CAL-101

Immune modulatory effects of Idelalisib in stromal cells of chronic lymphocytic leukemia

Sarah Handl, Franziska von Heydebrand, Simon Voelkl, Robert A. J. Oostendorp, Jochen Wilke, Anita N. Kremer, Andreas Mackensen & Gloria Lutzny-Geier

KEYWORDS
Idelalisib; CLL; stromal cells; PKCb; NF-jB; Notch

Introduction

The circular contact with the bone marrow is one of the hallmarks of chronic lymphocytic leukemia (CLL) [1]. The interaction between the microenvironment and malignant B-cell is a dynamic process and pro- vides protection from spontaneous apoptosis [2,3]. The expression of PKCb and the subsequent activation of NF-jB in bone marrow stromal cells (BMSCs) are prerequisites to support the survival of malignant B- cells [4]. Moreover, the resistance of CLL cells toward drug-induced apoptosis is also mediated by stromal contact, clinically recognized as a minimal-residual dis- ease (MRD) [5]. Recently, in vivo experiments demon- strated that the inhibition of PKCb in stromal cells enhances chemosensitivity in CLL and overcomes drug resistance [6]. It could be shown that stromal contact activates Notch signaling [7] indicating that Notch might play a distinct role in the communication between CLL cells and the microenvironment. Notably, stromal Notch2-dependent mechanisms control non-autonomous WNT-signaling in CLL cells. Pharmacological inhibition of the WNT pathway impairs stromal-mediated survival of the tumor cells [8].
Tyrosine kinase inhibitors (TKIs), targeting signals downstream B-cell receptor (BCR) have rapidly advanced clinical trials in patients with CLL. Their molecular targets are not restricted to the B-cell com- partment but regulate key functions in the microenvir- onment [9,10]. Idelalisib, an inhibitor of the phosphatidylinositol-3-kinase p110d subunit (PI3Kd), is approved for treating relapsed/refractory chronic lymphocytic leukemia, follicular lymphoma and small lymphocytic lymphoma [11]. The drug is associated with hematopoietic and pulmonary toxicities and can lead to CMV reactivations in patients, which limit its clinical use. However, the toxicity mechanisms are not completely elucidated [10,12]. Malignant B-cells modify infiltrating tissues and manipulate surrounding cells to generate a protective microenvironment. Moreover, they modify cells devoted to natural and adaptive immune response, inducing an immunosuppressive behavior [9,13]. TKIs reduce the lymphoid mass very efficiently, but can obtain a complete response only in a small minority of cases [9]. Analyzing the effects of TKIs on the microenvironment of B-cell malignancies is relevant to understand phenomena observed in the clinical setting and will help to define how protective niches are modified.
In the present study, we describe Idelalisib-induced effects on the PKCb/NF-jB pathway and the Notch regulation in stromal cells of CLL. The effect of Idelalisib shows deregulation of important immune- modulatory proteins in stromal cells. Additionally, findings of inhibitor combination studies in a new 3D- stroma/leukemia model could be exploited to gener- ate new approaches to therapeutically overcome stroma-mediated drug resistance.

Experimental procedures

Patient samples

After informed patients’ consent and in accordance with the Helsinki declaration, peripheral blood was obtained from untreated, infectious-free patients with a diagnosis of CLL. Studies were approved by the Ethics Committee of the University of Erlangen- Nu€rnberg (number: 219_14B, addendum 59_17 Bc). PBMC isolation was performed as previously described [4]. A list of patient samples used in each experiment is shown in Supplement Table.

Cell culture conditions

The culture conditions of CLL cells, EL08-1D2 and HS-5 cells (CRL-11882, ATCC) were previously described in detail [4]. Purified CD19þ CLL cells were cocultured on EL08-1D2 or HS-5 cells for 5 d. CLL cells were physic- ally removed from stromal cells by repeated washing resulting in minimal cross-contamination numbers. Inflow cytometry analysis CLL cells can easily be distin- guished using a CD19þ lymphocyte gate. Stromal cells were further purified using anti-CD19 magnetic beads to eliminate any minimal CLL contamination, especially in qPCR analysis.

3D cell culture

The scaffolds were initially prepared as suggested by the manufacturer (Alvetex, Reprocell Europe, Durham, UK). EL08-1D2 cell suspension was added to the center of the scaffold. After 3 h the medium was applied. After 24 h 3 × 106 CLL cells/ml were added in their appropriate medium to the EL08-1D2 cells on the scaf- fold for 5 d. The CLL cells were harvested by collecting the entire medium. With two additional washing steps in medium, the CLL cells sitting on stromal cells on the outside of the scaffold were released by gently pipetting. To retrieve the cells out of the inner core of the scaffold, we unclip the inserts, remove the scaffold using flat-ended forceps and cut the scaffold into pieces. The small pieces were transferred in 0.25% Trypsin/EDTA and incubated for 15 min at 37 ◦C, 5% CO2 on a tube rolling mixer. This CLL cell isolation step from the inner core of the scaffolds was repeated twice. CLL cells can be separated from stromal cells of this region using a CD19þ lymphocyte gate in flow cytometry.

RT2 assays

After coculture, CLL and stromal cells were separated with anti-CD19 magnetic beads and RNA of stromal cells was extracted. Stromal cDNA was prepared (RT2 First Strand Kit, Qiagen) and subjected to pathway- focused gene expression analyses of 84 genes involved in cancer inflammation and immunity cross- talk (RT2 Profiler PCR Arrays, Qiagen, Hilden, Germany).

CRISPR/Cas9 plasmids

Single-guide RNA sequences (sgRNA) were cloned into lentiCRISPRv2-GFP according to Zhang et al. LentiCRISPRv2GFP was a gift from David Feldser [14] (Addgene plasmid # 82,416; http://n2t.net/addg- ene:82,416; RRID:Addgene_82,416). Lentiviral infection of EL08-1D2 cells with two different Notch2-sgRNA sequences was performed (Table 3). Following the separation of GFP positive cells, EL08-1D2 cells were negatively sorted for Notch2 expression. The experi- ments were performed with EL08DNotch2 using sgRNA Notch2 #1.
HS-5 stromal cells were infected with lentiviral sgRNA (Edit-R Human NOTCH2 hEF1a-EGFP All-in-one Set, Horizon Discovery, Cambridge, UK) with three dif- ferent Notch2-sgRNA sequences (Table 3). Following the separation of GFP positive cells, HS-5 cells were negatively sorted for Notch2 expression. The experi- ments were performed with HS-5DNotch2 using sgRNA VSGH12180-249451941.

GM-CSF ELISA

After coculture of murine EL08-1D2 or human HS-5 cells with primary CLL cells, the supernatant was used for detection of GM-CSF in a mouse or human-specific enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, USA) according to manufac- turer’s protocol.

NF-jB assay

After coculture of EL08-1D2 and primary CLL cells for 5 d, nuclear-cytoplasmatic protein separation was per- formed of the stromal pellet (SP) using a Nuclear Extraction Kit (Abcam, Cambridge, UK). CLL cells were pretreated with the caspase inhibitor Z.vad.fmk (100 mM) for 30 min to suppress spontaneous apop- tosis. The nuclear protein pellet of the stromal cells was analyzed with the colorimetric NF-jB Transcription Factor Assay Kit (Abcam, Cambridge, UK) following the manufacturer’s manual.

Immunofluorescence staining of 3D-stroma/ leukemia cell culture

After 5 d of coculturing stromal cells with CLL cells on scaffolds in 8-well chamber slides (Ibidi, Gr€afelfing, Germany), the medium was removed. 3D cell culture was washed twice with PBS and fixed by incubation within 4% paraformaldehyde for 15 min at room tem- perature. The 3D culture was permeabilized in 1% Triton-X100 diluted in PBS for 15 min at room tem- perature. Specific binding of primary antibody (CD19- antibody, Table 1) and CellMask Green plasma mem- brane stain was achieved by incubating overnight at 4 ◦C. After washing, nuclei were counterstained with DAPI before the 3D cultures were mounted on glass slides within SlowFade antifade mountant medium (Invitrogen, Germany). Confocal imaging was per- formed on a Zeiss Spinning Disk Axio Z1 live-cell observer (Zeiss, Jena, Germany). Z-stack data of confocal images were reconstructed using Fiji [15] to obtain three-dimensional representa- tions of the stromal cell distribution within the 3D- stroma/leukemia model.

Statistical analysis

All experiments were repeated at least three times. The sample sizes for each experiment were provided in the figure legends. Statistical analyses were per- formed by one-way ANOVA for multiple comparisons. Two-tailed Student t-tests with unpaired or paired analysis were performed based on the distribution lev- els using GraphPad Prism Version 8 (GraphPad Prism Software Inc., La Jolla, CA). Throughout the manu- script, statistical significance was defined as p < 0.0001 (ωωωω), p < 0.001 (ωωω), p < 0.01 (ωω), p < 0.05 (ω) or ns (statistically non-significant). Results Idelalisib reduces the expression of distinct proteins of the immunity crosstalk in activated stromal cells We previously established a coculture system to study heterotypic cell-cell interactions between stromal cells and primary CLL cells [4]. The EL08-1D2 cells are pri- mary stromal cells derived from mouse embryonic liver (E11), which has been carefully characterized as stromal cells that are able to support human hemato- poietic stem cell activity [16]. We analyzed the relative gene expression of proteins that are related to inflam- mation and immunity in stromal cells to gain insight into a possible transcriptional regulation of Idelalisib treatment. We found Idelalisib-induced changes in the expression level of stromal genes like granulocyte- macrophage colony-stimulating factor 2 (GM-CSF), tumor necrosis factor (ligand) superfamily member 10 (Tnfsf10/CD253) and chemokine receptor 9 (Ccr9/ CD199). These genes were downregulated in EL08-1D2 stromal cells upon CLL contact due to incubation of Idelalisib (Figure 1(A,B)). To confirm the gene regula- tory data, we detected the GM-CSF levels in the super- natant of EL08-1D2 and HS-5 monocultures (Ctrl) in comparison to cocultures with CLL cells using a mouse- and human-specific GM-CSF ELISA. Idelalisib reduced the GM-CSF protein expression in murine EL08-1D2 stromal cells as well as in human HS-5 stro- mal cells (Figure 1(C)). The same effect was assessed by cell surface expression of stromal CD199 and CD253 on EL08-1D2 and HS-5 cells using FACS analysis (Figure 1(D,E)). Notch signaling in stromal cells after CLL contact is not affected by Idelalisib treatment Since inhibition of the Notch pathway diminishes the survival of stroma-protected CLL cells in vitro and dis- ease engraftment in vivo, we investigated the impact of Idelalisib on the stromal Notch pathway. First, we applied Idelalisib together with the Notch pathway inhibitor DAPT, which blocks the function of the c-sec- retase, on EL08-1D2/CLL and HS-5/CLL cocultures and detected the CLL survival rate using Annexin-V/PI staining. Interestingly, the combination of Idelalisib and DAPT did not induce a higher apoptosis rate in CLL cells compared to the single agents (Figure 2(A)). Additionally, we analyzed the gene expression of the treatment by FACS analysis (n 6). Bars indicate the standard error of the mean. ωp < 0.05; ωωp < 0.01; ωωωp < 0.001. Notch receptors in stromal cells in monoculture (white column) and after CLL contact (filled column) using qPCR studies (Figure 2(B,C), Table 2). Notch3 and Notch4 were not regulated after Idelalisib or DAPT treatment (Supplement Figure 1(A,B)). In line with Notch protein expression analysis published by Mangolini et al. [8], we detected a decrease in Notch1 and a clear increase in Notch2 gene expression in EL08-1D2 cells after CLL contact (Figure 2(B)). Moreover, CLL contact-induced an upregulation of the transcriptional factor Hes1 in EL08-1D2 and HS-5 stro- mal cells indicating activation of functional Notch tar- get genes (Figure 2(B,C) right panel). However, Idelalisib had no impact on the gene regulation of Notch receptors and Hes1 in stromal cells after CLL contact. This is in line with the protein expression of the Notch receptors on the cell surface of EL08-1D2 cells after coculture with CLL cells (Supplement Figure 1(C)). In agreement with a previous report, we could show that Notch1 and Notch2 had by far the highest surface expression in EL08-1D2 cells (Supplement PKCb inhibitor enzastaurin support the Idelalisib- induced CLL cell death despite stromal contact Direct contact of CLL cells activates stromal cells by inducing PKCb expression [4]. To assess to what extend Idelalisib manipulates the PKCb regulation in the stro- mal compartment of CLL, we first tested if Idelalisib has an effect on the expression of PKCb in EL08-1D2 and HS-5 stromal cells. The stromal cells were analyzed for PKCb protein expression after 5 d of coculture using an intracellular FACS staining. As expected, CLL contact- induced a PKCb expression in stromal cells. With the addition of Idelalisib in increasing concentrations, the PKCb expression was further increased in stromal cells (Figure 3(A), left panel). Similar to murine EL08-1D2 stromal cells, the PKCb expression in human HS-5 EL08-1D2 or HS-5 cells as control (white column). Bars indicate the standard error of the mean. ns (not significant); ωp < 0.05; ωωp < 0.01; ωωωωp < 0.0001. Abbreviations: CLL: chronic lymphocytic leukemia cells; DAPT: N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; p: p-value. the standard error of the mean ωp < 0.05; ωωp < 0.01; ωωωp < 0.001; ωωωωp < 0.0001. Abbreviations: CLL: chronic lymphocytic leukemia cells; Ctrl: control CLL cells with stromal contact without treatment; APC: Allophycocyanin; p: p-value. stromal cells was elevated after Idelalisib treatment dur- ing CLL contact (Figure 3(A), right panel). Next, we tested if Idelalisib could overcome the protective effect of stromal cells on CLL cells. We ana- lyzed apoptosis using Annexin-V/PI staining in primary CLL cells cultured as monoculture (Ctrl) for 24 h to detect the direct effect of Idelalisib avoiding cell death because of missing stromal contact (Figure 3(B)). Stromal cells were in contact with CLL cells for 5 d. The coculture was exposed to Idelalisib for 24 h before assessing CLL survival. As expected CLL cells cultured on EL08-1D2 or HS-5 cells had a clear survival benefit compared to suspension cells in monoculture (Figure 3(C); Ctrl). Notably, CLL cells were not protected from the cytotoxic effect of Idelalisib despite stromal con- tact (Figure 3(C)). Furthermore, the PKCb inhibitor Enzastaurin or pan-PKC inhibitor Sotrastaurin was applied separately or in combination to Idelalisib for 24 h on CLL cells in monoculture (Figure 3(B)) and cocultures (Figure 3(C)). Both compounds have the ability to inhibit the PKCb expression in stromal cells upon CLL contact (Figure 3(D)). Interestingly, only the PKCb-specific inhibitor Enzastaurin in combination with Idelalisib showed a significantly higher apoptotic CLL rate on protective EL08-1D2 and HS-5 stromal cells (Figure 3(C)). The enhanced PKCb expression seems not to be relevant for CLL survival during Idelalisib treatment. However, PKCb inhibition using Enzastaurin supported the Idelalisib-induced CLL cell death despite stromal contact. The Idelalisib-induced NF-jB activation in stromal cells in response to CLL contact is Notch2 dependent It is known that stromal PKCb activates NF-ŒB through the canonical pathway, which is indispensable for CLL survival [4]. Due to the fact that we detected an Idelalisib-dependent PKCb induction in stromal cells (Figure 3(A)), we assessed downstream NF-jB activa- tion in stromal cells. After 5 d of coculture with and without Idelalisib treatment for 24 h, the nuclear fractions from stromal cells were isolated and analyzed with an ELISA-based NF-jB Transcription Factor Activation Assay. CLL contact clearly induced NF-jB p50 and p65 subunits in stromal cells, which was fur- ther enhanced by Idelalisib treatment (Figure 4(A,B)). This NF-jB activation was not detected in PKCb knock- out stromal cells upon CLL contact and Idelalisib treat- ment (data not shown). Numerous reports also described regulation of NF-jB by Notch and vice versa through different mechanisms [17]. To investigate if Idelalisib can affect the NF-jB activation without regu- lating the Notch pathway, we generated Notch2 deleted EL08-1D2 (EL08DNotch2) and HS-5 cells (HS5DNotch2) using CRISPR/Cas9 technology (Figure 4C). Notably, the NF-jB activation of p50 and p65 in EL08DNotch2 and HS5DNotch2 cells was not altered due to CLL contact indicating that Notch2 signaling is required for NF-jB activation through the canonical pathway. Furthermore, Idelalisib treatment could not activate NF-jB in Notch2 deleted stromal cells sup- porting our data that the Notch pathway is not affected by Idelalisib (Figure 4(D,E)). Idelalisib treatment combined with PKCb inhibition can overcome stromal-mediated drug resistance in 3D cell culture We generate a 3D cell culture system as a stroma- based leukemia model cultivating stromal cells on a scaffold together with malignant B-cells to mimic the in vivo situation. Numerous key findings obtained from 2D cell cultures can be validated in these 3D stroma niches to rule out optional treatment approaches for patients. Particularly, the effectiveness of inhibitors could be evaluated directly in 3D cell cul- ture. Using this model, we aimed to analyze if inhibi- tor combinations with Idelalisib have the same effect on CLL survival and can overcome stromal-mediated protection as in standard 2D cell cultures. Tumor cells were exposed to different inhibitors alone (Supplement Figure 3(A,B)) and in combination with Idelalisib to assess their impact on viability in estab- lished cocultures. These experiments were also per- formed at lower doses of Idelalisib (Supplement Figure 4(A–E)). After adding Idelalisib in combination with Fludarabine (purine analog), ABT-199 (B cell lymphoma 2 (BCL2) inhibitor) or Ibrutinib (inhibitor of B cell receptor-induced kinase), CLL survival was investigated by Annexin-V/PI staining. As expected, the contact with EL08-1D2 or HS-5 stromal cells in 2D cell culture enhanced the survival of CLL cells in the medium con- trol (Ctrl) (Figure 5(A,B,C)) compared to CLL cells in monoculture (Supplement Figure 5(A,B,C)). Idelalisib treatment was able to overcome this protective stro- mal effect. Adding Idelalisib in combination with ABT-199 (Figure 5(A)), Fludarabine (Figure 5(B)) and Ibrutinib (Figure 5(C)), an additive effect on CLL cell death that was comparable to the CLL cell death in monocultures (Supplement Figure 5(A,B,C)) was detected. Interestingly, using the 3D leukemia model we can differentiate between two regions - the peripheral regions of the 3D stromal complex and the inside core region (Figure 5(D)). Importantly, stromal cells can migrate and grow in all regions of the scaffold (Figure 5(E)). Z-stack data of the confocal images were recon- structed using Fiji (3D script plugin) to obtain 3 D rep- resentations showing the distribution of the stromal cells upon CLL contact within the 3 D-stroma/leukemia model (Supplement Video 1, 2). We isolated the CLL cells that were in contact with stromal cells from the outside region of the scaffold and analyzed the CLL survival using Annexin-V/PI staining. As expected, the CLL cells profit from the stromal mediated protection (Figure (5F); Ctrl). The combination treatments of Idelalisib with Enzastaurin, Fludarabine or Ibrutinib were able to overcome stroma-mediated drug resist- ance resulting in increased CLL cell death (Figure 5(F)), which was comparable to the 2D situation (Figures 3(C) and 5(B,C)). The combination with ABT-199 exhib- ited a trend in elevated CLL cell death but was not as effective as in the 2D cell culture (Figure 5(F)). Notably, analysis of the CLL cells sitting in the inside core region of the 3D cell culture model, showed a different survival pattern. Only Idelalisib in combin- ation with the PKCb inhibitor Enzastaurin seems to be effective by diminishing the protective stromal effect on CLL cell survival in the more secure core region of the 3D cell culture (Figure 5(G)). Discussion TKIs can induce an immune-tolerant behavior in sur- rounding cells [9]. Interestingly, we monitored Idelalisib-induced changes in the immunity crosstalk in stromal cells after CLL contact. Stromal GM-CSF, Ccr9 and Tnfsf10 expression were reduced due to Idelalisib treatment upon CLL contact. GM-CSF has a profound role in regulating the immune response and maintain- ing immunological tolerance [18]. A lower level of GM- CSF could lead to lower activation of macrophages, a process crucial for fighting infections. This could be evidence for the patient’s side effects like pulmonary toxicity and susceptibility to infections, like mycosis. Figure 5. Idelalisib treatment combined with PKCb inhibition can overcome stromal-mediated drug resistance in 3D cell culture. Apoptotic CLL cells were quantified by Annexin-V/PI staining after contact to EL08-1D2 and HS-5 cells (Ctrl) for 5 d with 24 h exposure to (A) Idelalisib (5 mM) ± ABT-199 (10 nM) (B) Idelalisib (5 mM) ± Fludarabine (40 mg/ml), (C) Idelalisib (5 mM) ± Ibrutinib (10 mM) in a standard 2D cell culture (n 6). (D) Illustration of the scaffold-based 3D-model consisting of stromal cells and malig- nant B-cells with the core region and the peripheral regions. (E) CLL cell survival was measured using Annexin-V/PI staining after coculture in 3D cell culture conditions and 24 h inhibitor exposure to Idelalisib (5 mM) ± Enzastaurin (5 mM), ± ABT-199 (10 nM), ± Fludarabine (40 mg/ml) and ± Ibrutinib (10 mM). CLL cells were in direct contact to stromal cells (EL08-1D2, left panel; HS-5, right panel) and isolated from the peripheral regions of the scaffold (n 6). (F) Using confocal laser microscopy, the distribution of the stromal cells in the different regions of the scaffold was determined. Serial sections EL08-1D2 (left panel) and HS-5 stromal cells (right panel) in coculture with CLL cells were stained for CD19 (red) and CellMask plasma membrane stain (green). Nuclei were stained with DAPI (blue) (n 3). Scale bars: 100 lm. Z-stacks of stained 3D cultures were imaged by a confocal spinning disk microscope with an axial distance of 270 nm and reconstructed for 3D visualization. (G) CLL cell survival was measured using Annexin-V/PI staining after coculture in 3D cell culture conditions and 24 h inhibitor exposure to Idelalisib (5 mM) ± Enzastaurin (5 mM), ± ABT-199 (10 nM), ± Fludarabine (40 mg/ml) and ± Ibrutinib (10 mM). CLL cells were isolated after 5 d of coculture in direct contact to stromal cells (EL08-1D2, left panel; HS-5, right panel) from the core region of the scaffold (n 6). Bars indicate the standard error of the mean. ωp < 0.05; ωωp < 0.01; ωωωp < 0.001; ωωωωp < 0.0001. Abbreviations: CLL: chronic lymphocytic leukemia cells; Ctrl: control CLL cells with stromal contact without treatment; p: p-value. Ccr9 is involved in T-cell development in the thymus and in the gut-associated immune response, which might explain gastrointestinal symptoms in patients. Tnfsf10 activates the caspase 8-dependent apoptosis in tumor cells. Tnfsf10 receptor-targeted therapy has so far been disappointing in the clinic because only a small proportion of patients responded due to the development of resistance to Tnfsf10 [19]. However, targeting the Tnfsf10 receptor in combinational thera- pies or adding GM-CSF to compensate immune-modu- latory effects of Idelalisib to counterpart side effects might be helpful. Stromal cells get activated by direct CLL contact leading to stromal PKCb expression followed by NF-jB activation [4]. Additionally, CLL cells induce Notch2 in stromal cells that in turn activates the canonical WNT pathway in CLL cells [8]. The manipulation of both pathways in stromal cells leads to CLL survival demon- strating that CLL cells can change the function of sur- rounding cells. A recent study underlines the importance of PKCb expression in the stromal com- partment to protect malignant B-cells from cytotoxic therapies in vivo enrolling a PKCb-dependent effect in environment-mediated drug resistance of CLL cells [6]. The PI3K inhibitor Idelalisib demonstrated promising clinical efficiency in different B-cell malignancies [9]. Here, we describe that Idelalisib can stimulate the PKCb/NF-jB pathway in stromal cells after direct con- tact with CLL cells. Of note, the canonical NF-jB path- way was not activated in PKCb-deficient stromal cells (data not shown). Interestingly, the effect of Idelalisib can overcome the stroma-mediated protection despite a secondary increased PKCb/NF-jB expression in stro- mal cells. Notably, the stromal Notch pathway seems not to be affected by Idelalisib. However, adding the PKCb inhibi- tor Enzastaurin in combination with Idelalisib diminishes stroma protection and leads to CLL cell death. Cultivating primary CLL cells in a 3D leukemia model, where they are able to get in contact with stromal cells in multiple dimensions facilitating more natural interactions, can help us to get a defined look into the communication of different cell types. 3D cul- tures can reveal dramatic differences in cellular responses compared to monolayer culture representing the in vivo situation even better [20–23]. The combin- ation of Idelalisib with Enzastaurin, Fludarabine or Ibrutinib can overcome stromal protection in the 3D constellation (Figure 5(E)) comparable to the 2D cell culture. Interestingly, we could show that CLL cells sit- ting on stromal cells in the core region of the 3D cell culture model exhibit a higher drug resistance com- pared to CLL cells that were in direct contact with stro- mal cells located in the peripheral regions. Of note, stromal cells are evenly distributed in the scaffold (Figure 5(E)). CLL cells in the core region demonstrate a clear maintaining stromal protection visualized by low numbers of apoptotic cells despite ABT-199, Fludarabine or Ibrutinib treatment in combination with Idelalisib (Figure 5(G)). In contrast, Idelalisib in combin- ation with Enzastaurin can overcome the protective effect in the core area. This indicates that, on the one hand, CLL cells can frequent specially protected areas of stromal cells to survive the cytotoxic action of sev- eral drugs, which might lead to MDR. On the other hand, PKCb inhibition can overcome also stromal-medi- ated drug resistance in the more secure regions of the 3D cell culture. This symbolizes a clear difference to the 2D cell culture and might be an important point con- cerning the perspective of therapeutic efficiency and drug resistance of combination therapy (33). Moreover, this effect clearly shows that the drug diffusion into the whole high-porous scaffold is possible. Specifically, these results point to an essential role of the CLL cell distribution in a 3 D niche. 3D cell cultures and organo- ids have already been proven to be an excellent plat- form for drug screening and give the opportunity to mimic the complexity in signaling and tissue forma- tion [20–27]. In summary, our study provides new insight into the Idelalisib-mediated changes of the PKCb and NF- jB activation in stromal cells upon CLL contact. Additionally, we detected deregulation of immune- modulatory proteins in the stromal cells after contact to CLL, which can be responsible for specific side effects. Furthermore, PKCb inhibition can overcome the protective stromal effect on CLL survival in the core region of the 3D-stroma/leukemia model. The findings based on the 3D model point to an essential role of a more in vivo like the situation to improve therapies against the stromal microenvironment, espe- cially in the context of MRD in patients. References [1] Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med. 2005;352(8):804–815. [2] Lagneaux L, Delforge A, Bron D, et al. Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood. 1998;91(7): 2387–2396. [3] Ding W, Nowakowski GS, Knox TR, et al. Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression. Br J Haematol. 2009;147(4):471–483. [4] Lutzny G, Kocher T, Schmidt-Supprian M, et al. Protein kinase c-beta-dependent activation of NF- kappaB in stromal cells is indispensable for the sur- vival of chronic lymphocytic leukemia B cells in vivo. Cancer Cell. 2013;23(1):77–92. [5] Meads MB, Gatenby RA, Dalton WS. Environment- mediated drug resistance: a major contributor to min- imal residual disease. Nat Rev Cancer. 2009;9(9): 665–674. [6] Park E, Chen J, Moore A, et al. Stromal cell protein kinase C-beta inhibition enhances chemosensitivity in B cell malignancies and overcomes drug resistance. Sci Transl Med. 2020;12(526):eaax9340. [7] Jitschin R, Braun M, Qorraj M, et al. Stromal cell-medi- ated glycolytic switch in CLL cells involves Notch-c- Myc signaling. Blood. 2015;125(22):3432–3436. [8] Mangolini M, Gotte F, Moore A, et al. Notch2 controls non-autonomous Wnt-signalling in chronic lympho- cytic leukaemia. Nat Commun. 2018;9(1):3839. [9] Maffei R, Fiorcari S, Martinelli S, et al. Targeting neo- plastic B cells and harnessing microenvironment: the "double face" of ibrutinib and idelalisib. J Hematol Oncol. 2015;8:60. [10] Cheah CY, Fowler NH. Idelalisib in the management of lymphoma. Blood. 2016;128(3):331–336. [11] Zirlik K, Veelken H. Idelalisib. Recent Results Cancer Res. 2018;212:243–264. [12] George JA, Alshebli Z, Alneyadi A, et al. Idelalisib induces apoptosis in the lymphoid tissues and impairs lung function in mice. J Chemother. 2020;32(2):88–97. [13] ten Hacken E, Burger JA. Microenvironment depend- ency in chronic lymphocytic leukemia: the basis for new targeted therapies. Pharmacol Ther. 2014;144(3): 338–348. [14] Walter DM, Venancio OS, Buza EL, et al. Systematic in vivo inactivation of chromatin-regulating enzymes identifies setd2 as a potent tumor suppres- sor in lung adenocarcinoma. Cancer Res. 2017;77(7): 1719–1729. [15] Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–682. [16] Oostendorp RA, Harvey KN, Kusadasi N, et al. Stromal cell lines from mouse aorta-gonads-mesonephros sub- regions are potent supporters of hematopoietic stem cell activity. Blood. 2002;99(4):1183–1189. [17] Osipo C, Golde TE, Osborne BA, et al. Off the beaten pathway: the complex CAL-101 cross talk between Notch and NF-kappaB. Lab Invest. 2008;88(1):11.
[18] Bhattacharya P, Thiruppathi M, Elshabrawy HA, et al. GM-CSF: an immune modulatory cytokine that can suppress autoimmunity. Cytokine. 2015;75(2): 261–271.
[19] Dimberg LY, Anderson CK, Camidge R, et al. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based thera- peutics. Oncogene. 2013;32(11):1341–1350.
[20] Walsh AJ, Cook RS, Sanders ME, et al. Quantitative optical imaging of primary tumor organoid metabol- ism predicts drug response in breast cancer. Cancer Res. 2014;74(18):5184–5194
[21] Neal JT, Li X, Zhu J, et al. Organoid modeling of the tumor immune microenvironment. Cell. 2018;175(7): 1972–1988.
[22] Holloway EM, Capeling MM, Spence JR. Biologically inspired approaches to enhance human organoid complexity. Development. 2019; 146(8):dev166173.
[23] Wimmer RA, Leopoldi A, Aichinger M, et al. Human blood vessel organoids as a model of diabetic vascul- opathy. Nature. 2019;565(7740):505–510.
[24] Dekkers JF, Wiegerinck CL, de Jonge HR, et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med. 2013;19(7): 939–945.
[25] Seifert M, Lubitz A, Trommer J, et al. Crosstalk between immune cells and mesenchymal stromal cells in a 3D bioreactor system. Int J Artif Organs. 2012;35(11):986–995.
[26] Czerniecki SM, Cruz NM, Harder JL, et al. High- throughput screening enhances kidney organoid dif- ferentiation from human pluripotent stem cells and enables automated multidimensional phenotyping. Cell Stem Cell. 2018;22(6):929–940.
[27] Eliopoulos N, Francois M, Boivin MN, et al. Neo-organoid of marrow mesenchymal stromal cells secreting interleukin-12 for breast cancer therapy. Cancer Res. 2008;68(12):4810–4818.