Small cell and non small cell lung cancer form metastasis on cellular 4D lung model.

BACKGROUND: Metastasis is the main cause of death for lung cancer patients. The ex vivo 4D acellular lung model has been shown to mimic this metastatic process. However, the main concern is the model's lack of cellular components of the tumor's microenvironment. In this study, we aim to determine if the intact lung microenvironment will still allow lung cancer metastasis to form. METHODS: We harvested a heart-lung block from a rat and placed it in a bioreactor after cannulating the pulmonary artery, trachea and tying the right main bronchus for 10-15 days without any tumor cells as a control group or with NSCLC (A549, H1299 or H460), SCLC (H69, H446 or SHP77) or breast cancer cell lines (MCF7 or MDAMB231) through the trachea. We performed lobectomy, H&E staining and IHC for human mitochondria to determine the primary tumor's growth and formation of metastatic lesions. In addition, we isolated circulating tumor cells (CTC) from the model seeded with GFP tagged cells. RESULTS: In the control group, no gross tumor nodules were found, H&E staining showed hyperplastic cells and IHC showed no staining for human mitochondria. All of the models seeded with cancer cell lines formed gross primary tumor nodules that had microscopic characteristics of human cancer cells on H&E staining with IHC showing staining for human mitochondria. CTC were isolated for those cells labeled with GFP and they were viable in culture. Finally, all cell lines formed metastatic lesions with cells stained for human mitochondria. CONCLUSION: The cellular ex vivo 4D model shows that human cancer cells can form a primary tumor, CTC and metastatic lesions in an intact cellular environment. This study suggests that the natural matrix scaffold is the only necessary component to drive metastatic progression and that cellular components play a role in modulating tumor progression.

Breast cancer cells form primary tumors on ex vivo four-dimensional lung model.

BACKGROUND: Breast cancer mortality is most common in cancer in women, and there are no ex vivo models that can capture the primary growth of tumor with fidelity to the in vivo tumor growth. In this study, we grew human breast cancer cell lines in an acellular lung matrix of the ex vivo four-dimensional lung model to determine if they form primary tumor and the extent to which they mimic the histology and characteristics of the human tumors. MATERIALS AND METHODS: Rat lungs were harvested, decellularized, and placed in a bioreactor. To study the primary tumor growth, we seeded the lung via the trachea with human breast cancer cells SUM159, MCF7, or MDMB231 and perfused the pulmonary artery with oxygenated media. Lobectomies were performed and processed for hematoxylin and eosin, Ki-67, caspase-3, estrogen receptor, and progesterone receptor antibodies. RESULTS: All three cell lines grew in the ex vivo four-dimensional model and formed perfusable tumor nodules with similar histology and morphology as the primary tumors. SUM159 and MDAMB231 showed higher proliferation and apoptotic indices than MCF7. In addition, MCF7 retained its estrogen receptor and progesterone receptor positivity, whereas SUM159 and MDAMB 231 did not have any staining. CONCLUSIONS: Overall, our study showed that human breast cancer cells can be grown on the ex vivo four-dimensional lung model, which then form primary tumor nodules that mimic the morphology and histology of the original tumor.

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.

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.

Vitamin D receptor FokI gene polymorphisms may be associated with colorectal cancer among African American and Hispanic participants.

BACKGROUND: Vitamin D plays a role in cancer tumorogenesis and acts through the vitamin D receptor (VDR). Although African Americans have the lowest serum vitamin D levels, supplementation has not yielded a significant improvement in cancer. Gene polymorphisms in VDR may play a role. There is a dearth of information on VDR gene polymorphisms and colorectal cancer (CRC) among under-represented ethnic groups. In this study, the authors examined whether VDR gene single nucleotide polymorphisms (SNPs) were associated with CRC in predominately African American and Hispanic study participants. METHODS: Blood samples were collected from 378 participants, including a group of 78 patients with CRC (cases), a group of 230 noncancer participants without polyps (controls without polyps), and a group of 70 noncancer participants with polyps (controls with polyps). The 4 polymorphic SNPs in VDR (FokI, BsmI, TaqI, and ApaI) were assessed using the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: There was a significant association of the VDR-FokI FF genotype with CRC cases (odds ratio, 2.9; P= .036) compared with the controls without polyps. The most common VDR-FokI genotype in the overall study population was the FF genotype (46%). However, upon breakdown by ethnicity, the FF genotype was the most common in African American participants (61%), and the Ff genotype was the most common in Hispanic/Latino participants (49%). When the association was assessed in a multivariate model, there was no significant association with any VDR polymorphism and CRC cases (P> .05). The other 3 polymorphic variants of VDR (BsmI, TaqI, and ApaI) were not associated with CRC. CONCLUSIONS: The results from this study suggest that genetic variation of the VDR-FokI SNPs may influence CRC risk, particularly in African American cohorts.

Transcriptional activators YAP/TAZ and AXL orchestrate dedifferentiation, cell fate, and metastasis in human osteosarcoma.

Osteosarcoma (OS) is a molecularly heterogeneous, aggressive, poorly differentiated pediatric bone cancer that frequently spreads to the lung. Relatively little is known about phenotypic and epigenetic changes that promote lung metastases. To identify key drivers of metastasis, we studied human CCH-OS-D OS cells within a previously described rat acellular lung (ACL) model that preserves the native lung architecture, extracellular matrix, and capillary network. This system identified a subset of cells-termed derived circulating tumor cells (dCTCs)-that can migrate, intravasate, and spread within a bioreactor-perfused capillary network. Remarkably, dCTCs highly expressed epithelial-to-mesenchymal transition (EMT)-associated transcription factors (EMT-TFs), such as ZEB1, TWIST, and SOX9, which suggests that they undergo cellular reprogramming toward a less differentiated state by coopting the same epigenetic machinery used by carcinomas. Since YAP/TAZ and AXL tightly regulate the fate and plasticity of normal mesenchymal cells in response to microenvironmental cues, we explored whether these proteins contributed to OS metastatic potential using an isogenic pair of human OS cell lines that differ in AXL expression. We show that AXL inhibition significantly reduced the number of MG63.2 pulmonary metastases in murine models. Collectively, we present a laboratory-based method to detect and characterize a pure population of dCTCs, which provides a unique opportunity to study how OS cell fate and differentiation contributes to metastatic potential. Though the important step of clinical validation remains, our identification of AXL, ZEB1, and TWIST upregulation raises the tantalizing prospect that EMT-TF-directed therapies might expand the arsenal of therapies used to combat advanced-stage OS.

Acellular and Cellular Lung Model to Study Tumor Metastasis.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Immune cells inhibit the tumor metastasis in the 4D cellular lung model by reducing the number of live circulating tumor cells.

The immune system and tumor microenvironment play a decisive role in tumor progression. We developed a novel model to better understand tumor progression and interaction with immune cells and the cellular components. We grew 393 P non-metastatic and 344SQ metastatic murine cells in an acellular metastatic lung cancer model, where both cell lines formed circulating tumor cells (CTC) and metastatic lesions. When the CTC from this model were placed in the tail vein of nu/nu mice, both cell lines formed metastatic lesions. However, in syngeneic immune-competent mice, the CTC from the non-metastatic cell line did not metastasize while the CTC from the metastatic cell line metastasized. When we placed the activated immune cells in the cellular lung model, it decreased CTC and metastatic lesion formation for the non-metastatic cell line while it had no impact on metastatic cell line. The metastatic cell line had a significant increase in expression of programmed death-ligand 1 (PDL-1) compared to the non-metastatic cell line in the model. Overall, the immune cells showed an impact on viability of CTC for cell lines with a decreased expression of PDL-1 that leads to decreased metastatic lesion formation. Further studies are needed to understand the subtype of immune cells and mechanism of decreased CTC viability and metastasis inhibition.

Is there an inter-relationship between prostate specific antigen, kallikrein-2 and androgen receptor gene polymorphisms with risk of prostate cancer in north Indian population?

Androgen receptor (AR) and kallikrein (KLK-2) regulates the PSA (prostate specific antigen) transcription and activation, respectively. We investigated the individual and combined risk of KLK-2, PSA and AR gene polymorphism in histologically confirmed CaP patients and healthy controls from north India. DNA was extracted from peripheral blood leucocytes pellet of 277 subjects. AR repeats analysis was done by PCR-Genscan method. PSA and KLK-2 were genotyped by PCR-RFLP method. Kruskal-Wallis test and logistic regression was applied for mean comparison and risk determination. A significant association for CaP risk was observed with short AR-CAG repeats (OR=3.36, p<0.001) and CC genotype of KLK-2 (OR=2.78, p=0.031), however, no association was found with PSA and AR-GGN repeat polymorphism. PSA/GG genotype was significantly associated with higher Gleason score (> or =7) of tumor (OR=6.23, p<0.01). No association was observed with other confounding variables such as PSA and age with any of these polymorphisms. Thus, we hypothesize that these polymorphisms may influence the etiology of CaP and may have the probability to become appropriate marker either independently or in combination. The combined information on serum PSA level, PSA (G/A), KLK-2 (C/T) genotypes and AR (CAG; GGN repeat) may assist in the deterrence of unnecessary biopsies.

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.