Exosomes isolated from cancer patients’ sera transfer malignant traits and confer the same phenotype of primary tumors to oncosuppressor-mutated cells
© The Author(s). 2017
Received: 13 July 2017
Accepted: 24 August 2017
Published: 30 August 2017
Horizontal transfer of malignant traits from the primary tumor to distant organs, through blood circulating factors, has recently become a thoroughly studied metastatic pathway to explain cancer dissemination. Recently, we reported that oncosuppressor gene-mutated human cells undergo malignant transformation when exposed to cancer patients’ sera. We also observed that oncosuppressor mutated cells would show an increased uptake of cancer-derived exosomes and we suggested that oncosuppressor genes might protect the integrity of the cell genome by blocking integration of cancer-derived exosomes. In the present study, we tested the hypothesis that cancer patients’ sera-derived exosomes might be responsible for the malignant transformation of target cells and that oncosuppressor mutation would promote their increased uptake. We also sought to unveil the mechanisms behind the hypothesized phenomena.
We used human BRCA1 knockout (BRCA1-KO) fibroblasts as target cells. Cells were treated in vitro with cancer patients’ sera or cancer patients’ sera-derived exosomes. Treated cells were injected into NOD-SCID mice. Immunohistochemical analyses were performed to determine the differentiation state of the xenotransplants. Mass spectrometry analyses of proteins from cancer exosomes and the BRCA1-KO fibroblasts’ membrane were performed to investigate possible de novo expression of molecules involved in vesicles uptake. Blocking of the identified molecules in vitro was performed and in vivo experiments were conducted to confirm the role of these molecules in the malignant transformation carried out by cancer-derived exosomes.
Cells treated with exosomes isolated from cancer patients’ sera underwent malignant transformation and formed tumors when transplanted into immunodeficient mice. Histological analyses showed that the tumors were carcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression, on the plasma membrane of target cells, of receptors, responsible for the increased uptake of cancer-derived exosomes. The selective blocking of these receptors inhibited the horizontal transfer of malignant traits.
These findings strengthen the hypothesis that oncogenic factors transferred via circulating cancer exosomes, induce malignant transformation of target cells even at distance. Oncosuppressor genes might protect the integrity of the cell genome by inhibiting the uptake of cancer-derived exosomes.
KeywordsCancer patients’ serum Exosomes Malignant transformation Horizontal transfer Tumor suppressor genes Genometastasis Phenotypical differentiation
Metastasis is the leading cause of cancer-related mortality [1, 2]. It is traditionally described as a process involving detachment of cancer cells, from the primary tumors, travelling in the blood stream, and homing at metastatic sites . This paradigm has been challenged by recent studies reporting that cancer cells-derived factors could either prepare a niche to permit the engraftment of malignant cancer cell in distant organs or predispose target cells, located in distant organs, to their malignant transformation [4–9]. Horizontal transfer of malignant traits was first reported in immortalized mouse fibroblasts exposed to the serum of cancer patients’ plasma and was called “genometastasis” [10–12]. These observations were for the first time validated by our group in human-derived cell lines. We reported that immortalized human embryonic kidney (HEK293) cells as well as oncosuppressor gene-deficient human cells (BRCA1-KO fibroblasts) undergo malignant transformation when exposed to cancer patients’ sera [13, 14]. Histopathological analyses of the excised tumors, following injection of the treated HEK293 cells into immunodeficient mice, showed that the types of tumors grown were not dependent from the patient’s type of cancer. The histology confirmed that all tumors were poorly differentiated carcinomas as further attempts to characterize the tumors immunohistochemically, failed to show any more differentiating features . In contrast, when we treated BRCA1-KO fibroblasts with sera of patients with colon or pancreatic cancer, histological analyses of the tumors generated, showed that they were poorly differentiated adenocarcinomas with phenotypical characteristics related to the cancers of the blood donor patients, proving, for the first time, that complete metastatic transformation, through horizontal transfer, is possible .
Horizontal transfer of malignant traits involves factors (i.e. proteins or nucleic acids) that could be either circulating as free molecules or circulating as exosomes-packed cargo [5, 7, 15–21]. Exosomes form from cellular endosomal compartment under both physiological and pathological conditions [22–24], and contain in their lumen molecules that mirror the content of their cell of origin [25–27]: for instance, exosomes shed by cancer cells contain oncogenic drivers [9, 28–31]. By delivering their cargo into target recipient cells, either by autocrine, paracrine, or endocrine pathways, exosomes affects cells’ functions (i.e. cells growth and clonogenicity) [4, 8, 23, 32, 33].
Following our observation that cells carrying oncosuppressor mutations, display a significantly increased uptake of cancer-derived exosomes, we suggested that cancer-derived exosomes might carry the oncogenic information through the blood and be responsible for the malignant transformation of the target cells. We hypothesized that oncosuppressor genes might protect the integrity of the cell genome not only by repairing DNA damages and controlling cell cycle checkpoints, but also by blocking the uptake of oncogenic traits contained in cancer exosomes and thus preventing cell transformation . In this study, we sought to validate our hypotheses and therefore we aimed to establish if cancer patients’ sera-derived exosomes were responsible for the transfer of malignant traits and determine if a complete phenotypical transformation would be seen with sera from patients with types of cancers other than colon and pancreas. We also attempted to confirm both the role of oncosuppressor genes in cancer-derived exosomes uptake and unveil the mechanism of action behind this hypothesized function.
Cells treated with cancer patients’ sera-derived exosomes underwent malignant transformation as judged by their ability to form tumors in immunodeficient mice. Histological analyses showed that treated cells had changed completely their fate and the growing tumors were adenocarcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression of receptors, on the plasma membrane of target cells, responsible for the increased uptake of cancer-derived exosomes. The selective blocking of these receptors inhibited the horizontal transfer of malignant traits.
Patients’ recruitment and characteristics of cancers
Clinical features of patients recruited and the phenotype characterization of the xenografts obtained with cancer patients’ serum-treated cells
Xenograft Phenotype (Pathology Report)
Convincing differentiation toward intestinal adenocarcinoma
CEA, CK20, CDX-2, and AE1/AE3
HCC differentiation is pretty convincing
AFP, CK8/18, and CEA
Differentiation toward typical pancreatic ductal adenocarcinoma
CK7, CK19, and AE1/AE3
Ovarian cancer differentiation can be appreciated
CK20, WT-1, p53, and EMA
Convincing differentiation toward intestinal adenocarcinoma
CEA, CK20, CDX-2, and AE1/AE3
HCC differentiation can be appreciated although Hep par-1 is negative
AFP, CK8/18, and CEA (canalicular pattern)
Differentiation toward typical pancreatic ductal adenocarcinoma
CK7, CK19, and AE1/AE3
Ovarian cancer differentiation can be appreciated
CK20, WT-1, p53, and EMA
Blood collection and serum preparation from cancer patients and healthy subjects
Blood samples (20 ml) were collected from a peripheral vein in vacutainer tubes (Becton Dickinson) containing clot-activation additive and a barrier gel to isolate serum. Blood samples were incubated for 60 min at room temperature to allow clotting and subsequently were centrifuged at 1500 x g for 15 min. Serum was collected and a second centrifugation was performed on the serum at 2000 x g for 10 min, to clear it from any contaminating cells. Serum samples were aliquoted and stored at −80 °C until use.
Cell line and culture conditions
We used the CRISPR/Cas9 system to establish a stable BRCA1-KO in human fibroblasts as previously described . Cells were maintained as per supplier’s recommendations. When cells reached 30% confluence, they were treated with DMEM-F12 medium (Wisent, Saint-Bruno, Canada) supplemented with antibiotics and 10% cancer patient sera or control sera, which had been filtered through 0.2 μm filters. Otherwise, cells were exposed to an exosome load of 25–40 μg/ml (which corresponded to 2.6–4.1e + 07 particles/ml of culture medium. Cells were maintained in these conditions at 37 °C in humidified atmosphere containing 95% air and 5% CO2 with medium change every second day for 3 weeks. When cells reached 80–90% confluence, they were passaged 1 in 6 using 0.05% Trypsin-EDTA (Wisent, Saint-Bruno, Canada). To confirm that there was no contamination or carry-over of cells from human serum, aliquots of the culture medium were placed in a culture plate and incubated at 37 °C, 5% CO2 for 4 weeks.
Exosomes isolation and labeling
Exosomes were isolated from serum using the Total Exosome Isolation Kit according to the manufacturer’s protocol (Invitrogen, Burlington, Canada). Exosomes were labeled using the PKH26 dye following the manufacturer recommendations (Sigma, Oakville, Canada). Labeled exosomes were diluted in labeling stop solution (PBS/FBS) and pelleted by ultra-centrifugation for 80 min at 100,000 x g at 4 °C. The pellet was washed in Hank’s Balanced Salt Solution (HBSS) with an ultra-centrifugation using the same parameters. The pelleted exosomes were re-suspended in HBSS and stored at −80 °C. 10 μg of labeled exosomes was added to cells (~5 × 103) maintained in 8-well chamber slides (VWR, Mont-Royal, Canada). Cells were washed, fixed for 10 min with Paraformaldehyde 4%. Slides were mounted with coverslip in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlington, Canada). Stained cells were visualized using an LSM780 confocal microscope (Zeiss, Toronto, Canada). Exosomes internalization was quantified using ImageJ software. Where mentioned, a batch of cells was also analyzed by flow cytometry. Cells were acquired using a FACSCalibur flow cytometer (Becton-Dickinson) at a flow rate of ~300 cells/s. Dead cells and cell debris were excluded from acquisition by gating intact cells on a FCS and SSC biparametric plot.
Morphological examination of isolated exosomes was done using transmission electron microscope (JEM-2010, Jeol Ltd., Tokyo, Japan). Briefly, 20 μl of exosomes were loaded on a copper grid and stained with 2% phosphotungstic acid. Samples were dried by incubating them for 10 min under an electric incandescent lamp. Samples were examined under electron microscope and imaged using a Hitachi H-600 TEM operating at 60 kV. In parallel, an aliquot of exosome samples was run on a Nanosight NS500 system (Nanosight Ltd., Amesbury, UK), and size distribution was analyzed using the NTA 1.3 software.
Cells and pelleted exosomes were lysed in RIPA buffer containing protease inhibitors (Sigma, Oakville, Canada). Equal amounts of proteins were resolved on 10% SDS-PAGE and transferred to a nitrocellulose membrane (BioRad, CA, USA). Membranes were blocked in TBS (20 mM Tris, 150 mM NaCl, pH. 7.6) containing 5% non-fat dry milk and exposed overnight at 4 °C to rabbit-anti-GM130 (ab52649) and mouse-anti-TSG101 (ab83) (Abcam, MA, USA), and mouse-anti-Alix (2127, Cell Signaling, MA, USA). Membranes were washed in TBST (TBS-0.05% Tween-20) and incubated with either anti-rabbit or anti-mouse peroxidase-conjugated secondary antibody for 1 h at room temperature. After several washes in TBST, the blots were developed using Immobilon Western HRP Substrate (Millipore, Etobicoke, Canada).
Proteins preparation and mass spectrometry
Plasma membranes proteins were enriched from fibroblast lysates (control vs. BRCA1-KO) using the Plasma Membrane Protein Extraction Kit (ab65400, Abcam, MA, USA). Proteins were also prepared from pelleted exosomes. Samples were processed for mass spectrometry. Briefly, proteins were run on a stacking gel. The stacking gel bands were reduced with DTT, alkylated with iodoacetic acid and then digested with trypsin with re-solubilization in 0.1% aqueous formic acid/2% acetonitrile. Peptides were loaded onto a Thermo Acclaim Pepmap precolumn (Thermo, 75uM ID X 2 cm C18 3uM beads), and onto an Acclaim Pepmap Easyspray analytical column separation (Thermo, 75uM X 15 cm with C18 2uM beads) using a Dionex Ultimate 3000 uHPLC at 220 nl/min with a gradient of 2–35% organic (0.1% formic acid in acetonitrile) over 4 h. Peptides were analyzed using a Thermo Orbitrap Fusion mass spectrometer operating at 120,000 resolution (FWHM in MS1, 15,000 for MS/MS) with HCD sequencing all peptides with a charge of 2+ or greater. The raw data were converted into MGF format (Mascot Generic Format) searched using Mascot 2.3 against human sequences (Swissprot). The database search results were loaded onto Scaffold Q+ Scaffold_4.7.2 (Proteome Sciences) for spectral counting, statistical treatment and data visualization.
Exosomes uptake inhibition
Cells were blocked for 1 h. with different receptor antagonists: Anti-β4 Integrin (10 μg/ml, ASC-8, ab77801, abcam), Cytostatin (1.4 μg/ml, 19,602, Cedarlane), and Heparin (10 μg/ml, H3149, Sigma). In parallel, exosomes were treated for 2 h. with RGD (300 nM, 14,501–1, Cedarlane), and Collagenase I (500 μg/ml, C0130, Sigma). Afterwards, cells were washed, mixed with treated exosomes and incubated for 6 h. Exosomes internalization was analyzed as described in previous section.
In vivo tumor growth
Five-week-old female NOD-SCID mice (Jackson Laboratory) were used in compliance with McGill University Health Centre Animal Compliance Office (Protocol 2012–7280). Cells growing in log phase were harvested by trypsinization and washed twice with HBSS. Mice were injected subcutaneously with 2 million cells in 200 μl HBSS/Matrigel. Mice were euthanized one month post-injection. The resulting xenotransplants were photographed and processed as indicated below.
Immunohistochemistry labelling procedures and histological analyses
Mice xenotransplants were collected, fixed in 10% buffered formalin, embedded in paraffin, and stained with H&E (hematoxylin and eosin) according to standard protocols or processed for immunohistochemistry. Briefly, 5 μm tissue sections were dewaxed in xylene and rehydrated with distilled water. After antigen unmasking, and blocking of endogenous peroxidase (3% hydrogen peroxide), the slides were incubated with primary antibodies (Additional file 1: Table S1). Labeling was performed using iView DAB Detection Kit (Ventana) on the Ventana automated immunostainer. Sections were counterstained lightly with Hematoxylin before mounting. Histological analyses were performed by a certified pathologist.
Statistical differences were analyzed using Student’s t test for unpaired samples. An ANOVA followed by the Dunnett test was used for multiple comparisons with one control group. The criterion for significance (p value) was set as mentioned in figures.
Cells treated with cancer patient sera differentiated into the same lineages of the primary cancers.
We analyzed the expression of specific markers to further characterize these xenotransplants for differentiation patterns based on the primary tumor of the blood donors (Table 1 and Fig. 1). We observed that cancer sera-treated cells had completely changed their fate since all developing tumors stained negative for vimentin, which is normally expressed on fibroblasts (Fig. 1). Notably, xenotransplants obtained with cells exposed to the sera of patients with colorectal cancer, hepatocellular carcinoma, pancreatic cancer, and ovarian cancer displayed phenotypical characteristics of blood donors’ primary tumors (Table 1 and Fig. 1). Tumors, generated with colorectal cancer sera-treated cells, displayed epithelial features typical of colorectal adenocarcinomas, as they all stained negative for CK7, and expressed CEA, CK20, CDX-2 and AE1/AE3, which are universal markers of colorectal cancer (Fig. 1a). Tumors, generated with hepatocellular carcinoma sera-exposed cells, showed convincing hepatocellular carcinoma differentiation, although Hep par-1 staining was negative (Fig. 1b) in keeping with early stages of differentiation [34, 35]. The tumors, generated with cells treated with pancreatic cancer serum, stained strongly positive for CK7 and CK19, which are typical markers of pancreatic adenocarcinoma differentiation. Strikingly enough, different stages of differentiation could be visible in the xenotransplants with areas featuring poorly differentiated pancreatic cancer cells and areas containing cells displaying full differentiation (Fig. 1c). The tumors, produced with ovarian cancer sera-exposed cells, showed convincing differentiation toward ovarian carcinoma (Fig. 1d).
Altogether, these observations showed that treated cells had completely changed their fate and adopted features mimicking those of primary cancers of the blood donors. These findings suggest that cells in target organs, might have the potential to incorporate oncogenic factors delivered from primary cancers, acquire aberrant phenotypical traits identical or similar to the primary cancer and be therefore responsible for metastatic disease.
Cells treated with exosomes, isolated from cancer patient sera, differentiated into the same lineages of the primary cancers.
Increased exosomes uptake by BRCA1-KO cells is due to de novo expression or overexpression of plasma membrane receptors
Exosomes uptake involves a group of cell surface proteins that facilitate exosome/cell interactions and subsequent endocytosis [42, 43]. We scanned the MS outputs for proteins involved in exosomes uptake and compared their expression in the plasma membrane purified from BRCA1-KO and control fibroblasts. We found that BRCA1-KO cells, when compared to control fibroblasts, expressed de novo proteins involved in exosomes uptake such as flotillin, galectin, integrins, dynamin and afadin among others that were overexpressed (Fig. 4d, g, and Additional file 3: Table S3). This observation confirms our hypothesis that oncosuppressor gene mutations might induce expression of proteins on the membrane that would allow cancer exosomes to enter the cell, deliver their cargo and damage the genome [14, 44].
Transformed cells produced exosomes that overexpress ligands for receptors involved in vesicles internalization
In an attempt to determine whether exosomes shed by cancer cells are more prone than their normal counterpart to internalize into target cells, we isolated exosomes from normal fibroblasts exposed to the serum of healthy individuals (i.e. normal cells) and exosomes from BRCA1-KO fibroblasts after treatment with serum from CRC-LM patients (i.e. transformed cells). Cells were first treated for 3 weeks, then conditioned media from both cultures were collected and exosomes were isolated. Isolated exosomes were subjected to MS analyses that showed efficient exosomes isolation. Our MS analyses detected 62% of the top 100 exosomes proteins reported in the Exocarta protein list (http://www.exocarta.org) (Additional file 4: Table S4). Overall, we detected 1265 proteins: 216 were expressed only in control fibroblasts-derived exosomes, 100 were expressed only in BRCA1-KO fibroblasts-derived exosomes, and 949 were shared by both exosome populations (Fig. 4e and Additional file 5: Table S5).
We screened our MS data for exosomes-expressed ligands specific to the cell receptors involved in vesicles uptake (as shown in Additional file 3: Table S3). Notably, out of 41 sorted proteins, 39 proteins (96%) overlapped with the Exocarta protein list (Additional file 6: Table S6). Based on differential expression dataset, we found that transformed BRCA1-KO cells-derived exosomes overexpressed these ligands when compared to control fibroblasts-derived exosomes. These include laminin (integrins ligands), collagen, tenascin, cadherin, heparan-sulfate proteoglycan and filamin (Fig. 4f, h and Additional file 6: Table S6). Taken together these data show that cancer exosomes express proteins, which enable them to interact with receptors, de novo expressed on the membrane of BRCA1 mutated fibroblasts. These proteins are either not expressed or under-expressed in exosomes shed by non-cancerous cells.
Exosomes internalization blockage inhibited target cells transformation
To rule-out the possibility that the observed inhibitory effect was due to decreased cell viability or secondary to exosomes damage following antagonist treatments, we performed cell counting following trypan blue labeling and NanoSight analyses of exosomes (Fig. 5b, c). We found that the treatment with antagonists had a feeble effect on cell viability (Fig. 5b), and no effect on exosomes integrity (Fig. 5c). Altogether theses findings suggest that the newly expressed membrane proteins on both BRCA1-KO fibroblasts and cancer exosomes might have a role in exosomes uptake in target cells.
To determine if inhibition of the receptors involved in the exosomes uptake may inhibit cancer exosomes-induced cell transformation, we isolated exosomes from the serum of CRC-LM patients. Exosomes uptake was blocked using the same protocol shown in Fig. 5a. Antagonists-treated and non-treated cells were transplanted into NOD/SCID mice to analyze their tumorigenic behavior. Mice were followed up for tumor growth, and they were euthanatized 4 weeks after cell inoculation. Mice injected with non-treated cells displayed visible tumors as early as 10 days following inoculation, which continue growing until euthanasia. In contrast, mice injected with treated cells developed minimal palpable masses (0.73 +/− 0.09 cm3 vs. 0.26 +/− 0.07 cm3, P = 0.031; Fig. 5d, e). Histopathological analyses of tumors generated with non-treated cells showed a uniform sheet of poorly differentiated adenocarcinoma. In contrast, tumors obtained following injection of treated cells displayed zones of poorly differentiated adenocarcinomas with discrete areas of necrotic and non-proliferating cells (Fig. 5f).
Serum from patients with dysplastic lesions only, was able to transform BRCA1-KO fibroblasts into cancer cells
The results obtained in our experiments confirmed that horizontal transfer of malignant traits to target cells is not limited to colon and pancreatic cancer, as we previously demonstrated, but it is a concept applicable also to hepatocellular carcinoma and ovarian cancer. The evidence that BRCA1-KO fibroblasts can be reprogrammed and turn into several types of cancers by exposing them to only cancer sera, strengthens the hypothesis that metastasis is a pathological process that might not necessarily require transfer of cells. The evidence, herein demonstrated for the first time, that cancer exosomes are the main effectors of that cascade of events that lead a target cell to a full malignant transformation, is a fascinating discovery, which has important implications. Our data suggest a different role that cancer exosomes might have in the setting of metastatic disease and strengthen the evidence that cancer exosomes may be involved in cancer invasion and metastasis not simply by preparing the niche for the engraftment of circulating cells as it was thought until now [4–9, 23, 42]. Carcinogenesis steps such as initiation, promotion and progression may actually be a process reproducible through horizontal transfer of cancer factors, shed by primary tumors and carried in exosomes through the blood, to susceptible cells located at metastatic sites.
The metastatic process as described in the seed to soil hypothesis is a very inefficient and extraordinarily complex process, which requires a set of features that the cells must acquire to develop new foci of disease in other organs . In order to be able to metastasize, cancer cells must break intercellular junction, invade the basal membrane, acquire migratory capability, incorporate into the lymphatic and blood stream and ultimately home in the parenchyma of target organs . The inefficiency of this process is demonstrated by the evidence, shown in experimental models, that only 0.01% of cancer cells injected into the circulation form metastatic foci . The success of the seed to soil model to explain metastatic disease was nurtured by the evidence that metastatic lesions had immunohistochemical features similar to the cells of the primary tumor and therefore it was obvious to conclude, that metastases were secondary to cells, detaching from the primary tumor, travelling through the blood stream and invading other organs. Our results demonstrate that cancer exosomes can transfer oncogenic factors to cells and determine dramatic changes with acquisition of malignant characteristics and gain of immunohistochemical features similar or identical to the cancer cells that release the exosomes. This model of horizontal transfer offers an easier explanation to unexplained phenomena seen in the conventional model such as genomic differences between primary and metastatic cells [51–54], inefficiency of the process and patterns of metastatic spread. According to this model, exosomes would easily enter the blood stream, would be uptaken by target cells owing to newly expressed receptors and eventually the target cells would change their phenotype according to the type of cancer cell that released the “onco-information”. The metastatic cell would therefore be similar to the primary cancer cell; hence, the genomic differences would be a natural consequence of being alike but not the same cell . In other words, the molecular profiles of primary and metastatic lesions are not usually identical because metastases wouldn’t necessarily derive from cells detached from the primary tumor [51–54].
The full transformation of a fibroblast into such a variety of different cancers after exposure to cancer exosomes proves that the concept of horizontal transfer is scientifically sound and paves the way to a new understanding of the metastatic process, which deserves further study. Although these results are striking, it is still necessary to determine the nature of the exosome-carried factors involved and fully unveil the molecular mechanisms, which underlie the transfer of malignant phenotypical traits. While putative factors (i.e. DNA, mRNA, miRNA, proteins) were already described as serum-derived exosomal cargo, their respective role has not yet been fully defined [4, 5, 8, 9, 15, 16, 22, 28–32, 39] and, in light of the results of this study, further attention to this alternative pathway is certainly warranted.
The observation, already published by our group, that exosomes are uptaken more and faster by oncosuppressor mutated cells, prompted us to verify the hypothesis of a novel function of the oncosuppressor genes. In this model, oncosuppressor genes might protect the cell’s genome, not only by repairing DNA damages and controlling cell cycle checkpoints, but also by inhibiting the uptake of mutating extracellular oncogenic material. In order to confirm this view, we sought to verify if the mutation of the BRCA1 oncosuppressor would trigger some membrane changes, which would lead to an active uptake of cancer exosomes as opposed to passive penetration and selective membrane fusion [43, 49]. The discovery that the knock-out of the oncosuppressor BRCA1 is associated with the de novo expression of proteins already associated with metastasis and aggressiveness such as dynamin , integrins , galectin [56, 57] and EPCAM  is intriguing. According to the conventional theory, these molecules would facilitate cell migration, would promote metastasis and be a hallmark for aggressiveness. In the model that we hypothesized instead, these proteins enable the active uptake of cancer exosomes and their antagonistic blockage would inhibit the malignant transformation at distance and therefore the metastatic event. In other words, these results, once again, strengthen the concept that cell migration is not necessary to explain cancer dissemination and offer novel evidence that the molecules involved in cancer cell dissemination are the same molecules implicated in cancer exosome uptake, malignant cargo delivery and malignant transformation at distance.
In our experiments we used oncosuppressor mutated fibroblasts in the attempt to recreate in vitro the same conditions that characterize carcinogenesis in human beings. As a matter of fact, carcinogenesis is not a sudden process and it requires accumulation of several mutations that cause a normal cell to become first metaplastic, then anaplastic and eventually dysplastic. Therefore, a cell that turns into cancer is never a normal cell but it is a cell already abnormal and with a definite instability, of its genomic asset, that has matured over the years. As we already described , we postulate that in cancer patients, multiple or chronic cellular stresses due to several factors (metabolic, viral, environmental, etc.) might cause mutations that would favor the uptake of circulating cancer exosomes in cells located in distant organs with their subsequent malignant transformation.
To further corroborate the validity of this concept and demonstrate that truly cell migration might not be the only model to explain metastasis, we included in this work the exceptional results that were obtained, when we exposed BRCA1-KO fibroblasts to sera of patients who had only low and high-grade dysplastic lesions. Dysplastic lesions are precancerous lesions, which, by definition, have not invaded the basal membrane. They are not malignant cells and they have not metastatic potential since one of the hallmarks of cancer is invasion with subsequent ability to metastasize. Contrary to the predictions and dogmas of the conventional metastatic model, some dysplastic lesions have been found to have the capability to metastasize in clinical scenarios, raising suspicion of misdiagnosis or missed cancer lesions. The evidence, shown in this paper, that BRCA1-KO fibroblasts turn into gallbladder, colon and bile duct cancer after exposure to sera of patients with only dysplastic lesions is, in our opinion, the definitive proof that the metastatic process might be independent from cell migration and entirely reproducible at distance by malignant transformation mediated through onco-factors circulating in the serum.
The results of this study demonstrate that blood-circulating exosomes are the major contributing factors involved in the horizontal transfer of malignant traits to target cells. The evidence that BRCA1 mutation triggers the de novo expression or overexpression of proteins and receptors involved in vesicles uptake suggests a novel function that oncosuppressor genes might exhibit to protect the integrity of the genome. The observation that the serum from patients with dysplastic lesions was able to transform target cells strengthens the notion that circulating factors, originating from primary lesions, rather than circulating cells, might be the major players at metastatic sites.
We are grateful to Ayat Salman for her assistance with the Ethical Committee approvals, Laura Montermini for her assistance with Nanosight data acquisition, and Diane Gingras for her assistance with electron microscopy data acquisitions. MS analyses were performed at the Proteomics Platform at the Research Institute of the Mcgill University Health Centre. This work was financially supported by Giuseppe Monticciolo. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
GA supervised the study. MA, VA, MA and GA conceived and designed the study. MA, DH and GA developed the methodology. MA, DH, ZHG and GA acquired and analyze data, and managed patients. MA and GA drafted the manuscript. All authors read and approved the final draft of the manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Eccles SA, Welch DR. Metastasis: recent discoveries and novel treatment strategies. Lancet. 2007;369(9574):1742–57.View ArticlePubMedPubMed CentralGoogle Scholar
- Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.View ArticlePubMedGoogle Scholar
- Nguyen DX, Bos PD, Massagué J. Metastasis: from dissemination to organ specific colonization. Nat Rev Cancer. 2009;9:274–84.View ArticlePubMedGoogle Scholar
- Skog J, Wurdinger T, van Rijn S. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;10:1470–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Hood JL, San RS, Wickline SA. Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res. 2011;71:3792–801.View ArticlePubMedGoogle Scholar
- Grange C, Tapparo M, Collino F, Vitillo L, Damasco C, Deregibus MC, et al. Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. Cancer Res. 2011;71(15):5346–56.View ArticlePubMedGoogle Scholar
- Peinado H, Alečković M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, et al. Melanoma exosomes educate bone marrow progenitor cells toward a prometastatic phenotype through MET. Nat Med. 2012;18:883–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Abdel-Mageed ZY, Yang Y, Thomas R, Ranjan M, Mondal D, Moraz K, et al. Neoplastic reprogramming of patient-derived adipose stem cells by prostate cancer cell-associated exosomes. Stem Cells. 2014;32:983–97.View ArticlePubMedPubMed CentralGoogle Scholar
- Fujita Y, Yoshioka Y, Ochiya T. Extracellular vesicle transfer of cancer pathogenic components. Cancer Sci. 2016. [DOI: 10.1111Epub ahead of print].Google Scholar
- Garcıa-Olmo D, Garcıa-Olmo DC, Ontanon J, Martinez E, Vallejo M. Tumor DNA circulating in the plasma might play a role in metastasis. The hypothesis of the genometastasis. Histol Histopathol. 1999;14:1159–64.PubMedGoogle Scholar
- García-Olmo DC, Domínguez C, García-Arranz M, Anker P, Stroun M, García-Verdugo JM, et al. Cell-free nucleic acids circulating in the plasma of colorectal cancer patients induce the oncogenic transformation of susceptible cultured cells. Cancer Res. 2010;70:560–7.View ArticlePubMedGoogle Scholar
- Trejo-Becerril C, Perez-Cardenas E, Taja-Chayeb L, Anker P, Herrera-Goepfert R, Medina-Velazquez LA, et al. Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model. PLOS ONE. 2012;7(12):e52754.View ArticlePubMedPubMed CentralGoogle Scholar
- Abdouh M, Zhou S, Arena V, Arena M, Lazaris A, Onerheim R, et al. Transfer of malignant trait to immortalized human cells following exposure to human cancer serum. J Exp Clin Cancer Res. 2014;33(1):86.View ArticlePubMedPubMed CentralGoogle Scholar
- Hamam D, Abdouh M, Gao ZH, Arena V, Arena M, Arena GO. Transfer of malignant trait to BRCA1 deficient human fibroblasts following exposure to serum of cancer patients. J Exp Clin Cancer Res. 2016;35:80.View ArticlePubMedPubMed CentralGoogle Scholar
- Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9:654–9.View ArticlePubMedGoogle Scholar
- Runz S, Keller S, Rupp C, Stoeck A, Issa Y, Koensgen D. Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM. Gynecol Oncol. 2007;107:563–71.View ArticlePubMedGoogle Scholar
- Pisetsky DS, Gauley J, Ullal AJ. Microparticles as a source of extracellular DNA. Immunol Res. 2010;49:227–34.View ArticleGoogle Scholar
- Subra C, Grand D, Laulagnier K, Stella A, Lambeau G, Paillasse M, et al. Exosomes account for vesicle mediated transcellular transport of activatable phospholipases and prostaglandins. J Lipid Res. 2010;51:2105–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Gaiffe E, Pretet JL, Launay S, Jacquin L, Saunier M, Hetzel G. Apoptotic HPV positive cancer cells exhibit transforming properties. PLoS One. 2012;7:e36766.View ArticlePubMedPubMed CentralGoogle Scholar
- Balaj L, Lessard R, Dai L, Cho YJ, Pomeroy SL, Breakefield XO. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat Commun. 2011;2:180.View ArticlePubMedPubMed CentralGoogle Scholar
- Fleischhacker M, Schmidt B. Circulating nucleic acids (CNAs) and cancer--a survey. Biochim Biophys Acta. 2007;1775(1):181–232.PubMedGoogle Scholar
- Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2:569–79.PubMedGoogle Scholar
- Ogorevc E, Kralj-Iglic V, Veranic P. The role of extracellular vesicles in phenotypic cancer transformation. Radiol Oncol. 2013;47(3):197–205.View ArticlePubMedPubMed CentralGoogle Scholar
- Candelario KM, Steindler DA. The role of extracellular vesicles in the progression of neurodegenerative disease and cancer. Trends Mol Med. 2014;20(7):368–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Pant S, Hilton H, Burczynski ME. The multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker oppertunities. Biochem Pharmacol. 2012;83(11):1484–94.View ArticlePubMedGoogle Scholar
- Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30:255–89.View ArticlePubMedGoogle Scholar
- Falcone G, Felsani A, D’Agnano I. Signaling by exosomal microRNAs in cancer. J Exp Clin Cancer Res. 2015;34:32.View ArticlePubMedPubMed CentralGoogle Scholar
- D’Asti E, Chennakrishnaiah S, Lee TH, Rak J. Extracellular vesicles in brain tumor progression. Cell Mol Neurobiol. 2016;36(3):383–407.View ArticlePubMedGoogle Scholar
- Soldevilla B, Rodriguez M, San Millan C, Garcia V, Fernandez-Perianez R, Gil-Calderon B, et al. Tumor-derived exosomes are enriched in DNp73, which promotes oncogenic potential in acceptor cells and correlates with patient survival. Hum Mol Genet. 2014;23(2):467–78.View ArticlePubMedGoogle Scholar
- Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Kosh M, et al. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNS in the serum exosomes of patients with pancreatic cancer. J Biol Chem. 2014;289(7):3869–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 2014;24:766–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Antonyak MA, Li B, Boroughs LK, et al. Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells. Proc Natl Acad Sci U S A. 2011;108:4852–7.View ArticlePubMedPubMed CentralGoogle Scholar
- He M, Qin H, Poon TC, Sze SC, Ding X, Co NN, et al. Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs. Carcinogenesis. 2015;36(9):1008–18.View ArticlePubMedGoogle Scholar
- Fanz Z, van de Rijn M, Montgomery K, Rouse RV. Hep par 1 antibody stain for the differential diagnosis of hepatocellular carcinoma: 676 tumors tested using tissue microarrays and conventional tissue sections. Mod Pathol. 2003;16(2):137–44.View ArticleGoogle Scholar
- Kakar S, Muir T, Murphy LM, Lloyd RV, Burgart LJ. Immunoreactivity of Hep par 1 in hepatic and extrahepatic tumors and its correlation with albumin in situ hybridization in hepatocellular carcinoma. Am J Clin Pathol. 2003;119:361–6.View ArticlePubMedGoogle Scholar
- Lykke-Andersen S, Brodersen DE, Jensen TH. Origins and activities of the eukaryotic exosome. J Cell Sci. 2009;122(Pt 10):1487–94.View ArticlePubMedGoogle Scholar
- Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication. Leukemia. 2006;20(9):1487–95.View ArticlePubMedGoogle Scholar
- Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic M. Tumour exosome integrins determine organotropic metastasis. Nature. 2015;527(7578):329–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Kastelowitz N, Yin H. Exosomes and microvesicles: identification and targeting by particle size and lipid chemical probes. Chembiochem. 2014;15(7):923–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Hosseini-Beheshti E, Pham S, Adomat H, Li N, Tomlinson Guns ES. Exosomes as biomarker enriched microvesicles: characterization of exosomal proteins derived from a panel of prostate cell lines with distinct AR phenotypes. Mol Cell Proteomics. 2012;11(10):863–85.View ArticlePubMedPubMed CentralGoogle Scholar
- McKelvey KJ, Powell KL, Ashton AW, Morris JM, McCracken SA. Exosomes: mechanisms of uptake. J Circ Biomark. 2015;4:1–9.View ArticleGoogle Scholar
- Mulcahy LA, Pink RC, Carter DRF. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles. 2014;3:2464.View ArticleGoogle Scholar
- Arena G, Arena V, Arena M, Abdouh M. Transfer of malignant traits as opposed to migration of cells: a novel concept to explain metastatic disease. Med Hypotheses. 2017;100:82–6.View ArticlePubMedGoogle Scholar
- Kawada M, Amemiya M, Ishizuka M, Takeuchi T. Cytostatin, an inhibitor of cell adhesion to extracellular matrix, selectively inhibits protein phosphatase 2A. Biochim Biophys Acta. 1999;1452(2):209–17.View ArticlePubMedGoogle Scholar
- Christianson HC, Svensson KJ, van Kuppevelt TH, Li JP, Belting M. Cancer cell exosomes depend on cell-surface heparin sulfate proteoglycans for their internalization and functional activity. PNAS. 2013;110(43):17380–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Abdouh M, Hamam D, Arena V, Arena M, Alamri H, Arena G. Novel blood test to predict neoplastic activity in healthy patients and metastatic recurrence after primary tumor resection. Journal of Circulating Biomarkers. 2016;5:1–10.Google Scholar
- Barreto S, Pandanaboyana S, Ironside N, Windsor J. Does revision of resection margins based on frozen section improve overall survival following pancreaticoduodenectomy for pancreatic ductal adenocarcinoma? A meta-analysis. HPB (Oxford). 2017; (17): 30534–30548.Google Scholar
- Ribelles N, Santonja A, Pajares B, Llacer C, Alba E. The seed and soil hypothesis revisited: current state of knowledge of inherited genes on prognosis in breast cancer. Cancer Treat Rev. 2014;40:293–9.View ArticlePubMedGoogle Scholar
- Luzzi K, MacDonald I, Schmidt E, Kerkvliet N, Morris V, et al. Multistep nature of metastatic inefficiency. Am J Pathology. 1998;153(3):865–73.View ArticleGoogle Scholar
- Suzuki M, Tanin D. Gene expression profiling of human lynph node metastases and matched primary breast carcinomas: clinical implications. Mol Oncol. 2007;1(2):172–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Huang S, Chen Y, Podsypanina K, Li Y. Comparison of expression profiles of metastatic versus primary mammary tumors in MMTV-Wnt-1 and MMTV-Neu transgenic mice. Neoplasia. 2008;10(2):118–24.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu X, Zhang M, Go VLW, Hu S. Membrane proteomic analysis of pancreatic cancer cells. J Biomed Sci. 2010;17(1):74.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoshida A, Okamoto N, Tozawa-Ono A, KoizUmi H, Kiguchi K, Ishizuka B, et al. Proteomic analysis of differential protein expression by brain metastases of gynecological malignancies. Hum Cell. 2013;26(2):56–66.View ArticlePubMedPubMed CentralGoogle Scholar
- Qian W, Wang J, Van Houten B. The role of dynamin-related protein 1 in cancer growth: a promising therapeutic target? Expert Opin Ther Targets. 2013;17(9):997–1001.View ArticlePubMedPubMed CentralGoogle Scholar
- Sindrewicz P, Lian LY, Yu LG. Interaction of the Oncofetal Thomsen-Friedenreich antigen with galectins in cancer progression and metastasis. Front Oncol. 2016;6:79.View ArticlePubMedPubMed CentralGoogle Scholar
- Ahmed H, Al Sadek DM. Galectin-3 as a potential target to prevent cancer metastasis. Clin Med Insights Oncol. 2015;9:113–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Dai M, Yuan F, Fu C, Shen G, Hu S, Shen G. Relationship between epithelial cell adhesion molecule (EpCAM) overexpression and gastric cancer patients: a systematic review and meta-analysis. PLoS One. 2017;12(4):e0175357.View ArticlePubMedPubMed CentralGoogle Scholar