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It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEV). The tumor-supportive CAFs possess a distinct phenotypic profile, compared with normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5β1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Furthermore, we show that NetG1 expression in CAFs is required for the prosurvival properties of sEVs. In addition, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5β1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5β1 were detected in sEVs collected from plasma of patients with PDAC, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor–stroma interactions and pathogenic stroma detection. Significance: Results from this study identified two unique types of tumor-supporting CAF EVs, with evidence of these being detected in patients. Thus, this study facilitates a novel avenue to further dissect the subtleties of the tumor–stroma interactions responsible for PDAC homeostasis and progression, as well as the possibility of establishing future means to detect and monitor dynamic stroma staging.

Spread sheet including a single tab per Figure panel conveying each graph's statistical analyses. Note that tabs are named accordingly. The tabs include the comprehensive statistical analyses that were performed for each experiment in which statistical significance was given. Each tab contains the full statistical output for its labeled experiment. The statistical test performed for each experiment is identified within the corresponding tab. Statistical tests were carried out using the software, Prism, version 7.05.

Spreadsheet including the metabolomic analyses corresponding to the experiments indicated by the naming of the assorted tabs. Specific analysis parameters for each experiment are provided within the respective main figure legends.

Supplementary Figure 1. Confirmation that CAFs produce ECMs distinct from NLFs. Supplementary Figure 2. Direct co-culture of CAFs support PDAC cells during nutrient-deprivation. Supplementary Figure 3. Additional CAF cell lines generate sEVs that rescue PDAC cells from nutrient deprivation-induced apoptosis. Supplementary Figure 4. NetG1+ CAF-sEVs support PDAC cell survival in a NGL-1 dependent manner. Supplementary Figure 5. NetG1 expression in CAFs is necessary for sEV-mediated survival of nutrient-deprived PDAC cells. Supplementary Figure 6. NetG1 ablation does not affect the uptake of sEV cargo in PDAC cells. Supplementary Figure 7. sEV supernatant contains DNPs enriched with sub-exosome sized EVs. Supplementary Figure 8. Enriched gene ontology clusters from proteomic and metabolomic analysis comparing sEV and DNP fractions. Supplementary Figure 9. EV cargo requires transfer in intact vesicles to provide tumor-supportive effect.

Spreadsheet including the proteomic analyses corresponding to the experiments indicated by the naming of the assorted tabs. Specific analysis parameters for each experiment are provided within the respective main figure legends.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEV). The tumor-supportive CAFs possess a distinct phenotypic profile, compared with normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5β1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Furthermore, we show that NetG1 expression in CAFs is required for the prosurvival properties of sEVs. In addition, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5β1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5β1 were detected in sEVs collected from plasma of patients with PDAC, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor–stroma interactions and pathogenic stroma detection.Significance:Results from this study identified two unique types of tumor-supporting CAF EVs, with evidence of these being detected in patients. Thus, this study facilitates a novel avenue to further dissect the subtleties of the tumor–stroma interactions responsible for PDAC homeostasis and progression, as well as the possibility of establishing future means to detect and monitor dynamic stroma staging.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α(5)β(1) localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1(+) CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α(5)β(1) being enriched in exosomes. Finally, we found that NetG1 and Integrin α(5)β(1) were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection.

It is projected that, in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). This fibrous stroma, known as desmoplasia, causes the collapse of local blood vessels rendering a nutrient-deprived milieu. Hence, PDAC cells are nurtured by local CAF-secreted products, which include, among others, CAF-generated small extracellular vesicles (sEVs). It is well-accepted that upon culturing functionally tumor-promoting CAFs under pathophysiological-relevant conditions (e.g., within self-produced ECM), these cells express NetrinG1 (NetG1) and sustain endosomal pools rich in active α5β1-integrin; traits indicative of poor patient survival. We herein report that NetG1+ CAFs generate sEVs that rescue PDAC cells from nutrient-deprived induced apoptosis. Two unique sEVs, NetG1+ and α5β1-integrin+, were uncovered. The former constitutes cargo of CAF-generated exomeres, and the latter is detected in classic exosomes. Proteomic and metabolomic analyses showed that the sEV-dependent PDAC survival is, at least in part, dictated by the cargo packaged within sEVs in a NetG1-dependent manner. Indeed, despite producing a similar number of vesicles, selected key proteins and metabolites (e.g., glutamine) were incorporated within the unique sEVs. Finally, we found that NetG1 and α5β1-integrin were detected in sEVs collected from plasma of PDAC patients, while their concomitant levels were significantly lower in plasma of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF sEVs opens a new investigative avenue in tumor-stroma interactions and stroma staging detection.

Dendritic cell (DC) lectins mediate the recognition, uptake, and processing of antigens, but they can also be coopted by pathogens for infection. These distinct activities depend upon the routing of antigens within the cell. Antigens directed to endosomal compartments are degraded, and the peptides are presented on major histocompatibility complex class II molecules, thereby promoting immunity. Alternatively, HIV-1 can avoid degradation, as virus engagement with C-type lectin receptors (CLRs), such as DC-SIGN (DC-specific ICAM-3–grabbing nonintegrin) results in trafficking to surface-accessible invaginated pockets. This process appears to enable infection of T cells in trans . We sought to explore whether antigen fate upon CLR-mediated internalization was affected by antigen physical properties. To this end, we employed the ring-opening metathesis polymerization to generate glycopolymers that each display multiple copies of mannoside ligand for DC-SIGN, yet differ in length and size. The rate and extent of glycopolymer internalization depended upon polymer structure—longer polymers were internalized more rapidly and more efficiently than were shorter polymers. The trafficking, however, did not differ, and both short and longer polymers colocalized with transferrin-labeled early endosomes. To explore how DC-SIGN directs larger particles, such as pathogens, we induced aggregation of the polymers to access particulate antigens. Strikingly, these particulate antigens were diverted to the invaginated pockets that harbor HIV-1. Thus, antigen structure has a dramatic effect on DC-SIGN–mediated uptake and trafficking. These findings have consequences for the design of synthetic vaccines. Additionally, the results suggest strategies for targeting DC reservoirs that harbor viral pathogens.

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Pancreatic Ductal Adenocarcinoma (PDAC) has a five-year survival under 10%. Treatment is compromised due to a fibrotic-like stromal remodeling process, known as desmoplasia, which limits therapeutic perfusion, supports tumor progression, and establishes an immunosuppressive microenvironment. These processes are driven by cancer-associated fibroblasts (CAFs), functionally activated through transforming growth factor beta1 (TGFβ1). CAFs produce a topographically aligned extracellular matrix (ECM) that correlates with reduced overall survival. Paradoxically, ablation of CAF populations results in a more aggressive disease, suggesting CAFs can also restrain PDAC progression. Thus, unraveling the mechanism(s) underlying CAF functions could lead to therapies that reinstate the tumor-suppressive features of the pancreatic stroma. CAF activation involves the f-actin organizing protein palladin. CAFs express two palladin isoforms (iso3 and iso4) which are up-regulated in response to TGFβ1. However, the roles of iso3 and iso4 in CAF functions remain elusive. Using a CAF-derived ECM model, we uncovered that iso3/iso4 are required to sustain TGFβ1-dependent CAF activation, secrete immunosuppressive cytokines, and produce a pro-tumoral ECM. Findings demonstrate a novel role for CAF palladin and suggest that iso3/iso4 regulate both redundant and specific tumor-supportive desmoplastic functions. This study highlights the therapeutic potential of targeting CAFs to restore fibroblastic anti-tumor activity in the pancreatic microenvironment.

Short, strong hydrogen bonds (SSHBs) have been a source of interest and considerable speculation over recent years, culminating with those where hydrogen resides around the midpoint between the donor and acceptor atoms, leading to quasi-covalent nature. We demonstrate that X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy provide deep insight into the electronic structure of the short OHN hydrogen bond of 3,5-pyridinedicarboxylic acid, revealing for the first time distinctive spectroscopic identifiers for these quasi-symmetrical hydrogen bonds. An intermediate nitrogen (core level) chemical shift occurs for the almost centrally located hydrogen compared to protonated (ionic) and non-ionic analogues, and it reveals the absence of two-site disorder. This type of bonding is also evident through broadening of the nitrogen 1s photoemission and 1s → 1π* peaks in XPS and NEXAFS, respectively, arising from the femtosecond lifetimes of hydrogen in the potential wells slightly offset to either side of the centre. The line-shape of the core level excitations are thus related to the population occupancies, reflecting the temperature-dependent shape of the hydrogen potential energy well. Both XPS and NEXAFS provide a distinctive identifier for these quasi-symmetrical hydrogen bonds, paving the way for detailed studies into their prevalence and potentially unique physical and chemical properties.

Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide to metabolically “parasitic” PDAC cells with water-soluble metabolites such as glucose and amino acids, or bulk protein via micropinocytosis. To meet the elevated demands for cellular membrane lipids, cancer cells rely on uptake of the exogenous lipids. However, the mechanism by which PDAC cells obtain water-insoluble essential lipids, such as cholesterol, remains poorly understood. Here, we define CAFs as a main source of lipids for PDAC cells by direct “feeding” of the CAF plasma membrane (PM) to cancer cells via heterotypic cellular contacts, a phenomenon known as trogocytosis. To gain insights into the mechanisms of regulation of CAF trogocytosis, we engineered cholesterol-auxotrophic human and mouse PDAC cells. In the absence of exogenously provided cholesterol, these cancer cells undergo apoptosis, which is completely rescued in CAF co-cultures. Using CRISPRi-mediated depletion in CAFs of select genes involved in cholesterol trafficking, membrane fusion and membrane protrusions, we found that CAFs deficient in CD81, TMEM16F, or ARF6 exhibited markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing phosphatidylserine (PtdSer) on the outer leaflet of the PM. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures. Our overall model is that, to initiate trogocytosis, PDAC cells release paracrine “feed me” signals activating Ca2+-dependent phospholipid scramblase TMEM16F. As the result, increased outward expression of PtdSer on CAF PM is a hallmark “eat me” signal that is recognized by the trogocytic PDAC cells. We conclude that trogocytosis is the critical metabolic dependency in PDAC, and nominated TMEM16F as a plausible PDAC therapy target. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Ralph Francescone, Debora B. Barbosa Vendramini-Costa, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer-associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-015.

Radiation, combined with radio-sensitizing chemotherapy, is used preoperatively as neoadjuvant therapy (NT) for borderline resectable pancreatic ductal adenocarcinoma (PDAC) with the intent of facilitating a curative surgical intervention. PDAC is uniquely characterized by an extensive fibrous microenvironment, desmoplasia, which can unintendedly be aggravated by NT to foster its pro-tumoral function. Radiation, as part of NT, is aimed at providing a margin adjacent to un-resectable vessels and sterilizing regional lymph nodes. Yet, to avoid the unintended desmoplastic aggravation, and because of a risk of toxicities associated with high doses due to the radio-sensitivity of adjacent small bowel and stomach, the total dose of NT radiation delivered to PDAC patients is classically modest. Therefore, NT radiation in PDAC patients is often lower than optimal for effective tumor cell elimination. Pulsed low-dose-rate (PLDR) radiation improves the safety of radiation treatment as it allows time for DNA damage repair in non-tumorous cells/tissues while simultaneously remaining as effective as continuous dose rate (CDR) radiation in cancer cells. In theory, PLDR could be amendable for increased radiation dosage. Of interest, the use of PLDR in pre-clinical animal studies revealed a systemic lowering of transforming growth factor beta (TGFβ), a known immunosuppressive factor. Hence, we posit that since cancer-associated fibroblasts (CAFs) produce TGFβ and constitute one of the most abundant cells in PDAC desmoplasia, variations in TGFβ levels in response to NT will inform on desmoplastic dynamic changes. Our team has developed means to assess CAF functions and activation statuses. These efforts were guided by generating a desmoplastic biomarker signature obtained from patient-harvested CAFs during the production of extracellular matrix (ECM), using our well-established in vitro 3D system. We employed this in vivo-mimetic system to test the hypothesis that PLDR limits pro-tumoral desmoplastic CAF aggravation. As part of the CAF functional signature, we tested levels of immunosuppressive TGFβ secretion, expression of netrin-G1, palladin, and others; together with the biogenesis of unique extracellular vesicles, and the production of ECMs capable of nurturing PDAC cells under starvation. During a 5-day assay, human PDAC 3D ECM producing CAFs were treated with gemcitabine (Gem; 5nm) plus 4Gy or 8Gy PLDR vs. CDR. Results indicated that Gem alone or with CDR indeed aggravated CAF’s pro-PDAC phenotype and function, while Gem combined with PDLR limited and sometimes reverted this functional pro-tumor CAF signature. Based on these results, we are poised to test this functional CAF/desmoplasia signature, as laboratory correlatives, in an ongoing clinical trial at Fox Chase Cancer Center. The phase I trial is a dose-escalation study of PLDR radiation and chemotherapy at standard and intensified doses in preoperative pancreatic cancer patients with toxicity, histopathologic, and CAF-informing translational endpoints. Citation Format: Janusz Franco-Barraza, Tiffany Luong, Jessica K. Wong, Debora B. Vendramini-Costa, Ralph Francescone, Jaye C. Gardiner, Kristopher S. Raghavan, Joshua E. Meyer, Edna Cukierman. Pulsed low-dose-rate radiation (PLDR) limits pancreatic pro-tumor stroma aggravation: Pre-clinical basis for an ongoing PLDR trail [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-006.

Dendritic cell (DC) lectins mediate the recognition, uptake, and processing of antigens, but they can also be co-opted by pathogens for infection. These distinct activities depend upon the routing of antigens within the cell. Antigens directed to endosomal compartments are degraded and the peptides presented on MHC class II molecules thereby promoting immunity. Alternatively, HIV-1 can avoid degradation, as virus engagement with C-type lectin receptors (CLRs), such as DC-SIGN, results in trafficking to surface-accessible invaginated pockets. This process appears to enable infection of T cells intrans. We sought to explore whether antigen fate upon CLR-mediated internalization was affected by antigen physical properties. To this end, we employed the ring-opening metathesis polymerization to generate glycopolymers that each display multiple copies of mannoside ligand for DC-SIGN yet differ in length and size. The rate and extent of glycopolymer internalization depended upon polymer structure—longer polymers were internalized more rapidly and more efficiently than were shorter polymers. The trafficking, however, did not differ, and both short and longer polymers colocalized with transferrin-labeled early endosomes. To explore how DC-SIGN directs larger particles, such as pathogens, we induced aggregation of the polymers to access particulate antigens. Strikingly, these particulate antigens were diverted to the invaginated pockets that harbor HIV-1. Thus, antigen structure has a dramatic effect on DC-SIGN-mediated uptake and trafficking. These findings have consequences for the design of synthetic vaccines. Additionally, the results suggest new strategies for targeting DC reservoirs that harbor viral pathogens.Significance StatementDendritic cells (DCs) express cell-surface proteins (lectins) that bind to carbohydrates displayed on the surface of pathogens. The binding of pathogens to these lectins results in internalization to endosomal compartments where the pathogens are destroyed and an immune response is initiated. HIV-1 can subvert this process – lectin engagement routes HIV-1 to cellular compartments that allow the virus to evade destruction. We synthesized glycopolymers to test whether the size and physical properties of the antigens impact trafficking. Small polymers trafficked to endosomes, as expected. Alternatively, large particulate polymers localized in the non-endosomal compartments occupied by HIV. These data indicate that antigen structure affects routing by DC lectins. Our findings can be exploited to direct cargo to different compartments.

The extracellular matrix is a dynamic modulator of cellular function. Thus, to appropriately study matrix-induced cellular responses, cells' natural phenotypes and functions (in vivo) must be retained when cultured outside of the organism (in vitro). To this end, cell-derived matrices, known as CDMs, have become a valuable culturing system that enables researchers to elucidate numerous essential biological processes and multicellular functions regulated by cell–matrix interactions. This chapter highlights the historical perspective on the importance of the tumor microenvironment and focuses on the use of fibroblastic CDMs as a powerful culturing system for both basic and translational studies.