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. 2026 Jun 8;26:282. doi: 10.1186/s12935-026-04338-8

An herbal formula, SH003 alleviates colorectal cancer through dual targeting of adaptive and innate checkpoints PD-L1/CD47

Na-Ra Han 1,2, Hyun-Ha Hwang 3, Tae-Hyoun Kim 3, Hyeong-Chan Lee 3, Tae-Hoo Yi 4, Hi-Joon Park 5, Seong-Gyu Ko 2,6, Phil-Dong Moon 7,✉
PMCID: PMC13463639  PMID: 42260546

Abstract

Background

Colorectal cancer (CRC) is the third most common cancer. The use of immunotherapy for cancer has become widespread in recent decades and adaptive and innate checkpoints can be simultaneously targeted for enhanced immunotherapy. An herbal formula, SH003 has beneficial effects against multiple cancers. However, the anti-CRC efficacy of SH003 through dual targeting of adaptive and innate checkpoints, PD-L1/CD47 remain to be investigated.

Methods

We used two types of CRC patient-derived organoids (PDOs) and two types of CRC cell lines, and analyzed PD-L1 and CD47 expression levels with quantitative real-time PCR, Western blot, flow cytometry assays, and immunofluorescence staining. The cytotoxicity, proliferation, and apoptosis analysis were applied to clarify the regulatory effects on CRC growth. T cell immunity was analyzed through a co-culture system of CRC cell lines and T cells, as well as in in vivo CRC model.

Results

SH003 reduced the growth of CRC PDOs, leading to morphological changes and decreases in ATP and Ki67 levels. SH003 inhibited the mRNA and protein expression of both PD-L1 and CD47 though the regulation of c-Myc in CRC PDOs. In addition, SH003 suppressed VEGF, TNF-α, and IL-1β in CRC PDOs. This effect was validated in experiments of CRC cell lines, revealing that SH003 suppressed both PD-L1 and CD47 expression increased by IFN-γ stimulation via the regulation of c-Myc and reduced CRC progression. SH003 also enhanced the T cell-mediated killing of CRC cells with increased IL-2 production. In an in vivo CRC model, SH003 inhibited tumor growth while reducing both PD-L1, CD47, and c-Myc expression and increasing CD8+ T cell infiltration.

Conclusions

Our investigation demonstrated that SH003 served as a novel CRC immunotherapy by the dual targeting of adaptive and innate checkpoints, PD-L1/CD47 via the regulation of c-Myc signaling, highlighting that SH003 may be an effective herbal candidate drug for the treatment of CRC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-026-04338-8.

Keywords: SH003, Colorectal cancer, Patient-derived organoids, PD-L1, CD47, Immunotherapy

Background

In 2021, colorectal cancer (CRC) was the leading cause of cancer death in both men (3rd) and women (2nd) in Republic of Korea [1]. According to a 2024 report from the International Agency for Research on Cancer (IARC), CRC was the second leading cause of cancer death in 2022 [2]. Despite significant advances in CRC screening, surgical resection, and adjuvant treatment, the mortality rate among patients with CRC remains relatively high [3]. The effectiveness of surgery and adjuvant therapies remains limited, and recurrence, metastasis, and drug resistance remain major problems in cancer treatment [4]. It is necessary to search new targeted therapies for CRC and explore new drugs that can complement the limitations of adjuvant therapy.

Immunotherapy is a cancer treatment that activates and boosts the ability of the immune system to identify and eliminate cancerous cells [5]. CRC is one of several cancer types that may benefit from immunotherapy [6]. Activating both adaptive and innate immune responses is a more effective strategy for inducing sustained anti-cancer effects [7]. An adaptive checkpoint, PD-L1 binds to its receptor PD-1 and inhibits tumor-specific T cell function, leading a local immunodeficient environment and hindering tumor eradication through T cells [8]. CD47 pathway, an innate checkpoint molecule, is known to be an important mechanism by which cancer cells evade innate immune surveillance [9].

Patient-derived organoids (PDOs) are organ-like, a stem cell-derived, and three-dimensional self-organizing structures that mimic its corresponding cancers [10]. The development of cancer organoid culture systems has changed the paradigm of cancer research [11, 12]. PDOs have recently emerged as powerful preclinical models for cancers [13].

SH003 (a blend of Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes Kirilowii (Maxim.) has been used in traditional East Asian medicine. Astragalus membranaceus is a widely used traditional Korean/Chinese medicine and has the effect of replenishing energy (Qi tonifying) and preventing pathogens from entering the body [14]. Angelica gigas has been traditionally used to promote blood flow and increase the supply of oxygenated blood to nourish the brain and spleen [15]. Trichosanthes Kirilowii has traditionally been used to treat diabetes, breast abscesses, and cancer-related symptoms [16]. SH003 effectively inhibits several pathological processes including acute lung injury [17], inflammatory skin disorders [18], and neuropathic pain [19]. SH003 also has beneficial effects as an anti-cancer agent against oral cancer [20], lung cancer [21], and melanoma [22]. Nodakenin is an active compound of SH003 [20, 23]. Nodakenin has beneficial properties against inflammatory responses [24], ulcerative colitis ([25].), osteoporosis [26], and alcoholic liver disease [27]. Regarding CRC, there are reports of inhibition of CRC growth of SH003 [28] and colitis-associated CRC of nodakenin [29]. In addition, our previous study has reported that SH003 increases IL-2, IL-12, and IFN-γ via NF-κB pathways in macrophages or splenocytes and enhances immunostimulatory activity [30]. SH003 regulated the various immune-related genes during LPS-induced immune responses in human neutrophils [17]. SH003 exerted the anti-melanoma effect through PD-L1/STAT1 pathways [22]. Astragalus membranaceus enhanced immunomodulatory effects by regulating IL-1β, IL-6, TNF-α, Toll-like receptor 4 levels in chickens [31]. Angelica gigas enhanced immunity by modulating several immune-related cytokines, including IL-1β, IL-6, TNF-α, IL-2, IL-12, and IFN-γ via MAPK/NF-κB pathways in the immunosuppressed mouse model [32]. Based on these findings, we hypothesized that the anti-cancer efficacy of SH003 could be sufficiently associated with immunotherapy. There are no reports of CRC immunotherapy of SH003 and nodakenin though dual targeting of adaptive and innate checkpoints PD-L1/CD47. The present study investigated an anti-CRC effect of SH003 and nodakenin as an immunotherapy using CRC PDOs and CRC cell lines.

Materials and methods

SH003 preparation

SH003 was manufactured by Hanpoong Pharm and Foods Company (Jeonju, Republic of Korea) in compliance with Korea Good Manufacturing Practice. It was extracted using a standardized extraction process using a 1:1:1 (w/w) ratio of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii in 30% ethanol solvent [21]. Nodakenin (Ensol Biosciences Inc., Daejeon, Republic of Korea) was prepared with reference to a study of Choi et al., [20].

Organoids culture and treatment

CRC PDOs, SNU-4351S3-TO (#04351S3-TO) and SNU-4146S1-TO (#04146S1-TO, Korean Cell Line Bank, Seoul, Republic of Korea) were cultured with a complete organoid culture medium with reference to previously established protocols [12, 33, 34]. The culture medium consisted of a 1:1 mixture of L-WRN conditioned medium and basal culture medium supplemented with B-27 supplement, human FGF-10, human EGF, nicotinamide, prostaglandin E2, A83-01, SB202190, N-acetylcysteine, and primocin (Supplementary Table 1). The conditioned medium was obtained from culture medium of a L-WRN (Wnt-3a, R-spondin, and Noggin) cell line (#CRL-3276, ATCC, Manassas, VA, USA) which is consisted of DME/F-12, containing 10% FBS and 1% penicillin/streptomycin. The basal culture medium consisted of DME/F-12, containing 10% FBS and 1% penicillin/streptomycin. The organoids were cultured in basement membrane extracts (BME) dome in a humidified CO2 (5%) incubator at 37 ℃. After the organoids were cultured for 4 days, SH003 (500 µg/ml) or nodakenin (20 µg/ml) were treated for additional 5 days. Organoid diameter was analyzed using image analysis software (ToupView, Toup Tek Photonics Co., Ltd., Zhejiang, China). The BME dome was enzymatically and mechanically dissociated using TrypLE Express solution and the organoids were subjected to analysis.

Cell culture and treatment

CRC cell lines, HT-29 (#30038, Korean Cell Line Bank), DLD-1 (#10221, Korean Cell Line Bank), and HCT116 (#10247, Korean Cell Line Bank) and Jurkat T (#40152, Korean Cell Line Bank) were cultured in RPMI 1640 (Gibco) medium, containing 10% FBS and 1% penicillin/streptomycin. These cell lines were maintained in a humidified CO2 (5%) incubator at 37 ℃. HT-29 and DLD-1 cells were treated with SH003 (100, 200, 500 µg/ml) or nodakenin (20 µg/ml) and then stimulated with IFN-γ (10 ng/ml).

ATP quantification

ATP production in organoids were detected by a CellTiter-Glo® 3D Cell Viability Assay kit (#G9683, Promega, Madison, WI, USA). The organoids were treated with CellTiter-Glo 3D reagent to measure cellular ATP levels. After they were shaken vigorously, luminescence analysis was conducted using a luminometer (Molecular Device, San Jose, CA, USA).

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from CRC PDOs and cell lines according to the protocols of an TRIzol (Qiagen, Hilden, Germany) or easy-BLUE™ RNA extraction kit (iNtRON Biotech Inc., Seongnam, Republic of Korea). Reverse transcription was carried out using a cDNA synthesis kit (Bioneer Corporation, Daejeon, Republic of Korea). Subsequently, real-time qPCR was performed with Power SYBR® Green Master Mix (Thermo Fisher Scientific) using qPCR system (Applied Biosystems, Foster City, CA, USA). All data were normalized to GAPDH. The primer sequences are shown in Supplementary Table 2.

Western blot

PDOs, cell lines, and mouse tumor tissues were lysed with an EzRIPA Lysis kit (ATTO Co., Tokyo, Japan). Quantification and normalization were conducted using the BCA protein assay (Thermo Fisher Scientific). Then, the samples were separated by SDS-PAGE gel and transferred onto nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) by electrophoresis. The membranes were incubated with specific primary antibodies and then incubated with horseradish peroxidase-conjugated secondary antibodies. Detection was carried out with the ECL solution (DoGenBio Co., Seoul, Republic of Korea) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The used antibodies are listed in Supplementary Table 1.

Immunofluorescence

Each sample was fixed and permeabilized with BD Cytofix/Cytoperm (BD Science, Milpitas, CA, USA). After then incubated with bovine serum albumin, each sample was incubated with relative primary antibodies. The samples were then treated with fluorescein-conjugated secondary antibodies. The used antibodies are listed in Supplementary Table 1. Apoptosis in PDOs was detected with a terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) kit (In Situ Cell Death Detection Kit, TMR red, #12156792910, Roche, Basel, Switzerland). It was finally stained with DAPI staining solution and the fluorescence signals were examined using ZEISS confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

Differentially expressed genes analysis

Total RNA concentration of PDOs was measured with Quant-IT RiboGreen (#R11490, Invitrogen, Waltham, MA, USA). The samples were analyzed on the TapeStation RNA screentape to assess the integrity of total RNA (#5067–5576, Agilent, Santa Clara, CA, USA). Only high-quality RNA preparations with a RIN value of 7.0 or higher were used for RNA library construction. Libraries were constructed independently using an Illumina TruSeq Stranded mRNA Sample Prep Kit (#RS-122-2101, Illumina, Inc., San Diego, CA, USA). Indexed libraries were submitted to Illumina NovaSeqX (Illumina, Inc., San Diego, CA, USA) and paired-end (2 × 100 bp) sequencing was performed (Macrogen Inc., Seoul, Republic of Korea). Gene enrichment analysis for significant genes was performed using in-house Kyoto Encyclopedia of Genes and Genomes (KEGG) Viewer script for the KEGG pathway database (http://www.genome.jp/kegg/pathway.html). The statistical significance of differentially expressed genes (DEGs) was determined using DESeq2 nbinom WaldTest [35]. Fold change and p-value were extracted from WaldTest results and adjusted using the Benjamini-Hochberg algorithm. All data analyses and visualization of DEG were performed using R 4.2.2 (www.r-project.org).

Flow cytometry

Single-cell suspensions were stained with anti-human PD-L1 (PE-conjugated, #374511; isotype control, #400101, Biolegend, San Diego, CA, USA) and anti-human CD47 (FITC- conjugated, #323106; isotype control, #400110) in the dark. The washed suspensions were resuspended in FACS buffer. The suspensions were subjected to flow cytometry analysis using CytoFLEX (Beckman Coulter, Brea, CA, USA). Data analysis were performed with FlowJo™ Software (Tree Star, Inc., Ashland, OR, USA). The cell apoptosis was detected using a FITC Annexin V Apoptosis Detection Kit with PI (#640914, Biolegend) according to the manufacturer’s instructions.

Cytotoxicity

The cytotoxicity on CRC cells were assessed using an MTT assay. Adherent cells were treated with SH003 or nodakenin for 48 h. After adding MTT solution, the cells were placed in an incubator for another 4 h. Finally, the MTT crystals were dissolved in DMSO and absorbance at 540 nm was measured using a microplate reader (Molecular Device).

Proliferation

The proliferative ability of SH003 or nodakenin-treated CRC cells was analyzed using a Cell Proliferation ELISA, BrdU kit (#11647229001, Roche) according to the manufacturer’s instructions.

c-Myc inhibition

The CRC cell lines were pre-treated with a c-Myc inhibitor (10058-F4, 10 µg/ml, #HY-12702, MedChemExpress, Monmouth Junction, NJ, USA) for 72 h as described in previous reports [36–39]. The cells were then replaced with fresh media and treated with SH003, nodakenin, or IFN-γ.

T cell-mediated tumor cell killing assay

The T cell-mediated tumor cell killing assay was performed as previously described [40, 41]. CRC cell lines were seeded in 24-well plates and anti-CD3 antibody was coated in 6-well plates overnight. The CRC cell lines were treated SH003 or nodakenin and then stimulated with IFN-γ. Jurkat T cells were seeded in the CD3-coated plates and activated with anti-CD28 antibody. Following the removal of the culture medium of CRC cell lines, the cells underwent three washes with PBS. The activated Jurkat T cells were co-cultured with the CRC cell lines (10:1 ratio). The concentration of IL-2 in the culture medium was measured using ELISA according to the manufacturer’s instructions. The remaining CRC cell lines were subjected to an MTT assay to assess the cell viability.

In vivo studies

The animal experiment was approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHSASP-25-080). BALB/c nude mice (six-week-old male, NARA Biotech, Seoul, Republic of Korea) were housed in a specific pathogen-free facility at 22–23 °C and 40–60% humidity with a 12 h light/dark cycle. HCT116 cells (1 × 106 cells/mouse) were injected subcutaneously into the right flank of each mouse in a 100 µl mixture of PBS and Matrigel (1:1 ratio). After the average tumor size reached 100 mm3, the mice were randomly divided into two groups (n = 6): control (saline) and SH003 (300 mg/kg). Saline and SH003 were administered orally three times a week. Body weight and tumor volume were measured three times a week. Tumor volume was calculated using the formula: volume = (length × width²) × 1/2. The excised tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sectioned slides (4 μm) were deparaffinized, rehydrated, and stained with primary antibodies. The fluorescence signals were examined using ZEISS confocal laser scanning microscope (Carl Zeiss).

Statistical analysis

Statistical analysis was performed using SPSS statistical software package (SPSS Inc., Chicago, IL, USA) and Prism 9.0 software (Graphpad software, San Diego, CA). Data were expressed as mean ± SEM. For analyses among multiple groups, one-way ANOVA analysis was employed, followed by Tukey post hoc test. All data were adopted at a significance level of *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

SH003 inhibits the growth of CRC PDOs

We investigated the anti-CRC efficacy of SH003 using two established CRC PDOs, SNU-4351S3-TO and SNU-4146S1-TO [12, 33, 34]. We treated with SH003 and nodakenin and then monitored for growth and morphological changes. Control-PDOs had a smooth surface with a nearly circular shape and showed significant growth. In contrast, SH003 or nodakenin-treated organoids exhibited rough surfaces, apoptotic membrane blebbing, and condensation rather than expansion. In particular, morphological changes were evident in the SH003-treated organoids, including a reduction in size and shape alteration (Fig. 1A, B and Supplementary Fig. 1). In addition, SH003-treated organoids showed a significant decrease in diameter (Fig. 1B). ATP which is an important energy source, is a critical biomarker for assessing cell viability [42]. Thus, we investigated the effect of SH003 and nodakenin on PDOs viability by measuring ATP bioluminescence. SH003 and nodakenin led to decreases in ATP values within organoids (Fig. 1C). TUNEL staining revealed that the number of TUNEL-positive apoptotic cells was also higher in SH003 or nodakenin-treated organoids than in control-PDOs (Fig. 1D). We also evaluated the regulatory effect of SH003 on organoid proliferation by measuring the expression of Ki67 proliferation marker. Expectedly, SH003 and nodakenin significantly suppressed the mRNA and protein expression of Ki67 (Fig. 1E, F).

Fig. 1.

Fig. 1

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Inhibitory effects of SH003 on the growth of CRC PDOs. (A) The representative morphological comparison of SNU-4351S3-TO and SNU-4146S1-TO during 5 days of SH003 (500 µg/ml) and nodakenin (20 µg/ml) treatments. Scale bar, 80 μm. (B) The size quantification by measuring the diameters. (C) The ATP measurement for analysis of organoid viability. (D) Representative images from TUNEL assay. Scale bar, 20 μm. (E) The mRNA expression quantified by qPCR. (F) Representative images detected by immunofluorescence. Scale bar, 20 μm

SH003 regulates both PD-L1 and CD47 in CRC PDOs

Next, we investigated whether SH003 could regulate immune checkpoints, PD-L1 and CD47 in CRC PDOs. As illustrated in Fig. 2A and Supplementary Fig. 2A, SH003 and nodakenin effectively suppressed the mRNA expression of PD-L1 and CD47. The proto-oncogene Myc as a transcription factor regulates the expression of various gene products involved in cell proliferation, growth, and apoptosis including both PD-L1 and CD47 in cancers [43]. Thus, we analyzed the mRNA expression of c-Myc as well as PD-L1 and CD47. SH003 or nodakenin-treated CRC PDOs showed significant decreases in c-Myc levels (Fig. 2A). SH003 and nodakenin also considerably inhibited the protein levels of PD-L1, CD47, and c-Myc (Fig. 2B and Supplementary Fig. 2B). This inhibitory effect of SH003 and nodakenin on PD-L1, CD47, and c-Myc was further validated through an analysis of immunofluorescence in CRC PDOs (Fig. 2C).

Fig. 2.

Fig. 2

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Inhibitory effects of SH003 on PD-L1 and CD47 in CRC PDOs. CRC organoids, SNU-4351S3-TO and SNU-4146S1-TO were treated with SH003 (500 µg/ml) and nodakenin (20 µg/ml). (A) The mRNA expression levels quantified by qPCR. (B) The protein expression detected by Western blotting. Quantification is displayed as a bar chart on the right. (C) Representative images detected by immunofluorescence. Scale bar, 20 μm

SH003 regulates inflammatory mediators in CRC PDOs

Tumor-released inflammatory mediators play an important role in colorectal carcinogenesis [44]. VEGF is a critical angiogenic factor in primary and metastatic human CRC [45]. TNF-α and IL-1β in CRC promotes growth, invasion, and metastasis [44, 46]. In addition, VEGF, TNF-α, and IL-1β are involved in PD-L1/CD47 signaling pathways [47–49]. Thus, we investigated whether SH003 could regulate VEGF, TNF-α, and IL-1β expression in CRC PDOs. The results showed that SH003 and nodakenin markedly inhibited the mRNA and protein expression levels of VEGF, TNF-α, and IL-1β in both SNU-4351S3-TO and SNU-4146S1-TO (Fig. 3). Furthermore, we performed the transcriptome sequencing (RNA-seq) to analyze DEG and molecular mechanisms between SH003-treated and control samples. The DEG analysis revealed that 1007 and 475 genes were differentially expressed in SNU-4351S3-TO and SNU-4146S1-TO, respectively, relative to the control (Supplementary Fig. 3A). SH003 showed obvious regulation of inflammation/immune-related genes, sialophorin, immediate early response 3, tetraspanin 2, and FOS, CD8, and HPX in SNU-4351S3-TO and SNU-4146S1-TO (Supplementary Table 3). Cancer-related DEGs that showed significant differences compared to the control are also presented in Supplementary Tables 4 and 5. SH003-KEGG Pathway also revealed the activation of the immune system such as cytokine-cytokine receptor interaction, MAPK signaling pathway, and neutrophil extracellular trap formation as well as pathways in cancers (Supplementary Fig. 3B). We marked the decisive target genes of SH003 with blue boxes on the KEGG Pathway map of CRC, demonstrating that c-Myc would be a key mediator of the effects of SH003 on CRC (Supplementary Fig. 3C).

Fig. 3.

Fig. 3

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Inhibitory effects of SH003 on inflammatory mediators in CRC PDOs. CRC organoids, SNU-4351S3-TO and SNU-4146S1-TO were treated with SH003 (500 µg/ml) and nodakenin (20 µg/ml). (A) The mRNA expression levels quantified by qPCR. (B) The protein expression detected by Western blotting. Quantification is displayed as a bar chart on the right

SH003 inhibits the growth of CRC cells

Subsequently, in vitro experiments were further investigated the pharmacological effects of SH003 on CRC using HT-29 and DLD-1 cell lines. We first confirmed the regulatory effect of SH003 on CRC cell viability and apoptosis. Consistent with a previous report [28], this study also revealed that SH003 significantly decreased the CRC cell viability in a dose-dependent manner in both HT-29 and DLD-1 cells (Supplementary Fig. 4A). Supplementary Fig. 4B shows the higher proportions of Annexin V/PI double-positive cells and Annexin V-positive and PI-negative cells in SH003-treated groups compared to the control groups. Nodakenin also reduced the viability of CRC cells and induced apoptosis of CRC cells (Supplementary Fig. 4). The results of BrdU assay showed that cell proliferation was significantly suppressed by SH003 and nodakenin in both HT-29 and DLD-1 cells (Fig. 4A). In addition, SH003 and nodakenin significantly decreased the mRNA expression of Ki67 (Fig. 4B). Immunofluorescence staining of Ki67 revealed that SH003 and nodakenin effectively reduced the proliferation of CRC cells (Fig. 4C).

Fig. 4.

Fig. 4

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Effects of SH003 on proliferation in CRC cells. (A) The proliferation analysis by BrdU assay. (B) The mRNA expression levels quantified by qPCR. (C) Representative images detected by immunofluorescence. Scale bar, 20 μm

SH003 regulates both PD-L1 and CD47 in IFN-γ-stimulated CRC cells

IFN-γ is one of the most critical cytokines in the carcinogenesis [50]. We further assessed a regulatory effect of SH003 on proliferation of IFN-γ-stimulated HT-29 and DLD-1 cells. IFN-γ reduced proliferation of both cell lines, and SH003 further inhibited proliferation of IFN-γ-stimulated cells (Supplementary Fig. 5A). IFN-γ has dual functions in anti-tumor immunity and immune evasion. While IFN-γ potently inhibits tumor cell proliferation and activates cytotoxic T cells and macrophages, the prolonged exposure of IFN-γ can induce PD-L1/CD47 expression, as a feedback mechanism that aids immune escape [51, 52]. To confirm whether IFN-γ increases PD-L1 and CD47 levels in HT-29 and DLD-1 cells, we measured PD-L1 and CD47 expression by IFN-γ. We treated HT-29 and DLD-1 cells with IFN-γ (10 and 100 ng/ml) for 24 and 48 h and then performed qPCR and Western blot, as described in the previous studies [53, 54]. Figure 5A and Supplementary Fig. 5B show that both concentrations of IFN-γ significantly increased the mRNA and protein expression levels of PD-L1 and CD47 at 24 h and 48 h. SH003 and nodakenin substantially inhibited these levels in both HT-29 and DLD-1 cells (Fig. 5B). In addition, SH003 and nodakenin significantly decreased the protein expression levels of PD-L1 and CD47 in both HT-29 and DLD-1 cells (Fig. 5C). The results of flow cytometry also revealed that SH003 and nodakenin caused an effective attenuation of both cell surface PD-L1 and CD47 expression levels (Fig. 6A). This inhibitory effect of SH003 and nodakenin on PD-L1 and CD47 was further validated through an analysis of immunofluorescence in both HT-29 and DLD-1 cells (Fig. 6B).

Fig. 5.

Fig. 5

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Effects of SH003 on both PD-L1 and CD47 in IFN-γ-stimulated CRC cells. (A, B) The mRNA expression levels quantified by qPCR. (C) The protein expression detected by western blotting. Quantification is displayed as a bar chart on the right

Fig. 6.

Fig. 6

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Effects of SH003 on both cell surface PD-L1 and CD47. (A) Relative cell surface expression of PD-L1 and CD47 by flow cytometry. Mean fluorescent intensities (MFI) of each PD-L1 and CD47 staining is shown on the right side of the histogram. (B) Representative images detected by immunofluorescence. Scale bar, 20 μm

SH003 regulates c-Myc in IFN-γ-stimulated CRC cells

We continued to investigate whether SH003 regulates c-Myc in IFN-γ-stimulated HT-29 and DLD-1 cells. Figure 7A shows that IFN-γ (10 and 100 ng/ml) significantly increased the mRNA expression levels of c-Myc at 24 and 48 h in both HT-29 and DLD-1 cells. SH003 and nodakenin resulted in a significant decrease in the mRNA expression levels of c-Myc increased by IFN-γ (Fig. 7B). In the subsequent Western blot analysis with IFN-γ-stimulated HT-29 and DLD-1 cells, SH003 and nodakenin also suppressed the protein expression levels of c-Myc increased by IFN-γ (Fig. 7C). We further investigated whether c-Myc serves as an intermediate link in the regulation of tumor growth and PD-L1/CD47 by SH003 and nodakenin using a c-Myc inhibitor. The treatment with both c-Myc inhibitor and SH003 (500 µg/ml) significantly reduced the cell proliferation compared to the c-Myc inhibitor alone (Supplementary Fig. 6A). The PD-L1 and CD47 levels were significantly suppressed by the treatment with both c-Myc inhibitor and SH003 compared to the c-Myc inhibitor alone (Supplementary Fig. 6B). Thus, we examined the potential involvement of other known regulators of PD-L1 and CD47. The expression of PD-L1 and CD47 can also be regulated through STAT3 and NF-κB signaling pathways [55, 56]. We assessed whether SH003 and nodakenin would regulate the STAT3 and NF-κB as well as c-Myc. SH003 and nodakenin decreased the phosphorylation of STAT3 and NF-κB, indicating that SH003 could regulate PD-L1 and CD47 levels through STAT3/NF-κB pathways along with c-Myc pathways (Supplementary Fig. 6C).

Fig. 7.

Fig. 7

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Effects of SH003 on c-Myc in IFN-γ-stimulated CRC cells. (A, B) The mRNA expression levels quantified by qPCR in two human CRC cell lines (HT-29 and DLD-1). (C) The protein expression detected by western blotting. Quantification is displayed as a bar chart on the right

SH003 regulates cytotoxic T cell-mediated killing of CRC cells

To validate the impact of SH003 on immune cell-mediated killing of CRC cells, the PD-1-expressing activated Jurkat T cells were co-cultured with SH003-pre-treaed CRC cell lines. SH003 was shown to enhance cytotoxic T cell-mediated killing by inhibiting the viability of CRC cells (Fig. 8A). IL-2 promotes differentiation and cytotoxic effector cell formation in the CD8+ T cell and the inhibition of T cell proliferation and cytokine production by the interaction between PD-1 and PD-L1 can be overcome in an IL-2-rich environment ([57]; [58]). Figure 8B shows that IL-2 levels in medium from co-cultures were increased by SH003, indicating a positive effect on cytotoxic T cell activity [59, 60].

Fig. 8.

Fig. 8

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Effects of SH003 on the co-culture of CRC cells and T cells. (A) Viability of the remaining CRC cell lines (HT-29 and DLD-1) after the Jurkat T cells were removed. (B) IL-2 levels in culture supernatants measured by ELISA

SH003 regulates CRC in a mouse model

An HCT116 xenograft mouse model was constructed to evaluate the in vivo anti-tumor efficacy of SH003. From 7 days after treatment, the tumor volume in the SH003-treated group was smaller than that in the control, indicating tumor growth inhibition of SH003 (Fig. 9A). At endpoint, the SH003-treated group also showed decreases in the tumor sizes (Fig. 9B) and tumor weights compared to the control (Fig. 9C). During the in vivo experiment, the body weight remained unchanged, indicating no visible side effects (Fig. 9D). In addition, Western blot and immunofluorescence analyses showed that the expression levels of PD-L1, CD47, and c-Myc were reduced in the tumor tissues of SH003-treated group (Fig. 9E, F). The infiltration of CD8+ T cell was also increased in the tumor tissues of SH003-treated group (Fig. 9G).

Fig. 9.

Fig. 9

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Effects of SH003 on tumorigenesis in the HCT116 xenograft mouse model. (A) Tumor volume. (B) The representative images of excised tumors at endpoint. (C) Tumor weight at endpoint. (D) Body weight. (E) The protein expression detected by western blotting. Quantification is displayed as a bar chart on the right. (F, G) Representative images detected by immunofluorescence. Scale bar, 20 μm

Discussion

Building on earlier findings that SH003 regulates the apoptosis and proliferation of CRC cell lines, we further determined the efficacy of SH003 on adaptive and innate immune checkpoints in CRC PDOs as well as cell lines. The current findings showed that SH003 has anti-cancer effects by the dual targeting of PD-L1 and CD47 via the regulation of c-Myc signaling, simultaneously modulating inflammatory mediators in CRC. In addition, SH003 enhanced the cytotoxic T cell activity and infiltration. This study is the first to demonstrate that SH003 acts as a novel CRC immunotherapy (Fig. 10).

Fig. 10.

Fig. 10

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Proposed mechanism for the regulatory effect of SH003 on CRC growth

The co-expression of PD-L1 and CD47 were observed on several types of human cancer including CRC [61, 62]. Interestingly, simultaneous blockade of PD-L1 and CD47 augmented adaptive and innate tumor immune responses, indicating PD-L1 coordination with CD47 has more effective immunotherapy effect on cancers [63]. The simultaneous blockade of PD-L1 and CD47 with radiation therapy also resulted in clearance of primary tumors and robust abscopal effects on CRC [62, 64]. Wang et al., [62] suggested that targeting two different adaptive and innate checkpoints using bispecific molecules synergistically remodels the tumor microenvironment and promotes potent anti-tumor activity. In addition, lack of cytotoxic T cell infiltration and expansion of immunosuppressive factors within the tumor microenvironment are associated with tumor immune evasion mechanisms mediated by the PD-1/PD-L1 signaling pathway [65]. We found that SH003 inhibited both PD-L1 and CD47 expression in CRC PDOs and CRC cells and augmented cytotoxic T cell activity and infiltration, clarifying the immunological relevance of SH003-mediated PD-L1/CD47 regulation. However, co-culture studies of CRC organoids and T cells are further needed to provide direct evidence of how SH003-mediated downregulation of PD-L1/CD47 enhances T cell-mediated tumor cell killing.

c-MYC modulates the expression of PD-L1 and CD47, resulting in the immune evasion and immunosuppression, responsible the interaction between cancer cells and tumor microenvironment [66]. In the primary CRC of cohort 2, the protein and mRNA overexpression of c-Myc was detected in 57.6% and 77.4% of patients with CRC, respectively, suggesting that c-Myc plays an important role in the development of CRC [67]. Myc overexpression enforced relentless cellular proliferation and also accelerated cancer progression [68]. Small molecules that target Myc had suppressed cancer growth and synergized with immune checkpoint blockade therapy [69]. In addition, our previous study reported that SH003 inhibited the proliferation of several CRC cell lines through modulating RTK-STAT3 signaling pathways including ALK/c-Myc/cyclin D1, while inducing cell cycle arrest and apoptosis by regulating p-Rb, CDK2/4, PARP, caspase 3, caspase 8, and caspase 9 [28]. Additionally, the KEGG Pathway analysis confirmed that SH003 inhibits CRC proliferation through the c-Fos/c-Myc pathways. Our findings highlight the critical role of SH003 in decreasing c-Myc expression, thereby ultimately inhibiting both PD-L1/CD47 expression and CRC proliferation. Despite these promising results, further studies on clinicopathological feature and impact of Myc regulation are essential to validate the CRC immunotherapeutic efficacy of SH003.

Inflammation promotes cancer formation and numerous inflammatory factors significantly influence the PD-L1 and CD47 pathways [47, 70]. Chen et al., [70] suggested that combining anti-inflammatory agents with PD-1/PD-L1 inhibitors could be a target for immunotherapy with synergistic effects, as inflammatory factors influence tumor immune evasion. VEGF which is a known immunosuppressive factor, shapes the tumor microenvironment and drives tumor angiogenesis and immune evasion, and influences immune checkpoint activities [71]. Blocking VEGF signaling enhanced the anti-tumor efficacy of anti-PD-1/PD-L1 antibodies in preclinical models of several cancers including CRC [48]. The simultaneous inhibition of PD-L1 and VEGF showed superior inhibition of the proliferation of cancer cells than either alone [72]. IL-1β boosted PD-L1 expression in cancer cells, and IL-1β neutralizing inhibited cancer growth and showed synergistic anti-cancer efficacy with anti-PD-L1 antibodies in tumor-bearing in vivo models [49]. TNF-α blockade reduced CD47 expression and increased phagocytosis of cancer cells when combined with an anti-CD47 blocking antibodies [47]. The CD47 blockade combined with anti-VEGF therapy improved anti-cancer efficacy against gastric cancer [73]. We found that SH003 inhibited VEGF, TNF-α, and IL‐1β expression in CRC PDOs. These findings suggest that SH003 exerts greater anti-cancer efficacy against CRC by inhibiting PD-L1 and CD47, and simultaneously modulating inflammatory factors. Additionally, the KEGG Pathway analysis confirmed that SH003 regulates CRC through the activation of the immune system such as cytokine-cytokine receptor interaction. However, in-depth experiments on various inflammatory factors and their mechanisms of action are needed to determine a regulatory effect of SH003 on inflammation for CRC progression.

Several researchers have utilized CRC organoids to study biological mechanisms and pharmacological efficacy [74, 75]. Recently, the problems and limitations of animal testing have been raised with animal welfare and ethical concerns [76]. Organoid could be an alternative to animal testing [76, 77]. Several extracts from natural products have been demonstrated their anti-cancer effects using organoids [78, 79]. The strength of this study is that the critical role of SH003 is validated in CRC PDOs. Nevertheless, a limitation of the present study is the absence of research of SHOO3 on the complex cellular interactions and microenvironment using CRC PDOs. Additional studies are thus needed to clearly characterize SH003 as immunotherapy using CRC PDOs.

Conclusion

In conclusion, our study provides novel mechanistic insights demonstrating that SH003 has anti-cancer efficacy against CRC by dually targeting PD-L1 and CD47 through c-Myc pathways. Furthermore, this study is the first to verify the potential of SH003 as a potent modulator of both inflammatory mediators and immune checkpoints in CRC PDOs, providing a clinical approach that could significantly improve outcomes in CRC patients. However, further studies are needed to elucidate the complex molecular mechanisms underlying the efficacy of SH003 as an immunotherapy in the CRC microenvironment.

Electronic Supplementary Material

Supplementary Material 1 (2.2MB, docx)

Abbreviations

BrdU

Bromodeoxyuridine

CD47

Cluster of Differentiation 47

CRC

Colorectal cancer

IFN-γ

Interferon-γ

PD-L1

Programmed death-ligand

PDOs

Patient-derived organoids

TNF-α

Tumor-necrosis factor-α

VEGF

Vascular endothelial growth factor

Author contributions

N.R.H. and P.D.M. conceptualized and designed the study. N.R.H.wrote the paper draft. H.H.H., T.H.K., and H.C.L. performed the experiments.T.H.Y. provided technical assistance. H.J.P. and S.G.K. supervised the project and analyzed the data. P.D.M. reviewed the article.All authors have read and approved to the final version of the manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2020-NR049559).

Data availability

All data related to this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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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 1 (2.2MB, docx)

Data Availability Statement

All data related to this study are available from the corresponding author upon reasonable request.

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