e4b392e425ddf9c28200d6183f105680 pmc_10888954.pdf c88f00b98fe3da42d61816a4ee99cf8f2d294821 pmc_10888954.pdf 3a1380cfcd4ad4924bbf74603a2c7ad1605b4e2faea9261da2580dbb54638b1a pmc_10888954.pdf Title: Persistence of Chronic Lymphocytic Leukemia Stem-like Populations under Simultaneous In Vitro Treatment with Curcumin, Fludarabine, and Ibrutinib: Implications for Therapy Resistance Subject: Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy. Keywords: curcumin; CLL; LSCs; flow cytometry; stem-like; alkaline phosphatase Author: Àngel Bistué-Rovira, Laura G. Rico, Jorge Bardina, Jordi Juncà, Isabel Granada, Jolene A. Bradford, Michael D. Ward, Roser Salvia, Francesc Solé and Jordi Petriz Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Feb 7 07:27:26 2024 CET ModDate: Tue Feb 20 02:34:57 2024 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2148976 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- YZAJMJ+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 NWWNRU+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 HAWFJP+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 OAJCTO+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 BVCIWP+VnURWPalladioL Type 1 Custom yes yes yes 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 48 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 51 0 ZSBMBZ+EURM10 Type 1 Builtin yes yes yes 71 0 IHKFFE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 83 0 IHKFFF+PalatinoLinotype CID TrueType Identity-H yes yes yes 86 0 IHKFFG+PalatinoLinotype TrueType WinAnsi yes yes no 92 0 IHKFFI+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 95 0 IHKFNH+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 98 0 IHKFNI+PalatinoLinotype TrueType MacRoman yes yes no 101 0 IHKFFE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 126 0 IHKFFG+PalatinoLinotype TrueType WinAnsi yes yes no 129 0 IHKFFI+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 132 0 EUQOAW+EURM7 Type 1 Builtin yes yes yes 147 0 VORBJF+CMSY10 Type 1 Builtin yes yes yes 152 0 FBLJOH+TimesNewRoman CID TrueType Identity-H yes yes yes 166 0 FBLJPI+Arial CID TrueType Identity-H yes yes yes 172 0 FBKMOO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 178 0 FBKMPA+PalatinoLinotype TrueType WinAnsi yes yes no 181 0 FBKMPC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 184 0 FBKNOP+PalatinoLinotype TrueType MacRoman yes yes no 187 0 IHKFFE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 223 0 IHKFFF+PalatinoLinotype CID TrueType Identity-H yes yes yes 226 0 IHKFFG+PalatinoLinotype TrueType WinAnsi yes yes no 232 0 IHKFFI+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 235 0 IHKFNI+PalatinoLinotype TrueType MacRoman yes yes no 238 0 GIGFZE+CMR10 Type 1 Builtin yes yes yes 264 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-04-23 02:35:30 CEST RepresentationInformation: pmc_10888954.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-04-09 02:35:35 CEST Size: 2148976 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 488 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Limiting Dilution of the I83 Tetraploid Cell Line Reveals a Diploid Subpopulation That Can Spontaneously Regenerate the Tetraploid Main Population Destination: subsection.2.1 Item: Title: Diploid and Tetraploid I83 Presented Similar Susceptibility to Curcumin and Fludarabine Destination: subsection.2.2 Item: Title: ALP Activity Defines CLL Subpopulations with Higher Treatment Resistance Destination: subsection.2.3 Item: Title: Curcumin, Ibrutinib, and Fludarabine Show Efficacy to Reduce Population of CLL Cells with Highest ALP Activity Destination: subsection.2.4 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Lines Destination: subsection.4.1 Item: Title: Patient Sample Analysis and Primary Cultures Destination: subsection.4.2 Item: Title: Immunophenotyping Destination: subsection.4.3 Item: Title: Karyotyping Destination: subsection.4.4 Item: Title: Ploidy Analysis Destination: subsection.4.5 Item: Title: Comparative Cell Membrane Integrity Analysis on I83 Destination: subsection.4.6 Item: Title: Alkaline Phosphatase Activity Analysis Destination: subsection.4.7 Item: Title: Data Analysis Destination: subsection.4.8 Item: Title: References Destination: appendix.A. Info: Title: Persistence of Chronic Lymphocytic Leukemia Stem-like Populations under Simultaneous In Vitro Treatment with Curcumin, Fludarabine, and Ibrutinib: Implications for Therapy Resistance Author: Àngel Bistué-Rovira, Laura G. Rico, Jorge Bardina, Jordi Juncà, Isabel Granada, Jolene A. Bradford, Michael D. Ward, Roser Salvia, Francesc Solé and Jordi Petriz Subject: Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy. 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