Search

Legends to Supplementary Figures S1-S8.

Supplementary Materials and Methods

1.5 x 105 HeLa cells/6-well were either left untransfected or were transfected with (A) 0 µg, 0.025 µg or 0.1 µg of pCMV-Flag2-IRF1 or (B) 0 µg, 0.6 µg or 0.8 µg of pCMV-Flag2-IRF2.

HeLa cells were transfected with 10 pmol/1.5 x 105 cells of human IRF1-specific siRNA (#8) or mock siRNA.

Sections of human cervical SCCs were co-stained with the respective isotype control antibodies for CD4 and IL-17 or CCR6 and IL-17 by IF or with the respective isotype control antibody for CCL20 by IHC.

Cervical cancer is a consequence of persistent infection with human papillomaviruses (HPV). Progression to malignancy is linked to an inflammatory microenvironment comprising T-helper-17 (Th17) cells, a T-cell subset with protumorigenic properties. Neoplastic cells express only low endogenous levels of the Th17 chemoattractant CCL20, and therefore, it is unclear how Th17 cells are recruited to the cervical cancer tissue. In this study, we demonstrate that CCL20 was predominantly expressed in the stroma of cervical squamous cell carcinomas in situ. This correlated with stromal infiltration of CD4+/IL17+ cells and with advancing International Federation of Gynecology and Obstetrics (FIGO) stage. Furthermore, we show that cervical cancer cells instructed primary cervical fibroblasts to produce high levels of CCL20 and to attract CD4/IL17/CCR6-positive cells, generated in vitro, in a CCL20/CCR6-dependent manner. Further mechanistic investigations identified cervical cancer cell–derived IL6 as an important mediator of paracrine CCL20 induction at the promoter, mRNA, and protein level in fibroblasts. CCL20 was upregulated through the recently described CCAAT/enhancer-binding protein β (C/EBPβ) pathway as shown with a dominant-negative version of C/EBPβ and through siRNA-mediated knockdown. In summary, our study defines a novel molecular mechanism by which cervical neoplastic cells shape their local microenvironment by instructing fibroblasts to support Th17 cell infiltration in a paracrine IL6/C/EBPβ-dependent manner. Th17 cells may in turn maintain chronic inflammation within high-grade cervical lesions to further promote cancer progression. Cancer Res; 75(24); 5248–59. ©2015 AACR.

Legends to Supplementary Figures S1-S8.

Immunoreactive Score (IRS) according to Remmele & Stegner.

HeLa cells were transfected with 10 pmol/1.5 x 105 cells of human STAT3-specific siRNA (#8 or 9) or mock siRNA.

HeLa and SW756 cells were stimulated with medium or OSM.

IRF1 is not induced by OSM or IL-6/sgp80 stimulation in NECK.

79 human FFPE SCC were stained with anti-pTyr705-STAT3 Ab.

Neoadjuvant radio/chemotherapy regimens can markedly improve cervical cancer outcome in a subset of patients, while other patients show poor responses, but may encounter severe adverse effects. Thus, there is a strong need for predictive biomarkers to improve clinical management of cervical cancer patients. STAT3 is considered as a critical antiapoptotic factor in various malignancies. We therefore investigated STAT3 activation during cervical carcinogenesis and its impact on the response of cervical cancer cells to chemotherapeutic drugs. Tyr705-phosphorylated STAT3 increased from low-grade cervical intraepithelial neoplasia (CIN1) to precancerous CIN3 lesions. Notably, pTyr705-STAT3 activation significantly declined from CIN3 to invasive cancer, also when compared in the same clinical biopsy. pTyr705-STAT3 was also low or absent in cultured human cervical cancer cell lines, consistent with the in vivo expression data. Unexpectedly, IL6-type cytokine signaling inducing STAT3 activation rendered cervical cancer cells significantly more susceptible to chemotherapeutic drugs, that is, cisplatin or etoposide. This chemosensitization was STAT3-dependent and we identified IFN regulatory factor-1 (IRF1) as the STAT3-inducible mediator required for cell death enhancement. In line with these data, pTyr705-STAT3 significantly correlated with nuclear IRF1 expression in cervical cancer in vivo. Importantly, high IRF1 expression in pretreatment cervical cancer biopsy cells was associated with a significantly better response to neoadjuvant radio/chemotherapy of the patients. In summary, our study has identified a key role of the STAT3/IRF1 pathway for chemosensitization in cervical cancer. Our results suggest that pretherapeutic IRF1 expression should be evaluated as a novel predictive biomarker for neoadjuvant radio/chemotherapy responses. Cancer Res; 76(13); 3872–83. ©2016 AACR.

HeLa cells were co-transfected with 1.85 µg pEFBos-STAT3F or pEFBos control vector and 0.35 µg EGFP expression vector.

(A) NECF were stimulated with 50 µg/ml PMA or medium for 24h. Whole cell extracts were analyzed for C/EBPβ expression by Western blot. Shown is one experiment out of n=3, quantification represents n=3. (B) NECF were stimulated with 50 µg/ml PMA (lower panel) or medium (upper panel) for 24h and co-stained with DAPI (blue), anti-Vimentin (red) and anti-C/EBPβ (green) and analyzed in Immunofluorescence. (C) NECF were stimulated with 50 µg/ml PMA or medium for 24h. Supernatants were collected and analyzed for CCL20 protein expression. Shown are the values{plus minus}SD averaged from n=3 experiments performed in triplicates.

Low constitutive STAT3 activation in cervical cancer cell lines but not in primary cervical carcinoma cells or exocervical keratinocytes after prolonged exposure of Western blot analysis.

To achieve successful anti-cancer response with IL-2, it is necessary to develop a method delivering selective IL-2 signaling on CD8 T and NK cells than Tregs We report preclinical results with SLC-3010 that consist of IL-2 conjugate with anti-IL-2 antibody named TCB2 TCB2 is a humanized mouse antibody that specifically recognize the epitope on IL-2 where interact with CD25 as demonstrated with crystallography To develop SLC-3010, stable cell lines producing wild type human IL-2 or TCB2 were separately manufactured using mammalian cell system The pharmacokinetics analysis of SLC-3010 revealed 8-12h of half-life in mouse Although SLC-3010 dose not employ any artificial linker between IL-2 and TCB2, the half-life of SLC-3010 is comparable with other IL-2 based molecules thereby demonstrating stable binding of TCB2 against IL-2 under physiological condition In mouse model, single dosing of SLC-3010 preferentially stimulates CD8 T cells than Tregs in both secondary lymphoid organ and tumor microenvironment SLC-3010 exhibited strong anti-tumor activity which is demonstrated with circulating B cell lymphoma, metastatic solid tumor, and traditional solid tumor models Furthermore, synergistic anti-tumor response was observed by SLC-3010 with various anti-cancer drugs including chemotherapeutic reagents, targeted therapeutics, and checkpoint inhibitors such as anti PD-1 and anti CTLA-4 Once the transplanted tumor was completely cured by SLC-3010, the mice acquired strong memory response against the rejected tumor indicating strong immunological memory formation against recurrent tumors Non-human primate (NHP) efficacy test revealed that single dosing of SLC-3010 expanded CD8 T and NK cells in dose dependent manner, hence the number of CD8 T cells were increased up to 5 fold in the PBMC population with non-toxic dose of SLC3010 The peak of response in NHP was day 6-7 as analyzed the number of CD8 T cells in PBMC The strong activation of CD8 and NK cells with NHP model indicating that the binding of TCB2 and IL-2 is stable in the system as well The SLC-3010 induced selective stimulation of CD8 T cells was also demonstrated with human T cells using humanized NSG mice thereby providing an insight for developing future clinical trials with SLC-3010. Citation Format: Geon-A Kim, Da-Eun Kim, Yoo-Jin Kim, Kyung-Mi Han, Hee-Sung Kim, Soon-Hye Park, Jae-Hyun Park, Eun-Jihn Roh, Gil-Tae Wie, Jun-Young Lee. Strong anti-tumor activity and stability of IL-2/anti IL-2 conjugate SLC-3010 in preclinical experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 689.

Neoadjuvant radio/chemotherapy regimens can markedly improve cervical cancer outcome in a subset of patients, while other patients show poor responses, but may encounter severe adverse effects. Thus, there is a strong need for predictive biomarkers to improve clinical management of cervical cancer patients. STAT3 is considered as a critical antiapoptotic factor in various malignancies. We therefore investigated STAT3 activation during cervical carcinogenesis and its impact on the response of cervical cancer cells to chemotherapeutic drugs. Tyr705-phosphorylated STAT3 increased from low-grade cervical intraepithelial neoplasia (CIN1) to precancerous CIN3 lesions. Notably, pTyr705-STAT3 activation significantly declined from CIN3 to invasive cancer, also when compared in the same clinical biopsy. pTyr705-STAT3 was also low or absent in cultured human cervical cancer cell lines, consistent with the in vivo expression data. Unexpectedly, IL6-type cytokine signaling inducing STAT3 activation rendered cervical cancer cells significantly more susceptible to chemotherapeutic drugs, that is, cisplatin or etoposide. This chemosensitization was STAT3-dependent and we identified IFN regulatory factor-1 (IRF1) as the STAT3-inducible mediator required for cell death enhancement. In line with these data, pTyr705-STAT3 significantly correlated with nuclear IRF1 expression in cervical cancer in vivo. Importantly, high IRF1 expression in pretreatment cervical cancer biopsy cells was associated with a significantly better response to neoadjuvant radio/chemotherapy of the patients. In summary, our study has identified a key role of the STAT3/IRF1 pathway for chemosensitization in cervical cancer. Our results suggest that pretherapeutic IRF1 expression should be evaluated as a novel predictive biomarker for neoadjuvant radio/chemotherapy responses. Cancer Res; 76(13); 3872–83. ©2016 AACR.