PONE-D-24-53647Renal cancer cells acquire immune surface protein through trogocytosis and horizontal gene transferPLOS ONE

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The article was reviewed by two reviewers and both recommended major revisions. Please check the comments carefully and send the revised one in time-dependent manner.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Marcarian et al. discovered that human clear cell renal carcinoma can gain immune cell markers through both trogocytosis and horizontal gene transfer. Although trogocytosis was reported in several previous research, most of them focus on antigen transfer from tumor cells to immune cells. This finding provides a novel perspective in the field of cancer immunology and tumor microenvironment. However, the molecular mechanism of trogocytosis and how tumor benefit from gaining immune cell markers need further studied. I believe after moderate revision experiments and polishing to the text and figures, this could be a good fit for PLOS ONE.

Major concerns:

1. Trogocytosis discovered by previous studies were mediated by some level of interaction of the surface molecules between the two cells (TCR to MHC-peptide complex; CAR-antigen; CTLA4-B7 complex). What interacting molecules are responsible for trogocytosis of immune cells markers to tumor cells? Based on the result, trogocytosis discovered by the authors are stochastic and random.

2. To what extend this type of trogocytosis rely on the nature/characteristic of immune cells? Would you still observe trogocytosis if you coculture two tumor lines, with one of them overexpress immune cell marker like CD45?

3. Previous studies have shown that trogocytosis happens in both directions. Is this type of trogocytosis discovered by the authors also reciprocal (meaning that tumor markers like CA9 also found transferred to immune cells?

Minor concerns:

1. Figures should be citated/callout sequentially. However, Figure 3C was called out before Figure 3A and 3B.

2. Figure 2D:

a. N = 4 in the legend but there are only three data points for CD16. Please explain where the missing data point goes.

b. This figure is not very good at demonstrating the claim made in the result section: “Our data revealed that the vast majority of CAIXhi cells were positive for multiple immune cell markers.” It’s better to use one or multiple bar plots or pie charts to show the percent of quadrupole, triple, or double positive cells for each sample. For example, 30% CD45 positive and 30% CD14 positive doesn’t necessary mean 30% of the cells are double positive for these two markers. Very likely these 30% were not the same group of cells.

c. It would be better if use different symbol for different samples (or connected same sample with line). With this additional information, it would be interesting to see whether some sample have higher trogocytosis for all markers consistently.

3. Figure 3A only shows gating strategy for Lymphocytes, Non_trogocytic tumor, and Trogocytic tumor. It can be moved to supplemental.

4. Figure 3B Nanostring data would be better to include more immune markers. For example, Figure 3C and figure 3D both show upregulation of makers like CD45, CD14, and CD16.

5. The authors should cite more current research articles in the introduction part. Recommended high impact paper to cite:

a. Huang, J. F. et al. TCR-Mediated internalization of peptide-MHC complexes acquired by T cells. Science 286, 952–954 (1999).

b. Hamieh, M. et al. CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature 568, 112–116 (2019).

c. Zhou, X., Cao, H., Fang, SY. et al. CTLA-4 tail fusion enhances CAR-T antitumor immunity. Nat Immunol 24, 1499–1510 (2023).

d. Schriek, P. et al. Marginal zone B cells acquire dendritic cell functions by trogocytosis. Science 375, eabf7470 (2022).

Reviewer #2: Comments to manuscript PONE-D-24-53647

In their present work the authors showed that renal cancer cells were positive for lymphocyte/leukocyte markers, such as CD45, CD56, CD14 and CD16. The authors assumed that this finding was attributed to trogocytosis and horizontal gene transfer, which would be a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. In brief, this is an interesting finding, but the available data provide only limited support for the assumption that the process why lymphocyte/leukocyte markers can be found in renal cancer cells could be trogocytosis. Other mechanisms such as cell cannibalism, entosis and cell fusion must also be considered.

1) In addition to trogocytosis, other mechanisms have been identified that would explain the presence of lymphocyte/leukocyte proteins, such as CD45, CD56, CD14 and CD16 and lymphocyte/leukocyte DNA in cancer cells, which are cell cannibalism, entosis and cell fusion (e.g., Fais et al., 2018 Cell Death & Disease 9: 95; Kapsetaki et al., 2024 Scientific Reports 14: 7535; Shultes et al., 2024 The International Journal of Biochemistry & Cell Biology 175: 106649; Sieler et al., 2024 Results Probl Cell Differ 71:433). Thus, I would very much appreciate if the authors can thoroughly discuss what speaks for trogocytosis, cell cannibalism, entosis or cell fusion and what not.

As the authors know, trogocytosis is a process in which one cell physically extracts and ingests “bites” of cellular material from another cell (Bettadapur et al., 2020 doi.org/10.1128/iai.00930-19), but to my knowledge this cellular material rather comprises of parts of the plasma membrane and a certain cytosolic fraction, but not DNA. Thus, it remains unclear why leukocyte/lymphocyte DNA was found in renal cancer cells.

Cell fusion might be an explanation for this finding. In this regard, former studies of Yilmaz et al. and Rachkowsky et al. demonstrated that donor DNA was found in renal carcinoma cells of female BMT recipients (Bone Marrow Transplant 2005 Vol. 35 Issue 10 Pages 1021-4; Bone Marrow Transplant 2004 Vol. 34 Issue 2 Pages 183-6).

However, it cannot be ruled out that cancer cells have phagocytosed immune cells or that immune cells were taken up by cancer cells by entosis. For instance, in Fig. 4E A498 it seems that the A498 cell harbors a rather intact T-cell, which might be related to either cell cannibalism or entosis. However, it cannot be ruled out that this T-cell is simply attached on top of the A498 cell and not inside. Thus, 3D images with Z-stacks would be helpful. This also applies to the ACHN data in Fig. 4E. Unfortunately, the resolution is too low to conclude that the marked T cell is really inside the cancer cell or not. Nonetheless, the irregular form of the nucleus and the scattered CD45 staining pattern might indicate that lysis of the engulfed T-cell has already started, which would rather speak for cell cannibalism than for entosis. This might also be true for 768O data. Here, CD45 staining was not colocalized with EdU staining.

The assumption that rather cell cannibalism, entosis or cell fusion might be responsible for the presence of T-cell DNA in RCC cells (and possibly not trogocytosis) is further supported by data presented in Fig. 4A and 4C. As mentioned above, trogocytosis is rather the ingestion of small cellular parts of a donor cell, which usually did not contain DNA. Data presented in Fig. 4A and 4C indicate a partially high GFP-H2B/EdU fluorescence in single Caki-1 cells, which cannot be sufficiently explained by the uptake of small amounts of T-cell DNA. In this regard, it is more likely that whole GFP-H2B/EdU expressing/labeled T-cells were engulfed/ phagocytosed by Caki-1 cells. Moreover, appropriate FACS data for wildtype Jurkat cells and GFP-H2B Jurkat cells and Edu-stained Jurkat cells are missing.

2) In Fig 1A it is unclear why only CD14 was found in the membrane of renal cancer cells, but not CD16, CD45RA and CD56. CD16, CD45RA and CD56 are all membrane proteins and if parts of the macrophage membrane were actively transferred to the cancer cell membrane by trogocytosis one would expect that macrophage derived membrane protein would be found in the plasma membrane of the cancer cells. However, it cannot be ruled out that cancer cells have “eaten” small cellular parts of the macrophages, which would explain the cytosolic localization of CD16, CD45RA and CD56. But then CD14 should also be found in the cytosol of the cancer cells, which obviously is not seen here. Please explain.

3) The sentence “Using qRT-PCR analysis, we identified a significant induction of CD45 gene expression in RCC cells post coculture (Fig. 3C)” (line 203-204) should be revised as it suggests that RCC cells actively up-regulated CD45 expression. Instead, CD45 expression was rather attributed to the engulfed immune cells. By doing so, the heading “ RCC tumor cells express RNA for immune surface proteins” (line 189) must also be revised since it also implies that, e.g., CD14, etc. is expressed in RCC cells.

Similarly, statistical significance was not calculated. Thus, if the authors conclude that the observed higher CD45 expression was significant, this should be validated by an appropriate statistical test (like in Fig. 3D).

Moreover, Fig. 3C (line 204) was mentioned in the text before the Figure legend of Fig 3 (line 206 – 223) and Fig. 3A (line 227) and 3B (line 231). Please rearrange the order.

4) The description of nanostring analysis is missing in the Materials and Methods section. Please revise.

5) In Figure 4B monocultured Caki-1 cells showed a basal GFP-H2B fluorescence. I guess that this is the autofluorescenc/ background fluorescence of the cells.

6) Data presented in Fig. 5 also rather support the assumption that whole CD45+ cells were engulfed/ phagocytosed by renal cancer cells and that the presence of CD45+ cells was not attributed to trogocytosis. For instance, results in the first row likely indicate that an intact CD45+ immune cells was freshly engulfed/ phagocytosed. However, 3D images with Z-stacks would be more helpful to clarify whether these CD45+ immune cells were really engulfed/ phagocytosed by renal cancer cells. Data presented in the second and fourth row are less convincing. In the second row only a very faint Hoechst signal can be seen. Similarly, no and even not a faint Hoechst signal of the prospective CD45+ immune cell can be seen in the fourth row. Thus, it is difficult for me to conclude whether these Hoechst signals were really attributed to immune cell DNA in renal cancer cells. However, it cannot be ruled out that the weak Hoechst signal was lost due to data compression. Data presented in the third row also support the assumption that an intact CD45+ immune cells that was freshly engulfed/ phagocytosed by a renal cancer cell. However, the shape of the Hoechst and CD45 staining in Fig. 5B does not really fit with the appropriate data in Fig. 5A. It looks as if the picture has been rotated 90° to the left. For instance, a faint Hoechst and CD45 stain can be seen in the upper right corner, which, however, cannot be seen in the appropriate image of Fig. 5A. This must be checked and corrected.

7) Transmission images should be provided for all immunofluorescence data.

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Reviewer #2: Yes:  Thomas Dittmar

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Renal cancer cells acquire immune surface protein through trogocytosis and horizontal gene transfer

PONE-D-24-53647R1

Dear Dr. Bothwell,

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Additional Editor Comments (optional):

Dear Dr. Alfred Lester Meador Bothwell.

Thank you for resubmitting your manuscript. One reviewer and I agreed to the acceptance. I reviewed the manuscript instead of 2nd reviewer although the one did not take the re-review. I think the authors responded appropriately to all questions from the reviewer

Yours sincerely,

Kenji Fujiwara

Academic editor

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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Reviewer #2: No

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