Emerging nanomedicines for anti-stromal therapy against desmoplastic tumors

Journal Pre-proof Emerging nanomedicines for anti-stromal therapy against desmoplastic tumors

Xuexiang Han, Ying Xu, Marzieh Geranpayehvaghei, Gregory J. Anderson, Yiye Li, Guangjun Nie PII:

S0142-9612(19)30863-4

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119745

Reference:

JBMT 119745

To appear in:

Biomaterials

Received Date:

08 July 2019

Accepted Date:

25 December 2019

Please cite this article as: Xuexiang Han, Ying Xu, Marzieh Geranpayehvaghei, Gregory J. Anderson, Yiye Li, Guangjun Nie, Emerging nanomedicines for anti-stromal therapy against desmoplastic tumors, Biomaterials (2019), https://doi.org/10.1016/j.biomaterials.2019.119745

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

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Emerging nanomedicines for anti-stromal therapy against desmoplastic tumors

Xuexiang Hana, b, 1, Ying Xua, b, 1, Marzieh Geranpayehvagheia, Gregory J. Andersonc, Yiye Lia, b*, Guangjun Niea, b*

a

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, P.R. China b Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, P.R. China c

QIMR Berghofer Medical Research Institute, Royal Brisbane Hospital, Brisbane,

Queensland 4029, Australia

1These

authors contributed equally to this work.

*Corresponding

authors:

[email protected] (Y. Li) [email protected] (G. Nie)

1

Journal Pre-proof Abstract Solid tumors, especially desmoplastic tumors, are characterized by a dense fibrotic stroma composed of abundant cancer-associated fibroblasts and excessive extracellular matrix. These physical barriers seriously compromise drug delivery to tumor cells, leading to suboptimal treatment efficacy and resistance to current tumorcentric therapeutics. The need to overcome these problems has driven extensive investigations and sparked the flourish of anti-stromal therapy, particularly in the field of nanomedicines. In this paper, we firstly review the major components of the tumor stroma and discuss their impact on drug delivery. Then, according to the different stromal targets, we summarize the current status of anti-stromal therapy and highlight recent advances in anti-stromal nanomedicines. We further examine the potential of nano-enabled anti-stromal therapy to enhance the anti-tumor efficacy of other therapeutic modalities, including chemotherapy, immunotherapy, phototherapy and radiotherapy. Finally, the potential concerns and future developments of anti-stromal nanomedicines are discussed.

Keywords: Desmoplastic tumor, Cancer-associated fibroblast, Anti-stromal therapy, Nanomedicine, Drug delivery

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Journal Pre-proof 1. Introduction Despite the rapid development of new therapeutic approaches, malignant tumors are still one of the leading causes of human death globally. Currently, most clinical therapeutics aim at tumor cells while overlooking the surrounding tumor microenvironment (TME). The TME comprises all of the physiological and biochemical elements, including, but not limited to, the extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), cancer-associated immune cells, tumor vasculature system, and the hypoxic and acidic environment [1]. Solid tumors, especially desmoplastic tumors (e.g. pancreatic cancers [2], bladder cancers [3], and a subset of breast cancer [4]), consist of abundant CAFs and excessive ECM, which create a pathological barrier that impedes the delivery of tumor-centric therapeutics to tumor cells [5-7]. Moreover, accumulating evidences have emphasized the intrinsic impacts of the fibrotic TME on accelerating tumor progression and worsen therapeutic outcomes [8]. Since the rare lethal tumor cell “seed” is raised and protected by the abundant tumor stromal “soil” [9], it is crucial to develop stromalbased therapies to re-engineer the hostile TME.

Over the last two decades, nanomedicines have attracted tremendous attention and been

actively

investigated

for

the

delivery

of

chemotherapeutic

agents.

Nanomedicines have several advantages over conventional chemotherapeutic agents, such as improving the solubility of hydrophobic drugs, prolonging the blood circulation time of their cargo, preferentially accumulating within tumors via the enhanced permeability and retention (EPR) effect [10], enhancing cellular uptake, and alleviating off-target toxicities [11]. Up to now, the FDA has approved several nanomedicines for tumor therapy, including Doxil (liposomal doxorubicin), Onivyde (liposomal irinotecan) and Abraxane (albumin-bound paclitaxel, nab-paclitaxel). Despite the great hope for these marked nanomedicines, they only alleviate some dose-limiting toxicities but do not significantly improve overall survival compared to the traditional drugs [12]. Insufficient accumulation of these nanomedicines in tumor tissue is proposed to be the major reason for their limited clinical benefits [13, 14]. 3

Journal Pre-proof These nanomedicines rely heavily on the EPR effect to achieve passive tumor targeting and accumulation, however, this effect occurs merely to a limited extent in solid tumors due to the heterogeneous and desmoplastic TME [2, 15, 16]. In a typical scenario, these extravasated nanomedicines are restrictedly located to to the perivascular region and their further penetration into the tumor parenchyma is limited due to their bind and trap to the nearly impenetrable pathological barrier posed by the dense stroma [2]. This, in turn, results in the sublethal dose of nanomedicines in tumor cells and finally compromises treatment efficacy [17-21]. Unfortunately, this off-target effect is even more profound for active-targeting nanomedicines because of the high affinity between the targeting ligands and stromal receptors [22].

Recently, alternative to delivering nanomedicines to the tumor cells, increasing attention has been paid to harness the intrinsic off-target distribution of nanomedicines to achieve anti-stromal treatment, augment the EPR effect and ultimately potentiate other backbone therapies. In this review, we discuss the stromal barriers in desmoplastic tumors established by CAFs. We also summarize the potential stromal targets that can be utilized to design nanomedicines, and highlight emerging nano-enabled strategies for anti-stromal therapy. Finally, we discuss the combination therapy between anti-stromal therapy and other therapeutic modalities and provide a critical perspective on the future development of anti-stromal nanomedicines.

2. Stromal barriers A key characteristic of desmoplastic tumors is the presence of a dense fibrotic stroma, which is commonly accompanied by hyperactive CAFs and excessive ECM deposition. For example, pancreatic ductal adenocarcinoma (PDAC) is a highly stroma-rich and hard mass of which almost 90% is made up of stromal components [23]. The dense stroma can increase solid stress, elevate interstitial fluid pressure (IFP) and compress blood vessels [24], thus establishing a series of pathological barriers that substantially block the transport of circulating therapeutic agents to the 4

Journal Pre-proof tumor cells (Fig. 1). It should be noted that although endothelial cells, pericytes and tumor-associated macrophages substantially affect therapeutic agents delivery as well, they are beyond the scope of this review due to their less contribution to the desmoplastic reaction and are discussed in details elsewhere [18, 25, 26].

Fig. 1. Stromal barriers established by CAFs and their secreted ECM. CAFs are activated by paracrine pro-stromal cytokines from tumor cells and can maintain the activated phenotype in an autocrine manner. They produce a huge amount of ECM to isolate tumor cells into “nests”. Together, CAFs and ECM establish a series of physical barriers that limit the availability of therapeutic agents to tumor cells.

2.1 Extracellular matrix The ECM is the major acellular component of both normal tissues and tumors. It provides structure and support for the cellular components by creating an integrated three dimensional macromolecular network in the extracellular interstitium of tissues. The ECM consists of well-organized interactions of fibrous macromolecules, including

collagen,

fibronectin,

hyaluronic

acid

(HA),

glycoproteins

and

proteoglycans. It is a highly dynamic network, being constantly degraded by enzymes (e.g. matrix metalloproteinases (MMPs), and collagenases) and replenished by fibroblast secretions [8, 27, 28]. In normal tissues, the unique composition and structure of the ECM function as growth regulators, dynamically regulating tissue homeostasis and wound repair [8].

However, in malignant tumors, excessive ECM production and limited ECM 5

Journal Pre-proof turnover lead to fibrosis, which is a hallmark of many types of tumors. The dense ECM prevents drug penetration and uniform distribution in multiple ways [6, 29]. Firstly, the ECM compresses the tumor vessels, thus reducing blood perfusion and the delivery of therapeutic agents. Secondly, the ECM elevated IFP, which largely hinders extravasation of circulating therapeutic agents and impedes their penetration through the tumor interstitium via convective transport [30]. Thirdly, the mesh-like ECM network with its tortuous interstitial space establish a direct physical barrier for therapeutic agents and restrict their diffusion. Finally, positively charged collagen and negatively charged HA can further sequester charged therapeutic agents with high binding affinity and decrease their interstitial transport [31, 32]. Collectively, the abundant ECM forms the primary acellular barrier for drug delivery.

2.2 Cancer-associated fibroblasts Fibroblasts are quiescent, spindle-shaped cells in healthy tissues. During wound healing, they can be activated and become synthetically active to produce ECM components [33]. During tumorigenesis, a “wound” that never heals [34], fibroblasts are constantly activated and transdifferentiated into CAFs characterized by the expression of α-smooth muscle actin (α-SMA). So far, many pro-stromal factors have identified to be involved in CAFs activation and ECM production, including transforming growth factor-beta (TGF-β), Hedgehog (Hh), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) [33, 35]. CAFs are not “bystanders”, but “partners” in crime, for they actively facilitate tumor cell growth [36], invasion [37], and metastasis [38]. In desmoplastic tumors, CAFs are the major stromal cells and the key mediators for the stromal barriers. Usually, CAFs produce large amounts of ECM to isolate tumor cells into “nests” and contribute to the binding site barrier for interstitial transport of therapeutic agents [39]. Furthermore, the abundant CAFs not only increase solid stress to compress blood vessels to compromise the systemic delivery of therapeutic agents, but also elevate IFP to impair their interstitial transport [40]. Notably, CAFs themselves are able to entrap active therapeutic agents intracellularly and limit the availability of drugs for tumor cells 6

Journal Pre-proof [41]. Thus, the hyperactive CAFs form the major cellular barrier for drug delivery.

3. Nanomedicines-enabled anti-stromal therapy The dense stroma not only establishes a unique pathological obstacle for therapeutics delivery, but also offers a direct target for anti-stromal therapy. With the increased understanding of tumor-stromal biology, more and more stroma targets are discovered and actively investigated. In this section, we will discuss potential pharmacological targets and their corresponding stromal-targeting agents. In particular, recent advances in nanomedicines targeting ECM, CAFs and pro-stromal signaling are addressed (Fig. 2).

Fig. 2. A summary of nano-enabled anti-stromal strategies targeting ECM, CAFs and pro-stromal signaling. Based on their pharmacological targets, anti-stromal nanomedicines fall into three broad categories. The first one is targeting ECM by blocking ECM biogenesis, inhibiting ECM stiffness and facilitating ECM degradation. The second one is targeting CAFs by eliminating CAFs, reducing CAFs activity, inducing CAFs quiescent and turning CAFs against tumors. The last one is targeting prostromal signaling by scavenging pro-stromal cytokines, blocking receptor activation and inhibiting downstream signaling.

3.1 Targeting extracellular matrix The abundant ECM composition and its mesh-like structure prevent the access of therapeutic agents to tumor cells. As a consequence, various strategies targeting the ECM have been developed to enhance the transport and distribution of therapeutic agents. These include blocking ECM biogenesis, reducing ECM stiffness and facilitating ECM degradation. 7

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3.1.1 Blocking ECM biogenesis Excessive accumulation of ECM components, such as collagen, fibronectin, HA, proteoglycans and glycoproteins, leads to fibrotic stroma, which can be alleviated by interfering with ECM biogenesis. Collagen, the most abundant protein in the interstitial ECM, not only promotes tumor cell proliferation, invasion and metastasis, but also acts as a protective pathological barrier to hamper interstitial drug transport [42, 43]. So it is an important target for anti-stromal therapy. Collagen biosynthesis requires collagen prolyl-4-hydroxylase (P4H) activity to catalyze collagen proline hydroxylation, and inhibition of this enzyme by a P4H inhibitor in preclinical studies efficiently reduces collagen biogenesis and impairs tumor growth and metastasis. [44, 45]. Since P4H requires iron as cofactor for its enzymatic activity, Hartmann et al. successfully utilized immunoliposomes loaded with deferoxamine, an iron chelator, to interfere with the collagen synthesis in TGF-β stimulated fibroblasts [46]. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is also required for collagen biosynthesis by assisting the assembly and transportation of procollagen [47]. While there is no specific inhibitor for HSP47, highly specific siRNA nanotechnology-based strategies have been developed to block collagen biogenesis and reduce fibrosis [48-50].

HA, a non-sulfated linear glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid repeats of variable length [51], is another major component of the ECM that is involved in tumor progression, invasiveness and metastasis [52]. The space-filling, hydrogel-like HA mediates increased IFP, vascular collapse and drug unavailability. With a highly negative charge, it can also decrease the interstitial transport of macromolecules or nanoparticles (NPs) by forming aggregates [25, 53]. There are three types of HA synthases (HAS1, HAS2, and HAS3), which produce HA with various molecular sizes. The clinically approved choleretic and antispasmodic drug 4-methylumbelliferone (MU) can directly suppress HA synthesis by depleting uridine diphosphate glucuronic acid (UDP-GlcUA, one of the building blocks of HA) 8

Journal Pre-proof and decreasing the expression of HAS2 and HAS3 [54, 55]. Inhibition of HA biogenesis by MU has been shown to inhibit tumor progression and prolong survival time in tumor-bearing mice [56-59]. However, the low potency and poor water solubility of MU limit its applications [56]. To address these issues, Szoka et al. loaded water-soluble phosphorylated MU into liposomes [60]. This nanoformulation showed a stronger inhibitory effect on HA synthesis than conventional MU, leading to improved distribution and efficacy of Doxil in a 4T1 tumor model.

Therefore, blockage of ECM biogenesis by active ingredients shows great potential for anti-stromal therapy and nanoformulations may provide additional therapeutic benefits. However, the main drawback of this approach is that it has no effect on existing ECM components.

3.1.2 Inhibiting ECM stiffness The desmoplastic tumor is markedly stiffer than normal tissue, which is attributed to greater cross-linking of collagen fibers [61]. Mechanically, the highly cross-linked collagen fibers narrow the inter-fiber spacing, retard interstitial transport of therapeutics, limit their extravasation distance, and impair their tumor penetration [18]. Lysyl oxidase (LOX), an enzyme overexpressed in the hypoxic TME, is a key contributor to increased stromal stiffness by catalyzing cross-linkage of collagen and other ECM components [43]. In a murine model of breast cancer, LOX inhibition with a blocking antibody (LOXAb) prevented ECM stiffening and reduced malignancy [62]. In an autochthonous murine PDAC model, LOXAb treatment decreased ECM cross-linking, increased vasculature, and enhanced drug delivery and immune cells infiltration [63]. However, LOXAb requires a high dose to be effective and this is associated with significant side effects. To circumvent this dilemma, Ingber et al. developed a nanoformulated LOXAb [64]. They covalently linked LOXAb to the surface of poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-PEG) NPs to create LOX-targeting NPs (LOXAbNPs). Due to the passively targeted NPs and the ECM targeted LOXAb, LOXAbNPs preferentially concentrated within the TME of 9

Journal Pre-proof orthotopic mammary 4T1 tumors. As a result, LOXAbNPs exhibited a higher inhibition rate of collagen cross-linking and a greater therapeutic index than soluble LOXAb. Additionally, stromal stiffening is related to increased Rho kinase (ROCK) activity to some degree [65, 66]. Inhibition of ROCK activity with Fasudil reduced ECM cross-linking and enhanced the effects of chemotherapeutics in pancreatic cancer [67].

Taken together, altering the mechanical architecture of the ECM represents a promising anti-stromal strategy to overcome the stiffened ECM barrier. In principle, this strategy neither reduces ECM biogenesis nor affects established ECM network, so long-term treatment is necessary to reduce ECM stiffness in desmoplastic tumors.

3.1.3 Facilitating ECM degradation The dense stroma can be breached by eliminating existing ECM components and the most direct way is using ECM-degrading enzymes. Collagenase is an efficient ECM-degrading enzyme and multiple studies have found that intravenous injection of collagenase into tumor-bearing mice reduces collagen density and enhances the transportation of macromolecules or liposomes into tumors [68-71]. However, there are two shortcomings related to the systemic delivery of collagenase. The first one is that circulated collagenase can damage normal tissues [72]. To overcome this, nanoformulated collagenase has been applied to digest tumoral ECM. For example, Tsourkas et al. tethered collagenase to gold nanoparticles (AuNPs) [73]. Systemic delivery of this nanoformulation showed no signs of toxicity to liver and spleen, but significantly increased the accumulation of AuNPs. The second one is that the enzymatic activity of collagenase declines quickly once it is exposed to the plasma. So it would be preferential to embed collagenase inside a nanoparticle rather than anchor it to the surface. Schroeder et al. took this approach and encapsulated collagenase into liposomes [74]. This liposomal collagenase reduced collagen content from 12.8% to 5.6% in orthotopic pancreatic cancer, which significantly enhanced second-wave chemotherapy. 10

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Hyaluronidase is another ECM-degrading enzyme that can break down HA at specific sites. Pretreatment with hyaluronidase resulted in a 4-fold increase in the uptake of liposomal doxorubicin in osteosarcoma xenografts [75], and a 2-fold increase in the uptake of photosensitizer-loaded nanomicelles in breast tumors [76], due to the reduced IFP and enhanced EPR effect. In an autochthonous murine PDAC model, two independent groups confirmed that systemic administration of PEGPH20, a PEGylated recombinant human PH20 hyaluronidase, efficiently depleted stromal HA, re-expanded collapsed vessels, and increased the intratumoral delivery of therapeutic agents, which significantly enhanced the anti-tumor efficacy of gemcitabine and prolonged mouse survival [77, 78]. Based on these results, PEGPH20 has been tested in several clinical trials where it has shown promising results [79]. Despite this, some side effects, such as musculoskeletal pain and thromboembolism, have been reported [80]. To circumvent life-threatening side effects, Chen et al. modified the surface of PLGA-PEG NPs with hyaluronidase, then added an extra PEG layer to prolong the blood circulation time [81]. This elaborate design led to a 4-fold increase of NPs accumulation in 4T1 breast tumors. Mechanistically, these hyaluronidase-functionalized NPs only transiently degraded HA on their diffusion path, which might minimize unnecessary HA breakdown and avoid potential side effects. Above studies suggest that functionalization of NPs with enzymatic proteins can be a promising approach to degrade ECM and reduce stromal barriers, but the complexity of NPs fabrication and possible denaturing of enzyme should be taken into consideration.

In addition to the direct delivery of exogenous ECM-degrading enzymes, the induction of endogenous enzymes can also play an important role in ECM degradation [82]. MMPs are a family of zinc-dependent endopeptidases that can degrade nearly every component of the ECM [83]. The expression of various MMPs can be induced by relaxin, a hormone secreted during pregnancy [84]. Jain et al. proved that administration of relaxin could effectively degrade the matrix, modify 11

Journal Pre-proof collagen structure and improve macromolecule diffusion in tumors [85]. However, clinical application of relaxin is limited due to its short circulation half-life and systemic vasodilation. To overcome these, Prakash et al. conjugated the hormone to superparamagnetic iron oxide nanoparticles (SPION) [86]. This nanoformulation was more effective than free relaxin in reducing ECM density, retarding tumor growth and increasing the effectiveness of gemcitabine in stroma-rich pancreatic tumors.

Nitric oxide (NO) is an important secondary gas messenger with multiple biological activities, including neurotransmission, vasodilation, angiogenesis, immune responses and inhibition of platelet aggregation [87, 88]. Interestingly, Fang et al. revealed a new role of NO in depleting tumor ECM and presented a NO donorassisted strategy for enhanced drug delivery (Fig. 3) [89]. They first integrated Snitrosothiol (a NO donor) into PEGylated mesoporous silica nanoparticles (N@MSN) and then loaded doxorubicin (DN@MSN). The prepared NO-releasing DN@MSN increased both the content and activity of endogenous MMPs in 4T1 tumors, leading to reduced collagen content, increased NPs penetration and enhanced antitumor efficacy of doxorubicin. This work highlights the potential of the delivery of NO donor for depleting ECM, which might be more cost-effective than the delivery of high molecular weight enzymes or hormones. However, since the NO release is uncontrollable in this study, internal and external stimuli-responsive NO-releasing NPs that can realize spatiotemporally controlled NO generation and ECM manipulation are highly preferred in the future.

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Fig. 3. Nanomedicines-mediated ECM degradation by activating endogenous ECM-degrading enzymes. A) A scheme illustrating the synthesis of DN@MSN. B) Proposed mechanism for NO donormediated ECM depletion for enhanced NPs penetration in tumors. The released NO rapidly converts to ONOO−, which activates pro-MMPs into active MMPs via cysteine oxidation. Subsequently, MMPs degrade ECM to improve NPs penetration. C) Western blot analysis of MMPs induced by NOreleasing NPs. D) In situ zymography assay of MMPs activity (up) and immunofluorescence staining of Collagen I (down) in tumor tissues. E) Biodistribution (left) and penetration (right) profiles of iFluor 647-labled MSN (iFluor 647-MSN). The mice were i.v. injected with empty MSN or N@MSN (NO, 8 μmol/kg). After 48 h, the mice were i.v. administered with iFluor 647-MSN (iFluor 647, 0.2 mg/kg) for biodistribution and penetration analysis. F) Tumor growth curves (n = 5). The mice were i.v. administered with the indicated formulations (DOX, 3 mg/kg; NO, 8 μmol/kg) for four times. ***p < 0.001. (Adapted with the permission from Ref. [89]. Copyright 2019. American Chemical Society.).

3.2 Targeting cancer-associated fibroblasts 13

Journal Pre-proof CAFs are the predominant stromal cells, the major source of ECM components and the key mediators of the desmoplastic stroma. They can also promote tumor growth and mediate tumor chemoresistance and immune tolerance [90]. Therefore, they are attractive targets for anti-stromal therapy. CAFs are perivascularly localized, which makes them the direct targets of anti-stromal therapeutics after vascular intravasation. Currently, there are mainly four strategies for modulating CAFs: eliminating them, reducing their activity, inducing them quiescent and turning them against tumors.

3.2.1 Eliminating CAFs Direct depletion of CAFs as an anti-stromal strategy has the potential to reduce stromal barriers, limit tumor growth and facilitate second-wave therapy. For instance, nab-paclitaxel, a FDA approved drug, is posited to be concentrated in the TME after binding to secreted protein acidic and rich in cysteine (SPARC) and subsequently kill neighboring CAFs, thereby reducing stromal content and increasing gemcitabine accumulation [91, 92]. However, this hypothesis is still under debate, for further exploration demonstrated a lack of association between SPARC level and therapeutic efficacy [93].

In order to eradicate CAFs with a high specificity, it is of great importance to identify specific surface markers and harness potent strategy to deplete CAFs. Fibroblast activation protein (FAP), a membrane-bound serine protease, is selectively expressed by CAFs in over 90% of human epithelial tumors and is considered a pantumor antigen [94, 95], which is a promising target for CAFs depletion. Various FAPtargeting agents, including vaccines [96-98], bispecific antibodies [99, 100], antibodydrug conjugates [101-104], and chimeric antigen receptor (CAR) T cells [105-107], have been exploited to suppress tumor growth via the depletion of FAP+ stroma. However, since FAP+ cells also exist in some normal tissues (e.g. placenta, uterus, skeletal muscle, and bone marrow), systemic anti-FAP therapy could result in cachexia, muscle loss, bone toxicities and even death [90, 108-110]. To address this issue, Xie et al. developed a nano-enabled phototherapy approach to kill CAFs in a 14

Journal Pre-proof site-specific manner [111]. They conjugated the FAP single chain antibody variable fragment to the surface of photosensitizer-loaded ferritin nanocage. The prepared nanocage selectively bound to FAP+ CAFs in tumors and local irradiation resulted in photodynamic killing of these CAFs without damaging healthy tissues. This in turn reduced the CAFs-derived ECM and C-X-C motif chemokine ligand 12 (CXCL12, an immunosuppressive factor), leading to improved CD8+ T cells infiltration and tumor suppression. In another study, Nie et al. prepared a novel dual-mode nanomedicine integrating CAFs-targeting ability and cell-penetration ability for highly efficient CAFs eradication [112]. They designed a self-assembled peptide consisting of a cellpenetration peptide (CPP) and two cholesterols, and then encapsulated doxorubicin (PNP-D) inside and absorbed FAP monoclonal antibody (PNP-D-mAb) outside (Fig. 4). The formed PNP-D-mAb with both FAP-targeting and cell penetration features killed both CAFs and neighboring tumor cells, resulting in significantly enhanced drug delivery and anti-tumor efficacy with negligible side effects.

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Fig. 4. Nanomedicines-mediated CAFs depletion by targeted delivery of cytotoxic agents. A) Chemical structure of cholesterol-modified cell-penetration peptide. B) A scheme illustrating the preparation of PNP-D-mAb, including peptide assembling, drug loading and FAP antibody absorption. C) Proposed mechanism for PNP-D-mAb enabled CAFs targeting and NPs penetration. PNP-D-mAb can be actively targeted to tumors by recognizing FAP+ CAFs. Then, the CPP can enhance the penetration of NPs to CAFs and neighboring tumors cells. D) In vivo fluorescence imaging of tumor-bearing mice at 12 h post-treatment. The mice were i.v. injected with Cy5.5 labeled PNP-D-IgG or PNP-D-mAb. E) Tumor growth curves (n = 8). The mice were i.v. administered with the indicated formulations (DOX, 3 mg/kg) for four times. **p < 0.01. (Adapted with the permission from Ref. [112]. Copyright 2015. John Wiley &Sons, Inc.).

Interestingly, FAP also possesses proline-specific endopeptidase activity and this unique enzymatic activity has been widely utilized for specific activation of prodrugs 16

Journal Pre-proof to kill FAP+ cells [113]. Both natural toxins [114-117] and chemotherapeutics [118, 119] have been coupled to FAP-specific peptide substrates to generate inactivated prodrugs. These prodrugs maintain their nontoxic forms during the circulation until they are processed by FAP to release parent drugs. So they allow the selective killing of CAFs and neighboring cells in the TME, while minimizing the systemic toxicity associated with parent drugs [120]. Inspired by this, Nie et al. designed a novel amphiphilic peptide containing FAP-cleavable substrate and further loaded chemotherapeutics during its assembly. This co-assembled nanomedicine efficiently unloaded drugs in the TME, subsequently killed CAFs and ultimately enhanced the penetration and therapeutic efficacy of drugs in three types of tumor models [121].

Apart from above mentioned FAP-targeted nanomedicines, other CAFs-eradicating nanomedicines, including docetaxel-conjugate nanoparticles (Cellax) [122, 123], sigma receptor-targeted cisplatin nanoparticles [3, 124], and tenascin C-targeted liposomal Navitoclax (FH-SSL-Nav) [125, 126] have been reported to non-targeted or targeted kill CAFs. Even though these nanomedicines have been successfully applied to eradicate CAFs, their nonspecific tissue distribution and premature drug release may bring severe off-target toxicity and should not be overlooked.

It is worth mentioning that CAFs are not exclusively tumor-supportive and some may act to restrain tumor growth. Since 2014, several studies have shown that killing CAFs indiscriminately increases the risk of losing key stromal elements needed for tissue homeostasis and paradoxically accelerates tumor progression [127-130]. The heterogeneity of CAFs that comprise both cancer-supportive phenotype and cancerrestraining phenotype makes CAFs depletion a double-edged sword [131, 132]. Identification of the reliable marker of cancer-supportive CAFs will definitely benefit this area [133].Nevertheless, this radical anti-stromal strategy should be carried out with extreme care despite its high effectiveness.

3.2.2 Reducing CAFs activity 17

Journal Pre-proof Active CAFs proliferate excessively and produce a huge amount of ECM components. Correspondingly, inactivation of CAFs is proposed to reduce CAFs proliferation and ECM secretion. Many pharmacological compounds, possessing antifibrotic, anti-inflammatory or anti-oxidant properties have been demonstrated to reduce the activity of CAFs. These include pirfenidone [134, 135], minnelide (a water-soluble prodrug of triptolide) [136, 137], fraxinellone [138, 139], quercetin [140], curcumin [141-143], ellagic acid [144, 145], glycyrrhetinic acid [146], halofuginone [147], metformin [148], celecoxib [149], dexamethasone [150, 151], and losartan [72, 152, 153].

Since small molecule drugs often suffer from poor solubility, rapid clearance and low bioavailability, their nanoformulations have been rationally developed to improve their delivery. In one study, phosphorylated quercetin was encapsulated into lipidcalcium-phosphate NPs by Huang et al. Compared to free drug, this nanoformulation was more effective in inhibiting the proliferation of α-SMA+ CAFs and suppressing the secretion of Wnt family member 16 (Wnt16, a key contributor to chemoresistance) as well as collagen [140]. In turn, the chemotherapeutic effacacy of lipid-coated cisplatin NPs was enhanced in stroma-rich bladder tumor-bearing mice. In another study, Nie et al. loaded pirfenidone, a FDA-approved anti-fibrotic agent for treating idiopathic pulmonary fibrosis, into a matrix metalloproteinase-2 (MMP-2) responsive liposome to improve its performance in CAFs regulation [14]. This liposome could specifically unload pirfenidone in the TME and suppress the expression of multiple ECM components, thereby improving the therapeutic efficacy of gemcitabine against pancreatic tumor. Similarly, a β-cyclodextrin modified, MMP2 responsive liposome encapsulating pirfenidone and gemcitabine was constructed to combine anti-fibrotic therapy and chemotherapy by them. This integrated nanomedicine not only greatly weakened the stromal barriers by inhibiting the expression of collagen I and TGF-β, but also significantly suppressed the growth of pancreatic tumors [154]. 18

Journal Pre-proof These studies highlight the potential of inactivation of CAFs by anti-stromal nanomedicines. In general, this strategy is safer than CAFs depletion, but its antistromal effect is mild and transient. Once the treatment is halted, the recovery of CAFs activity is instant.

3.2.3 Inducing CAFs quiescent Since quiescent fibroblasts can transdifferentiate into CAFs to drive the desmoplastic reaction and tumor progression, reversal of activated CAFs back to the quiescent phenotype would be a straightforward approach for stromal modulation. The vitamin D receptor (VDR) is highly expressed in activated pancreatic stellate cells (PSCs; a resident fibroblast in pancreas) where it acts as a master transcriptional regulator of the phenotype of PSCs [155]. Evans et al. proved that treatment with calcipotriol (a VDR ligand) in autochthonous PDAC mice resulted in PSCs quiescence and stromal reprogramming without eliminating key stromal elements needed for tissue homeostasis, and this led to increased intratumoral gemcitabine delivery and enhanced chemotherapeutic efficacy [155]. For the first time, their work emphasizes the advantage of CAFs normalization over CAFs depletion [130]. Later, the same group found that bromodomain-containing protein 4 (BRD4) was highly enriched at the enhancers of multiple profibrotic genes in activated hepatic stellate cells (HSCs, a resident fibroblast in liver) where it acted as a global genomic regulator of the phenotype of HSCs [156]. Treatment with JQ-1 (a small molecule inhibitor of BRD4) transformed activated HSCs to a quiescent phenotype and thereby ameliorated liver fibrosis. Recently, JQ-1 was further proved to induce PSCs quiescence and alleviate PDAC-associated fibrosis by Munshi and colleagues [157]. Evidences presented above imply that JQ-1 have the capability to normalize CAFs of different tumor origins. To be noted, since JQ-1 is a pleiotropic drug with anti-tumor activity [158], it is expected to serve as both anti-stromal agent and anti-tumor agent for cooperative desmoplastic tumors management.

All-trans retinoic acid (ATRA) is an active metabolite of vitamin A that can 19

Journal Pre-proof maintain PSCs quiescence by regulating gene expression after binding to its nuclear receptors [159-162]. Several studies have reported that ATRA can normalize the desmoplastic stroma by inducing PSCs quiescence and this in turn increases the apoptosis of tumor cells [163], enhances the infiltration of CD8+ T cells [164], and potentiate chemotherapy [165]. Recently, Nie et al. developed a TME-responsive nanosystem based on PEGylated polyethylenimine-coated AuNPs, which was utilized to co-deliver ATRA and HSP47 siRNA (Au@PP/RA/siHSP47) to re-educate PSCs (Fig. 5) [50]. Treatment with this nanosystem simultaneously rendered PSCs quiescence and reduced ECM production, thereby significantly enhancing the therapeutic efficacy of gemcitabine in stroma-rich pancreatic tumors. This nanoenabled combinatorial strategy to restore homeostatic stromal function by targeting activated fibroblasts holds great promise for improving second-wave therapy in desmoplastic tumors.

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Fig. 5. Inducing CAFs quiescence using a TME-responsive nanomedicines. A) Schematic diagram of Au@PP/RA/siHSP47-induced PSCs quiescence and stroma modulation to enhance chemotherapy. Au@PP/RA/siHSP47 is activated in the acidic TME and efficiently engulfed by PSCs, leading to PSCs quiescence and HSP47 knockdown. This in turn homeostatically restores stroma and improves drug delivery. B) Trichrome staining of collagen and immunohistochemical staining of HSP47, α-SMA and fibronectin with quantitative analysis (n = 3). The mice bearing orthotopic pancreatic tumors were i.v. injected with different stroma modulation agents (ATRA, 2.4 mg/kg; siRNA, 0.97 mg/kg) every other day for a total of three times before tumors were harvested for stroma analysis. C) Tumor growth curves based on bioluminescence imaging (n = 3). The mice were treated with different stroma modulation agents for three times before they were i.v. injected with gemcitabine (GEM, 10 mg/kg) for five times. D) Photographs of excised tumors with spleens (n = 3). (Adapted with the permission from Ref. [50]. Copyright 2018. Springer Nature.).

Interestingly, Mukherjee et al. found that the 20 nm AuNPs had an innate ability to reprogram PSCs to a lipid-rich quiescent phenotype and inhibited matrix deposition [166]. Moreover, AuNPs could disturb the crosstalk between PSCs and tumor cells by 21

Journal Pre-proof altering their cellular secretome to slow the progression of orthotopic pancreatic tumors. In a recent study, they further unveiled that AuNPs transformed activated CAFs/PSCs to quiescence by inducing lipogenesis genes expression and enhancing lipid synthesis [167]. Their studies provide a novel point of view on harnessing the intrinsic properties of NPs to induce CAFs quiescence [168], and the relationship between the physiochemical properties (e.g. composition, size and shape) of the nanomaterials and CAFs activity is worth further investigation.

In principle, the potential risk for this strategy is much lower than CAFs depletion and its anti-stromal effect can last for a while once the treatment is terminated. Therefore, normalizing CAFs should be an appealing anti-stromal strategy to reverse desmoplasia and improve drug delivery, which might be more practical than eliminating CAFs or inactivating CAFs. However, tumor-centric therapeutics should always be administrated in time to achieve an effective therapeutic response. Indeed, recent years have witnessed the successful translation of this strategy into clinic with multiple ongoing clinical trials where anti-stromal agents are combined with potent chemotherapeutics [169].

3.2.4 Turning CAFs against tumors Generally speaking, eradication, inactivation or normalization of CAFs has limited impact on the activity of tumor cells and the vicious tumors cells are still able to recruit or reactivate fibroblasts. Inspired by the demonstration that fibroblasts can be transdifferentiated into macrophage-like cells [170], Huang et al. ingeniously converted tumor-promoting CAFs into tumor-killing players by in situ engineering, and dictated CAFs to attach tumors for the first time [171]. To achieve this, plasmids encoding secretable TNF-related apoptosis-inducing ligand (sTRAIL) were formulated into lipid-coated protamine DNA complexes and delivered to perivascular CAFs due to the intrinsic off-target effect of nanomedicines. sTRAIL secreted from the transfected CAFs successfully triggered apoptosis in adjacent tumor cells. In turn, the CAFs population and ECM content were reduced due to the lack of stimuli from 22

Journal Pre-proof tumor cells. As a result, this unique CAFs-redirecting therapy not only significantly retarded tumor growth, but also synergized with the second-wave chemotherapy in two desmoplastic xenograft models. It should be mentioning that this treatment was well tolerated in mice and no noticeable toxicity was observed owning to the low plasmid expression. Therefore, this study provided a promising paradigm of in situ generating tumoricidal CAFs for cooperative anti-tumor and anti-stromal therapies. However, the TRAIL resistance and the major differences of death receptors between mice and humans should be seriously considered if translating this technology into the clinic [172].

Notably, unlike depleting, inactivating or normalizing CAFs, which only weakens or terminates the intimate relationship between CAFs and tumor cells, turning CAFs against tumors establishes an adversarial relationship between them. Therefore, compared to other strategies, this one holds the potential to induce potent anti-tumor response. But, technically, it is the most challenging one.

3.3 Targeting pro-stromal signaling Quiescent fibroblasts can be activated into CAFs in response to paracrine cytokines secreted from tumor cells, including TGF-β, PDGF, FGF, sonic hedgehog (Shh), and interleukin 6 (IL-6) [173-176]. The activated CAFs further secrete various cytokines, including TGF-β, PDGF, FGF, CXCL12, IL-6 and connective tissue growth factor (CTGF), to maintain the activated phenotype via autocrine pathways, and also stimulate tumor cells via paracrine pathways [134, 177-179]. Interfering with these signaling pathways is regarded as a feasible strategy for anti-stromal therapy. Three main approaches have been taken to achieve this, including scavenging pro-stromal cytokines, blocking receptor activation and inhibiting downstream signaling.

3.3.1 Scavenging pro-stromal cytokines Neutralization with monoclonal antibodies (mAbs) and sequestration using ligand traps are two main strategies for removing pro-fibrotic cytokines to block the 23

Journal Pre-proof paracrine and autocrine pathways associated with CAFs activation, which have been actively investigated in preclinical and clinical studies [180]. For example, TGF-β is a potent pro-fibrotic factor, which can activate CAFs via TGF-β/SMAD pathway and mediate strong stromal reaction. The TGF-β neutralizing mAb ID11 has been used to block TGF-β signaling, which decreased collagen content and improved chemodrug delivery in two orthotopic breast tumor models [181]. Interestingly, this strategy can be integrated with other antibody-based therapies, such as immune checkpoint inhibitors. A novel bifunctional anti-PD-L1/TGF-β trap fusion protein M7824 that targets both PD-L1 and TGF-β has been developed, and it simultaneously reduced the stromal barriers and elicited potent anti-tumor immunity [182].

However, systemic delivery of mAbs and cytokine traps run the risk of disrupting immune balance and arousing multiple autoimmune diseases, which limit their therapeutic applications in the clinic [183]. In addition, the binding site barrier established by CAFs can prevent such agents penetrating deeply into tumors due to their relatively large size, leading to the compromised efficiency [39]. Similar to the delivery of sTRAIL plasmids, Huang et al. delivered plasmid DNA encoding small trapping proteins to perivascular cells to scavenge pro-fibrotic factors, including Wnt family member 5A (Wnt5a) [183] and CXCL12 [184]. The local and transient expression of small trapping proteins not only enhanced topical therapeutic concentrations with diminished systemic toxicity, but also allowed deeper diffusion into tumors. This in turn reduced stromal barriers and facilitated T-cell infiltration. When these agents were combined with PD-L1 trap, IL-10 trap or immunogenic chemotherapeutics, synergistic anti-tumor efficacy could be achieved in desmoplastic tumors [183, 185, 186]. Therefore, this NPs-enabled trap technology represents a promising approach for anti-stromal therapy and combinatorial immunotherapy against desmoplastic tumors. However, compared to mAbs, these plasmid-expressing nanomedicines are much more complex, so their scalability, reproducibility and stability should be taken into consideration. 24

Journal Pre-proof 3.3.2 Blocking receptor activation The activation of downstream signaling pathways by pro-fibrotic cytokines requires their binding to cell surface receptors, so targeted inhibition of those receptors by small molecule inhibitors or antibodies can interrupt this process. For example, TGFβ binds to type I receptor (TβR-I) and type II receptor (TβR-II) to activate downstream signaling and drive the evolution of CAFs, but this process can be blockaded by TβR-I inhibitors [179, 187-189]. IL-6 is also involved in CAFs activation [190], and IL-6 receptor blockade by anti-IL-6R antibodies has been reported to reduce the number of α-SMA+ stromal cells and thus enhance the efficacy of a PD-L1 checkpoint inhibitor in several preclinical models of PDAC [191]. CAFs are the major source of CXCL12, which interacts with its cognate receptor C-X-Cmotif chemokine receptor 4 (CXCR4) to initiate and maintain the transdifferentiation of normal fibroblasts into tumor-promoting CAFs via a self-sustaining autocrine signaling loop [179, 192]. Disruption of the CXCL12/CXCR4 axis by the CXCR4 antagonist AMD3100 blocked the evolution of CAFs and increased immune cell infiltration in desmoplastic tumors [179, 193].

Since antibodies and small molecule inhibitors have several drawbacks as mentioned above, developing nanomedicines to pharmacologically block receptor activation might provide great advantages. Several nanomedicines delivering CXCR4 or TβR-I inhibitors have been developed to modulate the TME [194, 195], but their biological effects on CAFs have not been fully studied. For example, Nel et al. used modified mesoporous silica NPs to delivered LY364947 (a potent TβR-I inhibitor) to decrease pericyte coverage via interfering the pericyte-recruiting TGF-β signaling, which enhanced vascular access and delivery of second-wave NPs to the PDAC tumor site [195]. It would be worthy to study if this nanomedicine also plays a role in reducing CAFs activation and secretion.

3.3.3 Inhibiting downstream signaling After pro-stromal cytokines bind with their receptors, downstream signaling 25

Journal Pre-proof pathways will be activated. Inhibiting intracellular pathways provides another method for anti-stromal therapy. For example, excess IL-6 stimulates hyperactivation of JAK/STAT3 signaling, which can be blocked by JAK inhibitors [196]. Alternatively, novel agents (e.g. peptidomimetics and antisense oligonucleotides) that inhibit either the function or expression of STAT3 can interrupt IL-6/JAK/STAT3 signaling [196, 197]. Hh signaling also plays an important role in establishing stromal barriers, as paracrine Shh from tumor cells can promote stromal desmoplasia by acting on CAFs [29, 198]. Binding of Shh to the Patched receptor on CAFs relieves repression of Smoothened (SMO) receptor, resulting in activation of glioma-associated oncogene (Gli) transcription factors. This pro-stromal Hh signaling pathway can be suppressed by a SMO inhibitor. In 2009, for the first time, Tuveson et al. demonstrated that inhibition of SMO by IPI-926, a semisynthetic derivative of cyclopamine, depleted stromal components, increased intratumoral vasculature and enhanced gemcitabine delivery in autochthonous murine PDAC [199]. Recently, both cyclopamine and vismodegib (a FDA-approved SMO inhibitor) were reported to significantly disrupt ECM, increase functional vessels, and improve the delivery and chemotherapeutic efficacy of nanomedicines in mouse models of pancreatic cancer [200, 201]. Despite the encouraging preclinical studies on Hh signaling blockade, clinical trials in human PDAC provided paradoxical results [202]. Further investigations revealed that the long-term administration of IPI-926 to PDAC-bearing mice resulted in an undifferentiated histology, increased vasculature, enhanced proliferation, and reduced survival [127]. Above results remind us that both the pharmacological target and the timing of drug administration should to be carefully considered to maximize the benefit and minimize the risk.

Generally speaking, it is more difficult to inhibit downstream signaling than to scavenge pro-stromal cytokines or to block receptor activation, because many therapeutic agents have to go across the cell membrane to interact with their intracellular targets and some compensatory signaling pathways may exist. From these aspects, nanomedicines with the abilities to overcome multiple biological 26

Journal Pre-proof barriers and deliver several small-molecule drugs or macromolecules should be helpful to inhibit downstream pathways.

4. Combination therapy As mentioned above, anti-stromal therapy alone may have no or weak anti-tumor effects, however, it can serve as an adjunct to greatly improve the efficacy of other backbone therapeutic modalities in desmoplastic tumors. In this section, some antistromal nanomedicines that are use to combined with anti-tumor therapeutics are summarized in Table 1, and a number of potential combination partners (e.g. chemotherapy, immunotherapy, phototherapy and radiotherapy) are discussed below.

Table 1. Summary of anti-stromal nanomedicines in combination with anti-tumor therapeutics. aCodelivered in the same nanocarrier

Anti-stromal strategies

Anti-stromal nanomedicines

Combination partners

Results

Ref.

Blocking ECM biogenesis

Phosphorylated methylumbelliferoneencapsulated liposome Collagenase encapsulated liposome

PEGylated liposomal doxorubicin Micellar paclitaxel

Reduce HA Improve tumor distribution of liposomes

[60] [74]

PEGylated hyaluronidase (PEGPH20) Hyaluronidase or PEGylated hyaluronidase IgG Fc fragment fused hyaluronidase

Gemcitabine

Degrade collagen Increase the uptake and efficacy of second-wave NPs Deplete HA and re-expands collapsed vessels Enhance the delivery and efficacy of gemcitabine

[76]

pH-responsive dextran modified hyaluronidase

Chlorine e6-loaded liposome and antiPD-L1 antibody

Hyaluronidase conjugated PLGA-PEG NPs Relaxin conjugated SPION Nitric oxide donor integrated MSN

aDoxorubicin

Nab-paclitaxel

Gemcitabine

Paclitaxel-loaded liposome

Heparin-coated lipid-siRNA NPs

Tenascin C-targeted liposomal Navitoclax

Transferrin receptor-targeted

Degrade HA and increase functional vessels Increase tumor oxygenation and drug delivery Improve the efficacy of PDT Deplete HA and decrease IFP Enhance intratumoral penetration and accumulation of various therapeutics Improve anti-tumor efficiency of various therapeutics Degrade HA and increase tumor oxygenation Facilitate the delivery of liposome and infiltration of T cells Enhance PDT and immunotherapy Degrade HA Facilitate NPs diffusion and distribution in tumor Inhibit tumor growth with a low drug dose Reduce CAFs and ECM Enhance anti-tumor effect of gemcitabine Activate MMPs and degrade collagen Enhance NPs/drug penetration Improve anti-tumor efficacy Decrease CAFs and collagen content Improve anti-tumor efficacy of gemcitabine Induce tumor cell apoptosis and decrease ECM abundance Promote NPs accumulation and penetration in tumor Significantly inhibit tumor growth and metastasis Eradicate CAFs and reduce ECM Decrease IFP and facilitate liposome penetration

Facilitating ECM degradation

Eliminating CAFs

Chlorine e6-loaded nanomicelles Doxorubicin, trastuzumab or gold nanorod

Gemcitabine aDoxorubicin

27

[77, 78]

[203]

[204]

[81] [86] [89] [91] [205]

[126]

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Reducing CAFs activity

Quercetin phosphateentrapped lipid calcium phosphate Pirfenidone encapsulated peptidehybrid liposome Pirfenidone entrapped β-cyclodextrin modified targeted liposome Albumin-ellagic acid complexes-encapsulated thermosensitive liposome Losartan-embedded peptide-based hydrogel Fraxinellone-loaded aminoethyl anisamidemodified nanoemulsion Fraxinellone-loaded CGKRK-modified PEG-PLA NPs

Glycyrrhetinic acidloaded dendrimerpeptide-modified PEGPCL NPs

liposomal doxorubicin Lipid-coated cisplatin NPs

Enhance chemotherapeutic efficacy

Gemcitabine aGemcitabine

aAlbumin-paclitaxel

complexes PEGylated liposomal doxorubicin Peptide vaccine

siKras-loaded lipid-coated calcium phosphate biomimetic highdensity lipoprotein NPs aGemcitabine prodrug

Inducing CAFs quiescent

ATRA and siHSP47 codelivered gold NPs

Gemcitabine

Turning CAFs against tumors

sTRAIL-expressing plasmid encapsulated lipid-coated protamine DNA NPs CXCL12 trapexpressing plasmid lipid-coated protamine DNA NPs

Lipid-coated cisplatin NPs

CXCL12 trapexpressing plasmid lipid-coated protamine DNA NPs Wnt5a trap-expressing plasmid lipid-coated protamine DNA NPs Cyclopamine-loaded polymeric micelle

aIL-10

Cyclopamine and paclitaxel co-delivered polymeric micelle

Anti-PD-1 antibody

Vismodegib-loaded polymeric micelle

amiR-let7b

Scavenging pro-stromal cytokines

Inhibiting downstream signaling

aPD-L1

trap plasmid

trap plasmid

Doxorubicin Co-delivery paclitaxel

28

of

Decrease α-SMA+ CAFs and collagen Suppress Wnt16 expression Increase the penetration and efficacy of secondwave NPs Decrease multiple ECM components Improve the penetration and efficacy of gemcitabine Decrease collagen I and TGF-β Enhance the penetration and efficacy of gemcitabine Reduce CAFs Improve drug distribution and matrix penetration Enhance chemotherapy against tumor

[140]

Decrease CAFs and collagen Improve chemotherapy Inhibit tumor growth and lung metastasis Decrease CAFs and stroma deposition Enhance T-cell infiltration and antigen-specific immune response with improved anti-tumor effect Interrupt TGF-β signaling and attenuate stromal barriers Enhance blood perfusion Improve the knockdown efficiency of KRAS and prolong animal survival

[206]

Reduce CAFs activity and collagen content Suppress Wnt16 expression Significantly inhibit tumor growth and metastasis

[146]

Induce CAFs quiescence and reduce ECM deposition Improve the penetration and efficacy of gemcitabine Trigger apoptosis in adjacent tumor cells Reduce CAFs and ECM Favor second-wave nanotherapy

[50]

Decrease CXCL12 and reduce α-SMA+ CAFs and collagen content Increase T-cell infiltration Enhance the anti-tumor immune response of PDL1 trap Decrease CXCL12 and reduce α-SMA+ CAFs and collagen content Enhance the anti-tumor immune response of IL10 trap Decrease Wnt5a and reduce collagen content Facilitate doxorubicin-mediated immunogenic chemotherapy Deplete CAFs and increases vessel density Alleviate hypoxia and reduce matrix stiffness without collagen depletion Simultaneously target stromal and tumor compartments with enhanced chemotherapy Decrease Shh ligand and FAP+ CAFs Increase vessel density without collagen depletion Promote the infiltration of CD8+ T cells Enhance anti-tumor immunity of anti-PD-1 therapy Synergistically Inhibit tumor growth

[185]

[14] [154] [145]

[138]

[139]

[171]

[186]

[183] [207]

[208]

[209, 210]

Journal Pre-proof 4.1 Combination of anti-stromal therapy and chemotherapy Chemotherapy is one of the most common therapeutic modalities. However, the desmoplastic stroma limits the efficacy of cytotoxic drugs by blocking their delivery to the tumor cells. Anti-stromal therapy can reduce the stromal barriers and facilitate drug delivery. In the last two decades, many anti-stromal nanomedicines have been developed to enhance the efficacy of chemotherapy in preclinical studies (Table 1). Moreover, several anti-stromal agents, such as nab-paclitaxel and PEGPH20, have already shown improved clinical benefits when used in combination with standard-ofcare treatment regimens [169].

It is worth mentioning that anti-stromal therapy leads to reprogramming tumor stroma and increasing functional vessels, which not only creates a therapeutic window for drug delivery, but also heightens the risk of tumor dissemination [155]. For example, genetic or long-term pharmacologic inhibition of Hh signaling depleted the stroma, but accelerated tumor growth and impaired survival of PDAC-bearing mice [127, 129]. To circumvent this dilemma, Li et al. proposed a nanomedicines-enabled anti-stromal approach with two key features: the first is the controlled reduction of the stroma by using a lower dose of the SMO inhibitor, while the other is the coadministration of a cytotoxic agent to kill invasive tumor cells [207]. They developed a polymeric micelle nanoformulation (M-CPA/PTX) containing cyclopamine and paclitaxel for complementary PDAC therapy (Fig. 6). M-CPA/PTX treatment successfully depleted CAFs and reduced matrix stiffness, but the tumor-restraining collagenous matrix was maintained. Furthermore, this treatment significantly lowered the percentages of poorly to moderately differentiated tumor phenotypes, suppressed PDAC growth and extended mice survival. This study underlines the promise of using multifunctional nanomedicines to simultaneously target stromal and tumor compartments to enhance the chemotherapeutic efficacy while reducing the risk of tumor progression, but the increased metabolic burden and adverse effects must be taken into account. 29

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Fig 6. Nanomedicines-mediated combination therapy by co-delivering a stroma modulation agent and a cytotoxic agent. A) Schematic diagram of simultaneously targeting stromal and tumor compartments by M-CPA/PTX. B) Proposed mechanism of generating a positive feedback loop for enhanced tumor therapy by multiple administration of M-CPA/PTX. C) Immunohistochemical staining of α-SMA and corresponding quantifications (n = 15). The autochthonous PDAC mice were i.v. injected with MCPA/PTX (5 mg/kg/drug/injection) for 6 times over 2 weeks before tumors were harvested for stroma analysis. D) Immunofluorescence staining of FAP-α and corresponding quantifications (n = 8). E) Picrosirius red staining of collagen and corresponding quantifications (n = 15). F) Kaplan-Meier survival analysis of autochthonous PDAC mice with different treatments. M-CPA/PTX was injected i.v. at 5 mg/kg/drug/injection at 3 injections/week for the first 2 weeks and then i.p. at the same schedule until mice became moribund. Gemcitabine was i.p. injected at 100 mg/kg twice per week. Abraxane was given at 5 mg/kg/injection with the same schedule of M-CPA/PTX. (Adapted with the permission from Ref. [207]. Copyright 2018. Elsevier Ltd.).

4.2 Combination of anti-stromal therapy and immunotherapy With the rapid development, immunotherapy is becoming the fourth pillar of tumor therapy, in addition to surgery, radiotherapy and chemotherapy. Among various immunotherapies, CAR-T cell therapy and immune checkpoint blockade (ICB) therapy are the two most promising therapeutic modalities and their principles as well as clinical applications have been comprehensively discussed elsewhere [211, 212]. CAR-T cell therapy has proved extremely successful in treating hematological malignancies (e.g. CD19 CAR-T cell therapy in leukemias), but this success has not yet been extrapolated to solid tumors [213]. ICB therapy targeting the cytotoxic T 30

Journal Pre-proof lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein-1 (PD1) pathways has resulted in long-lasting tumor responses in melanoma and non-small cell lung cancer [214]. However, only a small proportion of patients can benefit from this and patients bearing desmoplastic tumors (e.g. PDAC) barely show any therapeutic response towards ICB therapy [215].

Solid tumors harbor stromal barriers which are absent in hematological malignancies. The dense stroma can impede the penetration of immune checkpoint inhibitors and infiltration of T cells into tumor tissue [216, 217]. The CAFs-derived immunosuppressive cytokines can mediate the chemical exclusion of T cells [193, 218]. In addition, the desmoplastic stroma creates a hostile TME for T cells by worsening hypoxia and depriving nutrient, leading to T cell apoptosis, anergy, or exhaustion [219]. Anti-stromal therapy can reduce stromal barriers, increase tumor blood perfusion, facilitate transport of immune therapeutics and promote the infiltration of anti-tumor immune cells, resulting in enhanced anti-tumor immune responses [90, 105, 147, 164, 193, 218, 220, 221].

Recently, increasing attentions have made to develop anti-stromal nanomedicines to increase the efficacy of immunotherapy. For example, based on their previous study on M-CPA/PTX-enabled stromal modulation, Li et al. further combined it with anti-PD-1 antibodies for PDAC treatment [208]. This combination therapy increased intratumoral levels of CD8+ T cells and interferon-gamma and significantly prolonged mice survival. Huang and colleagues have contributed a lot to this area. They used their trap nanotechnology to remodel stroma by scavenging pro-fibrotic and immunosuppressive factors (e.g. Wnt5a, and CXCL12), which largely enhanced the anti-tumor immune responses of various therapeutic modalities [183, 185, 186]. In another study, they encapsulated fraxinellone into an aminoethyl anisamide-modified nanoemulsion and used it to relieve the fibrotic and immunosuppressive TME. This formulation improved the tumor-specific immune response of a BRAF peptide vaccine in BRAF-mutant desmoplastic melanoma [138]. 31

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These results suggest that anti-stromal nanomedicines could have great synergy with immunotherapeutics. Since the systemic toxicity of immunotherapy is generally lower

than

chemotherapy,

the

combination

of

anti-stromal

therapy

and

immunotherapy is more attractive and such studies are highly applauded.

4.3 Combination of anti-stromal therapy and other therapeutic modalities Anti-stromal therapy can also potentiate photothermal therapy (PTT) and photodynamic therapy (PDT) by increasing the delivery of photothermal agents and photosensitizers, respectively. For example, Jiang et al. developed a new PTT strategy by combining TME modulation and advanced design of erythrocyte membranecloaked gold nanorods. They used cyclopamine to disrupt stromal barriers, and this increased the accumulation of biomimetic gold nanorods and enhanced the efficacy of PTT in pancreatic tumors [222]. In another study, Liu et al. utilized hyaluronidase to break down HA, which greatly increased tumor oxygenation and tumor uptake of chlorine e6-loaded nanomicelles [76]. As a result, the efficacy of PDT was significantly improved. Since anti-stromal therapy can decompress blood vessels, increase blood perfusion and relieve tumor hypoxia, it can also sensitize the tumor to radiotherapy, another oxygen-dependent therapeutic modality. Currently, PEGPH20 combined with radiotherapy is under clinical investigation for pancreatic tumors [217].

It is worth mentioning that radiotherapy, PDT, PTT and chemotherapy can trigger immunogenic cell death and provoke strong immune responses [223]. Therefore, antistromal therapy could not only improve the direct tumor-killing effect of these immunogenic therapeutic modalities, but also facilitate the subsequent immune attack against tumors. For example, Liu et al. modified hyaluronidase with dextran via a pHresponsive traceless linker, which served as an effective adjuvant anti-stromal nanomedicine

to

assist

photodynamic-immunotherapy

[204].

The

masked

hyaluronidase was activated in the acidic TME to degrade HA, thereby enhancing the 32

Journal Pre-proof delivery of oxygen, chlorine e6-loaded liposomes and anti-PD-L1 mAbs. The reduced stromal barriers and improved PDT induced a strong anti-tumor immune response and promoted the infiltration of cytotoxic T cells, which further enhanced the immunotherapeutic efficacy of anti-PD-L1 mAbs against both local and abscopal tumors.

5. Conclusions and Perspectives ECM and CAFs are the major components of tumor stroma, which present physical barriers as well as direct targets in drug delivery. As discussed in this review, many anti-stromal strategies have been developed to remodel the TME aiming at improving the delivery of various therapeutics. The strategies have been summarized into three major categories according to their pharmacological targets (i.e. ECM, CAFs and prostromal signaling) and the recent advances of nano-enabled approaches in fighting desmoplastic stroma have been highlighted here. It should be noted that as a highly orchestrated TME, anti-stromal therapy mediated stroma reduction can further trigger chain reactions (e.g. decreasing solid stress and IFP, increasing functional vessels and tumor oxygenation, reducing immunosuppressive cytokines, and promoting T-cell infiltration), which prime the TME in favor of conventional therapeutic modalities. So anti-stromal agents can serve as a promising adjunct to improve the therapeutic outcomes of various drugs in desmoplastic tumors. Moreover, with the increased understanding of stroma biology and rapid advancements in nanotechnology, rationally designed anti-stromal nanomedicines with great promise to potentiate tumor-centric therapeutics are highly expected.

Despite many positive effects in anti-stromal therapy, there are also potential adverse effects (e.g. increasing the risk of tumor metastasis), suggesting that antistromal therapy can be a double-edged sword. From this aspect, combination treatment with potent tumor-killing therapeutics is not only a bonus to bring extra benefit, but also a prerequisite for risk reduction. Therefore, future attention should be focused on developing finely tuned treatment regimens targeting both stromal and 33

Journal Pre-proof tumor compartments for cooperative desmoplastic tumor therapy by taking full advantage of multifunctional nanomedicines.

Last but not least, it is of great importance that more advanced tumor models that closely resemble human desmoplastic tumors should be used to translate aforementioned anti-stromal studies to possible clinical therapies. One of successful paradigms is the depletion of HA by PEGPH20 in genetically engineered mouse model (GEMM) of PDAC, which has been translated into clinic with some encouraging results in HA-high PDAC patients [224]. Currently, most studies are proof-of-principle preclinical studies where cell line-derived allograft/xenograft is used. Although some of them (e.g. murine 4T1 breast tumor and human BxPC3 pancreatic tumor) have been reported to be a stroma-rich phenotype by a few groups [39, 225], the inoculated homogeneous cancer cells are generally unable to develop highly heterogeneous and fibrotic TME in a short growth period [226, 227]. Therefore, the patient-derived xenograft model or GEMM that recapitulates the stromal characteristics of human desmoplastic tumors are highly recommended to propel these anti-stromal strategies/nanomedicines from bench to bedside.

Acknowledgments This work was supported by grants from National Key R&D Program of China (Grant No. 2018YFA0208900), National Natural Science Foundation of China (Grant No. 31571021), Innovation Group of the National Natural Science Foundation of China (Grant No. 11621505), Frontier Research Program of the Chinese Academy of Sciences (Grant No. QYZDJ-SSW-SLH022) and Key Laboratory of Biomedical Effects of Nanomaterials and Nanosafety, CAS.

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Declaration of interests √The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: