PDF file 100K, Legends for the supplementary figures

PDF file 135K, Figure S1 contains data about Ceritinib specificity and potency on crizotinib na?ve EML4-ALK wild-type cells. Figure S2 contains the Cell survival curves of H3122 CR1, MGH021-4 and MGH045 cells following treatment with Crizotinib or ceritinib corresponding to Fig2A. Figure S3 contains the Cell survival curves of Ba/F3 cells following treatment with Crizotinib or ceritinib corresponding to Fig4A. Figure S4 contains tumor growth curves corresponding to the Development of crizotinib resistant H2228 xenograft tumor models used in Fig 5A-D

The Cancer Cell Line Encyclopedia presents the first results from a large-scale screen of some 947 cancer cell lines with 24 anticancer drugs, with the aim of identifying specific genomic alterations and gene expression profiles associated with selective sensitivity or resistance to potential therapeutic agents. Cancer cell lines are widely used as preclinical models to gain mechanistic and therapeutic insight. Two manuscripts in this issue describe the large-scale genetic and pharmacological characterization of human cancer cell lines. Each group characterized collections of several-hundred cell lines using different platforms and analytical methods. Their results are complementary, and confirm that many human cell lines capture the genomic diversity of their respective cancers. Initial findings include the identification of a number of potential markers of drug sensitivity and resistance. For example, Garnett et al. report an association between EWS-FLI1 gene translocations, frequently found in Ewing's sarcoma, and sensitivity to PARP inhibitors, a class of drug currently in clinical trials for other cancer types. Barretina et al. report a possible association between SLFN11 expression and sensitivity to topoisomerase inhibitors. The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available1. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of ‘personalized’ therapeutic regimens2.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described.

Abstract Non–small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Herein, we report the first preclinical evaluation of the next-generation ALK TKI, ceritinib (LDK378), in the setting of crizotinib resistance. An interrogation of in vitro and in vivo models of acquired resistance to crizotinib, including cell lines established from biopsies of patients with crizotinib-resistant NSCLC, revealed that ceritinib potently overcomes crizotinib-resistant mutations. In particular, ceritinib effectively inhibits ALK harboring L1196M, G1269A, I1171T, and S1206Y mutations, and a cocrystal structure of ceritinib bound to ALK provides structural bases for this increased potency. However, we observed that ceritinib did not overcome two crizotinib-resistant ALK mutations, G1202R and F1174C, and one of these mutations was identified in 5 of 11 biopsies from patients with acquired resistance to ceritinib. Altogether, our results demonstrate that ceritinib can overcome crizotinib resistance, consistent with clinical data showing marked efficacy of ceritinib in patients with crizotinib-resistant disease. Significance: The second-generation ALK inhibitor ceritinib can overcome several crizotinib-resistant mutations and is potent against several in vitro and in vivo laboratory models of acquired resistance to crizotinib. These findings provide the molecular basis for the marked clinical activity of ceritinib in patients with ALK-positive NSCLC with crizotinib-resistant disease. Cancer Discov; 4(6); 662–73. ©2014 AACR. See related commentary by Ramalingam and Khuri, p. 634 This article is highlighted in the In This Issue feature, p. 621

Abstract Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements are sensitive to the ALK tyrosine kinase inhibitor (TKI) crizotinib. However, these cancers invariably relapse due to the development of resistance, and approximately 1/3 of such cancers develop resistance mutations within the ALK tyrosine kinase domain. Here we report the preclinical evaluation of the next-generation ALK TKI, LDK378 in the setting of crizotinib resistance. Using EML4-ALK mutant Ba/F3 cellular models, in vivo models of acquired resistance to crizotinib, and novel cell lines established from biopsies of crizotinib-resistant NSCLC patients, we have examined the efficacy of LDK378 in crizotinib-naïve and -resistant ALK-positive cancers. These studies reveal that LDK378 is more potent than crizotinib and effectively overcomes resistance in vitro and in vivo. In particular, LDK378 inhibits ALK harboring crizotinib resistance mutations, including L1196M, G1269A, I1171T and S1206Y. Cell lines derived from crizotinib-resistant biopsies were sensitive to LDK378, including one that did not harbor an ALK resistance mutation and was also sensitive to crizotinib, suggesting that some crizotinib-resistant cancers with wildtype ALK are still sensitive to complete ALK inhibition. We observed that LDK378 did not effectively overcome two crizotinib-resistant ALK mutations, G1202R and F1174C ALK, and mutations in one of these residues was identified in 5 out of 11 biopsies from patients with acquired resistance to LDK378. Altogether our results demonstrate that LDK378 can overcome many mechanisms of crizotinib resistance, consistent with emerging clinical data showing marked efficacy of LDK378 in patients with crizotinib-resistant disease. Citation Format: Luc Friboulet, Nanxin Li, Ryohei Katayama, Christian C. Lee, Justin F. Gainor, Adam S. Crystal, Pierre-Yves Michellys, Mark M. Awad, Noriko Yanagitani, Sungjoon Kim, AnneMarie Pferdekamper, Jie Li, Shailaja Kasibhatla, Frank Sun, Xiuying Sun, Su Hua, Peter McNamara, Sidra Mahmood, Elizabeth L. Lockerman, Naoya Fujita, Makoto Nishio, Jennifer L. Harris, Alice T. Shaw, Jeffrey A. Engelman. The ALK inhibitor LDK378 overcomes crizotinib resistance in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 957. doi:10.1158/1538-7445.AM2014-957

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

The systematic translation of cancer genomic data into knowledge of tumor biology and therapeutic possibilities remains challenging.Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic

Abstract Comprehensive genomic characterization of cancer is proceeding at a rapidly accelerating pace, mainly due to the expanded use of massively parallel sequencing. Despite the promise of cancer genomics, many cancer drugs still fail in the clinic due to nonresponsive patients and this translates into a significant unmet medical need. Accurate predictions of which patients are more likely to respond to drugs in development could speed clinical trials and personalize treatments. Here we propose the use of a compendium of experimentally tractable cancer model systems, ∼1000 human genomically-annotated cancer cell lines (at the level of gene expression, DNA copy number alterations and mutations), coupled with pharmacological profiling, to systematically link genetic and transcriptional features to drug response. This resource, the Cancer Cell Line Encyclopedia (CCLE), is available online at www.broadinstitute.org/ccle. Through computational predictive modeling we have both rediscovered molecular features that predict response to several drugs and also uncovered a number of novel potential biomarkers of sensitivity and resistance to targeted agents and chemotherapy drugs. For instance, we have found that response to topoisomerase 1 inhibitors seem to be driven by expression of a single gene. We have also observed that tissue lineage is a key predictor for sensitivity to certain compounds, providing rationale for clinical trials of these drugs in particular cancer types. Our cell line-based platform provides a valuable tool for the development of personalized cancer medicine, revealing critical tumor dependencies and helping to stratify patients for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5455. doi:10.1158/1538-7445.AM2011-5455

Abstract A c-MET/ALK kinase inhibitor crizotinib has demonstrated significant activity in lung cancer patients with EML4-ALK in clinical studies. However relapse (or acquired resistance) has also been reported. We have developed crizotinib resistant tumor models and used the models to evaluate the ALK inhibitor LDK378. LDK378 is a potent and selective ALK inhibitor that does not cross react with c-MET. In a mouse xenograft tumor model derived from the EML4-ALK+ lung cancer cell line NCI-H2228, LDK378 caused complete tumor regression when dosed orally at 25 mg/kg/day. After tumor bearing animals had been treated with LDK378 at 50 mg/kg/day for 14 days, remission was maintained for more than 4 months. In several NCI-H2228 tumor models that were induced to become resistant to crizotinib, LDK378 demonstrated efficacy at 50 mg/kg/day. Based on 4-wk GLP toxicology studies the drug exposure associated with this dose is predicted to be below the exposure at the MTD in humans. ALK resistance mutations reported in crizotinib relapsed patients were also found in the crizotinib resistant NCI-H2228 tumor models. The results from these preclinical studies suggest that LDK378 may be active in crizotinib-relapsed patients. A phase I clinical study of LDK378 has recently begun in both crizotinib-relapsed and crizotinib-naïve patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B232.

The Cancer Cell Line Encyclopedia (CCLE) represents a collaborative effort to assemble a comprehensive resource of human cancer models for basic and translational research. Thus far, the CCLE contains high-density SNP array data, gene expression microarray data and selected cancer gene mutation data for approximately 1,000 human cancer cell lines spanning many tumor types. Additionally, we are assessing the sensitivity of these same cell lines using a series of pharmacological compounds that represent both conventional cytotoxic and targeted agents. Another goal of the CCLE collaboration involves systematic integration of the genomic and pharmacologic datasets in order to identify putative targets of prevalent genetic alterations as well as predictors and modifiers of pharmacologic sensitivity and resistance. The availability of high-quality data generated by uniform criteria across hundreds of cell lines markedly enhances the statistical power to discover genetic alterations involved in carcinogenesis and molecular predictors of pharmacologic vulnerability. As proof of principle, we have carried out systematic nomination of putative targets of genetic alterations using integrative analyses. Here, significant regions of genomic gains and losses have been linked to expression and mutation data to find significant correlations at both single-gene and pathway levels. We have also begun to assemble systematic algorithms that identify genetic predictors of sensitivity or resistance to particular pharmacological compounds, taking advantage of the fact that the CCLE is a comprehensive resource with extensive genomic characterization. Toward this end, we integrated a preliminary sensitivity dataset for 28 compounds accurately profiled against more than 400 cell lines with all genomic data available in the CCLE. To enhance the robustness of our method, we reduced the number of significant genomic features for each cell line to a number that allows properly determined prediction of sensitivity. Expression data was converted to cell line-specific readouts of gene set expression; and DNA gains and losses are reduced to statistically significant regions using the GISTIC algorithm. These values were combined with critical oncogene mutations as inputs to a multifaceted prediction model for pharmacological sensitivity, the accuracy of which was assessed using cross-validation. Our results suggest that this integrative approach applied to a robust cancer cell line collection has considerable power to discover novel associations that augment ongoing basic research into cancer biology and drug discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 105.

ALK (anaplastic lymphoma kinase) is an RTK (receptor tyrosine kinase) of the IRK (insulin receptor kinase) superfamily, which share an YXXXYY autophosphorylation motif within their A-loops (activation loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (insulin-like growth factor receptor kinase-1). Chromosomal translocations involving ALK were first identified in anaplastic large-cell lymphoma, a subtype of non-Hodgkin's lymphoma, where aberrant fusion of the ALK kinase domain with the NPM (nucleophosmin) dimerization domain results in autophosphosphorylation and ligand-independent activation. Activating mutations within the full-length ALK kinase domain, most commonly R1275Q and F1174L, which play a major role in neuroblastoma, were recently identified. To provide a structural framework for understanding these mutations and to guide structure-assisted drug discovery efforts, the X-ray crystal structure of the unphosphorylated ALK catalytic domain was determined in the apo, ADP- and staurosporine-bound forms. The structures reveal a partially inactive protein kinase conformation distinct from, and lacking, many of the negative regulatory features observed in inactive IGF1RK/IRK structures in their unphosphorylated forms. The A-loop adopts an inhibitory pose where a short proximal A-loop helix (alphaAL) packs against the alphaC helix and a novel N-terminal beta-turn motif, whereas the distal portion obstructs part of the predicted peptide-binding region. The structure helps explain the reported unique peptide substrate specificity and the importance of phosphorylation of the first A-loop Tyr1278 for kinase activity and NPM-ALK transforming potential. A single amino acid difference in the ALK substrate peptide binding P-1 site (where the P-site is the phosphoacceptor site) was identified that, in conjunction with A-loop sequence variation including the RAS (Arg-Ala-Ser)-motif, rationalizes the difference in the A-loop tyrosine autophosphorylation preference between ALK and IGF1RK/IRK. Enzymatic analysis of recombinant R1275Q and F1174L ALK mutant catalytic domains confirms the enhanced activity and transforming potential of these mutants. The transforming ability of the full-length ALK mutants in soft agar colony growth assays corroborates these findings. The availability of a three-dimensional structure for ALK will facilitate future structure-function and rational drug design efforts targeting this receptor tyrosine kinase.

Accurate prediction of patient drug response is required for successful personalized medicine. Cancer is a disease resulting from myriad genetic alterations. Accordingly, it should be possible to predict drug response of specific cancers using genetic and molecular signatures. To aid this effort, an ongoing collaboration established between Novartis and the Broad Institute called the Cancer Cell Line Encyclopedia (CCLE) has (i) comprehensively characterized genome-scale mRNA expression, copy number alteration and mutation profiles for nearly 1000 cancer cell line models spanning many tumor types and (ii) profiled ~500 of these cell lines against ~1000 anticancer compounds. Using these data, we are developing a scalable, extensible predictive modeling framework based on various supervised learning methods that include both categorical classification and linear regression approaches to predict compound sensitivity. By cross-validation of the generated models against test data sets, we quantitatively estimated model performance. Our initial results validate several preexisting genetic predictors of sensitivity. In addition, our models reported 70% or higher sensitivity and specificity for a number of compounds and yielded interesting potentially novel predictive features that, in some cases, outperform previously existing genetic predictors of sensitivity. Our integrative approach demonstrates that pharmacological profiling of large, genomically annotated cancer model systems, coupled with systematic predictive modeling, can uncover novel predictors of drug response and may ultimately aid patient stratification. This talk is also presented as Poster A4.

Abstract Cancer genome characterization efforts such as The Cancer Genome Atlas project are rapidly improving our knowledge of tumor genetic alterations. With the expanded use of massively parallel sequencing, the catalogue of known genetic alterations in cancer is expected to expand at an accelerating rate. In this context, the emphasis is shifting towards systematic identification of the genes and pathways targeted by recurrent genetic alterations, their functional impact in tumor biology, and the resulting cellular dependencies that might be exploited therapeutically. Anticipating the need for a companion resource to systematically probe tumor biology armed with cancer genomics knowledge, we have assembled a compendium of experimentally tractable cancer model systems consisting of ∼1000 human cancer cell lines and performed extensive genomic analysis (at the level of gene expression, DNA copy number and mutations) coupled with pharmacological profiling. This resource, which we call the Cancer Cell Line Encyclopedia (CCLE), is being used not only to identify the putative targets of prevalent genetic alterations, but also to systematically link the presence or absence of certain genetic alterations to drug sensitivity or resistance. To date, we have identified several previously unappreciated genomic predictors of response or intrinsic resistance to targeted anticancer agents. For instance, through integrative analysis, we have discovered additional mechanisms that may underlie sensitivity to MET inhibitors, beyond amplification of the MET receptor, highlighting the fact that response prediction in the clinic may require assessment of multiple variables. We have also broadened the potential relevance of known predictive biomarkers that might provide a rationale for future genotype-driven clinical trials. As an example, we have expanded on existing knowledge of resistance to receptor tyrosine kinase (RTK) inhibitors, showing that the presence of RAS mutations may predict lack of response to a broad spectrum of RTK inhibitors in addition to EGFR inhibitors. This work demonstrates that pharmacological profiling of large, genomically-annotated cancer model systems may uncover new tumor dependencies as well as positive and negative predictors of drug response. The results of this study are being made publicly available at a CCLE online portal, with the hope they will become a valuable resource for the cancer community to propel translation of the knowledge generated through in vitro integrative genomics into personalized cancer medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2620.

Clinical trials in melanoma patients carrying B-RAF gene mutations have shown promising results with the B-RAF kinase inhibitor PLX4032, but many patients go on to become resistant. Two papers now uncover possible mechanisms for this resistance. Nazarian et al. report that melanomas can acquire resistance due to mutations of N-RAS or increased expression of PDGFRβ, and Johannessen et al. report resistance due to upregulation of MAP3K8/COT. Each of these mechanisms seems to apply to some patients in the recent PLX4032 trial, yet surprisingly, no secondary B-RAF mutations were observed. Recent data from early clinical trials in melanoma patients carrying mutations in the B-RAF gene have shown promising results with the B-RAF kinase inhibitor PLX4032; however, many patients eventually develop resistance to this treatment. Two papers now uncover possible mechanisms of resistance to PLX4032. One paper shows that upregulation of MAP3K8 (which encodes COT) can confer resistance of melanoma cells to B-RAF inhibitors, whereas another paper found that melanomas can acquire resistance due to mutations of N-RAS or increased expression of PDGFRβ. Each of these resistance mechanisms seems to apply to at least some patients on recent PLX4032 trial, whereas, surprisingly, so far no secondary B-RAF mutations have been observed. Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50–70% of malignant melanomas1. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma2,3,4,5,6—an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials7,8,9. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance10,11,12. Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies13. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.

BIOPHYSICS. For the article ‘‘Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy,’’ by Ana Carolina Zeri, Michael F. Mesleh, Alexander A. Nevzorov, and Stanley J. Opella, which appeared in issue 11, May 27, 2003, of Proc Natl Acad Sci USA (100:6458– 6463; first published May 15, 2003; 10.1073 pnas.1132059100), the authors note that Fig. 4 appeared incorrectly. The corrected figure and its legend appear below. CELL BIOLOGY. For the article ‘‘Bcl10 plays a critical role in NFB activation induced by G protein-coupled receptors,’’ by Donghai Wang, Yun You, Pei-Chun Lin, Liquan Xue, Stephan W. Morris, Hu Zeng, Renren Wen, and Xin Lin, which appeared in issue 1, January 2, 2007, of Proc Natl Acad Sci USA (104:145–150; first published December 19, 2006; 10.1073 pnas.0601894104), the authors note that the affiliations for Donghai Wang and Xin Lin appeared incorrectly. The corrected author and affiliation lines, and related footnotes, appear below. The online version has been corrected.

As for the existence of epithelium barrier, accelerating the transport remains huge challenges for orally delivered protein and peptide drugs into blood circulation. Modifying nanopaticles (NPs) with targeting peptides can enhance the intestinal absorption of loaded macromolecular drugs. However, the transport process, which mainly means how the NPs pass through the apical membrane and the basolateral side and then enter into blood circulation, is needed comprehensive investigation. In this study, we systemically studied the transport mechanisms in Caco-2 cell model of trimethyl chitosan based NPs (TMC NPs) before and after modification of FQS, an integrin αvβ3 receptor targeting peptide. Our results showed FQS peptide mediated multiple endocytosis pathways and could activate integrin αvβ3 receptor by interacting with FAK and Src-family kinases to induce receptor-mediated endocytosis of the NPs. Then, both endocytosed NPs could transport from early endosome to lysososmes via late endosomes/lysosome pathway, as well as to recycling endosomes and Golgi apparatus through early endosome/recycling endosomes and Golgi apparatus/recycling endosomes/plasma membrane pathways, respectively. After FQS peptide modification, the endocytosis subpathways of NPs have been changed, and more pathways are involved in exocytosis process for FQS-modified NPs compared with non-modified NPs. Our study indicated the ligand modification could enhance the uptake and transport by altering some pathways in whole transport process of NPs.