Marrow cell genetic phenotype change induced by human lung cancer cells

Abstract

Microvesicles have been shown to mediate varieties of intercellular communication. Work in murine species has shown that lung-derived microvesicles can deliver mRNA, transcription factors, and microRNA to marrow cells and alter their phenotype. The present studies evaluated the capacity of excised human lung cancer cells to change the genetic phenotype of human marrow cells. We present the first studies on microvesicle production by excised cancers from human lung and the capacity of these microvesicles to alter the genetic phenotype of normal human marrow cells. We studied 12 cancers involving the lung and assessed nine lung-specific mRNA species (aquaporin, surfactant families, and clara cell-specific protein) in marrow cells exposed to tissue in co-culture, cultured in conditioned media, or exposed to isolated lung cancer-derived microvesicles. We assessed two or seven days of co-culture and marrow which was unseparated, separated by ficoll density gradient centrifugation or ammonium chloride lysis. Under these varying conditions, each cancer derived from lung mediated marrow expression of between one and seven lung-specific genes. Microvesicles were identified in the pellet of ultracentrifuged conditioned media and shown to enter marrow cells and induce lung-specific mRNA expression in marrow. A lung melanoma and a sarcoma also induced lung-specific mRNA in marrow cells. These data indicate that lung cancer cells may alter the genetic phenotype of normal cells and suggest that such perturbations might play a role in tumor progression, tumor recurrence, or metastases. They also suggest that the tissue environment may alter cancer cell gene expression.

Figure 1

Experimental design. After surgery, the lung tumor sample was placed into a 0.4 um impermeable filter insert and co-cultured with bone marrow cells. After 2 or 7 days, the bone marrow cells were harvested and their RNA was isolated and analyzed using reverse transcriptase polymerase chain reaction (RT-PCR).

Figure 2

Lung tumor samples co-cultured with human bone marrow cells for 2 or 7 days. Colors indicate fold increases of lung-specific mRNA levels as determined by reverse transcriptase polymerase chain reaction (RT-PCR) when compared to marrow cells cultured without lung tumor. Graph illustrates bone marrow cells that were co-cultured with lung tumor for (A) 2 days or (B) 7 days. The following bone marrow cell populations were used in co-culture: WBM = Whole bone marrow; Ficoll BM = Ficoll-separated bone marrow cells; ACL2 = bone marrow cells with red blood cells removed using an ammonium chloride-based lysis buffer.

Figure 3

Conditioned media or lung tumor-derived microvesicles co-cultured with human bone marrow cells for 7 days. (A) Bone marrow cells co-cultured with conditioned media, or (B) lung tumor-derived microvesicles. Colors indicate fold increases of lung-specific RNA levels as determined by reverse transcriptase polymerase chain reaction (RT-PCR) when compared to marrow cells cultured without conditioned media or microvesicles.

Figure 4

Digital images of bone marrow cells containing microvesicles. Examples of bone marrow cells containing PKH26 and CFSE labeled microvesicles are shown (A–C). (A) Composite image of one bone marrow cell taken through DAPI, FITC, and Texas Red filters. Blue color is DAPI (nuclear counter stain). (B, C) Images of the same cell taken through FITC and Texas Red filters, respectively. (D) Example of DAPI counterstained cell that has not taken up microvesicles image taken using DAPI, FITC, and Texas Red filters. Fluorescent images were taken with a Zeiss Axio Imager Z1 fluorescent microscope at 63 × magnification and Axiovision 4.6.3 software. Digital images were acquired using an AxioCam HRm. Three-dimensional images were taken utilizing a Zeiss ApoTome for structural imaging and a four-dimensional acquisition module.

Figure 5

Transmission electron microscope images of lung tumor-derived microvesicles. Lung tumor sample from patient #8 was cultured without bone marrow cells for 7 days, the conditioned media removed and microvesicles were isolated by high speed centrifugation. (A) Microvesicles, 44,000 × magnification, (B) 56,000 × magnification, (C) 71,000× magnification. Magnification bars are all equal to 100 nm. (D) Hematoxylin and eosin (H&E) stained pathology slide showing lung tumor cells from the same patient, 600 × magnification. Electron microscope sections were examined using a Morgagni 268-transmission electron microscope, and images were collected with an AMT Advantage 542 CCD camera system.