Growth Of Lewis Lung Carcinoma Research Articles

Influence of Polysaccharide from Helianthus tuberosus L. on Antiproliferative Activity of N-Glycoside Indolo[2,3-a]carbazole Derivative LCS-1269

The Lewis model of epidermoid carcinoma that developed in the lungs of F1(C57Bl/6 × DBA/2) hybrid mice has been used while investigating antitumor activity of the N-glycoside derivative indolo[2,3-a]carbazole (LCS-1269) with a polysaccharide from Helianthus tuberosus L. as an adjuvant agent. The antitumor effect of LCS-1269 together with the polysaccharide was evaluated by the inhibition of tumor growth in treated ani mals in comparison to the control group. As a result, it was found that combination of LCS-1269 with the polysaccharide from Helianthus tuberosus L. provided more pronounced therapeutic and longer-lasting effect than monotherapy showing a 53–64% decrease in growth of Lewis lung carcinoma for up to a 28-day obser vation period. Polysaccharide supplementation led to an increase in the number of blood cells leukocytes, lymphocytes and phagocytes responsible for antitumor immunity. The chemotherapy in combination with LCS-1269 and polysaccharide had a pronounced sustained antitumor effect on Lewis lung carcinoma in the peripheral blood system of mice in presence of a temporarily increased level of neutrophils and monocytes by the 12th day of tumor development. Apparently, the tested compounds stimulated proliferation of neutrophils and monocytes of certain phenotypes with antitumor activity at an earlier stage of Lewis lung carcinoma de velopment.

Structural Elucidation and Anti-Tumor Activity of a Polysaccharide (CP2-S) from Cordyceps militaris Fruit Bodies.

A polysaccharide (CP2-S), consisting of glucose with a weight average molecular weight of 5.9 × 106, was purified from the fruit bodies of Cordyceps militaris. In this work, the corresponding structure and anti-tumor activity in vivo were investigated. Methylation and NMR analysis revealed that CP2-S was composed of a →4)-α-D-Glcp-(1→ backbone with partial substitution occurring at O-6 by T-linked α-D-Glcp in every ten residues, which has not been reported in previous reports. In vivo anti-tumor experiments showed that CP2-S could inhibit the growth of Lewis lung carcinoma in mice. Tumor inhibition rates were 17.8%, 24.5%, and 29.5% at dosages of 12.5, 50, and 100 mg/kg/d, respectively. Compared with the cisplatin group, mice treated with CP2-S exhibited a significant increase in spleen index (increased 22.7-42.4%) and thymus index (increased 47.7-36.8%). Additionally, serum levels of IgM and IgG in tumor-bearing mice increased by approximately 6.11~10.75-folds and 1.31~1.38-folds, respectively. These findings prove that CP2-S significantly inhibited the growth of Lewis lung carcinoma through immune-enhancing activity in mice.

Study of lithium carbonate and ascorbate proliferative properties on transplantable Lewis lung carcinoma metastasis model

Objective: to study the antitumor effects of organic lithium salt (lithium ascorbate) in different doses in comparison with inorganic lithium salt (carbonate).Material and methods. Two series of experiments were carried out on the effect of lithium preparations on the dynamics of transplantable Lewis lung carcinoma (LLC) growth and metastasis in F1 mice (CBA × C57Bl/6j). In the first series, a comparative study of the effects of different lithium ascorbate doses (1 and 10 mg/kg/day based on elemental lithium) was performed, and in the second series, a comparison was made of the effects of lithium ascorbate and carbonate when used at the same dose (5 mg/kg/day).Results. Significant antitumor effects were found for lithium ascorbate lower doses (1 and 5 mg/kg/day). A statistically significant antitumor effect of lithium ascorbate was observed from Day 10 throughout the entire observation period (tumor growth inhibition index (TGII) 30–40%). The antitumor effect of lithium carbonate in this experiment was less pronounced and stable (TGII 20–30%). No antimetastatic effect was observed with both preparations.Conclusion. Subchronic intragastric administration of lithium ascorbate and carbonate to tumor-bearing animals at a daily dose of 5 mg/kg, an antitumor effect is observed, manifested by LLC growth inhibition. Effective and safe antitumor doses of lithium ascorbate are in the range of 1–5 mg/kg.

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.

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.

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.

Cancer aggravation due to persistent pain signals with the increased expression of pain-related mediators in sensory neurons of tumor-bearing mice

A growing body of evidence suggests that intractable pain reduces both the quality of life and survival in cancer patients. In the present study, we evaluated whether chronic pain stimuli could directly affect cancer pathology using tumor-bearing mice. For this purpose, we used two different models of chronic pain in mice, neuropathic pain and persistent postsurgical pain, with Lewis lung carcinoma (LLC) as tumor cells. We found that tumor growth was dramatically promoted in these pain models. As well as these pain models, tumor growth of LLC, severe osteosarcoma (AXT) and B16 melanoma cells was significantly promoted by concomitant activation of sensory neurons in AAV6-hM3Dq-injected mice treated with the designer drug clozapine-N-oxide (CNO). Significant increases in mRNA levels of vascular endothelial growth factor-A (Vegfa), tachykinin precursor 1 (Tac1) and calcitonin-related polypeptide alpha (Calca) in the ipsilateral side of dorsal root ganglion of AAV6-hM3Dq-injected mice were observed by concomitant activation of sensory neurons due to CNO administration. Moreover, in a model of bone cancer pain in which mice were implanted with AXT cells into the right femoral bone marrow cavity, the survival period was significantly prolonged by repeated inhibition of sensory neurons of AAV6-hM4Di-injected mice by CNO administration. These findings suggest that persistent pain signals may promote tumor growth by the increased expression of sensory-located peptides and growth factors, and controlling cancer pain may prolong cancer survival.

Influence of induced diabetes mellitus on hormonal profile of Lewis lung carcinoma in BALB/c Nude mice

Purpose of the study. The assessment of diabetes mellitus (DM) effect on levels of sex hormones in tumor and peritumoral tissues in BALB/c Nude mice with Lewis lung carcinoma (LLC).Materials and methods. The study included 42 male and female BALB/c Nude mice aged 8–9 weeks weighing 21–22 g. Alloxan-induced DM was reproduced in mice of the main group, and then LLC was transplanted. Levels of estrone (E1), estradiol (E2), testosterone (T), progesterone (P4) and prolactin (PRL), as well as steroid hormone receptors: estrogens (REα, REβ), androgens (RA), and progesterone (RP4) were measured by RIA and ELISA in samples of tumor and peritumoral tissues. Animals with LLC without DM were used as controls. The statistical analysis was performed using the Statistica 10 program; differences were considered significant at p &lt; 0.05.Results. DM in males was reproduced only after a double injection of alloxan, and was characterized by lower blood glucose levels compared to females. The growth of LLC in animals with alloxan-induced DM was possible only in female BALB/c Nude mice; in BALB/c Nude males, the tumor could not be transplanted either independently or in combination with DM. Females in the main group showed greater average tumor volumes throughout the experiment and reduced survival, compared to the control group. Tumor samples from females with LLC+DM were more saturated with sex steroids, but depleted in steroid hormone receptors, which probably contributed to the ability to avoid the body's regulatory signals.Conclusion. The growth of LLC in presence of induced DM was sex-dependent, since the tumor could not be transplanted to male mice. DM affected the levels of sex steroids and their receptors tumor tissues in female BALB/c Nude mice.

Design, synthesis and biological evaluation of a novel colchicine-magnolol hybrid for inhibiting the growth of Lewis lung carcinoma in Vitro and in Vivo.

Colchicine is a bioactive alkaloid originally from Colchicum autumnale and possesses excellent antiproliferative activity. However, colchicine-associated severe toxicity, gastrointestinal side effects in particular, limits its further therapeutic use. In the current study, we thus designed and synthesized a novel hybrid (CMH) by splicing colchicine and magnolol, a multifunctional polyphenol showing favorable gastrointestinal protection. The antitumor activity of CMH in Lewis lung carcinoma (LLC) was then evaluated in vitro and in vivo. Biologically, CMH inhibited the growth of LLC cells with an IC50 of 0.26μM, 100 times more potently than cisplatin (26.05μM) did. Meanwhile, the cytotoxicity of CMH was 10-fold lower than that of colchicine in normal human lung cells (BEAS-2B). In C57BL/6 mice xenograft model, CMH (0.5mg/kg) worked as efficacious as colchicine (0.5mg/kg) to inhibit tumor growth and 2 times more potently than cisplatin (1mg/kg). In terms of mortality, 7 out of 10 mice died in colchicine group (0.75mg/kg), while no death was observed in groups receiving CMH or cisplatin at 0.75mg/kg. Mechanistic studies using Western blot revealed that CMH dose-dependently suppressed the protein expression of phosphorylated ERK. Molecular docking analysis further indicated that CMH was well fitted in the colchicine binding site of tubulin and formed several hydrogen bonds with tubulin protein. These results enable our novel hybrid CMH as a potential antineoplastic agent with lower toxicity, and provide perquisites for further investigation to confirm the therapeutic potentiality of this novel hybrid.

Role of hypothalamic-pituitary-adrenal axis and catecholamine factors in independent and primary multiple types of tumor growth

Purpose of the study. To study the levels of adrenal axis factors in the hypothalamus, adrenal glands, blood serum of mice and catecholamines in the adrenal glands during the independent growth of B16 melanoma and Lewis lung carcinoma (LLC) and their combined growth in female mice, and in males – with independent growth of B16 and combined growth of B16 and LLC.Materials and methods. Male and female BALB/c Nude mice were divided into groups, n = 7 each: group 1 involved intact animals, group 2 involved mice with B16/F10 melanoma, group 3 – mice with LLC, group 4 – synchronous growth of melanoma and LLC. Levels of corticotropin releasing, noradrenaline and dopamine were determined in homogenates of the hypothalamus and adrenal glands and in the blood serum of all animals by ELISA, and levels of 17‑hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulfate (DHEA-S) and cortisol were determined by RIA. Statistical processing of results was performed using the Statistica 10.0 program.Results. All tumor-bearing females showed elevated corticotropin releasing in the hypothalamus together with an increase of all stress-characterizing parameters: cortisol, the cortisol/DHEA-S ratio, and noradrenaline. However, an increase in serum levels of cortisol was blocked by high levels of DHEA-S, and as a result, the cortisol/DHEA-S ratio was either within the normal range (B16 melanoma and B16+LLC combination) or reduced (LLC). Levels of corticotropin releasing in the hypothalamus of tumor-bearing males decreased, together with opposite changes in stress-characterizing parameters in the adrenal glands: cortisol increased, the cortisol/DHEA-S ratio did not differ significantly from the control values, and noradrenaline decreased. An increase in serum levels of cortisol was not blocked by high levels of DHEA-S, and as a result, the cortisol/DHEA-S ratio was sharply elevated in B16 melanoma and B16+LLC combination.Conclusion. At independent and primary multiple types of tumor growth, the sex-specific features of the functioning of the adrenal axis at the central and peripheral levels are observed, which determines a more pronounced stressful state of the body with B16+LLC combination growth, realized by various mechanisms.

Combination of STING agonist and CXCR3 antagonist disrupts immune tolerance to overcome anti-PD-L1 resistance in lung adenocarcinoma under oxidative stress

We investigated the role of the STING1–CXCR3 axis using database data and verified it in a mouse model bearing Lewis lung carcinoma (LLC) cells exposed to hydrogen peroxide (H2O2). Mice were treated with STING agonist liposomes (STING-Lip), anti-programmed death-ligand 1 (PD-L1), or STING-Lip + anti-PD-L1. The database data revealed that immune response pathways were enriched in patients with lung adenocarcinoma with upregulated STING1 signaling. Upregulated STING1 signaling was associated with a high abundance of immunoregulatory and effector molecules, cytokines, activated CD8+ T cells, and M1 macrophages in patients with lung adenocarcinoma. In this study, H2O2-treated LLC cells promoted an immunosuppressive microenvironment and enhanced tumor growth in mice. STING-Lip inhibited distant, untreated, and H2O2-induced LLC growth by activating systemic immunity. STING-Lip + anti-PD-L1 failed to slow distant and untreated LLC growth, whereas STING-Lip + anti-PD-L1 + CXCR3 antagonist inhibited distant tumor growth in mice. The combination of STING1 activation and CXCR3 inhibition may be a novel immunotherapeutic strategy to overcome immune checkpoint inhibitor resistance in lung adenocarcinoma by activating systemic immunity in the tumor microenvironment under oxidative stress.

Loss of apelin blocks the emergence of sprouting angiogenesis in experimental tumors.

Angiogenesis inhibitor drugs targeting vascular endothelial growth factor (VEGF) signaling to the endothelial cell (EC) are used to treat various cancer types. However, primary or secondary resistance to therapy is common. Clinical and pre-clinical studies suggest that alternative pro-angiogenic factors are upregulated after VEGF pathway inhibition. Therefore, identification of alternative pro-angiogenic pathway(s) is critical for the development of more effective anti-angiogenic therapy. Here we study the role of apelin as a pro-angiogenic G-protein-coupled receptor ligand in tumor growth and angiogenesis. We found that loss of apelin in mice delayed the primary tumor growth of Lewis lung carcinoma 1 and B16F10 melanoma when combined with the VEGF receptor tyrosine kinase inhibitor, sunitinib. Targeting apelin in combination with sunitinib markedly reduced the tumor vessel density, and decreased microvessel remodeling. Apelin loss reduced angiogenic sprouting and tip cell marker gene expression in comparison to the sunitinib-alone-treated mice. Single-cell RNA sequencing of tumor EC demonstrated that the loss of apelin prevented EC tip cell differentiation. Thus, apelin is a potent pro-angiogenic cue that supports initiation of tumor neovascularization. Together, our data suggest that targeting apelin may be useful as adjuvant therapy in combination with VEGF signaling inhibition to inhibit the growth of advanced tumors.

Resveratrol and Its Analogue 4,4'-Dihydroxy-trans-stilbene Inhibit Lewis Lung Carcinoma Growth In Vivo through Apoptosis, Autophagy and Modulation of the Tumour Microenvironment in a Murine Model.

Lung cancer is the most prevalent cancer worldwide. Despite advances in surgery and immune-chemotherapy, the therapeutic outcome remains poor. In recent years, the anticancer properties of natural compounds, along with their low toxic side effects, have attracted the interest of researchers. Resveratrol (RSV) and many of its derivatives received particular attention for their beneficial bioactivity. Here we studied the activity of RSV and of its analogue 4,4′-dihydroxystilbene (DHS) in C57BL/6J mice bearing cancers resulting from Lung Lewis Carcinoma (LLC) cell implantation, considering tumour mass weight, angiogenesis, cell proliferation and death, autophagy, as well as characterization of their immune microenvironment, including infiltrating cancer-associated fibroblasts (CAFs). C57BL/6J mice started treatment with RSV or DHS, solubilised in drinking water, one week before LLC implantation, and continued for 21 days, at the end of which they were sacrificed, and the tumour masses collected. Histology was performed according to standard procedures; angiogenesis, cell proliferation and death, autophagy, infiltrating-immune cells, macrophages and fibroblasts were assessed by immunodetection assays. Both stilbenic compounds were able to contrast the tumour growth by increasing apoptosis and autophagy in LLC tumour masses. Additionally, they contrasted the tumour-permissive microenvironment by limiting the infiltration of tumour-associated immune-cells and, more importantly, by counteracting CAF maturation. Therefore, both stilbenes could be employed to synergise with conventional oncotherapies to limit the contribution of stromal cells in tumour growth.

Immunonutritional Targeting of Cancer via Eicosanoids

Cancer causes widespread mortality via a systemic pro‐inflammatory and dysregulated immune response. Immunotherapy is the front‐line treatment in many cancers with a response rate of approximately 10–30%, although it remains unclear why the response rate is so diverse. Dietary intervention provides an opportunity to optimize the host immune response in cancer patients, however, mechanisms of immunonutritional regulation in cancer are poorly understood. Key components of immune surveillance include the action of cytotoxic T‐lymphocyte‐associated protein‐4 (CTLA4), as certain cancers can utilize CTLA4 to evade immune surveillance. Thus, several monoclonal antibodies targeting CTLA4 are FDA approved. Epidemiological and experimental studies provide evidence that omega‐3 polyunsaturated fatty acids (PUFAs) are beneficial for reducing the risk of cancer, whereas omega‐6 PUFAs can stimulate cancer. Cytochrome P450 epoxygenases represent one branch of PUFA metabolism, generating biologically active omega‐3 epoxy fatty acids (omega‐3 epoxides). Cytochrome P450 epoxygenases convert the omega‐6 PUFA arachidonic acid into epoxyeicosatrienoic acids, which suppress inflammation. Because the half‐life of fatty acid epoxides is rapid, drugs that stabilize their levels by inhibiting soluble epoxide hydrolase (sEHi) are in clinical trials for hyperinflammatory diseases. sEHi may synergize with omega‐3 fatty acids in reducing inflammation and cancer. Since immune checkpoint inhibitors (e.g. CTLA4) can augment cytotoxic T cell responses, we hypothesized that omega‐3 fatty acid supplementation in conjunction with sEHi would enhance immunotherapy in various cancer types. Mice were fed with standard diet, high omega‐6 diet, or high omega‐3 diet for 10 days prior to tumor inoculation and for the duration of the studies. One week following tumor injection, mice were randomized into treatment groups: control, sEHi TPPU (5 mg/kg/day via drinking water), immunotherapy (anti‐CTLA4; 200 µg initial dose, 100 µg Q3D), and TPPU + anti‐CTLA4. Here, we demonstrate in poorly immune responsive tumors (Lewis lung carcinoma (LLC) and B16F10 melanoma) that oral administration of TPPU enhances immunotherapy in mice on a high omega‐3 fatty acid diet to transform “cold” unresponsive tumors into “hot” responsive tumors. Indeed, the combination of TPPU and anti‐CTLA4 resulted in synergistic antitumor activity in mice on the high omega‐3 diet, with 52‐67% inhibition in LLC and B16F10 melanoma compared to control. Anti‐CTLA4 inhibited LLC primary tumor growth in mice on an omega‐3 fatty acid diet. In marked contrast, immune checkpoint blockade (anti‐CTLA4) accelerated LLC growth in mice on an omega‐6 fatty acid diet by 225% compared to control after 15 days of treatment. sEHis alone or in combination with an increased omega‐3 PUFA/omega‐6 PUFA dietary ratio may be a promising new approach to enhance immune checkpoint blockade in cancer. Together, these findings identify omega‐3 epoxides as important regulators of immunotherapy and sEH as a druggable target in cancer.

Differential action of pro-angiogenic and anti-angiogenic components of Danhong injection in ischemic vascular disease or tumor models

ObjectiveWe investigate the chemical basis and mechanism of angiogenesis regulation by a multicomponent Chinese medicine Danhong injection (DHI).MethodsDHI was fractionated and screened for angiogenesis activities by in vitro tube formation and migration assays. The composition of DHI components was determined by UPLC. The effects of the main active monomers on angiogenesis-related gene and protein expression in endothelial cells were determined by qPCR and Western blotting analyses. Mouse hind limb ischemia and tumor implant models were used to verify the angiogenesis effects in vivo by Laser Doppler and bioluminescent imaging, respectively.ResultsTwo distinct chemical components, one promoting (pro-angiogenic, PAC) and the other inhibiting (anti-angiogenic, AAC) angiogenesis, were identified in DHI. PAC enhanced angiogenesis and improved recovery of ischemic limb perfusion while AAC reduced Lewis lung carcinoma growth in vivo in VEGFR-2-Luc mice. Among the PAC or AAC monomers, caffeic acid and rosmarinic acid upregulated TSP1 expression and downregulated KDR and PECAM expression. Caffeic acid and rosmarinic acid significantly decreased while protocatechuic aldehyde increased CXCR4 expression, which are consistent with their differential effects on EC migration.ConclusionsDHI is capable of bi-directional regulation of angiogenesis in disease-specific manner. The pro-angiogenesis activity of DHI promotes the repair of ischemic vascular injury, whereas the anti-angiogenesis activity inhibits tumor growth. The active pro- and anti-angiogenesis activities are composed of unique chemical combinations that differentially regulate angiogenesis-related gene networks.

Environmental Enrichment Mitigates Age-Related Metabolic Decline and Lewis Lung Carcinoma Growth in Aged Female Mice.

Aging is a complex physiological process that leads to the progressive decline of metabolic and immune function, among other biological mechanisms. As global life expectancy increases, it is important to understand determinants of healthy aging-including environmental and genetic factors-and thus slow the onset or progression of age-related disease. Environmental enrichment (EE) is a housing environment wherein laboratory animals engage with complex physical and social stimulation. EE is a prime model to understand environmental influences on aging dynamics, as it confers an antiobesity and anticancer phenotype that has been implicated in healthy aging and health span extension. Although EE is frequently used to study malignancies in young mice, fewer studies characterize EE-cancer outcomes in older mice. Here, we used young (3-month-old) and aged (14-month-old) female C57BL/6 mice to determine whether EE would be able to mitigate age-related deficiencies in metabolic function and thus alter Lewis lung carcinoma (LLC) growth. Overall, EE improved metabolic function, resulting in reduced fat mass, increased lean mass, and improved glycemic processing; many of these effects were stronger in the aged cohort than in the young cohort, indicating an age-driven effect on metabolic responses. In the aged-EE cohort, subcutaneously implanted LLC tumor growth was inhibited and tumors exhibited alterations in various markers of apoptosis, proliferation, angiogenesis, inflammation, and malignancy. These results validate EE as an anticancer model in aged mice and underscore the importance of understanding environmental influences on cancer malignancy in aged populations. PREVENTION RELEVANCE: Environmental enrichment (EE) serves as a model of complex physical and social stimulation. This study validates EE as an anticancer intervention paradigm in aged mice and underscores the importance of understanding environmental influences on cancer malignancy in aged populations.

Abstract 2588: Oral administration of water extract from Euglena gracilis prevents lung carcinoma growth in mice by alteration of intestinal microbiota

Abstract Euglena gracilis, a single-celled alga, is rich in nutrients and thus, used as a nutritional dietary supplement. This alga can be found in both fresh and saltwater and possesses characteristics of both plants and animals. Euglena gracilis extracts have a wide range of medicinal properties including stimulation of anticancer immunity against multiple types of cancers; however, the anticancer mechanism has not yet been fully elucidated. Therefore, a water extract from Euglena gracilis devoid of water-insoluble mature paramylons was evaluated as an anticancer agent against lung carcinoma. Two different extracts were prepared using dried powder from whole Euglena gracilis. First, partially purified water extract (EWE) was prepared by suspending dry powder in PBS and centrifugation at 10,000g for 20 min followed by a filtration using a 0.22µm pore size membrane. Second, boiled EWE (bEWE) was prepared by immersing unfiltered EWE in boiling water for 12 min followed by centrifugation at 10,000g for 10 min and filtration using 0.22µm pore size membrane. Both EWE and bEWE treatments inhibited the growth of lung carcinoma cells in vitro in a dose- and time-dependent manner. Furthermore, both extracts significantly inhibited the three-dimensional growth of Lewis Lung Carcinoma (LLC) cells. Flow cytometry analysis showed that the EWE treatment attenuates granulocytic myeloid-derived suppressor cells (MDSCs) in bone marrow cell cultures. The in vivo study was conducted using a mouse LLC orthotopic allograft model. Oral administration of EWE and bEWE (100-200 mg/kg/day) three weeks prior to LLC cell inoculation attenuated the tumor growth in the lungs of immunocompetent mice while decreasing the peripheral granulocytes. However, this attenuation was not seen for the extract treatment initiated after LLC cell inoculation. The tumor growth attenuation was more efficient with the bEWE treatment than with EWE treatment. The fecal microbiomes of the mice were analyzed by 16s rRNA gene amplicon sequencing which revealed that alpha diversity in three groups (EWE, bEWE, and PBS control) was similar, however, the microbial compositions of the EWE- and bEWE-treated mouse groups were more diversified than in the PBS group. Specifically, an increase in the ratio of Bacteroidetes to Firmicutes was observed, and a significant increase in Akkermansia and Muribaculum was detected in EWE- and bEWE- treated mice compared to PBS treated mice. These studies suggest that oral administration of partially purified water extracts from Euglena gracilis altered the intestinal microbiome and the alteration may attenuate host MDSCs, thereby preventing lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Deepa Upreti, Susumu Ishiguro, Mayme Loyd, Nicole Robben, Paige Cote, Morgan Phillips, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Oral administration of water extract from Euglena gracilis prevents lung carcinoma growth in mice by alteration of intestinal microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2588.

Role of tumor/endothelial cell interactions in tumor growth and metastasis.

It is known that interactions between tumor and endothelial cells have a significant influence on the growth and metastasis of malignant tumors. To study the reciprocal effect of Lewis lung carcinoma (LLC) and endothelial cells on the growth rate of each other upon their co-cultivation in vitro and to assess the contribution of such tumor/endothelial cell crosstalk to in vivo LLC growth and metastasis. Two variants of Lewis lung carcinoma cells, high-metastatic (LLC) and low-metastatic (LLC/R9), and murine aorta endothelial cell line (MAEC) were used. Kinetics of tumor cell growth in vitro and in vivo, electrokinetic properties of tumor cells and their adhesion to endothelial monolayer, and the number of tumor and endothelial viable cells after 1-day contact or non-contact co-cultivation were estimated. LLC/R9 had significantly higher growth rate in vivo (as opposed to in vitro) than LLC. However, the number and volume of lung metastatic lesions in LLC/R9-bearing mice were 4.5-fold (p < 0.05) and 3.6-fold lower (p < 0.05), respectively, compared to those in LLC-bearing mice. Non-contact co-cultivation of LLC/R9 + MAEC caused more than a 34% (p < 0.05) LLC/R9-induced increase in the number of MAEC and a 60% (p < 0.05) MAEC-induced increase in the number of LLC/R9 cells as compared to those of corresponding controls (cells cultured alone). In contrast, in the case of LLC + MAEC, both the number of LLC and MAEC cells after their non-contact co-cultivation and cultivation alone did not differ significantly. Contact co-cultivation LLC+MAEC (in contrast to LLC/R9+MAEC) caused more than a 50% (p < 0.01) LLC-induced decrease in the number of MAEC and a 50% decrease (p < 0.05) MAEC-induced in the number of LLC cells as compared to the corresponding controls. Both tumor cell variants showed a bimodal distribution of cells by ζ-potential, but in the case of LLC there was observed a shift towards high values due to 52% of cells with a surface charge density > 10 C/m2, while in the case of LLC/R9 such a subpopulation was absent and 19% of cells had a surface charge < 5 C/m2. The number of LLC cells that adhered to the monolayer of endothelial cells was by 65% (p < 0.05) higher than that of LLC/R9 cells. Obtained data demonstrated that the tumor/endothelial cell relationships might reflect the features of tumor growth and metastasis of a malignant tumor.

Macrophage polarization in dynamics of Lewis lung carcinoma growth and metastasis.

To assess the functional state of macrophages based on various manifestations of their activity at the different stages of metastatic tumor growth in C57Bl mice. On days 7, 14, 21and 28after Lewis lung carcinoma transplantation to C57Bl mice, macrophages from various anatomic sites were isolated and tested on their cytotoxicity, metabolic activity, NO production and arginase activity. In the populations of peritoneal and splenic macrophages, on days 7and 21of tumor growth antitumor (M1) cells prevailed while on days 14and 28tumor-promoting (M2) macrophages predominated. In the population of lung macrophages, cells with M1phenotype were in the majority in the early stages of tumor growth. On days 21and 28, M1cells were gradually substituted by cells exhibiting M2phenotype. This shift correlated with metastasis to lungs. Lewis lung carcinoma growth is accompanied by the gradual change in macrophage polarization from antitumor (M1) towards tumor-promoting (M2) type. These changes were more evident in population of lung macrophages and correlated with the parameters of metastasis.

Traditional Chinese Medicine Xihuang Wan Inhibited Lewis Lung Carcinoma in a Syngeneic Model, Equivalent to Cytotoxic Chemotherapy, by Altering Multiple Signaling Pathways.

Xihuang Wan (XHW), a traditional Chinese medicine (TCM), has been used in China for a variety of cancers including lung cancer. The present study evaluated the efficacy of XHW on a Lewis lung mouse model and explored the potential mechanism via transcriptomics. The mice were randomized into 6 groups: 1) untreated control (n=10); 2) low-dose XHW; 3) medium-dose XHW; 4) high-dose XHW; 5) cisplatin; and 6) untreated blank (n=4). Lewis lung carcinoma (LLC) cells were injected subcutaneously except for the 4 mice in the blank group. The body weight and tumor length and width were measured every 3 days. RNA-sequencing was performed on tumors in the high-dose XHW group and the control group. XHW inhibited the growth of LLC in a syngeneic mouse model, without toxicity, with equivalent efficacy to cisplatin. RNA-sequencing demonstrated that many signaling pathways were involved in XHW-mediated inhibition of LLC, including tumor necrosis factor, estrogen, cyclic guanosine 3', 5'-monophosphate-protein kinase G, apelin and the peroxisome proliferator-activated receptor signaling pathways. XHW inhibited LLC carcinoma through different pathways and shows clinical promise for patients who cannot tolerate platinum-based drugs.