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Fibroblast-derived conditioned media promotes lung cancer progression

  • John C. Greenwell
    Affiliations
    Department of Pharmacology & Toxicology, University of Louisville Health Sciences Center, Louisville, KY, United States
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  • Edilson Torres-Gonzalez
    Affiliations
    Department of Medicine, Division of Pulmonary, Allergy, and Critical Care and The Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, United States
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  • Jeffrey D. Ritzenthaler
    Affiliations
    Department of Medicine, Division of Pulmonary, Allergy, and Critical Care and The Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, United States
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  • Jesse Roman
    Correspondence
    Corresponding author at: Jesse, Roman, MD, Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, 834 Walnut Street, Suite 650, Philadelphia, PA 19107, United States.
    Affiliations
    Department of Medicine, University of Louisville Health Sciences Center, Louisville, KY, United States

    Department of Medicine, Division of Pulmonary, Allergy, and Critical Care and The Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, United States
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Published:September 06, 2022DOI:https://doi.org/10.1016/j.amjms.2022.08.019

      Abstract

      Lung cancer is the leading cause of cancer death in men and women in the United States. Recent studies have implicated the tumor microenvironment as a new chemotherapeutic target by demonstrating the importance of tumor cell-stromal interactions in cancer progression. However, the exact mechanisms by which tumor cell-stromal interactions drive lung cancer progression remain undefined, particularly in the lung. We suspect host fibroblasts represent an important component of the tumor microenvironment that drives tumor progression. We found that human non-small cell lung carcinoma cell lines show alterations in cell morphology, proliferation, migration, and colony formation on soft agar when exposed to fibroblast-conditioned media (FCM). Interestingly, FCM also promoted tumor cell resistance to cisplatin-induced apoptosis. These effects varied depending on the cancer cell line used. Similar observations were made when exposing murine Lewis Lung Carcinoma cells to conditioned media harvested from primary murine lung fibroblasts. Certain effects of FCM, but not all, could be prevented by using a cMET inhibitor. In vivo, we observed enhanced growth of the primary tumors when treated with FCM, but no changes in metastatic behavior. Although the identity of the stimulating agent(s) in the fibroblast-conditioned media was not unveiled, further studies revealed that the activity is more than one factor with a high-molecular weight (over 100 kDa). These studies implicate lung fibroblast-derived factors in lung cancer progression. These data suggest that targeting the lung tumor stroma alone, or in combination with other interventions, is a promising concept that warrants further study in the setting of lung cancer.

      Key Indexing Terms

      Introduction

      Although the incidence of lung cancer has decreased over the past several decades, it still remains higher than that of breast cancer, prostate cancer, and colon cancer combined,
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      and remains a significant threat worldwide.
      World Cancer Research Fund /American Institute for Cancer Research
      Food, Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective.
      Tobacco use remains the number one risk factor, exposing an individual to a myriad of carcinogens, among other harmful compounds.
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      Tobacco smoke carcinogen and lung cancer.
      The prevailing paradigm for decades has been that cells exposed to these and other carcinogens, acquire mutations in vital genes that over time lead to the development of tumors.
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      However, it is now well-documented that mutations alone are not sufficient to account for tumor development and progression.
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      Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells.
      In fact, a decreased incidence of colon and breast cancer has been observed through the use of non-steroidal anti-inflammatory drugs,
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      which suggests that factors other than cellular transformation are needed to cause tumor growth. This concept was first proposed in 1889, when Stephen Paget revealed his seed and soil hypothesis, which described a non-random pattern of metastasis in which cancer cells (“seeds”) depend on crosstalk with the microenvironment (“soil”) for growth and progression. In other words, metastasis from a primary tumor to distant organs is site specific, where the soil produces molecular factors appropriate and necessary for the seed's survival. Since then, it has been shown that processes such as inflammation, angiogenesis, and paracrine signaling from stromal cells in the tumor microenvironment are vitally important for tumors to survive and progress.
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      Fibroblasts are the predominant cell type in the tissue microenvironment and are largely responsible for the production of the extracellular matrix, in which they are embedded. Much attention has been given to the role of cancer-associated fibroblasts, which have been termed activated fibroblasts, identified by their expression of alpha-smooth-muscle actin.
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      Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction.
      It is well-known that these activated fibroblasts can promote tumor progression. However, the role that normal lung fibroblasts play in the progression of lung cancer has been difficult to ascertain, with conflicting reports in the literature. For example, Yamauchi et al., showed that TIG-3 fibroblasts, when co-implanted with lung cancer cells, increased tumor growth at the site of injection and metastases to the lung, and that this effect was due, at least partly, to TGFβ-mediated interactions.
      • Yamauchi Y
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      • et al.
      Lewis lung carcinoma progression is facilitated by TIG-3 fibroblast cells.
      These experiments, however, were performed in NOG mice, which are highly immunodeficient and thus must be interpreted carefully. In contrast, others have shown that normal lung fibroblasts play a role in inhibiting lung cancer progression. For example, Mishra et al., showed significantly fewer metastatic lesions in an ex vivo 4D acellular lung model when H460 cells were seeded with normal lung fibroblasts compared to carcinoma-associated fibroblasts.
      • Mishra DK
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      • Thrall MJ
      • et al.
      Human lung fibroblasts inhibit non-small cell lung cancer metastasis in ex vivo 4D model.
      Others have shown an important role of fibroblast-derived hepatocyte growth factor (HGF) in lung cancer progression.
      • Kanaji N
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      • Nakano-Narusawa Y
      • et al.
      Hepatocyte growth factor produced in lung fibroblasts enhances non-small cell lung cancer cell survival and tumor progression.
      ,
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      Transgenic mice overexpressing hepatocyte growth factor in the airways show increased susceptibility to lung cancer.
      This has largely been shown in the setting of resistance to epidermal growth factor receptor tyrosine kinase inhibitors.
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      Dual inhibition of met kinase and angiogenesis to overcome HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.
      ,
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      Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations.
      These data, among others, point to the complicated nature of tumor-stromal cell interactions, and warrant further research in this area, particularly in the lung. With the emerging practice of personalized medicine based on a patient's genetic profile, perhaps the conflicting reports in the literature could be explained, at least in part, to differences in the genetic profile of the host and/or the various lung cancer cell lines used.
      Here, we attempt to assess the role that normal, untransformed fibroblasts of the lung play in lung cancer progression in vitro and in vivo in immunocompetent animals using human and murine lung fibroblast and lung cancer cell lines.

      Materials and methods

      Reagents and cell culture

      Lewis Lung Carcinoma (LLC) (CRL-1642), A549 (CCL-185), H1792 (CRL-5895), H460 (HTB-177), WI38 (CCL-75), and IMR90 (CCL-186) cells were purchased from ATCC and grown in DMEM supplemented with 10% fetal bovine serum (FBS), at 37 °C in a 5% CO2 incubator. Primary murine lung fibroblasts were isolated by mincing lungs from a three-month-old C57BL/6J mice. All chemical reagents were purchased from Tocris Bioscience unless otherwise specified.

      Fibroblast conditioned media (FCM)

      Primary murine lung fibroblasts (1.5E6) were plated in p150 dishes in 25 mL DMEM and allowed to grow for 3 days. For controls, 25 mL DMEM was added to p150 dishes with no cells and incubated for 3 days. Media was then collected into 50 mL conical tubes and spun at 200 x g for 5 min, filtered (0.2 μM), and transferred to new 50 mL conical tubes and stored at - 70°C. For IMR90 and WI38 fibroblasts, 1.5E6 and 1E6 cells were plated in p100 dishes in 15 mL DMEM, respectively.

      FCM characterization via antibody array

      A RayBio label-based (L-series) mouse antibody array L-308 membrane kit (# AAM-BLM-1-4) was used for FCM characterization, per the manufacturer's instructions. See supplementary material for full details.

      Western blot analysis

      See supplementary material for full details.

      Proliferation assay

      LLC (100/500 µL-well), A549 (1000/500 µL-well), H1792 (1500/500 µL-well) and H460 (1000/500 µL-well) cells were plated in 48-well plates and cultured in control DMEM or FCM, and allowed to grow up to 7 days. Media was replaced every 2-3 days. Cell proliferation was then evaluated using the CellTiter-Glo® Luminescent Cell Viability Assay. See supplementary material for full details.

      Colony formation assay

      The colony formation assay was performed according to Millipore's instructions (Cell Transformation Detection Assay, ECM570). See supplementary material for full details.

      Apoptosis assay

      LLC (3000/100 µL-well), A549 (5000/100 µL-well), H1792 (5000/100 µL-well) and H460 (5000/100 µL-well) cells were plated in white-walled, clear-bottom, 96-well plates and cultured in control DMEM or FCM for 24 h. Media was then aspirated and 50 µL of control DMEM or FCM with or without cisplatin was added. Apoptosis was then evaluated using the Caspase-Glo® 3/7 Assay. See supplementary material for full details.

      Transwell migration assay

      LLC, A549, H1792, and H460 cells (all at 50,000/200 µL) were plated into Transwell inserts (Corning, 3464) in serum free DMEM in 24-well plates. Complete DMEM or FCM (700 µL) was pipetted into the bottom of the well to act as a chemoattractant. See supplementary material for full details.

      Wound healing (Scratch) assay

      LLC (5E5/5 mL DMEM), A549 (7.5E5/5 mL DMEM), H1792 cells (1E6/5 mL DMEM) and H460 cells (1E6/5 mL DMEM) were plated into 6-well plates. After 24 h, cells were scratched with a sterile 1 mL pipette tip. See supplementary material for full details.

      Animal studies

      All experiments were approved by the Institutional Animal Care and Use Committee of the University of Louisville. See supplementary material for full details.

      Analysis of data

      Means plus standard deviations of the mean were calculated for all experimental values. Significance was assessed by using the Student's t test. All experiments were repeated a minimum of 3 times with each sample group containing a minimum number of three experimental groups.

      Results

      FCM alters morphology of lung cancer cells

      Lung cancer cells were cultured in complete DMEM versus fibroblast-conditioned media (FCM) for 5 days. FCM was obtained from IMR90 and WI38 human lung fibroblasts when testing human cancer cells, and from primary murine lung fibroblasts when testing LLC cells. Afterwards, cells were formalin fixed, permeabilized, and stained with phalloidin and DAPI. FCM altered the morphology of A549, H1792, and LLC cells, but not H460 cells (Fig. 1A-B). This change in morphology was characterized by a spindle shape appearance, and was accompanied by cell scattering, both of which suggested the possibility of epithelial-mesenchymal transition (EMT), a marker of increased metastatic potential. To test this, cells were serum starved for 24 h and then cultured in complete DMEM or FCM for 48 h. Protein was then isolated, followed by Western Blot analysis for vimentin, E-cadherin, alpha smooth-muscle actin, and fibronectin. Very small changes were noted in all cell lines, except for H1792 cells, were alpha smooth-muscle actin, vimentin, and fibronectin levels were all mildly increased in response to FCM (Fig. 1C-D).
      Fig. 1
      Fig. 1Effect of FCM on morphology, EMT, and proliferation.
      (A-B) A549, H1792, H460, and LLC cells were grown in 96-well plates in control DMEM or FCM for 5 days, formalin fixed and stained with phalloidin and DAPI. (C-D) A549, H1792, H460, and LLC cells were grown in 6-well plates in control DMEM or FCM for 24 h, serum starved for 24 h, and then grown in FCM for an additional 48 h. Protein was isolated, followed by Western Blot analysis (20 µg) for fibronectin (Fn, 1:1000), E-cadherin (Ecad, 1:500), Vimentin (1:500), alpha smooth muscle actin (aSMA, 1:1000), and GAPDH (loading control, 1:10,000). (E-H) A549, H1792, H460, and LLC cells were grown in 48-well plates in control DMEM or FCM for 7 days. Cells were refed every 2-3 days. Cell proliferation was quantified using Cell Titer-Glo Luminescent Cell Viability Assay Kit (Promega). Cell proliferation was increased in all cell lines tested in response to FCM. *P < .001.

      FCM has differential effects on proliferation, migration, and colony formation

      To examine the effects of FCM on lung cancer cell proliferation, cells were cultured in complete DMEM or FCM for up to 7 days. All cell lines cultured in FCM showed increased proliferation, as determined by ATP quantification, when compared to cells grown in complete DMEM (Fig. 1E-H). Likewise, transwell migration was increased in all cell lines (Fig. 2A-B), as determined by Boyden chamber migration assays, with the greatest effect being observed in H1792 cells. Additionally, colony formation of A549 cells was increased by the presence of FCM but was unchanged in H1792 and H460 cells (Fig. 2C), while a decrease was observed in LLC cells (Fig. 2D). Lastly, wound healing, as determined by the scratch assay, was increased in A549, H1792 and LLC cells cultured in the presence of FCM, again, with the greatest effect being observed in H1792 cells. (Fig. 2E). Interestingly, FCM had no effect on wound healing in H460 cells (Fig. 2E).
      Fig. 2
      Fig. 2Effect of FCM on Transwell migration, colony formation and wound healing.
      (A-B) A549, H1792, H460, and LLC cells were plated in a Transwell Boyden insert in serum- free media with control DMEM, IMR90 FCM, or WI38 FCM in the lower well. After 20-24 h, cells were fixed and stained using the Kwik-Diff kit (Thermo Scientific). Inserts were then rinsed in dH2O and non-adherent cells on the inside of the insert were removed with a cotton swab and photographed. Transwell migration was increased in all cell lines tested in response to FCM. (C-D) A549, H1792, H460, and LLC cells plated in 24-well plates were suspended in 0.4% top agar layer and plated on a 0.8% base agar layer, which was prepared according to Millipore's instructions. Control DMEM or FCM was then added. Media was replaced every 3-4 days. Colonies were followed for 14 days and then quantified using ImageJ software. Colony formation was increased in A549 cells and decreased in LLC cells in response to FCM, while no changed were observed in H1792 or H460 cells. (E) A549, H1792, H460, and LLC cells were plated in 6-well plates and allowed to grow overnight. A scratch was then created using a 1 mL pipette tip. Media was replaced with 5 mL control DMEM or FCM. Photographs taken at 0 and 48 h. Quantification was performed using ImageJ software. Wound healing was increased in all cell lines tested in response to FCM, except H460 cells. *P < .05, **P < .01

      FCM protects against cisplatin-induced death

      To evaluate the role of FCM in cisplatin-induced death, lung cancer cells were cultured in control DMEM or FCM for 24 h. Cells were then exposed to cisplatin for an additional 28 h. There was a significantly greater number of viable cells, after cisplatin exposure, when the cells were cultured in FCM compared to complete DMEM. (Fig. 3A-D). FCM induced cisplatin protection was accompanied by a decrease in the activity of caspases 3 and 7 in A549, H1792 and LLC cells (Fig. 3E, F and H), while no changes were observed in H460 cells (Fig. 3G). A summary of the in vitro findings is provided in Table 1.
      Fig. 3
      Fig. 3Effect of FCM on viability and caspase 3/7 activity in response to cisplatin.
      (A-D; Cell viability) A549, H1792, H460, and LLC cells were plated in white-walled, clear-bottom 96-well plates in control DMEM or FCM and allowed to grow overnight. Cisplatin (30 µM, 100 µM, 30 µM, and 10 µM, respectively) was then added and cells were allowed to grow an additional 24 h. The remaining viable cells were quantified using Cell Titer-Glo Luminescent Cell Viability Assay Kit (Promega). All cells lines tested showed resistance to cisplatin in response to FCM, with a greater effect being observed in H1792 and LLC cells. (E-H; Caspase activity) A549, H1792, H460, and LLC cells were plated in white-walled, clear-bottom 96-well plates in control DMEM or FCM and allowed to grow overnight. Cisplatin (30 µM, 100 µM, 30 µM, and 10 µM, respectively) was then added and cells were allowed to grow an additional 28 h. Caspase 3/7 activity was then detected using the Caspase-Glo 3/7 Assay Kit (Promega). Caspase 3/7 activation was decreased in all cells lines tested in response to FCM, except H460 cells. *P < .001.
      Table 1Effect of FCM on different parameters.
      ParameterA549H1792H460LLC
      MorphologyAlteredAlteredNo ChangeAltered
      ProliferationIncreasedIncreasedIncreasedIncreased
      Transwell MigrationIncreasedIncreasedIncreasedIncreased
      Colony FormationIncreasedNo ChangeNo ChangeDecreased
      Scratch MigrationIncreasedIncreasedNo ChangeIncreased
      Apoptosis ResistanceIncreasedIncreasedIncreasedIncreased

      Fibroblasts co-injected with LLC cells promote tumor growth, while FCM has small affect

      Having established the effects of FCM on lung cancer cells in vitro, we focused our attention on the role of fibroblasts in vivo. First, primary lung fibroblasts and LLC cells were grown separately until cells were in exponential growth phase. Cells were then co-injected into the hindflank of C57BL/6 mice. Tumor growth was followed until tumors reached a size of 15 mm in any direction, after which time, all animals were sacrificed, and lungs were harvested, and formalin fixed. Primary lung fibroblasts and LLC cells injected together caused larger tumor growth at the site of injection, compared to LLC cells alone (Fig. 4A). Primary lung fibroblasts injected alone did not cause tumors at the site of injection (data not shown). To evaluate lung metastasis, lungs were processed, paraffin embedded, sectioned, and H&E stained. As depicted in Fig. 4B, there were no differences in lung metastasis observed.
      Fig. 4
      Fig. 4Effect of PLF and FCM on tumor growth and lung metastasis in vivo.
      (A,B) 1E6 LLC cells (LLC, n = 6) or 1E6 LLC cells with 1E6 PLF cells (LLC+PLF, n = 7) were injected subcutaneously into the hind flank of WT C57BL/6 mice. (C,D) LLC cells were grown in control DMEM or FCM for 24 h and then injected subcutaneously into the hind flank of WT C57BL/6 mice. A tumor size of ≥15 mm in length or width was established as the endpoint according to IACUC regulations. Mice were then sacrificed and lungs harvested and processed for examination of metastases. Tumor volume was greater in the LLC+PLF group compared to LLC cells alone (A), while no differences in the number of lung metastases was observed (B). Tumor volume (C) and lung metastasis (D) was similar in animals injected with LLC cells grown in complete DMEM vs. FCM. *P < .05. (E) A RayBio label-based (L-series) mouse antibody array L-308 membrane kit (# AAM-BLM-1-4) was used for FCM characterization. Control DMEM or FCM samples were dialyzed and biotin labeled and then incubated with membranes containing 308 soluble mouse protein antibodies. Membranes were then washed and exposed to HRP-conjugated streptavidin, followed by ECL and then imaged using Biorad's ChemiDoc™ XRS+ System. Red boxes indicate proteins present in FCM that were not present in complete DMEM (see list).
      To evaluate the role that FCM plays in lung cancer metastasis, we grew LLC cells in FCM for 24 h and then injected them into the hindflank of C57BL/6J mice in FCM or complete DMEM. Tumor growth was again followed until tumors reached a size of 15 mm in any direction, after which time, all animals were sacrificed, and lungs were harvested, and formalin fixed. Although the number of metastases in the FCM group was greater in number than those in the complete DMEM group (2.6 vs 4.5), this was not statistically significant (Fig. 4D). Tumor size at the site of injection was also unchanged (Fig. 4C).

      FCM characterization

      In order to identify the soluble component(s) present in FCM responsible for the observed effects, we began by utilizing Corning® Spin-X® UF Concentrators to determine the relative size of the ‘active’ agent. Using columns with a molecular weight cutoff of 100 kDa, we were able to block the effects of FCM, with the exception of the protection from cisplatin-induced apoptosis, which indicates that most of the effects of FCM are due to factors greater than 100 kDa in size (not shown). Other studies suggested that the activity was not mediated by molecules capable of interacting with RGD-binding integrins, as RGD peptides (used at 500 µg/ml) did not block the effect. Heparin, boiling (100 °C, 10 min), and proteases (PMSF) also failed to inhibit the activity suggesting that the soluble factor(s) in FCM is either not a protein, or is a protein in a multiprotein complex, which protected it from degradation (not shown). Lastly, several antagonists for fibroblast growth factor receptors (SU 6668, PD161570, and FIIN1 HCl), a well characterized mitogen of fibroblasts, also failed to inhibit the activity of FCM on proliferation (not shown).
      For a more targeted approach, we used RayBio AAM- BLM-1 label-based mouse antibody array, which simultaneously measures the expression of 308 soluble murine proteins. Fifteen different proteins were identified in FCM that were not present in complete DMEM (Fig. 4E, red boxes). One of these proteins is hepatocyte growth factor (HGF). HGF is 105 kDa in size and is cleaved by serine proteases into a 69-kDa α-chain and a 34-kDa β-chain. To determine if HGF was responsible for the observed effects of FCM on lung cancer cells, we tested the effects of a commercially available hepatocyte growth factor receptor (cMet) antagonist, PF 04217903 mesylate. The presence of PF 04217903 prevented the change in tumor cell morphology induced by FCM, except in LLC cells, and blocked many, but not all, of the effects of FCM (Table 2). However, in all cell lines, it was unable to block the protective effect of FCM on cisplatin-induced apoptosis and the increase in proliferation. To further investigate the identity of the component in FCM responsible for the effects on proliferation and cisplatin resistance, we charcoal stripped the FCM. Interestingly, the effects of FCM on cisplatin resistance were abolished with charcoal-stripped FCM (not shown). These data suggest that the components in FCM responsible for increasing resistance to cisplatin are non-polar materials such as lipids.
      Table 2Effect of FCM and cMET antagonists.
      ParameterA549H1792H460LLC
      MorphologyBlockedBlockedNo ChangeNo Change
      ProliferationNo ChangeNo ChangeNo ChangeNo Change
      Transwell MigrationBlockedBlockedNo ChangeNo Change
      Colony FormationNo ChangeNo ChangeNo ChangeBlocked
      Scratch MigrationBlockedBlockedNo ChangeNo Change
      Apoptosis ResistanceNo ChangeNo ChangeNo ChangeNo Change

      Discussion

      The prevailing paradigm of cancer for decades has been that a cell acquires mutations over its lifespan that eventually lead to a malignant phenotype and the development of cancer. However, this paradigm does not tell the entire story, and we now know that stromal cells in the tumor microenvironment are not idle bystanders but coevolve with transformed cells and play a crucial role in cancer progression. To date, the vast majority of research in this area has focused on the role of tumor or carcinoma-associated fibroblasts.
      • Ding S
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      MRC-5 fibroblast-conditioned medium influences multiple pathways regulating invasion, migration, proliferation, and apoptosis in hepatocellular carcinoma.
      • Erez N
      • Truitt M
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      Cancer-associated fibroblasts are activated in incipient neoplasia to orchestrate tumor-promoting inflammation in an NF-??B-dependent manner.
      • Shiga K
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      Cancer-associated fibroblasts: their characteristics and their roles in tumor growth.
      We hypothesized that normal, unstimulated fibroblasts of the lung also represent an important component of the tumor microenvironment that helps drive tumor progression. Consistent with this hypothesis, our studies show that normal lung fibroblasts produce a soluble substance(s) capable of altering cancer cell morphology and stimulating proliferation, migration, and colony formation of several NSCLC lines, as well as protecting against cisplatin-induced apoptosis in vitro. We also observed an enhancement of primary tumor growth in vivo.
      Similar to our findings, others have reported that fibroblast-conditioned media may affect NSCLC, but the observations described in the literature are sometimes contradictory. This is likely due to the disparate experimental conditions tested. For example, much of the work present in the literature focuses on cell lines (mostly embryonic) or cancer-derived or ‘transformed’ fibroblasts rather than using unstimulated primary lung fibroblasts harvested from adult lungs. In some studies, the cells were cultured under serum-free conditions, which may enhance an effect, but is not very physiological. Furthermore, when testing these phenomena in vivo, many studies use immunosuppressed mice.
      • Kanaji N
      • Yokohira M
      • Nakano-Narusawa Y
      • et al.
      Hepatocyte growth factor produced in lung fibroblasts enhances non-small cell lung cancer cell survival and tumor progression.
      Although these models are useful, they are somewhat removed from physiological conditions. Our manuscript attempts to go further by testing primary lung fibroblasts cultured in the presence of serum and examining their effects in vivo using immunocompetent mice. Finally, many investigations focus on xenograft models where the size of tumors is measured at the site of injection. We, on the other hand, also examined for lung metastases.
      We first examined the effects of FCM on cell shape. Our observation that FCM promoted cell scattering and spreading, and a spindle-shape phenotype in three of four cell types suggested the breakdown of intercellular junctions and epithelial-mesenchymal transition (EMT). However, except for increased vimentin, fibronectin and alpha smooth muscle actin levels in H1792 cells, little to no changes were observed in A549, H460 and LLC cells. These data suggest that EMT, as it is classically defined, was not responsible for the observed changes in morphology. Interestingly, at least one group has shown that the behavioral and morphological changes that accompany EMT are not necessarily linked. For example, cadherin switching is necessary for increased motility, but not the morphological changes that accompany EMT.
      • Maeda M
      • Johnson KR
      • Wheelock MJ.
      Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition.
      FCM affected several other processes and, as presented in Table 1, we observed differential effects of FCM on A549, H1792, H460 and LLC cells in several functional assays. For example, FCM stimulated transwell migration in all cell lines tested, but this effect was much greater in H1792 cells. FCM increased migration across a wound in all cell lines, except H460 cells. Interestingly, colony formation was increased in A549 cells in response to FCM, but was unchanged in H1792 and H460 cells, and a decrease was observed in LLC cells. Lastly, FCM could protect all cell lines from cisplatin-induced death. This was accompanied by a decrease in the activity of caspases 3 and 7 in A549, H1792 and LLC cells, but not in H460 cells. There is precedent for human lung cancer cell lines behaving differently when studied in the laboratory. For example, Liang et al, demonstrated a decrease in cell proliferation in A549 cells, compared to a stimulation of cell proliferation in SK-MES-1 cells, when exposed to Budesonide, a PPARα and glucocorticoid receptor agonist. This differential effect was attributed to a mutation in TP53 in SK-MES-1 cells.
      • Liang H
      • Kowalczyk P
      • Junco JJ
      • et al.
      Differential effects on lung cancer cell proliferation by agonists of glucocorticoid and PPARa receptors.
      Additionally, Ling et al. showed that overexpression of MTSS1 enhanced the invasion and proliferation abilities of H920 and H1581 cells, while it inhibited invasion and proliferation in SW900 cells. These differential effects were shown to be due to differences in FAK phosphorylation and activity.
      • Ling DJ
      • Chen ZS
      • Liao Q De
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      Differential effects of MTSS1 on invasion and proliferation in subtypes of non-small cell lung cancer cells.
      Our observations that FCM displayed differential effects depending on cell line could explain differences observed in the literature when comparing normal fibroblasts to carcinoma-associated fibroblasts.
      The observation that FCM protected both human cell lines and LLC cells from cisplatin-induced apoptosis through effects on caspase activity is quite intriguing as it suggests that host fibroblast-derived soluble factors, and not only physical interaction, may influence the impact of chemotherapy on tumors. Cisplatin is a recognized cornerstone in the treatment of lung cancer. Unfortunately, some tumors are unresponsive, but more quickly become resistant. To date, there are limited strategies in the clinic that circumvent this problem. Cisplatin acts largely by promoting the formation of DNA adducts, mainly intrastrand crosslinks, as well as inducing oxidative stress.
      • Jamieson E
      • Lippard S.
      Structure, recognition, and processing of cisplatin − DNA adducts.
      • Santos NAG
      • Catão CS
      • Martins NM
      • et al.
      Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria.
      This triggers TP53 and MAPK signaling, among others, resulting in the induction of apoptosis. Thus, one must assume that the protective effects of FCM are due to inhibition of drug activity, drug uptake, or over expression of anti-apoptotic signals (e.g., bcl-2). Interestingly, our observations that FCM protected lung cancer cells from cisplatin-induced death is not in agreement with Bartling et al, who showed that conditioned media from WI38 fibroblasts could protect H358 cells from apoptosis induced by paclitaxel, but not by cisplatin.
      • Bartling B
      • Hofmann H-S
      • Silber R-E
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      Differential impact of fibroblasts on the efficient cell death of lung cancer cells induced by paclitaxel and cisplatin.
      It has been reported that sensitivity to cisplatin appears dependent on cell confluence,
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      Confluence-depenent resistance to cisplatin in lung cancer cells is regulated by transforming growth factor-beta.
      but how this explains the differences identified remains to be investigated. Either way, our data indicate that one possible approach to circumventing cisplatin resistance in the clinic would be to simultaneously target fibroblast signaling in the tumor stroma during chemotherapy. At least one group has shown that cisplatin resistance could be abrogated by inhibiting mTOR signaling.
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      Overcoming cisplatin resistance by mTOR inhibitor in lung cancer.
      As did others, we investigated the effects of HGF inhibitors, but we took a broader approach attempting to identify other potential drivers of cancer progression.  In doing so, we showed that HGF is not responsible for many of the effects of FCM and that other factors are likely involved in distinct ways depending on the tumor cell tested.  Of all the effects of FCM, stimulation of proliferation was the most consistent in all cell lines, and interestingly, this effect was not diminished by blocking HGFR (Table 2). This is intriguing because aberrant HGF/cMET signaling has been shown to promote an oncogenic phenotype in various tumor types, including lung cancers.
      • Trusolino L
      • Bertotti A
      • Comoglio PM.
      MET signalling: principles and functions in development, organ regeneration and cancer.
      There are many reasons why HGF/cMET inhibitors may act differently in different tumor cell lines including differences in the inhibitors used.
      • Ma PC
      • Jagadeeswaran R
      • Jagadeesh S
      • et al.
      Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer.
      ,
      • Matsubara D
      • Ishikawa S
      • Oguni S
      • et al.
      Molecular predictors of sensitivity to the MET inhibitor PHA665752 in lung carcinoma cells.
      What is important is that we demonstrated that HGF does not mediate many of the processes tested, which prompted more experiments. However, boiling the media, blocking FGFR, and blocking the PI3K and ERK pathways also had no effect (not shown). While this proliferative effect of FCM was not observed in vivo, co-injection of fibroblasts and tumor cells together increased primary tumor growth. This is likely due to the loss of the effects of transient exposure of cells to FCM in vivo, while fibroblasts co-injected with LLC cells persist and thus their effects are long lasting.
      The activity of FCM was impressive in certain assays, but was limited in its influence in vivo in our models. Nevertheless, identifying the agent(s) present in FCM responsible for these effects might unveil potential targets for intervention that could be used in the clinic. We found that FCM was characterized by altering cancer cell morphology and stimulating proliferation, migration, and colony formation of several NSCLC lines, as well as protecting against cisplatin-induced apoptosis in vitro. We also observed an enhancement of primary tumor growth in vivo, in immunocompetent animals. Many of these effects, but not all, appeared due to HGF.
      Altogether, these observations suggest that FCM contains not one but several factors capable of influencing tumor cell behavior. Our data showing differential effects of FCM, depending on the cell type, also indicate that the role that fibroblasts play in lung cancer progression is not as simple, and instead is rather complex. Thus, our data support the idea that targeting the tumor stroma, in combination with other therapies, is a promising concept that warrants further study.

      Contributions

      Concept and design: JCG, JDR, JR; Analysis and interpretation: JCG, ETG, JR; Drafting manuscript and important intellectual input: JCG, JR

      Acknowledgement

      The work was supported by research grants from the Department of Veterans Affairs and the National Institutes of Health.

      Appendix. SUPPLEMENTARY MATERIALS

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