Multiplex quantitative analysis of stroma-mediated cancer cell invasion, matrix remodeling, and drug response in a 3D co-culture model of pancreatic tumor spheroids and stellate cells

Abstract

Background Pancreatic ductal adenocarcinoma (PDAC) is a stroma-rich carcinoma, and pancreatic stellate cells (PSCs) are a major component of this dense stroma. PSCs play significant roles in metastatic progression and chemoresistance through cross-talk with cancer cells. Preclinical in vitro tumor model of invasive phenotype should incorporate three-dimensional (3D) culture of cancer cells and PSCs in extracellular matrix (ECM) for clinical relevance and predictability. Methods PANC-1 cells were cultured as tumor spheroids (TSs) using our previously developed minipillar chips, and co-cultured with PSCs, both embedded in collagen gels. Effects of PSC co-culture on ECM fiber network, invasive migration of cancer cells, and expression of epithelial-mesenchymal transition (EMT)-related proteins were examined. Conditioned media was also analyzed for secreted factors involved in cancer cell-PSC interactions. Inhibitory effect on cancer cell invasion was compared between gemcitabine and paclitaxel at an equitoxic concentration in PANC-1 TSs co-cultured with PSCs. Results Co-culture condition was optimized for the growth of TSs, activation of PSCs, and their interaction. Increase in cancer cell invasion via ECM remodeling, invadopodia formation and EMT, as well as drug resistance was recapitulated in the TS-PSC co-culture, and appeared to be mediated by cancer cell-PSC interaction via multiple secreted factors, including IL-6, IL-8, IGF-1, EGF, TIMP-1, uPA, PAI-1, and TSP-1. Compared to gemcitabine, paclitaxel showed a greater anti-invasive activity, which was attributed to suppresion of invadopodia formation in cancer cells as well as to PSC-specific cytotoxicity abrogating its paracrine signaling. Conclusions Here, we established 3D co-culture of TSs of PANC-1 cells and PSCs using minipillar histochips as a novel tumoroid model of PDAC. Our results indicate usefulness of the present co-culture model and multiplex quantitative analysis method not only in studying the role of PSCs and their interactions with tumor cell towards metastatic progression, but also in the drug evaluation of stroma-targeting drugs. Electronic supplementary material The online version of this article (10.1186/s13046-019-1225-9) contains supplementary material, which is available to authorized users.

Epithelial-mesenchymal transition: a hallmark in pancreatic cancer stem cell migration, metastasis formation, and drug resistance

“…These signaling factors cooperate in active cross-talk with cancer cells through paracrine signaling factors including chemokines, insulin-like growth factor, and proteases [52][53][54][55][56] . Furthermore, several pro-stemness paracrine factors are secreted by distinct CAFs [56][57][58][59][60][61][62] and support the self-renewal and the stemness properties of initial PCSCs in tumors or promote the conversion of cancer cells into PCSCs [63] . Additionally, chemotherapy (e.g., GEM), can affect CAFs in PDAC, which then acquire a senescence-like secretory phenotype and increase the production of pro-stemness chemokines which enhance tumorstemness and aggressiveness of PDAC after therapy [13] .…”

“…Interestingly, besides CAFs, the PDAC stroma also contains bone marrow-derived mesenchymal stem cells (MSCs) [65] . The MSCs significantly contribute to tumor progression and promote cancer stemness by secreting pro-stemness cytokines, chemokines, and growth factors or by differentiating into prostemness CAFs [62,63,66] . MSCs also produce pro-stemness niches in the stroma of PDAC, and infiltrating immune cells produce pro-stemness factors that form pro-PCSC niches [67][68][69][70] .…”

Pancreatic Adenocarcinoma Invasiveness and the Tumor Microenvironment: From Biology to Clinical Trials

“…In co-cultures of PSCs and pancreatic cancer cells, the frequency and distance of invasion increases when compared to mono-cultures of pancreatic cancer cell [ 82 ]. Pancreatic cancer cells growing as spheroids in the presence of PSCs exhibit increased invadopodia formation and ECM remodeling [ 83 ]. In addition, PSCs may lead invasion of PDAC cells by remodeling the ECM via collagen fibers to create a pathway for cancer cells to follow [ 82 ], and promote basement membrane destruction in PDAC organoid co-cultures through a mechanism involving the secretion of metalloproteinases by PSCs [ 84 ].…”

Ping-Pong—Tumor and Host in Pancreatic Cancer Progression

“…Increased radioresistance by aPSC/CAF relies on ß1 integrin-FAK activation and DNA damage response regulation (493,494). An impact on chemotherapy resistance hinges on accessibility (495), activation of the SDF1-CXCR4 axis with subsequent upregulation of IL6, increased HH expression, and IL1β-IRAK4 1 or mTOR/EIF4E 1 pathway activation (496)(497)(498)(499)(500)(501). Finally, aPSC/CAF support metastasis formation via the HGF/cMET/survivin pathway, which is regulated by TP53 1 /CDKN1A 1 (502) or through altered lipid metabolism, particularly oleic-, palmitoleic-, and linoleic-acid upregulation (503).…”