New Advances in the Study of CMTM6, a Focus on Its Novel Non-Canonical Cellular Locations, and Functions beyond Its Role as a PD-L1 Stabilizer
<p>Total CMTM6 and total PD-L1 in plasma from the healthy donors (HDs) group and the cervical cancer patients (CC) group. (<b>a</b>) Concentrations of CMTM6 in the plasma of HDs (<span class="html-italic">n</span> = 23) and CC (<span class="html-italic">n</span> = 23) were measured by enzyme-linked immunosorbent assay (ELISA). Data are shown as pg/mL of CMTM6. (<b>b</b>) PD-L1 levels in the plasma of HDs (<span class="html-italic">n</span> = 11) and CC (<span class="html-italic">n</span> = 21) are shown as pg/mL of PD-L1. Student’s <span class="html-italic">t</span>-test was used. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01.</p> "> Figure 2
<p>CMTM6 and PD-L1 were found in exosomes from the plasma of the healthy donor group (HD) and the group of patients with cervical cancer (CC). (<b>a</b>) CMTM6 is preferentially released in exosomes in both the HD (<span class="html-italic">n</span> = 10) and CC (<span class="html-italic">n</span> = 20) groups; data shown as pg/mL of CMTM6. (<b>b</b>) PD-L1 was elevated in the exosome-enriched plasma fractions from both the HDs (<span class="html-italic">n</span> = 6) and CC (<span class="html-italic">n</span> = 15); data shown as pg/mL. (<b>c</b>) Correlation between exosomal CMTM6 and PD-L1 in HD samples (<span class="html-italic">r</span> = 0.996). (<b>d</b>) Correlation between exosomal CMTM6 and PD-L1 in CC samples (<span class="html-italic">r</span> = 0.8346). (<b>e</b>) CMTM6 was found to increase in the exosomal fractions from HDs and CC patients by Western blot. CD63 was used as an exosomal marker. The uncropped blots are shown in <a href="#app1-cancers-16-03126" class="html-app">Figure S1</a>. Wilcoxon ranked test was used. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> < 0.005.</p> "> Figure 2 Cont.
<p>CMTM6 and PD-L1 were found in exosomes from the plasma of the healthy donor group (HD) and the group of patients with cervical cancer (CC). (<b>a</b>) CMTM6 is preferentially released in exosomes in both the HD (<span class="html-italic">n</span> = 10) and CC (<span class="html-italic">n</span> = 20) groups; data shown as pg/mL of CMTM6. (<b>b</b>) PD-L1 was elevated in the exosome-enriched plasma fractions from both the HDs (<span class="html-italic">n</span> = 6) and CC (<span class="html-italic">n</span> = 15); data shown as pg/mL. (<b>c</b>) Correlation between exosomal CMTM6 and PD-L1 in HD samples (<span class="html-italic">r</span> = 0.996). (<b>d</b>) Correlation between exosomal CMTM6 and PD-L1 in CC samples (<span class="html-italic">r</span> = 0.8346). (<b>e</b>) CMTM6 was found to increase in the exosomal fractions from HDs and CC patients by Western blot. CD63 was used as an exosomal marker. The uncropped blots are shown in <a href="#app1-cancers-16-03126" class="html-app">Figure S1</a>. Wilcoxon ranked test was used. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> < 0.005.</p> "> Figure 3
<p>CMTM6 and PD-L1 were found in the lysates of CC-derived cells. (<b>a</b>) CMTM6 expression was found to be higher in the total lysate of CaSki cells compared to those of HeLa and SiHa cells, shown as bands corresponding to the approximate molecular weight reported for CMTM6 and its densitometric analysis. (<b>b</b>) The bands corresponding to the molecular weight reported for PD-L1 were also found in the three cell lines, coinciding again to show that CaSki cells express the highest levels of this protein; the band pattern and its densitometric analysis are shown. In both cases, β-actin was used as a constitutive protein and loading control. The uncropped blots are shown in <a href="#app1-cancers-16-03126" class="html-app">Figure S2</a>. Data are shown as mean ± SD; three independent experiments were performed for each condition. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001, ns: no significance.</p> "> Figure 4
<p>CMTM6 was found in the cell membrane and intracellularly in cell lines derived from cervical cancer. (<b>a</b>) The percentage of CMTM6-positive cells was determined by flow cytometry. CMTM6 was found both intracellularly and associated with the plasma membranes of all cell lines. Interestingly, the highest percentages of CMTM6, both intracellular and on the membrane, were found in CaSki cells. Data are shown as mean ± SD; three independent experiments were performed for each condition. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001, ns: no significance. (<b>b</b>) Immunofluorescence staining verified that CMTM6 (AF-594, red stain) is found in different subcellular locations such as intracellular (cytoplasm shown with diamond-tipped arrow) and the plasma membrane (closed arrow). The nuclei (open arrow) are stained with DAPI (blue). Note the intracellular staining in SiHa cells, which obscures the nucleus, and the visible nucleus and membrane staining in CaSki cells. Images taken using the 10× objective (left side) and 30× (right side).</p> "> Figure 5
<p>PD-L1 is present in the cell membranes and cytoplasm of cell lines derived from cervical cancer. (<b>a</b>) The percentages of PD-L1-positive cells were determined by traditional flow cytometry. Interestingly, the highest percentages of membrane PD-L1 were in CaSki cells. Data are shown as mean ± SD; two independent experiments were performed for each condition. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, **** <span class="html-italic">p</span> ≤ 0.0001, ns: no significance. (<b>b</b>) Immunofluorescence was used to verify if PD-L1 (AF-488, green stain) was found in different subcellular locations, such as intracellular (cytoplasm marked with diamond-tipped arrow) and plasma membrane (closed arrow). Nuclei were stained with DAPI (blue). Images taken using the 10× objective (left side) and 30× (right side).</p> "> Figure 6
<p>CMTM6 released by cell lines derived from cervical cancer. ELISA of culture supernatants to quantify the total levels of supernatant CMTM6. Detectable levels of supernatant CMTM6 were seen in all cell lines; however, HeLa cells were found to release the highest concentration (11,015 pg/mL). Data are shown as mean ± SD; two independent experiments were performed for each condition. * <span class="html-italic">p</span> < 0.05; *** <span class="html-italic">p</span> < 0.001; **** <span class="html-italic">p</span> < 0.0001.</p> ">
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection and Preparation
2.2. Cell Culture
2.3. Isolation of Exosomes and Western Blot
2.4. ELISA
2.5. Immunofluorescence
2.6. Flow Cytometry
2.7. Statistical Analysis
3. Results
3.1. Plasma CMTM6 and PD-L1 Are Increased in Cervical Cancer
3.2. CMTM6 and PD-L1 Are Preferentially Released in Exosomes
3.3. CMTM6 and PD-L1 Are Present in CC-Derived Cell Line Lysates
3.4. CMTM6 Found in the Cell Membrane and Intracellularly in Cell Lines Derived from Cervical Cancer
3.5. PD-L1 Was Found in Its Typical Membrane-Associated Form in Cell Lines
3.6. CMTM6 Released by Cervical Cancer-Derived Cell Lines
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Healthy Donors | Cervical Cancer Patients | |
---|---|---|
(n = 23) | (n = 23) | |
Age | ||
Mean in years (range) | 45 (25–79) | 46 (25–72) |
Histopathology | ||
Squamous cell carcinoma | - | 12 (52%) |
Adenocarcinoma | - | 1 (4%) |
No information | - | 10 (44%) |
FIGO Stage | ||
I | 3 (13%) | |
II | 3 (13%) | |
III | 8 (35%) | |
IV | 5 (22%) | |
No information | 4 (17%) | |
Treatment scheme status | ||
Pre-treatment | - | 22 (96%) |
Post-treatment | - | 1 (4%) |
Chemotherapy | - | 1 (4%) |
Chemotherapy + Radiotherapy | - | 0 (0%) |
Disease free survival | - | 0 (0%) |
No information | 0 (0%) |
Healthy Donors | Cervical Cancer Patients | |
---|---|---|
(n = 11) | (n = 21) | |
Age | ||
Mean in years (range) | 43 (25–63) | 44 (25–79) |
Histopathology | ||
Squamous cell carcinoma | - | 13 (65%) |
Adenocarcinoma | - | 0 (0%) |
No information | - | 7 (35%) |
FIGO Stage | ||
I | 3 (14%) | |
II | 2 (10%) | |
III | 7 (33%) | |
IV | 5 (24%) | |
No information | 4 (19%) | |
Treatment scheme status | ||
Pre-treatment | - | 16 (76%) |
Post-treatment | - | 5 (24%) |
Chemotherapy | - | 1 (5%) |
Chemotherapy + Radiotherapy | - | 1 (5%) |
Disease free survival | - | 3 (14%) |
No information | - | 0 (0%) |
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Urciaga-Gutierrez, P.I.; Franco-Topete, R.A.; Bastidas-Ramirez, B.E.; Solorzano-Ibarra, F.; Rojas-Diaz, J.M.; Garcia-Barrientos, N.T.; Klimov-Kravtchenko, K.; Tellez-Bañuelos, M.C.; Ortiz-Lazareno, P.C.; Peralta-Zaragoza, O.; et al. New Advances in the Study of CMTM6, a Focus on Its Novel Non-Canonical Cellular Locations, and Functions beyond Its Role as a PD-L1 Stabilizer. Cancers 2024, 16, 3126. https://doi.org/10.3390/cancers16183126
Urciaga-Gutierrez PI, Franco-Topete RA, Bastidas-Ramirez BE, Solorzano-Ibarra F, Rojas-Diaz JM, Garcia-Barrientos NT, Klimov-Kravtchenko K, Tellez-Bañuelos MC, Ortiz-Lazareno PC, Peralta-Zaragoza O, et al. New Advances in the Study of CMTM6, a Focus on Its Novel Non-Canonical Cellular Locations, and Functions beyond Its Role as a PD-L1 Stabilizer. Cancers. 2024; 16(18):3126. https://doi.org/10.3390/cancers16183126
Chicago/Turabian StyleUrciaga-Gutierrez, Pedro Ivan, Ramon Antonio Franco-Topete, Blanca Estela Bastidas-Ramirez, Fabiola Solorzano-Ibarra, Jose Manuel Rojas-Diaz, Nadia Tatiana Garcia-Barrientos, Ksenia Klimov-Kravtchenko, Martha Cecilia Tellez-Bañuelos, Pablo Cesar Ortiz-Lazareno, Oscar Peralta-Zaragoza, and et al. 2024. "New Advances in the Study of CMTM6, a Focus on Its Novel Non-Canonical Cellular Locations, and Functions beyond Its Role as a PD-L1 Stabilizer" Cancers 16, no. 18: 3126. https://doi.org/10.3390/cancers16183126