Mini-review

Improving cancer therapies by targeting the physical and chemicalhallmarks of the tumor microenvironmentJill W. Ivey a,1, Mohammad Bonakdar b,1, Akanksha Kanitkar a, Rafael V. Davalos a,b,Scott S. Verbridge a,*a Department of Biomedical Engineering and Mechanics, Virginia Tech-Wake Forest University, Blacksburg, VA 24061, USAb Department of Mechanical Engineering, Virginia Tech, Blacksburg, VA 24061, USA

A R T I C L E I N F O

Keywords:Physical tumor microenvironmentChemcical tumor microenvironmentElectroporation therapyTumor microenvironment targeting

A B S T R A C T

Tumors are highly heterogeneous at the patient, tissue, cellular, and molecular levels. This multi-scaleheterogeneity poses significant challenges for effective therapies, which ideally must not only distin-guish between tumorous and healthy tissue, but also fully address the wide variety of tumorous sub-clones. Commonly used therapies either leverage a biological phenotype of cancer cells (e.g. high rateof proliferation) or indiscriminately kill all the cells present in a targeted volume. Tumor microenviron-ment (TME) targeting represents a promising therapeutic direction, because a number of TME hallmarksare conserved across different tumor types, despite the underlying genetic heterogeneity. Historically,TME targeting has largely focused on the cells that support tumor growth (e.g. vascular endothelial cells).However, by viewing the intrinsic physical and chemical alterations in the TME as additional therapeu-tic opportunities rather than barriers, a new class of TME-inspired treatments has great promise tocomplement or replace existing therapeutic strategies. In this review we summarize the physical andchemical hallmarks of the TME, and discuss how these tumor characteristics either currently are, or mayultimately be targeted to improve cancer therapies.

© 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction

Tumors aremarked by a high degree of heterogeneity bothwithinas well as between patients. This multi-scale heterogeneity dras-tically diminishes the treatment efficacy of many classical cancertherapies. The most commonly used chemotherapies target a bio-logical phenotype of cancer cells, specifically their highly proliferativenature. However, such therapies leave behind resistant sub-populations that repopulate the tumor, while also resulting in toxicityto healthy cells. Other therapies indiscriminately kill or remove allcells within a given tumor volume, such as surgical resection andradiation therapy, and these too may leave behind cells that causetumor relapse. Recent advancements in targeted therapy typicallyfocus on receptors that are upregulated in cancer. However, almostall of the receptors currently being targeted are also expressed innormal cell populations, leading to off-target effects and potentialtoxicity [1–4]. These targeted methods also drive the evolution ofresistant cells, causing eventual treatment failure. Often these re-maining cells are self-renewing progenitors known as cancer stemcells (CSC), which have been demonstrated to be responsible for

tumor initiation and growth maintenance in cancers of the brain[5], breast [6], colon [7], and the hematopoietic system [8,9]. Becausethese CSCs often share surfacemarkers with normal tissue stem cells,and furthermore do not exhibit the high proliferative tendency ofbulk tumor cells, they present several treatment challenges that arenot addressed by traditional methods.

With a current therapeutic focus on biological properties of neo-plastic cells, which tend to have a high degree of variance due tothe highly heterogeneous nature of tumors, tumor recurrence andmetastasis continue to present major challenges. However, the phys-ical and chemical hallmarks of malignancy, which often times aremore consistent across different tumors than biological markers, mayprovide effective and reliable targets that could complement moretraditional approaches. The dynamic process of tumor develop-ment results in a major restructuring of the entire tumormicroenvironment (TME). As this TME develops, there is a complexand dynamic feedback between the selective stimuli acting on thetumor cells and the surrounding profiles of hypoxia and acidity,growth factors, and mechanical forces. Not only is the surround-ing tumor milieu of importance in the development of cancer, it alsohas a profound effect on therapy efficacy. It is therefore critical toconsider the underlying TME in developing more effective treat-ment practices that are also effective against resistant cell clones.Exploitation of the physical and chemical properties of the TMEas therapeutic targets may greatly complement or enhance the

* Corresponding author. Tel.: 540-231-6908.E-mail address: sverb@vt.edu (S.S. Verbridge).

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.canlet.2015.12.0190304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

Cancer Letters 380 (2016) 330–339

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Cancer Letters

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efficacy of existing treatment options; however, these have onlyrecently begun to receive serious attention clinically. In thismini-review, we outline the major physical and chemical microen-vironmental alterations in cancer that we believe present the mostpromising targets for anti-tumor therapies. Wewill then discuss keyadvances in the development of this new class of therapies that con-siders these alterations in tumors as therapeutic opportunities ratherthan hurdles to effective treatment.

Physical and chemical alterations in cancer

Cancer development is a dynamic process characterized by a vastarray of alterations at the cellular and tissue level. This includes ab-normal growth and alterations in cells, the extracellular matrix, andblood vessels, and these alterations are all highly inter-related. Forexample, due to the high rate of cellular proliferation, solid tumorseventually become significantly less oxygenated than normal tissues,as the rapidly growing tumor mass exhausts the local supply ofoxygen (O2) [10]. The hypoxic microenvironment drives altera-tions in the behavior of cells, as they must adapt to enable survivalin a low O2 environment. Regulation of biological pathways byhypoxia-inducible genes is controlled by hypoxia inducible factor-1(HIF-1). Downstream signaling fromHIF-1 activation (resulting fromthe stabilization of HIF-1α in the absence of O2), regulates cell func-tions such as apoptosis, cell cycle arrest, angiogenesis, glycolysis andadaptation to low pH [11]. Newly formed vascular networks withintumors are in large part driven by HIF-1 tumor signaling in re-sponse to this low O2, however the vascular growth is rapid anddisordered, resulting in a network that does not deliver nutrientsefficiently [12]. Angiogenesis, the process of tumor vascular growthfrom the surrounding vasculature, greatly alters both the physicaland chemical TME. Leaky and uneven vasculature and poor lym-phatic drainage contribute to a complex environment with variableinterstitial pressure, hypoxic zones, and gradients of nutrients andgrowth factors within the tumor bulk [13]. Extracellular acidity isa primary characteristic of the TME as a result of metabolic alter-ation of hypoxic tumor cells, which rely on glycolysis for energyproduction, along with the poor perfusion associated with neo-plastic growth and angiogenesis.

The physical tumor microenvironment is altered from normaltissue due to matrix deposition and remodeling as well as in-creased proliferation of both cancerous and stromal cells. The rapiddivision and proliferation that characterizes cancer cells leads to in-creasedmass in a confined volume and causes increased intratumoralpressure and compression of lymphatic and blood vessels withinthe tumor [14]. Cancer associated fibroblasts (CAFs) form the majorsupporting structure for the tumorous tissue and exhibit in-creased proliferation, ECM production, and altered cytokine secretioncompared to normal fibroblasts [15]. The deposition of highly cross-linked and oriented collagen in the ECM causes tissue stiffening inthe case of liver [16], breast [17] and prostate tumors [18,19]. Tumortissue stiffening occurs despite higher deformability of individualcancer cells compared to normal cells, which is itself partly due tocytoskeletal reorganization [20,21]. Tumor stiffening results in thedevelopment of both increased tissue stress and interstitial fluid pres-sure (IFP) as seen in Fig. 1 [22]. These forces and resulting cellcompressions affect many aspects of cell behavior such as gene ex-pression, cell proliferation, invasiveness, and ECM organization[24–26]. Blood and lymphatic vessel compression resulting from theincreasing solid mass contributes to the hypoxic and acidic mi-croenvironment by obstructing the delivery of oxygen to or removalof waste products from tissue [27–29]. This demonstrates that thealterations in physical and chemical properties are actually inter-related, with bi-directional feedback.

While altered tissue mechanics and chemistry have received sig-nificant attention in the TME field, the electrical properties of tumor

tissues also become deregulated in a number of ways. Due to theincreased intracellular concentration of positively charged sodiumions and an increase in negatively charged glycocalyx on the cellmembrane of tumor cells, the electrochemical properties of normaland cancer cells differ [30]. Cancer cells exhibit decreased restingtransmembrane potential (TMP), in part because of these alteredion and charge compositions [31]. It has been shown that themitoticactivities of cells are related to the TMP, with cells having low TMP(referred to as depolarized) exhibiting increased proliferation [32].This alteration is largely attributed to electron and proton trans-port across the cell membrane, which results in accumulation ofnegative charges on the cell surface [31,33]. The increased surfacearea of the cancer cells as a result of membrane protrusions resultsin increased membrane capacitance [34]. An altered nuclear-to-cytoplasm ratio (NCR) has long been known to occur in a varietyof cancer cells including cancers of the breast [35], uterus and cervix[35], brain [36,37], prostate [38], colon, and lung, and this likely altersthe intracellular electrochemical properties of cells. Finally, the ECMalso undergoes alterations in electrical properties due to electri-cally charged semiconducting proteins and proteoglycans. Theseintegrated effects lead to an altered electrical TME, which eventu-ally yields higher electrical conductivity and permittivity within thetumor [39–41].

Several of these microenvironmental alterations are suffi-ciently universal to be used for diagnosis of cancer. Breast canceris diagnosed by changes in tissue stiffness detected by mammog-raphy. Microenvironmental electrical alteration between healthy andtumorous tissue is used in diagnosis procedures such as electricalimpedance spectroscopy (EIS) [42,43]. Clinical impedance mea-surements have detected an altered electrical environment in lungcancer patients, which demonstrated cancer patients to have areduced electrical reactance [44]. PET-CT scans commonly used indiagnosing cancer detect the increased glucose uptake that leadsto the acidic microenvironment. While a number of studies havesuggested the importance of these altered physical and chemicalproperties for detection and prognosis, few have translated thesealterations into effective therapy targets. In what follows we willreview the studies in which these microenvironmental alterationshave been leveraged toward targeted cancer therapy, as well aspropose future directions for this research.

Targeting the chemical tumor microenvironment

Targeting acidity

Because low pH has been implicated in enhancing malignancyin a number of tumor types, recent research has focused on tar-geting this chemical feature for the benefit of patients. In oneapproach, low pH insertion peptides (pHLIP) were used to selec-tively deliver drugs to the cells residing in acidic tissues [45]. ThesepHLIP peptides undergomembrane-associated foldingwhen exposedto acidic conditions, which causes them to change from a mem-brane surface state in neutral conditions, and then to be insertedinto the cell membrane in low pH environments [46]. These inser-tion peptides have been used to transport toxins, nucleic acids, andnanoparticles into the cytoplasm of cancerous cells resulting in re-duction of tumor growth in animal models [47–51]. Other drugdelivery designs have used low pH to activate the drug in order toreduce accumulation of drugs in normal tissues. One such designuses a small shielding molecule attached to the terminus of a tumorreceptor specific ligand. Upon exposure to acidic tissue, the shield-ing molecule detaches from the ligand, and exposes the targetingligand to bind to the targeted tumor cells [52]. Another approachhas been to use pH-sensitive coatings, such as the PEG coating de-veloped by Yang et al. for siRNA therapy. In this study the PEG coatingreduced non-specific interactions under normal pH, however within

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an acidic microenvironment this coating was degraded [53]. Tumoracidity has also been used to activate a charge-conversional nanogel.Triggered by slightly acidic conditions, the bonds that are stable inneutral and alkali pH degrade to expose positively charged aminogroups. Such a system has the benefit of having high drug-loadingefficiency at physiological pH, with enhanced cellular internaliza-tion triggered by nanogel–cell interaction that occurs only underlow pH [54]. These efforts, which have demonstrated improved se-lective targeting to the acidic microenvironment and subsequenttumor reduction, capitalize on the fact that extracellular acidity isa conserved property of the TME and a predictor of aggressive-ness. Therefore, targeting tumor acidity might represent a novel

approach for the delivery of therapeutic agents selectively to tumorcells most likely to resist standard therapies, as well as to tumorcells with the greatest metastatic potential.

To complement these acid-activated drugs, additional researchhas focused on actually buffering the tumor pH to alter tumor cel-lular dynamics. Because low pH is an active driver of malignancy,and tumor cells possess a competitive survival advantage under lowpH, the working hypothesis has been that pH buffering may reduceor reverse malignancy. In mouse models of metastatic breast cancer,oral administration of bicarbonate increased intratumoral pH andreduced the formation of spontaneous metastases [55]. A non-bicarbonate non-volatile buffer, IEPA has also been shown to reduce

Fig. 1. Major alterations in the mechanical tumor microenvironment. (a) The rapid proliferation of cancer cells and stromal cells along with the deposition of collagen createssolid forces within the tumor. These forces cause compression of blood vessels, which leads to areas of high interstitial fluid pressure in the tumor [22]. (b) A comparisonof the interstitial fluid pressure in aggregated data collected from a variety of human tumors as compared to normal human tissues shows an often drastic increase in IFPin the tumor microenvironment [23]. Data were collected from human patients using the wick-in-needle technique for measurement.

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metastasis in amousemodel when ingested orally [56]. These resultssuggest that oral administration of non-volatile buffers or bicar-bonate can be effective for reducing metastasis through themechanism of pH buffering, and suggest further research to trans-late and optimize this approach for human patients.

Targeting hypoxia

Due to the alterations in the TME caused by hypoxia, as well asthe links between hypoxia and therapy resistance, this chemical hall-mark has been considered as a prime therapeutic target. Oneapproach has been to develop bioreductive prodrugs wherein a non-toxic drug can be converted into a toxic molecule under low O2 [57].The general mechanism involves different chemical moieties thatcan be metabolized by enzymatic reduction under hypoxic condi-tions. This reduction produces a prodrug radical through one-electron reduction, which can participate in further reactions tobecome cytotoxic to cells thereby achieving hypoxia-selective killing[57–59]. Four different chemical groups: nitro groups, aromaticN-oxides, aliphatic N-oxides and transition metals have been shownto reduce to cytotoxic radicals under hypoxia, and have been usedas a structural basis to devise new hypoxia-activated drugs [58]. Mul-tiple different bioreductive drugs have demonstrated antitumorcytotoxicity against hypoxic cells [59–62].

A closely related approach to targeting hypoxia involves lever-aging cellular response to low O2 levels to enhance activation withinor delivery to the highlymalignant hypoxic tumor niches. Since HIF-1stimulates transcription of a large number of genes (hypoxia-responsive elements), constructs to obtain hypoxia-specifictranscription of a therapeutic gene would drive expression specif-ically in tumors [11,63]. One of the challenges associated with thistype of therapy is efficient delivery of vectors to hypoxic cells thatare located distant to blood vessels. Macrophages have been lev-eraged to overcome this challenge since they gather at tumor sitesand also express high levels of HIF-1α [64]. Hypoxia can inhibit mi-gration of these macrophages by inhibiting the monocytechemotactic protein (MCP-1) once they have infiltrated the hypoxicand necrotic areas of a tumor [65]. These macrophages can be trans-fected ex vivowith genes that encode for anti-cancer agents (tumorantigens, anti-angiogenic agents, etc.), resulting in significant killingof tumor cells [66,67].

Similar approaches have been developed using tissue stemcells, as well as bacterium that home to hypoxic tissues. Geneti-cally engineered strains of certain bacteria, such as Clostridium,Bifidobacterium, Salmonella,Mycobacterium, Bacillus, and Listeria areknown to localize and germinate in hypoxic regions of tumors,causing tumor cell lysis [68,69]. Clostridium spores and Clos-tridium strains genetically engineered to convert nontoxic drugs intocytotoxic drugs have been injected intomouse tumors causing tumorreduction [70–72]. In another study, bacterial tumor targeting wasdemonstrated to occur due to the dependence of E. coli reductaseactivity on hypoxia [57]. Bacterial strains are also beneficial for ther-apeutic delivery because they accumulate in areas of tumors distantfrom blood vessels that cannot be reached by other drugs [57]. Bac-terial therapy in combination with other more traditional treatmentshas the potential to be a promising approach for more effective com-binatorial targeting of the hypoxic tumor niche [69,73,74].

Targeting the physical tumor microenvironment

Exploiting the EPR effect

The Enhanced Permeability and Retention (EPR) effect de-scribes the phenomenon of tumor blood vessels facilitating transportof macromolecules into tumor tissues due to the leaky and highlypermeable nature of tumor vasculature. Macromolecules larger than

40 kDa selectively leak out from tumor vessels and accumulate intumor tissues [75]. Because this EPR effect does not occur in normaltissues, macromolecule therapies can be used to selectively targettumor tissues [76,77], representing one example by which thephysics of tumors has led the way to new therapeutic opportuni-ties. The EPR effect has been exploited for the selective delivery ofproteins, drug–polymer conjugates, micelles, liposomes,nanoparticles, DNA polyplexes and lipid particles [78–82]. By le-veraging this general characteristic of the TME in designing therapies,these macromolecular drugs have demonstrated prolonged reten-tion in the tumor and greater tumor selectivity, compared toconventional small molecule anticancer drugs, allowing for im-proved antitumor efficacy while limiting adverse reactions [82,83].

The EPR effect is mediated by factors that influence vascular per-meability such as angiotensin, nitric oxide (NO) and vascularendothelial growth factor (VEGF). Therefore, efforts have beenmadeto enhance the EPR effect by tuning these factors. Angiotensin, whichproduces systemic hypertension through vasoconstriction, causesan increase in blood pressure in normal vessels [84]. Tumor vessels,which lack the smooth muscle support needed for vasoconstric-tion remain open. This allows for an increase in blood flow to tumorsand a pressure buildup that forces the macromolecular drug intothe tumoral space [85,86]. Further efforts to enhance the EPR effecthave also capitalized on the altered hypoxic and acidic tumor mi-croenvironment. Nitroglycerin (NG) is an agent that liberates NOin hypoxic and acidic conditions [87]. NO, acting as a vasodilator,increases blood flow to the tumor. Because NO is selectively re-leased in the hypoxic and acidic environment by NG, blood flow isselectively increased in the TME, leading to an increase in drug de-livery to the tumor [88]. The simultaneous leveraging of multipleaspects of the altered microenvironment, such as leaky vascula-ture, underdeveloped blood vessel walls, hypoxia, and acidity, couldresult inmore effective therapies, as resistancemechanisms are likelyto differ between these.

Tumor ablation by pulsed electric fields

Application of pulsed electric fields (PEFs) across a tumor volumeinitiates a cascade of biophysical events at the cellular level that even-tually yield cell death. Depending on the pulse parameters, differentPEF therapies are categorizedas irreversible electroporation (IRE), highfrequency irreversible electroporation (H-FIRE), nanosecond-PEFs(nsPEFs), or tumor treatingfields (TTFields). Electroporation is thephe-nomenonof inducingnanoscalepores in the cellmembranedue to theapplicationof10–100micro-secondlongPEFsofhundredsofV/cmmag-nitude [89]. IRE is applied at a critical electric field so the cells cannotrepair the inducedpores, leading to cell death. This techniquehasbeenused extensively for non-thermal ablation of tumors [90]. Fig. 2 showsexamples of IRE treatments in vitro and in vivo. IRE enables killing cellswithin the targeted area while preserving the underlying structures.However, current IRE treatments do not discriminate between tumorsand healthy tissues, and rather ablate all cells within the range of theappliedelectricfield,highlighting thesignificanceofpretreatmentplan-ning techniques such as finite element modeling of electric fielddistribution. However, new studies that have been a focus of our ownresearchgroupshaveshownthat several keyphysicalpropertiesof cellsand their environmentaffect theirvulnerability in response to IRE treat-ment. The alterations in cellmembranemorphology and cell size havebeen used to electrically sort cells [95–97] and may be leveraged forselective treatment due to the known effects of these parameters onIREoutcome[98].H-FIREtreatment iscomprisedofburstsof about1kV/cmpulses that has been developed as an improvement to IREwithoutcausingmuscle contraction [99,100]. Our group has demonstrated thefeasibilityof selectively targeting tumorcellsbyH-FIREbasedonalteredNCR [101]. Because nuclear enlargement is a highly conserved char-acteristic of malignant cells, H-FIRE pulses tuned to target such a

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phenotypecanselectivelykill tumorouscellswhilesparinghealthycells.As opposed to IRE, H-FIRE is independent of tissue conductivity due tothe high frequencynature of pulses, andmore predictable lesionsmaybe attainable [102]. Recent studies have shown that electroporationchanges the electrical impedance spectrum of the TME [103], whichcould be used for monitoring the size of the ablated tissue [104]. Thiswould allow forprecise ablation confined to thepredetermined tumorarea, by continuedmonitoringof the electro-physical properties of theTME during treatment.

Owing to their short rise time, which is faster than cell mem-brane charging time, nanosecond pulsed electric fields (nsPEFs)penetrate thecellmembraneanddamage intracellular structures,withminimalmembrane electroporation [105,106]. In vitro studies on skincells have shown that tumor cells have a stronger response to nsPEFsthan normal cells [107,108], and suggest that an improved under-standing of the electrical differences among cells may lead to moretargeted therapies based on exploiting these differences.

Finally, tumor treating fields (TTFields) are a new class of elec-tric field therapies that allow for ablation of malignant cells bytargeting the highly proliferative phenotype of cancer cells. In-vented in 2004 by Kirson et al. [109], this treatment is comprisedof alternating electric fields with a frequency of 100-300 kHz andintensity of less than 2 V/cm. These fields interfere with mitosis byrotationally exciting the proteins involved in the formation of thecytokinetic cleavage furrow (CCF), and inducing apoptosis [110].These fields target the cells during the cytokinesis process and there-fore have specific inhibitory effects on dividing cells while leavingquiescent cells intact. The electric fields are applied locally so as toreduce the effect on quickly dividing healthy cells. This allows fora highly selective targeting of proliferative tumor cells. The optimalfrequency for TTfields varies among different cell types due to varia-tions in the cell membrane capacitance, which affects fieldpenetration into the cell [111]. Therefore, inherent differences inthe permittivity between healthy tissue and tumors could be ex-ploited for selective application of this treatment.

Tumor targeting via thermal ablation

Thermal ablation therapies expose cells to cytotoxic heat gen-erated from several different sources such as radiofrequency (RF),

laser, microwave and high intensity focused ultrasound (HIFU). Re-gardless of its source, hyperthermia damages both healthy and tumorcells. However, tumors are more vulnerable to hyperthermic damagethan normal tissue [112]. It has been shown that the acidic TMEmakes the cells more thermosensitive than the surrounding normaltissue, providing a therapeutic advantage for selective killing of tumorcells by hyperthermia [113]. The low extracellular pH in the tumorinduces a low intracellular pH, which is believed to be themain causeof this observed thermosensitivity [114,115]. Several transmem-brane antiport mechanisms regulate the intracellular pH (pHi) andprohibit its equilibrium with the extracellular pH (pHe), which isnecessary for its survival and proliferation. Therefore, the efficacyand selectivity of hyperthermia treatments can be improved by in-hibiting the pHi regulatory mechanisms resulting in a lower pHi inthe acidic TME [116–118].

Radiofrequency ablation (RFA) is a focal ablation therapy that usesthe cytotoxic heat generated from oscillating ions in the high fre-quency alternating electric field. Electrical and thermal properties ofthe TME have a significant influence on the outcome of the therapy[119,120]. In RFA, tumors are prone to more heat generation com-pared to normal tissue due to their higher concentration of ions. Therelatively low thermal conductivity of the tumor tissue, which is aresult of dysfunctional vasculature and inefficient bloodflow, hindersheat dissipation into the surrounding tissue and improves the local-ization of heating within the tumor [121]. Some tumors are alsosurrounded by low thermal conductivity tissues that act as addi-tional thermal insulators to the tumor [122,123]. Perfusion throughthe tissue decreases the extent and efficiency of RFA, because bloodflow acts as a means of heat dissipation [124,125]. Therefore, poorlyperfused tumors are more easily ablated than normal tissue [126].Experimentally it has been shown that normal tissues react to hy-perthermia by enhancing blood flow; on the contrary no significanteffect is observed in thebloodflowin tumors [127]. Theseeffects resultin ahigher temperature in tumors andevenmore selective cell killing.

High intensity focused ultrasound and tumor viscoelasticity

High intensity focused ultrasound (HIFU) works on the basis ofmechanical wave transmission and absorption, which is highly de-pendent on themechanical properties of the domain such as stiffness

Fig. 2. Irreversible electroporation from in vitro to in vivo (a) visualization of live/dead cells after IRE treatment in a 3D in vitro tumor model [91], (b) Sparing of major blood vesselsafter IRE treatment of canine brain [92], (c) Delineation between viable tissue (left) and reactive fibrosis and hemorrhage (right) is seen in human prostate IRE histology, adaptedfrom [93], (d) 7.0-T MRI of IRE-treated canine brain, adapted from [94], (e) dual probe insertion during the intracranial IRE procedure in canine brain [92].

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and damping. Depending on the frequency, magnitude, and dura-tion, HIFU can destroy the tissue by either thermal or purelymechanical means. The former occurs by viscous absorption of themechanical wave by the tissue and its subsequent conversion to heat[128]. Tissues with higher viscous damping are prone to more heatgeneration. Elastography of prostate cancer [129] and hepatocel-lular carcinoma [130] has shown that tumors have higher viscositythan normal tissue, which makes them more vulnerable toultrasound-induced hyperthermia, which means enhancement oftherapy targeting. On the other hand, it has been shown that tissueswith lower mechanical stiffness are more vulnerable to non-thermal ultrasound-induced cavitation and destruction (histotripsy)[131,132]. Despite the high tumor tissue stiffness, individual cancercells present lower stiffness compared to healthy cells. Therefore,given the complex dependencies of HIFU damage mechanisms onthe local tissue microenvironment, future studies are needed toexplore the use of HIFU in thermal andmechanical modes to achievepreferential killing of tumor cells while preserving ECM.

Challenges to targeting the TME

Though the alterations in the chemical and physical TME presentexciting targets for cancer therapy, there are many challenges as-sociated with such targeting. Some of these challenges may preventtranslation of individual targeting concepts; however, ongoing re-search is focused on overcoming these challenges for the benefitof cancer patients. As with all molecular targeted therapies, ther-apeutics directed to hypoxic and acidic microenvironments are onlyeffective if they physically reach those environments. Drugs acti-vated in acidic or hypoxic conditions may reduce side effects ascompared with their non-targeted analogs, however delivery to thetumor site is still a major hurdle. Because the delivery and reten-tion of drugs in the tumor is dependent on a large number of factorsand is influenced by the complex microenvironment, in many casesdelivery of targeted drugs results in only a few percent of the totaladministered dose reaching the desired location [133]. Similar limi-tations plague drugs designed to capitalize on the EPR effect fortargeting. In many cases the majority of macromolecule anti-cancer drugs accumulate in the liver or spleen, with the EPR effectonly resulting in very modest enhancements in tumor-selective de-livery [133]. Thus it becomes an ongoing challenge to improve theoverall delivery of therapeutics to the general tumor area so selec-tive targeting designs can take effect. One new approach recentlyleveraged electric fields to actively enhance drug penetration intoa tumor [134], and approaches such as this provide promising newavenues toward enhancement of tumor-targeted therapies broadly.

Focal ablation techniques, such as electrical, thermal and ultra-sound ablation, overcome the problem of delivery that faces targeteddrugs, as these ablation therapies are delivered directly at the tumorsite. However, these techniques do require surgery. The size and lo-cation of the tumor should be known prior to these treatments,which are usually facilitated by ultrasound or other imaging tech-niques. The invasiveness, althoughminimal, of these procedures dueto the insertion of the probe and lack of selectivity are drawbacksthat should be considered. Clinical IRE and RF protocols are con-sidered to be nonspecific ablation techniques that will ablate alltissue within a given treatment volume. In many cases the selec-tivity of these treatments may be improved by tuning them tocapitalize on different physical and chemical features. TTFields tar-geting dividing cells [109] and HFIRE targeting enlarged nuclei [105]improve the selectivity of electrical ablation methods. Thermal ab-lation methods may gain improved selectivity by inhibiting pHregulatory mechanisms in an acidic microenvironment. HIFU treat-ments may have increased efficacy if combined with modulationof tissue stiffness. Further work must be done into improving boththe delivery of targeted therapies and the selectivity of local

therapies. In both cases, the microenvironment seems bound to playa highly important role. These therapies may ultimately be com-bined, e.g. as is the case in new electrochemotherapy modalities[135], for synergistic enhancements in the efficacy of each approach.

Finally, the complexity and interconnectedness of these poten-tial physical and chemical features may result in adverse side effectsthat need to be considered during treatment design. Acidity, hypoxia,metabolism, and angiogenesis, for instance, are all very closelyrelated, and impacting one of these microenvironmental featuresmay have unintended consequences for the others. For instance, re-active oxygen species (ROS) produced as a result of aerobic glycolysisincrease cell proliferation and angiogenesis. Consistent with this fact,inhibition of ROS has been shown to decrease angiogenesis andtumor growth in vivo [136]. However, inhibition of ROS with an-tioxidants has conflictingly been shown to increase tumor growthin mice [137]. This inconsistency is attributed to the complex roleof ROS in regulating a variety of interconnected cellular processes[137]. The failure of many promising anti-angiogenesis drugs in clin-ical trials [138] has been attributed in part to the resulting hypoxiaand consequential outgrowth of resistant and invasive pheno-types that cause metastasis and treatment failure [139,140]. Thehypoxic microenvironment left as a result of anti-angiogenic therapymay also provide a suitable home for cancer stem cells [141]. Evenif anti-angiogenic therapy is effective at initially reducing growthof the primary tumor, residual cancer stem cells or evolved inva-sive phenotypes may lead to repopulation of a primary, or seedingof a metastatic tumor, resulting in poor therapy outcomes. There-fore, it remains an important area of future research to clarify thepotential consequences of any form of microenvironmental target-ing strategy, and the development of suitable model platforms forsuch studies remains a key priority. It may be found that multipleinterconnected features must be targeted in combination, such asangiogenesis and hypoxia, for high efficacy to be achieved in thecomplex environment found in vivo.

Conclusions

Chemical and physical characteristics of the TME act as a barrierto many traditional cancer therapies. However, there are intrigu-ing opportunities to leverage these same hallmarks to enhancetreatment efficacywith appropriately designed therapies. Fig. 3 sum-marizes various physical and chemical alterations of the TME, andprovides examples of therapies associated with these alterations.The aforementioned treatments either provide proof of concept fordevelopment or translation of newmethods (nsPEFs, H-FIRE), or areundergoing clinical trials (hypoxia selective drugs [59] [72], IRE [90],TTFields [142]). Despite these advancements, the area remains largelyunexplored, and more basic as well as translational work is neededto advance these approaches. The TME we have discussed is oftena general characteristic of tumors and does not display the samedegree of variance as observed for biological markers across dif-ferent tumor types. By targeting physical and chemical propertiesof the TME rather than the biology of neoplastic cells, the efficacyof these therapies is likely to depend on resistance processes thatdiffer from those leading to the failure of radiation, chemothera-py, or molecular targeted therapies. These approaches will thereforecomplement more traditional treatments. Therapies can then becombined so that the resistant sub-populations are non-overlapping,helping to reduce tumor recurrence and increase survival times forpatients. The physical and chemical hallmarks of the TME there-fore can pave the way to the design of more rational and effectivetherapeutic regimens, andwith potentially reduced side effects com-pared with some traditional therapies. However, further work willbe required to enhance their selectivity through an improved un-derstanding of the mechanism of individual as well as combinatorialtreatments.

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Acknowledgements

This work was supported by the R21 Award from the NationalCancer Institute of the National Institutes of Health (R21CA192042),the award from Center for Innovative Technology (MF13-037-LS),and the award from NIH (5R21 CA173092-01).

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