Orthotopic Lung Cancer Model Research Articles

Strategy to Enhance Dendritic Cell‐Mediated DNA Vaccination in the Lung

AbstractHere, a new paradigm is introduced to promote pulmonary DNA vaccination. Specifically, it is demonstrated that nanoparticles designed to rapidly penetrate airway mucus (mucus‐penetrating particle or MPP) enhance the delivery of an inhaled model DNA vaccine (i.e., ovalbumin‐expressing plasmids) to pulmonary dendritic cells (DC), leading to robust and durable local and trans‐mucosal immunity. In contrast, mucus‐impermeable particles are poorly taken up by pulmonary DC following inhalation, despite their superior ability to mediate DC uptake in vitro compared to MPP. In addition to the enhanced immunity achieved in mucosal surfaces, inhaled MPP unexpectedly provides significantly greater systemic immune responses compared to gold‐standard approaches applied in the clinic for systemic vaccination, including intradermal injection and intramuscular electroporation. It is also shown here that inhaled MPP significantly enhances the survival of an orthotopic mouse model of aggressive lung cancer compared to the gold‐standard approaches. Importantly, it is discovered that MPP‐mediated pulmonary DNA vaccination induces memory T‐cell immunity, particularly the ready‐to‐act effector memory‐biased phenotype, both locally and systemically. The findings here underscore the importance of breaching the airway mucus barrier to facilitate DNA vaccine uptake by pulmonary DC and thus to initiate full‐blown immune responses.

Abstract B32: Silencing of oncogenic KRAS by a mutant-favoring short interfering RNA

Abstract Oncogenic mutations in the KRAS gene are well-established drivers of cancer. Promising preclinical strategies including RNA interference (RNAi) have been developed to target oncogenic KRAS function, yet a clinically effective therapy to directly target KRAS remains to be achieved. While genetic knockdown of mutant KRAS (mKRAS) with RNAi is one promising approach, current methods are not mutant-selective, and thus they also decrease normal KRAS levels, raising concerns about normal cell toxicity. Here, we describe a custom short interfering RNA (siRNA) oligonucleotide (D1) designed to preferentially bind the most commonly occurring RAS missense mutations in codons 12 and 13. Our approach designed the siRNA to have three mismatches binding wild-type mRNA, but only two mismatches for the oncogenic mRNAs, thus having binding affinities more favorable for the mutant-specific sequences. Using NIH/3T3 mouse fibroblasts stably expressing human KRAS WT or mutant cDNA sequences and KRAS mutant lung cancer cell lines, we determined that D1 siRNA preferentially reduced mKRAS mRNAs versus the wild-type sequence at the transcriptional and protein levels. Phenotypically, D1 siRNA significantly reversed mKRAS-induced morphologic and growth transformation of NIH/3T3 cells. Furthermore, D1 siRNA significantly impaired the proliferation of several mKRAS lung cancer cells in 2D as well as 3D spheroids embedded in an extracellular matrix. To overcome clinical limitations of delivering the siRNA to lung tumors, we evaluated lipid nanoparticles (LNPs) as carriers of D1 siRNAs in orthotopic mKRAS lung cancer models and observed that Cy5.5-labeled LNPs are highly enriched in lung tumors and metastases. Compared with control siRNA-LNP treated mice, 344SQ (KRAS G12D) tumor-bearing mice injected once with D1 siRNA-LNPs had drastically decreased KRAS protein levels in tumors 72 h later (73% decrease, p=0.001). Using an orthotopically injected luciferase-labeled A549 (KRAS G12S) human lung cancer model, D1 siRNA-LNP treated mice had pronounced reductions in luciferase signal (56% reduction, p=0.045), chest wall metastases (67% reduction, p<0.001) and contralateral lung metastases (73% reduction, p<0.01). Interestingly, D1 siRNA-LNP did not impair tumor growth when applied in the 344SQ mKRAS orthotopic mouse lung model, consistent with the mKRAS-independent growth of this line. This result supports our conclusion that the effects of D1 siRNA-LNP in the A549 model are KRAS specific and highlight the importance of determining RAS-dependency a priori for KRAS-targeting therapeutics. Taken together, our data indicate a novel use of RNAi to specifically target oncogenic KRAS-driven cancers. Citation Format: Bjoern Papke, Amanda E.D. Van Swearingen, Anne Y. Feng, Salma H. Azam, Emily B. Harrison, Runying Yang, Adrienne D. Cox, Channing J. Der, Chad V. Pecot. Silencing of oncogenic KRAS by a mutant-favoring short interfering RNA [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B32.

Iodine-124 Labeled Gold Nanoclusters for Positron Emission Tomography Imaging in Lung Cancer Model.

This work reports the synthesis, radiolabeling and imaging studies of iodine-124 labeled peptide modified gold nanoclusters (AuNCs) as positron emission tomography (PET) tracer for lung cancer. The novel modified Au nanoclusters were successfully synthesized by conjugation of tumortargeting peptide luteinizing hormone releasing hormone (LHRH) to human serum albumin (HAS) as a scaffold, resulting in 73% labeling yield of 124I-LHRH-HSA AuNCs. After rapid purification, the radiochemical purity was above 98%. Dynamic PET study in normal rats showed high liver accumulation and rapid lung clearance. Both the PET and fluorescence imaging in A549 xenografted tumor model demonstrated certain amount of tumor uptake. In orthotopic lung cancer model, the tumor sites could be clearly visualized between 2 to 5 hours in PET images. The higher radioactivity concentration in the left lung which inoculated orthotopic tumor than right lung also exhibited the targeting properties. The biological properties of this iodine-124 labeled nanoclusters afford potential applications for early diagnosis of lung cancer with PET.

PLAG Exerts Anti-Metastatic Effects by Interfering with Neutrophil Elastase/PAR2/EGFR Signaling in A549 Lung Cancer Orthotopic Model.

The effectiveness of chemotherapy and radiotherapy to treat lung cancer is limited because of highly metastatic nature. Novel strategies and drugs to attenuate metastatic activity are urgently required. In this study, red fluorescence proteins (RFP)-labeled A549 human lung cancer cells were orthotopically implantation, where they developed primary tumors. Metastasis in brain and intestines were reduced by up to 80% by treatment with 100 mpk 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) compared with that in control mice. PLAG treatment also reduced the migration of the primary tumors. Interestingly, substantial neutrophil infiltration was observed in the tumors in control mice. The neutrophil contribution to A549 cell metastatic activity was examined in in vitro co-culture system. Metastatic activity could be achieved in the A549 cells through epidermal growth factor receptor (EGFR) transactivation mediated by protease activating receptor 2 (PAR2) receptor. Neutrophil elastase secreted from tumor-infiltrating neutrophils stimulated PAR2 and induced EGFR transactivation. However, this transactivation was inhibited by inducing PAR2 degradation following PLAG treatment and metastatic activity was effectively inhibited. PLAG attenuated cancer metastatic activity via modulated PAR2/EGFR transactivation by accelerating PAR2 degradation. These results suggest PLAG as potential therapeutic agent to combat tumor metastasis via regulating the activation signal pathway of PAR2 by tumor infiltrate-neutrophils.

5-Azacytidine inhaled dry powder formulation profoundly improves pharmacokinetics and efficacy for lung cancer therapy through genome reprogramming

BackgroundEpigenetic therapy through demethylation of 5-methylcytosine has been largely ineffective in treating lung cancer, most likely due to poor tissue distribution with oral or subcutaneous delivery of drugs such as 5-azacytidine (5AZA). An inhalable, stable dry powder formulation of 5AZA was developed.MethodsPharmacokinetics of inhaled dry powder and aqueous formulations of 5AZA were compared to an injected formulation. Efficacy studies and effect of therapy on the epigenome were conducted in an orthotopic rat lung cancer model for inhaled formulations.ResultsInhaled dry powder 5AZA showed superior pharmacokinetic properties in lung, liver, brain and blood compared to the injected formulation and for all tissues except lung compared to an inhaled aqueous formulation. Only dry powder 5AZA was detected in brain (~4-h half-life). Inhaled dry powder was superior to inhaled aqueous 5AZA in reducing tumour burden 70–95%. Superiority of inhaled 5AZA dry powder was linked to effectively reprogramming the cancer genome through demethylation and gene expression changes in cancer signalling and immune pathways.ConclusionsThese findings could lead to widespread use of this drug as the first inhaled dry powder therapeutic for treating local and metastatic lung cancer, for adjuvant therapy, and in combination with immunotherapy to improve patient survival.

Antisense Oligonucleotides: A 50‐nm‐Sized Micellar Assembly of Thermoresponsive Polymer‐Antisense Oligonucleotide Conjugates for Enhanced Gene Knockdown in Lung Cancer by Intratracheal Administration (Adv. Therap. 2/2020)

In article number 1900123, Kanjiro Miyata and co-workers develop a micellar assembly (ASOball) from a covalent conjugation between a thermoresponsive polymer, poly(2-n-propyl-2-oxazoline), and antisense oligonucleotide (ASO). The ASOball exhibits prolonged retention of ASO payloads in the lung and an orthotopic lung cancer model tissue, resulting in a superior gene knockdown effect in the lung cancer.

Inherently PET/CT Dual Modality Imaging Lipid Nanocapsules for Early Detection of Orthotopic Lung Tumors.

Accurate diagnosis of cancer at an early stage is the key to reduce cancer mortality and improve survival. PET imaging has high sensitivity but low spatial resolution, while CT imaging has good spatial location information. Therefore, the combination of PET and CT imaging can provide complementary advantages to achieve accurate early diagnosis of tumors. However, currently developed PET or CT imaging agents have only a single function. Here, we designed and constructed a self-assembled lipid nanocapsule encapsulated with iodixanol and labeled with self-chelated 64Cu for precise PET/CT imaging of tiny lung tumor. The lipid nanocapsule self-assembled in water using LPPC-Ce6, a conjugate of chlorin e6 (Ce6) and lysophosphatidylcholine (LPPC), to form a bilayer vesicular structure. 64Cu was embedded in the center of the tetrapyrrole ring of Ce6 by natural capture ability for Cu2+ ions. GLT21.T, the aptamer targeting lung cancer, was conjugated to the surface of the lipid nanocapsules. Iodixanol was loaded into the cavity of the lipid nanocapsule (64Cu@LCI-apt). In the nanostructure, the loading of iodixanol was sufficiently high, and the specific activity could be flexibly adjusted according to imaging requirements. The prepared 64Cu@LCI-apt achieves excellent radiolabeling efficiency, stability and effective targeting of lung tumor. In an early orthotopic lung cancer model, 64Cu@LCI-apt demonstrated the capabilities of sensitive PET imaging and enhanced contrast CT imaging to enable efficient high-quality PTE/CT imaging of tiny orthotopic lung tumor with a diameter of 500 μm. 64Cu@LCI-apt has great potential for early, sensitive, and accurate diagnosis of tumors through dual-mode PET/CT imaging.

Nanoparticles for the Treatment of Metastatic Non Small Cell Lung Cancer with 225Ac

Non small cell lung cancer (NSCLC) is the most common form of primary lung neoplasia with nearly 40% of patients having metastasis at the time of diagnosis, resulting in a 5year survival rate ranging from 13-36% in patients with nodal metastasis, and decreasing to as little as 2% for those with distant metastasis. Furthermore, pulmonary metastatic disease is the most common form of secondary lung tumors, being identified in 30-55% of all cancer patients. Targeted α-radiotherapy (TAT) agents have great potential for treating micro-metastatic disease, given their short, densely ionizing track length and high relative biologic effect (RBE). The use of in vivo alpha generators allows for multiple alpha decays from a single radioactive nucleus, but retaining the radioactive daughters at the target site throughout the decay process is a challenge. We present preclinical data describing a multilayered nanoparticle-antibody conjugate that can deliver multiple α radiations from the in vivo α-generator 225Ac at biologically relevant receptor sites while also containing the radioactive daughters at those sites. The layered nanoparticles (NP) consist of an 225Ac-doped (La0.5Gd0.5)PO4 core coated with four layers of GdPO4 and an outer layer of Au. These multi-shell particles combine the radiation resistance of crystalline lanthanide (Ln) phosphate to contain atoms of the therapeutic radionuclide and its radioactive daughters, the magnetic properties of gadolinium for facile separation during synthesis, and the chemistry of gold for attachment of targeting agents to the nanoparticle surface. In a proximity delivery model of cancer, 225Ac-NPs conjugated to mAb 201b resulted in a 73% decrease in the number of EMT6 colonies in the lung five days after treatment [1]. On biodistribution studies and SPECT/CT imaging, over 85% of the injected dose was delivered to the target tissue, and approximately 90% of the fourth daughter, 213Bi, was retained in the target 24 hours after injection. Competition assays demonstrated specific binding of the conjugated radiopharmaceutical to the target. Current studies are evaluating the application of these 225Ac nanoparticles to A549 NSCLC grown in a mouse orthotopic lung cancer model, while a spontaneous cancer model in canine patients is also being explored. [1]M.F. McLaughlin, J.D. Robertson, P.H. Pevsner, J.S. Wall, S. Mirzadeh, and J. Kennel, “LnPO4 Nanoparticles Doped with Ac-225 and Sequestered Daughters for Targeted Alpha Therapy,” Cancer Biotherapy and Radiopharmaceuticals 29(1), 34-41 (2014).

A 50‐nm‐Sized Micellar Assembly of Thermoresponsive Polymer‐Antisense Oligonucleotide Conjugates for Enhanced Gene Knockdown in Lung Cancer by Intratracheal Administration

AbstractFor intratracheal delivery of antisense oligonucleotides (ASOs) to lung cancer, a nano‐sized micellar assembly is fabricated from an ASO conjugate with thermoresponsive poly(2‐n‐propyl‐2‐oxazoline) (POX). POX‐ASO conjugate is prepared via a copper‐free click conjugation between azide‐terminal POX and dibenzocyclooctyne‐terminal ASO. Through the thermoresponsive transition of the POX segment from hydrophilic to hydrophobic at temperatures above a critical solution temperature, a 50‐nm‐sized micellar assembly with a narrow size distribution is successfully fabricated when the hydrophobicity of POX segment is strengthened by increasing the molecular weight to 30 kDa. The micellar assembly, termed “ASOball”, elicits more efficient cellular uptake of ASO and significantly higher gene knockdown, compared with non‐conjugated ASO, with negligible cytotoxicity in cultured human lung cancer cells. When the ASOballs are intratracheally administrated into an orthotopic lung cancer model mouse, they show enhanced retention in the tumor tissue 24 h post‐administration, compared with non‐conjugated ASO. Ultimately, the ASOballs significantly reduce the expression level of target long noncoding RNA (lncRNA) by ≈55% at a dose of 15 µg per mouse in a sequence‐specific manner 72 h post‐administration, whereas no significant knockdown effect is observed for non‐conjugated ASO. These results demonstrate the strong potential of ASOballs for intratracheal ASO delivery to lung cancers.

MA17.09 5-Azacytidine Inhaled Dry Powder Formulation Profoundly Improves Pharmacokinetics and Efficacy for Lung Cancer Therapy

Epigenetic therapy through its ability to activate hundreds of genes silenced by promoter hypermethylation in lung cancer could produce durable and sustained tumor regression. The demethylating agent 5-azacytidine (5AZA) is unstable in aqueous solution and subject to hydrolysis and catabolism by cytidine deaminase (CDA) in liver, thereby reducing drug concentration after systemic administration prior to reaching the lung. Delivering 5AZA by inhalation could mitigate these barriers. A stable, respirable (3.5 μM) dry powder formulation of 5AZA was generated. Pharmacokinetic (PK) studies in rats compared systemic dosing to inhaled delivery of a dry powder or aqueous formulation of 5AZA in the presence and absence of the CDA inhibitor tetrahyroduridine (THU). An orthotopic nude rat lung cancer model compared efficacy of inhaled dry powder versus aqueous 5AZA for treatment of engrafted human adenocarcinoma (Calu6, Calu3), adenocarcinoma in situ (H358), and squamous cell (RH2) tumor lines. Three weeks following engraftment (lungs contain multiple tumors), rats were treated 4 times weekly for 4 weeks, then sacrificed to assess tumor burden and genome-wide effects on the methylome in vehicle and treated tumors using the Illumina EPIC array. Plasma PK showed a ∼10-fold increase in area under the curve (AUC) and a 1.5 and 5-fold increase in maximum concentration (Cmax) comparing inhaled dry powder (0.6 mg/kg) to systemic (2 mg/kg, equivalent to the human injectable dose of 75 mg/m2) and inhaled aqueous (0.6 mg/kg) 5AZA that was augmented by THU. Inhaled dry powder and aqueous 5AZA PK in lung were similar and greatly exceeded systemic (30-fold Cmax; 47-fold AUC). PK in liver and brain were superior for dry powder with 7- and 26-fold increase in AUC and 7- and 3.3-fold increase in Cmax compared to systemic or aqueous dosing. The efficacy study comparing inhaled delivery of equivalent doses (0.6 mg/kg lung dose) showed dry powder was significantly better than aqueous with a 70–80% compared to 33–50% reduction in tumor burden for Calu6, Calu3, and RH2 and equally effective in largely curing H358 tumors. A significantly increased median number of genes (175–320 versus 25–270) exhibiting ≥30% demethylation of CpGs across their promoter region was seen for Calu6, Calu3, and H358 tumors exposed to dry powder versus aqueous 5AZA, with equivalent numbers of genes demethylated for RH2. Delivery of a dry powder formulation of 5AZA via an inhaler could be used to treat local and metastatic lung cancer (Support–CA196590).

Immunogenic Cell Death Induced by Tumor Treating Fields (TTFields) Enhances Efficacy When Combined with Anti-PD-1 Therapy in Lung and Colon Cancer Animal Models

Tumor Treating Fields (TTFields) therapy is an FDA approved anti-neoplastic treatment modality delivered via noninvasive application of low-intensity, intermediate-frequency, non-ionizing alternating electric fields. Previously it was shown that TTFields induce immunogenic cell death. In this study we evaluated whether TTFields-induced cell death can be further enhanced by the combination with anti-PD-1 therapy in murine models. Bone marrow derived dendritic cells (DCs) were co-incubated with TTFields treated cells and phagocytosis by DCs and DCs maturation were evaluated. The combination of TTFields and anti-PD-1 was evaluated in short duration treatment protocol in orthotopic lung cancer model and long duration treatment protocol in heterotopic subcutaneous colon cancer model. Tumor volume was monitored and analysis was performed for phenotypic characterization of infiltrating immune cells. We demonstrate that TTFields treated cells promote phagocytosis by DCs, DCs maturation in vitro, and promote immune cells recruitment in vivo. We also show that the combined treatment of lung tumor-bearing mice with TTFields plus anti-PD-1 led to a significant decrease in tumor volume compared to the other treatment groups. Significant increases in CD45+ tumor infiltrating cells were observed in the TTFields plus anti-PD-1 group in both models. In the orthotopic lung tumors, the CD45+ infiltrating cells, specifically macrophages and DCs, demonstrated upregulation of surface PD-L1 expression following short treatment duration. Correspondingly, cytotoxic T-cells isolated from these tumors have shown higher levels of IFN-γ production relative to untreated mice. In the heterotopic subcutaneous colon cancer tumors, significant increases in T-cell infiltration was observed following long treatment duration of heterotopic subcutaneous colon cancer tumors with TTFields plus anti-PD-1. These results demonstrate robust efficacy with concurrent application of TTFields and anti PD-1 therapy in mouse models of lung and colon cancers. These data suggest that combining TTFields with anti-PD-1 may achieve tumor control by further enhancing antitumor immunity.

The Hsp27-Mediated IkBα-NFκB Signaling Axis Promotes Radiation-Induced Lung Fibrosis.

Lung fibrosis is a major side effect experienced by patients after lung cancer radiotherapy. However, effective protection strategies and underlying treatment targets remain unclear. In an effort to improve clinical outcomes, pharmacologic treatment of fibrosis is becoming increasingly popular; however, no ideal therapeutic strategy is yet available. We used a mouse model to irradiate high focal (90 or 75 Gy) to 3-mm volume of the left lung. Lung tissues of mice were subjected to microarray, mRNA expression, and immunohistochemical analysis. Correlations of radiation (IR)-induced epithelial-mesenchymal transition (EMT) were validated in lung cell lines using appropriate treatments to activate or inhibit selected pathways. The expression of Hsp27 was increased during IR-induced lung fibrosis in a mouse model. Inhibition of functional Hsp27 using shRNA and a synthetic small molecule inhibitor (J2) in lung cells alleviated IR-mediated EMT. The activation of NFkB pathways via direct interaction between Hsp27 and IkBα resulted in increased expressions of Twist, IL-1β, and IL-6 and facilitated IR-mediated EMT, which was identified as an underlying mechanism of Hsp27-mediated fibrosis after IR. J2 also inhibited IR-induced lung fibrosis in an orthotopic lung cancer model, and IR-induced lung fibrotic tissues from patients showed higher expression of Hsp27 than unirradiated lungs. Collectively, IkBα-NFkB signaling activation by Hsp27, which resulted in the facilitation of Twist, IL1β, and IL6 expression, is involved in the EMT process that is tightly connected to the development of IR-induced lung fibrosis. Our findings also suggest that inhibition of Hsp27 has the potential to become a valuable therapeutic strategy for IR-induced lung fibrosis.

Abstract 1499: Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer

Abstract Mounting clinical and preclinical evidence demonstrates an important role for the intestinal microbiome in mediating efficacy of immune checkpoint inhibitor (ICI) antibody therapy across a number of tumor contexts. We sought to determine the effects of microbiome modulation on ICI therapy in a clinically recapitulative orthotopic murine lung cancer model. The microbiome of C57Bl/6NHsd mice was sterilized with antibiotic (ampicillin, streptomycin and colistin) for ten days, resulting in a 99.9% mean decrease in fecal aerobic and anaerobic bacterial load in comparison to naïve mice, handled under specific pathogen free (SPF) conditions. Luciferase-expressing murine Lewis lung carcinoma cells (LL/2-Red-FLuc) were surgically implanted into the left lung parenchyma of all animals. Animals were treated with locoregional radiotherapy (2x 9Gy fractions) targeted to the left lung. The gastrointestinal microbiome was reconstituted via oral gavage Q3D of ~1e09/dose commensal A. muciniphila (A. muc) and E. hirae (E. hir) over five doses or sterile saline as control and animals were randomized within commensal/saline treatment groups to equivalent mean tumor burden as measured by Lumina Series III In-Vivo Imaging System (IVIS). Animals were then administered anti-murine PD-1 or isotype control (0.25 mg) antibody (Ab) treatments Q3D over four doses via intraperitoneal injection. Tumor growth was monitored by IVIS over the course of the study, and ex-vivo IVIS was performed on lungs at endpoint (Day 12 post-tumor implantation). Tumor growth of microbiota non-reconstituted antibiotic-sterilized animals was only slightly inhibited by anti-PD-1 therapy. Animals administered commensal A. muc and E. hir and treatments also displayed slightly inhibited tumor growth kinetics, similar to those observed under saline/anti-PD-1 therapy. Fecal microbial sequencing and immunophenotypic analyses are ongoing. This study demonstrates the utility and ongoing development of a clinically recapitulative contextually accurate preclinical murine lung cancer model to assess the effects of specific microbiota in mediating the efficacy of anti-tumor immunotherapy. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Nicholas Rivelli, Alexandra Kury, Sallyann Vu, Gavin Gagnon, Veronica Ritchie, Kasey Reardon, Catarina Costa, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer [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 1499.

Abstract 3623: Mesenchymal stem cells engineered with TAT peptide functionalized nanoparticles improve therapeutic efficacy of paclitaxel in an orthotopic lung tumor model

Abstract Tumors are characterized by uneven vascular perfusion with near normal blood flow in the outer most regions, while the inner regions can be avascular. In addition, elevated interstitial fluid pressure and rigid extracellular matrix compromise intra-tumoral solute transport and hence limited drug efficacy. Mesenchymal stem cells (MSCs) have the unique advantage of being able to migrate specifically to both primary tumors and metastases following systemic administration. However, poor payload capacity of MSCs limits their use as drug delivery carriers. To address this issue, we investigated polymeric nanoparticles that were functionalized with transactivator of transcription (TAT) peptide. Paclitaxel loaded PLGA nanoparticles (15-16 % w/w paclitaxel; diameter of 225 ± 7 nm; and zeta potential of −15 ± 4 mV) were prepared by emulsion-solvent evaporation method, followed by TAT-conjugation to the surface of nanoparticles via maleimide-thiol chemistry. Nanoengineered MSCs were generated by incubating MSCs in suspension with 100 µg/mL paclitaxel loaded nanoparticles for 4 hours at 37°C. Our studies demonstrated that TAT functionalization improved the paclitaxel loading in MSCs. In addition, engineering MSCs with TAT functionalized nanoparticles did not affect the differentiation and migration properties of MSCs. Further, MSCs engineered with TAT peptide functionalized nanoparticles resulted in significant inhibition of tumor growth and overall improved survival in a mouse orthotopic model of lung cancer compared to that with free drug treatment. In summary, our results demonstrated that MSCs nanoengineered using TAT functionalized nanoparticles can serve as an efficient carrier for tumor specific delivery of anticancer drugs, resulting in greatly improved therapeutic efficacy. Citation Format: Swayam Prabha, Gopikrishna Moku, Buddhadev Layek, Jayanth Panyam. Mesenchymal stem cells engineered with TAT peptide functionalized nanoparticles improve therapeutic efficacy of paclitaxel in an orthotopic lung tumor model [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 3623.

Liver X receptor agonist T0901317 inhibits the migration and invasion of non-small-cell lung cancer cells in vivo and in vitro.

Liver X receptors are recognized as important regulators of cholesterol, fatty acid metabolism, inflammatory responses, and glucose homeostasis. The antineoplastic properties of synthetic liver X receptor (LXR) agonists (T0901317 and GW3965) have been reported in human carcinomas. Epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) is a first-line treatment for non-small-cell lung cancer patients with EGFR mutations. We used scratch and transwell assays to analyze cell migration and invasion. We evaluated tumor migration and invasion in vitro using a fluorescent orthotopic lung cancer model. An MMP9 (mouse) enzyme-linked immunosorbent assay kit was used to measure serum MMP9 concentrations. Protein expression was identified by western blot assays. In this study, we determined the effects of T0901317 and/or an EGFR-TKI on the lung cancer cell lines A549 and HCC827-8-1 in vitro and in vivo. We confirmed that the combination of the LXR agonist T0901317 and gefitinib can inhibit the migration and invasion of lung cancer both in vivo and in vitro, and this effect was possibly achieved by the inhibition of the ERK/MAPK signaling pathway. Our study showed that the combination of the LXR agonist T0901317 and gefitinib can inhibit the migration and invasion of lung cancer both in vivo and in vitro.

Improving Payload Capacity and Anti-Tumor Efficacy of Mesenchymal Stem Cells Using TAT Peptide Functionalized Polymeric Nanoparticles.

Mesenchymal stem cells (MSCs) accumulate specifically in both primary tumors and metastases following systemic administration. However, the poor payload capacity of MSCs limits their use in small molecule drug delivery. To improve drug payload in MSCs, we explored polymeric nanoparticles that were functionalized with transactivator of transcription (TAT) peptide. Paclitaxel loaded poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (15–16% w/w paclitaxel; diameter of 225 ± 7 nm; and zeta potential of −15 ± 4 mV) were fabricated by emulsion-solvent evaporation method, followed by TAT-conjugation to the surface of nanoparticles via maleimide-thiol chemistry. Our studies demonstrated that TAT functionalization improved the intracellular accumulation and retention of nanoparticles in MSCs. Further, nano-engineering of MSCs did not alter the migration and differentiation potential of MSCs. Treatment with nano-engineered MSCs resulted in significant (p < 0.05) inhibition of tumor growth and improved survival (p < 0.0001) in a mouse orthotopic model of lung cancer compared to that with free or nanoparticle encapsulated drug. In summary, our results demonstrated that MSCs engineered using TAT functionalized nanoparticles serve as an efficient carrier for tumor specific delivery of anticancer drugs, resulting in greatly improved therapeutic efficacy.

Baicalein Inhibits Orthotopic Human Non-Small Cell Lung Cancer Xenografts via Src/Id1 Pathway.

Non-small cell lung cancer (NSCLC) is one of the most lethal cancers worldwide. Inhibitor of differentiation 1 (Id1) is the member mostly linked to tumorigenesis in Id family and a potential molecular target in cancer therapy. In the current study, we established an orthotopic lung cancer model by injecting athymic nude mice with A549 cells and evaluated the antitumor effect of baicalein and expression of Id1-related proteins in vivo and in vitro. Micro-CT images showed that tumor volume in baicalein group was significantly reduced. Western blot analysis revealed that baicalein suppressed the expression of Id1 protein, epithelial-to-mesenchymal transition (EMT) related molecules (N-Cadherin, vimentin), and angiogenesis related protein (VEGF-A), accompanied by upregulation of epithelial markers (such as E-cadherin). In addition, phosphorylation of upstream molecular Src was significantly restrained after baicalein treatment. This study firstly demonstrates that baicalein inhibits tumor growth in orthotopic human NSCLC xenografts via targeting Src/Id1 pathway.

Small animal image-guided radiotherapy

Small animal image-guided radiotherapy

Nanoparticles for the Treatment of Metastatic Non Small Cell Lung Cancer with 225Ac

Non small cell lung cancer (NSCLC) is the most common form of primary lung neoplasia with nearly 40% of patients having metastasis at the time of diagnosis, resulting in a 5year survival rate ranging from 13-36% in patients with nodal metastasis, and decreasing to as little as 2% for those with distant metastasis. Furthermore, pulmonary metastatic disease is the most common form of secondary lung tumors, being identified in 30-55% of all cancer patients. Targeted α-radiotherapy (TAT) agents have great potential for treating micro-metastatic disease, given their short, densely ionizing track length and high relative biologic effect (RBE). The use of in vivo alpha generators allows for multiple alpha decays from a single radioactive nucleus, but retaining the radioactive daughters at the target site throughout the decay process is a challenge. We present preclinical data describing a multilayered nanoparticle-antibody conjugate that can deliver multiple α radiations from the in vivo α-generator 225Ac at biologically relevant receptor sites while also containing the radioactive daughters at those sites. The layered nanoparticles (NP) consist of an 225Ac-doped (La0.5Gd0.5)PO4 core coated with four layers of GdPO4 and an outer layer of Au. These multi-shell particles combine the radiation resistance of crystalline lanthanide (Ln) phosphate to contain atoms of the therapeutic radionuclide and its radioactive daughters, the magnetic properties of gadolinium for facile separation during synthesis, and the chemistry of gold for attachment of targeting agents to the nanoparticle surface. In a proximity delivery model of cancer, 225Ac-NPs conjugated to mAb 201b resulted in a 73% decrease in the number of EMT6 colonies in the lung five days after treatment [1]. On biodistribution studies and SPECT/CT imaging, over 85% of the injected dose was delivered to the target tissue, and approximately 90% of the fourth daughter, 213Bi, was retained in the target 24 hours after injection. Competition assays demonstrated specific binding of the conjugated radiopharmaceutical to the target. Current studies are evaluating the application of these 225Ac nanoparticles to A549 NSCLC grown in a mouse orthotopic lung cancer model, while a spontaneous cancer model in canine patients is also being explored. [1]M.F. McLaughlin, J.D. Robertson, P.H. Pevsner, J.S. Wall, S. Mirzadeh, and J. Kennel, “LnPO4 Nanoparticles Doped with Ac-225 and Sequestered Daughters for Targeted Alpha Therapy,” Cancer Biotherapy and Radiopharmaceuticals 29(1), 34-41 (2014).

An orthotopic non-small cell lung cancer model for image-guided small animal radiotherapy platforms.

An orthotopic non-small cell lung cancer model in NMRI-nude mice was established to investigate the complementary information acquired from 80 kVp microcone-beam CT (micro-CBCT) and bioluminescence imaging (BLI) using different angles and filter settings. Different micro-CBCT-based radiation-delivery plans were evaluated based on their dose-volume histogram metrics of tumor and organs at risk to select the optimal treatment plan. H1299 cell suspensions injected directly into the lung render exponentially growing single tumor nodules whose CBCT-based volume quantification strongly correlated with BLI-integrated intensity. Parallel-opposed single angle beam plans through a single lung are preferred for smaller tumors, whereas for larger tumors, plans that spread the radiation dose across healthy tissues are favored. Closely mimicking a clinical setting for lung cancer with highly advanced preclinical radiation treatment planning is possible in mice developing orthotopic lung tumors. BLI and CBCT imaging of orthotopic lung tumors provide complementary information in a temporal manner. The optimal radiotherapy plan is tumor volume-dependent.