In this study, both OS and RFS in the TBhigh group were significantly lower than those in the TBlow group. We confirmed that TB was one of the indications of poor prognosis in CRC patients. We could not show that a higher number of tumor buds was an independent poor prognostic factor in the multivariate analysis because of insufficient cases for statistical analysis in this study. However, a higher number of tumor buds has been demonstrated to be an independent poor prognostic factor in previous studies [3, 25]. Furthermore, tumor buds were demonstrated to have high metastatic potential [26]. Because CRC cells in the TB area may acquire a highly malignant phenotype, we examined the phenotype of CRC cells in the TB area.
A tumor bud has been defined as an isolated cancer cell or cluster comprising less than five cells [25] and has been recognized as one of the morphological phenotypes of CRC cells in the invasive front. In this study, we observed isolated CRC cells in the TB area (depth 5), as opposed to the clusters formed by many of those cells at depths 1–4. Because the number of CRC cells in the TB area (depth 5) differed from that at depths 1–4, TB would be involved in ITH in terms of morphological phenotype.
ITH has been shown to consist of subpopulations of cancer cells with different phenotypes [16]. These phenotypes are characterized by differences in gene expression (including protein expression), proliferation, metastatic potential, immunogenicity, and morphology [27]. Similarly, ITH has been shown to display diverse chemosensitivities. Chemotherapy diminishes the subpopulation with high sensitivity to chemotherapy so that the subpopulation with less sensitivity could be replaced. Selective pressure, including chemotherapy, metastatic cascade, and tumor-stromal interactions, often generates cancer cells with a highly malignant phenotype [28–30]. Because cancer cells with a highly malignant phenotype are involved in chemoresistance, selective pressure plays important roles in the acquisition of chemoresistance. Therefore, we examined the ITH of CRC in terms of the acquisition of chemoresistance in this study.
The acquisition of chemoresistance in cancer cells is involved in the malignant phenotype of cancers. CRC cells with higher expression of TOPO1 have been shown to be more sensitive to TOPO1 inhibitors, such as irinotecan [19]. The decreasing expression of TOPO1 in CRC cells implies resistance to irinotecan because irinotecan prevents the proliferation of CRC cells [19]. In this study, we demonstrated the heterogeneity of TOPO1 expression in CRC cells. Our IHC analysis revealed lower expression of TOPO1 in deeper CRC cells, especially CRC cells in the TB area (depth 5). These results indicated that the lowest expression of TOPO1 in CRC cells in the TB area would imply that those cells acquired strong chemoresistance, consistent with previous reports [17, 31–34]. Because the phenotype of CRC cells in the TB area differed from that at depths 1–4, the expression of TOPO1 would be involved in ITH in terms of the acquisition of chemoresistance. In this study, we examined the expression of CD205 to elucidate the phenotype associated with malignancy in CRC cells in the TB area. CD205 is expressed mostly in antigen-presenting cells and occasionally in cancer cells [21, 22]. The expression of CD205 in CRC cells has been shown to decrease with malignancy [23], although the expression of CD205 in ovarian cancer cells has been shown to increase with malignancy [35]. In this study, we demonstrated that the expression of CD205 in CRC cells was lost in CRC cells in the TB area (depth 5) of all cases regardless of staining intensity in the invasive front without TB (depth 4). These results indicated that the malignant phenotype of CRC cells in the TB area was higher than that at depth 4.
CRC cells, which transition to the mesenchymal state, acquire high motility and invasive potential [36], thereby inducing high metastatic potential [26]. CRC cells in the TB area were demonstrated to be positive for EMT markers represented by ZEB1 [31, 32]. EMT is a factor in the ITH of CRC.
ITH was demonstrated to contribute to chemoresistance in CRC patients [37]. In this study, in terms of the acquisition of chemoresistance, very low expression of TOPO1 in CRC cells in the TB area may show strong chemoresistance. In terms of the malignant phenotype, loss of CD205 expression in CRC cells in the TB area may indicate a highly malignant phenotype. In previous studies, in terms of gene expression, high expression of ZEB1 in CRC cells in the TB area was associated with high metastatic potential [26, 31, 32]. Taken together, the phenotypes of CRC cells in the TB area, such as very low TOPO1 expression, loss of CD205 expression, and high ZEB1 expression, were clearly distinct from those at other depths. Because tumor-stromal interactions play an important role in altering the phenotype of CRC cells in the TB area, we performed a histological spatial analysis of tumor-stromal interactions between CRC cells and Mφs.
Tumor-stromal interactions are often observed in the TME. The TME consists of cancer cells and subsequently stromal cells, such as Mφs and fibroblasts [30]. Mφs have been demonstrated to interact with cancer cells in the TME [8, 9, 11]. Many Mφs are often observed in the invasive front of CRC specimens [38, 39]. These findings indicated that Mφs might alter the phenotype of CRC cells in the invasive front. To demonstrate tumor-stromal interactions between CRC cells and Mφs in the invasive front, we focused on two histological findings: the number of Mφs per single CRC cell and the proximity between CRC cells and Mφs. Our CD68 IHC study revealed that the number of Mφs per single CRC cell in the TB area (depth 5) was 3.7 times higher than that in the invasive front without TB (depth 4); in particular, 2.6 Mφs interacted with a single CRC cell in the TB area (depth 5). Similar results were obtained by CD163 IHC. Our histological spatial analysis revealed that the number of neighboring Mφs within 20 µm surrounding CRC cells in the TB area (depth 5) was six times higher than that in the invasive margin without TB (depth 4). The distance of 20 µm corresponds to the size of one Mφ because the size of Mφs is known to be approximately 20 µm. Taken together, both the number of Mφs per single CRC cell and the proximity between CRC cells and Mφs induced the interaction. Next, we examined the cytokines produced by Mφs that altered the phenotype of CRC cells in the TB area.
To elucidate the signaling pathway activated in CRC cells in the TB area, we examined the cytokines produced by Mφs. IL-6, IL-8, and CCL2 secreted from neighboring Mφs have been shown to alter the phenotype of cancer cells [8, 9, 40–46]. Our preliminary IHC studies of IL-6, IL-8, and CCL2 showed that IL-6 was successfully stained in Mφs. IL-6-derived Mφs might regulate the IL-6R/STAT3/miR-204-5p axis in CRC cells, thereby inducing chemoresistance in CRC cells [9]. Our double-immunofluorescence staining revealed that many CD68+ Mφs produced IL-6 in the TB area (depth 5). Furthermore, our staining revealed that AE1/AE3-positive CRC cells in the TB area (depth 5) were positive for pSTAT3, indicating that the IL-6R/STAT3 signaling pathway was upregulated in CRC cells by neighboring Mφs. These results suggested that IL-6 derived from neighboring Mφs activates CRC cells in the TB area via the IL-6R/STAT3 signaling pathway. Although the action of irinotecan on CRC cells was demonstrated to be impaired by the activation of STAT3 [47], it is not known whether the nuclear translocation of pSTAT3 in CRC cells is directly involved in the decrease in TOPO1 expression in CRC cells. In future studies, we would like to show that TOPO1 expression is involved in activating the IL-6R/STAT3 signaling pathway in CRC cells in the TB area. Similarly, we would like to show that CD205 expression is involved in activating the IL-6R/STAT3 signaling pathway in CRC cells.