A549 Tumor Research Articles

A Novel Laser Fiberscope for Simultaneous Imaging and Phototherapy of Peripheral Lung Cancer

Phototherapy is an alternative treatment for patients with localized non-small cell lung cancer who are unable to undergo surgical resection. However, phototherapy is currently limited to treatment of centrally located lung cancer, with the much larger proportion of peripheral lesions remaining inaccessible. There are also concerns over the accuracy of targeted laser treatment because of the need to exchange visualization and irradiation fibers during therapy, preventing the operator from confirming the final location of the irradiation fiber. A newly developed parallel-type ultrasmall composite optical fiberscope (Laser-eYe Ultrathin fiberscope [LYU]), which enables simultaneous white-light imaging and phototherapy, was evaluated in preclinical lung cancer models. Three models were used: human lung cancer xenografts (A549) in mice, orthotopic VX2 lung tumors in rabbits, and exvivo pig lungs into which A549 tumor tissue was transplanted. A multifunctional porphyrin-phospholipid nanoparticle (porphysome) was used as a photosensitizer to evaluate fluorescence-guided photothermal therapy. The LYU's 0.97mm diameter and hydrophilic coating allowed easy passage through the working channel of all types of bronchoscopes and controlled guidance of the LYU tip in any desired direction. The LYU could visualize the peripheral bronchus and porphysome-laden peripheral tumors. The LYU could also perform photothermal therapy with simultaneous imaging. The LYU enables simultaneous imaging and phototherapy that allows accurate irradiation of peripheral lung cancers. This new laser device may enable ultraminimally invasive transbronchial treatment of peripheral lung cancer.

Measurement of Tumor Antioxidant Capacity and Prediction of Chemotherapy Resistance in Preclinical Models of Ovarian Cancer by Positron Emission Tomography.

Drug resistance is a major obstacle for the effective treatment of patients with high-grade serous ovarian cancer (HGSOC). Currently, there is no satisfactory way to identify patients with HGSOC that are refractive to the standard of care. Here, we propose the system xc - radiotracer (4S)-4-(3-[18F]fluoropropyl)-l-glutamate ([18F]FSPG) as a non-invasive method to measure upregulated antioxidant pathways present in drug-resistant HGSOC. Using matched chemotherapy sensitive and resistant ovarian cancer cell lines, we assessed their antioxidant capacity and its relation to [18F]FSPG uptake, both in cells and in animal models of human ovarian cancer. We identified the mechanisms driving differential [18F]FSPG cell accumulation and evaluated [18F]FSPG tumor uptake as predictive marker of treatment response in drug-resistant tumors. High intracellular glutathione (GSH) and low reactive oxygen species corresponded to decreased [18F]FSPG cell accumulation in drug-resistant versus drug-sensitive cells. Decreased [18F]FSPG uptake in drug-resistant cells was a consequence of changes in intracellular cystine, a key precursor in GSH biosynthesis. In vivo, [18F]FSPG uptake was decreased nearly 80% in chemotherapy-resistant A2780 tumors compared with parental drug-sensitive tumors, with nonresponding tumors displaying high levels of oxidized-to-reduced GSH. Treatment of drug-resistant A2780 tumors with doxorubicin resulted in no detectable change in tumor volume, GSH, or [18F]FSPG uptake. This study demonstrates the ability of [18F]FSPG to detect upregulated antioxidant pathways present in drug-resistant cancer. [18F]FSPG may therefore enable the identification of patients with HGSOC that are refractory to standard of care, allowing the transferal of drug-resistant patients to alternative therapies, thereby improving outcomes in this disease.

Highlights of This Issue

While trastuzumab-based chemotherapy has shown remarkable clinical benefits for HER2-positive breast cancer patients, a subset of patients (30%–40%) shows little or no effect. This highlights an important clinical need for biomarkers in addition to HER2 for better stratification of patients for therapy. In this multicenter retrospective study by Kang and colleagues, patients with HER2-positive breast cancer that also overexpress WBP2 had better pathologic complete response to trastuzumab-based neoadjuvant therapy. The potential to predict which patients would attain successful downstaging of their tumors from neoadjuvant therapy could help guide surgical decisions; for example, breast-conserving surgery versus mastectomy, consequently improving patients' outcome.Extending previous studies in more restricted populations, Nassar and colleagues examined 472 patients across the spectrum of urothelial carcinoma from nonmuscle-invasive bladder cancer (NMIBC) to upper-tract urothelial carcinoma (UTUC) for mutations in 237 cancer genes. Low-grade NMIBC has a distinct mutation spectrum from invasive disease, suggesting that it is truly a separate pathologic entity unrelated to invasive disease in most instances. Other key findings include enrichment of KDM6A mutations in women with low-grade NMIBC compared to men and identification of a subtype of high-grade UTUC with mutations in FGFR3, EP300, and PIK3CA associated with poorer survival.Drug-resistance is a major obstacle for the effective treatment of patients with high-grade serous ovarian cancer (HGSOC), often resulting in the selection of an ineffective treatment course. Using PET imaging with the radiotracer (4S)-4-(3-[18F]fluoropropyl)-L-glutamate ([18F]FSPG), Greenwood and colleagues showed decreased radiotracer uptake in chemotherapy-resistant A2780 tumors compared with parental drug-sensitive tumors, demonstrating the ability of [18F]FSPG to detect upregulated antioxidant pathways present in drug-resistant cancer. [18F]FSPG may, therefore, enable the identification of HGSOC patients who are refractory to standard-of-care, allowing the transferal of drug-resistant patients to alternative therapies and thereby improving outcomes in this disease.To understand the immunological nature of glioblastoma, Park and colleagues examined the expression of immune checkpoint inhibitory receptors in tumor-infiltrating CD8+ T cells from patients with primary glioblastoma and their differentiation status. In addition, they investigated the reinvigoration ability of tumor-infiltrating CD8+ T cells by ex vivo treatment with anti-PD-1 and anti-CTLA-4. They demonstrated that the differentiation status of tumor-infiltrating CD8+ T cells determines the reinvigoration ability. This study provides a rationale for developing optimal strategies for immune checkpoint inhibitor treatment in patients with glioblastoma.

Structurally diverse diterpenoids from Isodon ternifolius collected from three regions

Phytochemical investigation of the aerial parts of Isodon ternifolius from three regions in Yunnan Province afforded 9 new diterpenoids, ternifoliusins A–I (1–9), together with 26 known diterpenoids. Among them, ternifoliusins A–D possess rare 6,7:8,15-diseco-7,20-olide-6,8-cyclo-ent-kaurane diterpenoid scaffold. The structures of all compounds were determined by comprehensive spectroscopic analysis, as well as quantum chemical calculation methods. Moreover, the molecular constitutions and configurations of ternifoliusins A, E, and F (1, 5, 6) were confirmed by single-crystal X-ray diffraction analysis. Ten compounds were assayed for their cytotoxic activity against the HL-60, SMMC-7721, A-549, MCF-7, and SW-480 human tumor cell lines, and compounds 13, 17, 19, 20, 31 and 34 were demonstrated to be active against all of the five human tumor cell lines.

Local Delivery of Ox40l, Cd80, and Cd86 mRNA Kindles Global Anticancer Immunity.

Localized expression of effector molecules can initiate antitumor responses through engagement of specific receptors on target cells in the tumor microenvironment. These locally induced responses may also have a systemic effect, clearing additional tumors throughout the body. In this study, to evoke systemic antitumor responses, we utilized charge-altering releasable transporters (CART) for local intratumoral delivery of mRNA coding for costimulatory and immune-modulating factors. Intratumoral injection of the CART-mRNA complexes resulted in mRNA expression at the site of administration, transfecting a substantial proportion of tumor-infiltrating dendritic cells, macrophages, and T cells in addition to the tumor cells, resulting in a local antitumor effect. Using a two-tumor model, we further show that mRNA therapy locally administered to one tumor stimulated a systemic antitumor response, curing both tumors. The combination of Ox40l-, Cd80-, and Cd86-encoding mRNA resulted in the local upregulation of proinflammatory cytokines, robust local T-cell activation, and migration of immune cells to local draining lymph node or to an anatomically distant tumor. This approach delayed tumor growth, facilitated tumor regression, and cured tumors in both A20 and CT26 tumor models. These results highlight mRNA-CART therapy as a viable approach to induce systemic antitumor immunity from a single localized injection. SIGNIFICANCE: The mRNA-CART system is a highly effective delivery platform for delivering immunostimulatory genes into the tumor microenvironment for potential therapeutic development.

Preliminary study on the effect of lipopolysaccharide challenge on the genes expression of inflammatory factors in A549 cells

To explore the effects of lipopolysaccharide (LPS) on the expression of inflammatory genes in A549 cells line under different concentrations and different action time, this study laid the foundation for further establishment of acute respiratory distress syndrome (ARDS) cell model in the optimal concentration-time way. A549 cells line was incubated routinely in 5% CO2 incubator at 37 centigrade with high glucose DMEM medium which included 10% fetal calf serum. Cells in logarithmic phase was cultured for passage, the cells was count to adjust cell density to (5-7)×105 and tile evenly in six-hole plate. Cells were cultivated for 2 days and once the cells confluence to 50%-60%, serum-free medium DMEM was changed for 12 hours cultivation. 10 mg LPS was added to 10 mL DMEM for oscillated blending to prepare 1 g/L stock solution. 0.5, 1.0 and 2.5 mL LPS stock solution was taken respectively and diluted LPS stock solution for 50 mL constant volume to prepare 0, 10, 20 and 50 mg/L LPS working solution. Then 0, 10, 20 and 50 mg/L LPS solution was added to react for 0, 1, 3 and 5 hours respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine mRNA expression of A549 cells line interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α). LPS action of 10 mg/L for 0 hour was used as the time control group, LPS action of 0 mg/L for 1 hour was used as the concentration control group, and the gene expression was calculated with 2-ΔΔCt method. (1) As to the time factor, with the same action of LPS concentration, the relative expression levels of inflammatory genes (IL-6, IL-1β and TNF-α) in A549 cells line after being treated with 10 mg/L LPS for 1 hour were significantly higher than those for 0 hour respectively [IL-6 mRNA (2-ΔΔCt): 5.71±0.42 vs. 1.00±0.00, IL-1β mRNA (2-ΔΔCt): 5.63±0.30 vs. 1.00±0.00, TNF-α mRNA (2-ΔΔCt): 5.38±0.61 vs. 1.00±0.00, all P < 0.01], and there were no significant changes in the expression levels of inflammatory genes in A549 cells line of other time groups. (2) As to the concentration factor, with the same action time, the relative expression levels of inflammatory genes (IL-6, IL-1βand TNF-α) in A549 cells line after being treated with 10 mg/L LPS for 1 hour were significantly higher than with 0 mg/L for 1 hour respectively [IL-6 mRNA (2-ΔΔCt): 5.70±0.64 vs. 1.00±0.00, IL-1β mRNA (2-ΔΔCt): 6.25±0.25 vs. 1.00±0.00, TNF-α mRNA (2-ΔΔCt): 5.57±0.25 vs. 1.00±0.00, all P < 0.01], there were no significant changes in the expression levels of inflammatory genes in A549 cells line of other concentration groups. The LPS concentration of 10 mg/L and the action time of 1 hour are the most suitable concentration-time conditions for establishing ARDS cell models of A549 cells line.

Comparative analysis of chemical composition, antioxidant and anti-proliferative activities of Italian Vitis vinifera by-products for a sustainable agro-industry

Vitis vinifera leaves are wine industry wastes. In this study, the chemical composition, antioxidant and anti-proliferative activity of six Italian grapevine leaves extracts (Arvino, Gaglioppo, Greco Nero, Magliocco Canino, Magliocco Dolce, and Nocera) were evaluated. HPLC analyses revealed quercetin as dominant constituent (127.52–187.33 mg/kg) followed by rutin (55.99–143.67 mg/kg). The antioxidant activity was determined using DPPH, ABTS, FRAP and β-carotene bleaching tests. Gaglioppo showed the highest radical scavenging ability with IC50 of 7.2 and 19.1 μg/mL, for DPPH and ABTS, respectively. Magliocco Dolce showed a 1.6-times higher FRAP activity than that of the positive control BHT. The anti-proliferative activity was determined by SRB assay against MCF-7, MDA-MB-231, A549 and COR-L23 human tumor cells. Greco Nero showed the highest anti-proliferative activity against MDA-MB-231 with IC50 of 28.4 μg/mL. Based on the obtained results grape leaves should be considered an interesting ingredient for the development of functional food products.

Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies

PurposeRecent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs.ProceduresThe irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [111In]DOTA-NB/[177Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography.ResultsNo binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed.ConclusionsThis study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.

RRx-001 is a phase III aerospace-derived small molecule that immunonormalizes the tumor microenvironment.

156 Background: Tumor associated macrophages (TAMs) with M2-like phenotypes produce angiogenic factors and cytokines which lead to vascular immaturity. M2 cytokines also impair immune cell activity. CD47 and SIRP⍺ are innate immune checkpoints which inhibit macrophage phagocytosis and enhance M2 polarization. Disruption of the CD47/SIRP⍺ axis polarize TAMs away from an M2-like phenotype to normalize tumor vessels and stimulate antitumor immunity. Normalized vessels facilitate delivery of drugs and immune cells and also sensitizes tumors to therapy and promotes M1-like TAM phenotype. RRx-001 modulates TAM infiltration and phenotype via CD47/SIRP⍺ downregulation, which normalizes both the tumor vasculature, and the immune cell milieu. Methods: The effect of RRx-001 on intratumoral blood flow was evaluated both preclinically and clinically with dynamic contrast MRI. An in vitro phagocytotic assay was used to determine whether RRx-001 promoted engulfment of A549 tumor cells by macrophages. Transcriptional mRNA profiling in murine tumor associated macrophages (TAMs) was performed to analyze the cytokine profile of TAMs in the presence or absence of RRx-001. Athymic mice with intracranial GBM43 were administered RRx-001 (10 mg/kg) followed 4 hours later by 0.4 mg of irinotecan by tail vein on days 12, 18, 24 and 30 after implantation of tumor cells. Mice were euthanized 24 hours after last irinotecan administration, with brains immediately resected and tumor tissue dissected prior to snap-freezing by immersion in liquid nitrogen. Results: RRx-001 was shown to induce: vascular normalization with increased intratumoral perfusion, dual downregulation of CD47 and SIRP⍺, increased phagocytosis, improved delivery of chemotherapy and increased T cell infiltration. Conclusions: RRx-001 leads to vascular normalization and enhanced delivery of chemotherapy and immune cell infiltration. The increase in tumor perfusion diminishes hypoxia and also serves to polarize TAMs away from an M2-like state. These results also suggest and are borne out by clinical biopsies that the effect of RRx-001 depends on the number of TAMs present in the tumor microenvironment with higher numbers leading to better activity.

Pea-like nanocabins enable autonomous cruise and step-by-step drug pushing for deep tumor inhibition

Pea-like nanocabins (HA@APT§DOX) were designed for deep tumor inhibition. The AS1411 aptamer (APT) constituted “core shelf” which guaranteed DOX “beans” could be embedded, while the outer HA acted as “pea shell” coating. During the circulation (primary orbit), HA@APT§DOX could autonomously cruise until leak through tumor vasculature. Upon tumor superficial site, the “pea shell” could be degraded by highly expressed hyaluronic acid enzymes (HAase) and peel-off, resulting in orbit changing of released APT§DOX to reach the deep tumor tissue. Furthermore, APT§DOX could be specifically uptaken into A549 tumor cells (secondary orbit). Finally, DOX was released under the acidic environment of lysosome, and delivered into nuclear (targeting orbit) to achieve drug pushing for deep tumor inhibition. More importantly, the in vivo imaging and anti-tumor effects evaluations showed that these nanocabins could effectively enhance drugs accumulation in tumor sites and inhibit tumor growth, with reduced systemic toxicity in 4T1 tumor-bearing mice.

Chemical constituents of Patrinia heterophylla Bunge and selective cytotoxicity against six human tumor cells

Ethnopharmacological relevancePatrinia heterophylla Bunge, known as “Mu-Tou-Hui” in China, is distributed in most provinces and regions of China. As a traditional medicinal plant, which was first found in <Ben- Cao-Gang- Mu>. In many traditional herbal books, there are records of “Mu-Tou-Hui” of treatment for uterine bleeding, cancer, swelling pain, leukemia, etc. However, there are few studies on the chemical constituents of Patrinia heterophylla Bunge. Aim of the studyTo investigate the chemical constituents of P. heterophylla and the basis of their antitumor activity. Materials and methods15 compounds were isolated from roots and rhizomes of P. heterophylla by repeating various column chromatography techniques, whose structures were determined by organic spectrum analysis methods and compared with published data. The cytotoxicities were evaluated by MTT assay on six cancer cell lines: human melanoma cell (A375), human hepatocellular carcinoma cell (SMMC-7721), human gastric cancer cell (SGC-7901), human cervical cancer cell (HeLa), human colon cancer cell (HCT-116), and human breast cancer cell (MDA-MB-231). The apoptosis-inducing activities of compounds 1, 5, 12 and 15 in A375 tumor cell determined by flow cytometry. ResultFive phenylpropanoids, ethyl caffeate (1), coniferaldehyde (5), trans-p-coumaryl aldehyde (6), caffeic acid methyl ester (12), and 3,4-dihydroxycinnamic acid (15), four acetophenones, 1-(2,4-dihydroxyphenyl) ethanone (2), 2′,5′-Dihydroxyacetophenone (3), cynanchone A (8), and cynandione A (10), two phenols, vanillin (4) and catechol (9), two iridoids, sarracenin (7) and patriscabrol (11), one alkane, tetracosane (14), and one coumarin, scopoletin (13), were isolated from the EtOH extracts. Of them, compounds 1–10, 12 and 14–15 were isolated for the first time from the roots of P. heterophylla. Compounds 1 and 15 were reported for the first time with in vitro inhibitory activity against tumor cells. MTT assay showed that compounds 1, 5–9, 12–13 and 15 had selective cytotoxic activities (IC50 27.20–163.03 μM) against tumor cells. Apoptosis detected by flow cytometry revealed that compounds 1, 5, 12 and 15 can induce apoptosis for A375 at low concentrations when the concentrations of compounds 1, 5, 12 and 15 are the value of 14, 40, 34, 108 μM, the percentages of apoptotic cells were about 50%. ConclusionsCompounds 1–10, 12 and 14–15 were isolated for the first time from the P. heterophylla. This result enriches the previous studies on the chemical constituents of P. heterophylla. Compounds 1 and 15 were reported for the first time to have cytotoxic activities against tumor cells. Compounds 1, 5, 6, 7, 12, 15 showed cytotoxic activities against tumor cells. This result reveals that the active ingredient of P. heterophylla are composed of phenylpropanoids, iridoids and coumarins. This study provides some theoretical basis for the anti-tumor research of P. heterophylla.

Novel Bi-Functional 14-mer Peptides with Both Ovarian Carcinoma Cells Targeting and Magnetic Fe₃O₄Nanoparticles Affinity.

Fe3O4 magnetic nanoparticles (Fe3O4-MNPs) have attracted much interest for their potential medical applications due to their desirable magnetic properties. However, their potential cytotoxicity, high RES clearance in circulation, and nonspecific distribution in tissue might be the main obstacles in practice. In the present study, a novel bi-functional 14-mer peptide with both ovarian carcinoma cells targeting and magnetic Fe3O4 nanoparticles affinity was designed and synthesized, and then a facile and effective modification method was developed to bestow the Fe3O4-MNPs with tumor-targeting capability via modification, using the bi-functional peptides. First, on the basis of a tumor-targeting 7-mer peptide QQTNWSL (Q-L) and another Fe3O4-MNPs-targeting 7-mer peptide TVNFKLY (T-Y)—screened by phage-displayed peptide libraries—two bi-functional 14-mer peptides sequenced as LSWNTQQ-YLKFNVT (abbreviated as LQ-YT) and QQTNWSL-YLKFNVT (QL-YT) were synthesized through combining the Q-L peptide and T-Y peptide in predetermined configurations. Their specificity for bonding with A2780 tumor cells and affinity for Fe3O4-MNPs were verified. Then the bi-functional 14-mer peptides were applied to modify the Fe3O4-MNPs. Results showed that both bi-functional 14-mer peptides could be conjugated to the Fe3O4-MNPs surface with high affinity. Immunofluorescence and Prussian blue staining assays indicated that the LQ-YT-modified Fe3O4-MNPs could specifically bond to A2780 tumor cells. In addition to our findings suggesting that more β-turns and random coils are conducive to increasing polypeptide surface area for binding and exposing the target group and bonding sites on LQ-YT to external targets, we demonstrated that the bi-functional 14-mer peptide has affinity for Fe3O4-MNPs, and that Fe3O4-MNPs, which was modified with a 14-mer peptide, could be bestowed with a targeting affinity for ovarian carcinoma cells.

CD146-Targeted Multimodal Image-Guided Photoimmunotherapy of Melanoma.

For melanoma resistant to molecularly targeted therapy and immunotherapy, new treatment strategies are urgently needed. A molecularly targeted theranostic pair may thus be of importance, where the diagnostic probe facilitates patient stratification and the therapeutic companion treats the selected cases. For this purpose, flow cytometry is used to assess the CD146 level in melanoma cells. Based on YY146, a CD146‐specific monoclonal antibody, an imaging probe 89Zr‐Df‐YY146 is synthesized and its diagnostic performance is evaluated by positron emission tomography (PET) imaging. Furthermore, a photoimmunotherapy (PIT) agent IR700‐YY146 is developed and the therapeutic effect of IR700‐YY146 PIT is assessed comprehensively. CD146 is highly expressed in A375 and SK‐MEL‐5 cells. 89Zr‐Df‐YY146 PET readily detects CD146‐positive A375 melanomas. Tumor accumulation of 89Zr‐Df‐YY146 peaks at 72 h with an uptake value of 26.48 ± 3.28%ID g−1, whereas the highest uptake of the nonspecific 89Zr‐Df‐IgG is 4.80 ± 1.75%ID g−1. More importantly, IR700‐YY146 PIT effectively inhibits the growth of A375 tumors, owing to production of reactive oxygen species, decreased glucose metabolism, and reduced expression of CD146. To conclude, 89Zr‐Df‐YY146 and IR700‐YY146 are a promising theranostic pair with the former revealing CD146 expression in melanoma as a PET probe and the latter specifically treating CD146‐positive melanoma as an effective PIT agent.

Chemical constituents of the trunks and roots of Thuja sutchuenensis

Five new compounds including two stilbenes, designated thujasutchins A (1) and B (2), two phenolic compounds namely thujasutchins C (3) and D (4), as well as one sesquiterpene thujasutchin E (5), were isolated from the 95% ethanolic extract from the trunks and roots of Thuja sutchuenensis. Their structures were determined by means of extensively spectroscopic analysis including UV, IR, HRESIMS, 1H and 13C NMR (COSY, HSQC, HMBC). Moreover, compounds 1, 3–5 were evaluated for in vitro cytotoxic activities against SF-268, MCF-7, HepG-2, and A549 tumor cell lines.

Milk exosomes - Natural nanoparticles for siRNA delivery

Gene-silencing with targeted siRNAs has great potential as a therapeutic approach for various diseases including cancer. However, intracellular delivery of siRNA is challenging. We used bovine milk exosomes as a novel system for siRNA delivery. First, we demonstrated that exosomes can deliver endogenous RNA payloads into recipient cells. Next, we loaded siRNA against specific genes including VEGF, EGFR, AKT, MAPK, and KRAS. We utilized 5′−32P-labeled siKRAS as a tracer and found exosome loading with siRNA could be variable. We demonstrated that the siRNA of loaded exosomes is stable and resist degradation. Our results indicated that siRNAs against target genes ranged from 2 to 10-fold knockdown in expression levels in various cancers. Since mutated KRAS has been implicated in the development of various cancers including lung cancer, we tested a mutant-allele specific siRNA against KRASG12S, in A549 cells. We observed a dose-dependent anti-proliferative activity against A549 cells treated with exosomes carrying siKRASG12S. We observed significant inhibition of A549 tumor xenografts in animals treated with folic acid-functionalized exosomes carrying siKRAS. In summary, milk-derived exosomes represent a viable natural nano-carrier for the delivery of siRNA for therapeutic application against cancer.

Ultra-pH-sensitive indocyanine green-conjugated nanoprobes for fluorescence imaging-guided photothermal cancer therapy

Photothermal therapy (PTT) has been recognized as a promising approach for cancer treatment due to its minimal invasiveness and low systemic side effects. However, developing a photothermal agent with accurate tumor imaging capability is a prerequisite for the efficient PTT. Here, we developed a series of ultra-pH-sensitive indocyanine green (ICG)-conjugated nanoparticles for fluorescence imaging-guided tumor PTT. These nanoparticles exhibited high fluorescence activation ratio (~100-fold) with sharp pH transition (ΔpHon/off <0.25), and superior temperature response than free ICG. The in vivo imaging experiments demonstrated that the nanoparticles generated excellent tumor-to-normal tissue contrast through pH-triggered fluorescence activation in tumor sites, which provided information on tumor mass location, boundaries, and shape. Moreover, comparing to free ICG, the nanosystem had significantly longer blood circulation time and more accurate tumor targeting, providing efficient photothermal therapeutic effect against A549 tumor in living animals. In conclusion, this nanoplatform offers a potential strategy for imaging-guided cancer PTT.

Evaluation of Novel Imaging Devices for Nanoparticle-Mediated Fluorescence-Guided Lung Tumor Therapy

Nonsurgical and minimally invasive approaches for early-stage peripheral lung cancer are needed to avoid the known morbidity of surgical resection, particularly in high-risk patients. We previously demonstrated the utility of multifunctional porphyrin-phospholipid nanoparticles (porphysomes) for fluorescence imaging and phototherapy after preferential accumulation into tumors. The objective of this study was to demonstrate the feasibility ofporphysome-mediated imaging and photothermal therapy using a newly developed fiberscope and thoracoscope. To prepare this technology for clinical translation, we developed a porphysome-specific fiberscope (scanning fiber endoscope and porphysome-specific thoracoscope), both capable of detecting porphysome fluorescence, for image-guided transbronchial and transpleural photothermal therapy to treat endobronchial/peribronchial and subpleural tumors, respectively. These were tested in three animal models: human lung cancer xenografts (A549) in mice, orthotopic VX2 lung tumors in rabbits, and exvivo pig lung into which A549 tumor tissue was transplanted. The scanning fiber endoscope, with a 1.2-mm diameter, is small enough to pass through the working channel of a conventional bronchoscope and could visualize porphysome-laden tumors located inside or close to the peripheral bronchial wall. The porphysome-specific thoracoscope system had high sensitivity for porphysome fluorescence and enabled image-guided thoracoscopic resection of porphysome-accumulating tumors close to the pleura. Porphysomes also enhanced the efficacy of scanning fiber endoscope-guided transbronchial photothermal therapy and porphysome-specific thoracoscope-guided transpleural photothermal therapy, resulting in selective and efficient tumor tissue ablation in the rabbit and pig models. These results support the potential for clinical translation of this novel platform to affect nonsurgical and minimally invasive treatment options for early-stage peripheral lung cancer.

Abstract B128: Timing and sequence of CpG and anti-OX40 is critical for in situ vaccination

Abstract Immunotherapy seeks to enhance or manipulate host antitumor immunity by various approaches, among them, in situ vaccination. In situ vaccination is a local intervention in which immune enhancing agents are injected locally into one site of tumor, triggering a T-cell immune response locally that then travels to attack cancer throughout the body. We have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting systemic immune response, if any, is detected by the regression of the distant, untreated tumor. In this test for abscopal therapeutic effects, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG)—a TLR9 ligand—and agonist anti-OX40 antibody has provided the most impressive results. This combination lead to durable disease control and long-term treatment-free survival in multiple mouse models of cancer. CpG induced myeloid cells to secrete cytokines, which subsequently induced OX40 expression on T-cells. Thus, we hypothesized that administration sequence and timing may affect the antitumor responses of in situ vaccination. In order to screen for the best sequence and timing we implanted A20 lymphoma tumors bilaterally in opposite sides of the abdomen of Balb/C mice. After tumors were established, one tumor was injected at the selected sequence and timing with the test agents and the resulting immune response was monitored by the measuring growth of the distant, untreated tumor. The systemic antitumor response required the presence of both CD4+ and CD8+ T-cells, as mice treated with the corresponding depleting antibodies were unable to control tumor growth. Even a single injection of low-dose CpG (50µg) and anti-OX40 (8µg) resulted in a fully protective systemic immune response. In addition, the cured animals were protected from rechallenge with the same A20 tumor but not unrelated tumors. Decreasing the dose even further to 10µg CpG and 1µg anti-OX40 partially preserved the therapeutic response with a long-term survival of 60%. Concurrent administration of CpG and anti-OX40 resulted in eradication of both local and distant disease. Sequential administration of CpG followed by anti-OX40 preserved the therapeutic efficacy. However, sequential administration of anti-OX40 followed by CpG significantly attenuated the therapeutic effect. While CpG followed by a 24- or 48-hour-delayed anti-OX40 treatment preserved the therapeutic efficacy of concurrent therapy, 72h delay in anti-OX40 administration resulted in reduced therapeutic effect. These data demonstrate the importance of the administration sequence for fully protective and curative antitumor immune responses. Our data suggest that the anti-OX40 antibody should be administered at the same time as CpG or somewhat delayed but not the other way around. The combination of anti-OX40 and CpG is currently studied in a phase I trial. Our results here impact the planning of future clinical trials of in situ vaccination with these two agents. Citation Format: Idit Sagiv-Barfi, Debra K. Czerwinski, Ronald Levy. Timing and sequence of CpG and anti-OX40 is critical for in situ vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B128.

Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

Radiotherapy is a frequent mode of cancer treatment, although the development of radioresistance limits its effectiveness. Extensive investigations indicate the diversity of the mechanisms underlying radioresistance. Here, we aimed to explore the effects of extracellular signal-regulated kinase 5 (ERK5) on lung cancer radioresistance and the associated mechanisms. Our data showed that ERK5 is activated during solid lung cancer development, and ectopic expression of ERK5 promoted cell proliferation and G2/M cell cycle transition. In addition, we found that ERK5 is a potential regulator of radiosensitivity in lung cancer cells. Mechanistic investigations revealed that ERK5 could trigger IR-induced activation of Chk1, which has been implicated in DNA repair and cell cycle arrest in response to DNA double-strand breaks (DSBs). Subsequently, ERK5 knockdown or pharmacological inhibition selectively inhibited colony formation of lung cancer cells and enhanced IR-induced G2/M arrest and apoptosis. In vivo, ERK5 knockdown strongly radiosensitized A549 and LLC tumor xenografts to inhibition, with a higher apoptotic response and reduced tumor neovascularization. Taken together, our data indicate that ERK5 is a novel potential target for the treatment of lung cancer, and its expression might be used as a biomarker to predict radiosensitivity in NSCLC patients.

The synthesis, characterization and antitumor activities of A C-type arsenomolybdate

A new hetropolyoxometal compound (NH4)14Fe0.5[As2Fe6Mo22O85(H2O)]·16H2O (1) has been synthesized and characterized by elemental analysis, thermal analysis and single crystal X-ray diffraction. Crystal data for 1: Triclinic, P1, a = 12.6245(4) Å, b = 16.8421(5) Å, c = 27.1396(8) Å, α = 98.892(2)°, β = 91.161(2)°, γ = 104.129(3)°, V = 5518.5(3) Å3, Z = 2. The structural analysis has revealed that 1 represents a rare example of hexa-iron arsenomolybdate. The inhibitory effects of the compound 1 on A549 (human lung cancer) tumour cells were investigated by MTT assay. It is shown that compared with the reference polyoxometalate-based antitumor agent (NH3Pri)6[Mo7O24]·3H2O (PM-8), 1 present higher antitumor activities.