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Brief summary of the study - a sentence summarizing the study and general comments that apply across the full paper

• Here, the authors demonstrate how tumor-associated macrophages (TAMs) are able to phagocytose cancer cells and re-express cancer cell-derived proteins on their cellular surface. This may have influence on immune signaling, TAM reprogramming, tumor microenvironment, and clinical implications. 

• They utilize a combination of flow cytometry and several elegant in vitro co-culture assays, which include isotopic labelling to distinguish between cancer- and macrophage-native proteins via downstream proteomics as well as biotin-based labeling of only surface proteins, to illustrate how cancer cell-derived surface proteins need to be phagocytosed by macrophages in order to be re-deployed on the macrophage surface. 

• Intriguingly, many of those re-deployed proteins include transporters, which likely impact the macrophage’s metabolism. The authors validate one of those transporters, SLC1A5, to be expressed and functional on the surface of macrophages upon phagocytosis. 

• However, such a meaningful claim requires careful consideration and in-depth experimental validation. As other hypotheses explaining the authors’ findings cannot be fully excluded yet, further experiments might be required to strengthen their theory. Overall, the author’s discovery about this mechanism may help developing therapeutics by TAM manipulation.

Major comments  - Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions

• Overall a very neat study that devises a couple of creative co-culture approaches to argue that cancer cell protein re-deployment on macrophages depends on phagocytosis. Several open questions remain, though. 

  • Is the described process only applicable to cancer cell surface proteins? 

  • Is there some sort of selective pressure that determines which surface proteins get to be re-expressed on the macrophage surface or is it arbitrary?

  • Is it true that re-deployment of cancer cell proteins on the macrophage surface “dilute” the macrophage native surface protein landscape and thus impact its ability to phagocytose as proposed in the discussion? This is not necessarily reflected in the data and I’m wondering if this is a process that happens over longer periods of time?

  • What distinguishes eater from non-eater TAMs in vivo? Are they transcriptionally different, differentially localized etc?

  • How frequent are eater TAMs or in other words, how often does this process of protein re-deployment happen? Are higher frequencies of eater TAMs associated with a worse cancer prognosis?

• While the combined experiments support the authors’ hypotheses, they do not exclude other possible mechanisms explaining their findings. Most importantly, all findings could be explained by macrophages phagocytosing apoptotic or necrotic cancer cells (mentioned in the last paragraph, but not further discussed), or even protein biomass released by these. Macrophages continuously phagocytosing whole, intact cancer cells would to my knowledge be a novel concept for the field, which is why other explanations (like uptake of free protein) need to be considered and tested for. Comparison with uptake of free protein and the investigation of apoptosis rates in the experimental system would be desirable. The uptake of biotinylated protein seems to be a key experiment of the study, but the authors categorically exclude the previously shown trogocytosis as explanation for the transferred proteins, without testing for this mechanism in this setting. 

Minor comments - Clarifications to statements in the text, interpretation of the results, presentation of the data/figures

• Figure 1B could benefit by adding a WT control to ensure that with the reported gating strategy, no EpCAM positive macrophages are detected in healthy tissue (without 4T1).

• When looking into extended data figure 7, noticeably few events were reported in the FACS data. Please report how many cells were used and analyzed. Is there an experimental reason only so little material could be collected and investigated?

• Incubation of primary cells for 2 hours, as performed in several experiments throughout the manuscript, might lead to cell death. Reporting apoptosic rates before and after incubation would be desirable (potentially by re-analysis of presented FACS data).

• The authors claim that phagocytosis decreases diversity of native macrophage proteins on the cell surface. There appear to be some published datasets on e.g. transcriptional changes induced by phagocytosis that could be used for comparison, for instance https://www.nature.com/articles/s41598-020-70353-y 

• Figure 1 provides strong evidence for the acquisition of cancer cell surface proteins by TAMs. but further research is needed to fully understand the mechanisms and functional implications of this process.

Comments on reporting - information on the statistical analyses or availability of data.

• In Figs. 3B and 4B, the authors report to have used 2 biological replicates, in 2 technical replicates each. Nevertheless, 4 data points are found for most conditions, even 6 for naive SW620. Please clarify the number of replicates. If 2 biological replicates were used, I believe only 2 should be reported in the figure (mean of the two technical replicates per biological replicates), not 4 seemingly independent data points

• Please report the statistical test used for each analysis (neither found in main text nor supplementary data). 

Suggestions for future studies

• Compare the genomes of cancer cells and TAMs to identify genetic factors that may influence the efficiency or specificity of protein redeployment.

• employ single-cell RNA sequencing and proteomics to analyze the heterogeneity of TAMs and the variability in cancer cell protein redeployment within individual cells.

• Translate the findings of this study into clinical settings. Investigate the association between cancer cell protein redeployment and patient outcomes, tumor progression, or response to therapy. Develop potential therapeutic strategies targeting this process.

Conflicts of interest of reviewers

• There are no conflicts of interest to declare.

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• Extended Data Figure 3A and main text paragraph 3. They wrote “4T1 cells revealed myriad intracellular proteins from cell compartments including but not limited to the nucleus, cytosol, and mitochondria”

  • Do these proteins have any relation with tumor? What kind of these proteins are , TF, Kinases or something else ?

• Same paragraph. They wrote “our data show that macrophages that have phagocytosed target cells express a cell surface protein uniquely encoded by the phagocytosed cell, namely EpCAM”

  • I wonder if this is the Only Protein that is expressed, if so, then why ? How is this protein special? What could be the biological significance of expressing only this protein ?

• Paragraph 4. Starting from “ Established intracellular RBC proteins HBA and HBB1 were more detectable in Ter119+ macrophages compared to Ter119- macrophages (Figure 1E).” till the end of the paragraph

  • Here they talk about intracellular proteins of the cell-to-be-phagocytosed and then never really come back to it. 1) it supports their idea of actual phagocytosis as opposed to trogocytosis or protein secretion by the target cell and 2) it's interesting that target cell proteins seem to persist for a while in the macrophage. Can any target cell intracellular protein be expressed on the macrophage surface as well or is there some sort of signal peptide encoded in target cell surface proteins that only allows them to go to the cell membrane/surface of the macrophage?

• Paragraph 5. They write “Specifically, CSF1R and CATC expression have previously been implicated in phagocytic capacity and macrophage polarization, and LRP1 is an established receptor mediating macrophage phagocytosis of live or apoptotic red blood cells”

  • How can this be reconciled with what the authors say in their discussion ("It is not that TAMs are intrinsically poor phagocytes, rather it is that they have phagocytosed so many cancer cells that they have reengineered their cell surface, redeploying proteins that increase metabolic uptake while diluting out or losing surface proteins that promote phagocytosis.")?

• Figure 2. 

  • In panel C - what exactly do the authors mean by "light and heavy" white bars? Could those f.e. be cancer cell antigens assembled in macrophage MHC complexes (i.e. a mix of cancer and macrophage protein material)?

  • can the authors perhaps indicate a couple of examples of proteins or protein groups among the heavy surface proteins? To get a sense of which cancer cell proteins get re-expressed on the macrophage surface. Are they all exclusively surface proteins (as opposed to intracellular proteins)?

  • Figure 2 presents a co-culture experiment to directly demonstrate the acquisition of cancer cell surface proteins by macrophages. More research is needed to understand how these redeployed proteins affect macrophage behaviour and their interactions with other cells in the tumor microenvironment.

• Paragraph 8. The authors wrote: “Acquisition of heavy proteins on the cell surface was not observed for macrophages co-cultured with SW620 cancer cells when Eater and Non-Eater populations were not further purified, emphasizing the need to purify the less-abundant Eater population (Extended Data 9, Tables 7A and 7B)”

  • in clinical implications, further studies is needed to validate these findings in vivo and in different cancer models.

• Paragraph 9. The authors wrote:  “EpCAM is detectable on the surface of Eater macrophages but not Non-Eaters or naive macrophages in our MS data set”

  • Can author test- if becoming an eater macrophage is attributed to EpCam ?

• Paragraph 9. The authors wrote: “Pharmacologic inhibition of phagocytosis by Cytochalasin D prevented EpCAM detection on the surface of macrophages (+CytoD condition, Figure 3B)”

  • this (+ the subsequent use of cytochalasin D in the biotin experiment) is the strongest piece of evidence to support that macrophages actually have to phagocytose the cancer cell for surface protein re-expression!

• Paragraph 9. The authors wrote: “Imaging flow cytometry showed transfer of EpCAM to the cell surface is only observed when cancer cells have entered the macrophage lysosome”

  • As far as I can tell, the imaging flow cytometry data in 3C do no specifically show lysosomal location, but rather just general engulfment of a green cancer cell by a red macrophage

• Figure 3 

  • panel C lacks a scale bar.

  • Figure 3 provides strong evidence for the mechanism of cancer cell protein redeployment by macrophages and supports the generalizability of this phenomenon to different cancer cell types.

• Paragraph 11. The authors wrote: “If such proteins were functional following transfer, this could significantly alter the metabolic update of nutrients and metabolites by TAMs, which are known to be metabolically dysregulated”

  • While the study demonstrates the functional activity of some redeployed proteins, a more comprehensive analysis of the functional consequences for TAMs would be beneficial. Investigating the impact of redeployed proteins on TAM phagocytosis, cytokine production, migration, and interactions with other cells in the tumor microenvironment could reveal the full spectrum of their effects

• Paragraph 11. The authors wrote: “By MS and flow cytometry, the active transporter SLC1A5, an established temperature- and pH-sensitive glutamine transporter, was detected as one of the proteins transferred to the Eater surface”

  • SInce all images are low resolution images, ti was difficult to read the data. List other proteins as well, maybe classify with their family names. SLC1A5 is one example. This might need more proteins as proof-of concept

• Figure 4. 

  • while the SLC1A5 studies are very nice, a second example of a transporter would have been nice for validation purposes. Such studies should be relatively straightforward in vitro

• Paragraph 12. The authors wrote: “Importantly, this uptake was sensitive to temperature and glutamine concentration, a characteristic of SLC1A5 function”

  • Different expressed proteins could bring different characteristics. Strengthen your proof-of concept with using other proteins in the list

• Paragraph 13. The authors wrote - “Transporters are statistically over-represented in the dataset of transferred proteins detectable on the surface of macrophages that have phagocytosed, and these proteins appear to be functional”

  • the fact that The authors are functional was really just shown for one transporter (SLC1A5) and should not be generalized. A second example of a validated transporter (as mentioned in one of my earlier comments) would make a stronger case for this claim

• Paragraph 14. The authors wrote -  “Further work will be required to understand the precise mechanism of protein redeployment so that it can be perturbed both to understand the mechanistic intricacies of the process and to test its impact on innate immune control of cancer.”

  • Investigating the association between this process and patient outcomes, tumor progression, or response to therapy could highlight the potential clinical significance of these findings.

• Paragraph 15 The authors wrote - “The work described suggests a novel model in TAM etiology. It is not that TAMs are intrinsically poor phagocytes, rather it is that they have phagocytosed so many cancer cells that they have reengineered their cell surface, redeploying proteins that increase metabolic uptake while diluting out or losing surface proteins that promote phagocytosis”

  • Explore also too whether similar mechanisms of protein redeployment occur in other immune cell types, such as dendritic cells or neutrophils, could provide insights into the broader implications of this phenomenon and its potential therapeutic targets.

Competing interests

The authors declare that they have no competing interests.