Increased Tumor Cell Apoptosis Research Articles

Mechanical stretch promotes tumoricidal M1 polarization via the FAK/NF-κB signaling pathway.

Macrophages (Mφs) can be used as a part of cell-based cancer immunotherapy. However, they may be hampered by a failure to effectively and stably regulate their polarization state to enhance their tumoricidal effects. In this work, mechanical stretch (MS), as a biology-free modulatory method, was shown to enhance M1 polarization and tumoricidal effects. By using an in vitro Flexcell Tension system, we found that murine Mφ RAW264.7 cells showed higher M1 polarization-related mRNA expression and cytokine release after MS. Further molecular analyses found that focal adhesion kinase and NF-κB activation occurred in the MS-induced M1 polarization. Coculture of MS-preconditioned Mφ with B16F10 skin melanoma cells in vitro showed that the proliferation of B16F10 cells decreased, whereas caspase-3-induced apoptosis increased. Importantly, the injection of MS-preconditioned Mφ into murine skin melanomas in vivo impeded tumor growth; lesions were characterized by increased amounts of M1 Mφ, decreased tumor cell proliferation, and increased tumor cell apoptosis in the tumor microenvironment. Together, our results suggest that MS could be used as a simple preconditioning approach to prepare tumoricidal M1 Mφ for cancer immunotherapy.-Shan, S., Fang, B., Zhang, Y., Wang, C., Zhou, J., Niu, C., Gao, Y., Zhao, D., He, J., Wang, J., Zhang, X., Li, Q. Mechanical stretch promotes tumoricidal M1 polarization via the FAK/NF-κB signaling pathway.

Development of a Novel Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein-2 That Inhibits Triple-Negative Breast Cancer and Angiosarcoma Growth In Vivo.

We previously reported that secreted frizzled-related protein-2 (SFRP2) is expressed in a variety of tumors, including sarcoma and breast carcinoma, and stimulates angiogenesis and inhibits tumor apoptosis. Therefore, we hypothesized that a humanized SFRP2 monoclonal antibody (hSFRP2 mAb) would inhibit tumor growth. The lead hSFRP2 antibody was tested against a cohort of 22 healthy donors using a time course T-cell assay to determine the relative risk of immunogenicity. To determine hSFRP2 mAb efficacy, nude mice were subcutaneously injected with SVR angiosarcoma cells and treated with hSFRP2 mAb 4mg/kg intravenously every 3days for 3weeks. We then injected Hs578T triple-negative breast cells into the mammary fat pad of nude mice and treated for 40days. Control mice received an immunoglobulin (Ig) G1 control. The SVR and Hs578T tumors were then stained using a TUNEL assay to detect apoptosis. Immunogenicity testing of hSFRP2 mAb did not induce proliferative responses using a simulation index (SI) ≥ 2.0 (p < 0.05) threshold in any of the healthy donors. SVR angiosarcoma tumor growth was inhibited in vivo, evidenced by significant tumor volume reduction in the hSFRP2 mAb-treated group, compared with controls (n = 10, p < 0.001). Likewise, Hs578T triple-negative breast tumors were smaller in the hSFRP2 mAb-treated group compared with controls (n = 10, p < 0.001). The hSFRP2 mAb treatment correlated with an increase in tumor cell apoptosis (n = 11, p < 0.05). Importantly, hSFRP2 mAb treatment was not associated with any weight loss or lethargy. We present a novel hSFRP2 mAb with therapeutic potential in breast cancer and sarcoma that has no effect on immunogenicity.

Tumor-associated macrophages and individual chemo-susceptibility are influenced by iron chelation in human slice cultures of gastric cancer.

Purpose: Presence of tumor-associated macrophages (TAM) and high levels of ferritin and lipocalin 2 (Lcn2) in the tumor microenvironment are associated with poor prognosis in many types of cancer. Here we investigate whether iron deprivation influences TAM phenotype and chemotherapy resistance in tumor slice cultures (TSC) of gastric cancer. Results: TAM remained morphologically and functionally stable for four DIV. DFO treatment for 72 h decreased ferritin expression in TAM and in the tumor stroma but did not alter Lcn2 expression. TAM phenotype was altered after 72 h of cisplatin or DFO treatment compared with control conditions. Single DFO treatment and combined treatment with cytotoxic drugs significantly increased tumor cell apoptosis in TSC of gastric cancer. Methods: TSC were manufactured by cutting tissue of gastric cancer resection specimens in 350 μm thick slices and cultivating them under standard conditions on a filter membrane, at an air-liquid interface. After 24 h ex vivo, TSC were treated with irinotecan (100 nM) or cisplatin (10 μM) alone and in combination with deferoxamine (DFO; 10 μM, 100 μM), respectively, for 72 h. After four days in vitro (DIV) the TSC were fixated with paraformaldehyde, paraffin embedded and analyzed by immunohistochemistry for apoptosis (cPARP), proliferation (Ki67), TAM (CD68, CD163), ferritin, and Lcn2 expression. Conclusions: TAM are well preserved and can be studied in TSC of gastric cancer. Iron deprivation significantly increased tumor cell apoptosis.

Mobilization of CD8+ T Cells via CXCR4 Blockade Facilitates PD-1 Checkpoint Therapy in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy. Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization. mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis. Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA.See related commentary by Medina and Miller, p. 3747.

Abstract LB-135: Targeting the myeloid-cell specific SRC-kinase HCK improves anti-tumor immunity

Abstract Elevated expression of the myeloid cell-specific Src-family kinase HCK occurs in a majority of solid malignancies and correlates with poor patient survival [Poh et al., Frontiers Oncol 2018]. We have recently shown that genetic ablation, or therapeutic inhibition, of HCK suppresses the growth of primary tumors arising in the colon, stomach, lung and other mutagenized epithelia of mice. Mechanistically, we have attributed this to the capacity of HCK activity to support and retain the polarization of a wound-healing, Tie2+ (i.e. alternative activated) endotype of tumor-associated macrophages and concomitant tumor vascularization [Poh et al., Cancer Cell2017]. Here we extend these observations to MC38 colon cancer, B6/F10 melanoma and EO771 breast cancer allograft model, where the growth of the primary lesion and its metastatic spread is suppressed in HCK knockout hosts. More importantly, HCK gene deletion or therapeutic HCK inhibition with a small kinase inhibitor in mouse models further enhances the anti-tumor effects conferred by either chemotherapy or single agent immunotherapies based on anti-PD-L1, anti-CTLA4 or agonistic-CD40 antibodies. These observations are underpinned by increased tumour cell apoptosis that coincides with enhanced expression of ifng, gzmb, prf1 and other effector proteins in NK and infiltrating CD8+ cells. Likewise, HCK inhibition confers increased expression of il12 and ifng in tumor-associated macrophages, which adopt a more conventional activated endotype. Because HCK-deficiency of the host also associated with increased abundance of tumor infiltrating effectors cells, we propose that therapeutic targeting of HCK not only “makes tumors hotter”, but also improves host anti-tumor immunity. We are therefore developing novel HCK-specific small molecule inhibitors. Citation Format: Matthias R. Ernst, Ashwini Chand, Nicholas Huntington, Robert O'Donoghue, Ashleigh Poh. Targeting the myeloid-cell specific SRC-kinase HCK improves anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-135.

The FOXM1 Inhibitor RCM-1 Decreases Carcinogenesis and Nuclear β-Catenin.

The oncogenic transcription factor FOXM1 has been previously shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple cancer types. A small-molecule compound, Robert Costa Memorial drug-1 (RCM-1), has been recently identified from high-throughput screen as an inhibitor of FOXM1 in vitro and in mouse model of allergen-mediated lung inflammation. In the present study, we examined antitumor activities of RCM-1 using tumor models. Treatment with RCM-1 inhibited tumor cell proliferation as evidenced by increased cell-cycle duration. Confocal imaging of RCM-1-treated tumor cells indicated that delay in cellular proliferation was concordant with inhibition of FOXM1 nuclear localization in these cells. RCM-1 reduced the formation and growth of tumor cell colonies in the colony formation assay. In animal models, RCM-1 treatment inhibited growth of mouse rhabdomyosarcoma Rd76-9, melanoma B16-F10, and human H2122 lung adenocarcinoma. RCM-1 decreased FOXM1 protein in the tumors, reduced tumor cell proliferation, and increased tumor cell apoptosis. RCM-1 decreased protein levels and nuclear localization of β-catenin, and inhibited protein-protein interaction between β-catenin and FOXM1 in cultured tumor cells and in vivo Altogether, our study provides important evidence of antitumor potential of the small-molecule compound RCM-1, suggesting that RCM-1 can be a promising candidate for anticancer therapy.

Differential Effects of Depleting versus Programming Tumor-Associated Macrophages on Engineered T Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

Pro-Apoptotic and Anti-Angiogenesis Effects of Olive Leaf Extract on Spontaneous Mouse Mammary Tumor Model by Balancing Vascular Endothelial Growth Factor and Endostatin Levels

It has been proven that olive associated products such as olive leaf extract (OLE) causes significant reduction in cancer cells viability and proliferation. Female BALB/c adult mice were divided into four groups. Three days prior to oral treatments, tumors were transplanted. First group were treated with distilled water and other three groups were received, respectively, 75, 150, and 225 mg/kg/day of OLE for three weeks. For assessment of anti-angiogenesis and pro-apoptotic effect of OLE on tumor tissue, tumor volume, cell mitosis and apoptosis, and also vascular endothelial growth factor (VEGF) and endostatin levels were assessed. OLE treatment with 150 and 225 mg/kg/day lead to significant reduction in tumor volume and cell mitosis compared with the control group, while the same doses significantly increase tumor cell apoptosis. OLE treatment with 150 mg/kg/day increase endostatin levels, while the same dose did not significantly decrease VEGF levels. The VEGF level is significantly reduced by the treatment with OLE 225 mg/kg/day for three weeks. Although, further studies are needed to clarify anti-angiogenesis and anti-apoptotic mechanism of OLE, consumption of OLE polyphenols after tumor transplantation reduced spontaneous mouse mammary tumor growth.

The FOXM1 Inhibitor RCM‐1 Decreases Carcinogenesis and Nuclear β‐catenin

FOXM1, a member of the Forkhead box (FOX) family of transcription factors, has previously been shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple types of cancer when abnormally upregulated. Our lab previously identified a small molecule compound, Robert Costa Memorial drug‐1 (RCM‐1), through high‐throughput screening as a FOXM1 antagonist in vitro and in an in vivo mouse model of allergen‐mediated lung inflammation. Here, we examined antitumor activities of RCM‐1 using tumor models. Confocal imaging of RCM‐1‐treated tumor cells indicated that reduced cellular proliferation was concordant with inhibition of FOXM1 nuclear localization in these cells. RCM‐1 reduced the incidence and growth of tumor cell colonies in the colony formation assay. Migration assays in vitro demonstrated the ability of RCM‐1 to inhibit migration of tumor cells. In animal models, RCM‐1 treatment inhibited growth of mouse rhabdomyosarcoma Rd76‐9 and human lung adenocarcinoma H2122. RCM‐1 decreased FOXM1 protein levels in treated tumors, reduced tumor cell proliferation and increased tumor cell apoptosis in vivo as detected by immunohistochemical staining. Western blot analysis of cultured tumor cells showed that RCM‐1 decreased protein levels and nuclear localization of β‐catenin, an important constituent of the canonical Wnt signaling pathway, and inhibited protein‐protein interaction between β‐catenin and FOXM1. Altogether, our study provides important evidence of antitumor activity of the small molecule compound RCM‐1, suggesting that RCM‐1 has promising potential as a cancer therapy in the future.Support or Funding InformationPeter K. Lauf Award, Ohio Physiological Society; NIH funding awarded to TK.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ribociclib, a selective cyclin D kinase 4/6 inhibitor, inhibits proliferation and induces apoptosis of human cervical cancer in vitro and in vivo

Cervical cancer remains one of the main factors leading to tumor-related death worldwide. Many strategies of cancer treatment such as chemotherapy are developed and used nowadays. However, for the cancer chemotherapy resistance, reduction of the limitation of cancer chemotherapy efficacy is one of the aims of several oncology teams. Moreover, the cyclin-dependent kinase 4/6-cyclin D-retinoblastoma protein-E2F pathway is an important mechanism for cell cycle control and its dysregulation is one of the key factors for cancers development including cervical cancer. Ribociclib is one of the selective CDK4/6 inhibitors and is a new therapeutic approach showing promise as a good strategy of therapy in many human cancers. However, there are not the studies regarding the investigation of effects of Ribociclib in cervical cancer yet. In the present study, by western blotting and immunofluorescence assay, we found respectively that CDK4, CDK6 and cyclin D1 are highly expressed and are mostly localized in the nucleus with some localized in the cytoplasm of cervical cancer cell lines. Moreover, Ribociclib induced cell cycle arrest in G0-G1 phase and cell apoptosis, and inhibited C33A cell proliferation in dose – dependent manner following by decreased expression of certain related genes such as CDK4, CDK6, E2F1, P-Rb, and increased Bax expression. In C33A xenografts, Ribociclib inhibited tumor growth associated with decreased expressions of CDK4, CDK6, cyclin D1, Rb and Ki-67, and also significantly increased tumor cell apoptosis. However, we didn’t find side effect of Ribociclib concerning heart, liver and kidney perturbation and any Ribociclib anti-tumor effects on HeLa in vitro and in vivo which may be due to Hela cell infection by HPV. Based on our findings, the Rb-E2F pathway can be considered as an important factor in human cervical cancer pathogenesis and as a mechanism of Ribociclib, a potential strategy of treatment for the improvement of new therapeutic measures for the treatment of HPV-negative cervical cancer which application for HPV-positive cervical cancer is desired in further study.

Isoliquiritigenin inhibits the proliferation, apoptosis and migration of osteosarcoma cells

The overall survival rate of patients with osteosarcoma has remained unchanged for the last several decades. Therefore, novel drugs for osteosarcoma treatment are required. Isoliquiritigenin (ISL), a natural compound, has been demonstrated to inhibit the growth of various tumors. However, it is unclear whether ISL is able to inhibit the growth of osteosarcoma. In the present study, it was identified that ISL was able to inhibit the growth of the osteosarcoma cell line Saos‑2 cells in vitro and in xenograft tumors primarily by attenuating tumor cell proliferation and, cell migration and promoting tumor cell apoptosis. Decreased tumor cell proliferation induced by ISL was associated with downregulation of cyclin D1 and upregulation of p53, p21 and p27. Increased tumor cell apoptosis triggered by ISL was associated with downregulation of apoptosis regulator Bcl‑2, upregulation of apoptosis regulator Bax and damaged mitochondrial function evidenced by a low level of ATP‑synthesis. In addition, ISL was able to inhibit the migratory capacity of Saos‑2 cells by modulating the expression of matrix metalloproteinase (MMP)2 and MMP9. Mechanistic analysis revealed that the tumor growth‑inhibitory effect of ISL may depend on the action of ISL on the phosphorylation of PI3K and AKT. However, it remains to be investigated whether the inhibitory effect of ISL on the migration of Saos‑2 cells was associated with downregulated PI3K/AKT signaling. Overall, the present study provided evidence for the potential use of ISL against osteosarcoma.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured naïve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

A genetically engineered microRNA-34a prodrug demonstrates anti-tumor activity in a canine model of osteosarcoma.

Osteosarcoma (OSA) represents the most common primary bone tumor in humans and pet dogs. Little progress has been made with regard to viable treatment options in the past three decades and patients presenting with metastatic disease continue to have a poor prognosis. Recent mouse studies have suggested that microRNA-34a (miR-34a) may have anti-tumor activities in human OSA models. Due to the conservation of microRNA across species, we hypothesized that a bioengineered miR-34a prodrug (tRNA/miR-34a) would have similar effects in canine OSA, providing a valuable preclinical model for development of this therapeutic modality. Using a panel of canine OSA cell lines, we found that tRNA/miR-34a reduced viability, clonogenic growth, and migration and invasion while increasing tumor cell apoptosis. Furthermore, canine OSA cells successfully process the tRNA/miR-34a into mature miR-34a which reduces expression of target proteins such as platelet derived growth factor receptor alpha (PDGFRα), Notch1 and vascular endothelial growth factor (VEGF). Additionally, our subcutaneous OSA xenograft model demonstrated in vivo tumor growth delay, increased necrosis and apoptosis by tRNA/miR-34a, and decreased cellular proliferation ability. Taken together, these data support that this novel microRNA-based therapy may possess clinical utility in a spontaneously-occurring large animal model of OSA, which can then serve to inform the clinical development of this therapy for human OSA patients.

DNA-PK inhibition synergizes with oncolytic virus M1 by inhibiting antiviral response and potentiating DNA damage

Oncolytic virotherapy is a promising therapeutic strategy that uses replication-competent viruses to selectively destroy malignancies. However, the therapeutic effect of certain oncolytic viruses (OVs) varies among cancer patients. Thus, it is necessary to overcome resistance to OVs through rationally designed combination strategies. Here, through an anticancer drug screening, we show that DNA-dependent protein kinase (DNA-PK) inhibition sensitizes cancer cells to OV M1 and improves therapeutic effects in refractory cancer models in vivo and in patient tumour samples. Infection of M1 virus triggers the transcription of interferons (IFNs) and the activation of the antiviral response, which can be abolished by pretreatment of DNA-PK inhibitor (DNA-PKI), resulting in selectively enhanced replication of OV M1 within malignancies. Furthermore, DNA-PK inhibition promotes the DNA damage response induced by M1 virus, leading to increased tumour cell apoptosis. Together, our study identifies the combination of DNA-PKI and OV M1 as a potential treatment for cancers.

Abstract B07: Immunostimulatory gene therapy enhances PD-1 blockade antibody therapy in experimental lung cancer

Abstract This study aims to evaluate if immunostimulatory gene therapy is a good enhancer of checkpoint blockade therapy. Lung cancer is a deadly disease responding poorly to conventional therapy. Checkpoint blockade antibodies inhibiting PD-1/PD-L1 signaling have shown significant clinical benefit. Nevertheless, there is still room for improving overall survival rates. Since the success of PD-1 blockade therapy prerequisites the presence of tumor-reactive T cells, we hypothesized that an activating immunotherapy with high potency to stimulate T cell responses would be an ideal candidate to combine with anti-PD-1 antibody treatment. LOAd703 is a replication-restricted adenovirus serotype 5/35. It is armed with two immunostimulatory transgenes, 4-1BB ligand (4-1BBL) and a trimerized, membrane-bound human CD40 ligand (TMZ-CD40L) (PCT/EP2015/057489). Both molecules are key co-stimulatory molecules in the dendritic cell (DC)/T cell synapse. The capacity of LOAd703 was tested using the lung cancer cell lines A549 and H727. Infected tumor cells were killed by oncolysis in a 72hr MTS viability assay with high dose virus (100MOI) which also translated into tumor control in a xenograft mouse model. In tumor cell co-culture with peripheral blood mononuclear cells (PBMCs) (Incucyte), the PBMCs were rapidly tolerized by the tumor microenvironment and did not proliferate, nor kill, the allogeneic tumor. Addition of anti-PD-1 antibody did not increase tumor cell apoptosis as a monotherapy. If OKT3/IL2 was added to the co-culture, apoptosis was confirmed in the cultures peaking at day 5. Combination of OKT3/IL2 with anti-PD-1 antibody significantly enhanced the effect. LOAd703 was added to tumor cell/PBMC co-cultures at a low MOI to delay oncolysis but allow transgene expression. LOAd703 rapidly induced caspase activation in the tumor cells, starting already at day 2 and the activity was continuously rising throughout the experiment. Combining LOAd703 with anti-PD-1 antibody significantly increased apoptosis induction of tumor cells. A control virus lacking immunostimulatory transgenes was less efficient. At endpoint, LOAd703 plus anti-PD-1 was twice as effective as OKT3/IL2 plus anti-PD-1. Immune cells were analyzed by flow cytometry and supernatants taken for cytokine analysis. The T cells were increased in the same groups as tumor cell killing was previously noted. IFNg, TNF, IL2 were all enhanced by LOAd703 and OKT3/IL2. The LOAd703 virus does not infect murine cells since it targets human CD46. However, murine B cells can take up the virus by another route albeit with poor efficacy. Nevertheless, the murine A20 B cell lymphoma was used as a surrogate syngeneic model to test the ability of LOAd703 to potentiate aPD-1 therapy. In line with in vitro data, LOAd703 could enhance in vivo tumor control of anti-PD-1 therapy. IFNg and TNF were enhanced in serum of combination animals. In conclusion, LOAd703 is a potent stimulator of T cell responses and potentiates the effect of aPD-1 therapy. LOAd703 is currently in clinical phase I investigation prior initiating combination studies in suitable indications such as lung cancer. Citation Format: Jessica Wenthe, Emma Eriksson, Ann-Charlotte Hellström, Angelica Loskog. Immunostimulatory gene therapy enhances PD-1 blockade antibody therapy in experimental lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B07.

Mebendazole stimulates CD14+ myeloid cells to enhance T-cell activation and tumour cell killing.

Mebendazole (MBZ) was recently shown to induce a tumor suppressive M1 phenotype in THP-1 monocytes and macrophages. In the present study the immune effects of MBZ was further investigated using human peripheral blood mononuclear cells (PBMCs) co-cultured with tumour cells. The Biomap platform was used to screen for biomarkers induced from MBZ exposed co-cultures of T-cell receptor activated PBMCs, HT29 colon cancer cells and either human fibroblasts or human umbilical vein endothelial cells (HUVEC) cells. In these co-culture systems MBZ at 0.3-10 μM induced significant increases in TNFα and IFNγ indicating immune stimulation. PBMC cultures alone were subsequently tested for activation status and only in PBMCs activated by CD3/IL2 stimulation and MBZ, at a clinically achievable concentration, was able to increase PBMC clustering and release of pro-inflammatory IFNγ, TNFα, IL6 and IL1β cytokines. Moreover, when PBMC cultures were functionally tested for immune cell killing of lung cancer A549NucLightRed cells, MBZ significantly increased tumour cell apoptosis and reduced the number of surviving tumour cells. This effect was dependent on the presence of CD14 monocytes/macrophages in the co-culture. In summary, MBZ potentiated the immune stimulatory and anticancer effects of anti-CD3/IL2 activated PBMCs which could be relevant to explain the anticancer activity of MBZ observed in the clinic.

Abstract 4393: Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation

Abstract Introduction: Combining gemcitabine (gem) with irreversible electroporation (IRE) has been shown to result in increased tumor cell apoptosis in a murine model of pancreatic adenocarcinoma (PDAC). However, it remains unclear whether this effect can be augmented locally and systemically by utilizing more active chemotherapies. The objective of this study was to evaluate the efficacy of IRE+FOLFIRINOX (FOL: 5-FU, leucovorin, irinotecan, and oxaliplatin). Methods: Athymic nude mice underwent intrapancreatic injection with S2-013 PDAC cells. After 7-10 days, tumors were confirmed with ultrasound and mice were treated with chemotherapy (gem or FOL), IRE, or chemotherapy+IRE. Four hours after last chemotherapy, pancreas was evaluated histologically. Levels of irinotecan and its active metabolite (SN-38) were evaluated in organs of mice treated with FOL and IRE+FOL. Experiments were repeated and survival analyses performed to evaluate long-term efficacy. Pancreas was harvested for histology and evaluation of apoptotic machinery, cell cycle proteins, and tumor cell proliferation. Results: IRE+FOL (ECT) resulted in increased tumor cells apoptosis compared to gem, gem+IRE, or FOL (apoptotic index (AI) 34.6% vs. 3.8%, 7.3%, 5.8%, respectively, p&amp;lt;0.001). High performance liquid chromatography (HPLC) demonstrated a 1.5-2-fold increase in irinotecan and SN-38 in the pancreas and a 4-fold increase of both compounds in the liver in mice treated with ECT vs. FOL. ECT significantly improved overall survival when compared to mice treated with IRE or FOL (median 7 days ECT vs. 4 days IRE vs. 3 days FOL, p=0.03). Histology demonstrated increased tumor cell apoptosis at time of death (AI = 34.2% ECT vs. 13.2% IRE vs. 4.4% FOL, p=0.026). Western blot demonstrated an increase in active caspase 3 (0.14 ECT vs. 0.00 IRE vs. 0.03 FOL fold change/GAPDH, p=0.02). ECT resulted in lower cell proliferation compared to IRE or FOL (0.2% vs. 0.3% vs. 0.9, p&amp;lt;0.001). Conclusions: ECT with IRE and FOL causes significant tumor cell apoptosis, decreases tumor cell proliferation, enhances systemic FOL exposure, and improves overall survival. Human Phase 1 trials have been initiated to evaluate safety and efficacy of this therapeutic modality. Citation Format: Neal Bhutiani, Harshul Pandit, Qianqian Zheng, Suping Li, Yan Li, Robert C. Martin. Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation [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 4393.

Abstract 4790: YH25448, an irreversible 3rd generation EGFR TKI, exhibits superior anticancer effects with potent brain BBB penetration in NSCLC

Abstract EGFR mutated lung cancer shows approximately 10-15% of non-small cell lung cancer (NSCLC). Although the best therapeutic EGFR tyrosine kinase inhibitors (TKIs) targeting mutant EGFR, such as gefitinib and erlotinib, are used in the first line treatment of patients with advanced EGFR mutated NSCLC, the acquired resistance to the drugs usually appears in 10-12 months of therapy by the occurrence of a second EGFR mutation T790M. YH25448, a highly mutant-selective and irreversible 3rd generation EGFR TKI potently penetrating blood-brain barrier (BBB) penetration, targets both activating EGFR mutations Del19, L858R and T790M mutation while sparing wild type. In NSCLC cell lines and primary cancer cells from patients harboring EGFR mutations, YH25448 showed more potent inhibition of cancer cell growth and significantly increased tumor cell apoptosis compared to osimertinibs, which is one of 3rd generation EGFR TKIs. In vivo mouse model implanted with H1975 cells, YH25448 treatment at the once-daily showed a dramatic dose-dependent tumor regression in both subcutaneous and intracranial lesions with no abnormal signs such as skin keratosis shown in osimertinib-treated mice. Plasma half life of YH25448 was 5.9-6.8 hr and tumor to plasma AUC0-last ratio was 3.0-5.1 in tumor bearing mice. YH25448 also showed excellent penetration of the BBB, achieving CSF concentrations exceeding the IC50 value for pEGFR inhibition in the tumor-bearing mice. Taken together, these findings suggest important role for the further development of YH25448 as a novel therapeutic for the treatment of EGFR mutant-positive NSCLC patients with brain metastases. Citation Format: Jiyeon Yun, Min Hee Hong, Seok-Young Kim, Chae Won Park, So-Young Kim, Mi Ran Yun, Han Na Kang, Kyoung-Ho Pyo, Jong Sung Koh, Ho-Juhn Song, Young- Sung Lee, Se-Woong Oh, Soongyu Choi, Byoung-Chul Cho. YH25448, an irreversible 3rd generation EGFR TKI, exhibits superior anticancer effects with potent brain BBB penetration in NSCLC [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 4790.

Nrf2-activated expression of sulfiredoxin contributes to urethane-induced lung tumorigenesis

Lung cancer is the leading cause of cancer death worldwide. Cigarette smoking and exposure to chemical carcinogens are among the risk factors of lung tumorigenesis. In this study, we found that cigarette smoke condensate and urethane significantly stimulated the expression of sulfiredoxin (Srx) at the transcript and protein levels in cultured normal lung epithelial cells, and such stimulation was mediated through the activation of nuclear related factor 2 (Nrf2). To study the role of Srx in lung cancer development in vivo, mice with Srx wildtype, heterozygous or knockout genotype were subjected to the same protocol of urethane treatment to induce lung tumors. By comparing tumor multiplicity and volume between groups of mice with different genotype, we found that Srx knockout mice had a significantly lower number and smaller size of lung tumors. Mechanistically, we demonstrated that loss of Srx led to a decrease of tumor cell proliferation as well as an increase of tumor cell apoptosis. These data suggest that Srx may have an oncogenic role that contributes to the development of lung cancer in smokers or urethane-exposed human subjects.

Endogenous albumin-mediated delivery of redox-responsive paclitaxel-loaded micelles for targeted cancer therapy

Targeted delivery and accumulation of chemotherapeutics to tumor sites is in high demand and extremely challenging for anticancer therapy. Studies show that albumin is actively recruited into tumor tissue overexpressing albumin-binding proteins including secreted protein acidic and rich in cysteine (SPARC) and gp60. Herein, a novel redox-responsive paclitaxel-loaded micelle system with the modification of ABD035, which specifically and strongly binds to albumin, is designed to combine the strengths of albumin and micelles together for antitumor drug biomimetic delivery. The ABD035-modified micelle has a strong binding response to albumin and co-localization of BODIPY-labeled ABD035-modified micelle and SPARC is observed in tumor tissues. Furthermore, the ABD035-modified micelle significantly improves the therapeutic effect in the animal model bearing triple negative breast cancer by increasing drug accumulation in tumor tissue, enhancing cell uptake efficiency, rapidly releasing prototype drug under intracellular reductive conditions and increasing tumor cell apoptosis. These results together vote the biomimetic delivery of ABD035-modified micelle as a promising strategy for cancer therapy.