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Immunosuppressive tumor-associated macrophages expressing interlukin-10 conferred poor prognosis and therapeutic vulnerability in patients with muscle-invasive bladder cancer

Background Tumor-associated macrophages (TAMs) secreting IL-10 could be a specific functional cell subset with distinct polarization state and suppressive role in antitumor immune response. Here, we assessed the associations of clinical outcome, therapeutic responses and molecular features with IL-10+TAMs infiltration, and potential impact of IL-10+TAMs on the immune contexture in muscle-invasive bladder cancer (MIBC). Methods In this retrospective study, 128 patients and 391 patients with MIBC from Zhongshan hospital (ZS cohort) and The Cancer Genome Atlas cohort were included respectively. Immunohistochemistry was performed to quantify various immune cell infiltration in the ZS cohort. Single cell RNA sequencing and flow cytometry were performed to examine the functional status of IL-10+TAMs and its correlation with other immune cells. Survival analyses and assessment of the adjuvant chemotherapy (ACT) benefit analyses were also performed. Results High IL-10+TAMs infiltration was associated with inferior prognosis in terms of overall survival and recurrence-free survival, but superior chemotherapeutic response in MIBC. IL-10+TAMs with suppressive features were associated with immunoevasive tumor microenviroment characterized by exhausted CD8+ T cells, immature NK cells and increased immune checkpoint expression. Additionally, high IL-10+TAMs infiltration showed a strong linkage with basal-featured subtype and augmented EGF signaling. Conclusions Immunosuppresive IL-10+TAMs contributed to an evasive contexture with incapacitated immune effector cells and increased immune checkpoint expression, therefore, predicting unfavorable clinical outcomes despite better ACT responsiveness. IL-10+TAMs might provide guidance for customized selection of EGFR-targeted therapy, FGFR3-targeted therapy as well as immunotherapy. The potential of immunosuppressive IL-10+TAMs as a therapeutic target is worth further exploration.


Immunohistochemistry procedure
In brief, TMA was de-paraffinized and hydrated before being immersed and heated in sodium citrate buffer (0.01M sodium citrate buffer, pH=6) or Tris/EDTA buffer (pH=9.0) for antigen retrieval, and then blocked with PBS buffer containing 1% BSA and 5% serum. The slides were incubated with primary antibodies overnight in the humidified chamber at 4 °C, and visualized by 3,3′-diaminobenzidine (DAB) stain system. Five groups of double staining were performed for the following molecules: CD68 and IL10 to detect IL-10 + TAMs, HLA-DR and CD11c to detect DCs, CD4 and T-bet to detect Th1 cells, CD4 and GATA3 to detect Th2 cells. For double IHC staining, after deparaffinization, antigen retrieval and antigen blocking, the slides were incubated with the first primary antibodies overnight at 4 °C, before proceeding to an alkaline phosphatase-labeled secondary antibody incubation and Vector blue staining.
Subsequently, the TMA was incubated with the second primary antibodies at 37 °C for 2 hours, followed by horseradish peroxidase-labeled secondary antibody incubation and visualization using DAB reagent.
All slides were scanned by NanoZoomer-XR (Hamamatsu) and image Pro Plus 6.0.
Two pathologists who were blind to clinical information counted positive stained cells and scored all samples independently (Supplementary Table 2). Cell numbers and cell percentage were counted and evaluated under 200x microscopic field. IHC score was a combination of staining intensity and positive proportion. The staining intensity was graded as 0 (negative staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining), and the proportion was scored as percentage of positive cells (0%-100%).
IHC score was calculated by multiplication of the staining intensity and proportion, which generated an IHC score ranging from 0 to 300. The mean count of the two pathologists' evaluation was adopted. The cut-off values of TAMs and IL-10 + TAMs were 16 cells and 14 cells (200x) according to minimum log-rank P value method in

Flowcytometry Procedure
Firstly, collagenase IV was used to derive single-cell population from freshly dissected tumor tissues. Secondly, phorbol myristate acetate (50ng/mL) and ionomycin (1 μg/mL) were used in the presence of GolgiStop protein transport inhibition (1:1000) to stimulate cells for intracellular cytokine evaluation, and Fixable Viability Dye 510 (FVS510) was used to detect live cells. Thirdly, after being blocked by Human Fc Block (BD sciences) for 20 minutes at 4°C, cells were stained with interested surface marker.
Next, samples were pretreated by Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer's instructions for intracellular protein staining, after which cells were stained with antibodies for intracellular protein for 30 minutes at 4°C. Finally, cells were washed and re-suspended in phosphate-buffered saline/Cell staining buffer, tested with FACS Celesta flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star)

Single-cell Dissociation
The tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm 3 ) on ice and enzymatically digested with 100 U/mL collagenase I (Worthington) and 10 U/mL DNase I (Worthington) for 45 min at 37°C, with agitation. After digestion, samples were sieved through a 70µm cell strainer, and centrifuged at 300g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec) to lyse red blood cells. After washing with PBS containing 0.04% BSA, the cell pellets were resuspended in PBS containing 0.04% BSA and re-filtered through a 35μm cell strainer.
Dissociated single cells were then stained with AO/PI for viability assessment using Countstar Fluorescence Cell Analyzer. The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec). 3. min.pct>0.1. In order to identify the cell type detailed, the clusters of same cell type were selected for re-tSNE analysis, graph-based clustering and marker analysis.

Cell Communication Analysis.
To enable a systematic analysis of cell-cell communication molecules, we applied cell