# LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer

Joanne E Anstee, Karen T Feehan, James W Opzoomer, Isaac Dean, Henrike P Muller, Meriem Bahri, Tik Shing Cheung, Kifayathullah Liakath-Ali, Ziyan Liu, Desmond Choy, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Joanne E Anstee, Karen T Feehan, James W Opzoomer, Isaac Dean, Henrike P Muller, et al. LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer. Developmental Cell, 2023, 58 (17), pp.1548-1561.e10. 10.1016/j.devcel.2023.06.006. hal-04534358

HAL Id: hal-04534358

https://amu.hal.science/hal-04534358

Submitted on 5 Apr 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

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<figure><img src="image_2.png" /></figure>
# LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer

Joanne E Anstee, Karen T Feehan, James W Opzoomer, Isaac Dean, Henrike P Muller, Meriem Bahri, Tik Shing Cheung, Kifayathullah Liakath-Ali, Ziyan Liu, Desmond Choy, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Joanne E Anstee, Karen T Feehan, James W Opzoomer, Isaac Dean, Henrike P Muller, et al. LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer. Developmental Cell, 2023, 58 (17), pp.1548-1561.e10. 10.1016/j.devcel.2023.06.006. hal-04534358

HAL Id: hal-04534358

https://amu.hal.science/hal-04534358v1

Submitted on 5 Apr 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
# Developmental Cell

## LYVE-1⁺ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer

**Graphical abstract**

<figure><img src="image_3.png" /></figure>

**Authors**

Joanne E. Anstee, Karen T. Feehan,
James W. Opzoomer, ...,
David R. Withers, Toby Lawrence,
James N. Arnold

**Correspondence**

james.arnold@kcl.ac.uk

**In brief**

Lymphatic vessel endothelial hyaluronan receptor-1⁺ (LYVE-1⁺) tumor-associated macrophages (TAMs) form coordinated multi-cellular “nests” proximal to blood vasculature in cancer. In a murine breast cancer model, Anstee et al. show that LYVE-1⁺ TAMs develop in response to IL-6, promoting a CCR5-dependent signaling axis that guides their formation into a collaborative niche.

**Highlights**

* LYVE-1⁺ TAMs co-express HO-1 and form multi-cellular nests proximal to blood vessels

* LYVE-1⁺ TAMs develop in response to IL-6 within the tumor

* CCR5 drives LYVE-1⁺ TAMs' cross-communication to form dynamic nest arrangements

* The LYVE-1⁺ TAM niche facilitates CD8⁺ T cell exclusion and resistance to chemotherapy

<figure><img src="image_4.png" /></figure>
# Developmental Cell

## LYVE-1⁺ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer

### Graphical abstract

<figure><img src="image_3.png" /></figure>

### Authors

Joanne E. Anstee, Karen T. Feehan, James W. Opzoomer, ..., David R. Withers, Toby Lawrence, James N. Arnold

### Correspondence

james.arnold@kcl.ac.uk

### In brief

Lymphatic vessel endothelial hyaluronan receptor-1⁺ (LYVE-1⁺) tumor-associated macrophages (TAMs) form coordinated multi-cellular “nests” proximal to blood vasculature in cancer. In a murine breast cancer model, Anstee et al. show that LYVE-1⁺ TAMs develop in response to IL-6, promoting a CCR5-dependent signaling axis that guides their formation into a collaborative niche.

### Highlights

* LYVE-1⁺ TAMs co-express HO-1 and form multi-cellular nests proximal to blood vessels

* LYVE-1⁺ TAMs develop in response to IL-6 within the tumor

* CCR5 drives LYVE-1⁺ TAMs' cross-communication to form dynamic nest arrangements

* The LYVE-1⁺ TAM niche facilitates CD8⁺ T cell exclusion and resistance to chemotherapy

<figure><img src="image_4.png" /></figure>
## Article

# LYVE-1⁺ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer

Joanne E. Anstee,<fnref n="1" /> Karen T. Feehan,<fnref n="1" /> James W. Opzoomer,<fnref n="1" />,<fnref n="10" /> Isaac Dean,<fnref n="2" /> Henrike P. Muller,<fnref n="1" /> Meriem Bahri,<fnref n="1" /> Tik Shing Cheung,<fnref n="1" /> Kifayathullah Liakath-Ali,<fnref n="3" /> Ziyan Liu,<fnref n="1" /> Desmond Choy,<fnref n="1" /> Jonathan Caron,<fnref n="1" /> Dominika Sosnowska,<fnref n="1" /> Richard Beatson,<fnref n="1" />,<fnref n="11" /> Tamara Muliaditan,<fnref n="1" />,<fnref n="12" /> Zhengwen An,<fnref n="1" />,<fnref n="13" /> Cheryl E. Gillett,<fnref n="1" /> Guocheng Lan,<fnref n="4" />,<fnref n="14" /> Xiangang Zou,<fnref n="4" />,<fnref n="15" /> Fiona M. Watt,<fnref n="3" /> Tony Ng,<fnref n="1" />,<fnref n="5" /> Joy M. Burchell,<fnref n="1" /> Shahram Kordasti,<fnref n="1" />,<fnref n="6" /> David R. Withers,<fnref n="2" /> Toby Lawrence,<fnref n="7" />,<fnref n="8" />,<fnref n="9" /> and James N. Arnold<fnref n="1" />,<fnref n="16" />,*

## SUMMARY

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular “nest” structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1⁺ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1⁺ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8⁺ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.

## INTRODUCTION

Macrophages are a phenotypically and functionally diverse population of innate immune cells, which become exploited by tumors to facilitate disease progression and therapy resistance.<fnref n="1" />⁻<fnref n="8" /> Heterogeneity within the tumor-associated macrophage (TAM) population arises from the site of TAM origin<fnref n="9" /> and the influence of environmental cues within the tumor microenvironment (TME),<fnref n="3" />,<fnref n="10" />,<fnref n="11" /> which can be guided by both spatial<fnref n="12" />,<fnref n="13" /> and tem-

poral<fnref n="11" /> parameters. This results in the specialization of TAM subsets for executing specific functionality within the TME, which also extends to the co-existence of both pro- and anti-tumoral TAM subsets within the same TME.<fnref n="14" />,<fnref n="15" /> As such, it is vital to understand the respective functionality of TAM subsets and their niches, as optimal TAM-targeted therapeutic strategies could involve the targeting of pro-tumoral TAMs while concurrently preserving and enhancing their anti-tumoral TAM polarization states.

<figure><img src="image_5.png" /></figure>

<footnote n="1">¹School of Cancer and Pharmaceutical Sciences, King's College London, London SE1 1UL, UK</footnote>

<footnote n="2">²Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK</footnote>

<footnote n="3">³Centre for Stem Cells and Regenerative Medicine, King's College London, London SE1 9RT, UK</footnote>

<footnote n="4">⁴Cancer Research UK Cambridge Research Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 ORE, UK</footnote>

<footnote n="5">⁵UCL Cancer Institute, University College London, London WC1E 6DD, UK</footnote>

<footnote n="6">⁶Haematology Department, Guy's Hospital, London SE1 9RT, UK</footnote>

<footnote n="7">⁷Centre for Inflammation Biology and Cancer Immunology, School of Immunology & Microbial Sciences, King's College London, London SE1 1UL, UK</footnote>

<footnote n="8">⁸Aix Marseille University, CNRS, INSERM, CIML, Marseille, France</footnote>

<footnote n="9">⁹Henan Key Laboratory of Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China</footnote>

<footnote n="10">¹⁰Present address: UCL Cancer Institute, University College London, London WC1E 6DD, UK</footnote>

<footnote n="11">¹¹Present address: Respiratory Medicine, University College London, London WC1E 6JF, UK</footnote>

<footnote n="12">¹²Present address: Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, the Netherlands</footnote>

<footnote n="13">¹³Present address: Central Laboratory of Oral Biomedical Research, Dental Institute, Jilin University, Changchun, China</footnote>

<footnote n="14">¹⁴Present address: Stem Cell and Regenerative Consortium Centre, LKS Faculty of Medicine, The School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China</footnote>

<footnote n="15">¹⁵Present address: Chongqing Academy of Animal Science, Rongchang, Chongqing, China</footnote>

<footnote n="16">¹⁶Lead contact</footnote>

<footnote>*Correspondence: james.arnold@kcl.ac.uk</footnote>

<footnote>https://doi.org/10.1016/j.devcel.2023.06.006</footnote>
## Article

# LYVE-1⁺ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer

Joanne E. Anstee,<fnref n="1" /> Karen T. Feehan,<fnref n="1" /> James W. Opzoomer,<fnref n="1" />,<fnref n="10" /> Isaac Dean,<fnref n="2" /> Henrike P. Muller,<fnref n="1" /> Meriem Bahri,<fnref n="1" /> Tik Shing Cheung,<fnref n="1" /> Kifayathullah Liakath-Ali,<fnref n="3" /> Ziyan Liu,<fnref n="1" /> Desmond Choy,<fnref n="1" /> Jonathan Caron,<fnref n="1" /> Dominika Sosnowska,<fnref n="1" /> Richard Beatson,<fnref n="1" />,<fnref n="11" /> Tamara Muliaditan,<fnref n="1" />,<fnref n="12" /> Zhengwen An,<fnref n="1" />,<fnref n="13" /> Cheryl E. Gillett,<fnref n="1" /> Guocheng Lan,<fnref n="4" />,<fnref n="14" /> Xiangang Zou,<fnref n="4" />,<fnref n="15" /> Fiona M. Watt,<fnref n="3" /> Tony Ng,<fnref n="1" />,<fnref n="5" /> Joy M. Burchell,<fnref n="1" /> Shahram Kordasti,<fnref n="1" />,<fnref n="6" /> David R. Withers,<fnref n="2" /> Toby Lawrence,<fnref n="7" />,<fnref n="8" />,<fnref n="9" /> and James N. Arnold<fnref n="1" />,<fnref n="16" />,*

## SUMMARY

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular “nest” structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1⁺ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1⁺ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8⁺ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.

## INTRODUCTION

Macrophages are a phenotypically and functionally diverse population of innate immune cells, which become exploited by tumors to facilitate disease progression and therapy resistance.<fnref n="1" />⁻<fnref n="8" /> Heterogeneity within the tumor-associated macrophage (TAM) population arises from the site of TAM origin<fnref n="9" /> and the influence of environmental cues within the tumor microenvironment (TME),<fnref n="3" />,<fnref n="10" />,<fnref n="11" /> which can be guided by both spatial<fnref n="12" />,<fnref n="13" /> and tem-

poral<fnref n="11" /> parameters. This results in the specialization of TAM subsets for executing specific functionality within the TME, which also extends to the co-existence of both pro- and anti-tumoral TAM subsets within the same TME.<fnref n="14" />,<fnref n="15" /> As such, it is vital to understand the respective functionality of TAM subsets and their niches, as optimal TAM-targeted therapeutic strategies could involve the targeting of pro-tumoral TAMs while concurrently preserving and enhancing their anti-tumoral TAM polarization states.

<figure><img src="image_5.png" /></figure>

<footnote n="1">¹School of Cancer and Pharmaceutical Sciences, King's College London, London SE1 1UL, UK</footnote>

<footnote n="2">²Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK</footnote>

<footnote n="3">³Centre for Stem Cells and Regenerative Medicine, King's College London, London SE1 9RT, UK</footnote>

<footnote n="4">⁴Cancer Research UK Cambridge Research Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 ORE, UK</footnote>

<footnote n="5">⁵UCL Cancer Institute, University College London, London WC1E 6DD, UK</footnote>

<footnote n="6">⁶Haematology Department, Guy's Hospital, London SE1 9RT, UK</footnote>

<footnote n="7">⁷Centre for Inflammation Biology and Cancer Immunology, School of Immunology & Microbial Sciences, King's College London, London SE1 1UL, UK</footnote>

<footnote n="8">⁸Aix Marseille University, CNRS, INSERM, CIML, Marseille, France</footnote>

<footnote n="9">⁹Henan Key Laboratory of Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China</footnote>

<footnote n="10">¹⁰Present address: UCL Cancer Institute, University College London, London WC1E 6DD, UK</footnote>

<footnote n="11">¹¹Present address: Respiratory Medicine, University College London, London WC1E 6JF, UK</footnote>

<footnote n="12">¹²Present address: Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, the Netherlands</footnote>

<footnote n="13">¹³Present address: Central Laboratory of Oral Biomedical Research, Dental Institute, Jilin University, Changchun, China</footnote>

<footnote n="14">¹⁴Present address: Stem Cell and Regenerative Consortium Centre, LKS Faculty of Medicine, The School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China</footnote>

<footnote n="15">¹⁵Present address: Chongqing Academy of Animal Science, Rongchang, Chongqing, China</footnote>

<footnote n="16">¹⁶Lead contact</footnote>

<footnote>*Correspondence: james.arnold@kcl.ac.uk</footnote>

<footnote>https://doi.org/10.1016/j.devcel.2023.06.006</footnote>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 1. LYVE-1<sup>+</sup> macrophages co-express HO-1 and reside in nests within the Pv niche**</figcaption></figure>

(A) Representative image of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei;blue) and antibodies against F4/80 (magenta) and LYVE-1 (red). Functional vasculature was labeled *in vivo* using intravenous (i.v.) dextran-fluorescein isothiocyanate (FITC). Colocalizing pixels for F4/80 and LYVE-1 are shown in white. Scale bars: 25 µm.

(B and C) Uniform manifold approximation and projection (UMAP) plot of the scRNA-seq of F4/80<sup>+</sup> TAMs from n = 3 *MMTV-PyMT* tumors colored by their associated cluster identity, re-analyzed from Opzoomer et al.<sup>14</sup> (B). Violin plot of Lyve1 expression associated with the TAM cluster identity seen in (C).

(D–F) UMAP plot of scRNA-seq data from a publicly available human breast cancer dataset (gray)<sup>28</sup> identifying cells (red) that were deemed to display a tran- scriptional phenotype similar to that of the murine LYVE-1<sup>+</sup> TAM population,<sup>14</sup> in the full atlas (D) and within the myeloid cluster within the atlas (E). UMAP vi- sualizations of CD68 and selected genes (*LYVE1*, *MRC1*, and *HMOX1*) (F) that were preferentially expressed (p < 2.22 × 10<sup>-16</sup>) within the LYVE-1<sup>+</sup>-like TAM subset compared with other myeloid cells from the human breast cancer dataset.<sup>28</sup>

(G) Representative image of a frozen section of human invasive ductal carcinoma stained with DAPI (nuclei;blue) and antibodies against CD68 (magenta), HO-1 (red), and CD31 (green). Colocalizing pixels for F4/80 and HO-1 are shown in white. Scale bars: 50 µm.

(H) Representative image of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei;blue) and antibodies against F4/80 (magenta), LYVE-1 (yellow), and HO-1 (red). Functional vasculature was labeled *in vivo* using i.v. dextran-FITC. Colocalizing pixels for LYVE-1 and HO-1 are shown in white. Scale bars: 25 µm.

(I) Schematic depicting the cells in the Pv niche (left panel) and their relative expression of *Hmox1* and *Hmox2* from bulk RNA-seq (right panel).

(J) Representative image of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei;blue) and antibodies against F4/80 (magenta), HO-1 (red), and αSMA (green). Colocalizing pixels for F4/80 and HO-1 are shown in white. Scale bars: 25 µm.

(K–N) Schematic depicting the transgene of the HO-1-Luc-EGFP knockin mouse (HO-1<sup>Luc/EGFP</sup>) (lower panel) and the cross that is used in the subsequent tumor studies (upper panel) (K). Western blot of HO-1 and EGFP from BMDMs treated with and without *Hmox1* knockdown (KD) siRNA (n = 3 repeats) (L). Representative
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 1.** LYVE-1⁺ macrophages co-express HO-1 and reside in nests within the Pv niche</figcaption></figure>

(A) Representative image of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei; blue) and antibodies against F4/80 (magenta) and LYVE-1 (red). Functional vasculature was labeled *in vivo* using intravenous (i.v.) dextran-fluorescein isothiocyanate (FITC). Colocalizing pixels for F4/80 and LYVE-1 are shown in white. Scale bars: 25 µm.

(B and C) Uniform manifold approximation and projection (UMAP) plot of the scRNA-seq of F4/80⁺ TAMs from n = 3 *MMTV-PyMT* tumors colored by their associated cluster identity, re-analyzed from Opzoomer et al.¹⁴ (B). Violin plot of Lyve1 expression associated with the TAM cluster identity seen in (C).

(D–F) UMAP plot of scRNA-seq data from a publicly available human breast cancer dataset (gray)²⁸ identifying cells (red) that were deemed to display a tran- scriptional phenotype similar to that of the murine LYVE-1⁺ TAM population,¹⁴ in the full atlas (D) and within the myeloid cluster within the atlas (E). UMAP vi- sualizations of CD68 and selected genes (*LYVE1*, *MRC1*, and *HMOX1*) (F) that were preferentially expressed (p < 2.22 × 10⁻¹⁶) within the LYVE-1⁺-like TAM subset compared with other myeloid cells from the human breast cancer dataset.²⁸

(G) Representative image of a frozen section of human invasive ductal carcinoma stained with DAPI (nuclei; blue) and antibodies against CD68 (magenta), HO-1 (red), and CD31 (green). Colocalizing pixels for F4/80 and HO-1 are shown in white. Scale bars: 50 µm.

(H) Representative image of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei; blue) and antibodies against F4/80 (magenta), LYVE-1 (yellow), and HO-1 (red). Functional vasculature was labeled *in vivo* using i.v. dextran-FITC. Colocalizing pixels for LYVE-1 and HO-1 are shown in white. Scale bars: 25 µm.

(I) Schematic depicting the cells in the Pv niche (left panel) and their relative expression of *Hmox1* and *Hmox2* from bulk RNA-seq (right panel).

(J) Representative image of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei; blue) and antibodies against F4/80 (magenta), HO-1 (red), and αSMA (green). Colocalizing pixels for F4/80 and HO-1 are shown in white. Scale bars: 25 µm.

(K–N) Schematic depicting the transgene of the HO-1-Luc-EGFP knockin mouse (HO-1<sup>Luc/EGFP</sup>) (lower panel) and the cross that is used in the subsequent tumor studies (upper panel) (K). Western blot of HO-1 and EGFP from BMDMs treated with and without *Hmox1* knockdown (KD) siRNA (n = 3 repeats) (L). Representative
A collection of TAMs that reside proximal to blood vasculature (<15–20 µm) have emerged as a therapeutic target in cancer,¹⁶,¹⁷ as they support a variety of pro-tumoral functions, including neo-angiogenesis and¹⁴,¹⁷ metastasis,³,¹⁸,¹⁹ and facilitate tumor recurrence post chemotherapy.²⁰ However, they can also elicit immune-stimulatory functions,¹⁵ highlighting the need to further investigate the population and its heterogeneity. We recently characterized a subset of perivascular (Pv) TAMs expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1+) MHCII$^{lo}$CD206$^{hi}$, which orchestrated the expansion of pericyte-like α-smooth muscle actin (α-SMA)-expressing fibroblasts.¹⁴ LYVE-1+ macrophages have been demonstrated to populate a variety of healthy tissues in steady-state conditions²¹⁻²⁵ and accumulate in the TME,¹⁴,¹⁵,²⁶,²⁷ however, little is known about their development and function in cancer.¹⁴,¹⁵ In this study, we investigate the development of LYVE-1+ TAMs and their Pv niche in *MMTV-PyMT* tumors and define a collaborative function of these cells in forming immune-suppressive multi-cellular “nests,” which can influence the response of the tumor to chemotherapy. This study sheds light on the importance of the spatial arrangements of TAMs and their communication axes, which facilitate their pro-tumoral function in cancer.

## RESULTS

### LYVE-1+ TAMs accumulate in Pv nests within the TME

The LYVE-1+ TAM subset resides in the Pv niche of the TME (Figure 1A). We recently identified LYVE-1+ TAMs from the single-cell RNA sequencing (scRNA-seq) of the F4/80+ TAM population in the *MMTV-PyMT* murine model of breast cancer.¹⁴ To initially investigate whether an analogous TAM population exists in human cancer, we extracted the LYVE-1+ TAM cell cluster as previously described¹⁴ from a scRNA-seq dataset of 9,039 TAMs sorted from 3 individual *MMTV-PyMT* tumors (Figures 1B and 1C). We then mapped the murine LYVE-1+ population onto a recently published scRNA-seq atlas for human breast cancer.²⁸ The murine LYVE-1+ TAM population identified with cells within the human myeloid cell cluster (Figure 1D; Table S1). Focusing on the myeloid cells within the atlas, 1,444 of the 9,675 myeloid cells were judged to be LYVE-1+ TAM like in their phenotype (Figures 1E and 1F) and their expression of *LYVE1*, *MRC1* (CD206), and *HMOX1* (HO-1) were significantly associated with the identified cells (Figure 1F), demonstrating a conservation of this TAM subset between species. HO-1 is a marker that has previously been associated with PvTAMs in murine models of cancer,³,²⁰ and the HO-1 expressing TAM population was also observed in the Pv niche of human cancer (Figure 1G), highlighting a similar spatial location for these cells between species. HO-1 and LYVE-1 protein co-localized in tumor sections from *MMTV-PyMT* mice (Figure 1H), and LYVE-1+ TAMs were the major source of HO-1 within the Pv niche, which also includes, endothelial cells (ECs) and pericyte-like αSMA-expressing

cancer-associated fibroblasts (CAFs) (Figures 1I and 1J).¹⁴ Interestingly, from these analyses, it was evident that the LYVE-1+HO-1+ PvTAM subset was heterogeneously distributed along the endothelium and clustered to discrete regions where these cells were either lining or appearing in bunches proximal to the vasculature (Figures 1A, 1G, 1H, 1J, and S1A). These clusters, which we herein refer to as nests, highlighted an unappreciated multi-cellular PvTAM structure within the TME. As LYVE-1 is also expressed at high levels by the lymphatic endothelium,²⁹ and HO-1 expression is selectively expressed by this TAM subset (Figures 1H–1J), we generated a knockin reporter mouse for the *Hmox1* gene to facilitate the study of these cells (Figure 1K). The reporter consisted of Click Beetle luciferase (Luc) and enhanced green fluorescent protein (EGFP)³⁰ inserted before the stop codon of the genomic *Hmox1* gene locus. HO-1, Luc, and EGFP were separated by a self-cleaving P2A sequence³¹ to allow the equimolar expression of the three proteins (Figures 1K, 1L, and S1B) (mouse herein referred to as HO-1$^{Luc/EGFP}$). As HO-1 plays several important functional roles in the TME,³,³²⁻³⁴ the arrangement of the reporter construct ensured that native HO-1 expression was unaffected by the reporter elements. In HO-1$^{Luc/EGFP}$ mice, HO-1 expression could be detected in healthy tissues using bioluminescence imaging (Figure S1C). Analysis of healthy tissues from the HO-1$^{Luc/EGFP}$ mouse revealed the spleen, lung, mammary gland, visceral adipose, skin, and liver to be among the highest expressing tissues for HO-1 (Figure S1D), where its expression was restricted to F4/80$^{hi}$-tissue-resident macrophages (Figure S1E). The HO-1$^{Luc/EGFP}$ mouse was crossed onto the *MMTV-PyMT* background, and tumors were analyzed for their distribution of HO-1/EGFP expression (Figure 1K). TAMs (Figure S1F), specifically the LYVE-1+CD206$^{hi}$MHCII$^{lo}$ TAM subset (Figures 1M and S1G), were the major tumoral source of HO-1/EGFP. Further characterization of F4/80$^{hi}$HO-1/EGFP+ cells in tumors by immunofluorescence analysis also confirmed the localization of HO-1/EGFP to the Pv space within nest structures (Figure 1N). These data validate the HO-1$^{Luc/EGFP}$-reporter mouse as a tool for studying LYVE-1+ PvTAMs and identify a previously unappreciated multi-cellular nest structure for these cells in the Pv space.

### LYVE-1+ TAMs develop in the TME in response to IL-6

To investigate the development of LYVE-1+ TAMs within TME, we first considered whether they were a recruited or tissue-resident TAM population.⁹ To address this question, we utilized the photoconvertible *Kaede* mouse³⁵ crossed to the *MMTV-PyMT* model. When tumors reached 100 mm³, the entire TME was photoconverted from *Kaede*-green to *Kaede*-red using a UV-light source (Figure 2A). After 48 h post photoconversion of the TME, there was a clear *Kaede*-green (-red-negative) population of LYVE-1+ TAMs in the TME, indicative of their development from a recruited progenitor cell originating from outside the TME (Figure 2B). Although it is possible that highly replicating

<footnote>contour plot of fluorescence-activated cell sorting (FACs)-gated live (7AAD⁻) CD45⁺F4/80⁺ TAMs from enzyme-dispersed *MMTV-PyMT* HO-1*Luc/EGFP* tumors separated based on their respective expression of CD206 and MHCII (left panel) and then assessed for their expression of HO-1 and LYVE-1 (right panel; colored histograms) against that of the FMO staining control (gray histogram) (M). Representative of n = 10 mice. Representative image of a frozen section and quantification of a *MMTV-PyMT* HO-1*Luc/EGFP* tumor stained with antibodies against CD31 (red) are shown, and native EGFP fluorescence is shown in green (N). Scale bars: 25 µm (left panel). Proportion of HO-1/EGFP⁺ cells that are Pv (<20 µm from live vasculature) across n = 4 *MMTV-PyMT* tumors (right panel). Images in (I) and (K) were created using *BioRender* software. Bar charts represent the mean, and the dots show individual data points from individual tumors and mice. \* p < 0.05, \*\* p < 0.01.</footnote>
A collection of TAMs that reside proximal to blood vasculature (<15–20 µm) have emerged as a therapeutic target in cancer,¹⁶,¹⁷ as they support a variety of pro-tumoral functions, including neo-angiogenesis and¹⁴,¹⁷ metastasis,³,¹⁸,¹⁹ and facilitate tumor recurrence post chemotherapy.²⁰ However, they can also elicit immune-stimulatory functions,¹⁵ highlighting the need to further investigate the population and its heterogeneity. We recently characterized a subset of perivascular (Pv) TAMs expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1+) MHCII$^{lo}$CD206$^{hi}$, which orchestrated the expansion of pericyte-like α-smooth muscle actin (α-SMA)-expressing fibroblasts.¹⁴ LYVE-1+ macrophages have been demonstrated to populate a variety of healthy tissues in steady-state conditions²¹⁻²⁵ and accumulate in the TME,¹⁴,¹⁵,²⁶,²⁷ however, little is known about their development and function in cancer.¹⁴,¹⁵ In this study, we investigate the development of LYVE-1+ TAMs and their Pv niche in *MMTV-PyMT* tumors and define a collaborative function of these cells in forming immune-suppressive multi-cellular “nests,” which can influence the response of the tumor to chemotherapy. This study sheds light on the importance of the spatial arrangements of TAMs and their communication axes, which facilitate their pro-tumoral function in cancer.

## RESULTS

### LYVE-1+ TAMs accumulate in Pv nests within the TME

The LYVE-1+ TAM subset resides in the Pv niche of the TME (Figure 1A). We recently identified LYVE-1+ TAMs from the single-cell RNA sequencing (scRNA-seq) of the F4/80+ TAM population in the *MMTV-PyMT* murine model of breast cancer.¹⁴ To initially investigate whether an analogous TAM population exists in human cancer, we extracted the LYVE-1+ TAM cell cluster as previously described¹⁴ from a scRNA-seq dataset of 9,039 TAMs sorted from 3 individual *MMTV-PyMT* tumors (Figures 1B and 1C). We then mapped the murine LYVE-1+ population onto a recently published scRNA-seq atlas for human breast cancer.²⁸ The murine LYVE-1+ TAM population identified with cells within the human myeloid cell cluster (Figure 1D; Table S1). Focusing on the myeloid cells within the atlas, 1,444 of the 9,675 myeloid cells were judged to be LYVE-1+ TAM like in their phenotype (Figures 1E and 1F) and their expression of *LYVE1*, *MRC1* (CD206), and *HMOX1* (HO-1) were significantly associated with the identified cells (Figure 1F), demonstrating a conservation of this TAM subset between species. HO-1 is a marker that has previously been associated with PvTAMs in murine models of cancer,³,²⁰ and the HO-1 expressing TAM population was also observed in the Pv niche of human cancer (Figure 1G), highlighting a similar spatial location for these cells between species. HO-1 and LYVE-1 protein co-localized in tumor sections from *MMTV-PyMT* mice (Figure 1H), and LYVE-1+ TAMs were the major source of HO-1 within the Pv niche, which also includes, endothelial cells (ECs) and pericyte-like αSMA-expressing

cancer-associated fibroblasts (CAFs) (Figures 1I and 1J).¹⁴ Interestingly, from these analyses, it was evident that the LYVE-1+HO-1+ PvTAM subset was heterogeneously distributed along the endothelium and clustered to discrete regions where these cells were either lining or appearing in bunches proximal to the vasculature (Figures 1A, 1G, 1H, 1J, and S1A). These clusters, which we herein refer to as nests, highlighted an unappreciated multi-cellular PvTAM structure within the TME. As LYVE-1 is also expressed at high levels by the lymphatic endothelium,²⁹ and HO-1 expression is selectively expressed by this TAM subset (Figures 1H–1J), we generated a knockin reporter mouse for the *Hmox1* gene to facilitate the study of these cells (Figure 1K). The reporter consisted of Click Beetle luciferase (Luc) and enhanced green fluorescent protein (EGFP)³⁰ inserted before the stop codon of the genomic *Hmox1* gene locus. HO-1, Luc, and EGFP were separated by a self-cleaving P2A sequence³¹ to allow the equimolar expression of the three proteins (Figures 1K, 1L, and S1B) (mouse herein referred to as HO-1$^{Luc/EGFP}$). As HO-1 plays several important functional roles in the TME,³,³²⁻³⁴ the arrangement of the reporter construct ensured that native HO-1 expression was unaffected by the reporter elements. In HO-1$^{Luc/EGFP}$ mice, HO-1 expression could be detected in healthy tissues using bioluminescence imaging (Figure S1C). Analysis of healthy tissues from the HO-1$^{Luc/EGFP}$ mouse revealed the spleen, lung, mammary gland, visceral adipose, skin, and liver to be among the highest expressing tissues for HO-1 (Figure S1D), where its expression was restricted to F4/80$^{hi}$-tissue-resident macrophages (Figure S1E). The HO-1$^{Luc/EGFP}$ mouse was crossed onto the *MMTV-PyMT* background, and tumors were analyzed for their distribution of HO-1/EGFP expression (Figure 1K). TAMs (Figure S1F), specifically the LYVE-1+CD206$^{hi}$MHCII$^{lo}$ TAM subset (Figures 1M and S1G), were the major tumoral source of HO-1/EGFP. Further characterization of F4/80$^{hi}$HO-1/EGFP+ cells in tumors by immunofluorescence analysis also confirmed the localization of HO-1/EGFP to the Pv space within nest structures (Figure 1N). These data validate the HO-1$^{Luc/EGFP}$-reporter mouse as a tool for studying LYVE-1+ PvTAMs and identify a previously unappreciated multi-cellular nest structure for these cells in the Pv space.

### LYVE-1+ TAMs develop in the TME in response to IL-6

To investigate the development of LYVE-1+ TAMs within TME, we first considered whether they were a recruited or tissue-resident TAM population.⁹ To address this question, we utilized the photoconvertible *Kaede* mouse³⁵ crossed to the *MMTV-PyMT* model. When tumors reached 100 mm³, the entire TME was photoconverted from *Kaede*-green to *Kaede*-red using a UV-light source (Figure 2A). After 48 h post photoconversion of the TME, there was a clear *Kaede*-green (-red-negative) population of LYVE-1+ TAMs in the TME, indicative of their development from a recruited progenitor cell originating from outside the TME (Figure 2B). Although it is possible that highly replicating

<footnote>contour plot of fluorescence-activated cell sorting (FACs)-gated live (7AAD⁻) CD45⁺F4/80⁺ TAMs from enzyme-dispersed *MMTV-PyMT* HO-1*Luc/EGFP* tumors separated based on their respective expression of CD206 and MHCII (left panel) and then assessed for their expression of HO-1 and LYVE-1 (right panel; colored histograms) against that of the FMO staining control (gray histogram) (M). Representative of n = 10 mice. Representative image of a frozen section and quantification of a *MMTV-PyMT* HO-1*Luc/EGFP* tumor stained with antibodies against CD31 (red) are shown, and native EGFP fluorescence is shown in green (N). Scale bars: 25 µm (left panel). Proportion of HO-1/EGFP⁺ cells that are Pv (<20 µm from live vasculature) across n = 4 *MMTV-PyMT* tumors (right panel). Images in (I) and (K) were created using *BioRender* software. Bar charts represent the mean, and the dots show individual data points from individual tumors and mice. \* p < 0.05, \*\* p < 0.01.</footnote>
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 2.** LYVE-1⁺ PVTAMs are polarized by IL-6 in the tumor</figcaption></figure>

(A and B) Established tumors in *MMTV-PyMT* *Kaede* mice were photoconverted to *Kaede*-red (A). After 48 h, photoconverted tumors were analyzed for *Kaede*-red using flow cytometry, where cells negative for *Kaede*-red represent recruitment from outside the TME (B).

(C) Plot showing the significance value of IL-6 as a predicted upstream regulator of the gene expression profile of the respective TAM populations identified in Figure 1B generated by the IPA regulator analytic.

(D) Kaplan-Meier curve showing the tumor latency period for *II6⁺⁺* (WT; n = 23) and *II6⁻⁻* (n = 25) *MMTV-PyMT* mice; HR, hazard ratio; exact significance *p* = 0.0062.

(E–H) Abundance of live (7AAD⁻) CD45⁺F4/80⁺Ly6C⁻ TAMs in enzyme-dispersed tumors from *II6⁺⁺* and *II6⁻⁻* *MMTV-PyMT* mice assessed using flow cytometry (n = 6 per group) (E). Representative contour plot of FACs-gated live (7AAD⁻) F4/80⁺CD206⁺ TAMs from enzyme-dispersed *MMTV-PyMT* tumors showing their respective expression of CD206 and MHCII in *II6⁺⁺* (upper panel) and *II6⁻⁻* (lower panel) mice (F) and the quantification of the respective TAM populations (G) and abundance of LYVE-1⁺ TAMs as percentage of live cells (H) (n = 7 per group).

(I) Schematic depicting the cells in the PV niche (left panel) and their relative expression of *II6* mRNA from bulk RNA-seq (right panel).

(J and K) Representative images of formalin-fixed paraffin-embedded (FFPE) sections of *MMTV-PyMT* tumor (J) and healthy mammary gland (K) stained with DAPI (nuclei; blue) and antibodies against CD31 (green) and HO-1 (magenta) and probed for *II6* mRNA (red). Co-localization of *II6* and CD31 is displayed in yellow. Scale bars: 25 µm.

(L–N) BMDMs treated with and without IL-6 and the indicated knockdown (KD) siRNAs (n = 3 repeats). Western blots probing for STAT3 and c-MAF in cell lysates (L). Representative histograms of surface CD206 (left panel) and normalized (to Cntrl<sup>KD</sup>) marker expression across three individual experiments (right panel) (M) and representative histograms of surface LYVE-1 (left panel) and normalized (to Cntrl<sup>KD</sup>) marker expression across three individual experiments (right panel) (N); expression was assessed using a flow cytometry of BMDMs.

Image in (l) was created using *BioRender* software. Bar charts represent the mean, and the dots show individual data points from individual tumors and mice.
* *p* < 0.05, ** *p* < 0.01.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 2.** LYVE-1⁺ PVTAMs are polarized by IL-6 in the tumor</figcaption></figure>

(A and B) Established tumors in *MMTV-PyMT* *Kaede* mice were photoconverted to *Kaede*-red (A). After 48 h, photoconverted tumors were analyzed for *Kaede*-red using flow cytometry, where cells negative for *Kaede*-red represent recruitment from outside the TME (B).

(C) Plot showing the significance value of IL-6 as a predicted upstream regulator of the gene expression profile of the respective TAM populations identified in Figure 1B generated by the IPA regulator analytic.

(D) Kaplan-Meier curve showing the tumor latency period for *II6⁺⁺* (WT; n = 23) and *II6⁻⁻* (n = 25) *MMTV-PyMT* mice; HR, hazard ratio; exact significance *p* = 0.0062.

(E–H) Abundance of live (7AAD⁻) CD45⁺F4/80⁺Ly6C⁻ TAMs in enzyme-dispersed tumors from *II6⁺⁺* and *II6⁻⁻* *MMTV-PyMT* mice assessed using flow cytometry (n = 6 per group) (E). Representative contour plot of FACs-gated live (7AAD⁻) F4/80⁺CD206⁺ TAMs from enzyme-dispersed *MMTV-PyMT* tumors showing their respective expression of CD206 and MHCII in *II6⁺⁺* (upper panel) and *II6⁻⁻* (lower panel) mice (F) and the quantification of the respective TAM populations (G) and abundance of LYVE-1⁺ TAMs as percentage of live cells (H) (n = 7 per group).

(I) Schematic depicting the cells in the PV niche (left panel) and their relative expression of *II6* mRNA from bulk RNA-seq (right panel).

(J and K) Representative images of formalin-fixed paraffin-embedded (FFPE) sections of *MMTV-PyMT* tumor (J) and healthy mammary gland (K) stained with DAPI (nuclei; blue) and antibodies against CD31 (green) and HO-1 (magenta) and probed for *II6* mRNA (red). Co-localization of *II6* and CD31 is displayed in yellow. Scale bars: 25 µm.

(L–N) BMDMs treated with and without IL-6 and the indicated knockdown (KD) siRNAs (n = 3 repeats). Western blots probing for STAT3 and c-MAF in cell lysates (L). Representative histograms of surface CD206 (left panel) and normalized (to Cntrl<sup>KD</sup>) marker expression across three individual experiments (right panel) (M) and representative histograms of surface LYVE-1 (left panel) and normalized (to Cntrl<sup>KD</sup>) marker expression across three individual experiments (right panel) (N); expression was assessed using a flow cytometry of BMDMs.

Image in (l) was created using *BioRender* software. Bar charts represent the mean, and the dots show individual data points from individual tumors and mice.
* *p* < 0.05, ** *p* < 0.01.
<figure><img src="image_10.png" /><figcaption>Figure 3. LYVE-1+ TAMs communicate via a CCR5-dependent axis to maintain Pv nest formation</figcaption></figure>

(A and B) Schematic overview of cells in the Pv nest (A, left) and bar plot depicting normalized gene expression values for *Ccr5* in the bulk RNA-seq of the Pv niche populations (A, right) and *Ccl3*, *Ccl4*, and *Ccl5* (B) across n = 5 mice.

(C) Representative histograms of surface CCR5 expression in BMDMs treated with (red-filled histogram lower panel) and without (green-filled histogram upper panel) IL-6 against that of FMO staining (gray-filled histogram) (left) and quantitation of CCR5 expression (median fluorescence intensity [MFI]-subtracted FMO) across 5 biological repeats (right).

(D) Representative histograms of surface CCR5 expression (red histogram) for the indicated populations against that of the fluorescence minus one (FMO) staining control (blue histogram) in enzyme-dispersed *MMTV-PyMT* HO-1<sup>Luc/EGFP</sup> tumors where the Lyve-1<sup>+</sup> TAM population was gated based upon their GFP/ HO-1 expression.

(E) Schematic of the assay (left panel) and relative transendothelial migration (TEM) of M(0) and M(IL-6) BMDMs generated from Ccr5<sup>+/+</sup> and Ccr5<sup>-/-</sup> mice in the presence or absence of CCL4 after 24 h incubation (right panel). Each dot represents a paired ratio of CCL4/control (no CCL4) (n = 7).

(F) Relative change in the area of plated M(0) and M(IL-6) BMDMs spheroids over 72 h. Each point represents an individual spheroid.

(G) Kaplan-Meier curve showing the tumor latency for Ccr5<sup>+/+</sup> (WT; n = 23) and Ccr5<sup>-/-</sup> (n = 25) *MMTV-PyMT* mice; HR, hazard ratio; exact significance p = 0.004. (H–J) Tumors from Ccr5<sup>+/+</sup> and Ccr5<sup>-/-</sup> *MMTV-PyMT* mice were enzyme dispersed and assessed using flow cytometry for the abundance of live (7AAD<sup>-</sup>) CD45<sup>+</sup>F4/80<sup>+</sup>Ly6C<sup>-</sup> TAMs (H) and their phenotype as proportions of the TAM gate (I) and the abundance of LYVE-1<sup>+</sup> TAMs as the percentage of live cells (J) (n = 4–7 per group).

(legend continued on next page)
<figure><img src="image_10.png" /><figcaption>Figure 3. LYVE-1+ TAMs communicate via a CCR5-dependent axis to maintain Pv nest formation</figcaption></figure>

(A and B) Schematic overview of cells in the Pv nest (A, left) and bar plot depicting normalized gene expression values for *Ccr5* in the bulk RNA-seq of the Pv niche populations (A, right) and *Ccl3*, *Ccl4*, and *Ccl5* (B) across *n* = 5 mice.

(C) Representative histograms of surface CCR5 expression in BMDMs treated with (red-filled histogram lower panel) and without (green-filled histogram upper panel) IL-6 against that of FMO staining (gray-filled histogram) (left) and quantitation of CCR5 expression (median fluorescence intensity [MFI]-subtracted FMO) across 5 biological repeats (right).

(D) Representative histograms of surface CCR5 expression (red histogram) for the indicated populations against that of the fluorescence minus one (FMO) staining control (blue histogram) in enzyme-dispersed *MMTV-PyMT* HO-1<sup>Luc/EGFP</sup> tumors where the Lyve-1<sup>+</sup> TAM population was gated based upon their GFP/ HO-1 expression.

(E) Schematic of the assay (left panel) and relative transendothelial migration (TEM) of M(0) and M(IL-6) BMDMs generated from Ccr5<sup>+/+</sup> and Ccr5<sup>-/-</sup> mice in the presence or absence of CCL4 after 24 h incubation (right panel). Each dot represents a paired ratio of CCL4/control (no CCL4) (n = 7).

(F) Relative change in the area of plated M(0) and M(IL-6) BMDMs spheroids over 72 h. Each point represents an individual spheroid.

(G) Kaplan-Meier curve showing the tumor latency for Ccr5<sup>+/+</sup> (WT; n = 23) and Ccr5<sup>-/-</sup> (n = 25) *MMTV-PyMT* mice; HR, hazard ratio; exact significance p = 0.004. (H–J) Tumors from Ccr5<sup>+/+</sup> and Ccr5<sup>-/-</sup> *MMTV-PyMT* mice were enzyme dispersed and assessed using flow cytometry for the abundance of live (7AAD<sup>-</sup>) CD45<sup>+</sup>F4/80<sup>+</sup>Ly6C<sup>-</sup> TAMs (H) and their phenotype as proportions of the TAM gate (I) and the abundance of LYVE-1<sup>+</sup> TAMs as the percentage of live cells (J) (n = 4–7 per group).

(legend continued on next page)
cells can dilute their *Kaede*-red status post photoconversion, an analysis of the expression of the proliferation marker Ki-67 revealed that 40% ± 20% of the *Kaede*-green-gated LYVE-1⁺ TAMs did not express Ki-67. This highlights that a significant proportion of these cells were not replicating (Figures S2A and S2B). Furthermore, there was no difference in Ki-67 expression between *Kaede*-red and *Kaede*-green LYVE-1⁺ TAMs (Figures S2A and S2B). As such, these data suggest recruitment as the most likely origin of these cells. As LYVE-1⁺ TAMs were likely developing and polarizing within the TME, to explore the key signals directing this developmental program, we employed the QIAGEN Ingenuity Pathway Analysis software (QIAGEN IPA)³⁶ on a scRNA-seq data of TAMs from *MMTV-PyMT* tumors.¹⁴ Using this approach, IL-6 signaling was predicted to be an upstream polarization signal associated with the transcriptional programs active in the LYVE-1⁺ TAM subset (Figure 2C). To investigate the role of IL-6 in the polarization of the LYVE-1⁺ PVTAM subset, we crossed *MMTV-PyMT* mice onto an *Il6<sup>-/-</sup>* background and analyzed the tumors in these animals. In the absence of IL-6, the tumors were significantly slower to establish, with a median latency of 87 versus 100 days for *Il6<sup>+/-</sup>* and *Il6<sup>-/-</sup>* *MMTV-PyMT* mice, respectively (Figure 2D). When tumors reached ~500 mm³, they were enzyme dispersed and analyzed by flow cytometry. Despite no overall change in the abundance of TAMs within the TME (Figure 2E), there was a significant and selective loss of the LYVE-1⁺ TAM subset (Figures 2F-2H), highlighting that IL-6 was fundamental to the polarization of the TAM subset. Analysis of bulk RNA-seq data from sorted TME stromal populations within the Pv niche¹⁴ highlighted the αSMA⁺ pericyte-like CAF and ECs (but not the TAMs) to express *Il6* mRNA (Figure 2I). More widely, CAFs and ECs were the main tumoral sources of *Il6* mRNA expression, with no detectable expression in the tumor cells or other stroma subsets (Figure S2C). To identify the spatial location of the *Il6* mRNA in the TME, we used RNA-sequence (Figure 2J). *Il6* mRNA was detectable in nearly all ECs in the tumor, but the highest expression of *Il6* mRNA was evident in the ECs that were proximal to HO-1⁺ cells (LYVE-1⁺ TAMs) (Figures 2J and S2D-S2F); although αSMA⁺ CAFs could be found expressing *Il6* mRNA in the TME, as indicated by the bulk RNA-seq data, the cells expressing *Il6* mRNA were not proximal to the HO-1⁺ cells (Figures S2D and S2E). Interestingly, *Il6* was not expressed by the endothelium, nor were HO-1⁺ macrophage clusters present in the healthy mammary gland (Figure 2K), highlighting this axis to be associated specifically with the TME. These data suggest that LYVE-1⁺ TAMs develop within the Pv niche in response to IL-6 and that the proximal endothelium is the most likely source of IL-6 in the TME.

## LYVE-1⁺ macrophage phenotypic identity is dependent on STAT3/c-MAF pathways

To investigate whether IL-6 stimulation alone was sufficient to generate a LYVE-1⁺ TAM-like phenotype, in a gain-of-function experiment, bone-marrow-derived macrophages (BMDMs) were exposed to IL-6, and their phenotype was assessed. IL-6-stimulated BMDMs upregulated the expression of the three key phenotypic markers, HO-1 (Figures 2L, S2G, and S2H), CD206 (Figure 2M), and LYVE-1 (Figure 2N), which define the subset. These data suggested that IL-6 plays a dominant role in the polarization identity of LYVE-1⁺ TAMs, and the relative absence of the LYVE-1⁺ macrophage phenotype in *Il6<sup>-/-</sup>* *MMTV-PyMT* mice (Figures 2F-2H) was most likely directly due to the loss of IL-6 from the TME. Recent data demonstrated that the transcription factor cellular musculoaponeurotic fibrosarcoma (c-MAF) was vital to the development of LYVE-1⁺ Pv macrophages in healthy tissues,³⁷ and we sought to establish whether c-MAF signaling may account for the LYVE-1⁺ macrophage phenotype observed through IL-6 stimulation. IL-6 signaling is associated with the Janus kinase (JAK)/signal transducer and activator of transcription protein-3 (STAT3) pathway³⁸; however, STAT3 has been linked to the expression of c-MAF via the basic leucine zipper transcription factor activating transcription factor (ATF)-like Batf in T follicular helper cells.³⁹ To investigate whether c-MAF and STAT3 signaling play a role in the LYVE-1⁺ macrophage phenotype, we knocked down the expression of either c-MAF or STAT3 in BMDMs prior to stimulation by IL-6 (Figures 2L and S2G). Loss of either transcription factor in IL-6 stimulated BMDMs, indeed, compromised the phenotypic identity of the population, as assessed using the markers LYVE-1, CD206, and HO-1 (Figures 2L-2N and S2H). Interestingly, c-MAF appeared to be required for even basal HO-1 expression in BMDMs (Figures 2L and S2H). These data demonstrate that IL-6 is sufficient to upregulate the key phenotypic markers associated with the LYVE-1⁺ PVTAM subset through a STAT3- and c-MAF-dependent pathway.

## LYVE-1⁺ TAMs communicate via a CCR5-dependent axis to maintain nest formation

Having identified IL-6 as a driver of the LYVE-1⁺ TAM polarization program in the Pv niche (Figure 2), we investigated whether the polarization program might influence a communication axis between these cells required to maintain the formation of nests arrangements post polarization (Figure 1A). As such, to screen for IL-6-regulated chemokine receptors within the polarization program, we performed a microarray analysis on

(K and L) Representative images of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei;blue) and antibodies against CD31 (red) and HO-1 (green) in *Ccr5<sup>+/-</sup>* (K, upper left) and *Ccr5<sup>-/-</sup>* (K, lower left) *MMTV-PyMT* mice. Scale bars: 50 µm (left panel). Distance of individual HO-1⁺ TAMs to the nearest CD31⁺ cell or HO-1⁺ TAM as indicated, plotting individual cells (left panels, taken across 3 sections from each tumor and an average of 145 ± 65 and 175 ± 72 measurements per tumor between *CCR5<sup>+/-</sup>* and *CCR5<sup>-/-</sup>* groups, respectively) and average distances per tumor (right panels). Also plotted is the abundance of HO-1⁺ TAMs per unit area (L) in *Ccr5<sup>+/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* tumors (taken from n = 5 tumors).

(M and N) Schematic representing the dosing strategy for the CCR5 inhibitor maraviroc and vehicle in *MMTV-PyMT* mice (n = 3 per group) (M). Representative images of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei;blue) and antibodies against CD31 (red) and HO-1 (green) in vehicle-treated mice (N, upper left) and maraviroc-treated mice (N, lower left). Scale bars: 50 µm (left panel). Distance of individual HO-1⁺ TAMs to nearest CD31⁺ cell across vehicle- and maraviroc-treated *MMTV-PyMT* tumors, plotting individual cells (left panel, taken across 3 sections from each tumor and an average of 71 ± 36 and 128 ± 12 measurements per tumor between vehicle- and maraviroc-treated groups, respectively) and average distances per tumor (right panel) (N). Images in (A) and (M) were created using *BioRender* software. Bar charts represent the mean, and the dots show individual data points from individual tumors and mice. *p* < 0.05, **p** < 0.01, and ****p < 0.0001.
cells can dilute their *Kaede*-red status post photoconversion, an analysis of the expression of the proliferation marker Ki-67 revealed that 40% ± 20% of the *Kaede*-green-gated LYVE-1⁺ TAMs did not express Ki-67. This highlights that a significant proportion of these cells were not replicating (Figures S2A and S2B). Furthermore, there was no difference in Ki-67 expression between *Kaede*-red and *Kaede*-green LYVE-1⁺ TAMs (Figures S2A and S2B). As such, these data suggest recruitment as the most likely origin of these cells. As LYVE-1⁺ TAMs were likely developing and polarizing within the TME, to explore the key signals directing this developmental program, we employed the QIAGEN Ingenuity Pathway Analysis software (QIAGEN IPA)³⁶ on a scRNA-seq data of TAMs from *MMTV-PyMT* tumors.¹⁴ Using this approach, IL-6 signaling was predicted to be an upstream polarization signal associated with the transcriptional programs active in the LYVE-1⁺ TAM subset (Figure 2C). To investigate the role of IL-6 in the polarization of the LYVE-1⁺ PvTAM subset, we crossed *MMTV-PyMT* mice onto an *Il6<sup>-/-</sup>* background and analyzed the tumors in these animals. In the absence of IL-6, the tumors were significantly slower to establish, with a median latency of 87 versus 100 days for *Il6<sup>+/-</sup>* and *Il6<sup>-/-</sup>* *MMTV-PyMT* mice, respectively (Figure 2D). When tumors reached ~500 mm³, they were enzyme dispersed and analyzed by flow cytometry. Despite no overall change in the abundance of TAMs within the TME (Figure 2E), there was a significant and selective loss of the LYVE-1⁺ TAM subset (Figures 2F-2H), highlighting that IL-6 was fundamental to the polarization of the TAM subset. Analysis of bulk RNA-seq data from sorted TME stromal populations within the Pv niche¹⁴ highlighted the αSMA⁺ pericyte-like CAF and ECs (but not the TAMs) to express *Il6* mRNA (Figure 2I). More widely, CAFs and ECs were the main tumoral sources of *Il6* mRNA expression, with no detectable expression in the tumor cells or other stroma subsets (Figure S2C). To identify the spatial location of the *Il6* mRNA in the TME, we used RNA-scope (Figure 2J). *Il6* mRNA was detectable in nearly all ECs in the tumor, but the highest expression of *Il6* mRNA was evident in the ECs that were proximal to HO-1⁺ cells (LYVE-1⁺ TAMs) (Figures 2J and S2D-S2F); although αSMA⁺ CAFs could be found expressing *Il6* mRNA in the TME, as indicated by the bulk RNA-seq data, the cells expressing *Il6* mRNA were not proximal to the HO-1⁺ cells (Figures S2D and S2E). Interestingly, *Il6* was not expressed by the endothelium, nor were HO-1⁺ macrophage clusters present in the healthy mammary gland (Figure 2K), highlighting this axis to be associated specifically with the TME. These data suggest that LYVE-1⁺ TAMs develop within the Pv niche in response to IL-6 and that the proximal endothelium is the most likely source of IL-6 in the TME.

## LYVE-1⁺ macrophage phenotypic identity is dependent on STAT3/c-MAF pathways

To investigate whether IL-6 stimulation alone was sufficient to generate a LYVE-1⁺ TAM-like phenotype, in a gain-of-function experiment, bone-marrow-derived macrophages (BMDMs) were exposed to IL-6, and their phenotype was assessed. IL-6-stimulated BMDMs upregulated the expression of the three key phenotypic markers, HO-1 (Figures 2L, S2G, and S2H), CD206 (Figure 2M), and LYVE-1 (Figure 2N), which define the subset. These data suggested that IL-6 plays a dominant role in the polarization identity of LYVE-1⁺ TAMs, and the relative absence of the LYVE-1⁺ macrophage phenotype in *Il6<sup>-/-</sup>* *MMTV-PyMT* mice (Figures 2F-2H) was most likely directly due to the loss of IL-6 from the TME. Recent data demonstrated that the transcription factor cellular musculoaponeurotic fibrosarcoma (c-MAF) was vital to the development of LYVE-1⁺ Pv macrophages in healthy tissues,³⁷ and we sought to establish whether c-MAF signaling may account for the LYVE-1⁺ macrophage phenotype observed through IL-6 stimulation. IL-6 signaling is associated with the Janus kinase (JAK)/signal transducer and activator of transcription protein-3 (STAT3) pathway³⁸; however, STAT3 has been linked to the expression of c-MAF via the basic leucine zipper transcription factor activating transcription factor (ATF)-like Batf in T follicular helper cells.³⁹ To investigate whether c-MAF and STAT3 signaling play a role in the LYVE-1⁺ macrophage phenotype, we knocked down the expression of either c-MAF or STAT3 in BMDMs prior to stimulation by IL-6 (Figures 2L and S2G). Loss of either transcription factor in IL-6 stimulated BMDMs, indeed, compromised the phenotypic identity of the population, as assessed using the markers LYVE-1, CD206, and HO-1 (Figures 2L-2N and S2H). Interestingly, c-MAF appeared to be required for even basal HO-1 expression in BMDMs (Figures 2L and S2H). These data demonstrate that IL-6 is sufficient to upregulate the key phenotypic markers associated with the LYVE-1⁺ PvTAM subset through a STAT3- and c-MAF-dependent pathway.

## LYVE-1⁺ TAMs communicate via a CCR5-dependent axis to maintain nest formation

Having identified IL-6 as a driver of the LYVE-1⁺ TAM polarization program in the Pv niche (Figure 2), we investigated whether the polarization program might influence a communication axis between these cells required to maintain the formation of nests arrangements post polarization (Figure 1A). As such, to screen for IL-6-regulated chemokine receptors within the polarization program, we performed a microarray analysis on

(K and L) Representative images of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei;blue) and antibodies against CD31 (red) and HO-1 (green) in *Ccr5<sup>+/-</sup>* (K, upper left) and *Ccr5<sup>-/-</sup>* (K, lower left) *MMTV-PyMT* mice. Scale bars: 50 µm (left panel). Distance of individual HO-1⁺ TAMs to the nearest CD31⁺ cell or HO-1⁺ TAM as indicated, plotting individual cells (left panels, taken across 3 sections from each tumor and an average of 145 ± 65 and 175 ± 72 measurements per tumor between *CCR5<sup>+/-</sup>* and *CCR5<sup>-/-</sup>* groups, respectively) and average distances per tumor (right panels). Also plotted is the abundance of HO-1⁺ TAMs per unit area (L) in *Ccr5<sup>+/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* tumors (taken from n = 5 tumors).

(M and N) Schematic representing the dosing strategy for the CCR5 inhibitor maraviroc and vehicle in *MMTV-PyMT* mice (n = 3 per group) (M). Representative images of a frozen section of *MMTV-PyMT* tumor stained with DAPI (nuclei;blue) and antibodies against CD31 (red) and HO-1 (green) in vehicle-treated mice (N, upper left) and maraviroc-treated mice (N, lower left). Scale bars: 50 µm (left panel). Distance of individual HO-1⁺ TAMs to nearest CD31⁺ cell across vehicle- and maraviroc-treated *MMTV-PyMT* tumors, plotting individual cells (left panel, taken across 3 sections from each tumor and an average of 71 ± 36 and 128 ± 12 measurements per tumor between vehicle- and maraviroc-treated groups, respectively) and average distances per tumor (right panel) (N). Images in (A) and (M) were created using *BioRender* software. Bar charts represent the mean, and the dots show individual data points from individual tumors and mice. *p* < 0.05, **p** < 0.01, and ****p < 0.0001.
monocyte-derived macrophages exposed to IL-6, using basal (M0), IL-4 (M2), and IFNγ/lipopolysaccharide (LPS) (M1)⁴⁰ as comparator conditions (Figures S3A and S3B). The IL-6 polarization program was a distinct program from that of M1/M2 macrophages (Figures S3A and S3B). Interestingly, IL-6-polarized macrophages (M<sub>(IL-6)</sub>) were associated with higher *Pdgfc* expression (Figure S3C), a growth factor we recently demonstrated to play a role in the LYVE-1⁺ TAM-dependent expansion of αSMA CAFs within the Pv niche.¹⁴ In probing for IL-6-induced chemokine receptors, we identified a unique upregulation of the chemokine receptor gene *Ccr5* (Figure S3D). Analyzing the bulk RNA-seq data for cells in the Pv niche revealed both *Ccr5* (Figure 3A) and its cognate ligands *Ccl3* and *Ccl4* (Figure 3B) to be expressed by the LYVE-1⁺ TAMs within the Pv niche and identified a potential communication axis for these cells post IL-6 polarization. The expression of CCR5 was also regulated by IL-6 in BMDMs (Figure 3C), implicating the gene within the IL-6 polarization program. Within the TME, although all TAMs expressed some CCR5, the LYVE-1⁺ TAM subset was the highest expressor (Figures 3D and S3E). CCR5 was functional on the macrophages, and IL-6-stimulated BMDMs could migrate toward the CCR5 ligand, CCL4 (Figure 3E). Chemotaxis to CCL4 was CCR5 dependent, as migration was abolished when IL-6-stimulated BMDMs were derived from *Ccr5<sup>-/-</sup>* mice (Figure 3E). When IL-6-stimulated BMDMs were also placed in close contact *in vitro*, their ability to spread from one another was also reduced (Figure 3F). To test the role of CCR5 in maintaining LYVE-1⁺ TAMs in nests within the TME, we crossed *MMTV-PyMT* mice onto a *Ccr5<sup>-/-</sup>* background. In the absence of CCR5, tumors were significantly slower to establish, with a median latency of 87 versus 102 days for *Ccr5<sup>+/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice, respectively (Figure 3G). The loss of CCR5 did not affect the total prevalence of TAMs (Figure 3H) or the LYVE-1⁺ TAM subset (Figures 3I and 3J) in these tumors, demonstrating that CCR5 expression was not required for the maintenance of the polarization phenotype of these cells. However, immunofluorescence imaging of tissue sections from these tumors revealed an increase in the median distance between LYVE-1⁺ TAMs and from LYVE-1⁺ TAMs to the endothelium (Figure 3K), with no effect on their abundance overall (Figure 3L). This demonstrated that CCR5 was required to maintain the formation of the nest structures of LYVE-1⁺ TAMs and is a key functional protein of the IL-6 polarization program that spatially orientates these cells within the Pv niche. To explore whether the Pv niche was a dynamic structure and whether CCR5 also played an ongoing active role in maintaining the LYVE-1⁺ TAM nests post formation, rather than just the initial formation of the structure, we injected *MMTV-PyMT* mice with maraviroc (Figure 3M), a drug that is clinically used to inhibit CCR5.⁴¹ Therapeutically blocking CCR5 signaling using maraviroc in tumors with pre-formed LYVE-1⁺ PVTAM nests (Figures S3F and S3G) resulted in an observable dispersion of the LYVE-1⁺ TAM nests away from the Pv space (Figure 3N), highlighting that LYVE-1⁺ TAM nests are, indeed, dynamic structures and that CCR5 represents an ongoing communication axis for their maintenance. These data highlight a role for CCR5 in the collaborative formation and maintenance of the LYVE-1⁺ TAM nests that create a Pv niche.

## LYVE-1⁺ TAM nests are associated with immune exclusion in the TME

To understand how the absence of LYVE-1⁺ TAM nests may alter the wider TME, we analyzed the composition of the stroma in ~500 mm³ tumors from wild type (WT), *Il6<sup>-/-</sup>* (where LYVE-1⁺ TAMs could not develop), and *Ccr5<sup>-/-</sup>* (where their post-IL-6 polarization nesting was perturbed) *MMTV-PyMT* mice (Figure 4A). The stromal composition in enzyme-dispersed tumors were assessed using flow cytometry (Figures 4B and S4A). Broadly, the stromal composition of the tumors was highly similar despite the spontaneous nature of the tumor model (Figure 4B). However, the only consistent difference between both the *Il6<sup>-/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice and WT mice was a significant increase in tumor-infiltrating CD8⁺ T cells (Figure 4B). The CD8⁺ T cells had a similar overall proportion of cells that displayed effector function (Figures 4C, 4D, and S4B). The increase in tumor-infiltrating CD8⁺ T cells observed in the tumors from *Il6<sup>-/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice appeared independent from a change in the vasculature, as all tumors had equivalent numbers of CD31⁺ ECs (Figure 4B) and an equivalent vessel density in the tissue (Figure 4E). The endothelium expresses adhesion molecules, which permit leukocyte rolling, migration, and arrest prior to diapedesis into inflamed tissues,⁴² and it was possible that LYVE-1⁺ TAMs could modulate the endothelium due to their close proximity to one another. However, there was no evidence for a change in the expression of endothelial VCAM-1, ICAM-1, or PNA<sup>d</sup> in the absence of LYVE-1⁺ TAMs or their nests in the TME (Figure S5A). However, this highlighted that the key signaling axes, which are required for the polarization (IL-6) and spatial identity (CCR5) of LYVE-1⁺ TAMs, were associated with immune exclusion within the TME and were independent of the pro-angiogenic function of these cells.

## Loss of LYVE-1⁺ TAMs or their nests improves the immune-mediated effects of chemotherapy

There is a clear clinical link between the presence of tumor-infiltrating lymphocytes (TILs) and response to therapeutics that can modulate their effects through anti-tumor immune responses, such as immune checkpoint blockade⁴³,⁴⁴ and cytotoxic chemotherapies.⁴⁵,⁴⁶ We have demonstrated HO-1 activity to be a major immune-suppressive pathway preventing chemotherapy-elicited immune responses in the TME.⁴⁷ In this study, we have demonstrated that HO-1 expression is highly restricted to the LYVE-1⁺ TAM population (Figure 1). As such, we considered whether the presence of LYVE-1⁺ TAMs, or their nest structures, might influence the resistance to immune-modulating cytotoxic chemotherapeutics. Treatment of tumor-bearing *MMTV-PyMT* mice with 5-fluorouracil (5-FU), a chemotherapeutic that has been used in the clinic⁴⁸ and that has been demonstrated to have immune-stimulatory capabilities,⁴⁹,⁵⁰ did not significantly affect tumor growth in WT mice (Figures 4F and 4G). However, when 5-FU was administered to either *Il6<sup>-/-</sup>* or *Ccr5<sup>-/-</sup>* mice, a significant control of tumor growth was achieved (Figure 4G). To investigate whether the tumor control was immune dependent, we depleted CD8⁺ T cells using immune-depleting antibodies *in vivo* prior to the initiation of 5-FU treatment in *Il6<sup>-/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice in which the LYVE-1⁺ TAM subset was unable to polarize (*Il6<sup>-/-</sup>* or form Pv nest structures (*Ccr5<sup>-/-</sup>*) (Figures S5B–S5D). In the absence of CD8⁺ T cells,
monocyte-derived macrophages exposed to IL-6, using basal (M0), IL-4 (M2), and IFNγ/lipopolysaccharide (LPS) (M1)⁴⁰ as comparator conditions (Figures S3A and S3B). The IL-6 polarization program was a distinct program from that of M1/M2 macrophages (Figures S3A and S3B). Interestingly, IL-6-polarized macrophages (M<sub>(IL-6)</sub>) were associated with higher *Pdgfc* expression (Figure S3C), a growth factor we recently demonstrated to play a role in the LYVE-1⁺ TAM-dependent expansion of αSMA CAFs within the Pv niche.¹⁴ In probing for IL-6-induced chemokine receptors, we identified a unique upregulation of the chemokine receptor gene *Ccr5* (Figure S3D). Analyzing the bulk RNA-seq data for cells in the Pv niche revealed both *Ccr5* (Figure 3A) and its cognate ligands *Ccl3* and *Ccl4* (Figure 3B) to be expressed by the LYVE-1⁺ TAMs within the Pv niche and identified a potential communication axis for these cells post IL-6 polarization. The expression of CCR5 was also regulated by IL-6 in BMDMs (Figure 3C), implicating the gene within the IL-6 polarization program. Within the TME, although all TAMs expressed some CCR5, the LYVE-1⁺ TAM subset was the highest expressor (Figures 3D and S3E). CCR5 was functional on the macrophages, and IL-6-stimulated BMDMs could migrate toward the CCR5 ligand, CCL4 (Figure 3E). Chemotaxis to CCL4 was CCR5 dependent, as migration was abolished when IL-6-stimulated BMDMs were derived from *Ccr5<sup>-/-</sup>* mice (Figure 3E). When IL-6-stimulated BMDMs were also placed in close contact *in vitro*, their ability to spread from one another was also reduced (Figure 3F). To test the role of CCR5 in maintaining LYVE-1⁺ TAMs in nests within the TME, we crossed *MMTV-PyMT* mice onto a *Ccr5<sup>-/-</sup>* background. In the absence of CCR5, tumors were significantly slower to establish, with a median latency of 87 versus 102 days for *Ccr5<sup>+/+</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice, respectively (Figure 3G). The loss of CCR5 did not affect the total prevalence of TAMs (Figure 3H) or the LYVE-1⁺ TAM subset (Figures 3I and 3J) in these tumors, demonstrating that CCR5 expression was not required for the maintenance of the polarization phenotype of these cells. However, immunofluorescence imaging of tissue sections from these tumors revealed an increase in the median distance between LYVE-1⁺ TAMs and from LYVE-1⁺ TAMs to the endothelium (Figure 3K), with no effect on their abundance overall (Figure 3L). This demonstrated that CCR5 was required to maintain the formation of the nest structures of LYVE-1⁺ TAMs and is a key functional protein of the IL-6 polarization program that spatially orientates these cells within the Pv niche. To explore whether the Pv niche was a dynamic structure and whether CCR5 also played an ongoing active role in maintaining the LYVE-1⁺ TAM nests post formation, rather than just the initial formation of the structure, we injected *MMTV-PyMT* mice with maraviroc (Figure 3M), a drug that is clinically used to inhibit CCR5.⁴¹ Therapeutically blocking CCR5 signaling using maraviroc in tumors with pre-formed LYVE-1⁺ PVTAM nests (Figures S3F and S3G) resulted in an observable dispersion of the LYVE-1⁺ TAM nests away from the Pv space (Figure 3N), highlighting that LYVE-1⁺ TAM nests are, indeed, dynamic structures and that CCR5 represents an ongoing communication axis for their maintenance. These data highlight a role for CCR5 in the collaborative formation and maintenance of the LYVE-1⁺ TAM nests that create a Pv niche.

## LYVE-1⁺ TAM nests are associated with immune exclusion in the TME

To understand how the absence of LYVE-1⁺ TAM nests may alter the wider TME, we analyzed the composition of the stroma in ~500 mm³ tumors from wild type (WT), *Il6<sup>-/-</sup>* (where LYVE-1⁺ TAMs could not develop), and *Ccr5<sup>-/-</sup>* (where their post-IL-6 polarization nesting was perturbed) *MMTV-PyMT* mice (Figure 4A). The stromal composition in enzyme-dispersed tumors were assessed using flow cytometry (Figures 4B and S4A). Broadly, the stromal composition of the tumors was highly similar despite the spontaneous nature of the tumor model (Figure 4B). However, the only consistent difference between both the *Il6<sup>-/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice and WT mice was a significant increase in tumor-infiltrating CD8⁺ T cells (Figure 4B). The CD8⁺ T cells had a similar overall proportion of cells that displayed effector function (Figures 4C, 4D, and S4B). The increase in tumor-infiltrating CD8⁺ T cells observed in the tumors from *Il6<sup>-/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice appeared independent from a change in the vasculature, as all tumors had equivalent numbers of CD31⁺ ECs (Figure 4B) and an equivalent vessel density in the tissue (Figure 4E). The endothelium expresses adhesion molecules, which permit leukocyte rolling, migration, and arrest prior to diapedesis into inflamed tissues,⁴² and it was possible that LYVE-1⁺ TAMs could modulate the endothelium due to their close proximity to one another. However, there was no evidence for a change in the expression of endothelial VCAM-1, ICAM-1, or PNA<sup>d</sup> in the absence of LYVE-1⁺ TAMs or their nests in the TME (Figure S5A). However, this highlighted that the key signaling axes, which are required for the polarization (IL-6) and spatial identity (CCR5) of LYVE-1⁺ TAMs, were associated with immune exclusion within the TME and were independent of the pro-angiogenic function of these cells.

## Loss of LYVE-1⁺ TAMs or their nests improves the immune-mediated effects of chemotherapy

There is a clear clinical link between the presence of tumor-infiltrating lymphocytes (TILs) and response to therapeutics that can modulate their effects through anti-tumor immune responses, such as immune checkpoint blockade⁴³,⁴⁴ and cytotoxic chemotherapies.⁴⁵,⁴⁶ We have demonstrated HO-1 activity to be a major immune-suppressive pathway preventing chemotherapy-elicited immune responses in the TME.⁴⁷ In this study, we have demonstrated that HO-1 expression is highly restricted to the LYVE-1⁺ TAM population (Figure 1). As such, we considered whether the presence of LYVE-1⁺ TAMs, or their nest structures, might influence the resistance to immune-modulating cytotoxic chemotherapeutics. Treatment of tumor-bearing *MMTV-PyMT* mice with 5-fluorouracil (5-FU), a chemotherapeutic that has been used in the clinic⁴⁸ and that has been demonstrated to have immune-stimulatory capabilities,⁴⁹,⁵⁰ did not significantly affect tumor growth in WT mice (Figures 4F and 4G). However, when 5-FU was administered to either *Il6<sup>-/-</sup>* or *Ccr5<sup>-/-</sup>* mice, a significant control of tumor growth was achieved (Figure 4G). To investigate whether the tumor control was immune dependent, we depleted CD8⁺ T cells using immune-depleting antibodies *in vivo* prior to the initiation of 5-FU treatment in *Il6<sup>-/-</sup>* and *Ccr5<sup>-/-</sup>* *MMTV-PyMT* mice in which the LYVE-1⁺ TAM subset was unable to polarize (*Il6<sup>-/-</sup>* or form Pv nest structures (*Ccr5<sup>-/-</sup>*) (Figures S5B–S5D). In the absence of CD8⁺ T cells,
<figure><img src="image_11.png" /><figcaption>**Figure 4.** LYVE-1⁺ TAMs and their nests are associated with immune exclusion and resistance to chemotherapy</figcaption></figure>

(A-E) Tumors from WT, *Il6*⁻/⁻, or *Ccr5*⁻/⁻ *MMTV-PyMT* mice were analyzed for their stromal composition (n = 6 tumors per group). Tumor sizes on the day of analysis (A) and the relative proportions of live (7AAD⁻) stromal cell populations were assessed using flow cytometry (B). The markers used to differentiate cells can be found in the online *STAR Methods*. Gated CD8⁺CD3⁺ T cells were assessed for their expression of the indicated effector molecules post PMA/ionomycin treatment, shown as percentage of positive cells (C), and the MFI of the expression of the gated positive cells (D). CD31⁺ cell density was assessed in tumor sections by immunofluorescence and quantitated relative to DAPI staining (taken from multiple images from n = 5 tumors per group) (E).

(F and G) Schematic representing the dosing strategy for 5-FU and/or immune-depleting anti-CD8α antibodies in *MMTV-PyMT* mice. When mice received anti-CD8α antibodies, they were also given a day-2 loading dose, which is not depicted on the schematic (F). Growth curves of established spontaneous tumors in WT, *Il6*⁻/⁻, and *Ccr5*⁻/⁻ *MMTV-PyMT* mice that were given 5-FU (40 mg/kg/4 days) or their respective vehicles and immune-depleting anti-CD8α IgG where indicated (G). Indicated dosing started at day 0 (cohorts of n = 4-9 mice).

Image in (F) was created using *BioRender* software. Bar charts show the mean, and the dots show individual data points from individual tumors and mice. Line charts display the mean and SEM. \* p < 0.05.

5-FU had no effect on tumor growth in *Il6*⁻/⁻ or *Ccr5*⁻/⁻ tumors (Figures 4F and 4G), demonstrating that CD8⁺ T cells were directly implicated in the mechanism of tumor control observed in response to chemotherapy. As such, these data highlight an intriguing association between the LYVE-1⁺ PyTAM niche and the immune landscape of the TME, which can provide a resistance mechanism for the immune-mediated effects of cytotoxic chemotherapeutics.

## LYVE-1⁺ macrophages suppress CD8⁺ T cell transendothelial migration

To investigate whether LYVE-1⁺ TAMs may be playing a direct role in immune exclusion within the TME, we established an *in vitro* assay for creating artificial Pv nests (Figure 5A). In this assay, M<sub>(IL-6)</sub>, which are analogous to the LYVE-1⁺ TAMs (Figures 2L, 2M and 2N), were seeded onto the basolateral side of a transwell insert, and then a basement membrane and an endothelial layer were seeded on the apical side of the insert (Figure 5A). The presence of M<sub>(0)</sub> or M<sub>(IL6)</sub> on the apical side did not change the permeability or integrity of the endothelial layer (Figure 5B). However, when CD8⁺ T cells were placed in contact with the endothelial layer with a gradient of the T cell chemokine CXCL10, there was a significant reduction in T cell migration

across the endothelial layer in the presence of M<sub>(IL6)</sub> (Figure 5C). HO-1 is an enzyme that breaks down heme into the biologically active catabolites biliverdin, ferrous iron (Fe²⁺), and carbon monoxide (CO), and its activity has been demonstrated to play a role in vascular biology.<sup>33,51</sup> As such, we considered whether the enzyme might play a direct role in the mechanism of CD8⁺ T cell exclusion. Inactivation of the *Hmox1* gene in the M<sub>(IL-6)</sub> BMDMs (Figure S5E) improved T cell transendothelial migration in the *in vitro* assay (Figure 5D). These data suggest that HO-1 activity, at least in part, contributed to the mechanism of T cell exclusion by M<sub>(IL-6)</sub> macrophages.

To investigate whether HO-1 expression by LYVE-1⁺ TAMs played a direct role in the immune exclusion within the PV niche, we crossed *MMTV-PyMT* to a *Hmox1*<sup>fl/fl</sup> background with and without a *Lyve1*-promoter-driven Cre-recombinase (referred to as *Lyve1*<sup>Cre</sup> or *Lyve1*<sup>WT</sup> mice, respectively) to inactivate the HO-1 protein in the TAM subset (Figures 5E and S5F). Inactivation of HO-1 selectively in the LYVE-1⁺ TAM population resulted in a non-significant change in tumor latency, with a median latency of 104 versus 108 days for *Lyve1*<sup>WT</sup> and *Lyve1*<sup>Cre</sup> mice, respectively (Figure 5F). At the cellular level within the TME, the loss of HO-1 activity in this TAM subset did not affect the total prevalence of TAMs (Figure 5G) or the TAM subsets (Figures 5H
<figure><img src="image_11.png" /><figcaption>**Figure 4.** LYVE-1⁺ TAMs and their nests are associated with immune exclusion and resistance to chemotherapy</figcaption></figure>

(A-E) Tumors from WT, *Il6*⁻/⁻, or *Ccr5*⁻/⁻ *MMTV-PyMT* mice were analyzed for their stromal composition (n = 6 tumors per group). Tumor sizes on the day of analysis (A) and the relative proportions of live (7AAD⁻) stromal cell populations were assessed using flow cytometry (B). The markers used to differentiate cells can be found in the online *STAR Methods*. Gated CD8⁺CD3⁺ T cells were assessed for their expression of the indicated effector molecules post PMA/ionomycin treatment, shown as percentage of positive cells (C), and the MFI of the expression of the gated positive cells (D). CD31⁺ cell density was assessed in tumor sections by immunofluorescence and quantitated relative to DAPI staining (taken from multiple images from n = 5 tumors per group) (E).

(F and G) Schematic representing the dosing strategy for 5-FU and/or immune-depleting anti-CD8α antibodies in *MMTV-PyMT* mice. When mice received anti-CD8α antibodies, they were also given a day-2 loading dose, which is not depicted on the schematic (F). Growth curves of established spontaneous tumors in WT, *Il6*⁻/⁻, and *Ccr5*⁻/⁻ *MMTV-PyMT* mice that were given 5-FU (40 mg/kg/4 days) or their respective vehicles and immune-depleting anti-CD8α IgG where indicated (G). Indicated dosing started at day 0 (cohorts of n = 4-9 mice).

Image in (F) was created using *BioRender* software. Bar charts show the mean, and the dots show individual data points from individual tumors and mice. Line charts display the mean and SEM. \* p < 0.05.

5-FU had no effect on tumor growth in *Il6*⁻/⁻ or *Ccr5*⁻/⁻ tumors (Figures 4F and 4G), demonstrating that CD8⁺ T cells were directly implicated in the mechanism of tumor control observed in response to chemotherapy. As such, these data highlight an intriguing association between the LYVE-1⁺ PyTAM niche and the immune landscape of the TME, which can provide a resistance mechanism for the immune-mediated effects of cytotoxic chemotherapeutics.

## LYVE-1⁺ macrophages suppress CD8⁺ T cell transendothelial migration

To investigate whether LYVE-1⁺ TAMs may be playing a direct role in immune exclusion within the TME, we established an *in vitro* assay for creating artificial Pv nests (Figure 5A). In this assay, M<sub>(IL-6)</sub>, which are analogous to the LYVE-1⁺ TAMs (Figures 2L, 2M and 2N), were seeded onto the basolateral side of a transwell insert, and then a basement membrane and an endothelial layer were seeded on the apical side of the insert (Figure 5A). The presence of M<sub>(0)</sub> or M<sub>(IL6)</sub> on the apical side did not change the permeability or integrity of the endothelial layer (Figure 5B). However, when CD8⁺ T cells were placed in contact with the endothelial layer with a gradient of the T cell chemokine CXCL10, there was a significant reduction in T cell migration

across the endothelial layer in the presence of M<sub>(IL6)</sub> (Figure 5C). HO-1 is an enzyme that breaks down heme into the biologically active catabolites biliverdin, ferrous iron (Fe²⁺), and carbon monoxide (CO), and its activity has been demonstrated to play a role in vascular biology.<sup>33,51</sup> As such, we considered whether the enzyme might play a direct role in the mechanism of CD8⁺ T cell exclusion. Inactivation of the *Hmox1* gene in the M<sub>(IL-6)</sub> BMDMs (Figure S5E) improved T cell transendothelial migration in the *in vitro* assay (Figure 5D). These data suggest that HO-1 activity, at least in part, contributed to the mechanism of T cell exclusion by M<sub>(IL-6)</sub> macrophages.

To investigate whether HO-1 expression by LYVE-1⁺ TAMs played a direct role in the immune exclusion within the PV niche, we crossed *MMTV-PyMT* to a *Hmox1*<sup>fl/fl</sup> background with and without a *Lyve1*-promoter-driven Cre-recombinase (referred to as *Lyve1*<sup>Cre</sup> or *Lyve1*<sup>WT</sup> mice, respectively) to inactivate the HO-1 protein in the TAM subset (Figures 5E and S5F). Inactivation of HO-1 selectively in the LYVE-1⁺ TAM population resulted in a non-significant change in tumor latency, with a median latency of 104 versus 108 days for *Lyve1*<sup>WT</sup> and *Lyve1*<sup>Cre</sup> mice, respectively (Figure 5F). At the cellular level within the TME, the loss of HO-1 activity in this TAM subset did not affect the total prevalence of TAMs (Figure 5G) or the TAM subsets (Figures 5H
<figure><img src="image_12.png" /><figcaption>Figure 5. HO-1 expression by LYVE-1+ TAMs facilitates immune exclusion and resistance to chemotherapy</figcaption></figure>

(A–D) An *in vitro* Pv niche assay was established to investigate the role of Pv macrophages in CD8⁺ T cell transendothelial migration. A schematic of the assay (A). Permeability of the endothelial cell layer at the indicated time points to albumin in the presence or absence of M(I₀) or M(IL-6) cells (B). The relative transendothelial migration of CD8⁺ T cells in the presence or absence of M(I₀) or M(IL-6) cells on the basolateral surface (C), and the effect of genetic knockout of HO-1 (HO-1¹^KO) in M(IL-6) (using *Hmox1*fl/fl × *Lyz2*Cre mice) (D).

(E) Western blot for HO-1 and β-actin expressions in BMDMs under M(I₀) (MCF alone) and IL-6 polarization (M(IL-6)) conditions from MMTV-PyMT × *Hmox1*fl/fl mice with (*Lyve1*Cre) or without (*Lyve1*WT) Cre-recombinase driven from the *Lyve1* promoter.

(F) Kaplan-Meier curve showing the tumor latency for *Lyve1*WT (n = 10) and *Lyve1*Cre (n = 10) mice; HR, hazard ratio.

(G–J) Tumors from *Lyve1*WT and *Lyve1*Cre mice were enzyme dispersed and assessed using flow cytometry for the abundance of live (7AAD⁻) CD45⁺F4/80⁺Ly6C⁻ TAMs (G) and their phenotype as proportions of the TAM gate (H) and the abundance of LYVE-1⁺ TAMs as the percentage of live cells (I) and CD8⁺ and CD4⁺ T cells (J) (n = 4 per group).

(K and L) Schematic representing the dosing strategy for 5-FU in tumor-bearing *Lyve1*WT and *Lyve1*Cre mice (K). Growth curves of established spontaneous tumors that were given vehicle or 5-FU (40 mg/kg/4 days) where indicated. Indicated dosing started at day 0 (cohorts of n = 5–7 mice) (L).

(M) Schematic overview of the Pv niche and stepwise development and function of LYVE-1⁺ TAMs. Images in (E), (K), and (M) were created using BioRender software.

Bar charts show the mean, and the dots show individual data points from individual tumors and mice. Line charts display the mean and SEM. \* p < 0.05 and \*\* p < 0.01.

and 5l). However, in concordance with the *in vitro* observations and a wider role of LYVE-1⁺ TAMs in immune exclusion, the genetic inactivation of HO-1 in LYVE-1⁺ TAMs in the Pv niche in mice resulted in an improved and selective infiltration of CD8⁺ T cells into the TME (Figure 5J). Furthermore, the loss of HO-1 activity in the LYVE-1⁺ TAM subset rendered the tumors sensitive to chemotherapy treatment (Figures 5K and 5L). Collectively, these *in vitro* and *in vivo* data suggest that LYVE-1⁺ TAMs serve as gatekeepers in modulating CD8⁺ T cell entry into the tumor,

which dictates the sensitivity of the TME to the immune-stimulating effects of chemotherapy, and our data highlight HO-1 as a key effector molecule in this axis.

DISCUSSION

In this study, we describe a coordinated and collaborative role for LYVE-1⁺ PvTAMs in forming multi-cellular nest structures within the TME, which influence the immune-mediated
<figure><img src="image_12.png" /><figcaption>**Figure 5. HO-1 expression by LYVE-1+ TAMs facilitates immune exclusion and resistance to chemotherapy**</figcaption></figure>

(A–D) An *in vitro* Pv niche assay was established to investigate the role of Pv macrophages in CD8⁺ T cell transendothelial migration. A schematic of the assay (A). Permeability of the endothelial cell layer at the indicated time points to albumin in the presence or absence of M(I₀) or M(IL-6) cells (B). The relative transendothelial migration of CD8⁺ T cells in the presence or absence of M(I₀) or M(IL-6) cells on the basolateral surface (C), and the effect of genetic knockout of HO-1 (HO-1¹^KO) in M(IL-6) (using *Hmox1*fl/fl × *Lyz2*Cre mice) (D).

(E) Western blot for HO-1 and β-actin expressions in BMDMs under M(I₀) (MCF alone) and IL-6 polarization (M(IL-6)) conditions from *MMTV-PyMT × Hmox1fl/fl* mice with (*Lyve1*Cre) or without (*Lyve1*WT) Cre-recombinase driven from the *Lyve1* promoter.

(F) Kaplan-Meier curve showing the tumor latency for *Lyve1*WT (n = 10) and *Lyve1*Cre (n = 10) mice; HR, hazard ratio.

(G–J) Tumors from *Lyve1*WT and *Lyve1*Cre mice were enzyme dispersed and assessed using flow cytometry for the abundance of live (7AAD⁻) CD45⁺F4/80⁺Ly6C⁻ TAMs (G) and their phenotype as proportions of the TAM gate (H) and the abundance of LYVE-1⁺ TAMs as the percentage of live cells (I) and CD8⁺ and CD4⁺ T cells (J) (n = 4 per group).

(K and L) Schematic representing the dosing strategy for 5-FU in tumor-bearing *Lyve1*WT and *Lyve1*Cre mice (K). Growth curves of established spontaneous tumors that were given vehicle or 5-FU (40 mg/kg/4 days) where indicated. Indicated dosing started at day 0 (cohorts of n = 5–7 mice) (L).

(M) Schematic overview of the Pv niche and stepwise development and function of LYVE-1⁺ TAMs. Images in (E), (K), and (M) were created using BioRender software.

Bar charts show the mean, and the dots show individual data points from individual tumors and mice. Line charts display the mean and SEM. \* p < 0.05 and \*\* p < 0.01.

and 5I). However, in concordance with the *in vitro* observations and a wider role of LYVE-1⁺ TAMs in immune exclusion, the genetic inactivation of HO-1 in LYVE-1⁺ TAMs in the Pv niche in mice resulted in an improved and selective infiltration of CD8⁺ T cells into the TME (Figure 5J). Furthermore, the loss of HO-1 activity in the LYVE-1⁺ TAM subset rendered the tumors sensitive to chemotherapy treatment (Figures 5K and 5L). Collectively, these *in vitro* and *in vivo* data suggest that LYVE-1⁺ TAMs serve as gatekeepers in modulating CD8⁺ T cell entry into the tumor,

which dictates the sensitivity of the TME to the immune-stimulating effects of chemotherapy, and our data highlight HO-1 as a key effector molecule in this axis.

DISCUSSION

In this study, we describe a coordinated and collaborative role for LYVE-1⁺ PvTAMs in forming multi-cellular nest structures within the TME, which influence the immune-mediated
responses of cytotoxic chemotherapy. This study sheds light on the development of the LYVE-1⁺ TAM subset in cancer. Using photolabeling approaches, we demonstrate that LYVE-1⁺ TAMs develop from a progenitor cell recruited into the TME rather than a tissue-resident macrophage source, consistent with previous studies that have demonstrated that PvTAMs derive from a monocyte origin.<sup>11,25,52</sup> PvTAMs can develop through a sequential signaling program involving their upregulation of CXCR4 in response to tumor-cell-derived TGF-β, which guides their migration back to the endothelium on a gradient of CXCL12 expressed by a PvCAF population.<sup>11</sup> The importance of the CXCR4/CXCL12 axis in PvTAM accumulation at the vasculature has been demonstrated in several studies.<sup>20,53</sup> We propose that the mechanisms presented in this study provide insight on the subsequent developmental step of PvTAMs into the LYVE-1⁺ TAM population post reaching the endothelium. This is supported by the observation that the loss of IL-6 resulted in a striking and specific loss of the LYVE-1⁺ TAM population, leaving other TAM polarization states unchanged, suggesting that it is important for only the terminal step in polarization. The interaction of angiopoietin-2 expressed on ECs and Tie2 expressed on PvTAMs has also been demonstrated to play a role in their accumulation at the vasculature<sup>54,55</sup>; however, we did not find evidence of *Tie2/Tek* gene expression in LYVE-1⁺ TAMs, suggesting that these cells may be a discrete subset.

We demonstrate that a key feature of the LYVE-1⁺ TAM program was a high expression of the enzyme HO-1, a gene that we have previously linked to IL-6 signaling.<sup>3</sup> We identified *in vitro* that c-MAF signaling was required for LYVE-1, HO-1, and CD206 expressions on BMDMs in response to IL-6 in a STAT3/c-MAF-dependent pathway. This may explain the similarity of the population to tissue-resident macrophages, as these have recently been demonstrated to be dependent on c-MAF signaling.<sup>37</sup> Although the signal-inducing c-MAF in healthy tissues is unknown, it could explain the unexpected high expression of HO-1, which is generally considered as a stress- or inflammation-inducible enzyme.<sup>51,56</sup>

The identification of the immunological resistance mechanisms to cytotoxic chemotherapy responses are important, as it has become apparent that the immune-stimulating properties of these drugs may underlie a significant proportion of their anti-tumor efficacy.<sup>57-62</sup> This study also complements the wider association between TAMs playing a pivotal role in the resistance of the tumor to chemotherapeutic drugs<sup>6-8,47</sup> and facilitating relapse after the cessation of treatment.<sup>20</sup> Furthermore, this study provides further support for the link between IL-6 and the immune-stimulating effects of cytotoxic chemotherapies.<sup>63</sup> There are many studies that have described HO-1 as having pro-tumoral properties with important roles in immune suppression and cytoprotection.<sup>34,51,64-68</sup> Many of the effects of HO-1 have been attributed to its production of CO as a catabolite of heme degradation, which can modulate several signaling pathways, including p38 MAPK,<sup>69</sup> STAT1/3,<sup>70</sup> and NF-κB.<sup>71,72</sup> We previously demonstrated in the *MMTV-PyMT* model that HO-1 plays a pivotal role in the suppression of anti-tumor CD8<sup>+</sup> T cell responses elicited by chemotherapy in *MMTV-PyMT* mice.<sup>47</sup> This study builds on this previous observation to demonstrate the importance of CCR5 signaling in the chemotherapy sensitivity of the TME, a chemokine induced as part of

the IL-6 polarization program of Pv LYVE-1⁺ TAMs, which are the exclusive source of HO-1 in the TME. We show that in the absence of CCR5, the spatial arrangement and cluster of LYVE-1⁺ TAMs in the PV niche are hindered. Collectively, these data highlight a potential link between the spatial arrangement of this TAM subset and the maintenance of an immunologically "cold" TME through CD8⁺ T cell exclusion via an HO-1-dependent mechanism, which renders the TME resistant to the immune-stimulating effects of chemotherapy. This also highlights a potential biological function of TAMs that is associated with their ability to work in concert with one another to achieve this biological effect. It has been demonstrated that macrophage density can also relate to a "quorum licensing" of macrophage activation<sup>73</sup>; it would be interesting to understand how the nest structures might also influence or refine the effector function of LYVE-1⁺ TAMs in the context of cancer.

It is clear that PvTAMs reside in unique niche arrangements to support their function, such as the described TMEs of metastasis (TMEM), where a PvTAM, a tumor cell expressing a slice variant of mammalian-enabled protein "Mena,"<sup>74</sup> and an EC are in direct contact to facilitate the transendothelial migration of tumor cells into the blood from the tumor.<sup>18,75-78</sup> Recently, an elegant study by Nalio Ramos et al.<sup>15</sup> demonstrated in human breast cancer that a folate receptor beta (FOLR2)⁺cell adhesion molecule 1 (CADM1)⁻ human leukocyte antigen-DR (HLA-DR)⁺ PvTAM subset (which co-express *LYVE-1* and *MRC1*) was associated with CD8⁺ T cell infiltration into the TME and *ex vivo* had the potential to prime CD8⁺ T cell effector function. However, FOLR2⁺ TAMs have also been associated with immune-suppressive microenvironments in hepatocellular carcinoma.<sup>27</sup> Interestingly, in *MMTV-PyMT* tumors, we identified two CD206⁺ TAM subsets, which both express *Folr2* (Figure S5G). Taken together, this raises the possibility that LYVE-1⁺CD206⁺MHCII<sup>lo</sup> and LYVE-1⁻CD206⁺MHCII<sup>hi</sup> TAM subsets might play discrete, potentially opposing, roles in relation to modulating the immune landscape of the TME. As such, the heterogeneity of PvTAMs and their respective functions still requires further investigation.

In this study, we characterize the functionality associated with a multi-cellular LYVE-1⁺ PvTAM structure and define an unappreciated collaborative action of these cells to form a multi-cellular niche, which are associated with the immune exclusion of CD8⁺ T cells from the TME dependent on their expression of HO-1. We demonstrate that IL-6-driven CCR5 expression by LYVE-1⁺ TAMs supports a dynamic and ongoing TAM>TAM communication axis, which is pivotal to the formation and maintenance of their nest structures. Interestingly, the blockade of CCR5 using maraviroc has been explored in patients with metastatic colorectal cancer (NCT01736813), where 4/6 patients showed a trend toward an increase in CD8⁺ T cells within the TME.<sup>79</sup> Moreover, in this study, the authors identified a partial response (3/5 patients) and stable disease (1/5 patients) when maraviroc was combined with a chemotherapeutic agent.<sup>79</sup> Although tentative, it highlights intriguing key clinical parallels of our preclinical observations.

In summary (Figure 5M), we show that LYVE-1⁺ TAMs derive from an IL-6 polarization program in the TME and demonstrate that the LYVE-1⁺ TAM functions are not always autonomous but can be collaborative through their formation of nests within the Pv space using a CCR5-dependent communication axis.
responses of cytotoxic chemotherapy. This study sheds light on the development of the LYVE-1⁺ TAM subset in cancer. Using photolabeling approaches, we demonstrate that LYVE-1⁺ TAMs develop from a progenitor cell recruited into the TME rather than a tissue-resident macrophage source, consistent with previous studies that have demonstrated that PvTAMs derive from a monocyte origin.¹¹,²⁵,⁵² PvTAMs can develop through a sequential signaling program involving their upregulation of CXCR4 in response to tumor-cell-derived TGF-β, which guides their migration back to the endothelium on a gradient of CXCL12 expressed by a PvCAF population.¹¹ The importance of the CXCR4/CXCL12 axis in PvTAM accumulation at the vasculature has been demonstrated in several studies.²⁰,⁵³ We propose that the mechanisms presented in this study provide insight on the subsequent developmental step of PvTAMs into the LYVE-1⁺ TAM population post reaching the endothelium. This is supported by the observation that the loss of IL-6 resulted in a striking and specific loss of the LYVE-1⁺ TAM population, leaving other TAM polarization states unchanged, suggesting that it is important for only the terminal step in polarization. The interaction of angiopoietin-2 expressed on ECs and Tie2 expressed on PvTAMs has also been demonstrated to play a role in their accumulation at the vasculature⁵⁴,⁵⁵; however, we did not find evidence of *Tie2/Tek* gene expression in LYVE-1⁺ TAMs, suggesting that these cells may be a discrete subset.

We demonstrate that a key feature of the LYVE-1⁺ TAM program was a high expression of the enzyme HO-1, a gene that we have previously linked to IL-6 signaling.³ We identified *in vitro* that c-MAF signaling was required for LYVE-1, HO-1, and CD206 expressions on BMDMs in response to IL-6 in a STAT3/c-MAF-dependent pathway. This may explain the similarity of the population to tissue-resident macrophages, as these have recently been demonstrated to be dependent on c-MAF signaling.³⁷ Although the signal-inducing c-MAF in healthy tissues is unknown, it could explain the unexpected high expression of HO-1, which is generally considered as a stress- or inflammation-inducible enzyme.⁵¹,⁵⁶

The identification of the immunological resistance mechanisms to cytotoxic chemotherapy responses are important, as it has become apparent that the immune-stimulating properties of these drugs may underlie a significant proportion of their anti-tumor efficacy.⁵⁷⁻⁶² This study also complements the wider association between TAMs playing a pivotal role in the resistance of the tumor to chemotherapeutic drugs⁶⁻⁸,⁴⁷ and facilitating relapse after the cessation of treatment.²⁰ Furthermore, this study provides further support for the link between IL-6 and the immune-stimulating effects of cytotoxic chemotherapies.⁶³ There are many studies that have described HO-1 as having pro-tumoral properties with important roles in immune suppression and cytoprotection.³⁴,⁵¹,⁶⁴⁻⁶⁸ Many of the effects of HO-1 have been attributed to its production of CO as a catabolite of heme degradation, which can modulate several signaling pathways, including p38 MAPK,⁶⁹ STAT1/3,⁷⁰ and NF-κB.⁷¹,⁷² We previously demonstrated in the *MMTV-PyMT* model that HO-1 plays a pivotal role in the suppression of anti-tumor CD8⁺ T cell responses elicited by chemotherapy in *MMTV-PyMT* mice.⁴⁷ This study builds on this previous observation to demonstrate the importance of CCR5 signaling in the chemotherapy sensitivity of the TME, a chemokine induced as part of

the IL-6 polarization program of Pv LYVE-1⁺ TAMs, which are the exclusive source of HO-1 in the TME. We show that in the absence of CCR5, the spatial arrangement and cluster of LYVE-1⁺ TAMs in the PV niche are hindered. Collectively, these data highlight a potential link between the spatial arrangement of this TAM subset and the maintenance of an immunologically “cold” TME through CD8⁺ T cell exclusion via an HO-1-dependent mechanism, which renders the TME resistant to the immune-stimulating effects of chemotherapy. This also highlights a potential biological function of TAMs that is associated with their ability to work in concert with one another to achieve this biological effect. It has been demonstrated that macrophage density can also relate to a “quorum licensing” of macrophage activation⁷³; it would be interesting to understand how the nest structures might also influence or refine the effector function of LYVE-1⁺ TAMs in the context of cancer.

It is clear that PvTAMs reside in unique niche arrangements to support their function, such as the described TMEs of metastasis (TMEM), where a PvTAM, a tumor cell expressing a slice variant of mammalian-enabled protein “Mena,”⁷⁴ and an EC are in direct contact to facilitate the transendothelial migration of tumor cells into the blood from the tumor.¹⁸,⁷⁵⁻⁷⁸ Recently, an elegant study by Nalio Ramos et al.¹⁵ demonstrated in human breast cancer that a folate receptor beta (FOLR2)⁺cell adhesion molecule 1 (CADM1)⁻ human leukocyte antigen-DR (HLA-DR)⁺ PvTAM subset (which co-express *LYVE-1* and *MRC1*) was associated with CD8⁺ T cell infiltration into the TME and *ex vivo* had the potential to prime CD8⁺ T cell effector function. However, FOLR2⁺ TAMs have also been associated with immune-suppressive microenvironments in hepatocellular carcinoma.²⁷ Interestingly, in *MMTV-PyMT* tumors, we identified two CD206⁺ TAM subsets, which both express *Folr2* (Figure S5G). Taken together, this raises the possibility that LYVE-1⁺CD206⁺MHCIIlo and LYVE-1⁻CD206⁺MHCIIhi TAM subsets might play discrete, potentially opposing, roles in relation to modulating the immune landscape of the TME. As such, the heterogeneity of PvTAMs and their respective functions still requires further investigation.

In this study, we characterize the functionality associated with a multi-cellular LYVE-1⁺ PvTAM structure and define an unappreciated collaborative action of these cells to form a multi-cellular niche, which are associated with the immune exclusion of CD8⁺ T cells from the TME dependent on their expression of HO-1. We demonstrate that IL-6-driven CCR5 expression by LYVE-1⁺ TAMs supports a dynamic and ongoing TAM>TAM communication axis, which is pivotal to the formation and maintenance of their nest structures. Interestingly, the blockade of CCR5 using maraviroc has been explored in patients with metastatic colorectal cancer (NCT01736813), where 4/6 patients showed a trend toward an increase in CD8⁺ T cells within the TME.⁷⁹ Moreover, in this study, the authors identified a partial response (3/5 patients) and stable disease (1/5 patients) when maraviroc was combined with a chemotherapeutic agent.⁷⁹ Although tentative, it highlights intriguing key clinical parallels of our preclinical observations.

In summary (Figure 5M), we show that LYVE-1⁺ TAMs derive from an IL-6 polarization program in the TME and demonstrate that the LYVE-1⁺ TAM functions are not always autonomous but can be collaborative through their formation of nests within the Pv space using a CCR5-dependent communication axis.
We demonstrate that these multi-cellular nest structures are associated with immune exclusion in the TME, which is linked to the immune-mediated effects of cytotoxic chemotherapies. This study sheds light on the communication and niche arrangements of a TAM subset and their association with the outcome of chemotherapy, which could provide therapeutic opportunities for targeting their pro-tumoral functions in cancer.

## Limitations of the study

This study demonstrates that LYVE-1⁺ TAM expression of HO-1 facilitates immune exclusion in the tumor; however, the specific mechanism by which this exclusion occurs at the cellular/molecular level has not yet been deciphered, and additional experiments will be required to explore this. Additionally, although we validated that LYVE-1⁺ TAM nests exist in human breast cancer, the experiments in this study were primarily conducted in mice and in a single, albeit spontaneous, murine model. Therefore, further experiments utilizing further mouse models and human breast cancer tissue (as well as other cancer tissues) would be needed to better define the inter-tumor and inter-species heterogeneity of the LYVE-1⁺ TAM population. This study also used a range of knockout (KO) models; however, to further elucidate the biological implications, formation, and function of the LYVE-1⁺ TAM nests, the implementation of models that would enable the KO of IL-6R or CCR5 selectively on the LYVE-1⁺ TAM population would be a desirable next step.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **RESOURCE AVAILABILITY**

* Lead contact

* Material availability

* Data and code availability

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* Mice

* Cell lines

* Study approval

* **METHOD DETAILS**

* Tumor studies

* *In vitro* derived macrophage polarization and gene knockdown

* T-cell isolation

* *In vitro* macrophage transwell migration assay

* *In vitro* macrophage focal-point migration assay

* *In vitro* Pv nest transwell assay

* Western blot

* Immunofluorescence

* Bioluminescence imaging

* Flow cytometry

* Quantitative real time PCR

* Transcriptomic data and analysis

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.devcel.2023.06.006.

## ACKNOWLEDGMENTS

The authors thank Dr. Yasmin Haque (KCL) and the NIHR BRC flow cytometry platform at Guy's and St Thomas' Biomedical Research Centre for cell sorting and flow cytometry assistance and Dr. James Levitt and the Nikon Imaging Centre (KCL) for the use of their facilities and assistance with confocal microscopy analyses. The authors would like to thank Drs. Paul Lavender, Tracey Mitchell, Gilbert Fruhwirth (KCL), and Professor Awen Gallimore (University of Cardiff) for useful discussion/advice/support; Miss Rosamond Nuamah (KCL) for running the microarray; and Mr. Stuart Newman (KCL) for help with the rederivation of the HO-1<sup>Luc/EGFP</sup> reporter mouse. The authors thank Professor George Kollias (University of Athens) for the use of the *Hmox1<sup>fl/fl</sup>* allele mice. The authors would like to thank Miss Jiaying Yao for the initial optimization of the focal-point macrophage migration assay. This work was funded by the following grants: Cancer Research UK grant DCRPGF100009 (J.N.A.), European Research Council grant 335326 (J.N.A.), Cancer Research Institute/Wade F.B. Thompson CLIP grant (CRI3645) (J.N.A.), Medical Research Council grant MR/N013700/1 (J.E.A.), Medical Research Council grant MR/PO18823/1 (F.M.W.), Wellcome Trust grant 206439/Z/17/Z (F.M.W.), and Cancer Research UK grant C54019/A27535 (D.R.W.). The research was supported by the Cancer Research UK King's Health Partners Centre and Experimental Cancer Medicine Centre at King's College London and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health.

## AUTHOR CONTRIBUTIONS

J.E.A. and J.N.A. conceived the project, designed the approach, performed the experiments, interpreted the data, and wrote the manuscript. K.T.F., J.W.O., I.D., H.P.M., M.B., T.S.C., K.L.-A., Z.L., D.C., J.C., D.S., R.B., T.M., Z.A., and G.L. designed the approach, performed the experiments, and interpreted the data. C.E.G., X.Z., F.M.W., T.N., J.M.B., S.K., D.R.W., and T.L. designed the experiments, interpreted the data, and provided key expertise.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## INCLUSION AND DIVERSITY

We support inclusive, diverse, and equitable conduct of research.

Received: June 23, 2022

Revised: April 5, 2023

Accepted: June 20, 2023

Published: July 12, 2023

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2. DeNardo, D.G., Barreto, J.B., Andreu, P., Vasquez, L., Tawfik, D., Kolhatkar, N., and Coussens, L.M. (2009). CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell **16**, 91–102. https://doi.org/10.1016/j.ccr.2009.06.018.

3. Muliaditan, T., Caron, J., Okesola, M., Opzoomer, J.W., Kosti, P., Georgouli, M., Gordon, P., Lall, S., Kuzeva, D.M., Pedro, L., et al. (2018). Macrophages are exploited from an innate wound healing response to
We demonstrate that these multi-cellular nest structures are associated with immune exclusion in the TME, which is linked to the immune-mediated effects of cytotoxic chemotherapies. This study sheds light on the communication and niche arrangements of a TAM subset and their association with the outcome of chemotherapy, which could provide therapeutic opportunities for targeting their pro-tumoral functions in cancer.

## Limitations of the study

This study demonstrates that LYVE-1⁺ TAM expression of HO-1 facilitates immune exclusion in the tumor; however, the specific mechanism by which this exclusion occurs at the cellular/molecular level has not yet been deciphered, and additional experiments will be required to explore this. Additionally, although we validated that LYVE-1⁺ TAM nests exist in human breast cancer, the experiments in this study were primarily conducted in mice and in a single, albeit spontaneous, murine model. Therefore, further experiments utilizing further mouse models and human breast cancer tissue (as well as other cancer tissues) would be needed to better define the inter-tumor and inter-species heterogeneity of the LYVE-1⁺ TAM population. This study also used a range of knockout (KO) models; however, to further elucidate the biological implications, formation, and function of the LYVE-1⁺ TAM nests, the implementation of models that would enable the KO of IL-6R or CCR5 selectively on the LYVE-1⁺ TAM population would be a desirable next step.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **RESOURCE AVAILABILITY**

* Lead contact

* Material availability

* Data and code availability

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* Mice

* Cell lines

* Study approval

* **METHOD DETAILS**

* Tumor studies

* *In vitro* derived macrophage polarization and gene knockdown

* T-cell isolation

* *In vitro* macrophage transwell migration assay

* *In vitro* macrophage focal-point migration assay

* *In vitro* Pv nest transwell assay

* Western blot

* Immunofluorescence

* Bioluminescence imaging

* Flow cytometry

* Quantitative real time PCR

* Transcriptomic data and analysis

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.devcel.2023.06.006.

## ACKNOWLEDGMENTS

The authors thank Dr. Yasmin Haque (KCL) and the NIHR BRC flow cytometry platform at Guy's and St Thomas' Biomedical Research Centre for cell sorting and flow cytometry assistance and Dr. James Levitt and the Nikon Imaging Centre (KCL) for the use of their facilities and assistance with confocal microscopy analyses. The authors would like to thank Drs. Paul Lavender, Tracey Mitchell, Gilbert Fruhwirth (KCL), and Professor Awen Gallimore (University of Cardiff) for useful discussion/advice/support; Miss Rosamond Nuamah (KCL) for running the microarray; and Mr. Stuart Newman (KCL) for help with the rederivation of the HO-1<sup>Luc/EGFP</sup> reporter mouse. The authors thank Professor George Kollias (University of Athens) for the use of the *Hmox1<sup>fl/fl</sup>* allele mice. The authors would like to thank Miss Jiaying Yao for the initial optimization of the focal-point macrophage migration assay. This work was funded by the following grants: Cancer Research UK grant DCRPGF100009 (J.N.A.), European Research Council grant 335326 (J.N.A.), Cancer Research Institute/Wade F.B. Thompson CLIP grant (CRI3645) (J.N.A.), Medical Research Council grant MR/N013700/1 (J.E.A.), Medical Research Council grant MR/PO18823/1 (F.M.W.), Wellcome Trust grant 206439/Z/17/Z (F.M.W.), and Cancer Research UK grant C54019/A27535 (D.R.W.). The research was supported by the Cancer Research UK King's Health Partners Centre and Experimental Cancer Medicine Centre at King's College London and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health.

## AUTHOR CONTRIBUTIONS

J.E.A. and J.N.A. conceived the project, designed the approach, performed the experiments, interpreted the data, and wrote the manuscript. K.T.F., J.W.O., I.D., H.P.M., M.B., T.S.C., K.L.-A., Z.L., D.C., J.C., D.S., R.B., T.M., Z.A., and G.L. designed the approach, performed the experiments, and interpreted the data. C.E.G., X.Z., F.M.W., T.N., J.M.B., S.K., D.R.W., and T.L. designed the experiments, interpreted the data, and provided key expertise.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## INCLUSION AND DIVERSITY

We support inclusive, diverse, and equitable conduct of research.

Received: June 23, 2022

Revised: April 5, 2023

Accepted: June 20, 2023

Published: July 12, 2023

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17. Lewis, C.E., Harney, A.S., and Pollard, J.W. (2016). The multifaceted role of perivascular macrophages in tumors. Cancer Cell 30, 365. https://doi.org/10.1016/j.ccell.2016.07.009.

18. Wyckoff, J.B., Wang, Y., Lin, E.Y., Li, J.F., Goswami, S., Stanley, E.R., Segall, J.E., Pollard, J.W., and Condeelis, J. (2007). Direct visualization

of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res. 67, 2649–2656. https://doi.org/10.1158/0008-5472.CAN-06-1823.

19. Harney, A.S., Arwert, E.N., Entenberg, D., Wang, Y., Guo, P., Qian, B.Z., Oktay, M.H., Pollard, J.W., Jones, J.G., and Condeelis, J.S. (2015). Real-time imaging reveals local, transient vascular permeability, and tumor cell intravasation stimulated by TIE2hi macrophage-derived VEGFA. Cancer Discov. 5, 932–943. https://doi.org/10.1158/2159-8290.CD-15-0012.

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22. Cho, C.H., Koh, Y.J., Han, J., Sung, H.K., Jong Lee, H., Morisada, T., Schwendener, R.A., Brekken, R.A., Kang, G., Oike, Y., et al. (2007). Angiogenic role of LYVE-1-positive macrophages in adipose tissue. Circ. Res. 100, e47–e57. https://doi.org/10.1161/01 RES.0000259564.92792.93.

23. Pinto, A.R., Paolicelli, R., Salimova, E., Gospocic, J., Slonimsky, E., Bilbao-Cortes, D., Godwin, J.W., and Rosenthal, N.A. (2012). An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile. PLoS One 7, e36814. https://doi.org/10.1371/journal.pone.0036814.

24. Xu, H., Chen, M., Reid, D.M., and Forrester, J.V. (2007). LYVE-1-positive macrophages are present in normal murine eyes. Invest. Ophthalmol. Vis. Sci. 48, 2162–2171. https://doi.org/10.1167/iovs.06-0783.

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27. Sharma, A., Seow, J.J.W., Dutertre, C.A., Pai, R., Blériot, C., Mishra, A., Wong, R.M.M., Singh, G.S.N., Sudhagar, S., Khalilnezhad, S., et al. (2020). Onco-fetal reprogramming of endothelial cells drives immunosuppressive macrophages in hepatocellular carcinoma. Cell 183, 377–394.e21. https://doi.org/10.1016/j.cell.2020.08.040.

28. Wu, S.Z., Al-Eryani, G., Roden, D.L., Junankar, S., Harvey, K., Andersson, A., Thennavan, A., Wang, C., Torpy, J.R., Bartonicek, N., et al. (2021). A single-cell and spatially resolved atlas of human breast cancers. Nat. Genet. 53, 1334–1347. https://doi.org/10.1038/s41588-021-00911-1.

29. Jackson, D.G., Prevo, R., Clasper, S., and Banerji, S. (2001). LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol. 22, 317–321. https://doi.org/10.1016/s1471-4906(01)01936-6.

30. Kosti, P., Opzoomer, J.W., Larios-Martinez, K.I., Henley-Smith, R., Scudamore, C.L., Okesola, M., Taher, M.Y.M., Davies, D.M., Muliaditan, T., Larcombe-Young, D., et al. (2021). Hypoxia-sensing CAR T cells provide safety and efficacy in treating solid tumors. Cell Rep. Med. 2, 100227. https://doi.org/10.1016/j.ccrm.2021.100227.

31. Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park, J.H., Lee, K.Y., Kim, M.K., Shin, B.A., and Choi, S.Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, e18556. https://doi.org/10.1371/journal.pone.0018556.

32. Spranger, S., Dai, D., Horton, B., and Gajewski, T.F. (2017). Tumor-residing Batf3 dendritic cells are required for effector T cell trafficking
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4. Murray, P.J., Allen, J.E., Biswas, S.K., Fisher, E.A., Gilroy, D.W., Goerdt, S., Gordon, S., Hamilton, J.A., Ivashkiv, L.B., Lawrence, T., et al. (2014). Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41, 14–20. https://doi.org/10.1016/j.immuni.2014.06.008.

5. Noy, R., and Pollard, J.W. (2014). Tumor-associated macrophages: from mechanisms to therapy. Immunity 41, 866. https://doi.org/10.1016/j.immuni.2014.09.021.

6. Paulus, P., Stanley, E.R., Schäfer, R., Abraham, D., and Aharinejad, S. (2006). Colony-stimulating factor-1 antibody reverses chemoresistance in human MCF-7 breast cancer xenografts. Cancer Res. 66, 4349–4356. https://doi.org/10.1158/0008-5472.CAN-05-3523.

7. DeNardo, D.G., Brennan, D.J., Rexhepaj, E., Ruffell, B., Shiao, S.L., Madden, S.F., Gallagher, W.M., Wadhwani, N., Keil, S.D., Junaid, S.A., et al. (2011). Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov. 1, 54–67. https://doi.org/10.1158/2159-8274.CD-10-0028.

8. Ruffell, B., Chang-Strachan, D., Chan, V., Rosenbusch, A., Ho, C.M., Pryer, N., Daniel, D., Hwang, E.S., Rugo, H.S., and Coussens, L.M. (2014). Macrophage IL-10 blocks CD8+ T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells. Cancer Cell 26, 623–637. https://doi.org/10.1016/j.ccell.2014.09.006.

9. Franklin, R.A., Liao, W., Sarkar, A., Kim, M.V., Bivona, M.R., Liu, K., Pamer, E.G., and Li, M.O. (2014). The cellular and molecular origin of tumor-associated macrophages. Science 344, 921–925. https://doi.org/10.1126/science.1252510.

10. Colegio, O.R., Chu, N.Q., Szabo, A.L., Chu, T., Rhebergen, A.M., Jairam, V., Cyrus, N., Brokowski, C.E., Eisenbarth, S.C., Phillips, G.M., et al. (2014). Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 513, 559–563. https://doi.org/10.1038/nature13490.

11. Arwert, E.N., Harney, A.S., Entenberg, D., Wang, Y., Sahai, E., Pollard, J.W., and Condeelis, J.S. (2018). A unidirectional transition from migratory to perivascular macrophage is required for tumor cell intravasation. Cell Rep. 23, 1239–1248. https://doi.org/10.1016/j.celrep.2018.04.007.

12. Huang, Y.K., Wang, M., Sun, Y., Di Costanzo, N., Mitchell, C., Achuthan, A., Hamilton, J.A., Busuttil, R.A., and Boussioutas, A. (2019). Macrophage spatial heterogeneity in gastric cancer defined by multiplex immunohistochemistry. Nat. Commun. 10, 3928. https://doi.org/10.1038/s41467-019-11788-4.

13. Carmona-Fontaine, C., Deforet, M., Akkari, L., Thompson, C.B., Joyce, J.A., and Xavier, J.B. (2017). Metabolic origins of spatial organization in the tumor microenvironment. Proc. Natl. Acad. Sci. USA 114, 2934–2939. https://doi.org/10.1073/pnas.1700600114.

14. Opzoomer, J.W., Anstee, J.E., Dean, I., Hill, E.J., Bouybayoune, I., Caron, J., Muliaditan, T., Gordon, P., Sosnowska, D., Nuamah, R., et al. (2021). Macrophages orchestrate the expansion of a proangiogenic perivascular niche during cancer progression. Sci. Adv. 7, eabg9518. https://doi.org/10.1126/sciadv.abg9518.

15. Nalio Ramos, R., Missolo-Koussou, Y., Gerber-Ferder, Y., Bromley, C.P., Bugatti, M., Núñez, N.G., Tosello Boari, J., Richer, W., Menger, L., Denizeau, J., et al. (2022). Tissue-resident FOLR2(+) macrophages associate with CD8(+) T cell infiltration in human breast cancer. Cell 185, 1189–1207.e25. https://doi.org/10.1016/j.cell.2022.02.021.

16. Lapenna, A., De Palma, M., and Lewis, C.E. (2018). Perivascular macrophages in health and disease. Nat. Rev. Immunol. 18, 689–702. https://doi.org/10.1038/s41577-018-0056-9.

17. Lewis, C.E., Harney, A.S., and Pollard, J.W. (2016). The multifaceted role of perivascular macrophages in tumors. Cancer Cell 30, 365. https://doi.org/10.1016/j.ccell.2016.07.009.

18. Wyckoff, J.B., Wang, Y., Lin, E.Y., Li, J.F., Goswami, S., Stanley, E.R., Segall, J.E., Pollard, J.W., and Condeelis, J. (2007). Direct visualization

of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res. 67, 2649–2656. https://doi.org/10.1158/0008-5472.CAN-06-1823.

19. Harney, A.S., Arwert, E.N., Entenberg, D., Wang, Y., Guo, P., Qian, B.Z., Oktay, M.H., Pollard, J.W., Jones, J.G., and Condeelis, J.S. (2015). Real-time imaging reveals local, transient vascular permeability, and tumor cell intravasation stimulated by TIE2hi macrophage-derived VEGFA. Cancer Discov. 5, 932–943. https://doi.org/10.1158/2159-8290.CD-15-0012.

20. Hughes, R., Qian, B.Z., Rowan, C., Muthana, M., Keklikoglou, I., Olson, O.C., Tazzzyman, S., Danson, S., Addison, C., Clemons, M., et al. (2015). Perivascular M2 macrophages stimulate tumor relapse after chemotherapy. Cancer Res. 75, 3479–3491. https://doi.org/10.1158/0008-5472.CAN-14-3587.

21. Ensan, S., Li, A., Besla, R., Degousee, N., Cosme, J., Roufaiel, M., Shikatani, E.A., El-Maklizi, M., Williams, J.W., Robins, L., et al. (2016). Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth. Nat. Immunol. 17, 159–168. https://doi.org/10.1038/ni.3343.

22. Cho, C.H., Koh, Y.J., Han, J., Sung, H.K., Jong Lee, H., Morisada, T., Schwendener, R.A., Brekken, R.A., Kang, G., Oike, Y., et al. (2007). Angiogenic role of LYVE-1-positive macrophages in adipose tissue. Circ. Res. 100, e47–e57. https://doi.org/10.1161/01 RES.0000259564.92792.93.

23. Pinto, A.R., Paolicelli, R., Salimova, E., Gospocic, J., Slonimsky, E., Bilbao-Cortes, D., Godwin, J.W., and Rosenthal, N.A. (2012). An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile. PLoS One 7, e36814. https://doi.org/10.1371/journal.pone.0036814.

24. Xu, H., Chen, M., Reid, D.M., and Forrester, J.V. (2007). LYVE-1-positive macrophages are present in normal murine eyes. Invest. Ophthalmol. Vis. Sci. 48, 2162–2171. https://doi.org/10.1167/iovs.06-0783.

25. Chakarov, S., Lim, H.Y., Tan, L., Lim, S.Y., See, P., Lum, J., Zhang, X.M., Foo, S., Nakamizo, S., Duan, K., et al. (2019). Two distinct interstitial macrophage populations coexist across tissues in specific subtidal niches. Science 363. https://doi.org/10.1126/science.aau0964.

26. Dollt, C., Becker, K., Michel, J., Melchers, S., Weis, C.A., Schledzewski, K., Krewer, A., Kloss, L., Gebhardt, C., Utikal, J., et al. (2017). The shedded ectodomain of Lyve-1 expressed on M2-like tumor-associated macrophages inhibits melanoma cell proliferation. Oncotarget 8, 103682–103692. https://doi.org/10.18632/oncotarget.21771.

27. Sharma, A., Seow, J.J.W., Dutertre, C.A., Pai, R., Blériot, C., Mishra, A., Wong, R.M.M., Singh, G.S.N., Sudhagar, S., Khalilnezhad, S., et al. (2020). Onco-fetal reprogramming of endothelial cells drives immunosuppressive macrophages in hepatocellular carcinoma. Cell 183, 377–394.e21. https://doi.org/10.1016/j.cell.2020.08.040.

28. Wu, S.Z., Al-Eryani, G., Roden, D.L., Junankar, S., Harvey, K., Andersson, A., Thennavan, A., Wang, C., Torpy, J.R., Bartonicek, N., et al. (2021). A single-cell and spatially resolved atlas of human breast cancers. Nat. Genet. 53, 1334–1347. https://doi.org/10.1038/s41588-021-00911-1.

29. Jackson, D.G., Prevo, R., Clasper, S., and Banerji, S. (2001). LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol. 22, 317–321. https://doi.org/10.1016/s1471-4906(01)01936-6.

30. Kosti, P., Opzoomer, J.W., Larios-Martinez, K.I., Henley-Smith, R., Scudamore, C.L., Okesola, M., Taher, M.Y.M., Davies, D.M., Muliaditan, T., Larcombe-Young, D., et al. (2021). Hypoxia-sensing CAR T cells provide safety and efficacy in treating solid tumors. Cell Rep. Med. 2, 100227. https://doi.org/10.1016/j.ccrm.2021.100227.

31. Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park, J.H., Lee, K.Y., Kim, M.K., Shin, B.A., and Choi, S.Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, e18556. https://doi.org/10.1371/journal.pone.0018556.

32. Spranger, S., Dai, D., Horton, B., and Gajewski, T.F. (2017). Tumor-residing Batf3 dendritic cells are required for effector T cell trafficking
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80. Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W.M., 3rd, Hao, Y., Stoeckius, M., Smibert, P., and Satija, R. (2019). Comprehensive integration of single-cell data. Cell 177, 1888-1902.e21. https://doi.org/10.1016/j.cell.2019.05.031.

81. Pliner, H.A., Shendure, J., and Trapnell, C. (2019). Supervised classification enables rapid annotation of cell atlases. Nat. Methods 16, 983-986. https://doi.org/10.1038/s41592-019-0535-3.

82. Elso, C.M., Roberts, L.J., Smyth, G.K., Thomson, R.J., Baldwin, T.M., Foote, S.J., and Handman, E. (2004). Leishmaniasis host response loci (lmr1-3) modify disease severity through a Th1/Th2-independent pathway. Genes Immun. 5, 93-100. https://doi.org/10.1038/sj.gene.6364042.

83. Guy, C.T., Cardiff, R.D., and Muller, W.J. (1992). Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol. Cell. Biol. 12, 954-961.

84. Tzima, S., Victoratos, P., Kranidiot, K., Alexiou, M., and Kollias, G. (2009). Myeloid heme oxygenase-1 regulates innate immunity and autoimmunity by modulating IFN-beta production. J. Exp. Med. 206, 1167-1179. https://doi.org/10.1084/jem.20081582.

85. Skarnes, W.C., Rosen, B., West, A.P., Koutsourakis, M., Bushell, W., Iyer, V., Mujica, A.O., Thomas, M., Harrow, J., Cox, T., et al. (2011). A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337-342. https://doi.org/10.1038/nature10163.

86. Dutton, E.E., Gajdasik, D.W., Willis, C., Fiancette, R., Bishop, E.L., Camelo, A., Sleeman, M.A., Coccia, M., Didierlaurent, A.M., Tomura, M., et al. (2019). Peripheral lymph nodes contain migratory and resident innate lymphoid cell populations. Sci. Immunol. 4. https://doi.org/10.1126/sciimmunol.aau8082.

87. Kraman, M., Bambrough, P.J., Arnold, J.N., Roberts, E.W., Magiera, L., Jones, J.O., Gopinathan, A., Tuveson, D.A., and Fearon, D.T. (2010). Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha. Science 330, 827-830. https://doi.org/10.1126/science.1195300.

88. Qian, B.Z., Li, J., Zhang, H., Kitamura, T., Zhang, J., Campion, L.R., Kaiser, E.A., Snyder, L.A., and Pollard, J.W. (2011). CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature 475, 222-225. https://doi.org/10.1038/nature10138.

89. Bolstad, B.M., Irizarry, R.A., Astrand, M., and Speed, T.P. (2003). A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185-193.
of chemotherapy. Nat. Med. 20, 1301-1309. https://doi.org/10.1038/
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63. Bent, E.H., Millán-Barea, L.R., Zhuang, I., Goulet, D.R., Fröse, J., and Hemann, M.T. (2021). Microenvironmental IL-6 inhibits anti-cancer immune responses generated by cytotoxic chemotherapy. Nat. Commun. 12, 6218. https://doi.org/10.1038/s41467-021-26407-4.

64. Tan, Q., Wang, H., Hu, Y., Hu, M., Li, X., Aodengqimuge, M., Ma, Y., Wei, C., and Song, L. (2015). Src/STAT3-dependent heme oxygenase-1 induction mediates chemoresistance of breast cancer cells to doxorubicin by promoting autophagy. Cancer Sci. 106, 1023-1032. https://doi.org/10.1111/cas.12712.

65. Nuhn, P., Künzli, B.M., Hennig, R., Mitkus, T., Ramanauskas, T., Nobiling, R., Meuer, S.C., Friess, H., and Berberat, P.O. (2009). Heme oxygenase-1 and its metabolites affect pancreatic tumor growth in vivo. Mol. Cancer 8, 37. https://doi.org/10.1186/1476-4598-8-37.

66. Nowis, D., Bugajski, M., Winiarska, M., Bil, J., Szokalska, A., Salwa, P., Issat, T., Was, H., Jozkowicz, A., Dulak, J., et al. (2008). Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice. BMC Cancer 8, 197. https://doi.org/10.1186/1471-2407-8-197.

67. Di Biase, S., Lee, C., Brandhorst, S., Manes, B., Buono, R., Cheng, C.W., Cacciottolo, M., Martin-Montalvo, A., de Cabo, R., Wei, M., et al. (2016). Fasting-mimicking diet reduces HO-1 to promote T cell-mediated tumor cytotoxicity. Cancer Cell 30, 136-146. https://doi.org/10.1016/j.ccell.2016.06.005.

68. Berberat, P.O., Dambrauskas, Z., Gulbinas, A., Giese, T., Giese, N., Künzli, B., Autschbach, F., Meuer, S., Büchler, M.W., and Friess, H. (2005). Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment. Clin. Cancer Res. 11, 3790-3798. https://doi.org/10.1158/1078-0432.CCR-04-2159.

69. Otterbein, L.E., Bach, F.H., Alam, J., Fu, X.Y., and Lee, P.J. (2005). Carbon monoxide differentially modulates STAT1 and STAT3 and inhibits apoptosis via a phosphatidylinositol 3-kinase/Akt and p38 kinase-dependent STAT3 pathway during anoxia-reoxygenation injury. J. Biol. Chem. 280, 8714-8721. https://doi.org/10.1074/jbc.M408092200.

70. Zhang, X., Shan, P., Alam, J., Fu, X.Y., and Lee, P.J. (2005). Carbon monoxide differentially modulates STAT1 and STAT3 and inhibits apoptosis via a phosphatidylinositol 3-kinase/Akt and p38 kinase-dependent STAT3 pathway during anoxia-reoxygenation injury. J. Biol. Chem. 280, 8714-8721. https://doi.org/10.1074/jbc.M408092200.

71. Cepinskas, G., Katada, K., Bihari, A., and Potter, R.F. (2008). Carbon monoxide liberated from carbon monoxide-releasing molecule CORM-2 attenuates inflammation in the liver of septic mice. Am. J. Physiol. Gastrointest. Liver Physiol. 294, G184-G191. https://doi.org/10.1152/apgi.00348.2007.

72. Megías, J., Busserolles, J., and Alcaraz, M.J. (2007). The carbon monoxide-releasing molecule CORM-2 inhibits the inflammatory response induced by cytokines in Caco-2 cells. Br. J. Pharmacol. 150, 977-986. https://doi.org/10.1038/sj.bjp.0707184.

73. Muldoon, J.J., Chuang, Y., Bagheri, N., and Leonard, J.N. (2020). Macrophages employ quorum licensing to regulate collective activation. Nat. Commun. 11, 878. https://doi.org/10.1038/s41467-020-14547-y.

74. Goswami, S., Philippar, U., Sun, D., Patsialou, A., Avraham, J., Wang, W., Di Modugno, F., Nistico, P., Gertler, F.B., and Condeelis, J.S. (2009). Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo. Clin. Exp. Metastasis 26, 153-159. https://doi.org/10.1007/s10585-008-9225-8.

75. Roh-Johnson, M., Bravo-Cordero, J.J., Patsialou, A., Sharma, V.P., Guo, P., Liu, H., Hodgson, L., and Condeelis, J. (2014). Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation. Oncogene 33, 4203-4212. https://doi.org/10.1038/onc.2013.377.

76. Robinson, B.D., Sica, G.L., Liu, Y.F., Rohan, T.E., Gertler, F.B., Condeelis, J.S., and Jones, J.G. (2009). Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clin. Cancer Res. 15, 2433-2441. https://doi.org/10.1158/1078-0432.CCR-08-2179.

77. Rohan, T.E., Xue, X., Lin, H.M., D'Alfonso, T.M., Ginter, P.S., Oktay, M.H., Robinson, B.D., Ginsberg, M., Gertler, F.B., Glass, A.G., et al. (2014). Tumor microenvironment of metastasis and risk of distant metastasis of breast cancer. J. Natl. Cancer Inst. 106. https://doi.org/10.1093/jnci/dju136.

78. Sparano, J.A., Gray, R., Oktay, M.H., Entenberg, D., Rohan, T., Xue, X., Donovan, M., Peterson, M., Shuber, A., Hamilton, D.A., et al. (2017). A metastasis biomarker (MetaSite Breast™ Score) is associated with distant recurrence in hormone receptor-positive, HER2-negative early-stage breast cancer. NPJ Breast Cancer 3, 42. https://doi.org/10.1038/s41523-017-0043-5.

79. Halama, N., Zoernig, I., Berthel, A., Kahlert, C., Klupp, F., Suarez-Carmona, M., Suetterlin, T., Brand, K., Krauss, J., Lasitschka, F., et al. (2016). Tumoral immune cell exploitation in colorectal cancer metastases can be targeted effectively by anti-CCR5 therapy in cancer patients. Cancer Cell 29, 587-601. https://doi.org/10.1016/j.ccell.2016.03.005.

80. Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W.M., 3rd, Hao, Y., Stoeckius, M., Smibert, P., and Satija, R. (2019). Comprehensive integration of single-cell data. Cell 177, 1888-1902.e21. https://doi.org/10.1016/j.cell.2019.05.031.

81. Pliner, H.A., Shendure, J., and Trapnell, C. (2019). Supervised classification enables rapid annotation of cell atlases. Nat. Methods 16, 983-986. https://doi.org/10.1038/s41592-019-0535-3.

82. Elso, C.M., Roberts, L.J., Smyth, G.K., Thomson, R.J., Baldwin, T.M., Foote, S.J., and Handman, E. (2004). Leishmaniasis host response loci (lmr1-3) modify disease severity through a Th1/Th2-independent pathway. Genes Immun. 5, 93-100. https://doi.org/10.1038/sj.gene.6364042.

83. Guy, C.T., Cardiff, R.D., and Muller, W.J. (1992). Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol. Cell. Biol. 12, 954-961.

84. Tzima, S., Victoratos, P., Kranidiot, K., Alexiou, M., and Kollias, G. (2009). Myeloid heme oxygenase-1 regulates innate immunity and autoimmunity by modulating IFN-beta production. J. Exp. Med. 206, 1167-1179. https://doi.org/10.1084/jem.20081582.

85. Skarnes, W.C., Rosen, B., West, A.P., Koutsourakis, M., Bushell, W., Iyer, V., Mujica, A.O., Thomas, M., Harrow, J., Cox, T., et al. (2011). A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337-342. https://doi.org/10.1038/nature10163.

86. Dutton, E.E., Gajdasik, D.W., Willis, C., Fiancette, R., Bishop, E.L., Camelo, A., Sleeman, M.A., Coccia, M., Didierlaurent, A.M., Tomura, M., et al. (2019). Peripheral lymph nodes contain migratory and resident innate lymphoid cell populations. Sci. Immunol. 4. https://doi.org/10.1126/sciimmunol.aau8082.

87. Kraman, M., Bambrough, P.J., Arnold, J.N., Roberts, E.W., Magiera, L., Jones, J.O., Gopinathan, A., Tuveson, D.A., and Fearon, D.T. (2010). Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha. Science 330, 827-830. https://doi.org/10.1126/science.1195300.

88. Qian, B.Z., Li, J., Zhang, H., Kitamura, T., Zhang, J., Campion, L.R., Kaiser, E.A., Snyder, L.A., and Pollard, J.W. (2011). CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature 475, 222-225. https://doi.org/10.1038/nature10138.

89. Bolstad, B.M., Irizarry, R.A., Astrand, M., and Speed, T.P. (2003). A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185-193.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Antibodies</td><td></td><td></td></tr><tr><td>Rabbit anti-β-actin</td><td>Abcam</td><td>Cat# ab8227; RRID: AB_2305186</td></tr><tr><td>Rabbit anti-HO-1</td><td>Proteintech</td><td>Cat# 10701-1-AP; RRID: AB_2118685</td></tr><tr><td>Rabbit anti-c-MAF</td><td>Abcam</td><td>Cat# ab77071; RRID: AB_1951643</td></tr><tr><td>Rabbit anti-STAT3 (clone 79D7)</td><td>Cell Signaling Technology</td><td>Cat# 4904; RRID: AB_331269</td></tr><tr><td>Goat anti-rabbit immunoglobulins/HRP</td><td>Agilent Dako</td><td>Cat# P0448; RRID: AB_2617138</td></tr><tr><td>Rabbit anti-αSMA</td><td>AnaSpec</td><td>Cat# AS-29553; RRID: AB_2120181</td></tr><tr><td>Rabbit anti-CD31</td><td>Abcam</td><td>Cat# ab28364; RRID: AB_726362</td></tr><tr><td>Rat anti-mouse CD31 (clone ER-MP12)</td><td>Thermo Fisher Scientific</td><td>Cat# MA1-40074; RRID: AB_1072120</td></tr><tr><td>Rabbit anti-human CD31 (clone EP3095)</td><td>Abcam</td><td>Cat# ab134168; RRID: AB_2890012</td></tr><tr><td>Mouse anti-CD68 (clone KP1)</td><td>Invitrogen</td><td>Cat# MA5-13324; RRID: AB_10987212</td></tr><tr><td>Rat anti-mouse F4/80 (clone CI:A3-1)</td><td>Bio-RAD</td><td>Cat# MCA497; RRID: AB_323806</td></tr><tr><td>Goat anti-HO-1</td><td>R&amp;D systems</td><td>Cat# AF3776; RRID: AB_2295362</td></tr><tr><td>Rabbit anti-LYVE-1</td><td>Abcam</td><td>Cat# ab33682; RRID: AB_881387</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor® 488</td><td>Thermo Fisher Scientific</td><td>Cat# A32790; RRID: AB_2762833</td></tr><tr><td>Donkey anti-rat IgG (H+L) Alexa Fluor® 488</td><td>Thermo Fisher Scientific</td><td>Cat# A-21208; RRID: AB_2535794</td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor® 488</td><td>Thermo Fisher Scientific</td><td>Cat# A-11055; RRID: AB_2534102</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor® 568</td><td>Thermo Fisher Scientific</td><td>Cat# A10042; RRID: AB_2534017</td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor® 568</td><td>Thermo Fisher Scientific</td><td>Cat# A-11057; RRID: AB_2534104</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor® 647</td><td>Thermo Fisher Scientific</td><td>Cat# A-31573; RRID: AB_2536183</td></tr><tr><td>Donkey anti-mouse IgG (H+L) Alexa Fluor® 647</td><td>Thermo Fisher Scientific</td><td>Cat# A-31571; RRID: AB_162542</td></tr><tr><td>Goat anti-rat IgG NorthernLights™ NL637</td><td>R&amp;D systems</td><td>Cat# NL014; RRID: AB_884218</td></tr><tr><td>Donkey anti-sheep IgG (H+L) Cy™ 3</td><td>Jackson ImmunoResearch</td><td>Cat# 713-165-147; RRID: AB_2315778</td></tr><tr><td>Armenian hamster anti-mouse CCR5 PE (clone HM-CCR5 (7A4))</td><td>Thermo Fisher Scientific</td><td>Cat# 12-1951-82; RRID: AB_657684</td></tr><tr><td>Armenian hamster anti-mouse CD3ε APC (clone 145-2C11)</td><td>Thermo Fisher Scientific</td><td>Cat# MA1-10186; RRID: AB_11153519</td></tr><tr><td>Armenian hamster anti-mouse CD3ε PE (clone 145-2C11)</td><td>Thermo Fisher Scientific</td><td>Cat# A14714; RRID: AB_2534230</td></tr><tr><td>Rat anti-mouse CD3ε Brilliant Violet 421™ (clone 17A2)</td><td>Biolegend®</td><td>Cat# 100227; RRID: AB_10900227</td></tr><tr><td>Rat anti-mouse CD3ε PE/Cyanine 7 (clone 145-2C11)</td><td>Biolegend®</td><td>Cat# 100319; RRID: AB_312684</td></tr><tr><td>Rat anti-mouse CD4 APC (clone RM4-5)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-0042-82; RRID: AB_469323</td></tr><tr><td>Rat anti-mouse CD4 FITC (clone RM4-5)</td><td>Thermo Fisher Scientific</td><td>Cat# 11-0042-82; RRID: AB_464896</td></tr><tr><td>Rat anti-mouse CD4 PE (clone RM4-5)</td><td>BD Biosciences</td><td>Cat# 553048; RRID: AB_394584</td></tr><tr><td>Rat anti-mouse CD8α Brilliant Violet 421™ (clone 53-5.7)</td><td>Biolegend®</td><td>Cat# 100737; RRID: AB_10897101</td></tr><tr><td>Rat anti-mouse CD8α Brilliant Violet 711™ (clone 53-5.7)</td><td>Biolegend®</td><td>Cat# 100759; RRID: AB_2563510</td></tr><tr><td>Rat anti-mouse CD8α FITC (clone 53-5.7)</td><td>Biolegend®</td><td>Cat# 100705; RRID: AB_312744</td></tr><tr><td>Rat anti-mouse CD8β FITC (clone H35-17.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 11-0083-82; RRID: AB_657764</td></tr><tr><td>Rat anti-mouse CD8β eFluor™ 450 (clone H35-17.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0083-82; RRID: AB_11218504</td></tr><tr><td>Rat anti-mouse CD11b Brilliant Violet 510™ (clone M1/70)</td><td>Biolegend®</td><td>Cat# 101245; RRID: AB_2561390</td></tr><tr><td>Armenian hamster anti-mouse CD11c APC (clone N418)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-0114-82; RRID: AB_469346</td></tr></tbody></table>

(Continued on next page)
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Antibodies</td><td></td><td></td></tr><tr><td>Rabbit anti-β-actin</td><td>Abcam</td><td>Cat# ab8227; RRID: AB_2305186</td></tr><tr><td>Rabbit anti-HO-1</td><td>Proteintech</td><td>Cat# 10701-1-AP; RRID: AB_2118685</td></tr><tr><td>Rabbit anti-c-MAF</td><td>Abcam</td><td>Cat# ab77071; RRID: AB_1951643</td></tr><tr><td>Rabbit anti-STAT3 (clone 79D7)</td><td>Cell Signaling Technology</td><td>Cat# 4904; RRID: AB_331269</td></tr><tr><td>Goat anti-rabbit immunoglobulins/HRP</td><td>Agilent Dako</td><td>Cat# P0448; RRID: AB_2617138</td></tr><tr><td>Rabbit anti-αSMA</td><td>AnaSpec</td><td>Cat# AS-29553; RRID: AB_2120181</td></tr><tr><td>Rabbit anti-CD31</td><td>Abcam</td><td>Cat# ab28364; RRID: AB_726362</td></tr><tr><td>Rat anti-mouse CD31 (clone ER-MP12)</td><td>Thermo Fisher Scientific</td><td>Cat# MA1-40074; RRID: AB_1072120</td></tr><tr><td>Rabbit anti-human CD31 (clone EP3095)</td><td>Abcam</td><td>Cat# ab134168; RRID: AB_2890012</td></tr><tr><td>Mouse anti-CD68 (clone KP1)</td><td>Invitrogen</td><td>Cat# MA5-13324; RRID: AB_10987212</td></tr><tr><td>Rat anti-mouse F4/80 (clone CI:A3-1)</td><td>Bio-RAD</td><td>Cat# MCA497; RRID: AB_323806</td></tr><tr><td>Goat anti-HO-1</td><td>R&amp;D systems</td><td>Cat# AF3776; RRID: AB_2295362</td></tr><tr><td>Rabbit anti-LYVE-1</td><td>Abcam</td><td>Cat# ab33682; RRID: AB_881387</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor® 488</td><td>Thermo Fisher Scientific</td><td>Cat# A32790; RRID: AB_2762833</td></tr><tr><td>Donkey anti-rat IgG (H+L) Alexa Fluor® 488</td><td>Thermo Fisher Scientific</td><td>Cat# A-21208; RRID: AB_2535794</td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor® 488</td><td>Thermo Fisher Scientific</td><td>Cat# A-11055; RRID: AB_2534102</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor® 568</td><td>Thermo Fisher Scientific</td><td>Cat# A10042; RRID: AB_2534017</td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor® 568</td><td>Thermo Fisher Scientific</td><td>Cat# A-11057; RRID: AB_2534104</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor® 647</td><td>Thermo Fisher Scientific</td><td>Cat# A-31573; RRID: AB_2536183</td></tr><tr><td>Donkey anti-mouse IgG (H+L) Alexa Fluor® 647</td><td>Thermo Fisher Scientific</td><td>Cat# A-31571; RRID: AB_162542</td></tr><tr><td>Goat anti-rat IgG NorthernLights™ NL637</td><td>R&amp;D systems</td><td>Cat# NL014; RRID: AB_884218</td></tr><tr><td>Donkey anti-sheep IgG (H+L) Cy™ 3</td><td>Jackson ImmunoResearch</td><td>Cat# 713-165-147; RRID: AB_2315778</td></tr><tr><td>Armenian hamster anti-mouse CCR5 PE (clone HM-CCR5 (7A4))</td><td>Thermo Fisher Scientific</td><td>Cat# 12-1951-82; RRID: AB_657684</td></tr><tr><td>Armenian hamster anti-mouse CD3ε APC (clone 145-2C11)</td><td>Thermo Fisher Scientific</td><td>Cat# MA1-10186; RRID: AB_11153519</td></tr><tr><td>Armenian hamster anti-mouse CD3ε PE (clone 145-2C11)</td><td>Thermo Fisher Scientific</td><td>Cat# A14714; RRID: AB_2534230</td></tr><tr><td>Rat anti-mouse CD3ε Brilliant Violet 421™ (clone 17A2)</td><td>Biolegend®</td><td>Cat# 100227; RRID: AB_10900227</td></tr><tr><td>Rat anti-mouse CD3ε PE/Cyanine 7 (clone 145-2C11)</td><td>Biolegend®</td><td>Cat# 100319; RRID: AB_312684</td></tr><tr><td>Rat anti-mouse CD4 APC (clone RM4-5)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-0042-82; RRID: AB_469323</td></tr><tr><td>Rat anti-mouse CD4 FITC (clone RM4-5)</td><td>Thermo Fisher Scientific</td><td>Cat# 11-0042-82; RRID: AB_464896</td></tr><tr><td>Rat anti-mouse CD4 PE (clone RM4-5)</td><td>BD Biosciences</td><td>Cat# 553048; RRID: AB_394584</td></tr><tr><td>Rat anti-mouse CD8α Brilliant Violet 421™ (clone 53-5.7)</td><td>Biolegend®</td><td>Cat# 100737; RRID: AB_10897101</td></tr><tr><td>Rat anti-mouse CD8α Brilliant Violet 711™ (clone 53-5.7)</td><td>Biolegend®</td><td>Cat# 100759; RRID: AB_2563510</td></tr><tr><td>Rat anti-mouse CD8α FITC (clone 53-5.7)</td><td>Biolegend®</td><td>Cat# 100705; RRID: AB_312744</td></tr><tr><td>Rat anti-mouse CD8β FITC (clone H35-17.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 11-0083-82; RRID: AB_657764</td></tr><tr><td>Rat anti-mouse CD8β eFluor™ 450 (clone H35-17.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0083-82; RRID: AB_11218504</td></tr><tr><td>Rat anti-mouse CD11b Brilliant Violet 510™ (clone M1/70)</td><td>Biolegend®</td><td>Cat# 101245; RRID: AB_2561390</td></tr><tr><td>Armenian hamster anti-mouse CD11c APC (clone N418)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-0114-82; RRID: AB_469346</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Armenian hamster anti-mouse CD11c FITC (clone N418)</td><td>Biolegend®</td><td>Cat# 117305; RRID: AB_313774</td></tr><tr><td>Purified anti-mouse CD16 / CD32 (Fc Shield) (clone 2.4G2)</td><td>Tonbo Biosciences</td><td>Cat# 70-0161; RRID: AB_2621487</td></tr><tr><td>Rat anti-mouse CD19 Brilliant Violet 421™ (clone 6D5)</td><td>Biolegend®</td><td>Cat# 115537; RRID: AB_10895761</td></tr><tr><td>Rat anti-mouse CD19 APC (clone 6D5)</td><td>Biolegend®</td><td>Cat# 115511; RRID: AB_313646</td></tr><tr><td>Rat anti-mouse CD19 FITC (clone 1D3/CD19)</td><td>Biolegend®</td><td>Cat# 152403; RRID: AB_2629812</td></tr><tr><td>Rat anti-mouse CD31 FITC (clone 390)</td><td>Thermo Fisher Scientific</td><td>Cat# RM5201; RRID: AB_10373983</td></tr><tr><td>Rat anti-mouse CD31 PE (clone 390)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0311-82; RRID: AB_465632</td></tr><tr><td>Rat anti-mouse CD31 BD Horizon™ BV510 (clone MEC 13.3)</td><td>BD biosciences</td><td>Cat# 563089; RRID: AB_2737997</td></tr><tr><td>Rat anti-mouse CD45 APC (clone 30-F11)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-0451-82; RRID: AB_469392</td></tr><tr><td>Rat anti-mouse CD45 APC-eFluor® 780 (clone 30-F11)</td><td>Thermo Fisher Scientific</td><td>Cat# 47-0451-82; RRID: AB_1548781</td></tr><tr><td>Rat anti-mouse CD45 Brilliant Violet 605™ (clone 30-F11)</td><td>Biolegend®</td><td>Cat# 103139; RRID: AB_2562341</td></tr><tr><td>Rat anti-mouse CD45 Brilliant Violet 510™ (clone 30-F11)</td><td>Biolegend®</td><td>Cat# 103137; RRID: AB_2561392</td></tr><tr><td>Mouse anti-mouse CD90.1 (Thy-1.1) eFluor™ 450 (clone HIS51)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0900-82; RRID: AB_1272254</td></tr><tr><td>Mouse anti-mouse CD90.1 (Thy-1.1) Brilliant Violet 510™ (clone OX-7)</td><td>Biolegend®</td><td>Cat# 202535; RRID: AB_2562643</td></tr><tr><td>Rat anti-mouse CD90.2 (Thy-1.2) eFluor™ 450 (clone 53-2.1)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0902-82; RRID: AB_1272200</td></tr><tr><td>Rat anti-mouse CD90.2 (Thy-1.2) Brilliant Violet 510™ (clone 53-2.1)</td><td>Biolegend®</td><td>Cat# 140319; RRID: AB_2561395</td></tr><tr><td>Rat anti-mouse CD206 APC (clone C068C2)</td><td>Biolegend®</td><td>Cat# 141707; RRID: AB_10896057</td></tr><tr><td>Rat anti-mouse CD206 FITC (clone C068C2)</td><td>Biolegend®</td><td>Cat# 141703; RRID: AB_10900988</td></tr><tr><td>Rat anti-mouse CD206 Brilliant Violet 785™ (clone C068C2)</td><td>Biolegend®</td><td>Cat# 141729; RRID: AB_2565823</td></tr><tr><td>Goat anti-mouse CD206 APC</td><td>Bio-Techne</td><td>Cat# FAB2535A; RRID: AB_10971650</td></tr><tr><td>Rat anti-mouse F4/80 APC (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-4801-82; RRID: AB_2784648</td></tr><tr><td>Rat anti-mouse F4/80 APC-eFluor® 780 (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 47-4801-82; RRID: AB_2735036</td></tr><tr><td>Rat anti-mouse F4/80 PE (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-4801-82; RRID: AB_465923</td></tr><tr><td>Rat anti-mouse F4/80 eFluor™ 660 (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 50-4801-82; RRID: AB_11149361</td></tr><tr><td>Rat anti-mouse F4/80 Brilliant Violet 421™ (clone BM8)</td><td>Biolegend®</td><td>Cat# 123132; RRID: AB_11203717</td></tr><tr><td>Rat anti-mouse F4/80 Brilliant Violet 510™ (clone BM8)</td><td>Biolegend®</td><td>Cat# 123135; RRID: AB_2562622</td></tr><tr><td>Rat anti-mouse F4/80 FITC (clone BM8)</td><td>Biolegend®</td><td>Cat# 123107; RRID: AB_893500</td></tr><tr><td>Rat anti-mouse Gr-1 FITC (clone RB6-8C5)</td><td>Biolegend®</td><td>Cat# 108405; RRID: AB_313370</td></tr><tr><td>Mouse anti-human Granzyme-B PE (clone GB11)</td><td>Thermo Fisher Scientific</td><td>Cat# GRB04; RRID: AB_1500188</td></tr><tr><td>Rat anti-mouse ICAM-1 Brilliant Violet 421™ (clone YN1/1.7.4)</td><td>Biolegend®</td><td>Cat# 116141; RRID: AB_2876428</td></tr><tr><td>Rat anti-mouse IFN-γ APC (clone XMG1.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-7311-82; RRID: AB_469504</td></tr><tr><td>Rat anti-mouse Ki-67 Alexa Flour® 700 (clone SolA15)</td><td>Thermo Fisher Scientific</td><td>Cat# 56-5698-00 RRID: AB_2637480</td></tr><tr><td>Rat anti-mouse Ly6C APC (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-5932-82; RRID: AB_1724153</td></tr><tr><td>Rat anti-mouse Ly6C APC-eFluor® 780 (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 47-5932-82; RRID: AB_2573992</td></tr><tr><td>Rat anti-mouse Ly6C eFluor™ 450 (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5932-82; RRID: AB_10805519</td></tr><tr><td>Rat anti-mouse Ly6C PE (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5932-82; RRID: AB_10804510</td></tr><tr><td>Rat anti-mouse Ly6C FITC (clone HK1.4)</td><td>Biolegend®</td><td>Cat# 128005; RRID: AB_1186134</td></tr><tr><td>Rat anti-mouse Ly6G APC (clone 1A8)</td><td>Biolegend®</td><td>Cat# 127613; RRID: AB_1877163</td></tr><tr><td>Rat anti-mouse LYVE-1 Alexa Fluor® 488 (clone ALY7)</td><td>Thermo Fisher Scientific</td><td>Cat# 53-0443-82; RRID: AB_1633415</td></tr><tr><td>Rat anti-mouse LYVE-1 PE (clone ALY7)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0443-82; RRID: AB_2802179</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Armenian hamster anti-mouse CD11c FITC (clone N418)</td><td>Biolegend®</td><td>Cat# 117305; RRID: AB_313774</td></tr><tr><td>Purified anti-mouse CD16 / CD32 (Fc Shield) (clone 2.4G2)</td><td>Tonbo Biosciences</td><td>Cat# 70-0161; RRID: AB_2621487</td></tr><tr><td>Rat anti-mouse CD19 Brilliant Violet 421™ (clone 6D5)</td><td>Biolegend®</td><td>Cat# 115537; RRID: AB_10895761</td></tr><tr><td>Rat anti-mouse CD19 APC (clone 6D5)</td><td>Biolegend®</td><td>Cat# 115511; RRID: AB_313646</td></tr><tr><td>Rat anti-mouse CD19 FITC (clone 1D3/CD19)</td><td>Biolegend®</td><td>Cat# 152403; RRID: AB_2629812</td></tr><tr><td>Rat anti-mouse CD31 FITC (clone 390)</td><td>Thermo Fisher Scientific</td><td>Cat# RM5201; RRID: AB_10373983</td></tr><tr><td>Rat anti-mouse CD31 PE (clone 390)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0311-82; RRID: AB_465632</td></tr><tr><td>Rat anti-mouse CD31 BD Horizon™ BV510 (clone MEC 13.3)</td><td>BD biosciences</td><td>Cat# 563089; RRID: AB_2737997</td></tr><tr><td>Rat anti-mouse CD45 APC (clone 30-F11)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-0451-82; RRID: AB_469392</td></tr><tr><td>Rat anti-mouse CD45 APC-eFluor® 780 (clone 30-F11)</td><td>Thermo Fisher Scientific</td><td>Cat# 47-0451-82; RRID: AB_1548781</td></tr><tr><td>Rat anti-mouse CD45 Brilliant Violet 605™ (clone 30-F11)</td><td>Biolegend®</td><td>Cat# 103139; RRID: AB_2562341</td></tr><tr><td>Rat anti-mouse CD45 Brilliant Violet 510™ (clone 30-F11)</td><td>Biolegend®</td><td>Cat# 103137; RRID: AB_2561392</td></tr><tr><td>Mouse anti-mouse CD90.1 (Thy-1.1) eFluor™ 450 (clone HIS51)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0900-82; RRID: AB_1272254</td></tr><tr><td>Mouse anti-mouse CD90.1 (Thy-1.1) Brilliant Violet 510™ (clone OX-7)</td><td>Biolegend®</td><td>Cat# 202535; RRID: AB_2562643</td></tr><tr><td>Rat anti-mouse CD90.2 (Thy-1.2) eFluor™ 450 (clone 53-2.1)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0902-82; RRID: AB_1272200</td></tr><tr><td>Rat anti-mouse CD90.2 (Thy-1.2) Brilliant Violet 510™ (clone 53-2.1)</td><td>Biolegend®</td><td>Cat# 140319; RRID: AB_2561395</td></tr><tr><td>Rat anti-mouse CD206 APC (clone C068C2)</td><td>Biolegend®</td><td>Cat# 141707; RRID: AB_10896057</td></tr><tr><td>Rat anti-mouse CD206 FITC (clone C068C2)</td><td>Biolegend®</td><td>Cat# 141703; RRID: AB_10900988</td></tr><tr><td>Rat anti-mouse CD206 Brilliant Violet 785™ (clone C068C2)</td><td>Biolegend®</td><td>Cat# 141729; RRID: AB_2565823</td></tr><tr><td>Goat anti-mouse CD206 APC</td><td>Bio-Techne</td><td>Cat# FAB2535A; RRID: AB_10971650</td></tr><tr><td>Rat anti-mouse F4/80 APC (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-4801-82; RRID: AB_2784648</td></tr><tr><td>Rat anti-mouse F4/80 APC-eFluor® 780 (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 47-4801-82; RRID: AB_2735036</td></tr><tr><td>Rat anti-mouse F4/80 PE (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-4801-82; RRID: AB_465923</td></tr><tr><td>Rat anti-mouse F4/80 eFluor™ 660 (clone BM8)</td><td>Thermo Fisher Scientific</td><td>Cat# 50-4801-82; RRID: AB_11149361</td></tr><tr><td>Rat anti-mouse F4/80 Brilliant Violet 421™ (clone BM8)</td><td>Biolegend®</td><td>Cat# 123132; RRID: AB_11203717</td></tr><tr><td>Rat anti-mouse F4/80 Brilliant Violet 510™ (clone BM8)</td><td>Biolegend®</td><td>Cat# 123135; RRID: AB_2562622</td></tr><tr><td>Rat anti-mouse F4/80 FITC (clone BM8)</td><td>Biolegend®</td><td>Cat# 123107; RRID: AB_893500</td></tr><tr><td>Rat anti-mouse Gr-1 FITC (clone RB6-8C5)</td><td>Biolegend®</td><td>Cat# 108405; RRID: AB_313370</td></tr><tr><td>Mouse anti-human Granzyme-B PE (clone GB11)</td><td>Thermo Fisher Scientific</td><td>Cat# GRB04; RRID: AB_1500188</td></tr><tr><td>Rat anti-mouse ICAM-1 Brilliant Violet 421™ (clone YN1/1.7.4)</td><td>Biolegend®</td><td>Cat# 116141; RRID: AB_2876428</td></tr><tr><td>Rat anti-mouse IFN-γ APC (clone XMG1.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-7311-82; RRID: AB_469504</td></tr><tr><td>Rat anti-mouse Ki-67 Alexa Flour® 700 (clone SolA15)</td><td>Thermo Fisher Scientific</td><td>Cat# 56-5698-00 RRID: AB_2637480</td></tr><tr><td>Rat anti-mouse Ly6C APC (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-5932-82; RRID: AB_1724153</td></tr><tr><td>Rat anti-mouse Ly6C APC-eFluor® 780 (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 47-5932-82; RRID: AB_2573992</td></tr><tr><td>Rat anti-mouse Ly6C eFluor™ 450 (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5932-82; RRID: AB_10805519</td></tr><tr><td>Rat anti-mouse Ly6C PE (clone HK1.4)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5932-82; RRID: AB_10804510</td></tr><tr><td>Rat anti-mouse Ly6C FITC (clone HK1.4)</td><td>Biolegend®</td><td>Cat# 128005; RRID: AB_1186134</td></tr><tr><td>Rat anti-mouse Ly6G APC (clone 1A8)</td><td>Biolegend®</td><td>Cat# 127613; RRID: AB_1877163</td></tr><tr><td>Rat anti-mouse LYVE-1 Alexa Fluor® 488 (clone ALY7)</td><td>Thermo Fisher Scientific</td><td>Cat# 53-0443-82; RRID: AB_1633415</td></tr><tr><td>Rat anti-mouse LYVE-1 PE (clone ALY7)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0443-82; RRID: AB_2802179</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Rat anti-mouse LYVE-1 APC (clone 223322)</td><td>Bio-Techn</td><td>Cat# FAB2125A; RRID: AB_10972770</td></tr><tr><td>Rat anti-mouse MHCII PE (clone M5/114.15.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5321-82; RRID: AB_465928</td></tr><tr><td>Rat anti-mouse MHCII Brilliant Violet 421 <sup>TM</sup> (clone M5/114.15.2)</td><td>Biolegend®</td><td>Cat# 107631; RRID: AB_10900075</td></tr><tr><td>Rat anti-mouse MHCII Brilliant Violet 510 <sup>TM</sup> (clone M5/114.15.2)</td><td>Biolegend®</td><td>Cat# 107635; RRID: AB_2561397</td></tr><tr><td>Rat anti-mouse MHCII FITC (clone M5/114.15.2)</td><td>Biolegend®</td><td>Cat# 107605; RRID: AB_313320</td></tr><tr><td>Mouse anti-mouse NK1.1 APC (clone PK136)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-5941-82; RRID: AB_469479</td></tr><tr><td>Rat anti-mouse PNAlexa Fluor® 647 (clone MECA-79)</td><td>Biolegend®</td><td>Cat# 120807; RRID: AB_2783059</td></tr><tr><td>Rat anti-mouse TNFα PE (clone MP6-XT22)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-7321-82; RRID: AB_466199</td></tr><tr><td>Rat anti-mouse VCAM-1 PE (clone 429 (MVCAM.A))</td><td>Biolegend®</td><td>Cat# 105713; RRID: AB_1134166</td></tr><tr><td>Rat anti-mouse TER-119 PerCP-Cy5.5 (clone TER-119)</td><td>Thermo Fisher Scientific</td><td>Cat# 45-5921-82; RRID: AB_925765</td></tr><tr><td>Rat anti-mouse CD8α (clone 53-6.7)</td><td>Biolegend®</td><td>Cat# 100768; RRID: AB_2616823</td></tr><tr><td>Armenian hamster anti-mouse CD3ε (clone 145-2C11)</td><td>Thermo Fisher Scientific</td><td>Cat# 16-0031-82; RRID: AB_468847</td></tr><tr><td>Syrian hamster anti-mouse CD28 (Clone 37.51)</td><td>Thermo Fisher Scientific</td><td>Cat# 16-0281-82; RRID: AB_468921</td></tr></tbody></table>

Bacterial and virus strains

<table><tr><td>E. coli (DH10B competent cells)</td><td>Thermo Fisher Scientific</td><td>Cat# EC0113</td></tr></table>

Biological samples

<table><tr><td>Human breast adenocarcinoma tissue</td><td>King's Health Partners<br/>Cancer Biobank</td><td>REC reference 12/EE/0493</td></tr></table>

Chemicals, peptides, and recombinant proteins

<table><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>Cat# SAE0151</td></tr><tr><td>FCS</td><td>Thermo Fisher Scientific</td><td>Cat# 26170043</td></tr><tr><td>Spectinomycin</td><td>Sigma-Aldrich</td><td>Cat# S0692</td></tr><tr><td>LR Clonase™ II Plus enzyme mix</td><td>Thermo Fisher Scientific</td><td>Cat# 12538120</td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>Cat# T8154</td></tr><tr><td>Maraviroc</td><td>Cayman</td><td>Cat# 14641</td></tr><tr><td>5-Fluorouracil</td><td>Sigma-Aldrich</td><td>Cat# F6627</td></tr><tr><td>Collagenase I from Clostridium Histolyticum</td><td>Sigma-Aldrich</td><td>Cat# C0130</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>Cat# BSAV-RO</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>Cat# E8008</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma-Aldrich</td><td>Cat# P4333</td></tr><tr><td>Deoxyribonuclease I</td><td>AppliChem</td><td>Cat# A3778</td></tr><tr><td>RBC lysis buffer</td><td>Roche</td><td>Cat# 11814389001</td></tr><tr><td>Recombinant murine M-CSF</td><td>Bio-Techn</td><td>Cat# 416-ML</td></tr><tr><td>Recombinant murine IL-4</td><td>Bio-Techn</td><td>Cat# 404-ML</td></tr><tr><td>Recombinant murine IL-6</td><td>Bio-Techn</td><td>Cat# 406-ML</td></tr><tr><td>Recombinant murine IFN-γ</td><td>Bio-Techn</td><td>Cat# 485-ML</td></tr><tr><td>Recombinant murine CCL4</td><td>Bio-Techn</td><td>Cat# 451-MB</td></tr><tr><td>LPS from E.coli</td><td>Sigma-Aldrich</td><td>Cat# L2630</td></tr><tr><td>Lipofectamine™ RNAiMAX</td><td>Thermo Fisher Scientific</td><td>Cat# 13778075</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>Cat# M6250</td></tr><tr><td>Recombinant murine IL-2</td><td>Bio-Techn</td><td>Cat# 402-ML</td></tr><tr><td>Basement Membrane Extract</td><td>Cultrex</td><td>Cat# 3432-010-01</td></tr><tr><td>Recombinant murine CXCL10</td><td>Bio-Techn</td><td>Cat# 466-CR</td></tr><tr><td>SeeBlue™ Plus2 pre-stained makers</td><td>Thermo Fisher Scientific</td><td>Cat# LC5925</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>Cat# P1379</td></tr><tr><td>Luminata™ Crescendo Western HRP substrate</td><td>Millipore</td><td>Cat# WBLUR</td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>Cat# D9663</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>Cat# X100</td></tr><tr><td>FITC-conjugated dextran (2,000,000 MW)</td><td>Thermo Fisher Scientific</td><td>Cat# D7137</td></tr></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Rat anti-mouse LYVE-1 APC (clone 223322)</td><td>Bio-Techn</td><td>Cat# FAB2125A; RRID: AB_10972770</td></tr><tr><td>Rat anti-mouse MHCII PE (clone M5/114.15.2)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5321-82; RRID: AB_465928</td></tr><tr><td>Rat anti-mouse MHCII Brilliant Violet 421 <sup>TM</sup> (clone M5/114.15.2)</td><td>Biolegend®</td><td>Cat# 107631; RRID: AB_10900075</td></tr><tr><td>Rat anti-mouse MHCII Brilliant Violet 510 <sup>TM</sup> (clone M5/114.15.2)</td><td>Biolegend®</td><td>Cat# 107635; RRID: AB_2561397</td></tr><tr><td>Rat anti-mouse MHCII FITC (clone M5/114.15.2)</td><td>Biolegend®</td><td>Cat# 107605; RRID: AB_313320</td></tr><tr><td>Mouse anti-mouse NK1.1 APC (clone PK136)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-5941-82; RRID: AB_469479</td></tr><tr><td>Rat anti-mouse PNAlexa Fluor® 647 (clone MECA-79)</td><td>Biolegend®</td><td>Cat# 120807; RRID: AB_2783059</td></tr><tr><td>Rat anti-mouse TNFα PE (clone MP6-XT22)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-7321-82; RRID: AB_466199</td></tr><tr><td>Rat anti-mouse VCAM-1 PE (clone 429 (MVCAM.A))</td><td>Biolegend®</td><td>Cat# 105713; RRID: AB_1134166</td></tr><tr><td>Rat anti-mouse TER-119 PerCP-Cy5.5 (clone TER-119)</td><td>Thermo Fisher Scientific</td><td>Cat# 45-5921-82; RRID: AB_925765</td></tr><tr><td>Rat anti-mouse CD8α (clone 53-6.7)</td><td>Biolegend®</td><td>Cat# 100768; RRID: AB_2616823</td></tr><tr><td>Armenian hamster anti-mouse CD3ε (clone 145-2C11)</td><td>Thermo Fisher Scientific</td><td>Cat# 16-0031-82; RRID: AB_468847</td></tr><tr><td>Syrian hamster anti-mouse CD28 (Clone 37.51)</td><td>Thermo Fisher Scientific</td><td>Cat# 16-0281-82; RRID: AB_468921</td></tr></tbody></table>

Bacterial and virus strains

<table><tr><td>E. coli (DH10B competent cells)</td><td>Thermo Fisher Scientific</td><td>Cat# EC0113</td></tr></table>

Biological samples

<table><tr><td>Human breast adenocarcinoma tissue</td><td>King's Health Partners<br/>Cancer Biobank</td><td>REC reference 12/EE/0493</td></tr></table>

Chemicals, peptides, and recombinant proteins

<table><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>Cat# SAE0151</td></tr><tr><td>FCS</td><td>Thermo Fisher Scientific</td><td>Cat# 26170043</td></tr><tr><td>Spectinomycin</td><td>Sigma-Aldrich</td><td>Cat# S0692</td></tr><tr><td>LR Clonase™ II Plus enzyme mix</td><td>Thermo Fisher Scientific</td><td>Cat# 12538120</td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>Cat# T8154</td></tr><tr><td>Maraviroc</td><td>Cayman</td><td>Cat# 14641</td></tr><tr><td>5-Fluorouracil</td><td>Sigma-Aldrich</td><td>Cat# F6627</td></tr><tr><td>Collagenase I from Clostridium Histolyticum</td><td>Sigma-Aldrich</td><td>Cat# C0130</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>Cat# BSAV-RO</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>Cat# E8008</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma-Aldrich</td><td>Cat# P4333</td></tr><tr><td>Deoxyribonuclease I</td><td>AppliChem</td><td>Cat# A3778</td></tr><tr><td>RBC lysis buffer</td><td>Roche</td><td>Cat# 11814389001</td></tr><tr><td>Recombinant murine M-CSF</td><td>Bio-Techn</td><td>Cat# 416-ML</td></tr><tr><td>Recombinant murine IL-4</td><td>Bio-Techn</td><td>Cat# 404-ML</td></tr><tr><td>Recombinant murine IL-6</td><td>Bio-Techn</td><td>Cat# 406-ML</td></tr><tr><td>Recombinant murine IFN-γ</td><td>Bio-Techn</td><td>Cat# 485-ML</td></tr><tr><td>Recombinant murine CCL4</td><td>Bio-Techn</td><td>Cat# 451-MB</td></tr><tr><td>LPS from E.coli</td><td>Sigma-Aldrich</td><td>Cat# L2630</td></tr><tr><td>Lipofectamine™ RNAiMAX</td><td>Thermo Fisher Scientific</td><td>Cat# 13778075</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>Cat# M6250</td></tr><tr><td>Recombinant murine IL-2</td><td>Bio-Techn</td><td>Cat# 402-ML</td></tr><tr><td>Basement Membrane Extract</td><td>Cultrex</td><td>Cat# 3432-010-01</td></tr><tr><td>Recombinant murine CXCL10</td><td>Bio-Techn</td><td>Cat# 466-CR</td></tr><tr><td>SeeBlue™ Plus2 pre-stained makers</td><td>Thermo Fisher Scientific</td><td>Cat# LC5925</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>Cat# P1379</td></tr><tr><td>Luminata™ Crescendo Western HRP substrate</td><td>Millipore</td><td>Cat# WBLUR</td></tr><tr><td>Donkey serum</td><td>Sigma-Aldrich</td><td>Cat# D9663</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>Cat# X100</td></tr><tr><td>FITC-conjugated dextran (2,000,000 MW)</td><td>Thermo Fisher Scientific</td><td>Cat# D7137</td></tr></table>

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**Continued**

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>4',6-diamidino-2-phenylindole, dihydrochloride (DAPI)</td><td>Thermo Fisher Scientific</td><td>Cat# D1306</td></tr><tr><td>7-amino actinomycin D (7AAD)</td><td>Sigma-Aldrich</td><td>Cat# A9400</td></tr><tr><td>Fixable Viability Dye eFluor® 780</td><td>Thermo Fisher Scientific</td><td>Cat# 65-0865-14</td></tr><tr><td>Near-IR Dead cell staining kit</td><td>Thermo Fisher Scientific</td><td>Cat# L34976</td></tr><tr><td>Mm-IL6 probe</td><td>Bio-Techne</td><td>Cat# 315898</td></tr><tr><td>XenoLight D-luciferin</td><td>PerkinElmer</td><td>Cat# 122799</td></tr></tbody></table>

**Critical commercial assays**

<table><tbody><tr><td>Sequal PrepTM Long PCR kit</td><td>Thermo Fisher Scientific</td><td>Cat# A10498</td></tr><tr><td>MycoAlert Mycoplasma Detection Kit</td><td>Lonza</td><td>Cat# LT07-218</td></tr><tr><td>Anti-PE MicroBeads</td><td>Miltenyi Biotec</td><td>Cat# 130-048-801</td></tr><tr><td>MidiMacs separator</td><td>Miltenyi Biotec</td><td>Cat# 130-042-302</td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>Cat# 130-042-401</td></tr><tr><td>CD8a+ T-cell isolation Kit, mouse</td><td>Miltenyi Biotec</td><td>Cat# 130-104-075</td></tr><tr><td>AccuCheck counting beads</td><td>Thermo Fisher Scientific</td><td>Cat# PCB100</td></tr><tr><td>Pierce™ BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>Cat# 23225</td></tr><tr><td>RNAScope Multiplex Fluorescent Reagent Kit v2 Assay</td><td>Bio-Techne</td><td>Cat# 323100-USM</td></tr><tr><td>Opal™ 570 Reagent Pack</td><td>Akoya Biosciences</td><td>Cat# FP1488001KT</td></tr><tr><td>PureLink® RNA Mini Kit</td><td>Invitrogen</td><td>Cat# 12183020</td></tr><tr><td>Ovation® PicoSL WTA system V2</td><td>Tecan</td><td>Cat# 3312</td></tr><tr><td>Encore® BiotinIL Module</td><td>Tecan</td><td>Cat# 4200</td></tr><tr><td>Whole-Genome Gene Expression Direct Hybridisation Assay</td><td>Illumina</td><td>Cat# BD-901-1002</td></tr><tr><td>CellTracker™ Deep Red Dye</td><td>Thermo Fisher Scientific</td><td>Cat# C34565</td></tr><tr><td>Foxp3 Transcription Factor Staining Buffer Set</td><td>Thermo Fisher Scientific</td><td>Cat# 00-5523-00</td></tr></tbody></table>

**Deposited data**

<table><tbody><tr><td>Microarray dataset</td><td>This paper</td><td>GEO: GSE192911</td></tr><tr><td>Bulk RNA-seq dataset</td><td>Opzoomer et al.<sup>14</sup></td><td>GEO: GSE160561</td></tr><tr><td>scRNA-seq dataset</td><td>Opzoomer et al.<sup>14</sup></td><td>GEO: GSE160641</td></tr><tr><td>Microarray dataset</td><td>Muliaditan et al.<sup>3</sup></td><td>GEO: GSE113034</td></tr></tbody></table>

**Experimental models: Cell lines**

<table><tbody><tr><td>3B-11 murine endothelial cells</td><td>ATCC</td><td>Cat# CRL-2160; RRID: CVCL_5487</td></tr><tr><td>4T1 mammary adenocarcinoma cells</td><td>ATCC</td><td>Cat# CRL-2539; RRID: CVCL_0125</td></tr></tbody></table>

**Experimental models: Organisms/strains**

<table><tbody><tr><td>MMTV-PyMT (FVB/N background)</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:002374</td></tr><tr><td>WT Balb/c</td><td>Charles River</td><td>RRID: IMSR_CRL:028</td></tr><tr><td>WT C57BL/6</td><td>Charles River</td><td>RRID: IMSR_CRL:027</td></tr><tr><td>homozygous Il6<sup>-/-</sup> (B6.129S2-Il6tm1Kopf/J) C57BL/6</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:002650</td></tr><tr><td>Ccr5<sup>-/-</sup> (B6.129P2-Ccr5tm1Kuz/J) C57BL/6</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:005427</td></tr><tr><td>Lyz2-cre (B6.129P2-Lyz2tm1(cre)<sup>fl/fl</sup>) C57BL/6</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:004781</td></tr><tr><td>C57BL/6 homozygous Kaede</td><td>Professor Michio Tomura, Osaka Ohtani University, Osaka, Japan</td><td>N/A</td></tr><tr><td>Hmox1<sup>fl/fl</sup></td><td>Professor George Kollias, Biomedical Sciences Research Center "Alexander Fleming", Athens, Greece</td><td>N/A</td></tr><tr><td>HO-1<sup>Luc/EGFP</sup></td><td>This paper</td><td>N/A</td></tr></tbody></table>

**Oligonucleotides**

<table><tbody><tr><td>ON-TARGETplus SMARTpool siRNA targeting c-Maf</td><td>Horizon Discovery Ltd</td><td>Cat# J-040681-10-0005</td></tr><tr><td>ON-TARGETplus SMARTpool siRNA targeting Stat3</td><td>Horizon Discovery Ltd</td><td>Cat# J-040794-10-0005</td></tr></tbody></table>

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