Tumor-Associated Macrophages in Multiple Myeloma: Key Role in Disease Biology and Potential Therapeutic Implications

Round 1

Reviewer 1 Report

This is an interesting and overall well structured as well as comprehensive review of the contribution of macrophage polarisation to MM biology and treatment response.

As a reader I think it would be helpful particularly in the preclinical elements to break this down as clearly as possible, between mouse and human studies. While this is mostly quite clearly stated by the authors there are certain areas where this becomes a little unclear, for example line 169-175 reference 79 is clearly identified as a mouse study but reference 97 is in human MM patient samples.

Or in the section"immunohistochemical (IHC) studies showed an inconsistent evidence that total 130 TAM number is increased in the BM of MM-bearing mice if compared to monoclonal 131 gammopathy of uncertain significance (MGUS) [74-76]. Conversely, a significant increase 132 was demonstrated by studies using flow cytometry and macrophages were physically 133 associated with clonal PC [77-79]."Here reference 77 and 78 are in human but 79 is in mouse. 

It might be easier for the reader to balance the levels of evidence if the murine and human studies are separated with subheadings within in each conceptual component of the review.

Another minor point is to carefully recheck referencing. I note this only because checking for recent references I noted on Line 491-493 the references appear to have been switched around.

Line 491- 493 

Lenalidomide demonstrated the ability to drive TAM towards an  immunostimulatory M1 subtype through the cereblon-CRL4 E3 ligase to degrade by ubiquitination the transcription factor IKAROS family zinc finger 1 (IKZF1) [140]. Lenalidomide could also counteract the pro-angiogenic properties of M2 TAM through a  negative modulation of VEGF [141]. 

140. Quach, H.; Ritchie, D.; Stewart, A.K.; Neeson, P.; Harrison, S.; Smyth, M.J.; Prince, H.M. Mechanism of action of immuno- 939 modulatory drugs (IMiDS) in multiple myeloma. Leukemia 2009, 24, 22–32. doi: 10.1038/leu.2009.236 940 

141. Mougiakakos, D.; Bach, C.; Böttcher, M.; Beier, F.; Röhner, L.; Stoll, A.; Rehli, M.; Gebhard, C.; Lischer, C.; Eberhardt, M.; Vera, 941 J.; Büttner-Herold, M.; Bitterer, K.; Balzer, H.; Leffler, M.; Jitschin, S.; Hundemer, M.; Awwad, M.H.S.; Busch, M.; Stenger, S.; 942 Völkl, S.; Schütz, C.; Krönke, J.; Mackensen, A.; Bruns, H. The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myelo- 943 ma-Associated Macrophages. Cancer Immunol. Res. 2021, 9, 265–278. doi: 10.1158/2326-6066.CIR-20-0555 

I a few minor typographical errors that I noted as well:

Table 1

Immunosuppressione 

Line 401 GM-SCF, a pro-M1 cytokine  

Line 430 immunosspression

Line 443 methyl-tryptofan

Line 449 “Due to hematological malignancy” – consider replacing with “Since hematological malignancy” 

Author Response

Response to reviewer

Reviewer 1

The reviewer says this is an interesting and overall well structured review of the contribution of macrophages to MM biology and treatment response.

First of all, we would like to thank the reviewer for the positive comments.

The reviewer asks to break this down in the preclinical elements between mouse and human studies. For example line 169-175, references 79 and 97, or in the section “Immunohistochemical (IHC) studies showed an inconsistent evidence that total TAM number is increased in the BM of MM-bearing mice if compared to monoclonal gammopathy of uncertain significance (MGUS) [74-76]. Conversely, a significant increase was demonstrated by studies using flow cytometry and macrophages were physically associated with clonal PC [77-79].” The reviewer says it might be easier to separate with subheadings murine and human studies.

We agree with the reviewer. We have divided in subheadings each conceptual component of the review, if there were data about both human and mouse studies, as illustrated in Sections 3 and 5, except paragraphs 3.5, 5.4 (only human studies) and 5.2 (only mouse model studies). We have modified reference list accordingly.

The reviewer asks to carefully check referencing.

We agree with the reviewer. We have corrected the 2 switched references and we have rechecked the other references, as requested, both in the text and in the tables.

The reviewer says there are few minor typographical errors.

We agree with the reviewer. Table 1: we have changed from immunosoppressione to immunosuppression. Line 401: GM-SCF changed to GM-CSF. Line 430 immunosspression changed to immunosuppression. Line 443 Methyl-tryptofan changed to methyl-tryptophan. Line 449 Due to hematological malignancies changed to since hematological malignancies.

Reviewer 2 Report

1. As mentioned in your paper, the M1 type macrophages has anti-tumor activity and they produce IL-6 in addition to many other cytokines. It's well-known that IL-6 is an adverse prognostic factor in MM. How can this be explained?

2. Are there subtypes of M1 or M2 macrophages? If yes, any difference in their primary function?

3. Are there differences between the mature macrophages and TAM in regard to the CD cell surface markers?

4. What is the authors' suggestion on the differentiation between TAM and mature monocyte-macrophages?

5. What is the pathomechanism behind the proliferation of M2 macrophages?

6. What happens to MHCII expression in regard to APC function of macrophages?

7. What's the authors' opinion about reducing TAMs (e.g.; by bisphosphonates) and reprogramming them (by e.g.; CD40 agonists or JAK inhibitors)?

8. Any data about the correlation of TAM in MM and known cytogenetic abnormalities?

9. Will reducing TAM or reprogramming them prevent disease progression from MGUS to active MM? If yes, what is the best mechanism?

10. Any role for PD-1/PD-L1 checkpoint inhibitors in the treatment armamentarium of MM?

6. 

Author Response

Response to reviewer

Reviewer 2

1. M1 macrophage has anti-tumor activity and they produce IL-6, an adverse prognostic factor in MM. The reviewer asks how can this be explained?

We agree with the reviewer, it is a complex and not completely explained issue. We have illustrated in the paragraph 3.2.2 the possible explanation and we have confirmed IL-6 is a complex cytokine with different effect depending on level of expression and cell types, as showed below. We have updated reference list accordingly.

“IL-6 could stimulate c-Myc expression in MM cells due to an enhancement of c-Myc translation and it is a well known adverse prognostic factor in MM; however, it is produced by M1 TAM in addition to other cytokines [97-98]. A possible explanation is that IL-6 is a pleiotropic cytokine, with conflicting data about its role in inflammation [100]. Even if multiple studies demonstrated IL-6 was a pro-inflammatory cytokine in various settings, it could also promote the alternative activation of macrophages and exert an anti-inflammatory effect [99,100]. One possibility is that IL-6 could play a pro-inflammatory role in acute inflammation and an anti-inflammatory role at lower levels and/or in different cell subtypes. Finally, the binary division between M1 and M2 TAM is probably oversimplified and a broader spectrum of subtypes exists in humans [99,100].”

2. The reviewer says if there are subtypes of M1 and M2 macrophages and if there is any difference in their primary function.

We agree with the reviewer, we have added this information in the Introduction, page 3, as showed below

“However, the separation between M1/M2 subtypes was developed more than 20 years ago and it could represent an oversimplification of a broader spectrum, including at least 5 subsets: M1, M2a, M2b, M2c and M2d. M2a was induced by IL-4 and/or IL-13 and showed a primary anti-inflammatory and pro-wound healing function. M2b was induced by IL-1b and demonstrated an immuno-regulatory role. Conversely, M2c were induced by IL-10 and showed an increased expression of tissue remodeling and immune suppressive markers. Finally, M2d subtype could be induced by IL-6 and could express angiogenic markers.”

3. The reviewer asks if there are differences between mature macrophages and TAM in regard to CD cell surface markers.

We agree with the reviewer, we have added this information in the Introduction, at the beginning of page 3, as showed below.

“From a biological point of view, mature macrophages in humans are identifiable by some CD markers, including CD11b, CD11c, CD14, CD16, CD68, CD115, CD312. Interestingly, TAM M1 showed an elevated expression of CD38, CD40, CD64, CD80, CD86, while TAM M2 express high levels of CD163, CD204 and CD206.”

4. The reviewer says what is the author’s suggestion on the differentiation between TAM and mature macrophages.

We agree with the reviewer, we have added this information in the Introduction, page 3, as showed below.

“We suggest TAM represent a subpopulation of macrophages located in the tumor site, which is strongly influenced by cancer cells and TME. TAM originate from circulating monocytes after recruitment at tumor site by cancer cells and progressively acquire pro-tumor properties, making themselves similar to M2 macrophages that are present in the site of injury upon removal of damaged tissue (the so-called “wound that does not heal”).”

5. The reviewer asks what is the mechanism behind the proliferation of M2 macrophage.

We agree with the reviewer, we have added this information in the Introduction, page 3, after the paragraph of response to point 4, as showed below.

“In this field, during tissue reparation, there is a transition between classically activated M1 and alternatively activated M2 macrophages, which in turn coordinate the proliferation of cell subtypes useful for wound healing, such as vascular endothelial cells and fibroblasts. Tumor cells showed the capability to manipulate immune response with the aim of creating a pro-wound healing, anti-inflammatory macrophage phenotype. A possible explanation is that immune response against cancer is not effective, due to the immune-editing hypothesis, in which the inflammatory response could force tumor cells to reduce the expression of antigenic proteins and to limit the subsequent presentation of these antigens to immne cells of the host. According to this hypothesis, there is a limited macrophage responsiveness to neoplastic antigens and a reduced transition towards an M1 subtype. In addition, through the equilibrium/escape immune-editing processes and/or the capability of neoplastic cells to provide similar cues to those promoting a pro-wound healing response, there is an overall promotion of an M2 phenotype.”

6. The reviewer says what happens to MHC II expression in regard to APC function of macrophages.

We agree with the reviewere, we have added this information in the Introduction, at the end of page 2, as showed below.

“After phagocytosis, macrophages perform an antigen-presentin cell (APC) function, by the exposure on their surface of tumor antigen together with class II major histocompatibility complex (MHC II), thus permitting its recognition by T-lymphocytes [34,35]. Subsequent secondary signals include the engagement of costimulatory molecules, with CD40 ligand (CD40L) expressed by T cells engaging CD40 expressed by macrophages. After CD40 activation, macrophages release pro-inflammatory cytokines such as tumor necrosis factor (TNF)α and increase its expression of MHC II, which may in turn stimulate the anti-neoplastic activity of T-cell.”

7. The reviewer says what is the opinion of the authors about reducing or reprogramming TAM.

We agree with the reviewere and we have added this information, paragraphs 5.2 and 5.3.2, as showed below.

5.2

“In our opinion, TAM depletion, especially by using biphosphonates, could represent a promising therapy for MM patients. In particular, biphosphonates are already used in clinical daily practice to prevent skeletal complications in MM cases. These molecules could have an additional therapeutic role, due to their pro-apoptotic properties and the ability to reduce MVD, further suggesting a possible association with anti-VEGF molecules, such as lenalidomide. “

5.3.2

“In our opinion, TAM reprogramming represents an interesting research field for MM patients. Unfortunately, the anti-CD40 agonistic monoclonal antibody mitazalimab showed only a modest activity in patients with solid neoplasms with moderate toxicity in a phase I study. Conversely, ruxolitinib in association with methylprednisolone was investigated in R/R MM cases, including patients with high-risk cytogenetics, with an encouraging ORR of 31% and a median duration of response of 13.1 months. We suggest ruxolitinib should be investigated in association with other drugs with clinical activity against MM, such as steroids and lenalidomide.”

8. The reviewer asks if there are any data about the correlation between TAM and known cytogenetic abnormalities in MM.

We agree with the reviewer and we have added this information, at the beginning of Section 6, as showed below.

“Overall, there are limited available data about the role of TME, including TAM, in the natural history of MM. Adverse ISS stage, together with high-risk cytogenetics, were associated with distinct immune profiles in a cohort of MM cases receiving bortezomib, lenalidomide and dexamethasone (VRD). In this study, a unique profile characterized by higher T cells, with reduced erythroblasts and TAM could identify patients who achieve a CR after receiving VRD as induction therapy. Remarkably, there was a skewed ratio towards M2 subtype in patients who achieve worse ressponses and an increased TAM expression was demonstrated for patients with positive minimal residual disease (MRD+) if compared to MRD- MM cases.”

9. The reviewer asks if TAM depletion or reprogramming could prevent progression from MGUS to active MM and what is the mechanism.

We agree with the reviewer and we have added this information, paragraph 5.3.2, as showed below.

“TAM reprogramming has the aim to reduce the immunosuppressive M2 subtype, while promoting the immunostimulatory M1 subtype and represents a promising research field for MM therapy [135]. Interestingly, it could prevent disease progression from MGUS to active MM by the reduction of angiogenesis. A prospective study of circulating chemokines and angiogenic markers showed a significant association with future progression for MGUS cases who presented with elevated baseline levels of epidermal growth factor (EGF), fibroblast growth factor (FGF) and Ang-2. Due to M2 TAM play a pro-angiogenic role, we suggest an increased angiogenesis could represent the best mechanism to explain disease progression and TAM reprogramming towards an M1 subtype could counteract disease progression.”

10. The reviewer says if there is any role for PD-1/PD-L1 checkpoint inhibitors in the treatment of MM.

We agree with the reviewer, we have added this information in the paragraph 5.4, at the end of page 13, as showed below.

“The PD-L1/PD-1 axis was investigated by flow cytometry in the BM samples of 141 patients, including MGUS, smoldering MM (SMM) and active MM, either newly diagnosed or R/R cases. PD-L1 expression on MM cells was more elevated in SMM and MM cases if compared to MGUS. Even if a rational for the association with the anti-CD38 antibody daratumumab exists, clinical benefit was minimal and clinical trials were terminated for administrative reasons. ”

Reviewer 3 Report

In this review, Gozzetti and coworkers reported the role of tumor-associated macrophages on multiple myeloma progression, relapse or drug resistance.

The topic is one of the most interesting in current scientific literature and is herein presented systematically and clearly. Sections 5 and 6 are particularly significant, since they describe the impact of targeting TAMs with the cutting-edge therapeutic tools available.

Just two suggestions for the Authors are reported below.

-The figures' graphic impact should be enhanced. The text within them is hardly readable.

-A careful revision of English language throughout the manuscript is recommended. 

Author Response

Response to reviewer

Reviewer 3

The reviewer says the manuscript in interesting and well presented.

We would like to thank the reviewer for the positive comments.

The reviewer says the figures should be improved.

We agree with the reviewer, we have enlarged and re-formatted the figures, we do our best to improve their quality.

The reviewer says English language should be checked.

We agree with the reviewer, we have corrected some typographical errors.

Table 1: we have changed from immunosoppressione to immunosuppression. GM-SCF changed to GM-CSF. Developped changed to developed. Angiogenetic changed to angiogenic. Immunosspression changed to immunosuppression. Producted changed to produced. Methyl-tryptofan changed to methyl-tryptophan. Intrestingly changed to Interestingly. Due to hematological malignancies changed to since hematological malignancies.