Research ArticleExperimental Studies
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Inhibition of STAT3 Promotes Effector T Cell Infiltration But also Immunosuppression in the HCC Tumor Microenvironment

TAKETO SASAKI, KOHEI SHIGETA, SHUJI KITAHARA, YASUHIRO SUZUKI, SHIMPEI MATSUI, RYO SEISHIMA, KOJI OKABAYASHI, DAN G. DUDA and YUKO KITAGAWA
Anticancer Research November 2022, 42 (11) 5205-5215; DOI: https://doi.org/10.21873/anticanres.16027

Abstract

Background/Aim: STAT3 is involved in the progression of several cancers, and has been proposed as target for therapy. Indeed, the multitargeted tyrosine kinase inhibitor drug regorafenib, which indirectly inhibits STAT3, can significantly enhance the effects of anti-programmed death receptor (PD)-1 therapy in hepatocellular carcinoma (HCC) models. Here, we studied the impact of a direct STAT3 inhibitor on the tumor microenvironment and PD-1 blockade efficacy in HCC models. Materials and Methods: Orthotopic mouse models of HCC (RIL-175 and HCA-1 grafts in syngeneic mice) were used to test the efficacy of the selective STAT3 inhibitor STX-0119 alone or combined with anti-PD-1 antibodies. We evaluated the effects of therapy on tumor vasculature and the immune microenvironment using immunofluorescence (IF), cell viability assay and quantitative real-time (qRT)-PCR in tumor tissues. Results: Combining anti-PD-1 antibodies with a STX-0119 failed to show a growth delay or survival benefit compared to each agent alone or control in any of the HCC models. Interestingly, evaluation of intratumoral CD8+ T cell infiltration by IF showed a significant increase after one-week treatment with STX-0119 (p=0.034). However, STX-0119 treatment simultaneously promoted increased immunosuppression in the tumor microenvironment by increasing the proportion of Tregs, tissue hypoxia and α-SMA activated cancer-associated fibroblasts (CAFs) measured by IF. Consistent with these findings, we found increased immature tumor vessels by IF and VEGF, Tgf-β and Vash2 expression by qPCR. Conclusion: Pharmacologic STAT3 inhibition could significantly enhance CD8+ T cell infiltration in HCC but also significantly alter the immunosuppression and vascular abnormalization in the tumor microenvironment.

Key Words:

The incidence of hepatocellular carcinoma (HCC) is increasing worldwide, making it a leading cause of cancer-related death (1). Existing therapeutic options have limited efficacy. Immune checkpoint inhibitors (ICIs), such as anti-programmed cell death 1 (PD-1) drugs, have improved the overall survival (OS) of patients with various types of cancers (2, 3). However, despite promising response rates that range from 15% to 20% in clinical trials of single-agent treatment in first– and second–line setting, these drugs did not significantly improve OS (4). These results emphasize the need for combining ICIs with other agents to boost their efficacy.

In patients with unresectable HCC, the anti-PD ligand (PD-L) 1 antibody atezolizumab combined with the anti-VEGF antibody bevacizumab resulted in significant increase in OS and progression-free survival (PFS) rates over sorafenib in a randomized phase 3 trial (5). The combination of ICI with other available HCC drugs, such as multitargeted kinase inhibitors, is ongoing in phase 3 studies. Of note, regorafenib plus the PD-1 antibody nivolumab showed a manageable safety profile and encouraging anti-tumor activity in patients with gastric and colorectal cancers in the REGONIVO, EPOC1603 trial (6). These data argued that, in addition to inhibiting VEGF/VEGFR-driven angiogenesis, other targets may be relevant in advanced HCC.

We previously reported that rationally dosing regorafenib can significantly enhance PD-1 blockade effects in HCC models (7). The benefits were due to the concomitant normalization of HCC vasculature upon VEGFR inhibition and PD-1 blockade, and stimulation of anti-tumor immunity by indirect inhibition of STAT3, up-regulation of the chemokine CXCL10, and increased CXCR3+CD8+ T cell infiltration.

STAT3 has attracted attention for its involvement in promoting human cancer progression (8), and is constitutively activated in a wide variety of human tumors. STAT3 has also been reported to be involved in oncogenesis by up-regulating the transcription of several genes that control tumor cell survival, resistance to apoptosis, cell cycle progression, and angiogenesis. Targets of STAT3 include Bcl-2, Bcl-xl, c-myc, cyclin D1, vascular endothelial growth factor, and human telomerase reverse transcriptase (9-11). Several studies have shown that STAT3 activity in tumor cells plays a critical role in inducing immunosuppression and that targeted STAT3 inhibition results in tumor Treg cell reduction and heavy infiltration of CD8+ T cells in tumors, leading to effective antitumor immune responses (12, 13).

Here, we sought to determine the impact of direct STAT3 inhibition in the context of PD-1 blockade in HCC. We also examined the effect of a pharmacologic STAT3 inhibitor on CD8+ T cell infiltration as well as on tumor microenvironment using orthotopic immunocompetent HCC models.

Materials and Methods

Cells. Two murine cell lines were used in this study: HCA-1 (from C3H mice) (14, 15) and RIL-175 (p53/Hras mutant HCC cells from C57Bl/6 mice) (16). RIL-175 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% fetal bovine serum (FBS), pyruvic acid, and 1% of penicillin/streptomycin. HCA-1 cells were maintained in DMEM supplemented with 10% FBS, pyruvic acid, and 1% of penicillin/streptomycin. The cells were authenticated and tested for mycoplasma contamination prior to use in in vitro and in vivo experiments.

Orthotopic HCC mouse model. According to the protocol for establishing an orthotopic HCC mouse model (17), HCC cells (20 μl, 1 million 1:1 in Matrigel; Mediatech/Corning, Manassas, VA, USA) that matched their genetic background were grafted in the livers of 6–8-week-old male mice: HCA-1 cells in C3H mice and RIL-175 cells in C57Bl/6 mice. All experimental procedures were approved by our institution and were performed at least twice.

Agents and treatments. The STAT3 inhibitor STX-0119 (Sigma-Aldrich) was used in this study; STX-0119 is a cell-permeable oxadiazolyl-quinolinecarboxamide shown to selectively suppress STAT3, but not STAT1, STAT5a, or STAT5b (18). STX-0119 was diluted in a sterile 0.5% w/v methyl cellulose 400-cp solution (Wako, Tokyo, Japan) or dissolved in a mixture of 20% dimethyl sulfoxide (Wako) and 80% polyethylene glycol 300 (Wako) for use in vivo in the animal experiments. STX-0119 was administered to the mice at the dose of 40 mg/kg by daily oral gavage from days 0 to 4 (18).

Mouse anti-PD-1 antibody (clone 4H2) was supplied by the Ono Pharmaceutical Co., Ltd (Osaka, Japan). Anti-PD-1 antibody (aPD1) was given intraperitoneally at a dose of 10 mg/kg three times weekly on days 0, 3, and 6.

Immunohistochemistry. Tumor tissues were collected after 7 days of treatment and fixed in 4% paraformaldehyde and embedded in paraffin or optimal cutting temperature compound and frozen. Five random fields inside the tumor were selected from the center to the edge. Phospho-STAT3 (pSTAT3) was detected using Phospho-Stat3(Tyr705) antibody, dilution 1:200, Cell Signaling Technology, Danvers, MA, USA). CD8+ lymphocytes were detected using an anti-CD8 alpha antibody (dilution 1:200, Abcam, Cambridge, UK). Regulatory T cells (Tregs) were detected using an anti-FoxP3 antibody (Abcam). For analyses of M2 tumor-associated macrophages (TAMs), F4/80 (1:200, Abcam) and anti-CD163 antibody (dilution 1:100, Abcam) positive cells were considered as M2 macrophages. They were identified by scanning tumor sections under ×10 magnification and counted in five random fields under ×200 magnification. Hypoxia was detected and quantified by immunostaining with an anti-hypoxia-inducible factor (HIF)-1 alpha antibody (dilution, 1:100, Abcam). Activated cancer-associated fibroblasts (CAFs) were analyzed using anti-actin, α-smooth muscle (Sigma-Aldrich, 1:100). For analyses of endothelial and perivascular cells, tumor tissues containing the total areas of CD31 (BioLegend 1:200, San Diego, CA, USA) positive endothelial cells and desmin (dilution 1:100, Abcam) positive pericytes were identified by scanning tumor sections under ×10 magnification and counted in five random fields under ×200 magnification. Cell nuclei were identified using 4′,6-diamidino-2-phenylindole (DAPI; VECTASHIELD® Antifade Mounting Medium with DAPI VECTOR, Newark, CA, USA). Specimens were incubated with fluorescence (cyanine 3 and Alexa 647)-conjugated anti-hamster or anti-rabbit secondary antibodies, as appropriate (Jackson ImmunoResearch, West Grove, PA, USA). For quantification, three representative areas of vital tumor, which were defined as regions of interest, were analyzed in whole images.

These data were analyzed using ImageJ (US National Institutes of Health, Bethesda, MD, USA) and Photoshop (Adobe Systems Inc., San Jose, CA, USA) software. IF images were analyzed using a Keyence fluorescence microscope (Keyence, Osaka, Japan).

Cell viability assay. Cell viability was determined at 48 h and 72 h using a 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide inner salt (MTT) assay. HCA-1 (1×103 cells) and RIL-175 (5×102 cells) were seeded in 96-well plates (Corning Inc. Corning, NY, USA). After a 24-h incubation, cells were exposed to a range of drug concentrations. Viable cells were immediately stained using the Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan), and identified by measuring the absorbance at 450 and 600 nm with a spectrophotometer (Sunrise Basic Tecan Microplate Reader, Tecan, Männedorf, Switzerland) according to the manufacturer’s instructions. Cell viability was calculated according to the following equation: (mean absorbance of drug-treated well/mean absorbance of control wells) × 100%. The fraction of the control was calculated by dividing the fluorescence obtained from the drug-treated cells by the fluorescence obtained from the control (untreated cells).

Western blot analysis. Cells were lysed in RIPA lysis buffer, and protein concentrations were determined using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Thirty micrograms of proteins were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto a Immun-Blot® PVDF membrane (Bio-Rad). The membrane was blocked with 3% bovine serum albumin (BSA) in TBS-T for 1 h and then incubated with primary antibodies diluted 1:200 in TBS-T containing BSA overnight at 4°C. We used the following primary antibodies: anti-STAT3, anti-pSTAT3, and anti-beta (β)-actin (Actb). Subsequently, the membrane was washed three times in TBS-T and then incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. Finally, the protein bands were visualized with Immobilon Forte Western HRP substrate (Millipore, Darmstadt, Germany).

Enzyme-Linked Immunosorbent Assay (ELISA) for CXCL10 and Tgf-β. To determine if CXCL10 and transforming growth factor-β (Tgf-β) was produced by murine HCC cells in response to STAT3 inhibitor treatment, we cultured RIL-175 cells with various concentration of STAT3 inhibitor in vitro and measured the CXCL10 levels by ELISA (Abcam, Cambridge, UK) and Tgf-β levels by ELISA (Elabscience, Houston, TX, USA) according to the manufacturer’s instructions.

RNA extraction and quantitative real-time RT-PCR. Total RNA was extracted from mouse tissues using QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) and purified using the RNeasy Mini Kit (QIAGEN). First-strand cDNA was synthesized by reverse transcriptase using ReverTra Ace™ (TOYOBO, Osaka, Japan). Quantitative real-time RT-PCR was performed in a CFX96 real-time RT-PCR detection system (Bio-Rad Laboratories) according to the manufacturer’s instructions. PCR conditions consisted of an initial denaturation step at 95°C for 3 min, followed by 40 cycles of 10 s at 95°C, 10 s at 56°C, and 30 s at 72°C. Relative mRNA levels of target genes were normalized to the glyceraldehyde 3-phosphate dehydrogenase (Gapdh) mRNA levels. Primers used for quantitative real-time RT-PCR were Vash2 (forward: 5′-CTGTTCCACGTGAACAAGAG-3′ reverse: 5′-TCGTCATGGAC AACCTGTAG-3′), Vegf-A (forward: 5′-GCAGAAGTCCC ATGAAG TGA-3′ reverse: 5′-TCCAGGGCTTCATCGTTA-3′), PD-1 (forward: 5′-TGCACCCCAAGGCAAAAATC-3′ reverse: 5′-AGAGTGTCGTC CTTGCTTCC-3′), PD-L1 (forward: 5′-AGTCT CCTCGCCTGCA GATA-3′ reverse: 5′-GGGAATCTGCACTCC ATCGT-3′), Ctla-4 (forward: 5′-TTGTCGCAGTTAGCTTGGGG-3′ reverse: 5′-AACGG CCTTTCAGTTGATGG-3′), and Tgf-β (forward: 5′-CGTCAGACATT CGGGAAGCA-3′ reverse: 5′-TGCCGTACAACTCCAGTGAC-3′).

Statistical analysis. All statistical analyses were performed using Stata software, V.14.1 (StataCorp, College Station, TX, USA). Error bars indicate the standard error of the mean. Differences were considered significant when p-values were <0.05. Quantitative variables were compared using Student’s t-test. Experiments with more than two groups were analyzed using one-way analysis of variance with Scheffe’s correction for multiple comparisons. Median OS was estimated using the Kaplan–Meier method. Statistical analyses in the survival experiments were performed by Cox proportional hazard models; hazard ratios (HRs) and 95% confidence intervals (CIs) were also calculated.

Results

Evaluation of the effect of STAT3 inhibition on HCC cells. STAT3 and pSTAT3 levels were detectable in both RIL-175 and HCA-1 cells (Figure 1A). We examined the effect of the STAT3 inhibitor STX-0119 on HCC cells using a cell viability assay (Figure 1B and C). The cytotoxicity of STX-0119 in each cell type was evaluated after 48 h or 72 h of treatment. The IC50 values were 9.84 μM for HCA-1 and 24.14 μM for RIL-175 murine HCC cells at 72 h. These results indicate that STAT3 and pSTAT3 are expressed in RIL-175 cells and that STAT3 inhibitors exhibited cytotoxic properties at high doses. To reveal the effect of STX-0119 treatment on the RIL-175 models, we evaluated intratumoral pSTAT3 in RIL-175 tumors. The proportion of pSTAT3 within the tumor was evaluated by IF and was significantly decreased after 1 week of treatment in the tumors from mice that received STX-0119 therapy (p=0.033) (Figure 1D). By ELISA, treatment with STAT3 inhibitor (STX-0119) for 48 (left) and 72 (right) h significantly increased the levels of CXCL10 secreted by RIL-175 cells (Figure 1E).

Figure 1.
Figure 1.

Evaluation of STAT3 inhibition in an in vitro assay. A) Protein levels of STAT3 and phosphorylated STAT3 in RIL-175 and HCA-1 cells by western blotting. B-C, Cell viability after STX-0119 treatment of RIL-175 cells (B) an HCA-1 (C) murine HCC lines. The IC50 value calculated after 72 h was 24.14 μM (RIL-175) and 9.8421 μM (HCA-1). The intratumoral pSTAT3 in RIL-175 tumors. The proportion of pSTAT3 within the tumor was evaluated by IF and was significantly decreased after 1 week of treatment in tumors from mice that received STX-0119 therapy (S) compared with control (C) (p=0.033) (D). Treatment with STAT3 inhibitor (STX-0119) for 48 and 72 h significantly increased the levels of CXCL10 secreted by RIL-175 cells, assayed using ELISA (E).

Combination of a STAT3 inhibitor with anti-PD-1 therapy leads to reduced survival in mice bearing orthotopic HCC. The efficacy of combining an anti-PD-1 antibody and STX-0119 versus that of each agent alone and that of the control was tested in two different mouse models (orthotopic RIL-175 and HCA-1 grafts) (C; Control, P; anti-PD-1 therapy, S; STAT3 inhibitor, SP; STAT3 inhibitor plus anti-PD-1) (Figure 2A). Surprisingly, the combination treatment had no significant therapeutic effect on either survival or tumor growth compared with the control in both models. Furthermore, no significant effect of the combination treatment on the number of lung metastases counted using Bouin’s fluid (ScyTek) was found. STX-0119 treatment exerted no additional effect when combined with anti-PD-1 therapy (Figure 2B-H). Moreover, addition of STX-0119 compromised the benefit of anti-PD-1 antibody treatment, which was effective when used alone in the RIL-175 mouse model, in line with our previous study (7) (Figure 2B). Of note, we found no significant change in body weight of mice from each treatment group, which indicated that there were no severe treatment-related adverse effects (Figure 2D and G). These results suggest that although administration of the pharmacologic STAT3 inhibitor was safe, adding it to anti-PD-1 therapy had antagonistic rather than additive effects both in anti-PD-1 therapy sensitive and resistant models.

Figure 2.
Figure 2.

Combination of STAT3 inhibitor and anti-PD-1 treatment is ineffective. A) Treatment protocol. The STAT3 inhibitor STX-0119 was administered to mice at the dose of 40 mg/kg by daily oral gavage from days 0 to 4. Mouse anti-PD1 antibody (clone 4H2) was given intraperitoneally at a dose of 10 mg/kg three times per week on days 0, 3, and 6. B-D) Efficacy and safety study in RIL-175 orthotopic HCC model (n=7 mice): overall survival (B), tumor growth rate (C) and body weight changes post-treatment (D). E-G) Efficacy and safety study in HCA-1orthotopic HCC model (n=10 mice): overall survival (E), tumor growth rate (F) and body weight changes post-treatment (G). H. Spontaneous lung metastatic burden in the HCA-1 model. STAT3 inhibitor treatment had no additional effect when combined with anti-PD-1 therapy and compromised the efficacy of anti-PD-1 therapy in the RIL-175 HCC model. C: Control; P: anti-PD-1 therapy; S: STAT3 inhibitor; SP: STAT3 inhibitor plus anti-PD-1.

CD8+ T cells and regulatory T cells are increased by the STAT3 inhibitor. To reveal the negative effect of STX-0119 treatment on the tumor immune microenvironment in the RIL-175 model, we first evaluated intratumoral infiltration by CD8+ T cells in RIL-175 tumors. CD8+ T cell infiltration within the tumor was evaluated by IF and was significantly increased after 1 week of treatment in tumors from mice that received STX-0119 therapy (p=0.034) (Figure 3A). However, the intratumoral infiltration by Tregs was also significantly increased after STX-0119 therapy compared with the control group (p<0.001) (Figure 3B). Moreover, we found a non-significant trend towards increase in intratumoral infiltration by F4/80+CD163+ M2-type TAMs in the STX-0119-treated group (Figure 3C).

Figure 3.
Figure 3.

Effects of STAT3 inhibition on the tumor microenvironment in the RIL-175 hepatocellular carcinoma (HCC) model. A) CD8+ T cell infiltration into the tumor was evaluated using IF (in red) as the percent-positive surface area, and was significantly increased after 1 week of STAT3 inhibitor therapy compared to control (p=0.034) (C: 7 mice, S: 6 mice). B) Treg infiltration into the tumors was significantly increased after treatment with the STAT3 inhibitor compared with that in the control group (p<0.001) (C: 7 mice, S: 6 mice). C) The intratumoral distribution of TAMs using IF for F4/80 and CD163 (for M2 subset). The fraction of CD163+/F4/80+ area tended to be increased in the STAT3 inhibitor-treated group compared to the control group (p=0.25) (C:6 mice, S:5 mice). D) The expression levels of PD-1, PD-L1, CTLA-4, and Tgf-β were evaluated in the same tumor sample by qPCR. Only Tgf-β was significantly increased in the STAT3 inhibitor-treated group compared with the control group (p=0.028) (C: 7 mice, S: 6 mice). E) Treatment with the STAT3 inhibitor (STX-0119) significantly increased the levels of Tgf-β secreted by RIL-175 cells, as assayed by ELISA (p=0.019). C: Control; S: STAT3 inhibitor.

We also examined the expression levels of immune checkpoint molecules and Tgf-β, a key mediator of immunosuppression in tumors, by qPCR. We found no significant difference in the expression of PD-1, PD-L1 or CTLA-4 after treatment with the STAT3 inhibitor. However, Tgf-β expression was significantly increased in STX-0119-treatment group compared to the control treatment group (p=0.028) (Figure 3D). Also, by ELISA, STAT3 inhibitor (STX-0119) treatment resulted in a significant increase in the levels of Tgf-β secreted by RIL-175 cells (p=0.019) (Figure 3E).

Thus, while STAT3 inhibition could increase the infiltration by CD8+ T cells, this was counterbalanced by increased immunosuppression in the tumor microenvironment, particularly by increase in Treg infiltration and Tgf-β expression.

The STAT3 inhibitor promotes immunosuppression in the tumor microenvironment. Finally, to determine STAT3 inhibitor-induced changes in the tumor microenvironment, we evaluated the tumor vasculature, hypoxia, and activated CAFs. The fraction of pericyte covered (mature) tumor vessels, as determined by desmin and CD31 co-staining, was significantly decreased after STAT3 inhibition treatment (p=0.040) (Figure 4A). Moreover, the hypoxic tissue area in the tumor, as estimated by HIF-1α staining, was significantly increased in the STAT3 inhibitor group compared with the control (p<0.001) (Figure 4B). In addition, using qPCR, we evaluated the mRNA expression levels of Vegf-A and Vash2, two critical pro-angiogenesis factors mediating vascular maturation. Both Vegf-A and Vash2 were significantly increased in the tumor samples from the STAT3 inhibitor treated group compared to those from the control group (Vegf-A; p=0.013, Vash2; p=0.003) (Figure 4C). Furthermore, we evaluated activated CAFs by α-SMA staining and found that their number was significantly increased in the STAT3 inhibitor group compared with the control group (p<0.001) (Figure 4D). Altogether, these results show that the STAT3 inhibitor negatively alters tumor microenvironment towards vascular abnormalization, immunosuppression, and treatment resistance (Figure 5).

Figure 4.
Figure 4.

Promotion of a more immunosuppressive and abnormal tumor microenvironment after STAT3 inhibition. A) The proportion of pericyte covered vessels according to desmin and CD31 staining was significantly decreased after STAT3 inhibition (p=0.040) (C: 8 mice, S: 6 mice). B) The hypoxic area of the tumor, as estimated using IF for HIF-1α, was significantly increased in the STAT3 inhibitor group compared with the control group (p<0.001) (C:7 mice, S:3 mice). C) Expression levels of Vegf-A and Vash2 were significantly increased in the STAT3 inhibitor group compared with the control group (Vegf-A; p=0.013, Vash2; p=0.003) (C: 7 mice, S: 6 mice). D. Number of activated CAFs, identified using IF for α-SMA, showed a significant increase in the STAT3 inhibitor group compared with the control (p<0.001) (C: 5 mice, S: 5 mice). C: Control; S: STAT3 inhibitor.

Figure 5.
Figure 5.

Schematic diagram of the opposing mechanisms of STAT3 inhibitor treatment on hepatocellular carcinoma (HCC) immune microenvironment. Pharmacologic inhibition of STAT3 increases effector T cells but increases immunosuppression and vascular abnormalities of HCC microenvironment, compromising anti-tumor immunity.

Discussion

STAT3 has been proposed as a target for cancer therapy. Moreover, we previously found that decreased STAT3 activation by regorafenib increased the CD8+ T cell infiltration in HCC and had a synergic effect with anti-PD-1 therapy in HCC models (7). Combination treatment normalized tumor vascular structure and function, and increased intratumoral CD8+ T cell infiltration. This led us to investigate the effects of pharmacologic STAT3 inhibition with STX-0119, alone or with anti-PD-1 therapy.

Interestingly, we found that STX-0119 treatment increased tumor infiltration by CD8+ T cells and CXCL10 chemokine, However, this treatment had no significant survival benefit and compromised rather than enhance the benefit of anti-PD-1 therapy. To understand the mechanism of this antagonistic effect, we examined the effect of STAT3 inhibition on the tumor microenvironment of HCC, including the immune microenvironment.

We found that the STAT3 inhibitor promoted an immunosuppressive tumor microenvironment by multiple inter-related mechanisms. One involved increased intratumoral infiltration by Tregs and expression of Tgf-β, a mediator of immunosuppressive Treg functions. In addition, we found a trend for a shift in TAM phenotype to M2 (alternatively activated macrophages), considered as immunosuppressive and pro-angiogenic immune cells (19, 20). Finally, treatment with the STAT3 inhibitor increased the number of activated CAFs that are defined by α-SMA expression (21). CAFs constitute an important component of HCC stroma and can also promote the proliferation and invasion of cancer cells either directly via soluble signaling molecules or indirectly through the regulation of angiogenesis and immunity (22, 23). Indeed, the expression levels of Vegf-A and Vash2 were significantly increased in the treated tumors, which supports the finding that the establishment of abnormal tumor vasculature is enhanced by STAT3 inhibition. The role of VEGF involves not only angiogenic effects but also tumor suppression through activation of Tregs and recruitment of M2 TAMs (24). These results indicated that STAT3 inhibition could enhance CD8+ T cell infiltration into the tumor, promoted by CXCL10 up-regulation; however, the concomitant increases in abnormal/immature vessels and treatment-induced hypoxia aggravate immunosuppression in the microenvironment, leading to treatment resistance, as seen previously with high doses of the anti-VEGFR drug sorafenib (25). These data re-emphasize the role of vascular normalization, induced by judicious doses of anti-VEGF agents to prevent hypoxia and promote anti-HCC immunity (24, 26-29).

Pharmacologic STAT3 inhibitors have been already evaluated in phase 1 clinical studies in patients with relapsed and/or refractory hematological malignancies and treatment-refractory solid tumors (30, 31). Although potential anti-tumor activity was seen in patients with non-small-cell lung cancer, both studies revealed tolerability-related difficulties, including peripheral neuropathy and drug-induced pneumonitis. Several newer generation STAT3 inhibitors have been developed, some of which are also being tested in clinical trials, including in combination with ICI (32-35). Our preclinical results show that the effect of pharmacologic STAT3 inhibition may be antagonistic rather than additive or synergistic in HCC models, which warrants careful evaluation of the treatment effects in tumor-specific manner.

Several studies have shown that STAT3 inhibition results in tumor Treg cell reduction and infiltration of CD8+ T cells in tumors, leading to effective antitumor immune responses (12, 13), however, this study shows that STAT3 inhibition increases immune cell infiltration and also promotes an immunosuppressive tumor microenvironment. Non-significant decrease of intratumoral infiltration by Tregs after STX-0119 therapy was seen in HCA-1 (data not shown), however the HCA-1 orthotopic HCC model had severe lung and peritoneal metastases compared to RIL-175 model so that it was considered unsuitable for local evaluation. The tumor microenvironment may change depending on the type of carcinoma and treatment, which could affect the effect of STAT3 inhibition. Further investigation is needed to assess the relationship between the tumor microenvironment and various types of metastases.

In conclusion, we demonstrate that pharmacologic STAT3 inhibitor treatment is feasible and can significantly enhance CD8+ T cell infiltration by promoting CXCL10 up-regulation in orthotopic HCC models. However, this effect was counteracted by increased immunosuppression and vascular abnormalization in the tumor microenvironment. These opposing effects compromised rather than enhanced the efficacy of immunotherapy and should be further examined in other cancers where this combination therapy is being considered.

Acknowledgements

The Authors would like to thank Kaoru Koishikawa for the outstanding technical support.

Footnotes

  • Authors’ Contributions

    TS and KS designed the study and analyzed the data and wrote the manuscript. SK and YS analyzed the data and edited the manuscript. KO, RS, SM, DGD, and YK edited the manuscript. All Authors approved the final version of the manuscript.

  • Conflicts of Interest

    This research was funded by Japan Society for the Promotion of Science (20K16969). Dr. Yuko Kitagawa received lecture fees from CHUGAI PHARMACEUTICAL CO., LTD., TAIHO PHARMACEUTICAL CO., LTD, ASAHI KASEI PHARMA CORPORATION, ONO PHARMACEUTICAL CO., LTD and research grants from CHUGAI PHARMACEUTICAL CO., LTD., TAIHO PHARMACEUTICAL CO., LTD, Yakult Honsha Co. Ltd., ASAHI KASEI PHARMA CORPORATION, Otsuka Pharmaceutical Co., Ltd., ONO PHARMACEUTICAL CO., LTD., TSUMURA & CO., Eisai Co., Ltd., Otsuka Pharmaceutical Factory Inc., MEDICON INC., Takeda Pharmaceutical Co., Ltd. Dr. Dan Duda received research grants from Bayer, BMD, and Exelixis. Dr. Koji Okabayashi received research grants from CHUGAI PHARMACEUTICAL CO.

  • Received August 3, 2022.
  • Revision received August 27, 2022.
  • Accepted September 5, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Inhibition of STAT3 Promotes Effector T Cell Infiltration But also Immunosuppression in the HCC Tumor Microenvironment
TAKETO SASAKI, KOHEI SHIGETA, SHUJI KITAHARA, YASUHIRO SUZUKI, SHIMPEI MATSUI, RYO SEISHIMA, KOJI OKABAYASHI, DAN G. DUDA, YUKO KITAGAWA
Anticancer Research Nov 2022, 42 (11) 5205-5215; DOI: 10.21873/anticanres.16027
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