Neighboring macrophage-induced alteration in the phenotype of colorectal cancer cells in the tumor budding area

One hundred five tissue samples from 105 CRC patients were collected after diagnosis at Yamagata University Hospital between 2010 and 2023. Tissues were fixed with 10% neutral-buffered formalin for 48 h at room temperature and embedded in paraffin. Three-micrometer sections were prepared for hematoxylin–eosin (HE) staining and IHC. The CRC specimens were classified according to the TNM classification (Union for International Cancer Control, 2018). This study was approved by the Research Ethics Committee of Yamagata University Faculty of Medicine (2019-93) and was performed in accordance with the Declaration of Helsinki.

One hundred tissue samples with accompanying clinical data were used to evaluate TB. The criteria of the International Tumor Budding Consensus Conference 2016 recommend evaluating the number of tumor buds by HE stains [3]. Because the AE1/AE3-IHC specimen has been reported to help in recognizing CRC cells, particularly in TB area [24], we evaluated the number of tumor buds by AE1/AE3-IHC specimens (n = 100) using AE1/AE3 antibody (AE1/AE3, Abcam, Cambridge, UK). The number of tumor buds was confirmed by two board-certified pathologists (OR and FM) based on previous studies [3, 24]. For quantitative analysis of the number of tumor buds, AE1/AE3-IHC specimens were examined under a light microscope and assessed at a magnification of 200× for each specimen. Most of the significant differences in Kaplan‒Meier curves of OS and RFS were observed when the cutoff value was set to 14 buds/field at a magnification of 200×. “TB^high” was defined as 14 or more buds/field at a magnification of 200×, and “TB^low” was defined as less than 14 buds/field at a magnification of 200×.

We identified three lesions equidistant from the mucosal surface to the lesion above the invasive front and defined them as depth 1, depth 2, and depth 3. Because the invasive front includes TB, we defined the lesion of the invasive front without TB as depth 4, and we defined the lesion with TB as depth 5.

Single and double immunostaining were performed with BOND RX^m (Leica Biosystems, Nussloch, Germany) according to the manufacturer’s protocol. We used a TOPO1 antibody (EPR5375, Abcam) to estimate the acquisition of chemoresistance in CRC. For quantitative analysis, TOPO1-IHC samples were examined as whole-slide images. The positive ratio of TOPO1 on cancer cells was evaluated at each depth (depth 1–5) of CRC specimens (n = 20). We used a CD205 antibody (LY-75; EPR5233; Abcam) to estimate the malignant potential of CRC. For quantitative analysis, CD205-IHC samples were assessed at a magnification of 200× under a light microscope (n = 100). CD205 positivity was examined in two areas: the invasive front without TB (depth 4) and the TB area (depth 5). CD205-IHC was considered positive if more than 30% of CRC cells were positive. To compare proliferative potential of between CRC cells at depth 4 and that at depth 5, we performed double immunostaining for AE1/AE3 and Ki-67 (K-2, Leica Biosystems) (n = 5). In double immunostaining for AE1/AE3 and Ki-67, three areas were selected from each of the five cases and counted. We used antibodies of CD68 (514H12, Leica Biosystems), CD163 (10D6, Leica Biosystems), CD204 (SRA-E5, Trans Genic Inc, Fukuoka, Japan), and CD206 (5C11, Abnova, Taipei, Taiwan) to evaluate the induction of Mφs in CRC specimens (n = 20). Double immunostaining for CD68 or CD163 and interleukin-6 (IL-6) (1.2-2B11-2G10, Abcam) was performed using the Opal Multiplex IHC KIT (OPAL 4-COLOR AUTOMATION IHC KIT; AKOYA BIOSCIENCES, Marlborough, MA, USA) to estimate Mφ-produced IL-6 (n = 4). Similarly, double immunostaining for AE1/AE3 and phospho-STAT3 (pSTAT3) (D3A7, Cell Signaling Technology, Danvers, Massachusetts) was performed to estimate the upregulation of the IL-6 receptor (IL-6R)/STAT3 pathway in CRC cells (n = 4). An All-in-One Fluorescence Microscope (BZ-X810, KEYENCE, OSAKA, Japan) was used to assess the positivity according to double immunostaining.

Whole-slide imaging of the IHC slides of CD68 and CD163 was generated with a virtual scanner (NanoZoomer, HAMAMATSU, Hamamatsu, Japan). To demonstrate the distribution of CD68-positive (CD68⁺) or CD163-positive (CD163⁺) Mφs, histological spatial analysis with HALO software (Indica Labs) was used to allocate positive cells on a grid chart of CD68- or CD163-stained sections. The number of positive cells in a square lesion at each depth of CRC specimens was counted. Histological spatial analysis was used to count the number of cancer cells and Mφs and calculate the ratio of Mφs to surrounding CRC cells at depth 4 and depth 5. Histological spatial analysis identified CD68⁺ Mφs within 30 μm from a single CRC cell with a green color and CD68⁺ Mφs located more than 30 μm from a single CRC cell with a light green color. Furthermore, histological spatial analysis measured the distance between CRC cells and Mφs to evaluate the interaction between CRC cells and CD68⁺ or CD163⁺ Mφs in the selected areas (1 mm²) at depth 4 and depth 5, respectively. A histogram was drawn in which the number of CD68⁺ or CD163⁺ Mφs was shown in each class interval of the distance.

The human CRC cell lines, such as HCT116 and SW480, were used in this study. HCT116 were maintained in McCoy’s 5A (Modified) Medium (Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum supplemented with Antibiotic–Antimycotic Mixed Stock Solution (100×) (nacalai tesque, Kyoto, Japan) at 37 °C in a humidified atmosphere containing 5% CO₂. SW480 were maintained in Leibovitz’s L-15 Medium (Thermo Fisher Scientific) with 10% fetal bovine serum supplemented with Antibiotic–Antimycotic Mixed Stock Solution (100x) (nacalai tesque, Kyoto, Japan) at 37 °C in a humidified atmosphere without CO₂. HCT116 and SW480 cells were seeded in 96-well plates at the density (5000 cells/well). Following overnight adherence, cells were incubated with medium alone or media containing different concentrations of recombinant IL-6 (0, 0.01 ng/ml) (HumanKine® recombinant human IL-6 protein, Protein Tech, Rosemont, IL) for 24 h. Cell proliferation assay was evaluated by the absorbance value of Cell Counting Kit-8 (CCK-8, DOJINDO, Kumamoto, Japan). SW480 transfected with siRNA against IL-6 receptor (IL-6R) (s7314, Thermo Fisher Scientific) by electroporation (Neon Transfection System, Thermo Fisher Scientific), and we established mock cells (SW480^mock) and IL-6R knocked down SW480, SW480^IL-6R-ND and SW480^IL-6R-ND2. These cell lines were treated with recombinant human IL-6 (HumanKine® recombinant human IL-6 protein, Protein Tech) and were performed cell proliferation assay. All these cell proliferation assays were performed in 10 wells.

qRT-PCR was performed to quantify IL-6R mRNA relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Total RNA was extracted from CRC cell lines (HCT116, SW480, SW480^mock, SW480^IL-6R-ND, and SW480^IL-6R-ND2) using the ISOGEN system (NIPPON GENE, Tokyo, Japan). The cDNA was synthesized using TaKaRa RNA PCR™ Kit (AMV) Ver.3.0 (Takara, Otsu, Japan). The following primer sequences were designed by Primer3Plus (https://​www.​primer3plus.​com/​index.​html): 5′-AAACCAATGTTCTCTCTTCTCCAC-3′ and 5′-TATTCCACCCTCTCAAGAGAAAAC-3′ for human IL-6R (240 bp). The following primer sequences were used as the internal control: 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′-TGGTGAAGACGCCAGTGGA-3′ for human GAPDH (138 bp) [14]. IL-6R and GAPDH mRNA levels were assessed using PowerTrack™ SYBR Green Master Mix for qPCR (Thermo Fisher Scientific). Amplification was performed using the QuantStudio3 (Thermo Fisher Scientific). The PCR protocol consisted of initial heat activation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s, and dissociation was performed at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The relative gene expression levels were calculated relative to the levels of GAPDH mRNA using the comparative Ct (2^−ΔΔCt) method. All these qRT-PCR experiments were performed in triplicate.

The χ² test, Fisher’s exact test, or the Mann‒Whitney test was used to compare the clinicopathological characteristics of CRC patients. Kaplan‒Meier curves of OS and RFS were drawn and compared by the log-rank test. Univariate and multivariate Cox proportional hazards regression models were used to identify prognostic factors. The Kruskal–Wallis test was used for the TOPO1-positive ratios at all depths. If the ratios were significant, Bonferroni’s multiple comparison test was further applied. The Mann‒Whitney test was used to evaluation the IHC at depth 4 and that of depth 5. The t-test was used to compare relative expression of IL-6R mRNA. The Mann‒Whitney test was used to compare absorbance values of CCK-8. Differences with P values < 0.05 were considered significant in each analysis. These statistical analyses were performed using JMP version 16 (SAS Institute, Tokyo, Japan).

A typical example of TB was shown in Fig. 1A. To examine the relationship between the number of tumor buds and prognosis in CRC patients, we divided 100 CRC patients into TB^high (Fig. 1A) and TB^low groups (Fig. 1B). Kaplan‒Meier analysis revealed a statistically significant difference in OS (Fig. 1C) and RFS (Fig. 1D), including all stages (I-IV), when the cutoff value of the number of tumor buds was set to 14 at a magnification of 200×. At week 60, OS in the TB^high group was significantly lower than that in the TB^low group (Fig. 1C). Similarly, RFS in the TB^high group was significantly lower than that in the TB^low group (Fig. 1D). No significant difference was found in OS and RFS between the TB^high group and the TB^low group with matched patient backgrounds (stages I, II, III, or IV). Except for the number of tumor buds, no significant differences were demonstrated between the two groups in clinicopathological characteristics (Additional file 2: Table S1). Univariate analyses of OS and RFS revealed that poor prognosis was correlated with TB^high by Cox proportional hazards regression models (Additional file 3: Table S2). Multivariate analyses on OS and RFS revealed that only stage III/IV was an independent poor prognostic factor. Our results indicated that the number of tumor buds was one of the prognostic factors in CRC patients.

We observed TOPO1-positive findings in the nuclei of CRC cells (Fig. 2A). Among the TB^high cases, 20 cases were selected to elucidate the heterogeneity of TOPO1 positivity. We examined the expression of TOPO1 based on the depth of CRC specimens. The representative TOPO1-IHC showed that 98.5% of CRC cells were positive at depth 1 (Fig. 2A) and no CRC positive cells at depth 5 (Fig. 2B). Quantitative analysis revealed that the TOPO1-positive ratios at depth 1, 2, 3, 4, and 5 were 80.3 ± 12.7%, 60.0 ± 28.8%, 30.4 ± 33.4%, 27.5 ± 32.6%, and 5.7 ± 6.7%, respectively (n = 20) (Fig. 2C). The representative heatmap of the TOPO1-positive cells revealed that a higher density was observed in the mucosal area (depths 1–3), a lower density in the invasive front (depth 4), and a significantly lower density in the TB area (depth 5) (Fig. 2D). Furthermore, scatter plot between the positivity of TOPO1 and the depth revealed that the low TOPO1-positive ratio was negatively correlated with deeper depth (n = 20, r_s = − 0.6700, P < 0.01) (Fig. 2E). These results indicated the heterogeneity of TOPO1 positivity in CRC specimens, and the TOPO1-positive ratio was lowest in the TB area (depth 5).

To emphasize the difference in the phenotype of CRC cells at depth 4 and 5, we compared the TOPO1-IHC positive findings of CRC cells at depth 4 and 5. The TOPO1-positive ratios at depth 5 were statistically lower than those at depth 4 (depth 4, 27.5 ± 32.6%; depth 5, 5.7 ± 6.7%; P = 0.049) (Fig. 3A–C). We compared the expression of CD205 in CRC cells at depth 4 and 5 by IHC. In 14 of CD205 positive cases, the positive ratio of CD205 was 86.7 ± 14.4% at depth 4 (Fig. 3D), no CD205-positive CRC cells were observed at depth 5. (Fig. 3E). In 86 of CD205 negative cases, no CD205 positive cells were observed at depth 4 and 5. Furthermore, we compared the Ki-67 labeling indices (LI) of CRC cells at depth 4 and 5 to evaluate the proliferative potential. We found that the Ki-67 LI of CRC cells at depth 5 were statistically lower than those at depth 4 (Fig. 3F–H; depth 4, 24.2 ± 12.1%; depth 5, 5.3 ± 5.3%; P < 0.01). These results indicated that the phenotype of CRC cells at depth 5 (the TB area) was different from that at depth 4 (the invasive front without TB).

Because Mφs have been shown to alter the phenotype of cancer cells through tumor–stromal interactions, we hypothesized that Mφs may alter the phenotype of CRC cells in the TB area (depth 5). A higher number of Mφs near CRC cells would be involved in tumor–stromal interactions at depth 5; we compared the number of Mφs at depth 5 in 20 cases with that at depth 4 in the same cases. In histological spatial analysis of the representative case (Fig. 4A, B), we found that eight CD68⁺ Mφs infiltrated at depth 4 (Fig. 4A) and 50 Mφs infiltrated at depth 5 (Fig. 4B). Histological spatial analysis identified the nuclei of CRC cells and allocated CRC cells with a red color (Fig. 4C, D) on a grid chart. One hundred twenty-five CRC cells integrated into cancer tissue at depth 4 (Fig. 4C), and 40 isolated CRC cells were observed at depth 5 (Fig. 4D). The number of CD68⁺ Mφs per single CRC cell at depth 4 was 0.69 ± 0.45, and that at depth 5 was 2.56 ± 2.19. Similarly, the number of CD163⁺ Mφs per single CRC cell at depth 4 was 1.78 ± 1.57, and that at depth 5 was 5.31 ± 4.30. The number of CD204⁺ Mφs per single CRC cell at depth 4 was 0.22 ± 0.29, and that at depth 5 was 6.70 ± 4.48. The number of CD206⁺ Mφs per single CRC cell at depth 4 was 0.10 ± 0.24, and that at depth 5 was 1.34 ± 3.74. The number of Mφs per single CRC cell at depth 5 was higher than that at depth 4 in all the Mφ markers (P < 0.01) (Fig. 4E–H). In M2 makers, the number of CD206⁺ Mφs at depth 5 was very small compared to those of CD163⁺ Mφs and CD204⁺ Mφs (P < 0.01).

To estimate the proximity between CRC cells and Mφs, we compared the distance between CRC cells and Mφs in the invasive front without TB (depth 4) with that in the TB area (depth 5). The representative image of CD68-IHC identified 33 CRC cells as red dots and three CD68⁺ Mφs as green dots at depth 4 by HALO software (Fig. 5A). Histological spatial analysis clipped the colored dots from Fig. 5A and these extracted images could clarify the positioning of these colored dots (Fig. 5B). The histogram revealed that 98.3% of CD68⁺Mφs accumulated within 60 μm at depth 4 (Fig. 5C). Similar results were obtained by CD163-IHC (depth 4, 99.5%) (Fig. 5D). The highest accumulation of CD68⁺Mφs and CD163⁺Mφs was observed between 0 and 20 μm (Fig. 5C, D).

The representative image of CD68-IHC identified 13 CRC cells as red dots and 37 CD68⁺ Mφs as green and light green dots at depth 5 by HALO software (Fig. 5E). Histological spatial analysis clipped the colored dots from Fig. 5E and these extracted images could clarify the positioning of these colored dots (Fig. 5F). The histogram revealed that 98.2% of CD68⁺Mφs accumulated within 60 μm at depth 5 (Fig. 5G). Similar results were obtained by CD163-IHC (depth 5, 96.7%) (Fig. 5H). The highest accumulation of CD68⁺Mφs and CD163⁺Mφs was observed between 0 and 20 μm (Fig. 5G, H).

Thus, we found that a higher number of neighboring CD68⁺Mφs and CD163⁺Mφs were located within 60 µm surrounding CRC cells in the TB area (depth 5) than in the invasive margin without TB (depth 4). Our histological spatial analysis indicated that neighboring Mφs interact with CRC cells.

To identify cytokine involvement from Mφs in CRC specimens, we preliminarily examined IHC studies of IL-6, IL-8, and C-C motif chemokine ligand 2 (CCL2). IL-6 was successfully stained with Mφs. Representative images of double immunofluorescence of CD68 and IL-6 was shown in Fig. 6A. At higher magnification of the invasive margin without TB (depth 4), CD68⁺ cells were negative for IL-6 (Fig. 6B). At higher magnification of the TB area (depth 5), CD68⁺ Mφs were labeled with TRITC red (Fig. 6C), and IL-6-positive cells were labeled with FITC green (Fig. 6D). The merged yellow staining demonstrated that most CD68⁺ cells were positive for IL-6 (Fig. 6E). Similar results were obtained by double immunofluorescence of CD163 and IL-6 (Additional file 1: Fig. S1). These results indicated that IL-6 derived from CD68⁺ and CD163⁺ Mφs was involved in tumor–stromal interactions in the TB area.

To demonstrate the activation of the IL-6R/STAT3 pathway in CRC cells in the TB area, representative images of double immunofluorescence of AE1/AE3 and pSTAT3 was shown in Fig. 6F. At a higher magnification of the invasive margin without TB (depth 4), AE1/AE3-positive CRC cells were negative for pSTAT3 (Fig. 6G). At higher magnification of the TB area (depth 5), AE1/AE3-positive and pSTAT3-positive CRC cells were observed (Fig. 6H). Taken together, IL-6 derived from Mφs may activate the IL-6R/STAT3 pathway in CRC cells in the TB area.

To investigate altering the phenotype of CRC cells via IL-6/IL-6R, we chose two CRC cell lines, HCT116 and SW480, with different expression of IL-6R based on CellExpress (http://​cellexpress.​cgm.​ntu.​edu.​tw/​) [25]. We found mRNA expression of IL-6R in SW480 was higher than that in HCT116 (Relative expression of IL-6R; SW480, 1.00 ± 0.16; HCT116, 0.29 ± 0.13; P < 0.01) (Fig. 7A). Cell proliferation assay revealed that treatment of recombinant IL-6 suppressed the proliferation of SW480 cells (Absorbance value of CCK-8; IL-6 0 ng/ml, 100 ± 3.7%; IL-6 0.01 ng/ml, 77 ± 2.4%; P < 0.01), although that treatment did not suppress the proliferation of HCT116 cells (Absorbance value of CCK-8; IL-6 0 ng/ml, 100 ± 11.2%; IL-6 0.01 ng/ml, 91 ± 10.9%) (Fig. 7B). These results indicated that IL-6/IL-6R may suppress proliferation of SW480 with high-expression of IL-6R.

To confirm that involvement of IL-6/IL-6R on proliferation of SW480, we established SW480^IL-6R-ND1 and SW480^IL-6R-ND2. The mRNA expressions of IL-6R in SW480^IL-6R-ND1 and SW480^IL-6R-ND2 were lower than that in mock cells (SW480^mock) (Relative expression of IL-6R; SW480^mock, 1.00 ± 0.15; SW480^IL-6R-ND1, 0.27 ± 0.11, P < 0.05; SW480^IL-6R-ND2, 0.17 ± 0.06, P < 0.01) (Fig. 7C). Treatment of recombinant IL-6 did not suppress proliferation of SW480^IL-6R-ND1 (Absorbance value of CCK-8; IL-6 0 ng/ml, 100 ± 8.0%; IL-6 0.01 ng/ml, 99 ± 10.1%) and SW480^IL-6R-ND2 (Absorbance value of CCK-8; IL-6 0 ng/ml, 100 ± 4.65%; IL-6 0.01 ng/ml, 90 ± 4.4%), although that treatment suppressed proliferation of SW480^mock (Absorbance value of CCK-8; IL-6 0 ng/ml, 100 ± 9.3%; IL-6 0.01 ng/ml, 84 ± 7.6%; P < 0.01) (Fig. 7D). These results indicated that involvement of IL-6/IL-6R on proliferation of CRC cells in vitro.