An operation to treat cancer includes resection of the primary lesion, lymphadenectomy, reconstruction, and so on. To date, surgery has been considered to be the primary therapeutic regimen for most malignancies, even though sometimes the effect of surgery is not ideal, since the surgical process could increase the risk of metastases of residual cancer cells to other organs [1, 4, 21]. Experimental data suggest that increased surgical stress augments cancer metastasis via surgical stress-induced expression of proteinases in the target organ of metastasis [22]. The effect of surgery on metastasis may be attributed to a number of factors, including immunosuppression after surgical stress, action of cytokines or changes of tumor microenvironment [23]. To explain the presence of postoperative tumor metastasis, it is necessary to find the appropriate in vivo model, thereby gaining an in-depth understanding of the mechanism of tumor removal-induced metastasis in order to develop effective treatment options and to improve metastatic cancer prevention and treatment.
Our IVIS results showed that the surgical removal of tumor-bearing SiLN promoted the incidence of lung metastasis (Fig. 1). Further, immunohistochemistry showed that luciferase-positive tumor cells were sparsely spread in the control lung tissues and was significantly lower than in the SiLN removal group. Although there was no significant difference in the rate of PALN metastasis between the control and tumor-bearing SiLN removal group, the number of luciferase-positive tumor cells in PALN in the surgical SiLN removal group was significantly less than the control group (Fig. 2, Table 1).
The pre-metastatic niche is defined as microenvironment with a series of molecular and cellular changes, which forms a supportive and receptive pre-metastatic site and the fertile “soil” to prepare for the colonization of “seed”, that is metastatic tumor cells, thereby facilitating tumor cell adhesion and growth in the distant organs and promoting tumor metastasis [8]. Studies have shown that local and high-precision radiotherapy does not induce direct injury, but it directly kills or stimulates the tissue cells compared with surgery, which also accelerates the incidence of tumor metastasis during anti-cancer therapy [2, 24]. Previous studies have shown that local radiotherapy causes a stronger expression of angiogenic factors than anti-angiogenic factors and disturbs the configuration imbalance of VEGFs in the vascular bed of the metastatic site, which then results in outbreak growth of dormant metastatic tumors [24, 25]. Adjuvant applications of the exogenous VEFGR inhibitor cediranib after radiotherapy restored tumor inhibition, and studies have shown that using angiogenesis inhibitors to target the VEGF pathway or knocking out the VEGF gene in mouse models with pancreatic cancer or glioblastoma has anti-cancer therapeutic effects by reducing the tumor volume and prolonging survival, but the two actions change tumor phenotypes and enhance tumor invasion and metastasis [26,27,28]. The discontinuation of VEGF inhibitors still enhances the tumor invasion, suggesting that this treatment increases the persistence of tumor invasion. This implies that not only does the treatment-induced trauma lead to tumor-promoting responses, but the disappearance of the tumor itself also promotes tumor metastasis during anti-cancer therapy. For the tumor treatment targeting a single angle, tumors can derive responses of increased invasiveness and extended metastasis. Therefore, a comprehensive consideration of all characteristics of the tumor microenvironment may accomplish a radical anti-cancer treatment.
Studies have shown that LOX secreted from hypoxic primary tumor cells accumulates in the pre-metastatic sites; LOX is an indispensable factor for recruiting bone marrow-derived CD11b+ cells (i.e., immature myeloid progenitor cells) to metastatic sites [19, 20]. LOX expression in SiLN removal groups was significantly higher than in the control group and was positively correlated with luciferase expression. These findings are consistent with the results of previous studies [19, 20], suggesting that high LOX expression in the metastatic sites was closely associated with the colonization and metastasis of tumor cells. Moreover, the number of CD11b+ cells in the lung and liver of the mice in the SiLN removal groups was significantly increased, which was positively correlated with LOX expressions (Fig. 3, Tables 2, 3).
Since the activation of MMP increased the invasion of BMDCs [19, 29], we detected MMP-2 expression in the lung tissues of the surgical SiLN removal groups, which was interestingly higher than that in the control groups and it was positively correlated with CD11b expression (Fig. 3, Tables 2, 3).
Our results showed that the degradation of the collagen fibers around the pulmonary vessels in the lungs of mice with the surgical removal of tumor-bearing SiLN was quite significant and the quantity of collagen fibers was negatively correlated with the expression of MMP-2, leading to a decrease in barrier function, thereby providing a channel for the tumor cells to invade blood vessels and establishing helpful conditions for tumor cell metastasis. The fracture of reticular fibers in the SiLN removal groups facilitated tumor cell invasion, thereby accelerating lung metastasis (Fig. 4, Table 5).
The above results suggested that high LOX and MMP-2 expressions and a great number of CD11b+ cell facilitate the formation of the metastatic niche and the colonization, growth, and invasion of tumor cells in the lung. The accumulation of LOX at the metastatic site promoted CD11b+ BMDC adhesion, MMP-2 production, and degradation of intra-pulmonary vascular collagen. Moreover, the morphological changes of the extracellular matrix scaffold remodeled the local microenvironment and enhanced tumor cell invasion. Previous studies have shown that BMDC induced interstitial epithelial transformation of the disseminated tumor cells [30], secreted chemokines to enhance metastasis and nesting of tumor cells [31], and lowered in vivo immune surveillance by immunosuppression [7, 8, 32]. The current study also showed that VEGFA expression in the surgical removal groups was significantly higher than in the control group, and it is known that the secretion of VEGF and other angiogenic factors promote vascular angiogenesis, thereby possibly playing a synergistic role in promoting tumor metastasis.
The current study also identified LOX, CD11b, and luciferase expression in the liver and showed similar expression patterns of LOX and CD11b in the lung (Fig. 5). Interestingly, although the ex vivo IVIS results showed that the liver metastasis was only 5.9% in KM SiLN removal group, immunohistochemical staining showed that luciferase-positive tumor cells were scattered in liver sinusoids in all of the SiLN removal groups (Fig. 2), which might be associated with tumor cell types and organotropic targeting [33], unfavorable niche formation, or the short observation time in this study. Further studies will be necessary to address the above concerns.
Lymph nodes are the well-known routes for lymphatic metastasis and the primary place for anti-tumor immune responses. The current study observed the influx of tumor cells into the lymphoid sinus of PALN in the control group, which resulted in tumor nest formation, the retention of tumor cells, and the reduction of tumor cell metastasis to distant organs over time (Fig. 6). A study by Asano et al. [34] showed that subcutaneously injected tumor cells can be transported to lymph nodes (LNs) through the lymphatic flow and were phagocytosed by CD169+ macrophages in the LN subcapsular sinus, which then directly cross-present the dead cell-associated antigens to CD8+T cells. The current study found a large luciferase-positive cell influx into the subcapsular sinus in the PALN of the control group. In addition, the number of CD169+ and CD11c+ cells in the control group was significantly higher than in the SiLN removal groups (Fig. 6, Table 7), suggesting the production of anti-tumor immune responses, which, to a certain extent, reduced the chance of tumor cell metastasis to the distant organs, such as the lung and liver.
There is growing data to support that noncoding RNAs (ncRNAs) play a significant role in tumor metastasis [35, 36]. A diversity of ncRNAs was demonstrated to promote proliferation and metastasis of tumor cells [37,38,39]. We preformed additional microarray detection and indicated expression differences in both miRNAs and lncRNAs (data not shown) to clarify the specific mechanism associated with our findings. Our study will further demonstrate certain miRNAs and lncRNAs could be pre-metastatic biomarkers, prognostic tools and potential therapeutic targets.