The TENT5C/FAM46C locus on chromosome 1p12 is deleted or mutated in up to 20% of multiple myeloma (MM) patients, revealing a plasma cell (PC)-specific tumor suppressive activity.1-4 Unlike hitherto known oncosuppressors, TENT5C is not directly involved in DNA repair, cell cycle regulation or apoptosis. In fact, TENT5C is a non-canonical poly(A)polymerase that selectively polyadenylates and stabilizes mRNA encoding immunoglobulins (Ig) and other endoplasmic reticulum (ER)-targeted proteins, exerting a fundamental role in the physiology of PC as intensive antibody producing factories.5-9 We and others showed that TENT5C specificity for the secretory apparatus requires its association with ER transmembrane FNDC3 proteins, affording TENT5C localization where Ig are translated.9,10 In this way, TENT5C increases their synthesis, together with that of proteins involved in folding and trafficking along the secretory route, thereby operating as a potent booster of humoral immune response.9 Accordingly, we found that TENT5C is remarkably induced during PC differentiation (Online Supplementary Figure S1A), and that its high expression neatly discriminates MM lines from other tumors (Figure 1A). Further strengthening its suppressive role in MM pathobiology, analysis of publicly available genome-scale, pooled CRISPR-Cas9 loss-of viability screens (DepMap portal 23Q2 release) showed that TENT5C ablation results in an extremely significant, MM-specific proliferative advantage (Figure 1B). In line with a selective benefit for MM cells in reducing TENT5C expression, bone marrow-purified PC from MM patients display lower TENT5C expression than those obtained from healthy donors (Online Supplementary Figure S1B), with the subset of TENT5C-deleted MM patients showing a further decrease in TENT5C mRNA levels when compared with non-deleted MM (Online Supplementary Figure S1C). We propose that such advantage consists in restricting Ig production and the associated oxidative, metabolic, and degradative workload to save energy for proliferation. Indeed, we showed that TENT5C re-expression in mutated MM lines pushes secretory activity beyond sustainability, inducing ATP shortage, ROS accumulation, and a slower growth rate in vitro.9 However, whether TENT5C regulates the balance between MM growth and antibody secretion in vivo remains uninvestigated.
To assess TENT5C impact in vivo, we silenced or re-expressed TENT5C in ALMC-2 cells, a MM line expressing the wild-type protein and bearing a hemizygous loss in chromosome 1p,11 and then injected engineered cells into immunodeficient Rag2–/– IL2rg–/– mice. (Animal procedures were approved by the Italian Ministry of Health and the Institutional Animal Care and Use Committee [IACUC] under protocols 1057 and 1314). Expression of FLAG-tagged TENT5C in ALMC-2 cells significantly increased the abundance of ER mRNA and proteins, and decreased cell proliferation in vitro (Figure 1C-F, Online Supplementary Figure S1D), inducing cell cycle arrest in G0/G1 phase (Online Supplementary Figure S1E). Consistently, TENT5C re-expression had the same effects on proliferation and ER proteins in 3 other TENT5C-deleted MM lines (Figure 1C, Online Supplementary Figure S1F).
Conversely, TENT5C-silenced ALMC-2 cells displayed only slight reductions in ER-associated mRNA and proteins (Figure 1D-F, Online Supplementary Figure S1G) and no differences in cell growth, viability, and cell cycle distribution (Figure 1C, Online Supplementary Figure S1D, E). In general, we found smaller effects of TENT5C silencing in ALMC-2 cells if compared to previously investigated wildtype MM lines,9 consistent with a pre-existing significant suppression of TENT5C activity in ALMC-2 cells. Importantly, TENT5C was able to promote Ig production and secretion in a dose-dependent manner, with the lowest levels in silenced ALMC-2 cells and the highest in overexpressing counterparts (Figure 1G). Together, these data support a mechanistic link between TENT5C-mediated tumor suppression and its effects on the secretory activity, whereby TENT5C acts as a natural tuner of the secretory capacity of MM cells regulating the trade-off between Ig production and PC proliferation in vitro.
To challenge this role in vivo, we first injected control, TENT5C-silenced and overexpressing cells intravenously into Rag2–/– IL2rg–/– mice. Despite higher initial circulating Ig light chain (LC) levels (Online Supplementary Figure S2A), we found reduced tumor appearance and longer survival in recipients of TENT5C-overexpressing cells (Figure 2A). Indeed, half of the mice in the overexpressing group were still alive and without any sign of disease up to 330 days after injection, while 13 out of 16 mice in the other experimental groups (mock and TENT5C-silenced) had to be sacrificed within 150 days due to the appearance of bulky abdominal plasmacytomas (Figure 2A). Moreover, in line with negative effect of high TENT5C levels on MM proliferation, mRNA and protein analyses on excised plasmacytomas showed significant selection of low TENT5C-expressing cells in the overexpressing group (Figure 2B, Online Supplementary Figure S2B). Of note, the only tumor maintaining TENT5C overexpression (lane 11 in Figure 2B) displayed the highest levels of Ig and ER proteins, confirming in vivo a dose-dependent effect of TENT5C on the secretory compartment. On the contrary, no selection was detected in TENT5C-silenced tumors that maintained advantageous lower ER and Ig mRNA and protein patterns when compared with control plasmacytomas (Figure 2B, Online Supplementary Figure S2B). To timely evaluate the role of TENT5C in modulating the ratio between monoclonal protein and PC proliferation, we then injected TENT5C-overexpressing mice and control ALMC-2 subcutaneously to precisely follow tumor size in a shorter timeframe. We confirmed remarkably reduced tumor growth and longer survival in recipients of TENT5C-over-expressing cells, despite significantly higher circulating LC six days after injection (Figure 2C, D, Online Supplementary Figure S2C). At sacrifice, ex vivo qRT-PCR and immunoblot analyses showed that TENT5C-overexpressing tumors still displayed higher LC and ER mRNA and protein concentrations than control plasmacytomas (Figure 2E, Online Supplementary Figure S2D). Notably, TENT5C abundance significantly modified the ratio between tumor volume and the levels of monoclonal LC concurrently detected in sera (Figure 2F). This in vivo association of higher proliferation with decreased secretory activity clearly reveals that loss of TENT5C uncouples monoclonal protein levels from MM burden.
Figure 1.TENT5C is a myeloma-specific oncosuppressor that regulates endoplasmic reticulum protein expression and Ig production in a dose-dependent manner. (A) Dot-plot showing TENT5C expression in multiple myeloma (MM) versus other lymphoid versus other cancer cell lines derived from public data available in the DepMap portal (23Q2 release). The full list of 18 myeloma cell lines and of 1,001 non-MM cell lines are available at https://depmap.org/portal/download/all/; LP1 cells, known to harbor a homozygous deletion of TENT5C, are highlighted in red (median; ****P<0.0001; one-way ANOVA with Dunnett’s multiple comparison). (B) (Left) Volcano plot showing the dependencies enriched in MM cell lines versus non-MM cell lines, extracted from DepMap portal 23Q2 release. T statistic and P value for the Chronos dependency scores of the top significant genes (P<0.0005) are shown. A positive T statistic indicates a proliferative advantage of TENT5C CRISPR-KO specifically in MM cells. All genetic dependency data, statistical analyses and methods are publicly available in the DepMap portal (23Q2 release). (Right) Dot-plot showing the Chronos dependency score for TENT5C in MM versus other lymphoid versus other cancer cell lines (median; ****P<0.0001; one-way ANOVA with Dunnett’s multiple comparison). (C) Equal numbers of MM cells were seeded at 5x105 cells/ mL and counted with trypan blue staining after 2 (for ALMC-2, LP1 and MM.1S) or 3 days (for OPM2), (mean ± Standard Error of Mean [SEM]; N=3-6; *P<0.05; **P<0.01; unpaired t test vs. Mock). ALMC-2 cells were established at relapse as symptomatic MM of a patient initially diagnosed with AL amyloidosis and treated with oral dexamethasone and blood stem cell transplant. ALMC-2 cells bear a t(14;20) translocation, Myc amplification, and p53 deletion. LP1 cells bear a biallelic deletion of TENT5C while MM.1S and OPM2 cells a monoallelic deletion and a point mutation on the other allele. All the cell lines were genotyped and TENT5C gene was analyzed by Sanger sequencing to confirm wild-type TENT5C expression in ALMC-2 cells and M270V and E178A mutations in MM.1S and OPM2, respectively. Lentiviral viruses to stably express anti-TENT5C or a control shRNA were purchased by Mission shRNA (Sigma-Aldrich, SHC002 and TRCN0000166958). Human C-term FLAG-TENT5C cDNA was purchased by Genscript (OHu30151D) and was cloned in a plasmid with a hybrid bidirectional human PGK-miniCMV promoter co-expressing the protein of interest and truncated human CD271. (D) qRT-PCR analysis of mRNA of selected endoplasmic reticulum (ER)-resident proteins and Ig λ light chain (LC) in TENT5C silenced or overexpressing ALMC-2 cells (mean ± SEM normalized on H3 mRNA; N=4 for silenced, 5 for overexpressed; *P<0.05; Kruskal-Wallis one-way test with Dunn’s multiple comparison vs. Mock). Total RNA was extracted by lysis in TriFAST (Euroclone, EMR507100). 1000 ng of RNA were retro-transcribed with ImProm-II Reverse Transcriptase System (Promega, A3800). qPCR were performed using iTaq SYBR Green Supermix (Bio-Rad, 1725122) on Bio-Rad CFX96 PCR and analyzed on Bio-Rad CFX Maestro software. Primers are listed in Online Supplementary Table S1. (E) Representative immunoblots of selected ER-resident proteins and Ig λ LC in TENT5C silenced or overexpressing ALMC-2 cells. Immunoblots were performed as described by Fucci et al.9 Images were obtained using Uvitec Imager Mini HD9 (Uvitec Ltd.) for HRP-conjugated secondary Ab or FLA9000 (FujiFilm) for Alexa-Fluor conjugated secondary antibodies. Antibodies are listed in Online Supplementary Table S1. Band quantifications, performed using ImageJ software (http://rsbweb.nih.gov/ij/), are reported in Online Supplementary Figure S1G. (F) Immunoblot analyses of intracellular and secreted IgG λ light (LC) and heavy (HC) chains in TENT5C control, silenced or overexpressing ALMC-2 cells. (Top) Representative blots. (Bottom) Quantifications of intracellular and secreted IgG λ chains (N=3 normalized on actin; *P<0.05; **P<0.01; ****P<0.0001; one-way ANOVA with Dunnett’s multiple comparison vs. Mock). AU: arbitrary units.
Notably, the impact of TENT5C on secretory products is not restricted to Ig but also promotes the cell surface expression of calreticulin, an ER stress-induced “eat-me” signal, and CD38, an established target of monoclonal antibodies (Figure 3A, B, Online Supplementary Figure S3E), with relevant immunotherapeutic implications. Notably, attesting to biological relevance of TENT5C-mediated increase in surface calreticulin, TENT5C-overexpressing ALMC-2 cells underwent increased phagocytosis by primary macrophages in vitro (Figure 3C). Moreover, in line with an immunogenic role, qRT-PCR revealed higher expression of monocytic/macrophagic markers (i.e., CD68 and CD206) in TENT5C-overexpressing tumors, indicating larger myeloid infiltrates (Figure 3D), as further confirmed by CD68 immunoblot and immunohistochemistry analyses (Figure 3E, F). In conclusion, our data disclose a trade-off between the sustainable amount of antibodies that PC can produce and their proliferation. TENT5C acts as an “unselfish gene” whose distinctive expression, though inhibitory for PC proliferation, optimizes humoral immunity in favor of the entire organism. Conversely, MM tends to manipulate this equilibrium, blunting TENT5C expression to decrease proteosynthetic activity and favor its own growth. In keeping with this model, loss-of-function mutations have been identified in MM in key transcription factors regulating Ig production and TENT5C transcription, namely, XBP-1 and PRDM1.12 These observations indicate that MM may undergo selective pressure to lower, but not completely abolish Ig secretion, suggesting that maintaining Ig production may be beneficial through mechanisms that remain, however, elusive. Moreover, our demonstration that TENT5C modulation uncouples Ig secretion from tumor size may be extremely relevant in MM patients, where, in the presence of TENT5C inactivating mutations, monoclonal protein quantification may underestimate disease burden.
Figure 2.TENT5C regulates the trade-off between antibody secretion and multiple myeloma proliferation in vivo. (A) Kaplan-Meier survival curve of male and female 4-6-month-old BALB/c Rag2–/–Yc–/– mice intravenously injected in the tail with 10x106 control, TENT5C-silenced, overexpressing or control ALMC-2 cells. General health status of the mice was monitored every 3 days and mice were sacrificed at the appearance of lower limb paralysis or abdominal plasmacytomas (N=8 per group; Log-rank Mantel-Cox test). (B) Representative immunoblots of selected ER-resident proteins and IgG X in excised plasmacytomas formed after intravenous injection. (Left) Immunoblot images. (Right) Band quantifications (mean ± Standard Error of Mean [SEM] normalized on actin; N=5 Mock, 5 shTENT5C, 3 TENT5C; ordinary one-way ANOVA with Dunnett’s multiple comparison *P<0.05; **P<0.01; unpaired t test). (C) BALB/c Rag2–/–Yc–/– mice were subcutaneously implanted with 3x106 ALMC-2 multiple myeloma (MM) cells in the lateral abdominal region. Tumor growth was monitored every 3 days by caliper assessment and mice sacrificed when tumors reached the volume of 2,500 mm3. Growth of subcutaneous tumors represented individually (left) or as an average (right) in mm3 ± SEM (N=11 mock, 14 TENT5C; ****P<0.0001; two-way ANOVA with Bonferroni’s multiple comparisons). (D) Kaplan-Meier survival curve of mice subcutaneously injected with 3x106 control or TENT5C-overexpressing ALMC-2 cells (N=11 Mock, 14 TENT5C; ***P<0.01; Log-rank Mantel-Cox test). (E) Representative immunoblots of selected ER-resident proteins and IgG X in excised subcutaneous tumors. (Left) Immunoblot images. (Right) Band quantifications (mean ± SEM normalized on actin; N=7 Mock, 6 TENT5C; *P<0.05; unpaired t test). (F) Ratio between the tumor volume measured by caliper and Ig X LC levels measured by immunoblot in 1 [il of serum collected at the same timepoint (mean ± SEM; N=9 Mock, 10 TENT5C, *P<0.05; unpaired t test). AU: arbitrary units; LC: light chain; HC: heavy chain.
In line with our observation that TENT5C loss significantly increases proliferation, inactivating mutations are among the main drivers of evolution from smoldering to active MM.13 Moreover, deletions of locus 1p, that often include TENT5C, are an adverse prognostic factor in MM patients and predict worse responses even to the most active available regimens.2,14
Finally, our unprecedented evidence that TENT5C promotes the surface expression of calreticulin and CD38, increasing macrophagic infiltrates, suggests that TENT5C mutations may also contribute to reduce MM immunogenicity. In line with this possibility, Maura et al.15 recently found a significant correlation between low TENT5C expression and resistance to targeted immunotherapy with the anti-CD38 antibody daratumumab. However, since TENT5C may also promote the expression of other membrane molecules and cytokines, further investigations in immunocompetent preclinical models will be required to define the net impact of TENT5C loss on MM immunogenicity and sensitivity to immunotherapy.
Figure 3.TENT5C promotes the expression of calreticulin and CD38 on the cell surface. (A) Cytofluorimetric analysis of CALR and CD38 surface expression in control, TENT5C-overexpressing and silenced ALMC-2 cells (average median fluorescent intensity ± Standard Error of Mean [SEM]; N=18 for CALR and 9 for CD38; **P<0.01; ***P<0.001; Kruskal-Wallis one-way test with Dunn’s multiple comparison vs. Mock). See Online Supplementary Figure S2E for representative cytofluorimetric histograms. Data were obtained with Accuri C6 Flow Cytometer (BD Biosciences) and analyzed using the FCS Express 7 Flow Research Edition (De Novo Software). (B) Cytofluorimetric analysis of CALR and CD38 surface expression in control and TENT5C-overexpressing multiple myeloma (MM) cells (average median fluorescent intensity ± SEM; N=3; *P<0.05; **P<0.01; paired t test). (C) Cytofluorimetric analysis of in vitro phagocytosis of control, TENT5C-silenced or overexpressing ALMC-2 cells by primary macrophages. Macrophages were obtained by MCSF-induced differentiation of human monocyte purified from the peripheral blood of healthy donors with Pan Monocyte Isolation kit (Miltenyi, 130-096-537). After differentiation, macrophages were stained with 2 µM Cell-Trace™ Far Red Cell Proliferation kit (Thermo Scientific, C34564). In parallel, TENT5C manipulated ALMC-2 cells were stained with 2 µM CellTrace™ CFSE Cell Proliferation kit (Thermo Scientific, C34544). Stained macrophages and MM cells were mixed 1:1 and incubated at 37°C for 4 hours. The percentage of double positive macrophages was then assessed by BD FACSCanto II (mean ± SEM; N=6; *P<0.05; repeated measures one-way ANOVA with Dunnett’s multiple comparisons vs. Mock). (D) qRT-PCR analysis of murine markers in excised subcutaneous tumors (mean ± SEM normalized on GAPDH mRNA; N=7 Mock, 6 TENT5C; *P<0.05; unpaired t test). (E) Immunoblots of murine CD68 in excised subcutaneous tumors. (Top) Immunoblot images. (Bottom) Band quantifications (mean ± SEM normalized on actin; N=7 Mock, 6 TENT5C; *P<0.05; unpaired t test). (F) Immunohistochemistry of murine CD68 in excised subcutaneous tumors. (Left) Representative images: scale bar 200 µm. (Right) Quantifications of CD68 area (%) and counts. Quantification of CD68+ cells was performed with ImageJ software by applying a color threshold on the DAB signal to select positive pixels and analyzing particles (>15 pixels) for area. Area of CD68+ cells was calculated on 4 images/sample (20x magnification), (mean ± SEM; N=7 Mock, 6 TENT5C; *P<0.05; **P<0.01; unpaired t test). AU: arbitrary units.
To summarize, by disclosing the regulation in vivo of Ig secretion, MM growth, and the expression of PC surface markers by TENT5C our work advances the understanding of the myeloma-specific role of one of the most frequently mutated oncosuppressors in MM.
Footnotes
- Received September 18, 2023
- Accepted February 13, 2024
Correspondence
Disclosures
MN received honoraria from Janssen and research funding from Oncopeptides, Gate Bioscience and Pfizer, and is an inventor on a patent on immunoglobulin sequencing. MDV received funding from GSK and Jannsen (lectures and advisory boards) and Takeda and Amgen (advisory boards). NB received funding from GSK, Janssen, Amgen (honoraria for lectures) and Pfizer (advisory board). The other authors have no conflicts of interest to disclose.
Contributions
MN, SC and EM designed the research. MR, MM, UO, JB, RM, MDV, FL and NB contributed with crucial methodologies and resources. MR, SP, LV, TP and EM performed the experiments and analyzed the data. TP, SC and EM wrote the paper. MN, SC and EM provided the research funds. SC and EM supervised the research.
Funding
Acknowledgments
We are particularly grateful to Roberta Colzani for administrative assistance, Paolo Ghia, Diane F. Jelinek, Luca Rampoldi, Emilie Vénéreau, and Ineke Braakman for mice, cells, primers and antibodies, and all the members of the Cenci and Milan labs for fruitful discussions. We thank Amleto Fiocchi and the Animal Histopathology Service at IRCCS San Raffaele Scientific Institute for help with immunohistochemistry analyses. ALMC-2 cells were kindly provided by Diane F. Jelinek, Mayo Clinic, and LP1, MM.1S and OPM2 by Dr. Giovanni Tonon, San Raffaele Scientific Institute.
References
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Data Supplements
Figures & Tables
Figure 1.TENT5C is a myeloma-specific oncosuppressor that regulates endoplasmic reticulum protein expression and Ig production in a dose-dependent manner. (A) Dot-plot showing TENT5C expression in multiple myeloma (MM) versus other lymphoid versus other cancer cell lines derived from public data available in the DepMap portal (23Q2 release). The full list of 18 myeloma cell lines and of 1,001 non-MM cell lines are available at https://depmap.org/portal/download/all/; LP1 cells, known to harbor a homozygous deletion of TENT5C, are highlighted in red (median; ****P<0.0001; one-way ANOVA with Dunnett’s multiple comparison). (B) (Left) Volcano plot showing the dependencies enriched in MM cell lines versus non-MM cell lines, extracted from DepMap portal 23Q2 release. T statistic and P value for the Chronos dependency scores of the top significant genes (P<0.0005) are shown. A positive T statistic indicates a proliferative advantage of TENT5C CRISPR-KO specifically in MM cells. All genetic dependency data, statistical analyses and methods are publicly available in the DepMap portal (23Q2 release). (Right) Dot-plot showing the Chronos dependency score for TENT5C in MM versus other lymphoid versus other cancer cell lines (median; ****P<0.0001; one-way ANOVA with Dunnett’s multiple comparison). (C) Equal numbers of MM cells were seeded at 5x105 cells/ mL and counted with trypan blue staining after 2 (for ALMC-2, LP1 and MM.1S) or 3 days (for OPM2), (mean ± Standard Error of Mean [SEM]; N=3-6; *P<0.05; **P<0.01; unpaired t test vs. Mock). ALMC-2 cells were established at relapse as symptomatic MM of a patient initially diagnosed with AL amyloidosis and treated with oral dexamethasone and blood stem cell transplant. ALMC-2 cells bear a t(14;20) translocation, Myc amplification, and p53 deletion. LP1 cells bear a biallelic deletion of TENT5C while MM.1S and OPM2 cells a monoallelic deletion and a point mutation on the other allele. All the cell lines were genotyped and TENT5C gene was analyzed by Sanger sequencing to confirm wild-type TENT5C expression in ALMC-2 cells and M270V and E178A mutations in MM.1S and OPM2, respectively. Lentiviral viruses to stably express anti-TENT5C or a control shRNA were purchased by Mission shRNA (Sigma-Aldrich, SHC002 and TRCN0000166958). Human C-term FLAG-TENT5C cDNA was purchased by Genscript (OHu30151D) and was cloned in a plasmid with a hybrid bidirectional human PGK-miniCMV promoter co-expressing the protein of interest and truncated human CD271. (D) qRT-PCR analysis of mRNA of selected endoplasmic reticulum (ER)-resident proteins and Ig λ light chain (LC) in TENT5C silenced or overexpressing ALMC-2 cells (mean ± SEM normalized on H3 mRNA; N=4 for silenced, 5 for overexpressed; *P<0.05; Kruskal-Wallis one-way test with Dunn’s multiple comparison vs. Mock). Total RNA was extracted by lysis in TriFAST (Euroclone, EMR507100). 1000 ng of RNA were retro-transcribed with ImProm-II Reverse Transcriptase System (Promega, A3800). qPCR were performed using iTaq SYBR Green Supermix (Bio-Rad, 1725122) on Bio-Rad CFX96 PCR and analyzed on Bio-Rad CFX Maestro software. Primers are listed in Online Supplementary Table S1. (E) Representative immunoblots of selected ER-resident proteins and Ig λ LC in TENT5C silenced or overexpressing ALMC-2 cells. Immunoblots were performed as described by Fucci et al.9 Images were obtained using Uvitec Imager Mini HD9 (Uvitec Ltd.) for HRP-conjugated secondary Ab or FLA9000 (FujiFilm) for Alexa-Fluor conjugated secondary antibodies. Antibodies are listed in Online Supplementary Table S1. Band quantifications, performed using ImageJ software (http://rsbweb.nih.gov/ij/), are reported in Online Supplementary Figure S1G. (F) Immunoblot analyses of intracellular and secreted IgG λ light (LC) and heavy (HC) chains in TENT5C control, silenced or overexpressing ALMC-2 cells. (Top) Representative blots. (Bottom) Quantifications of intracellular and secreted IgG λ chains (N=3 normalized on actin; *P<0.05; **P<0.01; ****P<0.0001; one-way ANOVA with Dunnett’s multiple comparison vs. Mock). AU: arbitrary units.
Figure 2.TENT5C regulates the trade-off between antibody secretion and multiple myeloma proliferation in vivo. (A) Kaplan-Meier survival curve of male and female 4-6-month-old BALB/c Rag2–/–Yc–/– mice intravenously injected in the tail with 10x106 control, TENT5C-silenced, overexpressing or control ALMC-2 cells. General health status of the mice was monitored every 3 days and mice were sacrificed at the appearance of lower limb paralysis or abdominal plasmacytomas (N=8 per group; Log-rank Mantel-Cox test). (B) Representative immunoblots of selected ER-resident proteins and IgG X in excised plasmacytomas formed after intravenous injection. (Left) Immunoblot images. (Right) Band quantifications (mean ± Standard Error of Mean [SEM] normalized on actin; N=5 Mock, 5 shTENT5C, 3 TENT5C; ordinary one-way ANOVA with Dunnett’s multiple comparison *P<0.05; **P<0.01; unpaired t test). (C) BALB/c Rag2–/–Yc–/– mice were subcutaneously implanted with 3x106 ALMC-2 multiple myeloma (MM) cells in the lateral abdominal region. Tumor growth was monitored every 3 days by caliper assessment and mice sacrificed when tumors reached the volume of 2,500 mm3. Growth of subcutaneous tumors represented individually (left) or as an average (right) in mm3 ± SEM (N=11 mock, 14 TENT5C; ****P<0.0001; two-way ANOVA with Bonferroni’s multiple comparisons). (D) Kaplan-Meier survival curve of mice subcutaneously injected with 3x106 control or TENT5C-overexpressing ALMC-2 cells (N=11 Mock, 14 TENT5C; ***P<0.01; Log-rank Mantel-Cox test). (E) Representative immunoblots of selected ER-resident proteins and IgG X in excised subcutaneous tumors. (Left) Immunoblot images. (Right) Band quantifications (mean ± SEM normalized on actin; N=7 Mock, 6 TENT5C; *P<0.05; unpaired t test). (F) Ratio between the tumor volume measured by caliper and Ig X LC levels measured by immunoblot in 1 [il of serum collected at the same timepoint (mean ± SEM; N=9 Mock, 10 TENT5C, *P<0.05; unpaired t test). AU: arbitrary units; LC: light chain; HC: heavy chain.
Figure 3.TENT5C promotes the expression of calreticulin and CD38 on the cell surface. (A) Cytofluorimetric analysis of CALR and CD38 surface expression in control, TENT5C-overexpressing and silenced ALMC-2 cells (average median fluorescent intensity ± Standard Error of Mean [SEM]; N=18 for CALR and 9 for CD38; **P<0.01; ***P<0.001; Kruskal-Wallis one-way test with Dunn’s multiple comparison vs. Mock). See Online Supplementary Figure S2E for representative cytofluorimetric histograms. Data were obtained with Accuri C6 Flow Cytometer (BD Biosciences) and analyzed using the FCS Express 7 Flow Research Edition (De Novo Software). (B) Cytofluorimetric analysis of CALR and CD38 surface expression in control and TENT5C-overexpressing multiple myeloma (MM) cells (average median fluorescent intensity ± SEM; N=3; *P<0.05; **P<0.01; paired t test). (C) Cytofluorimetric analysis of in vitro phagocytosis of control, TENT5C-silenced or overexpressing ALMC-2 cells by primary macrophages. Macrophages were obtained by MCSF-induced differentiation of human monocyte purified from the peripheral blood of healthy donors with Pan Monocyte Isolation kit (Miltenyi, 130-096-537). After differentiation, macrophages were stained with 2 µM Cell-Trace™ Far Red Cell Proliferation kit (Thermo Scientific, C34564). In parallel, TENT5C manipulated ALMC-2 cells were stained with 2 µM CellTrace™ CFSE Cell Proliferation kit (Thermo Scientific, C34544). Stained macrophages and MM cells were mixed 1:1 and incubated at 37°C for 4 hours. The percentage of double positive macrophages was then assessed by BD FACSCanto II (mean ± SEM; N=6; *P<0.05; repeated measures one-way ANOVA with Dunnett’s multiple comparisons vs. Mock). (D) qRT-PCR analysis of murine markers in excised subcutaneous tumors (mean ± SEM normalized on GAPDH mRNA; N=7 Mock, 6 TENT5C; *P<0.05; unpaired t test). (E) Immunoblots of murine CD68 in excised subcutaneous tumors. (Top) Immunoblot images. (Bottom) Band quantifications (mean ± SEM normalized on actin; N=7 Mock, 6 TENT5C; *P<0.05; unpaired t test). (F) Immunohistochemistry of murine CD68 in excised subcutaneous tumors. (Left) Representative images: scale bar 200 µm. (Right) Quantifications of CD68 area (%) and counts. Quantification of CD68+ cells was performed with ImageJ software by applying a color threshold on the DAB signal to select positive pixels and analyzing particles (>15 pixels) for area. Area of CD68+ cells was calculated on 4 images/sample (20x magnification), (mean ± SEM; N=7 Mock, 6 TENT5C; *P<0.05; **P<0.01; unpaired t test). AU: arbitrary units.
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