Effect of Extracellular Survivin and Lymphoma Exosomes on Natural Killer Cells

Tumors alter their microenvironment to promote survival using methods such as angiogenesis promotion, growth signals, and immune suppression. The immune system becomes unresponsive to transformed neoplastic cells through a variety of methods including T cell suppression, increased myeloid-derived suppressor cells (MDSCs), and reduced natural killer (NK) cell activity. NK cells have inherent killing capabilities and thus are among the first responders in recognizing and destroying abnormal cells. However, many types of cancers inhibit the surveillance and cytotoxic abilities of NK cells by releasing exosomes, vesicles that can modulate the tumor microenvironment (TME) and intercellular communication for the purpose of enhancing tumor malignancy. These 30-150 nm sized lipid bound vesicles are secreted by many cell types, including immune cells and tumor cells, and the specific protein, lipid, mRNA and miRNA contents contribute to the complex intercellular communication occurring between malignant and normal cells. Cancer patients often have increased numbers of exosomes circulating through their body, including patients with hematological malignancies, such as lymphoma. The focus of this research was to determine the interactions between B cell lymphoma exosomes and NK cells, and characterize the resultant effects on NK cell function. A specific objective of this research was to determine whether Survivin, an Inhibitor of Apoptosis protein recently found to be localized within exosomes, has a role in modulating NK cells similar to previous findings of T cell modulations. We report that lymphoma exosomes have low levels of internalization into NK cells, and no detectable presence of immune modulating proteins MICA/B or TGF-β. Exposure of NK cells to lymphoma exosomes did not result in observable changes in degranulation or cytotoxic ability. However, treatment with recombinant Survivin protein was able to decrease NKG2D receptor levels in NK cells stimulated with target cells, and decrease protein levels of TNF-α, IFN-γ, perforin, and Granzyme B. A better understanding of the underlying processes by which Survivin or exosomes suppress immune cells in the TME may pave the way to more efficacious immunological therapies against cancer

The emerging role of exosomes in survivin secretion

The tumor microenvironment plays an integral part in the biology of cancer, participating in tumor initiation, progression, and response to therapy. Factors released by tumor cells themselves contribute in creating an environment mostly favorable but sometimes detrimental to the tumor. Survivin, one of the key members of the inhibitor of apoptosis (IAP) family of proteins, has been shown in the cytoplasm, mitochondria, nucleus, and most recently in the extracellular space, transported via small membrane bound vesicles called exosomes. Exosomes are secreted from hematopoietic, non-hematopoietic, tumor, and nontumor cells, shuttling essential molecules such as proteins, RNAs, and microRNAs, all believed to be important for cell-cell and cell-extracellular communication. In this review, we discuss exosomal Survivin and its role in modifying the tumor microenvironment

Curcumin Modulates Pancreatic Adenocarcinoma Cell-Derived Exosomal Function.

Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin's effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through exosomal facilitation

Natural Killer Cell Phenotype and Functionality Affected by Exposure to Extracellular Survivin and Lymphoma-Derived Exosomes

The inherent abilities of natural killer (NK) cells to recognize and kill target cells place them among the first cells with the ability to recognize and destroy infected or transformed cells. Cancer cells, however, have mechanisms by which they can inhibit the surveillance and cytotoxic abilities of NK cells with one believed mechanism for this: their ability to release exosomes. Exosomes are vesicles that are found in abundance in the tumor microenvironment that can modulate intercellular communication and thus enhance tumor malignancy. Recently, our lab has found cancer cell exosomes to contain the inhibitor of apoptosis (IAP) protein survivin to be associated with decreased immune response in lymphocytes and cellular death. The purpose of this study was to explore the effect of survivin and lymphoma-derived survivin-containing exosomes on the immune functions of NK cells. NK cells were obtained from the peripheral blood of healthy donors and treated with pure survivin protein or exosomes from two lymphoma cell lines, DLCL2 and FSCCL. RNA was isolated from NK cell samples for measurement by PCR, and intracellular flow cytometry was used to determine protein expression. Degranulation capacity, cytotoxicity, and natural killer group 2D receptor (NKG2D) levels were also assessed. Lymphoma exosomes were examined for size and protein content. This study established that these lymphoma exosomes contained survivin and FasL but were negative for MHC class I-related chains (MIC)/B (MICA/B) and TGF-β. Treatment with exosomes did not significantly alter NK cell functionality, but extracellular survivin was seen to decrease natural killer group 2D receptor (NKG2D) levels and the intracellular protein levels of perforin, granzyme B, TNF-α, and IFN-γ

Spectrophotometric detection of curcumin within exosomes from PANC-1 cells.

<p>Exosomes were isolated from untreated PANC-1 cells (curcumin-negative exosomes) or PANC-1 cells treated with 50 μM of curcumin for 24 hours (curcumin-positive exosomes). (A) Whole (non-lysed, blue) exosomes from curcumin-treated PANC-1 cells were subjected to spectral analysis compared to vehicle (1X PBS blank, red), in which optical density (OD) at 420 nm was measured. No peak in absorbance was detected at 420 nm from whole (non-lysed) exosomes. (B) Methanol and sonication were used to lyse exosomes from curcumin-treated PANC-1 cells (lysed curcumin-positive exosomes, green) or exosomes from untreated PANC-1 cells (lysed curcumin-negative exosomes, blue). A methanol-only blank (red) was used as a negative control for this assay. A characteristic peak in OD at 420 nm was detected in lysed curcumin-positive exosomes, but not in lysed curcumin-negative exosomes or the methanol-only blank. Data are representative of three independent experiments.</p

Validation of exosome isolation.

<p>(A) Acetylcholinesterase activity assays were used to detect exosomes in isolates from untreated PANC-1 cells (curcumin-negative exosomes) or PANC-1 cells treated with 50 μM of curcumin for 24 hours (curcumin-positive exosomes) compared to assay diluent, 1X PBS (control). (B) Nicomp dynamic light scattering (DLS) analysis was used to measure size distribution of particles in exosome isolates. (C) NanoSight nanoparticle tracking analysis (NTA) was used to confirm size distribution of particles in exosome isolates. (D) Particle concentration (particles/mL) was measured using NanoSight NTA. No significant differences were observed in acetylcholinesterase activity, size distribution, or particle concentration between curcumin-negative exosomes and curcumin-positive exosomes. Data are represented as mean ± SEM of three independent experiments, *p<0.05, **p<0.01, exosome fraction versus control.</p

Exosomal curcumin in recipient PANC-1 cells.

<p>Naïve PANC-1 cells were co-incubated with exosomes from untreated PANC-1 cells (curcumin-negative exosomes) or exosomes from PANC-1 cells treated with 50 μM of curcumin for 24 hours (curcumin-positive exosomes). In a separate culture, naïve recipient PANC-1 cells were treated with 10 μg/mL heparin to inhibit exosomal binding 30 minutes prior to and during co-incubation with curcumin-positive exosomes (heparin + curcumin-positive exosomes). After 24 hours, cells were washed and stained with DAPI for visualization of nuclei. Curcumin fluorescence (green) and DAPI (blue) were detected by fluorescence microscopy at 40X magnification. (B) Quantification of curcumin fluorescence was performed using the BZ II analyzer software. Data were collected in three separate images per independent experiment, three independent experiments. Data are represented as mean + SEM of three independent experiments, *p<0.05, **p<0.01, heparin + curcumin-positive exosomes versus curcumin-positive exosomes. ND = not detectable.</p

Exosomal curcumin reduces recipient pancreatic adenocarcinoma cell viability.

<p>Naïve recipient PANC-1 or MIA PaCa-2 cells were cultured for the indicated times with exosomes isolated from untreated cells (curcumin-negative exosomes) or exosomes isolated from cells treated with 50 μM of curcumin (curcumin-positive exosomes). In a separate experiment, naïve recipient cells were treated with 10 μg/mL heparin prior to and during incubation with curcumin-positive exosomes (heparin + curcumin-positive exosomes). Viability was determined via (A) AlamarBlue and (B) Trypan blue exclusion assays and exosome treatments were compared to naïve cells not exposed to exosomes or heparin (untreated). Data are represented as mean + SEM of three independent experiments, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Morphological features of recipient pancreatic adenocarcinoma cells following exposure to exosomal curcumin.

<p>PANC-1 and MIA PaCa-2 cells were exposed to curcumin-positive exosomes or exosome-free supplemented DMEM (untreated) for 24, 48 and 72 hours followed by imaging via Hoffman modulation contrast microscopy. White arrows indicate membrane blebs and cell shrinkage, morphological hallmarks of apoptosis. Results depicted represent findings from three independent experiments.</p