Intratumoral heterogeneity is a critical frontier in understanding how the tumor microenvironment (TME) propels malignant progression. Here, we deconvolute the human pancreatic TME through ...large-scale integration of histology-guided regional multiOMICs with clinical data and patient-derived preclinical models. We discover “subTMEs,” histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response. “Reactive” subTMEs rich in complex but functionally coordinated fibroblast communities were immune hot and inhabited by aggressive tumor cell phenotypes. The matrix-rich “deserted” subTMEs harbored fewer activated fibroblasts and tumor-suppressive features yet were markedly chemoprotective and enriched upon chemotherapy. SubTMEs originated in fibroblast differentiation trajectories, and transitory states were notable both in single-cell transcriptomics and in situ. The intratumoral co-occurrence of subTMEs produced patient-specific phenotypic and computationally predictable heterogeneity tightly linked to malignant biology. Therefore, heterogeneity within the plentiful, notorious pancreatic TME is not random but marks fundamental tissue organizational units.
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•PDAC regional heterogeneity stems from sub-tumor microenvironments (subTMEs)•SubTMEs exhibit distinct immune phenotypes and CAF differentiation states•SubTMEs execute distinct tumor-promoting and chemoprotective functions•Intratumoral subTME co-occurrence links stromal heterogeneity to patient outcome
Intratumoral heterogeneity in the human pancreatic tumor microenvironment is not random but originates in well-definable regional tissue states. The underlying sub-tumor microenvironments shape regional epithelial and immune phenotypes and influence key clinical metrics of disease progression.
Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide. There is an unmet need to develop novel clinically relevant models of NSCLC to accelerate ...identification of drug targets and our understanding of the disease.
Thirty surgically resected NSCLC primary patient tissue and 35 previously established patient-derived xenograft (PDX) models were processed for organoid culture establishment. Organoids were histologically and molecularly characterized by cytology and histology, exome sequencing, and RNA-sequencing analysis. Tumorigenicity was assessed through subcutaneous injection of organoids in NOD/SCID mice. Organoids were subjected to drug testing using EGFR, FGFR, and MEK-targeted therapies.
We have identified cell culture conditions favoring the establishment of short-term and long-term expansion of NSCLC organoids derived from primary lung patient and PDX tumor tissue. The NSCLC organoids recapitulated the histology of the patient and PDX tumor. They also retained tumorigenicity, as evidenced by cytologic features of malignancy, xenograft formation, preservation of mutations, copy number aberrations, and gene expression profiles between the organoid and matched parental tumor tissue by whole-exome and RNA sequencing. NSCLC organoid models also preserved the sensitivity of the matched parental tumor to targeted therapeutics, and could be used to validate or discover biomarker-drug combinations.
Our panel of NSCLC organoids closely recapitulates the genomics and biology of patient tumors, and is a potential platform for drug testing and biomarker validation.
Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway ...and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled 'dark state'.
Esophageal adenocarcinoma has few known recurrent mutations and therefore robust, reliable and reproducible patient-specific models are needed for personalized treatment. Patient-derived organoid ...culture is a strategy that may allow for the personalized study of esophageal adenocarcinoma and the development of personalized induction therapy. We therefore developed a protocol to establish EAC organoids from endoscopic biopsies of naïve esophageal adenocarcinomas. Histologic characterization and molecular characterization of organoids by whole exome sequencing demonstrated recapitulation of the tumors' histology and genomic (~ 60% SNV overlap) characteristics. Drug testing using clinically appropriate chemotherapeutics and targeted therapeutics showed an overlap between the patient's tumor response and the corresponding organoids' response. Furthermore, we identified Barrett's esophagus epithelium as a potential source of organoid culture contamination. In conclusion, organoids can be robustly cultured from endoscopic biopsies of esophageal adenocarcinoma and recapitulate the originating tumor. This model demonstrates promise as a tool to better personalize therapy for esophageal adenocarcinoma patients.
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human cancers, with 5-year patient survival rates of <5%. Activating mutations in KRAS are the predominant oncogenic drivers of ...PDAC but are accompanied by additional lower frequency genetic alterations. Our group previously identified the guanine nucleotide exchange factor ARHGEF10 in a genomic screen for genes with copy number alterations that may synergize with oncogenic KRAS to promote PDAC carcinogenesis. In the present study we show that ARHGEF10 possesses putative tumor suppressor function in PDAC. ARHGEF10 expression is reduced in over 70% of PDAC cell lines, and copy number loss is documented in more than 30% of PDAC patient-derived xenografts. Loss of ARHGEF10 expression enhanced subcutaneous tumor growth in mouse models, while its exogenous expression greatly impaired tumorigenesis. Loss of ARHGEF10 expression also increased in vitro proliferation, invasion, and motility of PDAC cell lines, and enhanced their metastatic spread in orthotopic mouse models. Treatment of ARHGEF10-depleted cells with the inhibitor dasatinib reduced levels of phospho Src kinase and attenuated motility and invasion in vitro. Together, our data indicate that ARHGEF10 may function as a tumor suppressor in PDAC.
Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and ...centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, β-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway.
The tumor microenvironment strongly influences cancer development, progression, and metastasis. The role of carcinoma-associated fibroblasts (CAFs) in these processes and their clinical impact has ...not been studied systematically in non-small cell lung carcinoma (NSCLC). We established primary cultures of CAFs and matched normal fibroblasts (NFs) from 15 resected NSCLC. We demonstrate that CAFs have greater ability than NFs to enhance the tumorigenicity of lung cancer cell lines. Microarray gene-expression analysis of the 15 matched CAF and NF cell lines identified 46 differentially expressed genes, encoding for proteins that are significantly enriched for extracellular proteins regulated by the TGF-β signaling pathway. We have identified a subset of 11 genes (13 probe sets) that formed a prognostic gene-expression signature, which was validated in multiple independent NSCLC microarray datasets. Functional annotation using protein-protein interaction analyses of these and published cancer stroma-associated gene-expression changes revealed prominent involvement of the focal adhesion and MAPK signaling pathways. Fourteen (30%) of the 46 genes also were differentially expressed in laser-capture-microdissected corresponding primary tumor stroma compared with the matched normal lung. Six of these 14 genes could be induced by TGF-β1 in NF. The results establish the prognostic impact of CAF-associated gene-expression changes in NSCLC patients.
Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the ...underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.
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