Dhruva K. Mishra

Human lung cancer cells grown in an ex vivo 3D lung model produce matrix metalloproteinases not produced in 2D culture.

We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture.

Human Lung Cancer Cells Grown on Acellular Rat Lung Matrix Create Perfusable Tumor Nodules

BACKGROUND Extracellular matrix allows lung cancer to form its shape and grow. Recent studies on organ reengineering for orthotopic transplantation have provided a new avenue for isolating purified native matrix to use for growing cells. Whether human lung cancer cells grown in a decellularized rat lung matrix would create perfusable human lung cancer nodules was tested. METHODS Rat lungs were harvested and native cells were removed using sodium dodecyl sulfate and Triton X-100 in a decellularization chamber to create a decellularized rat lung matrix. Human A549, H460, or H1299 lung cancer cells were placed into the decellularized rat lung matrix and grown in a customized bioreactor with perfusion of oxygenated media for 7 to 14 days. RESULTS Decellularized rat lung matrix showed preservation of matrix architecture devoid of all rat cells. All three human lung cancer cell lines grown in the bioreactor developed tumor nodules with intact vasculature. Moreover, the lung cancer cells developed a pattern of growth similar to the original human lung cancer. CONCLUSIONS Overall, this study shows that human lung cancer cells form perfusable tumor nodules in a customized bioreactor on a decellularized rat lung matrix created by a customized decellularization chamber. The lung cancer cells grown in the matrix had features similar to the original human lung cancer. This ex vivo model can be used potentially to gain a deeper understanding of the biologic processes involved in human lung cancer.

Gene expression profile of A549 cells from tissue of 4D model predicts poor prognosis in lung cancer patients

The tumor microenvironment plays an important role in regulating cell growth and metastasis. Recently, we developed an ex vivo lung cancer model (four dimensional, 4D) that forms perfusable tumor nodules on a lung matrix that mimics human lung cancer histopathology and protease secretion pattern. We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, a human lung cancer cell line, grown in a petri dish (two‐dimensional, 2D), and of the same cells grown in the matrix of our ex vivo model (4D). Furthermore, we obtained gene expression data of A549 cells grown in a petri dish (2D) and matrigel (three‐dimensional, 3D) from a previous study and compared the 3D expression profile with that of 4D. Expression array analysis showed 2,954 genes differentially expressed between 2D and 4D. Gene ontology (GO) analysis showed upregulation of several genes associated with extracellular matrix, polarity and cell fate and development. Moreover, expression array analysis of 2D vs. 3D showed 1,006 genes that were most differentially expressed, with only 36 genes (4%) having similar expression patterns as observed between 2D and 4D. Finally, the differential gene expression signature of 4D cells (vs. 2D) correlated significantly with poor survival in patients with lung cancer (n = 1,492), while the expression signature of 3D vs. 2D correlated with better survival in lung cancer patients with lung cancer. As patients with larger tumors have a worse rate of survival, the ex vivo 4D model may be a good mimic of natural progression of tumor growth in lung cancer patients.

Fibulin-2 is a driver of malignant progression in lung adenocarcinoma.

The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-of-function experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wild-type littermates, implying that malignant progression was dependent specifically upon tumor cell-derived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen.

Circulating tumor cells from 4D model have less integrin beta 4 expression.

BACKGROUND Currently, there is no in vitro or ex vivo model that can isolate circulating tumor cells (CTCs). Recently, we developed a four-dimensional (4D) lung cancer model that allows for the isolation of CTCs. We postulated that these cells have different properties than parental (2D) cells. MATERIALS AND METHODS We obtained CTCs by growing A549, H1299, 393P, and 344SQ cell lines on the 4D lung model. The CTCs were functionally characterized in vitro and gene expression of the cell adhesion molecules was compared with respective 2D cells. Integrin beta 4 (ITGB4) was further investigated by stably transfecting the A549 and H1299 cells. RESULTS We found that all cell lines produced CTCs, and that CTCs from the 4D model were less adherent to the plastic and have a slower growth rate than respective 2D cells (P < 0.01). Most of the cell adhesion molecules were downregulated (P < 0.05) in CTCs, and ITGB4 was the common molecule, significantly more underexpressed in CTCs from all cell lines than their respective 2D cells. The modulation of ITGB4 led to a differential function of 2D cells. CONCLUSIONS CTCs from the 4D model have different transcriptional, translational, and in vitro characteristics than the same cells grown on a petri dish, and these CTCs from the 4D model have the properties of CTCs that are responsible for metastasis.

Circulating tumor cells from a 4-dimensional lung cancer model are resistant to cisplatin

OBJECTIVE To determine the effect of cisplatin on circulatory tumor cells (CTC) and tumor nodules in a four-dimensional (4D) lung cancer model. METHODS CTCs from the 4D model seeded with H1299, A549, or H460 and respective cells that were grown under two-dimensional conditions in a Petri dish were treated with 50 μM cisplatin for 24 and 48 hours and cell viability was determined. The lung nodules in the 4D model were then treated with different continuous or intermittent doses of cisplatin and the nodule size, the number of CTCs, and the level of matrix metalloproteinase (MMP) were determined. RESULTS Cisplatin led to a significant decrease in the viability of tumor cells grown under 2D conditions (P < .01) but not in CTCs from the 4D model after both 24 hours and 48 hours. Cisplatin led to regression of tumor nodules with both the continuous and intermittent treatments. Moreover, there was a significantly higher number of CTCs per tumor area (P < .05) and MMP-2 production per tumor area (P = .007) for all human lung cancer cell lines grown in the 4D model when treated with cisplatin. CONCLUSIONS The 4D lung cancer model allows for the isolation of CTCs that are resistant to cisplatin treatment. The model may allow us to better understand the biology of cisplatin resistance.

Thy-1+ Cancer-associated Fibroblasts Adversely Impact Lung Cancer Prognosis

Cancer-associated fibroblasts (CAFs) regulate diverse intratumoral biological programs and can promote or inhibit tumorigenesis, but those CAF populations that negatively impact the clinical outcome of lung cancer patients have not been fully elucidated. Because Thy-1 (CD90) marks CAFs that promote tumor cell invasion in a murine model of KrasG12D–driven lung adenocarcinoma (KrasLA1), here we postulated that human lung adenocarcinomas containing Thy-1+ CAFs have a worse prognosis. We first examined the location of Thy-1+ CAFs within human lung adenocarcinomas. Cells that co-express Thy-1 and α-smooth muscle actin (αSMA), a CAF marker, were located on the tumor periphery surrounding collectively invading tumor cells and in perivascular regions. To interrogate a human lung cancer database for the presence of Thy-1+ CAFs, we isolated Thy-1+ CAFs and normal lung fibroblasts (LFs) from the lungs of KrasLA1 mice and wild-type littermates, respectively, and performed global proteomic analysis on the murine CAFs and LFs, which identified 425 proteins that were differentially expressed. Used as a probe to identify Thy-1+ CAF-enriched tumors in a compendium of 1,586 lung adenocarcinomas, the presence of the 425-gene signature predicted a significantly shorter survival. Thus, Thy-1 marks a CAF population that adversely impacts clinical outcome in human lung cancer.

Abstract A03: Lymphocytes lead to circulating tumor cell death from 4D model seeded with non-metastatic lung cancer cell line

Objective: Mouse lung cancer cell lines 393P and 344SQ are derived from Kras LA1/+ p53 R172HΔG mice and have different metastatic potential in immunocompetent mice. 393P cells have local primary tumor growth but do not form metastatic lesions when injected into the subcutaneous space of 129sv mice. However, 344SQ cells form a primary tumor and metastatic lesions when injected into the subcutaneous space. Our aim is to determine if lymphocytes play a role in this differential metastatic lung cancer formation between the two cell lines. Methods: Both 393P and 344SQ cells were grown in the ex vivo 4D lung cancer model. We determined the amount of primary tumor growth, the number of circulating tumor cells (CTCs), and metastatic lesion formation. The CTCs from the 4D model seeded with either cells were injected into the tail veins of immunocompetent 129sv mice (wt) or immunodeficient mice (nu/nu). We calculated the Kaplan Meier survival curve. Next, we added lymphocytes derived from a 129sv mouse spleen to an ex vivo 4D lung cancer model seeded with 393P or 344SQ and isolated the CTCs. We plated the CTCs on a petri dish and compared the survival of these cells. Results: Both 393P and 344SQ cells grew in the 4D model. There were no significant differences in the primary tumor growth and circulating tumor cell formation between the two cell lines. There were no significant differences in the number of metastatic lesion formation between the two cell lines (p=0.5). When CTCs from the 4D model seeded with 344SQ were injected into the tail vein of nu/nu mice or wt mice, there were no differences in the survival rates between the two types of mice (p=0.3). However, nu/nu mice that were injected with CTC from the 4D model seeded with 393P were less likely to survive compared to the wt mice (p=0.002). No metastatic lesions formed in the wt mice injected with 393P CTC for 8 months. The CTC from the 4D model seeded with 344SQ and treated with lymphocytes had no significant difference in cell growth when cultured on a petri dish compared to the control group (p=0.9). However, the CTC from the 4D model seeded with 393P treated with lymphocytes had significantly less live cells compared to the control group (p=0.01). Conclusions: Lymphocytes inhibit metastatic lesion formation in 393P cell lines but not 344SQ cell lines. The ex vivo 4D lung cancer model allows for the identification of the cell type that is responsible for inhibition of metastasis. The model can be used to study the role of different components of a tumor9s microenvironment in tumor metastasis. Citation Format: Dhruva K. Mishra, Humberto Juarez, Michael J. Thrall, Min P. Kim. Lymphocytes lead to circulating tumor cell death from 4D model seeded with non-metastatic lung cancer cell line. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A03.

Abstract B31: Lung fibroblast enhances MMP-1 secretion when co-cultured with human lung cancer cells in the 4D lung cancer model

Tumor microenvironment plays an important role in cancer development, progression and metastasis. However, it is difficult to determine the effect of fibroblast on lung cancer cells in patients with lung cancer. Recently, we have developed a novel ex vivo 4D lung cancer model that mimic the biology of human lung cancer growth. In order to determine the viability of the normal lung fibroblast, Wi-38, when grown in the 4D model, we grew Wi-38, alone or with A549, in the epithelial space of the acellular matrix of the 4D model with media that was continuously perfused through the vascular space. We performed a lobectomy on days 4, 6, 8, 11 and 14 from the 4D model. We performed HE however, when Wi-38 was grown with A549, there was a significantly less number of fibroblast (CK7 negative cells) on day 14 (2±1%) compared to day 4 (50±9%, p Citation Format: Dhruva Kumar Mishra, Michael J. Thrall, Jonathan M. Kurie, Min P. Kim. Lung fibroblast enhances MMP-1 secretion when co-cultured with human lung cancer cells in the 4D lung cancer model. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B31. doi:10.1158/1538-7445.CHTME14-B31

Abstract 1508: Gene expression analysis of perfusable nodules grown on ex vivo 3D model shows effects on extracellular matrix, cell signaling and immune response.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor microenvironment plays a very important role in regulating the cell behavior within a tissue, largely by interacting with the extracellular matrix (ECM). Recently, our lab has developed a novel ex vivo lung model that forms perfusable tumor nodules, which mimic human lung cancer histopathology and protease secretion patterns better than tumor cells grown on a petri dish. In this study, we have compared the genome-wide gene expression profiles (Affymetrix 430_2) and protein arrays (Reverse phase protein array or RPPA) of 2D (monolayer) culture versus our ex vivo 3D cell culture using 393p cells, derived from mice lung cancer. Expression array data showed a set 1314 gene probes differentially expressed between monolayer 2D culture and 3D ex vivo cell culture. Gene Ontology (GO) analysis of differentially expressed genes showed the upregulation of several genes associated with extracellular matrix (ECM1, BMP15, FMOD, cathepsin, MMP9, MMP10), cell signaling (VEGFA, TGFB2, FGF20), and immune response (C1, C3, MHC class II, IL1α, IL1RA, IL13, TNF receptor superfamily, CXCL2). Selected genes were also validated using real time PCR. We found 46 proteins with significant (p<0.01) differential levels in 2D vs 3D cell cultures of 393p. The proteins associated with extracellular matrix (Collagen VI, Caveolin1, Fibronectin) and cell signaling (mTOR, p38 (phosphorylated), Caspase 7 (Cleaved), BAD (phosphorylated), PI3K) were upregulated in 3D ex vivo cell culture compared to conventional 2D culture. Thus, our results showed the insightful influence of the 3D ex vivo model and its three-dimensional perfusable environment over cell growth and its behavior in terms of significant differences in gene expression and protein patterns as compared to the same cells grown on conventional 2D cell cultures. Citation Format: Dhruva K. Mishra, Chad Creighton, Yiqun Zhang, Don L. Gibbons, Jonathan M. Kurie, Min P. Kim. Gene expression analysis of perfusable nodules grown on ex vivo 3D model shows effects on extracellular matrix, cell signaling and immune response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1508. doi:10.1158/1538-7445.AM2013-1508