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Partial/complete pathologic response following neoadjuvant chemotherapy (NAC) in pancreatic cancer (PDAC) patients undergoing pancreatectomy is associated with improved survival. We sought to determine whether neutrophil-to-lymphocyte ratio (NLR) dynamics predict pathologic response following chemotherapy in PDAC, and if manipulating NLR impacts chemosensitivity in preclinical models and uncovers potential mechanistic underpinnings underlying these effects. Pathologic response in PDAC patients (n=94) undergoing NAC and pancreatectomy (7/2015-12/2019) was dichotomized as partial/complete or poor/absent. Bootstrap-validated multivariable models assessed associations between pre-chemotherapy NLR (%neutrophils÷%lymphocytes) or NLR dynamics during chemotherapy (ΔNLR = pre-surgery-pre-chemotherapy NLR) and pathologic response, disease-free survival (DFS), and overall survival (OS). To preclinically model effects of NLR attenuation on chemosensitivity, (PKT) mice and C57BL/6 mice orthotopically injected with (KPC) cells were randomized to vehicle, gemcitabine/paclitaxel alone, and NLR-attenuating anti-Ly6G with/without gemcitabine/paclitaxel treatment. In 94 PDAC patients undergoing NAC (median:4 months), pre-chemotherapy NLR (p<0.001) and ΔNLR attenuation during NAC (p=0.002) were independently associated with partial/complete pathologic response. An NLR score = pre-chemotherapy NLR+ΔNLR correlated with DFS (p=0.006) and OS (p=0.002). Upon preclinical modeling, combining NLR-attenuating anti-Ly6G treatment with gemcitabine/paclitaxel-compared with gemcitabine/paclitaxel or anti-Ly6G alone-not only significantly reduced tumor burden and metastatic outgrowth, but also augmented tumor-infiltrating CD107a -degranulating CD8 T-cells (p<0.01) while dampening inflammatory cancer-associated fibroblast (CAF) polarization (p=0.006) and chemoresistant IL-6/STAT-3 signaling in vivo. Neutrophil-derived IL-1β emerged as a novel mediator of stromal inflammation, inducing inflammatory CAF polarization and CAF-tumor cell IL-6/STAT-3 signaling in ex vivo co-cultures. Therapeutic strategies to mitigate neutrophil-CAF-tumor cell IL-1β/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC. Supported by KL2 career development grant by Miami CTSI under NIH Award UL1TR002736, Stanley Glaser Foundation, American College of Surgeons Franklin Martin Career Development Award, and Association for Academic Surgery Joel J. Roslyn Faculty Award (to J. Datta); NIH R01 CA161976 (to N.B. Merchant); and NCI/NIH Award P30CA240139 (to J. Datta and N.B. Merchant).

Pancreatic ductal adenocarcinoma (PDAC) is characterized by intratumoral abundance of neutrophilic/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) which exert anti-T-cell functions through multiple mechanisms, especially JAK2/STAT3-regulated arginase activity. To overcome limitations of systemic inhibition of PMN-MDSCs in cancer-bearing patients such as neutropenia and compensatory myelopoietic adaptations, we developed a nanoengineering strategy to disrupt cell-specific signaling exclusively in PMN-MDSCs without compromising viability or provoking neutropenia. Single-cell RNA sequencing in human/murine PDAC, and flow cytometry in peripheral blood cells from treatment-naïve PDAC patients, nominated surface receptor CXCR2 as a PMN-MDSC-exclusive target. We chemically modified a small-molecule CXCR2 inhibitor AZD5069 by conjugating it with polyethylene glycol (PEG) to enhance aqueous solubility. This AZD5069-PEG construct was then chemically grafted onto amphiphilic polysaccharide derivatives to engineer CXCR2-homing nanoparticles (CXCR2-NP). Cy5.5 dye-loaded CXCR2-NPs showed near-exclusive uptake in PMN-MDSCs compared with Kras LSL.G12D/ + ;p53 R172H/ + ;Pdx1 Cre/ + (KPC) PDAC tumor cells, KPC cancer-associated fibroblasts, macrophages, and dendritic cells. Encapsulation of JAK2/STAT3i Ruxolitinib (CXCR2-NP Ruxo ) resulted in more efficient and durable attenuation in STAT3-regulated arginase activity from PMN-MDSCs as well as robust induction of cytolytic T-cell activity compared with free Ruxolitinib in vitro and in orthotopic tumor cell-CAF co-injection models in vivo. Treatment of orthotopic tumor-bearing KPC mice with CXCR2-NP Ruxo reduced tumor burden compared with vehicle, free Ruxolitinib, or non-drug-loaded CXCR2-NP treatments, without causing appreciable neutropenia. Taken together, cell-specific delivery of payloads via CXCR2-homing immunonanoparticles represents a novel strategy to disrupt MDSC-mediated immunosuppression and invigorate antitumor immunity in PDAC.

Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS - TP53 co-alteration is associated with worse survival compared with either KRAS -alone or TP53 -alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non- TP53 mutated KRAS -altered tumors, KRAS - TP53 co-alteration engenders disproportionately innate immune-enriched and CD8 + T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS - TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS - TP53 co-altered and KRAS -altered/ TP53 WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS - TP53 co-altered PDAC. Immune subtyping of KRAS - TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS - TP53 co-altered “immunoregulatory program” predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.

BackgroundMajor pathologic response (MPR) following neoadjuvant therapy (NAT) in pancreatic ductal adenocarcinoma (PDAC) patients undergoing resection is associated with improved survival. We sought to determine whether racial disparities exist in MPR rates following NAT in patients with PDAC undergoing resection. MethodsPatients with potentially operable PDAC receiving at least 2 cycles of neoadjuvant FOLFIRINOX or gemcitabine/nab-paclitaxel ± radiation followed by pancreatectomy (2010-2019) at 7 high-volume centers were reviewed. Self-reported race was dichotomized as Black and non-Black, and multivariable models evaluated the association between race and MPR (i.e., pathologic complete response [pCR] or near-pCR). Cox regression evaluated the association between race and disease-free (DFS) and overall survival (OS). ResultsResults of 486 patients who underwent resection following NAT (mFOLFIRINOX 56%, gemcitabine/nab-paclitaxel 25%, radiation 29%), 67 (13.8%) patients were Black. Black patients had lower CA19-9 at diagnosis (median 67 vs. 204 U/mL; P = 0.003) and were more likely to undergo mild/moderate chemotherapy dose modification (40 vs. 20%; P = 0.005) versus non-Black patients. Black patients had significantly lower rates of MPR compared with non-Black patients (13.4 vs. 25.8%; P = 0.039). Black race was independently associated with worse MPR (OR 0.26, 95% confidence interval [CI] 0.10-0.69) while controlling for NAT duration, CA19-9 dynamics, and chemotherapy modifications. There was no significant difference in DFS or OS between Black and non-Black cohorts. ConclusionsBlack patients undergoing pancreatectomy appear less likely to experience MPR following NAT. The contribution of biologic and nonbiologic factors to reduced chemosensitivity in Black patients warrants further investigation.

Purpose Post-mastectomy breast reconstruction (PMBR) is an important component of breast cancer treatment, but disparities relative to insurance status persist despite legislation targeting the issue. We aimed to study this relationship in a large health system combining a safety-net hospital and a private academic center. Methods Data were collected on all patients who underwent mastectomy for breast cancer from 2011 to 2019 in a private academic center and an adjacent public safety-net hospital served by the same surgical teams. Multivariable logistic regression was used to assess the effect of insurance status on PMBR, controlling for covariates that included socioeconomic, demographic, and clinical factors. Results Of 1554 patients undergoing mastectomy for breast cancer, 753 (48.5%) underwent PMBR, of which 592 (79.9%) were privately insured, 50 (6.7%) Medicare, 68 (9.2%) Medicaid, and 31 (4.2%) uninsured. Multivariable logistic regression showed a significantly higher likelihood of not undergoing PMBR for uninsured (OR 6.0, 95% CI 3.7–9.8; p  < 0.0001), Medicare (OR 1.9, (95% CI 1.2–3.0; p  = 0.006), and Medicaid (OR 1.5, 95% CI 1.0–2.3; p  = 0.04) patients compared with privately insured patients. Age, stage, race and ethnicity, and hospital type confounded this relationship. Conclusion Patients without health insurance have dramatically reduced access to PMBR compared to those with private insurance. Expanding access to this important procedure is essential to achieve greater health equity for breast cancer patients.

BackgroundAlthough platinum-based chemotherapy is standard treatment for pancreatic ductal adenocarcinoma (PDAC) patients with either germline/somatic deficiencies in homologous recombination (mutHRD), a subset become platinum-resistant. We have shown that patients with mutHRD PDAC progressing on platinum chemotherapy respond to ICB. We sought to model these clinical findings in preclinical models to interrogate mechanisms that drive immune checkpoint blockade (ICB) sensitivity.MethodsmutHRD PDAC patients who received treatment with anti-PD-1+anti-CTLA4 ICB following resistance to platinum-based chemotherapy were selected. Cisplatin-sensitive and resistant Brca2-silenced (via shRNA knockdown) KPC cancer cells were generated and characterized in-vitro and in-vivo.ResultsIn 12 mutHRD PDAC patients treated with ICB after progression on platinum-based chemotherapy, duration of platinum exposure was associated with post-ICB disease response. Tumor-bearing syngeneic mice implanted with cisplatin-sensitive or resistant shBrca2 or wildtype KPC tumor cells treated with standard-of-care gemcitabine+cisplatin chemotherapy demonstrated expected rapid tumor growth of cisplatin-resistant shBrca2 tumors. However, we observed a profound reduction in tumor volumes of these chronically cisplatin-resistant KPC-shBrca2 tumors when subsequently treated with anti-PD-1+anti-CTLA4 ICB compared to wildtype-KPC or cisplatin-sensitive KPC-shBrca2 tumors. To understand mechanistic underpinnings of these novel observation, whole transcriptome sequencing revealed significant differential upregulation of type-1 interferon and cGAS-STING pathways in cisplatin-resistant KPC-shBrca2 compared to wildtype-KPC or cisplatin-sensitive KPC-shBrca2 controls. These transcriptomic changes manifested in a secretome enriched for T-cell trafficking cytokines CXCL10, CXCL9, and CCL5 from cisplatin-resistant KPC-shBrca2 cells. Indeed, cisplatin-resistant KPC-shBrca2 cells promoted increased transwell T-cell trafficking in-vitro as well as increased CD8+ T-cell infiltration in subcutaneous tumors in-vivo, compared with controls.ConclusionsChronicity of platinum exposure in mutHRD PDAC potentiates ICB sensitivity via induction of cell-autonomous type-1 interferon/cGAS-STING signaling and ensuing T-cell trafficking to the tumor microenvironment.AcknowledgementsI. Ogobuiro was supported by NIH/NCI T32 (to N. Merchant). This work was supported by KL2 career development grant by the Miami Clinical and Translational Science Institute (CTSI) under NIH Award UL1TR002736, American College of Surgeons Franklin H. Martin Research Fellowship, Association for Academic Surgery Joel J. Roslyn Faculty Award, Society of Surgical Oncology Young Investigator Award, and Elsa U. Pardee Foundation Award (to J. Datta). Research reported in this publication was supported by the NCI/NIH Award P30CA240139 to the Sylvester Comprehensive Cancer Center.

BACKGROUND AND AIMS: In vivo induction of alcoholic chronic pancreatitis (ACP) causes significant acinar damage, increased fibroinflammatory response, and heightened activation of cyclic response element binding protein 1 (CREB) when compared with alcohol (A) or chronic pancreatitis (CP) mediated pancreatic damage. However, the study elucidating the cooperative interaction between CREB and the oncogenic KrasG12D/+(Kras*) in promoting pancreatic cancer progression with ACP remains unexplored. METHODS: Experimental ACP induction was established in multiple mouse models, followed by euthanization of the animals at various time intervals during the recovery periods. Tumor latency was determined in these mice cohorts. Here, we established CREB deletion (Crebfl/fl) in Ptf1aCreERTM/+;LSL-KrasG12D+/-(KC) genetic mouse models (KCC-/-). Western blot, phosphokinase array, and qPCR were used to analyze the pancreata of Ptf1aCreERTM+/-, KC and KCC-/- mice. The pancreata of ACP-induced KC mice were subjected to single-cell RNA sequencing (scRNAseq). Further studies involved conducting lineage tracing and acinar cell explant cultures. RESULTS: ACP induction in KC mice had detrimental effects on the pancreatic damage repair mechanism. The persistent existence of acinar cell-derived ductal lesions demonstrated a prolonged state of hyperactivated CREB. Persistent CREB activation leads to acinar cell reprogramming and increased pro-fibrotic inflammation in KC mice. Acinar-specific Creb ablation reduced advanced PanINs lesions, hindered tumor progression, and restored acinar cell function in ACP-induced mouse models. CONCLUSIONS: Our findings demonstrate that CREB cooperates with Kras* to perpetuate an irreversible ADM and PanIN formation. Moreover, CREB sustains oncogenic activity to promote the progression of premalignant lesions toward cancer in the presence of ACP.Competing Interest StatementThe authors have declared no competing interest.

BackgroundA defining hallmark of immunosuppression in pancreatic ductal adenocarcinoma (PDAC) is the frequent trafficking of neutrophilic/granulocytic myeloid-derived suppressor cells (gMDSC) which exert their tolerogenic anti-T-cell functions through multiple mechanisms, particularly STAT3-mediated arginase-1 (Arg1) activity. Systemic inhibition of gMDSC trafficking and/or function (e.g., CXCR2, TGF-β, etc.) has been disappointing due to neutropenia, compensatory myelopoietic adaptations, and off-target effects. We sought to design a novel nanoengineering strategy to abrogate tolerogenic signaling in a gMDSC compartment-specific manner.MethodsWe chemically modified AZD5069—a small-molecule inhibitor of the gMDSC surface receptor CXCR2—by conjugating it with polyethylene glycol (PEG) to enhance aqueous solubility. This AZD5069-PEG construct was chemically grafted on an amphiphilic polysaccharide derivative to engineer AZD5069-decorated nanoparticles (NPCXCR2). We encapsulated hydrophobic STAT3i Ruxolitinib in NPCXCR2 nanoparticles and compared its effect on inhibition of Arg1 activity from gMDSCs and T-cell activation in-vitro and in-vivo.ResultsTo confirm CXCR2 as a gMDSC-specific target, we identified exclusive expression of CXCR2 in gMDSCs in human and murine PDAC via single-cell RNA sequencing and flow cytometric analysis in peripheral blood mononuclear cells derived from treatment-naïve PDAC patients (n=57). NPCXCR2 loaded with Cy5.5 dye showed dramatically higher uptake in gMDSC-like promyelocytic J774 cells compared with other PDAC-relevant cells in-vitro— KPC6694c2 tumor-cells, cancer-associated fibroblasts (CAF), and M0 macrophages RAW274.1. In in-vivo orthotopic K-rasLSL.G12D/+;p53R172H/+;Pdx1Cre/+ (KPC) tumor cell:CAF co-injection models, although Cy5.5 dye-loaded non-AZD5069 decorated NPCTL and NPCXCR2 both trafficked to tumor sites equally, NPCXCR2 but not NPCTL constructs preferentially concentrated in F4/80-Ly6G+ gMDSCs compared with F4/80+ macrophages, F4/80-Ly6C+ monocytic MDSCs, EpCAM+ tumor cells, PDPN+ CAFs, CD3+ T-cells, and Cd11c+ dendritic cells by flow cytometry. Encapsulation of STAT3i Ruxolitinib in NPCXCR2 and treatment of endogenous pSTAT3hi J774 cells in-vitro showed significantly more durable inhibition of pSTAT3 compared with Ruxolitinib drug treatment alone. Consequently, given that JAK2/STAT3 signaling is the major regulator of Arg1 activity in gMDSCs, Ruxolitinib-NPCXCR2 treatment of J774 cells significantly reduced gene expression and enzymatic activity of Arg1 compared with free Ruxolitinib treatment. Co-culture of splenocyte-derived murine CD8+ T-cells with J774 treated with Ruxolitinib-NPCXCR2 showed significant improvement in T-cell IFN-γ release compared with NPCTL or free Ruxolitinib-treated co-culture conditions. In orthotopic KPC tumor-bearing mice, intravenous delivery of NPCXCR2-encapsulated Ruxolitinib significantly augmented intratumoral trafficking of IFN-γ+CD107a+ CD8+ T-cells, compared with free Ruxolitinib treatment, without appreciable systemic neutropenia in-vivo.ConclusionsCell-specific delivery of payloads via CXCR2-homing nanoparticles represent a novel immunotherapeutic strategy to target tolerogenic signaling pathways in gMDSCs and invigorate antitumor immunity in PDAC.Ethics ApprovalAll animal experiments were performed in accordance with the NIH animal use guideline and protocol 21-176 approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Miami.

Background: Partial/complete pathologic response following neoadjuvant chemotherapy (NAC) in pancreatic cancer (PDAC) patients undergoing pancreatectomy is associated with improved survival. We sought to determine whether neutrophil-to-lymphocyte ratio (NLR) dynamics predict pathologic response following chemotherapy in PDAC, and if manipulating NLR impacts chemosensitivity in preclinical models and uncovers potential mechanistic underpinnings underlying these effects. Methods: Pathologic response in PDAC patients (n=94) undergoing NAC and pancreatectomy (7/2015-12/2019) was dichotomized as partial/complete or poor/absent. Bootstrap-validated multivariable models assessed associations between pre-chemotherapy NLR (%neutrophils÷%lymphocytes) or NLR dynamics during chemotherapy (ΔNLR=pre-surgery—pre-chemotherapy NLR) and pathologic response, disease-free survival (DFS), and overall survival (OS). To preclinically model effects of NLR attenuation on chemosensitivity, C57BL/6 mice (n=8-10/arm) were orthotopically injected with KrasG12D/+;Trp53fl/+;PdxCre(KPC) cells and 60 randomized to vehicle, NLR-attenuating anti-Ly6G, gemcitabine/paclitaxel, or gemcitabine/paclitaxel+anti-Ly6G treatments. Results: In 94 PDAC patients undergoing NAC (median:4 months), pre-chemotherapy NLR (P<0.001) and ΔNLR attenuation during NAC (P=0.002) were independently associated with partial/complete pathologic response. An NLR score=pre-chemotherapy NLR+ΔNLR correlated with DFS (P=0.006) and OS (P=0.002). Upon preclinical modeling, combining NLR-attenuating anti-Ly6G treatment with gemcitabine/paclitaxel—compared with gemcitabine/paclitaxel or anti-Ly6G alone—not only significantly reduced tumor burden and metastatic outgrowth, but also augmented tumor-infiltrating CD107a+-degranulating CD8+ T-cells (P<0.01) while dampening inflammatory cancer-associated fibroblast (CAF) polarization (P=0.006) and chemoresistant IL-6/STAT-3 signaling in vivo. Neutrophil-derived IL-1β emerged as a novel mediator of stromal inflammation, inducing inflammatory CAF polarization and CAF-tumor cell IL-6/STAT-3 signaling in ex vivo co-cultures. Conclusions: Therapeutic strategies to mitigate neutrophil-CAF-tumor cell IL-1β/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC. Competing Interest Statement The authors have declared no competing interest.