Research ArticleExperimental Studies
Open Access

DNA Ligase 4 Inhibition Sensitizes Prostate Cancer to Immune Checkpoint Blockade In Vivo

JIANCHUN WU, ANGELICA M. LAGUNAS and DAVID L. CROWE
Anticancer Research March 2025, 45 (3) 883-896; DOI: https://doi.org/10.21873/anticanres.17476

Abstract

Background/Aim: Prostate cancer is a common malignant tumor in men. DNA ligase IV (LIG4) expression correlated with poor prognosis in prostate cancer patients. LIG4 joins DNA double strand breaks and is essential for repair of these genetic lesions. Prostate cancers have not demonstrated clinically significant responses to anti-PD-1 immunotherapy. Prostate cancers express low PD-L1 levels and exhibit limited cytotoxic T lymphocyte infiltrates. To determine the effects of LIG4 inhibition on prostate tumorigenesis, we created a new genetically engineered in vivo model.

Materials and Methods: Lig4+/+;TAg and Lig4+/−;TAg prostate glands and tumors were processed for histopathology. Separate groups of prostate tumor-bearing mice were treated with anti-PD1 antibody or preimmune IgG. LIG4 and PD-L1 expression was determined by quantitative reverse transcription polymerase chain reaction. Expression of DNA damage repair proteins, cell senescence, and cell death markers was determined by immunohistochemistry and immunofluorescence microscopy. The prostate cancer stem cell fraction was analyzed by Sca1/CD49f flow cytometry and tumorsphere culture. PD-L1 protein expression was determined by western blot.

Results: LIG4 inhibition induced DNA double strand breaks and cellular senescence in prostate glands and cancers and significantly reduced prostate intraepithelial neoplasia and tumorigenesis. LIG4 inhibition reduced the prostate cancer stem cell fraction and proliferation in stem cell cultures. Prostate cancers resistant to LIG4 inhibition evaded anti-tumor immune response due to increased PD-L1 expression. PD-1 antibody treatment of these cancers induced CD8+ T lymphocyte infiltration and reduced tumor volume.

Conclusion: Inhibition of LIG4 sensitized prostate cancers to immune checkpoint inhibition.

Keywords:

Introduction

Prostate cancer is a common malignant tumor in men. Over 288,000 cases and 34,000 deaths are reported each year in the United States (1). Prostate cancer may start as lesions known as prostatic intraepithelial neoplasia (PIN; 2). Prostate cancer treatment may include surgery, radiation, chemotherapy, and hormonal therapy. Genomic studies classified prostate cancer into several molecular categories, characterized by fusions and specific genetic mutations (3).

DNA ligase IV (LIG4) joins DNA double strand breaks and is essential for repair of these lesions (4). LIG4 contains functional domains for DNA binding, nucleotide transfer, and oligonucleotide binding. LIG4 expression correlated with poor prognosis in prostate cancer patients (5). LIG4 coding variants have been associated with radiotherapy toxicity (6, 7). LIG4 expression was androgen dependent in human prostate cancer cells (8). Despite these important correlations between LIG4 expression and clinical outcomes, the role of this enzyme in prostate cancer progression has not been characterized.

Cytotoxic T lymphocyte infiltration in cancers has been associated with improved patient prognosis (9). Cytotoxic T lymphocytes express effector proteins which damage tumor cell membranes resulting in death. Cancer cells may express programmed death ligand 1 (PD-L1) which engages PD-1 receptor on T lymphocytes resulting in inhibited tumor immune response (10, 11). Primary prostate cancers express low PD-L1 levels and limited cytotoxic T lymphocyte infiltrates (12). PD-L1 was overexpressed in enzalutamide-resistant prostate cancers, and was associated with high Gleason score and lymph node metastasis (13-15). However, these advanced prostate cancers have not demonstrated clinically significant responses to anti-PD-1 immunotherapy (16-18).

To determine the effects of LIG4 inhibition on prostate tumorigenesis, we created a new genetically engineered in vivo model. LIG4 inhibition induced DNA double strand breaks and cell senescence in prostate glands and cancers and significantly reduced prostate tumorigenesis. Prostate cancers resistant to LIG4 inhibition evaded anti-tumor immune response due to increased PD-L1 expression. PD-1 antibody treatment of these cancers induced CD8+ T lymphocyte infiltration and reduced tumor volume. We concluded that inhibiting LIG4 sensitized prostate cancers to immune checkpoint inhibition.

Materials and Methods

Mouse breeding and procedures. B6;129S6-Lig4tm1Fwa/Kvm and C57BL/6-Tg(TRAMP)8247Ng/J mutant mouse strains were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). These mice were crossed to create 20 Lig4+/+;TAg and 20 Lig4+/−;TAg male offspring. The LIG4 null mutant mouse was not viable. Littermates were used to control for genetic background and assigned to experimental or control groups by genotype. An additional 20 prostate cancer bearing mice were generated for in vivo treatment with preimmune IgG or 25 μg anti-PD1 antibody. Prostate tumors were fixed in 4% buffered formaldehyde or trypsin dissociated for cryopreservation.

LIG4 expression in human prostate cancers. LIG4 mRNA expression data in human prostate glands and cancers were obtained from The Cancer Genome Atlas and Gene Expression Omnibus public databases. LIG4 gene copy number changes were obtained from The Cancer Genome Atlas public database.

qRT-PCR. RNA was extracted from prostate glands and tumors and reverse transcribed according to manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). cDNA was amplified using mouse primers for: LIG4 5′-GTGCTTTGTCCCAGCGTCAC-3′ and 5′-TGTGAGGAAGCC ATAGAAGCG-3′, PD-L1 5′-TGCTGTCACTTGCTACGGG-3′ and 5′- AAGGGCAGCATTTCCCTTCA-3′, and β-actin 5′-AAAAGCCACCCCCACTCCTAAG-3′ and 5′-TCAAGTCAGTGT ACAGGCCAGC-3′ according to our published protocol (19).

Histopathology, immunofluorescence, and immunohistochemistry. Fixed prostate glands and tumors were processed and analyzed according to our published protocol (19) using 53BP1 or H3K9me3 antibodies for immunofluorescence microscopy. Human prostate cancer tissue microarrays were purchased from TissueArray (Rockville, MD, USA). For immunohistochemical analysis, sections were incubated with Lig4, androgen receptor (AR), PCNA, p16INK4A, pATM, pATR, pChk1, pChk2, or CD8 antibodies according to our published protocol (19).

Cell death analysis. Apoptotic cells were analyzed in prostate gland and tumor sections by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) according to manufacturer’s recommendations (Roche Applied Sciences, Indianapolis, IN, USA).

Fluorescence activated cell sorting. Prostate tumor stem cells were analyzed by flow cytometry using phycoerythrin-conjugated Sca1 and AF488-conjugated CD49f antibodies according to our published protocol (19).

Tumorsphere culture. LIG4+/+;TAg and LIG4+/−;TAg prostate cancer cells were cultured as tumorspheres according to our published protocol (19).

Western blot. PD-L1 and β-actin expression was determined in LIG4+/+;TAg and LIG4+/−;TAg prostate cancers by western blot using our published protocol (19).

Antibodies. The following antibodies were used in this study: Abcam (Waltham, MA, USA) AR AB133273, CD8 AB217344, H3K9me3 AB8898, p16INK4A AB54210, pATM AB315019, pATR AB289363, pChk2 AB85743, pChk1 AB47318, PCNA AB92522; BioXCell (Lebanon, NH, USA) PD-1 BE0146; Cell Signaling (Danvers, MA, USA) β-actin 4970S; Novus Biologicals (Minneapolis, MN, USA) 53BP1 NB100-304; Stem Cell Technologies (Vancouver, BC, Canada) Sca1 60032PE, CD49f 60037AD.

Results

We compared LIG4 mRNA expression in human prostate glands and over 800 prostate cancer cases in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. There were no significant differences in LIG4 expression between prostate glands and primary prostate cancer subtypes in the TCGA database (Figure 1A). However, 12% of TCGA prostate cancer cases exhibited LIG4 gene copy number loss (Figure 1B). GEO datasets GDS1439 and GDS2545 demonstrated significant 3 fold reduction in metastatic prostate cancers compared to glands (p<0.02; Figure 1C). Lig4 protein expression was not detected by immunohistochemistry in 177/192 (92%) human prostate cancer pathology specimens (Figure 1D, E). LIG4-overexpressing human prostate cancer specimens did not correlate with age, stage, Gleason score, or prostate specific antigen expression. These results indicate that prostate cancers exhibit LIG4 copy number loss and decreased expression in metastatic prostate cancer.

Figure 1.
Figure 1.

LIG4 expression is reduced in metastatic prostate cancer. (A) LIG4 expression in normal prostate gland and prostate cancer genomic subtypes is shown using TCGA database. Log2 fragments of kilobase per million mapped reads (FPKM) is shown. (B) Percent TCGA prostate cancer cases (TCGA-PRAD) with LIG4 gene copy number loss (CNL) and copy number gain (CNG). (C) LIG4 expression in prostate gland and metastatic prostate cancer is shown from Gene Expression Omnibus datasets GDS1439 and GDS2545. Error bars indicate SEM. p-Value is shown. LIG4 protein expression is shown in human prostate cancer pathology specimens using immunohistochemistry. LIG4-positive (D) and -negative (E) prostate cancer specimens are shown. Nuclei were counterstained with hematoxylin and eosin. Scale bar=10 μm.

To examine the effects of LIG4 inhibition on prostate cancer in vivo, we created a novel genetically engineered model. LIG4 expression in LIG4+/+ and LIG4+/− prostate glands is shown by qRT-PCR in Figure 2A and in LIG4+/+;TAg and LIG4+/−;TAg prostate glands by immunohistochemistry in Figure 2J, K. The LIG4 homozygous null mutant mouse was not viable. Both LIG4+/+ and LIG4+/− prostate glands appeared histopathologically normal by H&E staining (Figure 2B, C). When crossed to prostate cancer prone TAg mice, 18/20 (90%) LIG4+/+;TAg animals developed prostate adenocarcinoma (Figure 2G). The remaining 2/20 (10%) LIG4+/+;TAg mice developed prostatic intraepithelial neoplasia (PIN; Figure 2E). In contrast, only 6/20 (30%) LIG4+/−;TAg developed prostate adenocarcinoma (Figure 2H). There were no significant differences in tumor latency between LIG4+/+;TAg and LIG4+/−;TAg mice (Figure 2I). Eight of 20 (40%) LIG4+/−;TAg mice developed PIN, and 6/20 (30%) exhibited histopathologically normal prostate glands (p<0.0002; Figure 2D, F). These results indicated that reduced LIG4 expression significantly inhibited prostate cancer development.

Figure 2.
Figure 2.

A LIG4 null allele inhibits prostate tumorigenesis in a genetically engineered in vivo model. (A) LIG4 expression in Lig4+/+ and Lig4+/− prostate glands is shown by qRT-PCR. Error bars indicate SEM. Histopathologic appearance of Lig4+/+ (B) and Lig4+/− (C) prostate glands is shown by H&E staining. Histopathologic classifications of Lig4+/+;TAg and Lig4+/−;TAg prostate glands and tumors are shown by H&E staining. Normal histology (D), PIN (E, F), and prostate adenocarcinoma (G, H) are shown. (I) Tumor latency in Lig4+/+;TAg and Lig4+/−;TAg prostate cancers is shown by Kaplan-Meier analysis. Percent tumor free mice is shown on the y-axis and days latency is shown on the x-axis. Lig4 protein expression is shown in Lig4+/+;TAg (J) and Lig4+/−;TAg (K) PIN is shown by immunohistochemistry. Nuclei were counterstained with hematoxylin. Scale bar=10 μm.

Given that LIG4 is responsible for DNA double strand break repair, we examined this type of DNA damage in LIG4+/+;TAg and LIG4+/−;TAg prostate glands by 53BP1 immunofluorescence microscopy. 53BP1 foci were significantly increased in LIG4+/−;TAg (39% vs. 5%; p<0.007; Figure 3A, B and M) compared to LIG4+/+;TAg prostate glands. The androgen receptor positive cell fraction was low and not significantly different between LIG4+/+;TAg and LIG4+/−;TAg prostate glands (1.2% vs. 1.8%; Figure 3C, D). We next examined expression of the cellular senescence markers H3K9me3 and p16INK4A in LIG4+/+;TAg and LIG4+/−;TAg prostate glands. The H3K9me3 positive cell fraction was significantly increased in LIG4+/−;TAg (36% vs. 7%; p<0.006; Figure 3E, F, M) compared to LIG4+/+;TAg prostate glands. The p16INK4A positive cell fraction was significantly increased in LIG4+/−;TAg (26% vs. 2%; p<0.009; Figure 3G, H, M) compared to LIG4+/+;TAg prostate glands. The proliferative PCNA positive cell fraction was low and not significantly different between LIG4+/+;TAg and LIG4+/−;TAg prostate glands (4% vs. 3%; Figure 3I, J). The apoptotic cell fraction was low and not significantly different between LIG4+/+;TAg and LIG4+/−;TAg prostate glands (0.1% vs. 0.1%; Figure 3K, L). We concluded that reduced LIG4 expression inhibited DNA double strand break repair and induced cellular senescence in prostate glands.

Figure 3.
Figure 3.

Increased DNA double strand breaks and cellular senescence in Lig4+/−;TAg PIN. 53BP1 foci in Lig4+/+;TAg (A) and Lig4+/−;TAg (B) PIN are shown by immunofluorescence microscopy. Nuclei were counterstained with DAPI. Scale bar=10 μm. AR expression in Lig4+/+;TAg (C) and Lig4+/−;TAg (D) PIN are shown by immunohistochemistry. Nuclei were counterstained with hematoxylin. SAHF in Lig4+/+;TAg (E) and Lig4+/−;TAg (F) PIN are shown by H3K9me3 immunofluorescence microscopy. Senescent cells in Lig4+/+;TAg (G) and Lig4+/−;TAg (H) PIN are shown by p16INK4A immunohistochemistry. Proliferating cells in Lig4+/+;TAg (I) and Lig4+/−;TAg (J) PIN are shown by PCNA immunohistochemistry. Programmed cell death in Lig4+/+;TAg (K) and Lig4+/−;TAg (L) PIN is shown by TUNEL analysis. (M) Quantitation of 53BP1, SAHF, and p16 positive cells in Lig4+/+;TAg and Lig4+/−;TAg PIN is shown. Percent positive cells is shown on the y-axis and genotypes are shown on the x-axis.

We next examined DNA damage signaling in LIG4+/−;TAg and LIG4+/+;TAg prostate cancers. 53BP1 foci were significantly increased in LIG4+/−;TAg (35% vs. 8%; p<0.01; Figure 4A, B, K) compared to LIG4+/+;TAg prostate cancers. We also examined activation of downstream DNA break signaling proteins in LIG4+/−;TAg and LIG4+/+;TAg prostate cancers. The pATM positive cell fraction was significantly increased in LIG4+/−;TAg (12% vs. 1%; p<0.03; Figure 4C, D, K) compared to LIG4+/+;TAg prostate cancers. The pATR positive cell fraction was significantly increased in LIG4+/−;TAg (24% vs. 5%; p<0.02; Figure 4E, F, K) compared to LIG4+/+;TAg prostate cancers. pChk1 and pChk2 expression were below detection limits for immunohistochemistry in LIG4+/+;TAg and LIG4+/−;TAg prostate cancers (Figure 4G-J). We concluded that reduced LIG4 expression results in increased DNA damage signaling in prostate cancers.

Figure 4.
Figure 4.

Increased DNA damage signaling in Lig4+/−;TAg prostate cancers. Expression of the DNA double strand break protein 53BP1 in Lig4+/+;TAg (A) and Lig4+/−;TAg (B) prostate cancer is shown by immunofluorescence microscopy. Nuclei were counterstained with DAPI. Expression of the activated double strand DNA break signaling protein pATM in Lig4+/+;TAg (C) and Lig4+/−;TAg (D) prostate cancers is shown by immunohistochemistry. Nuclei were counterstained with hematoxylin. Scale bar=10 μm. Expression of the activated single strand DNA break signaling protein pATR in Lig4+/+;TAg (E) and Lig4+/−;TAg (F) prostate cancers is shown by immunohistochemistry. Expression of the activated DNA damage signaling protein pChk1 in Lig4+/+;TAg (G) and Lig4+/−;TAg (H) prostate cancers is shown by immunohistochemistry. Expression of the activated DNA damage signaling protein pChk2 in Lig4+/+;TAg (I) and Lig4+/−;TAg (J) prostate cancers is shown by immunohistochemistry. (K) Quantitation of 53BP1, pATM and pATR positive cells in Lig4+/+;TAg and Lig4+/−;TAg prostate cancers is shown. Percent positive cells is shown on the y-axis and genotypes are shown on the x-axis.

We next examined the effects of increased DNA damage signaling on LIG4+/−;TAg and LIG4+/+;TAg prostate cancers. The androgen receptor positive cell fraction, a marker of prostate cancer differentiation, was significantly increased (18% vs. 2%; p<0.002; Figure 5A, B, K) in LIG4+/−;TAg compared to LIG4+/+;TAg prostate cancers. We examined expression of the cellular senescence markers H3K9me3 and p16INK4A in LIG4+/+;TAg and LIG4+/−;TAg prostate cancers. The SAHF positive cell fraction was significantly increased in LIG4+/−;TAg (25% vs. 5%; p<0.001; Figure 5C, D, K) compared to LIG4+/+;TAg prostate cancers. Similarly the p16INK4A positive cell fraction was significantly increased in LIG4+/−;TAg (17% vs. 1%; p<0.0004; Figure 5E, F, K) compared to LIG4+/+;TAg prostate cancers. The proliferative PCNA positive cell fraction was significantly reduced in LIG4+/−;TAg (28% vs. 49%; p<0.01; Figure 5G, H, K) compared to LIG4+/+;TAg prostate cancers. The apoptotic cell fraction was low and not significantly different between LIG4+/+;TAg and LIG4+/−;TAg prostate cancers (0.2% vs. 0.1%; Figure 5I, J). We concluded that reduced LIG4 expression produced increased differentiation, cellular senescence, and decreased proliferation in prostate cancer.

Figure 5.
Figure 5.

Increased differentiation, senescence, and decreased proliferation in Lig4+/−;TAg prostate cancers. AR expression in Lig4+/+;TAg (A) and Lig4+/−;TAg (B) prostate cancers is shown by immunohistochemistry. Nuclei were counterstained with hematoxylin. Scale bar=10 μm. SAHF in Lig4+/+;TAg (C) and Lig4+/−;TAg (D) prostate cancers are shown by H3K9me3 immunofluorescence microscopy. Senescent cells in Lig4+/+;TAg (E) and Lig4+/−;TAg (F) prostate cancers are shown by p16INK4A immunohistochemistry. Proliferating cells in Lig4+/+;TAg (G) and Lig4+/−;TAg (H) prostate cancers are shown by PCNA immunohistochemistry. Programmed cell death in Lig4+/+;TAg (I) and Lig4+/−;TAg (J) prostate cancers is shown by TUNEL analysis. (K) Quantitation of AR, SAHF, p16, and PCNA-positive cells in Lig4+/+;TAg and Lig4+/−;TAg prostate cancers is shown. Percent positive cells is shown on the y-axis and genotypes are shown on the x-axis.

To determine if reduced LIG4 expression affected the prostate cancer stem cell fraction, we sorted Sca1highCD49fhigh stem cells from LIG4+/−;TAg and LIG4+/+;TAg cancers. The prostate cancer stem cell fraction was significantly reduced (1.6% vs. 2.8%; p<0.02; Figure 6A, B) in LIG4+/−;TAg compared to LIG4+/+;TAg prostate cancers. We then cultured prostate cancer stem cells in tumorsphere conditions for 7 days. LIG4+/−;TAg prostate cancer stem cells grew significantly slower (31 vs. 89 μm mean diameter; p<0.0003; Figure 6C, D) than those from LIG4+/+;TAg tumors. We concluded that reduced LIG4 expression diminished the prostate cancer stem cell fraction and slowed growth of these cells in tumorsphere culture.

Figure 6.
Figure 6.

Decreased prostate cancer stem cells and tumorsphere formation in Lig4+/−;TAg prostate cancers. The Sca1highCD49fhigh stem cell fraction in Lig4+/+;TAg (A) and Lig4+/−;TAg (B) prostate cancers is shown by flow cytometry. Sca1 log and CD49f log scales are shown. Tumorsphere formation from Lig4+/+;TAg (C) and Lig4+/−;TAg (D) prostate cancer cells is shown by phase contrast microscopy. Scale bar=10 μm.

Given that the LIG4 null allele inhibited prostate tumorigenesis, we hypothesized that LIG4-null resistant cancers may evade immune surveillance in order to produce detectable tumors. LIG4+/−;TAg prostate cancers expressed 4 fold higher PD-L1 mRNA and protein levels compared to LIG4+/+;TAg tumors (Figure 7A, B). We treated LIG4+/+;TAg and LIG4+/−;TAg prostate cancers with PD-1 antibody or preimmune IgG to determine if this therapy could induce CD8+ cytotoxic T lymphocyte infiltration of these tumors. In LIG4+/+;TAg prostate cancers, treatment with preimmune IgG or PD-1 antibody did not affect prostate cancer histopathology (Figure 7C, D) nor CD8+ cytotoxic T lymphocyte infiltration (Figure 7G, H). However PD-1 antibody treatment resulted in hypocellular tumor histopathology in LIG4+/−;TAg prostate cancers (Figure 7E, F) which demonstrated prominent CD8+ cytotoxic T lymphocyte infiltration (Figure 7I, J). The volume of LIG4+/+;TAg prostate cancers treated with preimmune IgG or PD-1 antibody increased during the time course experiment (264 to 495 mm3 and 239 to 460 mm3 respectively; Figure 7K). The volume of LIG4+/−;TAg prostate cancers treated with preimmune IgG increased during the time course experiment (256 to 480 mm3; Figure 7K). However, PD-1 antibody treatment of LIG4+/−;TAg prostate cancers significantly reduced tumor volume (245 to 179 mm3; p<0.01; Figure 7K). We concluded that PD-1 antibody treatment inhibited LIG4+/−;TAg prostate tumorigenesis via CD8+ cytotoxic T lymphocyte infiltration.

Figure 7.
Figure 7.

Increased PD-L1 expression in Lig4+/−;TAg prostate cancers. (A) PD-L1 mRNA expression in Lig4+/+;TAg and Lig4+/−;TAg prostate cancers is shown by qRT-PCR. Relative PD-L1 expression is shown on the y-axis and genotypes are shown on the x-axis. p-Value is shown. (B) PD-L1 protein expression in Lig4+/+;TAg and Lig4+/−;TAg prostate cancers is shown by western blot.β-actin expression was used to control for protein loading in each lane. Molecular mass markers are shown. Increased CD8+ cytotoxic T lymphocyte infiltration in Lig4+/−;TAg prostate cancers treated with anti-PD1 antibody. Histopathologic appearance of Lig4+/+;TAg prostate cancers treated with preimmune IgG (C) or PD-1 antibody (D), and Lig4+/−;TAg tumors treated with IgG (E) or PD-1 antibody (F) is shown by H&E staining. Scale bar=10 μm. Cytotoxic T lymphocytes in Lig4+/+;TAg prostate cancers treated with preimmune IgG (G) or PD-1 antibody (H), and Lig4+/−;TAg tumors treated with IgG (I) or PD-1 antibody (J) are shown by CD8 immunohistochemistry. (K) Prostate cancer growth inhibition in Lig4+/+;TAg and Lig4+/−;TAg prostate cancers treated with preimmune IgG or PD-1 antibody. Tumor volume is shown on the y axis and time in days is shown on the x axis. Error bars indicate SEM.

Discussion

Our study demonstrated that LIG4 expression was reduced in metastatic prostate cancer. Reduced LIG4 expression was detected in colorectal and brain cancers (20, 21). Reduced LIG4 expression correlated with poor prognosis in lung cancer, myelodysplastic syndrome, ovarian cancer, and uveal melanoma (22-26). LIG4 expression predicted overall survival of nasopharyngeal cancer patients (27). LIG4 mutations were associated with increased lung cancer risk, radiation toxicity, and chemotherapy response (28-31). LIG4 single nucleotide polymorphisms (SNP) were associated with increased brain, colorectal, and ovarian cancer risk (32-37). These studies indicate that reduced LIG4 expression and activity are associated with increased risk of multiple cancers.

Our study demonstrated that LIG4 defective prostate cancer exhibited increased DNA double strand break signaling and cellular senescence. LIG4 defective cells exhibit increased DNA double strand breaks, translocations, ionizing radiation sensitivity, and reduced cell viability (38-41). Decreased LIG4 expression correlated with reduced DNA end joining activity in colorectal cancers (42).

Despite the fact that LIG4 deficiency induced DNA damage responses and inhibited tumor onset via cellular senescence in our genetically engineered in vivo model, 30% of mice in this group developed prostate cancers. Our study demonstrated that these prostate cancers resistant to LIG4 inhibition exhibited increased PD-L1 expression. We proposed that increased PD-L1 expression may allow these cancers to escape immune surveillance while increasing their sensitivity to immune checkpoint blockade. PD-1 antibody treatment has not been particularly effective in prostate cancer clinical trials (43, 44). Our study demonstrated that LIG4 deficient prostate cancers were sensitive to immune checkpoint blockade with PD-1 antibody. DNA damage induced by LIG4 deficiency may sensitize these tumors to PD-1 antibody treatment. Previous studies have demonstrated that cancers with DNA repair defects resulting in high mutational burden display increased sensitivity to immune checkpoint blockade (45). Immune checkpoint blockade allowed reduced cisplatin dose in a urothelial cancer model (46). Future studies will evaluate if LIG4 deficiency can predict response to immune checkpoint blockade in human prostate cancer.

Conclusion

DNA ligase 4 inhibition sensitizes prostate cancer to immune checkpoint blockade by promoting CD8+ T lymphocyte infiltration.

Acknowledgements

We thank Drs. Ke Ma, Balaji Ganesh, and Mark Maienschein-Cline for assistance with confocal microscopy, flow cytometry, and bioinformatics.

Footnotes

  • Authors’ Contributions

    JW and AML performed experiments, analyzed data, and wrote the manuscript. DLC conceived the study, analyzed data, and edited the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Funding

    This study was supported by the University of Illinois Cancer Center.

  • Received December 30, 2024.
  • Revision received January 22, 2025.
  • Accepted January 29, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. American Cancer Society
    . Key statistics for prostate cancer. Available at: www.cancer.org/cancer/types/prostate-cancer/about/key-statistics.html [Last accessed on January 28, 2025]
    1. Zhou M
    : High-grade prostatic intraepithelial neoplasia, PIN-like carcinoma, ductal carcinoma, and intraductal carcinoma of the prostate. Mod Pathol 31: 71-79, 2018. DOI: 10.1038/modpathol.2017.138
    OpenUrlCrossRefPubMed
  3. ↵
    1. Cancer Genome Atlas Research Network
    : The molecular taxonomy of primary prostate cancer. Cell 163(4): 1011-1025, 2015. DOI: 10.1016/j.cell.2015.10.025
    OpenUrlCrossRefPubMed
  4. ↵
    1. Williamson A,
    2. Leiros HS
    : Structural insight into DNA joining: from conserved mechanisms to diverse scaffolds. Nucleic Acids Res 48(15): 8225-8242, 2020. DOI: 10.1093/nar/gkaa307
    OpenUrlCrossRefPubMed
  5. ↵
    1. Grupp K,
    2. Roettger L,
    3. Kluth M,
    4. Hube-Magg C,
    5. Simon R,
    6. Lebok P,
    7. Minner S,
    8. Tsourlakis MC,
    9. Koop C,
    10. Graefen M,
    11. Adam M,
    12. Haese A,
    13. Wittmer C,
    14. Sauter G,
    15. Wilczak W,
    16. Huland H,
    17. Schlomm T,
    18. Steurer S,
    19. Krech T
    : Expression of DNA ligase IV is linked to poor prognosis and characterizes a subset of prostate cancers harboring TMPRSS2:ERG fusion and PTEN deletion. Oncol Rep 34(3): 1211-1220, 2015. DOI: 10.3892/or.2015.4080
    OpenUrlCrossRefPubMed
  6. ↵
    1. Damaraju S,
    2. Murray D,
    3. Dufour J,
    4. Carandang D,
    5. Myrehaug S,
    6. Fallone G,
    7. Field C,
    8. Greiner R,
    9. Hanson J,
    10. Cass CE,
    11. Parliament M
    : Association of DNA repair and steroid metabolism gene polymorphisms with clinical late toxicity in patients treated with conformal radiotherapy for prostate cancer. Clin Cancer Res 12(8): 2545-2554, 2006. DOI: 10.1158/1078-0432.CCR-05-2703
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Pugh TJ,
    2. Keyes M,
    3. Barclay L,
    4. Delaney A,
    5. Krzywinski M,
    6. Thomas D,
    7. Novik K,
    8. Yang C,
    9. Agranovich A,
    10. McKenzie M,
    11. Morris WJ,
    12. Olive PL,
    13. Marra MA,
    14. Moore RA
    : Sequence variant discovery in DNA repair genes from radiosensitive and radiotolerant prostate brachytherapy patients. Clin Cancer Res 15(15): 5008-5016, 2009. DOI: 10.1158/1078-0432.CCR-08-3357
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Munkley J,
    2. Maia TM,
    3. Ibarluzea N,
    4. Livermore KE,
    5. Vodak D,
    6. Ehrmann I,
    7. James K,
    8. Rajan P,
    9. Barbosa-Morais NL,
    10. Elliott DJ
    : Androgen-dependent alternative mRNA isoform expression in prostate cancer cells. F1000Res 7: 1189, 2018. DOI: 10.12688/f1000research.15604.1
    OpenUrlCrossRef
  9. ↵
    1. van der Leun AM,
    2. Thommen DS,
    3. Schumacher TN
    : CD8(+) T cell states in human cancer: insights from single-cell analysis. Nat Rev Cancer 20(4): 218-232, 2020. DOI: 10.1038/s41568-019-0235-4
    OpenUrlCrossRefPubMed
  10. ↵
    1. Micevic G,
    2. Bosenberg MW,
    3. Yan Q
    : The crossroads of cancer epigenetics and immune checkpoint therapy. Clin Cancer Res 29(7): 1173-1182, 2023. DOI: 10.1158/1078-0432.CCR-22-0784
    OpenUrlCrossRefPubMed
  11. ↵
    1. Gebhardt T,
    2. Park SL,
    3. Parish IA
    : Stem-like exhausted and memory CD8+ T cells in cancer. Nat Rev Cancer 23(11): 780-798, 2023. DOI: 10.1038/s41568-023-00615-0
    OpenUrlCrossRefPubMed
  12. ↵
    1. Patel D,
    2. McKay R,
    3. Parsons JK
    : Immunotherapy for localized prostate cancer: the next frontier? Urol Clin North Am 47(4): 443-456, 2020. DOI: 10.1016/j.ucl.2020.07.008
    OpenUrlCrossRefPubMed
  13. ↵
    1. Bishop JL,
    2. Sio A,
    3. Angeles A,
    4. Roberts ME,
    5. Azad AA,
    6. Chi KN,
    7. Zoubeidi A
    : PD-L1 is highly expressed in Enzalutamide resistant prostate cancer. Oncotarget 6(1): 234-242, 2015. DOI: 10.18632/oncotarget.2703
    OpenUrlCrossRefPubMed
    1. Li Y,
    2. Huang Q,
    3. Zhou Y,
    4. He M,
    5. Chen J,
    6. Gao Y,
    7. Wang X
    : The clinicopathologic and prognostic significance of programmed cell death ligand 1 (PD-L1) expression in patients with prostate cancer: a systematic review and meta-analysis. Front Pharmacol 9: 1494, 2019. DOI: 10.3389/fphar.2018.01494
    OpenUrlCrossRefPubMed
  15. ↵
    1. Sharma M,
    2. Yang Z,
    3. Miyamoto H
    : Immunohistochemistry of immune checkpoint markers PD-1 and PD-L1 in prostate cancer. Medicine (Baltimore) 98(38): e17257, 2019. DOI: 10.1097/MD.0000000000017257
    OpenUrlCrossRefPubMed
  16. ↵
    1. Graff JN,
    2. Alumkal JJ,
    3. Drake CG,
    4. Thomas GV,
    5. Redmond WL,
    6. Farhad M,
    7. Cetnar JP,
    8. Ey FS,
    9. Bergan RC,
    10. Slottke R,
    11. Beer TM
    : Early evidence of anti-PD-1 activity in enzalutamide-resistant prostate cancer. Oncotarget 7(33): 52810-52817, 2016. DOI: 10.18632/oncotarget.10547
    OpenUrlCrossRefPubMed
    1. Isaacsson Velho P,
    2. Antonarakis ES
    : PD-1/PD-L1 pathway inhibitors in advanced prostate cancer. Expert Rev Clin Pharmacol 11(5): 475-486, 2018. DOI: 10.1080/17512433.2018.1464388
    OpenUrlCrossRefPubMed
  18. ↵
    1. Vitkin N,
    2. Nersesian S,
    3. Siemens DR,
    4. Koti M
    : The tumor immune contexture of prostate cancer. Front Immunol 10: 603, 2019. DOI: 10.3389/fimmu.2019.00603
    OpenUrlCrossRefPubMed
  19. ↵
    1. Wu J,
    2. Crowe DL
    : Telomere DNA damage signaling regulates prostate cancer tumorigenesis. Mol Cancer Res 18(9): 1326-1339, 2020. DOI: 10.1158/1541-7786.MCR-19-1129
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Majerčíková Z,
    2. Dibdiaková K,
    3. Galanda M,
    4. Galanda T,
    5. Richterová R,
    6. Kolarovszki B,
    7. Račay P,
    8. Hatok J
    : Quantitative profiling of genes associated with cancer pathways in brain tumors. Klin Onkol 36(3): 224-233, 2023. DOI: 10.48095/ccko2023224
    OpenUrlCrossRefPubMed
  21. ↵
    1. Fukuda J,
    2. Sudo T,
    3. Kikuchi M,
    4. Kawahara A,
    5. Shigyo H,
    6. Kawamoto Y,
    7. Shimamura S,
    8. Koga F,
    9. Noguchi T,
    10. Nakane H,
    11. Shigaki T,
    12. Fujiyoshi K,
    13. Yomoda T,
    14. Yoshida N,
    15. Koushi K,
    16. Yoshida T,
    17. Akiba J,
    18. Fujita F
    : Identification of DNA repair-related genes as biomarkers reflecting MSI, TMB, and TIL in colorectal cancer. Anticancer Res 44(8): 3567-3575, 2024. DOI: 10.21873/anticanres.17179
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Ribeiro HL,
    2. Maia ARS,
    3. De Oliveira RTG,
    4. Costa MB,
    5. Farias IR,
    6. De Paula Borges D,
    7. De Sousa JC,
    8. Magalhães SMM,
    9. Pinheiro RF
    : DNA repair gene expressions are related to bone marrow cellularity in myelodysplastic syndrome. J Clin Pathol 70(11): 970-980, 2017. DOI: 10.1136/jclinpath-2016-204269
    OpenUrlAbstract/FREE Full Text
    1. Junior HLR,
    2. De Oliveira RTG,
    3. De Paula Borges D,
    4. Costa MB,
    5. Farias IR,
    6. Dos Santos AWA,
    7. Magalhães SMM,
    8. Pinheiro RF
    : Can synthetic lethality approach be used with DNA repair genes for primary and secondary MDS? Med Oncol 36(12): 99, 2019. DOI: 10.1007/s12032-019-1324-7
    OpenUrlCrossRefPubMed
    1. Ali R,
    2. Alabdullah M,
    3. Algethami M,
    4. Alblihy A,
    5. Miligy I,
    6. Shoqafi A,
    7. Mesquita KA,
    8. Abdel-Fatah T,
    9. Chan SY,
    10. Chiang PW,
    11. Mongan NP,
    12. Rakha EA,
    13. Tomkinson AE,
    14. Madhusudan S
    : Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers. Theranostics 11(17): 8350-8361, 2021. DOI: 10.7150/thno.51456
    OpenUrlCrossRefPubMed
    1. Peng L,
    2. Lidan H,
    3. Cuicui Z,
    4. Zhe Z,
    5. Sen Y,
    6. Xuan W,
    7. Ganghua L,
    8. Chao Z,
    9. Zhensheng L,
    10. Qiming W
    : DNA double-strand break repair capacity and its pathway gene variants predict the risk and prognosis of lung cancer. Lung Cancer 192: 107831, 2024. DOI: 10.1016/j.lungcan.2024.107831
    OpenUrlCrossRefPubMed
  26. ↵
    1. Zhang Y,
    2. Zheng J,
    3. Chen M,
    4. Zhao S,
    5. Ma R,
    6. Chen W,
    7. Liu J
    : Modulating DNA damage response in uveal melanoma through embryonic stem cell microenvironment. BMC Cancer 24(1): 519, 2024. DOI: 10.1186/s12885-024-12290-x
    OpenUrlCrossRefPubMed
  27. ↵
    1. Kim DH,
    2. Oh SY,
    3. Kim SY,
    4. Lee S,
    5. Koh MS,
    6. Lee JH,
    7. Lee S,
    8. Kim SH,
    9. Park HS,
    10. Hur WJ,
    11. Jeong JS,
    12. Ju MH,
    13. Seol YM,
    14. Choi YJ,
    15. Chung JS,
    16. Kim HJ
    : DNA Ligase4 as a prognostic marker in nasopharyngeal cancer patients treated with radiotherapy. Asian Pac J Cancer Prev 15(24): 10985-10989, 2015. DOI: 10.7314/apjcp.2014.15.24.10985
    OpenUrlCrossRef
  28. ↵
    1. Sakiyama T,
    2. Kohno T,
    3. Mimaki S,
    4. Ohta T,
    5. Yanagitani N,
    6. Sobue T,
    7. Kunitoh H,
    8. Saito R,
    9. Shimizu K,
    10. Hirama C,
    11. Kimura J,
    12. Maeno G,
    13. Hirose H,
    14. Eguchi T,
    15. Saito D,
    16. Ohki M,
    17. Yokota J
    : Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk. Int J Cancer 114(5): 730-737, 2005. DOI: 10.1002/ijc.20790
    OpenUrlCrossRefPubMed
    1. Jiang YH,
    2. Xu XL,
    3. Ruan HH,
    4. Xu WZ,
    5. Li D,
    6. Feng JG,
    7. Han QB,
    8. Mao WM
    : The impact of functional LIG4 polymorphism on platinum based chemotherapy response and survival in non-small cell lung cancer. Med Oncol 31(959): 959, 2014. DOI: 10.1007/s12032-014-0959-7
    OpenUrlCrossRefPubMed
    1. Li L,
    2. Tang S,
    3. Yin JC,
    4. Dong L,
    5. Yang Z,
    6. Liu Y,
    7. Ma J,
    8. Chang P,
    9. Pang J,
    10. Bao H,
    11. Mu D,
    12. Zheng X,
    13. Aishajiang R,
    14. He K,
    15. Zhang S,
    16. Ni M,
    17. Wu X,
    18. Wang X,
    19. Shao Y,
    20. Wang J,
    21. Ge H,
    22. Yu J,
    23. Yuan S
    : Comprehensive next-generation sequencing reveals novel predictive biomarkers of recurrence and thoracic toxicity risks after chemoradiation therapy in limited stage small cell lung cancer. Int J Rad Oncol Biol Phys 112(5): 1165-1176, 2022. DOI: 10.1016/j.ijrobp.2021.12.009
    OpenUrlCrossRefPubMed
  31. ↵
    1. Lee YH,
    2. Wang SC,
    3. Li CH,
    4. Chen LH,
    5. Shen TC,
    6. Liu YF,
    7. Wang YC,
    8. Tsai CW,
    9. Hsia TC,
    10. Bau DT,
    11. Chang WS
    : The contribution of DNA ligase 4 genetic variations to Taiwanese lung cancer. Anticancer Res 43(8): 3447-3453, 2023. DOI: 10.21873/anticanres.16520
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Liu Y,
    2. Zhou K,
    3. Zhang H,
    4. Shugart YY,
    5. Chen L,
    6. Xu Z,
    7. Zhong Y,
    8. Liu H,
    9. Jin L,
    10. Wei Q,
    11. Huang F,
    12. Lu D,
    13. Zhou L
    : Polymorphisms of LIG4 and XRCC4 involved in the NHEJ pathway interact to modify risk of glioma. Human Mutat 29(3): 381-389, 2008. DOI: 10.1002/humu.20645
    OpenUrlCrossRefPubMed
    1. Pearce CL,
    2. Near AM,
    3. Van Den Berg DJ,
    4. Ramus SJ,
    5. Gentry-Maharaj A,
    6. Menon U,
    7. Gayther SA,
    8. Anderson AR,
    9. Edlund CK,
    10. Wu AH,
    11. Chen X,
    12. Beesley J,
    13. Webb PM,
    14. Holt SK,
    15. Chen C,
    16. Doherty JA,
    17. Rossing MA,
    18. Whittemore AS,
    19. McGuire V,
    20. DiCioccio RA,
    21. Goodman MT,
    22. Lurie G,
    23. Carney ME,
    24. Wilkens LR,
    25. Ness RB,
    26. Moysich KB,
    27. Edwards R,
    28. Jennison E,
    29. Kjaer SK,
    30. Hogdall E,
    31. Hogdall CK,
    32. Goode EL,
    33. Sellers TA,
    34. Vierkant RA,
    35. Cunningham JM,
    36. Schildkraut JM,
    37. Berchuck A,
    38. Moorman PG,
    39. Iversen ES,
    40. Cramer DW,
    41. Terry KL,
    42. Vitonis AF,
    43. Titus-Ernstoff L,
    44. Song H,
    45. Pharoah PD,
    46. Spurdle AB,
    47. Anton-Culver H,
    48. Ziogas A,
    49. Brewster W,
    50. Galitovskiy V,
    51. Chenevix-Trench G, Australian Cancer Study, Australian Ovarian Cancer Study Group
    : Validating genetic risk associations for ovarian cancer through the international Ovarian Cancer Association Consortium. Br J Cancer 100(2): 412-420, 2009. DOI: 10.1038/sj.bjc.6604820
    OpenUrlCrossRefPubMed
    1. Zhao P,
    2. Zou P,
    3. Zhao L,
    4. Yan W,
    5. Kang C,
    6. Jiang T,
    7. You Y
    : Genetic polymorphisms of DNA double-strand break repair pathway genes and glioma susceptibility. BMC Cancer 13: 234, 2013. DOI: 10.1186/1471-2407-13-234
    OpenUrlCrossRefPubMed
    1. Su Y,
    2. Qi S,
    3. Dou C,
    4. Shuang L,
    5. Yan H
    : Association of LIG4 and XRCC4 gene polymorphisms with the risk of human glioma in a Chinese population. Int J Clin Exp Pathol 8(2): 2057-2062, 2015.
    OpenUrlPubMed
    1. Wang X,
    2. Sun CL,
    3. Hageman L,
    4. Smith K,
    5. Singh P,
    6. Desai S,
    7. Hawkins DS,
    8. Hudson MM,
    9. Mascarenhas L,
    10. Neglia JP,
    11. Oeffinger KC,
    12. Ritchey AK,
    13. Robison LL,
    14. Villaluna D,
    15. Landier W,
    16. Bhatia S
    : Clinical and genetic risk prediction of subsequent CNS tumors in survivors of childhood cancer: a report from the COG ALTE03N1 study. J Clin Oncol 35(32): 3688-3696, 2017. DOI: 10.1200/JCO.2017.74.7444
    OpenUrlCrossRefPubMed
  37. ↵
    1. Deng Y,
    2. Ke TW,
    3. Yueh TC,
    4. Chin YT,
    5. Wang YC,
    6. Hung YC,
    7. Mong MC,
    8. Yang YC,
    9. Wu WT,
    10. Chang WS,
    11. Gu J,
    12. Bau DT,
    13. Tsai CW
    : The contribution of DNA ligase 4 polymorphisms to colorectal cancer. In Vivo 38(1): 127-133, 2024. DOI: 10.21873/invivo.13419
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Soni A,
    2. Siemann M,
    3. Pantelias GE,
    4. Iliakis G
    : Marked contribution of alternative end-joining to chromosome-translocation-formation by stochastically induced DNA double-strand-breaks in G2-phase human cells. Mut Res Genet Toxicol Environ Mut 793: 2-8, 2015. DOI: 10.1016/j.mrgentox.2015.07.002
    OpenUrlCrossRefPubMed
    1. Kumari N,
    2. Antil H,
    3. Kumari S,
    4. Raghavan SC
    : Deficiency of ligase IV leads to reduced NHEJ, accumulation of DNA damage, and can sensitize cells to cancer therapeutics. Genomics 115(6): 110731, 2023. DOI: 10.1016/j.ygeno.2023.110731
    OpenUrlCrossRefPubMed
    1. Liberal FDCG,
    2. McMahon SJ
    : Characterization of intrinsic radiation sensitivity in a diverse panel of normal, cancerous and CRISPR-modified cell lines. Int J Mol Sci 24(9): 7861, 2023. DOI: 10.3390/ijms24097861
    OpenUrlCrossRefPubMed
  41. ↵
    1. Guerra Liberal FDC,
    2. Parsons JL,
    3. McMahon SJ
    : Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR edited cells. Med Phys 51(1): 591-600, 2024. DOI: 10.1002/mp.16764
    OpenUrlCrossRefPubMed
  42. ↵
    1. Kuhmann C,
    2. Li C,
    3. Kloor M,
    4. Salou M,
    5. Weigel C,
    6. Schmidt CR,
    7. Ng LW,
    8. Tsui WW,
    9. Leung SY,
    10. Yuen ST,
    11. Becker N,
    12. Weichenhan D,
    13. Plass C,
    14. Schmezer P,
    15. Chan TL,
    16. Popanda O
    : Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer. Hum Mol Genet 23(8): 2043-2054, 2014. DOI: 10.1093/hmg/ddt599
    OpenUrlCrossRefPubMed
  43. ↵
    1. De Velasco MA,
    2. Kura Y,
    3. Fujita K,
    4. Uemura H
    : Moving toward improved immune checkpoint immunotherapy for advanced prostate cancer. Int J Urol 31(4): 307-324, 2024. DOI: 10.1111/iju.15378
    OpenUrlCrossRefPubMed
  44. ↵
    1. Tsai AK,
    2. Kagalwalla S,
    3. Langer J,
    4. Le-Kumar T,
    5. Le-Kumar V,
    6. Antonarakis ES
    : Pembrolizumab for metastatic castration-resistant prostate cancer: trials and tribulations. Expert Opin Biol Ther 24(1-2): 51-62, 2024. DOI: 10.1080/14712598.2024.2311750
    OpenUrlCrossRefPubMed
  45. ↵
    1. Fumet JD,
    2. Limagne E,
    3. Thibaudin M,
    4. Truntzer C,
    5. Bertaut A,
    6. Rederstorff E,
    7. Ghiringhelli F
    : Precision medicine phase II study evaluating the efficacy of a double immunotherapy by durvalumab and tremelimumab combined with olaparib in patients with solid cancers and carriers of homologous recombination repair genes mutation in response or stable after olaparib treatment. BMC Cancer 20(1): 748, 2020. DOI: 10.1186/s12885-020-07253-x
    OpenUrlCrossRefPubMed
  46. ↵
    1. Sato R,
    2. Watanabe K,
    3. Matsushita Y,
    4. Watanabe H,
    5. Tamura K,
    6. Motoyama D,
    7. Otsuka A,
    8. Miyake H
    : Assessment of therapeutic effects of combined treatment with cisplatin and anti-mouse programmed death (PD)-1 antibody in a mouse urothelial cancer model. Anticancer Res 44(4): 1417-1423, 2024. DOI: 10.21873/anticanres.16938
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
DNA Ligase 4 Inhibition Sensitizes Prostate Cancer to Immune Checkpoint Blockade In Vivo
JIANCHUN WU, ANGELICA M. LAGUNAS, DAVID L. CROWE
Anticancer Research Mar 2025, 45 (3) 883-896; DOI: 10.21873/anticanres.17476
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords