TGFβ is a key regulator of oral squamous cell carcinoma (OSCC) progression, and its potential role as a therapeutic target has been investigated with a limited success. This study evaluates two novel TGFβ inhibitors as mono or combinatorial therapy with anti–PD-L1 antibodies (α-PD-L1 Ab) in a murine OSCC model. Immunocompetent C57BL/6 mice bearing malignant oral lesions induced by 4-nitroquinoline 1-oxide (4-NQO) were treated for 4 weeks with TGFβ inhibitors mRER (i.p., 50 μg/d) or mmTGFβ2-7m (10 μg/d delivered by osmotic pumps) alone or in combination with α-PD-L1 Abs (7× i.p. of 100 μg/72 h). Tumor progression and body weight were monitored. Levels of bioactive TGFβ in serum were quantified using a TGFβ bioassay. Tissues were analyzed by immunohistology and flow cytometry. Therapy with mRER or mmTGFβ2-7m reduced tumor burden (P < 0.05) and decreased body weight loss compared with controls. In inhibitor-treated mice, levels of TGFβ in tumor tissue and serum were reduced (P < 0.05), whereas they increased with tumor progression in controls. Both inhibitors enhanced CD8+ T-cell infiltration into tumors and mRER reduced levels of myeloid-derived suppressor cells (P < 0.001). In combination with α-PD-L1 Abs, tumor burden was not further reduced; however, mmTGFβ2-7m further reduced weight loss (P < 0.05). The collagen-rich stroma was reduced by using combinatorial TGFβ/PD-L1 therapies (P < 0.05), enabling an accelerated lymphocyte infiltration into tumor tissues. The blockade of TGFβ signaling by mRER or mmTGFβ2-7m ameliorated in vivo progression of established murine OSCC. The inhibitors promoted antitumor immune responses, alone and in combination with α-PD-L1 Abs.

This article is featured in Highlights of This Issue, p. 959

Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy and a major cause of cancer morbidity and mortality. Every year, approximately 500,000 new cases of oral and pharyngeal cancers are diagnosed worldwide (1). Current treatment modalities for OSCC include, among others, chemoradiotherapy, surgery, EGFR and COX-2 inhibitors, and photodynamic therapy (2). However, the five-year survival rate has improved only marginally in recent decades, which emphasizes the need for advancements in treatment of OSCC (3).

Most epithelial malignancies, including OSCC, are characterized by overexpression of oncogenes, growth factor receptors, enzymes, and various immunosuppressive factors, ultimately driving disease progression (1). Transforming growth factor-β (TGFβ) is a key regulator of OSCC progression. Although the prognostic relevance of TGFβ expression in OSCC and the correlation with clinicopathologic data have been controversial, the generally accepted view is that most oral carcinomas overexpress TGFβ and that TGFβ is essential for tumor progression (4, 5). Various promalignant functions of TGFβ have been reported and can be collectively attributed to its abilities to reshape the tumor microenvironment (TME; ref. 6). One major effect of TGFβ is the downregulation of immune cell functions, which facilitates tumor escape from immune surveillance (7). It was shown that TGFβ inhibits tumor clearance mediated by CD8+ cytotoxic T cells and downregulates natural killer (NK) cell cytotoxicity (8, 9). In addition to these systemic effects, TGFβ modulates the peritumoral stroma, reprogramming the extracellular matrix and promoting the formation of cancer-associated fibroblasts (CAF; ref. 10). The reprogrammed fibroblast- and collagen-rich peritumoral stroma prevents infiltration of immune cells into the tumor parenchyma, ultimately restraining antitumor immunity (11).

More recently, it was shown that TGFβ signaling might play an important role in patients who receive certain anticancer therapies. Zhu and colleagues demonstrated that chemotherapeutics can stimulate TGFβ production and consequently increase TGFβ signaling in the TME. The authors reported that combinatorial therapy with a TGFβ ligand trap alleviated this unintended side effect and enhanced therapeutic efficacy of chemotherapy (6). Similar findings were reported by Mariathasan and colleagues, who described enhanced TGFβ signaling in response to anti–PD-L1 antibody (α-PD-L1 Ab) therapy (11). These findings might explain why some cancer patients do not respond to immune-checkpoint inhibitors or manifest hyperprogression (12). Thus, inhibition of TGFβ signaling emerges as an urgent need in cancers refractory to available therapies. To date, various TGFβ inhibitors have been utilized alone or in combination with current cancer therapies in experimental human clinical trials with a variable success rate (13, 14). However, despite the known importance of TGFβ in promoting cancer progression, no TGFβ inhibitor has been approved by FDA for human use so far.

The 4-NQO oral carcinogenesis orthotopic murine model, which faithfully reproduces the initiation and progression of human OSCC, offers an opportunity for a variety of preclinical studies. The model has been used in studies of oral carcinogenesis (15), in cancer prevention research (16), and for screening of therapeutic efficacy of novel drugs (17). 4-NQO tumors express TGFβ and PD-L1, as previously shown in the literature (18, 19). The aim of this study was to evaluate therapeutic efficacy of two recently generated TGFβ inhibitors alone and in combination with α-PD-L1 Abs in the orthotopic and immunocompetent 4-NQO mouse model.

TGFβ inhibitors

TGFβ inhibitors were developed and produced in the laboratory of A.P.H. The trivalent TGFβ ligand trap RER was described previously and consists of the endoglin-like domain of the rat TGFβ coreceptor betaglycan (BGE, or E) fused to one domain of the human TGFβ type II receptor extracellular domains (RII or R) on the N- and C-terminus (6, 20). In this study, we used mRER, which is based on an all murine sequence, except for linkers, which are nonnatural. Compared with other types of TGFβ inhibitors, mRER has a near picomolar antagonistic potency and a size that is smaller than that of a neutralizing antibody, potentially enabling better penetration of dense tissues, such as the extracellular matrix, and effective sequestration, even at low concentrations of the inhibitor in tissues (6, 20). Daily intraperitoneal injections of mRER (50 μg) were performed starting in week 18 for 4 weeks (Fig. 1A).

Figure 1.

Characterization of the 4-NQO model. A, A schema is provided for 4-NQO oral administration in water for tumor initiation and for delivery of treatments beginning in week 18. Green, blue and red arrows indicate timing of the treatments. B, Experimental groups of this study. mRER was injected intraperitoneally daily in the dose of 50 μg. mmTGFβ2-7m was delivered by osmotic pumps in the dose of 10 μg per day. α-PD-L1 Abs or corresponding isotype control (100 μg) were injected intraperitoneally seven times every 72 hours starting in week 18. C, Representative images of sublingual and lingual tumors on gross observation, harvested at indicated time points and representative image of lingual tumor in situ. D, Marker expression of TGFβ and PD-L1 during 4-NQO carcinogenesis assessed by immunofluorescent stainings. Both markers are upregulated during the formation of oral carcinomas. Scale bars, 200 μm (1: basal layer; 2: epithelium; dotted line: tumor/stroma border; asterisk: tumor tissue).

Figure 1.

Characterization of the 4-NQO model. A, A schema is provided for 4-NQO oral administration in water for tumor initiation and for delivery of treatments beginning in week 18. Green, blue and red arrows indicate timing of the treatments. B, Experimental groups of this study. mRER was injected intraperitoneally daily in the dose of 50 μg. mmTGFβ2-7m was delivered by osmotic pumps in the dose of 10 μg per day. α-PD-L1 Abs or corresponding isotype control (100 μg) were injected intraperitoneally seven times every 72 hours starting in week 18. C, Representative images of sublingual and lingual tumors on gross observation, harvested at indicated time points and representative image of lingual tumor in situ. D, Marker expression of TGFβ and PD-L1 during 4-NQO carcinogenesis assessed by immunofluorescent stainings. Both markers are upregulated during the formation of oral carcinomas. Scale bars, 200 μm (1: basal layer; 2: epithelium; dotted line: tumor/stroma border; asterisk: tumor tissue).

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The other inhibitor is an engineered TGFβ monomer, designated mmTGFβ2-7m, which lacks the heel helix, a structural motif essential for binding the TGFβ type I receptor (TβRI) but dispensable for binding the other receptor required for TGFβ signaling, the TGFβ type II receptor (TβRII; ref. 21). mmTGFβ2-7m retains the same affinity and binding to TβRII as TGFβ1 and TGFβ3 dimers but is unable to recruit TβRI and signal. mmTGFβ2-7m has inhibitory activity against TGFβ1, -β2, and -β3 in the nanomolar range (20–60 nmol/L; refs. 21, 22). It has high specificity for TβRII and, therefore, has a high target selectivity (unlike small-molecule TGFβ receptor kinase inhibitors) and due to its small size (ca. 10 kDa) has great potential to penetrate dense tissues (like small-molecule TGFβ receptor kinase inhibitors, but unlike antibodies; ref. 22). mmTGFβ2-7m was delivered by osmotic pumps (Alzet NO 2004-0.25 μL/h 28 days pump; cat. #NC0836845; Durect Corporation), which were loaded and implanted subcutaneously in week 18 according to the manufacturer's recommendations. The delivery rate was 10 μg of mmTGFβ2-7m per day until experimental endpoint in week 22 (Fig. 1A).

Antibody treatment

The anti-mouse PD-L1 Ab (clone: 10F.9G2) and matching isotype control were purchased from Bio X Cell. Mice received seven intraperitoneal injections with 100 μg of Ab or isotype control every 72 hours starting in week 18 (Fig. 1A).

Murine oral carcinogenesis model

To establish the 4-nitroquinoline 1-oxide (4-NQO) model, female, immunocompetent C57BL/6J mice ages 8 weeks were purchased from The Jackson Laboratories. Protocols for animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) under reference #18042580. To induce the development of oral carcinomas, mice were administered the carcinogen 4-NQO (Tokyo Chemical Industry Co., Ltd.) in drinking water once a week. Therefore, 4-NQO was dissolved in propylene glycol (Sigma-Aldrich) as stock solution (4 mg/mL), which was immediately diluted to a concentration of 0.1 mg/mL in the drinking water. 4-NQO was protected from light at all times. Mice were treated with 4-NQO for 16 weeks followed by the provision of normal drinking water.

Experimental groups

A longitudinal study was performed in the first cohort of mice. Mice were treated with 4-NQO in drinking water for 16 weeks followed by normal drinking water until week 22. Mice were sacrificed at weeks 0, 16, 18, 20, and 22 (n = 4 for each time point), and tongue tissue was photographed and harvested for histopathologic analysis.

The second cohort of mice was randomly divided into experimental groups on week 18 and was treated until week 22, which was the experimental endpoint (Fig. 1A). The CTRL group either received intraperitoneal injections of PBS (vehicle of mRER and mmTGFβ2-7m) or rat IgG2b isotype control (n = 6). Mice were treated with mRER (n = 6), mmTGFβ2-7m (n = 5), and α-PD-L1 Abs (n = 7) as described above. Additionally, mice received combinatorial therapy of mRER and α-PD-L1 Abs (n = 6) or mmTGFβ2-7m and α-PD-L1 Abs (n = 5). Experimental groups are listed in Fig. 1B.

Pathologic analysis and tissue histology

At the experimental endpoint on week 22, the number of tumors was counted and sizes were measured by caliper. Volumetric caliper readings were cross validated with different formulas as previously described by us and most aligned with the volume of an ellipsoid: 4/3π(d1/2 × d2/2 × d3/2) (15). Quantification of tumor specimens was assessed blinded by an independent examiner (S.S.Y.).

For tissue histology, oral tumors were dissected, placed in 4% paraformaldehyde for 24 hours, and subsequently in 30% sucrose (Sigma-Aldrich) for 24 hours. Samples were embedded in OCT compound (Thermo Fisher Scientific) and stored at −80°C for subsequent sectioning. Cryostat sections (6 μm) were cut and stained with hematoxylin and eosin (H&E). Immunofluorescence staining was performed by incubating sections with a rat anti-mouse PD-L1 mAb (1:100, clone: 10F.9G2, BE0101, Bio X Cell), rabbit anti-mouse TGFβ1 (1:100, ab92486, abcam), rabbit anti-mouse collagen IV (1:100, ab6586, abcam), rat anti-mouse CD68 (1:200, ab53444, abcam), rat anti-mouse Ly-6G/Ly-6C (Gr-1; 1:100, MAB1037, R&D Systems), or rat anti-mouse CD8a (1:50, 550281, BD Biosciences) overnight at 4°C. After washing, tissue sections were incubated with Cy3-conjugated AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (1:400, 711-166-152, Jackson Immuno Research) or donkey anti-rat Alexa Fluor 488 (1:400, A-21208, Invitrogen) for 1 hour at room temperature. Negative controls were stained in parallel with the secondary antibodies alone. Sections were counterstained and mounted with DAPI Fluoromount-G (SouthernBiotech) and imaged using an Olympus BX61 microscope and the cellSens Dimension software (Version 1.17, Olympus). For image analysis, the fluorescent signals were captured using a fixed fluorescent scaling and signal intensities were quantified with the ImageJ software.

Flow-cytometric analysis of splenocytes

Spleens were harvested from all animals at experimental endpoint and placed in RPMI-1640 media (Lonza). Single-cell suspensions were prepared by mechanically pressing the cells through a 70-μm cell strainer. Red blood cells (RBCs) were lysed using RBC lysis buffer (Roche), and cells were washed twice with PBS. Splenocytes were frozen at −80°C in RPMI-1640 media supplemented with 20% (v/v) heat-inactivated FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin and 10% (v/v) DMSO (Sigma) until analysis by flow cytometry. Cells were stained with labeled antibodies for 60 minutes at room temperature in the dark to analyze the immune cell subpopulations. First, a gate on CD45+ cells (CD45-PE-Cy7, 1:50, #25-0451-82; Invitrogen) was used to isolate hematopoietic cells. Further gates were set on CD3+ (CD3-APC, 1:50, #47-0031-80; Invitrogen) and CD8a+ (CD8a-645, 1:50, #64-0081-80; Invitrogen) cells to study cytotoxic T cells. NK cells were studied by gating on NKp46+ cells (NKp46-FITC, 1:50, #560756; BD Biosciences). Myeloid-derived suppressor cells (MDSCs) were defined as CD11b+ (CD11b-PE, 1:3, #PNIM2581U; IOTest) and Gr-1+ (Gr-1-Alexa Fluor 700, 1:50, #56-5931-80; Invitrogen). Gating strategy is presented in Supplementary Fig. S1. The data were acquired on LSRFortessa Flow cytometer (BD Biosciences) and analyzed using the FlowJo software.

Quantification of bioactive TGFβ serum levels

Blood was drawn from mice by submandibular bleeding before treatment (week 18), during treatment (week 20), and at the experimental endpoint (week 22) and centrifuged at 1,000 × g for 10 minutes to obtain serum, which was frozen at −80°C until further use. A TGFβ bioassay was used to measure serum levels of bioactive TGFβ. Therefore, MFB-F11 reporter cells were used according to the protocols published by Tesseur and colleagues (23). Briefly, MFB-F11 cells were seeded at 4 × 104 cells/well in 96-well flat-bottom tissue culture plates (BD Falcon). After overnight incubation, cells were washed twice with PBS and incubated in 50 μL serum-free DMEM supplemented with penicillin/streptomycin for 2 hours before serum samples were added in 10 μL volume. For SEAP assay, a SEAP Reporter Gene Assay Chemiluminescent kit (Roche) was used according to the manufacturer's recommendations.

Statistical analysis

All data were analyzed using the GraphPad Prism software (v7.0). Values are expressed as mean ± SEM. Differences between groups were assessed by one-way ANOVA. To isolate differences between pairs of groups, Student–Newman–Keuls post hoc tests were performed. Differences were considered significant at P < 0.05.

4-NQO–induced oral carcinomas overexpress TGFβ and PD-L1

All mice receiving 4-NQO showed dysplastic lesions on the tongue on week 16, which transformed to invasive carcinomas on week 17 (Fig. 1A and C). During this period, tumor incidence was 100%. Tumors disrupted the basal layer of the tongue epithelium and infiltrated the neighboring tissue (Fig. 3A). Immunofluorescent staining showed expression of TGFβ and PD-L1 in the tumor tissue. Expression levels of these markers correlated with tumor progression in the 4-NQO model, and advanced carcinomas showed especially strong expression of TGFβ and PD-L1 (Fig. 1D). Interestingly, TGFβ was not only upregulated in tumor tissue, most stromal cells in advanced carcinomas were also positive for TGFβ, as illustrated in Fig. 1D. The 4-NQO model is ideal for evaluation of the effects of a targeted TGFβ and PD-L1 blockade, as it realistically recapitulates TGFβ and PD-L1 expression profiles of human oral carcinomas (4, 24, 25).

Blockade of TGFβ effectively inhibits tumor progression in 4-NQO–treated mice

Therapeutic efficacy of the TGFβ inhibitors was evaluated by measuring the number of primary tumors and the total sizes of tumors per animal. Treatment with mRER significantly reduced numbers and sizes of oral carcinomas (P < 0.05; Fig. 2A–C). Similarly, mmTGFβ2-7m reduced numbers and sizes of tumors; however, no significant differences compared with CTRL were detected. mmTGFβ2-7m significantly reduced the weight loss of mice from week 20 until the experimental endpoint (Fig. 2D). Until week 21, treatment with mRER resulted in no alterations of weight loss; however, at the experimental endpoint on week 22, the weight loss of mRER-treated mice was reduced (Fig. 2D). Additionally, serum was collected before treatment (week 18), during treatment (week 20), and after treatment (week 22), and bioactive TGFβ was measured in a TGFβ bioassay. The analysis showed that serum levels of bioactive TGFβ increased during 4-NQO carcinogenesis (Fig. 2E). This increase in TGFβ serum levels was completely blocked in all mice treated with mRER (P < 0.05; Fig. 2E). The treatment with mmTGFβ2-7m blocked the increase of TGFβ serum levels on week 20 (P < 0.05); however, increased values were present on week 22 (P < 0.05; Fig. 2E). Possibly, the delivery of mmTGFβ2-7m by osmotic pumps might have ended a few days before the experimental endpoint and thus a significant increase in TGFβ serum levels was observed at the experimental endpoint (P < 0.05; Fig. 2E). The use of osmotic pumps could also explain the outlier in each group of mice treated with mmTGFβ2-7m (Fig. 2A and B). All pumps were individually loaded and surgically implanted, thus creating a potential for individual variability.

Figure 2.

Effects of TGFβ inhibitors on tumor progression in the 4-NQO model. A, Number of tumors per mouse at the experimental endpoint. B, Aggregate volumes of tumors in mm3 per mouse as measured in groups of mice receiving indicated treatments at the experimental endpoint. C, Representative images of sublingual and lingual tumors on gross observation, harvested at the experimental endpoint. D, Rates of weight loss during 4-NQO carcinogenesis in mice treated with mRER or mmTGFβ2-7m compared with CTRL. E, Quantification of bioactive TGFβ in serum of mice before treatment (week 18), during treatment (week 20), and at experimental endpoint (week 22) using MFB-F11 reporter cells. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL.

Figure 2.

Effects of TGFβ inhibitors on tumor progression in the 4-NQO model. A, Number of tumors per mouse at the experimental endpoint. B, Aggregate volumes of tumors in mm3 per mouse as measured in groups of mice receiving indicated treatments at the experimental endpoint. C, Representative images of sublingual and lingual tumors on gross observation, harvested at the experimental endpoint. D, Rates of weight loss during 4-NQO carcinogenesis in mice treated with mRER or mmTGFβ2-7m compared with CTRL. E, Quantification of bioactive TGFβ in serum of mice before treatment (week 18), during treatment (week 20), and at experimental endpoint (week 22) using MFB-F11 reporter cells. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL.

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In 4-NQO–treated mice, the novel TGFβ inhibitors effectively inhibited tumor progression by ameliorating tumor burden, weight loss, and TGFβ serum levels.

TGFβ inhibition affects the peritumoral stroma and alters immune cell infiltration into the tumor

Tissue specimens were collected at the experimental endpoint and immunostaining was performed to evaluate density and phenotype of tumor-infiltrating cells. First, sections were stained for TGFβ and PD-L1, and as our results in the longitudinal cohort indicate (Fig. 1D), tumors in the CTRL group overexpressed TGFβ and PD-L1 (Fig. 3A). PD-L1 expression levels were not altered by TGFβ inhibitors (Fig. 3A and C). All mice that received daily injections of mRER showed reduced levels of TGFβ in the tumor tissue (P < 0.01; Fig. 3A and B). Mice treated with mmTGFβ2-7m also showed a significant decrease of TGFβ levels, however, to a smaller extent compared with mice treated with mRER (P < 0.05; Fig. 3A and B). This could be explained by either downregulation of TGFβ in the tumor tissue or blocking of the epitope by mRER. Immunostaining for collagen IV (Col IV) revealed no differences between treated or untreated mice (Fig. 3A and D). Treatment with mRER and mmTGFβ2-7m resulted in increased numbers of tumor-infiltrating CD8+ T cells (P < 0.05; Fig. 3A and E). Staining for CD68, a macrophage marker, showed that numbers of macrophages in the tongue tissue close to the tumor borders were reduced in mice receiving treatments with mRER (P < 0.05; Fig. 3A and F). Analogous to our observation for macrophage levels, numbers of MDSCs in the TME were significantly reduced in mice treated with mRER (P < 0.05) and slightly reduced in mice treated with mmTGFβ2-7m (Fig. 3A and G).

Figure 3.

Histopathologic analysis of 4-NQO tumors harvested from mice treated with TGFβ inhibitors. A, Representative images of sections of 4-NQO mice. The images show H&E staining and immunofluorescence staining for TGFβ, PD-L1, and collagen IV (Col IV; green fluorescence) with DAPI counterstaining (blue fluorescence) at 10× magnification. Immunofluorescence staining for CD8a, CD68, and Gr-1 (green fluorescence) with DAPI counterstaining (blue fluorescence) is presented in 20× magnification. Scale bars, 100 μm. PD-L1 staining presents the tumor tissue. CD68 and Gr-1 staining show surrounding stroma. Asterisks: tumor tissue; dotted lines: tumor/stroma border. B, Quantitative analysis of TGFβ+ signals by immunofluorescent staining. Staining intensity in tumor tissues was quantified by using ImageJ, and data are expressed as fold induction compared with CTRL. C, Quantitative analysis of PD-L1+ signals in the tumor tissue. D, Quantitative analysis of Col IV+ signals in the tumor stroma. E, Numbers of tumor-infiltrating CD8a+ cells per region of interest (ROI). The dotted lines in A indicate the tumor borders, and CD8a+ cells only within the tumor tissue were counted. F, Quantification of macrophage numbers in tongue tissues closely located to the 4-NQO tumors. Data are expressed as numbers of CD68+ cells per ROI. G, Quantification of MDSC numbers in the tumor tissue and associated stroma. Data are expressed as numbers of Gr-1+ cells per ROI. H, Analysis of splenocytes by flow cytometry at the experimental endpoint. Cytotoxic T cells were defined as CD45+CD3+CD8+. I, CD45+NKp46+ splenocytes were analyzed by flow cytometry to quantify NK cells. J, CD45+CD11b+Gr-1+ splenocytes were analyzed by flow cytometry to quantify MDSCs. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL; **, P < 0.01 vs. CTRL; ***, P < 0.001 vs. CTRL.

Figure 3.

Histopathologic analysis of 4-NQO tumors harvested from mice treated with TGFβ inhibitors. A, Representative images of sections of 4-NQO mice. The images show H&E staining and immunofluorescence staining for TGFβ, PD-L1, and collagen IV (Col IV; green fluorescence) with DAPI counterstaining (blue fluorescence) at 10× magnification. Immunofluorescence staining for CD8a, CD68, and Gr-1 (green fluorescence) with DAPI counterstaining (blue fluorescence) is presented in 20× magnification. Scale bars, 100 μm. PD-L1 staining presents the tumor tissue. CD68 and Gr-1 staining show surrounding stroma. Asterisks: tumor tissue; dotted lines: tumor/stroma border. B, Quantitative analysis of TGFβ+ signals by immunofluorescent staining. Staining intensity in tumor tissues was quantified by using ImageJ, and data are expressed as fold induction compared with CTRL. C, Quantitative analysis of PD-L1+ signals in the tumor tissue. D, Quantitative analysis of Col IV+ signals in the tumor stroma. E, Numbers of tumor-infiltrating CD8a+ cells per region of interest (ROI). The dotted lines in A indicate the tumor borders, and CD8a+ cells only within the tumor tissue were counted. F, Quantification of macrophage numbers in tongue tissues closely located to the 4-NQO tumors. Data are expressed as numbers of CD68+ cells per ROI. G, Quantification of MDSC numbers in the tumor tissue and associated stroma. Data are expressed as numbers of Gr-1+ cells per ROI. H, Analysis of splenocytes by flow cytometry at the experimental endpoint. Cytotoxic T cells were defined as CD45+CD3+CD8+. I, CD45+NKp46+ splenocytes were analyzed by flow cytometry to quantify NK cells. J, CD45+CD11b+Gr-1+ splenocytes were analyzed by flow cytometry to quantify MDSCs. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL; **, P < 0.01 vs. CTRL; ***, P < 0.001 vs. CTRL.

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These results demonstrate that treatments with mRER and mmTGFβ2-7m promoted the infiltration of lymphocytes into the tumor tissue, but reduced levels of macrophages or MDSCs, which are known to be activated by TGFβ.

TGFβ inhibitors induce systemic immunomodulatory effects in vivo

At the time of sacrifice (week 22), splenocytes were harvested from individual 4-NQO–treated mice and cryopreserved for analysis. Splenocytes were thawed, stained, and analyzed for selected immune cell subpopulations by flow cytometry. The analysis of CD8+ T cells revealed no significant differences compared with CTRL (Fig. 3H). mRER did not alter NK cell levels, but the treatment with mmTGFβ2-7m resulted in an increase of NK cell frequency (P < 0.05 vs. CTRL; Fig. 3I). Treatment with mRER reduced the frequency of MDSCs (P < 0.001; Fig. 3J). The data suggest that inhibition of TGFβ results in systemic immunoregulatory changes, ultimately creating a favorable immune landscape.

Specific inhibition of PD-L1 reduces tumor burden in 4-NQO–treated mice

Although the number of oral tumors was affected only slightly by α-PD-L1 Abs, the tumor burden was significantly decreased in mice that received PD-L1 monotherapy (P < 0.05; Fig. 4A, B, and C). The weight loss of mice was reduced after treatment with α-PD-L1 Abs throughout the observation period from weeks 19 to 22 (P < 0.05; Fig. 4D). The levels of soluble TGFβ in the serum of mice that received α-PD-L1 Abs were decreased in week 20 (P < 0.05), but increased more than 2-fold in week 22 compared with CTRL (P < 0.01; Fig. 4E).

Figure 4.

Effects of combinatorial PD-L1/TGFβ therapy on tumor progression in the 4-NQO model. A, Number of tumors per mouse at the experimental endpoint. B, Aggregate volumes of tumors in mm3 per mouse as measured in groups of mice receiving indicated treatments at the experimental endpoint. C, Representative images of sublingual and lingual tumors on gross observation, harvested at the experimental end-point. D, Rates of weight loss during 4-NQO carcinogenesis in mice treated with α-PD-L1 alone or in combination with mRER or mmTGFβ2-7m compared with CTRL. E, Quantification of bioactive TGFβ in serum of mice before treatment (week 18), during treatment (week 20), and at experimental endpoint (week 22) using MFB-F11 reporter cells. Data of CTRL group are also presented in Fig. 2. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL; #, P < 0.05 vs. CTRL; ##, P < 0.01 vs. CTRL.

Figure 4.

Effects of combinatorial PD-L1/TGFβ therapy on tumor progression in the 4-NQO model. A, Number of tumors per mouse at the experimental endpoint. B, Aggregate volumes of tumors in mm3 per mouse as measured in groups of mice receiving indicated treatments at the experimental endpoint. C, Representative images of sublingual and lingual tumors on gross observation, harvested at the experimental end-point. D, Rates of weight loss during 4-NQO carcinogenesis in mice treated with α-PD-L1 alone or in combination with mRER or mmTGFβ2-7m compared with CTRL. E, Quantification of bioactive TGFβ in serum of mice before treatment (week 18), during treatment (week 20), and at experimental endpoint (week 22) using MFB-F11 reporter cells. Data of CTRL group are also presented in Fig. 2. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL; #, P < 0.05 vs. CTRL; ##, P < 0.01 vs. CTRL.

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The analysis of tissue specimens that were harvested at the experimental endpoint by immunostainings revealed significantly downregulated levels of PD-L1 after treatment with α-PD-L1 Abs (P < 0.01; Fig. 5A and C), either by PD-L1 downregulation in the tumor tissue or by blocking of the epitope by the α-PD-L1 Ab treatment. No alterations of TGFβ levels in response to α-PD-L1 Abs were observed (Fig. 5B). However, the levels of Col IV were increased in the peritumoral stroma (P < 0.05; Fig. 5D), which is considered as one of the TGFβ-mediated effects in the TME. Similar to CTRL, mice that received α-PD-L1 Abs showed only small numbers of tumor-infiltrating CD8+ T cells (Fig. 5A and E). The levels of macrophages slightly increased and the levels of MDSCs significantly decreased in mice that received α-PD-L1 Abs (P < 0.05; Fig. 5A, F, and G). The analysis of splenocytes revealed increased frequencies of NK cells (P < 0.05), whereas no differences were observed for cytotoxic T cells and MDSCs (Fig. 6A–C).

Figure 5.

Histopathologic analysis of 4-NQO tumors harvested from mice treated with α-PD-L1 Abs alone or in combination with TGFβ inhibitors. A, Representative images of sections of 4-NQO mice. The images show HE-staining and immunofluorescence staining for TGFβ, PD-L1, and collagen IV (Col IV; green fluorescence) with DAPI counterstaining (blue fluorescence) at 10× magnification. Immunofluorescence staining for CD8a, CD68, and Gr-1 (green fluorescence) with DAPI counterstaining (blue fluorescence) is presented in 20× magnification. Scale bars, 100 μm. PD-L1 staining presents the tumor tissue. CD68 and Gr-1 staining show surrounding stroma. Asterisks: tumor tissue; dotted lines: tumor/stroma border. B, Quantitative analysis of TGFβ+ signals by immunofluorescent staining. Staining intensity in tumor tissues was quantified by using ImageJ, and data are expressed as fold induction compared with CTRL. C, Quantitative analysis of PD-L1+ signals in the tumor tissue. D, Quantitative analysis of Col IV+ signals in the tumor stroma. E, Numbers of tumor-infiltrating CD8a+ cells per ROI. The dotted lines in A indicate the tumor borders, and only CD8a+ cells within the tumor tissue were counted. F, Quantification of macrophage numbers in tongue tissues closely located to the 4-NQO tumors. Data are expressed as numbers of CD68+ cells per ROI. G, Quantification of MDSC numbers in the tumor tissue and associated stroma. Data are expressed as numbers of Gr-1+ cells per ROI. Data of CTRL group are also presented in Fig. 3. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL; **, P < 0.01 vs. CTRL; #, P < 0.05 vs. α-PD-L1.

Figure 5.

Histopathologic analysis of 4-NQO tumors harvested from mice treated with α-PD-L1 Abs alone or in combination with TGFβ inhibitors. A, Representative images of sections of 4-NQO mice. The images show HE-staining and immunofluorescence staining for TGFβ, PD-L1, and collagen IV (Col IV; green fluorescence) with DAPI counterstaining (blue fluorescence) at 10× magnification. Immunofluorescence staining for CD8a, CD68, and Gr-1 (green fluorescence) with DAPI counterstaining (blue fluorescence) is presented in 20× magnification. Scale bars, 100 μm. PD-L1 staining presents the tumor tissue. CD68 and Gr-1 staining show surrounding stroma. Asterisks: tumor tissue; dotted lines: tumor/stroma border. B, Quantitative analysis of TGFβ+ signals by immunofluorescent staining. Staining intensity in tumor tissues was quantified by using ImageJ, and data are expressed as fold induction compared with CTRL. C, Quantitative analysis of PD-L1+ signals in the tumor tissue. D, Quantitative analysis of Col IV+ signals in the tumor stroma. E, Numbers of tumor-infiltrating CD8a+ cells per ROI. The dotted lines in A indicate the tumor borders, and only CD8a+ cells within the tumor tissue were counted. F, Quantification of macrophage numbers in tongue tissues closely located to the 4-NQO tumors. Data are expressed as numbers of CD68+ cells per ROI. G, Quantification of MDSC numbers in the tumor tissue and associated stroma. Data are expressed as numbers of Gr-1+ cells per ROI. Data of CTRL group are also presented in Fig. 3. All values in this figure represent means ± SEM. *, P < 0.05 vs. CTRL; **, P < 0.01 vs. CTRL; #, P < 0.05 vs. α-PD-L1.

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Figure 6.

Analysis of splenocytes by flow cytometry at the experimental endpoint. A, Cytotoxic T cells were defined as CD45+CD3+CD8+. B, NK cells were defined as CD45+NKp46+. C, MDSCs were defined as CD45+CD11b+Gr-1+. Data of CTRL group are also presented in Fig. 3H–J. All values in this figure represent means ± SEM. **, P < 0.01 vs. CTRL; ****, P < 0.0001 vs. CTRL; #, P < 0.05 vs. α-PD-L1.

Figure 6.

Analysis of splenocytes by flow cytometry at the experimental endpoint. A, Cytotoxic T cells were defined as CD45+CD3+CD8+. B, NK cells were defined as CD45+NKp46+. C, MDSCs were defined as CD45+CD11b+Gr-1+. Data of CTRL group are also presented in Fig. 3H–J. All values in this figure represent means ± SEM. **, P < 0.01 vs. CTRL; ****, P < 0.0001 vs. CTRL; #, P < 0.05 vs. α-PD-L1.

Close modal

These results suggest that α-PD-L1 Abs effectively reduce tumor burden in 4-NQO–treated mice. However, the analysis of the peritumoral stroma shows several characteristics of a TGFβ signature, such as increased collagen production and an immune-excluded phenotype. In accordance with these findings, the TGFβ serum levels were significantly increased by α-PD-L1 Abs, indicating a potential benefit of the combinatorial blockade of PD-L1 and TGFβ.

Combinatorial therapy with α-PD-L1 Abs and TGFβ inhibitors enhances the antitumor immune response in 4-NQO–treated mice

To evaluate a potential benefit of a combined TGFβ/PD-L1 blockade, mice were simultaneously treated with α-PD-L1 Abs and novel TGFβ inhibitors as illustrated in Fig. 1A and B. This combination did not improve effects of PD-L1 monotherapy on the tumor burden. However, treatment with α-PD-L1 Abs and mmTGFβ2-7m further reduced the number of tumors (P < 0.05; Fig. 4A) as well as the weight loss of mice (P < 0.05; Fig. 4D). The increased TGFβ serum levels that were observed when mice were treated with α-PD-L1 Abs were significantly reduced when mice also received mRER (Fig. 4E). TGFβ levels were reduced not only in the serum, but also in the tumor tissue, as described above for monotherapy with TGFβ inhibitors (P < 0.05; Fig. 5B). The elevated Col IV levels in mice treated with α-PD-L1 Abs were significantly reduced when mice also received mRER or mmTGFβ2-7m (Fig. 4A and D). In comparison with PD-L1 monotherapy, the combined PD-L1/TGFβ blockade resulted in an increased number of infiltrating CD8+ T cells (P < 0.05; Fig. 4A and E). The increase of lymphoid cell infiltration might be explained by therapy-induced changes of the collagen-rich peritumoral stroma. The number of macrophages in the tumor stroma was decreased in mice receiving combinatorial therapy (P < 0.05; Fig. 4A and F). The number of MDSCs in the tumor stroma was further reduced when combining α-PD-L1 Abs with mRER (P < 0.05; Fig. 4A and G). The analysis of splenocytes revealed that mice treated with α-PD-L1 Abs and mRER had elevated NK cell frequencies, even further increased compared with the values of PD-L1 monotherapy (P < 0.05; Fig. 6B).

These results indicate that even without major differences in the tumor burden, the combinatorial PD-L1/TGFβ therapy improves the composition of the peritumoral stroma, as well as the systemic immune cell composition. It increases the infiltration of T cells into the tumor tissue and, therefore, enhances the potential of a response to α-PD-L1 Abs.

Accelerated development of TGFβ inhibitors in recent years has led to numerous clinical trials in patients with cancer (NCT03834662, NCT01291784, and NCT02452008). So far, no inhibitors have been approved by the FDA for use in humans. Among these evaluated TGFβ inhibitors are small-molecule TGFβ receptor kinase inhibitors, antisense oligonucleotides, and synthetic peptides, or protein-based biologics, such as TGFβ or TGFβ receptor neutralizing antibodies, TGFβ ligand traps, or antibodies that interfere with TGFβ maturation (22, 26). In this study, we demonstrated therapeutic efficacy of two novel TGFβ inhibitors, mRER and mmTGFβ2–7m, in an orthotopic and immunocompetent oral carcinoma mouse model. The tumor progression in this model was significantly reduced, and both inhibitors potently reduced levels of soluble TGFβ in the serum. One limitation of our study was the complicated drug delivery of mmTGFβ2-7m by osmotic pumps, which probably resulted in the observed outliers. The promising results of this study indicate that further improvements of the drug delivery should be considered. It might be possible to inject mmTGFβ2-7m either alone or as a conjugate with the Fc domain of an antibody or with albumin to diminish renal filtration and facilitate higher blood circulation half-life (22).

TGFβ was chosen as a target of our therapies because of its ability to modulate the TME and to suppress antitumor immune responses (7). In patients, high TGFβ levels are often associated with the signature of TGFβ signaling in CAFs in the fibroblast- and collagen-rich peritumoral stroma, where CD8+ T cells that were excluded from the tumor parenchyma are found (11). This “immune excluded” phenotype has previously been observed in various tumors (27). Inhibition of TGFβ signaling to enable effector T cells to come in contact with cancer cells is one of the therapeutic objectives expected to overcome this exclusion of T cells from the tumor parenchyma (28). In this study, we have demonstrated the presence of higher numbers of infiltrating CD8+ T cells in mice treated with mRER or mmTGFβ2-7m inhibitors. Additionally, TGFβ is expected to directly inhibit functions of Th1 helper and cytotoxic T cells, suppress NK cell functions, and activate MDSCs (29). The latter needs to be addressed in future studies by performing functional suppression assays to further show that suppression of T cell activation by MDSCs is blocked by mRER or mmTGFβ2-7m. Although we were able to show differences in the infiltration of immune cells after treatment with mRER or mmTGFβ2-7m, a further characterization of these infiltrating cells would be helpful to understand their functions. For instance, analysis of macrophage polarization toward M1 or M2 as well as analysis of Granzyme B+, Perforin-1+, or Ki-67+ CD8+ T cells should be included in future studies. In this study, although we did not observe changes in the numbers of CD8+ T cells in the spleen of mice, treatment with mRER or mmTGFβ2-7m altered the frequency of NK cells and MDSCs, indicating a shift to a favorable immune response.

The 4-NQO model is an immunocompetent model of OSCC and thus, naturally, we focused on immune suppression and its reversal by the combination of TGFβ inhibitors and α-PD-L1 Abs. However, TGFβ has effects on various other cancer-associated pathways and the antitumor response was unlikely solely to an activated immune response. One example would be the active change of the tumor cell plasticity by these inhibitors, which would make them more susceptible to immune cell lysis. These nonimmune-related effects need to be addressed in future studies.

Inhibitors for TGFβ have been developed for decades, resulting in promising candidates that have been introduced in recent years. The most advanced TGFβ signaling antagonists are large molecules, such as monoclonal antibodies, which have been developed for cancer therapy or fibrotic disorders (30). Both of these diseases are characterized by the presence of dense tissues with large amounts of the extracellular matrix, which can potentially compromise the infiltration of large inhibitors, such as monoclonal antibodies. mRER and mmTGFβ2-7m are both smaller than typical IgG monoclonal antibodies and may enable better tissue penetration, especially mmTGFβ2-7m, which is just 10 kDa in size (mRER is 70 kDa, whereas typical IgG monoclonal antibody is 150 kDa). The smaller size could also potentially facilitate their easier loading into nanoformulations such as nanoemulsions and liposomes. Other potential advantages of these inhibitors include their high potency, especially mRER, which has been shown to neutralize TGFβ3 at picomolar concentrations (20), and high specificity. High potency may contribute to the therapeutic efficacy of mRER by enabling it to efficiently sequester TGFβ, even at low tissue concentrations of both the inhibitor and TGFβ. High specificity is an especially significant and unique property of mmTGFβ2-7m compared with kinase inhibitors, as this provides a means of selectivity targeting TβRII, something that has not been possible using receptor kinase inhibitors (20, 22). In the concentrations used, both inhibitors showed antagonistic activity in treated mice by reducing levels of TGFβ in tumor tissue and in sera without any signs of toxicity. However, significant work on drug pharmacokinetic and pathway inhibition threshold using pharmacodynamic biomarkers will be necessary to evaluate potential toxicities at higher doses and over sustained treatment periods (31).

Recent papers by Mariathasan and colleagues (11) and Tauriello and colleagues (7) link poor responses to the PD-1/PD-L1 blockade with enhanced TGFβ signaling in the TME and demonstrate that the blockade of TGFβ together with α-PD-L1 Abs reduced TGFβ signaling in stromal cells, facilitated T cell infiltration into the tumor, and provoked vigorous antitumor immunity and tumor regression (7, 11). These findings were recently confirmed by showing the beneficial effects of α-PD-L1 Abs combined with different classes of TGFβ inhibitors (32, 33). In this study, we showed that monotherapy with α-PD-L1 Abs reduced disease progression in 4-NQO mice. These results are in agreement with other reports, which demonstrate that 4-NQO mice respond to the blockade of PD-1 (16, 34). However, the combinatorial therapy of α-PD-L1 Abs with TGFβ inhibitors was found to be more effective in stimulation of an antitumor immune response compared with treatment with α-PD-L1 Abs alone, indicating that the therapeutic efficacy of the PD-1/PD-L1 blockade can be further enhanced. Thus, lymphocyte infiltration into tumors was increased, whereas MDSC and macrophage frequency was reduced. However, in terms of tumor burden the combination of α-PD-L1 Abs with mRER was not leading to significant differences compared with α-PD-L1 Ab treatment alone. We showed slightly decreased numbers and sizes of 4-NQO tumors, reduction in TGFβ tumor tissue and serum levels, modulation of the collagen-rich stroma, higher numbers of tumor-infiltrating lymphocytes, and a drastic increase in NK cell frequency in the spleens. Further studies will be required to optimize timing to enable an effective therapeutic targeting, which might also lead to improvements in tumor burden. Future studies should also focus on the full characterization of the pharmacokinetics and pharmacodynamics of the TGFβ inhibitors and evaluate the most beneficial timing and drug delivery of a combinatorial TGFβ/PD-L1 blockade. Similar to the studies by Mariathasan and colleagues (11) and Zhu and colleagues (6), it would be of major interest to identify the convergences of TGFβ effects with other signaling pathways in the TME to ensure the use of TGFβ inhibitors in a most efficient way.

N. Ludwig reports grants from German National Academy of Sciences Leopoldina and Freier Verband Deutscher Zahnärzte e.V. (FVDZ). Ł. Wieteska reports grants from European Council during the conduct of the study. A.P. Hinck reports grants from NIH NCI (CA172886) and NIH NIGMS (GM58670) and personal fees from Precithera during the conduct of the study; in addition, A.P. Hinck has an issued patent for RER (US Patent 9,611,306) with royalties paid from Precithera and a pending patent for mmTGFb27M licensed to Western Oncolytics. T.L. Whiteside reports grants from the NIH during the conduct of the study. No disclosures were reported by the other authors.

N. Ludwig: Conceptualization, formal analysis, funding acquisition, investigation, visualization, methodology, writing–original draft, writing–review and editing. Ł. Wieteska: Investigation and methodology. C.S. Hinck: Investigation and methodology. S.S. Yerneni: Formal analysis and investigation. J.H. Azambuja: Formal analysis and investigation. R.J. Bauer: Resources, writing–review and editing. T.E. Reichert: Resources, supervision, writing–review and editing. A.P. Hinck: Resources, supervision, funding acquisition, writing–review and editing. T.L. Whiteside: Conceptualization, resources, supervision, funding acquisition, writing–review and editing.

This work was supported by NIH grant U01-DE029759 to T.L. Whiteside, RO1 CA172886 to A.P. Hinck and Dr. LuZhe Sun (University of Texas Health Science Center, San Antonio, TX), and RO1 GM58670 to A.P. Hinck. N. Ludwig was supported by the Leopoldina Fellowships LPDS 2017-12 and LPDR 2019-02 from German National Academy of Sciences Leopoldina and the young investigator award from Freier Verband Deutscher Zahnärzte e.V. (FVDZ). This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 893196 to Ł. Wieteska.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Leemans
CR
,
Snijders
PJF
,
Brakenhoff
RH
. 
The molecular landscape of head and neck cancer
.
Nat Rev Cancer
2018
;
18
:
269
82
.
2.
Gharat
SA
,
Momin
M
,
Bhavsar
C
. 
Oral squamous cell carcinoma: current treatment strategies and nanotechnology-based approaches for prevention and therapy
.
Crit Rev Ther Drug Carrier Syst
2016
;
33
:
363
400
.
3.
Howlader
N
,
Noone
AM
,
Krapcho
M
,
Garshell
J
,
Miller
D
,
Altekruse
SF
, et al
SEER Cancer Statistics Review, 1975–2011
.
Bethesda, MD
:
National Cancer Institute
; 
2013
.
4.
Nair
S
,
Nayak
R
,
Bhat
K
,
Kotrashetti
VS
,
Babji
D
. 
Immunohistochemical expression of CD105 and TGF-β1 in oral squamous cell carcinoma and adjacent apparently normal oral mucosa and its correlation with clinicopathologic features
.
Appl Immunohistochem Mol Morphol
2016
;
24
:
35
41
.
5.
Logullo
AF
,
Nonogaki
S
,
Miguel
RE
,
Kowalski
LP
,
Nishimoto
IN
,
Pasini
FS
, et al
Transforming growth factor beta1 (TGFbeta1) expression in head and neck squamous cell carcinoma patients as related to prognosis
.
J Oral Pathol Med
2003
;
32
:
139
45
.
6.
Zhu
H
,
Gu
X
,
Xia
Lu
,
Zhou
Y
,
Bouamar
H
,
Yang
J
, et al
A novel TGFβ trap blocks chemotherapeutics-induced TGFβ1 signaling and enhances their anticancer activity in gynecologic cancers
.
Clin Cancer Res
2018
;
24
:
2780
93
.
7.
Tauriello
DVF
,
Palomo-Ponce
S
,
Stork
D
,
Berenguer-Llergo
A
,
Badia-Ramentol
J
,
Iglesias
M
, et al
TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis
.
Nature
2018
;
554
:
538
43
.
8.
Thomas
DA
,
Massagué
J
. 
TGF-β directly targets cytotoxic T cell functions during tumor evasion of immune surveillance
.
Cancer Cell
2005
;
8
:
369
80
.
9.
Szczepanski
MJ
,
Szajnik
M
,
Welsh
A
,
Whiteside
TL
,
Boyiadzis
M
. 
Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-beta1
.
Haematologica
2011
;
96
:
1302
9
.
10.
Webber
J
,
Steadman
R
,
Mason
MD
,
Tabi
Z
,
Clayton
A
. 
Cancer exosomes trigger fibroblast to myofibroblast differentiation
.
Cancer Res
2010
;
70
:
9621
31
.
11.
Mariathasan
S
,
Turley
SJ
,
Nickles
D
,
Castiglioni
A
,
Yuen
K
,
Wang
Y
, et al
TGF-β attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells
.
Nature
2018
;
554
:
544
8
.
12.
Saâda-Bouzid
E
,
Defaucheux
C
,
Karabajakian
A
,
Coloma
VP
,
Servois
V
,
Paoletti
X
, et al
Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma
.
Ann Oncol
2017
;
28
:
1605
11
.
13.
Giannelli
G
,
Santoro
A
,
Kelley
RK
,
Gane
Ed
,
Paradis
V
,
Cleverly
A
, et al
Biomarkers and overall survival in patients with advanced hepatocellular carcinoma treated with TGF-βRI inhibitor galunisertib
.
PLoS One
2020
;
15
:
1
16
.
14.
Redman
JM
,
Steinberg
SM
,
Gulley
JL
. 
Quick efficacy seeking trial (QuEST1): a novel combination immunotherapy study designed for rapid clinical signal assessment metastatic castration-resistant prostate cancer 11 Medical and Health Sciences 1107 Immunology
.
J Immunother Cancer
2018
;
6
:
1
8
.
15.
Razzo
BM
,
Ludwig
N
,
Hong
CS
,
Sharma
P
,
Fabian
KP
,
Fecek
RJ
, et al
Tumor-derived exosomes promote carcinogenesis of murine oral squamous cell carcinoma
.
Carcinogenesis
2020
;
41
:
625
33
.
16.
Wang
J
,
Xie
T
,
Wang
B
,
William
WN
,
Heymach
JV
,
El-Naggar
AK
, et al
PD-1 blockade prevents the development and progression of carcinogen-induced oral premalignant lesions
.
Cancer Prev Res
2017
;
10
:
684
93
.
17.
Siddappa
G
,
Kulsum
S
,
Ravindra
DR
,
Kumar
VV
,
Raju
N
,
Raghavan
N
, et al
Curcumin and metformin-mediated chemoprevention of oral cancer is associated with inhibition of cancer stem cells
.
Mol Carcinog
2017
;
56
:
2446
60
.
18.
Gannot
G
,
Buchner
A
,
Keisari
Y
. 
Interaction between the immune system and tongue squamous cell carcinoma induced by 4-nitroquinoline N-oxide in mice
.
Oral Oncol
2004
;
40
:
287
97
.
19.
Wen
L
,
Lu
H
,
Li
Q
,
Li
Q
,
Wen
S
,
Wang
D
, et al
Contributions of T cell dysfunction to the resistance against anti-PD-1 therapy in oral carcinogenesis
.
J Exp Clin Cancer Res
2019
;
38
:
1
12
.
20.
Qin
T
,
Barron
L
,
Xia
Lu
,
Huang
H
,
Villarreal
MM
,
Zwaagstra
J
, et al
A novel highly potent trivalent TGF-beta receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands
.
Oncotarget
2016
;
7
:
86087
102
.
21.
Kim
SK
,
Barron
L
,
Hinck
CS
,
Petrunak
EM
,
Cano
KE
,
Thangirala
A
, et al
An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling
.
J Biol Chem
2017
;
292
:
7173
88
.
22.
Hinck
AP
. 
Structure-guided engineering of TGF-βs for the development of novel inhibitors and probing mechanism
.
Bioorganic Med Chem
2018
;
26
:
5239
46
.
23.
Tesseur
I
,
Zou
K
,
Berber
E
,
Zhang
H
,
Wyss-Coray
T
. 
Highly sensitive and specific bioassay for measuring bioactive TGF-β
.
BMC Cell Biol
2006
;
7
:
1
7
.
24.
Miranda-Galvis
M
,
Rumayor Piña
A
,
Sales de Sá
R
,
Almeida Leite
A
,
Agustin Vargas
P
,
Calsavara
VF
, et al
PD-L1 expression patterns in oral cancer as an integrated approach for further prognostic classification
.
Oral Dis
2020
. doi: .
25.
Takahashi
H
,
Sakakura
K
,
Arisaka
Y
,
Tokue
A
,
Kaira
K
,
Tada
H
, et al
Clinical and biological significance of PD-L1 expression within the tumor microenvironment of oral squamous cell carcinoma
.
Anticancer Res
2019
;
39
:
3039
46
.
26.
Syed
V
. 
TGF-β signaling in cancer
.
J Cell Biochem
2016
;
117
:
1279
87
.
27.
Gajewski
TF
. 
Next hurdle in cancer immunorapy: overcoming non-T-cell-inflamed tumor microenvironment
.
Semin Oncol
2015
;
42
:
663
71
.
28.
Ganesh
K
,
Massagué
J
. 
TGF-β inhibition and immunotherapy: checkmate
.
Immunity
2018
;
48
:
626
8
.
29.
Batlle
E
,
Massagué
J
. 
Transforming growth factor-β signaling in immunity and cancer
.
Immunity.
2019
;
50
:
924
40
.
30.
Yingling
JM
,
Blanchard
KL
,
Sawyer
JS
. 
Development of TGF-β signalling inhibitors for cancer therapy
.
Nat Rev Drug Discov
2004
;
3
:
1011
22
.
31.
de Gramont
A
,
Faivre
S
,
Raymond
E
. 
Novel TGF-β inhibitors ready for prime time in onco-immunology
.
Oncoimmunology
2017
;
6
:
1
5
.
32.
Sow
HS
,
Ren
J
,
Camps
M
,
Ossendorp
F
,
ten Dijke
P
. 
Combined inhibition of TGF-β signaling and the PD-L1 immune checkpoint is differentially effective in tumor models
.
Cells
2019
;
8
:
320
.
33.
David
JM
,
Dominguez
C
,
McCampbell
KK
,
Gulley
JL
,
Schlom
J
,
Palena
C
. 
A novel bifunctional anti-PD-L1/TGF-β Trap fusion protein (M7824) efficiently reverts mesenchymalization of human lung cancer cells
.
Oncoimmunology
2017
;
6
:
1
16
.
34.
Chen
Y
,
Li
Q
,
Li
X
,
Ma
Da
,
Fang
J
,
Luo
L
, et al
Blockade of PD-1 effectively inhibits in vivo malignant transformation of oral mucosa
.
Oncoimmunology
2018
;
7
:
e1388484
.

Supplementary data