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Ex vivo four-dimensional lung cancer model mimics metastasis.

ABSTRACT: We have developed a four-dimensional (4D) lung cancer model that forms perfusable tumor nodules. We determined if the model could be modified to mimic metastasis.We modified the 4D lung cancer model by seeding H1299, A549, or H460 cells through the trachea only to the left lobes of the acellular lung matrix. The model was modified so that the tumor cells can reach the right lobes of the acellular lung matrix only through the pulmonary artery as circulating tumor cells (CTC). We determined the gene expressions of the primary tumor, CTCs, and metastatic lesions using the Human OneArray chip.All cell lines formed a primary tumor in the left lobe of the ex vivo 4D lung cancer model. The CTCs were identified in the media and increased over time. All cell lines formed metastatic lesions with H460 forming significantly more metastatic lesions than H1299 and A549 cells. The CTC gene signature predicted poor survival in lung cancer patients. Unique genes were significantly expressed in CTC compared with the primary tumor and metastatic lesion.The 4D lung cancer model can isolate tumor cells in 3 phases of tumor progression. This 4D lung cancer model may mimic the biology of lung cancer metastasis and may be used to determine its mechanism and potential therapy in the future.

SUBMITTER: Mishra DK

PROVIDER: S-EPMC4387006 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Ex vivo four-dimensional lung cancer model mimics metastasis.

Mishra Dhruva K DK Creighton Chad J CJ Zhang Yiqun Y Chen Fengju F Thrall Michael J MJ Kim Min P MP

The Annals of thoracic surgery 20150218 4

<h4>Background</h4>We have developed a four-dimensional (4D) lung cancer model that forms perfusable tumor nodules. We determined if the model could be modified to mimic metastasis.<h4>Methods</h4>We modified the 4D lung cancer model by seeding H1299, A549, or H460 cells through the trachea only to the left lobes of the acellular lung matrix. The model was modified so that the tumor cells can reach the right lobes of the acellular lung matrix only through the pulmonary artery as circulating tumo ...[more]

PMID: 25701100

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Project description:BackgroundNon-small cell lung cancer (NSCLC) causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC.MethodsIn a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues) in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine) uptake as markers for proliferation and of cleaved (activated) effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines.ResultsViability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC) and human cell lines (CPC-N, HEK) proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC). Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas.ConclusionAlthough there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment. The results presented here shall serve as a base for multiple functional tests of novel chemotherapeutic approaches to NSCLC in the future.

Project description:Rationale: Continued reliance on preclinical animal models and traditional human cell culture experiments conducted on supraphysiologically stiff surfaces in two dimensions often limits the translation of cellular and molecular mechanistic findings into effective therapies for patients suffering from chronic obstructive pulmonary disease (COPD). COPD ranks as the fourth highest cause of mortality in the United States, and thus it is imperative to improve ex vivo models of this devastating disease. One emerging novel model of study involves using precision-cut lung slices as three-dimensional lung tissue culture (3D-LTC) platforms. Although this model retains the complex pulmonary architecture required for more precise ex vivo modeling, it is limited by short-term viability (?5–7 d). Objectives: We hypothesized that this decrease in viability is related to slices losing the extracellular matrix adhesion foundation critical for normal cellular functioning and that encapsulation of slices in custom hydrogel microenvironments would improve maintenance of a stable phenotype for longer-term investigational studies. Methods: The 3D-LTC slices tested here were created according to standard protocols from healthy mouse lungs and from the lungs of a patient with COPD. We synthesized poly(ethylene glycol)–based polymers to create custom hydrogels for encapsulation of 3D-LTCs with mechanical properties that match each type of lung tissue. The Young’s modulus (E) or stiffness of hydrogels was measured with a parallel-plate rheometer. Lung slices were analyzed for viability with a colorimetric assay that measures cellular metabolism (WST-1; Millipore Sigma) and by staining for cell membrane integrity (Invitrogen LIVE/DEAD viability kit; Thermo Fisher Scientific). All fluorescently labeled samples were imaged by spinning disk confocal microscopy, and cell viability at all time points was analyzed with ImageJ software. Results: The average percent viability of cells within hybrid 3D-LTCs was consistently higher than control 3D-LTCs on Day 1 (35% increase; P?<?0.001) and Day 7 (55% increase; P?<?0.001) according to LIVE/DEAD staining results. The WST-1 assay further showed no significant difference in viability between the two systems up to Day 18 in culture. Conclusions: We have demonstrated that it is possible to create customized hydrogel platforms that replicate the stiffness of healthy mouse and human COPD lung tissues. Precision cut lung slices embedded in these materials maintain higher levels of viability compared with controls in culture. Further optimization of this technology platform is ongoing and will facilitate the development of more organotypic ex vivo models of chronic pulmonary disease.

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Ex vivo four-dimensional lung cancer model mimics metastasis.

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Ex vivo four-dimensional lung cancer model mimics metastasis.

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