Nanotechnologies for the treatment of colon cancer: From old drugs to new hope

International Journal of Pharmaceutics 514 (2016) 24–40

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Nanotechnologies for the treatment of colon cancer: From old drugs to new hope Larissa Kotelevetsa,b,c,1, Eric Chastreb,c,1, Didier Desmaëlea , Patrick Couvreura,* a b c

Université Paris-Sud XI, Faculté de Pharmacie, UMR CNRS 8612, 92296 Châtenay-Malabry, France INSERM, U1149, CNRS ERL8252, Centre de Recherche sur l'Inflammation, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Laboratoire d'Excellence Inflamex, DHU FIRE, Faculté de Médecine, Site Bichat, Paris, France

A R T I C L E I N F O

Article history: Received 26 April 2016 Received in revised form 3 June 2016 Accepted 4 June 2016 Keywords: Nanomedicine Colorectal cancer Multidrug resistance Chemotherapy Nanoparticles Transporters Clinical trials

A B S T R A C T

Colorectal cancer is a wide-reaching health problem due to its incidence and to the high mortality rates. Adjuvant chemotherapies have considerably improved the prognosis and/or the overall survival of patients with locally advanced and metastatic cancers. Nevertheless, their efficiency remains limited due to intrinsic and emerging multidrug resistance (MDR) of cancer cells, and to major adverse effects and dose limiting toxicities. The present review discusses the knowledge of clinically relevant mechanisms of resistance to cytotoxic and targeted therapies for the treatment of colorectal cancer, and focuses on the benefit of nanomedicine approach to circumvent these processes. Nanomedicaments should allow extensive cancer cell drug loading independent on cell surface transporters,
thus overwhelming drug metabolism and efflux-, but also alleviate side-effects related to tissue-dependent drug uptake. Finally, we provide an outline of preclinical and clinical studies of nanoparticles formulations for colorectal cancer treatment, and briefly discuss strategies to optimize the selective delivery of these nanomedicines to colorectal cancer cells. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction: incidence of colorectal cancers, molecular characteristics, evolution Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality. Nearly 135,000 and 450,000 individuals are diagnosed

Abbreviations: ABC transporter, ATP-binding cassette transporter; CAF, cancerassociated fibroblasts; CSCs, cancer stem cells; CTR1, Copper transporter 1; CIMP, CpG island methylator phenotype; CRC, colorectal cancer; DPD, dihydropyrimidine dehydrogenase; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; EPR, enhanced permability and retension effect; HGF, hepatocyte growth factor; HNPCC, hereditary non polyposis colorectal cancer; mCRC, metastatic colorectal cancer; MMPs, matrix metalloproteinases; MRP2, multidrug resistance protein 2; MSI, microsatellite instability; MSS, microsatellite stable; NPs, nanoparticles; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; OHP, OXA, oxaliplatin; OPRT, orotate phosphoribosyltransferase; OS, overall survival; P-gp, P-glycoprotein; RCT, radiochemotherapy; SLC, solute carrier; TAM, tumor-associated macrophages; TGF-b, transforming growth factor-beta; 5-FU, 5-Fluorouracyl. * Corresponding author. Prof Patrick COUVREUR, Membre de l'Académie des Sciences Institut Galien, 5 rue Jean-Baptiste Clément, 92296 Chatenay-Malabry, France. E-mail addresses: [email protected], http://www.erc_ternanomed.u-psud.fr/, http://www.umr-cnrs8612.u-psud.fr (P. Couvreur). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2016.06.005 0378-5173/ã 2016 Elsevier B.V. All rights reserved.

annually in United-States and in Europe, where this cancer is responsible of approximately 50,000 and 215,000 related deaths, respectively (Ait Ouakrim et al., 2015; Siegel et al., 2016). In line with other malignancies, metastases are the main cause of colorectal cancer-related mortality. These distant metastases mainly develop in the liver, where they are evidenced in approximately 25% of patients at the time of diagnosis, whereas metachronous metastatic disease arises for 20–25% of patients, resulting in a relatively high overall mortality rate. When colorectal cancer is localized, the five-year survival rate is about 90% but it falls to nearly 12% once there are distant metastases (Siegel et al., 2014). The onset of CRC is linked to intrinsic risk factors, such as aging, gender
risk slightly higher for male-, and genetic disposition. About 75% of new cases of CRC
termed sporadic- occur in absence of known predisposing factors, whereas nearly 20% of new cases of CRC arise in a context of familial history of CRC without identified genetic defect. Hereditary non polyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP) are dominant inherited autosomic cancer syndromes that account for 5% and 1% of cases, and are related to the defect in DNA mismatch repair genes and the inactivation of the APC gene, respectively (Bronner et al., 1994; Kinzler et al., 1991; Nicolaides et al., 1994; Nishisho et al., 1991; Papadopoulos et al., 1994). Other rare inherited colorectal cancer

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syndromes characterized with a lower penetrance include the recessive MUTYH-associated polyposis caused by mutation in human Mut Y homologue MUTYH
a base excision repair system that protects against transversion G:C/A:T following oxidative DNA damage-, and dominant syndromes, e.g. Peutz-Jeghers (LKB1 inactivation), Juvenile Polyposis syndrome (BMPR1A or SMAD4 inactivation), Cowden syndrome (PTEN inactivation 85% cases, decreased activity of KILLIN and SDH B/D, or PIK3CA and AKT1 activation) and mutations in the proofreading domain of DNA polymerase epsilon and delta1. Some chronic diseases are associated with an increased risk of colon cancer, these include Inflammatory Bowell Disease (mainly ulcerative colitis), obesity and diabetes (Ma et al., 2013; Tsilidis et al., 2015; Ullman and Itzkowitz, 2011). The imbalance in intestinal microbiota and dysbiosis through the induction of inflammation, the release of reactive oxygen species and microbia-derived metabolism or toxins are also suspected to promote genetic and epigenetic alterations leading to CRC (Irrazabal et al., 2014). On the other hand, environmental and lifestyle factors, such as smoking, alcohol use, and high fat Western diet have been clearly linked to polyp development and CRC (Center et al., 2009; Chen et al., 2015; Cross et al., 2010; Bardou et al., 2002; Botteri et al., 2008a,b; Huxley et al., 2009). 2. Molecular mechanisms involved in colorectal carcinogenesis The evolution and heterogeneity of cancer cells might be depicted by two models. The clonal evolution model hypothesizes that all cancer cells are tumorigenic and accumulate stochastic genetic/ epigenetic alterations allowing the expansion of a subpopulations with growth advantage or better adaptation to their microenvironment. The cancer stem cells (CSCs) model postulates
by homology with the hierarchical organization of normal tissues-, the existence of a subset of progenitor cells with the ability to generate the diverse tumor cells. The characteristics of CSCs rely on their “infinite” lifespan, their self renewing, the generation of proliferative progenitors
with limited lifespan- that differentiate and might undergo apoptosis, their ability to disseminate, and their intrinsic resistance to chemotherapies (O’Brien et al., 2007; Cherciu et al., 2014). These two seemingly disparate models, are reconcilable, nevertheless based on the later model, an efficient strategy to cure for cancer implies eradicating CSCs. Colorectal cancers arise through the stepwise accumulation of genetic alterations leading from normal epithelia to aberrant crypt foci, adenoma, carcinoma and metastatic disease (Fearon and Vogelstein, 1990), and follow three molecular pathways characterized by i) chromosomal instability (CIN), ii) high microsatellite instability (MSI-H), or iii) CpG island methylator phenotype (CIMP). Colorectal cancers with chromosomal instability account for about 70% of colon cancers (Fearon and Vogelstein, 1990) and are associated with frequent chromosomic alterations (gain chromosome harms 7, 16, 17q 20 and X), and deletions of chromosome harms 1q, 4p, 5q (including APC, MMC), 6, 8p, 9q, 17p (including TP53), 18q (including DCC, SMAD2/4) and 22q (Fearon and Vogelstein, 1990; Muleris et al., 1990). These CRC preferentially arise in the left colon. Tumors MSI-H account for 15% of sporadic CRC, arise more frequently in the proximal colon and are characterized by the deficiency of the DNA mismatch repair system, e.g. through biallelic methylation/silencing of the promoter of hMLH1 (Thibodeau et al., 1998). The hypermutated phenotype of these tumors and the neo-antigenic presentation of numerous mutant proteins are associated with a marked leukocyte infiltration, and with a better prognosis in early stages. These mutations are inherited in patients with HNPCC.

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The CpG island methylator phenotype (CIMP) is characterized by the methylation of CpG islands in the promoters of certain genes leading to their silencing (Toyota et al., 1999). These tumors are more often located in the proximal colon. Tumors with CIMP-high are frequently associated with MSI as the result of the inactivation of hMLH1, as well as BRAFV600E activation, whereas tumors CIMPlow are microsatellite stable (MSS) and bear activating KRAS mutations (Fig. 1). Thus, some overlaps exist between these different tumors types. At the molecular levels, these 3 types of CRC are associated with a preferential stepwise accumulation of genetic alterations, including the activation/overexpression of a series of (proto)oncogenes and the deletion/inactivation of tumor suppressor genes. Among them, the phosphoinositide-3-kinase (PI3K)/protein kinase AKT signaling pathway which is important for proliferation of normal and transformed intestinal epithelial cells has been clearly implicated in the progression to the transformed phenotype leading to colorectal carcinoma (Kotelevets et al., 1998, 2001, 2005) (Fig. 1). Although the genetic defects in these 3 types of CRC differ, it is worth noting that they share similar signaling pathways dysregulation, e.g. APC inactivation vs. b-catenin and axin mutations; TP53 vs. Bax inactivation; Ki-ras activation vs. BRAF and PIK3CA activation, PTEN inactivation; SMAD2/4 inactivation vs. TGF-bRII, in the CIN and MSI CRC, respectively (Fig. 1). Taken together, these dysregulations can be classified into one or more of 12 pathways that confer a selective growth advantage, by governing cell survival, cell fate and genome maintenance (Vogelstein et al., 2013) Besides gene amplification, deletion and mutation, and epigenetic processes, including DNA methylation and histone modifications, other molecular mechanisms have also been linked to neoplastic progression. Alternative splicing allows increasing the biodiversity of proteins with a loss or gain of novel biological functions
as the result of mutation of acceptor or donor sites, or through the selective expression of splicing factors-. In this connection, we previously identified in CRC an activated and novel splice variant of the small GTPase Rac1, we designed Rac1b (Jordan et al., 1999) (Fig. 1). The balance in Rac1/Rac1b levels is regulated by splicing factors, which either induce skipping or favor inclusion of the alternative exon 3b (Goncalves et al., 2009, 2014). The ectopic expression of Rac1b was detected in inflammatory bowel diseases, and in human breast, pancreatic, thyroid, ovarian and lung cancers (Matos et al., 2013; Guo et al., 2015; Mehner et al., 2015; Schnelzer et al., 2000; Silva et al., 2013; Stallings-Mann et al., 2012; Ungefroren et al., 2013; Zhou et al., 2013). Experimental studies performed with transgenic mice revealed that Rac1b cooperated with activated K-Ras to promote lung cancer (StallingsMann et al., 2012; Zhou et al., 2013). Non-coding RNA, e.g. miRNA have also been implicated in neoplastic progression. These small RNA molecules (about 22 nucleotides long) negatively control target genes expression posttranscriptionally. Each miRNA is predicted to have several targets, and each mRNA may be regulated by more than one miRNA. Overexpression of some miRNA have been evidenced in CRC, e.g. mir21 and mir17-92 which target the mRNA of the PTEN tumor suppressor and of the BIM proapototic protein, respectively (Meng et al., 2007; Poulsen et al., 2014). Interestingly, the accumulation of mir17-92 is mediated by c-myc and b-catenin overexpression, and negatively controlled by APC and TP53 (Diosdado et al., 2009; Li Y. et al., 2016; Poulsen et al., 2014). 3. Cellular interplay in colorectal cancer progression The invasive phenotype and cancer cell dissemination is postulated to occur through the epithelial–mesenchymal

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transition (EMT), a reversible process characterized by the loss of epithelial properties, including the apico-basal polarity, cell–cell contacts (E-cadherins) and cytokeratins expression, and

concurrently, the acquisition of N-cadherin, vimentin, fibronectin expression, all events that favor cell mobility (Lim and Thiery, 2012). EMT is regulated by a series of transcription factors

Fig. 1. Schematic representation of the main signaling pathways involved in colorectal carcinogenesis. The receptors tyrosine kinase (RTK), e.g. EGF-R, c-met are frequently overexpressed in colorectal cancers. Furthermore, autocrine release of growth factors, e.g. TGF-a stimulate RTK. These activated receptors recruit adaptor molecules that stimulate the KRAS GTPase, which in turn activates the Ser/Thr kinase BRAF and the MAPK kinases cascade that supports cell growth and survival. Tyrosine kinase receptors also stimulate the phosphoinositide 3-kinase (PI3K) that is responsible for the production of phosphatidylinositol phosphorylated at position 3, e.g. PI(3,4,5)P3. The PI(3,4,5)P3 recruits to the plasma membrane proteins with pleckstrin homology domain, e.g. the Ser/ Thr kinases PDK1 and AKT, and GTP exchange factors for small GTPase, e.g. Rac1 involved in the cytoskelleton remodeling and cell migration but also ROS production and the activation of the b-catenin (b-ctn)/TCF4 pathway. The tumor suppressor PTEN counteracts PI3K activity by dephosphorylation of (PI(3,4,5)P3. PTEN might also dephoshorylates Tyr residues on adaptor molecules involved in RTK signaling. PTEN exerts tumor suppressor activity independent on its phosphatase activity through its multiple protein interactions, e.g. PTEN interaction strengthens TP53 transactivating activity. AKT plays a critical role in the control of the apoptotic process and cell survival. AKT induces nuclear translocation of NF-kB. Furthermore, once phosphorylated by AKT, FOXO3
a transcription factor involved in the apoptotic programme-, is sequestered in the cytoplasm by 14-3-3 proteins. Similarly, phosphorylation of BAD, a Bcl-2 family member involved in apoptosis initiation, causes its translocation from the mitochondrial membrane to the cytosol. AKT also phosphorylates Caspase-9 preventing a caspase cascade leading to cell death. Downstream of AKT, the activation of mTORC1 complex leads to the inhibition of 4EBP1 and activation of S6-kinase effectors, and the translation of selective transcripts, e.g. c-Myc, cyclin-D1 involved in cell cycle. S6-kinase also regulates VEGF expression by phosphorylating HIF1a. AKT also crosstalks with other signaling pathways involved in CRC, e.g. Wnt and TP53 pathways through the inactivation of GSK3, and the upregulation of the ubiquitin-ligase activity of murine double minute 2 (MDM2) that regulate TP53 accumulation (see below). As far as cell motility is concerned, AKT phosphorylates many proteins involved in polymerisation and stabilisation of the actin cytoskeleton. The activation of the Wnt signaling pathway has been proposed to trigger a crypt cell progenitor phenotype in intestine adenoma genesis, although a top-down model through a dedifferentiation process has been also recently evoked. The activation of the frizzled receptor and downstream effectors by the Wnt paracrine factors, lead to the b-ctn translocation to the nucleus. This allows formation of a transcriptional complex, and the expression of the Wnt target genes, e.g. cyclin D1 and c-myc involved in cell proliferation, and matrylisin/MMP7. In the absence of Wnt, the tumor suppressor APC shuttles from the nucleus to the cytosplasm to grab and recruit b-catenin, in a multiprotein complex including GSK3 and Axin, leading to b-catenin degradation by the proteasome. GSK3 also indirectly inhibits mTORC1. Besides the control of b-ctn/TCF pathway, APC is also involved in chromosome segregation. TGF-b binding to the Type II receptor (TGF-bRII) leads to recruitment and trans-phosphorylation of Type I receptor (TGF-bRI) that in turn phosphorylates its downstream targets, e.g. SMAD2 which forms a complex with SMAD4 and translocate to the nucleus, where they regulate gene expression, including the inhibitor of the cyclin-dependent kinases (CDKI) P15ink4b. TGF-b might also induce other non-SMAD signaling pathways, JNK, p38, and ERK1/2. In unstressed mammalian cells, the TP53 tumor suppressor is nonphosphorylated and undergoes ubiquitylation by MDM2 and degradation by the proteasome. When the cell is confronted with stress like DNA damage, hypoxia, cytokines, metabolic changes, viral infection, or oncogenes, TP53 ubiquitylation is suppressed and the tumor suppressor is stabilized and accumulates in the nucleus where it transactivates a wide variety of genes involved in apoptosis, growth arrest or senescence in response to genotoxic or cellular stress, e.g. the CDKI p21waf and the pro-apoptotic protein BAX. The CPG island phenotype is associated with the selective inhibition of gene expression, e.g. methyl-guanyl methyl transferase, a DNA repair protein, IGFBP7, and hMSH2/ hMSH6, hMLH1 involved in DNA mistmach repair. The proto-oncogenes preferentially activated in CIN tumors are represented in red, those preferentially activated in MSI tumors in orange. The tumor suppressors inactivated in CIN and MSI tumor are represented in deep and light green respectively.

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including the Snail zinc-finger family (SNAI1, SNAI2/SLUG), the distantly related zinc-finger E-box-binding homeobox family proteins ZEB1, ZEB2/SIP1, and the basic helix–loop–helix(bHLH) family of transcription factors, including TWIST1, TWIST2. The activity of these transcription factors is regulated by a combination of extracellular signals, including TGF-b, Wnt, hepatocyte growth factor (HGF)/Scatter factor which mobilize SMAD2/SMAD4, b-catenin/TCF4, MAPK, PI3K, and the small GTPase Rac1 and Rho. In this connection, the c-met oncogene which encodes the receptor for HFG is overexpressed in primarily colorectal tumors and their liver metastases (Di Renzo et al., 1995) (Fig. 1). Cancer cells might adopt an individual cell migration, amoeboid-like as a result of Rho activation, mesenchymal-like consecutively to Rac1 activation, or a multicellular in the case of moderately differentiated and differentiated carcinomas (Friedl and Alexander, 2011). It is proposed that after dissemination and the subsequent formation of distant metastases, cancer cells undergo the mesenchymalepithelial transition (MET), reversal to EMT. Nevertheless, carcinogenesis cannot be reduced to the stepwise accumulation of genetic and epigenetic defect in tumoral cells. Tumor cells transform and exploit their microenvironment by releasing cytokines and growth factors to activate normal, quiescent cells around them. Accordingly, tumor growth requires the angiogenic switch to be supplied in oxygen and nutrients (Weis and Cheresh, 2011). Cancer cells release angiogenic factors, e.g. vascular endothelial growth factor (VEGF) as the result of hypoxia or oncogenic activation, e.g. Ki-ras activation, TP53 or PTEN depletion. The induction the angiogenic switch occurs at an early stage of colorectal carcinogenesis through the increase in expression of multiple angiogenic factors and their receptors (André et al., 2000; Weis and Cheresh, 2011). The imbalance in the pro- and anti- angiogenic factors stimulates the sprouting and proliferation of endothelial cells on nearby blood vessels and lymphatics. The release of platelet-derived growth factor (PDGF) by platelets and activated endothelial cells recruit and activate perivascular cells. Nevertheless, the vascular stability and maturation devoted to pericytes are not fulfilled due to persistent stimulatory signals within the tumor microenvironment. This continuously remodeling leads to a leaky, tortuous and poorly organized tumor vasculature with structural and functional abnormalities. Cancer-associated fibroblasts (CAF) mainly contribute to the remodeling response by the aberrant deposit extracellular matrix (ECM) proteins, and the release stimulatory factors and matrix metalloproteinases (MMPs) (Alexander and Cukierman, 2016). Besides ECM remodeling, MMPs might also enhance the bioavailability of growth factor sequestered in the extracellular matrix, e.g. FGF, TGF-b, and generate bioactive peptides following proteolysis of ECM components, e.g. BM-40/SPARC. In this connection, we previously reported that fibroblast-like cells in colorectal cancers expressed high levels of BM-40/SPARC and MMP11/Stromelysin-3 (Porte et al., 1995). SPARC is a component of the extracellular matrix, involved in cell proliferation, cell motility and angiogenesis. The MMP11 expression occurs in the late phase of colorectal progression. The peculiarities of this MMP is its secretion as an active form after intracellular maturation in the Golgi apparatus, and its poorly documented selective substrates which might be insulin-like growth factor-binding protein 1 (IGFBP1) and Serine protease inhibitors. The tumor microenvironment is characterized by a chronic inflammation, and infiltration by immune cells, including myeloid cells (tumor-associated macrophages, TAM, and neutrophils) and lymphocytes. The infiltration of the tumor microenvironment by Tcytotoxic and memory cells has been correlated with a favorable outcome for patients with early-stage disease, especially when there is high infiltration of both subtypes at the tumor center, as well as at the invasion margin (Pagès et al., 2005).

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The macrophages undergo an M1 polarization after exposure to Th1 cytokines e.g.IFN-g. These macrophages efficiently produce effector molecules e.g. reactive oxygen and nitrogen intermediates and high production of pro-inflammatory cytokines e.g. Il1b, TNFa, Il6, Il12 and low expression of IL-10, and play a classic role in mediating resistance against microbes and tumor cells. On the other hand, Il4, Il10 and Il13 induce the alternative M2 macrophage polarization. M2 macrophages are characterized by a high production of IL-10, low expression of Il12 and a poor antigen presenting capacity. M2 macrophages produce chemokines such as CCL17, CCL22 and CCL24, involved in Treg cell, Th2, eosinophil and basophil recruitment. These macrophages promote wound healing, angiogenesis and tissue remodeling and might favor cancer progression (Erreni et al., 2010; Belgiovine et al., 2016). As stated above, except MMP7/matrilysin which is expressed by colorectal cancer cells as the result of the Wnt/b-catenin/TCF4 pathway activation (Fig. 1), most MMPs are produced by stromal cells. In this connection, an interesting cross-talk between cancer cells and myeloid cells has been discovered in Apc/Smad4 doubledeficient mice. These mice develop colon cancers which release the CCL9 chemokine
ligand of CCR1-, leading to the recruitment of CCR1+ immature myeloid cells from the bone marrow to the invasive front of the tumors. These myeloid cells produce MMP2 and MMP9 and promote tumor invasion (Kitamura et al., 2007). The involvement of primary tumor microenvironement in the metastatic cascade is further sustained by the identification of stroma from the tissue of origin in metastases (Duda et al., 2010). Another interesting point concerns the tissue specific implantation of metastasis, e.g. liver, lung, peritoneum for colorectal cancer; bone, brain, liver, lung for breast cancers; adrenal gland, bone, liver lung for prostate cancer; bone, brain, liver, lung, skin/ muscle for melanoma. These preferential implantations might be related to anatomy of vascular and lymphatic drainage from the site of the primary tumor and mechanical factors, e.g. liver microvessels emboli by aggregates of colorectal cancer cells and stromal cells downstream portal venous system or via the lymphatic drainage favoring cancer cells extravasation and implantation. Alternatively, Paget proposed in 1889 his “seed and soil” theory, that the organ-preference patterns of tumor metastasis is driven by favorable interactions between the seed (metastatic tumor cells) and the soil (microenvironment of a target organ) (Langley and Fidler, 2011). These hypotheses are not exclusive, the later being recently revisited on the basis of exosomes discovery. It has been proposed that the surface determinant on the extracellular vesicles released by tumor cells might support their selective tissue homing, and through their protein and nucleic acid content these exosomes trigger the formation of a preneoplastic niche (Syn et al., 2016). Nevertheless, the inadvertent transmission of a malignant tumor that occasionally occurred concomitant with organ transplantation indicates that metastatic cells may reside dormant in organs in the original host that are not usually the site of detectable secondary tumors, e.g. the kidneys and heart for melanoma (Friberg and Nyström, 2015). Thus, targeting the tumor microenvironment constitute complementary approaches to restrain cancer cell growth and dissemination through the control of the proliferation and migration of endothelial cells and angiogenesis, the activity of cancer-associated fibroblasts, the balance between the antitumor/ immune tolerance, reprogramming macrophages from M2 to M1, restraining the activity of MMPs. 4. Colorectal cancer treatments Several studies have taken into account the phenotypic, the genotypic, and the prognosis of CRC (Jass, 2007; Marisa et al.,

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2013). These molecular approaches have provided strong insight in the comprehension on CRC progression, in the identification of subgroups at high risk, and in the definition of novel therapeutic strategies. Nevertheless, management and clinical outcomes in CRC remain mainly determined by cancer stage by histological examination. As in any neoplastic disease, the stage of locoregional disease and the presence or absence of metastases, together with specific prognostic and predictive factors, are of utmost importance for individual patient management. The TNM staging system classifies the extent of cancer based on anatomical information about the size and the parietal invasion of primary tumor (T0-T4b), the regional lymph- node status (N0N2b) and the distant metastases (M0-M1b), grouping the cases with similar prognostic (Fig. 2). 4.1. Current colorectal cancer therapies The prognosis and the treatment of colorectal cancer are based on the curative surgical resection of the primary tumor (stages I and II), combined with adjuvant chemotherapy for patients with tumor stage II at high risk and tumor stage III (lymph node involvement). For patients with advanced cancer (distant metastasis, stage IV) the management consists of chemotherapy, and when feasible surgical resection of primary tumor and of liver or

lung metastases. In the case of rectal cancers, neoadjuvant radiation therapy and chemotherapy can be administrated in pre- or post- operative, as early as stage II to decrease the risks of local recurrence (Fig. 2). CRC treatment is currently based on the use of three cytotoxic chemotherapies, fluoropyrimidine (5-fluorouracile), oxaliplatin and irinotecan. The 5-fluorouracile(5-FU) and Capecitabine (an orally-administered prodrug of 5-FU, Xeloda1) act mainly through irreversible inhibition of thymidylate synthase through the FdUMP metabolite, thus depleting synthesis of the pyrimidine thymidine, a nucleoside required for DNA replication. Folinic acid (Leucovorin) is frequently co-administrated with 5-FU, since it stabilizes the FdUMP, thymidylate synthase complex, hence enhancing 5-FU's cytotoxicity. Other 5-FU metabolites, FdUTP and FUTP can incorporate in DNA and RNA, damaging these macromolecules. Oxaliplatin (Eloxatin1)
a third-generation of platinum derivatives- undergoes nonenzymatic biotransformation processes leading to reactive intermediates that form both inter- and intrastrand cross-links in DNA. These adducts prevent DNA replication and transcription, causing cell death. Oxaliplatin toxicity is also related to the production of reactive oxygen species (ROS) that might oxidize DNA, proteins and lipids. Irinotecan (CPT11, Camptosar1, a camptothecin analog) is a prodrug that is metabolized in the active metabolite SN-38 a

Fig. 2. Staging of CRC, treatment and prognosis. Staging of CRC according to the 7th edition (2009) of the Colon and Rectum Staging; International Union for Cancer Control (UICC) and the American Joint Committee for Cancer based on tumor extension and invasion (T, N, M stages). Stage 0 refers to in situ carcinomas (Tis); Stage I: tumors invading submucosa
muscularis propria (T1-T2); Stages IIa-IIc: tumor invades or is adherent to other organs or structures (T3-T4b), without lymph node involvement (N0) nor distant metastasis (M0); Stages IIIA-IIIC: correspond to tumors T1-T4b with lymph nodes involvement (N1 metastasis in 1–3 regional lymph nodes or tumor deposit in subserosa to N2b: metastasis in 7 or more regional lymph nodes); Stage IV: tumors with any T, any N, and M1- M1b: metastasis confined in one to several organs/sites. The 5 years overall survival according to tumor staging is from Siegel et al. (2014) and Li J. et al. (2016). The currently in use treatments involved surgery (stages I and stage II) in combination with chemotherapies (Stages  II) based on 5-FU/leucovorin or capecitabine alone or in combination with oxaliplatin and/irinotecan, and/or targeted therapies (see also Brandi, 2016; Murphy et al., 2015).

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topoisomerase-I inhibitor, causing DNA damage and inducing apoptosis. Combination therapy with these drugs are commonly used as first line treatments, e.g. 5-FU/leucovorin with oxaliplatin (FOLFOX, usually for stage III CRC); FOLFOX, 5-FU/leucovorin with irinotecan (FOLFIRI) or 5-FU/leucovorin, oxaliplatin and irinotecan (FOLFIRINOX) for stage IV, enabling synergistic cytotoxic effects and yielding a better response rate and progression-free survival compared with monotherapy (André et al., 1999; de Gramont et al., 2000; Ychou et al., 2013) (Fig. 2). Nevertheless, these therapies are frequently associated with common and major side effects, including nausea, vomiting, or diarrhea, neutropenia, fatigue, alopecia; and specific disorders, e.g. hand-foot syndrome for 5-FU; peripheral neurotoxicity and ototoxicity for oxaliplatin. More recently, therapies acting selectively on molecular targets that are involved in the growth, progression and spread of cancer, rather than targeting all rapidly dividing cells have been developed. These “targeted therapies” are often cytostatic, whereas standard chemotherapy agents are rather cytotoxic. These therapies are based on the use of either small inhibitory molecules (suffix
inib) that might impede intracellular effector systems or monoclonal antibodies (suffix
mab) that selectively interferes with extracellular or cell surface targets. Some of these compounds are in use in the treatment of colorectal cancers such as: Cetuximab (Erbitux1) and panitumumab (Vectibix1) that are humanized monoclonal antibodies

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directed against EGF receptors and inhibit the activation of the receptor. Antiangiogenic agents, such as Bevacizumab (avastin1) and Ramucirumab (Cyramza1), are two monoclonal antibodies which target VEGF and VEGF2 receptor, respectively. Ziv-aflibercept (Zaltrap1) is a protein comprising segments of the extracellular domains of human vascular endothelial growth factor receptors 1 (VEGFR1) and 2 (VEGFR2) fused to the constant region (Fc) of human IgG1, and acts as a soluble decoy receptor. Regorafenib (Stivarga1) is a multikinase inhibitor targeting VEGFR2-3, TIE-2, PDGFR, FGFR, RET and c-Kit. Trifluridine/tipiracil (TAS-102, Lonsurf1) is a combination of trifluridine, a nucleoside analog, and tipiracil hydrochloride, a thymidine phosphorylase inhibitor that prevents rapid metabolism of trifluridine, increasing its bioavailability. These targeted therapies are used either alone or in combination of the above-mentioned conventional chemotherapies in case of metastatic CRC. 4.2. Resistance to chemotherapies: intracellular mechanisms Drug resistance, whether intrinsic or acquired, is believed to cause treatment failure in over 90% of patients with metastatic cancer (Longley and Johnston, 2005). This resistance can occur at several levels, including drug influx/efflux; drug metabolism/ inactivation; alterations in drug target; processing of drug-induced damage; activation of alternative pathways independent on the target and evasion of apoptosis (Fig. 3).

Fig. 3. Schematic model depicting some mechanisms of resistance of currently in use chemotherapies for CRC. Mechanisms of resistance involve decrease influx, intracellular metabolism, drug efflux, changes in expression of effector systems and genetic alterations. Conventional chemotherapeutic agents have to cross the cell plasma membrane via selective transporters to reach their molecular targets. In general, resistance to traditional cytotoxic therapy is accomplished by decreasing the delivery of drug to the cancer cell, either by increased efflux out of the cell mediated by ATP-dependent transporters (ABCC5, ABCC11, ABCG2, ATP7A/B, MRP2), by decreased uptake into the cell (RFC, CTR1, OCT1-3, OATP2B1), or by a change in enzymes involved in metabolism. Furthermore, some side effects of these conventional therapies are related to the selective expression of drug transporters in healthy tissues. The rationale for nanomedicine approach is based to their accumulation at the vicinity of cancer cells, due to leaky angiogenic tumor vessels, and their massive intracellular loading, independently of any transporter. Alternatively, these nanodevices might be more selectively targeted to cancer cells using binding motifs of determinant expressed ectopically on cancer cell surface, e.g. EGF-R.

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Malignant colorectal tumors can exhibit a constitutive/intrinsic resistance, which is typically evidenced in early phase treatment. In this context, the use of personalized therapies requires the molecular typing of the tumors. Accordingly, the activation of effectors systems downstream of EGF-R, e.g. KRAS, BRAF, NRAS, or PIK3CA abrogate the efficiency of cetuximab treatment (Liu et al., 2016; Therkildsen et al., 2014). Nevertheless, due to tumor heterogeneity or spreading, pre-existing small subset of mutated cancers cells
bearing for instance KRAS mutation- can be missed using quantitative PCR and might explain some cases of early resistance to EGF-R targeted therapies (Diaz et al., 2013; Misale et al., 2014, 2013; Taly et al., 2013). Multiplex digital PCR (dPCR)/ picodroplet dPCR constitutes an alternative, noninvasive and highly sensitive approach, enabling the multiplex identification of mutated circulating DNA (Diaz et al., 2013; Taly et al., 2013). On the other hand, the mutations and amplification of KRAS have also been shown to be a mechanism of acquired resistance to EGF-R targeted therapy (Misale et al., 2013). Under treatment, cancer cells undergo various types of cellular stress (DNA damage, proteotoxic, mitotic, metabolic and oxidative stress) (Luo et al., 2009) and require stress support pathways for their survival. Beyond the activation of a crucial transcriptional programme required for mitotic progression (Fu et al., 2008), Polo like kinase 1 (PLK1) seems to improve the tolerance of cells to cancer associated cellular stress. It was shown that the overexpression of a hyperactive PLK1 mutant in cells harbouring damaged DNA overrides the DNA-damage checkpoint, and improves the tolerance towards the mitotic stress associated with chromosome instability (Yamamoto et al., 2006). Furthermore, cells in which mutant Ras acts as an oncogene are highly sensitive to PLK1 inhibition. Several inhibitors targeting PLKs are currently in development and are under investigation in a growing number of clinical trials. Recent data from preoperative radiochemotherapy (RCT) in rectal cancer indicated a broad variety in tumor responses, probably caused by the individual genetic accouterment of the tumor. It was shown that PLK1 was overexpressed at both the transcriptional and protein level in rectal cancer specimens as compared with normal rectal mucosa. For patients uniformly treated with a neoadjuvant RCT protocol, a higher PLK1 expression was significantly associated with poor tumor regression, and a higher likelihood of local recurrence indicating that tumor cells overexpressing PLK1 exhibit a more radioresistant phenotype (Fernandez-Acenero et al., 2015; Rödel et al., 2010; Tut et al., 2015). Concerning conventional cytotoxic therapy, resistance is conferred by decreasing the intracellular drug loading, either by i) a lower uptake, ii) an activation of the cellular detoxifying systems and drug metabolism, or iii) an increased efflux mediated by ATP-dependent or specific transporters (Table 1). Resistance to targeted therapies are based on multiple molecular mechanisms, including mutations, upregulation or activation of signaling effectors within specific bypass pathways that are highly dependent on the cellular context (Tejpar et al., 2012). As stated above, 5-fluorouracyl and capecitabine are members of fluoropyrimidine used as antimetabolite. Capecitabine is an orally available prodrug of 5-FU with lower side effects. Accordingly, capecitabine pass intact through the human intestinal mucosa, and is first converted to 50 -deoxy-5-fluorocytidine (DFCR) by carboxylesterase and then to 50 -deoxy-5-fluorouridine (DFUR) by cytidine deaminase, mainly in the liver. Systemic DFUR is converted to 5-FU by thymidine phosphorylase mainly in cancer cells, due to the increased activity in tumor tissue (Ishikawa et al., 1998; Verweij, 1999). The 5-FU undergoes metabolic pathways generating the main active compounds FdUMP a suicide inhibitor of the thymidylate synthase, as well as FdUTP and FUTP which integrate in DNA and RNA (Fig. 3). The first step in the activation of

5-FU is the phosphorylation by orotate phosphoribosyltransferase (OPRT), which directly metabolizes 5-FU to 5-fluorouridine monophosphate (FUMP) in the presence of 5-phosphoribosyl-1pyrophosphate. Therefore, OPRT expression correlates with 5-FU sensitivity (Table 1). The dihydropyrimidine dehydrogenase (DPD) is responsible for the detoxifying metabolism of fluoropyrimidines. Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase. On the other hand, genetic variations within the DPD gene can lead to reduced or absence of DPD activity, and individuals who are heterozygous or homozygous for these variations have increased risk of severe drug toxicity. Another well established mechanisms of resistance to 5-FU, and other antifolates, is the increased expression of thymidylate synthase, as reported in the meta analysis from Qiu et al. (Qiu et al., 2008). Concerning Thymidine Phosphorylase, which converts 5-FU to FUDR, the results remain controversial (Elamin et al., 2016). Irinotecan has a complex metabolic pathway where the first step is a rapid bioactivation to its active metabolite SN-38, by carboxylesterases (CES1 and CES2) expressed in the liver, as well as to a lesser extent in plasma, gastrointestinal (GI) tract, and tumor tissue. SN-38 is subsequently detoxified by uridine disphosphate glucuronosyltransferases (UGTs), mainly UGT1A1, to form the inactive metabolite SN-38 glucuronide (SN-38G) (Table 1) (Cummings et al., 2002). Resistance to Irinotecan and its active metabolite SN-38 might develop through a decrease in expression of topoisomerase I, mutation of Topo1 gene, posttrancriptional modifications inducing the relocalisation or the degradation of Topo I, and changes in downstream events such as suppression of apoptosis, cell cycle alterations, or enhancement of DNA repair (Rasheed and Rubin, 2003). Oxaliplatin has been shown to be active in cancer cell lines resistant to earlier generation platinum. This might result from the steric hindrance of the 1,2-diaminocyclohexyl ligand when oxaliplatin interacts with DNA. After cellular uptake, platinum drugs undergo aquation, and might interact with many biological molecules, including thiol-containing species, such as glutathione (GSH) and metallothioneins. Glutathione-S-transferases are enzymes involved in the cellular detoxification of electrophilic xenobiotica, including platinum derivatives, by catalyzing the conjugation with glutathione. GSTs favor inactivation and excretion of platinum compounds, and therefore prevent cells from DNA damage. Activation of DNA-repair pathways might reduce antitumor activity of oxaliplatin too. This includes genes of the nucleotide-excision repair pathway (NER)
removing DNA adducts- e.g., ERCC1, XPD and XPA, and genes of the base-excision repair (BER)
involved in the repair of strand breaks-pathway, e.g. XRCC1 and XRCC3 (Table 1). 4.3. Resistance to chemotherapies: involvement of transporters Drug influx/efflux processes by selective membrane transporters are critical determinants in regulating intracellular drug concentrations and their cytotoxic activities and resistance. These transporters belongs to i) the huge solute carriers (SLC) family
300 members organized into 52 families-, that either acts as facilitating transporters (solutes follow their electrochemical gradients) or secondary active transporters
symporter or antiporter- (solutes flow against the electrochemical gradient by coupling to transport of a second solute that follows its gradient). ii) the primary active ABC (ATP-Binding Cassette) transporters
encompassing seven subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White)- that allow the compounds to flow against the electrochemical gradient by coupling transport to ATP hydrolysis. The overexpression of ATP-binding cassette (ABC) efflux transporters in cancer cells such as P-glycoprotein (P-gp/

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Table 1 Reported mechanisms of resistance to chemotherapy agents. Chemotherapy

Enzyme/pathway/carrier

Thymidylate synthase (TS) 5-Fluorouracil (5FU), capecitabine (oral)

Methylene tetrahydrofolate reductase (MTHFR)

Thymidine phosphorylase (TP)

Orotate phosphoribosyl transferase (OPRT) Dihydropyridine dehydrogenase (DPD) ATP-dependent ABC transporters (encoded by the ABCB1 gene) Multidrug resistance proteins (MRPs) of the ABCC subfamily (encoded by ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC10, ABCC11), ABCG2 BCRP (breast cancer resistance protein, encoded by ABCG2)

Folinic acid (Leucovorin)

Reduced folate carrier 1 SLC46A1/PCFT and SLC19A1/RFC-1 OAT2 (Organic anion transporter) folylpolyglutamate synthase (FPGS)

Mechanism of resistance (MoR)

Reference

Increased expression leading to increased target of 5-FU inhibition

Popat et al. (2004), Qiu et al. (2008)

Decreased negative feedback by TS-FdUMP on its own expression Increased activity of MTHFR, decreasing CH2THF Zhao et al. (2014) availability required for inhibition of TS (postulated) Increased expression (postulated), may lead to Elamin et al. (2016) increased salvage pathway of nucleotides Low expression correlates with increased response to 5-FU therapy. Decreased expression (postulated), as high expression correlates with sensitivity Increased expression leading to increased degradation Increased cellular efflux Increased cellular efflux

Increased cellular efflux

Muhale et al. (2011) Salonga et al. (2000), Amstutz et al. (2011) Deeley (2006) Wilson et al. (2011), Hlavata et al. (2012) Lin et al. (2013), Noguchi et al. (2014)

Reduced expression or mutation of the RFC is a Odin et al. (2015) cause of primary resistance Nishino et al. (2013) High expression levels of OAT2 and RFC1 were significantly correlated with the antitumor effect of UFT/LV regimens

Irinotecan

Multidrug resistance protein ABCC2 Uridine diphosphate UGT1A1

Increased expression leading to increased efflux Increased expression leading to increased metabolism

Fujita et al. (2008) Cummings et al. (2002)

Oxaliplatin

Decresed expression of transporters: CTR1 OCT1 and OCT2

Kilari (2016) Zhang (2006)

miR-153,
143,
203 overexpression

Decreased intracellular drug influx Decreased intracellular drug influx (important determinants of oxaliplatin activity) Increased expression leading to increased efflux Increased levels of GSH inactivating the platinum and increasing export from cell Elevated expression of DNA repair genes: Increased DNA repair Epigenetic alteration

Increased levels of survivin, Bax expression

Cell death

Multidrug resistance protein (MDR1, ABCB1) Glutathione (GSH) Increased ERCC1 and XPF levels

ABCB1) represents the most common mechanisms of multi drug resistance (MDR). So far, all the effector molecules involved in the uptake of oxaliplatin, leucovorin, 5-FU/capecitabine and irinotecan belong to the solute carrier family of SLC (solute carriers) transporters. Most export proteins are members of the ABC transporter (ATP-binding cassette) superfamily with the exception of the MATE (multidrug and toxin extrusion) proteins, which are also members of the SLC transporter superfamily. Some major mechanisms are depicted in Fig. 3 and Table 1. The influx of 5-FU and capecitabine is performed by the anion transporter 2 (SLC22A7) and the equilibrative nucleoside transporters ENT1/2, whereas the efflux of the active 5-FU metabolites is mediated by active transporters such as ABCC5 and ABCC11 (Fig. 3). In addition, in a systematic approach correlating the expression of transporters and channels to drug resistance of 119 standard anticancer drugs in 60 human cancer cell lines of the National Cancer Institute (NCI-60 panel), further transporters and channels were identified to correlate with 5-FU resistance (e.g., SLC23A2, ATP1B3, ATP2A1, ATP2B4), which have not been explored yet

Zhang (2006) Kelland (1993) Hatch et al. (2014) Martínez-Balibrea et al. (2015) Van Houdt et al. (2011), Wen et al. (2013), Hayward et al. (2004)

(Huang et al., 2004). On the other hand, the disease-free interval of patients treated with adjuvant chemotherapy was significantly shorter in patients with low transcript levels of ABCA7, ABCA13, ABCB4, ABCC11 and ABCD4 (Hlavata et al., 2012). Moreover, the authors suggest that ABCC11 may be a promising candidate marker for a validation study on 5-FU therapy outcome in colon cancer patients. Cancer cells resistance to irinotecan/SN38 in CRC has been ascribed to the low intratumoral accumulation of the active metabolite. The increased active efflux by the multidrug resistance protein (MRP), an ATP-binding cassette (ABC) transporter protein, has been involved in this process (http://www.pharmgkb.org/do/ serve?objId=PA2001&objCls=Pathway) (Akiyama et al., 2012; Fujita et al., 2008; Zhao et al., 2013). However, pharmacogenetic studies have demonstrated mixed results regarding the role of drug transporters to adverse events and response (Teft et al., 2015; Trumpi et al., 2015). Copper transporter 1 (Ctr1, SLC31A1) is a membrane protein that
besides its physiological role in copper influx- plays a significant role in the cellular uptake of platinum derivatives

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(Holzer et al., 2004, 2006; Larson et al., 2009; Safaei and Howell, 2005). Down-regulation of Ctr1 markedly reduced cisplatin uptake in yeast and in mouse embryonic fibroblasts (Ishida et al., 2002; Lin et al., 2002). In this connection, clinical studies demonstrated that expression of CTR1 correlated with intratumoral platinum concentration and outcomes following Ptbased therapy. The organic cation transporters OCT1, OCT2, OCT3 (solute carrier 22A1, SLC22A2), and SLC22A3) have also been implicated in the intracellular influx of platinum derivatives. These carriers are known to mediate the intracellular uptake of a broad range of structurally diverse organic cations with molecular masses generally lower than 400 Da, such as choline, creatinine, and monoamine neurotransmitters, as well as a variety of xenobiotics, such as tetraethylammonium (prototypic organic cation), 1methyl-4-phenylpyridinium (neurotoxin), and clinically used drugs, e.g. metformin, cimetidine, and amantadine (Jonker, 2003; Wright, 2005). Expression of human OCT1 and OCT2 is mainly restricted to the liver and the kidney, respectively, whereas OCT3 (SLC22A3) is more widely distributed (Wright, 2005; Zhang, 2006). The selectivity of these transporters towards platinum derivatives remains controversial. In the human kidney epithelial HEK cells, the ectopic expression of OCT1 or OCT2 markedly increases oxaliplatin
but not cisplatin or carboplatin
accumulation and cytotoxicity in transfected cells, indicating that oxaliplatin is an excellent substrate of these transporters (Zhang, 2006). In contrast, the knockout of OCT1 in mice affects the uptake of cisplatin in the liver and intestine, but not the tissue accumulation of oxaliplatin, suggesting that OCT1 is not involved in oxaliplatin influx (Li et al., 2010). The accumulation of OCT3 transcript is about 10-fold higher in human colorectal than in control tissues (Yokoo et al., 2008). In colon cancer cell lines, gene transfer of OCT3 expression vector markedly enhances oxaliplatin uptake and cytotoxicity (Li et al., 2010). On the other hand, a recent retrospective study on the expression of oxaliplatin transporters OCT1, OCT2, OCT3, CTR1 and ATP7B in colorectal cancer and the subsequent response to FOLFOX-4 adjuvant therapy, revealed that high expression of OCT3 was related to resistance to this chemotherapy (Le Roy et al., 2016). These discrepancies might be explained by the distinct and selective biological function of OCTs carriers in influx/efflux of xenobiotics in vivo. OCT2 is located on the basolateral side of enterocytes, OCT3 on the apical side, whereas OCT1 is on both sides. Thus, these transporters might exert differential activities. After conjugation with glutathione, oxaliplatine can be excreted by MRP2/ABCC2 multidrug resistance protein. Other export mechanisms involved the copper-transporting ATPase 2 ATP7A/ B. These transporters are located in the trans Golgi network allowing platinum drugs accumulation in membrane vesicles followed by exocytosis. Noteworthy, the cross-talks of therapies have to be taken into account. The 5-FU suppressed ATP7B and OCT2 and increased MRP2 mRNA levels in the human colonic LS180 cell line (Theile et al., 2009). In metastatic CRC, the anti-EGFR monoclonal antibody cetuximab can overcome acquired resistance to irinotecan chemotherapy (Cunningham et al., 2004). Extensive pharmacogenetic approaches, e.g. based on singlenucleotide polymorphisms, and gene signatures have been devoted to identify some biomarkers of chemotherapy response, as well as to minimize side effects (De Mattia et al., 2015; Giacomini et al., 2013; Tripathi et al., 2016; Zheng et al., 2014). These studies offer the promise to identify gene sets and pathways related to drug resistance, and to propose more personalized treatments.

5. Use of nanomedicine to treat colorectal cancer 5.1. General aspects Nanotechnology coupled with novel efficacious therapeutic formulations can overcome many of the challenges associated with cancer drug development. Nanomaterials have been shown to exhibit properties deeply different from the bulk material from which they are synthesized. Moreover, the vast array of structural arrangements makes nanosystems incredibly versatile. In addition to improving aqueous solubility of antineoplastic drug molecules, the nanosize range allows for more rapid dissolution of the drug within the body, leading to better absorption and greater propensity to exert its effect. Drugs that are encapsulated into nanocarriers do not exhibit the same pharmacokinetic profiles as free drugs. The nanomaterial and the physical and chemical properties of the nanocarriers dictate the drug's biodistribution and pharmacokinetics upon systemic administration. This approach provides a convenient strategy for enhancing the accumulation and internalization of drugs within tumors. Nanodevices might also offer protection from unsuitable environments and metabolic and enzymatic degradation, as well as protection against early clearance from the body. Coating with compounds such as PEG allows nanosystems to navigate the body partly undetected by clearance systems, thereby increasing the circulation half-life (Vauthier, 2003). Furthermore, due to their size, NPs do interact directly with cell membrane and intracellular structures (Hillaireau and Couvreur, 2009). The application of nanotechnology has therefore an immense potential in overcoming or circumventing the extra- and intracellular mechanisms associated with drug resistance (de Verdière et al., 1997; Frank et al., 2014). Nanoparticles (NPs) have a critical role in reversing MDR by allowing enough drug to accumulate in the cytoplasm to be effective in both pump-dependent and pump-independent MDR. As mentioned in Table 1, members of the ABC superfamily including P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 2 (MRP2/ ABCC2), and breast cancer resistance protein (BCRP/ABCG2) function as ATP-driven drug efflux transporters, which are overexpressed in colon cancer cells, forming a unique defense against chemotherapeutics and a multitude of endogenous and exogenous cytotoxic agents. Nanomedicine enables the passive and/or the active targeting of cancer cells, taking advantage of the leaky tumor microvasculature, or of the presence of selective determinant on cancer cells (see below and Fig. 4). These properties favorably alter the biodistribution of antineoplastic drugs, simultaneously reducing adverse effects and increasing the concentration of drug within tumor tissue for the augmentation of therapeutic antineoplastic effect. Thus, the NPs-induced huge intracellular drug loading should circumvent the many resistance processes related to drug uptake, metabolism and efflux, and thus markedly improve chemotherapies efficiency. On the other hand, such approach should also alleviate sideeffects of chemotherapies that for a part are related to the selective and tissue-dependent expression of transporters. Accordingly, the neurotoxicity and the ototoxic activity of platinum derivatives have been ascribed to the expression of CTR1 and OCT2 in these tissues (Ciarimboli et al., 2010; More et al., 2010; Sprowl et al., 2013). Another point to consider concerns the way of administration of nanomedicine for CRC treatment. Nanomedicine might be administrated by inhalation, via rectal administration, per systemic route or orally. This later way of administration seems to be particularly well adapted in the treatment of gastrointestinal diseases, improving patient compliance and comfort. The oral colon specific drug delivery system (CDDS) should be capable of

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Fig. 4. Targeting strategy for nanodevices. In order to improve the intracellular delivery of the drug, nanoparticles could be functionalized with specific ligands that specifically bind receptors expressed primarily on malignant cells leading to receptor-mediated internalization, which is often necessary to release drugs inside the cells.

protecting the drug en route to the colon, i.e. drug release and absorption should not occur in the stomach as well as the small intestine, and neither the bioactive agent should be degraded in either of the dissolution sites but only released and absorbed once the system reaches the colon (Philip and Philip, 2010). Several studies reported that the delivery of 5-FU to the colon causes serious systemic side effects, such as mucositis, diarrhea, alteration in normal microflora structure, and translocation of bacteria. 5-FU may exert the cytotoxic effects on the colonic bacteria that are involved in the metabolism of some polysaccharides such as chitosan, xanthan gum [XG], amylose, and guar gum [GG]. Sitespecific delivery of 5-FU to the colon using GG and/or XG as a carrier (nanoparticles coated with natural gums) might allow drug release only in the colon, and might serve as nutrient for the probiotics enabling the normal colonic bacteria to thrive. Alternatively, the side effects of 5-FU nanoparticles on the gut microflora and gastrointestinal-related side effects could be overcome by the coadministration of probiotics (Singh et al., 2015). 5.2. Nanomedicine in cancer therapy Currently, most of the clinically approved cancer nanomedicines make use of the EPR effect, i.e., passive NPs accumulation into the tumor. The first nanoparticle developed in 1960s, Doxil, was a liposome, which encapsulated doxorubicin. This PEGylated liposomal doxorubicin particle of about 100 nm was approved for clinical use in 1995, for the treatment of HIV-related Kaposi's sarcoma, metastatic ovarian cancer and metastatic breast cancer (Barenholz, 2012). Since then, more cancer nanomedicines have entered the market, (Bourzac, 2012; Sheridan, 2012; Dawidczyk et al., 2014) which highlights the potential impact that nanomedicine can have on cancer therapy: DaunoXome
a 50 nm

33

liposomal daunorubicin particle
approved for HIV-related Kaposi's sarcoma; Myocet
an 150–180 nm non-PEGylated liposomal doxorubicin particle
approved in Europe and Canada for metastatic breast cancers; Abraxane
a 10 nm albumin-bound paclitaxel particle (following disintegration in plasma)
approved for metastatic breast cancer and recently for pancreatic ductal adenocarcinoma; Lipusu
a liposomal paclitaxel particle
approved in China for various cancers including breast cancers and non-small-cell lung cancer; Genexol-PM
a 20–50 nm Cremophor- free, polymeric micelle-formulated paclitaxel
approved in South Korea for metastatic breast cancer; MM-398– an 100 nm liposomal formulation of irinotecan
approved recently for the treatment of pancreatic ductal adenocarcinoma; and finally, PICN
a 100–110 nm formulation of paclitaxel stabilized with polymer and lipids
approved in India for metastatic breast cancer and currently in clinical trials in the US (Stylianopoulos and Jain, 2015). To be noted that we have developped a doxorubicin loaded poly (isohexyl cyanoacrylate) nanoparticle formulation (i.e., Livatag1) that showed considerable antitumor activity against multidrugresistant protein-overexpressing hepatocellular carcinoma in vivo model (Barraud et al., 2005). This nanomedicine which is currently in a multicentric phase III clinical trials for the treatment of hepatocellular carcinoma (HCC) recently received the “fast track” status from the FDA. In the phase II/III trial that compared the efficacy of those doxorubicin loaded poly(isohexyl cyanoacrylate) NPs (30 mg/m2 repeated doses), versus the current standard of care (transarterial chemoembolisation with a cytotoxic drug), 88.9% survival rate was observed after 18 months of nanoparticles treatment, against only 54.5% survival rate in patients treated by chemoembolization [http://www.onxeo.com/fr/doxorubicinetransdrug-amelioration-significative-de-la-duree-de-survie-despatients-atteints-dun-carcinome-hepatocellulaire-avance-dansun-essai-clinique-de-phase-ii/]. 5.3. Nanomedicines for colorectal cancer treatment The current researches focus on polymeric nanoparticles, dendrimers, liposomes, inorganic (silica, quantum dot core or iron), hybrid nanoparticles and polymeric micelles, some of them being stimuli-responsive (Brigger et al., 2012; Mura et al., 2013). These NPs have been first used ex vivo and in preclinical models as a proof of concept. More than one hundred studies concerning the preclinical evaluation of nanomedicine for CRC treatment have been published. Most of them concern the use of nanoparticles for imaging cancer cells or tumor angiogenesis, using radioisotopes, MRI, (near infra) fluorescence (fluorochrome, quantum dots) or luminescence (europium ions). These NPs were targeted towards tumor cells following several approaches, including carcinoembryonic antigen, RGD peptide or cetuximab (Fig. 4). Other nanoparticles were dedicated for the treatment of colorectal cancers, when loaded with conventional chemotherapies, including 5-FU, irinotecan and platinum derivatives, as well as targeted therapies (cetuximab, rapamycin, celecoxib). Nanoparticles offer also opportunities for other therapeutic approaches through the intra tumoral release of photosensizing or for thermal destruction of malignant cells. Concerning conventional chemotherapies, Oxaliplatin NPs were generated using stearic acid-g-chitosan oligosaccharide (CSO-SA) polymeric micelles as a drug-delivery system. These CSO-SA/oxaliplatin micelles showed excellent internalization ability, increasing oxaliplatin accumulation both in CRC cells and tissues. Furthermore, CSO-SA/oxaliplatin micelles exhibited an increased cytotoxicity against the bulk cancer cells, as compared to

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oxaliplatin as well as againts chemoresistance of CSC subpopulations in vitro (Wang et al., 2011). Targeting of oxaliplatin NPs towards cancer cells was also approached using EGF-liposomes as delivery systems (Zalba et al., 2016). These liposomes proved to efficiently target tumor cells in xenograft colon carcinoma model which represents an attractive nanosystem for cancer therapy. Other cell surface receptors, e.g. isoforms of Hyaluronic acid (HA) receptors, CD44 and RHAMM (a receptor for hyaluronan-mediated motility), are also overexpressed in colorectal carcinoma (Köbel et al., 2004; Wang et al., 1996), and constitute valuable determinant for targeting cancer cells. Working on this rationale, Hyaluronic acid (HA) coupled chitosan nanoparticles (HACTNP) loaded with either 5-flurouracil (5FU) or oxaliplatin, were prepared for the effective delivery of drugs to colon tumors (Jain and Jain, 2008; Jain, 2010). 5-FU-loaded HACTNP nanoparticles showed significant higher uptake by colonic cancer cells HT-29 and higher cytotoxicity as compared to the conventional 5-FU solution (Jain and Jain, 2008). Oral administration of HACTNPs bearing oxaliplatin showed relatively high local drug concentration in the colonic milieu as well as in colonic tumors with prolonged exposure time, which provides a potential to enhance antitumor efficacy with low systemic toxicity for the treatment of colon cancer (Jain, 2010). Another group has developed an oral formulation of oxaliplatin encapsulated in pH sensitive, mucoadhesive chitosan-coated alginate microspheres. These microparticles were formulated to release the chemotherapeutics after passing through the acidic gastric environment, thus targeting the intestinal tract. In vivo, these particles substantially reduced the tumor burden in an orthotopic mouse model of colorectal cancer, and reduced mortality (Urbanska et al., 2012). Other targeted therapy of colorectal neoplasia were developed using rapamycin (an inhibitor of the TORC1 signaling complex, downstream of receptors tyrosine kinase/PI3K/Akt pathway, see Figs. 1 and 3) in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia (LTTHYKL peptide). These NPs displayed comparable therapeutic efficacy than rapamycin free drug in mice, but with significantly less systemic toxicity (Khondee et al., 2015). Since PLK1 is considered as a predictive factor to identify patients likely to respond to preoperative RCT (Fernandez-Acenero et al., 2015; Rödel et al., 2010), several inhibitors targeting PLKs are currently in development and are under investigation in a growing number of clinical trials (Liu, 2015; Strebhardt, 2010). In preclinical studies, siRNA formulated in stable nucleic acid lipid particles (SNALP) induced Plk1 mRNA cleavage, silencing of Plk1 expression, and potent antiproliferative activity in various colon cancer cell lines ex vivo (Judge et al., 2009). An alternative to current strategies to improve the efficacy of conventional chemotherapy in the treatment of advanced or recurrent colon cancer might involve combined therapy such as use of 5-FU-loaded biodegradable poly(e-caprolactone) nanoparticles (PCL NPs) with the cytotoxic suicide gene E (Ortiz et al., 2015). On the other hand, a number of MDR reversal agents with P-gp inhibitory activity, also known as “chemosensitizers”, were identified (Szakács et al., 2006; Teodori et al., 2002). However, their unacceptable side effects (e.g., cyclosporine-A, verapamil) include unpredicted pharmacokinetic interaction with anticancer drugs and other transport proteins (e.g. PSC833, VX-710). Another concern with these inhibitors is that they may increase chemotherapeutic side effects by blocking physiological anticancer drug efflux from normal cells. For example, there are high levels of P-gp in the small intestine (Bebawy and Sze, 2008). These obstacles can be bypassed using a targeted nanocarrier delivery system to coencapsulate the anticancer drug and chemosensitizer (Song et al.,

2009). The functionalization with ligands and the pH-responsive technique could also be utilized to enhance the reversal of MDR. Curcumin, the major active principal of Curcuma longa, used for breast, colon and prostate cancers, was shown to suppress MRPs in cancer cells. Thus, Curcumin/5-fluorouracil loaded thiolated chitosan nanoparticles (CRC-TCS-NPs/5-FU-TCS-NPs) as well as Curcumin/5-fluorouracil N,O-carboxymethyl chitosan nanoparticles showed enhanced anticancer effects on colon cancer cells in vitro and improved the bioavailability of the drugs in vivo (Anitha et al., 2014). In vivo, administration of curcumin in liposomes (liposome-PEG-PEI complex) inhibited about 60
90% of tumor growth in mice bearing CT-26 or B16F10 cells (Lin et al., 2012). Drug delivery of combined cytotoxic and antivascular chemotherapies in “multidrug” nanoassemblies is another attractive way to improve the treatment of experimental cancers. The proof of concept of this approach has been done in a human colon carcinoma xenograft nude mice model by using the anticancer compound gemcitabine conjugated with squalene, together with isocombretastatin A-4, a new isomer of the antivascular combretastatin A-4. These molecules were able to spontaneously selfassemble as nanoparticles which induced complete tumor regression and were found superior to all the other treatments in human colon carcinoma xenograft model LS174-T (Maksimenko et al., 2014). 5.4. Clinical trials and colorectal cancer treatment using nanomedicines Concerning colorectal cancer, despite the abundance of preclinical studies, the number clinical trial involving nanomedicine (referenced at URL: https://clinicaltrials.gov/, accessed on 03/31/2016) remains quite limited (Table 2). These ongoing trials (phases I–II) concerns mainly the following nanoformulations: (i) irinotecan or derivatives loaded onto liposomes or cyclodextrin NPs, (ii) mitomycin-C encapsulated into pegylated liposomes, alone or in combination with LV/5-FU, (iii) siRNA targeting the polo-like kinase 1, (iv) a liposomal formulation of doxorubicin to be used in conjunction with thermal ablation, and (v) a radio-enhancer for rectal cancer. 5.4.1. Irinotecan and derivatives PEP02 (MM-398) is a highly stable nanoliposomal irinotecan that theoretically has therapeutic advantages over the free form of the drug (irinotecan and its active metabolite SN-38), due to its site-specific delivery and extended drug release. This formulation proved to be less toxic to healthy tissue, while maintaining or increasing antitumor potency (Drummond et al., 2006). In randomized phase II study, the efficacy of the combination of PEP02 with LV/5-FU was evaluated as a second line treatment in metastatic CRC (mCRC) patients who failed prior oxaliplatin-based first-line therapy (Table 2). The results of this study (termed PEPCOL) suggest that this regimen could be as active as the optimized FOLFIRI3 regimen, but more active than the standard FOLFIRI regimen in oxaliplatin-pretreated mCRC patients, with an acceptable safety profile and a best overall response rate. With further ongoing optimization, this regimen has the potential to provide a clinically useful treatment for post-oxaliplatin mCRC patients (Chibaudel et al., 2016). CPX-1 is being developed with the hypothesis that it will be more active than conventional irinotecan and fluoropyrimidine in the treatment of sensitive malignancies. CPX-1 is a novel, liposome-encapsulated formulation of irinotecan and floxuridine (FUDR, fluorodeoxyuridine) designed to deliver optimized synergistic molar ratios of both drugs postinfusion. An open-label, single-arm, dose-escalating phase I study was designed to

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Table 2 Examples of Nanoparticle formulations currently in clinical trials of colorectal cancer. Drug name and Therapy

Description of Drug Formulation

Indication

Official Title

PEP02 plus LV/5-FU (MM-398) as second-line therapy

Liposome-encapsulated irinotecan hydrochloride PEP02 with leucovorin/5-fluorouracil

Metastatic colorectal cancer previously treated with an oxaliplatin-based regimen Advanced Colorectal Carcinoma

A Randomized Phase II Study of PEP02 or Irinotecan NCT01375816 in Combination With Leucovorin and 5-Fluorouracil in Second Line Therapy of Metastatic Colorectal Cancer (PEPCOL) NCT00361842 Multicenter, Open-Label, Phase 2 Study Of CPX-1 (Irinotecan HCl: Floxuridine) Liposome Injection In Patients With Advanced Colorectal Carcinoma

Metastatic Colorectal Cancer

Phase II Trial of LE SN38 in Patients with Metastatic NCT00311610 Colorectal Cancer After Progression on Oxaliplatin

Metastatic Colorectal Cancer

NCT01705002 A Phase I, Dose-Escalating, Safety Study of an Intravenously Administered Pegylated Liposomal Mitomycin-C Lipid-based Prodrug (PL-MLP, PROMITIL) in Cancer Patients With Solid Tumors NCT01437007 A Phase 1 Dose Escalation Study of Hepatic IntraArterial Administration of TKM 080301 (Lipid Nanoparticles Containing siRNA Against the PLK1 Gene Product) in Patients With Colorectal, Pancreas, Gastric, Breast, Ovarian and Esophageal Cancers With Hepatic Metastases Phase Ib/II Study of Neoadjuvant Chemoradiotherapy NCT02010567 With CRLX-101 and Capecitabine for Locally Advanced Rectal Cancer

CPX-1 (Irinotecan HCl: Floxuridine) Liposome

CPX-1 Liposome Injection is a liposomal formulation of a fixed combination of the irinotecan HCl and floxuridine SN-38 Liposome (metabolite of Liposomal SN-38 irinotecan) as second-line therapy Pegylated Liposomal Mitomycin-C PL-MLP, PROMITIL Lipid-based Prodrug

TKM-080301

Lipid Nanoparticles Containing siRNA Against the PLK1 Gene Product

Colorectal Cancer With Hepatic Metastases

CRLX-101 plus Capecitabine, oral fluoropyrimidine prodrug, metabolically converted to 5-fluorouracil PEP503 (Radio-enhancer) plus 5-FU/Xeloda plus Radiotherapy

Nanoparticle conjugate: cyclodextrin-based polymer (CDP) and anti-cancer compound camptothecin (CPT) Nanoparticle formulation of hafnium oxide crystals

Locally Advanced Rectal Cancer

ThermoDox

Metastatic Colorectal Thermally sensitive liposomal doxorubicin designed to be used in Cancer (mCRC): Colon conjunction with thermal ablation Cancer Liver Metastasis

Unresectable Rectal Cancer

determine the maximum tolerated dose and pharmacokinetics of CPX-1 in patients with advanced solid tumors. Outpatient CPX-1 was well tolerated and antitumor activity was shown in patients with advanced solid tumors. This study represents the first step in the clinical testing of ratiometric dosing, a novel concept in combination chemotherapy for cancer, enabled by nanomedicine approach (Batist et al., 2009). LE-SN38 is a liposomal formulation of SN-38, the active metabolite of irinotecan, allowing the solubilisation of this compound, and enabling a more precise dosing of this metabolite, since the prodrug conversion rate of irinotecan in the liver shows important interindividual variations. Phase I clinical trial was completed during 2004, and demonstrated the safety and tolerability of LE-SN38 and established a maximum tolerated dose. A single arm, open label Phase II study was undertaken in 2006, aiming: i) to analyse the objective response rate following LE-SN38 treatment as a second-line treatment in patients with metastatic colorectal cancer, ii) to determine the toxicity profile of the drug, iii) to establish the proportion of patients who experience any grade 3 or greater toxicity, and iv) to follow the progressionfree survival and overall survival for patients treated with this liposomal formulation. This study performed on 30 patients did not evidence any complete or partial response, 11 patients having only stable disease. The authors of the study concluded that although LE-SN38 was associated with an acceptable toxicity profile, this formulation, dose and schedule did not deserve further evaluation (Ocean et al., 2008). CRLX101 is a nanoparticle formulation, consisting in a cyclodextrin-based polymer (CDP) and the anti-cancer compound camptothecin (CPT). CRLX101 was originally named “IT-101” and

Clinical Trials. gov Identifier

NCT02465593 A Phase Ib//II Study of PEP503 (Radio-enhancer) With Radiotherapy, in Combination With Concurrent Chemotherapy for Patients With Unresectable Rectal Cancer (5-FU or oral capecitabine) Phase II Open Label Trial of Thermal Ablation and NCT01464593 Lyso-Thermosensitive Liposomal Doxorubicin (Thermodox) for Metastatic Colorectal Cancer (mCRC) Liver Lesions (ABLATE)

was changed to CRLX101 after licensing to Cerulean Pharma Inc. CRLX101 is the official name in clinical trials (Davis, 2009). In CRLX101, CPT is linked covalently through a glycine link to the linear copolymer CDP, which in turn consists of alternating subunits of beta-cyclodextrin and polyethylene glycol (PEG) (Weiss et al. 2013; Clark et al., 2016). The CRLX101 nanoparticle is well dispersed in water. After intravenous injection, active CPT is slowly released as the linkage is hydrolysed. The size of the nanoparticle (20–50 nm in diameter) facilitates extravasation in the more leaky vessels of tumors via the so-called « enhanced permeability and retention (EPR) » effect, leading to increased tumor accumulation. The phase 1/2a showed CRLX101-related adverse events including fatigue, cystitis, anemia, neutropenia, leucopenia and thrombocytopenia in some patients, but these events have been far less in severity as compared to CPT or its derivatives (Weiss et al., 2013). An open label, single-arm, multi-center, Phase Ib/II study was designed at the end of 2013, to evaluate the maximum tolerated dose (Phase Ib) and the rate of pathological complete response (Phase II, time frame: 6 years) of CRLX101 combined with capecitabine and radiation therapy in patients with rectal cancer. The completion date of the study is estimated at the end of 2022. 5.4.2. Mitomycin C Mitomycin C (MMC) is an antineoplastic antibiotic isolated from the bacterium streptomyces caespitosus. MMC often retains activity against P-glycoprotein mediated multidrug resistant tumor cells. Resistance to MMC is more often related to the tumor levels of DT-diaphorase, which plays important role in bioreductive activation of MMC to reactive cytotoxic moieties. Despite its high potency as anti-cancer drug, the therapeutic use of MMC is

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hindered by undesirable dose limiting toxicities, such as cumulative bone marrow suppression, nephrotoxicity (hemolytic-uremic syndrome) and lung toxicity (interstitial pneumonitis). In addition, MMC is rapidly cleared and metabolized. A mitomycin-C lipidbased prodrug (MLP) formulated in pegylated liposomes (PL-MLP, PROMITIL) was previously reported to have significant antitumor activity and reduced toxicity in mouse tumor models (Gabizon, 2006). MLP is activated by thiolysis releasing mitomycin-C (MMC) which rapidly dissociates from liposomes (Gabizon et al., 2012). In recent years, lipid-based prodrug of MMC (MMC Lipid-based Prodrug, or MLP) has been formulated in long-circulating pegylated liposomes (PL-MLP), enabling high liposomal drug payload and resulting in a stable formulation with reduced toxicity (Gabizon et al., 2012; Gabizon, 2006), as recently confirmed in a phase 1 study in cancer patients with solid tumors, including metastatic CRC (Golan et al., 2015). Based on its robust stability profile, reduced toxicity, and broad spectrum of antitumor activity, PL-MLP is a promising candidate for clinical applications in a large variety of cancer indications (Patil et al., 2016). 5.4.3. Polo-like kinase I TKM-080301 (TKM-PLK1) is a lipid nanoparticle formulation of a small interfering RNA directed against Plk1. In preclinical studies, it was found that TKM-080301 down regulated Plk1 mRNA, silenced Plk1 expression in human cancer cell lines and exerted potent antiproliferative activity in a series of cancer cell lines and in subcutaneous and orthotopic xenografts of hepatocarcinoma cells in SCID/beige mice (Judge et al., 2009). TKM-080301 is being evaluated in a first-in-human, dose-escalation, phase I study in patients with lymphoma or advanced solid tumors, e.g. mCRC, which will include study of the pharmacodynamic effects of Plk1 inhibition in patient biopsy samples (Liu, 2015). 5.4.4. Radioenhancer PEP503 (also termed NBTXR3) is a nanoparticle formulation of hafnium oxide (HfO2). NBTXR3 NPs are prepared by mixing a solution of tetramethylammonium hydroxide (TMAOH) with hafnium chloride (HfCl4). Upon local injection of NBTXR3 into the tumor, the hafnium oxide-containing NPs accumulate in the tumor cells. Subsequent application of radiation beams to the tumor tissue causes HfO2 particles to emit huge amounts of electrons. This results in the formation of free radicals within the tumor cells, which in turn causes targeted destruction of the cancer cells (Weissig and Guzman-Villanueva, 2015). PEP503 is considered as a radioenhancer, not a radiosensitizer. A Phase Ib//II study of PEP503 with radiotherapy, in combination with chemotherapy has been designed in 2015 for patients with unresectable rectal cancer. Patients will receive PEP503, given as intratumor injection and followed by preoperative radiation therapy. Patients will have concurrent continuous IV intravenous infusion (CVI) of 5-FU or oral capecitabine. The estimated primary completion date of the study is expected for December 2017. 5.4.5. Thermally sensitive liposomal doxorubicin Using temperature-sensitive liposomes (TSLs) is one way to achieve specific delivery of a drug (e.g. doxocyclin, ThermoDox1) to a target site. The liposome acts as a protective carrier, allowing increased drug to flow through the bloodstream by minimizing clearance and non-specific uptake. On reaching microvessels within a heated tumor, the drug is released and quickly penetrates. A major advance in the field is ThermoDox1 (Celsion), demonstrating significant improvements to the drug release rates and drug uptake in heated tumors ( 41  C). Celsion has currently engaged a Phase II Study of ThermoDox(R) in combination with radiofrequency ablation (RFA) for the treatment of colorectal liver metastases (ABLATE, Table 2).

6. Conclusions This review has demonstrated the richness and inventivity of nanodevices for the treatment of colorectal cancers in preclinical assays. Most of the proposed strategies allow to reduce side effects and toxicity and to improve pharmacological efficacy through the control of the drug release and distribution. At the cellular level, nanoformulations are even capable to overcome resistance, due to drug efflux pumps, to downregulation of drug transporters or to excessive drug metabolization. Nevertheless, successful clinical trials remain limited, probably due to the important variability of patients and tumors physio-pathological characteristics, concerning nanocarriers opsonization, biodistribution and pharmacokinetics, as well as, tumor accessibility. Indeed, most of the therapeutic approaches are based on the EPR effect which dramatically vary from patient to patient and from tumor to tumor which is not the case in preclinical, well established rodent models. The difficulties in the design and scaling-up of nanodevices to meet drug regulatory approval is another important limitation which hinders the translation of nanomedicines from bench to bed side and to allow the treatment of colorectal cancer in the clinical reality. The development of new tools to allow the prediction of the patients eligibility for efficient nanomedicine treatment is probably one way to have more successful nanomedicines entering advanced clinical trials in the near future. Conflict of interest The authors have declared that no conflict of interest exists. Acknowledgements This work was supported by a funding from the European Research Council under the European Community’s Seventh Framework Programme FP7/2007-2013 Grant Agreement No. 249835, by Centre National de la Recherche Scientifique , and Institut National de la Santé et de la Recherche Médicale. References Ait Ouakrim, D., Pizot, C., Boniol, M., Malvezzi, M., Boniol, M., Negri, E., Bota, M., Jenkins, M.A., Bleiberg, H., Autier, P., 2015. Trends in colorectal cancer mortality in Europe: retrospective analysis of the WHO mortality database. BMJ h4970. Akiyama, Y., Fujita, K.-I., Ishida, H., Sunakawa, Y., Yamashita, K., Kawara, K., Miwa, K., Saji, S., Sasaki, Y., 2012. Association of ABCC2 genotype with efficacy of first-line FOLFIRI in Japanese patients with advanced colorectal cancer. Drug Metab. Pharmacokinet. 27, 325–335. Alexander, J., Cukierman, E., 2016. Stromal dynamic reciprocity in cancer: intricacies of fibroblastic-ECM interactions. Curr. Opin. Cell Biol. 42, 80–93. Amstutz, U., Froehlich, T.K., Largiadèr, C.R., 2011. Dihydropyrimidine dehydrogenase gene as a major predictor of severe 5-fluorouracil toxicity. Pharmacogenomics 12, 1321–1336. André, T., Louvet, C., Maindrault-Goebel, F., Couteau, C., Mabro, M., Lotz, J.P., GillesAmar, V., Krulik, M., Carola, E., Izrael, V., de Gramont, A., 1999. CPT-11 (irinotecan) addition to bimonthly, high-dose leucovorin and bolus and continuous-infusion 5-fluorouracil (FOLFIRI) for pretreated metastatic colorectal cancer. Gercor. Eur. J. Cancer 35, 1343–1347. André, T., Kotelevets, L., Vaillant, J.C., Coudray, A.M., Weber, L., Prévot, S., Parc, R., Gespach, C., Chastre, E., 2000. Vegf, Vegf-B, Vegf-C and their receptors KDR, FLT1 and FLT-4 during the neoplastic progression of human colonic mucosa. Int. J. Cancer 86, 174–181. Anitha, A., Sreeranganathan, M., Chennazhi, K.P., Lakshmanan, V.-K., Jayakumar, R., 2014. In vitro combinatorial anticancer effects of 5-fluorouracil and curcumin loaded N,O-carboxymethyl chitosan nanoparticles toward colon cancer and in vivo pharmacokinetic studies. Eur. J. Pharm. Biopharm. 88, 238–251. Bardou, M., Montembault, S., Giraud, V., Balian, A., Borotto, E., Houdayer, C., Capron, F., Chaput, J.-C., Naveau, S., 2002. Excessive alcohol consumption favours high risk polyp or colorectal cancer occurrence among patients with adenomas: a case control study. Gut 50, 38–42. Barenholz, Y., 2012. Doxil(R)
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