Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

  1. Alba Nicolas-Boluda
  2. Javier Vaquero
  3. Lene Vimeux
  4. Thomas Guilbert
  5. Sarah Barrin
  6. Chahrazade Kantari-Mimoun
  7. Matteo Ponzo
  8. Gilles Renault
  9. Piotr Deptula
  10. Katarzyna Pogoda
  11. Robert Bucki
  12. Ilaria Cascone
  13. José Courty
  14. Laura Fouassier
  15. Florence Gazeau
  16. Emmanuel Donnadieu

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Abstract

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

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  1. Version published to 10.7554/elife.58688 on eLife
  2. eLife

    ###Reviewer #3

    The focus of the manuscript by Nicolas-Boluda et al. is timely as it has been shown by this team and by others that dense collagen fibers and other features of the matrix architecture surrounding tumors may form a barrier for T cell infiltration into solid tumors. Despite the authors' claims, however, the data in this manuscript fall short of definitively demonstrating that response to anti-PD-1 therapy and T cell migration into tumors is improved upon reduction of collagen cross-linking. I have a number of concerns that would require additional substantive experiments to be adequately addressed. Below I list major and minor points that should be addressed before further consideration for publications.

    Major points:

    1. BAPN is used as a covalent inhibitor of LOX activity however the authors provide no evidence that the drug is having the expected effects in vivo. In order to draw specific conclusions about these studies the authors would need to provide measurements of collagen cross-links (DHLNL, PYP, DPD).

    2. Imbalance between the mechanical characterization of multiple tumor models with little space for defining the effect of tumor stiffness on anti-PD-1 efficacy and T cell distribution, motility and activation.

    3. Rationale for selected tumor models relative to human tumors was unclear.

    4. Sample sizes, # independent experiments and statistical analyses were inadequate across multiple figures.

    5. Measurements of stiffness, collagen structure and T cell speed should be provided for all treatment conditions (control, LOXi, PD1i and combo) rather than just for LOX inhibition.

    6. Lox inhibition was performed in a preventive setting. Do the authors think LOX inhibition would be as effective in changing tumor stiffness and matrix architecture if the treatment started at the same time point as anti-PD-1?

    7. In Figure 1 the correlation of tissue stiffness/collagen accumulation with tumor volume in clinical samples should be provided in order to attribute collagen cross-linking to tumor progression.

    8. The efficacy data in Figure 6 should be accompanied by survival data.

  3. eLife

    ###Reviewer #2

    In this manuscript, the authors provide a thorough analysis of the ECM architecture and stiffness in 4 murine tumor models. They then attempt to correlate ECM architecture and mechanics with T-cell migration and PD-1 efficacy. Substantive concerns are as follows:

    1. The study is highly correlative with inadequate sample size to be conclusive. The authors attempts to draw conclusions about when stiffness does and doesn't affect migration by attempting to interpret data across 4 very different tumor types. In two tumors the migration changes with BAPN and with 2 it does not. It is not possible to draw a conclusion based on 2 points.

    2. Data regarding the relationship between collagen organization and stiffness has been reported previously (as cited by the authors).

    3. Sirius Red staining is referred to and described in the text but no images are shown. Likewise, no SWE images are provided to show the relative heterogeneity described in the text. This is important since so much of the conclusions rests on this data.

    4. The results section discussing figure 1 emphasizes heterogeneity in stiffness, however none of the data shown depict spatial stiffness heterogeneities.

    5. The rationale for the choice of cancer models is not clear.

    6. Why is mPDAC measured and reported differently in figure 2A than the other tumor types?

    7. Why is 40kPa chosen as the cut-off for "stiff?"

    8. Mean-squared displacement is the more appropriate metric to describe cell path (and more conventional) rather than "straightness"

    9. How many cells were studied for each parameter in each condition in Table 2?

    10. The authors study migration of cells on slices, but isn't the more appropriate metric to study cell invasion into the tissue?

  4. eLife

    ###Reviewer #1

    In their article entitled "Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment" Alba Nicolas-Boluda and co-authors analyze the stiffness and collagen distribution in different tumor models implanted in mice. They show that treatment with an inhibitor of collagen crosslinking modifies the collagen network in these tumors and that this correlates with changes in their stiffness. They then analyze the motility of T cells in the different models and show that this motility is modified by the treatment and correlates with the stiffness of the tumor. In the last part of their study, the authors show that treatment of the mice with the inhibitor of collagen crosslinking changes the immune infiltrates in the tumors characterized by a more abundant presence of CD8+ T cells. They finally show that interfering with collagen stabilization leads to increased efficacy of anti-PD-1 blockade on tumor growth.

    Relevance of the study: T cells are excluded from a large proportion of solid tumor. This represents an obstacle to T-cell-based immunotherapies. The authors make the hypothesis that this can be, at least partly, due to the organization of the ECM in the tumor that would oppose physical resistance to the infiltration and migration of T cells. The results are sound and important for the community since 1) they describe thoroughly some of the mechanical aspects of several models used in the literature, 2) they thoroughly analyzed the effect of an inhibitor of collagen crosslinking on these mechanical properties 3) study the effects of these modifications in T cell motility and 4) test in one tumor model the effects of the combination of an inhibitor of collagen crosslinking with anti-PD1 immunotherapy. The results are convincing and I only have minor concerns.

    In the first part of their study, the authors analyze the structure heterogeneity of 5 different carcinomas, i.e. subcutaneous model of cholangiocarcinoma (EGI-1), subcutaneous (MET-1) and transgenic model (MMTV-PyMT) of mouse breast carcinoma, orthotopic (mPDAC) and subcutaneous (KPC) models of mouse pancreatic ductal adenocarcinoma.

    They measure the tumor stiffness during tumor growth using Shear Wave Elastography (SWE) and analyze the organization of the collagen fibers in these models. To my knowledge, this represents the first characterization of different tumor models classically used to study tumor immunity and is thus very useful for the scientific community. In particular, the authors show a correlation between high tumor stiffness and accumulation of thick and densely packed collagen fibers.

    Minor modifications: The authors should indicate more clearly the number of mice and tumors investigated.

    In the second part of their study, the authors treat the mice with beta-aminopropionitrile (BAPN), an inhibitor for LOX enzymatic activity in the drinking water and analyze the stiffness of tumors and collagen fiber organization in tumors. They show the heterogeneity of response in the different models in both stiffness modulation and collagen fibers remodeling. Mostly this treatment reduces the stiffness of tumors without affecting their growth.

    Minor modifications: The authors should clarify how "normalized tumor stiffness" indicated in the legend of figure 2 is calculated. Indeed, this is an important point since tumor stiffness is associated to the sizes of tumors. Moreover, they should also indicate more clearly the number of mice and tumors investigated. Concerning collagen fibers orientation, authors should use a dot plot representation instead of bar histograms in order to show the distribution in the different tumors.

    The authors then analyze how BAPN treatment modifies the migration of T lymphocytes in the tumors. Because of the different models used, the authors either added activated purified T cells from human donors (EGI-1model), or mouse activated T cells (MMTV-PyMT tumor model) or followed the motility of human resident T cells in mPDAC and KPC mice tumor models. Although the models are very different, the correlation between tumor stiffness and T cell speed and T cell displacement is specially striking in tumors from BAPN treated mice. It seems that T cell motility responds to two different regimens in tumor from untreated or BAPN treated mice. This might be due to difference of stiffness in untreated and treated mice but might also results from another parameter.

    Minor modifications: The authors should discuss this point. Indeed, the main conclusion of their work and short title of their study is that the main parameter involved in T cell motility and access to the tumor is tumor stiffness but then the slopes should be the same as in the spontaneous MMTV-PyMT tumor model. There are probably other parameters involved in the regulation.

    The authors then investigate the effect of BAPN treatment of tumor bearing mice on response to PD-1 immunotherapy. They perform experiments in KPC tumor bearing mice and show that BAPN treatment alone significantly decreases the number of neutrophils, increases the presence of MHCII+ TAMs. Yet, the combined therapy (BAPN and PD-1) is necessary to expand the percentage of GrzmB CD8+ T cells and the ratio of CD8+ to Treg cells and is associated with an increase in cytokine production. The combined treatment also leads to a decrease in the tumor sizes. Although these results are convincing as they are, confirmation of the results in another model would strengthen the results.

  5. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on July 12 2020, follows.

    Summary

    The work analyzes the stiffness and collagen distribution in different tumor models implanted in mice and shows that treatment with an inhibitor of collagen crosslinking correlates with changes in their stiffness. This results in a change in the motility of resident T cells. The inhibitor of collagen crosslinking increases the number of tumor-infiltrating CD8+ T cells and leads to increased efficacy of anti-PD-1 blockade on tumor growth. The reviewers have discussed the reviews with one another and the Reviewing Editor and their views concur. Although the work has potential for publication in eLife, it requires essential additional data and statistics to support the central claims of the paper. Each reviewer raised substantive concerns (see below) that need to be resolved experimentally. To quote a few, you should provide a measurement of the collagen crosslinking in mice treated by BAPN to confirm that this drug has the expected effects. The combined BAPN plus anti-PD-1 therapy needs also to be confirmed in another model. Measurements of stiffness, collagen structure and T cell speed should be provided for all treatment conditions (control, LOXi, PD1i and combo) rather than just for LOX inhibition. Importantly, several important conclusions are based on inadequate sample size to be conclusive (see below). Along that line, the number of mice and tumor cells plus corresponding statistics need to be indicated in all the figures.

  6. Version published to 10.1101/2020.05.19.104430v1 on bioRxiv