Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1
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eLife assessment
This study provides potentially important insights into the role of mesenchymal stem cells in CAR-T therapy, and suggest that the STC1 gene could be a key factor in influencing the efficacy of this treatment. This finding has the potential to improve current therapeutic strategies based on cell therapy and may indicate new biology related to how mesenchymal stem cells affect the immune state within the tumor microenvironment. Further research is necessary to clarify the signaling pathways, but the data presented by the authors are generally well-supported and convincing.
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Abstract
Stem cells play critical roles both in the development of cancer and therapy resistance. Although mesenchymal stem cells (MSCs) can actively migrate to tumor sites, their impact on chimeric antigen receptor modified T cell (CAR-T) immunotherapy has been little addressed. Using an in vitro cell co-culture model including lymphoma cells and macrophages, here we report that CAR-T cell-mediated cytotoxicity was significantly inhibited in the presence of MSCs. MSCs caused an increase of CD4 + T cells and Treg cells but a decrease of CD8 + T cells. In addition, MSCs stimulated the expression of indoleamine 2,3-dioxygenase and programmed cell death-ligand 1 which contributes to the immune-suppressive function of tumors. Moreover, MSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial reactive oxygen species release. Interestingly, all these suppressive events hindering CAR-T efficacy could be abrogated if the stanniocalcin-1 (STC1) gene, which encodes the glycoprotein hormone STC-1, was knockdown in MSC. Using xenograft mice, we confirmed that CAR-T function could also be inhibited by MSC in vivo, and STC1 played a critical role. These data revealed a novel function of MSC and STC-1 in suppressing CAR-T efficacy, which should be considered in cancer therapy and may also have potential applications in controlling the toxicity arising from the excessive immune response.
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Author Response
Reviewer #1 (Public Review):
Major points:
- How STC1 controls changes in MSCs' ability for hampering CAR-T cell-mediated anti-tumor responses is unclear.
In this study, we demonstrated that the presence of STC1 is critical for MSCs to exert their immunosuppressive role by inhibiting cytotoxic T cell subsets, activating key immune suppressive/escape related molecules such as IDO and PD-L1, and crosstalking with macrophages in the TME. These immunosuppressive functions of MSC could be significantly hampered when the STC1 gene was knockdown. Considering that staniocalcin-1 is glycoprotein hormone that is secreted into the extracellular matrix in a paracrine manner, we would conclude that the role of STC-1 is not to alter the function of MSCs intracellularly. Rather, it facilitates the immunosuppressive capabilities of …
Author Response
Reviewer #1 (Public Review):
Major points:
- How STC1 controls changes in MSCs' ability for hampering CAR-T cell-mediated anti-tumor responses is unclear.
In this study, we demonstrated that the presence of STC1 is critical for MSCs to exert their immunosuppressive role by inhibiting cytotoxic T cell subsets, activating key immune suppressive/escape related molecules such as IDO and PD-L1, and crosstalking with macrophages in the TME. These immunosuppressive functions of MSC could be significantly hampered when the STC1 gene was knockdown. Considering that staniocalcin-1 is glycoprotein hormone that is secreted into the extracellular matrix in a paracrine manner, we would conclude that the role of STC-1 is not to alter the function of MSCs intracellularly. Rather, it facilitates the immunosuppressive capabilities of MSCs through extracellular secretion into the TME as a pleiotropic factor, thus impacting the functioning of T cells, cancer cells and other immune cells.
The reviewer's question is well taken, and we have added the points mentioned above to the Discussion section to ensure a more comprehensive conclusion. Moreover, a recent study published in Cancer Cell, which was suggested by the other reviewer, is consistent with our results. It has provided further mechanistic information on how stanniocalcin-1 impacts immunotherapy efficacy and T cell activation. The reference has been cited and discussed as shown below.
"In this model, activated macrophages or stress signals during CAR-T therapy may prompt MSCs to secret staniocalcin-1 into the extracellular matrix of TME, serving as a pleiotropic factor to negatively impact the function of T cells and stimulate the expression of molecules that inactivate immune responses, ultimately providing an immunosuppressive effect of MSC." (page 22, highlighted). "In line with our study, it was recently reported that stanniocalcin-1 negatively correlates with immunotherapy efficacy and T cell activation by trapping calreticulin, which abrogates membrane calreticulin-directed antigen presentation function and phagocytosis [50]." (Page 20, highlighted)
- Is ROS important? It is not tested directly.
ROS plays an important role during immune response, which are released by neutrophils and macrophages. Not only do they act as key mediators of the adaptive immune response, but they also have the ability to modulate the activation of B-cells and T-cells. In our study, we suggest that ROS may be involved in NLRP3 inflammasome activation and the expression and secretion of STC1. Although we did not pursue this line of inquiry further as it was beyond the scope of our paper, we have included additional relevant research in Discussion and a reference is provided.
"It has been proved that the expression and secretion of STC1 in multiple cell lines can be stimulated by external stimuli, including cytokines and oxidative stress [26]." (Page 21, highlighted)
- The changes in CD8 and Treg are not convincing. Moreover, it is not tested how these changes can be elicited by the presence of MSCs.
We have included additional in vivo data to assess the levels of Treg cells and CD8+ in this revised manuscript. This not only confirms the alterations of CD8 and Treg, but also offers additional line of evidence to further analyze the influence of MSCs on CAR-T in vivo. The findings are presented in Figure 4B, and the corresponding discussion can be found on Page 17 (highlighted).
Reviewer #2 (Public Review):
Major points:
- STC-1 is expressed and secreted by many human cancer cells. This should be discussed in the introduction or discussion with more inter-related background info on both its regulation in cancer cells and secretion pattern into TME. It is important because you state that the STC-1 secreted by MSC has such strong functions, then how about those produced and secreted by cancer cells? Are those also stimulated by macrophages or other components in TME? Do they have possible functions in helping cancer cell to escape the immune surveillance mechanisms?
Thanks for the suggestion. We have added more details about the regulation and secretion of STC-1 in cancer cells (see below). The information is added to both the introduction and discussion (highlighted on pages 4 and 21), and all the above questions are addressed.
"It was proved that STC1 is involved in several oxidative and cancer-related signaling pathways such as NF-κB, ERK, and JNK pathways [26,27]. The expression and secretion of STC1 in cancer tissue can be stimulated by external stimulus including external cytokines and oxidative stress [26]. Under hypoxia conditions, STC1 could be modulated by HIF-1 to facilitate the reprogramming of tumor metabolism from oxidative to glycolytic metabolism [28]. STC1 was also reported to participate in the process of epithelial-to-mesenchymal transition (EMT), which is associated with tumor invasion and the reshape the tumor microenvironment, as well as increasing therapy resistance [29]." (Page 4)
"It has been proved that the expression and secretion of STC1 in multiple cell lines can be stimulated by external stimuli including cytokines and oxidative stress [26]." (Page 21)
- In Figure 4B, using a single marker of IL-1β to show the immune suppressive capability of MSC in vivo is not sufficient, staining for CD4+ and CD8+ should also be included to demonstrate whether MSC could modulate T cell compositions, which can give more direct evidence about MSC's impacts on CAR-T cell.
The above experiments were done as suggested, and the data were presented in figure 4B. Explanations of the results are shown on page 17 Results section and page 21 Discussion section (highlighted).
- One of the major risks associated with CAR-T therapy is an excessive immune response that causes cytokine release syndrome. MSCs have been used in clinics as a way to suppress immune response including post-CAR-T. What does the author think about using MSC with STC-1 knockout? Can it still help reduce toxicity while maintaining CAR-T efficacy? This might be a potential application.
This is definitely an interesting idea. Based on the data presented in the current study, it is clear that knockdown of STC-1 would abrogate the immune-suppressive impact of MSC, and therefore affect CAR-T efficacy. However, whether the presence of MSC can help reduce cytokine release syndrome when losing the function of STC-1 requires further study. We agree with the reviewer, and we had briefly discussed this possibility at the very end of the discussion as shown below (Page 22, highlighted).
"… the findings we presented here are no doubt that would have potential clinical applications toward improving the efficiency of CAR-T therapy as well as reducing the excessive toxicity by modulating the level of STC1 in TME".
- There was a recent study published in Cancer Cell (Lin et al. Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance. 2021), and they also reported that STC1 negatively correlates with immunotherapy efficacy and patient survival. It should be cited, and in fact, it provided support to the authors' present study with completely different experimental settings.
Thanks for providing this important information. It is an excellent study and consistent with our findings. The reference was added and discussed on page 20 (highlighted) as shown below.
"In line with our study, it was recently reported that stanniocalcin-1 negatively correlates with immunotherapy efficacy and T cell activation by trapping calreticulin, which abrogates membrane calreticulin-directed antigen presentation function and phagocytosis [50]"
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eLife assessment
This study provides potentially important insights into the role of mesenchymal stem cells in CAR-T therapy, and suggest that the STC1 gene could be a key factor in influencing the efficacy of this treatment. This finding has the potential to improve current therapeutic strategies based on cell therapy and may indicate new biology related to how mesenchymal stem cells affect the immune state within the tumor microenvironment. Further research is necessary to clarify the signaling pathways, but the data presented by the authors are generally well-supported and convincing.
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Reviewer #1 (Public Review):
This work aims to understand whether MSCs support the resistance in tumor cells upon CAR T cell treatment and whether the expression of STC1 in MSCs contributes to those changes. Overall, the in vivo data is interesting. However, the mechanistic understandings are correlated and based on many assumptions. Furthermore, the differences in Treg changes presented in Figure 2 are not convincing. It is also not clear the underlying mechanisms by which the presence of MSCs leads to these changes.
Major points:
1. How STC1 controls changes in MSCs' ability for hampering CAR T cell-mediated anti-tumor responses is unclear.
2. Is ROS important? It is not tested directly.
3. The changes in CD8 and Treg are not convincing. Moreover, it is not tested how these changes can be elicited by the presence of MSCs.
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Reviewer #2 (Public Review):
Zhang et al. addressed an intriguing question - whether the presence of mesenchymal stem cells (MSCs) could influence the efficacy of CAR-T therapy. After observing that CAR-T cytotoxicity was strongly inhibited by MSCs by modulating certain correlated immune response pathways, the authors sought to uncover the underlying mechanisms by examining the interaction between MSCs and macrophage, immune escaping mechanisms, and oxidative stress. Notably, the authors discovered that a single gene, STC1, played a major role in reversing the suppression when it was knocked down/out. Although more research is necessary to clarify the signaling pathways, the data presented by the authors were generally well-supported and convincing.
Major points:
1. STC-1 is expressed and secreted by many human cancer cells. This should …
Reviewer #2 (Public Review):
Zhang et al. addressed an intriguing question - whether the presence of mesenchymal stem cells (MSCs) could influence the efficacy of CAR-T therapy. After observing that CAR-T cytotoxicity was strongly inhibited by MSCs by modulating certain correlated immune response pathways, the authors sought to uncover the underlying mechanisms by examining the interaction between MSCs and macrophage, immune escaping mechanisms, and oxidative stress. Notably, the authors discovered that a single gene, STC1, played a major role in reversing the suppression when it was knocked down/out. Although more research is necessary to clarify the signaling pathways, the data presented by the authors were generally well-supported and convincing.
Major points:
1. STC-1 is expressed and secreted by many human cancer cells. This should be discussed in the introduction or discussion with more inter-related background info on both its regulation in cancer cells and secretion pattern into TME. It is important because you state that the STC-1 secreted by MSC has such strong functions, then how about those produced and secreted by cancer cells? Are those also stimulated by macrophages or other components in TME? Do they have possible functions in helping cancer cell to escape the immune surveillance mechanisms?
2. In Figure 4B, using a single marker of IL-1β to show the immune suppressive capability of MSC in vivo is not sufficient, staining for CD4+ and CD8+ should also be included to demonstrate whether MSC could modulate T cell compositions, which can give more direct evidence about MSC's impacts on CAR-T cell.
3. One of the major risks associated with CAR-T therapy is an excessive immune response that causes cytokine release syndrome. MSCs have been used in clinics as a way to suppress immune response including post-CAR-T. What does the author think about using MSC with STC-1 knockout? Can it still help reduce toxicity while maintaining CAR-T efficacy? This might be a potential application.
4. There was a recent study published in Cancer Cell (Lin et al. Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance. 2021), and they also reported that STC1 negatively correlates with immunotherapy efficacy and patient survival. It should be cited, and in fact, it provided support to the authors' present study with completely different experimental settings.
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