Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.

Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

Loss of Rnf43 Accelerates Kras-Mediated Neoplasia and Remodels the Tumor Immune Microenvironment in Pancreatic Adenocarcinoma.

BACKGROUND & AIMS: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been characterized sufficiently. In this study, we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. METHODS: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43flox/flox (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months, and at moribund status followed by analysis of pancreata by single-cell RNA sequencing. Comparative analyses between moribund KRC and a moribund Kras/Tp53-driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC sequencing, and in vitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. RESULTS: We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared with KC. Importantly, KRC mice have a significantly decreased survival compared with KC mice. Using single-cell RNA sequencing, we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes, with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine CXCL5 was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. CONCLUSIONS: The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared with previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

Pancreatic Cancer Metabolism: Molecular Mechanisms and Clinical Applications.

PURPOSE OF REVIEW: Pancreatic adenocarcinoma is a leading cause of cancer mortality in western countries with a uniformly poor prognosis. Unfortunately, there has been little in the way of novel therapeutics for this malignancy over the last several decades. Derangements in metabolic circuitry favoring excess glycolysis are increasingly recognized as a key hallmark of cancer. RECENT FINDINGS: The role of alterations in glutamine metabolism in pancreatic tumor progression has been elucidated in animal models and human cells lines, and there has been considerable interest in exploiting these aberrations for the treatment of pancreatic cancer. Other strategies targeting NQO1/GLS1 inhibition, NAD+ synthesis, and TCA cycle intermediates are being actively studied in the clinic. Aberrant metabolism in pancreatic cancer poses a unique therapeutic strategy. We review preclinical and clinical studies looking to exploit alterations in the metabolic circuitry of pancreatic cancer.

A functional in vitro model of heterotypic interactions reveals a role for interferon-positive carcinoma associated fibroblasts in breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in breast cancer pathogenesis by paracrine regulation of breast cancer cell biology. Several in vitro and mouse models have characterized the role of cell contact and cytokine molecules mediating this relationship, although few reports have used human CAFs from breast tumors. METHODS: Primary breast CAF cultures were established and gene expression profiles analysed in order to guide subsequent co-culture models. We used a combination of colorimetric proliferation assays and gene expression profiling to determine the effect of CAFs on the MCF-7 breast cancer cell in an indirect co-culture system. RESULTS: Using gene expression profiling, we found that a subgroup of breast CAFs are positive for a type one interferon response, confirming previous reports of an activated type one interferon response in whole tumor datasets. Interferon positive breast cancer patients show a poor prognostic outcome in an independent microarray dataset. In addition, CAFs positive for the type one interferon response promoted the growth of the MCF-7 breast cancer cell line in an indirect co-culture model. The addition of a neutralizing antibody against the ligand mediating the type one response in fibroblasts, interferon-ß, reverted this co-culture phenotype. CAFs not expressing the interferon response genes also promoted the growth of the MCF-7 breast cancer cell line but this phenotype was independent of the type one fibroblast interferon ligand. CONCLUSIONS: Primary breast CAFs show inter-patient molecular heterogeneity as evidenced by interferon response gene elements activated in a subgroup of CAFs, which result in paracrine pro-proliferative effects in a breast cancer cell line co-culture model.

The effect of valproic acid in combination with irradiation and temozolomide on primary human glioblastoma cells.

Glioblastoma multiforme (GBM) has nearly uniformly fatal with a median survival of less than 2 years. While there have not been any novel anti-GBM therapeutics approved for many years, there has been the gradual accumulation of clinical data suggesting that the widely used anti-convulsant agent, valproic acid (VPA) may significantly prolong survival in GBM patients. This pre-clinical study aimed to determine the potential clinical utility of VPA in the treatment of GBM. Primary GBM cells were treated with VPA as a monotherapy and in combination with temozolomide and irradiation. At clinically achievable concentrations, VPA was shown to be effective as a monotherapy agent in the five primary lines tested. VPA was then used as a sensitizing agent to in vitro radiation and showed significant augmentation of in vitro irradiation therapy. In addition, when VPA, radiation and temozolomide were combined an additive, rather than synergistic effect was noted. Gene expression profiling demonstrated close clustering of triple treated cells with VPA mono-treated cells while untreated cells clustered closer with TMZ-irradiation dual treated cells. These microarray data suggest a dominant role of VPA at the gene expression level when combining these different treatment options. Moreover, in an in vivo tumor transplantation model, we were able to demonstrate an increase in animal survival when cells were pre-treated with irradiation-VPA and when triple treated. These findings provide a significant rationale for the investigation of VPA in the treatment of GBM patients.

Evaluating the repair of DNA derived from formalin-fixed paraffin-embedded tissues prior to genomic profiling by SNP-CGH analysis.

Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression alterations can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing high-level gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immunohistochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where fresh frozen material is scarce.

Translational advances in pancreatic ductal adenocarcinoma therapy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that is most frequently detected at advanced stages, limiting treatment options to systemic chemotherapy with modest clinical responses. Here, we review recent advances in targeted therapy and immunotherapy for treating subtypes of PDAC with diverse molecular alterations. We focus on the current preclinical and clinical evidence supporting the potential of these approaches and the promise of combinatorial regimens to improve the lives of patients with PDAC.

Cellular heterogeneity during mouse pancreatic ductal adenocarcinoma progression at single-cell resolution.

Pancreatic ductal adenocarcinoma (PDA) is a major cause of cancer-related death with limited therapeutic options available. This highlights the need for improved understanding of the biology of PDA progression, a highly complex and dynamic process featuring changes in cancer cells and stromal cells. A comprehensive characterization of PDA cancer cell and stromal cell heterogeneity during disease progression is lacking. In this study, we aimed to profile cell populations and understand their phenotypic changes during PDA progression. To that end, we employed single-cell RNA sequencing technology to agnostically profile cell heterogeneity during different stages of PDA progression in genetically engineered mouse models. Our data indicate that an epithelial-to-mesenchymal transition of cancer cells accompanies tumor progression in addition to distinct populations of macrophages with increasing inflammatory features. We also noted the existence of three distinct molecular subtypes of fibroblasts in the normal mouse pancreas, which ultimately gave rise to two distinct populations of fibroblasts in advanced PDA, supporting recent reports on intratumoral fibroblast heterogeneity. Our data also suggest that cancer cells and fibroblasts may be dynamically regulated by epigenetic mechanisms. This study systematically describes the landscape of cellular heterogeneity during the progression of PDA and has the potential to act as a resource in the development of therapeutic strategies against specific cell populations of the disease.

Targeting TGFßR2-mutant tumors exposes vulnerabilities to stromal TGFß blockade in pancreatic cancer.

TGFß is important during pancreatic ductal adenocarcinoma (PDA) progression. Canonical TGFß signaling suppresses epithelial pancreatic cancer cell proliferation; as a result, inhibiting TGFß has not been successful in PDA. In contrast, we demonstrate that inhibition of stromal TGFßR2 reduces IL-6 production from cancer-associated fibroblasts, resulting in a reduction of STAT3 activation in tumor cells and reversion of the immunosuppressive landscape. Up to 7% of human PDA have tumor cell-specific deficiency in canonical TGFß signaling via loss of TGFßR2. We demonstrate that in PDA that harbors epithelial loss of TGFßR2, inhibition of TGFß signaling is selective for stromal cells and results in a therapeutic benefit. Our study highlights the potential benefit of TGFß blockade in PDA and the importance of stratifying PDA patients who might benefit from such therapy.

Breast carcinoma-associated fibroblasts rarely contain p53 mutations or chromosomal aberrations.

It has become increasingly clear that the cells within the tumor microenvironment play a critical role in cancer growth and metastasis. Studies in experimental models suggest that carcinoma-associated fibroblasts (CAF) differ from normal fibroblasts and are capable of promoting cancer progression through a variety of mechanisms. At present, a definitive view is lacking on whether genomic abnormalities are present and whether they might underlie the observed phenotypic differences. This study reports the molecular analysis of the largest series of breast CAFs reported to date, with an array comparative genomic hybridization-based DNA copy number analysis of cultured CAFs derived from 25 freshly resected human breast cancers. We found DNA copy number changes consisting of the whole arm of chromosomes 6p and 9p plus interstitial 4q loss in only one sample. No abnormalities were observed in non-tumor-associated fibroblast counterparts. Karyotyping of the same CAF revealed further chromosomal abnormalities, which included clonal loss of chromosomes, chromosomal duplications, and less frequent chromosomal rearrangements. These abnormalities were not associated with alterations in the global gene expression profile of this particular CAF, relative to its non-tumor-associated fibroblast counterpart. Moreover, this particular patient's CAF also displayed the only p53 mutation in the cohort, the first time such a mutation has been reported in freshly cultured human CAFs. These findings argue that the procancerous effects of CAFs are unlikely to be due to DNA copy number-type genomic abnormalities in the CAFs themselves. As such, breast CAFs should be mainly regarded as genomically stable cellular constituents that exist within complex cancer microenvironments.