Biology subjects

Olofsson Bagge, R.

Publications and source records attributed to Olofsson Bagge, R..

5 recordsLinked to original sources

bioRxiv2024

Extracellular vesicles isolated from frozen and fresh human melanoma tissue are similar in purity and protein composition

Extracellular vesicles (EVs) isolated from tumor tissues represent a precious source of EVs because they are enriched in disease-associated biomarkers and are highly concentrated. However, a fundamental question is whether freezing of tissues influences the EVs integrity and function and whether non-EVs are co-isolated with the EVs. In this work, we isolated EVs from metastatic melanoma tissue both immediately after tissue resection and after being frozen for at least two weeks. Specifically, the samples were divided into two parts: one was immediately processed for EV isolation (hereafter called fresh), and the other was frozen on dry ice and stored for two weeks before being processed for EV isolation (hereafter called frozen). Large and small EVs were isolated through ultracentrifugation, pooled, and further purified on an iodixanol density cushion. The EVs were analyzed by transmission electron microscopy, nanoparticle tracking analysis, and protein quantification as well as by quantitative mass spectrometry. The results did not show any significant difference between EVs isolated from fresh and frozen tissue. Importantly, there was no enrichment of either intracellular proteins or mitochondrial proteins in EVs isolated from frozen tissues vs. fresh tissues. Moreover, there were no significant differences in the quantity of proteins like MT-CO2, Cox6, SLC24A22, HLA-DR, and Erlin2, which were previously identified as potential markers of melanoma, ovarian cancer, and breast cancer. Overall, this study supports the use of frozen tissues as a source of EVs for research purposes because frozen tissue-derived EVs do not differ in purity or protein composition compared to their fresh counterparts.

cell biology↗

bioRxiv2023

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

Uveal melanoma (UM) is a rare melanoma originating in the eyes uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients succumb to recurrent disease after two years. This study aimed to provide a path towards enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA sequencing of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR sequencing, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

cancer biology↗

bioRxiv2023

Heat inactivation of foetal bovine serum causes protein contamination of extracellular vesicles

Both the release of extracellular vesicles (EVs) in cell cultures and the cargo that these EVs carry 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 on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and heat inactivation were evaluated, including FBS that was ultracentrifuged but not heat-inactivated, FBS that was heat inactivated before EV depletion, and FBS that was heat inactivated after EV depletion. The FBS samples were then added to the culture media of three melanoma cell lines, and after 72 h both large and small EVs were isolated by differential UC. We demonstrated by transmission electron microscopy, protein measurement, and quantification of the number of particles that heat inactivation performed after EV depletion reduced the purity of small EVs but had no effect on large EV purity. Quantitative mass spectrometry analysis of FBS-derived small EVs showed that the EV protein content was different when FBS was heat inactivated after EV depletion compared to EVs isolated from FBS that was not heat inactivated or that was heat inactivated prior to EV depletion. Moreover, several of the quantified proteins were wrongly attributed to be of human origin because the EVs were of obvious bovine origin. Our results demonstrated that proteins of bovine origin coming from FBS-derived EVs could mistakenly be attributed to human cell-derived EVs in EV proteomic studies. Moreover, we concluded that heat inactivation performed after EV depletion induced the release of proteins that might contaminate EV samples, and the recommendation is therefore to always perform heat inactivation prior to EV depletion.

cell biology↗

bioRxiv2022

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.

cancer biology↗

bioRxiv2019

Molecular profiling of driver events and tumor-infiltrating lymphocytes in metastatic uveal melanoma

Uveal melanoma (UM) is a rare form of melanoma with a genetics and immunology that is different from skin melanoma. Previous studies have identified genetic driver events of early stage disease when the tumor is confined to the eye. However due to lack of a clinical rationale to biopsy metastatic disease, access to tumor material to perform molecular profiling of metastases has been limited. In this study, we have characterized genomic events in UM metastases using whole-genome sequencing of fresh frozen biopsies from thirty-two patients and profiled the transcriptomes of individual tumor infiltrating lymphocytes in eight patients by single-cell sequencing. We find that 91% of the patients have metastases carrying inactivating events in the tumor suppressor BAP1 and this coincided with somatic alterations in GNAQ, GNA11, CYSLTR2, PLCB4, SF3B1 and/or CDKN2A. Mutational signature analysis revealed a rare subset of tumors with prominent signs of UV damage, associated with outlier mutational burden. We study copy number variations (CNV) and find overrepresented events, some of which were not altered in matched primary eye tumors. A focused siRNA screen identified functionally significant genes of some of the segments recurrently gained. We reintroduced a functional copy of BAP1 into a patient-derived BAP1 deficient tumor cell line and found broad transcriptomic changes of genes associated with subtype distinction and prognosis in primary UM. Lastly, our analysis of the immune microenvironments of metastases revealed a presence of tumor-reactive T cells. However, a majority expressed the immune checkpoint receptors TIM-3, LAG3 and TIGIT, and to a lesser extent PD-1. These results provide an updated view of genomic events represented in metastatic UM and immune interactions in advanced lesions.

cancer biology↗