B16F10 Melanoma Tumor Growth Research Articles

Dendritic Cell-Targeted Nanoparticles Enhance T Cell Activation and Antitumor Immune Responses by Boosting Antigen Presentation and Blocking PD-L1 Pathways.

Dendritic cells (DCs) within the tumor microenvironment (TME) have an insufficient capacity to activate T cells through antigen presentation. Furthermore, the programmed cell-death ligand 1 (PD-L1), abundantly expressed on tumor-associated DCs, binds the programmed cell-death 1 (PD-1)-positive T cells and suppresses their immune function. The binding of PD-L1 to CD80 (B7.1) on the same DC via cis-interactions further prevents T cell costimulation through CD28. Here, we present a strategy to simultaneously promote antigen cross-presentation and block the inhibitory interactions of PD-L1 on DCs to amplify T cell-mediated antitumor responses within the TME. Mesoporous silica nanoparticles (MSNPs) were loaded with clotrimazole (CLT) to boost MHC II-mediated antigen presentation by DCs, surface-modified with mannose to target CD206 on DCs, and then decorated with PD-L1 binding peptide (PDL1bp) to block PD-L1-mediated interactions. PDL1bp was cleaved from the mannosylated and CLT-loaded MSNPs (MSNP-MaN/CLT) under conditions simulating the TME and tethered to PD-L1 to reverse CD80 sequestration on DC2.4 cells. The blocking of PD-L1 by PDL1bp-decorated NPs (MSNP-MaN-PDL1bp) increased the cellular interactions between DC2.4 and EL4 T cells and the amount of IL-2 secretion. The MSNP-MaN/CLT were taken up rapidly by DC2.4 cells, promoted MHC II presentation of hen egg lysozyme (HEL), and increased IL-2 production from HEL antigen-primed 3A9 T cells, which was further enhanced by PDL1bp. In vivo investigation revealed that administration of the CLT-loaded and PDL1bp-functionalized MSNPs remarkably inhibited subcutaneous B16-F10 melanoma tumor growth when compared with anti-PD-L1 therapy. MSNP-MaN-PDL1bp/CLT treatment upregulated the levels of effector molecules such as granzyme B and proinflammatory cytokines (IFNγ and INFα) in the tumor tissue, indicating antitumoral T cell responses. This strategy of utilizing nanoparticles to trigger DC activation while promoting T cell stimulation can be used to amplify the antitumor T cell responses and represents a promising alternative to anti-PD-L1 immunotherapy.

The ω-3 Polyunsaturated Fatty Acid Docosahexaenoic Acid Enhances NK-Cell Antitumor Effector Functions.

ω-3 polyunsaturated fatty acids (PUFA) are known to directly repress tumor development and progression. In this study, we explored whether docosahexaenoic acid (DHA), a type of ω-3 PUFA, had an immunomodulatory role in inhibiting tumor growth in immunocompetent mice. The number of natural killer (NK) cells but not the number of T or B cells was decreased by DHA supplementation in various tissues under physiologic conditions. Although the frequency and number of NK cells were comparable, IFNγ production by NK cells in both the spleen and lung was increased in DHA-supplemented mice in the mouse B16F10 melanoma tumor model. Single-cell RNA sequencing revealed that DHA promoted effector function and oxidative phosphorylation in NK cells but had no obvious effects on other immune cells. Using Rag2-/- mice and NK-cell depletion by PK136 antibody injection, we demonstrated that the suppression of B16F10 melanoma tumor growth in the lung by DHA supplementation was dependent mainly on NK cells. In vitro experiments showed that DHA directly enhanced IFNγ production, CD107a expression, and mitochondrial oxidative phosphorylation (OXPHOS) activity and slightly increased proliferator-activated receptor gamma coactivator-1α (PGC-1α) protein expression in NK cells. The PGC-1α inhibitor SR-18292 in vitro and NK cell-specific knockout of PGC-1α in mice reversed the antitumor effects of DHA. In summary, our findings broaden the current knowledge on how DHA supplementation protects against cancer growth from the perspective of immunomodulation by upregulating PGC-1α signaling-mediated mitochondrial OXPHOS activity in NK cells.

Combined immunochemotherapy achieving targeted co-delivery of chlorogenic acid and doxorubicin by sialic acid-modified liposomes enhances anti-cancer efficacy.

Malignant melanoma is a high-grade aggressive skin tumor with an increasing incidence and mortality rates worldwide. Chemotherapeutic drugs such as doxorubicin have limited efficacy against melanoma due to their poor sensitivity, severe side effects, and drug resistance. Recent studies have shown that combinations of immunotherapy and chemotherapy have a synergistic effect in enhancing the anti-tumor effect. Here, we have developed liposomes co-loaded with chlorogenic acid (CA) and doxorubicin (DOX), modified with sialic acid-octadecylamine conjugate (SA-ODA), designated CA-DOX-SAL, that facilitate drug delivery by recognizing Siglec-1 receptor on TAMs. The physicochemical studies revealed the particle size and zeta potential of CA-DOX-SAL as 128.3 ± 0.8 nm and - 4.33 ± 0.50 mV, respectively. In vitro, CA-DOX-SAL demonstrated robust cellular uptake through SA receptor-mediated tumor-associated macrophages (TAM) targeting and exerted greater cytotoxicity on tumor cells. In vivo, targeted liposomes were found to accumulate in the tumor area, leading to an improvement in anti-tumor efficacy. In addition, CA-DOX-SAL effectively inhibited B16F10 melanoma tumor growth by stimulating the transition from tumor-promoting M2-type to anti-tumor M1-type and directly killing tumor cells. Overall, the co-delivery of immunomodulatory CA and chemotherapeutic DOX presents a promising therapeutic strategy to enhance clinical outcomes in the treatment of melanoma.

CD4 depletion enhances the efficacy of Super2 IL-33 armored CAR T cell against solid tumors and leads memory formation of both endogenous and CAR T cells

Abstract Chimeric antigen receptor (CAR) T cells exhibit tremendous efficacy in patients with relapsed and recurrent liquid cancers. However, CAR T cells fail to control solid tumors because of their inability to infiltrate the suppressive tumor environment and their poor persistence in vivo. The development of 4 thgeneration armored CAR T cells that employ direct tumor killing and release cytokines that can modulate the immune response to enhance tissue/tumor infiltration may improve CAR T cell persistence. Our lab has developed Super2 and IL-33 (S233) armored CAR T cells that significantly delayed B16F10 melanoma tumor growth and enhanced tumor infiltration of CARs and endogenous immune cells. Combination therapy of S233 CAR T Cells with anti-CTLA4 or anti-CD4 further enhanced this phenotype. S233 CAR T cells and anti-CD4 combination therapy promoted Melanoma-associated vitiligo (MAV), which correlates with development of tumor protective Tissue Resident Memory (TRM) cells. To assess whether CAR T cells differentiate into memory T cells, we treated tumor-bearing mice with S233 CAR T Cells with or without anti-CD4, resected their residual tumors and tracked memory T cell responses. We found that S233 CAR T cells persisted longer in the tumor draining lymph node and developed accelerated MAV when combined with anti-CD4. Both CAR T cells and endogenous T cells differentiated into central and effector memory but only endogenous T cells in the CD4 depleted group became TRM cells. While these findings provide insights on strategies to enhance long-term persistence of CAR T cells, additional studies are needed to induce CAR TRM cells and assess the durability of anti-tumor responses. NIH grant T32-AI007363 NIH grant P30-CA023108

Prosopis juliflora (Sw.) DC phytochemicals induce apoptosis and inhibit cell proliferation signaling pathways, EMT, migration, invasion, angiogenesis and stem cell markers in melanoma cell lines

Ethnopharmacological relevanceProsopis juliflora (Sw.), DC is a xerophytic plant species that extensively grow in Asia, Africa, Australia, and Brazil. From ancient time P. juliflora is being utilized in various folk remedies for example in wound healing, fever, inflammation, measles, excrescences, diarrhea and dysentery. Traditionally, gum, paste, and smoke obtained from the leaves and pods are applied for anticancer, antidiabetic, anti-inflammatory, and antimicrobial purposes. Aim of the studyOur previous studies have demonstrated the promising potential of Prosopis Juliflora leaves methanol extract (PJLME) against breast cancer, and suggested its possible integration as a complementary medicine for the effective management of breast cancer. However, evidence against how PJLME mechanistically target the cancer proliferative pathways and other targets is poorly understood. The basic aim of the present study was to understand the anti-melanoma potential of PJLME against B16f10 cells with possible mechanisms of action. Materials and methodsMTT assay was used to determine cell viability. Wound and transwell migration assay was performed to check migration potential of cells after PJLME treatment, while clonogenic assay was carried out to understand its colony inhibition actvity. Flow cytometry was used to perform annexin V/PI assay (apoptosis assay), ROS assay, cell cycle analysis. In-vitro angiogenesis assay was performed to check formation of capillary like vascular structure after PJLME treatment. Apoptotic genes, signaling pathways markers, EMT markers and stem cell markers were determined by western blotting. In-vivo BALB/C mice xenograft model study was performed to check the effect of PJLME on in-vivo melanoma tumor growth. ResultsThe experimental outcome of the present study has clearly demonstrated the inhibition of growth, migration, invasion, colony formation and apoptosis inducing potential of PJLME against mouse melanoma cancer cells. Treatment of B16F10 melanoma cells with PJLME resulted in arrest of cell cycle at G0/G1 phase. Annexin V-FITC/PI assay confirmed the apoptosis inducing potential of PJLME in B16F10 and A375 melanoma cells. Furthermore, Western blot experiments confirmed that the treatment of PJLME downregulates the expression of anti-apoptotic gene like Bcl2 and increase the expression profile of pro-apoptotic genes like Bax, Bad, and Bak in B16F10 melanoma cells. HUVEC (Human umbilical vein endothelial cells) tube formation assay clearly demonstrated the anti-angiogenic potential of PJLME. The study also revealed that PJLME has potential to inhibit the Akt and Erk signaling pathways which are participating in cancer cell proliferation, migration, invasion etc. The outcome of qRT-PCR and immunoblotting analysis clearly unveiled that PJLME treatment leads to downregulation of epithelial-mesenchymal transition (EMT) as well as stem cell markers. Finally, the in-vivo animal xenograft model study also revealed the anti-melanoma potential of PJLME by significantly inhibiting the B16F10 melanoma tumor growth in BALB/c mice model. The LC-ESI-MS/MS analysis of PJLME showed the presence of variety of bioactive molecules associated with anticancer effects. ConclusionThe outcome of the present investigation clearly demonstrated the anti-melanoma potential of PJLME against B16f10 melanoma cells. PJLME can be explored as an adjuvant or complementary therapy against melanoma cancer, however further studies are required to understand the clinical efficacy of PJLME. Nevertheless, it can be further explored as a promising resource for identification of novel anticancer candidate drug.

Abstract 1804: Durable eradication of tumors by single injections of a pHLIP-STING agonist

Abstract Purpose: pHLIPs (pH-Low Insertion Peptides) target cell surface acidity resulting from the metabolism of cells in a solid tumor. The acidity causes pHLIPs to insert as transmembrane helices, and attached cargoes can be delivered into targeted cells. STING agonists can trigger an immune response when they are inside cells. pHLIP targeted delivery of a STING agonist (STINGa) effectively triggers cytokine secretion to mobilize the immune system to eradicate tumors while avoiding side effects. Experimental Procedures: Constructs consisting of a pHLIP peptide (Var3) linked to a STINGa (diABZI) via a self-immolating linker were synthesized and characterized. Activation of the STING pathway by the agent was confirmed in cells. Small (100 mm3) and large (400-700 mm3) tumors were grown in female Balb/c mice following flank injections of CT26 colorectal cancer cells. Randomized groups of mice were injected i.p. or i.v. with pHLIP-STINGa made with L- or D- amino acid pHLIP peptides, STINGa alone, pHLIP alone or vehicle. Tumor size and body weight were then measured 3 times per week. The durability of the response to the agent was tested by a second injection of CT26 cells after 60 days. In separate experiments, the identities of targeted immune and stromal cells, the production of cytokines, the PK, tumor targeting and biodistribution were assessed. Results and Conclusions: pHLIP extends the lifetime of a STINGa in the blood 6-fold and delivers STINGa to acidic cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid derived suppressor cells (mMDSCs), and dendritic cells (DCs). The resulting activation of cytokines within the tumor microenvironment (TME) triggers the eradication of small and large CT26 tumors in mice after a single dose of pHLIP-STINGa. The tumor stroma was destroyed, intratumoral hemorrhage developed, and the TME pH increased, which is important for the efficient cytotoxic activity of immune cells. Further, no tumors developed in 20 out of 25 tumor-free mice re-challenged by an additional injection of cancer cells. The therapeutic effect on CT26 tumors was insignificant in nude mice lacking T-cells. Inhibition of MHC-I negative B16F10 melanoma tumor growth was also observed in Balb/c mice. Thus, targeted delivery of the STINGa to the tumor stroma and TAMs activates signaling, potentially resulting in the recruitment and infiltration of both T-cells and NK-cells, which gain access to the tumor core. The cytotoxic activity of immune cells is enhanced, and immune memory is developed. Citation Format: Donald M. Engelman, Yana K. Reshetnyak, Oleg A. Andreev. Durable eradication of tumors by single injections of a pHLIP-STING agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1804.

Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.

Cytokines are important immunotherapeutics with approved drugs for the treatment of human cancers. However, systemic administration of cytokines often fails to achieve adequate concentrations to immune cells in tumors due to dose-limiting toxicity. Thus, developing localized therapy that directly delivers immune-stimulatory cytokines to tumors may improve the therapeutic efficacy. In this study, we generated novel lipid nanoparticles (LNPs) encapsulated with mRNAs encoding cytokines including IL-12, IL-27 and GM-CSF, and tested their anti-tumor activity. We first synthesized ionizable lipid materials containing di-amino groups with various head groups (DALs). The novel DAL4-LNP effectively delivered different mRNAs in vitro to tumor cells and in vivo to tumors. Intratumoral injection of DAL4-LNP loaded with IL-12 mRNA was most potent in inhibiting B16F10 melanoma tumor growth compared to IL-27 or GM-CSF mRNAs in monotherapy. Furthermore, intratumoral injection of dual DAL4-LNP-IL-12 mRNA and IL-27 mRNA showed a synergistic effect in suppressing tumor growth without causing systematic toxicity. Most importantly, intratumoral delivery of IL-12 and IL-27 mRNAs induced robust infiltration of immune effector cells, including IFN-γ and TNF-α producing NK and CD8+ T cells into tumors. Thus, intratumoral administration of DAL-LNP loaded with IL-12 and IL-27 mRNA provides a new treatment strategy for cancer.

In Vivo Non-Thermal, Selective Cancer Treatment With High-Frequency Medium-Intensity Focused Ultrasound.

Focused ultrasound (FUS) has proven its efficacy in non-invasive, radiation-free cancer treatment. However, the commonly used low-frequency high-intensity focused ultrasound (HIFU) destroys both cancerous and healthy tissues non-specifically through extreme heat and inertial cavitation with low spatial resolution. To address this issue, we evaluate the therapeutic effects of pulsed (60 Hz pulse repetition frequency, 1.45 ms pulse width) high-frequency (20.7 MHz) medium-intensity (spatial-peak pulse-average intensity ISPPA < 279.1 W/cm2, spatial-peak temporal-average intensity ISPTA < 24.3 W/cm2) focused ultrasound (pHFMIFU) for selective cancer treatment without thermal damage and with low risk of inertial cavitation (mechanical index < 0.66), in an in vivo subcutaneous B16F10 melanoma tumor growth model in mice. The pHFMIFU with 104 μm focal diameter is generated by a microfabricated self-focusing acoustic transducer (SFAT) with a Fresnel acoustic lens. A three-axis positioning system has been developed for automatic scanning of the transducer to cover a larger treatment volume, while a water-cooling system is custom-built for dissipating non-acoustic heat from the transducer surface. Initial testing revealed that pHFMIFU treatment can be applied to a living animal while maintaining skin temperature under 35.6 °C without damaging normal skin and tissue. After eleven days of treatment with pHFMIFU, the treated tumors were significantly smaller with large areas of necrosis and apoptosis in the treatment field compared to untreated controls. Potential mechanisms of this selective, non-thermal killing effect, as well as possible causes of and solutions to the variation in treatment results, have been analyzed and proposed. The pHFMIFU could potentially be used as a new therapeutic modality for safer cancer treatment especially in critical body regions, due to its cancer-specific effects and high spatial resolution.

Altered Innate-like T Cell Development in Vα14-Jα18 TCRα Transgenic Mice.

CD1d-restricted invariant NKT (iNKT) cells are innate-like T cells that respond to glycolipids, a class of Ags that are invisible to conventional T cells. iNKT cells develop in the thymus where they receive strong "agonist" TCR signals. During their ontogeny, iNKT cells differentiate into discrete iNKT1, iNKT2, and iNKT17 effector subsets akin to helper CD4 T cells. In this study, we found that transgenic (Tg) expression of the canonical Vα14-Jα18 TCRα-chain at the double-positive thymocyte stage led to premature iNKT cell development and a cell-intrinsic bias toward iNKT2 cells, due to increased TCR signaling upon selection. Consistent with the strong iNKT2 bias, innate memory CD8+ T cells were found in greater numbers in Vα14 Tg mice, whereas the prevalence of mucosa-associated invariant T cells was reduced. iNKT cells from Vα14 Tg mice were hyporesponsive to stimulation by their cognate Ag α-galactosylceramide. Finally, Vα14 Tg mice displayed increased B16F10 melanoma tumor growth compared with wild-type mice. This study reveals some of the limitations of Vα14 Tg mice and warrants the cautious interpretation of past and future findings using this mouse model.

A heat-shocked melanoma cell lysate vaccine enhances tumor infiltration by prototypic effector T cells inhibiting tumor growth

BackgroundImmune checkpoint blocker (ICB) therapy has shown survival benefits for some patients with cancer. Nevertheless, many individuals remain refractory or acquire resistance to treatment, motivating the exploration of complementary immunotherapies....

Natural product corynoline suppresses melanoma cell growth through inducing oxidative stress.

Natural product corynoline is a unique isoquinoline alkaloid extracted from traditional Chinese medicine Corydalis bungeana Turcz, whereas its anticancer properties have not been investigated. In this study, we found that corynoline potently impairs the growth of melanoma cells, B16F10, and A375 in a concentration-dependent manner. Treatment of melanoma cells with corynoline results in G2 cell arrest accompanied by reduced cdc2 activation. Furthermore, corynoline triggers apoptosis of melanoma cells, which is associated with increased expression of Bax and cleaved caspase-3. Mechanistic study indicates that corynoline strongly induces reactive oxygen species (ROS) generation and subsequent DNA damage as evidenced by γ-H2AX accumulation. Notably, the effect of corynoline on melanoma cell cycle and apoptosis is abolished by a ROS scavenger N-acetyl cysteine (NAC), indicating a ROS-dependent mechanism. Finally, corynoline significantly inhibits in vivo B16F10 melanoma tumor growth accompanied by reduced expression of Ki-67 in tumor tissue. Taken together, our data suggest that corynoline suppresses melanoma cell growth in vitro and in vivo by inducing oxidative stress and represents a potential therapeutic agent for melanoma patients.

Utilizing 808nm laser for sensitizing of melanoma tumors to megavoltage radiation therapy.

Melanotic melanoma has high content of melanin and laser can destroy melanin-containing cells through thermal effect. In this study, the therapeutic effect of 808nm laser therapy was investigated on B16-F10 melanoma tumor growth and tumor-bearing mice survival time. In addition, as laser can destroy melanin as the main cause of melanoma radioresistance, the effect of laser administration to enhance radiation therapy efficacy at B16-F10 cancer cells was evaluated in vitro and in vivo. Laser therapy (1W/cm2 × 4min) could cause significant (P < 0.05) inhibition of melanoma tumors' growth (~ 61%) and about three times increase of the tumor-bearing mice survival time in comparison with no-treatment group. In addition, the mice which were treated with 1W/cm2 × 4min laser administration plus 6Gy megavoltage radiation therapy exhibited ~ 68% lesser tumors' volume and 27days increase of survival time in comparison with 6Gy irradiated tumor-bearing mice. Also, significantly higher (P < 0.05) tumor necrosis percentage was observed at the histopathological slides of 1W/cm2 × 4min laser + RT treated mice tumors (57 ± 12%) in comparison with radiation therapy group (31 ± 10%). Therefore, not only laser therapy can inhibit melanoma tumors' growth per se but also its combination with radiation therapy can cause a significant enhancement of radiation therapy efficacy. The laser administration can be used as a radiosensitizing method for melanotic melanoma radiation therapy.

Abstract 196: The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth

Abstract The objective of the present study was to examine the effect of modulated electro-hyperthermia (mEHT) on B16-F10 mouse melanoma cell line in vitro and in vivo. mEHT induces tumor-selective heat shock at ~42°C and is used as adjuvant to conventional cancer therapy. For in vitro comparison of the effect of mEHT to conventional hyperthermia (convHT), cells grown on coverslips were treated using mEHT or convHT in water-bath at 42°C for 1x60 min. Changes in gene expression, viability and invasiveness were measured. For in vivo studies, B16-F10 cells mixed with Matrigel were injected subcutaneously into C57Bl/6 mice as the tumors were exposed to 42°C for 30 minutes with mEHT every second day for three times. Tumors were removed 48 hours post treatment, weighed and digested to single-cell suspension. The supernatant of tumor cells and the plasma of tumor-bearing mice were used to screen a membrane-based antibody array representing a panel of 111 cytokines. The tumor cells were stained for flow cytometry to determine the changes induced on melanoma cells and tumor-infiltrating leukocytes. As a marker of in vitro treatment we measured HSPA2 expression with qPCR. Similar expression patterns were induced both by mEHT and convHT. Three hours after treatment the peak levels of HSPA2 were 3 times higher as compared to the untreated control in a time-course experiment. Next, we showed that in parallel to the upregulation of proapoptotic genes Puma, Bak-1 and Bax, downregulation of prosurvival genes XIAP, Bcl-2, Bcl-XL was induced both by convHT and mEHT with similar kinetics and magnitude. Furthermore, cell viability decreased and the invasivness was impaired as measured by scratch assay by both treatment types. The in vivo mEHT of primary melanoma tumors resulted in considerable tumor size reduction. Flow cytometry analysis revealed the increase of MHC class I on the surface of melanoma cells and concomitant activation of CD8+ cytotoxic T lymphocytes in response to treatment. Increased number of CD4+ T cells and Gr1+ granulocytes was detected, although changes in the various subpopulations need further analysis. F4/80+CD11b+ macrophages and mature DC-s were slightly elevated in the treated tumors. The cytokine array analysis shows that several cytokines responsible for leukocyte recruitment were enriched in the treated tumor (SDF-1, osteopontin, IL-1). Complement activation was induced by mEHT both in the tumor microenvironment and the plasma, indicating an acute inflammatory response to treatment. Furthermore, along the short pentraxins, CRP and SAP, the long pentraxin PTX3 was elevated in the tumor and plasma of the treated mice, suggesting that mEHT induced the tumor clearance. Taken together, our results demonstrate that in vitro mEHT and convHT produce comparable effects, whereas the in vivo mEHT treatment was effective in reducing the tumor size by enhancing immune recognition of B16-F10. This study was supported by NVKP 16-1- 2016-0042 grant. Citation Format: Balázs Besztercei, Enikő Major, Anett Benedek, Zoltán Benyó, Andrea Balogh. The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 196.

Enhanced anti-metastatic and anti-tumorigenic efficacy of Berbamine loaded lipid nanoparticles in vivo

Research on metastasis is gaining momentum for effective cancer management. Berbamine (BBM) has the potency to act as a therapeutic in multiple cancers and cancer metastasis. However, the major limitation of the compound includes poor bioavailability at the tumor site due to short plasma half-life. Here, our major objective involved development of lipid based nanoparticles (NPs) loaded with BBM with an aim to circumvent the above problem. Moreover its, therapeutic potentiality was evaluated through various in vitro cellular studies and in vivo melanoma primary and experimental lung metastatic tumor model in C57BL/6 mice. Results of different cellular experiments demonstrated enhanced therapeutic efficacy of BBM-NPs in inhibiting metastasis, cell proliferation and growth as compared to native BBM in highly metastatic cancer cell lines. Further, in vivo results demonstrated suppression of primary B16F10 melanoma tumor growth in C57BL/6 mice model treated with BBM-NPs than that of native BBM. Importantly, a moderately cytotoxic dose of BBM-NPs was able to significantly suppress the incidence of B16F10 cells lung metastasis in vivo. Results indicated development of an effective approach for aggressive metastatic cancer.

Release of bioactive peptides from polyurethane films in vitro and in vivo: Effect of polymer composition

Thermoplastic polyurethanes (TPUs) are widely used in biomedical applications due to their excellent biocompatibility. Their role as matrices for the delivery of small molecule therapeutics has been widely reported. However, very little is known about the release of bioactive peptides from this class of polymers. Here, we report the release of linear and cyclic peptides from TPUs with different hard and soft segments. Solvent casting of the TPU at room temperature mixed with the different peptides resulted in reproducible efflux profiles with no evidence of drug degradation. Peptide release was dependent on the size as well as the composition of the TPU. Tecoflex 80A (T80A) showed more extensive release than ElastEon 5-325, which correlated with a degree of hydration. It was also shown that the composition of the medium influenced the rate and extent of peptide efflux. Blending the different TPUs allowed for better control of peptide efflux, especially the initial burst effect. Peptide-loaded TPU prolonged the plasma levels of the anti-inflammatory cyclic peptide PMX53, which normally has a plasma half-life of less than 30min. Using a blend of T80A and E5-325, therapeutic plasma levels of PMX53 were observed up to 9days following a single intraperitoneal implantation of the drug-loaded film. PMX53 released from the blended TPUs significantly inhibited B16-F10 melanoma tumor growth in mice demonstrating its bioactivity in vivo. This study provides important findings for TPU-based therapeutic peptide delivery that could improve the pharmacological utility of peptides as therapeutics. Statement of SignificanceTherapeutic peptides can be highly specific and potent pharmacological agents, but are poorly absorbed and rapidly degraded in the body. This can be overcome by using a matrix that protects the peptide in vivo and promotes its slow release so that a therapeutic effect can be achieved over days or weeks. Thermoplastic polyurethanes are a versatile family of polymers that are biocompatible and used for medical implants. Here, the release of several peptides from a range of polyurethanes was shown to depend on the type of polymer used in the polyurethane. This is the first study to examine polyurethane blends for peptide delivery and shows that the rate and extent of peptide release can be fine-tuned using different hard and soft segment mixtures in the polymer.

Systemic Platelet-activating Factor Receptor Activation Augments Experimental Lung Tumor Growth and Metastasis.

Pro-oxidative stressors including cigarette smoke (CS) generate novel lipids with platelet-activated factor-receptor (PAF-R) agonistic activity mediate systemic immunosuppression, one of the most recognized events in promoting carcinogenesis. Our previous studies have established that these oxidized-PAF-R-agonists augment murine B16F10 melanoma tumor growth in a PAF-R-dependent manner because of its effects on host immunity. As CS generates PAF-R agonists, the current studies sought to determine the impact of PAF-R agonists on lung cancer growth and metastasis. Using the murine Lewis Lung Carcinoma (LLC1) model, we demonstrate that treatment of C57BL/6 mice with a PAF-R agonist augments tumor growth and lung metastasis in a PAF-R-dependent manner as these findings were not seen in PAF-R-deficient mice. Importantly, this effect was because of host rather than tumor cells PAF-R dependent as LLC1 cells do not express functional PAF-R. These findings indicate that experimental lung cancer progression can be modulated by the PAF system.

Antitumor Effects of Recombinant Antivascular Protein ABRaA-VEGF121 Combined with IL-12 Gene Therapy

Development and neoplastic progression strongly rely on tumor microenvironment cells. Various kinds of cells that form such tumor milieu play substantial roles in angiogenesis and immunosuppression. Attempts to inhibit tumor vascularization alter tumor milieu and enhance immune response against the tumor. Anticancer therapeutic strategy bringing together antiangiogenic and immunostimulating agents has emerged as a promising approach. We here investigated whether therapy directed against preexisting vessels, combined with an immunomodulatory factor would be equally effective in arresting tumor growth. To this goal, we investigated the effectiveness of ABRaA-vascular endothelial growth factor isoform 121 (VEGF121), an antivascular drug constructed by us. It is a fusion protein composed of VEGF121, and abrin A chain (translation-inhibiting toxin). We used it in combination with interleukin (IL-12) gene therapy and tried to inhibit B16-F10 melanoma tumor growth. ABRaA-VEGF121 is a chimeric recombinant protein capable of destroying tumor vasculature and triggering necrosis in the vicinity of damaged vessels. IL-12 cytokine, in turn, activates both specific and non-specific immune responses. Our results demonstrate that combination of ABRaA-VEGF121 antivascular agent with immunostimulatory cytokine IL-12 indeed inhibits tumor growth more effectively than either agent alone, leading to complete cure of ca. 20 % mice. Post-therapeutic analysis of tumors excised from mice treated with combination therapy showed decreased numbers of blood microvessels in the tumor microenvironment, lowered numbers of regulatory T lymphocytes, as well as showed higher levels of CD4+ and CD8+ as compared to control mice. It seems that bringing together antivascular strategy and the action of immunostimulating agents indeed inhibits growth of tumors.

‘Reduced malignancy as a mechanism for longevity in mice with adenylyl cyclase type 5 disruption’

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age-related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV-HER-2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9-β-D-arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan.

Inhibitory effect of antidepressants on B16F10 melanoma tumor growth

BackgroundAntidepressant drugs, like fluoxetine, a selective serotonin reuptake inhibitor, desipramine, a nonselective noradrenaline reuptake inhibitor, and mirtazapine, an antagonist of noradrenaline α2 auto- and heteroreceptors, are widely used for the treatment of depressive symptoms in cancer patients. Since these antidepressants have different activities targeting the immune system, they might also modulate tumor growth in cancer patients. MethodsIn the present study, we investigated the effects of administration of antidepressant drugs: fluoxetine, desipramine and mirtazapine on B16F10 melanoma tumor growth. These drugs were administered intraperitoneally (ip) for 17 days after subcutaneous injection of B16F10 melanoma cells to male C57BL/6J mice. ResultsFluoxetine significantly inhibited melanoma solid tumor growth and desipramine tended to decrease this parameter whereas mirtazapine had no effect. ConclusionThe inhibitory effect of fluoxetine on melanoma growth was associated with an increased mitogen-induced T cell proliferation which may at least partly participate in the mechanism of the antitumor effect of this antidepressant. It appears that the inhibitory effect of fluoxetine on tumor growth is not related with changes in cytokine levels except for IL-10.

Abstract LB-337: Protection against tumor growth by disruption of adenylyl cyclase type 5 (AC5).

Abstract Adenylyl cyclase (AC) catalyzes the conversion of ATP to the second messenger cAMP, which in turn activates protein kinase A (PKA). AC/cAMP/PKA signaling pathway is known to mediate tumor growth but the results of previous studies have been controversial. More importantly, little is known about how specific AC isoforms affect tumor growth. Based on our previous data showing that knocking out the isoform AC type 5 in mice (AC5KO) extends longevity and that the major cause of premature mortality in mice is neoplastic diseases, we hypothesized that AC5KO mice can be protected against tumor growth. We previously demonstrated that AC5KO mice are protected against oxidative stress and obesity, which should also provide protection against neoplastic disease. In order to elucidate whether AC5 plays a role in the tumor microenvironment, we initially investigated the growth of B16F10 melanoma in this model. We have found that AC5KO mice exhibit protection against B16F10 melanoma tumor growth (p&amp;lt;0.01). Tumor latency, wet tumor weights and tumor volume were also significantly reduced in AC5KO mice (p&amp;lt;0.01). At day 27, tumor volumes were significantly lower (p&amp;lt;0.01) (AC5KO mice: 500.30±119 vs. WT: 2692.28±508 mm3). Tumors from AC5KO mice had a 68% decrease in cell proliferation index and AC5HET mice had a 37% both with respect to the WT (p&amp;lt;0.01 and p&amp;lt;0.05 respectively). Also, tumors from AC5KO mice displayed a significantly larger apoptotic rate compared to the AC5WT (2.30±0.48% vs. 1.00±0.20 %, p&amp;lt;0.05). Ablation of AC5 in mice also leads to a significantly lower intra-tumor microvessel density (AC5HET: 8±0.4 and AC5KO: 6.2±0.6 compared to WT: 11.5±0.9; p&amp;lt;0.05 and p&amp;lt;0.01).In addition, AC5KO mice had significantly lower VEGF serum levels than WT mice (AC5KO: 2.5±0.1 vs. AC5WT: 3.3±0.2 ng/ml; p&amp;lt;0.05). Tumors in AC5KO mice induced significantly lower angiogenic response than in WT mice, as measured by total vessel length (AC5KO: 58.7±6.2 vs. WT: 100± 8.5, p&amp;lt;0.001). Obesity is a risk factor for the development of breast cancer. In accord with the fact that AC5KO are protected against obesity, we have found that circulating levels of leptin were significantly reduced in AC5KO: 1.99±0.28 vs. WT: 3.23±0.35 ng/ml (p&amp;lt;0.05). In an attempt to elucidate the role of the stromal AC5 isoform and the connection between the AC5KO protection against obesity and tumor incidence; we crossbred AC5 mice with MMTV Her 2 neu mice; a model prone to develop mammary tumors. We have found that AC5KO x MMTV HER-2 neu mice weighed significantly less (p&amp;lt;0.001) and had a significantly lower mammary tumor incidence (p&amp;lt;0.05). All these results together show that understanding the mechanism underlying the role of AC5 in regulating tumor growth and its anti-tumor properties will have a profound impact on its potential therapeutic application as a target for anticancer drug development. Citation Format: Mariana S. De Lorenzo, Wen Y. Chen, Erdene Baljinnyam, Krista La Perle, Sanford P. Bishop, Thomas E. Wagner, Patricio Abarzua, Arnold B. Rabson, Dorothy E. Vatner, Lydia I. Puricelli, Stephen F. Vatner. Protection against tumor growth by disruption of adenylyl cyclase type 5 (AC5). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-337. doi:10.1158/1538-7445.AM2013-LB-337