BACKGROUND: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. METHODS: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. RESULTS: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).

• Keywords
• 3D culture, CLL, CMC, Carboxymethyl cellulose, Chronic lymphocytic leukemia, Collagen, Gel, PEG, Polyethylene glycol, Scaffolds,
• MeSH
• Cell Culture Techniques methods   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * pathology   metabolism   MeSH
• Gels chemistry   MeSH
• Collagen * chemistry   pharmacology   MeSH
• Humans MeSH
• Polyethylene Glycols * chemistry   MeSH
• Receptors, CXCR4 metabolism   MeSH
• Carboxymethylcellulose Sodium * chemistry   pharmacology   MeSH
• Cell Culture Techniques, Three Dimensional methods   MeSH
• Tissue Scaffolds * chemistry   MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CXCR4 protein, human MeSH Browser
• Gels MeSH
• Collagen * MeSH
• Polyethylene Glycols * MeSH
• Receptors, CXCR4 MeSH
• Carboxymethylcellulose Sodium * MeSH

Bruton tyrosine kinase (BTK) inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL), which lasts for several months. It remains unclear whether nongenetic adaptation mechanisms exist, allowing CLL cells' survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70% of CLL cases, ibrutinib treatment in vivo increases Akt activity above pretherapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of Forkhead box protein O1 (FoxO1) transcription factor, which induces expression of Rictor, an assembly protein for the mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knockout or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. The FoxO1/Rictor/pAktS473 axis represents an early nongenetic adaptation to B cell receptor (BCR) inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically and its inhibition induces CLL cells' apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T cell factors (CD40L, IL-4, and IL-21).

• Keywords
• Drug therapy, Hematology, Leukemias, Oncology, Signal transduction,
• MeSH
• Adenine * analogs & derivatives   pharmacology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * drug therapy   metabolism   genetics   pathology   MeSH
• Forkhead Box Protein O1 * metabolism   genetics   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins metabolism   genetics   MeSH
• Piperidines * pharmacology   MeSH
• Rapamycin-Insensitive Companion of mTOR Protein * genetics   metabolism   MeSH
• Agammaglobulinaemia Tyrosine Kinase metabolism   genetics   antagonists & inhibitors   MeSH
• Proto-Oncogene Proteins c-akt * metabolism   genetics   MeSH
• Pyrazoles * pharmacology   MeSH
• Pyrimidines * pharmacology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenine * MeSH
• BTK protein, human MeSH Browser
• Forkhead Box Protein O1 * MeSH
• FOXO1 protein, human MeSH Browser
• ibrutinib MeSH Browser
• Neoplasm Proteins MeSH
• Piperidines * MeSH
• Rapamycin-Insensitive Companion of mTOR Protein * MeSH
• Agammaglobulinaemia Tyrosine Kinase MeSH
• Proto-Oncogene Proteins c-akt * MeSH
• Pyrazoles * MeSH
• Pyrimidines * MeSH

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

• MeSH
• Stromal Cells * metabolism   pathology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * pathology   genetics   drug therapy   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Interleukin-21 MeSH
• Interleukins genetics   metabolism   MeSH
• Coculture Techniques * MeSH
• Humans MeSH
• CD40 Ligand * metabolism   genetics   MeSH
• Mice MeSH
• Cell Proliferation * MeSH
• T-Lymphocytes immunology   metabolism   MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protein Kinase Inhibitors MeSH
• Interleukin-21 MeSH
• Interleukins MeSH
• CD40 Ligand * MeSH