Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells

Round 1

Reviewer 1 Report

The authors written the manuscript schematically. However, address the following for the improvement.

First of all, slightly modify the title of the manuscript. It reflects the some review model. discussion of the manuscript is very simple. Write or elaborate with more published literature for the results in discussion part. "One way to induce such an immunogenic environment" what are the different types for this and include in the manuscript. "An increasing number of mammalian gene products, including intracellular proteins, are now available as purified recombinant proteins". Specify the available products with tabular format. B16 melanoma tumor cells - what type of cells and which type of tumor is involved with these cells - unclear. what are the advantages of vapor nanotubule technology. What is the commercial significance compared with conventional methods. stability of protein through VNT is not discussed. How the protein purification and structure integrity is maintained in VNT is not explained. In figure 2, C and D sections are very poor quality. Improve the resolution and scale of her figures also. What is the proof of concept if error - source not found statements.

Author Response

Response to reviewers’comments:

First, we would like to thank both reviewers for their time, remarks and concerns about our manuscript. Below, please find out point-by-point response to the questions that were raised on the work reported in our initial submission (ijms-578901).

 

Reviewer #1 (remarks to the Authors)

The authors written the manuscript schematically. However, address the following for the improvement.

First of all, slightly modify the title of the manuscript. It reflects the some review model. discussion of the manuscript is very simple. Write or elaborate with more published literature for the results in discussion part. "One way to induce such an immunogenic environment" what are the different types for this and include in the manuscript. "An increasing number of mammalian gene products, including intracellular proteins, are now available as purified recombinant proteins". Specify the available products with tabular format. B16 melanoma tumor cells - what type of cells and which type of tumor is involved with these cells - unclear. what are the advantages of vapor nanotubule technology. What is the commercial significance compared with conventional methods. stability of protein through VNT is not discussed. How the protein purification and structure integrity is maintained in VNT is not explained. In figure 2, C and D sections are very poor quality. Improve the resolution and scale of her figures also. What is the proof of concept if error - source not found statements.

Specific comments:

Sligtly modify the title of the manuscript. It reflects the some review model.

Author response:

We have changed the title as follows: “Delivery of Mixed Lineage kinase domain-like protein by vapor nanobubble photoporation induces necroptotic-like cell death in tumor cells”

 

Discussion of the manuscript is very simple. Write or elaborate with more published literature for the results in discussion part.

2.1) “one way to induce such an immunogenic environment” what are the different types for this and include in the manuscripts.

Author response:

We agree that our manuscript, which communicates a straightforward yet new finding, could be improved by elaborating more on the implications of our experimental data in the discussion. The following text was added to the manuscript line 50 to 55 (introduction):  

“To reshape the tumor microenvironment immune stimulatory molecules (1), blockade of inhibitory cytokines (2) as well as immune checkpoint blockade (3-5) can be introduced at the tumor site to restore immunological fitness. The direct intratumoral injection of mRNA encoding such immune modulatory proteins is an attractive approach. Hewitt and colleagues, for example, delivered IL-23/IL-36γ/OX40L triplet mRNA mixture combined with checkpoint blockade to successfully reshape the tumor microenvironment (1).”

In addition, the  following text was added to the manuscript line 244 to 260 (discussion):

“Tumors often comprise a network of both malignant and non-malignant cells. Although various immune effector cells are recruited to the tumor site, in many cases their anti-tumor functions are downregulated, largely due to tumor-derived immunosuppressive signals. As a consequence, immune cells in the tumor microenvironment fail to exert antitumor effector functions and the tumor escapes from an attack by the host immune system. The immunosuppressive tumor microenvironment is a major obstacle in cancer immunotherapy. Thanks to growing insights into the suppressive mechanism of the tumor microenvironment, possible ways to block tumor escape are currently under investigation, including the delivery at the site of the tumor of immune stimulatory molecules (e.g. OX40 ligand and IL-23(1)), blockade of inhibitory cytokines (e.g. TGF-β receptor II to capture TGF-β(2)) as well as immune checkpoint blockade (e.g. PD-1 or CTLA-4(3-5)). Recently it became clear that by eliciting immunogenic cell death (ICD) an immunogenic tumor environment can be created. ICD is a common denominator for different cell death modalities that result in thet release of damage-associated molecular patterns (DAMPs). These DAMPs can, together with other immune-stimulatory componencts, recruit and activate DCs in the tumor microenvironment (6-8). It is shown that tumor cells succumbing from irradiation, chemotherapeutics and hypericin-based photodynamic therapy undergo immunogenic apoptosis (9-13). Also necroptosis is considered as a type of cell death with immunogenic properties (14, 15).”

 

2.2) “An increasing number of mammalian gene products, including intracellular proteins, are now available as purified recombinant proteins.” Specifiy the available products with tabular format

Author response:

We agree that this is a generalizing statement that should be specified more precisely. We propose to add a reference (McKenzie et al.; Methods; 2018) to a recent review on purified recombinant proteins at the end of the sentence. We felt that the suggestion to specificy the available recombinant proteins in a tabular format would require too much of an effort because it would require us to scrutinize product catalogues of many Life Sciences companies.

 

2.3) B16 melanome tumor cells-what type of cells and which type of tumor is involved with these cells- unclear.

Author response:

B16 melanoma cells are transformed cells that originate from melanin-producing epithelia of mice. Upon subcutaneous injection, B16 cells will form a palpable tumor. B16 cells are frequently used as a model for human melanoma.

In the abstract, “B16 cells” is now replaced by “murine B16 melanoma cells”. In the discussion (line 262 to line 266) we added the following sentence to clarify the used cell line:

“In this study, we show that caspase-3 or -8, or MLKL protein delivery by VNB photoporation induces respectively apoptotic and necroptotic cell death in murine B16 melanoma tumor cells. The B16 tumor cell line is frequently used as a model for human melanoma.”

 

 

 

2.4) What are the advantages of vapor nanotubule technology. What is the commercial significance compared with conventional methods.

Author response:

As briefly discussed in the introduction, current commercially available transfection methods in general work poorly for intracellular protein delivery, or are associated with high cell toxicity. This is why so much effort goes into developing more refined intracellular delivery methods, as nicely reviewed by Stewart and colleagues (16). Vapor nanobubble (VNB) photoporation is one of the most promising upcoming new physical transfection technologies of which we and other have repeatedly demonstrated in the past years that it offers gentle intracellular delivery of different types of macromolecules, including proteins, in a wide variety of cell types, such as cancer cell lines (17, 18), primary lymphocytes (19, 20) and neuronal cells (21). Based on our extensive expertise with this technology, and considering its efficiency and safety, we opted to use this one in the present study, which resulted in a very postive outcome indeed. The technology is, to the best of our knowledge, not yet commercially available, but we are aware of efforts in this direction so that it hopefully will become more widely available in the near future.

Some of the most important advantages of photoporation are now mentioned in the discussion of the revised manuscript lines 266-272:

“Vapor nanobubble (VNB) photoporation represents a very promising physical transfection technology, already shown to enable gentle intracellular delivery of different types of macromolecules (e.g. siRNA) in a wide variety of cell types, such as cancer cell lines (17, 18), primary lymphocytes (19, 20) and neuronal cells (21). By the combination of absorbing gold nanoparticles and a weakly focused laser beam, the technology also allows simultaneous intracellular delivery of macromolecules in a large number of cells, while maintaining a high level of spatial control (21).

 

2.5) Stability of protein through VNT is not discussed. How the protein purification and structure integrity is maintained in VNT is not explained. 

Author response:

We understand the reviewer’s concern that protein integrity might be affected upon vapor nanobubble (VNB) formation. However, it should be noted that only few gold nanoparticles (AuNPs) remain attached to each cell after washing away the excess of unbound AuNPs. These few AuNPs will very locally damage surrounding structures, such as the cell membrane, upon laser irradation and only affect a very small fraction of proteins in close proximity of the formed VNB. Therefore, even though it cannot be excluded that some of the nearby proteins become damaged upon VNB formation, on the protein ensemble level effects are negligible since no VNB are formed within the cell medium.

We have briefly commented on this in the discussion of the revised manuscript lines 273– 277:

“For this, B16 cells were incubated with different concentrations of gold nanoparticles (AuNPs), followed by VNB generation by pulsed laser irradiation in presence of FITC-dextrans or FITC-BSA molecules. Considering that only few gold nanoparticles adsorb to each cell and the arising VNBs only affect neighboring structures very locally, damage to nearby molecules is possible but negligible on the total molecule ensemble level.

 

3) In figure 2, C and D sections are very poor quality. Improve the resolution and scale of her figures also.

Author response:

We acknowledge that some parts of this figure could be improved. For this, we enhanced the resolution of figures 2C and 2D, and enlarged the scales of figures 2A and 2B.

 

What is the proof of concept if error - source not found statements. 

Author response:

We are confused by this remark. We noticed that in the Word document that was returned to us after the review, the wording “Error! Reference source not found” appeared several times in the manuscript, where normally a number reference should have been typed. We regret these errors, which may have erroneously been introduced upon final formatting of our manuscript. However, we note that the generated and approved pdf version of our submitted manuscript did not contain these errors.”

 

 

Reviewer #2 (remarks to the Authors)

Authors have designed essential experiments to demonstrate intra-cellular delivery of proteins through vapor nanobubble photoporation technique. The paper is of interest and the content fits well under the scope of this journal. Manuscript can be accepted after minor revisions.

Modify the abstract by adding experiments and quantitative results in brief.

Check the references throughout the manuscript. There are many ‘reference unfound errors’.

Number all the sub-sections in section 2 and 4. In section 2, discuss all results with respect to photoporation mechanism at molecular level.

 

Specific comments:

Modiy the abstract by adding experiments and quantitative results in brief.

Author response:

We added the following quantitative results of our experiment to the abstract:

Line 21-24: Here, we show the possibility to deliver a model polysaccharide (dextran of 70 kDa) and a model protein (bovine serum albumin) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively.

Line 27-29: A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery.

 

Check the references throughout the manuscript. There are many’reference unfound errors’

Author response:

We apologize for this error. The problems with the references are solved in the revised manuscript. The problem was with references to figures.

 

Number all the sub-sections in section 2 and 4.

Author response:

All the sub-sections in the revised manuscript are now numbered accordingly.

 

In Section 2, discuss all results with respect to photoporation mechanism at molecular level

Author response:

We must admit that this question was not entirely clear to us. The principle and mechanism of photoporation was explained in the introduction and Figure 1. Perhaps the reviewer shares the concern of Reviewer 1 in question 2.5 that proteins may perhaps be damaged by VNB formation. As pointed out this may possibly happen to proteins that happen to be in the immediate vicinity of AuNP when VNB are formed, but since only very few AuNP are present per cell and no AuNP are present in the cell medium, this potential effect is in any case negligible on the protein ensemble level. This was further clarified in the discussion of the revised manuscript in line 273-277: For this, B16 cells were incubated with different concentrations of gold nanoparticles (AuNPs), followed by VNB generation by pulsed laser irradiation in presence of FITC-dextrans or FITC-BSA molecules. Considering that only few gold nanoparticles adsorb to each cell and the arising VNBs only affect neighboring structures very locally, damage to nearby molecules is possible but negligible on the total molecule ensemble level.

References

Hewitt SL, Bai A, Bailey D, Ichikawa K, Zielinski J, Karp R, et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36gamma, and OX40L mRNAs. Sci Transl Med. 2019;11(477). Van der Jeught K, Joe PT, Bialkowski L, Heirman C, Daszkiewicz L, Liechtenstein T, et al. Intratumoral administration of mRNA encoding a fusokine consisting of IFN-beta and the ectodomain of the TGF-beta receptor II potentiates antitumor immunity. Oncotarget. 2014;5(20):10100-13. Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, et al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell. 2016;165(1):35-44. Ribas A, Dummer R, Puzanov I, VanderWalde A, Andtbacka RHI, Michielin O, et al. Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy. Cell. 2017;170(6):1109-19 e10. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515(7528):568-71. Hackl H, Charoentong P, Finotello F, Trajanoski Z. Computational genomics tools for dissecting tumour-immune cell interactions. Nat Rev Genet. 2016;17(8):441-58. Krysko O, Love Aaes T, Bachert C, Vandenabeele P, Krysko DV. Many faces of DAMPs in cancer therapy. Cell Death Dis. 2013;4:e631. Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P. Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer. 2012;12(12):860-75. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007;13(1):54-61. Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, Marysael T, et al. A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J. 2012;31(5):1062-79. Galluzzi L, Kepp O, Kroemer G. Enlightening the impact of immunogenic cell death in photodynamic cancer therapy. EMBO J. 2012;31(5):1055-7. Galluzzi L, Kepp O, Kroemer G. Immunogenic cell death in radiation therapy. Oncoimmunology. 2013;2(10):e26536. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy. Annu Rev Immunol. 2013;31:51-72. Yatim N, Jusforgues-Saklani H, Orozco S, Schulz O, Barreira da Silva R, Reis e Sousa C, et al. RIPK1 and NF-kappaB signaling in dying cells determines cross-priming of CD8(+) T cells. Science. 2015;350(6258):328-34. Aaes TL, Kaczmarek A, Delvaeye T, De Craene B, De Koker S, Heyndrickx L, et al. Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity. Cell Rep. 2016;15(2):274-87. Stewart MP, Langer R, Jensen KF. Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts. Chem Rev. 2018;118(16):7409-531. Xiong R, Joris F, Liang S, De Rycke R, Lippens S, Demeester J, et al. Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging. Nano Lett. 2016;16(10):5975-86. Xiong R, Raemdonck K, Peynshaert K, Lentacker I, De Cock I, Demeester J, et al. Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells. ACS Nano. 2014;8(6):6288-96. L. Raes CVH, J. Michiels, S. Stremersch, J.C. Fraire, T. Brans, R. Xiong, S. De Smedt, L. Vandekerckhove, K. Raemdonck, K. Braeckmans,. Gold Nanoparticle-Mediated Photoporation Enables Delivery of Macromolecules over a Wide Range of Molecular Weights in Human CD4+ T Cells. Crystals. 2019;9. Wayteck L, Xiong R, Braeckmans K, De Smedt SC, Raemdonck K. Comparing photoporation and nucleofection for delivery of small interfering RNA to cytotoxic T cells. J Control Release. 2017;267:154-62. Xiong R, Verstraelen P, Demeester J, Skirtach AG, Timmermans JP, De Smedt SC, et al. Selective Labeling of Individual Neurons in Dense Cultured Networks With Nanoparticle-Enhanced Photoporation. Front Cell Neurosci. 2018;12:80.

 

Reviewer 2 Report

Review Report-IJMS 578901

Protein delivery by vapor nanobubble photoporation controls cell death modalities in tumor cells

Authors have designed essential experiments to demonstrate intra-cellular delivery of proteins through vapor nanobubble photoporation technique. The paper is of interest and the content fits well under the scope of this journal. Manuscript can be accepted after minor revisions.

Modify the abstract by adding experiments and quantitative results in brief.

Check the references throughout the manuscript. There are many ‘reference unfound errors’.

Number all the sub-sections in section 2 and 4. In section 2, discuss all results with respect to photoporation mechanism at molecular level.

 

Author Response

Reviewer #2 (remarks to the Authors)

Authors have designed essential experiments to demonstrate intra-cellular delivery of proteins through vapor nanobubble photoporation technique. The paper is of interest and the content fits well under the scope of this journal. Manuscript can be accepted after minor revisions.

Modify the abstract by adding experiments and quantitative results in brief.

Check the references throughout the manuscript. There are many ‘reference unfound errors’.

Number all the sub-sections in section 2 and 4. In section 2, discuss all results with respect to photoporation mechanism at molecular level.

 

Specific comments:

Modiy the abstract by adding experiments and quantitative results in brief.

Author response:

We added the following quantitative results of our experiment to the abstract:

Line 21-24: Here, we show the possibility to deliver a model polysaccharide (dextran of 70 kDa) and a model protein (bovine serum albumin) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively.

Line 27-29: A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery.

 

Check the references throughout the manuscript. There are many’reference unfound errors’

Author response:

We apologize for this error. The problems with the references are solved in the revised manuscript. The problem was with references to figures.

 

Number all the sub-sections in section 2 and 4.

Author response:

All the sub-sections in the revised manuscript are now numbered accordingly.

 

In Section 2, discuss all results with respect to photoporation mechanism at molecular level

Author response:

We must admit that this question was not entirely clear to us. The principle and mechanism of photoporation was explained in the introduction and Figure 1. Perhaps the reviewer shares the concern of Reviewer 1 in question 2.5 that proteins may perhaps be damaged by VNB formation. As pointed out this may possibly happen to proteins that happen to be in the immediate vicinity of AuNP when VNB are formed, but since only very few AuNP are present per cell and no AuNP are present in the cell medium, this potential effect is in any case negligible on the protein ensemble level. This was further clarified in the discussion of the revised manuscript in line 273-277: For this, B16 cells were incubated with different concentrations of gold nanoparticles (AuNPs), followed by VNB generation by pulsed laser irradiation in presence of FITC-dextrans or FITC-BSA molecules. Considering that only few gold nanoparticles adsorb to each cell and the arising VNBs only affect neighboring structures very locally, damage to nearby molecules is possible but negligible on the total molecule ensemble level.

 

 

References

Hewitt SL, Bai A, Bailey D, Ichikawa K, Zielinski J, Karp R, et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36gamma, and OX40L mRNAs. Sci Transl Med. 2019;11(477). Van der Jeught K, Joe PT, Bialkowski L, Heirman C, Daszkiewicz L, Liechtenstein T, et al. Intratumoral administration of mRNA encoding a fusokine consisting of IFN-beta and the ectodomain of the TGF-beta receptor II potentiates antitumor immunity. Oncotarget. 2014;5(20):10100-13. Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, et al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell. 2016;165(1):35-44. Ribas A, Dummer R, Puzanov I, VanderWalde A, Andtbacka RHI, Michielin O, et al. Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy. Cell. 2017;170(6):1109-19 e10. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515(7528):568-71. Hackl H, Charoentong P, Finotello F, Trajanoski Z. Computational genomics tools for dissecting tumour-immune cell interactions. Nat Rev Genet. 2016;17(8):441-58. Krysko O, Love Aaes T, Bachert C, Vandenabeele P, Krysko DV. Many faces of DAMPs in cancer therapy. Cell Death Dis. 2013;4:e631. Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P. Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer. 2012;12(12):860-75. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007;13(1):54-61. Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, Marysael T, et al. A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J. 2012;31(5):1062-79. Galluzzi L, Kepp O, Kroemer G. Enlightening the impact of immunogenic cell death in photodynamic cancer therapy. EMBO J. 2012;31(5):1055-7. Galluzzi L, Kepp O, Kroemer G. Immunogenic cell death in radiation therapy. Oncoimmunology. 2013;2(10):e26536. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy. Annu Rev Immunol. 2013;31:51-72. Yatim N, Jusforgues-Saklani H, Orozco S, Schulz O, Barreira da Silva R, Reis e Sousa C, et al. RIPK1 and NF-kappaB signaling in dying cells determines cross-priming of CD8(+) T cells. Science. 2015;350(6258):328-34. Aaes TL, Kaczmarek A, Delvaeye T, De Craene B, De Koker S, Heyndrickx L, et al. Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity. Cell Rep. 2016;15(2):274-87. Stewart MP, Langer R, Jensen KF. Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts. Chem Rev. 2018;118(16):7409-531. Xiong R, Joris F, Liang S, De Rycke R, Lippens S, Demeester J, et al. Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging. Nano Lett. 2016;16(10):5975-86. Xiong R, Raemdonck K, Peynshaert K, Lentacker I, De Cock I, Demeester J, et al. Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells. ACS Nano. 2014;8(6):6288-96. L. Raes CVH, J. Michiels, S. Stremersch, J.C. Fraire, T. Brans, R. Xiong, S. De Smedt, L. Vandekerckhove, K. Raemdonck, K. Braeckmans,. Gold Nanoparticle-Mediated Photoporation Enables Delivery of Macromolecules over a Wide Range of Molecular Weights in Human CD4+ T Cells. Crystals. 2019;9. Wayteck L, Xiong R, Braeckmans K, De Smedt SC, Raemdonck K. Comparing photoporation and nucleofection for delivery of small interfering RNA to cytotoxic T cells. J Control Release. 2017;267:154-62. Xiong R, Verstraelen P, Demeester J, Skirtach AG, Timmermans JP, De Smedt SC, et al. Selective Labeling of Individual Neurons in Dense Cultured Networks With Nanoparticle-Enhanced Photoporation. Front Cell Neurosci. 2018;12:80.

Round 2

Reviewer 1 Report

The comments addressed are good.