siRNA-mediated silencing of placenta-specific protein 1 (PLAC1) alters CD4+, CD8+, and regulatory T cells in a murine colon cancer model

Abstract

PLAC1 has been found to be upregulated in colorectal cancer (CRC). However, its precise role and the molecular mechanisms driving CRC progression remain unclear. This study aimed to elucidate the role of PLAC1 in CRC progression and to evaluate its immunomodulatory effects using an in vivo syngeneic mouse model. Small interfering RNA (siRNA) was employed to silence PLAC1 expression in the CT26 murine colorectal cancer cell line. Gene silencing efficiency was confirmed by quantitative real-time PCR (qRT-PCR). CT26 cells were subcutaneously injected into BALB/c mice to establish an in vivo tumor model. Tumor growth was monitored, and immune responses were assessed by analyzing tumor-infiltrating lymphocytes (TILs) and splenic immune cell populations. Flow cytometry was used to characterize T cell subsets (CD3, CD4, CD8, FoxP3) and PLAC1 expression. PLAC1 knockdown was associated with a reduction in the frequency of regulatory T cells (Tregs) in both tumor and spleen. It was also associated with an increased proportion of CD3⁺CD4⁺ T cells in the spleen and CD3⁺CD8⁺ T cells in the tumor. Intratumoral administration of PLAC1-targeting siRNA was associated with reduced tumor volume, extended survival, and was associated with changes consistent with enhanced antitumor immunity in tumor-bearing mice. Silencing of PLAC1 was associated with decreased Treg frequency and increased effector T cell populations, suggesting a shift toward a less immunosuppressive tumor microenvironment. These findings may support further investigation of PLAC1 as a potential target for immunotherapeutic strategies in CRC and other malignancies expressing PLAC1.

Introduction

Colorectal cancer (CRC), accounting for approximately 10% of all new cancer cases, is the third leading cause of cancer-related deaths worldwide^1,2. In most sporadic cases, CRC starts as a benign polyp and progresses to cancerous stages over time. This transformation of normal colon mucosa into adenocarcinoma is driven by a combination of genetic and epigenetic alterations, influenced by aging and lifestyle-related risk factors. Approximately 2–5% of CRC cases are hereditary^3,4. Screening and early detection efforts have contributed to reduced CRC mortality rates. However, diagnostic approaches such as biopsy and imaging are associated with limitations, including high cost and the need for sufficient tumor cell content to ensure accurate detection⁵. Despite advancements in cancer treatment modalities, including surgery, chemotherapy, radiotherapy, and immunotherapy, a considerable proportion of patients with CRC experience limited clinical benefit, treatment resistance, or disease recurrence^6,7. Therefore, researchers are increasingly investigating combination therapies incorporating targeted agents as adjunctive strategies to enhance therapeutic efficacy and improve patient outcomes^3,7. Tumor-specific antigens, including cancer-testis antigens (CTAs), hold great promise for the development of effective cancer immunotherapies due to their restricted expression in normal tissues and elevated levels in tumors. Among these, placenta-specific 1 (PLAC1) is particularly noteworthy. PLAC1 is located on the X chromosome and plays a role in placental embryonic development. Although PLAC1 has been found to be upregulated in CRC, its precise functional role in tumor progression and immune modulation remains poorly understood. Targeted PLAC1 inhibition leads to decreased proliferation, migration, and invasion of breast cancer cells. In addition, silencing PLAC1 suppresses both cyclin D1 expression and protein kinase B (Akt) phosphorylation. Research indicates that PLAC1 is expressed in various human malignancies, including liver carcinoma, kidney carcinoma, prostate adenocarcinoma, and CRC. PLAC1 protein expression correlates closely with tumor differentiation, stage, and lymph node metastasis in CRC. Notably, elevated PLAC1 expression has been observed in most CRC cell lines and in 12.8% of CRC patient samples⁸.Concerning the immune response, PLAC1-expressing tumor cells can activate both cellular and humoral arms of the immune system⁹. Antigen-presenting cells (APCs) internalize apoptotic bodies derived from cancer cells and present PLAC1-derived peptides to T cells, potentially leading to the induction of PLAC1-specific cytotoxic T lymphocytes (CTLs) and antibody responses. Given the insights into PLAC1’s role in cancer development and its expression across malignancies of diverse tissue origins, PLAC1 has emerged as a promising CTA for targeted immunotherapy. In addition to its therapeutic effects on tumor cells, targeting PLAC1 may also help restore immune cell function within the tumor microenvironment^10,11. In this study, small interfering RNA (siRNA) was employed to achieve specific and sustained silencing of PLAC1 in the CT26, a murine colorectal cancer cell line. Specifically, we evaluated tumor-infiltrating lymphocytes and splenocytes in mice, focusing on CD3, CD4, CD8, and FoxP3 markers. This study aimed to elucidate the role of PLAC1 in CRC development and explore its potential immunomodulatory effects in vivo. Our findings provide novel in vivo evidence that PLAC1 silencing modulates the tumor immune microenvironment in a syngeneic mouse model of CRC.

Materials and methods

Mice

All animal experiments were conducted in accordance with the ARRIVE guidelines and approved by the Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.AEC.1402.007). Female BALB/c mice (6–8 weeks old, weighing 16–22 g) were obtained from the Royan Research Institute, Tehran, Iran. Mice were housed under specific pathogen-free (SPF) conditions with free access to water and standard laboratory diet for at least one week prior to the experiments. Female BALB/c mice were used to minimize stress-related variability associated with male aggression. Sex-dependent immune differences were not specifically evaluated in this study. Animals designated for tissue collection, mice were anesthetized with ketamine/xylazine (80/10 mg/kg, intraperitoneally) prior to sacrifice, and tissues were immediately harvested following cervical dislocation. Remaining animals intended for survival studies were euthanized at the end of the experiment by CO₂ inhalation followed by cervical dislocation to ensure death, in accordance with institutional ethical standards.

Cell culture

The CT26 murine colorectal cancer cell line was obtained from the Pasteur Institute of Iran (Tehran, Iran) and cultured in RPMI-1640 medium (Denazist, Iran) supplemented with 10% fetal bovine serum (FBS; Biosera, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Pen-Strep; Aminsan, Iran). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. (Fig. 1, Step 1,2).

Fig. 1.

Schematic representation of the experimental design.

Transfection of CT26 cells with PLAC1-specific siRNA

CT26 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well in 2 ml of antibiotic-free RPMI-1640 medium supplemented with 10% FBS. The cells were incubated at 37 °C in a CO₂ incubator until they reached 60–80% confluence, which was typically achieved within 18–24 h. Transfection of siRNA oligonucleotides (sc-61364, Santa Cruz Biotechnology) was performed using the Santa Cruz Transfection Reagent (sc-29528), according to the manufacturer’s instructions. PLAC1 siRNA (mouse; sc-61364, Santa Cruz Biotechnology) is a commercially validated pool of three target-specific siRNAs, each 19–25 nucleotides in length, designed to knock down murine PLAC1. Individual sequences are proprietary and not disclosed. Each vial contained 3.3 nmol of lyophilized siRNA, which was resuspended in 330 µl of RNase-free water to obtain a 10 µM stock solution. PLAC1 siRNA was added at 8 µl (80 pmols) per well. The commercially validated siRNA pool minimizes off-target effects. Efficient PLAC1 silencing was confirmed by qRT-PCR, and conclusions were made accordingly without relying solely on siRNA specificity. Non-transfected CT26 wild-type (WT) cells served as negative controls (Fig. 1, Step1).

Quantitative real-time PCR (qRT-PCR)

To assess the temporal pattern of gene expression following transfection, quantitative real-time PCR (qRT-PCR) was performed on transfected cells at 3, 6, 12, 24, and 48 h post-transfection. Total RNA was extracted from CT26 cells (~ 1 × 10⁷) using RNX-Plus reagent (Sinaclon, Tehran, Iran) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop 2000c UV–Vis spectrophotometer (Thermo Scientific, USA), with the OD260/OD280 ratio used to determine nucleic acid purity. First-strand cDNA was synthesized using the DENA ZIST cDNA Synthesis Kit (DENA ZIST, Mashhad, Iran) following the manufacturer’s protocol. qRT-PCR was performed using the Corbett Rotor-Gene 6000 system (QIAGEN) with Green High Rox Master Mix (Ampliqon, Denmark). The cycling settings were adjusted for an initial 15-min denaturation at 95 °C and 40 repeated cycles (15 s at 95 °C, 35 s at 59 °C, and 45 s at 72 °C). At the end of each run, a melting curve analysis was performed to confirm that only the target product was amplified and to verify specificity. Relative gene expression levels were quantified using the 2^−ΔΔCT method (Fig. 1, Step1). Fold changes in expression were calculated by normalizing the expression of the target gene (PLAC1) to the reference gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and comparing treated samples to controls. All qRT-PCR data are presented as mean ± standard deviation (SD) from three independent experiments (n = 3). Primer sequences used for real-time PCR were as follows: For gene PLAC1, forward: 5′-CATCCGCATCAAGGCTGTCTCT-3′, Reverse: 5′-TTGCTAGGTGCTTTCGCGGAGT-3′, for gene GAPDH Forward:5′-CATCACTGCCACCCAGAAGACTG-3′, Reverse: 5′-ATGCCAGTGAGCTTCCCGTTCAG-3ʹ.

Animal studies

Forty mice were randomly allocated into four groups (10 mice per group): Group I (Normal control)—tumor-free, untreated mice; Group II (WT control)—mice injected with wild-type CT26 cells and left untreated; Group III (WT + siRNA)—mice bearing wild-type CT26 tumors and treated with siRNA; and Group IV (TR + siRNA)—mice injected with PLAC1-transfected CT26 cells and treated with siRNA. The TR group was included as a confirmatory group to account for the transient nature of siRNA-mediated gene silencing and to evaluate whether short-term PLAC1 knockdown at the time of tumor establishment would influence subsequent tumor growth or immune profiles, compared with tumors treated with intratumoral siRNA alone.

To induce localized tumors, 1 × 10⁶ wild-type CT26 cells or 1 × 10⁶ CT26 cells transfected with PLAC1-specific siRNA suspended in 100 µL phosphate-buffered saline (PBS) were subcutaneously injected into the right flank of each mouse. (Fig. 1, Step2). Fourteen days after tumor inoculation, when tumors reached approximately 100–150 mm³, intratumoral siRNA injections were administered every 48 h. Initial injections used lower concentrations (50–70 pmol), followed by doses at 100 pmol per injection of PLAC1 siRNA. PLAC1 siRNA was complexed with transfection reagent at a 1:2 ratio, and then incubated at room temperature for 20 min prior to injection. For the intratumoral injection, we used a fine (27G) needle to deliver a minimal volume of 20 µL per tumor. Mice were randomized into groups to ensure age and body weight were balanced; tumor measurements were conducted by the investigators without blinding; flow cytometry analysis was performed by a blinded operator. All treated animals received the same formulation, injection volume, and dosing schedule, ensuring reproducibility. Tumor dimensions were measured with a digital caliper, and volumes were calculated as: tumor volume (mm³) = 0.5 × (short diameter)² × (long diameter), (Fig. 1, Step3). On day 26 post-tumor inoculation, five mice per group (n = 5) were euthanized, and spleens and tumor tissues were collected for flow cytometry analyses. The remaining mice (n = 5 per group) were monitored for up to 2 months to assess survival (Fig. 1, Step4). For survival analysis, mice were monitored every two days for tumor growth, body weight, mobility, and food intake. The Mean of tumor volume and body weight in Table 1 correspond to the same time points, ensuring direct comparability between the data in the table and the tumor growth curve. Predefined humane endpoints included excessive tumor burden (maximum allowable tumor burden according to institutional animal care guidelines), tumor ulceration, or marked clinical deterioration manifested by substantial impairment in mobility or feeding behavior. Animals reaching these humane endpoints were humanely euthanized using carbon dioxide inhalation followed by cervical dislocation. No animals were euthanized prior to reaching the predefined humane endpoints. Mice that died spontaneously before meeting these criteria were recorded as natural deaths and included in the survival analysis.

Table 1.

Mean of body weight and tumor volume at selected time points during tumor progression.

Time (days)	Control (WT)	WT+siRNA	TR+siRNA
12	19.17 g 248.37 mm3	18.70 g 154.80 mm3	19.72 g 103.30 mm3
14	18.97 g 404.36 mm3	18.97 g 224.57 mm3	19.59 g 152.40 mm3
16	19.86 g 487.88 mm3	19.11 g 409.30 mm3	20.12 g 318.21 mm3
18	20.06 g 806.41 mm3	18.98 g 568.78 mm3	20.43 g 402.04 mm3
20	20.32 g 1346.13 mm3	18.93 g 1147.99 mm3	20.46 g 588.96 mm3
22	20.55 g 1881.12 mm3	19.24 g 1205.35 mm3	21.12 g 649.62 mm3
The full dataset was used for analysis and Table 1 shows representative time points for visualization.

Generation of single-cell suspensions of spleen and tumor tissues

Spleen and tumor tissues were collected on day 26 after tumor inoculation. Tumors were excised and mechanically dissociated into small fragments, followed by enzymatic digestion in serum-free RPMI-1640 medium containing 2 mg/mL collagenase type I and 10 U/mL DNase I for 60 min at 37 °C to obtain single-cell suspensions. The digested tissue was passed through a 70 μm cell strainer, pooled, and centrifuged at 300×g for 10 min. Spleens were aseptically harvested, immediately placed in serum-free RPMI-1640 medium, and mechanically dissociated by gently pressing through a 70 μm cell strainer. The resulting single-cell suspensions were centrifuged at 300×g for 10 min, followed by two rounds of red blood cell lysis (5 min each) using RBC lysis buffer. Pellets were washed twice with PBS, and viable cells were counted by trypan blue exclusion (0.4%, w/v), (Fig. 1, Step5).

Flow cytometry

Flow cytometry was performed to evaluate immune cell frequencies in tumor tissue and spleen, in order to assess local and systemic immune responses induced by treatment. PLAC1 staining and FoxP3 intracellular staining were performed in separate staining panels to avoid fluorophore overlap, as both antibodies were conjugated to PE. Cells were initially gated based on FSC/SSC characteristics to exclude debris and non-viable events, followed by singlet discrimination prior to downstream analysis. For surface staining of T cell subsets, cells were incubated for 30 min at room temperature in the dark with CD3-APC, CD4-PerCP, and CD8-AF488. In a separate staining panel, PLAC1 expression was evaluated specifically in the tumor cell population using rabbit anti-mouse PLAC1 IgG followed by goat anti-rabbit IgG-PE (BioLegend, San Diego, CA), in order to assess the efficiency of PLAC1 silencing. Tumor cell populations for PLAC1 analysis were operationally gated based on FSC/SSC characteristics, excluding lymphocytes to ensure specificity to tumor cells. For intracellular FoxP3 staining, cells from a dedicated panel were fixed and permeabilized using True-Nuclear™ 10X Perm Buffer according to the manufacturer’s instructions, followed by incubation for 30 min in the dark with mouse anti-FoxP3-PE antibody. Cells were washed by centrifugation (250×g, 5 min) in staining buffer (PBS containing 2% FBS), resuspended in 200 µL staining buffer, and analyzed by flow cytometry. CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, CD3⁺CD4⁺FoxP3⁺ regulatory T cells (Tregs) were identified within the CD3⁺ gate. Unstained, single-color, and fluorescence-minus-one (FMO) controls were included to minimize background noise, ensure proper compensation, and reliably identify immune cell subsets as well as PLAC1⁺ tumor cells (Fig. 1, Step 6). Data were analyzed using FlowJo, and statistical analyses were performed using GraphPad Prism 8. All results are presented as percentages of total analyzed single-cell populations. Absolute cell counts were not determined.

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). The normality of data distribution was assessed using the Shapiro–Wilk and Kolmogorov–Smirnov (KS) tests. Differences between two groups were evaluated using the unpaired Student’s t-test. Differences among multiple groups were analyzed by one-way ANOVA followed by Tukey’s and Dunnett’s multiple comparisons tests. Survival analysis was conducted using Kaplan–Meier curves, and statistical differences in survival rates among groups were determined using the log-rank (Mantel–Cox) test. Data are presented as mean ± standard deviation (SD). A p-value < 0.05 was considered statistically significant, with significance levels denoted as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Result

siRNA-mediated inhibition of PLAC1 expression in the CT26 cell line

PLAC1 gene expression in the CT26 cell line was assessed. Gene silencing efficiency was evaluated by quantitative real-time PCR at 3, 6, 12, 24, and 48 h post-transfection with PLAC1-specific siRNA. PLAC1 expression decreased at 12, 24, and 48 h compared to untreated controls (Fig. 2a), with the most pronounced suppression occurring at 48 h post-transfection (p < 0.0001).

Fig. 2.

Effects of siRNA treatment on PLAC1 expression at the mRNA and protein levels. (a) PLAC1 mRNA expression levels were quantified by real-time PCR and normalized to GAPDH. Data are presented as mean ± SD from three independent experiments. (b) Flow cytometry analysis of PLAC1 protein expression, shown as the percentage of PLAC1⁺ tumor cells, was performed in tumor tissues from WT, WT+siRNA, and TR+siRNA tumor-bearing mice. PLAC1 staining was conducted in a dedicated staining panel, and PLAC1 expression was evaluated specifically within the tumor cell population. (c) Representative flow cytometry histograms showing PLAC1⁺ tumor cell populations in the WT, WT+siRNA, and TR+siRNA groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

In vivo downregulation of PLAC1 protein following siRNA-mediated gene silencing

CT26 tumors were established in BALB/c mice, followed by local administration of PLAC1-specific siRNA at the tumor site. PLAC1 protein expression was analyzed in all three tumor-bearing groups, with the wild-type (WT) group as control. Flow cytometric analysis demonstrated a lower proportion of PLAC1⁺ tumor cells in both siRNA-treated groups compared with the WT group (p < 0.01) (Fig. 2b).

In vivo antitumor effects of PLAC1 silencing

Following favorable in vitro results, in vivo studies were conducted. A schematic overview of the in vivo evaluation steps is provided in Fig. 3a. Tumor growth reduction was observed in both siRNA-treated groups compared to controls, indicating that intratumoral siRNA injection significantly inhibited tumor growth (Fig. 3b–c). No significant changes in body weight were detected across all groups during the treatment period, suggesting minimal toxicity of siRNA administration (Fig. 3d). Median survival was prolonged in both siRNA-treated groups compared with the WT control group (Fig. 3e).

Fig. 3.

Assessment of the in vivo antitumor efficacy of PLAC1-specific siRNA in tumor-bearing mice. (a) Schematic illustration of the in vivo experimental design related to b–e. (b) Representative tumor images from each group on day 26 post-inoculation. (c) Tumor volume. (d) Changes in body weight. (e) Survival rates of treated mice up to 2 months post-inoculation. Data are presented as mean ± SD (n = 5 mice per group); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Alterations of T cell subsets in the spleen and tumor following PLAC1 knockdown via siRNA

Flow cytometry analyses were performed using spleen and tumor samples from five mice per group (n = 5). In the spleen, the frequency of total CD3⁺ T cells was significantly reduced in all tumor-bearing mice compared with normal controls. within the CD3⁺ T-cell gate, CD3⁺CD4⁺ T cells were significantly increased in the WT (p < 0.05), WT+siRNA (p < 0.01), and TR+siRNA (p < 0.001) groups, whereas CD3⁺CD8⁺ T cells were significantly decreased in all tumor-bearing mice. CD3⁺CD4⁺ FoxP3 ⁺ Tregs was reduced in both siRNA-treated groups (p < 0.01), consistent with diminished systemic immunosuppression. In contrast, the WT group showed an increased Treg frequency (p < 0.01) (Fig. 4a–d).

Fig. 4.

Evaluation of immune cell population changes in spleen and tumor following PLAC1 silencing. (a–d) Frequencies of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and CD3⁺CD4⁺ FoxP3 ⁺ regulatory T (Treg) cells in the spleen. (e–h) Corresponding frequencies of these immune cell populations in tumor tissues. Immune cell frequencies were calculated as percentages of total CD3⁺ T cells within each gate, to reflect relative abundance of specific subsets within the T-cell compartment. (i, j) Representative flow cytometry histograms from one mouse per group. Data are presented as mean ± SD (n = 5 mice per group); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant.

In tumor tissues, the overall frequency of CD3⁺ T cells were reduced in both siRNA-treated groups compared with the WT group (p < 0.0001). However, within the CD3⁺ T-cell gate, CD3⁺CD8⁺ T cells was increased in the WT+siRNA (p < 0.05) and TR+siRNA (p < 0.0001) groups, indicating a shift toward a CD8⁺ T-cell–enriched intratumoral T-cell population within the tumor microenvironment. Consistent with the spleen findings, the proportion of CD3⁺CD4⁺ FoxP3 ⁺ Tregs was reduced in both siRNA-treated groups (p < 0.01) (Fig. 4e–h). These immune alterations should be interpreted with caution, as non-specific inflammatory responses induced by siRNA delivery or transfection reagents cannot be fully excluded in the absence of appropriate control siRNAs.

Discussion

PLAC1 is a CTA with an approximate molecular weight of 23 kDa, predominantly localized on the cell surface. Its expression is restricted in normal tissues but is markedly upregulated in various malignancies, including CRC^8,12,13. Overexpression of PLAC1 has been consistently associated with aggressive tumor behavior and poor clinical outcomes. It promotes cell proliferation by upregulating cyclin D1 and activating the Akt signaling pathway, as reported in previous studies. Moreover, PLAC1 contributes to epithelial–mesenchymal transition (EMT) through inflammatory mediators such as CXCL1^14,15. Additionally, it has been reported that PLAC1 enhances angiogenesis and promotes invasion and metastasis in colorectal cancer cell models^16,17. siRNAs have demonstrated considerable potential as both research tools and therapeutic agents across a broad spectrum of diseases^18,19. In our in vivo study, siRNA-mediated silencing of PLAC1 in CT26 cells not only reduced tumor growth and prolonged survival but was associated with measurable changes in immune cell phenotypes within the tumor microenvironment in BALB/c mice. These findings suggest an association between PLAC1 expression and immune modulation in CRC, extending its known functions beyond tumor growth and metastasis. In this study, we investigated PLAC1 expression at the mRNA level in the CT26 cell line. A reduction in PLAC1 mRNA levels was detected as early as 12 h post-siRNA transfection, with maximal suppression observed at 48 h, demonstrating the efficiency and time-dependence of PLAC1 gene silencing. To evaluate PLAC1 protein expression, flow cytometric analysis was conducted on tumor-derived cell suspensions isolated from mice. A rabbit anti-mouse PLAC1 IgG was used as the primary antibody, followed by a PE-conjugated goat anti-rabbit IgG as the secondary antibody. The analysis revealed a substantial reduction in PLAC1 surface expression, further confirming the effectiveness of siRNA-mediated knockdown at both transcriptional and translational levels. Considering the important role of the PLAC1 in tumor progression, we examined immune cell alterations in the spleen and tumor tissues of siRNA-treated mice compared with untreated tumor-bearing controls. In the spleen, the frequencies of total CD3⁺ T cells, CD3⁺CD8⁺ T cells, and CD3⁺ CD4⁺ FoxP3 ⁺ Tregs were reduced, while the frequency of CD3⁺CD4⁺ T cells was elevated in tumor-bearing mice. In contrast, tumor tissues exhibited an increased frequency of CD3⁺CD8⁺ T cells and a reduced frequency of CD3⁺CD4⁺ FoxP3 ⁺ Tregs, which is consistent with a shift toward a less immunosuppressive immune phenotype within the tumor microenvironment. The observed changes in Treg frequencies may reflect alterations in immune cell composition or FoxP3 -associated regulatory processes, rather than direct mechanistic regulation by PLAC1. Previous studies have reported that PLAC1 may contribute to the recruitment of immunosuppressive cell populations, including Tregs and myeloid-derived suppressor cells (MDSCs), through the upregulation of chemokines such as CCL5 and CXCL1, thereby facilitating the development of an immunosuppressive tumor microenvironment^10,20–22. Although these pathways were not directly assessed in the present study, and no mechanistic conclusions regarding chemokine-mediated immune recruitment can be drawn, PLAC1 inhibition was associated with immune changes consistent with a less immunosuppressive phenotype. Notably, a reduction in the total CD3⁺ T cell population was observed in both the spleen and tumor tissues, which may reflect shifts in relative T-cell subset composition rather than a global reduction in T-cell immunity¹⁰. These findings suggest that PLAC1 downregulation may be associated with immune alterations consistent with reduced tumor-induced immunosuppression and alterations adaptive immune responses, particularly reflected by increased T CD8⁺ cell frequencies. Therefore, the observed immune changes should be viewed as associations observed in the context of confirmed PLAC1 downregulation, rather than as definitive evidence of PLAC1-specific immune regulation. It is important to note that the present study focused exclusively on T-cell subsets within the tumor and spleen. Innate immune populations, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), were not evaluated. Given the critical roles of these cells in shaping the tumor microenvironment and modulating antitumor immunity, future studies incorporating these populations will be essential to fully elucidate PLAC1’s immunoregulatory potential. Such findings are in agreement with previous studies showing that tumors actively modulate immune responses within secondary lymphoid organs^22–25. Notably, PLAC1 may contribute to immunosuppression through mechanisms that extend beyond conventional immune checkpoint pathways. Our findings in the CT26 model are consistent with a potential association between PLAC1 expression and immune resistance, suggesting a multifaceted role for PLAC1 at both structural and cellular levels²⁶. The potential of siRNA-based therapies to overcome tumor-induced immune evasion has been supported by evidence from multiple preclinical studies. For instance, CpG–STAT3 siRNA conjugates²⁷, 4-1BB aptamer–CD25 siRNA constructs²⁸ and co-silencing of STAT3 and PD-L1²⁹ have each demonstrated efficacy in restoring CD8⁺ T cell functionality in immune-resistant tumors.

Our findings demonstrating that PLAC1 silencing is associated with changes in effector T cell frequencies are aligned with previously reported siRNA-based strategies and underscore the therapeutic promise of RNA interference in cancer immunotherapy. Although the results are encouraging, several limitations warrant further investigation. Functional analyses of T cell subsets—such as cytokine secretion, proliferation, and cytotoxicity—were not performed in the current study and should be included in future work to yield a more comprehensive understanding of the immune mechanisms involved. Moreover, this study was restricted to the CT26 syngeneic model in BALB/c mice; further validation in additional preclinical models, including orthotopic and metastatic settings, is essential to enhance the translational relevance of these findings. In addition, only female BALB/c mice were used in this study; therefore, potential sex-dependent immune differences were not evaluated and should be addressed in future investigations.

Conclusion

In conclusion, our data demonstrate that PLAC1 silencing is associated with reduced tumor progression and immune microenvironment changes—including decreased frequencies of regulatory T cells and increased effector T cell populations—suggesting a shift toward a less immunosuppressive profile. These findings indicate that PLAC1 may serve as a potential target for further investigation, particularly in combination with immune checkpoint blockade (e.g., anti–PD-1/PD-L1 or anti–CTLA-4) or T cell–activating strategies. While the present study does not establish direct mechanistic links, it supports continued exploration of PLAC1-targeted approaches in colorectal cancer and other PLAC1-expressing malignancies³⁰.

Author contributions

Mojgan Esparvarinha: Conceptualization, Methodology, Data Curation, Validation, Investigation Software, Formal analysis, Preparation of the original draft. Hamid Nickho: Methodology, Visualization, Investigation, Preparation of the graphical abstract, Resources. Alireza Najafi: Methodology, Visualization, Resources. Ali Zarezadeh Mehrabadi: Methodology, Validation. Maryam Keykhaee: Software, Validation, Formal analysis. Leili Aghebati-Maleki: Methodology, Investigation, Visualization. Reza Falak: Supervision, Conceptualization, Funding acquisition. Reviewing and editing the manuscript. Mehdi Yousefi: Supervision, Conceptualization, Funding acquisition. Reviewing and editing the manuscript.

Funding

This study is the main part of the Ph.D. thesis of the first author and was supported by Tabriz University of Medical Sciences, through grant # 70937.

Data availability

All data for this article can be obtained from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council) and approved by the Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.AEC.1402.007).

Consent to participate

Not applicable.

Consent of publication

Not applicable.

Clinical trial number

Not applicable.

Informed consent

Not applicable.