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Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

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Supplementary Figures 1-4

Table S2

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

Supplementary Methods

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to most therapies, including immune checkpoint blockade. To elucidate mechanisms of immunotherapy resistance, we assessed immune parameters in resected human PDA. We demonstrate significant interpatient variability in T-cell number, localization, and phenotype. CD8+ T cells, Foxp3+ regulatory T cells, and PD-1+ and PD-L1+ cells were preferentially enriched in tertiary lymphoid structures that were found in most tumors compared with stroma and tumor cell nests. Tumors containing more CD8+ T cells also had increased granulocytes, CD163+ (M2 immunosuppressive phenotype) macrophages, and FOXP3+ regulatory T cells. PD-L1 was rare on tumor cells, but was expressed by CD163+ macrophages and an additional stromal cell subset commonly found clustered together adjacent to tumor epithelium. The majority of tumoral CD8+ T cells did not express molecules suggestive of recent T-cell receptor (TCR) signaling. However, 41BB+PD-1+ T cells were still significantly enriched in tumors compared with circulation. Tumoral CD8+PD-1+ T cells commonly expressed additional inhibitory receptors, yet were mostly T-BEThi and EOMESlo, consistent with a less terminally exhausted state. Analysis of gene expression and rearranged TCR genes by deep sequencing suggested most patients have a limited tumor-reactive T-cell response. Multiplex immunohistochemistry revealed variable T-cell infiltration based on abundance and location, which may result in different mechanisms of immunotherapy resistance. Overall, the data support the need for therapies that either induce endogenous, or provide engineered, tumor-specific T-cell responses, and concurrently relieve suppressive mechanisms operative at the tumor site. Cancer Immunol Res; 5(11); 978–91. ©2017 AACR.

S1. TLS composition and immunological features of resected PDA. S2. Gating strategy for analyzing CD244 (2B4) and Tim3 expression on CD8 T cells in blood of normal individuals (Nl), in blood of PDA patients (PDA blood) and in tumors. S3. Representative human PDA section stained with the multiplex immunohistochemical panel. S4. Representative images of human PDA from the multiplex without (top row, A and B) and with (bottom, C and D), the stromal vs. tumor regions demarcated and cell numbers quantified. S5. PD-L1+ stromal cells in PDA include CD163+ macrophages (Figure 6A) as well as an additional CD163- cell subset shown here. Supplementary Table 1. Antibodies used for tissue multiplex in Figure 6. Supplementary Table 2. Patient characteristics. Supplementary Table 3. TCRβ CDR3 sequence data in blood and tumors from PDA patients.

Col6A1 and Lcn2 promote the migration of PDA cells.

1) Supplementary Table 1. Summary of imaging assessment. 2) Supplementary Table 2. Efficacy results by HA content and dose level of PEGPH20. 3) Supplementary Figure 1. Change from baseline in serum CA19-9 concentration for treated patients. 4) Supplementary Figure 2. Representative DCE-MRI images of 2 patients with liver metastases prior to and 24 hours following treatment with intravenous PEGPH20.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related death in the United States, and its incidence is on the rise. Advanced disease is nearly uniformly lethal, emphasizing the need to identify PDA at its earliest stages. To discover early biomarkers of PDA, we evaluated the circulating proteome in murine preinvasive and invasive plasma samples and human prediagnostic and diagnostic samples.Experimental Design: Using a customized antibody microarray platform containing >4,000 features, we interrogated plasma samples spanning preinvasive and invasive disease from a highly faithful mouse model of PDA. In parallel, we mined prediagnostic plasma from women in the Women's Health Initiative (WHI) who would later succumb to PDA together with matched, cancer-free control samples. Samples collected after an establishing diagnosis of PDA were also interrogated to further validate markers.Results: We identified ERBB2 and TNC in our cross-species analyses, and multiple antibodies identified ESR1 in prediagnostic plasma from people that succumb to PDA. This 3-marker panel had an AUC of 0.86 (95% confidence interval [CI], 0.76–0.96) for the diagnostic cohort that increased to 0.97 (95% CI, 0.92–1.0) with CA19-9 included. The 3-marker panel also had an AUC of 0.68 (95% CI, 0.58–0.77) for the prediagnostic cohort.Conclusions: We identified potential disease detection markers in plasma up to 4 years before death from PDA with superior performance to CA19-9. These markers might be especially useful in high-risk cohorts to diagnose early, resectable disease, particularly in patients that do not produce CA19-9. Clin Cancer Res; 21(7); 1764–71. ©2015 AACR.

KPC and KPTC Secretomes

Purpose: This phase Ib study evaluated the safety and tolerability of PEGylated human recombinant hyaluronidase (PEGPH20) in combination with gemcitabine (Gem), and established a phase II dose for patients with untreated stage IV metastatic pancreatic ductal adenocarcinoma (PDA). Objective response rate and treatment efficacy using biomarker and imaging measurements were also evaluated.Experimental Design: Patients received escalating intravenous doses of PEGPH20 in combination with Gem using a standard 3+3 dose-escalation design. In cycle 1 (8 weeks), PEGPH20 was administrated twice weekly for 4 weeks, then once weekly for 3 weeks; Gem was administrated once weekly for 7 weeks, followed by 1 week off treatment. In each subsequent 4-week cycle, PEGPH20 and Gem were administered once weekly for 3 weeks, followed by 1 week off. Dexamethasone (8 mg) was given pre- and post-PEGPH20 administration. Several safety parameters were evaluated.Results: Twenty-eight patients were enrolled and received PEGPH20 at 1.0 (n = 4), 1.6 (n = 4), or 3.0 μg/kg (n = 20), respectively. The most common PEGPH20-related adverse events were musculoskeletal and extremity pain, peripheral edema, and fatigue. The incidence of thromboembolic events was 29%. Median progression-free survival (PFS) and overall survival (OS) rates were 5.0 and 6.6 months, respectively. In 17 patients evaluated for pretreatment tissue hyaluronan (HA) levels, median PFS and OS rates were 7.2 and 13.0 months for “high”-HA patients (n = 6), and 3.5 and 5.7 months for “low”-HA patients (n = 11), respectively.Conclusions: PEGPH20 in combination with Gem was well tolerated and may have therapeutic benefit in patients with advanced PDA, especially in those with high HA tumors. Clin Cancer Res; 22(12); 2848–54. ©2016 AACR.

Histologic features of liver metastases in KPTC, KPC, KTC and KC mice.

Purpose: TP53 and the TGFβ pathway are major mediators of pancreatic cancer metastasis. The mechanisms by which they cause hematogenous metastasis have not been fully explored.Experimental Design: KPC (LSL-KRASG12D/+;LSL-Trp53R172H/+; Ptf1aCre/+) mice were generated, and the frequency and morphology of organ-specific hematogenous metastases compared with that seen in KPTC and KTC littermates (Tgfbr2+/−). Key findings were validated in primary cells from each genotype and samples of human pancreatic cancer liver metastases.Results: The frequency of hematogenous metastasis in KPTC mice was significantly lower than for KPC mice (41% vs. 68%, P < 0.05), largely due to a reduction in liver metastases. No differences were found between KPC and KPTC lung metastases, whereas liver metastases in KPTC mice showed a profound extravasation deficiency characterized by sinusoidal growth and lack of desmoplastic stroma. Analogous findings were confirmed in liver samples from patients indicating their clinical relevance. Portal vein colonization as a direct mode of access to the liver was observed in both mice and humans. Secretome analyses of KPC cells revealed an abundance of secreted prometastatic mediators including Col6A1 and Lcn2 that promoted early steps of metastatic colonization. These mediators were overexpressed in primary tumors but not metastases, suggesting that the ability to colonize is, in part, developed within the primary site, a phenomenon we refer to as the “Cinderella effect.”Conclusions: These findings establish a novel paradigm for understanding pancreatic cancer metastasis and the observed clinical latencies of liver versus lung metastases specifically. Clin Cancer Res; 23(6); 1607–20. ©2016 AACR.

Characterization of lung metastases in KPC and KPTC mice.

Supplementary Tables S1-S3, Figures S1-S2. Supplementary Table S1: Candidate biomarkers for KPC preinvasive and invasive plasma drawn between 8-10 weeks and midway through disease progression, respectively. Supplementary Table S2: Candidate down-regulated plasma biomarkers from analysis of WHI pre-diagnostic plasma antibody microarray interrogation. Supplementary Table S3: A comparison of receiver operator characteristic (ROC) area under the curve values for the 3-marker panel of ESR1, ERBB2 and TNC in pre-diagnostic plasma to those for previously reported serum and plasma biomarkers discovered in diagnostic plasma samples. Supplementary Figure S1: Plasma M-values for TNC, ERBB2 and ESR1 do not show significant change as the time between blood draw and death decreases. Supplementary Figure S2: Dot plot of individual case and control diagnostic plasma composite values for (A) 3 marker panel (TNC, ERBB2 and ESR1) and (B) 3 marker panel plus CA19-9. Mean and standard deviations are indicated.

Patterns of Metastatic Spread by Genotype

Genotyping of Mice and Features of PanINs.

Supplementary Figure 1 from Dynamics of the Immune Reaction to Pancreatic Cancer from Inception to Invasion

Supplementary Figure Legends. Legends for Supplementary Figures S1-S2.

Supplementary Figure Legends - PDF file 90K, Figure legends for Supplementary Material

Supplementary Table S2. Estimated regression coefficients from equation 2.

Figure S2 - PDF file 2023K, IHC images for HIF-1� and SHH in patient matched tumor TMA cores

Figures S3 to S7 and Tables S1 and S2 - PDF file 532K, S3 Kaplan-Meier patient survival analyses reveals no correlation between survival and PTCH levels. S4 Survival plots based on the univariate table S5 Increased expression of SHH during hypoxia in pancreatic cancer cell lines. S6 A HIF1-�- and SHH-dependent activation of hedgehog signaling in fibroblasts grown in 3D co-cultures with Panc-1 cells. S7, SHH formation by pancreatic cancer cells in hypoxia is mediating the desmoplastic reaction in fibroblasts Table S1 Univariate table showing that high histologic grade Table S2 A pathway array profiling the expression of genes involved in hedgehog signaling in MiaPaca 2 cells transfected with shHIF-1� in normoxia and hypoxia

Supplementary Figure S1. A multispectral image of an in vivo murine subcutaneous pancreatic tumor treated with pHIFU with varying levels of peak-rarefactional pressure.

The dynamics of cancer immunosurveillance remain incompletely understood, hampering efforts to develop immunotherapy of cancer. We evaluated the evolving in vivo immune response to a spontaneous tumor in a genetically defined mouse model of pancreatic ductal adenocarcinoma from the inception of preinvasive disease to invasive cancer. We observed a prominent leukocytic infiltration even around the lowest grade preinvasive lesions, but immunosuppressive cells, including tumor-associated macrophages, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg), dominated the early response and persisted through invasive cancer. Effector T cells, however, were scarce in preinvasive lesions, found in only a subset of advanced cancers, and showed no evidence of activation. The lack of tumor-infiltrating effector T cells strongly correlated with the presence of intratumoral MDSC with a near mutual exclusion. In vitro, we found that MDSC suppressed T-cell proliferation. Overall, our results show that suppressive cells of the host immune system appear early during pancreatic tumorigenesis, preceding and outweighing antitumor cellular immunity, and likely contribute to disease progression. Thus, in contrast to the hypothesis that an early “elimination phase” of cancer immunosurveillance is eventually overwhelmed by a growing invasive tumor, our findings suggest that productive tumor immunity may be undermined from the start. Efforts to test potent inhibitors of MDSC, tumor-associated macrophages, and Treg, particularly early in the disease represent important next steps for developing novel immunotherapy of cancer. [Cancer Res 2007;67(19):9518–27]

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.

Supplementary Figure 2 from Dynamics of the Immune Reaction to Pancreatic Cancer from Inception to Invasion

Supplementary Table 2 from Dynamics of the Immune Reaction to Pancreatic Cancer from Inception to Invasion

Supplementary Table 1 from Dynamics of the Immune Reaction to Pancreatic Cancer from Inception to Invasion

Pancreatic cancer is characterized by extensive stromal desmoplasia, which decreases blood perfusion and impedes chemotherapy delivery. Breaking the stromal barrier could both increase perfusion and permeabilize the tumor, enhancing chemotherapy penetration. Mechanical disruption of the stroma can be achieved using ultrasound-induced bubble activity—cavitation. Cavitation is also known to result in microstreaming and could have the added benefit of actively enhancing diffusion into the tumors. Here, we report the ability to enhance chemotherapeutic drug doxorubicin penetration using ultrasound-induced cavitation in a genetically engineered mouse model (KPC mouse) of pancreatic ductal adenocarcinoma. To induce localized inertial cavitation in pancreatic tumors, pulsed high-intensity focused ultrasound (pHIFU) was used either during or before doxorubicin administration to elucidate the mechanisms of enhanced drug delivery (active vs. passive drug diffusion). For both types, the pHIFU exposures that were associated with high cavitation activity resulted in disruption of the highly fibrotic stromal matrix and enhanced the normalized doxorubicin concentration by up to 4.5-fold compared with controls. Furthermore, normalized doxorubicin concentration was associated with the cavitation metrics (P < 0.01), indicating that high and sustained cavitation results in increased chemotherapy penetration. No significant difference between the outcomes of the two types, that is, doxorubicin infusion during or after pHIFU treatment, was observed, suggesting that passive diffusion into previously permeabilized tissue is the major mechanism for the increase in drug concentration. Together, the data indicate that pHIFU treatment of pancreatic tumors when resulting in high and sustained cavitation can efficiently enhance chemotherapy delivery to pancreatic tumors. Cancer Res; 75(18); 3738–46. ©2015 AACR.

Cancer-associated fibroblast (CAF) heterogeneity is increasingly appreciated, but the origins and functions of distinct CAF subtypes remain poorly understood. The abundant and transcriptionally diverse CAF population in pancreatic ductal adenocarcinoma (PDAC) is thought to arise from a common cell of origin, pancreatic stellate cells (PSC), with diversification resulting from cytokine and growth factor gradients within the tumor microenvironment. Here we analyzed the differentiation and function of PSCs during tumor progression in vivo. Contrary to expectations, we found that PSCs give rise to a numerically minor subset of PDAC CAFs. Targeted ablation of PSC-derived CAFs within their host tissue revealed nonredundant functions for this defined CAF population in shaping the PDAC microenvironment, including production of specific extracellular matrix components and tissue stiffness regulation. Together, these findings link stromal evolution from distinct cells of origin to transcriptional heterogeneity among PDAC CAFs and demonstrate unique functions for CAFs of a defined cellular origin. Significance: By tracking and ablating a specific CAF population, we find that a numerically minor CAF subtype from a defined cell of origin plays unique roles in establishing the pancreatic tumor microenvironment. Together with prior studies, this work suggests that mesenchymal lineage heterogeneity and signaling gradients diversify PDAC CAFs. See related commentary by Cukierman, p. 296. This article is highlighted in the In This Issue feature, p. 275

BackgroundAchieving robust responses with adoptive cell therapy for the treatment of the highly lethal pancreatic ductal adenocarcinoma (PDA) has been elusive. We previously showed that T cells engineered to express a mesothelin-specific T cell receptor (TCRMsln) accumulate in autochthonous PDA, mediate therapeutic antitumor activity, but fail to eradicate tumors in part due to acquisition of a dysfunctional exhausted T cell state.MethodsHere, we investigated the role of immune checkpoints in mediating TCR engineered T cell dysfunction in a genetically engineered PDA mouse model. The fate of engineered T cells that were either deficient in PD-1, or transferred concurrent with antibodies blocking PD-L1 and/or additional immune checkpoints, were tracked to evaluate persistence, functionality, and antitumor activity at day 8 and day 28 post infusion. We performed RNAseq on engineered T cells isolated from tumors and compared differentially expressed genes to prototypical endogenous exhausted T cells.ResultsPD-L1 pathway blockade and/or simultaneous blockade of multiple coinhibitory receptors during adoptive cell therapy was insufficient to prevent engineered T cell dysfunction in autochthonous PDA yet resulted in subclinical activity in the lung, without enhancing anti-tumor immunity. Gene expression analysis revealed that ex vivo TCR engineered T cells markedly differed from in vivo primed endogenous effector T cells which can respond to immune checkpoint inhibitors. Early after transfer, intratumoral TCR engineered T cells acquired a similar molecular program to prototypical exhausted T cells that arise during chronic viral infection, but the molecular programs later diverged. Intratumoral engineered T cells exhibited decreased effector and cell cycle genes and were refractory to TCR signaling.ConclusionsAbrogation of PD-1 signaling is not sufficient to overcome TCR engineered T cell dysfunction in PDA. Our study suggests that contributions by both the differentiation pathways induced during the ex vivo T cell engineering process and intratumoral suppressive mechanisms render engineered T cells dysfunctional and resistant to rescue by blockade of immune checkpoints.

A defining characteristic of pancreatic ductal adenocarcinoma (PDA) is its robust desmoplastic reaction that promotes tumor progression and protects PDA from treatment. As a major component of the extreme microenvironment, hyaluronan (HA) is synthesized by the hyaluronan synthase (Has) family of enzymes and secreted in excess by both epithelial cells and cancer-associated fibroblasts (CAFs). We have previously described the role of HA in binding water and swelling to create vessel-crushing interstitial gel-fluid pressures and its function as a biophysical barrier. However, HA also acts by signaling through a wide range of specific receptors that can drive proliferation, migration and invasion. In an effort to understand the role of HA in PDA development and pathogenesis, we developed a KPC mouse model of autochthonous pancreas cancer in which Has2 is conditionally deleted in tumor epithelial cells (TECs). We demonstrate that Has2-deficient epithelial cells produce little to no HA and altered the dense microenvironment. We also found that deletion of Has2 leads to an increase in overall survival and a striking reduction in metastasis. We have further investigated the cell autonomous functions of Has2, associated with 3D invasion and activation of oncogenic signaling pathways in TECs. These data implicate a potential Achilles’ heel in PDA, whereby HAS2 is required to maintain a highly metastatic phenotype involving both non-cell and cell autonomous mechanisms. Citation Format: Bo Tu, Heather Wright T. Wright, Christopher C. Dufort, Martin C. Whittle, Libing Feng, Shelley M. Thorsen, Sunil R. Hingorani. Epithelial hyaluronan synthase 2 expression promotes pancreas cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A056.

The robust desmoplasia of PDA is its distinguishing feature and non-malignant components constitute the bulk of the tumor mass. This stromal reaction includes myofibroblasts, inflammatory fibroblasts, endothelial cells, pericytes, and various immune-cell subsets, all embedded within a dense and complex extracellular matrix (ECM). Dominated by non-malignant cells, the mutated epithelium must therefore combat, cooperate with, or coopt the surrounding cells and signaling processes in its microenvironment. It is proposed that an invasive PDA represents the coordinated evolution of malignant and non-malignant cells and mechanisms that subvert and repurpose the dictums of normal tissue composition, architecture, and physiology to foster tumorigenesis. The detailed kinetics and stepwise construction of the pancreas cancer neo-organ suggest that it is governed by a discrete set of organizing rules and principles – and repeated attempts to target its specific components reveal self-regulating mechanisms of resistance. Indeed, many of these cells arise and operate at the behest of oncogenic KRAS, the signature genetic mutation in PDA. These cells, in turn, support and shape the evolving malignancy. It is frequently debated in pancreas and other cancers whether the stroma is “good” or “bad”, tumor-constraining or tumor-promoting. However, because the stroma is neither static nor monolithic, the question is misguided. The stroma is more nuanced: some elements overtly accelerate, and others attenuate, disease progression but most of the cells and processes found in an evolving pancreas cancer are adaptive at some point during its development. In the end, these elements collectively conspire to create a drug- and immune-privileged sanctuary for PDA progression. To fully overcome the extraordinary resistance of this formidable cancer will require parsing the myriad interactions and interdependencies among the various stromal elements and the epithelial PDA cell. Understanding how to combine strategies against these distinct components and processes – which ones to target, in what order and for how long – will be critical, and perhaps necessary, for success and to avoid making matters worse. Citation Format: Sunil R. Hingorani. Stromal-epithelial co-evolution and complicity in pancreas cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA019.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

Bright long-wavelength-excitable (green and beyond) semiconducting polymer dots (LWE-Pdots) are highly desirable for in vivo fluorescent imaging, as well as applications of multiple- wavelength-excitation based in vitro bioassays. Conventional LWE-Pdots have been obtained previously by incorporating a small amount of near-infrared (NIR) emitter into the backbone of a polymer host to develop a binary donor-acceptor (D-A) system. They usually suffer from severe concentration quenching and a trade-off between fluorescence quantum yield (Φ(f)) and absorption cross-section (σ), limiting the improvement in brightness. In this communication, we describe a ternary component (D(1)/D(2)-A) strategy to achieve ultrabright, green laser (532 nm)-excitable Pdots with narrow-band NIR emission by introducing a BODIPY-based assistant polymer donor as D(1). The novel use of BODIPY as predominant absorber in a Pdot allows strong, narrow-band absorption at 532 nm. The D(1)/D(2)-A Pdots possess simultaneously improved Φ(f) and σ compared to corresponding binary D(2)-A Pdots. Their Φ(f) is as high as 40.2%, one of the most efficient NIR Pdots reported. The ternary D(1)/D(2)-A Pdots show ultrahigh single-particle brightness, ~83 times brighter than Qdot 705 when excited by a 532 nm laser. Intravenously injected Pdots accumulate in implanted tumors in mice through the enhanced permeability and retention effect. Higher contrast in vivo tumor imaging was achieved using the ultrabright ternary Pdots versus the binary D-A Pdots. What’s more, this D(1)/D(2)-A design strategy supplies us a promising way to pursue ultrabright Pdots with even longer absorption and emission.

PURPOSE Pegylated recombinant human hyaluronidase (PEGPH20) degrades hyaluronan (HA) and, in combination with chemotherapy, prolongs survival in preclinical models. The activity of PEGPH20 with modified fluorouracil, leucovorin, irinotecan, and oxaliplatin (mFOLFIRINOX) was evaluated in patients with metastatic pancreatic cancer (mPC). MATERIALS AND METHODS Patients had untreated mPC, a performance status of 0 to 1, and adequate organ function. Tumor HA status was not required for eligibility. After a phase Ib dose-finding study of mFOLFIRINOX plus PEGPH20, the phase II open-label study randomly assigned patients (1:1) to the combination arm or to mFOLFIRINOX alone (n = 138). The primary end point was overall survival (OS). RESULTS PEGPH20 dosages of 3 µg/kg every 2 weeks were more tolerable than twice-weekly dosages used in the phase I study, so 3 µg/kg every 2 weeks was the phase II dosage. An amendment instituted enoxaparin prophylaxis in the PEGPH20 combination arm as a result of increased thromboembolic (TE) events. The planned interim futility analysis when 35 deaths (of 103 analyzable patients) occurred resulted in an OS hazard ratio (HR) of 2.07 that favored the control arm, and the study was closed to accrual. The treatment-related grade 3 to 4 toxicity was significantly increased in the PEGPH20 combination arm relative to control (odds ratio, 2.7; 95% CI, 1.1 to 7.1). The median OS in the mFOLFIRINOX arm was 14.4 months (95% CI, 10.1 to 15.7 months) versus 7.7 months (95% CI, 4.6 to 9.3 months) in the PEGPH20 combination arm. CONCLUSION Addition of PEGPH20 to mFOLFIRINOX seems to be detrimental in patients unselected for tumor HA status. This combination caused increased toxicity (mostly GI and TE events) and resulted in decreased treatment duration compared with mFOLFIRINOX alone. The median OS in the mFOLFIRINOX control arm (14.4 months) is, to our knowledge, the longest yet reported and can be considered for patients with good PS.

Cancer-associated fibroblast (CAF) heterogeneity is increasingly appreciated, but the origins and functions of distinct CAF subtypes remain poorly understood. The abundant and transcriptionally diverse CAF population in pancreatic ductal adenocarcinoma (PDAC) is thought to arise from a common cell of origin, pancreatic stellate cells (PSCs), with diversification resulting from cytokine and growth factor gradients within the tumor microenvironment. Here we analyzed the differentiation and function of PSCs during tumor progression in vivo. Contrary to expectations, we found that PSCs give rise to a numerically minor subset of PDAC CAFs. Targeted ablation of PSC-derived CAFs within their host tissue revealed non-redundant functions for this defined CAF population in shaping the PDAC microenvironment, including production of specific components of the extracellular matrix. Together, these findings link stromal evolution from distinct cells of origin to transcriptional heterogeneity among PDAC CAFs, and demonstrate unique functions for CAFs of a defined cellular origin.Statement of significanceBy tracking and ablating a specific CAF population, we find that a numerically minor CAF subtype from a defined cell of origin plays unique roles in establishing the pancreatic tumor microenvironment. Together with prior studies, this work suggests that mesenchymal lineage heterogeneity as well as signaling gradients diversify PDAC CAFs.

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by Kras(G12D) expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1 enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests activation of TGFβ signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote basal type of PDAC conferring poor outcomes in patients.

PURPOSE To evaluate the efficacy and safety of pegvorhyaluronidase alfa (PEGPH20) plus nab-paclitaxel/gemcitabine (AG) in patients with hyaluronan-high metastatic pancreatic ductal adenocarcinoma (PDA). PATIENTS AND METHODS HALO 109-301 was a phase III, randomized, double-blind, placebo-controlled study. Patients ≥ 18 years of age with untreated, metastatic, hyaluronan-high PDA were randomly assigned 2:1 to PEGPH20 plus AG or placebo plus AG. Treatment was administered intravenously in 4-week cycles (3 weeks on, 1 week off) until progression or intolerable adverse events: PEGPH20 3.0 µg/kg twice per week for cycle 1 and once per week thereafter; nab-paclitaxel 125 mg/m2 once per week; and gemcitabine 1,000 mg/m2 once per week. The primary end point was overall survival (OS); secondary end points included progression-free survival (PFS), objective response rate (ORR), and safety. Response was independently assessed per RECIST v1.1. RESULTS At data cutoff, 494 patients were randomly assigned, with 492 (327 for PEGPH20 and 165 for placebo) included in intention-to-treat analyses. Baseline characteristics were balanced for PEGPH20 plus AG versus placebo plus AG. There were 330 deaths, with a median OS of 11.2 months for PEGPH20 plus AG versus 11.5 months for placebo plus AG (hazard ratio [HR], 1.00; 95% CI, 0.80 to 1.27; P = .97); median PFS was 7.1 months versus 7.1 months (HR, 0.97 [95% CI, 0.75 to 1.26]); ORR was 47% versus 36% (ORR ratio, 1.29 [95% CI, 1.03 to 1.63]). Grade ≥ 3 adverse events with a ≥ 2% higher rate with PEGPH20 plus AG than with placebo plus AG included fatigue (16.0% v 9.6%), muscle spasms (6.5% v 0.6%), and hyponatremia (8.0% v 3.8%). CONCLUSION The addition of PEGPH20 to AG increased the ORR but did not improve OS or PFS. The safety profile of PEGPH20 plus AG was consistent with that found in previous studies. These results do not support additional development of PEGPH20 in metastatic PDA.

Pancreatic ductal adenocarcinoma (PDA) is aggressive, highly metastatic and characterized by a robust desmoplasia. Connexin proteins that form gap junctions have been implicated in tumor suppression for over 30 years. Cx43, the most widely expressed connexin, regulates cell behaviors, including migration and proliferation. Thus, we hypothesized that Cx43 could regulate PDA progression. Phosphorylation of Cx43 by Casein Kinase 1 (CK1) regulates gap junction assembly. We interbred the well-established KrasLSL-G12D/+;p48Cre/+ (KC) mouse model of PDA with homozygous “knock-in” mutant Cx43 mice bearing amino acid substitution at CK1 sites (Cx43CK1A) and found profound and surprising effects on cancer progression. Crossing the Cx43CK1A mouse onto the KC background (termed KC;CxCK1A) led to significant extension of lifespan, from a median of 370 to 486 days (p = 0.03) and a decreased incidence of metastasis (p = 0.045). However, when we examined early stages of disease, we found more rapid onset of tissue remodeling in the KC;CxCK1A mouse followed by divergence to a cystic phenotype. During tumorigenesis, gap junctions are increasingly present in stromal cells of the KC mice but are absent from the KC;Cx43CK1A mice. Tail vein metastasis assays with cells derived from KC or KC;CxCK1A tumors showed that KC;CxCK1A cells could efficiently colonize the lung and downregulate Cx43 expression, arguing that inhibition of metastasis was not occurring at the distal site. Instead, stromal gap junctions, their associated signaling events or other unknown Cx43-dependent events facilitate metastatic capacity in the primary tumor.

576 Background: Stromal HA poses a physical barrier and protects tumor cells from immune surveillance. PEGPH20 is a pegylated, human recombinant PH20 hyaluronidase that remodels tumor stroma. Preclinical studies of PEGPH20 showed improved infiltration of cytotoxic T-lymphocytes and delivery of chemotherapy and PD1/PD-L1 antibodies. This study aimed to evaluate the efficacy, safety, and translational biomarkers of PEGPH20 plus pembrolizumab in pts with HA-high refractory mPC. Methods: mPC pts with HA-high expression, ECOG PS 0-1, ≤ 2 prior therapies for metastatic disease, life expectancy ≥ 12 weeks were treated with PEGPH20 3 µg/kg iv on D1, D8, D15 and pembrolizumab 200 mg iv on D1 in 21-day cycles. Primary endpoint was progression-free survival (PFS). Secondary endpoints were safety, overall survival (OS), and objective response rate (ORR). Blood and tumor biopsies were collected at baseline and on-study. Translational endpoints included flow cytometry and IHC for immune subsets, T-cell receptor sequencing, immune transcriptome, circulating cytokines, and plasma and tumor HA levels. Assuming a one-sided α-level of 0.05 and power of 80%, 31 evaluable pts were needed to detect an improvement of median PFS from 3 to 6 months. Results: Between May 2019 to Nov 2019, 38 pts were screened for HA expression, and 8 pts were enrolled, with median age 68 years (range 60-73), 7 males, and median 2 prior therapies (range 1-4). The accrual was stopped early by Halozyme Pharmaceuticals due to lack of benefit from PEGPH20 added to chemotherapy in the HALO-301 study. Treatment exposure median was 2 cycles (range 1-6). Reasons for study discontinuation were disease progression (n = 4), termination by sponsor (n = 3), patient withdrawal to enroll in hospice (n = 1). Treatment related toxicities were musculoskeletal (n = 6, grade 1/2), edema (n = 2, grade 1), fatigue (n = 1, grade 3), dyspnea (n = 1, grade 2), hypothyroidism (n = 1, grade 2). Median OS was 7.2 months (95% CI 1.2-11.8), and median PFS was 1.5 months (95% CI 0.9-4.4). Best response was stable disease (n = 2, 25%) lasting 2.2 and 9 months, respectively, and no responses were noted. Patients with available molecular sequencing data had MSS tumors. Translational biomarkers will be presented. Conclusions: Pembrolizumab and PEGPH20 did not increase PFS compared to historical data among heavily pretreated mPC pts, but the median OS of 7.2 months is encouraging. Translational analyses will provide insights into immune modulatory effects from PEGPH20 that could inform future studies with stroma targeted therapies and immune checkpoint blockade in mPC. Clinical trial information: NCT03634332.