Immune escape and tolerance in the tumor microenvironment are closely involved in tumor progression, and are caused by T‐cell exhaustion and mediated by the inhibitory signaling of immune checkpoint molecules including programmed death‐1 (PD‐1), cytotoxic T‐lymphocyte associated protein 4, and T‐cell immunoglobulin and mucin domaincontaining molecule‐3. In the present study, we investigated the expression of the PD‐1 ligand 1 (PD‐L1) in a lymphoma microenvironment using paraffin‐embedded tissue samples, and subsequently studied the detailed mechanism of upregulation of PD‐L1 on macrophages using cultured human macrophages and lymphoma cell lines. We found that macrophages in lymphoma tissues of almost all cases of adult T‐cell leukemia/lymphoma (ATLL), follicular lymphoma and diffuse large B‐cell lymphoma expressed PD‐L1. Cell culture studies showed that the conditioned medium of ATL‐T and SLVL cell lines induced increased expression of PD‐L1/2 on macrophages, and that this PD‐L1/2 overexpression was dependent on activation of signal transducer and activator of transcription 3 (Stat3). In vitro studies including cytokine array analysis showed that IL‐27 (heterodimer of p28 and EBI3) induced overexpression of PD‐L1/2 on macrophages via Stat3 activation. Because lymphoma cell lines produced IL‐27B (EBI3) but not IL‐27p28, it was proposed that the IL‐27p28 derived from macrophages and the IL‐27B (EBI3) derived from lymphoma cells formed an IL‐27 (heterodimer) that induced PD‐L1/2 overexpression. Although the significance of PD‐L1/2 expressions on macrophages in lymphoma progression has never been clarified, an IL‐27‐Stat3 axis might be a target for immunotherapy for lymphoma patients.

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Although CD138 expression is a hallmark of plasma cells and myeloma cells, reduced CD138 expression is occasionally found. However, the mechanisms underlying CD138 downregulation in myeloma cells remain unclear. Previous reports suggest that the bone marrow microenvironment may contribute to CD138 downregulation. Among various factors in the tumor microenvironment, hypoxia is associated with tumor progression, poor clinical outcomes, dedifferentiation and the formation of cancer stem cell niches in solid tumors. Since recent findings showed that progression of multiple myeloma (MM) delivers hypoxia within the bone marrow, we hypothesized that CD138 expression may be regulated by hypoxia. In the present study, we examined whether the expression of CD138 and transcription factors occurred in myeloma cells under hypoxic conditions. MM cell lines (KMS-12BM and RPMI 8226) were cultured under normoxic or hypoxic conditions for up to 30 days. Changes in the phenotype and the expression of surface antigens and transcription factors were analyzed using flow cytometry, RT-PCR and western blotting. All-trans retinoic acid (ATRA) was used to examine the phenotypic changes under hypoxic conditions. The expression levels of CD138, CS1 and plasma cell-specific transcription factors decreased under hypoxic conditions, while those of CD20, CXCR4 and B cell-specific transcription factors increased compared with those under normoxic conditions. Stem cell-specific transcription factors were upregulated under hypoxic conditions, while no difference was observed in ALDH activity. The reduced CD138 expression under hypoxic conditions recovered when cells were treated with ATRA, even under hypoxic conditions, along with decreases in the expression of stem cell-specific transcription factor. Interestingly, ATRA treatment sensitized MM cells to bortezomib under hypoxia. We propose that hypoxia induces immature and stem cell-like transcription phenotypes in myeloma cells. Taken together with our previous observation that decreased CD138 expression is correlated with disease progression, the present data suggest that a hypoxic microenvironment affects the phenotype of MM cells, which may correlate with disease progression.

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5–5 μM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 μM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10–20 μM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

BackgroundLactate levels within tumors are correlated with metastases, tumor recurrence, and radioresistance, thus apparently contributing to poor outcomes in patients with various cancers. We previously reported that high-level production of lactate by multiple myeloma (MM) cell lines is associated with high-level LDH activity within such MM cells. However, the kinetics of lactate remains to be studied. In the present study, we attempted to elucidate the mechanism of lactate incorporation into MM cells.MethodsSix MM cell lines and stromal cells obtained through long-term culture of bone marrow samples from MM patients were employed. Incorporation of lactate was quantified using C14-labeled lactate. The role of MCT1, a member of the monocarboxylate transporters (MCTs), expressed on MM cells, was examined in the presence of its inhibitor (α-cyano-4-hydroxycinnamic acid: CHC) and by using gene-silencing technique.ResultsMM cell lines as well as stromal cells were found to produce lactate. Incorporation of C14-labeled lactate into MM cells occurred in all 6 MM cell lines analyzed. Inhibition of MCT1 by using CHC or MCT1-targeting siRNA reduced lactate incorporation and caused apoptosis in MM cells. This apoptosis was enhanced when the activity of pyruvate dehydrogenase kinase was blocked by dichroloacetate. Survival of normal peripheral blood mononuclear cells was not influenced by MCT1 inhibition.ConclusionsThe present data suggest that lactate is produced by MM cell lines and stromal cells, and contributes to the survival of such MM cells in autocrine or paracrine manners. Suppression of lactate incorporation by targeting MCT1 may provide a novel therapeutic strategy for MM which may be applicable for other B-cell neoplasms.