Soluble factors released from carcinoma-associated fibroblasts that induce the invasion of oral squamous cell carcinoma

Abstract

[한글]암 발생 과정은 단순히 암 유발유전자의 활성과 암 억제 유전자의 비활성에 의한 돌연변이 세포의 증식이라기 보다는, 암 실질세포와 그 주위 기질간의 상호작용의 결과로 볼 수 있다. 기질 세포는 암세포의 영향으로 인해 형태적 기능적으로 변질되며, 그 중에서도 섬유모세포가 암종의 개시와 진행, 침윤과정에 핵심적인 역할을 담당한다. 또한 이러한 암세포와 섬유모세포의 상호작용은 다양한 수용성 인자들에 의해 매개된다. 따라서 본 연구에서는 구강편평세포암종의 침윤과정을 좀더 명확히 이해하기 위한 일환으로, 암종에서 유래한 섬유모세포(Carcinoma Associated Fibroblasts: CAF)의 생물학적 특성과 암세포와 섬유모세포의 상호작용을 매개하여 암세포의 침윤에 영향을 미치는 수용성 인자(soluble factors)들을 규명하고자 하였다.

먼저 CAF 와 정상섬유모세포 (Normal Fibroblasts: NF)의 조직학적인 차이를 비교하기 위하여 면역형광염색을 시행한 결과, CAF와 NF에서 vimentin과 α- smooth muscle actin (SMA)의 발현을 확인하였으며, CAF의 경우 NF보다 더 높은 발현 율을 보였다.

In vitro 에서 섬유모세포가 구강편평세포암종 (Oral Squamous Cell Carcinoma: OSCC)의 침윤을 촉진하는 지의 여부를 확인하기 위하여 Transwell invasion assay를 실시하였다. 그 결과 암세포의 침윤성 성장은 섬유모세포와 공동 배양했을 때 유의적으로 증가하였으며, 특히 CAF가 NF에 비해 암세포의 침윤성 성장을 더 촉진하였다.

OSCC 와 공동 배양한 섬유모세포가 단독 배양한 섬유모세포에 비해 chemokine의 분비가 더 증가하는지의 여부를 확인하기 위해 효소결합면역흡수 분석법 (Enzyme-Linked Immunosorbent Assay: ELISA) 을 시행한 결과 OSCC 와 공동배양한 섬유모세포에서 chemokine(IL-8, CXCL1, CCL7)의 분비가 증가하였으며, OSCC와 공동 배양한 CAF가 NF에 비해 chemokine의 분비가 유의적으로 증가하였다. 이로 인해 OSCC의 자극에 대해 CAF의 반응성이 NF보다 높음을 알 수 있었다.

CAF의 cytokine 분비를 유도하는 OSCC세포의 soluble factor를 확인하기 위해 cytokine detection kit을 사용하여 antibody array를 실시하였다. VEGF와 IL-1α는 Ca 9.22 와 YD-38에서 발현량의 차이가 크게 나타났다.

VEGF와 IL-1α가 CAF의 cytokine 분비를 조절하는 지의 여부를 확인하기 위해 CAF 에 VEGF 와/또는 IL-1α를 첨가하여 분비되는 CCL7, IL-8, CXCL1을 측정한 결과, IL-1α는 CAF에서 CCL7, IL-8, CXCL1의 발현을 농도 의존적으로 유도하였으나 VEGF 는 CCL7, IL-8, CXCL1의 발현을 유도하지 못했다. 또한 VEGF는 IL-1α 에 의해 발현이 유도된 CCL7, IL-8, CXCL1에 상승효과를 보였다. 이러한 현상은 IL-1α 에 대한 중화항체에 의해 완전히 감소하였다. 또한 IL-1α를 중화함으로써 CAF의 자극에 의한 OSCC의 침윤성도 현저하게 감소되었다.

paracrine IL-1α자극에 반응하여 CAF가 CCL7, IL-8, CXCL1등의 chemokine을 분비하는데 IL-1α에 대한 수용체(IL-1RI )가 중요한 역할을 담당하는지의 여부를 분석하기 위해 IL-1RI siRNA를 CAF에 주입하였다. 그 결과 IL-1α 가 섬유모세포의 IL-1RI를 통해 CAF의 chemokine 분비를 촉진함을 알 수 있었다.

이상의 결과를 통해 암세포에서 유래한 섬유모세포가 정상섬유모세포에 비해 CCL7, IL-8, CXCL1 의 분비가 증가하였으며, 이러한 차이는 암세포와 공동 배양한 섬유모세포에서 더 현저하게 나타남을 알 수 있었다. 또한 암세포의 침윤성은 CAF를 암세포와 공동배양 하였을 때가 NF를 공동배양 하였을 때보다 현저하게 증가함도 확인되었다. cytokine 분비는 섬유모세포를 암세포와 공동배양 하였을 때 증가했기 때문에, 암세포에서 섬유모세포의 cytokine 분비를 자극하는 soluble factor를 확인한 결과 IL-1α가 가장 유력한 cytokine candidate임을 알게 되었다. 본 연구는 암종 주위 섬유모세포의 생물학적 특성을 확인하고, 구강편평세포암종에서 암세포와 섬유모세포의 상호작용을 매개하는 수용성인자들을 규명하였다. 이는 암의 진행과정을 이해하기 위한 기초 연구로, 후속 연구를 위한 초석이 되고자 한다.

[영문]Cancer development is not merely a result of clonal expansion of mutant cells, but a product of the evolving interactions between cancer parenchymal cells and their surrounding stroma. Among the stromal cells, the fibroblasts play an active role in the process of cancer development such as initiation, progression and invasion which are changed morphologically and functionally by cancer cells.

Therefore, better understanding of the characteristics of carcinoma associated fibroblasts (CAF) and the soluble factors which are influencing on CAF and cancer cells in cancer invasion might offer a promising point of departure for the development of new therapies. With this hope, the purpose of this study was to characterize CAF and to investigate the mediating soluble factors between cancer cells and fibroblasts in oral squamous cell carcinoma (OSCC).

From the immunofluorescence staining, the expression of vimentin and α-smooth muscle actin was found in both CAF and normal fibroblasts (NF), and CAF showed stronger expression pattern as compared to NF.

To determine whether fibroblasts facilitate OSCC cell invasion in vitro, a transwell invasion assay employing collagen type-I coated filters was established. OSCC cell invasion was significantly increased by co-culturing with fibroblasts as compared to that without co-culturing with fibroblasts. Also CAF enhanced cell invasion at significantly higher levels than NF.

To evaluate whether the chemokines released from fibroblasts increases by co-culturing with OSCC cells as compared to those from mono-cultured fibroblasts, sandwich ELISAs was established that is specific to IL-8, CXCL1, and CCL7. Co-culturing the OSCC cells and fibroblasts resulted in significant increase of the chemokine release into the culture media within 24 h of incubation. In addition, when comparing CAF to NF for their abilities to release the chemokines into the co-cultured media, chemokine release from the co-cultured CAF was significantly higher than that from the co-cultured NF, indicating that CAF were more responsive to OSCC cell stimulation for enhancing chemokine release than were NF.

To find the soluble factors released from OSCC cells that induce cytokine secretion by CAF, antibody array using 36 cytokine detection kit was carried out. VEGF and IL-1? showed remarkable intensity difference between Ca9.22 and YD-38 cells. To confirm whether VEGF and IL-1? regulate cytokine secretion released from CAF, each cytokine such as CCL7, IL-8 and CXCL1 was measured by adding VEGF and/or IL-1? to CAF culture. Findings were that IL-1? induced CCL7, IL-8 and CXCL1 expression dose dependently in CAF, while VEGF failed to enhance CCL7 , IL-8 and CXCL1 expression. Interestingly, VEGF has a synergic effect on CCL7, IL-8 and CXCL1 secretion induced by IL-1?. This phenomenon was completely diminished by applying anti-IL-1? monoclonal antibody. When comparing the effect of invasiveness of IL-1? by neutralization, the invasive potential of OSCC cells by stimulation of CAF was markedly decreased by neutralization of IL-1?.

This study investigated whether IL-1RI on CAF plays an important role in releasing chemokines, such as IL-8, CXCL1, and CCL7, in response to paracrine IL-1? stimulation. To do so, IL-1RI siRNA into CAF was introduced to knockdown the protein expression. The down-regulated IL-1RI protein expression on CAF was confirmed by western blot analysis. The chemokine release from the transfected CAF stimulated by IL-1? was determined by ELISAs specific to IL-8, CXCL1, and CCL7. Inhibiting IL-1RI expression in CAF significantly reduced IL-8, CXCL1, and CCL7 secretion, when compared to control siRNA-transfected CAF. In addition, the invasion of OSCC cells co-cultured with siRNA- transfected CAF was markedly reduced as compared to them co-cultured with control siRNA-transfected CAF. These observations indicate that IL-1? acts to enhance the chemokine release from CAF through the IL-1RI on fibroblasts.

To sum up, the present study attempted to identify the role of fibroblasts in cancer invasion by investigating the difference of cytokine expression between CAF and NF. And it was found that CAF secreted CCL7, IL-8 and CXCL1 more than NF did when co-cultured with cancer cells. In addition, the invasiveness of cancer cells was prominently increased when cancer cells were co-cultured with CAF than with NF. Since cytokine expression was increased when fibroblasts were co-cultured with cancer cells, this study tried to figure out which soluble factor secreted from the cancer cells stimulates fibroblasts, and found that IL-1α was the most prime candidate of cytokines.

The results of this study clarified the role of CAF by characterizing them morphologically and functionally and addressed cytokines that interact between CAF and cancer cells. Although this study may be an early step to understand the interaction process between cancer cells and fibroblasts, it could be a new branch point for research of cancer signaling and cancer development.