RESUMEN
We have shown that KRAS-TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC. SIGNIFICANCE: By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell-excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer. This article is highlighted in the In This Issue feature, p. 1275.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neutrófilos , Receptores Tipo II del Factor de Necrosis Tumoral/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Inflamación/genética , Microambiente Tumoral/fisiología , Quimiocina CXCL1/genética , Neoplasias PancreáticasRESUMEN
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, Ptf1aCre/+; KrasLSL-G12D/+;Tgfbr2flox/flox (PKT) mice and C57BL/6 mice orthotopically injected with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1Cre(KPC) cells were randomized to vehicle, gemcitabine/paclitaxel alone, and NLR-attenuating anti-Ly6G with/without gemcitabine/paclitaxel treatment. 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-1ß 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-1ß/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC. Funding: 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).
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Linfocitos T CD8-positivos , Carcinoma Ductal Pancreático/patología , Fibroblastos/patología , Humanos , Interleucina-6 , Linfocitos/patología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/patología , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras) , Receptor Tipo II de Factor de Crecimiento Transformador beta , Neoplasias PancreáticasRESUMEN
Chondrosarcoma is a malignant bone neoplasm that is refractory to chemotherapy and radiation. With no current biological treatments, mutilating surgical resection is the only effective treatment. Proline rich polypeptide 1 (PRP1), which is a 15amino acid inhibitor of mammalian target of rapamycin complex1 (mTORC1), has been indicated to exert cytostatic and immunomodulatory properties in human chondrosarcoma cells in a monolayer. The aim of the present study was to evaluate the effects of PRP1 on an in vitro 3D chondrosarcoma tumor model, known as spheroids, and on the cancer stem cells (CSCs) which form spheroids. JJ012 cells were cultured and treated with PRP1. An ALDEFLUOR™ assay was conducted (with N,Ndiethylaminobenzaldehyde as the negative control) to assess aldehyde dehydrogenase (ALDH) activity (a recognized CSC marker), and bulk JJ012, ALDHhigh and PRP1 treated ALDHlow cells were sorted using flow cytometry. Colony formation and spheroid formation assays of cell fractions, including CSCs, were used to compare the PRP1treated groups with the control. CSCs were assessed for early apoptosis and cell death with a modified Annexin V/propidium iodide assay. Western blotting was used to identify mesenchymal stem cell markers (STRO1, CD44 and STAT3), and spheroid selfrenewal assays were also conducted. A clonogenic doseresponse assay demonstrated that 20 µg/ml PRP1 was the most effective dose for reducing colony formation capacity. Furthermore, CSC spheroid growth was significantly reduced with increasing doses of PRP1. Annexin V analysis demonstrated that PRP1 induced CSC cell death, and that this was not attributed to apoptosis or necrosis. Western blot analysis confirmed the expression of mesenchymal markers, and the spheroid selfrenewal assay confirmed the presence of selfrenewing CSCs. The results of the present study demonstrate that PRP1 eliminates anchorage independent CSC growth and spheroid formation, indicating that PRP1 likely inhibits tumor formation in a murine model. Additionally, a decrease in nonCSC bulk tumor cells indicates an advantageous decline in tumor stromal cells. These findings confirm that PRP1 inhibits CSC proliferation in a 3D tumor model which mimics the behavior of chondrosarcoma in vivo.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/metabolismo , Condrosarcoma/metabolismo , Células Madre Neoplásicas/citología , Antígenos de Superficie/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrosarcoma/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Humanos , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismoRESUMEN
Chondrosarcoma is the second most common primary malignant bone tumor and is resistant to chemotherapy and radiation. Inadequate treatment response and poor prognosis requires novel therapeutic approaches. Prolinerich polypeptide1 (PRP1), synthesized by brain neurosecretory cells, has demonstrated antitumor properties in JJ012cells; however, its underlying molecular mechanism remains unclear. The present study aimed to investigate the epigenetic regulation by which PRP1 inhibits chondrosarcoma cancer stem cell (CSC) proliferation and to elucidate additional CSC biomarkers in human chondrosarcoma other than ALDH1A1. Human chondrosarcoma JJ012cells were treated with PRP1 prior to performing an Aldefluor™ assay and fluorescenceactivated cell sorting in order to determine aldehyde dehydrogenase (ALDH) expression levels and isolate ALDHhigh and ALDHlow cell populations. ALDH is an established marker of CSCs in several neoplasms, including chondrosarcoma. The cells were collected and lysed for gel electrophoresis, followed by western blot analysis. The Aldefluor™ assay was used to assess the expression levels of wellestablished CSC biomarkers, including CD133, CD4, CD10, CD144, CD177, CD221, CD271, leucinerich repeatcontaining G proteincoupled receptor 5, SOX2 and B lymphoma MoMLV insertion region 1 homolog (BMI1), within the ALDHhigh population of JJ012 cells. The results confirmed that ALDHA1 was the biomarker for chondrosarcoma CSCs. PRP1 was demonstrated to inhibit the ALDHhigh population colony and sarcosphere formation; 5 µg/ml PRP1 was indicated to be the optimum concentration in eliminating colonies formed by JJ012 cells (92%, P<0.001) and by the ALDHhigh CSCpopulation (80.5%, P<0.001) in the clonogenic doseresponse assay. Spheroid growth unequivocally decreased with an increase in PRP1 dose. In order to determine the molecular mechanism by which PRP1 decreased the CSC population, the regulation of the mammalian Switch/sucrose nonfermenting (SWI/SNF) complex, also referred to as BRG1associated factor (BAF) complex, which either activates or represses transcription, thus acting as an oncogene or tumor suppressor in human cells, was analyzed. PRP1 was demonstrated to decrease the expression levels of BRG, BAF170 and BRM; therefore, in JJ012 cells, these key players of the SWI/SNF (BAF) complex served an oncogenic role. The results of the present study demonstrated that PRP1 targets chromatinremodeling complexes; therefore, future efforts will be directed towards determining the interconnection between CSC maintenance, selfrenewal capacity and BAF complexes.