Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

Red blood cell extracellular vesicles deliver therapeutic siRNAs to skeletal muscles for treatment of cancer cachexia.

Cancer cachexia is a multifactorial syndrome characterized by a significant loss of skeletal muscle, which negatively affects the quality of life. Inhibition of myostatin (Mstn), a negative regulator of skeletal muscle growth and differentiation, has been proven to preserve muscle mass in muscle atrophy diseases, including cachexia. However, myostatin inhibitors have repeatedly failed clinical trials because of modest therapeutic effects and side effects due to the poor efficiency and toxicity of existing delivery methods. Here, we describe a novel method for delivering Mstn siRNA to skeletal muscles using red blood cell-derived extracellular vesicles (RBCEVs) in a cancer cachectic mouse model. Our data show that RBCEVs are taken up by myofibers via intramuscular administration. Repeated intramuscular administrations with RBCEVs allowed the delivery of siRNAs, thereby inhibiting Mstn, increasing muscle growth, and preventing cachexia in cancer-bearing mice. We observed the same therapeutic effects when delivering siRNAs against malonyl-CoA decarboxylase, an enzyme driving dysfunctional fatty acid metabolism in skeletal muscles during cancer cachexia. We demonstrate that intramuscular siRNA delivery by RBCEVs is safe and non-inflammatory. Hence, this method is useful to reduce the therapeutic dose of siRNAs, to avoid toxicity and off-target effects caused by systemic administration of naked siRNAs at high doses.

ETC-159, an Upstream Wnt inhibitor, Induces Tumour Necrosis via Modulation of Angiogenesis in Osteosarcoma.

There is an increasing urgency in the search for new drugs to target high-grade cancers such as osteosarcomas (OS), as these have limited therapeutic options and poor prognostic outlook. Even though key molecular events leading to tumorigenesis are not well understood, it is widely agreed that OS tumours are Wnt-driven. ETC-159, a PORCN inhibitor that inhibits the extracellular secretion of Wnt, has recently progressed on to clinical trials. In vitro and in vivo murine and chick chorioallantoic membrane xenograft models were established to examine the effect of ETC-159 on OS. Consistent with our hypothesis, we noted that ETC-159 treatment not only resulted in markedly decreased ß-catenin staining in xenografts, but also increased tumour necrosis and a significant reduction in vascularity-a hereby yet undescribed phenotype following ETC-159 treatment. Through further understanding the mechanism of this new window of vulnerability, therapies can be developed to potentiate and maximize the effectiveness of ETC-159, further increasing its clinical utility for the treatment of OS.

αvß1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin-3.

Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour microenvironment and promote metastasis. To identify EV-bound proteins related to metastasis, we used mass spectrometry to profile EVs from highly and poorly metastatic breast cancer lines of human and mouse origins. Comparative mass spectrometry indicated that integrins, including αv and ß1 subunits, are preferentially enriched in EVs of highly metastatic origin over those of poorly metastatic origin. These results are consistent with our histopathological findings, which show that integrin αv is associated with disease progression in breast cancer patients. Integrin αv colocalizes with the multivesicular-body marker CD63 at a higher frequency in the tumour and is enriched in circulating EVs of breast cancer patients at late stages when compared with circulating EVs from early-stage patients. With a magnetic bead-based flow cytometry assay, we confirmed that integrins αv and ß1 are enriched in the CD63+ subsets of EVs from both human and mouse highly metastatic cells. By analysing the level of integrin αv on circulating EVs, this assay could predict the metastatic potential of a xenografted mouse model. To explore the export mechanism of integrins into EVs, we performed immunoprecipitation mass spectrometry and identified members of the galectin family as potential shuttlers of integrin αvß1 into EVs. In particular, knockdown of galectin-3, but not galectin-1, causes a reduction in the levels of cell surface integrins ß1 and αv, and decreases the colocalization of these integrins with CD63. Importantly, knockdown of galectin-3 leads to a decrease of integrin αvß1 export into the EVs concomitant with a decrease in the metastatic potential of breast cancer cells. Moreover, inhibition of the integrin αvß1 complex leads to a reduction in the binding of EVs to fibronectin, suggesting that integrin αvß1 is important for EV retention in the extracellular matrix. EVs retained in the extracellular matrix are taken up by fibroblasts, which differentiate into cancer associated fibroblasts. In summary, our data indicate an important link between EV-bound integrin αvß1 with breast cancer metastasis and provide additional insights into the export of integrin αvß1 into EVs in the context of metastasis.

CD33-targeting extracellular vesicles deliver antisense oligonucleotides against FLT3-ITD and miR-125b for specific treatment of acute myeloid leukaemia.

INTRODUCTION: Acute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%-65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs). METHODS: In this study, we harness red blood cell-derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33-binding monoclonal antibody onto the EV surface. RESULTS: The conjugation of RBCEVs with the CD33-binding antibody significantly increases the uptake of RBCEVs by CD33-positive AML cells, but not by CD33-negative cells. We also load CD33-targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3-ITD or miR-125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML. CONCLUSION: Targeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.

Robust delivery of RIG-I agonists using extracellular vesicles for anti-cancer immunotherapy.

The RIG-I pathway can be activated by RNA containing 5' triphosphate, leading to type I interferon release and immune activation. Hence, RIG-I agonists have been used to induce immune responses against cancer as potential immunotherapy. However, delivery of 5' triphosphorylated RNA molecules as RIG-I agonists to tumour cells in vivo is challenging due to the susceptibility of these molecules to degradation. In this study, we demonstrate the use of extracellular vesicles (EVs) from red blood cells (RBCs), which are highly amenable for RNA loading and taken up robustly by cancer cells, for RIG-I agonist delivery. We evaluate the anti-cancer activity of two novel RIG-I agonists, the immunomodulatory RNA (immRNA) with a unique secondary structure for efficient RIG-I activation, and a 5' triphosphorylated antisense oligonucleotide with dual function of RIG-I activation and miR-125b inhibition (3p-125b-ASO). We find that RBCEV-delivered immRNA and 3p-125b-ASO trigger the RIG-I pathway, and induce cell death in both mouse and human breast cancer cells. Furthermore, we observe a significant suppression of tumour growth coupled with increased immune cell infiltration mediated by the activation of RIG-I cascade after multiple intratumoral injections of RBCEVs loaded with immRNA or 3p-125b-ASO. Targeted delivery of immRNA using RBCEVs with EGFR-binding nanobody administrated via intrapulmonary delivery facilitates the accumulation of RBCEVs in metastatic cancer cells, leading to potent tumour-specific CD8+ T cells immune response. This contributes to prominent suppression of breast cancer metastasis in the lung. Hence, this study provides a new strategy for efficient RIG-I agonist delivery using RBCEVs for immunotherapy against cancer and cancer metastasis.