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2010;70:560-567. Published OnlineFirst January 12, 2010.Cancer ResDolores C. Garca-Olmo, Carolina Domnguez, Mariano Garca-Arranz, et al.Susceptible Cultured CellsCancer Patients Induce the Oncogenic Transformation ofCell-Free Nucleic Acids Circulating in the Plasma of Colorectal

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Molecular and Cellular Pathobiology

Cell-Free Nucleic Acids Circulating in the Plasma of

Colorectal Cancer Patients Induce the Oncogenic

Transformation of Susceptible Cultured Cells

Dolores C. Garca-Olmo1, Carolina Domnguez2, Mariano Garca-Arranz2, Phillipe Anker4,

Maurice Stroun4, Jos M. Garca-Verdugo5, and Damin Garca-Olmo3

Abstract

It has been proposed that cell-free nucleic acids in the plasma participate in tumorigenesis and the devel-

opment of metastases via transfection-like uptake of such nucleic acids by susceptible cells. This putative

phenomenon is tentatively referred to as genometastasis. In the present study, we examined the effects

on cultured cells of plasma from healthy individuals and from patients with colon cancer. Cultures of NIH-

3T3 cells and human adipose-derived stem cells (hASC) were supplemented with samples of plasma from

patients with K-rasmutated colorectal tumors or from healthy subjects using two different protocols: direct

addition of plasma to cultures in standard plates and addition in the absence of contact between plasma and

cells, which were separated by a membrane with 0.4-m pores. In plasma-treated hASCs, no K-rasmutated

sequences were detected by real-time PCR. In contrast, in most cultures of plasma-treated NIH-3T3 cells(murine cells), the transfer of human DNA occurred, as verified by the detection of human K-ras sequences,

p53 sequences, and -globinencoding sequences. Moreover, NIH-3T3 cells that had been cultured with

plasma from patients with colon cancer were oncogenically transformed, as shown by the development of

carcinomas in nonobese diabeticsevere combined immunodeficient mice after the injection of such cells.

Microscopic analysis of membranes that had separated plasma from cultured cells confirmed the complete

absence of cells in the plasma. We only observed noncell particles, having diameters of


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The present study was performed to show the biologicalfeasibility of gene transfer and of the transformation of cellsby cell-free tumor-derived nucleic acids in the plasma ofcancer patients. In our analysis, we examined the effects oncultured cells of plasma from healthy individuals and frompatients with colon cancer.

Materials and Methods

Patients and healthy subjects. We recruited 13 patientswith colon cancer who had undergone resection of primarycolorectal cancer in the Department of General Surgery at LaPaz University Hospital (Madrid, Spain) according to a pro-tocol approved by the Ethics Committee of the hospital. Clin-ical profiles are shown in Table 1.

In all cases, histopathologic analysis revealed that tumors

were adenoca rcin omas. Heal thy subj ects were recruitedaccording to normal protocols for such studies.

Detection ofK-ras mutations in tumors. In this study,

mutatedK-rassequences wereusedfor detection of genetrans-

fer phenomena. Therefore, we selected only those patientswhose tumors had such mutations. We examined samples of

individual tumors by real-time PCR and later confirmed our re-sults by sequencing. The purpose of the first test was to obtainresults in a short time so that plasma from the identified pa-

tients could be added to cell cultures within 24 h of collection.After the extraction of DNA with a DNeasy Tissu e kit

(Qiagen), samples were analyzed by real-time PCR with fluo-rescent hybridization probes to examine the presence ofmutations at codon 12 of the exon 1 of the K-ras gene (Glyto Asp, to Cys, or to Val). Briefly, in addition to specific pri-mers for amplification of a 164-bp fragment, we used twohybridization probes for each amplification. One of the two

probes (the sensor probe) was designed to be complemen-tary to a point mutation of interest and labeled at its 5 endswith LC-Red705 dye and phosphorylated at its 3 ends. Theanchor probe was labeled at its 3 end with fluorescein.Probes were manufactured by Tib Mobiol. In addition, weadded a peptide nucleic acid (PNA-oligomer) probe, as a

PCR-clamp, to suppress the amplification of the wild-typeK-ras sequence such that only mutated K-ras sequenceswould be amplified. PNA-oligomer encompassed codons 10to 14 and was obtained from Panagene.

We per formed all PCRs wit h the LightC ycl er Sys tem

(Roche Diagnostics) using LightCycler Software (version 3.5).When a tumor yielded positive results that corresponded to

one of the mutations of interest, the result was validated by

sequencing, and simultaneously, a sample of the correspond-ing plasma was added to cultures of human adipose-derivedstem cells (hASC) and NIH-3T3 cells, as noted below.

For sequencing, we first amplified a 286-bp sequence of theK-ras gene. Then, we prepared the reaction mixtures for se-quencing of the products of PCR using the BigDye Terminator

Cycle Sequencing v3.1 Ready Reaction kit (Applied Biosys-tems) following the manufacturer's instructions. Sequenceswere analyzed with a Genetic Analyzer (model 3130xl) and Se-quencing Analysis Software v5.2 from Applied Biosystems.

Isolation of human plasma. Samples of blood were col-lected, in tubes with EDTA, in the operating room immedi-ately before surgical resection of tumors. We obtained 15 mLof blood from each patient. All samples were subjected to cen-trifugation immediately after extraction at 1,800 gfor 10 min.Plasma was collected and subjected to a second centrifugationat 3,000 g for 10 min.

Det ectio n ofK-ras mutations in plasma. DNA wasextracted from plasma with a QIAamp DNA Blood Mini kit

Table 1. Main clinical characteristics of the patients with colorectal cancer whose plasma was added to

cultures of nontumor cells

Code Age (y) Gender Tumor location TNM Differentiation

Patients whose plasma was in contact with cultured cells

342 78 Male Sigmoid colon Not analyzed (irresectable tumor)

346 64 Male Rectum T3N2M0 Moderate

353 67 Female Sigmoid colon T4N1M0 Moderate

360 91 Female Right colon T3N1M0 Moderate

362 78 Male Sigmoid colon T3N1M0 Moderate

364 76 Female Left colon T3N1M0 Moderate

369 53 Male Right colon T3N2M0 Moderate378 70 Male Sigmoid colon T3N0M0 Colloid


399 83 Male Rectum T3N1M0 RT+CHT Interrupted


480 69 Male Recto-sigmoid T4N2M1 Moderate

Patient whose plasma was separated from cultured cells by a membrane


Abbreviations: TNM, tumor-node-metastasis; RT+CHT, radiotherapy and chemotherapy.

Circulating DNA Induces Oncogenic Transformation

Cancer Res; 70(2) January 15, 2010www.aacrjournals.org 561


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(Qiagen) according to the manufacturer's protocol for a sam-ple volume of 1 mL. Samples were analyzed by two real-timePCRs with fluorescent hybridization probes by the same pro-tocol as described for the analysis of DNA from tumors.

First, we amplified the total (mutated and nonmutated)K-ras sequences. For the second PCR, we added the PNA-

oligomer probe so that only mutated K-ras sequences wereamplified. When no mutated K-raswas detected, we assumedthat amplification of K-ras corresponded exclusively to theamplification of nonmutated DNA.

Culture of cells with human plasma. We used standard

NIH-3T3 cells and hASCs that had been isolated from lipoas-pirates from noncancer patients, as described previously (15).

The NIH-3T3 and hASCs cells were usually cultured in

monolayer in a mixture of 89% DMEM, 10% fetal bovine se-rum, and 1% penicillin plus streptomycin mixture (10,000units/mL plus 10,000 g/mL; Life Technologies). Cells werepassaged after dispersion in 0.05% (w/v) trypsin in EDTA.

We used two protocols for culturing of cells with plasma:direct addition of plasma to cultures in standard plates and

addition of plasma in the absence of direct contact with cellsin Transwell plates.

For direct culture of cells with human plasma, we usedplasma from 12 patients with colon cancer (Table 1) andfrom 9 healthy subjects. Each sample of plasma was addedto cultures of NIH-3T3 cells and of hASCs as follows. Cellswere dispersed and counted. Then, 100,000 NIH-3T3 cellsor hASCs were mixed, in culture medium, with 1.5 mL ofplasma and CaCl2 (0.7 mg/mL), which was added to promoteclotting so that cells would be trapped in a clot. Every 3 to4 d, we replaced the medium with fresh medium, without re-moving the plasma clot. After 10 d, clots were removed andcells were dispersed. Then, we maintained the cultures understandard conditions for 20 d more, testing cells for the pres-ence of mutated human K-rassequences by real-time PCR, asdescribed above, at each dispersion (every 34 d).

The plasma from one cancer patient and from one healthysubject was also used when we cultured NIH-3T3 cells andhASCs in Transwell plates (Corning) by the same protocolas described above. Plasma was placed above the insertand the cells were placed, in culture medium, on the bottom

of the plate such that a membrane with 0.4-m pores sepa-rated the plasma from the cells. Ten days later, each insertcontaining plasma was fixed for electron microscopy. Cells

were cultured for a further 20 d, with the presence of mutat-ed K-ras sequences examined at each dispersion.

In parallel with each experiment, we set up control cul-

tures with no added plasma (untreated cells).Detection of human p53 sequences by PCR. Murine cells

cultured with human plasma were subjected to an allele-specific PCR amplification test for detection of human

p53 sequences, as described previously (16). The test wasspecific for detection of the ARG/PRO polymorphism at co-don 72 of exon 4. In brief, all amplifications by PCR wereperformed with a minimum of 20 ng of template DNA in atotal volume of 20 L using specific primers for ARG- or PRO-encoding sequences. The products of amplification weresubjected to electrophoresis on a 3% agarose gel in Tris-

borate EDTA buffer (Pronadisa) that contained 2 g/mL ethi-dium bromide.

Detection of human -globinencoding sequences by

PCR. A PCR amplification test was performed by a previouslydescribed protocol to detect human -globinencoding se-quences (17) in murine cells that hadbeen cultured with human

plasma. Amplifications by PCR of a 249-bp fragment were per-formed with a minimum of 20 ng of template DNA in a total

volume of 50 L. The products of amplification were subjectedto electrophoresis on a 2% agarose gel, as described above.

Detection of mutated murine k-ras sequences by mutant

allele-specific amplification. We performed mutant allele-specific amplification (MASA) by PCR, as previously describedfor rat sequences (11) and modified for the detection of mouse

DNA. Using this technique, we examined the presence of theGly12Asp mutation in exon 1 of the k-rasoncogene. We am-plified a fragment of 140 bp. In brief, the reaction mixture forPCR, with a total volume of 50 L, contained a minimum of 20ng of template DNA. To avoid unspecific reactions (false-pos-itive results), the amount of DNA did not exceed 250 ng. The

products of amplification were subjected to electrophoresison a 3% agarose gel, as described above.

To ensure the validity of the technique, all samples of DNAwere also amplified to examine the presence of nonmutatedk-ras under the same conditions.

Electron microscopy. Inserts of Transwells were fixed in3.5% glutaraldehyde for 30 min at 37C, postfixed in 2% os-mium tetroxide for 2 h, rinsed, dehydrated, and embedded inAraldite (Durcupan, Fluka BioChemika). We cut serial 1.5-msemithin sections with a diamond knife and stained themwith 1% toluidine blue for localization of pores. For the iden-tification of cells and particles within membranes, we cut ul-trathin (0.05 m) sections with a diamond knife, stainedthem with lead citrate, and examined them under an FEITecnai spirit electron microscope.

Assessment in vivo of the tumorigenicity of cells that

had been cultured with human plasma. Nine-week-old

male nonobese diabeticsevere combined immunodeficient(NOD-SCID) mice (Charles River Laboratories) were used.Breeding and care of animals were undertaken in compliancewith European Community Directive 86/609/CEE for the useof laboratory animals and with Spanish law (Real Decreto1201/2005).

Two million cells were injected s.c. into each mouse. Fouranimals were injected with untreated NIH-3T3 cells; two withNIH-3T3 cells that had previously been directly cultured withplasma from healthy subjects; four with NIH-3T3 cells that

had previously been directly cultured with plasma from can-cer patients; and two with NIH-3T3 cells that had previouslybeen cultured with plasma from a cancer patient and ahealthy subject, respectively, in Transwell plates. We also in-jected three mice with human colon adenocarcinoma cells(SW-480 cells) as a positive control. The SW-480 cells havea point mutation (Gly to Val) at codon 12 of exon 1 of the

K-ras oncogene.The growth of s.c. tumors was monitored in all animals and

recorded weekly. We measured the greatest diameter of eachtumor with electronic calipers. Animals were sacrificed 32 to

Garca-Olmo et al.

Cancer Res; 70(2) January 15, 2010 Cancer Research562




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36 d after injection of cells. Before sacrifice, mice were anes-thetized with 2.5% to 3% isoflurane. Then, blood (approxi-mately 0.71.0 mL per mouse) was withdrawn by cardiacpuncture. All samples of blood were collected in tubes withEDTA and subjected to two centrifugations, as described forhuman samples, for the preparation of plasma. Blood and tu-

mors were analyzed as described above.

Results

Analysis by PCR of cells cultured with human plasma.

All samples of plasma from patients with colorectal cancerand used for culturing cells were positive for mutations incodon 12 of the exon 1 of the K-ras oncogene. The amount

of K-rasmutated DNA in the plasma ranged from 0.7 to5.5 fg/mL (2.3 1.5 fg/mL; mean SD).

In hASCs to which human plasma had been added directly,

no mutated K-rassequences were detected by real-time PCR.In contrast, in 7 of 12 cultures of NIH-3T3 cells (murine cells),we detected human mutated sequences from the first to the

third dispersion until the end of the experiment (Fig. 1). In 4of the 12 cultures, mutated DNA was detected intermittently,and in 1 of the 12 cultures, cells died before the first disper-sion. We repeated this last culture with the same result. NIH-3T3 cells that had been cultured with plasma from healthysubjects were positive, in all cases, for human K-ras se-quences and negative for mutated K-ras sequences. Thus,they harbored only nonmutated K-rassequences. No cultureof NIH-3T3 cells was positive for mutated murine K-ras se-quences when examined by the MASA technique.

We detected human p53 sequences and human -globinencoding sequences in six of eight samples of NIH-3T3 cellsthat had been cultured with plasma from cancer patients. Weinvestigated the presence of p53 sequences in untreatedhASCs and found only the ARG-encoding triplet at codon72 of exon 4. Thus, we were able to examine whether thePRO-encoding sequence, which was present in the DNA inthree of the samples of plasma added to cells, had beentransferred to cells. We performed PCR to analyze the DNAfrom hASCs that had been cultured with patients' plasma

and we failed to detect any PRO-encoding sequence.Analysis of humanK-rassequences in cells cultured with hu-

man plasma in Transwell plates yielded similar results to thosedescribed above for direct cultures. Specifically, we detected

mutated K-ras sequences in NIH-3T3 cells but not in hASCs.Microscopic analysis of membranes of Transwell that

had contained plasma. Microscopic analysis of the inserts

of Transwell plates that had contained plasma confirmedthe complete absence of cells. We observed only particles(Fig. 2), many of which seemed to be passing through thepores of the membrane of the insert. Thus, such particleshad diameters of


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As descr ibed for tis sue sample s, we detec ted hum ansequences in the buffy coat from all animals, with the excep-tion of the animals injected with untreated NIH-3T3 cells.Moreover, mutated K-rassequences (to derived, presumably,from circulating tumor cells) were found only in the bloodfrom mice that had been injected with SW-480 cells.

In an analysis of plasma samples, we detected mutated hu-man K-rassequences in the mice that had been injected withSW-480 cells and in the mice that had been injected withNIH-3T3 cells that had previously been cultured with plasmafrom cancer patients (Fig. 4). No human sequences were de-tected in the plasma from the animals that had been injectedwith cells cultured with plasma from healthy subjects.

We also found a tumor in the mouse that had been in-jected with NIH-3 T3 cells that had been cultu red withplasma from a cancer patient in a Transwell plate. Thistumor, with a final maximum diameter of 1 cm, was pos-itive for mutated human K-ras sequences. No mass wasfound in the mouse injected with NIH-3T3 cells that hadbeen cultured with plasma from a healthy subject in aTranswell plate. Identical to above described results, we

detected human K-ras sequences in all samples of tissuesfrom the two animals, but mutated K-ras sequences wereonly detected in the samples obtained from the mouse

that was injected with cells that had previously been cul-tured with plasma from a cancer patient.

Discussion

In the present study, two lines of cells (i.e., NIH-3T3 cellsand hASCs) were cultured with plasma from patients withcolorectal cancer and from healthy subjects. NIH-3T3 cells,derived from mouse embryo fibroblasts, are p16-deficientcells that have frequently been used in studies of oncogenictransformation (18, 19). Disruption of the expression of thep16/Rb pathway in rodent cells is extremely efficient in pre-venting Ras-induced growth arrest (18) . hASCs are well-established lines of pluripotent stem cells that are often used

in clinical trials6 as a consequence of their excellent safetyprofile (20).

In the cultures of hASCs treated with plasma from patientswith K-rasmutated colorectal tumors, we never detectedmutated K-ras sequences by real-time PCR. In contrast, inmost such cultures of NIH-3T3 cells, we detected mutatedhuman sequences soon after the start of incubation (fromthe first to the third dispersion) and these sequences werestill detectable at the end of the experiment, 3 weeks afterremoval of human plasma from the culture medium. NIH-3T3 cells that had been cultured with plasma from healthysubjects were negative, in all cases, for mutated human

K-ras sequences and positive for nonmutated K-ras se-quences. Thus, it seemed that, whereas hASCs were resistantto transformation by cell-free tumor-derived DNA in patients'plasma, NIH-3T3 cells (p16-deficient cells) were able to stablyincorporate such foreign DNA during simple incubation withplasma from cancer patients.

As positive controls for transfection of cells with tumorDNA, we set up cultures of hASCs and NIH-3T3 cells supple-mented with a solution of DNA that had been extracted from

K-rasmutated tumors and CaCl2. The results were similarto those obtained when cells were cultured with plasma:we detected mutated K-ras sequences in NIH-3T3 cells but

not in hASCs (data not shown). However, when we quantifiedthe amounts of mutated K-rassequences in the samples usedfor the various incubations, we found that the amount was

almost 1 order of magnitude higher in the case of DNA fromtumors (30.7 19.3 fg; mean SD) than in plasma (3.5 2.3 fg). The low concentrations of DNA in the plasma andthe fact that such sequences were incorporated into cellsas a result of the simple addition of plasma to cell cultures,without prior extraction of DNA, suggest the considerableefficiency of the transfection phenomenon when the DNAis added and originates in plasma.

Figure 2. Electron micrographs of the insert of a Transwell plate. Plasma was placed above the insert, and NIH-3T3 cells were placed in the bottom

of the plate so that there was a membrane with 0.4-m pores between the plasma and the cells. Ten days later, the insert was removed, fixed, and sectioned

for electron microscopy. No cells were detected in the plasma, but many unidentified particles of


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When we incubated six plates of NIH-3T3 cells with DNAthat had been extracted from buffy coat samples fromhealthy subjects, we detected human K-rassequences in onlythree of the cultures (data not shown). Thus, the rate oftransfection with the purified DNA seemed to be lower thanafter the addition of human plasma.

We detected the p53 human sequences and human -globinencoding sequences in five of six samples of cells thathad been cultured with plasma from cancer patients. In con-trast, DNA from none of the genes examined was transferredto hASCs during culture with plasma from cancer patients.

Thus, the transfer of DNA to murine NIH-3T3 cells was notrestricted to tumor-related DNA sequences. Moreover, itseemed that humanization of murine cells had occurred

during incubation with plasma from cancer patients. Theseresults are coherent with those of a previous study in whichauthors showed that mouse fibroblasts, which were trans-formed by transfection with human tumor DNA, acquirednot only the transformation genes but also a large array ofother cotransfected human DNA fragments (21). This simili-

tude also supports the idea that transfection provoked by thesimple addition of plasma to cell cultures is strongly similar,although more efficient, to that obtained by transfection withDNA extracted from tumors.

Having shown the apparent transfer of human DNA toNIH-3T3 cells, we examined whether this DNA could recom-bine with the DNA of the host cell to generate a similar mu-tation within the DNA of the host. Using a MASA technique(11), we examined the presence of mutated murine K-rassequences in the NIH-3T3 cells that had been cultured withplasma from K-rasmutated cancer patients. No cultureswere positive for such K-ras mutation.

NIH-3T3 cells that had previously been cultured with plasmafrom cancer patients generated undifferentiated carcinomaswhen injected into NOD-SCID mice (Figs. 3 and 4). Moreover,mutated human K-ras sequences were detected in distantparenchyma (liver and lungs) and in plasma from such mice,as also observed when human colon adenocarcinoma cells(SW-480 cells), as positive controls, were injected into NOD-SCID mice. Thus, our results indicated that, when NIH-3T3 cellswere cultured with plasma from patients with colon cancer,

the cells acquired the potential for oncogenic activity in vivo.Moreover, this activity was similar to that of a standard lineof colon cancer cells. NIH-3T3 cells are normally unable to pro-duce tumors spontaneously in NOD-SCID mice (as confirmedhere), but they do have a genetic alteration (p16 deficiency)that confers elevated susceptibility to transformation by ras,

as reported previously (18). Incubation with plasma fromcancer patients provoked a definitive change in the cells, medi-ated perhaps by gene transfer, which resulted in oncogenic

Figure 3. Photographs of a mouse that had been injected s.c. with

NIH-3T3 cells that had been cultured with plasma from a cancer

patient. The mouse was sacrificed 36 d after inoculation (when the

photographs A and B were taken). C and D, sections of the tumor

were stained with H&E and examined by light microscopy. Original

magnification, 10 (C) and 40 (D). Light micrographs show that

the tumor was an epithelial carcinoma in which undifferentiated regions

were mixed with necrotic tissue.






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transformation.Thus, previous reportsthat plasmafrom cancerpatients can transform susceptible cells (9, 10) were confirmed.

The oncogenic transformation of NIH-3T3 cells occurredin an identical manner no matter whether cells were in directcontact with plasma or were separated from the plasma by amembrane with 0.4-m pores. Moreover, electron microsco-py failed to reveal any cells on or in the membrane of theinsert. We observed only particles (Fig. 2), many of whichseemed to be passing through the pores of the membraneof the insert and had, therefore, diameters of


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