Name: endobronchial ultrasound-guided intratumoral injection of cisplatin for the treatment of isolated mediastinal recurrence of lung cancer
Uploaded: 2017-02-12T14:00:00
Description: Watch this Scientific Journal Video about Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer at JoVE.com

The authors have no acknowledgements.

Data on all patients treated with EBUS-guided cisplatin were reviewed. The institutional review board at the University of Florida approved this study (#IRB201400823). All data was prospectively collected and retrospectively analyzed. Use the following enrollment criteria.
1. Patient Selection
<ol>
	<li>Select patients between the ages of 18 and 80 years who have a biopsy-confirmed non-small cell lung cancer (NSCLC) or small cell lung carcinoma (SCLC) and have a pathologically-confirmed recu...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Agency for Research on Cancer. , (2012).</li><li>Siegel, R., Ma, J., Zou, Z., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2014.">Cancer statistics, 2014.</a> CA Cancer J Clin. 64 (1), 9-29 (2014).</li><li>Jeremic, B., Videtic, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chest+reirradiation+with+external+beam+radiotherapy+for+locally+recurrent+non-small-cell+lung+cancer:+a+review.">Chest reirradiation with external beam radiotherapy for locally recurrent non-small-cell lung cancer: a review.</a> Int J Radiat Oncol Biol Phys. 80 (4), 969-977 (2011).</li><li>Perez, C. 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Our manuscript focuses on the management of isolated mediastinal and hilar recurrences of lung cancer for patients in whom more radiation is not an option. The incidence of IMHR is approximately 9%11. Different treatment options have been tried in these patients, including palliative chemotherapy, more external beam radiation therapy, or supportive care and surveillance. At our institution, we treat these patients with intratumoral cisplatin guided by bronchoscopy.
Ther...

Lung cancer is the most prevalent cancer worldwide in both men and women and accounts for 1 in 5 of all cancer-related deaths. It has a case fatality rate of 0.871,2. Recurrence after initial treatment accounts for most deaths in patients with lung cancer. Therapeutic options for recurrent lung cancer are limited. Recurrent lung cancer also leads to significant impairment in quality of life and to significant caregiver burnout and requires additional supportive care, all of which decrease the likelihood of acceptance and tolerance to further interventions3.
Nearly 30% of patients with NSCLC have locoregional recurrence after radiation4. Treatment options for these patients are limited and include systemic chemotherapy, which has an objective response rate of only 10%5,6. Although repeat chest radiation is feasible, it has been studied mainly for palliative purposes and symptom relief and has not been used for disease remission3.
At our institution, all patients with isolated mediastinal and hilar recurrence (IMHR) accessible through bronchoscopy were treated with an endobronchial ultrasound (EBUS)-guided transbronchial intratumoral injection of cisplatin (ITC). Concurrent treatment with systemic chemotherapy and/or external beam radiation therapy (EBRT) was allowed as per the treating oncologists&#39; discretions. This manuscript highlights the safety, efficacy, feasibility, and detailed methods of the protocol used at our institution.

<table border="0" cellpadding="0" cellspacing="0" width="261">
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			<td colspan="2" height="20" style="height:20px;width:261px;">Bronchoscope</td>
		</tr>
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			<td colspan="2" height="20" style="height:20px;">22 Guage olympus EBUS needle</td>
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			<td colspan="2" height="20" style="height:20px;">40 g of cisplatin in 40 mls of normal saline</td>
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	</tbody>
</table>

endobronchial ultrasound-guided intratumoral injection of cisplatin for the treatment of isolated mediastinal recurrence of lung cancer

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients do not have many treatment options and are usually offered palliative chemotherapy or best supportive care. Endobronchial ultrasound (EBUS)-guided intratumoral injection of cisplatin (ITC) is a novel approach for these patients. The procedure is performed under conscious sedation. The lesion is located with a bronchoscopy using EBUS, and a 22-gauge EBUS needle is advanced through the working channel of the scope and locked in position. Under ultrasound guidance, the wall of the tracheobronchial tree is punctured and the needle is moved into the target lesion. The needle stylet is then removed and cisplatin (40 mg/40 mL) is injected into the lesion. One to two sites are treated per session. Details of the procedure are described in the protocol section of paper. At our center, 50 sites were treated in 36 patients (19 males, 17 females). The mean age of our cohort was 61.9 &#177;8.5 years. We performed final analyses on 35 patients and 41 sites. 24/35 (69%) had complete or partial response (responders), whereas 11/35 (31%) had stable or progressive disease (non-responders). Overall, survival in our group was 8 months (95% CI of 6-11 months), with patients who responded having significantly better survival than the ones who did not.

The response was measured by follow-up imaging 8-12 weeks after therapy. The response was classified as complete remission (CR), partial remission (PR), stable disease (SD), progressive disease (PD), or unable to assess response based on RECIST 1.1 criteria8. Patients with CR and PR were considered responders, and the others were classified as non-responders. Secondary outcomes included response based on tumor histology, size of recurrence, and concurrent systemic ...

Watch this Scientific Journal Video about Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer at JoVE.com

Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field.

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients ...

The overall goal of this procedure is to describe an endobronchial ultrasound or EBUS-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously radiated field. This method can help answer key questions in the field of lung cancer treatment about the safety and efficacy of EBUS-guided trans-bronchial intratumoral injection of cisplatin for recurrence of lung cancers in the post-radiated field. The main advantage of this technique is that it provides an alternative treatment option to systemic chemotherapy for patients with isolated, hilar, and mediastinal recurrence of lung cancer.For real-time visualization of hilar, mediastinal, and peribronchial structures for intratumoral cisplatin injection, first intravenously administer dexamethasone and ondansetron to a patient who has abstained from oral food and fluids for at least six hours. Next, induce conscious sedation with intravenous Midazolam and Fentanyl treatment, coupled with the topical application of five milliliters of Lidocaine. Use standard monitoring, including a continuous 3-lead electrocardiogram for heart rate monitoring, continuous pulse oximetry, and respiratory rate measurements.Now locate the target lesion using a convex probe EBUS with a built-in ultrasound probe on a flexible bronchoscope to advance the scope through the vocal cords. When the scope reaches the airways, turn on the EBUS processor and use one to two milliliters of normal saline to inflate the balloon at the tip of the scope. Flex the scope across the airway wall to locate the lesion.Then deploy the 22-gauge EBUS needle through the working channel of the scope and lock the needle into position. Under real-time ultrasound guidance, puncture the tracheobronchial wall and insert the needle into the target lesion. Remove the stylet within the needle and inject 10 milliliters of cisplatin into the lesion.Then retract the needle back into the sheath and inject the needle into a separate area of the lesion for the injection of another 10 milliliters of cisplatin. Remove the needle from the working channel of the scope and suction any additional drug from the distal airways. Then remove the bronchoscope.In this representative study, 8 to 12 weeks after therapy, 69%of the patients were classified as responders and 31%as non-responders, with a median survival of 8 months, 10 months for the responders, and six months for the non-responders, with a significantly higher progression-free survival rate observed in the responders group. While attempting this procedure, it is important to select appropriate patients, and to perform all of the steps in the protocol in a systematic manner. After its development, this technique paved the way for us and other researchers in the field of lung cancer to explore intratumoral chemo and other immunotherapies in animal models and in humans.After watching this video, you should have a good understanding of how to treat mediastinal and hilar recurrence of lung cancer with EBUS-guided intratumoral cisplatin injection.

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Research

JoVE Journal

Cancer Research

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...