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1.
Stem Cell Reports ; 19(5): 639-653, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38608679

ABSTRACT

Sepsis survivors exhibit immune dysfunction, hematological changes, and increased risk of infection. The long-term impacts of sepsis on hematopoiesis were analyzed using a surgical model of murine sepsis, resulting in 50% survival. During acute disease, phenotypic hematopoietic stem and progenitor cells (HSPCs) were reduced in the bone marrow (BM), concomitant with increased myeloid colony-forming units and extramedullary hematopoiesis. Upon recovery, BM HSPCs were increased and exhibited normal function in the context of transplantation. To evaluate hematopoietic responses in sepsis survivors, we treated recovered sham and cecal ligation and puncture mice with a mobilizing regimen of granulocyte colony-stimulating factor (G-CSF) at day 20 post-surgery. Sepsis survivors failed to undergo emergency myelopoiesis and HSPC mobilization in response to G-CSF administration. G-CSF is produced in response to acute infection and injury to expedite the production of innate immune cells; therefore, our findings contribute to a new understanding of how sepsis predisposes to subsequent infection.


Subject(s)
Granulocyte Colony-Stimulating Factor , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cells , Myelopoiesis , Sepsis , Animals , Sepsis/complications , Granulocyte Colony-Stimulating Factor/pharmacology , Mice , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Disease Models, Animal , Mice, Inbred C57BL , Male , Survivors
2.
Pharmacol Res Perspect ; 10(2): e00898, 2022 04.
Article in English | MEDLINE | ID: mdl-35257504

ABSTRACT

Previously published digital autoradiography of 3 H-labeled capecitabine reveals a near-uniform distribution of activity throughout a murine pancreatic model. This is in contrast both to 14 C-labeled gemcitabine, and established expectations, as the dense stroma of pancreatic cancer is understood to inhibit drug penetration. Capecitabine is a pro-drug for 5 FU. The positioning of the radiolabel on capecitabine leaves open the possibility that much of the autoradiographic signal is generated by nontoxic compounds. Studies were performed on tumors derived via organoid culture from a murine KPC tumor. As before, we performed autoradiography comparing 3 H capecitabine to the gemcitabine analog 18 F-FAC. The metabolism of capecitabine in this model was studied through LC-MS of tumor tissue. The autoradiographs confirmed that the 3 H label from capecitabine was much more uniformly distributed through the tumor than the 18 F from the gemcitabine analog. LC-MS revealed that approximately 75% of the molar mass of capecitabine had been converted into 5 FU or pre-5 FU compounds. The remainder had been converted into nontoxic species. Therapeutically relevant capecitabine metabolites achieve a relatively even distribution in this pancreatic cancer model, in contrast to the gemcitabine analog 18 F-FAC. In a human xenograft model, (BxPC3), the 3 H label from capecitabine was also uniformly spread across the tumor autoradiographs. However, at 2 h post-administration the metabolism of capecitabine had proceeded further and the bulk of the agent was in the form of nontoxic species.


Subject(s)
Pancreatic Neoplasms , Prodrugs , Animals , Autoradiography , Capecitabine , Disease Models, Animal , Humans , Mice , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms
3.
Nature ; 599(7886): 679-683, 2021 11.
Article in English | MEDLINE | ID: mdl-34759319

ABSTRACT

Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.


Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm/genetics , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Acetonitriles/pharmacology , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line , Cohort Studies , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Humans , MAP Kinase Signaling System/drug effects , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Piperazines/pharmacology , Piperazines/therapeutic use , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Pyridines/pharmacology , Pyridines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Xenograft Model Antitumor Assays
4.
Cancer Cell ; 39(11): 1479-1496.e18, 2021 11 08.
Article in English | MEDLINE | ID: mdl-34653364

ABSTRACT

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.


Subject(s)
Gene Expression Profiling/methods , Lung Neoplasms/genetics , Phospholipase C gamma/genetics , Small Cell Lung Carcinoma/genetics , Cell Plasticity , Humans , Neoplasm Metastasis , Prognosis , Sequence Analysis, RNA , Single-Cell Analysis , Survival Analysis
5.
J Nucl Med ; 62(2): 195-200, 2021 02.
Article in English | MEDLINE | ID: mdl-32646874

ABSTRACT

18F-FAC (2'-deoxy-2'-18F-fluoro-ß-d-arabinofuranosylcytosine) has close structural similarity to gemcitabine and thus offers the potential to image drug delivery to tumors. We compared tumor 18F-FAC PET images with 14C-gemcitabine levels, established ex vivo, in 3 mouse models of pancreatic cancer. We further modified tumor gemcitabine levels with injectable PEGylated recombinant human hyaluronidase (PEGPH20) to test whether changes in gemcitabine would be tracked by 18F-FAC. Methods:18F-FAC was synthesized as described previously. Three patient-derived xenograft (PDX) models were grown in the flanks of NSG mice. Mice were given PEGPH20 or vehicle intravenously 24 h before coinjection of 18F-FAC and 14C-gemcitabine. Animals were euthanized and imaged 1 h after tracer administration. Tumor and muscle uptake of both 18F-FAC and 14C-gemcitabine was obtained ex vivo. The efficacy of PEPGPH20 was validated through staining with hyaluronic acid binding protein. Additionally, an organoid culture, initiated from a KPC (Pdx-1 Cre LSL-KrasG12D LSL-p53R172H) tumor, was used to generate orthotopically growing tumors in C57BL/6J mice, and these tumors were then serially transplanted. Animals were injected with PEGPH20 and 14C-gemcitabine as described above to validate increased drug uptake by ex vivo assay. PET/MR images were obtained using a PET insert on a 7-T MR scanner. Animals were imaged immediately before injection with PEGPH20 and again 24 h later. Results: Tumor-to-muscle ratios of 14C-gemcitabine and 18F-FAC correlated well across all PDX models and treatments (R2 = 0.78). There was a significant increase in the tumor PET signal in PEGPH20-treated PDX animals, and this signal was matched in ex vivo counts for 2 of 3 models. In KPC-derived tumors, PEGPH20 raised 14C-gemcitabine levels (tumor-to-muscle ratio of 1.9 vs. 2.4, control vs. treated, P = 0.013). PET/MR 18F-FAC images showed a 12% increase in tumor 18F-FAC uptake after PEGPH20 treatment (P = 0.023). PEGPH20-treated animals uniformly displayed clear reductions in hyaluronic acid staining. Conclusion:18F-FAC PET was shown to be a good surrogate for gemcitabine uptake and, when combined with MR, to successfully determine drug uptake in tumors growing in the pancreas. PEGPH20 had moderate effects on tumor uptake of gemcitabine.


Subject(s)
Deoxycytidine/analogs & derivatives , Pancreatic Neoplasms/diagnostic imaging , Pancreatic Neoplasms/metabolism , Positron-Emission Tomography , Animals , Deoxycytidine/chemistry , Deoxycytidine/metabolism , Deoxycytidine/therapeutic use , Disease Models, Animal , Drug Carriers/chemistry , Drug Carriers/metabolism , Hyaluronoglucosaminidase/metabolism , Mice , Pancreatic Neoplasms/drug therapy , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Gemcitabine
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