Extracellular vesicles from Nurse-Like Cells and THP1-derived Type-2 macrophages transfer aggressiveness to chronic lymphocytic leukemia cells

doi:10.21203/rs.3.rs-3578709/v1

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Extracellular vesicles from Nurse-Like Cells and THP1-derived Type-2 macrophages transfer aggressiveness to chronic lymphocytic leukemia cells

https://doi.org/10.21203/rs.3.rs-3578709/v1

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published 25 Jun, 2024

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The resistance of leukemic B cells to cell death is mainly associated with interactions within the tumor microenvironment, where they interact with various types of cells. Within this microenvironment, CLL B cells produce and bind cytokines, growth factors, and microvesicles. In the present study, extracellular vesicles (EVs) purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL B cell cultures. The vesicles were rapidly internalized by B cells, leading to a decrease in spontaneous apoptosis and an increase in proliferation. Additionally, the vesicles induced an increase in the resistance of CLL B cells to Bruton kinase inhibitor Ibrutinib. A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene Bcl-2 but not Mcl-1 and an increase in the expression of proliferation-inducing gene APRIL following treatment of CLL B cells with EVs derived from both NLCs and M2-THP1 cells. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2, CD40, p53 and Bcl-2 following EVs treatment. Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of B CLL cells treated or not with NLCs EVs show various differentially expressed genes.

Biological sciences/Cancer/Cancer microenvironment

Biological sciences/Cell biology/Mechanisms of disease

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of monoclonal CD5⁺/CD19⁺/IgM^low/IgD^low mature B cells in the blood, bone marrow, lymph nodes and spleen with a resistance to apoptosis and a low proliferation rate¹. The CLL B cells proliferation rate is high when the cells reside in lymphoid organs such as the bone marrow, lymph nodes, and spleen, but is low in the peripheral blood². This characteristic highlights that interactions between malignant B cells and the tumor microenvironment (TME) play a major role in the physiopathology of CLL³. Within the TME, CLL B cells receive signals from various populations of accessory cells such as mesenchymal stromal cells, monocyte-derived nurse-like cells (NLCs), follicular dendritic cells, and T cells. They communicate through the interaction of various surface and secreted proteins, B CLL cells also receive signals from extracellular vesicles (EVs) notably produced by stromal cells⁴.

EVs are small vesicles known to mediate intercellular communications in local and distant microenvironments under physiological and pathological conditions. They are separated into two main categories; exosomes that are 30–150 nm vesicles generated through the formation of multivesicular bodies and microvesicles (previously referred to as ectosomes or microparticles) that are 150–1000 nm vesicles resulting from the blebbing of the plasma membrane⁵. Crompot et al. highlighted the impact of bone marrow MSC EVs on CLL B cells in vitro⁴. These authors showed that MSC-derived EVs are rapidly incorporated in CLL B cells and that they increase CLL cell migration, suggesting that these EVs could give CLL cells survival advantages in vivo. Recently, Gargiulo et al. also underscored the functional importance of EVs produced in the tumor microenvironment in promoting CLL progression by showing that TME EVs impair CD8⁺ T-cell mediated antitumor immune response and are indispensable for leukemia progression in vivo in murine models⁶.

Although the impact of TME-derived EVs remains largely unknown, the impact of EVs from B CLL cells onto the TME is well documented. For instance, Ghosh evidenced that CLL B cells exosomes activate bone marrow stromal cells (BMSCs) via transfer of bioactive molecules including Axl receptor tyrosine kinase⁷. B CLL-derived exosomes are actively incorporated by endothelial cells and mesenchymal stem cells ex vivo and in vivo and the transfer of exosomal proteins and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts. EVs released by activated CLL B cells are also reported to increase immune synapse activity while downregulating CD69 expression levels on CD4⁺ T cells. Altogether, it is likely that CLL EVs target diverse cell types in the TME to reprogram their function and promote disease progression.

Here, we investigated the role of EVs derived from Nurse-Like Cells (NLCs) on different biological processes critical for CLL cell survival. NLCs are tumor associated macrophages that promote CLL B cells survival notably through the secretion of APRIL, CXCL12 and soluble CD14 or LFA-3/CD2 interaction³ and BDNF ^8,9.

Ethical approval and consent to participate

The study was approved by the Limoges University Hospital Institutional Review Board, AC 72-2011-18. All patients and healthy donors provided informed consent to participate, all experiments were performed in accordance with relevant named guidelines and regulation.

Primary culture

Isolation of peripheral blood mononuclear cells (PBMC) by density gradient was performed on blood samples from patients and healthy donors. The viability and quantity of isolated B-CLL are assessed and they are then frozen in decomplemented serum supplemented with 10% DMSO. After thawing, the cells were placed at 37°C in RPMI 1640 medium (ThermoFisher) supplemented with 10% decomplemented fetal calf serum (FBS) and 1% antibiotics (penicillin/streptomycin) for 1 h before starting any experiments.

Cell line culture

The THP1 cell line was cultured in RPMI 1640 medium (ThermoFisher) supplemented with 10% decomplemented FBS, 1% antibiotics (penicillin/streptomycin) and 0.1% β-mercaptoethanol (50nM) (ThermoFisher). Cultures were performed at 37°C, 5% CO₂.

Cell treatments with EVs

CLL-B cells were seeded either in a 6-well plate at 2.10⁶ cells if they were destined for western blot analysis or in a 96-well plate at 25 000 cells for apoptosis and proliferation analyses. They were then treated at a ratio of 1.10^− 3 µg of EVs/cell resuspended in an “exo-free” culture medium. Exo-free medium is a conventional culture medium with EVs-deprived FBS by ultracentrifugation at 120 000 x g for 16h.

Monocyte differentiation and polarization

To differentiate THP1 into M2-like tumor-associated macrophages, 4.5.10⁶ cells were seeded in a T75 flask and treated with 50 ng/ml of Phorbol 12-myristate 13-acetate (PMA) for 24h. Cells were then washed with PBS and the medium switched with fresh one, cells were incubated for 72h. Medium was then replaced and supplemented with 20 ng/ml of IL4 and IL13 for 48h^10,11. Cells were then rested in fresh medium for 24h. Finally, the medium was switched with exo-free medium and cells were incubated for 48h before supernatant collection.

NLCs differentiation

NLCs were derived by culturing approximately 5x10⁶ cells of PBMCs from CLL patients in 2 ml of complete medium in 6-well plates. After 14 days CLL B cells were removed by vigorously washing each well with culture medium¹².

Isolation of EVs by ultracentrifugation

Culture supernatants were collected and filtrated using a 0.2 µm filter, they were then ultracentrifuged at 120 000 x g for 90 min twice to pellet and wash vesicles¹³. Then, EVs were resuspended in PBS for nanoparticle tracking analysis (NTA) or in culture medium for cell treatment or in RIPA buffer (ThermoFisher) for western blot analysis.

Nanoparticle tracking analysis (NTA)

NTA was performed using NanoSight NS300 (Malvern Panalytical Ltd.). Captures and analysis were achieved using the NanoSight Software NTA3.3.301. Samples were diluted in PBS and their concentration was adjusted by observing a particles/frame rate of around 50 (30–100 particles/frame). For each measurement, five consecutive 60 s videos were recorded (cell temperature: 25^◦C, syringe speed: 22 µl/s).

Labeling and internalization of exosomes

To check for the ability of exosomes to penetrate cells, we labeled them using a PKH67 labelling kit (Merck) according to the manufacturer’s instructions. We then treated B CLL cells with the labelled exosomes and checked for their internalization using a CytoFLEX flow cytometer (Beckman Coulter), results were analyzed using the Kaluza software (Beckman Coulter). We also observed treated cells using confocal microscopy (LSM880, Zeiss).

Evaluation of cell proliferation and apoptosis

Apoptosis and proliferation are monitored using the IncucyteS3 (Sartorius). Labeling of apoptotic cells is performed with Incucyte Annexin V Dye for Apoptosis (Sartorius) and proliferation is evaluated either through nuclei labeling with NucLight (Sartorius) or using the Incucyte cell-by-cell tracking module. Analysis is performed with the Incucyte Base Analysis Software.

Western blot assay

Both cells and EVs samples were lysed with RIPA cell lysis buffer (Sigma-Aldrich) supplemented with 1% protease inhibitor cocktail (Cell Signaling). Lysates were then sonicated and centrifuged at 16,000 x g / 4°C for 20 minutes before being subjected to western blot analysis.

Antibody array

200 µg of cell lysates treated or not with NLCs EVs were analyzed using the Human Apoptosis Antibody Array (Abcam) according to the manufacturer’s instructions.

RNA extraction and RTqPCR analysis

Total RNA were extracted using the Quick-RNA™ Microprep Kit (Zymo Research) and quantified by NanoDrop 2000 (Thermo Fischer). For RT-qPCR analyses, 2 µg of RNA were reverse-transcribed into cDNA using the cDNA Archive kit (Applied Biosystems). Quantitative gene expression was performed using SensiFAST Probe Hi-ROX kit (Bioline, London) on a QuantStudio 5 (Thermo Fischer). Results were normalized to actin expression and analyzed using the ∆∆Ct method.

Transcriptomic profiling by RNASeq

mRNA from B cells from five CLL patients that were either treated with NLCs EVs or left untreated (paired control samples), were sequenced by Novogene Company (UK) according to the manufacturer’s protocol. Quality control of 150 bp paired-end reads were checked by FastQC software, low-quality end reads were trimmed (Phred quality score < 30), and adapter removal was performed using Cutadapt¹⁴. The clean reads of each sample were mapped to the human reference genome (GRCh38 assembly) using STAR¹⁵ splice-mapper and read counts for each gene were obtained using the featureCounts¹⁶ counting tool. Mapping quality analysis revealed that two matched patients’ samples (EVs-treated and control) failed to obtain a good mapping rate (< 70%) and were discarded for subsequent analysis¹⁷.

Differential gene expression was conducted pair-wise using DESeq2¹⁸ to identify differentially expressed genes (DEGs) according to the following selection criteria: absolute log₂ fold change superior to 1 and p value inferior to 0.05. The Benjamini-Hochberg (BH) multiple testing correction was applied but none of the genes had a p-adjusted inferior to 0.05, certainly due to the small number of analyzed samples.

Sample preparation for LC MSMS and whole proteome analysis

Sample preparation and whole proteome analysis of exosomes was performed by the PISSARO proteomics platform (Rouen, France). Exosomal proteins (n = 3) were resuspended in a dedicated solubilization buffer and protein concentration was estimated by Bradford assay (Biorad). An “in gel” tryptic digestion on 10 µg of the total protein extract was performed to obtain the subsequent peptide fraction used for mass spectrometry experiments. Peptides fractions were solubilized in Formic Acid 0.1% and analyzed on a Q-Exactive Plus Orbitrap™ mass spectrometer (Thermo Scientific) coupled to a chromatographic system, Easy nanoLCII system (Thermo Scientific) All spectra obtained were exported in “raw” format to identify peptides and proteins with Proteome Discoverer 1.4 Software (Thermo Scientific). Peak lists were searched using MASCOT search software (Matrix Science) against the human Swissprot database.

To analyze the data, we started by using Venny¹⁹ to generate a Venn diagram using our three replicates, we kept the proteins found in common, which represented 56.6% of the total amount. We then used DAVID²⁰ to perform functional enrichment according to REACTOME terms and only retained entries with p-values inferior to 0.05 using the Benjamini-Hochberg correction to determine the false discovery rate.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 9.0. Data was compared using either a t-test or a one-way ANOVA followed by pairwise comparisons. P values ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 were considered significant.

Patient-derived NLCs and M2-THP1 cells produce EVs which are rapidly incorporated into CLL B cells.

Co-culture of CLL B cells with NLCs induced a decrease of B cells apoptosis of 76% after 8 days of culture (Supplementary Fig. 1A). In the same way, co-culture with M2-THP1 cells, induced a diminution of apoptosis of 57% (Supplementary Fig. 1B). As previously evidenced, these data show that both NLCs and M2-THP1 cells enable the survival of leukemic B cells^21,22. This ability could notably be associated with the secretion of EVs, as shown on Supplementary Figs. 1B-1F.

We hypothesized that both NLCs and M2-THP1 are able to send survival signals to leukemic cells through EVs. To investigate, we first verified the presence of EVs in the culture supernatants of NLCs and M2-THP1 cells. EVs were isolated by ultracentrifugation and their size distribution was analyzed by nanoparticle tracking analysis (NTA). A mode of 103 ± 10 nm was obtained for the size of both types of EVs (Figs. 1A and B), signifying that the isolated vesicles are mainly the size of exosomes²³. EVs were also analyzed by immunoblots directed against CD9 and Flotillin-1. These two exosomal markers²⁴ were highly expressed in the protein extracts from vesicles, further confirming the possibility that these EVs are exosomes (Fig. 1C).

Next, to check for the ability of these exosomes to penetrate CLL B cells, they were incubated for 30 min, 2h, 12h or 24h with PKH67 green-labeled EVs obtained from NLCs or M2-THP1 cells, fluorescence was analyzed by flow cytometry. The uptake of exosomes was a rapid process as observed on Figs. 1D and 1E, with more than 93% of CLL-B cells having integrated fluorescent exosomes after 30 min. The integration of exosomes was also confirmed by confocal microscopy imaging of CLL B cells after 24h of incubation (Fig. 1F).

EVs from NLCs and M2-THP1 protect CLL B cells from spontaneous apoptosis ex vivo.

The addition of EVs from NLC and M2-THP1 cells to CLL-B cells resulted in a decrease of spontaneous apoptosis of CLL B cells. Rapidly after addition, the apoptosis level of CLL B cells decreased and reached a maximum after 16h (Supplementary Fig. 3). Very surprisingly, the decrease of apoptosis level was equivalent for all the EVs quantities tested (10 to 100 ng of EVs proteins).

The addition of EVs also affects the proliferation of CLL B cells ex vivo. The number of cells still increased after 48h of culture. Conversely to the data observed for apoptosis, the proliferation rate responded in a dose-dependent manner to the EVs concentration (Supplementary Fig. 3). This data underlines the strong anti-apoptotic effect of EVs, even at relatively low dosage, as well as their proliferative effect.

The analyses realized with the IncucyteS3 device allowed us to monitor apoptosis and proliferation levels over a period of 48h. Figures 2A represents the global measurements of apoptosis and proliferation levels, realized over the 48h of incubation. As observed in Figs. 2A and B, the addition of EVs (25 ng proteins per 25 000 cells) resulted in a significant decrease of apoptosis regardless of their origin (NLCs or M2-THP1 cells). For both EVs origins, the proliferation of CLL B cells was increased (Fig. 2B).

As resistance to apoptosis is a hallmark of B CLL clonal lymphocytes, which are characterized by an elevated expression of anti-apoptotic Bcl-2 family proteins²⁵, we measured the expression level of Bcl-2 and Mcl-1 genes, two genes encoding anti-apoptotic proteins, in CLL B cells incubated with EVs from M2-THP1 cells or NLCs (Figs. 2C and D). Mcl-1 gene expression was unaffected by EVs (Fig. 2D), but Bcl-2 was significantly overexpressed in CLL B cells incubated with EVs (Fig. 2C). In the same way, APRIL (a proliferation inducing ligand) was already described as overexpressed in CLL B-cells²⁶, so we evaluated the expression level of this growth factor in CLL B cells incubated with EVs from M2-THP1 cells or NLCs (Fig. 2E) and observed that EVs induced an overexpression of APRIL in CLL B cells.

EVs from NLCs activate anti-apoptotic pathways in B CLL cells.

We previously observed that when incubated with EVs derived from NLCs and M2-THP1 cells, CLL B cells demonstrated enhanced survival, a process already described as dependent on the overexpression of Bcl-2⁸. To gain a deeper understanding of the anti-apoptotic pathways triggered by those EVs, we conducted a comprehensive analysis of apoptotic pathways activation using a protein array approach (Supplementary Fig. 4). The array we employed allowed us to quantify the relative levels of 43 proteins associated with apoptosis. Comparative analysis between untreated CLL B cells and those incubated with EVs from patient-derived NLCs revealed a significant increase in the levels of IGFBP-2 and CD40 proteins following EVs treatment. There was also an increase in the amount of Bcl-2 and p53 proteins, whose involvement in CLL has already been well documented^27,28, which was however not significant. In order to better confirm these variations in protein levels, we analyzed B CLL cells from three different patients treated or not with NLCs EVs (Fig. 3). Results show a significant increase in the levels of proteins IGFBP-2, CD40, p53 as well as Bcl-2.

EVs from NLCs affect gene expression in CLL patients.

The elevated levels of anti-apoptotic proteins observed in CLL B cells when incubated with NLCs EVs prompted us to explore the transcriptome alterations in the treated B cells in comparison to untreated cells. To achieve this, we performed an RNAseq analysis on B cells obtained from five distinct CLL patients that were either treated with NLCs EVs or left untreated. Unfortunately, the analysis was inconclusive for two out of the five patients, so we were only able to analyze the data for three patients. Due to the small size of the cohort and high inter-individual variability (Fig. 4A), we analyzed results using a paired-analysis method. A list of the 121 differentially expressed genes, 21 upregulated and 100 downregulated, between the treated versus the control conditions, is available in Supplementary Table 3. Interestingly, 44% of DEGs were protein-coding genes and 40% were lncRNA.

Among the upregulated genes, we found: RGS1, Regulator of G protein Signaling 1; BCL2A1, Bcl2-related protein A1; TNFRSF18, TNF Receptor Superfamily Member 18 (also known as GITR); and CARD16, Caspase Recruitment Domain Family Member 16, as shown on Fig. 4D.

RGS1 has been found to be upregulated in several cancers, it is an unfavorable prognostic marker in renal and stomach cancer²⁹, high immunohistochemical expression of RGS1 has also been linked to poor overall survival in diffuse large B cell lymphoma (DLBCL) patients³⁰. In multiple cancers, RGS1 has been found to promote T-cell exhaustion³¹, in breast cancer for instance, its upregulation reduced trafficking of anti-tumor lymphocytes to tumors and was associated with shorter survival of patients³².

BCL2A1 is upregulated in various cancers as well, notably in CLL, where it has been shown to induce significant resistance to Bcl-2 inhibitor ABT-737³³.

As for TNFRSF18, it has been found to have both coinhibitory and costimulatory effects on anti-tumor T-cell responses³⁴. It is a favorable prognostic marker in renal and endometrial cancers but an unfavorable one in head and neck cancer³⁵.

Finally, CARD proteins have been established as key regulators of cell death and cytokine production³⁶. While the involvement of CARD16 specifically in pathogenesis remains largely unknown, it is a known activator of Caspase 1³⁷, whose high levels of expression have been linked to poor prognosis in acute myeloid leukemia (AML)³⁸.

RNASeq results also revealed various downregulated genes (Fig. 4B). Among them, IRF9, Interferon Regulatory Factor 9 and miRNA 4539 (Fig. 4D).

Interestingly, IRF9 was shown to be downregulated in samples from AML patients compared to healthy donors. IRF9 knockdown promoted proliferation, colony formation and survival in AML cell lines, whereas IRF9 overexpression led to the opposite. IRF9 also increased the acetylation of p53, and promoted the expression of p53 target genes in those same cell lines³⁹.

Regarding miRNA 4539, a study comparing miRNA expression in colorectal cancer (CRC) tissue versus healthy tissue in 1945 individuals found that miRNA 4539 was significantly downregulated in CRC. Its dysregulation was identified as being important in CRC pathogenesis based on a random forest assessment method of analysis⁴⁰.

Overall, these results suggest that EVs from patient-derived NLCs are able to induce gene expression modifications in B CLL cells that promote CLL progression through the activation of inhibition of diverse signaling pathways involved in oncogenesis. However, it is important to note that due to the small number of individuals included in the study and high inter-individual variability, these results remain preliminary. Although they provide valuable insight into the potential of NLCs EVs to alter gene expression in order to transfer aggressiveness to B CLL cells, there is a need for further experimental work both to confirm and deepen our understanding of the observed changes.

M2-THP1 EVs contain proteins involved in various oncogenic pathways.

In order to identify which protein actors could be responsible for transferring aggressiveness to CLL B cells, we conducted a whole proteome analysis on EVs using mass spectrometry. We were unable, due to technical difficulties, to perform this analysis on EVs derived from NLCs, so we used EVs from M2-THP1 cells, as they were easier to obtain in quantities sufficient for subsequent mass spectrometry analysis.

Proteomics analysis identified more than 1000 proteins in the 3 replicates, from which 608 proteins were common to all samples (Supplementary Table 2). The whole proteome analysis of M2-THP1 EVs identified several pathways that have been linked to hematological malignancies, and in some cases, specifically to chronic lymphocytic leukemia. In Fig. 5A, we present a Venn diagram of the proteins that were identified in each replicate by mass spectrometry in M2-THP1 EVs against the human protein repertoire of the database ExoCarta⁴¹, which lists every protein that has been identified within human exosomes. This shows that 93.6% of the proteins we identified are within the ExoCarta database and that 71% of the most-found proteins within exosomes (ExoCarta top 100) are present within the samples we analyzed, further confirming that they are in fact exosomes.

In Fig. 5B, we generated a REACTOME enrichment dotplot using several identified pathways that had significative p-values and that we deemed the most interesting in the context of our study. The list of all identified pathways and associated genes as well as the list of all the proteins identified by mass spectrometry are available in the supplementary data appendix (Supplementary Table 2). Among the pathways identified, we unsurprisingly found pathways relating to the immune system, T cell receptor (TCR) signaling, interleukin signaling and BCR signaling. Dysregulation of the immune system is a key factor in the development of hematological malignancies, as it can lead to the loss of tolerance to self-antigens and the accumulation of malignant cells⁴². These results are in accordance with previous work which evidenced that proteins involved in those same pathways are overexpressed in EVs from the TME of murine CLL models compared with healthy controls⁶. Meanwhile, BCR signaling plays a crucial role in the survival and proliferation of B cells, dysregulation of its signaling has been implicated in the pathogenesis of CLL and many other B cell malignancies.

In accordance with results obtained within this study, many proteins involved in the regulation of apoptosis were identified. Additionally, we found actors involved in the activation of transcription factors such as NF-κB and RUNX3, which have both been implicated in the development of hematological malignancies^43,44.

Furthermore, many proteins involved in the stabilization of p53 were found. The p53 pathway is a critical tumor suppressor pathway that prevents the accumulation of unrepaired DNA damage. In CLL, p53 alterations are associated with poor clinical outcomes and treatment failures^45,46.

In summary, the pathways identified in the proteomic analysis of EVs produced by M2-THP1 cells are involved in various aspects of hematological malignancies, including immune regulation, cell survival, and cell death. These pathways have been linked to the development and progression of CLL and other hematological malignancies, highlighting the potential importance of EVs in the pathogenesis of these diseases.

EVs from NLCs cells increase resistance to Ibrutinib of CLL B cells.

As seen before, EVs induce resistance to apoptosis, but we wondered whether they were implicated in CLL B cells drug resistance, notably resistance to Ibrutinib. We treated B CLL cells from three different patients with either Ibrutinib alone, or Ibrutinib in combination with EVs from NLCs. Following treatments, B CLL cells treated with both Ibrutinib and NLCs EVs showed a significantly lower (p-value < 0,0001) apoptosis level compared to those treated with Ibrutinib alone with a decrease of almost 20% in apoptosis level. These results therefore suggest that EVs derived from NLCs are able to induce Ibrutinib resistance in patients leukemic B cells in vitro.

Extracellular vesicles are membrane-encapsulated particles that mediate cell-to-cell communication by transferring regulatory molecules such as proteins, lipids, and nucleic acids between cells⁴⁷. In the context of hematological malignancies, EVs have been shown to contribute to the development and progression of the disease by promoting cancer cell survival, growth, and invasion⁴⁸. They have been shown to carry oncogenic molecules, modulate the immune system, promote angiogenesis, and induce stroma remodeling⁴⁹. Furthermore, EVs have potential applications in cancer diagnosis and therapy, as their molecular cargo reflects the nature and status of the cells of origin, making them interesting tools to identify new specific therapeutic targets, improve prognosis assessment⁵⁰ or even be used as therapeutic carriers⁵¹. EVs therefore play a crucial role in hematological malignancies by mediating intercellular communication and influencing various aspects of cancer development and progression.

In the present study, we investigated the role of EVs as communication tools between the microenvironment, specifically nurse-like cells, and leukemic B cells. We showed that EVs derived from NLCs, as well as their in vitro model equivalent, M2-polarized THP1 cells, protect B CLL cells from apoptosis and enhance their proliferation ex vivo notably through the overexpression of anti-apoptotic gene Bcl-2 and proliferation-inducing gene APRIL.

We also showed that following EVs treatment, there was an increase in the expression of several proteins involved in apoptosis-related pathways in B CLL cells, and that, regardless of the EVs source. Among them, Bcl-2, whose involvement in CLL has already been extensively described⁵². There was also an increase in IGFBP-2 levels, while there is no direct evidence of the relationship between IGFBP-2 and CLL, it is highly expressed in certain human acute leukemia cells, and it supports the activity of hematopoietic stem cells (HSCs)⁵³. Therefore, it is possible that IGFBP-2 may play a role in the development and progression of CLL, but more research is needed to confirm this hypothesis. Conversely to IGFBP-2, the relationship between CD40 and CLL is well documented. For example, CLL cells strongly depend on interactions with bystander T cells and monocyte-derived cells (MDCs) within the lymph node (LN) microenvironment for their survival and resistance to therapy⁵⁴. An increased level of p53 protein was also observed, it has already been linked to an advanced clinical stage, more progressive forms of the disease, poor response to therapy and an overall lower survival²⁸. Furthermore, a study by Samuel et al. describes that a 30- to 40-fold induction of wild-type p53 protein was observed in 50 distinct human CLL patients tested, without the induction of either cell-cycle arrest or apoptosis, however, the mRNA levels for p53 did not increase, indicating that its elevation occurred post-transcriptionally⁵⁵.

To assess the potential impact of NLCs EVs on B CLL cells gene expression, we evaluated mRNA expression levels of B CLL cells treated or not with NLCs EVs through RNAseq analysis. We found that EVs treatment induced changes in the expression of various genes, some already known for their involvement in pathogenesis. These results show that NLCs are in fact able to modulate gene expression in leukemic cells through the production of EVs but remain preliminary and need further investigation to precisely identify the actors involved.

Upon the observation of the changes induced by EVs, we pondered their potential content. To investigate it, we performed a whole proteome analysis of EVs derived from M2-THP1 cells. Results highlighted the presence of many proteins involved in pathways critical to pathogenesis. Among them, pathways relating to cell death, transcriptional regulation, and various signaling pathways linked to oncogenesis. This analysis offers valuable insight regarding the protein cargo of EVs, but it remains a study of an in vitro model and not actual patient-derived NLCs. It would be of interest to perform the same analysis on NLCs EVs if we are ever able to obtain them in sufficient quantities.

Finally, in addition to their effect on B CLL cells apoptosis and proliferation rate, we tested the effect of EVs on treatment resistance. EVs from NLCs increased the resistance of B CLL cells to Ibrutinib.

Overall, these results show that EVs from the microenvironment, and not only EVs from malignant B cells⁷, are able to modulate their recipient’s cell activity, sending oncogenic signals to promote leukemic cell aggressiveness. NLCs had previously been identified as key actors in the TME²¹ but they had never been shown to transfer information through EVs before. This work underlines the potential of NLCs as therapeutic targets and the pertinence of exosomes as biomarkers in this pathology.

Acknowledgements

This work was supported and funded by grants from the Nouvelle Aquitaine region and La Ligue Nationale Contre le Cancer (Comité Départemental de La Haute-Vienne). The PISSARO platform thanks the Rouen Normandy University and Normandy region for their support. Europe gets involved in Normandy with European Regional Development Fund.

We are grateful to the Genotoul bioinformatics platform Toulouse Occitanie⁵⁶ for providing computing and storage resources.

Data availability statement

Data will be made available upon reasonable request to the corresponding author.

Conflict of interest statement

The authors declare no conflict of interest in relation to this work.

Author contributions

MOJ, PFG and LI contributed to the conception and design of the study and the study protocol. LI performed the experimental work and performed data analysis. NR and RF performed research. CG and PC performed the mass spectrometry experiments. CG, PC, HD and MB performed proteomics data analyses. SD performed RNAseq data analysis. NG and ND provided patients samples and information. All authors helped with data interpretation. LI wrote this manuscript with input from all co-authors. All authors read and approved the final version of the manuscript.

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GENOTOUL – Un ensemble de ressources au service de la R&D. https://www.genotoul.fr/.

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Extracellular vesicles from Nurse-Like Cells and THP1-derived Type-2 macrophages transfer aggressiveness to chronic lymphocytic leukemia cells

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Abstract

Figures

Introduction

Methods

Primary culture

Cell line culture

Cell treatments with EVs

Monocyte differentiation and polarization

NLCs differentiation

Isolation of EVs by ultracentrifugation

Nanoparticle tracking analysis (NTA)

Labeling and internalization of exosomes

Evaluation of cell proliferation and apoptosis

Western blot assay

Antibody array

RNA extraction and RTqPCR analysis

Transcriptomic profiling by RNASeq

Sample preparation for LC MSMS and whole proteome analysis

Statistical analyses

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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