We hypothesized that via extracellular vesicles (EVs), chronic lymphocytic leukemia (CLL) cells turn endothelial cells into CLL-supportive cells. To test this, we treated vein-derived (HUVECs) and artery-derived (HAOECs) endothelial cells with EVs isolated from the peripheral blood of 45 treatment-naïve patients. Endothelial cells took up CLL-EVs in a dose- and time-dependent manner. To test whether CLL-EVs turn endothelial cells into IL-6-producing cells, we exposed them to CLL-EVs and found a 50% increase in IL-6 levels. Subsequently, we filtered out the endothelial cells and added CLL cells to this IL-6-enriched medium. After 15 min, STAT3 became phosphorylated, and there was a 40% decrease in apoptosis rate, indicating that IL-6 activated the STAT3-dependent anti-apoptotic pathway. Phospho-proteomics analysis of CLL-EV-exposed endothelial cells revealed 23 phospho-proteins that were upregulated, and network analysis unraveled the central role of phospho-β-catenin. We transfected HUVECs with a β-catenin-containing plasmid and found by ELISA a 30% increase in the levels of IL-6 in the culture medium. By chromatin immunoprecipitation assay, we observed an increased binding of three transcription factors to the IL-6 promoter. Importantly, patients with CLL possess significantly higher levels of peripheral blood IL-6 compared to normal individuals, suggesting that the inducers of endothelial IL-6 are the neoplastic EVs derived from the CLL cells versus those of healthy people. Taken together, we found that CLL cells communicate with endothelial cells through EVs that they release. Once they are taken up by endothelial cells, they turn them into IL-6-producing cells.

Abstract It is now well accepted that cancer cells change their microenvironment from normal to tumor-supportive state to provide sustained tumor growth, metastasis and drug resistance. These processes are partially carried out by exosomes, nano-sized vesicles secreted from cells, shuttled from donor to recipient cells containing a cargo of nucleic acids, proteins and lipids. By transferring biologically active molecules, cancer-derived exosomes may transform microenvironmental cells to become tumor supportive. Telomerase activity is regarded as a hallmark of cancer. We have recently shown that the transcript of human telomerase reverse transcriptase (hTERT), is packaged in cancer cells derived- exosomes. Following the engulfment of the hTERT transcript into fibroblasts, it is translated into a fully active enzyme [after assembly with its RNA component (hTERC) subunit]. Telomerase activity in the recipient, otherwise telomerase negative cells, provides them with a survival advantage. Here we show that exosomal telomerase might play a role in modifying normal fibroblasts into cancer associated fibroblasts (CAFs) by upregulating $$\mathrm{\alpha }$$ α SMA and Vimentin, two CAF markers. We also show that telomerase activity changes the transcriptome of microRNA in these fibroblasts. By ectopically expressing microRNA 342, one of the top identified microRNAs, we show that it may mediate the proliferative phenotype that these cells acquire upon taking-up exosomal hTERT, providing them with a survival advantage.

Polycythemia vera (PV) is a classical "myeloproliferative neoplasm" characterized by clonal proliferation of myeloid progenitor cells caused by their acquisition of a mutation in the JAK2 gene. Its common complications are thrombosis and second primary malignancies. Secreted from virtually all cell types, exosomes are extracellular vesicles that carry bioactive cargo derived from their cells of origin. When engulfed, their cargo may alter the phenotype of the recipient cells. For example, neoplastic exosomes modulate the cellular makeup of bystander cells. Therefore, since mutated JAK2 is present in virtually all PV-patients, we hypothesize that PV-derived exosomes (hereafter PV-exo) carry the mutated JAK2 oncogene and are taken up by non-clonal cells, thus contributing to the thrombotic manifestations and oncogenic potential of patients with PV. We isolated and characterized exosomes from PV-patients and from the Human erythroleukemia (HEL) cell line (hereafter HEL-exo). We exposed normal cells implicated in thrombosis and skin cells to these exosomes and examined the cells' phenotypic changes and activation after exposure. Using Sanger sequencing we discovered that both PV-exo and HEL-exo, like their parental cells, carry the mutated JAK2 transcripts, allowing clonal cells to export oncogenes to distant, non-clonal sites. Our preliminary findings suggest that these exosomes promote pro-coagulant and malignant phenotypes in multiple ways. Thrombin generation assay pointed to a significantly higher generation of thrombin in the presence of PV-exo than those derived from healthy donors. Similarly, flow cytometry analysis revealed that PV-exo increased the expression of platelet activation markers. Moreover, Trans Endothelial Electrical Resistance (TEER) assay results suggest that HEL-exo induces endothelial dysfunction. Furthermore, using the Sulforhodamine B assay, we found that exposure to HEL-exo increased the proliferation of normal keratinocytes. In summary, we developed the concept of exosomes as “mini metastasis” that might promote disease manifestations either directly or indirectly. Since unlike solid tumors, hematological malignancies do not metastasize, exosomes derived from neoplastic hematological cells may fulfill this function. Such exosomes may spread oncogenes (e.g., mutated JAK2), damage non-clonal tissues (e.g., endothelial dysfunction) or act as a miniature reflection of their cells of origin (e.g., exosomes as mini platelets) in a parallel way to "real" metastasis; this concept may affect treatment related decisions. For example, by reducing clonal burden, early treatment even in “low risk” patients may prevent exosomal spread and exosomal-dependent damage to distant tissues. The results of our study highlight PV-exo as promoters of PV-complications, thrombosis and malignant transformation. Citation Format: Orit Uziel, Adi Shacham-Abulafia, Galia Spectre, Ester Ziv, Karyn Revital Geiger, Zinab Sarsor, Neria Ron, Einat Beery, Pia Raanani, Shoshana Revel-Vilk, Mira Naamad, Uri Rozovski. Exosomes in polycythemia vera: "mini platelets" with oncogenic and thrombogenic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3849.

We hypothesized that via exosomes, CLL cells turn endothelial cells into "CLL-supportive cells." To test this hypothesis, we transfected vein-derived (HUVECs) and arterial-derived (HAOEC) endothelial cells with exosomes that we isolated from the peripheral blood of 45 treatment-naïve patients. We found that endothelial cells take up CLL exosomes in a dose- and time-dependent manner. Since CLL cells are protected from apoptosis in an IL-6-rich environment, we wondered whether CLL exosomes turn endothelial cells into IL-6-producing cells. To test this, we exposed endothelial cells to CLL exosomes and found a 50% increase in IL-6 levels, suggesting that the endothelial cells exposed to CLL exosomes produced and secreted IL-6. Subsequently, we filtered out this growth medium and added CLL cells to this IL-6-enriched medium. After 15 minutes, STAT3 became phosphorylated, and there was a 40% decrease in the rate of apoptosis among these cells, indicating that IL-6 activated the STAT3-dependent anti-apoptotic pathway. Phosphor-proteomic analysis of endothelial cells that were loaded with CLL exosomes revealed 23 phosphor-proteins that were upregulated. Annotation analysis unraveled the central role of phosphor-β-catenin. To test whether β-catenin enhances the generation of IL-6 in these cells, we transfected HUVECs with a β-catenin-containing plasmid. We found, by ELISA, a 30% increase in the levels of IL-6 in the culture medium, and, by ChIP, the increased binding of 3 transcription factors (NF-κB, LEF/TCF, and C/EBP) to the IL-6 promoter. Taken together, we found that CLL cells communicate with endothelial cells through the exosomes that they release. Once these exosomes are taken up by endothelia, they turn them into IL-6-producing cells. We hypothesized that via exosomes, CLL cells turn endothelial cells into "CLL-supportive cells." To test this hypothesis, we transfected vein-derived (HUVECs) and arterial-derived (HAOEC) endothelial cells with exosomes that we isolated from the peripheral blood of 45 treatment-naïve patients. We found that endothelial cells take up CLL exosomes in a dose- and time-dependent manner. Since CLL cells are protected from apoptosis in an IL-6-rich environment, we wondered whether CLL exosomes turn endothelial cells into IL-6-producing cells. To test this, we exposed endothelial cells to CLL exosomes and found a 50% increase in IL-6 levels, suggesting that the endothelial cells exposed to CLL exosomes produced and secreted IL-6. Subsequently, we filtered out this growth medium and added CLL cells to this IL-6-enriched medium. After 15 minutes, STAT3 became phosphorylated, and there was a 40% decrease in the rate of apoptosis among these cells, indicating that IL-6 activated the STAT3-dependent anti-apoptotic pathway. Phosphor-proteomic analysis of endothelial cells that were loaded with CLL exosomes revealed 23 phosphor-proteins that were upregulated. Annotation analysis unraveled the central role of phosphor-β-catenin. To test whether β-catenin enhances the generation of IL-6 in these cells, we transfected HUVECs with a β-catenin-containing plasmid. We found, by ELISA, a 30% increase in the levels of IL-6 in the culture medium, and, by ChIP, the increased binding of 3 transcription factors (NF-κB, LEF/TCF, and C/EBP) to the IL-6 promoter. Taken together, we found that CLL cells communicate with endothelial cells through the exosomes that they release. Once these exosomes are taken up by endothelia, they turn them into IL-6-producing cells.

In all patients with polycythemia-vera (PV) a point mutation in JAK2 is present in hematopoietic progenitors. Patients develop thrombotic complications, leukemic transformation, and may also develop solid tumors. Preliminarily, we identified the mutated transcripts of JAK2 in PV patients-derived exosomes. This discovery led us to explore the contribution of clonal- exosomes which carry the driver mutation away from their cell-of-origin to the systemic manifestation characterizing PV, thus contributing to the thrombotic and neoplastic complications in non-hematopoietic tissues. Endothelial dysfunction may lead to those complications, either directly by introducing pro-thrombotic factors on their surface or indirectly by inducing chronic inflammatory milieu that promotes thrombosis and oncogenic transformation. Our working hypothesis was that PV-exosomes lead to endothelial dysfunction. First, we showed that similar to their parent cells, PV-exosomes also carry mutated JAK2 transcripts in all patients and the presence of wild-type JAK2 in all healthy individuals. Next, we found that FM-1-43 labelled JAK2mutated-exosomes were taken up by endothelial cells (HUVECs, by flow cytometry) in a dose- and time- dependent manner and induced endothelial dysfunction (by measuring membrane resistance). The resistance was dramatically decreased in the presence of PV-exosomes, indicating that the tight junction between adjacent endothelial cells was impaired and in line with our working hypothesis. To study whether PV-exosomes also have direct effect, we measured the rate of thrombin generation in platelets-poor-plasma to assess the thrombogenic potential of PV-exosomes. An increase in all parameters of the thrombin concentration in the presence of PV-exosomes compared with exosomes from healthy individuals was obtained. We also detected an upregulation of three markers of platelet aggregation in response to PV- exosomes compared with their healthy-derived counterparts. Finally, we found that PV-exosomes have the capacity to increase the proliferation of normal cells from various tissues. We conclude that like their parental cells, PV-derived exosomes carry mutant JAK2 transcripts. These exosomes are pro-thrombotic and have transforming potential both directly and indirectly through endothelial dysfunction. In all patients with polycythemia-vera (PV) a point mutation in JAK2 is present in hematopoietic progenitors. Patients develop thrombotic complications, leukemic transformation, and may also develop solid tumors. Preliminarily, we identified the mutated transcripts of JAK2 in PV patients-derived exosomes. This discovery led us to explore the contribution of clonal- exosomes which carry the driver mutation away from their cell-of-origin to the systemic manifestation characterizing PV, thus contributing to the thrombotic and neoplastic complications in non-hematopoietic tissues. Endothelial dysfunction may lead to those complications, either directly by introducing pro-thrombotic factors on their surface or indirectly by inducing chronic inflammatory milieu that promotes thrombosis and oncogenic transformation. Our working hypothesis was that PV-exosomes lead to endothelial dysfunction. First, we showed that similar to their parent cells, PV-exosomes also carry mutated JAK2 transcripts in all patients and the presence of wild-type JAK2 in all healthy individuals. Next, we found that FM-1-43 labelled JAK2mutated-exosomes were taken up by endothelial cells (HUVECs, by flow cytometry) in a dose- and time- dependent manner and induced endothelial dysfunction (by measuring membrane resistance). The resistance was dramatically decreased in the presence of PV-exosomes, indicating that the tight junction between adjacent endothelial cells was impaired and in line with our working hypothesis. To study whether PV-exosomes also have direct effect, we measured the rate of thrombin generation in platelets-poor-plasma to assess the thrombogenic potential of PV-exosomes. An increase in all parameters of the thrombin concentration in the presence of PV-exosomes compared with exosomes from healthy individuals was obtained. We also detected an upregulation of three markers of platelet aggregation in response to PV- exosomes compared with their healthy-derived counterparts. Finally, we found that PV-exosomes have the capacity to increase the proliferation of normal cells from various tissues. We conclude that like their parental cells, PV-derived exosomes carry mutant JAK2 transcripts. These exosomes are pro-thrombotic and have transforming potential both directly and indirectly through endothelial dysfunction.

CLL is characterized by gradual accumulation of mature appearing long-lived lymphocytes that travel in blood and reside in lymph nodes, spleen and bone marrow. In these sites, pro inflammatory humoral factors support the survival and proliferation of the neoplastic cells. Previous studies showed that levels of the proinflammatory cytokine IL-6 are at least 10 folds higher in patients with CLL compared with healthy individuals. Yet, which cells produce and secrete IL-6 and what triggers this cellular activity in CLL is unknown.