
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00221-3
10.1016/j.tranon.2024.102094
102094
Original Research
Niraparib plays synergistic antitumor effects with NRT in a mouse ovarian cancer model with HRP
Lu Jiefang acg
Liu Haiying ac
Wang Binming a
Chen Chengcheng f
Bai Fumao d
Su Xiaoping bloooge@163.com
ef⁎⁎
Duan Ping dppddpp@wmu.edu.cn
ab⁎
a Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, China
b Oncology Discipline Group, The Second Affiliated Hospital of Wenzhou Medical University, China
c Department of Obstetrics and Gynecology, Lishui People's Hospital, China
d Department of clinical laboratory, The First Affiliated Hospital of Wenzhou Medical University, China
e School of Basic Medicine, Wenzhou Medical University, China
f Department of Gastrointestinal Surgery, The Second Afliated Hospital of Wenzhou Medical University, China
g Department of Obstetrics and Gynecology, The First Affiliated Hospital of Lishui College, China
⁎ Corresponding author at: Department of Obstetrics and Gynecology, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, China. dppddpp@wmu.edu.cn
⁎⁎ Corresponding author at: The Teaching and Research Section of Microbiology and Immunology, Wenzhou Medical University, China. bloooge@163.com
19 8 2024
11 2024
19 8 2024
49 10209424 4 2024
22 7 2024
11 8 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Niraparib can remodel the tumor immune microenvironment in the HRP ovarian cancer mouse model.

• Niraparib can promote M1 polarization of TAMs and CD8 + T cell infiltration in tumor.

• The synergistic anti-tumer mechanism of NRT combined with Niraparib may be related to CCL5, CXCL9 and CXCL10.

Objective

PARPi offers less clinical benefit for HRP patients compared to HRD patients. PARPi has an immunomodulatory function. NRT therapy targets tumor neoantigens without off-target immune toxicity. We explored the synergy between Niraparib and NRT in enhancing antitumor activity in an HRP ovarian cancer mouse model.

Methods

In the C57BL/6 mouse ID8 ovarian cancer model, the effect of Niraparib on reshaping TIME was evaluated by immune cell infiltration analysis of transcriptomic data. The antitumor effects of Niraparib, NRT, and their combined use were systematically evaluated. To corroborate alterations in TILs, TAMs, and chemokine profiles within the TIME, we employed immunofluorescence imaging and transcriptome sequencing analysis.

Results

Niraparib increased the M1-TAMs and activated CD8+ T cells in tumor tissues of C57BL/6 mice with ID8 ovarian cancer. GSEA showed that gene set associated with immature DC and INFα, cytokines and chemokines were significantly enriched in immune feature, KEGG and GO gene sets, meanwhile CCL5, CXCL9 and CXCL10 play dominant roles together. In the animal trials, combined group had a tumor growth delay compared with Niraparib group (P < 0.01) and control group (P < 0.001), and longer survival compared with the single agent group (P<0.01) .

Conclusions

Niraparib could exert immune-reshaping effects, then acts synergistic antitumor effects with NRT in HRP ovarian cancer model. Our findings provide new ideas and rationale for combined immunotherapy in HRP ovarian cancer.

Keywords

Ovarian cancer
Neoantigen-reactive T cells
PARPi
Homologous recombination
Tumor microenvironment
==== Body
pmcOvarian cancer is a highly deceptive and lethal disease, about 80 % of which are advanced stages (stage III/IV) when diagnosed, with a 5-year survival rate of approximately 50 % [1,2]. Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective as maintenance treatment after initial complete response (CR) or partial response (PR) to frontline debulking surgery and platinum-based chemotherapy in patients with advanced homologous recombination-deficient(HRD) ovarian cancer, as shown in the clinical trial SOLO1/GOG 3004 (NCT01844986) after a 5-year and 7-year follow-up and the PRIMA trial [3,4] (PRIMA/ENGOT-OV26/GOG-3012, NCT02655016) . However, for approximately 50 % of ovarian cancer patients who have Homologous Recombination Proficiency (HRP) [5], the benefits of maintenance treatment with PARPis are significantly lower compared to HRD-positive patients [[6], [7], [8]]. The PRIMA trial released its data in 2019, demonstrated that the median progression-free survival was 8.1 months in the HRP subgroup treated with Niraparib, significantly shorter than 22.1 months in the HRD subgroup [6].

PARPis play an crucial role in the treatment of ovarian cancer, not only because they could induce synthetic lethality in HRD ovarion cancer cells [9], but also could reshape the tumor immune microenvironment (TIME), including increased activation of the intratumorally recruitment of CD4+ and CD8+T cells, production of immune-stimulatory cytokines, recruitment of antigen-presenting dendritic cells, polarization of M1-tumor-associated macrophages, and inhibited activation of myeloid-derived suppressor cells and cancer-associated fibroblasts present in the extracellular matrix [10,11]. Moreover, by inhibiting tumor angiogenesis, PARPi can impede the formation of new blood vessels that support tumor growth. These can trigger an anti-tumor immune response that does not depend on the DNA Damage Response (DDR) status. Ultimately, PARPi can transform "cold tumors" into "hot tumors" - tumors that are unresponsive to immunotherapy into ones that are more susceptible to it. This transformation significantly increases the sensitivity of the tumor to immunotherapy, enhancing the chances of successful treatment [12].

Although immunotherapy has shown promising prospects, only a small proportion of ovarian cancer patients have responded well to date. Programmed death receptor-1 (PD-1) / programmed death ligand 1 (PD-L1) blockade is the most studied immunotherapy for ovarian cancer, but the response rate was only 8.0 to 22.2 % as a monotherapy trial [10,13]. The JAVELIN PARP MEDLEY trial (NCT03330405) which investigated the utility of a combination of immune checkpoint blockade (ICB) with PARPi have currently published that in a group of platinum-sensitive recurrent ovarian cancer patients without BRCA-mutation, the objective response rate (ORR) of the combination with Avelumab and Talazoparib was 20.0 %, and the median duration of responses is 3.9 months, similar to using PARPi or immune checkpoint inhibitors (ICIs) alone [14]. Given these results, it is necessary to explore new approaches by combining PARPi with other immunotherapies.

Tumor neoantigens which are specifically expressed in tumor cells, making them an attractive target for cancer therapy and a new research hotspot as they avoid the occurrence of extra-tumor targeted toxicity in recent years. Tumor neoantigen vaccines (Neo-VAC) and tumor neoantigen reactive T lymphocytes (NRT) therapies have shown exciting results in several types of solid tumors, including melanoma, nonsmall-cell lung cancer (NSCLC) , and colorectal cancer [12,15]. It has been observed that, after cultivation, NRT cells can infiltrate into tumors directly and can overcome the inhibition from the tumor immune microenvironment by modifying signaling molecules genetically [16]. However, only a limited group of patients might experience a clinical benefit from the NRT therapy, and its effectiveness is hindered by factors such as the tumor immune microenvironment, depletion of tumor-specific neoantigens, and other mechanisms [17]. Some studies have reported synergistic effects when combining PARPi with tumor Neo-VAC [[18], [19], [20]], offering valuable insights for the combination of PARPi and NRT therapy.

Thus, we investigated in this study the effects of PAPRi on the tumor-microenvironment of HRP ovarian cancer and the antitumor effects in combination with NRT. Our results found that Niraparib could increase the M1-TAMs and activated CD8+ T cells in tumor tissues of HRP ovarian cancer, and combination of Niraparib and NRT had a synergistic antitumor effect on HRP tumors.

Materials

The reagents and antibodies used in this study are listed in Supplementary Table 1.

Cell culture

The ID8 cell line is derived from Beijing Beinan Chuanglian Biotechnology Research Institute and has been validated as an HRP cell line undergone HRD scoring. The cells were cultured at 37 °C in 5 % CO2 in RPMI1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 U/mL penicillin (complete RPMI).

Mice

6–8 week-old female C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co. Ltd. Beijing, China. All animal experiments were carried out under specific pathogen-free conditions. The animal experiments were approved by the Animal Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University.

Tumour-bearing mouse model

At 6–8 weeks of age, female C57BL/6 mice were subcutaneously injected with 2 × 105 ID8 cells on the right lower abdomen. The mice were monitored daily, and tumor volume was measured daily or every other day. Since the tumor can be palpated subcutaneously, the tumor's longest diameter (D) and shortest diameter (d) were measured using a caliper to monitor tumor growth. The formula for calculating tumor volume is as follows: Tumor volume = (D) x (d)² x (π/6).

Tissue sequencing and neoantigen prediction

In order to profiled potential neoantigen, whole-exome sequencing (WES), RNA sequencing (RNA-seq) and transcriptomic sequencing were performed on ID8 cells and C57BL/6 mouse tail tissue, with a coverage depth of 200 × . In brief, according to manufacturer's instructions, RNA/DNA was extracted from ID8 cells, and DNA was extracted from the mouse tail tissue. RNA-seq expression was calculated using the HISAT2 and StringTie pipeline on the Illumina Novaseq 6000 platform (paired-end, 150 bp) to obtain FPKM/TPM values. Somatic mutation analysis of whole-exome sequencing data was performed using GATK (Mutect2) and Sentieon (TNscope): DNA somatic mutations with variant allele frequency (VAF) ≥ 0.005 were selected (referring to the minimum detectable mutation frequency of 0.005 in mutation analysis), and for each missense mutation, the binding affinity between 8 and 11 mer peptides and mouse MHC class I alleles (H-2-Db and H-2-Kb) was predicted by NetMHCpan4.0, while the binding affinity between corresponding 15-mer peptides and MHC class II allele (H-2-IAb) was predicted by NetMHCpanII3.2. The detected peptide segments were then scored using the final formula: priority-score = affinity-mutant-score * expression-score * float(allele-frequency) * normal-match. Finally, the mutations detected or peptides with gene expression in RNA-seq data were ranked based on the priority-score and considered as neoantigen candidates.

Personalized neoantigen long-peptide vaccine synthesis

To generate personalized neoantigen vaccines, researchers opted to select 10 potential neoantigen mutations and employ standard chemical solid-phase peptide synthesis techniques adhering to GMP-like standards (purity > 98 %, endotoxin concentration < 0.01EU/g, residual TFA < 1 %) to prioritize the synthesis of clinical-grade long peptides consisting of 27 amino acids. During the peptide preparation process, challenges related to sequence composition and hydrophobicity may lead to aggregation of fragments, resulting in difficulties in synthesis and purification. Consequently, only 8 out of the 10 peptides synthesized were successfully prepared for the development of new antigen vaccines. The peptides were grouped into 2 to 4 pools (designated pools 1 to 4, each containing 3 to 5 peptides at a concentration of 0.3 mg/peptide), and underwent rigorous quality control measures, including sterility, mycoplasma, pyrogen, and abnormal toxicity testing.

Generation of NRT

To acquire bone marrow-derived dendritic cells (BMDCs), euthanasia was performed on 6–8 week-old female C57BL/6 mice based on the Chinese Animal Care Guidelines. Initial dendritic cells (DCs) were cultured in complete RPMI at 37 °C and 5 % CO2, supplemented with 20 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) during the first 48 h, 10 ng/mL of GM-CSF during the following 6 days, 5 ng/mL of GM-CSF during the following 2 days. And 10 ng/mL of interleukin (IL)−4 was supplemented from the 6th day to the last day. 8 neoantigen peptides we prepared (25 µM per peptide) were added on the 10th day and 20 ng/mL of tumor necrosis factor (TNF) were supplemented during the following days. On day 12, Neo-DCs which were loosely adherent or floating in the culture were harvested.

Fresh spleens were obtained from the tumor-bearing C57BL/6 mice 10 days after subcutaneous inoculation of ID8 cells which were euthanized based on the Chinese Animal Care Guidelines. A single cell suspension was prepared from the spleen of tumor-bearing mice using Miltenyi gentle MACS™ Dissociator. The lymphocytes were either used fresh or immediately stored at −80 °C for future use.

The mature Neo-DCs were co-cultured with T cells at a ratio of 1:4 (DC:T) in complete RPMI supplemented 100 U/ml of IL-2 and 20 ng/ml of IL-7 at 37 °C and 5 % CO2 during the first 4 days, 30 ng/ml of OKT3 (CD3 monoclonal antibody) was added on day 6, and 3000 U/ml of IL-2 was supplemented from day 6 to the last day. On day 15, NRT were harvested either for freshly injected or stored at −80 °C.

Interferon‑γ (IFN-γ) ELIspot assay

According to the manufacturer's instructions, the IFN-γ Mabtech ELISPOT assay kit was used to measure the IFN-γ secretion by NRT cells through the ELISpot assay. Briefly, 2.5 × 105 conventional T cells or NRT cells were plated with 2.5 × 104 conventional DC or Neo-DC (DC was stimulated separately by each neoantigen-peptide, 200 µl per well) for 24 h without exogenously added cytokines at 37 °C. After, The cells were removed by emptying the plate and washing with PBS 5 times, then diluted detection antibody R4–6A2-biotin (1 µg/ml) were added into PBS containing 0.5 % FBS (100 µl/well), and incubated at room temperature for 2 h. After washed with PBS 5 times, diluted enzyme conjugate (streptavidin-hrp) was added at a 1:1000 ratio (100 µl/well) to the plate, and incubated at room temperature for 1 h. Substrate solution (BCIP/NBT, 100 µl/well) was added and incubated until the distinct spots appear. Then to stop color development, deionized water was added to the plate. Finally, the spots were imaged and analyzed. ImageJ software was used for counting analysis. Spots that are more than twice the spots in the control group were considered positive T cell reactivity.

Flow cytometry

The contents of CD3+ and CD137+ T cells in NRT were detected by flow cytometry after co-culturing with mature neo-DCs for 8 h. The anti-mouse monoclonal antibodies used for cell surface staining were CD3-APC and CD137-PE (clone: 41BB), which were all from BioLegend (San Diego, CA). In brief, cell particles were washed with FACS buffer and then incubated with anti-mouse CD16/32 at room temperature for 10 min to block the FC receptors. Surface antibodies were stained in the dark for approximately 20 min. To determine the intracellular levels of IFN-γ, the aforementioned procedure was used to stimulate 1–2 × 106 NRT/T cells. After stimulation, LIVE/DEAD fixable dye was used to exclude dead cells, followed by blocking of FC receptors and cell surface staining. Then, fixed and permeabilized cells were stained using anti-mouse monoclonal antibodies for CD3-APC and CD137-PE with the help of the Fixation/Permeabilization kit. Before collecting cells on an Arial II-Optics flow cytometer, cells were washed twice with FACS staining buffer or Perm/Wash™ buffer. All data were performed on live and single cells, and the measurements were performed in triplicate. All data were analyzed using FlowJo software.

Antitumor effects of Niraparib combinding with NRT in vivo

2 × 105 ID8 cells were injected subcutaneously into the right lower abdomen of female C57BL/6 mice. When the average tumor volume reaches approximately 20–30 mm3, the mice in Niraparib group and combined group are injected with Niraparib tosylate (50 mg kg-1 per day) on days 1–3. On day 4, 1.1 × 107 NRT is injected into the tail vein, followed by intraperitoneal injection of IL-2 (180,000 units) for three consecutive days (days 4–6). The mice were monitored and tumor volume was measured. Mice are euthanized when the tumor exceeds 20 mm in any dimension or a volume of 2000mm3, or when signs of distress such as ulceration, necrosis, or infection occur, or when there is interference with eating or walking. When the maximum tumor volume reached or closed to 2000mm3 in control group (day17), the experiment was terminated and all mice were euthanized, whole blood, plasma, tumor tissue, heart, lungs, liver, and kidneys were collected for relevant laboratory examination. In addition, we also performed the survival analysis experiments, the endpoints were set to reach a tumor size of 1500mm3 or the mice showing significant signs of sickness due to cumulative tumor burden.

Immunocyte infiltration analysis

Utilized a reference dataset (LM22) consisting of gene expression characteristics of 22 known immunocyte subtypes, the relative scores and proportions of different immune cell types were calculated for the FPKM expression files of transcriptome sequencing by Cibersort (R package). Visualization of the results can be achieved using online bioinformatics analysis platforms, such as those dedicated to metagenomics (http://bioinformatics.com.cn/) , or through the utilization of R packages.

Gene set enrichment analysis (GSEA)

To determine the enrichment of specific cellular functions and pathways, gene set enrichment analysis (GSEA) was performed including immune signature gene sets from MsigDB and HALLMARK, GO, KEGG gene sets. The analysis were used the free online data analysis platform, OmicShare tool (www.omicshare.com/tools), allowing for the identification of enriched gene sets and subsequent visualization.

Immunofluorescence analysis and histopathological evaluation

Subcutaneous mouse tumor blocks were perfused with 0.1 M PBS (pH 7.4) and embedded in optimal cutting temperature compound for cryosectioning. Cryosections were fixed in 4 % PFA at room temperature for 15 min. After washing with PBS, cryosections were incubated at room temperature with blocking solution (PBS containing 3 % donkey serum, 1 % BSA, 0.3 % Triton X-100, pH 7.4) for 30 min. Primary antibodies for immunofluorescence analysis included anti-CD3 antibody, anti-CD137 antibody, anti-HMGB1 antibody, anti-CRT antibody, anti-CXCL9 antibody, anti-CXCL10 antibody, and anti-CCL5 antibody. Secondary antibodies used were Cy3-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-rabbit IgG. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence microscopy images were obtained after incubating with fluorescently labeled secondary antibodies and DAPI, and were observed using ECLIPSE C1 confocal fluorescence microscope (Nikon, Japan).

Tissue pathology examination was carried out by Sevier Biotech Co., Ltd., in which the tumor samples was fixed in 3.5 % neutral buffered formaldehyde and H&E staining were performed. The H&E stained images were evaluated and photographed using an upright bright-field microscope.

Statistical analysis

The tumor volume data obtained from in vivo experiments did not meet the assumption of homogeneity of variances. Therefore, a mixed effects model analysis using SPSS software (IBM SPSS Statistics 27) was performed for statistical analysis. For the remaining data that met the assumptions of normality and homogeneity of variances, one-way analysis of variance was conducted using GraphPad Prism 9.0 software, and the data were expressed as mean ± standard error of the mean. Statistical significance was considered at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Graphs were generated using GraphPad Prism 9.0 software.

Results

Niraparib promotes polarization of M1 phenotype in tumor-associated macrophages (TAMs) and infiltration of CD8+ T cells within the immune competent mouse HRP ovarian cancer model (ID8 cell line)

We verified that the ID8 cell line was a HRP cell line using the HRD-related genomic scar score (scaHRD), with a total score of 20 (including LOH 2, LST 18, TAI 0). Then the C57BL/6 mouse subcutaneous tumor model with ID8 ovarian cancer were established to assess the changes of TIME before and after Niraparib administration by transcriptome sequencing and immune cell infiltration analysis. The results confirmed that compared to the control group, Niraparib treatment leading to an increased proportion of M1-TAMs and activated CD8+ T cell, as well as a decreased proportion of M2-TAMs (Fig. 1a & b). Furthermore, gene set enrichment analysis (GSEA) reveals that upon Niraparib treatment, out of 961 gene sets showing confident enrichment (P < 0.05), 567 displayed significant enrichment (P < 0.01). Among these, 351 gene sets, including those associated with immature dendritic cells and interferon alpha, were upregulated, whereas 216 were downregulated (Fig. 1c). In addition, cytokines-related and chemokine-related gene sets were up-regulated in both KEGG pathway and GO (Fig. 1d-f), indicating that chemokines may play an important role in the regulation of TIME. Interaction analysis of the leading subsets of chemokine-related enriched pathways by Wayne diagram found that C—C motifs (including CCL5) and C-X-C motif (including CXCL9 and CXCL10) play a major role after Niraparib treatment (Fig. 1g). These results suggest that in HRP ovarian cancer, Niraparib may increase the expression and secretion of chemokines such as CCL5, CXCL9, CXCL10 in tumor, and then change TIME by induce the activation and infiltration of CD8 + T cells, TAMs polarization away from the M2-phenotype to the M1-phenotype, thereby synergizing with immunotherapy.Fig. 1 Effect of Niraparib on infiltration of TAMs and CD8 + T cells in ID8 ovarian cancer TIME and enrichment of GSEA

a&b. Box plot(a) and bubble plot(b)of immune cell infiltration distribution analyzed using CIBERSORT for transcriptomic data. The results showed that the proportion of M1 macrophage phenotype was increased, and the proportion of M2 was reduced, and meanwhile an the proportion of activated CD8+ T cells was increased. c. GSEA of immune signature gene sets, showing significant enrichment of inflammation-related gene sets such as GSE7509_UNSTIM_VS_IFNA_STIM_IMMATURE_DC_DN(NES=1.8941579, FDR=0, P = 0).d&e.The GSEA of GO gene sets, showing an enrichment of chemokines and cytokine-related pathways (FDR<0.1, P = 0) .f. GSEA of KEGG pathway gene sets, showing a significant enrichment in the cytokine-cytokine receptor response pathway (NES=1.8385506, FDR=0.0058, P = 0) . g. Interaction analysis of the leader subset of chemokine-related gene sets showed that chemokines promoting immune responses, including CCL 5, CXCL 9, and CXCL10, played a dominant role in all subsets. n = 3.FDR, false discovery rate.FDR 〈0.25 for credible enrichment, | NES |〉 1 and P < 0.05/<0.01 indicate statistical significant results.

Fig. 1:

The neoantigens in the mouse ovarian cancer cell line ID8 was predicted, and the neoantigen-reactive T cells was successfully prepared

Adoptive cell transfer therapy is an important tumor immunotherapy method, and Neoantigen-reactive T cell transfer therapy provides a new option for patients who lack suitable CAR-T therapy targets. In this study, we conducted WES and RNA-seq analysis on ID8 cell lines and C57BL/6 mouse tail tissues, selected the top10 neoantigens and successfully prepared 8 NeoVAC (Supplemental Table 2), and then prepared NRT with good immunogenicity.

Activated NRTs were obtained by co-culture of Neo-DCs, which were obtained by stimulating dendritic cells (DCs) with successfully prepared neoantigen peptides, and tumor-bearing mouse T cells, and then expanded in vitro. The percentage of CD3+CD137+ T cells in the prepared NRTs, which were co-cultured with Neo-DCs for 8 h, was analyzed by flow cytometry to evaluate the quantity of activated T cells. The results showed that the percentage of CD3+/CD137+ T cells in the prepared NRTs was 8.48 %, significantly higher than the control group's 0.23 % (Fig. 2a & b).Fig. 2 Presents the preparation, function, and immune response evaluation of NRT cells: a. and b. After co-culturing with DCs for 8 h, T cells were collected, and activated T-cell percentage (CD3+/CD137+ T) in NRT preparation was significantly higher compared to the conventional T-cell. c and d. The ELISPOT detection image of conventional T cells and new antigen-specific T cells stimulated by various peptides, respectively, showing that the number of spots generated after stimulation with each peptide was more than twice as high as the negative control. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

Fig. 2

In addition, we separately induced the in vitro activation of NRTs by stimulating DCs with each neoantigen peptide. The immune reactivity of the live cells was evaluated by ELISpot assay to detect IFN-γ release. The spots were counted using ImageJ software, and the results showed that the number of spots generated after stimulation with each neoantigen peptide was more than twice as high as that of conventional T cells. The increase in spot numbers was particularly evident in response to ID8-Mkrn1–5, ID8-Hp1bp3–6, ID8-Lrrc45–7, ID8-Mrpl2–9, and ID8-Ecm1–10 (Fig. 1c & d). This result confirmed that the NRT we prepared exhibited excellent immunogenicity.

Niraparib and nrt treatment had synergistic antitumor effects in the C57BL/6 mouse ID8 ovarian cancer model

Previous preclinical (in mouse tumor model) and clinical studies of NRT treatment administered by our team have not shown ideal anti-tumor effects, and we need to explore combination strategies that can enhance efficacy. Our results provided a theoretical basis for the combination of PARPi (Niraparib) with NRT, but it is still unclear that whether there is a synergistic effect in in-vivo experiments. Therefore, we performed the validation in the C57BL/6 mice. When the tumor became palpable, the mice were randomly divided into four groups as follows: control group (inject PBS), Niraparib group (inject Niraparib tosylate), NRT group, and combined group (inject Niraparib tosylate+ NRT) (Fig. 3a). Statistical analysis of tumor volume and survival analysis showed that the tumor growth of the combined group was delayed compared with the single agent group (Fig. 3b & d), and the survival was prolonged (Fig. 3c). These results demonstrate that the combination of Niraparib and NRT therapy produced synergistic anti-tumor effects in the C57BL/6 mouse with ID8 ovarian cancer.Fig. 3 In vivo antitumor assay in ID8 mouse model.

a. ID8 cells were transplanted into C57BL/6 mice. The mice were randomly divided into four groups after successful modeling(n = 6). From day 7 to 9, Nilaparib was injected intraperitoneally into mice at a dose of 50 mg kg-1 per day in Niraparib group and the combined group. On day 10, 1.1 × 10^7 NRT cells were injected into tail venous in NRT group and combined group, and meanwhile IL-2 was injected intraperitoneallyata dose of 180,000 units per day for three consecutive days. On day 17, samples and dataes were collected according to the pre-planned sampling scheme when the maximum volume is up to 2000 mm3 in the control group. All mice were euthanized under specified conditions at the end of the trial. b. Lograms of tumor growth showed that Niraparib combined with adoptive treatment of NRT significantly suppressed tumor volume (p<0.01, n = 6) . c. The Kaplan-Meier survival curves: survival of mice in the combined group was prolonged (n = 6, P < 0.01). d. Representative views of tumor tissue from each experimental and control group. e. Body weight comparison of mice in each group: there was no significant difference compared with the control group. f. There were no significant differences in major indicators such as white blood cells, hemoglobin, platelets, liver and kidney function, blood lipids, and blood glucose among the groups compared to the normal reference values. g. Representative images of histopathological evaluation of major organs using H&E staining analysis: the tissue structures of heart, lungs, liver, and kidneys, removed from the mice on the 17thday, remained normalin all groups. And although some organs showed mild chronic granulocyte infiltration, no significant damage was observed. H&E microscopic examination: 40 ×, 200 × magnification. ns indicates no statistically significant difference, ** indicates P < 0.01, and **** indicates P < 0.0001.

Fig. 3

Biosafety is a crucial consideration in biomedical applications. The results showed that the body weight of mice in all groups increased over time, and there were no statistically significant differences between groups (Fig. 3e). Although individual indicators such as white blood cells (WBC) and aspartate aminotransferase (ALT) were slightly elevated compared to the normal reference range, there were no statistically significant differences between groups in major biochemical indicators (Fig. 3f). Histopathological analysis of major organs (heart, lungs, kidneys, and liver) using H&E staining showed that the tissue structures in all groups of mice remained normal, although some organs showed mild chronic granulocyte infiltration but no significant damage (Fig. 3g). These findings suggest that the use of Niraparib combined with NRT therapy would not induce immunologically related adverse events in organs, during the observation period. All of these indicate that the combination treatment regimen of Niraparib and NRT is associated with low toxicity.

Compared with NRT group, the combined group showed increased M1 polarization and infiltration of CD8+ T cells within the tumor tissue, as well as enrichment of immune-related chemokines and signaling pathways

To further validate our findings regarding the regulation of TME by Niraparib, we performed immunofluorescence staining analysis of CD3/CD137 on tumor tissue obtained from the in-vivo experiments. The results showed increased infiltration of CD3+ T cells and CD137+ T cells (activated T cells) in all the experimental group, and most pronounced in the combined group (Fig. 4a & b). Additionally, immunofluorescence staining analysis of M1 marker CD86 and M2 marker CD206 showed increased expression of CD86 in the Niraparib group and combined group compared to control group and NRT group respectively (Fig. 4c). Furthermore, we performed transcriptome sequencing analysis of tumor tissue from the NRT group and combined group. The analysis of immune cell infiltration showed a similar change to that treated by Niraparib previously, with an increased proportion of M1 phenotype and a decreased proportion of M2 phenotype in combined group, compared to NRT group, and the increase of the activated CD8+ T cell proportion as well (Fig. 4d & e).GSEA of the KEGG pathways showed that, in addition to the same enrichment of cytokines-related and chemokine-related gene sets (Fig. 4g & h), there was also a credible upregulation enrichment of gene sets including JAK-STAT pathway, T cell receptor response and antigen processing and presentation pathway (P < 0.001) (Fig. 4i & k), unlike Niraparib treated alone. To understand the functional enrichment after combined treatment, further analysis of Hallmark gene sets showed that in the top 10 gene sets, IL6/JAK/STAT3 pathway, INF α, INF γ, TNF α, and inflammation-related gene sets significant upregulated enriched (Fig. 4l & p), and hypoxia and angiogenesis related gene sets were upregulated enriched too (Fig. 4f).Fig. 4 Immunofluorescence staining analysis and transcriptome sequencing results of each group.a. and b. Representative images of CD3 (a. red) and CD137 (b. red) immunostaining and DAPI nuclear staining (blue) of tumors in control group and the three experimental groups, as indicated; scale bar = 50 mm. Increased expression of CD3 and CD137 was observed after Niraparib treatment, with the most significant increase in the combination group. c. Representative images of immunofluorescence staining of CD86 (M1, red) and CD206 (M2, green), and DAPI nuclear staining (blue) . Increased expression of M1 in the Niraparib group compared to the control group, and increased expression of M1 in the combination group compared to the NRT group. d and e. Bubble plot (d) and stacking diagram (e) of immune infiltration analysis of tumor tissue transcriptome sequencing. Increased proportion of M1 phenotype and activated CD8+ T cell, and decreased proportion of M2 phenotype in combined group compared to NRT group. f. In the GSEA of HALLMARK gene set, hypoxia and angiogenesis-related gene sets were upregulated enriched (P 0.05, FDR <0.1). g and h. GSEA of KEGG pathway gene sets showed enrichment of KEGG_CHEMOKINE_SIGNALING_PATHWAY(FDR=0.0167,P = 0), KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION(FDR=0, P = 0). i-p. The GSEA of KEGG pathway and HALLMARK gene sets showed significant upregulation of immune-related and inflammation-related gene sets, including T cell receptor pathway, JAK-STAT signaling pathway, antigen processing and presentation pathway, TNF- α and IFN- γ / α signaling pathways

Fig. 4:

In conclusion, our study confirms that Niraparib can induce M1 phenotype polarization of TAMs, promote the recruitment and activation of CD8+T cells, thereby transforming the immunosuppressive tumor microenvironment of HRP ovarian cancer into a "hot" anti-tumor immune microenvironment, and enhancing the synergistic anti-tumor effect of NRT treatment combined with Niraparib on mice with HRP ovarian cancer. Moreover, this novel treatment strategy is safe and warrants further investigation.

Discussion

Ovarian cancer is the deadliest gynecologic malignancy [1], with a mere 50 % 5-year survival rate following diagnosis [2]. Monotherapy or combination treatments involving PARPi, PD-1/PD-L1 inhibitors, and anti-angiogenic agents have shown some promise in improving the prognosis of advanced ovarian cancer patients [3,4,6,21,22]. However, for patients with HRP advanced ovarian cancer, clinical outcomes remain discouraging, and the need for effective treatment options remains largely unmet [23]. This study proposes a innovation scientific hypothesis to combined Niraparib with NRT, aimed at enhancing the antitumor effects of NRT in a HRP ovarian cancer mouse model that is immunocompetent, and further analysis the underlying mechanisms.

In the past decade, immunotherapy has fundamentally transformed the landscape of cancer treatment. Research into immunotherapy for advanced and recurrent ovarian cancer has been on the rise, but the efficacy has fallen short of expectations. Tumor heterogeneity and the complex tumor microenvironment, poor pro-inflammatory immune cells infiltration and active immune suppression within immunosuppressive tumor microenvironment have been significant obstacles in ovarian cancer immunotherapy. The antitumor effects of immunotherapy rely on the infiltration of antitumor immune cells, such as tumor-infiltrating lymphocytes (TILs), into the tumor [24] . In advanced ovarian cancer, PARPi, anti-angiogenic agents, and other therapies have been combined with ICIs to enhance the response rate and treatment outcomes of immunotherapy [25]. Ovarian cancer is one of the first malignancies in which TIL density was positively correlated with overall survival (OS) [26], providing a theoretical basis for TIL adoptive transfer therapy in ovarian cancer. NRT adoptive transfer therapy, as a form of specific targeting of tumor neoantigens within adoptive cell therapy (ACT), has been explored in recent years for various solid tumors, including melanoma [15], renal cell carcinoma [20], gastrointestinal malignancies [19], and lung cancer [27]. However, its effectiveness remains limited, especially in low-mutational burden ovarian cancer. The underlying reasons are linked to the exhaustion of effector T cells, the absence of new antigens, and the presence of a tumor immune-suppressive microenvironment [12]. The effectiveness of NRT largely depends on the infiltration and activation of tumor-killing T cells, the persistence of NRT in the body, and the formation of an antitumor immune microenvironment [28]. Therefore, exploring a combined treatment approach that integrates NRT therapy with strategies to reshape the TIME for synergistic antitumor effects may offer a promising avenue to improve response rates.

In order to enhance the antitumor efficacy of NRT, current approaches often involve pre-treatment strategies, such as low-dose cyclophosphamide, sub-lethal radiotherapy, and other agents that deplete regulatory T cells and suppress the tumor immune status, to reinvigorate and activate the antitumor activity of tumor-specific CD8+ T cells [12,29]. However, these strategies work insensitivity to ovarian cancer, and their side effects limits their clinical use. Therefore, there is a pressing need for a more clinically viable pre-treatment or combination therapy approach to advance the clinical application of NRT in ovarian cancer. Most previous studies have confirmed the immunomodulatory effects of PARPi on HRD ovarian cancer through the cGAS-STING pathway, but unknow on HRP’s. Our research validates that, in HRP ovarian cancer, Niraparib induces a "hot" tumor state favorable for the antitumor effects of NRT as well. In a sense, this offers a novel approach to expand the therapeutic window for Niraparib in HRP ovarian cancer. Analysis of immune cell infiltration in tumor tissues post-Niraparib treatment revealed increased M1-TAMs, activated memory CD8+ T cells, alongside decreased M2-TAMs. These findings suggest that Niraparib promotes an antitumor TIME. To confirm the results obtained from sequencing analysis, we performed immunofluorescence staining for CD3, CD137, CD86, and CD206 in tumor tissues, which further validated the increased infiltration of the M1 phenotype and activated T cells. Recent studies have reported the impact of PARPi on TAMs polarization and phagocytic function [[30], [31], [32]], which aligns with our research findings. In summary, we have explored the feasibility of combining Niraparib and NRT as a treatment strategy to enhance antitumor effects in advanced HRP ovarian cancer. We have also provided initial insights into how Niraparib may reshape TIME by promoting M1-TAMs and the infiltration of CD8+ T cells, potentially explaining the mechanisms underlying their synergistic antitumor effects with NRT.

Olaparib, Niraparib, and Rucaparib are recommended for the treatment of ovarian cancer. Large-scale clinical studies have confirmed that clinical benefit of PARPi has also been observed in HRP ovarian cancer, but still falls short of the advantages seen in HRD individuals [6,8,33,34], highlighting the urgent need for exploration of novel combination therapy approaches. Current research efforts aimed at enhancing the antitumor effects of PARPi in HRP ovarian cancer primarily focus on two main avenues. One approach involves inducing DNA damage and increase sensitivity of tumors to PARPi [9,[35], [36], [37], [38], [39], [40], [41], [42]]. The other is to reshape the TIME to promote a stronger antitumor immune response [43]. In our research, transcriptome sequencing data of tumor specimens did not show enrichment of gene sets related to homologous recombination repair and cell death pathways in GSEA enrichment analysis, suggests that the effects of Niraparib in our study may not primarily involve synthetic lethality. After Niraparib treatment, increased intratumorally infiltration of M1-TAMs and activated CD8+ T cells have been observed. This induction resulted in a tumor-microenvironment with good infiltration of cytotoxic T cells, elucidating a potential mechanism through which Niraparib enhances the antitumor effects of immunotherapy by improving the tumor immune infiltration microenvironment. This discovery is similar to results of the research conducted by Skelin, M. et al. [33]. Their study have revealed that the antitumor effect of Olaparib in HRP ovarian cancer patients is not primarily related to the direct cytotoxic effect on tumor cells through synthetic lethality. Instead, it is attributed to their regulation of tumor-associated-macrophages, which enhances macrophage phagocytic function and reshapes the pro-inflammatory antitumor tumor microenvironment, resulting in antitumor effects [33].

The infiltration density of CD8+ T cells is a key prognostic factor for long-term survival in patients with different types of cancer. Ovarian cancer patients with high expression of CCL5, CXCL9, CXCL10 have a favorable prognosis [44,45]. Moreover, the high expression of CCL5, CXCL9, CXCL10 in tumor cells is closely related to the recruitment of cytotoxic T cells and the M1 polarization of TAMs [46,47], and induced the recruitment and infiltration of CD8+ T cells into the tumor in ovarian cancer and colon cancer, thereby reducing the level of cancer metastasis and improving the survival rate [45]. Meanwhile, the infiltration of T lymphocytes and the tumor microenvironment have a significant impact on the efficacy of immunotherapy [11]. In order to understand whether the treatment of Niraparib has a similar effect on the expression of chemokines in the tumor in our experiment, we conducted a GSEA analysis of the transcriptome sequencing data of tumor tissue samples after Niraparib treatment. The results showed enrichment of the gene sets related to the activation of cytokines and chemokines, including CXCL9, CXCL10, CCL5 and their receptors, in the leading subset. In addition, the sequencing results verified the high expression of chemokines such as CCL5, CXCL9, CXCL10 in the tumor, combined with the increased M1 polarization in the tumor. We speculate that Niraparib can promote the secretion of chemokines such as CXCL9 and CXCL10, thereby inducing the M1 polarization of tumor-associated macrophages and the recruitment and activation of CD8+ T cells in the tumor, creating a "hot" tumor state that is conducive to the effectiveness of NRT. However, this hypothesis needs further experimental verification.

In contrast to these findings, other studies have demonstrated that within regions of the tumor affected by chronic hypoxia, chemokines like CCL5 and CCL2 can induce the recruitment of TAMs and Tregs, ultimately promoting pro-cancer effects [44]. Furthermore, CXCL9 and CXCL10 serve as endogenous angiogenesis inhibitors [48]. However, in the combination group of our study, the expressions of CCL2 and CCL5 were significantly elevated, and the angiogenesis gene set was upregulated, but the in-vivo experiments ultimately revealed antitumor characteristics in our study. It can be inferred that incorporating anti-angiogenic agents into our approach may further enhance the antitumor effects. In the future of cancer immunotherapy, the use of a "cocktail" approach that combines various mechanisms to bolster its antitumor effects offers a broader, more "panoramic" specificity in combating tumors. This approach is not constrained by antigen evasion or HR status and may well become a new treatment trend.

However, targeted therapy and immunotherapy are the two major research directions in ovarian cancer treatment. Due to the characteristics of different cell lines and models, usually different experimental systems are used for both [49,50]. In our study, we used the ID8 mouse-derived cell line to study subcutaneous tumor growth in C57BL/6 mice, which to some extent restricts the study of PARPi targeted therapy in human cell line molecular mechanisms. Therefore, further research and verification are needed to determine whether the expected effects can be achieved in humans. We look forward to more related studies in other human ovarian cancer cell lines in the future. We can also use syngeneic organoid models for relevant research to further verify the synergistic effect of PARPi and NRT and eventually apply this combination therapy strategy to clinical research.

In conclusion, our study suggests that Niraparib may modulate the TIME in ovarian cancer by regulating the chemokines CCL5 and CXCL9/10. This enriches our understanding of Niraparib's mechanisms of action in HRP ovarian cancer models and lays a theoretical foundation for the clinical application of combined therapy involving Niraparib and NRT. Through the optimization of treatment strategies and further exploration, we aim to take another small step forward in advancing the goal of improving clinical outcomes for advanced HRP ovarian cancer patients.

Fund numbers

1. The Major Science and Technology Special Project of Wenzhou Science and Technology Bureau (ZY2020009 )

2. 2019 Clinical Research project of the Second Affiliated Hospital of Wenzhou Medical University (1010293 )

3. The National Natural Science Foundation of China (82272967 )

4. National Health Commission Science Research Fund of 2024—Major Science and Technology Program of Health in Zhejiang Province (WKJ-ZJ-2410)

CRediT authorship contribution statement

Jiefang Lu: Writing – original draft, Software, Methodology, Formal analysis, Data curation, Conceptualization. Haiying Liu: Methodology, Data curation, Conceptualization. Binming Wang: Data curation. Chengcheng Chen: Data curation. Fumao Bai: Supervision, Funding acquisition. Xiaoping Su: Validation, Supervision, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization. Ping Duan: Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests

Ping Duan reports equipment, drugs, or supplies was provided by Shanghai Zai Lab Co., Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102094.
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