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. 2026 Mar 2;15(1):2640261. doi: 10.1080/2162402X.2026.2640261

Steroid receptor coactivator 3-deficient regulatory T cells eradicate multiple solid tumors in syngeneic mouse models

Nuri Sung a,‡, Eunsu Kim a,‡, Yosef Gilad a, Yuri Park a, Adam M Dean a, Yan Xia a, Jianming Xu a,b, Clifford C Dacso a,b,c, David M Lonard a,b,*, Sang Jun Han a,b,*
PMCID: PMC12959200  PMID: 41772940

ABSTRACT

Steroid receptor coactivator 3 (SRC-3) is highly expressed in regulatory T cells (Tregs) and is important for their immunosuppressive activity. Recently, we demonstrated that disrupting SRC-3 expression in Tregs eliminates triple-negative breast cancer (TNBC) and prostate cancer in syngeneic animal models by generating an anti-tumor immune microenvironment without inducing immune-related adverse events (irAEs). Further analysis of these mice revealed that SRC-3 knockout (KO) Tregs infiltrated breast tumors and facilitated the infiltration of CD8+, CD4+, and natural killer (NK) immune cells into the tumor microenvironment (TME). Given the anti-tumor effects of SRC-3KO Tregs in two different solid cancers, we sought to extend our studies to additional cancer types. Here, we showed that SRC-3KO Tregs exerted a potent antitumor immunity-like effect, capable of eradicating glioblastoma, melanoma, and lung cancer in their respective syngeneic mouse models by generating an anti-tumor immune environment. These results support the translational development of SRC-3-targeted Treg modulation as a safe and effective immunotherapy platform for treatment-refractory cancers.

Keywords: Regulatory T cells, steroid receptor coactivator 3, syngeneic murine cancer models, lung cancer, glioblastoma, melanoma

Introduction

Although they are not abundant, regulatory T cells (Tregs) are a critical component of the immune system that prevent autoimmune disease.1 his central role of Tregs in suppressing pathological immune responses is frequently co-opted by solid cancers to evade immune surveillance, thereby hindering the activity of effector immune cells within the tumor microenvironment (TME).2,3 Consequently, Tregs have become important therapeutic targets in cancer, driving the development of immune checkpoint blockade (ICB) therapies that have demonstrated significant efficacy in certain cancers such as melanoma, renal clear cell carcinoma, and lung cancer.4-6 However, the therapeutic benefits of ICB remain limited for other malignancies, including triple-negative breast cancer (TNBC), while some solid tumors exhibit minimal or no response to currently available ICB treatments.7 As a result, the estimated eligibility for ICB therapy has increased from 1.54% in 2011 to 56.55% in 2023.8 Moreover, ICB therapies are often associated with serious immune-related adverse events (irAEs), including organ-specific toxicities and chronic inflammation, with approximately 14% of patients experiencing grade 3 or 4 events.9,10

Steroid Receptor Coactivator-3 (SRC-3) functions as a coactivator for nuclear receptors and other transcription factors, dynamically modulating cellular physiology in response to external stimuli such as hormonal changes.11-13 SRC-3 activity partially overlaps with that of the other steroid receptor coactivator family members, SRC-1 and SRC-2, such that genetic disruption of the SRC-3 gene can alter cellular function without necessarily causing cell death.14 Due to its pivotal physiological role, alterations in SRC-3—such as gene amplification or elevated expression—are predominantly associated with oncogenic processes, promoting cancer cell proliferation, invasion, and metastasis by modulating nuclear receptor signaling.15

Beyond its function in cancer cells, SRC-3 also plays an essential role in the immune system. For instance, genetic disruption of SRC-3 results in B- and T-cell hyperproliferation and reduced cytokine production from macrophages.16,17 Pharmacological inhibition of SRC-3 with the small-molecule inhibitor SI-2 generates an anti-tumor immune environment by enhancing the recruitment of cytotoxic T cells and natural killer (NK) cells, thereby suppressing breast cancer progression.18 In regulatory T cells (Tregs), SRC-3 is critical for maintaining their proliferation and immunosuppressive functions.17 Remarkably, deletion of SRC-3 in Tregs reprograms their molecular properties, converting them from pro-tumorigenic to anti-tumor phenotypes.19 As a result, SRC-3 knockout (KO) Tregs actively infiltrate breast and prostate tumors and completely eradicate them in syngeneic mouse models by establishing a tumor-suppressive immune microenvironment at the tumor borders without inducing irAEs.19

Given the evidence that SRC-3 knockout (KO) Tregs can eradicate tumors by generating a tumor-suppressive immune microenvironment, we sought to investigate their effects on solid cancers beyond breast cancer. Herein, using an established genetically engineered Treg cell–specific SRC-3 knockout (KO) mouse model combined with syngeneic cancer cells on a C57BL/6 background, we demonstrate that engrafted lung, melanoma, and glioblastoma tumors are likewise eradicated. Furthermore, during extended follow-up, no tumor recurrence was observed, highlighting the durable nature of SRC-3 KO Treg–mediated anti-cancer activity and recapitulating the effects previously reported in triple-negative breast and prostate cancer. These findings underscore the broad therapeutic potential of SRC-3 KO Tregs for treating diverse solid tumors without inducing irAEs.

Materials and methods

Cell lines

CT-2A (MilliporeSigma, Catalog number: SCC194), B16F10 (American Type Culture Collection, Catalog number: CRL-6475), and LL/2 cells (American Type Culture Collection, Catalog number: CRL-1642) were used to generate syngeneic mouse models of glioblastoma, melanoma, and lung cancer, respectively. All cell lines were cultured in full Dulbecco's Modified Eagle Medium/F-12 (DMEM/F-12) and maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

Generation of luciferase-labeled CT-2A, B16F10, and LL/2 cells

The firefly luciferase cDNA was cloned into the PSMPUW-Hygro lentiviral vector (Cell Biolabs, catalog number VPK-214). Lentiviruses containing the luciferase expression cassette were produced in 293 TN cells (System Bioscience) by transient transfection using the PSMPUW-Hygro lentiviral vector, ViraSafe™ Lentiviral Packaging System (Cell Biolabs), and Lipofectamine 2000 (Thermo Fisher Scientific). The recombinant lentivirus titer was measured with Lenti-X™ GoStix™ Plus (ClonTech). Transduction of cancer cell lines with the lentiviral vector carrying the luciferase expression cassette was performed using Transude MAX™ (System Bioscience). Luciferase-labeled CT-2A, B16F10, and LL/2 cell lines were then selected by exposure to hygromycin (300 µg/mL). Luciferase gene expression was validated using a luciferase activity assay kit (Promega). Finally, luciferase-labeled cells were maintained in full DMEM supplemented with 300 µg/mL hygromycin.

Mouse lines

C57BL/6J female mice (6 weeks old) were purchased from Jackson Laboratory. C57BL/6J, SRC-3f/f, Foxp3ERT2Cre/Y, and SRC-3f/f:Foxp3ERT2Cre/Y mice were maintained in a designated animal care facility at Baylor College of Medicine in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of laboratory animals. All animal experiments in this study were conducted under an IACUC-approved protocol (Assurance number D16-00475). All animal experiments were conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Our previous study established that tamoxifen treatment induces specific deletion of the SRC-3 gene in Foxp3⁺ regulatory T cells in Treg cell–specific SRC-3 knockout (SRC-3f/f:Foxp3ERT2Cre/Y) mice, relative to control mice.19

In vivo study

In female mice, one of the two X chromosomes is randomly inactivated to form a Barr body, a transcriptionally inactive structure. This can result in a mosaic pattern of Cre expression, where some cells express Cre while others do not, depending on which X chromosome is inactivated.20 In this study, we will use male bigenic mice (SRC-3f/f:Foxp3ERT2Cre/Y) to achieve a more effective and consistent knockout compared to female bigenic mice (SRC-3f/f:Foxp3ERT2Cre/Y). All animal experiments were conducted in triplicate for statistical analysis. All investigators will be blinded to mouse genotype information.

Animal numbers and power calculations

The required number of animals per group was determined using a power calculation to ensure adequate statistical power (α = 0.05, power = 80%) to detect a biologically significant difference. Sample size estimation was performed using GPower21 based on preliminary data. The calculations were designed to achieve a statistical significance threshold of p < 0.05.

Tamoxifen treatment schedule in each tumor mouse model

The proliferation rate varies among tumors. When the total luciferase flux of an individual tumor, measured as photons per second with a 1-minute exposure, reaches approximately 1,000, tamoxifen is administered to induce SRC-3 gene deletion in Tregs.

Glioblastoma syngeneic mouse model

Eight-week-old male SRC-3f/f and SRC-3f/f:Foxp3ERT2CRE/Y male mice were anesthetized using isoflurane (3–5% for induction, 1–2% for maintenance) and secured in a stereotactic frame while maintaining anesthesia throughout the procedure. A 1 cm midline scalp incision was made to expose the skull, and a 0.5 mm burr hole was created in the skull using a microsurgical drill. A Hamilton syringe was used to inject 2–5 μL of 10⁴ luciferase-labeled CT-2A cell suspension per mouse to a depth of 3.0 mm into the brain. Cell injection was carried out slowly to prevent pressure buildup, and the needle was held in place for 2 minutes before slow retraction to minimize reflux. The surgical site was cleaned with sterile saline before closing using sutures or surgical glue. Tamoxifen was intraperitoneally injected (75 mg/kg per day) on the 6th day after cancer cell injection, for five consecutive days. Bioluminescence was measured twice a week until all mice succumbed.

Histology of glioblastoma

For histological analysis of glioblastoma, brains from SRC-3f/f and SRC-3f/f:Foxp3ERT2CRE/Y male glioblastoma-bearing mice were harvested on day 25 following tamoxifen treatment. In brain tumor–bearing mice, the skull was opened, and the entire brain was carefully removed and fixed in 10% neutral buffered formalin.

Lung cancer syngeneic mouse model

3 × 10⁵ LL/2 cells per mice were suspended in 100 μL DMEM/F-12 supplemented with 5 μM EDTA (LL/2 suspension media). Eight-week-old SRC-3f/f (n = 5) and SRC-3f/f:Foxp3 ERT2Cre/Y (n = 5) old male mice were anesthetized as described above, intubated using a Mouse Intubation Kit (Kent Scientific), and injected with the LL/2 cell suspension into the lungs via a catheter, followed by administration of an additional 50 μL LL/2 suspension media to clear residual cancer cells. On the 3rd day after cancer cell injection, tamoxifen was administered as described above. Bioluminescence was measured twice a week until all mice succumbed.

Histology of lung cancer

For histological analysis of lung cancers, lungs from SRC-3f/f and SRC-3f/f:Foxp3ERT2CRE/Y male lung cancer-bearing mice were harvested on day 18 following tamoxifen treatment. Mice were euthanized by cervical dislocation under deep isoflurane anesthesia. A midline laparotomy followed by thoracotomy was performed to incise the diaphragm and rib cage, thereby exposing the thoracic cavity and lungs. For vascular perfusion, the renal artery was incised, and 3 mL of phosphate-buffered saline (PBS) was slowly injected into the right ventricle using a 5-mL syringe fitted with a 21-gauge needle until the lungs blanched. The trachea was then exposed, and 10% neutral buffered formalin was gently instilled through a 21-gauge needle inserted into the tracheal lumen until the lungs were fully inflated and fixative reflux was observed at the tracheal opening.

Melanoma syngeneic mouse model

Eight-weeks-old male SRC-3f/f (n = 4) and SRC-3f/f:Foxp3 ERT2Cre/Y (n = 4) mice were anesthetized as described above and slowly injected intradermally with 3 × 10⁴ B16F10 cells per mice suspended in 20 μL DMEM/F-12, followed by careful needle withdraw. On the 7th day after cancer cell injection, tamoxifen was administered as described above. Bioluminescence was measured twice a week until melanoma reached ~2.0 cm in any dimension. For histological analysis of melanoma, melanoma from SRC-3f/f and SRC-3f/f:Foxp3ERT2CRE/Y male melanoma-bearing mice were harvested on day 14 following tamoxifen treatment. In melanoma-bearing mice, the primary tumor mass, along with adjacent skin margins, was excised and fixed in 10% neutral buffered formalin.

Quantification of in vivo bioluminescence data

Mice were anesthetized with a 1.5% isoflurane/air mixture using an Inhalation Anesthesia System (VetEquip), followed by intraperitoneal injection of d-Luciferin (ThermoFisher) at a dose of 300 mg/kg. Ten minutes after the D-luciferin injection, the mice were imaged using an IVIS Imaging System (Xenogen) with continuous 1% to 2% isoflurane exposure. Imaging variables were maintained for comparative analysis. Grayscale-reflected and pseudo colorized images reflecting bioluminescence were superimposed and analyzed using Living Image software (Version 4.4, Xenogen). A region of interest (ROI) was manually selected over the relevant regions of signal intensity. The ROI area was kept constant across experiments, and the intensity was recorded as total photon counts per second per cm2 within the ROI.

Immunohistochemistry

After tissue fixation, samples were processed, embedded in paraffin, and sectioned at a thickness of 7 μm. Antigen retrieval was performed using heat-induced epitope retrieval in Tris buffer (pH 9) (Vector Laboratories, Cat. No. H-3301-250) for 40 minutes with a steam-based system. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Sections were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CD4 (Abcam, Cat. No. ab183685; 1:1000), rabbit anti-CD8α (Abcam, Cat. No. ab217344; 1:2000), and rabbit anti-CD49b (Abcam, Cat. No. ab133557; 1:500). After washing, sections were incubated with the ImmPRESS™-HRP Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratories, Cat. No. MP745150) for 30 minutes, followed by development with ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories, Cat. No. SK-4105). Nuclei were counterstained with hematoxylin. All immunohistochemistry procedures were performed identically across experimental groups to allow direct comparison. Images were acquired and analyzed using a Keyence BZ-X810 microscope with BZ-X800 camera software (Keyence Corporation of America, Raleigh, NC, USA).

Immunofluorescence

Paraffin-embedded sections were routinely deparaffinized and rehydrated through a graded ethanol series, followed by thorough washing in PBS. Antigen retrieval was performed in a Tris-based solution (Vector Laboratories, Cat. No. H-3301-250) for 40 minutes in a heating chamber. After washing, sections were permeabilized with 0.25% Triton X-100 for 10 minutes. Sections were then washed in PBS and blocked with 5% goat serum for 1 hour at room temperature. Primary antibodies against CD4 (Abcam, Cat. No. ab183685, 1:200), CD8 (Abcam, Cat. No. ab217344, 1:200), and CD49b (Abcam, Cat. No. ab181548, 1:200) were diluted in blocking solution, and sections were incubated overnight at 4 °C. The following day, sections were washed with PBS and incubated with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen, Cat. No. A-11008, 1:200) for 1 hour at room temperature. After washing, nuclei were counterstained with Hoechst 33342, and sections were mounted using an anti-fade mounting medium (Invitrogen, Cat. No. P36930). Secondary-only controls were included for each antibody. For image acquisition, three to four representative images per section were captured using a Keyence BZ-X800 microscope.

Quantification of immunohistochemistry (IHC) and immunofluorescence (IF)

IHC and IF images were quantified using QuPath (version 0.6.0).22

Statistical analyzes will be performed using GraphPad Prism 8. P values < 0.05 will be considered statistically significant.

Results

SRC-3KO Treg eradicates glioblastoma in a syngeneic mouse model

CT-2A glioblastoma cell line represents a well-established immunocompetent system for investigating the molecular etiology of high-grade gliomas and for evaluating new therapeutic strategies for this type of cancer.23 Previous studies have shown that luciferase activity reflects the proliferation of luciferase-labeled tumors.24 Therefore, the loss of luciferase activity indicates tumor eradication. To noninvasively track glioblastoma progression in mice, therefore, luciferase-labeled CT-2A cells were generated using a lentiviral transduction system carrying a luciferase expression unit, as luciferase expression does not significantly alter cancer cell metabolism or growth in mice.25 To define the role of SRC-3KO Tregs in glioblastoma progression in mice, we employed both tamoxifen-inducible Treg-cell specific SRC-3KO bigenic mice (SRC-3f/f:Foxp3ERT2Cre/Y) as an experimental model and floxed SRC-3 (SRC-3f/f) mice as a control.19 To mimic the glioblastoma microenvironment in vivo, luciferase-labeled CT-2A cells were orthotopically injected into the brains of recipient mice (Figure 1A). Luciferase activity was monitored for 52 d after tamoxifen treatment revealing robust development of glioblastoma in SRC-3f/f not SRC-3f/f:Foxp3ERT2Cre/Y mice (Figure 1B, C and D). These findings suggest that SRC-3KO Tregs are crucial for the effective suppression of glioblastoma. Furthermore, all glioblastoma-bearing SRC-3f/f mice died within 41 d following glioblastoma cell inoculation, whereas all SRC-3f/f:Foxp3ERT2Cre/Y counterparts survived for at least 52 d, suggesting a sustained therapeutic effect associated with SRC-3KO Tregs (Figure 1E). To determine whether SRC-3 knockout (KO) in Tregs alters immune cell profiling in glioblastoma, we assessed CD4⁺ and CD8⁺ T-cell levels in glioblastoma tissues from SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice using immunohistochemistry (IHC) (Figure 1F and G). For histological analysis, brains from male glioblastoma-bearing SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice were harvested on day 25 following tamoxifen treatment, prior to glioblastoma eradication in SRC-3f/f:Foxp3ERT2Cre/Y mice. IHC revealed a large glioblastoma mass in the brain regions of SRC-3f/f mice compared to SRC-3f/f:Foxp3ERT2Cre/Y mice (arrowheads in Figure 1F and G). Furthermore, CD4⁺ and CD8⁺ T-cell levels were markedly elevated in glioblastoma tissues from SRC-3f/f:Foxp3ERT2Cre/Y mice compared to SRC-3f/f controls (Figure 1F and G). These findings suggest that SRC-3 KO in Tregs promotes anti-tumor immunity, leading to glioblastoma eradication.

Figure 1.

Figure 1.

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The loss of SRC-3 in Treg leads to immune suppression of glioblastoma in mice: (A) Experimental design for the orthotopic injection of glioblastoma cells (luciferase-labeled CT-2A) into the mouse brain, followed by tamoxifen treatment to induce SRC-3 KO Tregs. (B) In vivo imaging analysis of luciferase activity in glioblastoma-bearing SRC-3f/f (n = 2) and SRC-3f/f:Foxp3ERT2Cre/Y (n = 3) male mice treated with tamoxifen. (C) Quantification of luciferase activity in glioblastoma-bearing SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice from panel B. Each line represents a single mouse. (D) Tumor luciferase activity was measured across three independent experiments. The total number of mice used for the SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y groups was 5 and 7, respectively. Luciferase activity was recorded for each mouse at the conclusion of the experiment. (E) Survival curve of SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y male mice with glioblastoma. (F and G) IHC analysis of CD4⁺ (F) and CD8⁺ (G) T cells in glioblastoma from SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice at day 25 following tamoxifen treatment. T-cell levels were quantified using QuPath. Arrowheads indicated the glioblastoma mass in the brain. Statistical significance was determined using an unpaired two-tailed Student’s t-test in GraphPad Prism.

SRC-3KO Tregs suppress tumor progression in a melanoma mouse model.

The B16-F10 orthotopic melanoma model is a well-established platform for studying melanoma progression, metastasis, and therapeutic strategies in an immunocompetent system.26 B16-F10 cells were labeled with luciferase using a lentiviral vector, to enable non-invasive monitoring of melanoma growth, and utilized in conjunction with our syngeneic genetic mouse model. Luciferase-labeled B16-F10 cells were implanted intradermally into SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice (Figure 2A). Luciferase activity analysis showed significant melanoma progression in three out of four SRC-3f/f mice 23 d after tamoxifen treatment, whereas only one out of four mice in the SRC-3f/f:Foxp3ERT2Cre/Y group exhibited substantial melanoma growth during the same period (Figure 2B, C, and E). In addition to measuring tumor luciferase activity, we quantified tumor volumes in these mice to validate the bioluminescence results. Consistent with the tumor luciferase activity, SRC-3 KO Tregs significantly reduced melanoma tumor mass compared with controls (Figure 2D). All melanoma-bearing SRC-3f/f mice were euthanized within 23 d of melanoma cell injection, upon reaching the maximum allowed tumor size, while only one out of four SRC-3f/f:Foxp3ERT2Cre/Y melanoma-bearing mice had to be euthanized by day 23 (Figure 2F). Overall, these data show that induction of SRC-3KO in Tregs is associated with a 75% survival rate in melanoma, compared to 0% survival rate of the control cohort. Moreover, the surviving SRC-3f/f:Foxp3ERT2Cre/Y mice exhibited no relapse for at least 50 d following tamoxifen treatment. To determine whether SRC-3 knockout (KO) in Tregs alters the immune landscape within melanoma, we evaluated CD4⁺ T cells, CD8⁺ T cells, and natural killer (NK) cells in tumors from SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice using Immunofluorescence (IF). For histological analyzes, melanomas were harvested on day 14 after tamoxifen treatment—prior to complete tumor eradication in SRC-3f/f:Foxp3ERT2Cre/Y mice. Hematoxylin and eosin (H&E) staining revealed markedly smaller melanoma lesions in SRC-3f/f:Foxp3ERT2Cre/Y mice compared with controls (Figure 2G). IF analysis further demonstrated significantly increased infiltration of CD4⁺ T cells (Figure 2H), CD8⁺ T cells (Figure 2I), and NK cells, as indicated by CD49b staining (Figure 2J), in melanomas from SRC-3f/f:Foxp3ERT2Cre/Ymice relative to SRC-3f/f controls. Collectively, these data indicate that SRC-3 deletion in Tregs enhances anti-tumor immunity, resulting in melanoma eradication in mice.

Figure 2.

Figure 2.

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The loss of SRC-3 in Treg suppress melanoma progression in mice: (A) Experimental design for the orthotopic injection of melanoma cells (luciferase-labeled B16-F10) into the skin of the mouse, followed by tamoxifen treatment to induce SRC-3 KO Tregs. (B) In vivo imaging analysis of luciferase activity in melanoma-bearing SRC-3f/f (n = 4) and SRC-3f/f:Foxp3ERT2Cre/Y (n = 4) male mice treated with tamoxifen. (C) Quantification of luciferase activity in melanoma-bearing SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y male mice from panel B. Each line represents a single mouse. (D) The melanoma tumor volume for each mouse shown in panel B was quantified. The formula used to determine the volume of endometriotic lesions was 0.5 × length × width².20 (E) Tumor luciferase activity was measured across three independent experiments. The total number of mice used for the SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y groups was 8 and 9, respectively. Luciferase activity was recorded for each mouse at the conclusion of the experiment. (E) Survival curve of SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y male mice with melanoma. (G) Representative H&E staining of melanomas harvested 14 d after tamoxifen treatment in SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice. Arrowheads indicated the melanoma mass. (H–J) Levels of CD4⁺ T cells (H), CD8⁺ T cells (I), and NK cells, identified by CD49b staining (J), were assessed in melanomas harvested 14 d after tamoxifen treatment in SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice using IF and quantified with the QuPath software. Statistical significance was determined using an unpaired two-tailed Student’s t-test in GraphPad Prism.

SRC-3KO Treg cells suppress tumor progression in a lung cancer mice model.

The LL/2 (Lewis Lung Carcinoma 2) cell line was derived from a spontaneous epidermoid carcinoma that developed in the lung of a C57BL/6J mouse27 and is used as an orthotopic lung cancer model.26 For noninvasive monitoring of lung cancer progression, luciferase-labeled LL/2 cells were intratracheally injected into the lungs (Figure 3A).28 Lung cancer cells were successfully deposited in SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice (Figure 3B). Eleven days following tamoxifen treatment, a regression in tumor development was observed in both cohorts, which was reflected by disappearance of the luciferase signal. However, within the subsequent 14-day period, luciferase signals intensified in four out of five SRC-3f/f lung cancer-bearing mice, while remaining absent in the SRC-3f/f:Foxp3ERT2Cre/Y cohort (Figure 3B, D and E). All SRC-3f/f lung cancer bearing mice died within 32 d following tamoxifen treatment, while three out of five SRC-3f/f Foxp3ERT2Cre/Y counterparts survived (Figure 3F). Importantly, two out of five SRC-3f/f Foxp3ERT2Cre/Y lung cancer-bearing mice (#2 and #5 in Figure 3B) were died on the 38th and 49th d after tamoxifen treatment without increasing luciferase signal (Figure 3B and D). H&E staining further confirmed the absence of detectable tumor masses in the lungs of these two mice (Figure 3C). Accordingly, the death of these mice in the absence of a luciferase signal was not attributable to tumor-related toxicity. Collectively, these results indicate that SRC-3KO Tregs effectively suppress lung cancer progression and extend survival in a syngeneic mouse model. To determine whether SRC-3 knockout (KO) in Tregs alters the immune landscape within lung cancers, we evaluated CD4⁺ T cells, CD8⁺ T cells, and natural killer (NK) cells in tumors from SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice using IHC. For histological analyzes, lung cancers were harvested on day 18 after tamoxifen treatment—prior to complete tumor eradication in SRC-3f/f:Foxp3ERT2Cre/Y mice. H&E staining revealed markedly smaller melanoma lesions in SRC-3f/f:Foxp3ERT2Cre/Y mice compared with controls (arrowheads in Figure 3G). IHC analysis further demonstrated significantly increased infiltration of CD4⁺ T cells (Figure 3H), CD8⁺ T cells (Figure 3I), and NK cells, as indicated by CD49b staining (Figure 3J), in lung cancers from SRC-3f/f:Foxp3ERT2Cre/Ymice relative to SRC-3f/f controls. Collectively, these data indicate that SRC-3 deletion in Tregs enhances anti-tumor immunity, resulting in lung cancer in mice.

Figure 3.

Figure 3.

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The loss of SRC-3 in Treg suppress lung cancer progression in mice: (A) Experimental design for the orthotopic injection of lung cancer cells (luciferase labeled LL/2) into lung of mouse, followed by tamoxifen treatment to induce SRC-3 KO Tregs. (B) In vivo imaging analysis of luciferase activity in lung cancer-bearing SRC-3f/f (n = 5) and SRC-3f/f:Foxp3ERT2Cre/Y (n = 5) male mice treated with tamoxifen. The lung tumor luciferase signal was very weak before tamoxifen treatment. Therefore, the luciferase image was overexposed to determine whether lung cancer cells had established in the lung. (C) H&E staining of lung from #2 and #5 SRC-3f/f:Foxp3ERT2Cre/Y in panel B. (D) Quantification of luciferase activity in lung cancer-bearing SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y male mice from panel B. Each line represents a single mouse. (E) Tumor luciferase activity was measured across three independent experiments. The total number of mice used for the SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y groups was 10 and 10, respectively. Luciferase activity was recorded for each mouse at the conclusion of the experiment. (F) Survival curve of SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y male mice with lung cancer. (G) Representative H&E staining of lung cancers harvested 18 d after tamoxifen treatment in SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice. Arrowheads indicated the lung cancer mass. (H–J) Levels of CD4⁺ T cells (H), CD8⁺ T cells (I), and NK cells, identified by CD49b staining (J), were assessed in melanomas harvested 18 d after tamoxifen treatment in SRC-3f/f and SRC-3f/f:Foxp3ERT2Cre/Y mice using IHC and quantified with the QuPath software. Statistical significance was determined using an unpaired two-tailed Student’s t-test in GraphPad Prism.

Discussion

ICB therapy has substantially improved outcomes across a limited range of solid cancers. The total number of cancer patients receiving ICB treatment continues to rise each year, even while the proportion of newly diagnosed advanced, metastatic cancers are declining.29 However, in most cancers, the efficacy of ICB therapy is very limited. The primary factor determining the success of an ICB therapy is the tumor's immunological status, specifically whether it is “cold” or “hot”.30 Immunologically hot tumors are characterized by high presence of tumor infiltrating lymphocytes (TILs), elevated PD-L1 expression, and high tumor mutational burden (TMB), which is associated with tumors being more responsive to ICB. In contrast, immunologically cold tumors lack these features and are often dominated by immunosuppressive cell populations.31 Increasing TILs infiltration into the TME of cold tumors can transform their immunological status into a hot status, providing potential opportunities for improving therapeutic outcomes of ICB therapy.32 Glioblastoma is considered an “immune-cold” tumor, characterized by generally low levels of T-cell infiltration compared with immunogenic tumors such as melanoma and lung cancer. Despite this overall paucity of T cells, the relative abundance of regulatory T cells (Tregs) is often higher in glioblastoma than in normal brain tissue, driven in part by active recruitment through tumor-secreted chemokines such as CCL2.33 Leveraging this intrinsic Treg-recruiting property of glioblastoma, SRC-3–deficient (SRC-3 KO) Tregs are expected to be efficiently recruited into the tumor microenvironment. Notably, SRC-3 KO Tregs exhibit significantly higher proliferative capacity than wild-type Tregs19 allowing them to robustly expand after tumor infiltration. Once established within glioblastoma, SRC-3 KO Tregs promote conversion of the tumor microenvironment from an immune-cold to a more immune-hot state by actively recruiting cytotoxic T cells. In addition to glioblastoma, SRC-3 KO Treg cells also generated the tumor-immune microenvironment in lung cancer, and melanoma by elevating T cells (CD4 + /CD8+) and NK cells in the tumor environment to change “immune cold” to “immune hot” to suppress the tumor progression.

In a recently published study, we demonstrated that systemic pharmacological inhibition of SRC-3 effectively suppresses BC tumor growth in an immune-dependent manner.18 These findings suggested a crucial role for SRC-3 in the immune system regulation and shaping of the TME. In a follow up study, we have shown a more specific role of SRC-3 in shaping the anti-tumor immune response; dominant presence of SRC-3KO Tregs in the TME, leads to the complete elimination of TNBC tumor by a mechanism that involves infiltration of cytotoxic immune cells through the elevation of the interferon gamma (IFNγ)/Chemokine (C-X-C motif) ligand 9 (CXCL9) axis in the TME.19 Unlike ICB therapy, which relies on a pre-existing hot TME for effectiveness, SRC-3KO Tregs activate the IFN-γ/CXCL9 axis to recruit C-X-C motif chemokine receptor 3 (CXCR3)s cytotoxic immune cells, potentially transforming tumors into an immune-hot status. This underlying mechanism suggests that the anti-cancer effects of SRC-3KO Tregs may be broadly effective across multiple cancer types. While the activation of the IFN-γ/CXCL9 axis is likely a core mechanism driving the anti-tumor activity of SRC-3KO Tregs, it remains unclear whether this process is accompanied by distinct features in different cancer types. This question is currently being investigated in our laboratory, aiming to uncover the full range of tumor-specific signaling pathways involved in SRC-3KO Treg-mediated cancer suppression.

Tumor recurrence is one of the main problems in current cancer treatment.34-36 Our prior study showed that cancer cells injected into TNBC tumor-cured mice with SRC-3KO Tregs were rejected, demonstrating a long-lasting therapeutic effect.19 Therefore, SRC-3KO Treg therapy for glioblastoma, lung cancer, and melanoma is expected to significantly reduce tumor recurrence when compared to current therapies. Moreover, therapy with SRC-3KO Tregs offers a significant benefit to cancer patients, as it were not associated with any observed side effects, including irAEs.19 To assess the potential of SRC-3 KO Tregs as a cancer vaccine in glioblastoma, lung cancer, and melanoma, tumor rechallenge studies in mice are planned.

Supplementary Material

Supplementary Material

ARRIVE_Sang_12212025

KONI_A_2640261_SM3318.pdf (155.6KB, pdf)

Acknowledgments

We dedicate this work to the memory of Dr. Bert W. O’Malley, whose visionary leadership, scientific insight, and unwavering support were instrumental in shaping this study. We are deeply grateful for his guidance and mentorship throughout this work. His legacy will live on through the countless lives and scientific endeavors he influenced.

SJH, CCD, DML conceptualized this work. NS, EK, YP curated data. SJH, NS, EK, YG, YP, AMD, YX, DML investigated this project. JX provided floxed SRC-3 mice. SJH, CCD, DML supervised this work. SJH, NS, EK, YP, YG, YX validated the results in this work. SJH, YG, DML prepared the original draft. SJH, YG, CCD, DML reviewed and edited this manuscript. All authors have read and approved the final version of manuscript.

Funding Statement

This work is partly supported by funding from the Adrienne Helis Malvin Medical Research Foundation to DML (DML), CoRegen Inc. (SJH and DML), Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01-HD098059, SJH), and Department of Defense's Congressionally Directed Medical Research Programs (HT9425-24-1-0254, SJH).

Disclosure of potential conflicts of interest

Drs Han, Lonard, and Dacso are founding members of a new non-public company, called CoRegen, Inc. Laboratory research support exists for Drs Han, Lonard, and Dacso from CoRegen. Dr Gilad is a paid consultant by and disclose an equity position in CoRegen, Inc.

Data availability statement

The data that support the findings of this study are available from the corresponding authors (DML and SJH) upon reasonable request.

Ethical approval

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and institutional guidelines. The Assurance number of the animal protocol is D16-00475.

Supplemental Material

Supplemental data for this article can be accessed at https://doi.org/10.1080/2162402X.2026.2640261.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material

ARRIVE_Sang_12212025

KONI_A_2640261_SM3318.pdf (155.6KB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding authors (DML and SJH) upon reasonable request.

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