**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><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>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>ON-TARGETplus SMARTpool siRNA targeting Hmox1</td><td>Horizon Discovery Ltd</td><td>Cat# J-040543-12</td></tr><tr><td>ON-TARGETplus non-targeting siRNA</td><td>Horizon Discovery Ltd</td><td>Cat# (D-001810-01-05</td></tr><tr><td>II6 gene expression assay</td><td>Thermo Fisher Scientific</td><td>Cat# 4331182; Assay ID Mm00446190_m1</td></tr><tr><td>Tbp gene expression assay</td><td>Thermo Fisher Scientific</td><td>Cat# 4331182; Assay ID Mm01277045_m1</td></tr></tbody></table>

Recombinant DNA

<table><tr><td>Flp mRNA</td><td>Miltenyi Biotech</td><td>Cat# 130-106-769</td></tr></table>

Software and algorithms

<table><tr><td>NIS Elements Advanced Research software</td><td>Nikon</td><td>https://www.microscope.healthcare.nikon.com/products/software/nis-elements/nis-elements-advanced-research</td></tr><tr><td>Cell counter</td><td>ImageJ-plugin</td><td>https://imagej.nin.gov/ij/plugins/cell-counter.html</td></tr><tr><td>Analyze Spheroid Cell Invasion In 3D Matrix</td><td>ImageJ-macro</td><td>https://github.com/MontpellierRessourcesImagerie/imagej-macros_and scripts/wiki/Analyze-Spheroid-Cell-Invasion-In-3D-Matrix</td></tr><tr><td>FlowJo v.10 Software</td><td>Tree Star, Inc; BD biosciences</td><td>https://www.flowjo.com/</td></tr><tr><td>Living Image Software</td><td>PerkinElmer</td><td>https://www.perkinelmer.com/product/spectrum-200-living-image-v4series-1-128113</td></tr><tr><td>QIAGEN IPA software</td><td>QIAGEN Inc; Krämer et al.<sup>36</sup></td><td>https://digitalinsights.qiagen.com/IPA</td></tr><tr><td>R version 4.0.2</td><td>The R Project for Statistical Computing</td><td>https://www.r-project.org/</td></tr><tr><td>Seurat v3 R package</td><td>Stuart et al.<sup>80</sup></td><td>https://satijalab.org/seurat/</td></tr><tr><td>Garnett package</td><td>Pliner et al.<sup>81</sup></td><td></td></tr><tr><td>GraphPad Prism 8 software</td><td>GraphPad</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr><tr><td>Partek® Genomics Suite® software</td><td>Partek</td><td>https://www.partek.com/partek-genomics-suite/</td></tr><tr><td>“CompareGrowthCurves” function of the statmod software package</td><td>Elso et al.<sup>82</sup></td><td>https://rdrr.io/cran/statmod/man/growthcurve.html</td></tr></table>

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Dr James Arnold (james.arnold@kcl.ac.uk).

Material availability

Mice and reagents generated in this study will be made available from the Lead Contact for academic/non-commercial research purposes on request under a Material Transfer Agreement.

Data and code availability

The RNA-seq transcriptomic and microarray datasets that support the findings of this study are available through the Gene Expression Omnibus; GSE160561, GSE160641, GSE113034. The microarray datasets are available at GSE192911. The authors declare that all other data supporting the findings of this study are available within the paper and its supplementary information files. Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request.
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>ON-TARGETplus SMARTpool siRNA targeting Hmox1</td><td>Horizon Discovery Ltd</td><td>Cat# J-040543-12</td></tr><tr><td>ON-TARGETplus non-targeting siRNA</td><td>Horizon Discovery Ltd</td><td>Cat# (D-001810-01-05</td></tr><tr><td>II6 gene expression assay</td><td>Thermo Fisher Scientific</td><td>Cat# 4331182; Assay ID Mm00446190_m1</td></tr><tr><td>Tbp gene expression assay</td><td>Thermo Fisher Scientific</td><td>Cat# 4331182; Assay ID Mm01277045_m1</td></tr></tbody></table>

Recombinant DNA

<table><tr><td>Flp mRNA</td><td>Miltenyi Biotech</td><td>Cat# 130-106-769</td></tr></table>

Software and algorithms

<table><tr><td>NIS Elements Advanced Research software</td><td>Nikon</td><td>https://www.microscope.healthcare.nikon.com/products/software/nis-elements/nis-elements-advanced-research</td></tr><tr><td>Cell counter</td><td>ImageJ-plugin</td><td>https://imagej.nin.gov/ij/plugins/cell-counter.html</td></tr><tr><td>Analyze Spheroid Cell Invasion In 3D Matrix</td><td>ImageJ-macro</td><td>https://github.com/MontpellierRessourcesImagerie/imagej-macros_and scripts/wiki/Analyze-Spheroid-Cell-Invasion-In-3D-Matrix</td></tr><tr><td>FlowJo v.10 Software</td><td>Tree Star, Inc; BD biosciences</td><td>https://www.flowjo.com/</td></tr><tr><td>Living Image Software</td><td>PerkinElmer</td><td>https://www.perkinelmer.com/product/spectrum-200-living-image-v4series-1-128113</td></tr><tr><td>QIAGEN IPA software</td><td>QIAGEN Inc; Krämer et al.<sup>36</sup></td><td>https://digitalinsights.qiagen.com/IPA</td></tr><tr><td>R version 4.0.2</td><td>The R Project for Statistical Computing</td><td>https://www.r-project.org/</td></tr><tr><td>Seurat v3 R package</td><td>Stuart et al.<sup>80</sup></td><td>https://satijalab.org/seurat/</td></tr><tr><td>Garnett package</td><td>Pliner et al.<sup>81</sup></td><td></td></tr><tr><td>GraphPad Prism 8 software</td><td>GraphPad</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr><tr><td>Partek® Genomics Suite® software</td><td>Partek</td><td>https://www.partek.com/partek-genomics-suite/</td></tr><tr><td>“CompareGrowthCurves” function of the statmod software package</td><td>Elso et al.<sup>82</sup></td><td>https://rdrr.io/cran/statmod/man/growthcurve.html</td></tr></table>

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Dr James Arnold (james.arnold@kcl.ac.uk).

Material availability

Mice and reagents generated in this study will be made available from the Lead Contact for academic/non-commercial research purposes on request under a Material Transfer Agreement.

Data and code availability

The RNA-seq transcriptomic and microarray datasets that support the findings of this study are available through the Gene Expression Omnibus; GSE160561, GSE160641, GSE113034. The microarray datasets are available at GSE192911. The authors declare that all other data supporting the findings of this study are available within the paper and its supplementary information files. Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request.
## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Mice

Mice used in this study were housed under specific pathogen-free conditions in individually ventilated cages at a temperature of 21°C with water and food *ad libitum*. Wild type mice used in this study were female and 4-6 weeks-old on either a Balb/c or C57BL/6 background and obtained from Charles River. Transgenic mice used in the study included homozygous Il6/- (B6.129S2-Il6tm1-Kopf/J), Ccr5/- (B6.129P2-Ccr5tm1Kuz/J), *Lyz2*-Cre (B6.129P2-Lyz2tm1(cre)lf6/J) and *Lyve-1-cre* (B6;129P2-Lyve1tm1.1(EGFP/cre)Cys/J) were on a C57BL/6 background and *MMTV-PyMT* mice were on FVB/N background<sup>83</sup> and obtained from The Jackson Laboratory. Where indicated, female KO mice were crossed with male *MMTV-PyMT* mice and the F2 homozygous or F2 WT offspring used experimentally. Female C57BL/6 homozygous *Kaede* mice<sup>35</sup> were crossed with male *MMTV-PyMT* (FVB background) mice and the F1 offspring were used experimentally. Cohort sizes were informed by prior studies.<sup>3,47</sup> The *Hmox1fl/fl* and *Lyz2* (Lysozyme M) or *Lyve-1* driven Cre-recombinase were crossed for the *MMTV-PyMT/Hmox1fl/fl* *Lyz2Cre*<sup>84</sup> or *MMTV-PyMT/Hmox1fl/fl* *Lyve1Cre* (referred to as *Lyve1Cre* mice in the manuscript). *Hmox1fl/fl* mice were a gift from Professor George Kollias, Biomedical Sciences Research Center "Alexander Fleming", Athens, Greece. All mice used for experiments were female and randomly assigned to treatment groups. Mice were approximately 21-26 g when tumors became palpable. Experiments were performed in at least duplicate and for spontaneous *MMTV-PyMT* tumor studies individual mice were collected on separate days and all data points are presented. End points for tumor studies were assessed on a welfare assessment, however, were typically when the primary tumor reached 1500mm<sup>3</sup> (unless stated otherwise). For generation of HO-1-Luciferace-EGFP- knock-in mouse (HO-1<sup>Luc/EGFP</sup>) we have used BAC (Bacterial Artificial Chromosomes) recombineering strategy.<sup>85</sup> A synthetic cassette containing P2A-Luciferase-P2A-EGFP-Stop-FRT-βAct-Neo-pA-FRT sequence was inserted before the endogenous stop codon of *Hmox1* in BACs that correspond to *Hmox1* locus. The insert-containing BAC was further subcloned into pR3R4ccdB plasmid that contains gateway sites. The resulting "intermediate" vector contains ~5kb 5' and 3' homology arms and was used for generation of the final vector by modular vector assembly by the gateway method. The gateway reaction was performed using LR Clonase™ II Plus enzyme mix (Thermo Fisher Scientific) according to the manufacturers' protocol. The intermediate targeting vector was combined with pL3/L4 (DTA selection cassette) and incubated at 25°C overnight (O.N.). After treatment with Proteinase K (Sigma-Aldrich), the reaction mix was transformed into chemically competent *E. coli* (DH10B, Thermo Fisher Scientific) and plated onto YEG (yeast extract with glucose) agar plates containing 4-chlorophenylalanine and spectinomycin antibiotic (Sigma-Aldrich) (25 µg/mL). Individual colonies were picked and verified with restriction digestion quality control and sequenced across all recombineered junctions. A positive final targeting vector was linearized at *Asisl* restriction site and electroporated to C57BL/6J embryonic stem (ES) cells. Clones were selected and picked under G418 antibiotic selection. Genomic DNA from positively selected ES cell clones were further screened with long range and short-range PCR for target recombination, 5' and 3' homology arms using the Sequal Prep™ Long PCR kit (Thermo Fisher Scientific). The neomycin selection cassette, which was flanked by flippase (Flp) recombination target sites, was removed by fertilizing WT C57BL/6 oocytes with sperm from HO-1<sup>Luc/EGFP</sup> mice. Six hours after insemination, fertilized zygotes were identified by the presence of pronuclei (87%) and received a cytoplasmic injection of Flp mRNA (Miltenyi Biotech). Injected zygotes were surgically transferred to CD-1 0.5dpc pseudopregnant recipient females. Long range PCR confirmed successful recombination.

### Cell lines

3B-11 murine endothelial cells and 4T1 mammary adenocarcinoma were obtained from ATCC. Cell lines were confirmed to be mycoplasma free using the MycoAlert Mycoplasma Detection Kit (Lonza) and were cultured in RPMI (Gibco) supplemented with 10% FCS (Thermo Fisher Scientific).

### Study approval

All experiments involving animals were approved by the Animal and Welfare and Ethical Review Board of King's College London or the University of Birmingham and the Home Office UK. Human breast adenocarcinoma tissue was obtained with informed consent under ethical approval from the King's Health Partners Cancer Biobank (REC reference 12/EE/0493).

## METHOD DETAILS

### Tumor studies

4T1 (Balb/c) cells were orthotopically implanted for tumors (to generate splenic tumor-derived, but TME naive, monocytes for *in vitro* studies). A total of 2.5 x 10<sup>5</sup> cells in 100 µL RPMI were injected subcutaneously into the mammary fat pad of syngeneic female mice. In *MMTV-PyMT* mice, tumors arose spontaneously. When tumors became palpable, volumes were measured every 2-4 days using digital caliper measurements of the long (L) and short (S) dimensions of the tumor. Tumor volume was established using the following equation: Volume= (S<sup>2</sup>xL)/2. *MMTV-PyMT* *Kaede* mice were photolabeled under anesthesia. Individual tumors were exposed to a violet light (405nm wavelength) through the skin for a total of nine 20 second exposure cycles with a short 5 second break interval between each cycle. Black cardboard was used to shield the rest of the mouse throughout the photoconversion procedure. Mice for 0 h time points were culled immediately after photoconversion. This photoconversion approach was adapted from that used to label peripheral lymph nodes<sup>86</sup> and was optimized for *MMTV-PyMT* tumors.<sup>14</sup> Tumor tissue for flow cytometry analyses was enzyme-digested to release single cells as previously described.<sup>47,87</sup> In brief, tissues were minced using scalpels, and then single
## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Mice

Mice used in this study were housed under specific pathogen-free conditions in individually ventilated cages at a temperature of 21°C with water and food *ad libitum*. Wild type mice used in this study were female and 4-6 weeks-old on either a Balb/c or C57BL/6 background and obtained from Charles River. Transgenic mice used in the study included homozygous Il6/- (B6.129S2-Il6tm1-Kopf/J), Ccr5/- (B6.129P2-Ccr5tm1Kuz/J), *Lyz2*-Cre (B6.129P2-Lyz2tm1(cre)lf6/J) and *Lyve-1-cre* (B6;129P2-Lyve1tm1.1(EGFP/cre)Cys/J) were on a C57BL/6 background and *MMTV-PyMT* mice were on FVB/N background<sup>83</sup> and obtained from The Jackson Laboratory. Where indicated, female KO mice were crossed with male *MMTV-PyMT* mice and the F2 homozygous or F2 WT offspring used experimentally. Female C57BL/6 homozygous *Kaede* mice<sup>35</sup> were crossed with male *MMTV-PyMT* (FVB background) mice and the F1 offspring were used experimentally. Cohort sizes were informed by prior studies.<sup>3,47</sup> The *Hmox1fl/fl* and *Lyz2* (Lysozyme M) or *Lyve-1* driven Cre-recombinase were crossed for the *MMTV-PyMT/Hmox1fl/fl* *Lyz2Cre*<sup>84</sup> or *MMTV-PyMT/Hmox1fl/fl* *Lyve1Cre* (referred to as *Lyve1Cre* mice in the manuscript). *Hmox1fl/fl* mice were a gift from Professor George Kollias, Biomedical Sciences Research Center "Alexander Fleming", Athens, Greece. All mice used for experiments were female and randomly assigned to treatment groups. Mice were approximately 21-26 g when tumors became palpable. Experiments were performed in at least duplicate and for spontaneous *MMTV-PyMT* tumor studies individual mice were collected on separate days and all data points are presented. End points for tumor studies were assessed on a welfare assessment, however, were typically when the primary tumor reached 1500mm<sup>3</sup> (unless stated otherwise). For generation of HO-1-Luciferace-EGFP- knock-in mouse (HO-1<sup>Luc/EGFP</sup>) we have used BAC (Bacterial Artificial Chromosomes) recombineering strategy.<sup>85</sup> A synthetic cassette containing P2A-Luciferase-P2A-EGFP-Stop-FRT-βAct-Neo-pA-FRT sequence was inserted before the endogenous stop codon of *Hmox1* in BACs that correspond to *Hmox1* locus. The insert-containing BAC was further subcloned into pR3R4ccdB plasmid that contains gateway sites. The resulting "intermediate" vector contains ~5kb 5' and 3' homology arms and was used for generation of the final vector by modular vector assembly by the gateway method. The gateway reaction was performed using LR Clonase™ II Plus enzyme mix (Thermo Fisher Scientific) according to the manufacturers' protocol. The intermediate targeting vector was combined with pL3/L4 (DTA selection cassette) and incubated at 25°C overnight (O.N.). After treatment with Proteinase K (Sigma-Aldrich), the reaction mix was transformed into chemically competent *E. coli* (DH10B, Thermo Fisher Scientific) and plated onto YEG (yeast extract with glucose) agar plates containing 4-chlorophenylalanine and spectinomycin antibiotic (Sigma-Aldrich) (25 µg/mL). Individual colonies were picked and verified with restriction digestion quality control and sequenced across all recombineered junctions. A positive final targeting vector was linearized at *Asisl* restriction site and electroporated to C57BL/6J embryonic stem (ES) cells. Clones were selected and picked under G418 antibiotic selection. Genomic DNA from positively selected ES cell clones were further screened with long range and short-range PCR for target recombination, 5' and 3' homology arms using the Sequal Prep™ Long PCR kit (Thermo Fisher Scientific). The neomycin selection cassette, which was flanked by flippase (Flp) recombination target sites, was removed by fertilizing WT C57BL/6 oocytes with sperm from HO-1<sup>Luc/EGFP</sup> mice. Six hours after insemination, fertilized zygotes were identified by the presence of pronuclei (87%) and received a cytoplasmic injection of Flp mRNA (Miltenyi Biotech). Injected zygotes were surgically transferred to CD-1 0.5dpc pseudopregnant recipient females. Long range PCR confirmed successful recombination.

### Cell lines

3B-11 murine endothelial cells and 4T1 mammary adenocarcinoma were obtained from ATCC. Cell lines were confirmed to be mycoplasma free using the MycoAlert Mycoplasma Detection Kit (Lonza) and were cultured in RPMI (Gibco) supplemented with 10% FCS (Thermo Fisher Scientific).

### Study approval

All experiments involving animals were approved by the Animal and Welfare and Ethical Review Board of King's College London or the University of Birmingham and the Home Office UK. Human breast adenocarcinoma tissue was obtained with informed consent under ethical approval from the King's Health Partners Cancer Biobank (REC reference 12/EE/0493).

## METHOD DETAILS

### Tumor studies

4T1 (Balb/c) cells were orthotopically implanted for tumors (to generate splenic tumor-derived, but TME naive, monocytes for *in vitro* studies). A total of 2.5 x 10<sup>5</sup> cells in 100 µL RPMI were injected subcutaneously into the mammary fat pad of syngeneic female mice. In *MMTV-PyMT* mice, tumors arose spontaneously. When tumors became palpable, volumes were measured every 2-4 days using digital caliper measurements of the long (L) and short (S) dimensions of the tumor. Tumor volume was established using the following equation: Volume= (S<sup>2</sup>xL)/2. *MMTV-PyMT* *Kaede* mice were photolabeled under anesthesia. Individual tumors were exposed to a violet light (405nm wavelength) through the skin for a total of nine 20 second exposure cycles with a short 5 second break interval between each cycle. Black cardboard was used to shield the rest of the mouse throughout the photoconversion procedure. Mice for 0 h time points were culled immediately after photoconversion. This photoconversion approach was adapted from that used to label peripheral lymph nodes<sup>86</sup> and was optimized for *MMTV-PyMT* tumors.<sup>14</sup> Tumor tissue for flow cytometry analyses was enzyme-digested to release single cells as previously described.<sup>47,87</sup> In brief, tissues were minced using scalpels, and then single
cells were liberated by incubation for 60 mins at 37°C with 1 mg/mL Collagenase I from *Clostridium Histolyticum* (Sigma-Aldrich) and 0.1 mg/mL Deoxyribonuclease I (AppliChem) in RPMI. Released cells were then passed through a 70 µm cell strainer prior to staining for flow cytometry analyses. Viable cells were numerated using a hemocytometer with trypan blue (Sigma-Aldrich) exclusion. For drug treatments, drugs were freshly prepared on the day of injection and administered by intraperitoneal (i.p.) injection using a 26 G needle. Maraviroc (Cayman) was solubilized in ethanol and diluted with saline and administered to mice i.p. using a bi-daily dose of 10mg/kg. 5-Fluorouracil (Sigma-Aldrich) was prepared fresh and dissolved in saline at 6 mg/mL and injected to mice i.p. at 40 mg/kg/4 days. Immune-depleted mice were injected i.p. every 4 days, starting 48 h prior to the commencement of treatment, with 400 µg of anti-CD8α (53-6.7) (Biolegend®).

**In vitro derived macrophage polarization and gene knockdown**

Murine bone marrow (BM) was flushed from the femur and tibia of non tumor bearing WT C57BL/6 mice using a syringe and needle. Splenocytes for monocyte isolation were acquired from spleens of 4T1 tumor bearing mice by crushing through a 70 µm pore strainer. RBC were lysed using RBC lysis buffer (Roche). Ly6C⁺ monocytes were isolated from the splenocytes by blocking Fc receptors using 5 µg/mL anti-CD16/32 (2.4G2, Tonbo Biosciences) prior to staining with Ly6C PE (HK1.4; Thermo Fisher Scientific) in MACs buffer (DPBS (Thermo Fisher Scientific), 0.5% BSA (Sigma-Aldrich), 2mM EDTA (Sigma-Aldrich)) at 1 µg/mL, followed by anti-PE MicroBeads (Miltenyi Biotec) and isolated using a MidiMacs separator and LS columns (Miltenyi Biotec) according to the manufacturers' protocol. BM cells or isolated Ly6C⁺ monocytes were plated in RPMI, 10% FCS, 1 x penicillin/streptomycin (Sigma-Aldrich), 10 ng/mL recombinant murine M-CSF (Bio-Techne) at 1 x 10⁶ cells/well on 6 well plates for 72 h prior to subsequent downstream mRNA and protein analyses. Where viable macrophages were required for ongoing experiments, 5.5 x 10⁶ BM cells were plated at day 0 on 6 cm non tissue culture-treated plates in the above macrophage culture media. Additional murine cytokines, IL-4, IL-6, IFN-γ (Bio-Techne) and/or LPS from *Escherichia Coli* (Sigma-Aldrich) were added where indicated in the figure legends at 50 ng/mL unless stated otherwise. After 72 h in culture, macrophage purity was assessed by flow cytometry. Macrophages differentiated in the presence of M-CSF alone were referred to as M<sub>(0)</sub> cells, and macrophages differentiated in the presence of M-CSF and IL-6 were labelled M<sub>(IL-6)</sub> cells. For siRNA knock down experiments, M<sub>(0)</sub> macrophages had their media changed to IMDM, 10% FCS and 10 ng/mL M-CSF. In an Eppendorf, ON-TARGET<sub>plus</sub> SMARTpool siRNA (Horizon Discovery Ltd) targeting c-Maf (J-040681-10-0005) Stat3 (J-040794-10-0005), Hmox1 (J-040543-12) or ON-TARGET<sub>plus</sub> non-targeting siRNA (D-001810-01-05), were added to 250 µL of Opti-MEM (Thermo Fisher Scientific) at a concentration of 100 pmol. To each respective tube, an equal volume of Opti-MEM mixed with 5 µL Lipofectamine™ RNA<sub>IMAX</sub> (Thermo Fisher Scientific) was added and incubated for 20 mins at room temperature (RT). The transfection mixture was then drop-wise added to M<sub>(0)</sub> BMDMs. The wells were gently mixed until the siRNA transfection buffer distributed evenly and incubated for 96 h to allow for protein knock down in the presence or absence of polarizing cytokines at 25 ng/mL as indicated.

**T-cell isolation**

For isolating murine T-cells, spleens were excised from WT C57BL/6 mice and placed in RPMI, 10% FCS, 20 µM 2-mercaptoethanol (Sigma-Aldrich), 1X penicillin/streptomycin. Spleens were crushed through a 70 µm pore strainer and washed through using RPMI. Liberated splenocytes were centrifuged at 500 x g for 3 mins and the cell pellet was re-suspended in 1 mL of red blood cell lysis buffer (Roche) for 2 mins at RT. Cells were then re-centrifuged at 500 x g for 3 mins and the pellet was resuspended in RPMI. Live cells were numerated using Trypan blue exclusion on a hemocytometer. CD8⁺ T-cells were purified using the CD8a⁺ T-cell isolation Kit, mouse (Miltenyi Biotec) and isolated using a MidiMacs separator and LS columns (Miltenyi Biotec) according to the manufacturers' protocol. T-cells were resuspended in T-cell culture media that was further supplemented with 2 ng/mL recombinant murine IL-2 (Bio-Techne) and purified CD8⁺ T-cells were plated at a density of 0.1x10⁶ cells/well in 200 µL onto a high binding 96-well plate (Sigma-Aldrich) that had been pre-coated O.N. with a mix of anti-mouse CD3ε (145-2C11, 5 µg/mL) and anti-mouse CD28 (37.51, 3 µg/mL) antibodies in sterile DPBS (100 µL/well) at 4°C. After 48 h CD8⁺ T-cells were transferred to a fresh uncoated plate and rested for at least 48 h before being numerated and used for down-stream *in vitro* assays.

**In vitro macrophage transwell migration assay**

$M_{(0)}$ and $M_{(IL-6)}$ BMDMs were prepared as described above from WT or Ccr5<sup>-/-</sup> C57BL/6 mice. Macrophages were removed from the plate using 1 mL of enzyme-free dissociation media (Thermo Fisher Scientific). $M_{(0)}$ or $M_{(IL-6)}$ cells were fluorescently labelled using CellTracker™ deep red dye (Thermo Fisher Scientific) and suspended at 1x10<sup>6</sup>/mL in RPMI, 10% FCS (complete media) and 100 µL was transferred onto a 8 µm pore transwell insert (Corning) sitting in 24-well plate. After 10-minute incubation at 37°C, 5% $CO_2$, 600 µL of complete media with/without 50 ng/mL recombinant murine CCL4 (Bio-Techne) was added to the well. After 24-hour incubation at 37°C, 5% $CO_2$, the transwell insert was removed and the medium in the well was transferred to a FACS tube. The wells were washed with PBS 0.5% (w/v) BSA to ensure all the cells were collected. Collected cells were centrifuged and the cell pellet was resuspended in 200 µL PBS 0.5% (w/v) BSA. 20 µL of AccuCheck counting beads (Thermo Fisher Scientific) was added to each tube prior to FACS analysis. Counts of labelled viable cells and beads were determined using FlowJo software. Migrated cells concentration (cell/µL) was then calculated based on the manufacturers' manual. Relative migration was normalized using the CCL4 sample against the paired control.
cells were liberated by incubation for 60 mins at 37°C with 1 mg/mL Collagenase I from *Clostridium Histolyticum* (Sigma-Aldrich) and 0.1 mg/mL Deoxyribonuclease I (AppliChem) in RPMI. Released cells were then passed through a 70 µm cell strainer prior to staining for flow cytometry analyses. Viable cells were numerated using a hemocytometer with trypan blue (Sigma-Aldrich) exclusion. For drug treatments, drugs were freshly prepared on the day of injection and administered by intraperitoneal (i.p.) injection using a 26 G needle. Maraviroc (Cayman) was solubilized in ethanol and diluted with saline and administered to mice i.p. using a bi-daily dose of 10mg/kg. 5-Fluorouracil (Sigma-Aldrich) was prepared fresh and dissolved in saline at 6 mg/mL and injected to mice i.p. at 40 mg/kg/4 days. Immune-depleted mice were injected i.p. every 4 days, starting 48 h prior to the commencement of treatment, with 400 µg of anti-CD8α (53-6.7) (Biolegend®).

**In vitro derived macrophage polarization and gene knockdown**

Murine bone marrow (BM) was flushed from the femur and tibia of non tumor bearing WT C57BL/6 mice using a syringe and needle. Splenocytes for monocyte isolation were acquired from spleens of 4T1 tumor bearing mice by crushing through a 70 µm pore strainer. RBC were lysed using RBC lysis buffer (Roche). Ly6C⁺ monocytes were isolated from the splenocytes by blocking Fc receptors using 5 µg/mL anti-CD16/32 (2.4G2, Tonbo Biosciences) prior to staining with Ly6C PE (HK1.4; Thermo Fisher Scientific) in MACs buffer (DPBS (Thermo Fisher Scientific), 0.5% BSA (Sigma-Aldrich), 2mM EDTA (Sigma-Aldrich)) at 1 µg/mL, followed by anti-PE MicroBeads (Miltenyi Biotec) and isolated using a MidiMacs separator and LS columns (Miltenyi Biotec) according to the manufacturers' protocol. BM cells or isolated Ly6C⁺ monocytes were plated in RPMI, 10% FCS, 1 x penicillin/streptomycin (Sigma-Aldrich), 10 ng/mL recombinant murine M-CSF (Bio-Techne) at 1 x 10⁶ cells/well on 6 well plates for 72 h prior to subsequent downstream mRNA and protein analyses. Where viable macrophages were required for ongoing experiments, 5.5 x 10⁶ BM cells were plated at day 0 on 6 cm non tissue culture-treated plates in the above macrophage culture media. Additional murine cytokines, IL-4, IL-6, IFN-γ (Bio-Techne) and/or LPS from *Escherichia Coli* (Sigma-Aldrich) were added where indicated in the figure legends at 50 ng/mL unless stated otherwise. After 72 h in culture, macrophage purity was assessed by flow cytometry. Macrophages differentiated in the presence of M-CSF alone were referred to as M<sub>(0)</sub> cells, and macrophages differentiated in the presence of M-CSF and IL-6 were labelled M<sub>(IL-6)</sub> cells. For siRNA knock down experiments, M<sub>(0)</sub> macrophages had their media changed to IMDM, 10% FCS and 10 ng/mL M-CSF. In an Eppendorf, ON-TARGET<sub>plus</sub> SMARTpool siRNA (Horizon Discovery Ltd) targeting *c-Maf* (J-040681-10-0005) *Stat3* (J-040794-10-0005), *Hmox1* (J-040543-12) or ON-TARGET<sub>plus</sub> non-targeting siRNA (D-001810-01-05), were added to 250 µL of Opti-MEM (Thermo Fisher Scientific) at a concentration of 100 pmol. To each respective tube, an equal volume of Opti-MEM mixed with 5 µL Lipofectamine™ RNA<sub>IMAX</sub> (Thermo Fisher Scientific) was added and incubated for 20 mins at room temperature (RT). The transfection mixture was then drop-wise added to M<sub>(0)</sub> BMDMs. The wells were gently mixed until the siRNA transfection buffer distributed evenly and incubated for 96 h to allow for protein knock down in the presence or absence of polarizing cytokines at 25 ng/mL as indicated.

**T-cell isolation**

For isolating murine T-cells, spleens were excised from WT C57BL/6 mice and placed in RPMI, 10% FCS, 20 µM 2-mercaptoethanol (Sigma-Aldrich), 1X penicillin/streptomycin. Spleens were crushed through a 70 µm pore strainer and washed through using RPMI. Liberated splenocytes were centrifuged at 500 x g for 3 mins and the cell pellet was re-suspended in 1 mL of red blood cell lysis buffer (Roche) for 2 mins at RT. Cells were then re-centrifuged at 500 x g for 3 mins and the pellet was resuspended in RPMI. Live cells were numerated using Trypan blue exclusion on a hemocytometer. CD8⁺ T-cells were purified using the CD8a⁺ T-cell isolation Kit, mouse (Miltenyi Biotec) and isolated using a MidiMacs separator and LS columns (Miltenyi Biotec) according to the manufacturers' protocol. T-cells were resuspended in T-cell culture media that was further supplemented with 2 ng/mL recombinant murine IL-2 (Bio-Techne) and purified CD8⁺ T-cells were plated at a density of 0.1x10⁶ cells/well in 200 µL onto a high binding 96-well plate (Sigma-Aldrich) that had been pre-coated O.N. with a mix of anti-mouse CD3ε (145-2C11, 5 µg/mL) and anti-mouse CD28 (37.51, 3 µg/mL) antibodies in sterile DPBS (100 µL/well) at 4°C. After 48 h CD8⁺ T-cells were transferred to a fresh uncoated plate and rested for at least 48 h before being numerated and used for down-stream *in vitro* assays.

**In vitro macrophage transwell migration assay**

$M_{(0)}$ and $M_{(IL-6)}$ BMDMs were prepared as described above from WT or Ccr5<sup>-/-</sup> C57BL/6 mice. Macrophages were removed from the plate using 1 mL of enzyme-free dissociation media (Thermo Fisher Scientific). $M_{(0)}$ or $M_{(IL-6)}$ cells were fluorescently labelled using CellTracker™ deep red dye (Thermo Fisher Scientific) and suspended at 1x10⁶/mL in RPMI, 10% FCS (complete media) and 100 µL was transferred onto a 8 µm pore transwell insert (Corning) sitting in 24-well plate. After 10-minute incubation at 37°C, 5% $CO_2$, 600 µL of complete media with/without 50 ng/mL recombinant murine CCL4 (Bio-Techne) was added to the well. After 24-hour incubation at 37°C, 5% $CO_2$, the transwell insert was removed and the medium in the well was transferred to a FACS tube. The wells were washed with PBS 0.5% (w/v) BSA to ensure all the cells were collected. Collected cells were centrifuged and the cell pellet was resuspended in 200 µL PBS 0.5% (w/v) BSA. 20 µL of AccuCheck counting beads (Thermo Fisher Scientific) was added to each tube prior to FACS analysis. Counts of labelled viable cells and beads were determined using FlowJo software. Migrated cells concentration (cell/µL) was then calculated based on the manufacturers' manual. Relative migration was normalized using the CCL4 sample against the paired control.
**In vitro macrophage focal-point migration assay**

$M_{(0)}$ macrophages were generated as described above and removed from the plate using 1 mL of enzyme-free dissociation media (Thermo Fisher Scientific) and mechanically detaching the cells using a cell scrapper. The resultant solutions were centrifuged at 2000 x g for 3 mins and resuspended at a concentration of 10,000 cells/mL in 80% RPMI (inc.10%FCS) and 20% methylcellulose with 10 ng/mL M-CSF. To prepare a methylcellulose stock solution; autoclaved methylcellulose (Sigma-Aldrich) was dissolved at 24 g/L in pre-heated serum-free RPMI media for 2 mins at 60°C. After this, the solution was diluted with 2 volumes of RT serum-free RPMI and then mixed O.N. at 4°C. The final solution was cleared by centrifugation at 5000 x g for 2 h at RT. To the lid of an inverted petri dish, 25 µL drops of macrophages were placed, the lid was carefully inverted and placed on top of a DPBS filled petri dish. The dish was incubated for 24 h to allow the formation of macrophage spheroids within the hanging drop. Subsequently, media containing spheroids were collected in Eppendorf tubes using DPBS. The spheroids were allowed to briefly settle in the bottom of the Eppendorf tubes after which the DPBS was carefully removed and the spheroids were placed in wells containing RPMI, 10% FCS, 10 ng/mL M-CSF with or without 25 ng/mL IL-6 and placed in a 37°C 5% $CO_2$ incubator to allow attachment and eventual spreading. Images of spheroid cultures was performed using the live cell Eclipse Ti-2 inverted microscope in the Nikon Imaging Centre at King's College London. NIS Elements Advanced Research software (Nikon) was used to process the images. Total area of spheroids was measured using the "Analyze Spheroid Cell Invasion In 3D Matrix" macro in ImageJ, and cell number was counted using the "Cell counter" plugin in ImageJ.

**In vitro Pv nest transwell assay**

Transwell assays were conducted with transwell inserts with 8 µm pores (Corning) for migration studies and 0.4 µm pores (Corning) for permeability studies. Inserts were coated with Basement Membrane Extract (Cultrex) diluted 1:100 in RPMI for 1 h at RT. Excess Basement Membrane Extract was aspirated and 2 x $10^4$ 3B-11 endothelial cells were seeded onto the apical side of the transwell insert in RPMI supplemented with 10% FCS and left to attach for 24 h. Media was removed, the whole plate inverted and $10^5 M_{(0)}$ or $M_{(IL-6)}$ BMDMs were seeded onto the basolateral side of the transwell membrane in RPMI supplemented with 10% FCS and left to attach for 2 h at 37°C. Subsequently, the plate was reinverted to its original position and RPMI supplemented with 10% FCS, 10 ng/mL M-CSF with or without 10 ng/mL IL-6 added to the apical and basolateral space. After cells were left to interact for 24 h at 37°C, $4 x 10^5 CD8^+$ T cells (which had been prior incubated on anti-CD3 and -CD28 coated plated to develop effector function) were added to the inserts in RPMI, 10% FCS and 100 ng/mL with murine CXCL10 (Bio-Techne) spiked into the wells. After 16 h, migrated cells were collected from the well and stained for flow cytometry analysis and quantification with AccuCheck counting beads (Thermo Fisher Scientific). The permeability assay was performed as described previously.⁸⁸ In brief, permeability of the *in vitro* Pv niche was measured using 4% (w/v) Evans Blue-conjugated Bovine Serum Albumin (BSA) diluted in DPBS was placed into the apical transwell chamber while phenol red-free RPMI, 10% FCS was added to the basolateral chamber. Presence of Evans Blue-BSA in the basolateral chamber was assessed at indicated time-points by absorbance at 620 nm on a NanoDrop™ spectrophotometer (Thermo Fisher Scientific). These experiments were performed in the presence or absence of 10 ng/mL IL-6 and M-CSF.

**Western blot**

Cells were lysed and SDS PAGE/western blots were conducted as previously described.³ In brief, cells were lysed in the well using western blot lysis buffer 0.1M Tris-hydrochloride pH 6.8, with 20% glycerol and 4% sodium dodecyl sulphate containing 1X protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). All tubes were heated at 95°C for 15 mins to break down DNA. Protein concentration was then determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) using the manufacturers' protocol. Samples were then run under reducing conditions on 12% bis-tris sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels alongside SeeBlue™ Plus2 pre-stained makers (Thermo Fisher Scientific). SDS-PAGE gels were then transferred onto polyvinyl-difluoride (PVDF) membranes which were subsequently blocked in 100 mM Tris, 140 mM NaCl, 0.1% Tween 20 (Sigma-Aldrich), pH7.4 (TBS-T) containing 5% skimmed milk at RT for 1 h. Primary antibodies were applied at 4°C O.N. and secondary antibodies for 1 h at RT. Wash steps to remove unbound antibodies were 3 x 20 mins in TBS-T. The following primary antibodies were used: rabbit anti-β-actin, 1:5,000 (ab8227, Abcam), rabbit anti-HO-1 1:1,000 (10701-1-AP, Proteintech), rabbit anti-c-MAF 1:1,000 (ab77071, Abcam), rabbit anti-STAT3 1:2000 (clone 79D7, Cell Signaling Technology). These antibodies were detected using goat anti-rabbit immunoglobulins/HRP secondary antibody 1:2,000 (Agilent Dako). Protein bands were detected using Luminata™ Crescendo Western HRP substrate (Millipore) and CL-XPosure™ Film (Thermo Fisher Scientific).

**Immunofluorescence**

Mouse mammary tumor tissue or human in invasive ductal carcinoma tissue was fixed O.N. in 4% paraformaldehyde, followed by O.N. dehydration in 30% sucrose prior to embedding in OCT and snap freezing absolute ethanol and dry ice. Sections from the embedded tumors ($10 \mu m$) were placed onto microscope slides were incubated further in 4% paraformaldehyde in DPBS for 10 mins at RT prior to washing in TBS-T and blocked using TBS-T, 10% donkey serum (Sigma-Aldrich), 0.2% Triton X-100 (Sigma-Aldrich). Immunofluorescence staining was performed as previously described.³ Antibodies against the following targets and their dilutions were used as follows; αSMA 1:100 (AS-29553, AnaSpec), CD31 1:100 (ab28364 Abcam), CD31 1:100 (MA1-40074, Thermo Fisher Scientific), CD31 1:100 (EP3095, Abcam), CD68 1:100 (KP1, Invitrogen), F4/80 1:100 (C1:A3-1, Bio-Rad), HO-1 1:100 (AF3776, R&D systems), HO-1 1:100 (10701-1-AP, Proteintech), LYVE-1 1:100 (ab33682, Abcam). Primary antibodies were detected using antigen specific donkey IgG, used at 1:200: AlexaFluor® 488 anti-rabbit IgG, AlexaFluor® 488 anti-rat IgG,
**In vitro macrophage focal-point migration assay**

$M_{(0)}$ macrophages were generated as described above and removed from the plate using 1 mL of enzyme-free dissociation media (Thermo Fisher Scientific) and mechanically detaching the cells using a cell scrapper. The resultant solutions were centrifuged at 2000 x g for 3 mins and resuspended at a concentration of 10,000 cells/mL in 80% RPMI (inc.10%FCS) and 20% methylcellulose with 10 ng/mL M-CSF. To prepare a methylcellulose stock solution; autoclaved methylcellulose (Sigma-Aldrich) was dissolved at 24 g/L in pre-heated serum-free RPMI media for 2 mins at 60°C. After this, the solution was diluted with 2 volumes of RT serum-free RPMI and then mixed O.N. at 4°C. The final solution was cleared by centrifugation at 5000 x g for 2 h at RT. To the lid of an inverted petri dish, 25 µL drops of macrophages were placed, the lid was carefully inverted and placed on top of a DPBS filled petri dish. The dish was incubated for 24 h to allow the formation of macrophage spheroids within the hanging drop. Subsequently, media containing spheroids were collected in Eppendorf tubes using DPBS. The spheroids were allowed to briefly settle in the bottom of the Eppendorf tubes after which the DPBS was carefully removed and the spheroids were placed in wells containing RPMI, 10% FCS, 10 ng/mL M-CSF with or without 25 ng/mL IL-6 and placed in a 37°C 5% $CO_2$ incubator to allow attachment and eventual spreading. Images of spheroid cultures was performed using the live cell Eclipse Ti-2 inverted microscope in the Nikon Imaging Centre at King's College London. NIS Elements Advanced Research software (Nikon) was used to process the images. Total area of spheroids was measured using the "Analyze Spheroid Cell Invasion In 3D Matrix" macro in ImageJ, and cell number was counted using the "Cell counter" plugin in ImageJ.

**In vitro Pv nest transwell assay**

Transwell assays were conducted with transwell inserts with 8 µm pores (Corning) for migration studies and 0.4 µm pores (Corning) for permeability studies. Inserts were coated with Basement Membrane Extract (Cultrex) diluted 1:100 in RPMI for 1 h at RT. Excess Basement Membrane Extract was aspirated and 2 x $10^4$ 3B-11 endothelial cells were seeded onto the apical side of the transwell insert in RPMI supplemented with 10% FCS and left to attach for 24 h. Media was removed, the whole plate inverted and $10^5 M_{(0)}$ or $M_{(IL-6)}$ BMDMs were seeded onto the basolateral side of the transwell membrane in RPMI supplemented with 10% FCS and left to attach for 2 h at 37°C. Subsequently, the plate was reinverted to its original position and RPMI supplemented with 10% FCS, 10 ng/mL M-CSF with or without 10 ng/mL IL-6 added to the apical and basolateral space. After cells were left to interact for 24 h at 37°C, $4 x 10^5 CD8^+$ T cells (which had been prior incubated on anti-CD3 and -CD28 coated plated to develop effector function) were added to the inserts in RPMI, 10% FCS and 100 ng/mL with murine CXCL10 (Bio-Techne) spiked into the wells. After 16 h, migrated cells were collected from the well and stained for flow cytometry analysis and quantification with AccuCheck counting beads (Thermo Fisher Scientific). The permeability assay was performed as described previously.⁸⁸ In brief, permeability of the *in vitro* Pv niche was measured using 4% (w/v) Evans Blue-conjugated Bovine Serum Albumin (BSA) diluted in DPBS was placed into the apical transwell chamber while phenol red-free RPMI, 10% FCS was added to the basolateral chamber. Presence of Evans Blue-BSA in the basolateral chamber was assessed at indicated time-points by absorbance at 620 nm on a NanoDrop™ spectrophotometer (Thermo Fisher Scientific). These experiments were performed in the presence or absence of 10 ng/mL IL-6 and M-CSF.

**Western blot**

Cells were lysed and SDS PAGE/western blots were conducted as previously described.³ In brief, cells were lysed in the well using western blot lysis buffer 0.1M Tris-hydrochloride pH 6.8, with 20% glycerol and 4% sodium dodecyl sulphate containing 1X protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). All tubes were heated at 95°C for 15 mins to break down DNA. Protein concentration was then determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) using the manufacturers' protocol. Samples were then run under reducing conditions on 12% bis-tris sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels alongside SeeBlue™ Plus2 pre-stained makers (Thermo Fisher Scientific). SDS-PAGE gels were then transferred onto polyvinyl-difluoride (PVDF) membranes which were subsequently blocked in 100 mM Tris, 140 mM NaCl, 0.1% Tween 20 (Sigma-Aldrich), pH7.4 (TBS-T) containing 5% skimmed milk at RT for 1 h. Primary antibodies were applied at 4°C O.N. and secondary antibodies for 1 h at RT. Wash steps to remove unbound antibodies were 3 x 20 mins in TBS-T. The following primary antibodies were used: rabbit anti-β-actin, 1:5,000 (ab8227, Abcam), rabbit anti-HO-1 1:1,000 (10701-1-AP, Proteintech), rabbit anti-c-MAF 1:1,000 (ab77071, Abcam), rabbit anti-STAT3 1:2000 (clone 79D7, Cell Signaling Technology). These antibodies were detected using goat anti-rabbit immunoglobulins/HRP secondary antibody 1:2,000 (Agilent Dako). Protein bands were detected using Luminata™ Crescendo Western HRP substrate (Millipore) and CL-XPosure™ Film (Thermo Fisher Scientific).

**Immunofluorescence**

Mouse mammary tumor tissue or human in invasive ductal carcinoma tissue was fixed O.N. in 4% paraformaldehyde, followed by O.N. dehydration in 30% sucrose prior to embedding in OCT and snap freezing absolute ethanol and dry ice. Sections from the embedded tumors ($10 \mu m$) were placed onto microscope slides were incubated further in 4% paraformaldehyde in DPBS for 10 mins at RT prior to washing in TBS-T and blocked using TBS-T, 10% donkey serum (Sigma-Aldrich), 0.2% Triton X-100 (Sigma-Aldrich). Immunofluorescence staining was performed as previously described.³ Antibodies against the following targets and their dilutions were used as follows; αSMA 1:100 (AS-29553, AnaSpec), CD31 1:100 (ab28364 Abcam), CD31 1:100 (MA1-40074, Thermo Fisher Scientific), CD31 1:100 (EP3095, Abcam), CD68 1:100 (KP1, Invitrogen), F4/80 1:100 (C1:A3-1, Bio-Rad), HO-1 1:100 (AF3776, R&D systems), HO-1 1:100 (10701-1-AP, Proteintech), LYVE-1 1:100 (ab33682, Abcam). Primary antibodies were detected using antigen specific donkey IgG, used at 1:200: AlexaFluor® 488 anti-rabbit IgG, AlexaFluor® 488 anti-rat IgG,
AlexaFluor® 488 anti-goat IgG, AlexaFluor® 568 anti-rabbit IgG, AlexaFluor® 568 anti-goat IgG, AlexaFluor® 647 anti-rabbit IgG (Thermo Fisher Scientific), AlexaFluor® 647 anti-mouse IgG, NL637 anti-rat goat IgG (R&D Systems) and Cy3® anti-sheep IgG (Jackson ImmunoResearch). Viable blood vessels were visualized in mice through intravenous (i.v.) injection of FITC-conjugated dextran (2,000,000 MW, Thermo Fisher Scientific) 20 mins prior to sacrifice. Nuclei were stained using 1.25 µg/mL 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Thermo Fisher Scientific). RNA scope was performed on formalin-fixed paraffin-embedded (FFPE) MMTV-PyMT tumor sections as per manufacturers' instructions using the RNAscope® Multiplex Fluorescent Reagent Kit v2 Assay (Bio-Techne; 323100-USM). The Mm-IL6 (Bio-Techne; 315898) probe was used and was detected using the Opal™ 570 Reagent Pack (FP1488001KT, Akoya Biosciences). Following RNAscope, immunofluorescence imaging was performed as previously described above. Images were acquired using a Nikon Eclipse Ti-E Inverted spinning disk confocal. For 5-color confocal microscopy, images were acquired on a Nikon A1R spectral deconvolution confocal microscope. Using a 32-channel A1-GasAsP-detector unit, fluorochrome emission can be split up in up to 32 bands from 400 to 750 nm with a spectral discrimination of 10 or 20 nm bandwidth when excited by a laser-box with 4 solid state lasers 405, 488, 561 and 640 nm. The acquisition signals were then clearly and reliably distinguished by a process called "spectral unmixing". Images were analyzed using the NIS-Elements software.

## Bioluminescence imaging

For assessing Luc bio-distribution *in vivo* mice were injected i.p. with 3 mg XenoLight D-luciferin (PerkinElmer) in sterile DPBS 10 mins prior to imaging. For whole-body imaging animals were anesthetized and placed in the *in vivo* Imaging System (IVIS®) Lumina Series III (PerkinElmer). For imaging the Luc bio-distribution of different tissues, the mice were injected with D-luciferin and sacrificed after 10 mins and the dissected tissues were then imaged 15 mins later. To quantify luminescence, a region of interest (ROI) was drawn around a specific area and total photon flux (PF) (photon/second; p/s) was measured. All data was analyzed using the Living Image Software (PerkinElmer).

## Flow cytometry

Flow cytometry was performed as previously described.³⁴ The following antibodies against the indicated antigen were purchased from Thermo Fisher Scientific and were used at 1 µg/mL unless stated otherwise: CCR5 PE (HM-CCR5(7A4)), CD3ε APC, PE, PE-Cy7 (145-2C11) and Brilliant Violet (BV)421 (17A2; Biolegend®), CD4 APC, FITC and PE (RM4-5), CD8α BV421, BV711 and FITC (53-6.7; Biolegend®), CD8β FITC and eFluor®450 (H35-17.2), CD11b BV510 (M1/70; Biolegend®), CD11c APC (N418) and FITC (N418; Biolegend®), CD16/32 (2.4G2; Tonbo Biosciences), CD19 BV421, APC (6D5; Biolegend®) and FITC (1D3/CD19; Biolegend®), CD31 FITC, PE (390) and BV510 (MEC 13.3; BD biosciences), CD45 BV605 (30-F11; Biolegend®), APC, APC-eFluor® 780 (30-F11), BV510 and BV785 (30-F11; Biolegend®), CD90.1 eFluor® 450 (HIS51) and BV510 (OX-7; Biolegend®), CD90.2 eFluor® 450 and BV510 (53-2.1; Biolegend®), CD206 APC, FITC and BV785 (C068C2; Biolegend®) and APC (FAB2535A; Bio-Techne), F4/80 APC, APC-eFluor® 780, PE and eFluor® 660, BV421 and FITC (BM8; Biolegend®), Gr-1 FITC (RB6-8C5; Biolegend®), Granzyme-B PE (GB11), ICAM-1 BV421 (YN1/1.7.4, Biolegend®), IFN-γ APC (XMG1.2), Ki-67 AF700 (SolA15), Ly6C APC, APC-eFluor® 780, eFluor®450 and FITC (HK1.4; Biolegend®), Ly6G APC (1A8; Biolegend®), LYVE-1 Alexa Fluor® 488, PE (ALY7) and APC (FAB2125A; Bio-Techne), MHCII PE, BV421, BV510 and FITC (M5/114.15.2; Biolegend®), NK1.1 APC (PK136), PNAd AF647 (MECA-79, Biolegend®), TNF-α PE (MP6-XT22), VCAM-1 PE (429 (MVCAM.A), Biolegend®). Positive stains were compared to fluorescence minus one (FMO) controls. Intracellular stains were performed as previously described³⁴ using the Foxp3 Transcription factor staining buffer set (Thermo Fisher Scientific). Dead cells and red blood cells were excluded using 1 µg/mL 7-amino actinomycin D (7AAD; Sigma-Aldrich), Fixable Viability Dye eFluor® 780 or Near-IR Dead cell staining kit (Thermo Fisher Scientific) or DAPI alongside anti-Ter-119 PerCP-Cy5.5 (Ter-119). Data were collected on a BD FACS Canto II (BD Biosciences). Cells were sorted on a BD FACS Aria (BD biosciences). Data was analyzed using FlowJo software (BD biosciences). Immune cells (CD4⁺) were separated based upon the following surface characteristics: CD11c⁺F4/80⁻ (dendritic cells), CD11b⁺F4/801¹² (macrophages), F4/80⁻/lowLy6G⁻Ly6C⁺ (monocytes), CD11b⁺Ly6G⁺ (neutrophils), NK1.1⁺ (NK/NKT-cells), CD3ε⁺ (T cells), CD3ε⁺CD4⁺ (CD4⁺ T cells), CD3ε⁺(or FSC-A⁻low)CD8α/β⁺ (CD8⁺ T cells), CD3ε⁻CD19⁺ (B cells). Cancer associated fibroblasts (CAFs) were identified as CD45⁻Thy1⁺ cells and tumor cells were identified as CD45⁻Thy1⁻CD31⁻.

## Quantitative real time PCR

mRNA was extracted and quantitative reverse transcription PCR was performed as previously described³⁴ using the following primers/probes purchased from (Thermo Fisher Scientific): II6 Mm00446190_m1 and Tbp Mm01277045_m1. Expression is represented relative to the house-keeping gene Tata-binding protein (Tbp). Gene expression was measured using an ABI 7900HT Fast Real Time PCR instrument (Thermo Fisher Scientific).

## Transcriptomic data and analysis

TAM, CAF and endothelial Bulk RNAseq and human and mouse TAM scRNA-seq datasets were previously published and described¹⁴,²⁸ and datasets are publicly accessible (see ‘Data Availability’ section). Downstream analysis was performed using the Seurat v3 R package⁸⁰ and analysis pipeline outlined in.¹⁴ For upstream regulator analysis we used the QIAGEN IPA (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA).³⁶ When comparing scRNA-seq datasets between human and mouse TAMs, the Garnett package was used⁸¹ which has previously been employed to perform mouse-human cross-species comparative analysis.²⁷ Murine data and a marker file specifying LYVE1 were provided to Garnett and the model was trained (train_cell Classfier()) with default
settings, using the same 2000 genes with highest variance chosen for clustering previously.¹⁴ Publicly available human data²⁸ were then classified ( classify cells() ) with default settings. Results were projected and plotted on the associated UMAP coordinates from the same data using a customised R script. For Illumina microarray analysis purified mRNA for the respective polarized splenocyte-derived macrophages were cultured for isolated using the PureLink® RNA Mini Kit (Invitrogen) according to the manufacturers' pro- tocol. The purity of the isolated mRNA was assessed using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific) and the quality and integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA was converted to cDNA, then subsequently amplified using the Ovation® PicoSL WTA system V2 (Tecan), biotinylated using the Encore® BiotinIL Module (Tecan), and then hy- bridized to MouseWG-6 V2.0 Beadchip microarray (Illumina). Following hybridization, the arrays were washed, blocked, and stained with streptavidin-Cy3 using the Whole-Genome Gene Expression Direct Hybridisation Assay (Illumina). Microarrays were run on an Illumina iScan system, raw fluorescence signals were collected using GenomeStudio (Illumina), and the data imported into Partek Genomics Suite for analysis. Background was subtracted from the raw data and fluorescence signals were normalized using the quantiles method.⁸⁹ All p-values were adjusted for multiple testing using the procedure of Benjamini and Hochberg.

QUANTIFICATION AND STATISTICAL ANALYSIS

When comparing groups for establishing statistical significance, firstly normality of the data was determined using a Shapiro-Wilk
normality test and homogeneity of variance was assessed using an F-test. For normally distributed (parametric) data (where also
there was also least n=6 data points in the dataset), a two-sided unpaired Students t test was conducted. A Welch's correction
was applied when comparing groups with unequal variances. For non-normally distributed (non-parametric) data (or there was <6
datapoints in the dataset) a Mann-Whitney test was performed. Statistics was performed using GraphPad Prism 8 software. For mi-
croarray gene analysis, significance of differences (fold change) between the groups were assessed with Partek® Genomics Suite®
software (Partek®) using an ANOVA test. Correction for multiple hypotheses was applied to p values by controlling the percentage
of false discovery rate. Adjusted p values of < 0.01 were considered significant. Statistical analysis of tumor growth curves was
performed using the “CompareGrowthCurves” function of the statmod software package.⁸² The Figures legends state the cohort
sizes, definitions of center (mean and median) and dispersion and precision measures (SD and SEM) and degree of significance using
*p* < 0.05, **p** < 0.01, ***p** < 0.001 differentiations. The text also presents mean +/- SD information for indicated cohorts. No outliers
were excluded from any data presented.
settings, using the same 2000 genes with highest variance chosen for clustering previously.¹⁴ Publicly available human data²⁸ were then classified ( classifyCells() ) with default settings. Results were projected and plotted on the associated UMAP coordinates from the same data using a customised R script. For Illumina microarray analysis purified mRNA for the respective polarized splenocyte-derived macrophages were cultured for isolated using the PureLink® RNA Mini Kit (Invitrogen) according to the manufacturers' pro- tocol. The purity of the isolated mRNA was assessed using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific) and the quality and integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA was converted to cDNA, then subsequently amplified using the Ovation® PicoSL WTA system V2 (Tecan), biotinylated using the Encore® BiotinIL Module (Tecan), and then hy- bridized to MouseWG-6 V2.0 Beadchip microarray (Illumina). Following hybridization, the arrays were washed, blocked, and stained with streptavidin-Cy3 using the Whole-Genome Gene Expression Direct Hybridisation Assay (Illumina). Microarrays were run on an Illumina iScan system, raw fluorescence signals were collected using GenomeStudio (Illumina), and the data imported into Partek Genomics Suite for analysis. Background was subtracted from the raw data and fluorescence signals were normalized using the quantiles method.⁸⁹ All p-values were adjusted for multiple testing using the procedure of Benjamini and Hochberg.

QUANTIFICATION AND STATISTICAL ANALYSIS

When comparing groups for establishing statistical significance, firstly normality of the data was determined using a Shapiro-Wilk
normality test and homogeneity of variance was assessed using an F-test. For normally distributed (parametric) data (where also
there was also least n=6 data points in the dataset), a two-sided unpaired Students t test was conducted. A Welch's correction
was applied when comparing groups with unequal variances. For non-normally distributed (non-parametric) data (or there was <6
datapoints in the dataset) a Mann-Whitney test was performed. Statistics was performed using GraphPad Prism 8 software. For mi-
croarray gene analysis, significance of differences (fold change) between the groups were assessed with Partek® Genomics Suite®
software (Partek®) using an ANOVA test. Correction for multiple hypotheses was applied to p values by controlling the percentage
of false discovery rate. Adjusted p values of < 0.01 were considered significant. Statistical analysis of tumor growth curves was
performed using the “CompareGrowthCurves” function of the statmod software package.⁸² The Figures legends state the cohort
sizes, definitions of center (mean and median) and dispersion and precision measures (SD and SEM) and degree of significance using
*p* < 0.05, **p** < 0.01, ***p** < 0.001 differentiations. The text also presents mean +/- SD information for indicated cohorts. No outliers
were excluded from any data presented.