Intercellular Protein Transfer Research Articles

Shotgun Proteomics of Co-Cultured Leukemic and Bone Marrow Stromal Cells from Different Species as a Preliminary Approach to Detect Intercellular Protein Transfer

Cellular interactions within the bone marrow microenvironment modulate the properties of subsets of leukemic cells leading to the development of drug-resistant phenotypes. The intercellular transfer of proteins and organelles contributes to this process but the set of transferred proteins and their effects in the receiving cells remain unclear. This study aimed to detect the intercellular protein transfer from mouse bone marrow stromal cells (OP9 cell line) to human T-lymphoblasts (CCRF-CEM cell line) using nanoLC-MS/MS-based shotgun proteomics in a 3D co-culture system. After 24 h of co-culture, 1513 and 67 proteins from human and mouse origin, respectively, were identified in CCRF-CEM cells. The presence of mouse proteins in the human cell line, detected by analyzing the differences in amino acid sequences of orthologous peptides, was interpreted as the result of intercellular transfer. The transferred proteins might have contributed to the observed resistance to vincristine, methotrexate, and hydrogen peroxide in the co-cultured leukemic cells. Our results suggest that shotgun proteomic analyses of co-cultured cells from different species could be a simple option to get a preliminary survey of the proteins exchanged among interacting cells.

Tunneling Nanotubes Facilitate Intercellular Protein Transfer and Cell Networks Function.

The past decade witnessed a huge interest in the communication machinery called tunneling nanotubes (TNTs) which is a novel, contact-dependent type of intercellular protein transfer (IPT). As the IPT phenomenon plays a particular role in the cross-talk between cells, including cancer cells as well as in the immune and nervous systems, it therefore participates in remodeling of the cellular networks. The following review focuses on the placing the role of tunneling nanotube-mediated protein transfer between distant cells. Firstly, we describe different screening methods used to study IPT including tunneling nanotubes. Further, we present various examples of TNT-mediated protein transfer in the immune system, cancer microenvironment and in the nervous system, with particular attention to the methods used to verify the transfer of individual proteins.

The Role of Trogocytosis in the Modulation of Immune Cell Functions.

Trogocytosis is an active process, in which one cell extracts the cell fragment from another cell, leading to the transfer of cell surface molecules, together with membrane fragments. Recent reports have revealed that trogocytosis can modulate various biological responses, including adaptive and innate immune responses and homeostatic responses. Trogocytosis is evolutionally conserved from protozoan parasites to eukaryotic cells. In some cases, trogocytosis results in cell death, which is utilized as a mechanism for antibody-dependent cytotoxicity (ADCC). In other cases, trogocytosis-mediated intercellular protein transfer leads to both the acquisition of novel functions in recipient cells and the loss of cellular functions in donor cells. Trogocytosis in immune cells is typically mediated by receptor–ligand interactions, including TCR–MHC interactions and Fcγ receptor-antibody-bound molecule interactions. Additionally, trogocytosis mediates the transfer of MHC molecules to various immune and non-immune cells, which confers antigen-presenting activity on non-professional antigen-presenting cells. In this review, we summarize the recent advances in our understanding of the role of trogocytosis in immune modulation.

Tunneling nanotube-mediated intercellular vesicle and protein transfer in the stroma-provided imatinib resistance in chronic myeloid leukemia cells

Intercellular communication within the bone marrow niche significantly promotes leukemogenesis and provides protection of leukemic cells from therapy. Secreted factors, intercellular transfer of mitochondria and the receptor–ligand interactions have been shown as mediators of this protection. Here we report that tunneling nanotubes (TNTs)—long, thin membranous structures, which have been identified as a novel mode of intercellular cross-talk—are formed in the presence of stroma and mediate transfer of cellular vesicles from stroma to leukemic cells. Importantly, transmission of vesicles via TNTs from stromal cells increases resistance of leukemic cells to the tyrosine kinase inhibitor, imatinib. Using correlative light-electron microscopy and electron tomography we show that stromal TNTs contain vesicles, provide membrane continuity with the cell bodies and can be open-ended. Moreover, trans-SILAC studies to reveal the non-autonomous proteome showed that specific sets of proteins are transferred together with cellular vesicles from stromal to leukemic cells, with a potential role in survival and adaptation. Altogether, our findings provide evidence for the biological role of the TNT-mediated vesicle exchange between stromal and leukemic cells, implicating the direct vesicle and protein transfer in the stroma-provided protection of leukemic cells.

Global Analysis of Intercellular Homeodomain Protein Transfer

SUMMARYThe homeodomain is found in hundreds of transcription factors that play roles in fate determination via cell-autonomous regulation of gene expression. However, some homeodomain-containing proteins (HPs) are thought to be secreted and penetrate neighboring cells to affect the recipient cell fate. To determine whether this is a general characteristic of HPs, we carried out a large-scale validation for intercellular transfer of HPs. Our screening reveals that intercellular transfer is a general feature of HPs, but it occurs in a cell-context-sensitive manner. We also found the secretion is not solely a function of the homeodomain, but it is supported by external motifs containing hydrophobic residues. Thus, mutations of hydrophobic residues of HPs abrogate secretion and consequently interfere with HP function in recipient cells. Collectively, our study proposes that HP transfer is an intercellular communication method that couples the functions of interacting cells.

Intercellular transfer of HLA-G: its potential in cancer immunology.

Intercellular protein transfer between cancer cells and immune cells is a very common phenomenon that can affect different stages of host antitumor immune responses. HLA‐G, a non‐classical HLA class I antigen, has been observed to be widely expressed in various malignancies, and its immune‐suppressive functions have been well recognised. HLA‐G expression in cancer cells can directly mediate immune tolerance by interacting with inhibitory receptors such as ILT2 and ILT4 expressed on immune cells. Moreover, a network of multiple directional intercellular transfers of HLA‐G among cancer cells and immune cells through trogocytosis, exosomes and tunnelling nanotubes provides malignant cells with an alternative ploy for antigen sharing and induces more complex heterogeneity, to modulate immune responses, ultimately leading to immune evasion, therapy resistance, disease progression and poor clinical outcome. Herein, we discuss the relative aspects of the intercellular transfer of HLA‐G between tumor cells and immune cells and its potential use in tumor immunology research and translational cancer therapy.

Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer.

Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101-encoded proteins, the pilus-tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface-displayed TraC and Pep are required for an efficient conjugative transfer, 'extracellular complementation' potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the β-barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS-encoded, pilus-independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.

Exosomes mediate interepithelial transfer of functional P-glycoprotein in chronic rhinosinusitis with nasal polyps.

P-glycoprotein (P-gp) drives type-2 helper T-cell inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) through unknown posttranslational mechanisms of overexpression. A recent randomized clinical trial demonstrated that inhibition of P-gp was as effective as oral steroids and biologics in treating CRSwNP. Exosomes are 30- to 150-nm vesicles capable of intercellular membrane protein transfer. The aims of this study were 1) to determine whether CRSwNP mucus exosomes are enriched with P-gp, and 2) whether exosomal P-gp can be functionally transferred to autologous epithelial cells as a putative mechanism for the proinflammatory overexpression of P-gp in CRSwNP. Institutional review board-approved study in CRSwNP and control patients (n = 10 per group). P-gp content of purified mucus exosomes was characterized by transmission electron microscopy and enzyme-linked immunosorbent assay. Epithelial transfer of exosomal P-gp was determined by time-lapse fluorescent microscopy and calcein acetoxymethylester functional P-gp assay. CD63+/P-gp+ exosomes were detected in both groups. P-gp was significantly enriched in CRSwNP exosomes relative to control (median 198.5; interquartile range 123.6-270.5 vs. 74.4; 41.3-95.0 pcg P-gp/109 exosomes, P = 0.002). Exosomes were absorbed by epithelial cells within 10 minutes, resulting in a significant increase in P-gp activity in CRSwNP patients relative to control (P = 0.006). Here we demonstrate the presence and P-gp enrichment of mucus-derived exosomes, or rhinosomes, in CRSwNP. These rhinosomes are capable of rapid intercellular transfer of P-gp, leading to increased P-gp function within recipient cells. This represents a novel mechanism for maintaining P-gp overexpression in CRSwNP, and more generally for interepithelial transfer of other proteins between mucosal epithelial cells. NA. Laryngoscope, 127:E295-E300, 2017.

Abstract 1649: Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation

Abstract Despite surgery or chemo radiotherapy the five-year survival rate of esophageal carcinoma (eSCC) patients remains around 15% indicating the need for new therapy regimes. Recently the role of the tumor microenvironment has been recognized as an important determinant of radiation responses. Here we investigate the influence of stromal fibroblasts (hF) on radiation induced tumor cell death. To evaluate functional consequences of ionizing radiation hF and eSCC cells were either cultured separately and irradiated (2Gy) or co-cultured after irradiation to investigate radiation dependent tumor-stroma interactions. Subsequently cells were monitored by time-lapse microscopy and cell death was assessed visually as well as by PARP cleavage, live cell staining with Hoechst33342/propidium iodide and quantification of sub-G1 cell cycle. The rate of cell death was barely altered in monocultures. Of note significantly increased cell death of eSCC was observed in co-culture with irradiated hF compared to co-cultures with mock-irradiated fibroblasts (16.10±4.25 fold of control). Microscopic evaluation of co-cultures revealed that hF and cancer cells are connected via tunnelling nanotubes (TNT). Furthermore, TNT formation was found to be dependent on the extracellular matrix component hyaluronan. eSCC cell death was prevented by pharmacological inhibition of TNT formation by cytochalasin B as well as inhibition of hyaluronan by 4-MU. As TNTs are known to mediate intercellular protein or mitochondrial transfer we investigated which stromal proteins are shuttled into eSCC cells by trans-SILAC experiments. For this purpose the proteome of fibroblasts was labeled with heavy amino acids. After co-culturing hF and eSCC were separated by FACS-sorting and labeled stromal proteins were identified in the cancer cells by mass spectrometry. Four consistently shuttled proteins were identified in the cancer cells, which were exclusively transferred after irradiation of both cell lines. Knock-down experiments of these proteins in hF were performed and SHANK1, which is not expressed in eSCC cells, was found to be responsible for the hF induced cell death of eSCC cells in response to radiation. In conclusion, by usage of a novel trans-SILAC approach, we provide evidence that in response to ionizing radiation stromal hF transfer SHANK1 to eSCC cells, thereby inducing cellular death. SHANK1 might therefore serve as potential new pharmacological target to sensitize cancer cells to radiation therapy. Citation Format: Katharina Röck, Alexandra Schütze, Janna Morawitz, Verena Jendrossek, Klaus Pantel, Jens W. Fischer. Deadly fuel: Fibroblasts mediate cancer cell death through tunneling nanotubes in response to ionizing radiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1649.

Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells.

Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Senescent cells talk frankly with their neighbors

Communication between cells is a vital process that governs cellular organization and coordination in an organism. Thus, multiple mechanisms of cell communication have evolved to respond to all the needs of an organism. The modes of cell-cell communication range from the ones that require direct contact between cells, such as cytoplasmic bridges (CBs) and gap junctions, to others that may take place over long distances throughout the organism, such as hormone signaling. Precise integration of data acquired from all these different modes of communication maintains tissue homeostasis and allows optimal adaptation of an organism to various stresses. Cellular senescence represents one of the outcomes of cellular response to stress.1 Senescent cells execute essential functions in different physiological and pathophysiological conditions. They are present in pre-malignant lesions, sites of tissue damage, aging tissues and even during embryonic development. In all these places senescent cells communicate with cells in their surroundings and modulate the function of these cells.2-5 The effect of senescent cells on nearby cells is commonly attributed to the secretion of cytokines, chemokines and matrix metalloproteinases.4,6 We have recently demonstrated that in addition to secretion, senescent cells affect neighboring cells by direct intercellular protein transfer (IPT).2 Proteins from senescent cells are directly transferred to recipient neighboring cells, such as immune and cancer cells, triggering activation of signaling pathways in these cells, ultimately leading to changes in cellular behavior. We have detected IPT from senescent cells and demonstrated that these cells form CBs with other cells, including immune cells, cancer and non-cancer epithelial cells.2 CBs, which in some conditions are called tunneling nanotubes, are open on both edges and allow transfer of cytoplasmic content to neighboring cells. Interestingly, we also identified mitochondria and lysosomes in CBs, implying that organelles might be transferred between the cells. Transfer of these organelles was indeed identified in several other systems. Since dysfunctional mitochondria are thought to play a role in cellular senescence, it can be speculated that transfer of the dysfunctional mitochondria to healthy neighboring cells may induce senescence in these cells. Transfer of cytoplasmic content or whole organelles might serve therefore, for communication of cellular stress. The functional consequences of cell communication of senescent cells through IPT are not completely understood and could be cell type dependent. For instance, we have reported that IPT facilitates elimination of senescent cells by NK cells since transfer of proteins to NK cells correlated with NK cell activation and cytotoxicity. Remarkably, inhibition of CDC42 in senescent cells resulted in decreased IPT and impaired elimination of the senescent cells by NK cells.2 In addition to interaction with NK cells senescent cells also communicate with other components of the innate and adaptive immune systems.2,5,6 Thus, it is possible that senescent cells transfer proteins to other types of immune cells and alter their behavior. To fulfill their function immune cells respond to cytokines and chemokines using receptor-ligand interactions to activate cellular responses (Fig. 1). IPT can serve as an additional mechanism regulating activity of immune cells following contact mediated interaction with resident cells and thereby fine-tune the immune response. Figure 1. Senescent cells communicate with NK cells by multiple mechanisms. Senescent cells influence NK cells by (i) senescent associated secretory phenotype (SASP); (ii) specific receptor ligand interactions; (iii) cytoplasmic bridges. These diverse mechanisms ... Senescent cells can impact tumorigenesis, mainly by secretion of pro-inflammatory cytokines.3,5 Our data demonstrate that senescent cells form CBs with cancer cells, thereby implementing IPT in interaction of senescent and cancer cells. Since the interaction of these cells is long lasting, in contrast to the interaction with NK cells, higher amount and number of proteins could potentially be transferred. The functional impact of IPT from senescent cells to cancer cells is unknown, as it could potentially promote or restrain cancer cells. Senescent cells could transfer cell cycle inhibitors (i.e. p16, p15 and p21) to cancer cells, thereby inhibiting their proliferation. Conversely, cancer cells may also receive organelles and proteins to support their rapid proliferation. In addition to pathological conditions, senescent cells modulate physiological processes, including embryonic development.4 During embryonic development human placental syncytiotrophoblast exhibit features and molecular markers of cellular senescence.7 The syncytiotrophoblast is a multinucleate epithelium which supports fetal growth by creating an interface between maternal and fetal circulation. The syncytiotrophoblast cells communicate with subjacent layer of mono-nucleated cytotrophoblasts to promote division and fusion of cytotrophoblasts into the syncytiotrophoblast. These cells also communicate with immune cells to control immune tolerance at the maternal-fetal interface. Therefore, senescent syncytiotrophoblast cells might also form CBs to facilitate these non-cell autonomous effects which are essential for embryonic development. Overall, IPT from senescent cells represents a pivotal mode of cellular communication with possible physiological outcomes on cancer progression, tissue repair, embryonic development, aging and immune modulation.

Senescent cells communicate via intercellular protein transfer.

Mammalian cells mostly rely on extracellular molecules to transfer signals to other cells. However, in stress conditions, more robust mechanisms might be necessary to facilitate cell-cell communications. Cellular senescence, a stress response associated with permanent exit from the cell cycle and the development of an immunogenic phenotype, limits both tumorigenesis and tissue damage. Paradoxically, the long-term presence of senescent cells can promote tissue damage and aging within their microenvironment. Soluble factors secreted from senescent cells mediate some of these cell-nonautonomous effects. However, it is unknown whether senescent cells impact neighboring cells by other mechanisms. Here we show that senescent cells directly transfer proteins to neighboring cells and that this process facilitates immune surveillance of senescent cells by natural killer (NK) cells. We found that transfer of proteins to NK and T cells is increased in the murine preneoplastic pancreas, a site where senescent cells are present in vivo. Proteomic analysis and functional studies of the transferred proteins revealed that the transfer is strictly dependent on cell-cell contact and CDC42-regulated actin polymerization and is mediated at least partially by cytoplasmic bridges. These findings reveal a novel mode of intercellular communication by which senescent cells regulate their immune surveillance and might impact tumorigenesis and tissue aging.

Human mesenchymal stroma/stem cells exchange membrane proteins and alter functionality during interaction with different tumor cell lines.

To analyze effects of cellular interaction between human mesenchymal stroma/stem cells (MSC) and different cancer cells, direct co-cultures were performed and revealed significant growth stimulation of the tumor populations and a variety of protein exchanges. More than 90% of MCF-7 and primary human HBCEC699 breast cancer cells as well as NIH:OVCAR-3 ovarian adenocarcinoma cells acquired CD90 proteins during MSC co-culture, respectively. Furthermore, SK-OV-3 ovarian cancer cells progressively elevated CD105 and CD90 proteins in co-culture with MSC. Primary small cell hypercalcemic ovarian carcinoma cells (SCCOHT-1) demonstrated undetectable levels of CD73 and CD105; however, both proteins were significantly increased in the presence of MSC. This co-culture-mediated protein induction was also observed at transcriptional levels and changed functionality of SCCOHT-1 cells by an acquired capability to metabolize 5'cAMP. Moreover, exchange between tumor cells and MSC worked bidirectional, as undetectable expression of epithelial cell adhesion molecule (EpCAM) in MSC significantly increased after co-culture with SK-OV-3 or NIH:OVCAR-3 cells. In addition, a small population of chimeric/hybrid cells appeared in each MSC/tumor cell co-culture by spontaneous cell fusion. Immune fluorescence demonstrated nanotube structures and exosomes between MSC and tumor cells, whereas cytochalasin-D partially abolished the intercellular protein transfer. More detailed functional analysis of FACS-separated MSC and NIH:OVCAR-3 cells after co-culture revealed the acquisition of epithelial cell-specific properties by MSC, including increased gene expression for cytokeratins and epithelial-like differentiation factors. Vice versa, a variety of transcriptional regulatory genes were down-modulated in NIH:OVCAR-3 cells after co-culture with MSC. Together, these mutual cellular interactions contributed to functional alterations in MSC and tumor cells.

A cell culture model for monitoring α-synuclein cell-to-cell transfer

The transfer of α-synuclein (α-syn) between cells has been proposed to be the primary mechanism of disease spreading in Parkinson's disease. Several cellular models exist that monitor the uptake of recombinant α-syn from the culture medium. Here we established a more physiologically relevant model system in which α-syn is produced and transferred between mammalian neurons. We generated cell lines expressing either α-syn tagged with fluorescent proteins or fluorescent tags alone then we co-cultured these cell lines to measure protein uptake. We used live-cell imaging to demonstrate intercellular α-syn transfer and used flow cytometry and high content analysis to quantify the transfer. We then successfully inhibited intercellular protein transfer genetically by down-regulating dynamin or pharmacologically using dynasore or heparin. In addition, we differentiated human induced pluripotent stem cells carrying a triplication of the α-syn gene into dopaminergic neurons. These cells secreted high levels of α-syn, which was taken up by neighboring neurons. Collectively, our co-culture systems provide simple but physiologically relevant tools for the identification of genetic modifiers or small molecules that inhibit α-syn cell-to-cell transfer.

Modeling Intercellular Transfer of Biomolecules Through Tunneling Nanotubes

Tunneling nanotubes (TNTs) have previosly been observed as long and thin transient structures forming between cells and intercellular protein transfer through them has been experimentally verified. It is hypothesized that this may be a physiologically important means of cell-cell communication. This paper attempts to give a simple model for the rates of transfer of molecules across these TNTs at different distances. We describe the transfer of both cytosolic and membrane bound molecules between neighboring populations of cells and argue how the lifetime of the TNT, the diffusion rate, distance between cells, and the size of the molecules may affect their transfer. The model described makes certain predictions and opens a number of questions to be explored experimentally.

Exosomes: vesicular carriers for intercellular communication in neurodegenerative disorders

The intercellular transfer of misfolded proteins has received increasing attention in various neurodegenerative diseases characterized by the aggregation of specific proteins, as observed in Alzheimer’s, Parkinson’s and Huntington’s disease. One hypothesis holds that intercellular dissemination of these aggregates within the central nervous system results in the seeded assembly of the cognate soluble protein in target cells, similar to that proposed for transmissible prion diseases. The molecular mechanisms underlying the intercellular transfer of these proteinaceous aggregates are poorly understood. Various transfer modes of misfolded proteins including continuous cell-cell contacts such as nanotubes, unconventional secretion or microvesicle/exosome-associated dissemination have been suggested. Cells can release proteins, lipids and nucleic acids by vesicular exocytosis pathways destined for horizontal transfer. Encapsulation into microvesicular/exosomal vehicles not only protects these molecules from degradation and dilution in the extracellular space but also facilitates delivery over large distances, e.g. within the blood flow or interstitial fluid. Specific surface ligands might allow the highly efficient and targeted uptake of these vesicles by recipient cells. In this review, we focus on the cell biology and function of neuronal microvesicles/exosomes and discuss the evidence for pathogenic intercellular protein transfer mediated by vesicular carriers.

Abstract LB-7: Protein crosstalk in tumor microenvironment: direct identification of intercellular protein transfer in retinoblastoma after topotecan or radiation treatment

Abstract Current work on the tumor microenvironment has shown that crosstalk between cancer and normal cells can be of significant importance. Here we show direct intercellular communication between different cancer cell populations can provide insights into the tumor microenvironment and the bystander effect. Retinoblastoma is the primary intraocular cancer in childhood and resistance to current treatments such as chemotherapy or radiation often results in enucleation. We seek to improve retinoblastoma treatments by elucidating the diversity of and communication between retinoblastoma tumors. Here we provide the first direct evidence of intercellular protein transfer between retinoblastoma cells after topotecan or radiation treatment using stable isotope labeling and tandem mass spectrometry-based protein identification. Retinoblastoma Y79 cells were grown in media containing 13C, 15N lysine and arginine analogues (Y79S) so that all the proteins had full incorporation of these stable isotopes. The Y79S cells were untreated, treated with 0.02 micromolar topotecan, or treated with 4 Gy of radiation before being co-cultured with either Y79 or CHLA215 retinoblastoma cells for 48 hr. Protein populations within each cell type and the concentrated media were identified using mass spectrometry-based sequencing. Direct intercellular transfer was monitored using the unique 13C, 15N protein tags. Changes in the tumor microenvironment and the protein communication between retinoblastoma cells with chemotherapy and radiation treatment were monitored using a transwell co-culture system. As expected, irradiation resulted in a significant decrease in Y79S cell number and viability. Additionally, irradiated Y79S cells caused a significant decrease in the viability of the non-irradiated Y79 bystander cells. Studies of the proteins directly transferred from Y79S to the other retinoblastoma cells identified heat shock protein 90 (HSP90) in both the topotecan and radiation studies. Significantly, time point data provided insights into the protein transfer dynamics. We show significant protein transfer by 24 hours with a decrease in isotopically labeled proteins by 48 hours. We are currently working on elucidating the functional roles of HSP90 and the other proteins transferred during chemotherapy and radiation treatment. In summary, we have shown the first evidence for direct protein crosstalk between retinoblastoma cell populations in response to chemotherapy and radiation. We anticipate that this work will lead to greater insights into how tumor heterogeneity contributes to treatment response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-7. doi:1538-7445.AM2012-LB-7

Mechanisms of cellular communication through intercellular protein transfer

In a multicellular system, cellular communication is a must for orchestration and coordination of cellular events. Advent of the latest analytical and imaging tools has allowed us to enhance our understanding of the intercellular communication. An intercellular exchange of proteins or intact membrane patches is a ubiquitous phenomenon, and has been the subject of renewed interest, particularly in the context of immune cells. Recent evidence implicates that intercellular protein transfers, including trogocytosis is an important mechanism of the immune system to modulate immune responses and transferred proteins can also contribute to pathology. It has been demonstrated that intercellular protein transfer can be through the internalization/pathway, dissociation-associated pathway, uptake of exosomes and membrane nanotube formations. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. In this review, we will discuss the important findings concerning intercellular protein transfers, possible mechanisms and highlight their physiological relevance to the immune system, with special reference to T cells such as the stimulatory or suppressive immune responses derived from T cells with acquired dendritic cell membrane molecules.

Intercellular exchange of proteins: The immune cell habit of sharing

The recent recognition of new types of cell–cell communication pathways challenges classic theories of cell autonomy. Evidence of functional “proteome mixing” among interacting cells, particularly immune cells, supports the notion that no cell is an island, and that even these “unsplittable” units are actually non-autonomous. We summarize various mechanisms of intercellular transfer of proteins—trans-endocytosis, trogocytosis, exosomal transport, shuttle through nanotubes, and cell-contact-dependent intercellular transfer of intracellular proteins including oncogenic Ras. These phenomena suggest exciting new possibilities for proteome research, focusing on system-level proteomics that characterize cell contents and functions in the context of intercellular protein transfer.