Abstract

Background

   Resistance to existing therapies is a major cause of treatment failure
   in patients with refractory and relapsed B-cell non-Hodgkin’s lymphoma
   (r/r B-NHL). Therapy-induced senescence (TIS) is one of the most
   important mechanisms of drug resistance.

Methods

   This study used single-cell RNA sequencing to analyze
   doxorubicin-induced senescent B-NHL cells. C-C chemokine receptor 7
   (CCR7) expression in patients with aggressive B-NHL was assessed using
   immunohistochemistry and flow cytometry. Lentiviral transfection was
   used to target CCR7 expression in Raji and SU-DHL-2 cells. Protein
   localization was visualized through immunofluorescence, while western
   blotting and co-immunoprecipitation were used to analyze protein
   expression and interactions. Cell proliferation was measured with the
   Cell Counting Kit-8 assay, and senescent cells were detected using
   senescence-associated β-galactosidase staining. The stemness of cells
   was evaluated through colony and sphere formation assays. Transwell
   assays assessed cell migration and invasion. Finally, inhibitors GS143
   and Y27632 were used to examine the effect of IKBα and ARHGAP/RhoA
   inhibition on B-NHL-TIS.

Results

   Here we identified a distinct group of TIS, composed of memory B-cell
   population characterized by strong positive expression of CCR7, which
   was significantly elevated in TIS population compared with normal
   proliferating and autonomously senescent lymphoma cell populations.
   Additionally, CCR7 expression was significantly upregulated in patients
   with r/r B-NHL, and was an independent prognostic factor in B-NHL, with
   high CCR7 expression being strongly associated with poor prognosis. In
   vitro results indicated that CCL21 induced migration and invasion of
   B-NHL cells via CCR7, while blocking CCR7 reduced doxorubicin-induced
   migration and invasion of these cells. Furthermore, B-NHL-TIS regulated
   by CCR7 and exhibited enhanced phenotypic and functional stemness
   features, including the upregulation of stemness markers, increased
   colony-forming, invasive and migratory capabilities. Mechanistically,
   blocking CCR7 reversed the stemness characteristics of senescent B-NHL
   cells by inhibiting the activation of ARHGAP18/IKBα signaling.

Conclusions

   Together, TIS promotes the stemness of B-NHL cells via
   CCR7/ARHGAP18/IKBα signaling activation and targeting CCR7/ARHGAP18
   might overcome the chemoresistance of senescent B-NHL cells by
   inhibiting stemness acquisition and maintenance.

   Keywords: B cell, Cytokine, Stem cell, Chemotherapy, Hematologic
   Malignancies
     __________________________________________________________________

WHAT IS ALREADY KNOWN ON THIS TOPIC

     * Therapy-induced senescence (TIS) contributes to drug resistance in
       B-cell non-Hodgkin’s lymphoma (B-NHL), with C-C chemokine receptor
       7 (CCR7) implicated in cancer progression.

WHAT THIS STUDY ADDS

     * This study demonstrates that CCR7 enhances the stemness of TIS
       B-NHL cells through the ARHGAP18/IKBα pathway, which contributes to
       chemoresistance.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

     * Targeting the CCR7/ARHGAP18 signaling pathway may offer a new
       approach to overcoming chemoresistance in relapsed B-NHL by
       reducing stemness in senescent cells.

Introduction

   B-cell non-Hodgkin’s lymphoma (B-NHL) is the most common type of
   lymphoid malignancy in adults and is highly heterogeneous in clinical
   manifestation and prognosis. Although rituximab-based
   immunochemotherapy has been applied for several decades in B-NHL and
   the remission rate is dramatically improved, treatment failure with
   disease recurrence remains a major problem.[53]^1 2 Thus, the molecular
   mechanisms of B-NHL recurrence and drug resistance need further
   elucidation.

   Cellular senescence has been reported as a potent tumor-suppressive
   mechanism, mediated by the cell cycle arrest program and the
   senescence-associated secretory phenotype (SASP), which induces
   immuno-surveillance of pre-malignant cells.[54]^3 4 However, senescent
   tumor cells can re-enter the cell cycle and resume proliferation when
   external stressors are removed or environmental conditions
   change.[55]^5 6 Studies show that conventional chemotherapy often
   induces senescence in tumor cells, and this therapy-induced senescence
   (TIS) correlates strongly with poor clinical outcomes.[56]7,[57]9 TIS
   is responsible for tumor relapse and distant metastasis in
   post-treatment stage, by conferring the resistance and fueling the
   repopulation of remaining cancerous cells.[58]^10 Moreover, a study by
   Milanovic et al in Nature reveals that TIS promotes stem-cell-related
   properties of malignant cells via the activation of Wnt
   signaling.[59]^11 Exploring the mechanisms driving senescent tumor
   cells to acquire therapeutic resistance and stemness is crucial for
   understanding clinical resistance and recurrence in patients and for
   improving treatment strategies.

   C-C chemokine receptor 7 (CCR7) is the first identified
   lymphocyte-specific G protein-coupled receptor, and it has only two
   specific ligands: C-C chemokine ligand 21 (CCL21) and CCL19.[60]^12 The
   core function of the CCL19/CCL21/CCR7 axis is to induce dendritic cells
   carrying CCR7 to migrate directionally to draining lymph nodes, a
   process closely related to the occurrence and development of
   lymphoma.[61]^13 CCR7 is also highly expressed in various tumor cells,
   including B-NHL cells, and its expression is associated with a high
   risk of tumor metastasis through the induction of angiogenesis, lymph
   angiogenesis, and epithelial-mesenchymal transition (EMT).[62]13,[63]15
   Currently, research on the involvement of chemokine receptors in the
   regulation of senescence mainly focuses on the well-studied receptors
   CXCR2 and CXCR4, with fewer studies on other receptors. CXCR2 is
   transactivated by p53 and significantly induces p38-mediated cellular
   senescence, exhibiting strong protumor activity.[64]^16 Both CXCR2 and
   CXCR4 have been reported to promote renal tubular cell senescence and
   renal fibrosis through β-catenin, a key molecule in Wnt
   signaling.[65]^17 18 Canonical Wnt and Notch pathways are significantly
   enriched in TIS and play central roles in stem-cell renewal in
   hematological malignancies.[66]^11 Notably, CCR7 has been reported to
   promote cancer cell stemness by activating the Notch[67]^19 and
   JAK/STAT[68]^20 pathways. However, the role of CCR7 in tumor TIS,
   particularly in altering the stemcell-related properties of lymphoma
   senescence, remains unclear.

   In this work, we perform single-cell RNA sequencing (ScRNA-seq) on
   chemotherapeutic drug doxorubicin (DOX) induced B-NHL-TIS model and
   identify a lymphoma TIS cell subset which consists of a type of memory
   B-cell population characterized by the CCR7+. We report here that
   chemotherapy-induced cellular senescence enhances the stemness of
   aggressive B-NHL by activating CCR7 signaling, promoting resistance and
   relapse.

Methods and materials

Human cell lines

   The human B-NHL cell lines Raji, Namalwa, SU-DHL-4, OCI-LY3, SU-DHL-2
   and acute leukemia cell lines HL-60, MOLM-13 were obtained from the
   China Center for Type Culture Collection. Cells were maintained in
   Roswell Park Memorial Institute-1640 (Hyclone, Logan, Utah, USA;
   SH30809.01) with the addition of 10% fetal bovine serum (Lonsera,
   Uruguay; S711-001S). Cell cultures were maintained and incubated at
   37°C in humidified air with 5% CO[2].

Patients

   From March 2021 to October 2023, a total of 112 patients with
   aggressive B-NHL, including 56 with diffuse large B-cell lymphoma
   (DLBCL), 3 with Burkitt lymphoma, and 53 with other types of aggressive
   B-NHL, as well as 6 patients with reactive lymph node hyperplasia (RLN)
   and 29 healthy controls, were enrolled in this study at the Second
   Affiliated Hospital of Anhui Medical University. Healthy individuals
   and RLN served as controls. The clinical characteristics of patients
   with B-NHL and controls are presented in [69]online supplemental table
   S1. Initial evaluation, staging, and response assessment of all
   patients with B-NHL were according to The Lugano International
   Conference on Malignant Lymphoma. All healthy volunteers included in
   this study demonstrated normal liver and kidney function, were free
   from autoimmune diseases, and had no history of using immunosuppressive
   drugs.

TIS model constructing and ScRNA-seq

   Human B-NHL cell lines Raji (2×10^5 cells/mL) were treated with 20 nM,
   40 nM and 80 nM DOX (Beyotime, Shanghai, China, SC0159) by repeated
   pressure in vitro and was induced to senescence as previously
   reported.[70]^21 The optimal concentration for induction was determined
   through staining with senescence-associated β-galactosidase kit
   (SA-β-Gal, Beyotime, Shanghai, China, C0602). Subsequently, single-cell
   suspensions (2×10^5 cells/mL) were loaded onto microfluidic devices and
   ScRNA-seq libraries were constructed according to the Singleron
   GEXSCOPETM protocol by GEXSCOPETM Single-Cell RNA Library Kit
   (Singleron Biotechnologies, Nanjing, China, 4180011). Libraries were
   diluted to 4 nM, pooled, and sequenced on an Illumina HiSeq X using
   150 bp paired-end reads.

Lentiviral transfection

   Raji and SU-DHL-2 cells were seeded at 1×10^5 cells per well in a
   24-well plate. Human shCCR7 and CCR7 overexpression lentiviral vector
   plasmids (eRFP-puro, Gene ID 1236 and [71]NM_001838.4, constructed by
   Hanbio Tech, Shanghai, China) were added at an optimal multiplicity of
   infection of 50 for shCCR7 and 100 for CCR7 overexpression, based on
   pilot experiments. Lipofectamine 3,000 (Thermo Fisher Scientific,
   Waltham, USA, L3000075) was prepared at 5 µg/mL in Opti-MEM medium
   (Thermo Fisher Scientific, Waltham, USA, 31985062). Then, it was mixed
   with the virus and incubated at room temperature for 20 min. The
   mixture was added to the cells and incubated for 24 hours. After 24
   hours, the medium was replaced with a complete medium and the cells
   were cultured for 72 hours. Cells were observed for fluorescence under
   a fluorescence microscope (Zeiss, Germany). Puromycin (Thermo Fisher
   Scientific, Waltham, USA, A1113803) was added at a concentration of
   1.5 µg/mL to select successfully transfected cells. The surviving cells
   were subsequently expanded and cryopreserved for further use.

Inhibitors treatment

   GS143 (IKBα inhibitor, MedChemExpress, New Jersey, USA, HY-110261) and
   Y27632 (RhoA/Rock inhibitor, MedChemExpress, New Jersey, USA, HY-10071)
   lyophilized powders were dissolved in dimethyl sulfoxide (Sangon
   Biotech, Shanghai, China, ST038) to prepare 10 mM stock solutions. The
   final working concentrations of the inhibitors GS143 and Y27632 were 0,
   0.1, 1, 10, and 100 µM, and they were incubated with Raji cells (2×10⁶
   cells/mL) for 1 hour. After incubation, the inhibitor-containing medium
   was replaced with DOX for induction as described previously.

Statistical analysis

   Statistical analyses were conducted using SPSS V.19.0 software (IBM,
   Chicago, Illinois, USA) and GraphPad Prism V.8.0.2 (GraphPad Software,
   La Jolla, California, USA). For quantitative data following a normal
   distribution, the t-test was used for comparisons between two groups,
   while one-way analysis of variance (ANOVA) was applied for comparisons
   among multiple independent samples. For quantitative data that did not
   meet normal distribution criteria, the Mann-Whitney U test was used for
   comparing two independent samples, and non-parametric ANOVA was
   employed for comparisons among multiple samples. Categorical variables
   were presented as counts and percentages (n (%)), with comparisons made
   using either the χ^2 test or Fisher’s exact test. Overall survival (OS)
   was defined as the duration from the date of diagnosis to either death
   or the last follow-up, and progression-free survival (PFS) was defined
   as the time from the start of treatment to disease progression. Groups
   with high and low CCR7 expression were formed based on a predetermined
   cut-off value, and differences between these groups were assessed using
   the log-rank test. Survival analysis was performed using the
   Kaplan-Meier method, with between-group differences in survival curves
   evaluated via the log-rank test. Univariate and multivariate Cox
   proportional hazards regression models were used to evaluate the
   influence of various factors on OS. A p value of <0.05 was considered
   statistically significant.

Results

Identification of senescent cell subpopulations in the chemotherapy-induced
senescent B-NHL cells model

   Various concentrations of DOX (20, 40, and 80 nM) were used to
   construct the TIS model of Raji cells in vitro. It was found that 40 nM
   DOX had significant advantages in constructing the TIS model of Raji
   cells, hence, it was used for subsequent experiments ([72]figure 1A,B).
   Subsequently, a total of 5,330 cells were subjected to ScRNA-seq
   analysis, comprising 3,261 cells from the control group and 2,069 cells
   from the DOX-induced TIS group. Using t-distributed stochastic neighbor
   embedding (t-SNE), the combined samples (comprising TIS and control
   groups) were categorized into eight primary B-cell subpopulations
   ([73]figure 1C). Clusters 1, 4, 5, and 7 were defined as plasma
   cell-like (MZB1, IGKC, JCHAIN), cluster 2 as memory B cells (CD27,
   CD19, PAX5, CD86), and clusters 3, 6, and 8 as undistinguishable B
   cells (MS4A1, CD27, PAX5, IGHM, MZB1, IGKC, JCHAIN) ([74]online
   supplemental figure S1A–K). Samples from the control group
   predominantly clustered in clusters 1, 4, 5, 6, 7, and 8, while those
   from the DOX-induced group primarily clustered in clusters 2, 3, and 8
   ([75]figure 1D,E). Substantial variances in gene expression profiles
   were observed between the two groups, suggesting that
   chemotherapy-induced markedly modified the gene expression profile in
   B-NHL cells. Subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathway enrichment analysis revealed notable enrichment of cell cycle
   and DNA replication pathways in clusters 1, 3, 4, 5, 7, and 8
   (non-senescent cell clusters), whereas proteins involved in cell cycle
   and DNA replication in clusters 2 and 6 (senescent cell clusters)
   showed significant downregulation ([76]figure 1F–H and [77]online
   supplemental figure S2A–P). It suggested that cluster 2 depicted a
   DOX-induced senescent cell population in the chemotherapy-induced
   senescent B-NHL cells model (TIS group), whereas cluster 6 represented
   an autonomous senescent cell population (control group). Meanwhile,
   chemotherapy-induced senescent B-NHL cells exhibited significant
   enrichment of Nuclear Factor kappa B(NF-κB) pathway, whereas NF-κB
   signaling was significantly downregulated in non-senescent cells
   (cluster 1) ([78]online supplemental figure S2B,J). Gene expression
   profile analysis using ScRNA-seq revealed the expression of chemokine
   factor family including CCR7, NF-κB family including NFKBIA, and
   lymphocyte survival protective factor including BCL2A1, were
   significantly higher in cluster 2 of senescent B-NHL cells compared
   with other non-senescent clusters or autonomous senescent clusters
   ([79]figure 1I). The violin plot results showed the distribution of the
   top nine marker genes of the senescent cluster 2 in various cell
   subpopulations. Notably, CCR7 exhibited the most significant expression
   difference between the senescent cluster and the non-senescent clusters
   ([80]figure 1J). These results suggested that activation of CCR7 and
   NF-κB pathway might play a crucial role in the formation and function
   of senescent B-NHL cells.

Figure 1. Identification of senescent cell population in a DOX-induced
B-NHL-TIS model. (A) Chemical staining showing SA-β-Gal positive senescent
cells after induction of Raji cells with 20, 40 and 80 nM DOX. (B) The
proportion of SA-β-Gal positive senescent cells in Raji cells induced by 20,
40 and 80 nM DOX. (C) ScRNA-seq identified major senescent cell
subpopulations in the senescence model. t-SNE dimensionality reduction
analysis showing the clustering of cells in the combined sample (colored by
cell group). (D) t-SNE dimensionality reduction analysis joint sample cell
clustering situation (colored by sample). (E) The proportion of each cell
cluster in two groups of samples. (F) Violin plots of cell cycle protein
enrichment for cluster 1–8. (G) Violin plots of DNA replication protein
enrichment for cluster 1–8. (H) Normalized Enrichment Scores (NES) of the
cell cycle and DNA replication pathways are presented for clusters 1–8. NES
values indicate the degree of enrichment for these pathways within each
cluster, with positive values representing significant upregulation. Pathways
with NES values above 1.5 are considered highly enriched, while those below
−1.5 indicate downregulation. (I) Heatmap displaying the most significantly
expressed marker genes in each cell population from the combined samples. (J)
Violin plots show the distribution of significantly different genes in each
cell population. Validation of marker gene expression in senescent cell
populations in the B-NHL-TIS in vitro model. **p<0.01, ***p<0.001,
****p<0.0001. B-NHL, B-cell non-Hodgkin’s lymphoma; DOX, doxorubicin;
SA-β-Gal, senescence-associated β-galactosidase; TIS, therapy-induced
senescence; t-SNE, t-distributed stochastic neighbor embedding.

   [81]Figure 1
   [82]Open in a new tab

Stemness characteristics analysis of chemotherapy-induced senescent B-NHL
cells

   In pseudotime trajectory analysis, we found that clusters 1, 4, 5, and
   7, which concentrated on the left side of the trajectory, were
   identified as terminally differentiated plasma cell types. Cluster 2,
   which concentrated on the right side of the trajectory, was identified
   as early-stage differentiating cells, with differentiation direction
   from right to left ([83]figure 2A). These results indicated the
   chemotherapy-induced senescent B-NHL cell population was distributed in
   the earliest stage of cell differentiation, suggesting that senescent
   B-NHL cells might possess the potential capability of stemness. We then
   performed cluster analysis of the significantly differentially
   expressed genes and senescence-related genes. CCR7 was downregulated
   along the pseudotime trajectory, reflecting dynamic changes in various
   gene modules ([84]figure 2B,C). Next, the stemness characteristics of
   chemotherapy-induced senescent cells were investigated in this study.
   The expression levels of stemness markers CD34, CD44, CD150, and LGR5
   were significantly higher in the senescent cell group compared with the
   control group ([85]figure 2D). Moreover, the TIS group demonstrated a
   significantly enhanced ability for stemness colony formation compared
   with the control group on day 3 ([86]figure 2E), and exhibited strong
   positive expression of CCR7, as well as co-expression with the stemness
   marker CD44 and the senescence marker H3K9me3, as observed using
   fluorescence confocal microscopy ([87]figure 2F). This indicates that
   chemotherapy-induced cellular senescence enhances the phenotypic and
   functional stemness features of B-NHL cells.

Figure 2. Analysis of the phenotypic and functional stemness features in
B-NHL-TIS cell populations. (A) Cell trajectory plot colored according to
cell clusters. (B) Changes in expression modules of differentially expressed
genes during the pseudo time process. (C) Expression changes of
differentially expressed genes from the start to the end of the pseudo time
process. Expression profile of stemness markers in the TIS model. (D)
Expression levels of stemness markers CD34/CD44/CD150/LGR5 in the control
group and TIS group detected using qRT-PCR. (E) Clonogenic capacity of cells
in the control group and TIS group. (F) Co-expression of CCR7 and
CD44/H3K9me3 in cells detected using immunofluorescence in the control group
and TIS group. ****p<0.0001. B-NHL, B-cell non-Hodgkin’s lymphoma; CCR7, C-C
chemokine receptor 7; DOX, doxorubicin; mRNA, messenger RNA; qRT-PCR,
quantitative Reverse Transcription Polymerase Chain Reaction; TIS,
therapy-induced senescence.

   [88]Figure 2
   [89]Open in a new tab

Abnormally high expression of CCR7 is related to poor prognosis in patients
with B-NHL

   To investigate the expression levels of CCR7, CCL21, and CCL19,
   Immunohistochemistry (IHC) analysis was performed on lymph node tissue
   from patients with B-NHL and RLN. The results showed that CCL21 and
   CCR7 exhibited strong positive expression in the lymph nodes of
   patients with B-NHL, while CCL19 expression was negative. Additionally,
   CCR7 expression was significantly higher in patients with B-NHL
   compared with those with RLN, while CCL21 showed significant expression
   in both groups ([90]figure 3A). IHC analysis was also performed on bone
   marrow biopsy samples from patients with newly diagnosed (ND) B-NHL
   with or without clinically confirmed bone marrow involvement. The
   results demonstrated that the levels of CCL21 and CCR7 were higher in
   the bone marrow involvement group compared with the non-involvement
   group. Furthermore, EMT-related markers, vimentin and N-cadherin, were
   elevated, while E-cadherin was reduced in the bone marrow involvement
   group compared with the non-involvement group ([91]figure 3B). These
   findings suggest that abnormally high expression of CCL21 and CCR7 is
   associated with malignant bone marrow invasion in patients with ND
   B-NHL.

Figure 3. The abnormally high expression of CCR7 is associated with bone
marrow invasion and poor prognosis of patients with B-NHL. (A) The expression
of CCL21/CCL19/CCR7 in serial sections of lymph node tissue from patients
with reactive lymph node hyperplasia, DLBCL, and Burkitt lymphoma was
assessed by Immunohistochemistry, scale bar, 20 µm (×40). (B) The expression
of CCL21/CCR7 and epithelial-mesenchymal transition-related indicators
vimentin, N-cadherin, E-cadherin in bone marrow biopsy samples of patients
with B-NHL with and without bone marrow invasion was detected by
Immunohistochemistry, scale bar, 50 µm (×20). The expression of CCR7 (C) and
CCL21 (D) in peripheral blood of healthy controls (HC, n=9) and patients with
B-NHL with newly diagnosed (ND, n=15), completely remission (CR, n=10) and
relapsed/refractory (r/r, n=23) was analyzed by qRT-PCR. (E) The percentage
of CCR7+cells on the peripheral blood B lymphocyte membrane surface in HC
(n=29), ND (n=32), CR (n=39) and r/r (n=41) groups was detected by flow
cytometry. (F) The percentage of CCR7+cells on the membrane surface of
abnormal B lymphocytes and T lymphocytes in the peripheral blood of patients
with B-NHL (n=97) was detected by flow cytometry. (G) The receiver operating
characteristic curve for CCR7 expression in patients with B-NHL (n=112). (H)
Kaplan-Meier survival curve of OS according to CCR7 expression in B
lymphocytes of patients with B-NHL (n=112). (I) Kaplan-Meier survival curve
of PFS according to CCR7 expression in B lymphocytes of patients with B-NHL
(n=82). *p<0.05, ***p<0.001, ****p<0.0001. CCR7, C-C chemokine receptor 7;
B-NHL, B-cell non-Hodgkin’s lymphoma; CCL21, C-C chemokine ligand 21; CCL19,
C-C chemokine ligand 19; BMI, bone marrow involvement; qRT-PCR, quantitative
Reverse Transcription Polymerase Chain Reaction; AUC, area under the curve;
OS, overall survival; PFS, progression-free survival.

   [92]Figure 3
   [93]Open in a new tab

   To further analyze the differences in CCR7 expression among patients
   with B-NHL at different disease stages, quantitative Reverse
   Transcription Polymerase Chain Reaction (qRT-PCR) and flow cytometry
   were used to detect CCR7 levels in peripheral blood from patients with
   ND, complete remission (CR), relapsed or refractory (r/r) B-NHL, and
   healthy individuals. The levels of CCL21/CCR7 in the ND and r/r groups
   were significantly higher than those in healthy individuals and the CR
   group. Simultaneously, the expression of CCR7 in the r/r group was
   markedly elevated compared with the ND group ([94]figure 3C,D). Flow
   Cytometry (FCM) results revealed that the expression of CCR7 on B
   lymphocytes in the peripheral blood of patients with B-NHL was
   significantly higher than that in healthy controls. Additionally, r/r
   patients also exhibited significantly higher CCR7 levels compared with
   ND patients ([95]figure 3E). Further analysis of CCR7 expression on the
   membrane surface of different cell subpopulations in patients with
   B-NHL revealed that CCR7 was scarcely expressed in the monocytes and
   granulocytes, but was significantly expressed in lymphocytes. Notably,
   it was significantly expressed in the B lymphocyte subpopulation,
   showing markedly higher levels than in T lymphocyte subsets ([96]figure
   3F).

   To further evaluate the association between CCR7 expression and
   survival in patients with B-NHL, we followed the survival status of 112
   patients with B-NHL. A receiver operating characteristic curve was
   generated based on the percentage of CD197+B lymphocytes in patients
   with B-NHL. The area under the curve was 0.8876 (95% (CI): 0.8269 to
   0.9483, p<0.0001) and the cut-off value is 88.24% ([97]figure 3G). The
   relationship between CCR7 expression in peripheral blood and survival
   of patients with B-NHL was further evaluated. Patients with B-NHL were
   divided into a high CCR7 expression group and a low expression group
   according to the cut-off value. OS (median survival time 6 vs 33
   months, p<0.001) and PFS (median survival time 4 months vs 6 months,
   p<0.001) of patients with B-NHL in the CCR7 high expression group were
   significantly shorter than those in the low expression group by
   Kaplan-Meier survival analysis (p<0.001) ([98]figure 3H,I), suggesting
   that patients with B-NHL with low CCR7 expression have a significant
   survival advantage over patients with high expression levels. Patients
   were further stratified into groups based on relevant indicators that
   may affect survival, including gender, age, white blood cell count,
   hemoglobin level, platelet count, International Prognostic Index,
   Eastern Cooperative Oncology Group scores and CCR7 expression. COX
   multivariate regression was used to analyze the association between the
   above indicators and survival time, and it found that CCR7 (p=0.032) is
   a prognostic indicator for patients with B-NHL ([99]online supplemental
   table S2). High CCR7 expression is strongly associated with poor
   prognosis in patients with B-NHL, indicating it as a potential
   prognostic marker.

CCR7 promoted the invasion and migration of chemotherapy-induced senescent
B-NHL cells

   This study investigated seven hematological malignancy cell lines,
   comprising two leukemia cell lines (HL-60 and MOLM-13), two Burkitt
   lymphoma cell lines (Raji and Namalwa), and three DLBCL cell lines
   (SU-DHL-2, SU-DHL-4, and OCI-LY3), suggested that CCR7 expression in
   Burkitt lymphoma and DLBCL was significantly higher than that in the
   leukemia group ([100]online supplemental figure S3A,B). B-NHL cells
   were stimulated in vitro with various concentrations of the exogenous
   ligand CCL21. It was observed that at concentrations of 50 ng/mL and
   100 ng/mL, CCL21 significantly enhanced the migration and invasion
   abilities of Raji and SU-DHL-2 cells, respectively ([101]online
   supplemental figure S3C,D). We analyzed the impact of CCR7 on the
   migration and invasion abilities of Raji and SU-DHL-2 cells induced by
   CCL21 using CAP-100, a novel humanized IgG1-blocking monoclonal
   antibody directed against the ligand-binding site of hCCR7. The results
   revealed that CCL21 induces migration and invasion of B-NHL cells
   through CCR7 ([102]online supplemental figure S3E–G).

   Lentiviral-mediated short hairpin RNA (shRNA) was employed to interfere
   with CCR7 expression in Raji cells. It was observed that the virus
   successfully transfected B-NHL cells ([103]figure 4A), with the sh2
   groups having the strongest inhibition rate in Raji cells ([104]figure
   4B). The cell proliferation ability was reduced in the shCCR7 group
   compared with the NC group ([105]figure 4C). Inducing the NC group with
   40 nmol DOX resulted in a significant increase in the proportion of
   cells in G0/G1 phase, while the proportion of cells in the S phase and
   G2/M phase decreased, suggesting that the cell cycle was arrested in
   the G0/G1 phase ([106]figure 4D), while the shCCR7 group did not show
   similar results. Using 40 nmol DOX to induce NC and shCCR7 groups, it
   enhanced the invasion and migration ability of Raji cells ([107]figure
   4E,F). However, compared with the NC group, the invasion and migration
   ability of cells in the shCCR7 group significantly reduced ([108]figure
   4E,F). Similar results were obtained in SU-DHL-2 cells ([109]figure
   4G–L). These findings suggest that inhibiting CCR7 expression
   significantly reduces the invasion and migration of B-NHL cells, which
   are induced by sublethal doses of the chemotherapeutic DOX.

Figure 4. Knockdown of CCR7 suppresses the invasive and migratory
capabilities induced by sublethal doses of DOX in B-cell non-Hodgkin’s
lymphoma. (A) Fluorescence images of stably transfected CCR7 shRNA in Raji
cells, with red fluorescence indicating virus-infected cells. (B) Verify the
transfection efficiency of stable CCR7 shRNA in Raji cells using qRT-PCR. (C)
CCK-8 detection of the proliferation ability of Raji cells stably transfected
with CCR7 shRNA. (D) The effect of CCR7 expression on the cell cycle
distribution of stable CCR7 shRNA in Raji cells analyzed by flow cytometry.
The effect of CCR7 expression on the migration (E) and invasion (F) ability
of sublethal doses of DOX-induced Raji cells stably transfected with CCR7
shRNA detected by Transwell (×20). (G) Fluorescence images of stably
transfected CCR7 shRNA in SU-DHL-2 cells, with red fluorescence indicating
virus-infected cells. (H) Verify the transfection efficiency of stable CCR7
shRNA in SU-DHL-2 cells using qRT-PCR. (I) CCK-8 detection of the
proliferation ability of SU-DHL-2 cells stably transfected with CCR7 shRNA.
The effect of CCR7 expression on the migration (J) and invasion (K) ability
of sublethal doses of DOX-induced SU-DHL-2 cells stably transfected with CCR7
shRNA detected by Transwell (×20). *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001. CCR7, C-C chemokine receptor 7; DOX, doxorubicin; mRNA,
messenger RNA; shRNA, short hairpin RNA; qRT-PCR, quantitative Reverse
Transcription Polymerase Chain Reaction; CCK8, Cell Counting Kit-8.

   [110]Figure 4
   [111]Open in a new tab

   Raji cells were infected with lentiviral vectors and Green Fluorescent
   Protein (GFP) fluorescence expression was successfully observed 72
   hours after viral infection ([112]figure 5A). qRT-PCR validation
   demonstrated a significant increase in CCR7 expression in the
   overexpression group compared with the empty vector group ([113]figure
   5B). Using DOX to induce Vec and overexpression groups, respectively,
   the proliferation ability of CCR7 overexpressed Raji cells was enhanced
   ([114]figure 5C). DOX-induced Raji cells showed a significant increase
   in the proportion of G1 phase cells compared with untreated cells. This
   was coupled with a significant reduction in the percentage of cells in
   the G2 phase observed in both DOX-induced vector and overexpression
   groups. Notably, the proportion of G1 phase cells in the overexpression
   induction group was significantly higher than that in the vector
   induction group ([115]figure 5D). In stably transfected Raji cells, the
   CCR7 overexpression group showed significantly enhanced migration and
   invasion abilities after DOX induction, compared with the uninduced
   group or the induced or uninduced empty plasmid group ([116]figure
   5E,F). Stable transfection of CCR7 overexpression plasmids into
   SU-DHL-2 cells ([117]figure 5G,H) did not show significant differences
   in cell proliferation ability among the groups ([118]figure 5I). The
   results for cell cycle, migration, and invasion were similar to those
   observed in Raji cells ([119]figure 5J,K). High expression of CCR7
   enhances the invasive and migratory capabilities induced by sublethal
   doses of chemotherapeutics DOX in B-NHL cells.

Figure 5. High expression of CCR7 enhances the invasive and migratory
capabilities induced by sublethal doses of DOX in B-cell non-Hodgkin’s
lymphoma. (A) Fluorescence images of stably transfected human CCR7 lentivirus
in Raji cells, with red fluorescence indicating virus-infected cells. (B)
Verify the transfection efficiency of stable transfection CCR7 lentivirus in
Raji cells using qRT-PCR. (C) CCK-8 detection of the proliferation ability of
Raji cells stably transfected with CCR7 lentivirus. (D) The effect of CCR7
expression on the cell cycle distribution of sublethal doses of DOX-induced
Raji cells stably transfected with CCR7 lentivirus analyzed by flow
cytometry. The effect of CCR7 expression on the migration (E) and invasion
(F) ability of sublethal doses of DOX-induced Raji cells stably transfected
with CCR7 lentivirus detected by Transwell (×20). (G) Fluorescence images of
stably transfected human CCR7 lentivirus in SU-DHL-2 cells, with red
fluorescence indicating virus-infected cells. (H) Verify the transfection
efficiency of stable transfection CCR7 lentivirus in SU-DHL-2 cells using
qRT-PCR. (I) CCK-8 detection of the proliferation ability of SU-DHL-2 cells
stably transfected with CCR7 lentivirus. (J) The effect of CCR7 expression on
the cell cycle distribution of sublethal doses of DOX-induced SU-DHL-2 cells
stably transfected with CCR7 lentivirus analyzed by flow cytometry. The
effect of CCR7 expression on the migration (K) and invasion (L) ability of
sublethal doses of DOX-induced SU-DHL-2 cells stably transfected with CCR7
lentivirus detected by Transwell (×20). *p<0.05, **p<0.01, ***p<0.01,
****p<0.0001.CCR7, C-C chemokine receptor 7; DOX, doxorubicin; mRNA,
messenger RNA; qRT-PCR, quantitative Reverse Transcription Polymerase Chain
Reaction; CCK8, Cell Counting Kit-8.

   [120]Figure 5
   [121]Open in a new tab

CCR7 enhanced the stemness characteristics of senescent B-NHL cells by
activating ARHGAP18/IKBα/p65 pathway activation

   To explore the effect of silencing CCR7 on TIS formation in B-NHL
   cells, DOX was used to induce cells in NC-CCR7 and shCCR7 groups. The
   proportion of SA-β-Gal positive cells in the DOX-induced group was
   significantly higher than in the untreated group. However, CCR7
   knockdown significantly reduced SA-β-Gal-positive cells, indicating
   that silencing CCR7 expression in Raji cells significantly inhibited
   DOX-induced cellular senescence ([122]figure 6A). We detected CCR7
   expression in the DOX-induced NC/shCCR7 group and found that the CCR7
   expression levels corresponded with the proportion of SA-β-gal positive
   cells ([123]figure 6B). To further evaluate the reasons that knockdown
   of CCR7 inhibited the DOX-induced cellular senescence of B-NHL cells,
   we tested the stemness function of DOX-induced B-NHL cells. It was
   observed that the spheroid formation ability and colony formation
   ability of DOX-induced Raji cells in the shCCR7 group were
   significantly lower than those in the NC group, and similar results
   were obtained in the phenotypic stemness features test ([124]figure
   6C–E). Similar results were obtained in SU-DHL-2 cells ([125]figure
   6G–K). These findings indicate that silencing CCR7 expression
   significantly inhibited the stemness characteristics and altered the
   phenotype of B-NHL cells. On the contrary, upregulated expression of
   CCR7 promoted the stemness characteristics and phenotype of senescent
   B-NHL cells ([126]figure 7A–E and L–P).

Figure 6. Knockdown of CCR7 inhibits the phenotypic and functional stemness
features of senescent B-cell non-Hodgkin’s lymphoma cells by suppressing
NF-κB pathway activation. (A) Silencing CCR7 expression in Raji cells
significantly inhibits the formation of DOX-induced senescence. (B) CCR7
expression in DOX-induced Raji cells which stably transfected with shCCR7.
(C) Silencing CCR7 expression significantly suppresses the stemness phenotype
of senescent Raji cells. (D) Silencing CCR7 expression in Raji cells
significantly inhibits the abilities of DOX-induced sphere formation. (E)
Silencing CCR7 expression in Raji cells significantly inhibits the ability of
DOX-induced colony formation. (F) Silencing CCR7 expression significantly
inhibits the activation of NF-κB pathway in senescent Raji cells. (G)
Silencing CCR7 expression in SU-DHL-2 cells significantly inhibits the
formation of DOX-induced senescence. (H) CCR7 expression in DOX-induced
SU-DHL-2 cells which stably transfected with shCCR7. (I) Silencing CCR7
expression significantly suppresses the stemness phenotype of senescent
SU-DHL-2 cells. (J) Silencing CCR7 expression in SU-DHL-2 cells significantly
inhibits the abilities of DOX-induced sphere formation. (K) Silencing CCR7
expression in SU-DHL-2 cells significantly inhibits the ability of
DOX-induced colony formation. (L) Silencing CCR7 expression significantly
inhibits the activation of NF-κB pathway in senescent SU-DHL-2 cells.
*p<0.05, **p<0.01, ***p<0.01, ****p<0.0001. CCR7, C-C chemokine receptor 7;
DOX, doxorubicin; mRNA, messenger RNA; SA-β-Gal, senescence-associated
β-galactosidase; Nuclear Factor kappa B, NF-κB.

   [127]Figure 6
   [128]Open in a new tab

Figure 7. Overexpression of CCR7 enhances the phenotypic and functional
stemness features of senescent B-cell non-Hodgkin’s lymphoma cells by
activating NF-κB pathway activation. (A) Upregulated CCR7 expression
significantly enhances the formation of DOX-induced senescence in Raji cells.
(B) CCR7 expression in DOX-induced Raji cells stably transfected with CCR7
lentivirus. (C) Upregulated CCR7 expression significantly increases the
stemness phenotype of senescent Raji cells. (D) Upregulated CCR7 expression
significantly promotes the abilities of DOX-induced sphere formation in Raji
cells. (E) Upregulated CCR7 expression significantly promotes the ability of
DOX-induced colony formation in Raji cells. (F) Upregulated CCR7 expression
significantly enhances the activation of Wnt/β-catenin and NF-κB pathway in
senescent Raji cells. (G) Co-IP assay and immunoblotting with ARHGAP18/CCR7
antibody (or IgG) in Raji cells. (H) The effect of the IKBα inhibitor GS143
treatment on ARHGAP18 activation in DOX-induced Raji cells. (I) The effect of
RhoA/ROCK inhibitor Y27632 treatment on IKBα activation in DOX-induced Raji
cells. (J) The effect of IKBα inhibitor GS143 treatment on the stemness and
sphere-forming ability of DOX-induced Raji cells. (K) The effect of RhoA/Rock
inhibitor Y27632 treatment on sphere-forming ability of DOX-induced Raji
cells. (L) Upregulated CCR7 expression significantly increases the formation
of DOX-induced senescence in SU-DHL-2 cells. (M) CCR7 expression in
DOX-induced SU-DHL-2 cells which stably transfected with CCR7 lentivirus. (N)
Upregulated CCR7 expression significantly increases the stemness phenotype of
senescent SU-DHL-2 cells. (O) Upregulated CCR7 expression significantly
inhibits the abilities of DOX-induced sphere formation in SU-DHL-2 cells. (P)
Upregulated CCR7 expression significantly promotes the ability of DOX-induced
colony formation in SU-DHL-2. (Q) Upregulated CCR7 expression significantly
enhances the activation of Wnt/β-catenin and NF-κB pathway in senescent
SU-DHL-2 cells. **p<0.01, ****p<0.0001. CCR7, C-C chemokine receptor 7; DOX,
doxorubicin; mRNA, messenger RNA; SA-β-Gal, senescence-associated
β-galactosidase; Co-IP, Co-immunoprecipitation; NF-κB, Nuclear Factor kappa
B.

   [129]Figure 7
   [130]Open in a new tab

   Further analysis of the downstream pathways revealed that CCR7
   significantly activated the expression of ARHGAP18 and IKBα while
   inhibiting p65 expression ([131]figures6F,L [132]7F,Q). Meanwhile, we
   found that CCR7 could significantly activate the canonical stemness
   pathway, Wnt/β-catenin, including the activation of Wnt1 and β-catenin
   ([133]figures6F,L [134]7F,Q). To further investigate the potential
   interaction between ARHGAP18 and CCR7, we conducted a
   Co-immunoprecipitation(Co-IP) assay. Both ARHGAP18 and CCR7 were
   identified in the input group, confirming their presence in the sample.
   The IgG group within the IP set exhibited no bands, ruling out
   non-specific binding. Conversely, the anti-CCR7 group showed reduced
   bands for ARHGAP18 and CCR7. Both proteins were found in the
   precipitated complex, providing evidence of an interaction between
   ARHGAP18 and CCR7 ([135]figure 7G). Next, we performed rescue
   experiments using the IKBα inhibitor GS143[136]^22 and the RhoA/ROCK
   inhibitor Y27632[137]^23 to further explore the underlying mechanisms.
   First, GS143 and Y27632 significantly inhibited the expression of IKBα
   and ARHGAP18, respectively, in a dose-dependent manner. Inhibition of
   the IKBα pathway did not significantly alter ARHGAP18 expression.
   However, when ARHGAP18 expression was significantly suppressed, the
   activated IKBα signaling in TIS was notably inhibited ([138]figure
   7H1). Notably, inhibiting either IKBα or ARHGAP18 resulted in varying
   degrees of suppression of DOX-induced sphere formation in Raji cells
   ([139]figure 7J,K). These results indicate that CCR7 promotes the
   stemness characteristics and phenotype of senescent B-NHL cells by
   activating the ARHGAP18/IKBα/p65 signaling pathway.

Discussion

   Cellular senescence is an essential tumor suppressive mechanism, but
   TIS plays an important role in drug resistance of tumor cells because
   it is a reversible cellular behavior.[140]^5 6 Pharmacological
   targeting of TIS cells could mitigate the risk of cancer recurrence and
   thereby improve the therapeutic efficacy of cancer
   chemotherapy,[141]^24 25 but is still limited by the high heterogeneity
   of senescent cells. Currently, the recognized senescent cell marker to
   identify senescent cells is the measurement of SA-β-Gal
   activity,[142]^26 which can reflect the increase of lysosomal quality
   in senescent cells. But in fact, non-senescent cells such as
   macrophages stain positive for SA-β-Gal, and tissue sections from
   preclinical mouse models and patient samples can only be detected using
   frozen sections, to a certain extent. This limits the future clinical
   application of SA-β-Gal as a specific marker of senescence.[143]^27
   Except for SA-β-gal, senescent cells exhibit elevated expression of
   cell cycle regulators, including Cdkn2a (p16) and Cdkn1a (p21), which
   play roles in regulating cell cycle arrest. However, p16/p21 may not be
   used as a specific or sensitive indicator of cellular senescence
   because not all cells with elevated p16 levels are necessarily in a
   senescent state, and some senescent tumor cells often lack the
   expression of p16/p21.[144]^28 29 Therefore, due to a combination of
   technical issues and the complexity and high heterogeneity of
   senescence, no clinically universal senescence markers have yet to be
   truly promoted.[145]^27 Research is crucial to precisely characterize
   cellular senescence phenotypes and functions in different tumor types.
   This will help identify specific features of senescence that can be
   targeted to either inhibit or enhance, without adversely affecting
   normal cells. In the present study, we used doxorubicin to construct
   B-NHL-TIS, and then identified the distinctive chemotherapy-induced
   senescent cell subpopulation using ScRNA-seq analysis. Compared with
   the non-senescent and autonomous senescent cell subpopulations, the TIS
   cell subpopulation not only characterized by typical cell cycle
   proteins deficiency and cellular senescence signaling proteins
   enrichment, but also had a high expression of chemokine family and the
   anti-apoptotic BCL2 family and the NF-κB family. The chemokine receptor
   CCR7 exhibited significant differential expression in the senescent
   cell population and the non-senescent cell population. This is the
   first ScRNA-seq report on CCR7 is uniquely overexpressed in
   chemotherapy-induced senescent B-NHL subpopulation, which may be
   crucial for understanding the molecular mechanism of TIS and
   chemoresistance in B-NHL.

   CCR7 is an important signaling molecule in tumorigenesis and tumor
   development.[146]1320 30,[147]35 In our study, the expression levels of
   CCR7 and CCL21 were notably higher in patients with aggressive B-NHL
   compared with those with RLN. However, CCL19 exhibited almost no
   expression in the lymph nodes of patients with most ND DLBCL and
   Burkitt lymphoma. A recent large-scale multicenter survival study on
   patients with cancer found that in breast and ovarian cancers, patients
   with high CCL19 expression had a significantly better prognosis
   compared with those with low CCL19 expression. Furthermore, inhibition
   of CCL19 expression led to significant cancer progression and increased
   apoptotic activity,[148]^36 supporting our findings on CCL19 expression
   in patients with aggressive B-NHL. Further exploration reveals that
   CCL21 significantly enhances DOX-induced migration and invasion of
   B-NHL cells through interaction with CCR7. When CCR7 was blocked by
   CAP-100 (a novel humanized IgG1 blocking monoclonal antibody),[149]^37
   38 the CCL21-induced migration and invasion abilities were
   significantly inhibited. Additionally, CCR7 expression was
   significantly elevated in patients with r/r B-NHL compared with ND
   patients, suggesting its potential role in disease progression. High
   CCR7 expression is an independent prognostic factor for patients with
   B-NHL, consistent with findings reported in primary DLBCL.[150]^39 We
   analyzed CCR7 expression across various hematological malignancies and
   observed significant upregulation at both the transcriptional and
   post-translational levels in DLBCL and Burkitt lymphoma. However, CCR7
   expression was low in two leukemia cell lines, indicating that CCR7 may
   serve as a differential target in B-cell lymphoma subtypes as opposed
   to leukemia subtypes. Currently, chemotherapy is the primary treatment
   for B-NHL. Our findings suggest that CCR7 is not only an independent
   prognostic factor but also a novel therapeutic target for patients with
   r/r B-NHL. Targeting CCR7 could enable the development of new treatment
   strategies combining chemotherapy with agents that exploit
   senescence-inducing mechanisms, offering significant clinical
   potential.

   In the pseudotime analysis, we found that the TIS population was
   distributed at the earliest stage of the pseudotime trajectory,
   suggesting that senescent B-NHL cells possess self-renewal capability
   and multipotent differentiation potential. We further investigated
   whether chemotherapy-induced senescence could enhance the stemness of
   B-NHL cells and the role that CCR7 plays in this process. Senescent
   B-NHL cells exhibited typical stem cell characteristics, including the
   upregulation of stem cell molecular markers, enhanced tumorsphere and
   colony formation capabilities, and increased invasion and migration
   abilities. This is consistent with reports that chemotherapy-induced
   senescence enhances stem cell characteristics and increases tumor
   initiation potential,[151]^11 promoting stemness and
   tumorigenicity.[152]^40 Furthermore, the formation of B-NHL-TIS is
   strictly regulated by CCR7. Targeted inhibition of CCR7 not only
   suppresses chemotherapy-induced senescence in lymphoma cells but also
   significantly inhibits the acquisition of phenotypic and functional
   stemness features. Mechanistically, CCR7 enhances stemness in oral
   squamous cell carcinoma and MMTV-PyMT mammary cancer cells by
   activating the JAK/STAT[153]^20 and Notch[154]^19 pathways.
   Additionally, Wnt signaling plays a central role in the renewal of stem
   cells and is essential for the development of cancer stem cells in
   hematologic malignancies.[155]^11 41 42 CCR7 activation leads to a
   significant enrichment of the wnt1 and β-catenin, while targeted
   inhibition of CCR7 restricts the expression of wnt1 and β-catenin,
   supporting the typical Wnt/β-catenin signaling pathway enrichment in
   B-NHL-TIS.

   We provide the first direct evidence of NFKBIA and NF-κB pathway
   activation in the CCR7+B-NHL-TIS population, while NF-κB is
   significantly inhibited in non-senescent, proliferating B-NHL cells.
   Second, by further blocking CCR7 and screening for senescence-related
   proteins and the NF-κB pathway, we found a significant inhibition of
   RhoA GTPase-activating protein ARHGAP18 and NFKBIA-encoded protein IKBα
   expression. They exhibited a synergistic trend in the
   chemotherapy-induced senescent B-NHL cells stage regulated by CCR7.
   When the TIS process was reversed, ARHGAP18 and IKBα expression was
   significantly inhibited, while the expression of NF-κB p65 was the
   opposite. ARHGAP18 is a GTPase-activating protein for RhoA that
   controls cell migration, proliferation, and survival,[156]^43 and
   regulates stress-induced senescence in endothelial cells via p16 and
   retinoblastoma activation.[157]^44 We previously reported that ARHGAP18
   is significantly enriched in B-NHL-TIS.[158]^21 The regulatory
   relationship between CCR7 and ARHGAP18 is unknown, and our results
   suggest that activated CCR7 binds to its ligand, it is internalized and
   activates ARHGAP18, which in turn binds to RhoA to promote its
   inactivation, then acts on the NF-κB pathway[159]^45 as reported. It is
   worth noting that the loss of RhoA function reported by Hiroki Sasaki
   et al leads to increased invasiveness and stemness in gastric
   cancer,[160]^46 which is consistent with our observation of enhanced
   ARHGAP and increased stemness in lymphoma cells. NF-κB signaling is
   chronically activated during senescence to promote the SASP.[161]^47 48
   NFKBIA is transcriptionally induced as part of a negative feedback
   mechanism to regulate NF-κB signaling.[162]^49 When NF-κB is activated,
   one of the target genes it upregulates is NFKBIA, which produces IKBα
   binding to the p65 DNA-binding sites to inhibit further NF-κB
   activation.[163]^50 In senescent cells, the SASP lead to persistent,
   low-level NF-κB activation and constant expression of IKBα to prevent
   excessive activation, resulting in both NF-κB and IKBα being elevated
   simultaneously. The activation of CCR7 can enhance NF-κB signaling,
   leading to the upregulation of cancer stem cell markers such as
   Wnt1/β-catenin signaling, as well as CD34 and CD44, thereby promoting
   the stemness of B-NHL-TIS. Herein, our studies provide evidences for a
   key role of CCR7 in B-NHL-TIS, contributing to the reprogramming of
   cellular senescence and promote the senescence-associated stemness of
   B-NHL cells, which has not been reported yet. The activation of the
   CCR7/ARHGAP18/IKBα signaling cascade is one of the crucial mechanisms
   by which TIS lymphoma acquires stemness features.

   In conclusion, this study demonstrated that TIS promotes the stemness
   of B-NHL cells via CCR7/ARHGAP18/IKBα signaling activation, which might
   elucidate a potential mechanism in chemoresistance for B-NHL. Targeting
   the signaling might overcome the chemoresistance of B-NHL cells by
   inhibiting stemness acquisition and maintenance.

supplementary material

   online supplemental file 1
   [164]jitc-13-1-s001.pdf^ (877KB, pdf)
   DOI: 10.1136/jitc-2024-009356
   online supplemental file 2
   [165]jitc-13-1-s002.pdf^ (14.4MB, pdf)
   DOI: 10.1136/jitc-2024-009356

Acknowledgements