Transfer of malignant traits as opposed to migration of cells: A novel concept to explain metastatic disease

Abstract Metastatic disease is believed to develop following dissemination of cells to target organs. Inability of this theory to effectively explain certain phenomena such as patterns of metastatic spread, late metastasis formation, different gene patterns between primary cancer and metastasis have brought forward the need for alternative models. Recent discoveries have strengthened the validity of theories supporting a humoral transfer of malignant traits as opposed to migration of malignant cells to explain metastatic disease in cancer patients. In light of this new evidence, we would like to highlight a model that offers a new perspective to explain cancer metastasis. In the system that we theorize, genetic material released by cancer cells would travel, either free or packed in exosomes, through the blood. Target cells located in organs deriving from the same embryological layer might uptake this genetic material due to expression of specific receptors. Interplay with the immune system would determine the fate of these oncofactors and would regulate their ability to circulate in the blood, integrate in the genome and be transcribed. We also hypothesize that the expression of cell membrane receptors such as integrins, to which cancer exosomes ligate might be mediated by inherited or acquired oncosuppressor mutations

Novel blood test to predict neoplastic activity in healthy patients and metastatic recurrence after primary tumor resection

We reported that single oncosuppressor-mutated (SOM) cells turn malignant when exposed to cancer patients’ sera. We tested the possibility to incorporate this discovery into a biological platform able to detect cancer in healthy individuals and to predict metastases after tumor resection. Blood was drawn prior to tumor resection and within a year after surgery. Blood samples from healthy individuals or metastatic patients were used as negative and positive controls, respectively. Patients at risk for cancer were included in the screening cohort. Once treated, cells were injected into nonobese diabetic/severe combined immunodeficiency mice to monitor tumor growth. All samples of sera coming from metastatic patients transformed SOM cells into malignant cells. Four samples from screened patients transformed SOM cells. Further clinical tests done on these patients showed the presence of early cancerous lesions despite normal tumor markers. Based on the xenotransplants size, we were able to predict metastasis in three patients before diagnostic tests confirmed the presence of the metastatic lesions. These data show that this serum-based platform has potentials to be used for cancer screening and for identification of patients at risks to develop metastases regardless of the Tumor Node Metastasis (TNM) stage or tumor markers level

Reprogramming Malignant Cancer Cells toward a Benign Phenotype following Exposure to Human Embryonic Stem Cell Microenvironment

<div><p>The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity <i>in vitro</i> and <i>in vivo</i>. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells.</p></div

hESCs-Exo decreased cancer cell proliferation and increased cancer cell death.

<p>MDA-MB231 and HT29 cells were cultured for 3 days in control medium (5%FBS), or with exosomes derived from fibroblast-CM (Fibro-Exo) or hESCs-CM (hESCs-Exo), and cells were analyzed for their growth potential (A-E), and apoptosis (F). (A) Bright field pictures of cell cultures at 3 days post-treatments. Note the significant dose-dependent reduction in cell density in cultures maintained in hESCs-Exo. Scale bar: 50 μm. (B and C) note that the legend is the same for all graphs: (B) 100,000 cells were plated and their number was counted after 2 and 3 days of culture. Values are presented as mean ± SD (n = 3 independent cultures, *P < 0.05, **P < 0.01, ***P < 0.001). (C) The metabolic activity following treatment for 2 and 3 days. Cultures were incubated for 5 h with Alamar Blue and data acquired by spectrofluorometry. Data are presented as mean ± SD and are representative of 3 independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). (D and E) CFSE load dilution in cultures at 3 days post-treatments. Full refer to CFSE loading at the beginning of the culture period. Numbers in brackets are the percentages of fully CFSE-loaded cells (cells that did not divide yet). Data are mean ± SD (n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001). (F) Cell apoptosis analyses following labeling with Annexin V and loading of propidium iodide (PI). Apoptotic cells (Annexin V positive and PI negative) were scored and their percentages were shown (n = 3 independent cultures, **P < 0.01, ***P < 0.001).</p

hESCs-CM inhibited cell cycle progression of cancer cells and initiated a pro-apoptotic program.

<p>MDA-MB231 and HT29 cells were cultured for 3 days in control medium or hESCs-CM, and cells were analyzed for their proliferation (A-C) and cell viability (D). (A) Control and hESCs-CM-treated cells were loaded with propidium iodide and analyzed for their progression in the cell cycle. Barre plots display the data of 3 independent experiments. Note that hESCs-CM-treated cells progress slowly through the G1 phase. (B) Cells were analyzed by Western blot for the expression of cell cycle regulatory proteins. alpha-Tubulin (α-Tub.) was used as a proteins loading control. (C) Cells were analyzed by immunocytofluorometry for the expression of Ki67 and phosphor-histone 3 (PH3). The graphs display the raw data. Values are mean ± SD of positive cells (n = 3 independent cultures, **P < 0.01, ***P < 0.001). Scale bars: 50 and 15 μm in low and high magnification, respectively. (D) (Left) Cells were analyzed for apoptosis following labeling with Annexin V and loading of propidium iodide (PI). Apoptotic cells (Annexin V positive and PI negative) were scored and their percentages were shown (bottom right corner, n = 3 independent cultures, *P < 0.05). (Right) Parallel cultures were plated on chamber slides and analyzed for the cleavage of caspase 3. Values are mean ± SD of positive cells (n = 3 independent cultures, **P < 0.01). Scale bars: 50 and 10 μm in low and high magnification, respectively.</p

hESCs-CM inhibited the oncogenic potential of cancer cells in vitro and in vivo.

<p>MDA-MB231 and HT29 cells were cultured for 2 weeks in control medium or hESCs-CM. (A and B) Cells were grown in soft agar for another 2 weeks to analyze their anchorage-independent growth. (A) Bright field pictures. Note the decrease of colony sizes and numbers when cells were exposed to hESCs-CM. Scale bar: 200 μm. (B) (Left) The graph represents the number of colonies counted per field. (Right) The graph represents the size of the colonies obtained. Colonies were measured using ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, **P < 0.01, ***P < 0.001). (C and D) Cells treated as in (A) were injected subcutaneously in NOD/SCID mice. (C) 4 weeks after injection, xenograft were photographed and their volumes were calculated. Values are mean ± SD, (n = 4–6 xenotransplants, **P < 0.01). Scale bar: 1 cm. (D) Formalin-fixed paraffin-embedded xenotransplant samples were processed for H&E staining, or immunolabeled with antibodies against cytokeratin-7 and Ki67 (tumors obtained with MDA-MB231 cells are shown). Scale bar: 40 μm.</p

hESCs-CM decreased cancer cells growth.

<p>MDA-MB231 and HT29 cells were cultured for 3 days in control medium or hESCs-CM, and cells were analyzed for their growth potential. (A) Bright field pictures of cell cultures at 3 days post-treatments. Note the significant reduction in cell density in cultures maintained in hESCs-CM. Scale bar: 50 μm. (B) 100,000 cells were plated and their number was followed for the 3 days of culture period. Values are cells counts presented as mean ± SD (n = 3 independent cultures, *P < 0.05, **P < 0.01, ***P < 0.001). (C) The metabolic activity following 2 and 3 days treatment duration. Cultures were incubated for 5 h with Alamar Blue and data acquired by spectrofluorometry. Data are presented as mean ± SD and are representative of 3 independent experiments (**P < 0.01). (D) CFSE load dilution in cultures at 3 days. Full refer to CFSE loading at the beginning of the culture period. Numbers in brackets are the percentages of fully CFSE-loaded cells (cells that did not divide yet). Data are mean ± SD (n = 3 independent experiments, P < 0.05 in MDA-MB231 cultures and P < 0.01 in HT29 cultures).</p

hESCs-Exo induced the expression of hESCs markers in cancer cells.

<p>Cells were treated with exosome-free medium (CTL) or hESCs-Exo medium, and analyzed for the expression of hESCs markers. (A) qPCR analyses for the expression of SOX2 and OCT4 transcripts. Data were normalized to GAPDH, and the level of transcripts expression in control medium was set at 1. Results are presented as mean ± SD (n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001). (B and C) Parallel cultures were analyzed for the expression of hESCs protein markers by Western blot (B) and cytofluorometry (C). In (B), β-actin is used as calibrator for proteins loading. Scale bar: 20 μm.</p

Cancer cells internalized efficiently hESCs-Exo.

<p>(A) Exosomes were isolated as described under Material and Methods. Representative micrographs of TEM show small vesicles of approximately 50–120 nm in diameter. Scale bar: 100 nm. (B) NanoSight analyses of samples prepared as in (A). The size was centered around 101 nm in diameter. Data are expressed as concentration average (red line) of 3 exosome preparations. (C) qPCR analyses for the expression of pluripotency transcription factors SOX2, OCT4 and NANOG transcripts in hESCs and hESCs-Exo. Upper panel; data are expressed as threshold cycle (Mean Ct). Lower panel; data were normalized to the level of GAPDH, and the levels of SOX2, OCT4 and NANOG transcripts expression in hESCs were set at 1. Results are presented as mean ± SD (n = 2 independent experiments repeated in triplicates). (D) Proteins isolated from cells (hESCs and fibroblasts) or exosomes (hESCs-Exo and fibro-Exo) were analyzed by Western blot for the expression of specific hESCs and exosomes markers. (E) Confocal microscopy monitoring of PKH-26-labeled (red dots) exosome uptake <i>in vitro</i> into MDA-MB231 cells (12 h incubation). Note that exosomes are uniformly dispersed in the cytoplasm and tended to form aggregates in the perinuclear regions. Similar results were obtained with HT29 cells. Scale bar: 10 μm.</p