Human lung-on-a-chip technology is used to study behavior and drug responses of lung cancer in its natural environment, uncovering key mechanical dependencies
By Benjamin Boettner
(BOSTON) — Cancer researchers have come to understand that generating human tumors in mice by injecting cancer cell lines under the skin does not recapitulate how tumors normally emerge and spread to specific organs in the human body, nor how they respond to anti-cancer drugs. So, they turned to injecting tumor cells at the organ sites where they originated from in humans, so-called ‘orthotopic’ sites. Orthotopic tumors, such as those created by injecting breast cancers into the mammary fat pads of mice, exhibit growth and metastatic behaviors more like those seen in patients, however, these organ environments are still not human. It is also not possible to visualize how tumor cells grow, move and respond to therapeutics in these orthotopic animal models, which restricts our ability to understand how different organ microenvironments influence tumor behavior and thereby develop better drugs.
As reported in Cell Reports, a team at the Wyss Institute for Biologically Inspired Engineering led by Founding Director and Wyss Core Faculty member Donald Ingber, now has leveraged its human Organs-on-Chips technology to confront this challenge. In previous work, the team successfully modelled two different regions of the lung — the air-conducting small airway and the oxygen and carbon dioxide-exchanging alveoli at the tips of the small airways — in microfluidic devices that are created with microchip manufacturing methods. The different lung cells inhabit one of two microchannels that run parallel through the chip, separated by a thin porous membrane from a microvessel lined by human lung endothelium in the second channel. Like in the human lung, the resulting small airway epithelium is thicker, stiffer and covered with moving cilia, while the thinner alveolar epithelium is more permeable to enable efficient gas exchange and it is exposed to cyclic mechanical deformations to mimic breathing motions in the chip. The researchers continuously stream cell culture medium through the vascular channel to support the epithelial and endothelial cell layers over many weeks as occurs with blood-flow in living lung. In addition to having engineered the basic tissue architecture and functionalities of these two lung regions on chips, the team previously showed that they can successfully model lung diseases, including chronic obstructive pulmonary disease (COPD), asthma and pulmonary edema.
In this new study, the team developed human orthotopic lung cancer models using these two lung chips. Approximately 85% of all lung cancers are diagnosed as non-small cell lung cancer (NSCLC), and the team focused on the adenocarcinoma form of this cancer which roughly accounts for 40% of all NSCLCs. In the human body, NSCLC adenocarcinoma cells are known to arise at the interface between the lung’s small airways and alveoli, but the tumor then primarily grows within the alveolar structures.
Ingber’s team showed that when NSCLC adenocarcinoma cells are grown in the Lung Airway and Alveolus Chips, the tumor cells grow rampantly in the microengineered alveolar microenvironment whereas they remain quiescent in the Airway Chip, just as is observed in human patients. “Our lung cancer-on-chip platforms can model central aspects of orthotopic NSCLC in real time and high-resolution, and much more closely than other in vivo and in vitro approaches. They offer a literal window on the biological tumor complexities,” said Ingber. Ingber also is the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).
To achieve this, the researchers developed co-plating and injection strategies that enabled them to stably integrate a small number of NSCLC cells into the two lung chips. “This approach allows us to recreate key hallmarks of this cancer, including its growth and invasion patterns, and to determine how they are influenced by cues from surrounding normal cells. In the Airway Cancer Chips, cancer cells remained dormant for up to 12 days before they started to grow, while in Alveolar Cancer Chips they commence their growth much more rapidly, and once they reach a critical mass, they separate themselves and invade the endothelium as part of their metastatic process,” said first author Bryan Hassell, Ph.D., who as a SEAS Graduate Student on Ingber’s team developed the lung cancer-on-a-chip platform.
The team next asked whether physiological breathing motions within alveoli might affect cancer cell behaviors. Suprisingly, they discovered that when cyclic mechanical forces are applied to the lung epithelial channel to mimic breathing motions, both cancer cell growth and invasion were potently inhibited. “This is the first time that a clear impact of cyclical breathing motions on cancer cell growth and invasion has been demonstrated in any in vitro system modeling NCSLC behaviors,” said Hassell. The researchers think that when lung cancer cells grow and fill patients’ lung alveoli, this interferes with their natural motions, which in turn could speed up tumor growth and facilitate invasive behavior.
Finally, they took their approach another step further by investigating whether breathing mechanics could affect the sensitivity of NSCLC cells to a clinically used anti-cancer drug, known as a tyrosine kinase inhibitor (TKI). TKIs target frequently mutated enzymes, like the so-called epidermal growth factor receptor (EGFR), which unleashes unfettered growth in NSCLC and other cancers. Because early-generation TKIs can lose their efficiencies when cancer cells become resistant to them by producing new EGFR variants, cancer researchers are trying to design ever smarter TKIs. However, the constantly morphing cancer cells learn to deal even with those, by genetically rewiring themselves so that they can activate alternative cancer mechanisms.
Importantly, Ingber’s team found that in the Alveolar Cancer Chip, NSCLC cells that are already resistant against a first-generation TKI can still be stopped cold in their tracks by a third-generation TKI in the absence of breathing motions, as might occur within large tumors that fill the lung’s alveoli and stops their motion. However, in breathing mode, they become impervious to the drug and continue to survive and grow slowly, essentially creating cancer ‘persister’ cells that are known to be the nemesis of cancer therapy.
The researchers also observed that the levels of cytokines, proteins involved in cell communication that are produced by epithelial, endothelial, and cancer cells, and known to be important prognostic indicators for NSCLC, reflect cancer growth in the Alveolus Cancer Chip and are modulated by breathing motions and drug treatment. “The effects of breathing motions on cancer cell behavior in our models could explain how tumor cells, which remain from a shrinking tumor after therapy, could become persister cells, able to defy drug therapy, linger and eventually cause the cancer to relapse. Our orthotopic in vitro platform thus could be well-suited for dissecting how these persister cells arise, and they may be a useful tool in future drug development efforts that aim to eradicate them,” said Ingber.
Other authors on the study include Wyss Institute Associate Faculty member Christopher Chen, Ph.D., Wyss Postdoctoral Fellows Girja Goyal, Alexandra Sontheimer-Phelps, Ph.D., and Esak Lee, Ph.D., who is mentored by Chen and Ingber, as well as Wyss Staff Scientist Oren Levy, Ph.D. The study was funded by the Wyss Institute for Biologically Inspired Engineering at Harvard University, Defense Advanced Research Projects Agency (DARPA), National Institutes of Health (NIH), and fellowships from the International Foundation for Ethical Research (IFER) and the Lymphatic Education and Research Network to Hassell and Lee, respectively.
Teknolojia ya mapafu ya binadamu kwenye chip hutumiwa kusoma tabia na majibu ya dawa ya saratani ya mapafu katika mazingira yake ya asili, kufunua utegemezi muhimu wa mitambo. - Benjamin Boettner, PhD, Mtaalamu wa Saratani: Watafiti wa saratani wamegundua kwamba kuzalisha uvimbe wa binadamu katika panya kwa kuingiza mistari ya seli za saratani chini ya ngozi haionyeshi jinsi uvimbe unavyoibuka na kuenea kwa viungo maalum katika mwili wa binadamu. Kwa hiyo, walianza kuingiza chembe za uvimbe katika viungo ambavyo vilitokana na viungo vya wanadamu, vinavyoitwa "vituo vya orthotopic". Tumors za orthotomy, kama vile zile zilizotengenezwa kwa kuingiza saratani ya matiti katika pads ya mafuta ya mama ya panya, zinaonyesha ukuaji na tabia za metastatic zaidi kama zile zilizoonekana kwa wagonjwa, hata hivyo, mazingira haya ya viungo bado si ya binadamu. Pia haiwezekani kuona jinsi seli za uvimbe zinavyoongezeka, kusonga na kujibu matibabu katika mifano hii ya wanyama ya orthotopic, ambayo inazuia uwezo wetu wa kuelewa jinsi mazingira tofauti ya viungo vinavyoathiri tabia ya uvimbe na hivyo kukuza dawa bora. "Kama ilivyoelezwa katika Ripoti za Seli, timu ya Taasisi ya Wyss ya Uhandisi wa Kuchochewa na Biolojia iliyoongozwa na Mkurugenzi wa Mwanzilishi na mwanachama wa Kitivo cha Wyss Donald Ingber sasa imetumia teknolojia yake ya ""viungo vya binadamu kwenye chips"" kukabiliana na changamoto hii." Katika kazi ya awali, timu hiyo ilifanikiwa kuunda maeneo mawili tofauti ya mapafu - njia ndogo ya hewa na alveoli ya kubadilishana oksijeni na kaboni dioksidi kwenye ncha za njia ndogo za hewa - katika vifaa vya microfluidic vinavyoundwa na mbinu za utengenezaji wa microchip. Chembe za mapafu huishi katika moja ya microchannels mbili ambazo zinaendesha sambamba kupitia chip, kutenganishwa na membrane nyembamba porous kutoka microvascular lined na endothelium ya mapafu ya binadamu katika channel ya pili. Kama ilivyo katika mapafu ya binadamu, epithelium ndogo ya njia ya hewa ni nene, ngumu na imefunikwa na cilia inayoweza kuhamishwa, wakati epithelium nyembamba ya alveolar ni rahisi zaidi kuwezesha kubadilishana kwa gesi na inaonyeshwa kwa deformations ya mitambo ya mzunguko ili kuiga harakati za kupumua. Watafiti wanaendelea kusambaza chembe za kukuza kupitia njia ya mishipa ya damu ili kuunga mkono tabaka za seli za epithelial na endothelial kwa wiki nyingi kama inavyotokea na mtiririko wa damu katika mapafu ya hai. Pamoja na kuunda usanifu wa msingi wa tishu na utendaji wa maeneo haya mawili ya mapafu kwenye chips, timu hiyo ilionyesha hapo awali kuwa wanaweza kuunda kwa mafanikio magonjwa ya mapafu, pamoja na ugonjwa wa mapafu wa kudumu (COPD), pumu na edema ya mapafu. Katika utafiti huu mpya, timu hiyo ilitengeneza mifano ya kansa ya mapafu ya binadamu kwa kutumia vipande hivi viwili vya mapafu. Karibu asilimia 85 ya saratani zote za mapafu huchukuliwa kuwa saratani ya mapafu isiyo ya seli ndogo (NSCLC), na timu hiyo ililenga aina ya adenocarcinoma ya saratani hii ambayo inawakilisha karibu asilimia 40 ya saratani zote za mapafu. Katika mwili wa binadamu, seli za NSCLC adenocarcinoma zinajulikana kutokea kwenye kiunganishi kati ya njia ndogo za hewa za mapafu na alveoli, lakini uvimbe huo hukua hasa ndani ya miundo ya alveolar. Timu ya Ingber ilionyesha kwamba wakati seli za adenocarcinoma za NSCLC zinapokua katika Chip ya Njia ya Pumu na Alveolus, seli za uvimbe hukua kwa kasi katika mazingira ya microengineered alveolar, wakati zinabaki kimya katika Chip ya Njia ya Pumu, kama inavyoonekana kwa wagonjwa wa binadamu. <unk>Platform zetu za kansa ya mapafu kwenye chip zinaweza kuiga mambo makuu ya orthotopic NSCLC kwa wakati halisi na azimio la juu, na kwa karibu zaidi kuliko mbinu zingine za in vivo na in vitro. Wao kutoa dirisha halisi juu ya utata wa kibiolojia uvimbe, <unk> alisema Ingber. Ingber pia ni Profesa wa Judah Folkman wa Biolojia ya Vascular katika HMS na Programu ya Biolojia ya Vascular katika Hospitali ya Watoto ya Boston, na pia Profesa wa Bioengineering katika Shule ya Uhandisi na Sayansi ya Harvard John A. Paulson (SEAS). Kwa hivyo, watafiti waliendeleza mbinu za co-plating na sindano ambazo ziliwawezesha kuunganisha idadi ndogo ya seli za NSCLC katika vipande viwili vya mapafu. "Hiyo inatuwezesha kuunda alama muhimu za saratani hii, ikiwa ni pamoja na ukuaji wake na mifumo ya uvamizi, na kuamua jinsi wanavyoathiriwa na ishara kutoka kwa seli za kawaida zinazozunguka.""" "Katika Airway Cancer Chips, seli za saratani zilibaki katika hali ya kupumzika kwa siku 12 kabla ya kuanza kukua, wakati katika Alveolar Cancer Chips zinaanza ukuaji wao haraka zaidi, na mara tu zinapofikia wingi muhimu, zinajitenga na kuvamia endothelium kama sehemu ya mchakato wao wa metastatic, ""alisema mwandishi wa kwanza Bryan Hassell, PhD, ambaye kama mwanafunzi wa shahada ya juu wa SEAS kwenye timu ya Ingber." Kisha, kikundi hicho kiliuliza ikiwa harakati za kupumua za kisaikolojia ndani ya alveoli zaweza kuathiri tabia za chembe za kansa. Kwa kushangaza, waligundua kwamba wakati nguvu za kimakani za mzunguko zinapotumiwa kwenye njia ya epithelial ya mapafu kuiga harakati za kupumua, ukuaji wa seli za kansa na uvamizi zilizuiliwa kwa nguvu. "Hii ni mara ya kwanza kwamba athari ya wazi ya harakati za kupumua za mzunguko juu ya ukuaji wa seli za saratani na uvamizi umeonyeshwa katika mfumo wowote wa in vitro wa kuiga tabia za NCSLC, "alisema Hassell." Watafiti wanafikiri kwamba wakati seli za saratani ya mapafu zinapokua na kujaza alveoli za mapafu ya wagonjwa, hii huingilia harakati zao za asili, ambazo kwa upande wake zinaweza kuharakisha ukuaji wa uvimbe na kuwezesha tabia ya uvamizi. Kwa hivyo, kwa sababu ya hali ya hewa, wanasayansi waliamua kuamua ikiwa kuna uwezekano wa kuathiri athari za chembe za NSCLC kwa dawa ya kupambana na saratani inayojulikana kama tyrosine kinase inhibitor (TKI). TKIs huelekea kwa enzymes zilizobadilika mara nyingi, kama vile epidermal growth factor receptor (EGFR), ambayo huanzisha ukuaji usio na kikomo katika NSCLC na saratani nyingine. Kwa sababu TKIs za kizazi cha mapema zinaweza kupoteza ufanisi wao wakati seli za saratani zinapokuwa sugu kwa kuzizalisha tofauti mpya za EGFR, watafiti wa saratani wanajaribu kubuni TKIs za akili zaidi. Hata hivyo, chembe za kansa zinazobadilika daima hujifunza kushughulika na hata hizo, kwa kujipanga upya kwa maumbile ili ziweze kuamsha utaratibu mbadala wa kansa. Kwa mujibu wa utafiti huo, kwa mfano, seli za NSCLC ambazo tayari zimepinga TKI ya kizazi cha kwanza zinaweza kusimamishwa na TKI ya kizazi cha tatu wakati hakuna harakati za kupumua, kama ilivyo katika uvimbe mkubwa ambao hujaza alveoli ya mapafu. Hata hivyo, katika hali ya kupumua, wao kuwa impervious kwa dawa na kuendelea kuishi na kukua polepole, kimsingi kujenga kansa "kuendelea" seli ambayo ni inayojulikana kuwa nemesis ya tiba ya kansa. "Wanafunzi pia waligundua kuwa viwango vya cytokines, protini zinazohusika katika mawasiliano ya seli zinazozalishwa na epithelial, endothelial, na seli za saratani, na zinazojulikana kuwa viashiria muhimu vya utabiri wa NSCLC, zinaonyesha ukuaji wa saratani katika ""Alveoli"" na zinaongozwa na harakati za kupumua na matibabu ya dawa." "Madhara ya harakati za kupumua juu ya tabia ya seli za saratani katika mifano yetu inaweza kuelezea jinsi seli za uvimbe, ambazo zinabaki kutoka kwa uvimbe unaopungua baada ya matibabu, zinaweza kuwa seli za kudumu, zinazoweza kupinga matibabu ya dawa, kudumu na hatimaye kusababisha saratani kurudi.""" "Kwa hivyo, jukwaa la orthotopic in vitro linaweza kuwa sawa kwa kuchunguza jinsi seli hizi za kudumu zinavyoibuka, na zinaweza kuwa chombo muhimu katika juhudi za maendeleo ya dawa za baadaye ambazo zinalenga kuzikomesha. """ Wanasayansi wengine wa utafiti huo ni pamoja na Christopher Chen, Ph.D., Wyss Postdoctoral Fellows, Girja Goyal, Alexandra Sontheimer-Phelps, na Esak Lee, Ph.D., ambaye ni mshauri wa Chen na Inger, pamoja na Wyss Staff Scientist Oren Levy. Utafiti huo ulifadhiliwa na Taasisi ya Wyss ya Uhandisi wa Kibiolojia katika Chuo Kikuu cha Harvard, Shirika la Utafiti wa Maendeleo ya Ulinzi (DARPA), Taasisi ya Kitaifa ya Afya (NIH), na ushirika kutoka kwa Shirika la Kimataifa la Utafiti wa Maadili (IFER) na Mtandao wa Elimu na Utafiti wa Lymphatic kwa Hassell na Lee.