Expression of the transmembrane protein PD-L1 is frequently upregulated in cancer. Because PD-L1–expressing cells can induce apoptosis or anergy of T lymphocytes through binding to the PD1 receptor, the PD-L1–mediated inhibition of activated PD1+ T cells is considered a major pathway for tumor immune escape. However, the mechanisms that regulate the expression of PD-L1 in the tumor microenvironment are not fully understood. Analysis of organotypic tumor tissue slice cultures, obtained from mice with implanted syngeneic tumors (MBT2 bladder tumors in C3H mice, Renca kidney, and CT26 colon tumors in BALB/c mice), as well as from patients with cancer, revealed that tumor-associated hyaluronan (HA) supports the development of immunosuppressive PD-L1+ macrophages. Using genetically modified tumor cells, we identified epithelial tumor cells and cancer-associated mesenchymal fibroblast-like cells as a major source of HA in the tumor microenvironment. These HA-producing tumor cells, and particularly the vimentin-positive fibroblast-like cells of bone marrow origin, directly interact with tumor-recruited myeloid cells to form large stromal congregates/clusters that are highly enriched for both HA and PD-L1. Furthermore, similar cell clusters composed of HA-producing fibroblast-like cells and PD-L1+ macrophages were detected in tumor-draining, but not in distant, lymph nodes. Collectively, our findings indicate that the formation of multiple large HA-enriched stromal clusters that support the development of PD-L1–expressing APCs in the tumor microenvironment and draining lymph nodes could contribute to the immune escape and resistance to immunotherapy in cancer.

The immunosuppressive ligand PD-L1 plays an important role in the regulation of T cell–mediated immune response and tumor-associated immune tolerance (1). Recent studies have demonstrated that PD-L1 expression by the host’s myeloid APCs is essential for PD-L1–mediated immune evasion and immunotherapy (24). Multiple types of cancer are associated with upregulated expression of inhibitory ligand PD-L1 (59) and the strong presence of immunosuppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) (79).

Tumor cells can stimulate PD-L1 expression in TAMs (6); however, the mechanism of tumor-mediated regulation of PD-L1 expression in myeloid cells remains poorly understood. In addition, high expression of PD-L1 has been detected among stromal cells in several cancer types (10, 11).

The tumor stroma plays a major role in tumor growth and is composed of both cellular and extracellular components. It is enriched with both cancer-associated fibroblasts (CAFs) and hyaluronan (HA), one of the major components of the extracellular matrix (11, 12). CAFs play diverse roles in tumor development and progression, including stimulation of tumor-promoting inflammation, tumor proliferation, invasion, neovascularization, and tumor-associated immune suppression (1315). Several lines of evidence suggest a strong interplay between CAFs and myeloid cells. It has been shown that fibroblasts play a pivotal role in the recruitment of CCR2-expressing monocytic myeloid cells and polarization of recruited cells to M2 macrophages or MDSCs (16, 17). The roles of CAFs in the recruitment of monocytes/macrophages to the tumor tissue is supported by the fibroblast’s production of monocyte-macrophage chemotactic factor CCL2. Furthermore, HA deficiency in tumor stroma resulted in a marked reduction of macrophage recruitment and tumor neovascularization (17, 18).

In this study, we demonstrate that tumors stimulate the gathering of stromal cells into large cell clusters. The major cellular components of these clusters include HA-producing vimentin-positive fibroblast-like cells, tumor epithelial cells, and F4/80+PD-L1+ macrophages. These stromal structures with HA-producing fibroblasts and PD-L1+ macrophages were also detected in tumor-draining lymph nodes (TDLNs), but not in distant lymph nodes (LNs). Our results support the idea that HA produced by stroma and tumor cells contributes to the development of immunosuppressive PD-L1–expressing macrophages, thus leading to the formation of the tolerogenic tumor microenvironment.

Murine recombinant fibroblast growth factor 2 (FGF-2) and IL-1β were acquired from R&D Systems (Minneapolis, MN); Proteinase K and Benzonase were purchased from Sigma-Aldrich. HA with molecular masses 10, 20, 200, 500, 700, and 1000 kDa was obtained from Lifecore Biomedical (Chaska, MN). Biotinylated HA-binding protein was supplied by Millipore-Sigma. Hyaluronidase 2 (Hyal2) polyclonal Ab conjugated with biotin or Alexa 488 was obtained from Bioss Abs. The Abs used for immune fluorescence and flow cytometric analysis were acquired from BioLegend (San Diego, CA) or BD Biosciences (Franklin Lakes, NJ). In vitro experiments were conducted using RPMI 1640 medium (Hyclone or Corning), supplemented with 20 mM HEPES, 200 U/ml penicillin, 50 μg/ml streptomycin (all from Hyclone), and 10% FBS from American Type Culture Collection (Manassas, VA).

Female 6- to 8-wk-old C3/He, BALB/c, and C57/BL6 mice were obtained from the Taconic or Envigo. The murine bladder carcinoma cell line MBT-2 was purchased from JCRB Cell Bank (Japan), murine colon carcinoma CT26/GFP from GeneCopoiea (Rockville, MD), and murine kidney carcinoma Renca and bladder tumor MB49 from American Type Culture Collection. Tumor cells were maintained at 37°C in a 5% CO2 humidified atmosphere in complete culture media. To establish s.c. tumors, we injected mice with 1 × 106 tumor cells into the right flank of syngeneic mice.

Lentivirus-encoded GFP/luciferase (Luc) was acquired from GeneCopoiea (Rockville, MD). To generate the GFP/Luc-expressing tumors, we cultured murine bladder MBT2 tumor cells in RPMI-1640 medium supplemented with heat-inactivated FBS (10%), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C and 5% CO2 for 2 d. The tumor cells were transduced then with GFP/Luc by adding 5 µl (2 × 108 IU/µl) viral solution together with 5 µg/ml polybrene (EMD Millipore) and culturing for 5–7 d. The GFP/Luc-expressing stable tumor cell line was established by the selection of GFP+ cells cultured in a puromycin-supported culture medium.

The precision-cut 300-μm tissue slices (bladder tumors, draining, and distant LNs) were prepared using a Compresstome tissue slicer VF-300-0Z. After tissue cutting, slices were placed into 24-well cell culture plates in complete RPMI-1640 medium supplemented with 10% FBS and antibiotics and cultured in a humidified CO2 incubator for 1–2 wk. Cell viability of cultured tissue slices was tested using the Live/Dead kit (Invitrogen).

Tumor tissue slices were cultured in a humidified CO2 incubator at 37°C to allow for the production of HA. At 7–10 d after tissue culture initiation, tissue-produced HA was found settled at the bottom of the culture plate wells. To monitor and visualize the accumulation of tissue-produced HA fragments on the plastic surface, we removed the tissue slices and culture medium at different time points. The empty wells were washed with warm PBS and fixed with 4% formaldehyde for 30 min. After fixation, plates were washed with PBS containing 2% FBS and incubated overnight with biotinylated HA-binding protein (3 μg/ml; Calbiochem-EMD Millipore) at 4°C (19). The next day, after washing the wells with PBS containing 2% FBS, streptavidin conjugated with fluorochrome was added to the wells and incubated for 30 min at 4°C. After careful washing with PBS, the tissue-produced HA was visualized using an immunofluorescent imaging microscope.

Analysis of HA m.w. was done using PAGE as described previously (20). In brief, conditioned medium tissue slices were centrifuged, aliquoted, and stored at −80°C. To prepare samples for HA size analysis, thawed samples were digested with Proteinase K to remove proteins, Benzonase for the depletion of nucleic acids (RNA, DNA), and ethanol to extract lipids. Samples along with HA standards were then subjected to polyacrylamide electrophoresis. The tissue-produced HA was visualized on the gel by staining with “Stains All” dye (Sigma-Aldrich).

Immunofluorescent staining and flow cytometric analysis were performed as follows. Flow cytometric analyses were performed on a BD Fortessa (BD Biosciences, San Jose, CA) flow cytometer with accompanying analytical software (BD FACSDiva). The secondary analysis was performed in FlowJo software version 10.8. In brief, we incubated 106 cells with Fc block for 15 min at 4°C to block nonspecific binding. Cells were stained with anti-CD16/CD32 mAbs. Cells were then incubated for 30 min on ice in 50 µl of PBS with 1 µg of relevant fluorochrome-conjugated or matched isotype control Abs. The expression of PD-L1 and other markers was assessed using the fluorochrome-conjugated mAbs from BioLegend (PE mouse anti-human CD45, BV650 mouse anti-human PD-L1, and FITC mouse anti-human Hyal-2) and AmCyan for Live/Dead from Thermo Fisher. Cells were then kept in the dark and on ice until flow analysis. The compensation was run before the samples. Immunofluorescence was evaluated using EVOS fluorescent imaging microscope (Life Technology) or Lionheart (BioTek Instruments).

Tissue slices were seeded in a 24-well glass-bottom Grenier plate with 500 μl of complete culture media. The surrounding wells were filled with 1000 μl of sterile PBS to provide adequate humidification. Slices were incubated for 2 h before imaging. Plates were transferred to the BioTek Lionhart FX chamber, which was preheated to 37°C and supported with 5% CO2. Using ×20 magnification, 5–10 beacons were chosen per well. To compensate for good variation, z-stacks were of 15 slices of 4.2 μM thickness. Images were taken at 20-min intervals for 96 h. Image processing and video rendering were done using Gen5 Image Prime 3.10 (BioTek Instruments).

Gr-1+ MDSCs were isolated from the spleen or bone marrow of MBT2 tumor-bearing mice using MACS columns according to the manufacturer's instruction (Miltenyi Biotec, Auburn, CA). Briefly, splenic cells were incubated with biotin-conjugated anti–Gr-1 Abs for 15 min. After washing with cold MACS buffer, stained cells were incubated with anti-biotin microbeads (Miltenyi Biotec) for an additional 15 min and subsequently subjected to positive selection of Gr-1+ cells on MACS LS columns.

Silencing of murine Hyal2 gene in isolated MDSCs was performed using Accel small interfering RNA (siRNA) provided by Dharmacon (Horizon Discovery). Briefly, 5 × 105 myeloid cells were seeded in a 24-well Corning plate with 500 μl of Accell media without FBS or antibiotics. Cells were treated with Hyal2 siRNA or scrambled siRNA per the manufacturer's protocol for the first 24 h. After 24 h, 5 × 105 MBT2 tumor cells were suspended in 500 µl of media in which 250 µl was RPMI with 10% FBS plus 5% MEM, and 250 µl of Accell media was added to each well that contained myeloid cells with Accell/siRNA. On day 6, cells were fixed and stained for PD-L1 and pan-hematopoietic marker CD45. Cells were imaged with a BioTek Lionheart FX using ×10 magnification; six beacons were chosen per well. Image processing and analysis were done using Gen5 Image Prime 3.10 software (BiTtek). Graphing and statistical analysis were done with JMP Pro 16 using Dunnett's test.

Total RNA was collected using Monarch Total RNA Miniprep kit (NEB #T2010s). RT-PCR was performed with SimpliAmp Thermal Cycler (ABI) using LunaScript RT SuperMix Kit (NEB #E3010) per the manufacturer’s instructions. Quantitative PCR was performed on Rotor-Gene Q (Qiagen) using LunaScript RT SuperMix Kit (NEB #E3010) per the manufacturer’s protocol. Hyal2 PrimeTime probe primers (IDT) and 18sRNA probe primers (ABI) as normalizers were used in the quantitative PCR to determine the fold change with the δ-δ cycle threshold method. The assay was conducted in quadruplets, and a t test was used to determine significance.

Tumor tissues from 10 patients diagnosed with bladder urothelial carcinomas were collected during transurethral resection of a bladder tumor or radical cystectomy at the Department of Urology, University of Florida. Surgically removed tumor tissues were obtained from patients with bladder cancer after informed consent and approval by the University of Florida institutional review board.

The statistical significance between values was determined by the Student t test. All data were expressed as the mean ± SD. The p values ≥0.05 were considered nonsignificant. Significant p values ≤0.05 were expressed with an asterisk (*). The flow cytometry data shown are representative of at least three separate determinations.

To explore the development of PD-L1+ macrophages in the tumor microenvironment, we used the organoid tumor tissue slice technique. First, we injected MBT2, the murine bladder tumor cells, into syngeneic C3/He mice, surgically excised the developed tumors, and prepared precision-cut 300-μm tumor tissue slices using tissue slicer. Prepared tissue slices were cultured in 24-well plates in complete culture medium.

At 6–12 h after the initiation of tumor tissue slice cultures, we noticed the development of multiple stromal cell conglomerates/clusters firmly attached to the plastic (Fig. 1A, Supplemental Fig. 1). Staining for PD-L1 revealed that these cell clusters were highly positive for this marker. Furthermore, these stromal cell clusters were highly enriched for HA. The majority of the PD-L1+ cells were also positive for pan-hematopoietic marker CD45 (Supplemental Fig. 2A). These data implicate the potential involvement of tumor stroma–associated HA in the development of PD-L1+ cells. In addition to tumors, we also analyzed the tissue slices prepared from TDLNs and distant LNs as described previously (21). A few days later, plates were fixed and stained for the presence of PD-L1 and HA. To our surprise, the TDLN tissue slices developed the HA-enriched adherent stromal cell clusters with incorporated PD-L1+ macrophages, similarly to the tumor tissues. In contrast, the distant LNs did not show development of any HA-enriched PD-L1+ stromal cell clusters. Costaining of LN tissue cultures with Abs against PD-L1, HA, and F4/80 revealed that cluster-associated PD-L1+ cells coexpress macrophage marker F4/80 (Supplemental Fig. 2B). Notably, LN tissue cultures produced fewer PD-L1+ cell clusters than tumor tissues. Similar stromal HA-enriched PD-L1–expressing clusters were observed in cancer tissue samples obtained from patients with bladder cancer (Supplemental Fig. 3A). Taken together, these data indicate that PD-L1+ macrophages can be detected among HA-enriched clusters in tumor tissues and TDLNs.

FIGURE 1.

Stroma-associated HA supports the development of PD-L1+ cells in tumor tissue and TDLNs. MBT2 tumor cells were injected into C3/He mice. Two weeks later, mice were sacrificed, and tumors were surgically excised. Tissue slices were prepared using freshly collected tumor tissue pieces (A) or TDLNs (B). Plates were fixed with 4% formaldehyde and stained for the expression of PD-L1 (magenta) and HA (green). Representative images of tumor stroma are shown. Scale bar, 100 µm.

FIGURE 1.

Stroma-associated HA supports the development of PD-L1+ cells in tumor tissue and TDLNs. MBT2 tumor cells were injected into C3/He mice. Two weeks later, mice were sacrificed, and tumors were surgically excised. Tissue slices were prepared using freshly collected tumor tissue pieces (A) or TDLNs (B). Plates were fixed with 4% formaldehyde and stained for the expression of PD-L1 (magenta) and HA (green). Representative images of tumor stroma are shown. Scale bar, 100 µm.

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CAFs represent one of the major components of tumor stroma. CAFs exert diverse functions, including matrix deposition and remodeling, extensive reciprocal signaling interactions with tumor cells, and infiltrating immune cells (11, 12). The origin of these cells is not fully understood; however, some CAFs demonstrate a bone marrow origin (22). In addition, it has been reported that bone marrow–derived mesenchymal cells with fibroblast-like appearance constitutively produce HA (23). In this study, we show that bone marrow–derived cells obtained from normal naive mice and stimulated with tumor-conditioned medium (Fig. 2A) or recombinant FGF-2 and IL-1β give a rise to HA-producing fibroblast-like cells and PD-L1+ macrophages in the absence of tumor cells (Fig. 2B). It appears that IL-1β and FGF-2 have a synergistic effect on the development of HA-enriched PD-L1+ clusters because stimulation of bone marrow cells with these cytokines individually results in much weaker effects. Furthermore, similar clusters comprising PD-L1+ macrophages and fibroblast-like HA-producing cells were also detected in TDLNs (Supplemental Fig. 3B), but not in distant LNs.

FIGURE 2.

IL-1β and FGF contribute to the formation of stromal PD-L1 clusters in bone marrow cell cultures. (A) Freshly obtained naive bone marrow cells were cultured in a 24-well plate in the presence of an MBT2 TCM. Seven days later, cell cultures were stained with DAPI (blue), anti-vimentin (green), and PD-L1 (magenta) Abs. (B) Naive bone marrow cells were cultured in a 24-well plate in the presence of recombinant murine IL-1β and basic FGF. On day 7, the expression of PD-L1 (red) and CD44 (green) were evaluated using IF microscopy. Representative images are shown.

FIGURE 2.

IL-1β and FGF contribute to the formation of stromal PD-L1 clusters in bone marrow cell cultures. (A) Freshly obtained naive bone marrow cells were cultured in a 24-well plate in the presence of an MBT2 TCM. Seven days later, cell cultures were stained with DAPI (blue), anti-vimentin (green), and PD-L1 (magenta) Abs. (B) Naive bone marrow cells were cultured in a 24-well plate in the presence of recombinant murine IL-1β and basic FGF. On day 7, the expression of PD-L1 (red) and CD44 (green) were evaluated using IF microscopy. Representative images are shown.

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To elucidate the formation of stromal cell clusters in the tumor microenvironment, we took a 96-h time-lapse video after initiation of tumor-slice culture. Data presented in Supplemental Fig. 4A demonstrate that the formation of macrophage-fibroblast clusters is a highly dynamic and interactive process where both large irregularly shaped fibroblast-like cells and smaller round-shaped macrophages move around each other during the formation of stromal cell clusters.

Vimentin is a marker for both CAFs and mesenchymal cells (24, 25). We show that bone marrow–derived fibroblast-like cells express vimentin (Fig. 2A). In separate experiments using tumor tissue slices, we confirmed that vimentin-positive fibroblast-like cells can be frequently detected near the F4/80+ macrophages (Fig. 3A). Furthermore, data presented in (Fig. 3B show that tumor-associated HA (green) directly interacts with PD-L1+ macrophages (red). Collectively, these data support the idea that fibroblast-like vimentin-expressing cells and HA are involved in the development of PD-L1+ macrophages.

FIGURE 3.

Tumor-associated HA directly interacts with PD-L1+ macrophages. Freshly prepared MBT2 tumor tissue slices were cultured for 24 h. Then cultures were fixed with 4% formaldehyde and stained with DAPI (blue) and anti-vimentin (green) and F4/80 (red) Abs (A) or PD-L1 (red) and HA (green) (B). Representative IF images are shown.

FIGURE 3.

Tumor-associated HA directly interacts with PD-L1+ macrophages. Freshly prepared MBT2 tumor tissue slices were cultured for 24 h. Then cultures were fixed with 4% formaldehyde and stained with DAPI (blue) and anti-vimentin (green) and F4/80 (red) Abs (A) or PD-L1 (red) and HA (green) (B). Representative IF images are shown.

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Both CAFs and tumor cells produce HA (18, 26). To delineate the involvement of fibroblast-like cells and epithelial tumor cells in the development of PD-L1+ cells, we transfected the murine MBT2 tumor cell line with GFP using lentivirus. GFP-expressing tumor cells were injected into mice, and harvested tumor was used for the preparation of tumor tissue slices (Fig. 4A). (Fig. 4B shows that initially PD-L1+ clusters (magenta) and GFP+ epithelial tumor cells (green) are localized separately. However, a few days later, as the tumor stroma became denser, the PD-L1+ cells, fibroblasts, and tumor cells started mixing, forming cell clusters (Supplemental Fig. 4B). Analysis of small whole GFP+ MBT2 tumor pieces (Fig. 4C) supports the idea that those cells are mixed.

FIGURE 4.

Identification of HA-producing cells in tumor stroma. GFP-expressing MBT-2 tumor cells (green) were injected into mice. Tumors were collected 2 wk later after tumor cell inoculation. Prepared tumor tissue slices were cultured for 72 h, fixed, and stained with PD-L1–allophycocyanin (PD-L1-APC; magenta) and HA-PE (red) Abs. Representative images of the whole GFP+ tumor piece (A) or tumor stroma (B) are shown. (C) GFP+ tumor tissue slices were stained with DAPI (blue) and PD-L1 (red) Abs. The whole tumor piece is shown.

FIGURE 4.

Identification of HA-producing cells in tumor stroma. GFP-expressing MBT-2 tumor cells (green) were injected into mice. Tumors were collected 2 wk later after tumor cell inoculation. Prepared tumor tissue slices were cultured for 72 h, fixed, and stained with PD-L1–allophycocyanin (PD-L1-APC; magenta) and HA-PE (red) Abs. Representative images of the whole GFP+ tumor piece (A) or tumor stroma (B) are shown. (C) GFP+ tumor tissue slices were stained with DAPI (blue) and PD-L1 (red) Abs. The whole tumor piece is shown.

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In addition to the bladder tumor model, we also observed the development of PD-L1–expressing cell clusters in murine colorectal CT26-GFP+ tumors (Supplemental Fig. 4C). Similar to the MBT2-GFP tumor model, tissue slice cultures prepared from CT26-GFP tumors produced the HA-enriched tumor stroma (red) composed of GPF+ epithelial tumor cells (green) and PD-L1+ round-shaped macrophages (magenta). Taken together, our data indicate that HA-producing fibroblasts play initiating and supporting roles in the development of PD-L1+ macrophages by forming macrophage-fibroblast cell congregates, followed by further incorporation of epithelial tumor cells, leading to the formation of larger HA-enriched cell clusters.

It has been demonstrated that the coculture of tumor cells with Gr-1+ MDSCs leads to the development of immunosuppressive PD-L1+F4/80+ macrophages (6). To examine the potential involvement of tumor-produced HA in the tumor-induced upregulation of PD-L1 expression in myeloid cells, we cocultured MBT2 tumor cells and murine Gr-1+ cells with an inhibitor of HA synthase, 4-methylumbelliferone, added. Data presented in (Fig. 5A demonstrate that inhibition of HA synthesis results in a dose-dependent reduction of PD-L1 expression.

FIGURE 5.

Inhibition of HA synthesis abolishes the expression of PD-L1. (A) Gr-1+ MSDCs were isolated from MBT2 tumor-bearing mice using MACS columns, mixed with MBT-2 tumor cells, and cultured for 7 d. HA synthase inhibitor 4-methylumbelliferone was added at 0.2 and 0.4 mM concentrations immediately after the plating of myeloid and tumor cells. On day 7, cells were collected and stained with anti–PD-L1-PE (red) Abs. Average means ± SD are shown; *p < 0.05. (B) Tumor-induced PD-L1+ myeloid cells coexpress Hyal2. Gr-1+ cells were isolated from MBT2 tumor-bearing mice. Myeloid cells were mixed with tumor cells and cultured in 24-well plates. On day 5, all cells were collected and stained with Abs against CD45 (red), PD-L1 (magenta), and Hyal2 (green). Representative images are shown. (C) Cocultured myeloid and tumor cells were stained with anti-CD45, PD-L1, and Hyal2 Abs, and the proportion of PD-L1+Hyal2+ cells among CD45+ cells was measured by flow cytometry.

FIGURE 5.

Inhibition of HA synthesis abolishes the expression of PD-L1. (A) Gr-1+ MSDCs were isolated from MBT2 tumor-bearing mice using MACS columns, mixed with MBT-2 tumor cells, and cultured for 7 d. HA synthase inhibitor 4-methylumbelliferone was added at 0.2 and 0.4 mM concentrations immediately after the plating of myeloid and tumor cells. On day 7, cells were collected and stained with anti–PD-L1-PE (red) Abs. Average means ± SD are shown; *p < 0.05. (B) Tumor-induced PD-L1+ myeloid cells coexpress Hyal2. Gr-1+ cells were isolated from MBT2 tumor-bearing mice. Myeloid cells were mixed with tumor cells and cultured in 24-well plates. On day 5, all cells were collected and stained with Abs against CD45 (red), PD-L1 (magenta), and Hyal2 (green). Representative images are shown. (C) Cocultured myeloid and tumor cells were stained with anti-CD45, PD-L1, and Hyal2 Abs, and the proportion of PD-L1+Hyal2+ cells among CD45+ cells was measured by flow cytometry.

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MDSCs may affect HA metabolism in tumor tissue through the membrane-bound enzyme hyaluronidase 2 (Hyal2) (27). Hyal2 is a rate-limiting enzyme, which on activation degrades extracellular HA into small fragments with low m.w. (LMW-HA), and its expression is increased in both tumor-associated and blood-derived myeloid cells in patients with bladder cancer.

To examine whether Hyal2-expressing cells could potentially contribute to the HA-mediated development of PD-L1+ macrophages, we cocultured Gr-1+ MDSCs and MBT2 tumor cells for 5 d and then costained with CD45 (pan-hematopoietic marker), PD-L1, and Hyal2. Data presented in (Fig. 5B indicate that PD-L1+ cells coexpress the membrane-bound enzyme Hyal2. This finding was confirmed by flow cytometry (Fig. 5C). Next, we examined whether the tumor-associated myeloid cells also express both PD-L1 and Hyal2. To this end, we isolated CD11b cells from murine MBT2 bladder tumor tissue and stained for those markers. Data presented in (Fig. 6A–C show that the majority of tumor-infiltrating PD-L1+ cells also coexpress the Hyal2 enzyme. Taken together, the earlier provided data suggest that Hyal2-expressing MDSCs are direct precursors of PD-L1+ macrophages and potentially Hyal2 itself could contribute to the development of PD-L1+ macrophages from MDSCs in the tumor microenvironment. To address this question, we examined next whether deletion of the Hyal2 gene in MDSCs could affect the development of PD-L1+ macrophages. To this end, we have isolated Gr-1+ MDSCs and incubated them for 24 h with murine Hyal2-siRNA or scrambled control in Accel media according to the manufacturer’s protocol. The reduction of Hyal2 expression in siRNA-treated myeloid cells was confirmed by quantitative RT-PCR (qRT-PCR) (Fig. 6D). After 24 h of treatment with siRNA, similar to experiments described in (Fig. 5A, 5B and a previously published study (6), the myeloid cells were mixed with tumor cells. Cocultures of myeloid and tumor cells were analyzed for the expression of PD-L1 in hematopoietic CD45+ cells. Data presented in (Fig. 6E demonstrate that deletion of Hyal2 in MDSCs results in significant reduction in numbers of PD-L1+ macrophages as compared with controls.

FIGURE 6.

Tumor-associated CD11b myeloid cells coexpress PD-L1 and Hyal2. (A) MBT2 tumors are infiltrated with CD11b myeloid cells. MBT2 tumor single-cell suspension was prepared using enzymatic tissue digestion and stained with anti-CD45 and CD11b Abs. The number of tumor-infiltrating CD11b+CD45+ cells was measured using flow cytometry. (B) MBT2 tumor-infiltrating CD11b cells were purified with magnetic beads and cultured overnight. The next day, cultured cells were fixed and stained for PD-L1 (magenta, left panel) and Hyal-2 (red, right panel). (C) Freshly isolated tumor-infiltrating CD11b cells were costained with anti–PD-L1 (green, left panel) and Hyal2 (red, right panel) Abs. Images were taken using an EVOS FL imaging system. Representative images are shown. (D) Gr-1+ cells were isolated from MBT2 tumor-bearing mice. Silencing of murine Hyal2 gene in MDSCs was conducted using Accel Hyal2 siRNA or scrambled siRNA according to the manufacturer’s protocol (Dharmacon). Reduction of Hyal2 expression in siRNA-treated myeloid cells was confirmed by qRT-PCR. (E) Twenty-four hours later, treated and untreated cells were mixed with MBT2 tumor cells and cultured in 24-well plates. On day 6, all cells were collected and stained with Abs against PD-L1 and CD45. Cells were imaged with a BioTek Lionheart FX automated microscope, and quantification of PD-L1 expression was done using Gen5 Image Prime 3.10 software (BioTek).

FIGURE 6.

Tumor-associated CD11b myeloid cells coexpress PD-L1 and Hyal2. (A) MBT2 tumors are infiltrated with CD11b myeloid cells. MBT2 tumor single-cell suspension was prepared using enzymatic tissue digestion and stained with anti-CD45 and CD11b Abs. The number of tumor-infiltrating CD11b+CD45+ cells was measured using flow cytometry. (B) MBT2 tumor-infiltrating CD11b cells were purified with magnetic beads and cultured overnight. The next day, cultured cells were fixed and stained for PD-L1 (magenta, left panel) and Hyal-2 (red, right panel). (C) Freshly isolated tumor-infiltrating CD11b cells were costained with anti–PD-L1 (green, left panel) and Hyal2 (red, right panel) Abs. Images were taken using an EVOS FL imaging system. Representative images are shown. (D) Gr-1+ cells were isolated from MBT2 tumor-bearing mice. Silencing of murine Hyal2 gene in MDSCs was conducted using Accel Hyal2 siRNA or scrambled siRNA according to the manufacturer’s protocol (Dharmacon). Reduction of Hyal2 expression in siRNA-treated myeloid cells was confirmed by qRT-PCR. (E) Twenty-four hours later, treated and untreated cells were mixed with MBT2 tumor cells and cultured in 24-well plates. On day 6, all cells were collected and stained with Abs against PD-L1 and CD45. Cells were imaged with a BioTek Lionheart FX automated microscope, and quantification of PD-L1 expression was done using Gen5 Image Prime 3.10 software (BioTek).

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Membrane-bound enzyme Hyal2 contributes to the degradation of extracellular HA. The HA-degrading activity of Hyal2 in myeloid cells can be stimulated with a tumor-conditioned medium (TCM) or IL-1β (27). To examine whether this activation is associated with the upregulation of PD-L1 expression, we used the murine CD11b cells isolated from normal bone marrow. Data presented in (Fig. 7A demonstrate that similarly to its human counterpart, the TCM-activated murine myeloid cells can degrade extracellular HA. A similar extent of HA degradation was detected while analyzing HA produced by MBT2 tumor tissue slices, but not by the MBT2 tumor cell line (Fig. 7B). These findings were confirmed by gel electrophoresis (Fig. 7C). Specifically, the electrophoretic analysis confirmed that HA produced by the MBT2 tumor cell line mostly consisted of 10- to 20-kDa fragments, whereas tumor tissue slice–derived HA showed lower molecular mass (<10 kDa). Because the Hyal2 enzyme specifically degrades HA to fragments with a molecular mass of 20 kDa (28, 29), other types of hyaluronidase could have been involved.

FIGURE 7.

Myeloid cell–mediated HA degradation. Twenty-four-well cell culture plates were precoated with sterile commercial HA (200 kDa). (A) Myeloid cells were plated on precoated HA and cultured in the absence (left image) or presence of TCM (right image). On day 7, cell cultures were fixed and stained for HA (red). (B) Comparative analysis of tumor-associated HA degradation in tumor tissue and tumor cell line. (C) Electrophoretic analysis of HA degradation. Commercial HA samples with molecular masses of 20, 200, and 500 kDa were used as controls. HA was visualized by staining with the dye “Stains All.” (D) Analysis of Hyal1 expression in MBT2 tumor cell line (MBT2), a whole tumor from MBT2 tumor-bearing mice (tumor), and tumor-infiltrating CD11b myeloid cells. The expression of Hyal1 mRNA was measured using qRT-PCR. Average means ± SD are shown; n = 3; *p < 0.05.

FIGURE 7.

Myeloid cell–mediated HA degradation. Twenty-four-well cell culture plates were precoated with sterile commercial HA (200 kDa). (A) Myeloid cells were plated on precoated HA and cultured in the absence (left image) or presence of TCM (right image). On day 7, cell cultures were fixed and stained for HA (red). (B) Comparative analysis of tumor-associated HA degradation in tumor tissue and tumor cell line. (C) Electrophoretic analysis of HA degradation. Commercial HA samples with molecular masses of 20, 200, and 500 kDa were used as controls. HA was visualized by staining with the dye “Stains All.” (D) Analysis of Hyal1 expression in MBT2 tumor cell line (MBT2), a whole tumor from MBT2 tumor-bearing mice (tumor), and tumor-infiltrating CD11b myeloid cells. The expression of Hyal1 mRNA was measured using qRT-PCR. Average means ± SD are shown; n = 3; *p < 0.05.

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To address this question, we measured the expression of Hyal1 and Hyal3 using qRT-PCR in the MBT2 tumor cell line, whole-tumor tissue from tumor-bearing mice, and CD11b cells isolated from the tumor. Levels of Hyal3 were very low, whereas the expression of Hyal1 was upregulated in myeloid cells when compared with the MBT2 tumor cell line (Fig. 7D). Taken together, our data indicate that it is very likely that both the Hyal2 and Hyal1 enzymes are involved in the degradation of tumor-associated HA.

A better understanding of stroma–immune interactions in the tumor microenvironment could elucidate the roles of the tumor stroma in the regulation of antitumor immune response. However, most of the classical research approaches to studying the tumor microenvironment have significant limitations. One of the popular methods is the preparation of single tumor suspension using mechanical and enzymatic tissue digestion. The resultant single-tumor cell suspension is suitable for the analysis of immune tumor-infiltrating cells using flow cytometry or immune fluorescence, isolation of certain cell subsets, etc. However, because mechanical and enzymatic tissue digestion leads to the disruption of naturally occurring cell–cell interactions, this approach does not work for studies focused on tumor stroma and stroma–immune interaction. Another research method to study tumor tissue is immunohistochemistry of formalin-fixed or frozen tissues. This method works in many cases; however, because of the high complexity of the tumor microenvironment, it is very challenging to study the mechanisms of stroma–immune interactions using immunohistochemistry.

In contrast, the cultures of precision-cut tissue slices prepared from freshly excised experimental or clinical tumors create nearly ideal conditions to explore the interaction between tumor stroma and immune cells. Once tumor slices are placed in a culture flask or plate, they start the formation of an adherent stroma that includes an extracellular matrix with attached fibroblasts, macrophages, and other immune cells. Using the GFP-expressing tumor models, we noticed that initial stromal clusters were composed mainly of HA-producing fibroblasts and myeloid cells. However, a few days later, tumor cells migrated from tissue slices and were incorporated into the stroma. Both epithelial tumor cells and CAFs were able to produce HA, which directly interacted with myeloid cells and supported the development of PD-L1+F4/80+ macrophages. These stromal cell clusters are dynamic structures that quickly grow over time in size and numbers. We also noticed that more vascularized tumors produced higher numbers of stromal clusters. Multiple cell structures with incorporated HA-producing fibroblasts and PD-L1+ round-shaped myeloid cells were detected in several tumor types, including bladder carcinoma MBT2, murine colon carcinoma CT26 (Supplemental Fig. 4C), and kidney carcinoma Renca (Supplemental Fig. 4D). These findings indicate that the formation of multiple stromal clusters enriched for HA-producing fibroblasts, tumor cells, and PD-L1+ APCs may represent a mechanism of immune escape in cancer.

HA has been implicated in regulating a variety of cellular functions in both tumor cells and tumor-associated stromal cells, suggesting that altered HA levels can influence tumor growth and malignancy at multiple levels. Previously published studies have demonstrated that HA increases the proliferation rate of tumor cells in vitro and promotes cell survival under anchorage-independent conditions (26, 28). At the molecular level, HA activates the PI3K/Akt pathway and influences the expression of cell-cycle regulators. Furthermore, HA also can inhibit tumor cell apoptosis, as demonstrated by experimentally modifying HA levels. Importantly, increased HA production in cancer is frequently associated with enhanced HA degradation because of high levels of expression/activity of hyaluronidases (29, 30). Increased HA degradation leads to the accumulation of LMW-HA (3133). LMW-HA seems to have specific protumoral functions that promote inflammation, tumor angiogenesis, and metastasis by stimulating the production of cytokines, chemokines, and growth factors in a TLR2/TLR4-dependent manner (34). In contrast with the LMW-HA, high m.w. HA shows anti-inflammatory and antioncogenic effects (28, 35).

Data presented in this study suggest that both major mammalian hyaluronidases Hyal2 and Hyal1 are involved in enhanced HA degradation observed in tumor tissue. It has been proposed that the membrane-bound enzyme Hyal2, which initiates degradation by breaking down the extracellular HA, is working in concert with Hyal1 to produce the LMW-HA (36, 37). Hyal2 is a membrane-bound, rate-limiting enzyme that is expressed in both epithelial tumor cells and tumor-recruited myeloid cells such as MDSCs and TAMs (27, 28, 36, 37). The Hyal1 is an intracellular lysosomal enzyme that degrades the internalized HA to very small fragments with a low molecular mass of <5 kDa (LMW-HA). Tumor-infiltrating myeloid cells show significantly higher expression of Hyal1 than tumor cells (Fig. 7D) and have the ability to degrade HA to much smaller fragments (27). Taking into consideration that major sources of HA in tumor tissues are epithelial tumor cells and CAFs, we propose that tumor-recruited myeloid cells such as Hyal2+ MDSCs may further degrade the tumor-associated HA into small fragments with low m.w., thus fueling cancer inflammation and angiogenesis. Furthermore, tumor-associated HA is also involved in the development of PD-L1+F4/80+ macrophages (Figs. 5, 6), which may contribute to the formation of the immunosuppressive and tolerogenic tumor microenvironment. A recently published study demonstrated that PD-L1–expressing TAMs are critical for suppression of CD8 T cell–mediated immune response in tumor host (38). Taken together, our data indicate that in addition to cancer-related inflammation and tumor angiogenesis, the tumor-associated HA is also involved in tumor-associated immune suppression because HA contributes to the development of immunosuppressive PD-L1+ macrophages in both tumor tissue and TDLNs.

TDLNs are essential for the initiation of an effective antitumor T cell immune response. However, tumors may affect the immune-initiating function of TDLNs (39). A recently published study demonstrated that TDLNs in tumor-bearing mice are enriched for both PD-L1+ APCs and tumor-specific PD1+ T cells (40). TDLN-targeted PD-L1 blockade induced enhanced antitumor T cell immunity by seeding the tumor site with T cells, resulting in improved tumor control. Moreover, abundant PD-1/PD-L1 interactions in TDLNs were also observed in patients with nonmetastatic melanoma. Our data demonstrate that similarly to the tumor tissue, PD-L1+ APCs in the TDLNs are associated with HA-producing fibroblasts and characterized by enhanced degradation of TDLN-associated HA. Taking into account the prominent role of PD-L1+ cells in immune tolerance and tumor-associated immunosuppression, it is plausible that observed cell clusters comprising HA-producing cells and PD-L1+ APCs may directly contribute to the formation of the immunosuppressive and immune tolerogenic microenvironment in both tumor tissue and TDLNs.

This work was supported by Grant 8JK05 from the James and Esther King Biomedical Research Program (to S.K.) and 1923 Fund (to S.K. and P.L.C.).

P.R.D.-G.: acquisition of data, data analysis, methodology, and technical support editing of the manuscript; E.P.K.: acquisition of data, data analysis, methodology, and technical support; W.D.: acquisition of data, data analysis, technical support, and editing of the manuscript; M.M. and A.D.: methodology, acquisition of data, data analysis, an editing of the manuscript; P.O.M.: administrative, technical, or material support; P.L.C.: administrative, technical, or material support and writing, review, and editing of the manuscript; S.K.: conception and design, study supervision, methodology, writing, review, and editing of the manuscript.

The online version of this article contains supplemental material.

Abbreviations used in this article

     
  • CAF

    cancer-associated fibroblast

  •  
  • FGF-2

    fibroblast growth factor 2

  •  
  • HA

    hyaluronan

  •  
  • Hyal2

    hyaluronidase 2

  •  
  • LMW-HA

    hyaluronan small fragments with low m.w.

  •  
  • LN

    lymph node

  •  
  • Luc

    luciferase

  •  
  • MDSC

    myeloid-derived suppressor cell

  •  
  • qRT-PCR

    quantitative RT-PCR

  •  
  • siRNA

    small interfering RNA

  •  
  • TAM

    tumor-associated macrophage

  •  
  • TCM

    tumor-conditioned medium

  •  
  • TDLN

    tumor-draining lymph node

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The authors have no financial conflicts of interest.

Supplementary data