1. Human Lung Fibroblasts Inhibit Non-Small Cell Lung Cancer Metastasis in Ex Vivo 4D Model 
Dhruva K. Mishra, PhD, Steven D. Compean, BS, Michael J. Thrall, MD, Xin Liu, MS, Erminia Massarelli, MD, PhD, Jonathan M. Kurie, MD, Min P. Kim, MD 
The Annals of Thoracic Surgery 
Volume 100, Issue 4, Pages (October 2015)
DOI: /j.athoracsur
Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

2. Fig 1
An ex vivo four-dimensional (4D) lung bioreactor. The acellular lung matrix is placed in a 500 mL bottle. The trachea is connected to a trachea cannula that is connected to a one-way valve connector on the cap, and the pulmonary artery cannula is connected to 3-way stopcock, which is connected to the oxygenator and the pump. There is a third connector on the cap with tubing to the bottom of the bottle that is connected to the pump and the oxygenator that completes a closed circuit for perfusion of the media to the acellular lung matrix.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

3. Fig 2
Human lung fibroblast (LF) and cancer-associated fibroblast (CAF) cell culture on the ex vivo four-dimensional (4D) metastatic model. (A) LFs and (B) CAFs both grew on the ex vivo 4D lung scaffold for 15 days. Hematoxylin and eosin (H&E) staining of (C) LFs and (D) CAFs shows the fibroblast morphology such as thin, wavy, and spindle-shaped cells with distinct nuclei. No difference in morphology was observed between LFs and CAFs. Immunohistochemistry of (E) LFs and (F) CAFs on the 4D model with α-smooth muscle actin marker show positive staining. (G and H) H&E staining of the contralateral lung showed no metastatic lesion formation on day 15. (Original magnification 40×.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

4. Fig 3
Coculture of human lung fibroblast (LFs) with lung cancer cell lines on the ex vivo four-dimensional (4D) model. Formation of perfusable tumor nodule on ex vivo lung by day 15 with (A) H460 tumor cell alone, (B) H460+LFs, and (C) H460 and cancer-associated fibroblasts (H460+CAFs). No difference is seen in the gross appearance of the primary tumor nodule or pathologic hematoxylin and eosin images on day 15 of (D) H460 alone, (E) H460+LFs, and (F) H460+CAFs. There are no cells stained for α-smooth muscle actin (SMA) for (G) H460 alone, but there are α-SMA–stained cells for (H) H460+LFs and (I) H460+CAFs. (J) Similarly, there are no α-SMA–stained cells for A549 cells alone. There are more α-SMA cells in the peripheral pattern for (K) A549+LFs than for (L) A549+CAFs. (Original magnification 40×.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

5. Fig 4
The effect of lung fibroblast (LF) and cancer-associated fibroblast (CAF) coculture on metastasis with H460 tumor cells on the ex vivo four-dimensional model. Hematoxylin and eosin images of metastatic tumor emboli formation by day 15 on right lower lobes (A) without fibroblasts, (B) with LFs, and (C) with CAFs (original magnification 40×). The presence of LF cells shows fewer cells and thus reduced metastatic tumor growth. (D) A comparative graph with number of cells (H460, black bars; H460+LFs, gray bars; H460+CAFs, white) per high-power field (HPF, at original magnification ×40) on day 9 right upper (RU), day 12 right middle (RM), and day 15 right lower (RL) shows a significant inhibition of metastatic tumor growth in the presence of LF cells. The error bars show the standard error of the mean.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

6. Fig 5
The effect on metastasis by the addition of conditioned media from lung fibroblast (LF) and cancer-associated fibroblast (CAF) cells growing on the ex vivo four-dimensional (4D) model. The primary tumor nodule formation by H460 tumor cell growth is shown in the presence of (A) LFs and (B) CAFs from patient 1 conditioned media along with hematoxylin and eosin staining of the primary tumor (C) LF tumor and (D) CAF tumor, showing robust growth with no effect of conditioned media. The effect of (E) LF and (F) CAF conditioned media from patient 1 on metastatic tumor formation by day 15 is shown on right lower lobes. (E) The presence of LF-conditioned media inhibits the metastatic tumor growth compared with (F) CAF. (G) There was no difference in the primary tumor size on day 15 and (H) the total number of live circulating tumor cells for H460 cells grown on the 4D model treated with LF (black bars) and CAF (white bars) from all 3 patients. (I) A comparative graph with the number of cells per high-power field (HPF, original magnification ×40) on day 9 (RU), day 12 (RM), and day 15 (RL) shows a significant inhibition of metastatic tumor growth in the presence of LF-conditioned media from all 3 patients. The error bars show the standard error of the mean.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions