirfenidone, an approved drug for the treatment of lung fibrosis. Five human lung cancer cell lines and one murine line were investigated for transforming growth factor beta inhibition via Pirfenidone by using flow cytometry, In-Cell western analysis, proliferation assays as well as comprehensive analyses of the transcriptome with subsequent bioinformatics analysis. Overall, Pirfenidone induced cell cycle arrest, down-regulated SMAD expression and reduced proliferation in lung cancer. Furthermore, cell stress pathways and pro-apoptotic signaling may be mediated by reduced expression of Survivin. A murine subcutaneous model was used to assess the in vivo drug efficacy of Pirfenidone and showed reduced tumor growth and increased infiltration of T cells and NK cells. This data warrant further clinical evaluation of Pirfenidone with advanced non-small cell lung cancer. The observed in vitro and in vivo effects point to a substantial benefit for using Pirfenidone to reactivate the local immune response and possible application in conjunction with current immunotherapies.

In this study, we investigate the use of multifunctional smart radiotherapy biomaterials (SRBs) loaded with immunoadjuvants for boosting the abscopal effect of local radiotherapy (RT). SRBs were designed similar to currently used inert RT biomaterials, incorporating a biodegradable polymer with reservoir for loading payloads of the immunoadjuvant anti-CD40 monoclonal antibody. Lung (LLC1) tumors were generated both on the right and left flank of each mouse, with the left tumor representing metastasis. The mice were randomized and divided into eight cohorts with four cohorts receiving image-guided RT (IGRT) at 5 Gy and another similar four cohorts at 0 Gy. IGRT and Computed Tomography (CT) imaging were performed using a small animal radiation research platform (SARRP). Tumor volume measurements for both flank tumors and animal survival was assessed over 25 weeks. Tumor volume measurements showed significantly enhanced inhibition in growth for the right flank tumors of mice in the cohort treated with SRBs loaded with CD40 mAbs and IGRT. Results also suggest that the use of polymeric SRBs with CD40 mAbs without RT could generate an immune response, consistent with previous studies showing such response when using anti-CD40. Overall, 60% of mice treated with SRBs showed complete tumor regression during the observation period, compared to 10% for cohorts administered with anti-CD40 mAbs, but no SRB. Complete tumor regression was not observed in any other cohorts. The findings justify more studies varying RT doses and quantifying the immune-cell populations involved when using SRBs. Such SRBs could be developed to replace currently used RT biomaterials, allowing not only for geometric accuracy during RT, but also for extending RT to the treatment of metastatic lesions.

Tumor vessels with resistance to anti-angiogenic therapy are characterized by the normalization of the vascular structures through integration of mature pericytes and smooth muscle cells (SMC) into the vessel wall, a process termed vessel stabilization. Unfortunately, stabilization-associated vascular remodeling can result in reduced sensitivity to subsequent anti-angiogenic therapy. We show here that blockade of VEGF by bevacizumab induces stabilization of angiogenic tumor blood vessels in human tumor specimen by recruiting Nestin-positive cells, whereas mature vessels down-regulated Nestin-expression. Using xenograft tumors growing on bone-marrow (BM) chimera of C57Bl/6 wildtype and Nestin-GFP transgenic mice, we show for first time that Nestin(+) cells inducing the maturation of tumor vessels do not originate from the BM but presumably reside within the adventitia of adult blood vessels. Complementary ex vivo experiments using explants of murine aortas revealed that Nestin(+) multipotent stem cells (MPSCs) are mobilized from their niche and differentiated into pericytes and SMC through the influence of tumor-cell-secreted factors. We conclude that tissue-resident Nestin(+) cells are more relevant than BM-derived cells for vessel stabilization and therefore have to be considered in future strategies for anti-angiogenic therapy. The identification of proteins mediating recruitment or differentiation of local Nestin(+) cells with potential stem cell character to angiogenic blood vessels may allow the definition of new therapeutic targets to reduce tumor resistance against anti-angiogenic drugs.

Neem Leaf Glycoprotein (NLGP) is a natural immunomodulator, have shown sustained tumor growth restriction as well as angiogenic normalization chiefly by activating CD8+ T cells. Here, we have investigated the direct role of NLGP as a regulator of tumor microenvironmental hypoxia and associated vascular endothelial growth factor (VEGF) production. We observed a significant reduction in VEGF level in both in vivo murine tumor and in vitro cancer cells (B16Mel, LLC) and macrophages after NLGP treatment. Interestingly, NLGP mediated VEGF downregulation in tumor cells or macrophages within hypoxic chamber was found at an early 4 h and again at late 24 h in mRNA level. Our data suggested that NLGP prevented hypoxia-induced strong binding of HIF1α with its co-factors, CBP/p300 and Sp3, but not with Sp1, which eventually limit the binding of HIF1α-transcriptional complex to hypoxia responsive element of VEGF promoter and results in restricted early VEGF transcription. On the otherhand, suppressed phosphorylation of Stat3 by NLGP results reduction of HIF1α at 24 h of hypoxia that further support sustained VEGF down-regulation. However, NLGP fails to regulate VHL activity as observed by both in vivo and in vitro studies. Therefore, this study for the first time reveals a mechanistic insight of NLGP mediated inhibition of angiogenesis by suppressing VEGF, which might help in vascular normalization to influence better drug delivery.

Background: Physical exercise is suspected to reduce cancer risk and mortality. So far, little is known about the underlying mechanisms. Although limited, murine models represent a promising attempt in order to gain knowledge in this field. Objective: A systematic review and meta-analysis examining various treatment protocols was conducted in order to determine the impact of exercise on tumor growth in rodents. Methods: PubMed, Google scholar and System for information on Gray literature in Europe were screened from inception to October 2017. Risk of bias within individual studies was assessed using the Office of Health Assessment and Translation risk of bias rating tool for human and animal trials. The effect of exercise on tumor growth over and above non-exercise control was pooled using random-effects model. Subgroup analyses were conducted to identify potential moderators. Results: The quality of the included 17 articles ranged between "probably low" and "high risk of bias." A significant reduction in tumor growth in exercising animals compared to controls was detected (Hedges' g = -0.40; 95% CI -0.66 to -0.14, p < 0.01) with between-study heterogeneity (τ2 = 0.217, I 2 = 70.28%, p < 0.001). The heterogeneity was partially explained by three moderators representing the in-between group differences of "maximum daily exercise" R 2 = 33% (p < 0.01), "type of cancer administration" R 2 = 28% (p < 0.05), and "training initiation" R 2 = 27% (p < 0.05). Conclusion: This meta-analysis suggests that physical exercise leads to reduction of tumor size in rodents. Since "maximum daily exercise" was found to have at least modest impact on tumor growth, more clinical trials investigating dose-response relationships are needed.