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Keywords:

  • mantle cell lymphoma;
  • therapy-resistant tumor;
  • GLI;
  • bortezomib;
  • immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5–7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.

Mantle cell lymphoma (MCL) is one of the most aggressive lymphomas among the B-cell malignancies, with a median survival of only 5–7 years.1 MCL is characterized genetically by the t(11;14)(q13;q32) translocation, which leads to overex pression of cyclin D1.2 Although existing therapies using different combinations of chemotherapeutic agents such as cyclo phosphamide, vincristine, doxorubicin, prednisone (CHOP), cyclophosphamide, vincristine, doxorubicin, dexamethasone (hyper-CVAD), Rituximab and Bortezomib (BTZ) increase disease-free survival, relapse due to residual therapy-resistant MCL is the main roadblock to cure of this disease.3, 4 Therefore, an effective treatment strategy is needed to target the biological basis of therapy resistance.

Current evidence suggests that single or multiple agents are often inadequate for the treatment of aggressive lymphomas like MCL.3–7 Therefore, effort should be put into developing new approaches such as strategies that sequentially administer targeted agents.5, 8 We have developed an innovative therapeutic strategy that combines CHOP chemotherapy (to reduce the tumor burden) and inhibition of hedgehog signaling or tumor microenvironment (to target therapy-resistant MCL) followed by treatment using transplantation of MCL-specific cytotoxic T lymphocytes (CTLs). Despite the known role of hedgehog signaling in the pathogenesis of various cancers,9–11 its role has only recently been demonstrated in leukemia and lymphoma.12–16 Hedgehog signaling is important in the maintenance of cancer progenitor cells and is known to promote drug resistance.17–21 We have recently demonstrated that targeting Glioma (GLI) family of transcription factors, mediators of hedgehog signaling, increased the susceptibility of MCL to chemotherapy in vitro.13 Irrespective of how the hedgehog signaling is deregulated in tumor cells, GLI transcription factors are ultimately overexpressed. Therefore, targeting of GLI in vivo, in combination with chemotherapy, may inhibit the growth of MCL cells and make them susceptible to other therapies. The use of proteosome inhibitors, such as BTZ, also has excellent potential in MCL.22 BTZ has multiple effects on cancer cells: it inhibits two major pathways that lead to NF-κB activation, it stabilizes proapoptotic members of the BCL-2 family and it causes the build-up of misfolded proteins.23 BTZ may also disrupt tumor–stromal interactions that drive NF-κB activation and angiogenesis.23 Because of these multiple effects, it may be useful in combination therapies. These modalities can also be combined with immunotherapy to bypass some of the characteristics of chemoresistance.24–27 We have reported the efficacy of CTLs against MCL cells in vitro and in vivo.28, 29 These findings suggest that a strategy combining CHOP chemotherapy plus targeting GLI or proteosome inhibition followed by MCL-specific CTLs may improve the outcome in therapy-resistant MCL. This research evaluated this proposition.

For our in vivo model, NOD/SCID mice were transplanted with Granta 519 MCL cells, an extensively used patient-derived MCL cell line,30 and treated with CHOP chemotherapy. Relapsing MCL cells were isolated from the liver (Granta resistant [GR]) of the mice and characterized using cytomorphology, flow cytometry and growth assays. Our studies indicated that these therapy-resistant tumor cells were not only resistant to chemotherapeutics but also overexpressed molecules such as GLI transcription factors and survivin (BIRC5). Combination chemotherapy with CHOP decreased tumor burden in NOD/SCID mice bearing MCL. Prior targeting of MCL with GLI antisense oligonucleotides or proteosome inhibition with BTZ-senstiized the tumors to killing. Furthermore, MCL-specific CTLs significantly decreased the remaining therapy-resistant tumors and increased disease-free survival.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cell culture

The MCL cell line Granta 519 used in this study was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). This cell line was maintained in DMEM media (Invitrogen, CA) containing 10% fetal bovine serum (U.S. Bio-Technologies, PA), 1× penicillin/streptomycin (Invitrogen, CA) and 1× L-glutamine (Invitrogen, CA). MDA-MB-231, a breast cancer cell line, which is matched with one human leukocyte antigen (HLA) allele (HLA-A2) to Granta 519 cells, was purchased from ATCC (Manassas, VA) and used as an irrelevant tumor target. This cell line was maintained in RPMI-1640 (Invitrogen, CA) instead of Dulbecco's modified eagle medium (DMEM) (Invitrogen, CA) media.

Isolation of therapy-resistant MCL cells

NOD.CB17-Prkdcscid/J mice (6–8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME) and housed in the University of Nebraska Medical Center (UNMC) animal facility. The mice were exposed to 215 Gy of total body ionizing radiation prior to MCL cell transplantation. Two million Granta 519 cells suspended in 100 μl of RPMI were injected intravenously via a lateral tail vein. After 10 days of tumor growth, the mice were treated with three consecutive daily doses of CHOP chemotherapy, which included cyclophosphamide (30 mg/kg i.v.), vincristine (0.375 mg/kg i.v.), doxorubicin (2.5 mg/kg i.v.) and prednisone (0.15 mg/kg p.o.) (all purchased from Sigma, MO). After 3 days, the mice were treated with four doses of BTZ (0.5 mg/kg i.v.) on alternative days. When symptoms of disease such as weight loss, ruffled skin or a hunched back were observed, the mice were euthanized and the liver was removed. The liver was washed with DMEM media and chopped using scalpels. Then pieces of these tissues were minced using fine scissors and aspirated repeatedly using a syringe. Each cell suspension was allowed to settle for 10 min, and the supernatant cell suspension was centrifuged, washed and cultured in DMEM media containing 50 μg/ml gentamycin (Sigma, MO). The nontumorigenic cells derived from mouse died out after 6–8 days and only therapy-resistant MCL cells grew in these cultures. These therapy-resistant tumor cells were described as GR and compared to parental Granta (GP) 519 cells.

Characterization of therapy-resistant MCL cells

The GP cells and the therapy-resistant MCL cells (GR) were stained with Wright–Giemsa in the UNMC pathology core laboratory and the cytomorphology was compared by light microscopy. The presence of surface markers for MCL cells, including CD19, CD20 and CD45, was determined by flow cytometry. Briefly, 2 × 105 cells in 100 μl RPMI media were incubated with flurochrome-conjugated antibodies (BD Pharmingen, CA) specific for these markers for 30 min at 4°C. Cells were washed three times with phosphate buffered saline (PBS) and transferred to Fluorescence-activated cell sorting (FACS) fix solution. The percent positive cells were enumerated using a FACStar PLUS flow cytometer (Becton Dickinson, NJ) and analyzed using Cellquest-Pro software.

In vitro growth assay

(3-(4,5-Di-Methyl Thiazole-2-yl)-2,5-diphenyl Tetrazolium bromide (MTT) and 3[H]-thymidine assays were performed as described previously.13, 14 Briefly, 5,000 GP and therapy-resistant GR cells were cultured in 96-well plates in 200 μl RPMI media, and the growth of these cells was determined at 72 hr by MTT and 3[H]-thymidine assays. To determine the chemoresistant nature of these tumor cells, 5,000 cells were cultured in the presence of 25 nM doxorubicin and 5 μM vincristine for 72 hr. The growth rate was measured by MTT and 3[H]-thymidine assays as above. The fold change in growth of GR cells was determined relative to that of GP cells.

Growth and chemoresistant properties of GR and GP cells in vivo

NOD/SCID mice were prepared for the in vivo study as explained above. One million each of GR and GP cells were transplanted via a tail vein and the survival of the mice was determined by the Kaplan–Meier method and analyzed for significance using the log-rank test. In another set of experiments, tumor-bearing mice were treated with CHOP chemotherapy and survival was analyzed as above.

Real-time polymerase chain reaction (PCR)

RNA was isolated from fresh passages of GP and GR cells using Trizol reagent (Invitrogen, CA), cDNA was synthesized from DNase-treated RNA, and real-time PCR was performed for molecules associated with the hedgehog signaling pathway including GLI1 and GLI2, and for BIRC5, FRAP1 and PI3K using an ABI PRISM 7000 Sequence Detection System, as described previously.14

Generation of MCL-specific CTLs

MCL-specific CTLs were prepared as described previously.28, 29 Briefly, normal human peripheral blood mononuclear cells (PBMCs) were harvested by apheresis from healthy HLA-A2+ donors, that is, matching Granta 519 cells. Dendritic cell (DC) precursors were adhered to tissue culture flasks, and nonadherent cells were separated and used for T-cell expansion. To generate immature DCs, the adherent mononuclear cells were cultured in RPMI media containing 10% human AB serum (Atlanta Biologicals, GA) supplemented with 800 U/ml of GM-CSF (Immunex Corporation, WA) and 400 U/ml of Interleukin-4 (IL-4) (Peprotech, NJ) for 6 days, with one feeding in between. Hybrid cells of immature DCs and MCL cells were generated by electrofusion using a BioRad GenePulser II (Bio-Rad, CA) as described previously.28, 29 These hybrid cells were cultured in maturation media supplemented with GM-CSF (800 U/ml), IL-4 (400 U/ml), TNF-α (50 ng/ml), IL-1β (25 ng/ml), Interferon-gamma (IFN-γ) (1,000 U/ml), polyinosinic:polycytidylic acid (20 μg/ml; Amersham Biosciences, NJ) and IFN-α (3,000 U/ml). The mature DCs presenting MCL-associated antigens were harvested on Day 7 for CTL generation. To generate MCL-specific CTLs, the T-cells stimulated with IL-2 (50 U/ml) were cocultured with mature DCs for 14 days in media supplemented with IL-2 (50 U/ml). The cultures were restimulated with freshly primed and matured DCs 7 days after the first addition. The tumor-specific cytotoxicity of CTLs was determined by a 51Cr release assay as described previously.28, 29 The tumor-specific CTLs and 51Cr-labeled MCL cells (GP 519 cells or GR) were cocultured in a 96-well round-bottomed plate to obtain effector:target ratios (E:T) of 1:1, 10:1, 25:1 and 50:1. 51Cr-labeled MDA-MB-231 cells, transfected with Epstein–Barr virus (EBV), were used as an irrelevant HLA-matched tumor-target control.

Targeting therapy-resistant MCL with GLI-ASO/BTZ followed by CTLs in vivo

Granta 519 cells (1 × 106) were transplanted into irradiated NOD/SCID mice by tail vein injection. After 10 days, mice other than the control group were treated with three consecutive doses of CHOP chemotherapy as described above. The mice were then divided into different groups after excluding a CHOP-only control group (six mice). One set of mice (12 mice) was treated with four doses of GLI1-ASO13 + GLI2-ASO13 (5 mg/kg i.v.) on alternate days, and another set of mice (12 mice) was treated with four doses of BTZ (0.5 mg/kg i.v.) on alternate days. From these mice, six mice in each group and six mice from CHOP chemotherapy group were further treated with four weekly injections of Granta 519-specific CTLs (5 × 106 per mice, i.v.). We began administering CTLs 1 week after the last CHOP chemotherapy in the CHOP + CTL group, and 1 week after the last GLI-ASO therapy in the CHOP + GLI-ASO + CTL group or last BTZ therapy in the CHOP + BTZ + CTL group. The survival of the mice was monitored and analyzed as described above. The liver, spleen, kidneys, lungs, intestines and heart were removed at the time of death from either tumor progression or toxicity and fixed in 10% formalin for histological analysis.

Immunohistochemistry

The Histology Core Facility of the Nebraska Medical Center performed staining of the mouse tissue sections with hematoxylin and eosin stain (H&E stain) and immunohistochemical analysis using CD20 antibody according to the manufacturer's protocol. The images were scanned using automated “virtual” digital microscopy and the extent and patterns of staining quantitated using Neuroinformatica software.

Statistical analysis

Data were collected from triplicates of at least three independent experiments or as otherwise mentioned in the respective figure legend. Data were represented as average ± SEM. Statistically significant differences between groups were determined using Student's t-test. The median survival of mice with different treatments was calculated by the Kaplan–Meier method, and the statistical significance was analyzed using the log-rank test. A p value of <0.05 was considered as significant.

Study approval

All animal experiments were approved by the Institutional Animal Care and Use Committee and the human blood mononuclear cells were obtained using a protocol approved by the Institutional Review Board of the University of Nebraska Medical Center, with informed consent of the donors.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In vivo model of therapy-resistant MCL cells

To validate our in vivo model system for studying the biological basis of therapy resistance, we transplanted Granta 519 MCL cells, a well-characterized patient-derived human MCL cell line,30 intravenously into NOD/SCID mice by tail vein injection. Ten days later, mice were treated with CHOP chemotherapy (Fig. 1a), a commonly used chemotherapy regimen for MCL patients to reduce tumor burden.31 The injected tumor cells colonized in the major organs such as the liver, kidney and lungs of these mice. Following CHOP chemotherapy, the tumor burden was significantly reduced as observed by immunohistological analyses (Fig. 1b). In addition, the sizes of tumor nodules in the livers of chemotherapy-treated mice were significantly (p < 0.0001) reduced compared to those in livers of vehicle-treated mice liver (Fig. 1c). We also determined the survival of mice in these two groups by Kaplan–Meier analysis with the log-rank test. CHOP-treated mice demonstrated significantly (p < 0.0001) increased survival with a median survival of ∼60 days compared with vehicle-treated mice with an average survival of only ∼30 days (Fig. 1d). Although CHOP-treated mice had almost normal livers and significantly increased survival compared with vehicle-treated mice, they had very few, but still detectable tumor cells remaining after the chemotherapy, in the liver as identified by H&E staining (Fig. 1b). These results suggested that a small population of tumor cells may survive chemotherapy and cause tumor relapse as seen in humans. Thus, this optimized in vivo model mimics MCL-patient relapses and was used in these studies to develop a therapeutic strategy to target these cells by inhibiting overexpressed molecules in resistant MCL cells followed by tumor-specific killing with T-cell immunotherapy.

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Figure 1. In vivo model of therapy-resistant MCL cells. (a) Granta 519 bearing mice were treated with combination chemotherapy. Once relapsing tumor was detected, the mice were euthanized and the therapy-resistant relapsing MCL cells were isolated from the liver. (b) Representative image of tumors and H&E staining of the liver sections from mice treated with vehicle or CHOP chemotherapy. (c) Quantitative analysis of tumor nodule size in vehicle or CHOP-treated mice using virtual microscopy. (d) The mice bearing Granta 519 were treated with either vehicle or CHOP chemotherapy, and the survival was determined by Kaplan–Meier analysis with a log-rank test of significance. Total number of mice, N = 12.

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Isolation and characterization of therapy-resistant MCL cells

To isolate therapy-resistant MCL cells, we transplanted Granta 519 MCL cells into NOD/SCID mice and treated them with combination chemotherapy (Fig. 1a), as described above. Therapy-resistant MCL cells were then isolated from the liver as described in the Material and Methods section. This cell line was called GR, indicating their resistant property. The human origin of the therapy-resistant MCL cells was confirmed by immunophenotyping (Fig. 2). Examination of these cells after Wright–Giemsa staining revealed the characteristics of typical MCL cells (Fig. 2a). Immunophenotypic analysis of GR cells using antibodies CD19, CD20 and CD45 showed the typical MCL phenotype with more than 98% of the cells being positive for these markers, similar to those of GP 519 cells (Figs. 2b2d), thus confirming the MCL characteristics of the therapy-resistant cells. Together, these results indicate that the therapy-resistant GR cells were a immunophenotypically homogenous population with typical MCL characteristics.

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Figure 2. Cytomorphological and immunophenotypic characterization of therapy-resistant MCL cells. (a) Cytomorphological analysis of therapy-resistant MCL cells was performed using Wright–Giemsa staining and compared with GP 519 cells. (bd) Immunophenotypic analysis of therapy-resistant MCL cells (GR) was performed for CD45, CD19 and CD20 and compared to GP 519 cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Growth of therapy-resistant MCL cells in vitro and in vivo

In MCL patients, therapy-resistant tumor cells are known to exhibit aggressive growth.3, 6 Therefore, we hypothesized that the isolated therapy-resistant MCL cells from our mouse model might also exhibit increased growth. To determine the growth of therapy-resistant MCL cells in vitro, GR and GP cells were cultured in growth media; growth was measured by MTT and 3[H]-thymidine uptake assays after 72 hr. We observed an increased absorbance and 3[H]-thymidine incorporation into the GR cells as compared to control GP cells (Figs. 3a and 3b), indicating the increased growth of therapy-resistant MCL cells in vitro. To determine the growth potential of these cells in vivo, we transplanted 1 million GR and GP cells into NOD/SCID mice via a tail vein and monitored the survival of the mice. We found a significantly shorter survival (p < 0.001) for mice bearing GR cells compared to those bearing GP cells (Fig. 3c), indicating an increased in vivo growth rate of therapy-resistant MCL cells. Together, these results demonstrate in the mouse model the aggressive disease progression by therapy-resistant tumor cells as observed in patients,3, 6 thereby validating our model system.

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Figure 3. Growth of therapy-resistant MCL cells in vitro and in vivo. Relapsing, therapy-resistant (GR) MCL cells and parental (GP) MCL cells (5,000) were cultured in RPMI media in 96-well plates and the growth rate was measured using MTT (a) and 3[H]thymidine (b). (c) One million GR or GP cells were transplanted intravenously into NOD/SCID mice via a tail vein. Survival was monitored, analyzed using Kaplan–Meier survival analysis and tested for significance using the log-rank test. Total number of mice, N = 12.

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Chemoresistant properties of therapy-resistant MCL cells in vitro and in vivo

In MCL patients, the treatment of relapsed disease is often difficult because of therapy-resistant residual tumor cells.3 Therefore, we decided to evaluate whether the therapy-resistant MCL cells have chemoresistance properties. To test this premise, we cultured GR or GP cells in the presence of chemotherapeutic drugs vincristine or doxorubicin and measured their growth. The growth rates were determined by MTT (Fig. 4a) and 3[H]-thymidine uptake (Fig. 4b) assays. The growth of GR cells cultured in media containing vincristine or doxorubicin increased by twofold to fourfold relative to GP cells as determined by MTT assay (Fig. 4a) and 3[H]thymidine-uptake assay (Fig. 4b), indicating chemoresistant properties of therapy-resistant MCL cells compared to parental cells. Furthermore, to confirm the chemoresistant nature of these MCL cells in vivo, we transplanted GR or GP cells into NOD/SCID mice and treated them with CHOP chemotherapy. The mice bearing GR cells had a significantly shorter survival (p < 0.001) compared to mice bearing GP cells (Fig. 4c), confirming the chemoresistance properties of these cells in vivo. Thus, both our in vitro and in vivo studies demonstrated that relapsing therapy-resistant MCL cells have chemoresistance properties compared to parental MCL cells.

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Figure 4. Chemoresistant properties of therapy-resistant MCL cells in vitro and in vivo. Therapy-resistant (GR) and parental (GP) MCL cells (5,000) were cultured in RPMI media containing 5 μM vincristine or 25 nM doxorubicin in 96-well plates for 72 hr. (a) The growth rate of the cells was measured using MTT reagent and fold change was calculated with respect to the absorbance exhibited by parental cells. The results presented are mean (±SEM) from three independent experiments. (b) The growth rate was also measured using 3[H]thymidine uptake and fold change was calculated with respect to the CPM in parental cells. The results presented are mean (±SEM) from three independent experiments. (c) One million GR or GP cells were transplanted intravenously into NOD/SCID mice. After 10 days, the mice were treated with three daily doses of CHOP chemotherapy. The survival was determined using Kaplan–Meier analysis and significance tested by the log-rank test. Total number of mice, N = 8.

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Expression of potential therapeutic targets in therapy-resistant MCL cells

To identify potential therapeutic targets in therapy-resistant MCL cells, quantitative real-time PCR was performed for hedgehog-signaling target molecules GLI1 and GLI2 and for some of the molecules that have previously been reported to play a role in the aggressiveness and/or progression of lymphomas such as survivin (BIRC5), aurora-A kinase and PI3K-mTOR-Akt (PI3K and FRAP1). The expression of GLI1, GLI2 and BIRC5 (survivin) transcripts increased by twofold to eightfold in GR cells compared to GP cells (Supporting Information Fig. S1). Thus, confirming recent reports that GLI transcription factors are overexpressed in MCL cells compared to normal B cells. Targeting GLI transcription factors with antisense oligonucleotides decreased the proliferation and increased the susceptibility of MCL cells to chemotherapy by downregulating antiapoptotic BCL-2 and Cyclin D1.13 Therefore, our current and previous studies indicated that targeting GLI transcription factors using these optimized antisense oligonucleotides could be a promising strategy to target therapy-resistant MCL cells in vivo.

Targeting therapy-resistant MCL with GLI-ASO/BTZ followed by MCL-specific CTLs

Based on the above observations, we hypothesized that a small subset of tumor cells would survive after chemotherapy and result in the relapse of MCL. Therefore, targeting these therapy-resistant cells would be an ideal strategy to improve the outcomes of MCL.21, 32 Therefore, as a novel therapeutic strategy, we targeted therapy-resistant MCL cells after chemotherapy using hedgehog-signaling or proteosome inhibitors in combination with transplantation of MCL-specific CTLs. Before using the CTLs in vivo, we determined their efficacy and specificity of killing against GP and GR cells in vitro by a 51Cr release assay (Fig. 5a). In addition, we measured the specificity of these CTLs against an HLA-matched irrelevant breast cancer cell line, MDA-MB-231. These CTLs demonstrated significant cytotoxicity against parental GP and therapy-resistant GR MCL cells to a similar extent, which suggests that CTLs generated against parental MCL cells would target therapy-resistant MCL cells. There was a significantly less cytotoxicity against MDA-MB-231 (Fig. 5a) or EBV-infected MDA-MB-231 cells (data not shown) compared to either GP or GR MCL targets. These results demonstrated a significant increased cytotoxic effect of MCL-specific CTLs against therapy-resistant MCL cells compared to an irrelevant tumor target.

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Figure 5. Targeting of therapy-resistant MCL cells by GLI-ASO/BTZ followed by CTL transplantation in vivo. (a) MCL-specific CTLs were cocultured for 4 hr with 51Cr-labeled GP, GR or MDA-MB-231 cells as a control. The cytotoxicity of the CTLs was determined by released 51Cr in the culture supernatants. Target cells in media and 1% Triton X-100 were used for spontaneous and total 51Cr release to calculate percent cytotoxicity. The results presented are mean (±SEM) from triplicate wells of four independent experiments. (b) Granta 519 MCL cells (1 × 106 i.v.) were transplanted intravenously into NOD/SCID mice. After 10 days, the mice were treated with three daily doses of CHOP chemotherapy followed by four doses of GLI-ASO (5 mg/kg i.v.) on alternate days. After 1 further week, mice were treated with four weekly doses of MCL-specific CTLs (5 × 106 i.v.). For the CHOP + CTLs group, the mice were treated with CTLs 1 week after the last CHOP chemotherapy. The survival of the mice was determined by Kaplan–Meier analysis with a log-rank test of significance. Appropriate control groups: MCL cells without treatment, MCL cells with CHOP, CHOP + GLI-ASO and CHOP + CTLs were included in the study. Total number of mice, N = 30. (c) Mice bearing Granta 519 MCL cells (1 × 106 i.v.) treated with CHOP chemotherapy and BTZ as above, after 1 further week, treated with four weekly doses of MCL-specific CTLs (5 × 106 i.v). The survival of the mice was determined as above. Appropriate control groups of mice: MCL cells without treatment, MCL cells with CHOP, CHOP + BTZ and CHOP + CTLs were included in the study. Total number of mice, N = 30. Control groups were common for (b) and (c).

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To develop an effective therapeutic strategy for MCL, we used our optimized in vivo model (Fig. 1). We transplanted Granta 519 cells intravenously into mice via tail vein, which resulted in tumors in multiple organs including the liver, kidneys and lungs as previously reported.28 Then, the mice were treated with CHOP chemotherapy to reduce the tumor burden. As expected, CHOP chemotherapy eliminated the bulk of the tumors; however, a few MCL cells remained, which we have shown to be aggressive, chemoresistant and responsible for relapse. The mice were further treated with either GLI-ASO or BTZ in an attempt to kill therapy-resistant tumor cells. This treatment was followed by transplantation of MCL-specific CTLs. The mice treated with CHOP + GLI-ASO + CTLs had a significantly increased disease-free survival (p < 0.0001) with a mean survival of 86 days. The mean survival for untreated mice was only 30 days, with CHOP it was 55 days, with CHOP + GLI-ASO 58 days and with CHOP + CTLs 69 days (Fig. 5b). In addition, the mice treated with CHOP + BTZ + CTLs had a significantly increased disease-free survival (p < 0.0001) with a mean survival of 89 days compared to the mean survival for untreated mice of 30 days. As noted above, survival with CHOP was 55 days, but with CHOP + BTZ, it increased to 61 days and with CHOP + CTLs was 69 days (Fig. 5c). In addition, tumor burden in different organs, collected at the time of death from tumor progression or toxicity in the above experiment was determined by immunohistochemical staining for CD20. Almost all the MCL cells were eliminated from the liver, kidneys and lungs of mice treated with either CHOP + GLI-ASO + CTLs or CHOP + BTZ + CTLs compared to mice treated with CHOP + GLI-ASO, CHOP + BTZ or CHOP alone (Fig. 6). These results suggest that the mice treated with either CHOP + GLI-ASO + CTLs or CHOP + BTZ + CTLs might have died due to toxicity but not due to tumor burden. Together, these results demonstrated that inclusion of BTZ and GLI-ASO in the treatment regimen resulted in better tumor elimination, so that using this strategy, we were able to achieve a significantly prolonged disease-free survival in our preclinical model of CHOP resistant MCL.

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Figure 6. Immunohistochemical analysis of the liver, kidney and lungs of mice under going different treatments. NOD/SCID mice bearing MCL cells were treated with CHOP chemotherapy only, CHOP + GLI-ASO therapy, CHOP + BTZ therapy, CHOP + GLI-ASO + MCL-specific CTLs or CHOP + BTZ + MCL-specific CTLs. Organs were removed at the time mouse death from tumor progression or toxicity from the same experiment as in Figure 5. The tissue sections were stained for CD20, imaged digitally and analyzed using a digital scanning microscope using Neuroimformatica software.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The treatment of therapy-resistant residual tumors is a major clinical problem in MCL.3 Although relapse is a common occurrence in MCL patients,3 there is no reliable model system to study the behavior of therapy-resistant MCL cells. Therefore, we developed a murine model by transplanting human MCL cells into NOD/SCID mice followed by treatment with chemotherapeutic agents (Fig. 1). From these mice, we isolated and characterized a small subset of tumor cells that remained after chemotherapy and defined them as therapy-resistant tumor cells. Using cytomorphology and immunophenotypic analyses, we confirmed the human origin and MCL characteristics of these cells (Fig. 2). Recently, a similar approach has been used to identify genes related to cisplatin resistance in squamous cell carcinoma of the head and neck using chemoresistant sublines generated in vitro.33 As observed in refractory MCL patients, the growth of therapy-resistant MCL cells was significantly increased in comparison with parental cells in vitro (Fig. 3). This result also correlated well with a shorter survival time of mice bearing relapsing MCL cells (GR) in vivo compared to parental (GP) cells (Fig. 3). Emerging evidence in various cancers34–37 indicates that the subset of tumor cells that remain after chemotherapy has chemoresistant properties. Therefore, we also confirmed the chemoresistant properties of these GR cells (Fig. 4). In addition, the mice transplanted with GR cells survived for a significantly shorter time compared to mice bearing GP cells.

Various signaling pathways, including hedgehog signaling, are known to be active in cancer stem/progenitor cells and are involved in sustained cell growth, survival and drug resistance.20, 21, 38, 39 We have also recently reported that MCL and B-CLL cells express molecules associated with hedgehog signaling.13, 14 Furthermore, we demonstrated that GLI, transcription factors of hedgehog signaling, are overexpressed in MCL cells compared to normal B cells.13 In addition, targeting GLI decreased MCL proliferation and increased the susceptibility of MCL cells to chemotherapy. In this study, we determined the expression of hedgehog signaling molecules PTCH, SMO, SUFU, GLI1 and GLI2 in therapy-resistant relapsing MCL cells compared to GP cells. Interestingly, expression of GLI1 and GLI2 increased more than twofold in GR MCL cells compared to parental cells (Supporting Information Fig. S1). However, expressions of the other molecules of the hedgehog pathway were not altered (data not shown). These results suggest that hedgehog signaling is likely deregulated in therapy-resistant MCL cells by an unknown mechanism. This needs to be explored further. Indeed, targeting of GLI transcription factors in patients with therapy-resistant tumors may be an excellent strategy to improve the outcomes of treatment. This notion is also strengthened by the recent reports on the role of hedgehog signaling in diffuse large B-cell lymphoma40 and myeloid leukemia41 and in promoting chemoresistance. In addition, Gli1 is also reported to play a dominant role in chemoresistance of glioma cells.42 In addition, we observed increased expression of survivin (BIRC5) in therapy-resistant MCL cells that are known to be present in aggressive MCL.43, 44 Another strategy to target therapy-resistant MCL cells is to target the tumor microenvironment, a strategy that is known to improve the outcomes.39, 45 An effective agent is BTZ, a proteosome inhibitor and food and drug administration (FDA) approved drug for MCL, which affects cancer cells by various mechanisms: it stabilizes proapoptotic members of the BCL-2 family, inhibits the pathways leading to NF-κB activation, increases misfolded proteins and disrupts stromal–cancer cell interactions.23

With anticipated heterogeneity of therapy-resistant tumor cells, targeting GLI with GLI-ASO or targeting the tumor microenvironment with BTZ might improve treatment but may not completely eliminate MCL cells. Therefore, we targeted residual therapy-resistant MCL cells with another putative non-cross-reactive approach to achieve greater success. Emerging evidence indicates that antigen-specific immunotherapy is effective in combination with other therapies.24–27, 32 As specific target antigens for MCL are not known, we developed a strategy to load all surface antigens of MCL cells onto immature DCs by generating tumor-DC hybrid cells and to obtain CTLs.28, 29 In addition, we have demonstrated the specificity of CTLs toward MCL by their cytokine analysis in vitro and in vivo.29 The CTLs generated by this method demonstrated significant cytotoxicity against parental and therapy-resistant MCL cells in vitro indicating that these CTLs may also be effective against relapsing tumor cells in vivo (Fig. 5a). The CTLs exhibited higher cytotoxicity against MCL than an HLA-matched irrelevant breast cancer cell line MDA-MB-231 cells or EBV-infected MDA-MB-231 cells. However, we cannot completely exclude the contribution of allogenic T cells. Although we used PBMCs with one HLA match, there could be other mismatches.

We tested our novel therapeutic strategy for the treatment of MCL in a novel preclinical mouse model of NOD/SCID for MCL. In this combination approach, CHOP + GLI-ASO + CTLs or CHOP + BTZ + CTLs, resulted in significantly increased disease-free survival (p < 0.0001) and eliminated most of the tumor burden compared to mice treated with either CHOP, CHOP + GLI-ASO, CHOP + BTZ, CHOP + CTLs or controls without any treatment (Figs. 5 and 6). Although, there was not a clear advantage of GLI-ASO or BTZ alone with CHOP, there was a major advantage of these treatments when combined with CTLs compared to CHOP+CTLs suggesting an increased elimination of tumor cells by targeting GLI or the proteosome. Whether this is from direct cytoreduction of the MCL cells or sensitization of the cells to CTLs or other mechanisms needs to be explored. A similar approach in which targeting AKT signaling sensitizes cancer to cellular immunotherapy has been reported by Hahnel et al.32 Also, these in vivo studies used an NOD/SCID mouse model that lacks human immune cells. Tumor relapse in patients is likely to be more complex, resulting not only from cell-intrinsic and epigenetic changes but also from mechanisms of immune escape. These factors in the tumor microenvironment that may not be recapitulated in the mouse model especially interactions with immune cells, regulatory T cells, regulatory T cells, immune-suppressive cytokines and stromal interactions. Also, unlike in other groups, mice in the multiagent therapy groups did not die due to tumor burden but rather due to toxicity induced by therapy. Despite the apparent eradication of tumor, the mechanisms of this toxicity leading to the death of the mice receiving CHOP + BTZ + CTL and CHOP + GLI + CTL needs to be explored because it raises cautions for human studies. This study demonstrates proof of principle of the potential advantages of combination chemotherapy with molecular targeting, followed by cellular immunotherapy, over single therapies. However, as noted, this needs to be carefully investigated in additional models before attempting translation into the clinical setting. This approach may have even better outcomes for MCL patients if combined with autologous stem cell transplantation to increase reductions in tumor burden. Importantly, this model system also has potential for incorporation of various new compounds generated by drug discovery programs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank the flow cytometry core facility, virtual microscopy core facility at UNMC for their help in these studies. The authors also thank Kathryn Hyde for help in preparing this manuscript.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_27602_sm_SuppFig1.tif7238KSupporting Information Figure 1

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