[Research Progress on the Role of Tumor-Associated Macrophages in Multiple Myeloma --Review].

Bone marrow microenvironment is a highly complex environment surrounding tumor, which plays an important role in the survival, proliferation, drug resistance and migration of multiple myeloma (MM) cells. As an important cellular component in tumor microenvironment, tumor-associated macrophages(TAM) has attracted attention due to its key role in tumor progression and drug resistance. Targeting TAM has shown potential therapeutic value in cancer treatment. In order to clarify the role of macrophages in MM progression, it is necessary to understand the differentiation of TAM and its characteristics of promoting MM. This paper reviews the research progress on how TAM is programmed in MM and the mechanism of TAM promoting tumor development and drug resistance.

[Influence of rosiglitazone and all-trans-retinoic acid on angiogenesis and growth of myeloma xenograft in nude mice].

OBJECTIVE: To observe the effect of rosiglitazone (RGZ) and all-trans-retinoic acid (ATRA) on the growth of myeloma xenograft in nude mice and to explore the influence of RGZ and ATRA on VEGF expression and angiogenesis in the tumor. METHODS: VEGF gene expression in myeloma cell line U266 cells was analyzed by semi-quantitative RT-PCR after incubation with RGZ, ATRA, or RGZ + ATRA for 24 h. Myeloma xenograft was established by subcutaneous injection of 10(7) U266 cells in the scapula area of 4-week old nude mice. 7 days later, the nude mice were administered with RGZ, ATRA or RGZ + ATRA, respectively, by intraperitoneal injection once every day for 21 days. The control mice were given equal volume of normal saline instead of the drug. On the 21(st) day of treatment, the mice were sacrificed and the tumors were taken off, and the tumor volume and weight were measured. The tumors were examined by histopathology with HE staining, and microvessel density (MVD), CD34 and VEGF expression in the tumors were analyzed by immunohistochemical staining. RESULTS: VEGF mRNA was highly expressed in U266 cells and was decreased in a dose-dependent manner after incubation with RGZ. The VEGF mRNA level was further more decreased after RGZ + ATRA treatment. Xenografts of U266 cells were developed in all nude mice. The volume and weight of xenografts in the RGZ group were (785 ± 262) mm(3) and (1748 ± 365) mg, respectively, significantly lower than those of the control group (both P < 0.01). More significant inhibition was in the RGZ + ATRA group, (154 ± 89) mm(3) and (626 ± 102) mg, respectively, both were P < 0.05 vs. the RGZ group. RGZ inhibited the angiogenesis in U266 xenografts and immunohistochemical staining showed that the tumor MVD and VEGF expression were significantly decreased by RGZ treatment, and further more inhibited in the RGZ + ATRA group. VEGF protein was expressed in all xenografts in the nude mice. Its immunohistochemical staining intensity was 2.20 ± 0.40 in the control group, significantly higher than that of 1.48 ± 0.37 in the RGZ group (P < 0.01), and that of RGZ + ATRA group was 0.58 ± 0.26, further significantly lower than that of the RGZ group (P < 0.01). CD34 was expressed in all xenografts, most highly in the control group and lowest in the RGZ + ATRA group. The microvessel density (MVD) was highest in the control group (56.4 ± 15.2), significantly lower in the RGZ group (44.6 ± 11.2) (P < 0.05), and lowest in the RGZ + ATRA group (21.5 ± 8.6, P < 0.01). CONCLUSIONS: The growth of myeloma cells can also be inhibited by RGZ and ATRA in nude mice in vivo. In addition to differentiation and apoptosis induction, RGZ can inhibit the formation of myeloma xenograft probably also through the downregulation of VEGF expression and subsequent angiogenesis.

Multiple myeloma presenting with amyloid arthropathy as the first manifestation: Two case reports.

BACKGROUND: Multiple myeloma (MM) can be accompanied by amyloidosis, which occurs in a small number of patients and is characterized by deposition of light chains in the joints, leading to multiple myeloma-associated amyloid arthropathy (MAA). As a rare complication of MM, clinical manifestations of MAA are often similar to those of rheumatoid arthritis, and the two are easily confused. CASE SUMMARY: In recent years, our center treated two patients of MM with amyloid arthropathy as the first manifestation, both of whom presented with polyarthritis. After treatment for MM, both patients achieved complete remission. However, subsequently, the two patients underwent hip arthroplasty for femoral neck fractures. Congo red staining and immunofluorescence of the joint tissues confirmed MAA after surgery. Eventually, one of the patients died of MM recurrence, while the other survived. CONCLUSION: MAA should be regarded as an initial symptom of MM and should be taken seriously.

[Clinical Significance of CD28 Expression in Newly Diagnosed Multiple Myeloma].

OBJECTIVE: To explore the expression of CD28 in multiple myeloma and its correlation with tumor burden and clinical prognosis. METHODS: Flow cytometry was adopted to analyze bone marrow specimens of 91 newly diagnosed patients with multiple myeloma. According to CD28 expression, the patients were divided into CD28+ group and CD28- group, and the differences between the two groups in clinical features, genetic abnormalities, and treatment response were compared. Staging was carried out in accordance with the International Staging System (ISS). RESULTS: Among 91 newly diagnosed patients, there were 31 cases in CD28+ group and 60 cases in CD28- group. The proportion of ISS-Ⅲ patients in the CD28+ group was 70.97%, which was higher than 50.00% in the CD28- group (P<0.05). The median of bone marrow plasma cells in the CD28+ group was 41.78(2.00-77.00), which was higher than 26.92(2.00-92.00) in the CD28- group (P<0.05). ß2-microglobulin level in the CD28+ group was 6.53(2.11-36.50) mg/L, which was higher than 5.76(2.00-31.34) mg/L in the CD28- group (P<0.05). The positive rate of poor karyotype in the CD28+ group was 70.00% (21/30), which was higher than 45.00% (27/60) in the CD28- group (P=0.025). After 4 cycles of chemotherapy, the total effective rate of CD28- group was 86.27%, which was higher than 60.00% of CD28+ group (P<0.05). After a median follow-up of 10 months, the progression-free survival (PFS) time of CD28+ group was 10.7 months, which was lower than 14 months of CD28- group (P<0.05). Univariate analysis showed that age ≥ 65 years old, hemoglobin < 60 g/L, ISS-III, CD28+ expression and ≥ 2 genetic abnormalities were not risk factors for PFS, while further multivariate analysis showed that induction effect < partial response (PR) and CD28+ expression and were independent risk factors for PFS. CONCLUSION: CD28+ is associated with clinical characteristics and prognosis of newly diagnosed multiple myeloma patients, and can be used as a reference index to evaluate the prognosis.

Treatment and outcome patterns of patients with Waldenström's macroglobulinemia: a large, multicenter retrospective review in China.

In this study, we aimed to investigate treatment options and the prognosis of patients with WM in China. This retrospective study included 1141 patients diagnosed with symptomatic WM between January 2003 and December 2019 at 35 tertiary hospitals in 22 provinces of China. Fifty-four patients (7.3%) received monotherapy, 264 (36.0%) received chemoimmunotherapy, 395 (53.8%) received other combination regimens without rituximab, and 21 (2.9%) received ibrutinib. Using a multivariable Cox regression model, age > 65 years old, platelets <100 × 109/L, serum albumin <3.5 g/dl, ß2 microglobulin concentration ≥4 mg/L and LDH ≥250 IU/L predicted poor OS. In summary, our study showed that frontline treatment choices for WM are widely heterogeneous. We validated most of the established prognostic factors in the rIPSS (age >65 years, LDH ≥250 IU/L, ALB <3.5 g/dl and ß2 microglobulin ≥4 mg/L) together with PLT ≤ 100 × 109/L indicate a poor prognosis for patients with WM.

[The effect of TNF-alpha on TAZ expression and osteogenic potential of mesenchymal stem cells from myeloma patients].

OBJECTIVE: To explore the relationship between TNF-alpha, transcriptional co-activator with PDZ-binding motif (TAZ) and bone disease of multiple myeloma. METHODS: The biological characteristics, especially the osteogenic potential of marrow MSCs from myeloma patients and normal subjects were studied. Real-time RT-PCR and Western-blot were employed to detect mRNA and protein expression of TAZ in MSCs. The concentration of TNF-alpha in the marrow plasma was detected using ELISA method. CD138(+) myeloma cells were cocultured with normal MSCs with or without anti-human TNF-alpha monoclonal antibody in the Transwell system. Real-time RT-PCR was employed to detect the mRNA expressions of ALP, Cbfa1 and TAZ in MSCs two weeks later. von Kossa staining was used to detect the mineral deposition. TNF-alpha was added into the culture media of normal marrow MSCs and real-time RT-PCR and Western-blot were employed to detect mRNA and protein expression of TAZ in MSCs one week later. RESULTS: Real-time RT-PCR revealed that the mRNA of osteogenic markers was decreased in comparison with that of normal controls after cultured in the osteogenic medium. von Kossa staining showed weakened mineral deposition in MSCs from multiple myeloma patients compared with that in normal subjects after osteogenic differentiation for two weeks. The mRNA and protein levels of TAZ in the MSCs from myeloma patients were decreased. TNF-alpha concentration in the marrow plasma of myeloma patients was higher than that in the normal controls [(355.4 +/- 49.1) vs. (92.3 +/- 17.2) pg/ml]. CD138(+) myeloma cells inhibited mRNA expressions of ALP, Cbfal1 and TAZ in MSCs, which could be partially reversed by anti-human TNF-alpha monoclonal antibody. CONCLUSION: The osteogenic potential of MSCs from myeloma patients is significantly decreased in comparison with that in normal subjects, which may play an important role in the pathology of myeloma bone disease. TAZ expression inhibited by TNF-alpha may play an important role in this inhibition effect.

[Curcumin Increases the Chemosensitivity of Multiple Myeloma to Bortezomib by Inhibiting the Notch1 Signaling Pathway].

OBJECTIVE: To evaluated the effect of curcumin on the bortezomib-resistant myeloma cells and the expression of Notch1 signaling pathway, in order to further explore its potential mechanism. METHODS: Curcumin, bortezomib, and curcumin combined bortezomib were added into RPMI 8266, U266, 5 nmol/L bortezomib-resistant RPMI 8266 (RPMI 8226-V5R), 5 nmol/L bortezomib-resistant U 266 (U266-V5R) and CD138+ plasma cells respectively. The cell proliferation was measured by MTT assay. the apoptotic rate was determined by flow cytometry, and the Western blot was used to detect the expression of apoptosis-related proteins. Then, the expression of Notch1 in cells was inhibited by notch1 inhibitor DAPT and RNA interference, the above-motioned experiments should be repeated. RESULTS: Compared with single drug-treated groups, the treatment with 2 drugs could further inhibit cell proliferation, induce apoptosis and enhance the inhibition effect on notch1 signaling pathway (P<0.05), while the inhibiting Notch1 signaling pathway could reduce cell proliferation and increase the expression of cleaved caspase-3. CONCLUSION: Curcumin can increase chemosensitivity of myeloma cells to bortezomib, this effect may be related to the inhibition of Notch1.

Tight Junction Protein 1 Modulates Proteasome Capacity and Proteasome Inhibitor Sensitivity in Multiple Myeloma via EGFR/JAK1/STAT3 Signaling.

Proteasome inhibitors have revolutionized outcomes in multiple myeloma, but they are used empirically, and primary and secondary resistance are emerging problems. We have identified TJP1 as a determinant of plasma cell proteasome inhibitor susceptibility. TJP1 suppressed expression of the catalytically active immunoproteasome subunits LMP7 and LMP2, decreased proteasome activity, and enhanced proteasome inhibitor sensitivity in vitro and in vivo. This occurred through TJP1-mediated suppression of EGFR/JAK1/STAT3 signaling, which modulated LMP7 and LMP2 levels. In the clinic, high TJP1 expression in patient myeloma cells was associated with a significantly higher likelihood of responding to bortezomib and a longer response duration, supporting the use of TJP1 as a biomarker to identify patients most likely to benefit from proteasome inhibitors.

[Effects of RNA interference targeting hypoxia-inducible factor-1alpha (HIF-1alpha) on chemosensitivity of leukemia K562 cells towards homoharringtonine].

BACKGROUND & OBJECTIVE: Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcription factor under anoxic circumstances. Little is known about changes in biological characters of hematological malignancies, especially leukemia. This study was to explore the influence of RNA interference (RNAi) targeting HIF-1alpha on sensitivity of human chronic myelogeneous leukemia (CML) K562 cells towards homoharringtonine (HHT). METHODS: HIF-1alpha short hairpin RNA (shRNA) was constructed using pSilencer 2.1-U6 hygro vector and transfected into K562 cells. Positive clones were screened using hygromycin. After inhibition of HIF-1alpha, expressions of its target genes such as vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), phosphoglycerate kinase (PGK), and P-glycoprotein (P-gp) were detected by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Sensitivity of K562 cells to HHT was detected by MTT assay. RESULTS: HIF-1alpha expression was inhibited at both mRNA and protein levels after transfection of RNAi HIF-1alpha, which subsequently caused a dramatic decrease in VEGF, Glut-1, PGK, and P-gp under hypoxic conditions. In addition, HIF-1alpha inhibition was found to increase drug sensitivity of K562 cells to HHT. CONCLUSION: HIF-1alpha inhibition may result in a decrease of genes related to angiogenesis and glycolysis metabolism and an increase of drug sensitivity to HHT in K562 cells.

[Inhibition of hypoxia-inducible factor-1 alpha in RPMI8226 myeloma cells results in reduced tumor growth in nude mice].

OBJECTIVE: To explore the influence of inhibition of hypoxia-inducible factor-1 alpha (HIF-1 alpha) by RNA interference (RNAi) on tumorigenesis of human myeloma cell line (HMCL) RPMI8226 cells in nude mice. METHOD: RNAi vector of HIF-1 alpha was constructed with commercial shRNA expression vector pSilencer 2. 1-U6 hygro. RT-PCR and western blot were used to detect HIF-1 alpha mRNA and protein expression respectively. Vascular endothelial growth factor (VEGF) secretion and cell cycle changes were detected by ELISA and flow cytometry respectively. Expression of target gene of HIF-1 alpha, VEGF and Glut-1 were tested under hypoxia condition. Tumorigenesis was observed after transfected cells were injected subcutaneously in nude mice. RESULTS: After interference, expression of HIF-1 alpha decreased significantly at both mRNA and protein level. Under normoxia condition, VEGF concentrations in HIF-la inhibited cells (RPMI8226-il and RPMI8226-i2) and non-inhibited cells (RPMI8226-c and RPMI8226) showed no differences. While under hypoxia condition, VEGF concentration in the above four cells was (506.0 +/- 53.2), (494.7 +/- 63.1), (984.4 +/- 61.9) and (938.2 +/- 62.2) pg/ml, respectively, being significantly lower in RPMI8226-il and RPMI8226-i2 cells than in RPMI8226-c and RPMI8226 cells (P <0.05). HIF-1 alpha interference was found to suppress the cells shift from S-phase to G1 induced by hypoxia. VEGF and Glut-1 expressions were markedly attenuated (P <0.05). The growth rate of HIF-1 alpha inhibition tumors in subcutaneous xenograft model decreased drastically. CONCLUSIONS: RNAi inhibits HIF-1 alpha expression. Reduced tumor growth by HIF-1 alpha inhibition may partly through inhibiton of angiogenesis and glycolysis metabolism.

[Effect of total body irradiation on cellular senescence related indexes of bone marrow mesenchymal stem cells].

The aim of this study was to explore the changes in cellular senescence related indexes of bone marrow mesenchymal stem cells (BMMSCs) after total body irradiation (TBI). At different time points after 4 Gy irradiation, BMMSCs were isolated from male C57BL/6 mice and cultured. Morphology, senescence-associated beta-galactosidase (SA-beta-gal) staining and cell cycle analysis were used to evaluate the changes in BMMSCs at cellular level while real-time RT-PCR was used to detect the alterations in senescence related gene expression including p16INK4a, p21Cip1/Waf1, p53 and TGF-beta1. The results showed that within 4 weeks after exposure to 4 Gy TBI, the morphology of BMMSCs and the expression level of SA-beta-gal were not significantly changed, the cellular senescence-related cell cycle arrest was not occurred and the senescence related gene expression level was not increased. It is concluded that at the early stage after 4 Gy TBI, the related molecular level of cellular senescence in BMMSCs is not changed.

[Reduction of bone marrow mesenchymal stem/progenitor cells pool and alteration of their osteogenesis potential caused by total body irradiation].

To investigate the effect of irradiation on the quantity and osteogenesis potential of BMMSCs and to explore the response of them in the irradiation stress and its contribution to long-term effects of radiation-induced bone and hematologic injury, a total body irradiation (TBI) murine model was adopted. The number of CFU-F and cell cycle profile of BMMSCs were analyzed at different time points before and after TBI. Osteogenic differentiation was evaluated by Von Kossa staining, expressions of osteogenesis-related genes and transcriptional coactivator with PDZ-binding motif (TAZ) were detected by real-time RT-PCR. The results showed that the number of CFU-F decreased greatly at day 28 after TBI. At day 3 after TBI, more cells entered cell cycle and the osteogenesis potential was greatly enhanced followed by recovery of cell cycle distribution and significant defect in osteoblast differentiation respectively, meanwhile the expression of TAZ was changed. It is concluded that TBI results in the reduction of bone marrow mesenchymal stem/progenitor cell pool and alters the osteogenesis potential of BMMSCs, which is related to the change of TAZ expression.