CSCs have been extensively studied in many cancers [1, 2, 6,7,8,9,10,11,12,13,14, 16, 25,26,27,28,29,30,31,32,33,34,35,36,37,38], with ALHD1, CD44, and CD133 among the most investigated CSC markers [39, 40]. Studies using flow cytometry or IHC have reported that CSC comprise from 0.1 to 20% of cells in some tumors . Our study examined ALHD1, CD44, and CD133 expression before and after preoperative chemotherapy in a cohort of STS cases enrolled on a prospective clinical trial to assess whether FDG PET-CT imaging predicts response to neoadjuvant PLD and ifosfamide . Our results did not support the initial hypothesis that IHC staining for ALHD1, CD44, and CD133 would detect significant changes in CSC between pre-and post-treatment STS samples. Detection of CSCs with ALDH1 and CD44 was not straightforward because tumor-infiltrating macrophages were often a prominent component of the tumor and they also expressed CD44 and ALDH1.
The concept of CSCs is further complicated in that CSCs and non-CSCs are not necessarily “hardwired”; in some cases, non-CSCs may become CSCs, and CSCs may convert to non-CSCs . In addition, while CSCs do not have to be resting or G0 cells , if “non-CSCs” were more resistant to a treatment when in G0 phase, they might functionally appear as “true CSCs.”
In many systems, the inflammatory response may have a dramatic effect on tumor growth [41,42,43]. In our study, CD68-positive macrophages were found to represent a prominent component of the viable tumor regions of many of the STS cases. Macrophages express CD44 [23, 24, 44], and inflammation up-regulates ALDH1 expression in some macrophages . Our finding of many macrophages in the necrotic tumor regions was not surprising, as monocyte-derived macrophages can sense multiple signals reflecting tumors and rapidly accumulate in inflamed tissue (reviewed in  and ). Tumor associated macrophages (TAMs) exist on a spectrum, from a pro-inflammatory M1 to a pro-tumor M2 state and are not static [45, 47, 48]. For example, macrophages can vary in phenotype from producing growth-inhibiting nitric oxide to growth-promoting ornithine, both derived from arginine via inducible nitric oxide synthase or arginase, respectively . CSF1 recruits macrophages, and a “CSF response signature” was seen in a subset of breast cancer that was associated with higher grade and decreased estrogen receptor expression . Therefore, macrophages could play an important role in STS biology.
Macrophages in sarcomas
Many characteristics of tumors are derived in varying degrees from infiltrating normal host cells, either stromal or blood-derived. Chronic inflammation has been linked to cancer development since the time of Virchow [48, 51], and tumors have been described as “wounds that do not heal” ; normal infiltrating inflammatory cells, including TAMs and even myofibroblasts, could contribute to tumor growth (reviewed in [18, 53,54,55]). In fact, infiltration of tumors by macrophages, in some cases representing 50% of the cells in the tumor, was recognized by Virchow in the nineteenth century, terming them lymphoreticular cells [2, 51]. In some tumors, the number and/or distribution of TAMs correlated with prognosis . While lymphocytes were sometimes seen in the samples, there was no clear correlation with macrophage density.
Macrophage infiltration of STS has been previously reported [57,58,59,60,61,62], and CD68 expression has been reported to have prognostic significance in leiomyosarcoma [58, 59]; in addition, some leiomyosarcoma cells were shown to produce M-CSF in vitro. Prominent infiltration of many STS tumors was also recently reported in a phase II trial of PD-1 inhibition . Thus, the prominent macrophage infiltrate of many sarcomas suggests a potential role for targeting tumor macrophages in some sarcomas, as has been suggested in some other tumors [18, 58,59,60,61, 63]. In our study, both pre- treatment and post-treatment CD68 staining in viable tumor correlated positively with the baseline SUVmax and negatively with percent viable tumor cells at resection (after chemotherapy). Post-treatment CD68 staining also correlated weakly positively with the post-treatment SUVmax. While the negative correlation of post-treatment CD68 staining with percent viable tumor cells in the resection specimen could reflect a treatment effect, the negative correlation of the pre-treatment CD68 staining with percent viable tumor cells after chemotherapy further suggests that infiltrating macrophages may play an important role in STS biology.
The role of paracrine signaling in tumors is dramatically evident in giant cell tumor of bone where RANKL secretion by tumor cells recruits normal osteoclasts to the tumor from circulating precursors . Elimination of osteoclasts by a monoclonal antibody to RANKL results in dramatic effects on the neoplastic tumor cells as well, with associated clinical benefit, presumably by eliminating factors produced by the osteoclasts that are beneficial for growth and maintenance of the tumor phenotype of the neoplastic cells [64,65,66]. Analogous to these observations, interfering with signaling from CSF1 produced by the tumor cells in pigmented villonodular synovitis can markedly decrease macrophage infiltration and have striking beneficial effects [67,68,69]. TAM infiltration in some other sarcomas could possibly affect tumor cell biology in a similar manner, and targeting macrophages has shown anti-tumor activity in a model of leiomyosarcoma [70,71,72]. Thus, it is possible that infiltrating CD68 positive macrophages in some STS could produce factors that have a trophic effect on the tumor cells, and that chemotherapy effects on these macrophages could result in less production of tumor supporting factors. Another possibility could reflect primary damage to the tumor resulting in new antigens then recognized by the macrophages resulting in augmented immune destruction.
Similarly, TAMs and other stromal cells may be involved in a variety of processes critical to tumor development including immuno-suppression, angiogenesis, invasion, metastasis, response to chemotherapy and radiation therapy (reviewed in [73, 74]), and macrophage content has been correlated with prognosis in several cancers . For example, TAMs could contribute to tumor growth by several mechanisms, including releasing factors that promote tumor cell growth and inhibiting the immune response to the tumor (reviewed in ).
Macrophages can secrete a variety of cytokines capable of paracrine signaling and potentially affecting tumor biology ; one study suggested that CSF-1 production with macrophage infiltration may promote neovascularization , and angiogenesis plays an important role in tumor growth . In our study, CD31 staining was similar before and after chemotherapy in most cases evaluated. Pre-treatment CD31 staining was negatively correlated with post-chemotherapy SUVmax and weakly with percent viable tumor cells in the resection specimen, and it was positively correlated with change in tumor size (i.e., more tumor growth and higher baseline CD31).
In addition to the possible effects of inflammatory cells in the tumor microenvironment on tumor growth and angiogenesis, tumor macrophages may influence response to chemotherapy [56, 61, 76,77,78]. Depending on the model, depletion of TAMs increased or decreased the efficacy of chemotherapy . In fact, there is evidence that some of the anti-tumor effects of trabectedin may be related to a reduction in inflammatory mediator production by tumor cells and by reducing infiltrating inflammatory cells such as macrophages [76,77,78].
The presence of large numbers of macrophages in some tumors also raises the question of their role in the findings of our study correlating changes in PET scan measurements after chemotherapy with outcome . The positive correlation of both pre-and post-treatment CD68 staining with the pre- and post-treatment SUVmax, respectively, suggests that intra-tumor macrophages may contribute significantly to the observed SUVmax on PET imaging in some cases. An analogous case occurs in giant cell tumor of bone, where normal osteoclasts are recruited by RANKL secreted by tumor cells [64, 79]; osteoclasts contain large amounts of H+ transporting ATPase and may avidly take up FDG [80, 81]. PET activity falls rapidly following treatment with denosumab, an antibody to RANKL, which may reflect the change in energy use by osteoclasts rather than reflecting a change in activity of neoplastic cells . Similarly, FDG uptake by tumor macrophages may play a role in changes in PET activity in some sarcomas and other solid tumors, and this may contribute to less correlation between PET findings and pathologic findings of tumor viability. This possibility is supported by the positive correlation between CD68 staining and SUVmax in both the pre-and post-chemotherapy specimens. Macrophage infiltration also may have a significant effect on tumor volume and thereby contribute to traditional measures of tumor size and response to therapy such as RECIST.
Cancer stem cells in sarcomas
Our findings suggest that while CD133, ALDH1, and CD44 may be useful markers of CSC in epithelial tumors, their clinical utility in STS seems limited. CD133 was reported to be expressed in synovial sarcoma , where CD133 expression was found in 5/5 tumor samples and 3/3 cell lines with the percentage of cells expressing CD133 ranging from ~ 2 to ~ 20%. A recent study found that a CD133+ subpopulation of a synovial sarcoma cell line had several CSC properties, including self-renewal and resistance to chemotherapy . CD133 expression has also been reported in other sarcomas [35, 37], and correlated with lung metastases and poor prognosis in osteosarcoma , and poor survival in embryonal rhabdomyosarcoma . In the current study, no CD133 staining was observed in 5 synovial sarcomas. While only 2 STS cases in the current study stained for CD133, the changes in CD133 staining following chemotherapy were compatible with a decrease in “stemness” after treatment.
ALDH1 has been proposed to be a marker of both normal and cancer stem cells, and expression in breast cancer has been correlated with survival in some studies . However, the utility of ALDH1 as a marker of CSCs is not clear in some solid tumors such as ovarian cancer [12,13,14]. Liu et al. found ALDH1 expression in ~ 1% of cells in the synovial sarcoma cell line SW982 . In the current study, ALDH1 staining of tumor cells was detected in 11 of 30 pre-treatment biopsy samples and was similar before and after chemotherapy in half of the cases, while in some cases staining was higher and in some lower. Overall, patients whose pre-treatment ALDH1 staining intensity was high had worse survival outcomes, and patients whose post-treatment ALDH1 staining intensity was high had increased tumor growth; however, these correlations were weak. Thus, ALDH1 expression does not seem to be a useful marker of CSC in STS.
CD44 has been described as a marker of CSC in some carcinomas [6, 8]. In our study, CD44 expression was detected in tumor cells in 26 of 30 pre-treatment samples and was similar before and after chemotherapy. As with ALDH1, CD44 does not appear to be a useful CSC marker in STS.