Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers
Abstract
:1. Introduction
2. Results
2.1. Plan of the Study
2.2. PD-L1/PD-L2 and PD-1 Protein Expression Dominated in Tumor Tissues as Compared to the Paired Samples of Tumor-Adjacent Normal Endometrial Tissues
2.3. PD-L1 Dominated TME Landscape in Endometrial Tumor Tissues
2.4. Establishment of Patient Tissue-Derived Primary Culture of CAF Ex Vivo
2.5. Marker-Based Validation of CAFs in Ex Vivo Culture of the Patient’s Tumor Tissue and Tumor-Adjacent Normal Tissue by Flowcytometry
2.6. Stromal PD-L1 and PD-L2 Expressions in Tumor (TCAF from TME) and Tumor-Adjacent Normal (NCAF from SME)
2.6.1. PD-L1/PD-L2 mRNA Expression Status in Cultured CAF from Paired Samples of Tumor and Tumor-Adjacent Normal Tissues by qRT-PCR
2.6.2. PD-L1 Protein Expression Status in Cultured CAF from Paired Samples of Tumor and Tumor-Adjacent Normal Tissues by Flow Cytometry
2.6.3. Cellular Distribution of PD-L1 Protein in Primary Cultured CAF from Paired Samples of Tumor and Tumor-Adjacent Normal Tissues by ICC Staining
2.7. Plasma sPD-L1 and sPD-L2 Protein Expression Status from Peripheral Blood Samples of Patients with EC by ELISA
2.8. Relationship between the Pathological Parameters and Tumor-TME Landscape of PD-L1/PD-L2 and PD-1 from Paired Samples of Tumor and Tumor-Adjacent Normal Tissues in EC
2.9. MMR Status of PD-L1+ Tumor Cells (by IHC) Versus PD-L1+ TCAFs in Patients with EC
3. Discussion
4. Methods and Materials
4.1. Patient Consent and Enrollment
4.2. Tissue and Blood Collection
4.3. IHC Expression of PD-L1, PD-L2, and PD-1 in FFPE Sections from Tumor and Tumor-Adjacent Normal Tissues
4.4. Primary Culture of Cancer-Associated Fibroblasts from Tumor and Tumor-Adjacent Normal Tissues
4.5. Flow Cytometric Expression of PD-L1 in Primary Culture of CAF from Tumor and Tumor-Adjacent Normal Tissues
4.6. Relative Gene Expression of PD-L1 and PD-L2 in Primary Culture of CAF from Tumor and Tumor-Adjacent Normal Tissues by qRT-PCR
- ○
- PD-L1 Forward: ACC TAC TGG CAT TTG CTG AAC G;
- ○
- PD-L1 Reverse: ATA GAC AAT TAG TGC AGC CAG GT;
- ○
- PD-L2 Forward: TGG AAT TGC AGC TTC ACC AGA;
- ○
- PD-L2 Reverse: TGG CTG TTA TTG CTC CAA GGT;
- ○
- GAPDH Forward: TGC ACC ACC AAC TGC TTA GC;
- ○
- GAPDH Reverse: GGC ATG GAC TGT GGT CAT GAG.
4.7. ICC Expression of SMA, S100A4, TE-7, EpCAM, CK 8, 18, and PD-L1 in Primary Culture of CAFs from Tumor and Tumor-Adjacent Normal Tissues
5. Patent Status
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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PD-L1 | ||||
T-test: Type 2, and Two Tail; n = 26 patients in each group | Difference in PD-L1 expression: Epithelium vs. SME | Difference in PD-L1 expression: Tumor vs. TME | Difference in PD-L1 expression: Epithelium vs. Tumor | Difference in PD-L1 expression: TME vs. SME |
p Value | p = 0.0496 | p = 0.936 | p = 0.00015 | p = 0.00000027 |
PD-L2 | ||||
T-test: Type 2, and Two Tail; n = 26 patients in each group | Difference in PD-L2 expression: Epithelium vs. SME | Difference in PD-L2 expression: Tumor vs. TME | Difference in PD-L2 expression: Epithelium vs. Tumor | Difference in PD-L2 expression: TME vs. SME |
p Value | p = 0.82 | p = 0.5971 | p = 0.0110 | p = 0.0000069 |
PD-1 | ||||
T-test: Type 2, and Two Tail; n = 26 patients in each group | Difference in PD-1 expression: Epithelium vs. SME | Difference in PD-1 expression: Tumor vs. TME | Difference in PD-1 expression: Epithelium vs. Tumor | Difference in PD-1 expression: TME vs. SME |
p Value | p = 0.261 | p = 0.003045 | p = 0.03486 | p = 0.0023 |
Proteins | Correlation between Expression(s) of PD-L1, PD-L2 and PD-1: Grade 3 vs. Grade 1 | |||||
PD-L1 | PD-L2 | PD-1 | ||||
Expression Site of the Proteins | Expression in Tumor Cells | Expression in TME/ Tumor Stroma (Lymphocyte/Macrophage/Blood Vessel) | Expression in Tumor Cells | Expression in TME/ Tumor Stroma (Lymphocyte/Macrophage/Blood Vessel) | Expression in Tumor Cells | Expression in TME/ Tumor Stroma (Lymphocyte/Macrophage/Blood Vessel) |
r | 0.3964 | 0.4745 | 0.4294 | 0.3961 | −0.19 | 0.444 |
R2 | 0.1571 | 0.2252 | 0.1844 | 0.1569 | 0.03609 | 0.1971 |
p (Two-tailed test) | 0.1034 | 0.0466 | 0.0753 | 0.1037 | 0.4502 | 0.0649 |
Significant (alpha = 0.05) | Not Significant | Significant | Not Significant | Not Significant | Not Significant | Not Significant |
# of Pairs | 18 | 18 | 18 | 18 | 18 | 18 |
Proteins | Correlation between expression(s) of PD-L1, PD-L2 and PD-1: Grade 2 vs. Grade 1 | |||||
PD-L1 | PD-L2 | PD-1 | ||||
Expression site of the proteins | Expression in Tumor Cells | Expression in TME/ Tumor Stroma (Lymphocyte/Macrophage/Blood Vessel) | Expression in Tumor Cells | Expression in TME/ Tumor Stroma (Lymphocyte/Macrophage/Blood Vessel) | Expression in Tumor Cells | Expression in TME/ Tumor Stroma (Lymphocyte/Macrophage/Blood Vessel) |
r | 0.3141 | 0.2148 | −0.1605 | 0.077 | −0.0548 | 0.503 |
R2 | 0.0987 | 0.0461 | 0.0258 | 0.0059 | 0.003 | 0.253 |
p (Two-tailed) | 0.2195 | 0.4077 | 0.5383 | 0.7689 | 0.8344 | 0.0396 |
Significant (alpha = 0.05) | Not Significant | Not Significant | Not Significant | Not Significant | Not Significant | Significant |
# Pairs | 17 | 17 | 17 | 17 | 17 | 17 |
Proteins | Correlation between “Tumor + TME Combined Scores” for PD-L1, PD-L2 and PD-1: Tumor Grades 1, 2 and 3 | |||||
PD-L1 | PD-L2 | PD-1 | ||||
Expression site of the proteins | Expression in Tumor Cells and Cells of TME | Expression in Tumor Cells and Cells of TME | Expression in Tumor Cells and Cells of TME | |||
r | 0.4609 | 0.4573 | 0.2918 | |||
R2 | 0.2124 | 0.2091 | 0.08513 | |||
p (Two-tailed test) | 0.0178 | 0.0188 | 0.1481 | |||
Significant (alpha = 0.05) | Significant | Significant | Not Significant | |||
# Pairs | 26 | 26 | 26 |
Site | Correlations | R-Value | R2 Value | p Value | Significance | # of Patients (N) |
---|---|---|---|---|---|---|
TUMOR | Tumor PD-L1 by IHC vs. Percentage of Myometrial Invasion | 0.2085 | 0.04345 | 0.3068 | Not Significant | 26 |
TME | TME PD-L1 by IHC vs. Percentage of Myometrial Invasion | 0.4524 | 0.2047 | 0.0203 | Significant | 26 |
TUMOR + TME | Tumor + TME combined PD-L1 by IHC vs. Percentage of Myometrial Invasion | 0.3688 | 0.1360 | 0.0637 | Not Significant | 26 |
Tumor + TME combined PD-L1 by IHC Cutoff = 15 vs. Percentage of Myometrial Invasion | 0.3665 | 0.1343 | 0.0656 | Not Significant | 26 | |
TUMOR | Tumor PD-L1 by IHC vs. Presence of TILs | 0.1560 | 0.02433 | 0.4467 | Not Significant | 26 |
TME | TME PD-L1 by IHC vs. Presence of TILs | 0.4901 | 0.2402 | 0.0110 | Significant | 26 |
TUMOR + TME | Tumor + TME combined PD-L1 by IHC vs. Presence of TILs | 0.3480 | 0.1211 | 0.0815 | Not Significant | 26 |
Tumor + TME combined PD-L1 by IHC Cutoff = 15 vs. Presence of TILs | 0.4501 | 0.2025 | 0.0211 | Significant | 26 |
Figure # | Sub-Figure | Data Presented | Summary of Data |
---|---|---|---|
1 | A | Overall Plan of the Study | Data generated from patients’ tumor tissue, tumor-adjacent normal tissue, and blood |
B | PD-L1 status of tumor cells | PD-L1 +ve Tumor cells | |
C | PD-L2 status of tumor cells | PD-L2 +ve Tumor cells | |
D | PD-1 status of tumor cells | PD-1 −ve Tumor cells | |
E | PD-L1 status of cells of TME | PD-L1 +ve Lymphocytes | |
F | PD-L1 status of cells of TME | PD-L1 +ve Macrophage | |
G | PD-L2 status of cells of TME | PD-L2 −ve Tumor Cells and Lymphocyte | |
H | PD-L2 status of cells of TME | PD-L2 +ve Macrophage and Lymphocyte | |
I | PD-1 status of cells of TME | PD-1 +ve Lymphocytes | |
2 | A | PD-L1 status of cells of SME in tumor-adjacent normal tissue | PD-L1 +ve Lymphocyte |
B | PD-L2 +ve epithelium of tumor-adjacent normal tissue | PD-L2 −ve normal Epithelium and Macrophage | |
C | PD-1 status of tumor-adjacent normal tissue | PD-1 −ve Epithelium and PD-1 +ve Lymphocytes | |
D | PD-L1 +ve SME of tumor-adjacent normal tissue | PD-L1 +ve Lymphocyte and PD-L1 −ve Epithelium | |
E | PD-L2 +ve SME of tumor-adjacent normal tissue | PD-L2 +ve Mesenchyme | |
F | PD-1 +ve SME of tumor-adjacent normal tissue | PD-1 +ve Lymphocyte and PD-1 −ve Epithelium | |
3 | A | Flowcytometric expression of positive and negative CAF markers in NCAF and TCAF | Positive expression of FAP-A, SMA, and negative expression of S100A4, CD31, EpCAM |
B | Flowcytometric expression of positive and negative CAF markers in NCAF and TCAF | Positive expression of FAP-A, SMA, S100A4, CD90 and negative expression of CD31, EpCAM | |
C | Flowcytometric expression of positive and negative CAF markers in NCAF and TCAF | Positive expression of FAP-A, SMA, CD90 and negative expression of CD31, EpCAM, S100A4 | |
D | Percentage of expression of epithelial (EpCAM), fibroblast (SMA, S100A4, FAP, and CD90), and endothelial (CD31) marker proteins by flow cytometry | Both TCAFs and NCAFs are positive for SMA, CD90, and FAP-A and negative for CD31 and EpCAM. S100A4 is differentially expressed. | |
E | Expression of CK 8, 18, and EpCAM in NCAF from passages 1, 2, and 3 | Negative expression of CK 8, 18, and EpCAM in NCAF | |
F | Expression of CK 8, 18, and EpCAM in TCAF from passages 1, 2, and 3 | Negative expression of CK 8, 18, and EpCAM in TCAF | |
G | Expression of SMA, S100A4, TE-7, and PD-L1 in NCAF from passages 1, 2, and 3 | Positive expression of SMA, TE-7 and negative expression of S100A4, PD-L1 in NCAF | |
H | Expression of SMA, S100A4, TE-7, and PD-L1 in TCAF from passages 1, 2, and 3 | Positive expression of SMA, TE-7 and negative expression of S100A4, PD-L1 in TCAF | |
I | Percentage of expression of epithelial (EpCAM and CK 8,18) and fibroblast (SMA, S100A4, and TE-7) marker proteins by ICC | Both TCAFs and NCAFs are positive for SMA and TE-7 and negative for CK 8, 18, and EpCAM. S100A4 is differentially expressed. | |
4 | A | Relative Ratio of PD-L1 by qRT-PCR | TCAFs tend to express higher levels of PD-L1 mRNA than NCAFs |
B | Relative Ratio of PD-L2 by qRT-PCR | TCAFs tend to express higher levels of PD-L2 mRNA than NCAFs | |
C | Heatmap of PD-L1 and PD-L2 mRNA expressions in different patients | Ratios of PD-L1 and PD-L2 mRNA expressions in TCAFs/NCAFs pairs are patient-specific. | |
5 | A | Flow cytometric expression of PD-L1 in NCAFs and TCAFs pairs | PD-L1 expression tends to be higher in TCAFs compared to TCAFs. |
B | |||
C | |||
6 | A | ICC expression of PD-L1 in TCAF-NCAF pair | PD-L1 is expressed at a comparable level in both NCAFs and TCAFs |
B | PD-L1 expressed in NCAFs is higher than TCAFs | ||
C | PD-L1 expressed in TCAFs is higher than NCAFs | ||
D | Conditional formatting of the IHC (FFPE of tumor-adjacent normal epithelium/mesenchyme) vs. ICC (NCAFs) expression of PD-L1 in normal epithelium and mesenchyme in the same patient | The epithelium is rarely positive for PD-L1 compared to NCAFs in the same patient. | |
E | Conditional formatting of the IHC (FFPE of tumor/TME) vs. ICC (TCAFs) expression of PD-L1 in tumor cells and TME in the same patient | Expression of PD-L1 in TCAFs is higher than tumor/TME in the same patient. | |
7 | A | Standardization of sPD-L1 from plasma of blood from patients with EC as compared to the normal subjects | sPD-L1 from the plasma of blood from patients with EC is higher than sPD-L1 from the blood of healthy normal subjects |
B | Standardization of sPD-L2 from plasma of blood from patients with EC as compared to the normal subjects | sPD-L2 from the plasma of blood from patients with EC is comparable to sPD-L2 from the blood of healthy normal subjects | |
C | The ratio of sPD-L1 to sPD-L2 in the Plasma of patients with EC | The average sPD-L1 is lower than the average sPD-L2 in the Plasma of patients with EC. | |
8 | A | Expression pattern of PD-L1, PD-L2, and PD-1 in tumor cells/TME of patients with grade 1, 2, and 3 diseases as obtained from tumor samples | Expression of PD-L1 is correlated to the highest grade of the disease. |
B | Expression patterns of PD-L1, PD-L2, and PD-1 in epithelium/SME of patients with grade 1, 2, and 3 diseases were obtained from tumor-adjacent normal tissue samples. | No correlation was observed between the expression of PD-L1 with the grade of the disease. | |
9 | Diagram presenting the expression pattern-based tumor–TME interaction of PD-L1, PD-L2, and PD-1 in endometrial cancers. | Expression landscape-based tumor–TME interactions of PD-L1, PD-L2 and PD-1 in endometrial cancers |
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Sulaiman, R.; De, P.; Aske, J.C.; Lin, X.; Dale, A.; Koirala, N.; Gaster, K.; Espaillat, L.R.; Starks, D.; Dey, N. Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers. Int. J. Mol. Sci. 2023, 24, 11079. https://doi.org/10.3390/ijms241311079
Sulaiman R, De P, Aske JC, Lin X, Dale A, Koirala N, Gaster K, Espaillat LR, Starks D, Dey N. Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers. International Journal of Molecular Sciences. 2023; 24(13):11079. https://doi.org/10.3390/ijms241311079
Chicago/Turabian StyleSulaiman, Raed, Pradip De, Jennifer C. Aske, Xiaoqian Lin, Adam Dale, Nischal Koirala, Kris Gaster, Luis Rojas Espaillat, David Starks, and Nandini Dey. 2023. "Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers" International Journal of Molecular Sciences 24, no. 13: 11079. https://doi.org/10.3390/ijms241311079