Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

Background The proliferation of cancer-associated fibroblasts (CAFs) hampers drug delivery and anti-tumor immunity, inducing tumor resistance to immune checkpoint blockade (ICB) therapy. However, it has remained a challenge to develop therapeutics that specifically target or modulate CAFs. Methods We investigated the involvement of Meflin+ cancer-restraining CAFs (rCAFs) in ICB efficacy in patients with clear cell renal cell carcinoma (ccRCC) and urothelial carcinoma (UC). We examined the effects of Am80 (a synthetic retinoid) administration on CAF phenotype, the tumor immune microenvironment, and ICB efficacy in cancer mouse models. Results High infiltration of Meflin+ CAFs correlated with ICB efficacy in patients with ccRCC and UC. Meflin+ CAF induction by Am80 administration improved ICB efficacy in the mouse models of cancer. Am80 exerted this effect when administered prior to, but not concomitant with, ICB therapy in wild-type but not Meflin-deficient mice. Am80-mediated induction of Meflin+ CAFs was associated with increases in antibody delivery and M1-like tumor-associated macrophage (TAM) infiltration. Finally, we showed the role of Chemerin produced from CAFs after Am80 administration in the induction of M1-like TAMs. Conclusion Our data suggested that Am80 administration prior to ICB therapy increases the number of Meflin+ rCAFs and ICB efficacy by inducing changes in TAM phenotype.


Supplementary Table
Supplementary Fig. S3.Marginal effects of Am80 on the proliferation of MB49 cells in vitro.
After treatment, cells were assessed with the WST-1 assay.
(B) Proliferation of MB49 cells was evaluated each day using WST-1 assays, followed by quantification.
The statistical methods used are 1-way ANOVA with the Tukey test (B).FBS: fetal bovine serum.(A) C57BL/6 wild-type female mice were subcutaneously transplanted with MB49 cells (1 × 10 6 cells/mouse), followed by oral administration of either Am80 (2.0 mg/kg) or DMSO and intraperitoneal injection of anti-PD-L1 antibodies at the indicated schedules.
(B) Time courses of the volume of tumors developed in mice treated by the indicated regimens.
(D) Time courses of the volumes of tumors of the indicated groups.
Differences between groups were analyzed using 1-way ANOVA with the Tukey test (B and D).PD-L1: programmed cell death ligand 1.Statistical analysis was performed using 1-way ANOVA with Tukey test (B and D).(A) C57BL/6 wild-type female mice were subcutaneously transplanted with mT5 cells (1 × 10 6 cells/mouse), followed by oral administration of DMSO or Am80 every day for the indicated period.Single-cell suspensions prepared from the developed tumors were subjected to single-cell RNA sequencing using the 10× single cell sequencing platform, followed by analysis using BBrowser software from BioTurning.Shown in the top right panel is a combined uniform manifold approximation and projection visualization of transcriptomes of all cells prepared from the DMSO and Am80 groups.T cell and macrophage clusters are indicated by circles.
(B) Expressions levels of Cd3e, Cd8a, and Cd4 in T cell clusters of the DMSO and Am80 groups were analyzed and quantified by BBrowser.For the analysis of Cd4 + Il2ra + FoxP3 + cells, cells that express all three genes the levels arbitrarily determined by BBrowser were counted and quantified.
(C) Expressions levels of the indicated genes in macrophage clusters of the DMSO and Am80 groups were analyzed by BBrowser, followed by quantification.
Statistical analysis was performed using the Welch's t-test (B, C).    (B, C, D) Tissue sections prepared from the MB49 tumors were stained for the indicated markers by IHC.The stained positive cells found in the intratumoral, but not peritumoral, areas were evaluated, followed by quantification.
The statistical methods used are 1-way ANOVA with the Tukey test (B-E).

Fluorescent immunostaining
Fluorescent immunostaining was performed on frozen sections prepared from mouse tumor tissue samples.After incubation with 10% neutral buffered formaldehyde solution (cat.no.
37152-51; Nakalai) for 5 min at RT, the slides were washed twice.For staining with anti-mPD-L1, the slides were incubated with Alexa 594-conjugated donkey anti-rat IgG (cat.no. A21209; Thermo Fisher Scientific) for 1 h at RT. Nuclei were visualized using DAPI staining.Images were acquired and quantified with a fluorescent microscope equipped with a CCD camera (BZ-X710; KEYENCE).

Quantitative polymerase chain reaction (qPCR)
Total RNA was purified from TGC-induced peritoneal macrophages and cultured cells using the RNeasy Mini Kit (Cat.no.74104; QIAGEN) according to the manufacturer's instructions.Purified RNA samples were reverse-transcribed using ReverTra Ace (Cat.no. TRT-101; Toyobo) with oligo dT and random primers.qPCR of the generated cDNA was Patients with ccRCC (left) or UC (right) not treated with ICB therapy were stratified by the numbers of Meflin + CAFs in all stromal cells.(B) Overall survival of Meflin-high and -low ccRCC (left) or UC (right) cases who were not treated with ICB therapy.Differences between groups were assessed with the log-rank (Mantel-Cox) test (B).CAF: cancer-associated fibroblast; ccRCC: clear cell renal cell carcinoma; ICB: immune checkpoint blockade; UC: urothelial carcinoma.
αPD-L1 therapy started on Day 8 Prior to αPD-L1 therapy started on Day 17 Simultaneously with αPD-L1 therapy started on Day 17 Following αPD-L1 therapy started on Day 8 αPD-L1 therapy from Day 17 to Day 23 αPD-L1 therapy from Day 8 to Day 14 Oral Am80 administration (+) S4.A priori oral Am80 administration enhances the efficacy of anti-PD-L1 antibody treatment in the MB49 UC mouse models.
C57BL/6 wild-type female mice were subcutaneously transplanted with Ex-3LL/CMV-Luc#1, a derivative of LLC (Lewis Lung Carcinoma) cells transduced with Luciferase (1 × 10 6 cells/mouse), followed by oral administration of DMSO or Am80 and subsequent intraperitoneal injection of anti-PD-L1 antibodies (A) and the measurement of tumor volume (B).(C,D) C57BL/6 wild-type female mice were subcutaneously transplanted with YTN5c cells (5 × 10 6 cells/mouse), which is a subline of the mouse gastric cancer cell line YTN5, followed by oral administration of DMSO or Am80 and subsequent intraperitoneal injection of anti-PD-1 antibodies (C) and the measurement of tumor volume (D).Differences were assessed using the Welch's t-test (B and D).PD-1: programmed cell death protein 1; PD-L1: programmed cell death ligand 1.
Tissue sections prepared from MB49 tumors developed in wild-type (WT) or Meflin knockout (KO) mice which received orally administration of DMSO or Am80 were stained for the indicated markers.Representative images of each staining are shown.The stained positive cells found in the intratumoral, but not peritumoral, areas were selectively evaluated for quantification.PD-1: programmed cell death protein 1; PD-L1: programmed cell death ligand Evaluation of the infiltration of immune cells in the mT5 model by single-cell transcriptomic analysis.
C57BL/6 wild-type or Meflin KO female mice were subcutaneously transplanted with MB49 cells (1 × 10 6 cells/mouse) on Day 1, followed by oral administration of DMSO or Am80 and intratumoral injection (subcutaneous injection) of PBS or chemerin-9 (0.2 mg/kg) every day during Day 5 to Day 11.The developed tumors were harvested on Day 12 and subjected to IHC to evaluate the infiltration of immune cells.

. Hazard ratios and P values for multivariate Cox proportional hazard regression model analysis in patients with ccRCC and UC who did not receive ICB therapy. (a)
Hazard ratios and P values for multivariate Cox proportional hazard regression model analysis in patients with ccRCC who did not receive ICB therapy