The dark side of foetal bovine serum in extracellular vesicle studies

Extracellular vesicles (EVs) have been shown to be involved in cell‐cell communication and to take part in both physiological and pathological processes. Thanks to their exclusive cargo, which includes proteins, lipids, and nucleic acids from the originating cells, they are gaining interest as potential biomarkers of disease. In recent years, their appealing features have been fascinating researchers from all over the world, thus increasing the number of in vitro studies focused on EV release, content, and biological activities. Cultured cell lines are the most‐used source of EVs; however, the EVs released in cell cultures are influenced by the cell culture conditions, such as the use of foetal bovine serum (FBS). FBS is the most common supplement for cell culture media, but it is also a source of contaminants, such as exogenous bovine EVs, RNA, and protein aggregates, that can contaminate the cell‐derived EVs and influence their cargo composition. The presence of FBS contaminants in cell‐derived EV samples is a well‐known issue that limits the clinical applications of EVs, thus increasing the need for standardization. In this review, we will discuss the pros and cons of using FBS in cell cultures as a source of EVs, as well as the protocols used to remove contaminants from FBS

Heat inactivation of fetal bovine serum increases protein contamination of extracellular vesicles

Introduction: Extracellular vesicles (EVs) released in cell cultures are influenced by the cell culture conditions, such as the use of fetal bovine serum (FBS). FBS contains EVs and it is usually depleted of EVs by ultracentrifugation (UC) and/or heat inactivated (HI). Several studies have evaluated the effect of different UC protocols for FBS by evaluating both cells and EVs. However, less is known about the effect of HI on the cells and the released EVs. The aim of this study was therefore to evaluate the effect of HI on EV purity. Methods: To determine the effect of heat inactivation, three different protocols were applied based on different combinations of: 1) UC at 118,000 × g for 18h and 2)HI at 56◦C for 30 min. The three conditions tested were: FBS ultracentrifuged but not heat inactivated (no-HI), FBS heat inactivated before UC (HI-before EV-dep), and FBS heat inactivated after EV depletion (HI-after EV-dep). The FBS was add to themedia of threemelanoma cell lines (MML1, UM22Ctr and UM22BAP1) at a final concentration of 10%. After 72h, large and small EVs were isolated by differential UC. The EV purity was determined by protein quantity, electron microscopy (EM) and nanoparticle tracking analysis (NTA). Results: The protein quantity (μg/μl) of large EVs was similar in the three conditions analyzed. On the contrary for small EVs, the protein amount was higher when the HI was performed after EV depletion as compared to HI before the UC and UC alone. However, significantly more particles were not detected in the HI-after EV-dep which resulted in a lower purity of small EVs in HI-after EV-dep illustrated by calculating the ratio of number of particles/μg proteins. Presence of contaminants (indicated by strong background) was observed in EM pictures of small EVs isolated in HI-after EV-dep condition differently from large EV samples. Summary/Conclusion: The HI of FBS induces release of contaminating elements that end up in small EVpellets if not previously removed

Three-dimensional reconstruction of interstitial extracellular vesicles in human liver as determined by electron tomography

Extracellular vesicles (EVs) are lipid bilayer nanoparticles involved in cell-cell communication that are released into the extracellular space by all cell types. The cargo of EVs includes proteins, lipids, nucleic acids, and metabolites reflecting their cell of origin. EVs have recently been isolated directly from solid tissues, and this may provide insights into how EVs mediate communication between cells in vivo. Even though EVs have been isolated from tissues, their point of origin when they are in the interstitial space has been uncertain. In this study, we performed three-dimensional (3D) reconstruction using transmission electron tomography of metastatic and normal liver tissues with a focus on the presence of EVs in the interstitium. After chemical fixation of the samples and subsequent embedding of tissue pieces in resin, ultrathin slices (300 nm) were cut and imaged on a 120 ekV transmission electron microscopy as a tilt series (a series of subsequent images tilted at different angles). These were then computationally illustrated in a 3D manner to reconstruct the imaged tissue volume. We identified the cells delimiting the interstitial space in both types of tissues, and small distinct spherical structures with a diameter of 30-200 nm were identified between the cells. These round structures appeared to be more abundant in metastatic tissue compared to normal tissue. We suggest that the observed spherical structures in the interstitium of the metastatic and non-metastatic liver represent EVs. This work thus provides the first 3D visualization of EVs in human tissue.Three-dimensional (3D) reconstruction of both liver metastases and normal liver, using transmission electron tomography, identified spherical structures with a diameter of 30-200 nm in the interstitium of both types of tissues.imag

Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion

Extracellular vesicle DNA from human melanoma tissues contains cancer-specific mutations

Liquid biopsies are promising tools for early diagnosis and residual disease monitoring in patients with cancer, and circulating tumor DNA isolated from plasma has been extensively studied as it has been shown to contain tumor-specific mutations. Extracellular vesicles (EVs) present in tumor tissues carry tumor-derived molecules such as proteins and nucleic acids, and thus EVs can potentially represent a source of cancer-specific DNA. Here we identified the presence of tumor-specific DNA mutations in EVs isolated from six human melanoma metastatic tissues and compared the results with tumor tissue DNA and plasma DNA. Tumor tissue EVs were isolated using enzymatic treatment followed by ultracentrifugation and iodixanol density cushion isolation. A panel of 34 melanoma-related genes was investigated using ultra-sensitive sequencing (SiMSen-seq). We detected mutations in six genes in the EVs (BRAF, NRAS, CDKN2A, STK19, PPP6C, and RAC), and at least one mutation was detected in all melanoma EV samples. Interestingly, the mutant allele frequency was higher in DNA isolated from tumor-derived EVs compared to total DNA extracted directly from plasma DNA, supporting the potential role of tumor EVs as future biomarkers in melanom

Isolated Limb Perfusion With Melphalan Triggers Immune Activation in Melanoma Patients

Hyperthermic isolated limb perfusion with melphalan (M-ILP) is a treatment option for melanoma patients with metastases confined to the limbs. This study aimed at defining the role of cellular immunity for the clinical response to M-ILP in melanoma patients. It was observed that patients with enhanced cytotoxic CD8+ T cell reactivity to common antigens (HCMV/EBV/influenza virus) prior to M-ILP were more likely to achieve a complete disappearance of macroscopic tumors (complete response). Following M-ILP treatment, the proportions of CD16+ intermediate and non-classical monocytes in peripheral blood were significantly enhanced along with induction of HLA-DR on CD4+ and CD8+ T cells. For further studies of the mechanism behind melphalan-induced immune activation an in vitro model, aiming at mimicking the clinical M-ILP protocol, was established, where PBMCs were co-cultured with melanoma cells, which had been pre-exposed to melphalan under mild hyperthermia. Upon exposure to melphalan, melanoma cells showed increased expression of immune-related markers including MHC class I and Hsp70. Moreover, when the melphalan-treated melanoma cells were co-cultured with PBMCs, this triggered an increased proportion of CD33+CD14+CD16++ non-classical monocytes among the PBMCs. Furthermore, the melphalan-treated melanoma cells stimulated the expansion of CD8+ T cells in the co-cultured PBMCs. These cells produced enhanced levels of IFN-γ and granzyme B and were capable of killing melanoma cells. To further verify an immunogenic role of melphalan, mice were vaccinated with melphalan-exposed murine melanoma cells. When challenged with live melanoma cells, vaccinated mice showed reduced tumor growth and enhanced infiltration of tumor-specific T cells into tumors. We conclude that melphalan-exposed melanoma cells trigger expansion of CD16+ monocytes and activate cytotoxic T cells and that these events may contribute to the antitumoral efficacy of M-ILP

BRAF(V600) inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAF(V600) mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.1114sciescopu

Treatment with Anti-HER2 Chimeric Antigen Receptor Tumor-Infiltrating Lymphocytes (CAR-TILs) Is Safe and Associated with Antitumor Efficacy in Mice and Companion Dogs

Patients with metastatic melanoma have a historically poor prognosis, but recent advances in treatment options, including targeted therapy and immunotherapy, have drastically improved the outcomes for some of these patients. However, not all patients respond to available treatments, and around 50% of patients with metastatic cutaneous melanoma and almost all patients with metastases of uveal melanoma die of their disease. Thus, there is a need for novel treatment strategies for patients with melanoma that do not benefit from the available therapies. Chimeric antigen receptor-expressing T (CAR-T) cells are largely unexplored in melanoma. Traditionally, CAR-T cells have been produced by transducing blood-derived T cells with a virus expressing CAR. However, tumor-infiltrating lymphocytes (TILs) can also be engineered to express CAR, and such CAR-TILs could be dual-targeting. To this end, tumor samples and autologous TILs from metastasized human uveal and cutaneous melanoma were expanded in vitro and transduced with a lentiviral vector encoding an anti-HER2 CAR construct. When infused into patient-derived xenograft (PDX) mouse models carrying autologous tumors, CAR-TILs were able to eradicate melanoma, even in the absence of antigen presentation by HLA. To advance this concept to the clinic and assess its safety in an immune-competent and human-patient-like setting, we treated four companion dogs with autologous anti-HER2 CAR-TILs. We found that these cells were tolerable and showed signs of anti-tumor activity. Taken together, CAR-TIL therapy is a promising avenue for broadening the tumor-targeting capacity of TILs in patients with checkpoint immunotherapy-resistant melanoma

The generalisability of randomised clinical trials : an interim external validity analysis of the ongoing SENOMAC trial in sentinel lymph node-positive breast cancer

Purpose None of the key randomised trials on the omission of axillary lymph node dissection (ALND) in sentinel lymph-positive breast cancer have reported external validity, even though results indicate selection bias. Our aim was to assess the external validity of the ongoing randomised SENOMAC trial by comparing characteristics of Swedish SENOMAC trial participants with non-included eligible patients registered in the Swedish National Breast Cancer Register (NKBC). Methods In the ongoing non-inferiority European SENOMAC trial, clinically node-negative cT1-T3 breast cancer patients with up to two sentinel lymph node macrometastases are randomised to undergo completion ALND or not. Both breast-conserving surgery and mastectomy are eligible interventions. Data from NKBC were extracted for the years 2016 and 2017, and patient and tumour characteristics compared with Swedish trial participants from the same years. Results Overall, 306 NKBC cases from non-participating and 847 NKBC cases from participating sites (excluding SENOMAC participants) were compared with 463 SENOMAC trial participants. Patients belonging to the middle age groups (p = 0.015), with smaller tumours (p = 0.013) treated by breast-conserving therapy (50.3 versus 47.1 versus 65.2%, p < 0.001) and less nodal tumour burden (only 1 macrometastasis in 78.8 versus 79.9 versus 87.3%, p = 0.001) were over-represented in the trial population. Time trends indicated, however, that differences may be mitigated over time. Conclusions This interim external validity analysis specifically addresses selection mechanisms during an ongoing trial, potentially increasing generalisability by the time full accrual is reached. Similar validity checks should be an integral part of prospective clinical trials. Trial registration: NCT 02240472, retrospective registration date September 14, 2015 after trial initiation on January 31, 2015Peer reviewe

1 Versus 2-cm Excision Margins for pT2-pT4 Primary Cutaneous Melanoma (MelMarT): A Feasibility Study

Abstract Background There is a lack of consensus regarding optimal surgical excision margins for primary cutaneous melanoma &gt; 1 mm in Breslow thickness (BT). A narrower surgical margin is expected to be associated with lower morbidity, improved quality of life (QoL), and reduced cost. We report the results of a pilot international study (MelMarT) comparing a 1 versus 2-cm surgical margin for patients with primary melanoma &gt; 1 mm in BT. Methods This phase III, multicentre trial [NCT02385214] administered by the Australia &amp; New Zealand Medical Trials Group (ANZMTG 03.12) randomised patients with a primary cutaneous melanoma &gt; 1 mm in BT to a 1 versus 2-cm wide excision margin to be performed with sentinel lymph node biopsy. Surgical closure technique was at the discretion of the treating surgeon. Patients’ QoL was measured (FACT-M questionnaire) at baseline, 3, 6, and 12 months after randomisation. Results Between January 2015 and June 2016, 400 patients were randomised from 17 centres in 5 countries. A total of 377 patients were available for analysis. Primary melanomas were located on the trunk (56.9%), extremities (35.6%), and head and neck (7.4%). More patients in the 2-cm margin group required reconstruction (34.9 vs. 13.6%; p &lt; 0.0001). There was an increased wound necrosis rate in the 2-cm arm (0.5 vs. 3.6%; p = 0.036). After 12 months’ follow-up, no differences were noted in QoL between groups. Discussion This pilot study demonstrates the feasibility of a large international RCT to provide a definitive answer to the optimal excision margin for patients with intermediate- to high-risk primary cutaneous melanoma. </jats:sec