Stephen Kalscheuer

Differential expression of microRNAs in early-stage neoplastic transformation in the lungs of F344 rats chronically treated with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

While numerous microRNAs (miRNAs) have been reported to alter their expression levels in human lung cancer tissues compared with normal tissues, the function of these miRNAs and their contribution to the long process of lung cancer development remains largely unknown. We applied a tobacco-specific carcinogen-induced cancer model to investigate the involvement of miRNAs in early lung cancer development, which could also provide information on potential, early biomarkers of lung cancers. Male F344 rats were first chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogen present in tobacco products, for up to 20 weeks. The expression profiles of miRNAs in rat lungs were then determined. As measured by miRNA microarrays and confirmed by Northern blot and real-time polymerase chain reaction analyses, NNK treatment reduced the expression of a number of miRNAs, such as miR-101, miR-126*, miR-199 and miR-34. Significantly, these miRNAs overlap with previously published reports on altered miRNA expression in human lung cancer samples. These miRNAs might, therefore, represent early-response miRNAs that signify the molecular changes associated with pulmonary tumorigenesis. Moreover, we identified cytochrome P450 (CYP) 2A3, a critical enzyme in rat lungs that activates NNK to render it carcinogenic, as a potential target of miR-126*. NNK treatment in rats repressed miR-126* but induced CYP2A3 expression, a mechanism that may potentiate the oncogenic effects of NNK.

Exploiting nanotechnology to overcome tumor drug resistance: Challenges and opportunities☆

Tumor cells develop resistance to chemotherapeutic drugs through multiple mechanisms. Overexpression of efflux transporters is an important source of drug resistance. Efflux transporters such as P-glycoprotein reduce intracellular drug accumulation and compromise drug efficacy. Various nanoparticle-based approaches have been investigated to overcome efflux-mediated resistance. These include the use of formulation excipients that inhibit transporter activity and co-delivery of the anticancer drug with a specific inhibitor of transporter function or expression. However, the effectiveness of nanoparticles can be diminished by poor transport in the tumor tissue. Hence, adjunct therapies that improve the intratumoral distribution of nanoparticles may be vital to the successful application of nanotechnology to overcome tumor drug resistance. This review discusses the mechanisms of tumor drug resistance and highlights the opportunities and challenges in the use of nanoparticles to improve the efficacy of anticancer drugs against resistant tumors.

Folic Acid Functionalized Nanoparticles for Enhanced Oral Drug Delivery

The oral absorption of drugs that have poor bioavailability can be enhanced by encapsulation in polymeric nanoparticles. Transcellular transport of nanoparticle-encapsulated drug, possibly through transcytosis, is likely the major mechanism through which nanoparticles improve drug absorption. We hypothesized that the cellular uptake and transport of nanoparticles can be further increased by targeting the folate receptors expressed on the intestinal epithelial cells. The objective of this research was to study the effect of folic acid functionalization on transcellular transport of nanoparticle-encapsulated paclitaxel, a chemotherapeutic with poor oral bioavailability. Surface-functionalized poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles loaded with paclitaxel were prepared by the interfacial activity assisted surface functionalization technique. Transport of paclitaxel-loaded nanoparticles was investigated using Caco-2 cell monolayers as an in vitro model. Caco-2 cells were found to express folate receptor and the drug efflux protein, p-glycoprotein, to high levels. Encapsulation of paclitaxel in PLGA nanoparticles resulted in a 5-fold increase in apparent permeability (Papp) across Caco-2 cells. Functionalization of nanoparticles with folic acid further increased the transport (8-fold higher transport compared to free paclitaxel). Confocal microscopic studies showed that folic acid functionalized nanoparticles were internalized by the cells and that nanoparticles did not have any gross effects on tight junction integrity. In conclusion, our studies indicate that folic acid functionalized nanoparticles have the potential to enhance the oral absorption of drugs with poor oral bioavailability.

Inhibition of vinyl carbamate-induced pulmonary adenocarcinoma by indole-3-carbinol and myo-inositol in A/J mice

In previous studies, we reported that indole-3-carbinol (I3C) and myo-inositol (MI) inhibit lung adenoma induced by tobacco smoke carcinogens in A/J mice. In this paper, we extended our work and examined the effects of I3C (70 or 30 micromol/g diet) and MI (56 micromol/g diet) against vinyl carbamate (VC)-induced lung adenocarcinoma by administering the agents from 1 week after the second of two injections of VC until termination of the study at week 18. The higher dose of I3C decreased multiplicities of tumors on the surface of the lung (26%, P = 0.0005), carcinoma incidence (38%), multiplicity (67%, P < 0.0001) and size (complete abolition of carcinoma with an area of >1.0 cm(2)) as well as adenoma with cellular pleomorphism (46%, P < 0.0001). The lower dose of I3C was less effective. MI decreased multiplicities of pulmonary surface tumors (20%, P = 0.0005), adenoma with cellular pleomorphism (40%, P < 0.0001) and lung adenoma (52%, P < 0.0001) and the proportion of the biggest carcinoma (carcinoma with an area of >1.0 cm(2), P < 0.05). Immunoblot analyses of lung tissues for potential target identification showed that I3C (70 micromol/g diet) inhibits IkappaBalpha degradation, nuclear factor-kappaB activation, expression of cyclooxygenase-2, phospho-Akt and fatty acid synthase (FAS) and activates caspase-3 and poly ADP ribose polymerase cleavage. The effect of MI was limited to inhibition of phospho-Akt and FAS expression. Our data show that I3C and MI inhibit lung carcinoma and provide a basis for future evaluation of these compounds in clinical trials as chemopreventive agents for current and former smokers.

Silicate Esters of Paclitaxel and Docetaxel: Synthesis, Hydrophobicity, Hydrolytic Stability, Cytotoxicity, and Prodrug Potential

We report here the synthesis and selected properties of various silicate ester derivatives (tetraalkoxysilanes) of the taxanes paclitaxel (PTX) and docetaxel (DTX) [i.e., PTX-OSi(OR)3 and DTX-OSi(OR)3]. Both the hydrophobicity and hydrolytic lability of these silicates can be (independently) controlled by choice of the alkyl group (R). The synthesis, structural characterization, hydrolytic reactivity, and in vitro cytotoxicity against the MDA-MB-231 breast cancer cell line of most of these derivatives are described. We envision that the greater hydrophobicity of these silicates (vis-à-vis PTX or DTX itself) should be advantageous from the perspective of preparation of stable aqueous dispersions of amphiphilic block-copolymer-based nanoparticle formulations.

Nanoparticles Containing High Loads of Paclitaxel-Silicate Prodrugs: Formulation, Drug Release, and Anticancer Efficacy.

We have investigated particle size, interior structure, drug release kinetics, and anticancer efficacy of PEG-b-PLGA-based nanoparticles loaded with a series of paclitaxel (PTX)-silicate prodrugs [PTX-Si(OR)3]. Silicate derivatization enabled us to adjust the hydrophobicity and hydrolytic lability of the prodrugs by the choice of the alkyl group (R) in the silicate derivatives. The greater hydrophobicity of these prodrugs allows for the preparation of nanoparticles that are stable in aqueous dispersion even when loaded with up to ca. 75 wt % of the prodrug. The hydrolytic lability of silicates allows for facile conversion of prodrugs back to the parent drug, PTX. A suite of eight PTX-silicate prodrugs was investigated; nanoparticles were made by flash nanoprecipitation (FNP) using a confined impingement jet mixer with a dilution step (CIJ-D). The resulting nanoparticles were 80-150 nm in size with a loading level of 47-74 wt % (wt %) of a PTX-silicate, which corresponds to 36-59 effective wt % of free PTX. Cryogenic transmission electron microscopy images show that particles are typically spherical with a core-shell structure. Prodrug/drug release profiles were measured. Release tended to be slower for prodrugs having greater hydrophobicity and slower hydrolysis rate. Nanoparticles loaded with PTX-silicate prodrugs that hydrolyze most rapidly showed in vitro cytotoxicity similar to that of the parent PTX. Nanoparticles loaded with more labile silicates also tended to show greater in vivo efficacy.

Abstract 368: Phage display derived antibodies for the detection of mesenchymal CTCs in TNBC

Enumeration of circulating tumor cells (CTCs) in peripheral blood of patients allows clinicians to obtain immediate evidence of metastatic dissemination. CELLSEARCH® is the only method currently approved for CTC detection (1). The critical assumption of the CELLSEARCH® platform is that CTCs will express EpCAM, based on the fact that the cell of origin in carcinoma is epithelial. However, the key cellular event that gives rise to CTCs is epithelial-to-mesenchymal transition (EMT) and the acquisition of the mesenchymal phenotype within the primary tumor. Recent studies have demonstrated EMT marker gene expression in CTCs, especially during disease progression. (2, 3). Thus, the full complement of CTCs may not be effectively captured with existing CTC technologies that rely solely on epithelial marker expression. We have developed a competition based cell panning procedure which employs a commercially available single chain Fv (scFv) phage display library to identify antibodies with selective affinity to EMT phenotypic cells. HMLE cells as well as a sub-clone stably expressing Twist1 (HMLE-Twist1; a transcription factor known to induce EMT) were used for the panning. A two-color fluorescent scheme was employed to selectively sort the target cells from mixed populations containing the control cells, in the presence of phage library, via fluorescence activated cell sorting. Repeated iterations of this procedure yielded a scFv (‘clone 6′) with selective affinity to HMLE-Twist1cells. We then engineered a full length human IgG1 antibody in which the variable region sequences were derived from clone 6 scFv. The clone 6 IgG showed high affinity for metastatic cell lines (MDA-MB-231-LM2, LCC6, and HS578T). The antibody was also able to detect the presence of CTCs in an orthotopic MDA-MB-231-LM2 mouse tumor model. Intra-sample comparison showed distinct cell populations captured with anti-EpCAM antibody and Clone 6 IgG. Unlike those captured by anti-EpCAM antibody, the cells captured by Clone 6 were negative for EpCAM expression and were mesenchymal in phenotype. Future studies will examine the usefulness of clone 6 IgG in recognizing CTCs in patient samples. References 1. M. Cristofanilli et al., New Engl J Med 351, 781 (2004). 2. B. Aktas et al., Breast Cancer Res : BCR 11, R46 (2009). 3. G. Kallergi et al., Breast Cancer Research : BCR 13, R59 (2011). Citation Format: Stephen Kalscheuer, Jayanth Panyam. Phage display derived antibodies for the detection of mesenchymal CTCs in TNBC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 368. doi:10.1158/1538-7445.AM2015-368

Abstract 2181: Antibody-conjugated nanoparticles for targeting metastatic triple-negative breast cancer

Early detection and the availability of new treatments have improved the survival rates of patients presenting with local or regional breast cancer to as high as 99% and 85%, respectively. On the contrary, patients with metastatic disease have a dismal 5-year survival rate of 17%. Thus, there is an urgent need for treatment strategies directed towards metastasis. Our lab has developed antibodies (Clone 6 and AM6) capable of recognizing tumor cells that have undergone epithelial-to-mesenchymal transition (EMT), a key step in the generation of circulating tumor cells and metastasis. The goal of the current study was to determine whether we use these antibodies as targeting ligands for directing anticancer drug-loaded polymeric nanoparticles to metastatic triple negative breast cancer cells as a novel therapeutic option. Polymeric PLGA nanoparticles loaded with paclitaxel, a chemotherapeutic agent, were functionalized with the antibodies using thiol-maleimide chemistry. We optimized the conjugation reaction in order to achieve maximal cell uptake of nanoparticles without compromising antibody binding. In vitro studies were carried out in an MDA-MB-231 derivative cell line with enhanced lung metastatic potential as well as a melanoma metastatic cell line M12. Clone 6 nanoparticles and AM6 nanoparticles showed significant improvement in cellular uptake as well as retention. A competition experiment confirmed target-mediated uptake of nanoparticles. Cytotoxicity studies showed improved cell kill using Clone 6 nanoparticles and AM6 nanoparticles. Based on these promising in vitro results, we are currently carrying out in vivo studies in mice. The development of a targeted drug delivery system for the treatment of metastatic triple negative breast cancer can significantly enhance the survival rate for patients who often have a life-expectancy of less than one year. Citation Format: Vidhi Khanna, Stephen Kalscheuer, Ameya Kirtane, Jayanth Panyam. Antibody-conjugated nanoparticles for targeting metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2181. doi:10.1158/1538-7445.AM2017-2181

Abstract 4593: Glycoengineered antibodies for click chemistry applications

Antibody-drug conjugates (ADC) hold considerable promise as anticancer agents. A critical determinant of the effectiveness of ADCs is the chemistry that is used to conjugate the payload. Currently used approaches include primarily conjugation to either side-chain amine or carboxylic acid groups or conjugation to thiols. Because these reactions are not site specific and not easily controlled, these chemistries can result in reduced affinity for the target antigen. Further, these conjugation reactions lack selectivity and can result in heterogeneous mixtures of products that differ in the sites and stoichiometry of modification. We investigated a glycoengineering strategy that enables the introduction of artificial azide groups in the antibody without affecting their antigen affinity. This is based on the observation that glycosyltransferases can incorporate non-natural sugars (e.g., azido mannose) at different sites on an IgG molecule. The azide groups inthese artificial sugars are then available to react with alkynes through copper-catalyzed ‘click’ chemistry or with strained alkynes such as dibenzyl cyclooctyne (DBCO) allowing for biorthogonal, copper-free ‘click’ chemistry. Because the sugars are added reproducibly and at asite that does not affect antigen binding, the glycoengineering technology would overcome problems associated with traditional conjugation strategies. Using this approach, azide groups were introduced in anti-CD133 and anti-perlecan antibodies. Further, the azide groups were available to react with various DBCO conjugates including fluorophores, drug molecules and nanoparticles. Importantly, the addition of artificial sugar and subsequent azide-alkyne reactiondid not affect the affinity of the antibody for the target antigen. Conjugation of nanoparticles to antibodies using this approach resulted in enhanced cellular uptake of the nanoparticles. Similarly, conjugation of a cytotoxin to the antibody resulted in enhanced cell kill in vitro. Weexpect that this glycoengineering strategy will prove to be a unique platform technology that will have a significant impact on antibody-based therapeutics. Citation Format: Drishti Sehgal, Stephen Kalscheuer, Tanmoy Sadhukha, Jayanth Panyam. Glycoengineered antibodies for click chemistry applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4593. doi:10.1158/1538-7445.AM2017-4593

Abstract A42: The dietary agents indole-3-carbinol and myo-inositol prevent lung cancer induced by vinyl carbamate in A/J mice

Abstracts: Frontiers in Cancer Prevention Research 2008 A42 Because of their expected safety, food-derived products could be highly interesting for development as chemopreventive agents. Among promising cancer preventive dietary agents are indole-3-carbinol (I3C) and myo -inositol (MI). I3C is derived from the enzymatic hydrolysis of indolyl glucosinolates contained in cruciferous vegetables. MI is found in a variety of animal and plant foods in its free form, as an inositol-containing phospholipid, and inositol hexaphosphate (IP6, phytic acid,). In earlier studies, we found that both I3C and MI prevent lung adenoma induced by tobacco smoke carcinogens in A/J mice. In the present study, we examined the efficacy of I3C and MI to prevent lung adenocarcinoma. Female A/J mice were injected i.p. with two doses of vinyl carbamate (VC, 0.32 mg per mouse) dissolved in physiological saline, 1 week apart, and then randomized into the different treatment groups. After waiting 1 week following the second injection of the carcinogen, mice were maintained either on untreated control diet, or diets supplemented with I3C (70 µmol/g diet) or MI (56 µmol/g diet) for 15 weeks. At necropsy, lungs were removed and grossly visible lesions on the surface of all lobes of the lung were counted and the size of the tumors determined using a dissecting microscope. Then, the left lobe of the lung from each mouse was preserved in buffered formalin for histopathological analysis. Mice treated with VC and fed conventional diet had 42.6 ± 11.9 lung tumors/mouse. Compared to mice treated with VC alone, carcinogen-treated mice given I3C or MI-supplemented diet had a significantly lower tumor multiplicity, 31.6 ±4.8 and 33.9 ± 6.1 lung tumors, corresponding to a reduction by 26% and 20% respectively (p = 0.0003 for VC + I3C group and p = 0.0005 for VC + MI group compared using Wilcoxon non-paramteric test). Classification of the tumors into three different size groups ( 2 mm) showed that the multiplicity of only the larger tumors was reduced by the chemopreventive agents (25.4 ± 6.8, 16.4 ± 8.3 and 0.69 ±1.3 tumors/mouse for 2 mm size groups, respectively, in mice treated with VC alone versus 28.8 ± 4.9, 2.8 ± 2.0, and 0.05 ± 0.22 tumors/mouse in the group treated with VC + I3C and 26.8 ± 5.1, 6.7 ± 2.3, and 0.40 ± 0.6 tumors/mouse in VC + MI-treated mice). Furthermore, we determined the effect of the chemopreventive agents on the incidence and multiplicity of the different microscopic lesions (hyperplasia, adenoma, adenoma with cellular pleomorphism and adenocarcinoma). Preliminary results showed that I3C reduced the incidence (71% in the VC group versus 36% in the VC + I3C group) and multiplicity (1.1 /mouse in the VC group versus 0.4 /mouse in the VC + I3C group) of adenocarcinoma. I3C also reduced the multiplicity (3.6/mouse in the VC group versus 2.4 /mouse in the VC + I3C group) of adenoma with cellular pleomorphism lesions. In mice maintained on MI-supplemented diet, there was no change either in the incidence or multiplicity of adenocarcinoma but the multiplicity of adenoma with cellular pleomorphism lesions was markedly reduced (3.6/mouse in the VC group versus 1.5/mouse in the VC + MI group). The chemopreventive agents did not affect the incidence or multiplicity of hyperplastic or adenoma lesions. In conclusion, our results indicate the potential lung cancer chemopreventive effects of I3C and MI in former smokers. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A42.