An oral heme oxygenase inhibitor targets immunosuppressive perivascular macrophages in preclinical models of cancer

Abstract

A subset of perivascular tumor-associated macrophages (PvTAMs) expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) relies on heme oxygenase-1 (HO-1) activity to maintain an immunologically cold tumor microenvironment, which suppresses the efficacy of chemotherapy. Consequently, HO-1 inhibition represents a strategy to target immunosuppressive LYVE-1⁺ PvTAMs and improve therapeutic responses. We developed and characterized KCL-HO-1i, a small-molecule, orally bioavailable HO-1 inhibitor. In chemotherapy-resistant spontaneous murine MMTV-PyMT breast cancer and subcutaneous MN/MCA1 sarcoma models, targeting the PvTAM function with KCL-HO-1i enhanced chemotherapy effects and sensitized tumors to treatment. KCL-HO-1i combined with chemotherapy promoted an immunologically hot tumor microenvironment characterized by increased infiltration of CD8⁺ T cells exhibiting effector function. These findings identify KCL-HO-1i as a nontoxic, orally bioavailable small-molecule immunotherapeutic targeting a key subset of protumoral PvTAMs, offering a combinatorial strategy to enhance chemotherapy efficacy in cancer.

Figure 1. LYVE-1⁺ PvTAMs modulate the immune landscape and resistance to chemotherapy in cancer through their expression of HO-1.

(A) Representative image of a frozen section of MMTV-PyMT tumor stained with DAPI (nuclei; blue) and antibodies against F4/80 (magenta), HO-1 (red), CD31 (green). Colocalizing pixels for F4/80 and HO-1 are shown in yellow. Scale bar is 50 µm. (B-E) Schematic depicting the transgene of the HO-1 reporter mice (HO-1^Luc/eGFP) (top panel) and representative dot plot of FACs-gated live (7AAD^-) cells from enzyme-dispersed MMTV-PyMT HO-1^Luc/eGFP tumors separated based on their respective expression of HO-1 (eGFP) and CD45 (bottom panel) (B). HO-1/eGFP normalized median fluorescent intensity (MFI) of the indicated tumoral cell populations with background MFI subtracted from WT mice (n=5) (C). Representative contour plot of FACs-gated live (7AAD^-), CD45⁺F4/80⁺ TAMs separated based on their expression of CD206 and MHCII (left) and histograms showing the HO-1/eGFP expression of the subsets alongside the background fluorescence in WT mice (right) (D). Representative histograms of the TAM populations identified in (D) for their surface expression of LYVE-1 alongside their respective fluorescence minus one (FMO) staining (E). (F) Quantitation of the respective TAM populations identified in (D) across MMTV-PyMT tumors (n=10). (G) Quantitation of T-cell populations (left) in FACs-gated live (7AAD^-) cells from enzyme-dispersed MMTV-PyMT tumors (n=10) and representative image of a frozen section of MMTV-PyMT tumor (right) stained with DAPI (nuclei;blue) and antibodies against CD3 (magenta), HO-1 (red) and CD31 (green). Scale bar is 50 µm. (H) Western blot for HO-1 and β-actin expression in bone marrow derived macrophages (BMDMs) under M₍₀₎ (CSF-1 alone) and IL-6 polarization (M_(IL-6)) conditions from MMTV-PyMT x Hmox1^fl/fl mice with (Lyve1^Cre) or without (Lyve1^WT) cre-recombinase driven from the Lyve1 promoter. (I) Tumors from Lyve1^WT and Lyve1^Cre MMTV-PyMT Hmox1^fl/fl mice were enzyme-dispersed and assessed using flow cytometry for the abundance of live (7AAD^-) CD8⁺ and CD4⁺ T-cells (n=4 per group) relative to the Lyve1^WT animals. (J-K) Schematic representing the dosing strategy for gemcitabine in Lyve1^WT and Lyve1^Cre MMTV-PyMT Hmox1^fl/fl (J). Growth curves of established spontaneous tumors that were given vehicle or gemcitabine (6.6mg/kg/7days) where indicated. Indicated dosing started at day zero (cohorts of n=5-8 mice) (K). (L) Representative image of a frozen section of human invasive ductal carcinoma stained with SYTO (nuclei; blue) and antibodies against CD68 (magenta), HO-1 (red), and CD31 (green). Colocalizing pixels for CD68 and HO-1 are displayed in orange. Scale bars represent 200 μm (left panel) and 50 μm (right panel). (M) Correlation of the association between LYVE-1⁺ PvTAMs and ratio CD8⁺:CD8^- T-cell infiltration from ScRNAseq data (described in methods). Images in panels (B), (H) and (J) were created using BioRender software. Bar charts represent the mean, error bars SD, and the dots show individual data points from individual tumors in mice or human patients as indicated. Gemc.; gemcitabine. * P<0.05

Figure 2. KCL-HO-1i is an orally bioavailable HO-1 inhibitor.

(A) Chemical structure of KCL-HO-1i. (B-C) In vitro HO-1 activity in response to a dose escalation of KCL-HO-1i or SnMP using either rat splenic microsomes (B) or human hemin-induced HO-1 expressing HEK293T cell lysates (C) (n=3 biological repeats). (D-F) Schematic representing the dosing strategy and experimental protocol for the PK analysis (D) and plasma concentrations of KCL-HO-1i and SnMP at the indicated times post-delivery of 25µMol/kg via i.p (E) or gavage feeding (F) routes of administration (n=3 mice at each time point). (G-I) Schematic depicting the transgene of the HO-1 reporter mice (HO-1^Luc/eGFP) (upper panel) and the experimental outline (below) (G). Luciferase expression was measured in HO-1^Luc/eGFP reporter mice at time 0 (baseline) and 24 h post administration with either KCL-HO-1i (25µMol/kg), SnMP (25µMol/kg) or vehicle control. Whole mouse total photon flux at each timepoint and condition was quantitated (H). Heat map displaying luciferase expression across the indicated dissected tissues (left panel) and their percentage change relative to vehicle treated animals (right panel) (I) (cohorts of n=6 mice). Images in panel (D) and (G) were created using BioRender. Bar charts show the mean, error bars SD, and the dots show individual data points from individual mice. Line charts display the mean and SEM. * P<0.05, ** P<0.01, **** P<0.0001.

Figure 3. KCL-HO-1i synergizes with chemotherapy to achieve immunological control of tumor growth.

(A) Schematic representing the i.p. dosing strategy for KCL-HO-1i (25 µMol/kg/day) or SnMP (25 µMol/kg/day) and/or 5-FU (40 mg/kg/4 days) or gemcitabine (6.6 mg/kg/7 days) and/or vehicle in MMTV-PyMT mice bearing established tumors (left) and the individual tumor growth curves for the respective treatment (right). (B) Schematic representing the i.p. dosing strategy for non-immune IgG and immune-depleting anti-CD8α antibodies that were also given alongside KCL-HO-1i (25 µMol/kg/day) and gemcitabine (6.6 mg/kg/7 days) and in MMTV-PyMT mice. Vertical dashed black lines mark the start of treatment (day 0) and horizontal red line marks 250 mm³. Solid lines represent individual tumors and mice. Images in panel (A) and (B) were created using BioRender. ** P<0.01.

Figure 4. KCL-HO-1i and chemotherapy create an immunologically hot TME.

KCL-HO-1i and/or 5-FU or gemcitabine and/or vehicle were administered to MMTV-PyMT mice bearing 500-700 mm³ tumors. At 36 h post treatment initiation of treatment mice were sacrificed and tumors were harvested and analyzed. (A) Schematic representing the dosing strategy. (B) The fold change of tumor growth over 36 h post initiation of treatment. (C-G) Tumors were enzyme-digested to release single cells which were analyzed for their live (7AAD^-) stromal cell composition using flow cytometry (n=5 tumors per group) (C) and CD8⁺ T-cells (D). Gating strategy (left panel) and quantitation of CD8⁺ T-cell subsets based on CD62L and CD44 expressions (right panel; TCM; T central memory cells, TEM; T effector memory cells, Teff; T effectors cells) (E), and the ability of the tumoral CD8⁺ T-cells to secrete IFNγ post ex vivo exposure to PMA/ionomycin treatment (F). (G) Representative image of a frozen section of MMTV-PyMT tumor treated with KCL-HO-1i and gemcitabine stained with DAPI (nuclei;blue) and antibodies against CD8 (red) and CD31 (green). Scale bar is 100 µm and white arrows indicate infiltrating CD8⁺ T-cells. (H) Schematic of the perivascular niche assay (left) and the relative transendothelial migration of CD8⁺ T-cells in the presence or absence of M₍₀₎ or M_(IL-6) cells on the basolateral surface with or without 25 µM KCL-HO-1i as indicated. Image in panel (A) was created using BioRender. Bar charts show the mean, error bars SD, and the dots show individual data points from individual tumors and mice. Gemc.; gemcitabine. * P<0.05, ** P<0.01, *** P<0.001.

Figure 5. KCL-HO-1i rewires the TME of breast cancer.

(A) Schematic representing the i.p. dosing strategy for mice bearing established MMTV-PyMT tumors which were treated with KCL-HO-1i (25µMol/kg/day) and/or 5-FU (40mg/kg) or gemcitabine (6.6mg/kg) and/or vehicle. Tumor tissue (cohorts of n=5 mice and tumors),) was analyzed at 36 h post treatment initiation by bulk RNAseq. (B) Venn diagram showing all upregulated DEGs in the tumor TME for the respective treatments against vehicle-treated mice and their intercepts between groups. (C-E) Venn diagram showing all upregulated tumor tissue DEGs for KCL-HO-1i treated mice against vehicle-treated animals and their intercepts between groups (C), heatmap of hierarchical clustered common upregulated DEGs between treatment groups (419 genes) that are secreted genes (91 genes (Figure S7A)) across treatments (D), and heatmap of hierarchical clustered common upregulated DEGs for KCL-HO-1i/chemotherapy (384 genes) that are secreted genes (60 genes (Figure S7D)) (E). (F-H) Venn diagrams showing all upregulated DEGs for chemotherapy treated animals (F) and chemokine and upregulated DEGs associated dual therapy treated animals (G) against vehicle-treated animals and their intercepts between groups. (H) Heatmap of hierarchical clustered chemokine and cytokine upregulated DEGs associated with KCL-HO-1i treatment. (I) Volcano plot showing DEGs from bulk RNAseq of FACs-sorted LYVE-1⁺ TAM from MMTV-PyMT tumors treated for 36 h with Vehicle or KCL-HO-1i (25µMol/kg/day) (cohort of n=3 mice; red dots show significantly regulated gene changes). Image in panel (A) was created using BioRender. Gemc.; gemcitabine.

Figure 6. Orally delivered KCL-HO-1i is non-toxic and synergizes with gemcitabine to control tumor growth.

(A-C) Schematic representing the p.o. dosing strategy for KCL-HO-1i (25µMol/kg/day) and/or the i.p. dosing for gemcitabine (6.6 mg/kg/7 days) and/or vehicle in MMTV-PyMT mice bearing established spontaneous tumors or C57Bl/6 mice bearing ectopic MN-MCA1 tumors (cohorts of n=5-6 mice) (A) and tumor growth curves (vertical black dashed line represents treatment initiation) for MMTV-PyMT (B) and MN-MCA1 (C) respectively. (D-G) The MMTV-PyMT mice in (B) were evaluated for evidence of toxicity, including weight change from treatment initiation (D), end of treatment (day 21) plasma ratio of AST:ALT enzymes (E), blood circulating immune cells assessed using flow cytometry (F) and pathology assessment of inflammatory cell infiltrate in the lungs alongside an additional cohort or WT non-tumor bearing mice given dual treatment for 21 days and then assessed at treatment cessation or 30 days after treatment cessation (G). Image in panel (A) was created using BioRender. Bar charts show the mean, error bars SD, and the dots show individual data points from individual tumors and mice. Line charts display the mean and SEM. Gemc.; gemcitabine. * P<0.05, ** P<0.01.