cells, tissues, and organs on chips challenges and opportunities for the cancer tumor...

LI

ISSN 1757-9694

www.rsc.org/ibiology Volume 5 | Number 9 | September 2013 | Pages 1089–1198

REVIEW ARTICLEEdmond W. K. YoungCells, tissues, and organs on chips: challenges and opportunities for the cancer tumor microenvironment

Interdisciplinary approaches for molecular and cellular life sciences

1096 Integr. Biol., 2013, 5, 1096--1109 This journal is c The Royal Society of Chemistry 2013

Cite this: Integr. Biol.,2013,5, 1096

Cells, tissues, and organs on chips: challenges andopportunities for the cancer tumor microenvironment

Edmond W. K. Young*

The transition to increasingly sophisticated microfluidic systems has led to the emergence of ‘‘organ-on-

chip’’ technology that can faithfully recapitulate organ-level function. Given the rapid progress at the

interface between microfluidics and cell biology, there is need to provide a focused evaluation of the

state-of-the-art in microfluidic systems for cancer research to advance development, accelerate discovery

of novel insights, and facilitate cooperation between engineers, biologists and oncologists in the

clinic. Here, we provide a focused review of microfluidics technology from cells- and tissues- to

organs-on-chips with application toward studying the tumor microenvironment. Key aspects of the tumor

microenvironment including angiogenesis, hypoxia, biochemical gradients, tumor–stromal interactions,

and the extracellular matrix are summarized for both solid tumors and non-solid hematologic malignancies.

An overview of microfluidic systems designed specifically to answer questions related to different

aspects of the tumor microenvironment is provided, followed by an examination of how these

systems offer new opportunities to study outstanding challenges related to the major cancer hallmarks.

Challenges also remain for microfluidics engineers, but it is hoped that cooperation between engineers

and biologists at the intersection of their respective fields will lead to significant impact on the utility of

organs-on-chips in cancer research.

Insight, innovation, integrationOrgan-on-chip technologies are attracting significant interest with existing reviews focusing mostly on technical aspects of the field. This review focuses insteadon cells, tissues, and organs-on-chips specifically for studying cancer biology. By discussing the advancement of microfluidic cell-based systems from thestandpoint of the disease and the key elements within the tumor microenvironment, this review offers a different perspective that may lead to new organ-on-chipinnovations as well as unexpected insights on how we approach cancer research in the future.

1. Introduction

The integration of microfluidics and cell biology research hasrecently reached another significant milestone with develop-ment of ‘‘organ-on-chip’’ technologies. What began at the turnof the millennium as simple demonstrations of biological cellsbeing transported and manipulated in microchannels for basicshort-term analysis1,2 has now advanced to the point where wecan engineer living cellular microsystems with controllablemicroenvironments that behave and function – with organ-levelcomplexity – like their counterparts in vivo.3–5 While advance-ment from cell- to tissue- to organ-level function in vitro hasbeen impressive from an engineering perspective, what is truly

compelling is the potential impact this technology will have onthe study of human diseases, and on clinical and therapeuticapplications that may ultimately improve overall human health.

Cancer research in particular has potential to reap immensebenefits from the application of organ-on-chip technologies.A major challenge in cancer research has been the need todevelop more accurate, more informative, and more predictiveexperimental models of human tumor development than thosecurrently available. Traditional in vitro systems and in vivoanimal models have both made major contributions to ourcurrent understanding of the disease, and to the discovery andpractical application of oncotherapies, but these models alsohave shortcomings that limit their overall effectiveness towardpredicting the success or failure of many candidate drugcompounds during the screening process, as well as that ofselected therapeutic strategies.6 Ineffectiveness in the formercase has led to a disappointingly high attrition rate of potential

Department of Mechanical & Industrial Engineering, University of Toronto,

5 King’s College Road, MC314B, Toronto, ON, M5S 3G8, Canada.

E-mail: [email protected]; Fax: +1 (416) 978-7753; Tel: +1 (416) 978-1521

Received 16th April 2013,Accepted 6th June 2013

DOI: 10.1039/c3ib40076j

www.rsc.org/ibiology

Integrative Biology

REVIEW ARTICLE

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http://dx.doi.org/10.1039/c3ib40076j

http://pubs.rsc.org/en/journals/journal/IB

http://pubs.rsc.org/en/journals/journal/IB?issueid=IB005009

This journal is c The Royal Society of Chemistry 2013 Integr. Biol., 2013, 5, 1096--1109 1097

candidates late in the drug discovery process that is both costlyand time consuming. Organ-on-chip technologies have potentialto offer significant improvements in in vitro models of thedisease, with better physiological mimicry of the tumor micro-environment than existing in vitro assays, and more speciesrelevance than in vivo animal models (if primary human cellsare employed). Importantly, the role of the tumor micro-environment in cancer initiation and progression, and therecognition that a tumor itself should be considered a complexorgan,7 suggests that physiological microenvironments withimproved relevance to humans and perhaps even organ-levelcomplexity are integral to our understanding of the disease,and must be given more consideration in future methodologiesof experimental research. The purpose of developing improvedexperimental models through organ-on-chip technologies is toaccelerate progress in fundamental research, to increase effi-ciency in drug discovery, and to advance the translation of newknowledge into clinical outcomes. To accelerate the potentialimpact of this technology on cancer research, it is importantand necessary to review the pertinent literature to date, parti-cularly work that has already shown the promise of microfluidicchip technologies in meeting the needs of cancer research.

This review discusses the current state-of-the-art in cells-,tissues-, and organs-on-chips technology specifically for cancermicroenvironments. While other recent reviews on this technol-ogy have provided more general overviews of technical advancesin device functionality and complexity,8,9 this review focuseson cancer, and categorizes various microsystems based on keyfactors relevant to the tumor microenvironment. The potentialfor these microsystems to contribute to cancer research isproposed in relation to major cancer hallmarks, and a discus-sion of major challenges facing those working at the interfacebetween microfluidics and cancer is provided. The combinationof these different aspects distinguishes this review from otherrecent articles summarizing the application of microfluidics forcancer research.10,11 For convenience and completeness, a briefoverview is provided on the essential elements of the tumormicroenvironment for both solid tumors and nonsolid malig-nancies. Regarding nomenclature, where appropriate, differentcellular microsystems are referred to in specific terms as cells-on-chips, tissues-on-chips, or organs-on-chips, depending onbiological complexity. However, for brevity, these microsystemsare also collectively referred to here as either microfluidicsystems or ‘‘biochips’’ to cover all three hierarchical levels.

2. Essentials of the tumor microenvironment

It has become increasingly evident that cancer development, inits remarkable complexity, is not only a matter of individualtumor cells evolving as a result of multiple genetic mutations,but also involves the complex interactions between the tumorcells and the many physical, biological, and molecular factorsof the surrounding tumor microenvironment. From a historicalperspective, the well-known seed-soil hypothesis by Paget in 1889,as a proposed explanation for site-specific metastases, was one ofthe first signs of recognition that the local microenvironment

(although distant from the primary site in this case) may con-tribute to tumorigenicity, enabling metastatic lesions to thrive inan otherwise foreign and hostile environment.12,13 Since then, anextensive body of literature has provided strong support for therole of the tumor microenvironment,14,15 leading to majorfields of research ranging from angiogenesis and hypoxia todimensionality and cancer mechanobiology. The tumor micro-environment as a whole has thus emerged as a central player incancer research, and as a consequence has become a majorpotential target for cancer therapy.

In experimental biology, our inability to accurately model thehuman tumor microenvironment in experimental systems is oneof the major and perhaps most important reason why many ofour results in the lab do not translate directly to improvedoutcomes in the clinic. The question of whether current in vitrosystems and in vivo animal models are sufficiently representativeof human physiological conditions to be predictive is a majorsource of debate, and stems from the recognition that thesemodels are limited in physiological context, either lacking impor-tant spatial and temporal cues in the case of simplistic planar(2D) in vitro models, or lacking the genotype specific to humansin the case of animal models. Thus, for decades, while bothexperimental paradigms provided significant contributions toour understanding of the disease, and resulted in novel dis-coveries in therapeutics, there remained an ongoing concernrelated to the limitations of our experimental repertoire.

The potential for organ-on-chip technology to provide arealistic, unconventional and potentially disruptive alternativeto existing in vitro and in vivo methods is therefore an excitingproposition. We now have the ability through microfabricationtechniques and microengineered control to construct withvarying levels of complexity living microsystems that recapitulatedifferent aspects of the microenvironment using human cells,perhaps even those derived directly from diseased individuals.This level of physiological relevance to humans is not achievablevia existing methodologies. Given such power to tailor themicroenvironment as desired for each experiment or applica-tion, the question is no longer whether we can do it. Instead, thequestion is how we do it, and what we do with the technology tobest serve the future of cancer research.

To begin to answer these questions, we briefly examine thevarious aspects of the developing tumor microenvironment(Fig. 1), first in the context of an epithelial-derived carcinoma.Carcinomas represent over 80% of all cancers, including thoseof the breast, colon, liver, lung, pancreas, and prostate, amongothers, and are collectively considered the largest class ofneoplasias. Subsequently, we will also examine aspects of themicroenvironment for non-solid hematologic malignancies, asecond major class of cancer that has unique features distinctfrom the microenvironment of solid tumors. For convenience,the discussion will proceed in sequence through the stepsleading from normal health to diseased state.

Solid tumors

Carcinomas derived from epithelial tissue originate from singlecell clones, which proliferate and aggregate into a mass of

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mutant cells that form the basis of a tumor. Because tumorcells continue to accumulate mutations during tumor progres-sion, the population of cells becomes increasingly hetero-geneous, with successive generations of mutant cells mixedwith the initial clonal population. Accumulating evidence sug-gests that this amalgam also consists of tumor initiating cells,more popularly known as cancer stem cells, which are capableof spawning new tumors when injected into previously healthymice.16 Tumor cells are surrounded by the stroma, whichconsists of the three-dimensional structural framework ofextracellular matrix (ECM) components and the various othercell types that support the associated connective tissue. TheECM allows anchorage of tumor and stromal cells via cell–surface integrins that transduce mechanical signals andmediate various mechanobiological responses. Non-tumorstromal cells make up an estimated 80% of the cells in the

tumor microenvironment, and are arranged in specific posi-tions and in specific proportions, creating a complex networkof heterotypic interactions that plays a central role in tumorprogression.17 Notable cell types in the stroma include: thevarious types of fibroblasts (e.g., normal stromal fibroblasts,myofibroblasts, cancer-associated fibroblasts), which are closelyrelated, yet display distinct phenotypic markers, and mayoriginate from different precursors;18 immune cells such asmacrophages, neutrophils, and T and B lymphocytes that sensepro-inflammatory cytokines and chemokines secreted by thetumor and infiltrate the microenvironment; adipocytes such asthose found in the mammary gland;19 and endothelial cells andsupporting pericytes, which are recruited into the micro-environment by the tumor-associated inflammatory cells thatsecrete soluble factors involved in inducing angiogenesis.20

Angiogenesis is the formation and extension of new blood

Fig. 1 The cancer tumor microenvironment consists of a heterogeneous mix of cells in complex spatial arrangement. Cell types include: tumor cells (e.g., carcinoma),cancer stem cells, invasive phenotypes, inflammatory immune cells, assorted fibroblasts, pericytes, and endothelial cells of angiogenic blood vessels. The cells aresupported by extracellular matrix. Regions far from blood supply are hypoxic and subject to oxygen gradients (dark shaded region). Invasive phenotypes may undergoepithelial–mesenchymal transition (EMT), migrate away from the tumor core toward the margins (dotted line), and intravasate into neighboring blood vessels wherethey circulate to ectopic locations and form metastases. (a) Prostate cancer coculture spheroids in microchannels.33 (b) 3D side-by-side coculture of human mammaryfibroblasts and ductal carcinoma in situ (DCIS) cells to study role of stromal interactions in transition to invasive ductal carcinoma.48 (c) Microfluidic chip for generatingperfusable microvessel networks.58 HUVECs stained for nuclei (blue), f-actin (green), and CD31 (red). (d) Microfluidic system for creating oxygen gradient by oxygen-generating and oxygen-scavenging chemical reactions.63 For (a) to (d), images reproduced in part from ref. 33, 48, 58, and 63, respectively, with permission of TheRoyal Society of Chemistry. (e) Microfluidic system for studying cancer cell (HT1080, red) invasion and intravasation through endothelium (human microvascularendothelial cells, MVECs, green). Adapted from ref. 71 Copyright 2012 National Academy of Sciences, USA.

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vessels from existing vasculature that provides oxygen andnutrients to the tumor that are necessary for its growth. Asthe primary tumor grows and blood vessels infiltrate the tumor,internal oxygen and chemical gradients form in relation to theangiogenic network, creating regions of hypoxic and necroticcells. The complex set of microenvironmental cues contributeto the differentiation of some tumor cells into more migratoryand invasive phenotypes, possibly via epithelial–mesenchymaltransition (EMT), which gives rise to cells that escape the tumormass via intravasation into nearby blood vessels. These circulat-ing tumor cells (CTCs) then travel via the circulatory system todistant ectopic locations where they have the potential to reside,colonize, and form secondary tumors called metastases. Fig. 1illustrates these major elements of the tumor microenvironment.

Liquid tumors

A second major class of cancers is hematologic malignancies,or cancers related to the blood, bone marrow, and lymphnodes (i.e., ‘‘liquid tumors’’).21 While certain elements of themicroenvironment exist in both solid and liquid tumors (e.g.,angiogenesis), elements unique to hematologic cancers areimportant enough to warrant mention here. For example, interms of cell types, hematopoietic stem cells reside in the bonemarrow and have been implicated in the pathogenesis andprogression of hematologic malignancies,22 but have notbeen implicated in solid tumors. Other cell types such asmesenchymal stromal cells, bone marrow stromal cell (BMSCs),and monocyte-derived nurse-like cells belong to the hemato-poietic lineage, and thus are also only found in the micro-environments of hematologic malignancies. The presence oflymphoma-associated macrophages (LAMs) suggests a similarlyimportant role for inflammatory cells in liquid tumors as insolid tumors. Secondary lymphoid organs are characterized bypresence of T cells responsible for assisting in antigen recogni-tion. Naturally, the bone milieu also includes osteoblasts andosteoclasts that reside in the bone matrix. Whereas solidtumors can be described by the location of the primary site,hematologic cancers exist in the lymphatic and circulatorytransport systems, and are thus characterized by their presencein circulation. Of importance is the notion that the role of themicroenvironment in blood-related cancers is highly variablebetween cancer types, ranging from a very limited role inBurkitt lymphoma to a highly involved, coexisting role inmultiple myeloma (MM) and chronic lymphocytic leukemia(CLL), which has implications on the likely responsiveness ofa blood cancer type to therapeutic strategies that target themicroenvironment.23

3. Microfluidic technology for tumormicroenvironments

From a molecular and cell biology perspective, the briefoverview of tumor microenvironments presented above isadmittedly too simple to capture the inherent complexity of amulticellular system that can be influenced by so many

environmental factors. The important point to take from thesummary above is that development of the tumor micro-environment progresses through a series of major events (e.g.,tumor initiation; angiogenesis) that involve key features (tumornodule formation; new blood vessels) with specific functionalroles, and that the coordinated interplay of these features leadsto tumor progression. Advances in microfluidic cell-basedsystems or biochips have led to a new class of in vitro toolswith both demonstrated utility in recapitulating the variousaspects of the developing tumor microenvironment (Fig. 1), andthe potential to offer insight into unprecedented spatio-temporal dynamics of these events. Microfluidic systems havevarying levels of complexity, ranging from simple 2D cellculture microchips to more physiologically relevant 3D tissuemicrochips. Importantly, microfluidic systems offer variousadvantages over traditional in vitro systems, including morecomplex geometries, better spatiotemporal control of micro-environmental factors and stimuli, added functionality, andincreased throughput.24 In this section, recent research studiesemploying microfluidic systems to study aspects of the tumormicroenvironment are reviewed, with an attempt to organizethe studies in order of increasing physiologic complexity andmore advanced stage of tumor development (Table 1).

Tumor spheroids

To model a tumor, a popular method is to employ multicellulartumor spheroids (MCTSs), which are derived from a collectionof cells that aggregate under non-adherent culture conditionsto form three-dimensional cellular masses. The morphology,growth kinetics, and cell–cell and cell–matrix interactionswithin these spheroids resemble tumor nodules, making themexcellent models of tumor initiation and growth, and a usefultool for testing drug delivery and efficacy.25 Various methodshave been developed over decades to achieve efficient spheroidformation, including rotating cultures in roller tubes andspinner flasks, as well as stationary cultures in hanging drops,96-well plates, and related organotypic cultures.26 Techniquesinvolving 96-well plates have been adapted to enable high-throughput spheroid-based drug screens, with protocolsbecoming increasingly standardized.27 Although microtiterplates have many advantages, such as enabling testing andmonitoring of spheroids in independent wells, and allowingconsiderable reduction in reagent consumption compared torotating cultures, the static, diffusion-dominant conditions inwells lead to accelerated nutrient depletion and waste accumu-lation, which can affect spheroid formation and function, andpotentially lead to false identification of drug candidates.27

Microfluidic systems have recently been shown to be effective atfacilitating the formation of tumor spheroids on chip. High-throughput arrays of hanging drops can be generated in well-plate format with the use of microfluidics to create uniform andindependently addressable spheroids.28–30 Microscale geo-metries can be designed to hydrodynamically trap cells to createspheroids with highly uniform size distributions, and cansimultaneously allow continuous perfusion to maintain long-term spheroid culture.31,32 In addition, porous membranes


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Tab

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Tab

le1

(con

tinu

ed)

Can

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type

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res

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mor

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inte

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ion

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reas

tca

nce

r–

Hu

man

brea

stca

rcin

oma

(MB

-MD

A-2

31)

–3D

Typ

eI

coll

agen

orM

atri

gel

mat

rix

–C

ell

mig

rati

onin

3Dge

lH

uan

get

al.

(200

9)4

1

–M

ouse

mac

roph

age

(RA

W26

4.1)

–3D

co-c

ult

ure

sw

ith

2ce

llty

pes

–E

CM

rem

odel

ing

–B

reas

tca

nce

r–

Hu

man

mam

mar

ygl

and

epit

hel

ial

(MC

F-D

CIS

.com

)–

3DT

ype

Ico

llag

en+M

atri

gel

mix

edge

l–

DC

IStr

ansi

tion

toin

vasi

ved

uct

alca

rcin

oma

Sun

get

al.

(201

1)4

8

–H

um

anm

amm

ary

fibr

obla

st(H

MF)

–3D

co-c

ult

ure

sw

ith

2ce

llty

pes

–Lu

ng

can

cer

–H

um

anlu

ng

fibr

obla

st(M

RC

-5)

–D

irec

tion

alpa

racr

ine

sign

alin

gvi

ad

ynam

icva

lve

con

trol

–T

GF-b

sign

alin

gH

suet

al.

(201

1)3

7

–H

um

anlu

ng

aden

ocar

cin

oma

(CL1

-0)

–M

yofi

brob

last

acti

vati

on

An

giog

enes

is–

Bre

ast

can

cer,

glio

blas

tom

a–

Rat

mam

mar

yad

enoc

arci

nom

a(M

TLn

3)–

3DT

ype

Ico

llag

enm

atri

x–

En

dot

hel

ial

mon

olay

erm

igra

tion

Ch

un

get

al.

(200

9)5

3

–H

um

angl

iobl

asto

ma

(U87

MG

)–

VE

GF

grad

ien

t–

Hu

man

der

mal

mic

rova

scu

lar

end

oth

elia

l(H

MV

EC

)–

Hor

izon

tal

2Dm

onol

ayer

–M

ouse

fibr

obla

st(1

0T1/

2)

–N

/Aa

–H

um

anu

mbi

lica

lco

rden

dot

hel

ial

(HU

VE

C)

–3D

coll

agen

/fib

ron

ecti

nm

atri

x–

Flow

-in

du

ced

angi

ogen

icsp

rou

tin

gSo

ng

and

Mu

nn

(201

1)5

4

–C

ontr

olle

dsh

ear

stre

ssan

din

ters

titi

alfl

ow–

VE

GF

grad

ien

ts–

Hor

izon

tal

and

vert

ical

2Dm

onol

ayer

s

–N

/A–

Hu

man

der

mal

mic

rova

scu

lar

end

oth

elia

l(H

MV

EC

)–

3DT

ype

Ico

llag

enm

atri

x–

An

giog

enic

spro

uti

ng

and

tip

cell

mig

rati

onin

mu

ltip

legr

adie

nts

Shin

etal

.(2

011)

55

–V

EG

Fan

dA

NG

-1gr

adie

nts

–V

erti

cal

2Dm

onol

ayer

–N

/A–

Hu

man

um

bili

cal

cord

end

oth

elia

l(H

UV

EC

)–

3DT

ype

Ico

llag

en+M

atri

gel

mix

edge

l–

An

giog

enic

spro

uti

ng

in3D

co-c

ult

ure

Bis

chel

etal

.(2

012)

56

–M

ouse

fibr

obla

st(1

0T1/

2)–

VE

GF

grad

ien

ts–

Cir

cula

r3D

lum

ens

–N

/A–

Hu

man

um

bili

cal

cord

end

oth

elia

l(H

UV

EC

)–

3DT

ype

Ico

llag

enm

atri

x–

En

dot

hel

ial-p

eric

yte

inte

ract

ion

and

mic

rova

scu

lar

form

atio

n

Zhen

get

al.

(201

2)5

7

–H

um

anbr

ain

vasc

ula

rpe

ricy

tes

–G

ravi

ty-d

rive

npe

rfu

sion

–C

ircu

lar

3Dlu

men

s–

Bar

rier

perm

eabi

lity

–Le

uke

mia

,gl

iobl

asto

ma

–H

um

anle

uke

mia

cell

s(H

L-60

)–

3Dco

llag

en/f

ibri

nm

atri

x–

An

giog

enic

spro

uti

ng

Kim

etal

.(2

013)

58

–H

um

angl

iobl

asto

ma

cell

s(U

87M

G)

–3D

co-c

ult

ure

sbe

twee

nen

dot

hel

ial

cell

s,st

rom

alfi

brob

last

s,pe

ricy

tes

and

can

cer

cell

s–

Perf

usi

onof

mic

robe

ads

thro

ugh

pate

nt

mic

rove

ssel

net

wor

k–

Hu

man

um

bili

cal

cord

end

oth

elia

l(H

UV

EC

)–

Hu

man

plac

enta

peri

cyte

s–

Perf

usi

onin

mic

rove

ssel

s–

Hu

man

lun

gfi

brob

last

s(L

F)

Oxy

gen

Gra

die

nts

–N

/A–

Rat

myo

fibr

obla

sts

(C2C

12)

–2D

mon

ocu

ltu

reof

myo

fibr

obla

sts

onm

icro

elec

trod

es–

Hyp

erox

ia-i

nd

uce

dap

opto

sis

Park

etal

.(2

006)

62

–O

xyge

ngr

adie

nt

byel

ectr

olys

is

–N

/A–

Rat

myo

fibr

obla

sts

(C2C

12)

–2D

mon

ocu

ltu

re–

Oxy

gen

con

sum

ptio

nby

myo

fibr

obla

sts

Meh

taet

al.

(200

7)6

1

–C

onti

nu

ous

perf

usi

onvi

ad

ynam

icva

lve

con

trol

–O

xyge

ngr

adie

nt

bypa

ssiv

eco

nsu

mpt

ion

Tab

le1

(co

nti

nu

ed)


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Tab

le1

(con

tinu

ed)

Can

cer

mod

elC

ell

type

sN

otab

lem

icro

envi

ron

men

tfe

atu

res

Focu

sof

stu

dy

Ref

.

–Lu

ng

can

cer

–C

arci

nom

ich

um

anal

veol

arba

sal

epit

hel

ial

cell

s(A

549)

–2D

mon

ocu

ltu

re–

Oxy

gen

grad

ien

tby

oxyg

en-g

ener

atin

gan

dox

ygen

-sca

ven

gin

gre

acti

ons

–H

yper

oxia

-in

du

ced

apop

tosi

s–

Hyp

oxia

-in

du

ced

cyto

toxi

city

byti

rapa

zam

ine

Ch

enet

al.

(201

1)6

3

Inva

sion

and

Met

asta

sis

–B

reas

tca

nce

r–

Hu

man

brea

stca

nce

r(M

CF-

7;n

on-m

etas

tati

c)–

2Dm

onoc

ult

ure

–C

ell

mig

rati

on,

inva

sion

thro

ugh

mic

roga

psC

haw

etal

.(2

007a

)70

–H

um

anbr

east

carc

inom

a(M

B-M

DA

-231

)–

3Dm

atri

gel

mat

rix

coat

ing

inm

icro

gaps

–B

reas

tca

nce

r–

Hu

man

brea

stca

rcin

oma

(MD

A-M

B-4

35S)

–2D

mon

ocu

ltu

re–

Cel

lm

igra

tion

and

inva

sion

thro

ugh

mic

roga

ps

Ch

awet

al.

(200

7b)6

9

–Li

ver

can

cer

–H

um

anli

ver

carc

inom

a(H

epG

-2)

–3D

mat

rige

lm

atri

xco

atin

gin

mic

roga

ps–

Cer

vica

lca

nce

r–

Hu

man

cerv

ical

can

cer

(HeL

a)–

Subc

onfl

uen

ten

dot

hel

ial

lin

ing

acro

ssm

icro

gaps

–H

um

anm

icro

vasc

ula

ren

dot

hel

ial

(HM

EC

)

–B

reas

tca

nce

r–

Hu

man

brea

stca

rcin

oma

(MD

A-2

31)

–3D

hyd

roge

lm

atri

x–

Tu

mor

–en

dot

hel

ial

inte

ract

ion

san

d3D

intr

avas

atio

n

Zerv

anto

nak

iset

al.

(201

2)7

1

–Fi

brob

last

icsa

rcom

a–

Hu

man

fibr

osar

com

a(H

T10

80)

–3D

cocu

ltu

res

betw

een

end

oth

elia

lce

lls,

mon

ocyt

esan

dca

nce

rce

lls

–H

um

anu

mbi

lica

lco

rden

dot

hel

ial

(HU

VE

C)

–H

um

anm

icro

vasc

ula

ren

dot

hel

ial

(MV

EC

)–

Hor

izon

tal

and

vert

ical

2Dm

onol

ayer

s–

Mou

sem

onoc

yte

(RA

W26

4.7)

Com

bin

atio

n–

Col

onca

nce

r–

Hu

man

colo

nca

rcin

oma

(LS1

74T

)–

Lon

g-te

rmsp

her

oid

cult

ure

(4d

)–

Com

bin

edsp

her

oid

sw

ith

grad

ien

tsW

alsh

etal

.(2

008)

60

–D

iffu

sed

grad

ien

tsof

dox

oru

bici

n

–Sa

liva

rygl

and

aden

oid

cyst

icca

rcin

oma

–H

um

anad

enoi

dcy

stic

carc

inom

a(A

CC

-M)

–3D

mat

rix

subs

titu

te(b

asem

ent

mem

bran

eex

trac

t)–

CA

F-m

edia

ted

tum

orce

llin

vasi

onLi

uet

al.

(201

0)3

5

–H

um

anem

bryo

nic

lun

gfi

brob

last

(HFL

-1)

–Pr

imar

yis

olat

edca

rcin

oma-

asso

ciat

edfi

brob

last

s(C

AFs

)–

3Dsp

her

oid

sin

co-c

ult

ure

wit

hn

eigh

bori

ng

CA

Fs–

Com

bin

edsp

her

oid

sw

ith

stro

mal

inte

ract

ion

–B

reas

tca

nce

r–

Mou

sem

amm

ary

tum

or(4

T1)

–3D

Mat

rige

lm

atri

x–

Hyp

oxia

-dep

end

ent

mig

rati

onof

tum

oran

den

dot

hel

ial

cell

s

Gao

etal

.(2

011)

40

–H

um

and

erm

alm

icro

vasc

ula

ren

dot

hel

ial

(HD

ME

C)

–C

onst

ant

med

iape

rfu

sion

–In

du

ced

hyp

oxia

byC

oCl 2

–A

ttem

pted

mod

elof

com

bin

edh

ypox

iaan

dan

giog

enes

is

aN

/A–

not

appl

icab

leor

not

spec

ifie

d.

Tab

le1

(co

nti

nu

ed)


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can be incorporated to create additional channel compartmentsthat permit indirect perfusion, which has been applied to yieldformation of coculture spheroids with as many as three celltypes to model tumor heterogeneity within metastatic prostatecancer (Fig. 1a).33 Besides providing perfusion and physiologicalflow,32 microfluidic systems offer the potential for more advancedin vitro models of the microenvironment, where spheroids can beembedded within a 3D matrix,34 and can even support the cultureof surrounding carcinoma-associated fibroblasts (CAFs) to allowstudies on tumor–stromal interactions.35 These examples offerevidence that we are beginning to witness more integration oftumor spheroid cultures into microfluidic systems.

Tumor–stromal interactions

A large body of work within the area of microfluidic systems hasfocused on leveraging the high spatial resolution afforded bymicrofabrication techniques to create precise structures andarrangements for more complex in vitro coculture models tostudy tumor–stromal interactions. Coculture models havetraditionally been achieved by (i) placing one cell type in directphysical contact to the second cell type, with one forming amonolayer of underlying ‘‘feeder’’ cells, or (ii) using Transwellmembrane inserts to compartmentalize two cell types whileallowing soluble factor signaling through the membrane. WhileTranswell inserts are useful for decoupling paracrine andmechanical signals that are coupled in cultures having directcell–cell contacts, Transwell-based assays are static, are limitedto two compartments, and the typical culture volumes anddiffusion distances of these assays often lead to significantdilution of factors, and loss of effectiveness of factors withshort half-lives. Microfluidic systems, even with the most basicdesigns, can achieve improved spatial organization, increasedlevel of compartmentalization, and more controlled diffusion offactors than Transwell inserts, leading to improved sensitivity,36

and importantly, more control over the dynamics of the micro-environment, including fluid flow. Examples of microfluidiccoculture models for tumor–stromal interactions are quitevaried, and include: lung cancer37 and prostate cancer cells38

cocultured with myofibroblasts; cervical cancer cells withfibroblasts;39 breast cancer cells with either endothelial cells40

or macrophages;41 and for hematologic cancers in the bonemarrow microenvironment, multiple myeloma cells with bonemarrow stromal cells.42 While these examples demonstrateimproved compartmentalization, including a case with threeseparated cell types,43 as well as improved dynamic control viamicrovalves,37 the majority (except for ref. 41) have focused onparacrine signaling at the cellular level in 2D, neglecting theinfluence of dimensionality and 3D cell–matrix interactions atthe tissue and organ levels. Nevertheless, microfluidic coculturesystems offer an alternative to existing coculture assays withadvantages that motivate continued interest in this area.

ECM and three-dimensionality

The addition of ECM components to create a 3D structuralframework for cells in culture adds mechanical context anddimensionality to the in vitro microenvironment. While 2D

compartmentalized cocultures are advantageous because theyare simple to use, have straightforward readouts, and areamenable to high-throughput applications, there is little doubtas to the importance of the third dimension in culture,26

and the role of mechanobiology in tumorigenesis.44,45 Tumorgrowth is associated with biomechanical alterations in themicroenvironment, including increased solid stresses in theneighboring tissue, increased matrix stiffness, and aberrantinterstitial fluid flow.46 In addition, matrix remodeling andother mechanobiological cues affect the invasiveness and meta-static potential of tumor cells.47 Tumor spheroids, discussedabove, is one approach to achieve three-dimensionality, but themorphology of spheroids does not necessarily apply to alltumor microenvironments, nor does it represent the optimalform for testing and experimentation. Various microfluidicsystems have incorporated 3D matrix components and hydrogelsinto compartments, and thus advanced 2D cells-on-chip technol-ogy toward 3D tissues-on-chip. By incorporating collagen-basedgels, cancer cell invasion can be monitored in 3D, and matrixremodeling can be visualized by second harmonic generationimaging of the changing collagen structure.41 This can now beachieved quite easily with pipette-based passive pumping into asimple Y-shaped microchannel (Fig. 1b).48 The gel has the capa-city to support stromal cells such as mammary fibroblasts, andadditional microchannel geometries can also be incorporated tomodify diffusion distances, allowing greater spatial controlbetween cell types, even in 3D. Given the mounting evidencetoward the importance of 3D culture, and the continued advance-ment of technology to facilitate its adoption, future microfluidicsystems will likely shift even more toward 3D cultures.

Angiogenesis

One feature of the tumor microenvironment that requiresspecialized 3D tissue structure beyond the simple addition ofa third dimension is the network of angiogenic blood vesselsthat supply oxygen and nutrients to the tumor. While stromalfibroblasts and immune cells can be simply embedded into 3Dgels by re-suspending in the matrix solution prior to loading,endothelial cells must form confluent monolayers, withappropriate formation of cell–cell junctions and basementmembrane layers, in tubular lumen structures in order toresemble the actual structure of blood vessels. 2D endothelialmonolayers have been popular for decades for studying flow-induced shear stress on vascular endothelial cells. Thisapproach has been applied to enable shear stress studies onendothelial cells in microfluidic systems that promised toincrease throughput, lower reagent consumption, and reducesample quantities.49,50 Recently, microfluidic cell culturesystems have advanced toward endothelial-lined blood vesselmimics that are potentially useful for cancer-related angiogen-esis studies.51 Many of these microsystems employed simple 2Dendothelial monolayers as before, but in a vertical orientationto allow visualization of horizontal angiogenic vessel growthinto hydrogels.52–55 One design in particular superposed VEGFgradients and shear flow over the vertical monolayers to studyflow-induced angiogenic sprouting.54 To create more advanced


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3D lumens with cylindrical cross-section, a simple viscousfingering method via micropipetting was employed in onedesign,56 while an elaborate microfluidic assembly was con-structed in another design to house a slab of collagen gel withmolded microfeatures for the seeding and remodeling ofendothelial-lined microvessels.57 Interestingly, by embeddingthe collagen gel with pericytes in this latter design, endothelialcells were able to recruit pericytes to the microvessel walls, andan appropriate basal lamina between pericytes and endothelialcells was allowed to form. This was also accompanied byangiogenic sprouting into the collagen gel slab. Altogether, thisexample demonstrates how complex interplay between distinctmicroenvironmental cues can lead to natural remodeling of amicrovessel network that displays many in vivo-like characteri-stics, some of which can be applied to the engineering of atumor microenvironment. Others have since been able to createfull angiogenic microvessel networks in a 3D chip (Fig. 1c) thatcan be cocultured with different cell types and be perfused withmicrobeads,58 showing further advancement in complexity formodels involving angiogenesis.

Gradients

In addition to aspects of the microenvironment related tocellular organization, dimensionality, ECM architecture, andmorphological properties, microfluidic systems are also naturallywell suited for generating and maintaining spatiotemporal gradi-ents of biochemical and physicochemical components. Becauseof well-recognized laminar flow characteristics in low-Reynoldsnumber microchannel flows, microfluidic gradient generatorswere one of the first and most popular types of systems developedin the field.59 Several microfluidic angiogenesis systems haveemployed gradients of VEGF to induce sprouting,53,54,56 includingone in particular that used a combination of VEGF and ANG-1gradients to examine cooperative effects between the two solublefactors.55 Furthermore, it is possible to superpose gradients ontumor spheroids to further add complexity to the spheroidmodel.60 Aside from biochemical gradients, the control of oxygengradients have also been demonstrated as a way to model hypoxicconditions within the tumor. Oxygen gradients can either beproduced passively by allowing oxygen consumption by cellscultured in the microchannels,61 or actively by controlling waterelectrolysis via patterned electrodes,62 or by oxygen-generatingand oxygen-scavenging chemical reactions (Fig. 1d), the latter ofwhich was used to investigate oxygen tension on alveolar basalcarcinoma cells.63 The power of microfluidics will become evenmore evident as we continue to create more complex micro-environments through superposition of multiple gradients, aswell as other microenvironment factors.

Metastases

While the focus of this section is to highlight advances specificto the tumor microenvironment, it should be noted thatmicrofluidic systems have also been valuable for studies relatedto invasion, intravasation, extravasation, and metastasis, stagesof tumor progression where the events have begun to differ-entiate, or are differentiated, from the primary tumor region.

Steps of the invasion-metastasis cascade have thus far beenchallenging to examine in detail with in vitro systems, withTranswell inserts again being a popular choice as an invasionassay.64,65 Metastasis studies have almost exclusively beenconducted with in vivo mouse models because of the lack ofin vitro methods.66–68 Recently, a few microfluidic systems weredeveloped to study specific dynamic events within the metastasiscascade, including migration through gels and transmigrationthrough subconfluent endothelial linings,69,70 and real-timeimaging of invasion and extravasation steps with insight intotumor cell–endothelial cell interactions (Fig. 1e).71 Given theenormous challenges that remain in elucidating mechanismsof metastasis, these examples are likely just the beginning formicrofluidic metastasis systems, with much research needed tofurther improve models.

In summary, numerous studies of microfluidic systems,from cells-on-chips to tissues-on-chips, have been performedto date to tackle one specific aspect of the tumor micro-environment. Only a few systems have attempted to tackle morethan one aspect in the same device (Table 1, ‘‘Combination’’).35,40,60

Much research is necessary to further advance cells-, tissues-,and organs-on-chips toward even more complex micro-environments. Doing so will likely allow us to pursue our goalof acquiring more knowledge and answering more outstandingquestions in cancer research.

4. Cancer hallmarks: longstandingchallenges meet new opportunities

Our understanding of the biology of cancer has been crystal-lizing gradually over the years with the recognition that amidstall its complexity, cancer can be described in terms of basicunderlying principles that revolve around six essential acquiredcapabilities of cancer cells. These so-called ‘‘hallmarks of cancer’’,namely (i) the self-sufficiency in growth signals, (ii) insensitivity toanti-growth signals, (iii) ability to evade apoptosis, (iv) sustainedangiogenesis, (v) limitless replicative potential, and (vi) tissueinvasion and metastasis, were first summarized in 2000,72

and then subsequently revisited a decade later in an updatedreview in 2011.15 The two landmark reviews have not onlyserved as a guide to cancer research, but have also providedtwo mileposts on the cancer research timeline that offers avaluable perspective of the progress made in the interveningyears. Since 2000, new insights on cancer stem cells have beendiscovered; the importance of tumor heterogeneity has beenrecognized; major progress has been made in understandingEMT, invasion, and metastasis; and two new ‘‘emerging hall-marks’’ and ‘‘enabling characteristics’’ have been identified.15

Of particular relevance to this review is the clearly emergentrole of the tumor microenvironment, a topic which seemed topervade the discussion across all the different hallmarks. Thefact that these recognized hallmarks are essential for tumordevelopment, yet remain inadequately understood, providesmajor challenges on one hand, but may also present newresearch opportunities on the other. To provide a glimpse of


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the types of opportunities that may be available, a brief journeyis made through some outstanding problems associated withthese well-known hallmarks.

The ability of cancer cells to simultaneously (i) sustain theirown growth and proliferation, (ii) evade anti-growth signals,and (iii) resist cell death, three distinct yet connected acquiredhallmarks of cancer, demonstrates their commitment to breakthe delicate balance of cell population demographics that iscrucial to homeostasis. Notably, such population demo-graphics, which encompass different cell types, their numbers,and their locations, are important to the spatial and temporalpatterns of regulatory signals governing proliferation, apoptosisand necrosis, and vice versa. The fact that our knowledge andunderstanding of the precise signals, their interactions, and themechanisms regulating those interactions remains incompletemay be attributed to difficulties in reconstructing and testingin vitro cellular microenvironments that capture the properdemographics found in vivo. Furthermore, it is suggested thatthe phenomenon of contact inhibition commonly observed in2D culture may be related to mechanisms in vivo that controlcell numbers. These problems may be difficult to address withconventional cultures, but the ability to use 3D cultures withimproved spatial patterning of cell populations in microfluidicsystems provides a method that can potentially resolve thesecomplex interactions, and shed light on the importance ofpopulation demographics.

The acquired capability of cancer cells to avoid senescenceand effectively become immortalized with ‘‘limitless replicativepotential’’ may involve molecular factors of the tumor milieuthat have yet to be fully identified. Experimentally, it has alsobeen suggested that culture conditions may be tuned in amanner that can influence the onset of cell senescencein vitro.15 Microscale systems can be applied either (i) to createtumor microenvironments suitable for testing different bio-chemical factors on normal cells in an accurate physiologiccontext and measuring their replicative potential, possibly bymonitoring cell divisions within the engineered micro-environments, or (ii) to test various culture conditions withdifferent microchannel dimensions, media components, serumand glucose levels, in a high-throughput, physiologically rele-vant manner to elucidate how culture conditions affect cellsenescence. In both cases, the flexibility of microfluidic systemscan be leveraged to help elucidate the role of the micro-environment, and the culture conditions within it.

One cancer hallmark that has already received considerableattention in the microfluidics community is angiogenesis.Paradoxically, in vitro models of angiogenesis are becomingincreasingly sophisticated in some cases,57 yet also becomingmore routine and accessible in others.56 Both of these 3Dcoculture models have potential to elucidate important rolesfor stromal and perivascular cells in angiogenic sprouting.Given that the proper microvascular structure composed ofprecise apposition between endothelial cells and pericytes canbe faithfully recapitulated in vitro as demonstrated, new ques-tions can be posed regarding the effect of pericytes on endo-thelial barrier function, leakiness, and the susceptibility of the

vessel wall to intravasation and extravasation. Real-time moni-toring of engineered angiogenesis systems may allow us to tracethe origin, progression, and/or transdifferentiation of normalendothelial cells to tumor-associated endothelial cells, or trackthe interactions between endothelial tip and stalk cells withinangiogenic sprouts. As more complex in vitro models become areality, it may be possible to combine tumor spheroids andangiogenic microvessels on the same microchip, allowing thesimultaneous monitoring of tumor growth and neovascularrecruitment, and investigations into vascular normalization atthe cellular level.73

While much progress has been made over the past decade inunderstanding invasion and metastasis, it is notable that manychallenging questions remain unanswered.15 Appropriately,these questions present interesting opportunities for micro-fluidic systems to serve as novel experimental platforms thatcan offer fresh perspectives on the multistep metastatic pro-cess. For example, paracrine signals from stromal cells of thetumor microenvironment may be involved in activating a panelof transcription factors that coordinate EMT. Moreover, aspatial pattern exists where cancer cells that have undergoneEMT appear more frequently near invasive margins of thetumor rather than near the tumor core, suggesting that spatialposition and the local microenvironment are important factorsof the EMT process. In both cases, spatial control offered bymicrofluidic systems may be exploited. Since dissemination oftumor cells from the primary site often occurs via the lymphaticsystem, research is also needed to create in vitro models oflymphatic vessels for potential application in studies of lymphaticmetastasis. Additionally, a major phase within metastasis thatis particularly fraught with experimental challenges is coloniza-tion, the development of micrometastases into secondarytumors. Interesting and unanswered questions related to dormancyand mechanisms of the seed-soil hypothesis must inherentlyinvolve both the seed (i.e., circulating tumor cell (CTC) originat-ing from the primary tumor) and the soil (i.e., the tissue bed ofthe distant organ on which the CTC settles). While isolationand handling of CTCs may be the subject of other microfluidicmanipulations,74,75 development of engineered tissue beds thatserve as the soil in colonization experiments may be attainablewith advances in organ-on-chip technologies. This work willlikely be essential to the successful modeling of metastaticcolonization in vitro.

An important topic that has been identified as both anemerging hallmark and an enabling characteristic of canceris inflammation.15 The incorporation of inflammatory cells intoin vitro microfluidic coculture systems was reviewed for severalexamples involving macrophages (Table 1), providing a glimpseof the potential utility of biochips to understanding tumor–stromal interactions involving other inflammatory cell typessuch as neutrophils and lymphocytes. In fact, other micro-fluidic systems developed to study neutrophil migration forother diseases76 can potentially be modified for cancer studies.Using these systems, the signals between tumor cells andimmune cells can be monitored in real-time, and used toelucidate the temporal dynamics of the inflammatory response.


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The other emerging hallmark is the ability for cancer cells toreprogram metabolism, and this is an area that can benefitsignificantly from the application of microfluidic systems.Much research is currently underway on the development ofanalytical methods for metabolomics in microfluidic systems,with the goal of being able to sample, detect, and measurequantities of metabolites in high throughput.77–79 Within thesesystems, the front-end cultures are so far basic cell cultureswithout elaborate microenvironment features, but potentialdefinitely exists to incorporate more sophisticated physiologicmodels that can then be sampled downstream for metabolitesin the sample media. Since the most commonly used materialfor microfluidic devices is poly(dimethylsiloxane) (PDMS), andit is well-known to absorb hydrophobic molecules from themedia,80 other device materials such as polystyrene (PS) andother thermoplastics may need to be considered for any studyinvolving the measure of metabolites. In addition, recentresearch has shown that cells in microculture are underincreased cell stress due to metabolic constraints,81 indicatingthat improved understanding of cellular metabolism in micro-fluidic cultures is fundamentally important to the future micro-environment studies on the effects of metabolism in cancer.

5. Remaining technical challenges

While many outstanding questions await cancer researchers, asoutlined above, it should be emphasized that outstandingchallenges also remain for microfluidic technologists. Someof these challenges are more technical, and apply to micro-fluidic technologies in general, while others are more prag-matic, and apply specifically to chips designed for cancer tumormicroenvironments. Regardless, these challenges must first beovercome at the technology end, if biochips are to realize theirfull potential in providing a new and useful platform forresearch in cancer and the tumor microenvironment. Thefollowing discussion aims to synthesize the various challengesfacing engineers, biologists, and those working at the interface,and importantly start the dialogue needed to further advancethe application of biochip technologies.

Starting from a technical standpoint, what remains at thecore of engineered microsystems is microfabrication. Con-tinued research in microfabrication methods is necessary forfuture advancements in biochips, and it simply cannot beneglected. The fact that soft lithography and PDMS offer manymore pros than cons has been demonstrated time and again,but this fact should not limit us from pursuing innovativefabrication methods that may be faster, cheaper, more reliable,and/or more amenable to high volume manufacturing.Although polystyrene (PS) offers familiarity to biologists, andis gaining recognition in the microfluidics world,82,83 PS has itsshortcomings as well, as its high modulus of elasticity pre-cludes its use in applications that require deformations in thedevice structure, such as for a breathing lung-on-a-chip.4 Never-theless, more research on how to fabricate microdevices in PSand other thermoplastics will still be valuable, both to serve asa practical alternative to PDMS in academic research, and to

offer insights on how to potentially enable mass manufacturingcapabilities for future commercialization. Besides selection ofdevice material, other technical issues such as throughput,world-to-chip interfacing, and the cost-benefit ratio of inte-grating additional functionality are engineering challenges thatmust be considered for each specific application, and will nodoubt be an important research focus if organs-on-chip plat-forms, particularly for cancer drug screening and personalizedmedicine, are to become a reality.

While engineers will need to commit some effort to advan-cing device fabrication, they must also commit to designing for(i) user operability and (ii) endpoint measurements, aspectsthat are likely of greater concern to the biologists and clinicianswho will actually be using the tools to acquire useful data. Themajority of the microfluidic systems reviewed here were proof-of-concept designs, many with primitive prototyping setupsthat do not translate immediately and conveniently into biologylaboratories. Operability may not be a high priority duringprototyping and proof-of-concept stages, but because itbecomes critical during translation and adoption stages, it isprudent to design operability directly into the system as early aspossible during development.

Also, more research is needed toward more endpointmeasurements. The majority of microsystems discussed so farhave relied heavily on microscopy to acquire endpoint readouts.This is reasonable and desirable given that direct visualization ofcells and their behavior, perhaps with different dyes and labels,offers the most reliable and direct proof that cells are viable andfunctioning as expected in these systems. Furthermore, func-tional readouts such as the number and length of angiogenicsprouts, or the migration rates of cells or monolayer fronts, canonly be detected and measured via microscopy. However, muchinformation at the molecular level that cannot be acquired bymicroscopy has yet to be uncovered in many microfluidic cellculture systems. Some microscale platforms allow cell lysis sothat contents can be collected for qRT-PCR and western blottingoff-chip;38,84,85 and a combination of immunocytochemicalstaining and protein localization analysis can be employed toquantify nuclear translocation as a substitute for electrophoreticmobility shift assays (EMSAs) used to study transcription factoractivation;42 Moreover, other platforms have achieved single-cellresolution qPCR performed completely on-chip.86 However,it remains a challenge to harvest cells cultured from micro-fluidic systems with more complex 3D microenvironments. Forexample, it would be useful and powerful to have the ability toisolate and independently harvest the endothelial cells fromangiogenic vessels without contaminating the sample withneighboring cocultured stromal cells, but this has yet to bedemonstrated. In addition, an ongoing challenge has been todetect and quantify secreted factors from microcultures. While thishas been shown as proof-of-concept with simple microcultures,87,88

more research is necessary to demonstrate this for complextumor microenvironments.

The interdisciplinary nature of our current discussionmeans inherently that the challenge not only exists from atechnical perspective, but also from a pragmatic perspective.


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Even if the technical hurdles are overcome, many questionsawait regarding the application of the technology in cancerresearch. For example, assuming the technology has reached alevel of maturity and robustness sufficient for serious applica-tions in biology beyond the proof-of-concept, what tumors andtumor microenvironments are the most interesting and imme-diately important to develop in vitro? Lung, breast, prostate,colon, and liver cancers appear to be excellent candidates tostart given that they have the highest mortality rates, and alsobecause lung and gut-on-a-chip devices have already beenreported.4,5 The bone marrow microenvironment is also ofinterest because, as mentioned above, it is responsible forproviding the niche within which the class of hematologiccancers are developed. Furthermore, the bone microenvironmentalso serves as a major site for metastases of the lung, breast,and prostate,89 and thus will be an important model for on-chipmetastasis studies.

Another question is how much complexity is required alongthe cell-, tissue-, and organ-level spectrum to yield useful,functional readouts that can offer new insights in humandiseases like cancer. There is clearly a compromise betweenhaving more complex physiologic microenvironments withmore convoluted interactions and less straightforward readoutsversus less physiologic context, and thus less accuracy, in themicroenvironment model, but more control over interactionsand more basic but similarly useful readouts. This question canonly be addressed by rigorous validation and comparisonbetween in vitro systems offering varying levels of complexity,in addition to comparisons with in vivo animal models thatserve as positive controls.

Regardless of physiologic complexity, the basic units of anybiochip are the basic units of life itself: the cells. While themajority of microdevices have demonstrated feasibility usingimmortalized cell lines or commercially available primary cellslike HUVECs, the promise that organs-on-chips can signifi-cantly improve drug screens and provide a future involvingpersonalized medicine depends on our ability to integrate intothe chip technologies primary human cells obtained directlyfrom individuals. This issue, and the others described before it,may not be specific to cancer research, but they should beemphasized for their importance to the successful technologicaladvancement of these systems for cancer research, and otherresearch areas.

Summary

The ability for cells-, tissues-, and organs-on-chips to recapitulatecomplex and controlled microenvironments enables them tooffer new and innovative approaches to cancer research and thestudy of the role of the tumor microenvironment. As evidencedby the extensive literature already amassed in the last severalyears on microfluidic systems for tumor microenvironmentstudies, the field is growing at a remarkable rate, and keepingpace is a challenge itself. Both cancer research and micro-fluidics technology development continue to face numerouschallenges, but the intersection of these fields provides a major

opportunity for significant impact in both areas, with potentialfor scientific breakthroughs. The impact of organs-on-chips oncancer research may not be apparent in the next few years, butif the current rate of progress is any indication, interestingdevelopments are certainly on the horizon.

Acknowledgements

The author acknowledges financial support from the NaturalSciences and Engineering Research Council of Canada (NSERC)through a Discovery Grant. The author also apologizes for anyunintended omissions of pertinent literature in the currentdiscussion.

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cells, tissues, and organs on chips challenges and opportunities for the cancer tumor...

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