A novel machine learning algorithm selects proteome signature to specifically identify cancer exosomes.

Non-invasive early cancer diagnosis remains challenging due to the low sensitivity and specificity of current diagnostic approaches. Exosomes are membrane-bound nanovesicles secreted by all cells that contain DNA, RNA, and proteins that are representative of the parent cells. This property, along with the abundance of exosomes in biological fluids makes them compelling candidates as biomarkers. However, a rapid and flexible exosome-based diagnostic method to distinguish human cancers across cancer types in diverse biological fluids is yet to be defined. Here, we describe a novel machine learning-based computational method to distinguish cancers using a panel of proteins associated with exosomes. Employing datasets of exosome proteins from human cell lines, tissue, plasma, serum, and urine samples from a variety of cancers, we identify Clathrin Heavy Chain (CLTC), Ezrin, (EZR), Talin-1 (TLN1), Adenylyl cyclase-associated protein 1 (CAP1), and Moesin (MSN) as highly abundant universal biomarkers for exosomes and define three panels of pan-cancer exosome proteins that distinguish cancer exosomes from other exosomes and aid in classifying cancer subtypes employing random forest models. All the models using proteins from plasma, serum, or urine-derived exosomes yield AUROC scores higher than 0.91 and demonstrate superior performance compared to Support Vector Machine, K Nearest Neighbor Classifier and Gaussian Naive Bayes. This study provides a reliable protein biomarker signature associated with cancer exosomes with scalable machine learning capability for a sensitive and specific non-invasive method of cancer diagnosis.

Engineered immunomodulatory extracellular vesicles derived from epithelial cells acquire capacity for positive and negative T cell co-stimulation in cancer and autoimmunity.

Extracellular vesicles (EVs) are generated by all cells and systemic administration of allogenic EVs derived from epithelial and mesenchymal cells have been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cells derived EVs can be modified to acquire the capacity to induce immune response, we engineered 293T EVs to harbor the immunomodulatory CD80, OX40L and PD-L1 molecules. We demonstrated abundant levels of these proteins on the engineered cells and EVs. Functionally, the engineered EVs efficiently elicit positive and negative co-stimulation in human and murine T cells. In the setting of cancer and auto-immune hepatitis, the engineered EVs modulate T cell functions and alter disease progression. Moreover, OX40L EVs provide additional benefit to anti-CTLA-4 treatment in melanoma-bearing mice. Our work provides evidence that epithelial cell derived EVs can be engineered to induce immune responses with translational potential to modulate T cell functions in distinct pathological settings.

A novel machine learning algorithm selects proteome signature to specifically identify cancer exosomes.

Non-invasive early cancer diagnosis remains challenging due to the low sensitivity and specificity of current diagnostic approaches. Exosomes are membrane-bound nanovesicles secreted by all cells that contain DNA, RNA, and proteins that are representative of the parent cells. This property, along with the abundance of exosomes in biological fluids makes them compelling candidates as biomarkers. However, a rapid and flexible exosome-based diagnostic method to distinguish human cancers across cancer types in diverse biological fluids is yet to be defined. Here, we describe a novel machine learning-based computational method to distinguish cancers using a panel of proteins associated with exosomes. Employing datasets of exosome proteins from human cell lines, tissue, plasma, serum and urine samples from a variety of cancers, we identify Clathrin Heavy Chain (CLTC), Ezrin, (EZR), Talin-1 (TLN1), Adenylyl cyclase-associated protein 1 (CAP1) and Moesin (MSN) as highly abundant universal biomarkers for exosomes and define three panels of pan-cancer exosome proteins that distinguish cancer exosomes from other exosomes and aid in classifying cancer subtypes employing random forest models. All the models using proteins from plasma, serum, or urine-derived exosomes yield AUROC scores higher than 0.91 and demonstrate superior performance compared to Support Vector Machine, K Nearest Neighbor Classifier and Gaussian Naive Bayes. This study provides a reliable protein biomarker signature associated with cancer exosomes with scalable machine learning capability for a sensitive and specific non-invasive method of cancer diagnosis.

Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic function.

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

Extracellular vesicles as central regulators of blood vessel function in cancer.

Blood vessels deliver oxygen and nutrients that sustain tumor growth and enable the dissemination of cancer cells to distant sites and the recruitment of intratumoral immune cells. In addition, the structural and functional abnormalities of the tumor vasculature foster the development of an aggressive tumor microenvironment and impair the efficacy of existing cancer therapies. Extracellular vesicles (EVs) have emerged as major players of tumor progression, and a growing body of evidence has demonstrated that EVs derived from cancer cells trigger multiple responses in endothelial cells that alter blood vessel function in tumors. EV-mediated signaling in endothelial cells can occur through the transfer of functional cargos such as miRNAs, lncRNAs, cirRNAs, and proteins. Moreover, membrane-bound proteins in EVs can elicit receptor-mediated signaling in endothelial cells. Together, these mechanisms reprogram endothelial cells and contribute to the sustained exacerbated angiogenic signaling typical of tumors, which, in turn, influences cancer progression. Targeting these angiogenesis-promoting EV-dependent mechanisms may offer additional strategies to normalize tumor vasculature. Here, we discuss the current knowledge pertaining to the contribution of cancer cell-derived EVs in mechanisms regulating blood vessel functions in tumors. Moreover, we discuss the translational opportunities in targeting the dysfunctional tumor vasculature using EVs and highlight the open questions in the field of EV biology that can be addressed using mass spectrometry-based proteomics analysis.

Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer.

CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant Kras G12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.

Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker.

Exosomes are extracellular vesicles derived from the endosomal compartment that are potentially involved in intercellular communication. Here, we found that frequently used biomarkers of exosomes are heterogeneous, and do not exhibit universal utility across different cell types. To uncover ubiquitous and abundant proteins, we used an unbiased and quantitative proteomic approach based on super-stable isotope labeling with amino acids in cell culture (super-SILAC), coupled to high-resolution mass spectrometry. In total, 1,212 proteins were quantified in the proteome of exosomes, irrespective of the cellular source or isolation method. A cohort of 22 proteins was universally enriched. Fifteen proteins were consistently depleted in the proteome of exosomes compared to cells. Among the enriched proteins, we identified biogenesis-related proteins, GTPases and membrane proteins, such as CD47 and ITGB1. The cohort of depleted proteins in exosomes was predominantly composed of nuclear proteins. We identified syntenin-1 as a consistently abundant protein in exosomes from different cellular origins. Syntenin-1 is also present in exosomes across different species and biofluids, highlighting its potential use as a putative universal biomarker of exosomes. Our study provides a comprehensive quantitative atlas of core proteins ubiquitous to exosomes that can serve as a resource for the scientific community.

Multifunctional Applications of Engineered Extracellular Vesicles in the Treatment of Cancer.

Extracellular vesicles (EVs) are key players of intercellular communication in the physiological and pathological setting. In cancer, EVs mediate complex signaling mechanisms between cancer cells and the tumor microenvironment (TME), and can influence tumor progression and the response to existing therapies. Importantly, EVs can be loaded with therapeutic agents and modified to display tumor-targeting molecules. In the field of nanomedicine, EVs have been engineered to serve as therapeutic delivery vehicles for several anticancer agents, including antibodies, chemotherapy, compounds, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated endonuclease 9), and small interfering RNA (siRNA). Notably, the engineered EVs were shown to suppress malignant features of cancer cells, to elicit antitumor immunity, and to decrease tumor angiogenesis. Here, we review the EV-based therapies designed to target cancer cells and to educate components of the TME to drive antitumor responses. These studies illustrate the multifunctional applications of EVs in the development of anticancer therapies and their translational potential for cancer treatment.

Exosomes as mediators of immune regulation and immunotherapy in cancer.

Exosomes are nanosized extracellular vesicles of endosomal origin that enclose a multitude of functional biomolecules. Exosomes have emerged as key players of intercellular communication in physiological and pathological conditions. In cancer, depending on the context, exosomes can oppose or potentiate the development of an aggressive tumor microenvironment, thereby impacting tumor progression and clinical outcome. Increasing evidence has established exosomes as important mediators of immune regulation in cancer, as they deliver a plethora of signals that can either support or restrain immunosuppression of lymphoid and myeloid cell populations in tumors. Here, we review the current knowledge related to exosome-mediated regulation of lymphoid (T lymphocytes, B lymphocytes, and NK cells) and myeloid (macrophages, dendritic cells, monocytes, myeloid-derived suppressor cells, and neutrophils) cell populations in cancer. We also discuss the translational potential of engineered exosomes as immunomodulatory agents for cancer therapy.

B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Hypoxic cancer-associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling.

Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro. Mass spectrometry-based proteomic analysis of the CAF secretome unraveled that hypoxic CAFs contributed to blood vessel abnormalities by altering their secretion of various pro- and anti-angiogenic factors. Hypoxia induced pronounced remodeling of the CAF proteome, including proteins that have not been previously related to this process. Among those, the uncharacterized protein NCBP2-AS2 that we renamed HIAR (hypoxia-induced angiogenesis regulator) was the protein most increased in abundance in hypoxic CAFs. Silencing of HIAR abrogated the pro-angiogenic and pro-migratory function of hypoxic CAFs by decreasing secretion of the pro-angiogenic factor VEGFA and consequently reducing VEGF/VEGFR downstream signaling in the endothelial cells. Our study has identified a regulator of angiogenesis and provides a map of hypoxia-induced molecular alterations in mammary CAFs.

Mitogen-Activated Protein Kinase Kinase 5 Regulates Proliferation and Biosynthetic Processes in Procyclic Forms of Trypanosoma brucei.

The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated protein kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light on the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acids biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acids biosynthesis in procyclic T. brucei.

Cancer Associated Fibroblasts: The Architects of Stroma Remodeling.

Fibroblasts have exceptional phenotypic plasticity and capability to secrete vast amount of soluble factors, extracellular matrix components and extracellular vesicles. While in physiological conditions this makes fibroblasts master regulators of tissue homeostasis and healing of injured tissues, in solid tumors cancer associated fibroblasts (CAFs) co-evolve with the disease, and alter the biochemical and physical structure of the tumor microenvironment, as well as the behavior of the surrounding stromal and cancer cells. Thus CAFs are fundamental regulators of tumor progression and influence response to therapeutic treatments. Increasing efforts are devoted to better understand the biology of CAFs to bring insights to develop complementary strategies to target this cell type in cancer. Here we highlight components of the tumor microenvironment that play key roles in cancer progression and invasion, and provide an extensive overview of past and emerging understanding of CAF biology as well as the contribution that MS-based proteomics has made to this field.

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity.

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.

The MAP kinase MAPKLK1 is essential to Trypanosoma brucei proliferation and regulates proteins involved in mRNA metabolism.

Protein phosphorylation and dephosphorylation events regulate many cellular processes. The identification of all phosphorylation sites and their association to a respective protein kinase or phosphatase is a challenging and crucial step to have a deeper understanding of the effects of signaling networks on cells. Pathogenic trypanosomatids have a large number of protein kinases and phosphatases in comparison to other organisms, which reinforces the relevance of the phosphorylation process in these early eukaryotes, nevertheless little is known about protein phosphorylation in these protozoa. In this context, the role of a MAP kinase-like kinase (MAPKLK1), observed to be essential to proliferation of procyclic Trypanosoma brucei, was studied. After silencing MAPKLK1 expression by RNAi, the cells were evaluated by SILAC MS-based proteomics and RNA-Seq. We identified 1756 phosphorylation sites of which 384 were not previously described in T. brucei. Despite being essential, few modulations were observed at the phosphorylation patterns and gene expression levels of MAPKLK1 knockdown. These indirect targets and potential substrates of MAPKLK1 are related to key cellular processes enriched to mRNA processing and stability control. SIGNIFICANCE: The field of cell signaling is a promising topic of study for trypanosomatids, since little is known about this topic and the gene expression regulation occurs at post-transcriptional level. In this sense, the present work increases the knowledge on protein phosphorylation process in Trypanosoma brucei. We depleted one MAP kinase (MAPKLK1) of T. brucei and evaluated the effects on the cell. We showed that MAPKLK1 is essential to the cell, while few modulations on phosphoproteome, proteome and transcriptome are observed with its depletion. Although in low number, the changes in phosphoproteome were significant, presenting possible substrate candidates of MAPKLK1 and indirect targets related to mRNA processing and stability control, metabolic pathways, among others. This result provides insights in the phosphorylation network of T. brucei, a model organism that impacts human and animal health.

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway^® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation.

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.

LM14 defined medium enables continuous growth of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between distinct morphological and functional forms during its life cycle. Axenic multiplication and differentiation processes of this protozoan parasite can be reproduced in vitro, enabling the isolation and study of the different evolutionary forms. Although there are several publications attempting the cultivation of T. cruzi under chemically defined conditions, in our experience none of the published media are capable of maintaining T. cruzi in continuous growth. RESULTS: In this work we modified a known chemically defined medium for Trypanosoma brucei growth. The resulting LM14 and LM14B defined media enabled cultivation of five different strains of T. cruzi for more than forty passages until now. The parasite's biological characteristics such as morphology and differentiation to metacyclic trypomastigotes were maintained when defined media is used. CONCLUSIONS: The establishment of a defined medium for T. cruzi cultivation is an important tool for basic biological research allowing several different approaches, providing new perspectives for further studies related to cell biology of this parasite.

Towards the phosphoproteome of trypanosomatids.

The identification and localization of protein phosphorylation sites provide clues to what proteins or pathways might be activated in a given condition, helping to improve our understanding about signaling networks. Advances in strategies for enrichment of phosphorylated peptides/proteins, mass spectrometry (MS) instrumentation, and specific MS techniques for identification and quantification of post-translational modifications have allowed for large-scale mapping of phosphorylation sites, promoting the field of phosphoproteomics. The great promise of phosphoproteomics is to unravel the dynamics of signaling networks, a layer of the emerging field of systems biology. Until a few years ago only a small number of phosphorylation sites had been described. Following large-scale trends, recent phosphoproteomic studies have reported the mapping of thousands of phosphorylation sites in trypanosomatids. However, quantitative information about the regulation of such sites in different conditions is still lacking. In this chapter, we provide a historical overview of phosphoproteomic studies for trypanosomatids and discuss some challenges and perspectives in the field.