Pericyte stem cells induce Ly6G+ cell accumulation and immunotherapy resistance in pancreatic cancer.

We report the identification of a cell population that shares pericyte, stromal and stemness features, does not harbor the KrasG12D mutation and drives tumoral growth in vitro and in vivo. We term these cells pericyte stem cells (PeSCs) and define them as CD45- EPCAM- CD29+ CD106+ CD24+ CD44+ cells. We perform studies with p48-Cre;KrasG12D (KC), pdx1-Cre;KrasG12D ;Ink4a/Arffl/fl (KIC) and pdx1-Cre;KrasG12D ;p53R172H (KPC) and tumor tissues from PDAC and chronic pancreatitis patients. We also perform single-cell RNAseq analysis and reveal a unique signature of PeSC. Under steady-state conditions, PeSCs are barely detectable in the pancreas but present in the neoplastic microenvironment both in humans and mice. The coinjection of PeSCs and tumor epithelial cells leads to increased tumor growth, differentiation of Ly6G+ myeloid-derived suppressor cells, and a decreased amount of F4/80+ macrophages and CD11c+ dendritic cells. This population induces resistance to anti-PD-1 immunotherapy when coinjected with epithelial tumor cells. Our data reveal the existence of a cell population that instructs immunosuppressive myeloid cell responses to bypass PD-1 targeting and thus suggest potential new approaches for overcoming resistance to immunotherapy in clinical settings.

Stiffness-induced cancer-associated fibroblasts are responsible for immunosuppression in a platelet-derived growth factor ligand-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a vast stromal reaction that arises mainly from cancer-associated fibroblasts (CAFs) and promotes both immune escape and tumor growth. Here, we used a mouse model with deletion of the activin A receptor ALK4 in the context of the KrasG12D mutation, which strongly drives collagen deposition that leads to tissue stiffness. By ligand-receptor analysis of single-cell RNA-sequencing data, we identified that, in stiff conditions, neoplastic ductal cells instructed CAFs through sustained platelet-derived growth factor (PDGF) signaling. Tumor-associated tissue rigidity resulted in the emergence of stiffness-induced CAFs (siCAFs) in vitro and in vivo. Similar results were confirmed in human data. siCAFs were able to strongly inhibit CD8+ T-cell responses in vitro and in vivo, promoting local immunosuppression. More importantly, targeting PDGF signaling led to diminished siCAF and reduced tumor growth. Our data show for the first time that early paracrine signaling leads to profound changes in tissue mechanics, impacting immune responses and tumor progression. Our study highlights that PDGF ligand neutralization can normalize the tissue architecture independent of the genetic background, indicating that finely tuned stromal therapy may open new therapeutic avenues in pancreatic cancer.

ßig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer.

Macrophages play an important role in immune and matrix regulation during pancreatic adenocarcinoma (PDAC). Collagen deposition massively contributes to the physical and functional changes of the tissue during pathogenesis. We investigated the impact of thick collagen fibers on the phenotype and function of macrophages. We recently demonstrated that the extracellular protein ßig-h3/TGFßi (Transforming growth factor-ß-induced protein) plays an important role in modulating the stiffness of the pancreatic stroma. By using atomic force microscopy, we show that ßig-h3 binds to type I collagen and establishes thicker fibers. Macrophages cultured on ßig-h3-structured collagen layers display a different morphology and a pro-tumoral M2 phenotype and function compared to those cultured on non-structured collagen layers. In vivo injection of those instructed CD206+CD163+ macrophages was able to suppress T cell responses. These results reveal for the first time that the collagen structure impacts the phenotype and function of macrophages by potentiating their immunosuppressive features.

A Promising Biomarker and Therapeutic Target in Patients with Advanced PDAC: The Stromal Protein ßig-h3.

With an overall survival rate of 2-9% at 5 years, pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related deaths in the industrialized world and is predicted to become the second by 2030. Owing to often late diagnosis and rare actionable molecular alterations, PDAC has not yet benefited from the recent therapeutic advances that immune checkpoint inhibitors (ICI) have provided in other cancer types, except in specific subgroups of patients presenting with tumors with high mutational burden (TMB) or microsatellite instability (MSI). The tumor microenvironment (TME) plays a substantial role in therapeutic resistance by facilitating immune evasion. An extracellular stromal protein, ßig-h3/TGFßi, is involved in the pathogenesis of PDAC by hampering T cell activation and promoting stiffness of the TME. The study BIGHPANC included 41 patients with metastatic PDAC, and analyzed ßig-h3 levels in serum and tumor samples to assess the ßig-h3 prognostic value. ßig-h3 serum levels are significantly associated with overall survival (HR 2.05, 95%CI 1.07-3.93; p = 0.0301). Our results suggest that ßig-h3 serum levels may be considered a prognostic biomarker in patients with metastatic PDAC.

Oncogene-Induced Senescence Limits the Progression of Pancreatic Neoplasia through Production of Activin A.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions. SIGNIFICANCE: This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

Syndecan-1 interaction with the LG4/5 domain in laminin-332 is essential for keratinocyte migration.

Laminin 5/laminin 332 (LN332) is an adhesion substrate for epithelial cells. After secretion of LN332, a regulated cleavage occurs at the carboxy-terminus of its alpha3 subunit, which releases a tandem of two globular modules named LG4/5. We show that the presence of the LG4/5 domain in precursor LN332 decreases its integrin-mediated cell adhesion properties in comparison with mature LN332. Whereas cell adhesion to the recombinant LG4/5 fragment relies solely on the heparan sulfate proteoglycan (HSPG) receptor syndecan-1, we reveal that both syndecan-1 and the alpha3beta1 integrin bind to precursor LN332. We further demonstrate that syndecan-1 mediated cell adhesion to the LG4/5 fragment and pre-LN332 allows the formation of fascin-containing protrusions, depending on the GTPases Rac and Cdc42 activation. Reducing syndecan-1 expression in normal keratinocytes prevents cell protrusions on pre-LN332 with subsequent failure of the peripheral localization of the alpha3beta1 integrin. We finally show that cell migration on pre-LN332 requires syndecan-1. Therefore, the LG4/5 domain in precursor LN332 appears to trigger intracellular signaling events, which participate in keratinocyte motility.

Matrilysin 1 influences colon carcinoma cell migration by cleavage of the laminin-5 beta3 chain.

Matrilysin 1 [matrix metalloproteinase 7 (MMP7)] is one of the most important metalloproteinases expressed in human tissues. This enzyme is generally not expressed by normal differentiated epithelial colon cells, but has been shown to be up-regulated in human colon adenomas and adenocarcinomas. Little is known about the role of MMP7 in cell invasion and its involvement in proteolytic processes. By searching the ligands of MMP7 in the colonic carcinoma cells HT29, we identified laminin-5/laminin-332 (LN5) as a specific target for MMP7 enzymatic activity. LN5, composed of alpha3, beta3, and gamma2 chains, is an important component of epithelial basement membranes where it induces firm adhesion and hemidesmosome formation. In this study, we show that LN5 and MMP7 are coexpressed in HT29 cells as well as in HT29 xenograft tumors and human colorectal adenocarcinomas. We provide evidence that human LN5 is a ligand for MMP7 and that a specific cleavage occurs in its beta3 chain, giving rise to a carboxyl-terminal beta3 chain fragment of 90 kDa. We have identified the MMP7 cleavage site at position Ala(515)-Ile(516) in the beta3 chain. Videomicroscopic analysis of HT29 cells plated on LN5 substrates reveals that the MMP7-processed LN5 significantly enhances cell motility. Moreover, the delayed migration of HT29 cells obtained after specific inhibition of MMP7 reinforces the hypothesis supporting its involvement in cell migration. Altogether, our results show that MMP7 is likely to play a crucial role in the regulation of carcinoma cell migration by targeting specific proteolytic processing of the LN5 beta3 chain.

Normal human keratinocytes bind to the alpha3LG4/5 domain of unprocessed laminin-5 through the receptor syndecan-1.

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, we recombinantly expressed the human alpha3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.