Research ArticleExperimental Studies
Open Access

Hypoxia-adapted Multiple Myeloma Stem Cells Resist γδ-T-Cell-mediated Killing by Modulating the Mevalonate Pathway

YUSUKE SANO, NAOKO KUWABARA, SAORI NAKAGAWA, YUKI TODA, SHIGEKUNI HOSOGI, SHINJI SATO and EISHI ASHIHARA
Anticancer Research February 2023, 43 (2) 547-555; DOI: https://doi.org/10.21873/anticanres.16191

Abstract

Background/Aim: The prognosis of patients with multiple myeloma (MM) has recently improved due to the emergence of new molecular targeting agents. However, MM remains incurable because MM stem cells are resistant to these agents. Therefore, it is essential to develop strategies to eradicate MM stem cells. We have previously demonstrated that MM cells cultured under prolonged hypoxic conditions (1% O2) (i.e., hypoxia-adapted MM cells; MM-HA cells) exhibited stem-cell-like characteristics. γδ T cells attack tumor cells by recognizing butyrophilin (BTN) 3A1 and BTN2A1, which are activated by the intracellular accumulation of isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway. In the present study, we investigated the cytotoxicity of γδ T cells against MM-HA stem-like cells. Materials and Methods: We used a combination of flow cytometry, liquid chromatography-tandem mass spectrometry, and western blotting methods to investigate the cytotoxicity of γδ T cells against MM-HA cells and measured the amounts of IPP in MM-HA cells and their supernatants. Results: The cytotoxicity of γδ T cells against MM-HA cells was significantly lower than that against MM cells cultured under normoxic conditions (20% O2; MM-Normo). Furthermore, the concentration of IPP in MM-HA cells was lower than that in MM-Normo cells. The expression of mevalonate decarboxylase and farnesyl diphosphate synthase proteins were decreased in MM-HA-cells. Conclusion: The cytotoxicity of γδ T cells against MM-HA cells was suppressed by the reduced IPP accumulation by modulating the mevalonate pathway in MM-HA cells.

Key Words:

Multiple myeloma (MM) is a cancer of plasma cells. MM cells proliferate in the bone marrow (BM), suppressing hematopoiesis and causing immune dysfunction. MM cells produce and secrete the M protein, which induces kidney damage. The recent application of new molecular targeting agents against MM has improved the prognosis of this disease (1-4). However, despite the development of novel therapies, MM remains incurable. One of the reasons for the difficulty in curing MM is that MM stem cells are resistant to existing therapeutic agents (5). Therefore, it is essential to develop strategies aimed at eradicating these cells. MM cells reside in the BM (<10% O2) (6) and preferentially accumulate in the BM niche (<1.3% O2), which is characterized by hypoxia (7). We have previously demonstrated that MM cells, which reside in the hypoxic BM niche (MM-HA cells), are adapted to prolonged hypoxic conditions (1% O2) and exhibit stem-cell-like characteristics (7).

T cells are classified into two categories: αβ T and γδ T cells. αβ T cells are the dominant T cell population in the peripheral blood and recognize cancer cells in a major histocompatibility complex (MHC)-restricted manner. By contrast, γδ T are much less abundant in the blood and perform their antitumor cytotoxic functions independently of MHCs (8, 9). It was recently revealed that γδ T cells attach to cancer cells by recognizing butyrophilin (BTN) 3A1 and BTN2A1 (10). Moreover, the intracellular accumulation of isopentenyl pyrophosphate (IPP), which is an intermediate in the mevalonate pathway and acts as a phosphoantigen for γδ T cells, induces conformational changes in BTN3A1. The conformationally transformed BTN3A1 gains the ability to bind the γδ T cell receptor (TCRγδ), ultimately inducing γδ T cell activation (11, 12). Nitrogen-containing aminobisphosphonates, such as zoledronic acid (ZOL), inhibit farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, leading to the accumulation of IPP in target cells (13, 14). Consequently, ZOL treatment increases the cytotoxicity of γδ T cells. In this study, we investigated the γδ-T-cell-mediated cytotoxicity against stem-like MM-HA cells to test the potential mechanisms of MM resistance to γδ-T-cell-mediated killing, looking towards the development of future therapeutic strategies against MM.

Materials and Methods

Cell lines and reagents. Human MM (RPMI8226, EJM and U266) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were maintained in a humidified incubator at 37°C, with 20% O2, 5% CO2, and 75% N2 (MM-Normo). The MM cells were then cultured in 1.0% O2, 5% CO2, and 94% N2 for over 1 month, to establish the hypoxia-adapted RPMI8226 (RPMI8226-HA), EJM (EJM-HA), and U266 (U266-HA) cell lines. RPMI8226-Normo and RPMI8226-HA were cultured in RPMI1640 (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 20% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (PS, Wako Pure Chemical Industries). EJM-Normo and EJM-HA were cultured in Iscove’s modified Dulbecco’s medium (Sigma-Aldrich), containing 4 mM L-glutamine (Nacalai Tesque, Inc., Kyoto, Japan), 10% FBS and 1% PS. U266-Normo and U266-HA were cultured in RPMI1640, supplemented with 10% FBS and 1% PS. All cell lines were tested negative for mycoplasma contamination. ZOL was purchased from Novartis Pharma AG (Basel, Switzerland) and recombinant human (rh) interleukin (IL)-2 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Carboxyfluorescein diacetate succinimidyl ester (CFSE) (#341-06443) was purchased from Dojindo Corporation (Kumamoto, Japan). IPP, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP) and ammonium bicarbonate (NH4HCO3) were purchased from Sigma-Aldrich. Acetonitrile, methanol, 2-propanol and 28% ammonia solution were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Ex vivo expansion of human γδ T cells. Informed consent for peripheral blood collection was obtained from a healthy donor, in accordance with the Declaration of Helsinki and with approval from the Kyoto Pharmaceutical University Review Board (E21-006-02, 20-30). Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque (GE Healthcare, Little Chalfont, Bucks, UK) density gradient centrifugation. The γδ T cells were expanded from cultured PBMCs using ZOL and rhIL-2, as described previously (15). We ensured that the proportion of γδ T cells within the expanded PBMC preparations exceeded 90% prior to use in experiments.

In vitro cytotoxicity assay. The cytotoxic function of γδ T cells was evaluated by mixing γδ T cells [with or without ZOL (1 μM)] and CFSE-labeled MM cells (1.0×105 cells/well) in a 10:1 effector (E) to target (T) ratio, under normoxic or hypoxic conditions, as previously described (16). MM cell killing (%) was calculated using the following formula: cytotoxicity (%)=[CFSE+ propidium iodide (PI)+ cells/CFSE+ cells] ×100.

Flow cytometry. For γδ T cell staining during flow cytometry, the antibodies used were: anti-CD3-fluorescein (FITC; #555332, BD Biosciences, Franklin Lakes, NJ, USA), anti- TCRαβ-phycoerythrin (PE; #564728, BD Biosciences), anti- TCRγδ-allophycocyanin (APC; #555718, BD Biosciences), antilymphocyte function-associated antigen (LFA)-1-FITC (#555379, BD Biosciences), antinatural killer group 2 member D (NKG2D)-FITC (#11-5878, eBioscience, San Diego, CA, USA), anti-DNAX accessory molecule-1 (DNAM-1)-PE (#338305, Biolegend, San Diego, CA, USA), anti-CD107a-PE (#555801, BD Biosciences), antiperforin-Alexa Fluor 488 (#563764, BD Biosciences), antigranzyme B-PE (#561142, BD Biosciences), and anti-interferon (IFN)-γ-PE (#506506, Biolegend). For MM cell staining during flow cytometry, the antibodies used were: anti-intercellular adhesion molecule (ICAM)-1-PE (#560971, BD Biosciences), anti-MHC class I chain related gene (MIC)A/B-PE (#12-5788, eBioscience), and antipoliovirus receptor (PVR)-PE (#337609, Biolegend). To induce the expression of the cytotoxicity marker CD107a (17), γδ T cells were stimulated with the Cell Stimulation Cocktail (eBioscience) for 4 h. For intracellular staining, γδ T cells were stimulated with the Cell Stimulation Cocktail (eBioscience) for 4 h in the presence of Golgistop Protein Transport Inhibitor (BD Biosciences), and then fixed and permeabilized with 4% paraformaldehyde and 0.2% Tween 20. Staining for intracellular markers was performed at 4°C for 20 min in the dark. All samples were acquired on a FACSCalibur (BD Biosciences) or an LSR Fortessa X-20 (BD Biosciences) flow cytometer and analyzed using FlowJo software (BD Biosciences).

Measurement of IPP, GPP, FPP and GGPP in culture supernatants and cells. The concentrations of IPP, GPP, FPP and GGPP in culture supernatants and cells were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, as described previously (8, 18), with slight modifications. In brief, culture supernatants or cell samples were mixed with 2-propanol and 0.1 M NH4HCO3 (1:1 v/v), and acetonitrile, and then vortexed. After centrifugation, cell supernatants or pellets were dried under a stream of nitrogen. The residues were dissolved in 40 μl of 0.01 M NH4OH (for supernatant samples) or 80 μl of methanol and 0.01 M NH4OH (7:3 v/v) (for cell samples), and then analyzed by LC-MS/MS. The LC-MS/MS conditions were described previously (8). In this study, the LC-MS/MS method was not able to separate and identify the isoforms of IPP and dimethylallyl pyrophosphate (DMAPP) as had been previously reported (8, 18). Therefore, the IPP concentration was expressed as the amount of IPP out of the total amount of IPP and DMAPP (IPP + DMAPP).

Western blotting. Preparation of MM cells lysates was performed as described previously (15). Samples were blotted with primary antibodies against the following proteins: mevalonate decarboxylase (MVD; #sc-376975, Santa Cruz Biotechnology, Dallas, TX, USA), FDPS/FPS (#ab189874, Abcam, Cambridge, UK), and β-actin (#A5441, Sigma-Aldrich). Horseradish peroxidase-coupled anti-rabbit and anti-mouse immunoglobulin (Ig)G monoclonal antibodies were used as secondary antibodies. Bands corresponding to immunoreactive proteins were detected by ImageQuant™ LAS 4000 with the ECL Prime Western Blotting Detection Kit (Cytiva, Tokyo, Japan). To normalize for loading differences, β-actin was used as an internal control.

Statistical analysis. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were analyzed using unpaired t-tests. Multiple comparisons between groups were analyzed using two-way ANOVA followed by Bonferroni’s test. p-Values <0.05 were considered as a measure of statistical significance.

Results

Ex vivo expansion of human γδ T cells was achieved using ZOL and rhIL-2. γδ T cells are present at very low frequencies in human peripheral blood. However, they can be expanded by treating PBMCs with ZOL and rhIL-2. In this ex vivo culture system, ZOL activates γδ T cells by inducing the accumulation of IPP within PBMC-derived monocytes (19). We found that over the 11-day expansion period, the frequency of γδ T cells increased from 0.9% (on day 0) to 91.3% (on day 11) (Figure 1A), and the number of γδ T cells increased approximately 2400-fold (Figure 1B). These results indicate that γδ T cells can be selectively expanded using this protocol.

Figure 1.
Figure 1.
Figure 1.

Ex vivo expansion of γδ T cells with ZOL (5 μM) and rhIL-2 (100 IU/ml). (A) Flow cytometry analysis of the percentage of γδ T cells on day 0 (upper panels) and day 11 (lower panels), expanded from healthy-donor-derived peripheral blood mononuclear cells. γδ T cells were defined as the CD3+/TCRγδ+ population. (B) The number of expanded γδ T cells after 11 days of in vitro culture. The number of γδ T cells was calculated by multiplying the overall cell count by the percentage of CD3+/TCRγδ+ cells. Data are representative of three independent experiments.

The cytotoxicity of γδ T cells against MM cells diminished under hypoxic conditions. To examine the ability of γδ T cells to kill MM cells grown under normoxic or hypoxic conditions, we established MM-HA stem-like cell lines (using the three MM cells lines: RPMI8226-HA, EJM-HA and U266-HA) by culturing MM-Normo cells (RPMI8226-Normo, EJM-Normo and U266-Normo) under hypoxic conditions for over 1 month, in accordance with our previous protocols (7). The γδ T cells were then cultured for 4 h with CFSE-labeled MM-Normo cells or MM-HA cells. We found that the cytotoxicity of γδ T cells against RPMI8226-HA cells and EJM-HA cells was lower than their cytotoxicity against the same cells grown under normoxic conditions. However, the γδ T cells exerted similar levels of cytotoxicity against the U266-HA cells and U266-Normo cells (Figure 2).

Figure 2.
Figure 2.
Figure 2.

Cytotoxicity of γδ T cell against multiple myeloma cell lines cultured under normoxic conditions (MM-Normo) and prolonged hypoxic conditions (MM-HA). (A) MM cells were stained with 0.5 μM CFSE and either treated with ZOL (1 μM) overnight or left untreated. The MM cells were then co-cultured with γδ T cells for 4 h in normoxic or hypoxic conditions. Results of the flow cytometric analysis of γδ T cell cytotoxicity against RPMI8226-Normo cells are shown. (B) Cytotoxicity (on the y-axis) was calculated by subtracting the cytotoxicity value of the control condition (i.e., without γδ T cells). Comparison of the γδ-T-cell-mediated killing of MM-Normo and MM-HA cells. Data represent the mean±standard error of the mean (SEM) of three independent experiments. The statistical significance of differences was determined by two-way ANOVA with the Bonferroni post-hoc test (**p<0.01, MM-Normo vs. MM-HA).

The expression levels of adhesion molecules and cytokines remained stable under hypoxic conditions. γδ T cells exert a cytotoxic response against MM cells by recognizing specific adhesion molecules (20-22). Therefore, we examined the expression levels of adhesion molecules on MM cells and γδ T cells to investigate barriers to the killing of MM-HA cells. We found that the expression levels of adhesion molecules (i.e., MIC A/B, ICAM-1, PVR in MM cells, NKG2D, LFA-1, and DNAM-1 in γδ T cells) were similar between the expression levels under normoxic and hypoxic conditions (Figure 3A). These results indicate that the decrease in γδ-T-cells-mediated cytotoxicity against MM-HA cells was not related to the expression of adhesion molecules on MM cells. Next, we examined perforin, granzyme B, IFN-γ, and CD107a expression by γδ T cells, and found that the expression of these markers was also similar between normoxic and hypoxic conditions (Figure 3B). Taken together, these observations suggest that hypoxia did not alter adhesion molecule expression on MM and γδ T cells or γδ T cell activation.

Figure 3.
Figure 3.

Expression levels of adhesion molecules and cytokines. (A) Flow cytometry analysis of cell surface molecule expression by MM-Normo and MM-HA cells (MIC A/B, ICAM-1 and PVR; top panel) and by γδ T cells (NKG2D, LFA-1, DNAM-1 and CD107a; bottom panel). (B) Flow cytometry analysis of intracellular perforin, granzyme B, and IFN-γ expression by γδ T cells. γδ T cells were stimulated with Cell Stimulation Cocktail (500×). White histograms represent normoxic conditions, black histograms represent hypoxic conditions. Dotted lines indicate negative controls, and solid lines indicate stained samples. Data are representative of three independent experiments.

γδ-T-cell-mediated cytotoxicity against MM-HA cells was reduced by modification of the mevalonate pathway. We next considered whether the MM-HA cells may employ other mechanisms to reduce the cytotoxicity of γδ T cells in the hypoxic environment. Hwang et al. demonstrated that the mevalonate pathway is suppressed under hypoxic conditions (23). We thus hypothesized that the mevalonate pathway could be suppressed in MM-HA cells, leading to a decrease in intracellular IPP levels in these cells. To validate our hypothesis, we examined IPP levels in MM cells grown under normoxic or hypoxic conditions, either with or without ZOL. When cultured with ZOL (0.5 or 1 μM), the IPP concentrations in the RPMI8226 cells and their supernatants were significantly lower under hypoxic conditions than under normoxic ones. However, there was no significant difference between the intracellular IPP levels of ZOL-untreated RPMI8226-Normo and RPMI8226-HA cells. Similarly, the IPP levels of ZOL-treated EJM-HA cells and their supernatants were significantly lower than those of the EJM-Normo cells and supernatants. However, there was no significant difference between the IPP concentrations of U266 cells (and their supernatants) grown under hypoxic or normoxic conditions, irrespective of ZOL treatment (Figure 4A, B).

Figure 4.
Figure 4.

IPP content of multiple myeloma (MM) cells and their culture supernatants. ZOL was applied to 5×106 MM cells at the indicated concentrations (0.5 or 1 μM) in 5 ml of medium. The MM-Normo and MM-HA cells were then cultured for 24 h. Concentrations of IPP in MM cells (A) and their culture supernatants (B) were measured using LC-MS/MS. White histograms represent MM-Normo cells, and black histograms represent MM-HA cells. Statistical significance of differences was determined by two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, MM-Normo vs. MM-HA).

We next assessed the expression of MVD (an IPP synthase) and FDPS (an IPP metabolic enzyme) proteins in MM cells. We found that the expression of both proteins was lower in MM-HA cells than in MM-Normo cells (Figure 5B). Collectively, these results suggest that MM-HA cells resist γδ-T-cells-mediated attack by altering their mevalonate pathway.

Figure 5.
Figure 5.
Figure 5.

Expression levels of mevalonate-pathway-related proteins in multiple myeloma (MM) cells. (A) An outline of the mevalonate pathway and the implicated enzymes. (B) Western blot analysis of MVD and FDPS expression in MM cells with or without ZOL treatment. β-actin was used as a loading control. Normo, MM cells cultured under normoxic conditions; HA, MM cells cultured under prolonged hypoxic conditions.

Discussion

Many studies, including our own, have shown that γδ T cells exert cytotoxicity against MM cells, and that MM is a suitable target for γδ T cell immunotherapy (22, 24-28). Therefore, γδ T cell immunotherapy has the potential to improve the outcomes of patients with MM. In spite of the emergence of the novel MM-targeting therapeutic agents, MM remains incurable because MM stem cells are resistant to these existing agents. In the present study, we investigated the cytotoxicity of γδ T cells against MM-HA cells, which possess stem-cell characteristics. ZOL treatment leads to the accumulation of IPP inside MM cells. Therefore, we saw that the killing of RPMI8226-Normo and EJM-Normo cells by γδ T cells was enhanced by ZOL. However, despite ZOL treatment, the intracellular IPP levels of both the RPMI-HA and EJM-HA cell lines were significantly lower than the IPP levels of their normoxic counterparts, resulting in their resistance to γδ-T-cell-mediated killing (Figure 2A and Figure 4A, B). Meanwhile, the intracellular accumulation of IPP in U266 cells was considerably lower than that in the other two MM cell lines, irrespective of ZOL treatment. Consequently, the cytotoxicity of γδ T cells against U266-Normo and U266-HA cells was also low. These observations indicate that the cytotoxicity of γδ T cells is dependent on IPP production by their target MM cells. Moreover, the hypoxic adaptation of MM cells decreased these intracellular IPP levels and reduced γδ T cell cytotoxicity.

We have previously demonstrated that MM cells secrete IPP into their environment, which may induce the chemotaxis of γδ T cells toward ZOL-treated target MM cells (29). The IPP levels in the culture supernatants of RPMI8226-HA and EJM-HA cells were significantly lower than in the supernatants of their normoxic equivalents. We therefore speculate that γδ T cell migration might also decrease under hypoxic conditions.

To clarify the mechanism of IPP production in MM-HA cells, we evaluated the expression of MVD and FDPS proteins in MM cells. The expression of these mevalonate pathway enzymes was lower in MM-HA cells than in MM-Normo cells. This implies that under hypoxic conditions, MM cells became resistant to γδ-T-cell-mediated cytotoxicity by reducing the expression of MVD and FDPS and thus lowering their IPP production.

We have previously demonstrated that the cytotoxicity of γδ T cells decreases in the presence of prostate cancer (PC) stem cells generated by prostasphere methods (30). The data from the present study of MM stem cells support these previous findings; however, unlike in the present study, the mechanism responsible for the decrease in γδ-T-cell-mediated cytotoxicity against PC stem cells has not been clarified.

Although ZOL treatment increased IPP production in MM-HA cells (Figure 4A, B), the increase in γδ-T-cell-mediated cytotoxicity against MM-HA cells after ZOL treatment was minimal (Figure 2B). This indicates that ZOL treatment alone is not sufficient to eradicate MM stem cells, and that it is necessary to discover additional strategies to enhance γδ T cell cytotoxicity. The programmed cell death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway induces T cell immunosuppression. In this study, we found that the expression of PD-L1 on RPMI8226-HA cells was lower than that of RPMI8226-Normo cells (data not shown). Smith et al. demonstrated that PD-L1 expression in bladder cancer was decreased under hypoxic conditions (31). These observations are in agreement with our data. In our study, the reduced expression of PD-1 on RPMI8226-HA did not promote more potent antitumor killing by γδ T cells. Moreover, we have previously reported that γδ T cells demonstrate antitumor effects independently of PD-L1 expression (16). Based on the above results, we speculate that blockade of the PD-1/PD-L1 axis might not enhance γδ-T-cell-mediated cytotoxicity against MM stem-like cells.

Conclusion

In conclusion, the cytotoxicity of ex vivo expanded γδ T cells against MM-HA cells was attenuated by the suppression of IPP production via the mevalonate pathway. To improve the cytotoxicity of γδ T cells against MM-HA cells, we need further investigation for the recognition of γδ T cells against CSCs. We hope that this work will contribute to the development of strategies aimed at the eradication of MM stem cells.

Acknowledgements

We thank Dr. Mako Tomogane (Kyoto Pharmaceutical University) for helpful discussion of our work. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (grant number 19K08826, to E. Ashihara), and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2015-2019 (grant number S1511024L, to E. Ashihara).

Footnotes

  • Authors’ Contributions

    Y.S., N.K., and S.N. performed experiments and analyzed data; S.S., Y.T., S.H., and E.A. provided intellectual guidance; Y.S., S.N., and E.A. drafted the manuscript; and Y.S. and E.A. conceptualized the study.

  • Conflicts of Interest

    The Authors have no conflicts of interest to disclose with regards to this study.

  • Received November 13, 2022.
  • Revision received December 2, 2022.
  • Accepted December 5, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Björkstrand B and
    2. Gahrton G
    : High-dose treatment with autologous stem cell transplantation in multiple myeloma: past, present, and future. Semin Hematol 44(4): 227-233, 2007. PMID: 17961721. DOI: 10.1053/j.seminhematol.2007.08.010
    OpenUrlCrossRefPubMed
    1. Richardson PG,
    2. Mitsiades C,
    3. Schlossman R,
    4. Munshi N and
    5. Anderson K
    : New drugs for myeloma. Oncologist 12(6): 664-689, 2007. PMID: 17602058. DOI: 10.1634/theoncologist.12-6-664
    OpenUrlAbstract/FREE Full Text
    1. Vij R,
    2. Wang M,
    3. Kaufman JL,
    4. Lonial S,
    5. Jakubowiak AJ,
    6. Stewart AK,
    7. Kukreti V,
    8. Jagannath S,
    9. McDonagh KT,
    10. Alsina M,
    11. Bahlis NJ,
    12. Reu FJ,
    13. Gabrail NY,
    14. Belch A,
    15. Matous JV,
    16. Lee P,
    17. Rosen P,
    18. Sebag M,
    19. Vesole DH,
    20. Kunkel LA,
    21. Wear SM,
    22. Wong AF,
    23. Orlowski RZ and
    24. Siegel DS
    : An open-label, single-arm, phase 2 (PX-171-004) study of single-agent carfilzomib in bortezomib-naive patients with relapsed and/or refractory multiple myeloma. Blood 119(24): 5661-5670, 2012. PMID: 22555973. DOI: 10.1182/blood-2012-03-414359
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Lacy MQ,
    2. Hayman SR,
    3. Gertz MA,
    4. Dispenzieri A,
    5. Buadi F,
    6. Kumar S,
    7. Greipp PR,
    8. Lust JA,
    9. Russell SJ,
    10. Dingli D,
    11. Kyle RA,
    12. Fonseca R,
    13. Bergsagel PL,
    14. Roy V,
    15. Mikhael JR,
    16. Stewart AK,
    17. Laumann K,
    18. Allred JB,
    19. Mandrekar SJ and
    20. Rajkumar SV
    : Pomalidomide (CC4047) plus low-dose dexamethasone as therapy for relapsed multiple myeloma. J Clin Oncol 27(30): 5008-5014, 2009. PMID: 19720894. DOI: 10.1200/JCO.2009.23.6802
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Magee JA,
    2. Piskounova E and
    3. Morrison SJ
    : Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21(3): 283-296, 2012. PMID: 22439924. DOI: 10.1016/j.ccr.2012.03.003
    OpenUrlCrossRefPubMed
  6. ↵
    1. Hu J,
    2. Van Valckenborgh E,
    3. Menu E,
    4. De Bruyne E and
    5. Vanderkerken K
    : Understanding the hypoxic niche of multiple myeloma: therapeutic implications and contributions of mouse models. Dis Model Mech 5(6): 763-771, 2012. PMID: 23115205. DOI: 10.1242/dmm.008961
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Nakagawa Y,
    2. Ashihara E,
    3. Yao H,
    4. Yokota A,
    5. Toda Y,
    6. Miura Y,
    7. Nakata S,
    8. Hirai H and
    9. Maekawa T
    : Multiple myeloma cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-β/Smad pathway activation. Biochem Biophys Res Commun 496(2): 490-496, 2018. PMID: 29309790. DOI: 10.1016/j.bbrc.2018.01.034
    OpenUrlCrossRefPubMed
  8. ↵
    1. Ashihara E,
    2. Munaka T,
    3. Kimura S,
    4. Nakagawa S,
    5. Nakagawa Y,
    6. Kanai M,
    7. Hirai H,
    8. Abe H,
    9. Miida T,
    10. Yamato S,
    11. Shoji S and
    12. Maekawa T
    : Isopentenyl pyrophosphate secreted from Zoledronate-stimulated myeloma cells, activates the chemotaxis of γδT cells. Biochem Biophys Res Commun 463(4): 650-655, 2015. PMID: 26047704. DOI: 10.1016/j.bbrc.2015.05.118
    OpenUrlCrossRefPubMed
  9. ↵
    1. Tanaka Y,
    2. Morita CT,
    3. Tanaka Y,
    4. Nieves E,
    5. Brenner MB and
    6. Bloom BR
    : Natural and synthetic non-peptide antigens recognized by human gamma delta T cells. Nature 375(6527): 155-158, 1995. PMID: 7753173. DOI: 10.1038/375155a0
    OpenUrlCrossRefPubMed
  10. ↵
    1. Rigau M,
    2. Ostrouska S,
    3. Fulford TS,
    4. Johnson DN,
    5. Woods K,
    6. Ruan Z,
    7. McWilliam HEG,
    8. Hudson C,
    9. Tutuka C,
    10. Wheatley AK,
    11. Kent SJ,
    12. Villadangos JA,
    13. Pal B,
    14. Kurts C,
    15. Simmonds J,
    16. Pelzing M,
    17. Nash AD,
    18. Hammet A,
    19. Verhagen AM,
    20. Vairo G,
    21. Maraskovsky E,
    22. Panousis C,
    23. Gherardin NA,
    24. Cebon J,
    25. Godfrey DI,
    26. Behren A and
    27. Uldrich AP
    : Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells. Science 367(6478): eaay5516, 2020. PMID: 31919129. DOI: 10.1126/science.aay5516
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Sebestyen Z,
    2. Scheper W,
    3. Vyborova A,
    4. Gu S,
    5. Rychnavska Z,
    6. Schiffler M,
    7. Cleven A,
    8. Chéneau C,
    9. van Noorden M,
    10. Peigné CM,
    11. Olive D,
    12. Lebbink RJ,
    13. Oostvogels R,
    14. Mutis T,
    15. Schuurhuis GJ,
    16. Adams EJ,
    17. Scotet E and
    18. Kuball J
    : RhoB mediates phosphoantigen recognition by Vγ9Vδ2 T cell receptor. Cell Rep 15(9): 1973-1985, 2016. PMID: 27210746. DOI: 10.1016/j.celrep.2016.04.081
    OpenUrlCrossRefPubMed
  12. ↵
    1. Yang Y,
    2. Li L,
    3. Yuan L,
    4. Zhou X,
    5. Duan J,
    6. Xiao H,
    7. Cai N,
    8. Han S,
    9. Ma X,
    10. Liu W,
    11. Chen CC,
    12. Wang L,
    13. Li X,
    14. Chen J,
    15. Kang N,
    16. Chen J,
    17. Shen Z,
    18. Malwal SR,
    19. Liu W,
    20. Shi Y,
    21. Oldfield E,
    22. Guo RT and
    23. Zhang Y
    : A structural change in butyrophilin upon phosphoantigen binding underlies phosphoantigen-mediated Vγ9Vδ2 T cell activation. Immunity 50(4): 1043-1053.e5, 2019. PMID: 30902636. DOI: 10.1016/j.immuni.2019.02.016
    OpenUrlCrossRefPubMed
  13. ↵
    1. Gober HJ,
    2. Kistowska M,
    3. Angman L,
    4. Jenö P,
    5. Mori L and
    6. De Libero G
    : Human T cell receptor gammadelta cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med 197(2): 163-168, 2003. PMID: 12538656. DOI: 10.1084/jem.20021500
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Morita CT,
    2. Jin C,
    3. Sarikonda G and
    4. Wang H
    : Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol Rev 215: 59-76, 2007. PMID: 17291279. DOI: 10.1111/j.1600-065X.2006.00479.x
    OpenUrlCrossRefPubMed
  15. ↵
    1. Tomogane M,
    2. Omura M,
    3. Sano Y,
    4. Shimizu D,
    5. Toda Y,
    6. Hosogi S,
    7. Kimura S and
    8. Ashihara E
    : Expression level of BTN3A1 on the surface of CD14(+) monocytes is a potential predictor of γδ T cell expansion efficiency. Biochem Biophys Res Commun 588: 47-54, 2022. PMID: 34952469. DOI: 10.1016/j.bbrc.2021.12.060
    OpenUrlCrossRefPubMed
  16. ↵
    1. Tomogane M,
    2. Sano Y,
    3. Shimizu D,
    4. Shimizu T,
    5. Miyashita M,
    6. Toda Y,
    7. Hosogi S,
    8. Tanaka Y,
    9. Kimura S and
    10. Ashihara E
    : Human Vγ9Vδ2 T cells exert anti-tumor activity independently of PD-L1 expression in tumor cells. Biochem Biophys Res Commun 573: 132-139, 2021. PMID: 34407491. DOI: 10.1016/j.bbrc.2021.08.005
    OpenUrlCrossRefPubMed
  17. ↵
    1. Aktas E,
    2. Kucuksezer UC,
    3. Bilgic S,
    4. Erten G and
    5. Deniz G
    : Relationship between CD107a expression and cytotoxic activity. Cell Immunol 254(2): 149-154, 2009. PMID: 18835598. DOI: 10.1016/j.cellimm.2008.08.007
    OpenUrlCrossRefPubMed
  18. ↵
    1. Henneman L,
    2. van Cruchten AG,
    3. Denis SW,
    4. Amolins MW,
    5. Placzek AT,
    6. Gibbs RA,
    7. Kulik W and
    8. Waterham HR
    : Detection of nonsterol isoprenoids by HPLC-MS/MS. Anal Biochem 383(1): 18-24, 2008. PMID: 18782552. DOI: 10.1016/j.ab.2008.08.023
    OpenUrlCrossRefPubMed
  19. ↵
    1. Roelofs AJ,
    2. Jauhiainen M,
    3. Mönkkönen H,
    4. Rogers MJ,
    5. Mönkkönen J and
    6. Thompson K
    : Peripheral blood monocytes are responsible for gammadelta T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP. Br J Haematol 144(2): 245-250, 2009. PMID: 19016713. DOI: 10.1111/j.1365-2141.2008.07435.x
    OpenUrlCrossRefPubMed
  20. ↵
    1. Liu Z,
    2. Guo B and
    3. Lopez RD
    : Expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 is critical in determining sensitivity of pancreatic cancer cells to cytolysis by human gammadelta-T cells: implications in the design of gammadelta-T-cell-based immunotherapies for pancreatic cancer. J Gastroenterol Hepatol 24(5): 900-911, 2009. PMID: 19175829. DOI: 10.1111/j.1440-1746.2008.05668.x
    OpenUrlCrossRefPubMed
    1. Märten A,
    2. von Lilienfeld-Toal M,
    3. Büchler MW and
    4. Schmidt J
    : Soluble MIC is elevated in the serum of patients with pancreatic carcinoma diminishing gammadelta T cell cytotoxicity. Int J Cancer 119(10): 2359-2365, 2006. PMID: 16929491. DOI: 10.1002/ijc.22186
    OpenUrlCrossRefPubMed
  22. ↵
    1. Niu C,
    2. Jin H,
    3. Li M,
    4. Zhu S,
    5. Zhou L,
    6. Jin F,
    7. Zhou Y,
    8. Xu D,
    9. Xu J,
    10. Zhao L,
    11. Hao S,
    12. Li W and
    13. Cui J
    : Low-dose bortezomib increases the expression of NKG2D and DNAM-1 ligands and enhances induced NK and γδ T cell-mediated lysis in multiple myeloma. Oncotarget 8(4): 5954-5964, 2017. PMID: 27992381. DOI: 10.18632/oncotarget.13979
    OpenUrlCrossRefPubMed
  23. ↵
    1. Hwang S,
    2. Nguyen AD,
    3. Jo Y,
    4. Engelking LJ,
    5. Brugarolas J and
    6. DeBose-Boyd RA
    : Hypoxia-inducible factor 1α activates insulin-induced gene 2 (Insig-2) transcription for degradation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase in the liver. J Biol Chem 292(22): 9382-9393, 2017. PMID: 28416613. DOI: 10.1074/jbc.M117.788562
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Uchida R,
    2. Ashihara E,
    3. Sato K,
    4. Kimura S,
    5. Kuroda J,
    6. Takeuchi M,
    7. Kawata E,
    8. Taniguchi K,
    9. Okamoto M,
    10. Shimura K,
    11. Kiyono Y,
    12. Shimazaki C,
    13. Taniwaki M and
    14. Maekawa T
    : Gamma delta T cells kill myeloma cells by sensing mevalonate metabolites and ICAM-1 molecules on cell surface. Biochem Biophys Res Commun 354(2): 613-618, 2007. PMID: 17250803. DOI: 10.1016/j.bbrc.2007.01.031
    OpenUrlCrossRefPubMed
    1. von Lilienfeld-Toal M,
    2. Nattermann J,
    3. Feldmann G,
    4. Sievers E,
    5. Frank S,
    6. Strehl J and
    7. Schmidt-Wolf IG
    : Activated gammadelta T cells express the natural cytotoxicity receptor natural killer p 44 and show cytotoxic activity against myeloma cells. Clin Exp Immunol 144(3): 528-533, 2006. PMID: 16734623. DOI: 10.1111/j.1365-2249.2006.03078.x
    OpenUrlCrossRefPubMed
    1. Saitoh A,
    2. Narita M,
    3. Watanabe N,
    4. Tochiki N,
    5. Satoh N,
    6. Takizawa J,
    7. Furukawa T,
    8. Toba K,
    9. Aizawa Y,
    10. Shinada S and
    11. Takahashi M
    : Anti-tumor cytotoxicity of gammadelta T cells expanded from peripheral blood cells of patients with myeloma and lymphoma. Med Oncol 25(2): 137-147, 2008. PMID: 18488155. DOI: 10.1007/s12032-007-9004-4
    OpenUrlCrossRefPubMed
    1. Kunzmann V,
    2. Bauer E,
    3. Feurle J,
    4. Weissinger F,
    5. Tony HP and
    6. Wilhelm M
    : Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 96(2): 384-392, 2000. PMID: 10887096.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Mariani S,
    2. Muraro M,
    3. Pantaleoni F,
    4. Fiore F,
    5. Nuschak B,
    6. Peola S,
    7. Foglietta M,
    8. Palumbo A,
    9. Coscia M,
    10. Castella B,
    11. Bruno B,
    12. Bertieri R,
    13. Boano L,
    14. Boccadoro M and
    15. Massaia M
    : Effector gammadelta T cells and tumor cells as immune targets of zoledronic acid in multiple myeloma. Leukemia 19(4): 664-670, 2005. PMID: 15744346. DOI: 10.1038/sj.leu.2403693
    OpenUrlCrossRefPubMed
  29. ↵
    1. Ashihara E,
    2. Munaka T,
    3. Kimura S,
    4. Nakagawa S,
    5. Nakagawa Y,
    6. Kanai M,
    7. Hirai H,
    8. Abe H,
    9. Miida T,
    10. Yamato S,
    11. Shoji S and
    12. Maekawa T
    : Isopentenyl pyrophosphate secreted from Zoledronate-stimulated myeloma cells, activates the chemotaxis of γδT cells. Biochem Biophys Res Commun 463(4): 650-655, 2015. PMID: 26047704. DOI: 10.1016/j.bbrc.2015.05.118
    OpenUrlCrossRefPubMed
  30. ↵
    1. Miyashita M,
    2. Tomogane M,
    3. Nakamura Y,
    4. Shimizu T,
    5. Fujihara A,
    6. Ukimura O and
    7. Ashihara E
    : Sphere-derived prostate cancer stem cells are resistant to γδ T cell cytotoxicity. Anticancer Res 40(10): 5481-5487, 2020. PMID: 32988870. DOI: 10.21873/anticanres.14559
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Smith V,
    2. Mukherjee D,
    3. Lunj S,
    4. Choudhury A,
    5. Hoskin P,
    6. West C and
    7. Illidge T
    : The effect of hypoxia on PD-L1 expression in bladder cancer. BMC Cancer 21(1): 1271, 2021. PMID: 34819027. DOI: 10.1186/s12885-021-09009-7
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Hypoxia-adapted Multiple Myeloma Stem Cells Resist γδ-T-Cell-mediated Killing by Modulating the Mevalonate Pathway
YUSUKE SANO, NAOKO KUWABARA, SAORI NAKAGAWA, YUKI TODA, SHIGEKUNI HOSOGI, SHINJI SATO, EISHI ASHIHARA
Anticancer Research Feb 2023, 43 (2) 547-555; DOI: 10.21873/anticanres.16191
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords