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. 2024 May 24;23(6):682–692. doi: 10.1080/15384101.2024.2355825

Pumilio RNA binding family member 1 deficiency activates anti-tumor immunity in hepatocellular carcinoma via restraining M2 macrophage polarization

Yang Yu a,*, Gang Nie b,*, Yi-Wei Ren c, Liu Ouyang b,c,✉, Chen-Ming Ni b,✉
PMCID: PMC11229713  PMID: 38794797

ABSTRACT

Pumilio RNA-binding family member 1 (PUM1) has been implicated in both the progression of colorectal cancer and the regulation of inflammation. The role of PUM1 in the polarization of tumor-associated macrophages (TAMs) into the M2 phenotype has not yet been reported in hepatocellular carcinoma. Using the PUM1-knockout mice model, flow cytometry, and IHC, we validated the role of PUM1 in hepatocellular carcinoma (HCC) TAMs. One-way analysis of variance (ANOVA) or student’s t-tests was used to compare the experimental groups. We found that PUM1 inhibited anti-tumor immunity in HCC through TAM-mediated inhibition of CD8+ T cells. We also showed that PUM1 promotes the transformation of TAMs into pro-tumorigenic M2-like phenotypes by activating cAMP signaling pathway. This study emphasized the potential of PUM1 as a target for immunotherapy in HCC through TAMs. The present study revealed the molecular mechanism underlying the pro-tumor role of PUM1 in HCC.

KEYWORDS: Pumilio RNA-binding family member 1 (PUM 1), tumor-associated macrophages (TAMs), tumor microenvironment (TME), hepatocellular carcinoma (HCC), immunotherapy

Background

The incidence and mortality of HCC rank fifth and third among different cancers, respectively [1]. The majority of patients are diagnosed in the later stages, which worsens the overall prognosis of HCC. In addition to hepatitis C virus and hepatitis B virus infection, inflammation and several molecular mechanisms and signaling pathways have been linked to HCC progression [2]. Drug resistance, tumor recurrence, and metastasis significantly undermine the efficacy of numerous treatment options in HCC, including targeted therapy and immunotherapeutic medications [3].

Recently, it has been shown that the tumor microenvironment (TME) plays an essential role in the growth of tumors and can be a potential target for cancer treatment [4]. TAMs, as crucial immunosuppressive cellular components, are widely distributed in the TME [5]. TAM activation is a complicated process that manifests during tumor progression [6]. Typically, early-stage tumor-initiating macrophages have an M1 phenotype and engage in the anti-tumor response [7], while pro-tumorigenic TAMs induce an immunosuppressive state and encourage the growth of the malignant cells by secreting various anti-inflammatory cytokines or blocking the cytotoxic activity of CD8+ T cells, thus helping cancer cells to evade the immune surveillance [8]. TAMs have anti-tumorigenic and pro-tumorigenic phenotypes; thus, they are regarded as a potential target for treating malignancies [9]. Several preclinical investigations have shown that preventing macrophage recruitment or inhibiting M2 polarization can prevent tumor growth and improve the therapeutic response [10–14]. The identification of novel compounds capable of interfering with macrophage polarization may provide novel targets for TAM-based cancer immunotherapy.

Eukaryotic RNA-binding proteins (RBPs) are highly conserved in nature. The members of the PUF family, including FBF and Pumilio, bind to the sequence regions post-transcriptionally, thereby repressing gene expression. By binding to targets specifically on the 3’UTR region of mRNAs, the Pumilio-binding elements (PBEs) control cell differentiation [15–17]. Recent studies on genes encoding RNA-binding proteins showed that Pumilio-1 (Pum1) and Pumilio-2 (Pum2), members of the mammalian PUF family, post-transcriptionally control the growth and development of the eukaryotic brain and malignant tumors [18–20]. An earlier study revealed that PUM1 is overexpressed in colon cancer cells with cetuximab resistance and is associated with TAM abundance [21,22]. However, the mechanistic effect of PUM1 in HCC is still largely unknown.

In the current study, we investigated the processes by which PUM1 promotes the development of HCC. Using PUM1-knockout mice, we found that PUM1 inhibits anti-tumor immunity in HCC and is necessary for M2 polarization of TAMs. Our findings show the function of PUM1 in the microenvironment of HCC.

Material and methods

Mice and tumor model

Wild-type (WT) and PUM1-knockout (PUM1-/-) C57BL/6J mice were acquired from Service Bio-Co., Ltd (Wuhan, China). The Experimental Animal Ethics Committee of Service Bio Co., Ltd (Wuhan, China) approved the animal experiments, and this study was conducted following guidelines. MHCC97H cells were subcutaneously injected into the right flanks of female WT and PUM1-/- mice (6–8 weeks old) to reproduce the subcutaneous tumor model. Tumor tissues were removed at the end of the experiment for further measurements. We used bone marrow-derived macrophages (BMDMs) in this study. C57BL/6J mice lost macrophages after the intraperitoneal injection of 200 mL of clodronate liposomes (YEASEN, Shanghai, China) 48 hours before the inoculations. The combination of MHCC97H cells and macrophages obtained from WT or PUM1-/- mice were subcutaneously injected into macrophage-depleted C57BL/6J mice. After measuring the diameter of the tumor, mice were sacrificed according to protocols, and tumor-infiltrating lymphocytes were measured following previous studies [23].

Cell lines and preparation of tumor-conditioned medium

Hep3B, HepG2, HCCLM3, Huh7, and MHCC-97 H HCC cell lines were used in this study. Moreover, U937 and THP-1 were the two human monocyte cell lines used in this study. All cell lines were kept and used following corresponding guidelines from the American Type Culture Collection (Maryland, USA). By adding phorbol-12-myristate-13-acetate (PMA; 200 nmol/L, Sigma Aldrich, Taufkirchen, Germany) to human U937 and THP-1 cell lines, these cells were transformed into mature macrophages after 48 hours. A complete growth medium was used to culture cancer cells. The medium was replaced by a serum-free medium when cells reached about 80% confluence. A fresh batch of conditioned medium (CM) was obtained after 24 hours, centrifuged for 5 minutes at 500 g, filtered through 0.22 µm filters, and then stored at −80°C.

Extraction and stimulation of mouse primary cells

Cells obtained from the bone marrow of female WT and PUM1-/- mice were cultured for 7 days in Iscove’s modified Dulbecco’s medium (Gibco, Grand Island, NY, USA) supplemented with FBS (concentration 10%) and M-CSF (50 ng/mL; PeproTech, NJ, USA) to obtain BMDMs [23]. BMDMs were cultured for 48 hours with IL4/IL13 (20 ng/mL, PeproTech) to induce M2 macrophage differentiation. To obtain TAMs, a half volume of tumor CM was mixed with the culture medium, and the mixture was incubated for 48 hours.

In vitro phagocytosis assay

First, several phenotypes of macrophages were added to 96-well low-attachment plates. These plates were then co-cultured with Carboxyfluorescein succinimidyl ester (CFSE)-labeled MHCC-97 H cells. All cells were collected after two hours and stained with F4/80 antibody for 30 minutes on ice. This antibody targets a murine macrophage surface marker. Then, samples were subjected to flow cytometry. The proportion of F4/80+ cells with fluorescence from CFSE was used to calculate the efficiency of phagocytosis.

CD8+ T cell suppression assay

To isolate CD8+ T cells of mice from C57BL/6J mice, we used a CD8+ T cell isolation kit specific for mice (Stem Cell Technologies, Vancouver, Canada). After stimulation with aCD3/aCD28 and IL-2 (PeproTech) and co-culturing with various phenotypes of macrophages for 72 hours, CD8+ T cells supplemented with eBioscience™ Cell Stimulation Cocktail (Invitrogen, Carlsbad, CA, USA) were incubated for 4 hours. Cells were then collected and subjected to flow cytometry to determine whether they proliferated and expressed functional markers.

Immunohistochemical staining (IHC)

All paraffin sections were routinely deparaffinized and rehydrated in xylene and graded ethanol solutions, and IHC was performed as previously described in the literature [23]. Anti-CCR2 antibody (1:100 dilution; Abcam, Cambridge, UK) and anti-CCL2 antibody (1:100 dilution; Abcam, Cambridge, UK) were used as primary antibodies.

Statistical analysis

One-way analysis of variance (ANOVA) or student’s t-test was used for comparing the experimental groups. p < 0.05 was considered statistically significant. Statistical Package for the Social Sciences (SPSS) v17.0 (IBM, USA) and GraphPad Software (La Jolla, CA, USA) were used for all statistical analyses.

Results

PUM1 knockout reduced the growth of MHCC-97 H cells and promoted the polarization of TAMs to the M2-like phenotype

MHCC-97 H cells were subcutaneously injected into WT and PUM1 knockout mice to explore the role of PUM1 in the tumor microenvironment. PUM1 expression was considerably low in the primary BMDMs of PUM1-/- mice (Figure S1A). The growth of MHCC-97 H tumors in PUM1-/- mice was noticeably slower than that in the WT group (Figure 1(a,b)). We also investigated immune cell infiltration into MHCC-97 H tumors. Tumors from PUM1-/- mice exhibited reduced TAM infiltration compared with tumors from WT mice. There was no discernible variation in the frequency of all hematopoietic cells, such as CD3e, CD11b, dendritic cells (DC), CD4+T, and CD8+T cells (Figure 1(c,d). Further experiments showed that PUM1 deficiency reduced the abundance of CD206+ TAM cells (Figure 1(e)) but increased M1 to M2 macrophage ratio and the number of IFN-γ+CD8+T cells compared with MHCC-97 H-WT animals (Figure 1(f,g), suggesting that PUM1 knockout reduced the differentiation of TAMs to the M2-like immunosuppressive phenotype. Although the difference was not significant, we showed that PUM1-/- mice had a lower abundance of monocytes in their blood (Figure S1B). Additionally, PUM1-/- mice displayed lower serum levels of IL-10 (Figure S1C), and decreased protein levels of TAM-associated markers CCR2 and CCL2 (Figure S1D). These findings suggest that the controlled phenotypic change of TAMs to an M2-like state may help decelerate tumorigenesis in PUM1-/- mice.

Figure 1.

Figure 1.

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PUM1 deficiency inhibited the formation of MHCC-97 H tumors and induced the transformation of TAMs to M2-like macrophages. 2 × 106 (n = 3) MHCC-97 H cells were subcutaneously implanted into 8-week-old female WT (n = 3; triplicate) and PUM1 knockout (PUM1-/-) mice (n = 3). Experimental mice were sacrificed after 21 days. (a) Growth curve obtained for each tumor. (b) The weight of each tumor was measured. (c)–(g) Flow cytometry of various immune subsets in each tumor. Infiltration of (c) CD11b+, DC, CD3e, CD4+ T cells, and CD8+ T cells, and (d) TAMs (CD11b+ F4/80+) were examined by flow cytometry. (e) M2-like TAMs (CD11b+ F4/80+ CD206+), (F) M1 (CD11b+ F4/80+ CD86+)/M2 ratio, and (g) IFN-γ+ CD8+T cells were also measured using flow cytometry. Data are reported as mean and standard deviation (SD) (n = 3). p-values (*p < 0.05) were calculated using a two-tailed student’s t-test. #non-significant results compared with the control (WT) group.

PUM1-deficient macrophages were less likely to polarize toward M2-like phenotypes

The effects of PUM1 deficiency on macrophages were then verified in vitro. Firstly, BMDMs were isolated and incubated with IL-4 and IL-13 from PUM1-/- mice (PUM1-/-BMDMs) or WT mice (WT-BMDMs). PUM1-/- BMDMs produced lower levels of ARG-1 and IL10, two typical M2 markers, compared with WT-BMDMs. In line with these findings, the number of macrophages (CD206+M2) was lower in PUM1-/-BMDMs (Figure 2(a,b)). These findings suggest that PUM1 is necessary for macrophage polarization. To better represent the role of PUM1 in the TME, WT-BMDMs were cultivated with CM isolated from MHCC-97 H cell culture (MHCC-97 H-CM), which upregulated PUM1 in TAMs (Figure 2(c)). CM obtained from other human tumor cell cultures produced comparable results (Figure 2(d)). In addition, we measured the phenotypic differences of BMDMs derived from wild-type and PUM1-/- animals. Compared with their WT counterparts, PUM1-/- BMDMs co-cultured with MHCC-97 H-CM showed enhanced gene expression, which was related to the M1 type of macrophages (Figure 2(e)) and reduced expression of genes related to M2 type macrophages (Figure 2(f)). Flow cytometry indicated that PUM1-/- macrophages less likely polarized to the M2 phenotype, evidenced by higher expression of CD86 and a lower expression of CD206 (Figure 2(g,h)).

Figure 2.

Figure 2.

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PUM1 deficiency in the macrophages resistant to M2 polarization. (a) BMDMs from WT and PUM1-/- mice were treated with or without IL4/IL13 (6 hours). We performed qPCR of ARG-1 and IL-10 to measure their expression. (b) For 48 hours, we cultured BMDMs with IL4/IL13. Flow cytometry was used to determine the percentage and mean fluorescence intensity of F4/80+ CD206+ M2 phagocytes. (c) The expression of PUM1 in BMDMs cultured with CM from MHCC-97 H cells for 48 hours (Western blot bands are shown in the left panel) and 6 hours (qPCR is shown in the right panel). (d) Western blotting (48 hours) and qPCR (6 hours) measuring PUM1 expression in U937 macrophages cultured with CM from HCC cells. WT and PUM1/BMDMs were co-cultured with MHCC-97 H-CM for 6 hours. qPCR was used to detect the expression of genes associated with (e) inflammation or (f) immunosuppressive macrophages. β-actin was considered as the control. Flow cytometry (48 hours) was used to measure the expression of (g) CD86 and (h) CD206. All of our data are represented as mean ± SD (n = 3). As previously mentioned, we used a two-tailed student’s t-test to calculate statistical significance (*p < 0.05).

PUM1 deficiency increased phagocytosis and decreased tumor-promoting activity

We evaluated how PUM1 affects the ability of macrophages to phagocytose tumor cells. WT- or PUM1-/- BMDMs were co-cultured with CFSE-labeled MHCC-97 H cells. Compared with WT mice, M2 macrophages from PUM1-/- mice showed enhanced phagocytosis of MHCC-97 H cells (Figure 3(a)). Similar outcomes were also seen in BMDMs treated with MHCC-97 H-CM. After exposure to MHCC-97 H-CM, PUM1-/- BMDMs exhibited increased phagocytosis ability than WT-BMDMs (Figure 3(b)). Next, we explored how PUM1 affected tumor cell proliferation. CM from WT macrophage increased the proliferation of MHCC-97 H cells. In contrast, the proliferation ability of MHCC-97 H cells treated with CM from PUM1−/− macrophages was decreased compared with that of WT-BMDMs (Figure 3(c,d)).

Figure 3.

Figure 3.

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PUM1 deficiency enhanced the phagocytic property of macrophages and attenuated its tumor-promoting properties. 1 × 105 BMDMs were taken (WT and PUM1-/- mice) and cultured with IL-4/IL-13 or CM-treated MHCC-97 H for 48 hours. 2 × 105 CFSE- fluorescently labeled MHCC-97Hs were used for an additional 2 hours. Phagocytosis data are represented as (a) M2- macrophages or (b) CM-macrophages and were determined using the flow cytometry technique. (c) and (d) show the effect of PUM1 on the proliferation and migration of tumor cells. BMDMs (PUM1-/- and WT) were exposed to IL-4/IL-13 or MHCC-97 H-CM for 48 hours, and macrophage-specific CM was harvested. MHCC-97 H cells were used to cultured either (C) M2- CM or (D)TAM- CM. Two-tailed student’s t-test was employed to determine p-values. *p < 0.05, compared with the WT group.

PUM1 deficiency attenuated TAM-mediated inhibition of CD8+ T cells

TAMs had a suppressive effect on the proliferation ability and anti-tumor activity of T-cells. Thus, we investigated the immunomodulatory effects of PUM1 on phagocytes by co-culturing M2 or CM-treated macrophages with CD8+ T cells. Co-culturing CD8+ T cells with M2 macrophages derived from PUM1-/- mice increased activated CD8+ T cell/total CD8+ T cell ratio. In addition, the abundance of CD8+ T cells that were positive for granzyme B, IFN-γ, or TNF-α also increased compared with WT controls (Figure 4(a–c)). CD8+ T cells co-cultured with tumor CM-treated macrophages from PUM1-/- mice exhibited similar characteristics. After co-culturing with CM-treated PUM1-/- phagocytes, the cytotoxic cytokines of CD8+T cells were upregulated (Figure 4(d–f)). These findings highlight the critical role of PUM1 in the immunosuppressive properties of TAMs.

Figure 4.

Figure 4.

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PUM1 deficiency attenuated the suppressive effect of TAM on CD8+ T cells. PUM1-deficient macrophages enhanced the function of co-cultured CD8+ T cells. the number of seeded macrophages was 1 × 105 taken from either wild-type or PUM1-/- animals. They were exposed to (a–c) IL- 4/IL-13 or (d)-(f) MHCC-97 H-CM for 48 hours, and then co-cultured with CD8+ T cells (number of cells = 5–10 × 105) for 72 hours. Flow cytometry was utilized to analyze the three most prevalent cytotoxic cytokines produced by CD8+ T cells, including IFN- γ, TNF- α, and Granzyme B (GzmB). Data are reported as mean and SD (n = 3). Two-tailed student’s t-test was used to determine statistical significance (*p < 0.05).

Activation of cAMP signaling by PUM1 induced the M2 polarization of macrophages

We also measured cAMP level and found that it was increased in WT-BMDMs treated with IL4/IL13 or MHCC-97 H-CM but was reduced in PUM1-/- BMDMs (Figure 5(a,b)). Forskolin also significantly increased IL-10 and ARG-1 expression in PUM1-/- macrophages (Figure 5(c–f)). These data indicate that PUM1 induced cAMP signaling to promote macrophage M2 polarization.

Figure 5.

Figure 5.

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Activation of the cAMP signaling pathway by PUM1 is responsible for the M2 polarization of macrophages. (a&b) IL4/IL13 (a) or MHCC-97 H-CM (b) stimulated WT and PUM1-/-BMDMs for 2 hours, and cAMP levels were measured. (c–F) WT and PUM1-/- macrophages were pre-treated with Forskolin. Next, they were treated with IL4/IL13 (c&d) or MHCC-97 H-CM (e&f) for 6 hours. The expression of IL-10 (d&f) and ARG-1 (c&e) was determined. Data are presented as mean ± SD (n = 3). Statistically significant findings (p-value) were determined using one-way analysis of variance (ANOVA). *p < 0.05.

PUM1-/- BMDM inhibited MHCC-97 H tumor growth and enhanced antitumor immunity

We investigated whether PUM1-/- BMDMs can reshape TME and demonstrate anti-tumor effects in vivo. Herein, MHCC-97 H cells and PUM1-/- or WT BMDMs were combined before implantation into macrophage-depleted mice (C57BL/6J). PUM1-/- macrophage-containing MHCC-97 H xenografts showed slower tumor development than those with WT macrophages (Figure 6(a,b)). Thereafter, we measured the effect of PUM1-/- macrophages on the turnover of TAMs and tumor infiltration of different groups of immune cells, specifically CD8+T cells. The abundance of CD45+ cells, a group of hematopoietic cells, did not change in the TME (Figure 6(c)). The abundance of TAMs decreased in PUM1-/- macrophages co-cultured MHCC-97 H group (Figure 6(c)). In addition, the number of CD86+M1 macrophages increased, whereas the abundance of CD206+M2 macrophages significantly decreased (Figure 6(e,f)). These findings imply that PUM1 deficiency shifted TAM polarization from the M2-like phenotype to the “anti-tumor” M1-like phenotype. The balance between “don’t eat me” and “eat me” signals influences tumor cell recognition and phagocytosis. In the PUM1-/- macrophage co-cultured MHCC-97 H group, the “don’t eat me” signaling molecule CD47 was downregulated (Figure S1E), indicating that PUM1 prevented macrophage-mediated phagocytosis of tumor cells. The phenotypic change of TAMs was accompanied by noticeable upregulation of MHC class II on DCs (Figure 6(g)) and increased abundance of IFN-γ+ CD4+T cells (Figure 6(h)), suggesting the activation of DCs and Th1-dominant response. Additionally, the PUM1-/- macrophage group had an increased abundance of granzyme B+CD8+ T cells (Figure 6(i)) and IFN-γ+CD8+T cells (Figure 6(j)) and decreased number of PD-1+CD8+T cells (Figure 6(k)). The results showed that PUM1-/- BMDMs modified the immunosuppressive TME to enhance the anti-tumor immune response in the TME.

Figure 6.

Figure 6.

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PUM1/BMDM inhibited MHCC-97 H tumor growth and enhanced anti-tumor immunity. MHCC-97 H cells (2 × 106 cells) were subcutaneously injected into C57BL/6J mice whose macrophage levels were previously decreased and were injected with WT or PUM1-/- phagocytes (5 × 105 cells). (a) The volume and (b) weight of the tumor are presented. (C)–(K) Analysis of immune subsets in the TME by flow cytometry. (c) CD45+ cell infiltration among tumor cells. The observed infiltration data of (d) TAMs (CD11b+F4/80+), (e) CD86+TAMs, (f) CD206 + TAMs, (g) MHC on DC, (H) IFN-γ+CD4 +T cells, (i) IFN-γ+CD8+T cells, (j) GzmB + CD8+T cells, and (k) PD1+ CD8+T cells were measured. Data are provided as mean and SD (n = 3). The p-values were computed using a two-tailed student’s t-test. *p < 0.05, # non-significant result, compared with the WT-BMDMs group.

Discussion

Established tumors with TAMs are typically biased in the direction of the pro-tumorigenic M2 polarization, encouraging tumor growth and suppressing the anti-tumor immune response. High abundance of M2 macrophages results in a poor prognosis in most malignancies [24]. Thus, TAMs are regarded as crucial targets for cancer immunotherapy. In this study, we identified an essential regulatory function of PUM1 in TAM polarization in HCC. PUM1 induced the immunosuppressive phenotype of macrophages and blocked the anti-tumor immune response in TME. Therefore, we concluded that PUM1 is a promising target for HCC immunotherapy.

Our findings suggest that PUM1 is necessary for M2 polarization of macrophages and induction of an immunosuppressive phenotype of macrophages in the TME. Furthermore, PUM1 inhibition or knockdown in macrophages increased the number of pro-inflammatory macrophages. This study explored the role of PUM1 in remodeling TAMs and immunosuppression in HCC. Compared with MHCC-97 H-WT mice, PUM1-/- animals had reduced levels of CD206+TAM and TAM and higher levels of IFN± CD8+T cells. These findings suggest that PUM1 exerts an immunosuppressive effect on the TME and inhibits tumorigenesis. Additionally, our in vitro data showed that PUM1 induced TAMs to polarize into a pro-tumoral state. PUM1-/-macrophage inhibited tumor growth and enhanced the immune response against cancer in vivo, evidenced by a marked decline in the abundance of M2-like TAMs and increased abundance of anti-tumor lymphoid cells, such as CD4 +T cells, mature DCs, and CD8+ cells. The immunological microenvironment of a carcinoma determines how various immunotherapeutic approaches will work [25]. Reprogramming immune cells and boosting anti-tumor immunity with the use of immunosuppressive TAMs and other similar agents can overcome treatment resistance [26–29]. PUM1 might be a promising target for improving response to immunotherapy.

Various extracellular stimuli stimulate the second messenger cAMP through GPCRs [30–35]. Furthermore, cAMP and IL-4 synergistically increase the expression of STAT6 and reprogram macrophage polarization. We further verified that cAMP contributes to the differentiation of TAMs in the TME via PUM1. Additionally, in PUM1-deficient macrophages, inducing cAMP synthesis restored M2-like behaviors. Our findings suggest that PUM1 deficiency in TAMs prevents the polarization of macrophages toward pro-tumor phenotypes by reducing cAMP signaling.

Conclusions

Our findings demonstrate the significant regulatory role of PUM1 in TAMs. It enhances the immunosuppressive phenotype of macrophages, suppresses anti-tumor immunity, and accelerates the growth of HCC. Thus, PUM1 is a target for anti-tumor immunotherapy in HCC. It may help break the resistance to immune checkpoint blockade (ICB) therapies.

Supplementary Material

supplement-data.docx
KCCY_A_2355825_SM6848.docx (905.8KB, docx)

Acknowledgements

We sincerely thank editsprings company (https://www.editsprings.com) for their professional linguistic services provided.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Liu Ouyang and Chen-ming Ni designed the study. All of the authors were involved in conducting the experiment, data analysis, and writing the manuscript. All of the authors revised the manuscript. All of the authors studied and approved the final manuscript.

Availability of data and materials

The data used for the current study will be available from the corresponding authors upon reasonable request.

Supplemental material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2024.2355825

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Associated Data

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

Supplementary Materials

supplement-data.docx
KCCY_A_2355825_SM6848.docx (905.8KB, docx)

Data Availability Statement

The data used for the current study will be available from the corresponding authors upon reasonable request.

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