Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance.

Tolerance of mouse kidney allografts arises in grafts that develop regulatory Tertiary Lymphoid Organs (rTLOs). scRNAseq data and adoptive transfer of alloreactive T cells post-transplant showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required since adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8 KO recipients resulted in acceptance and not rejection. Analysis of scRNAseq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call "defensive tolerance." This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.

Allysine-Targeted Molecular MRI Enables Early Prediction of Chemotherapy Response in Pancreatic Cancer.

Neoadjuvant therapy (NAT) is routinely used in pancreatic ductal adenocarcinoma (PDAC), but not all tumors respond to this treatment. Current clinical imaging techniques are not able to precisely evaluate and predict the response to neoadjuvant therapies over several weeks. A strong fibrotic reaction is a hallmark of a positive response, and during fibrogenesis allysine residues are formed on collagen proteins by the action of lysyl oxidases (LOX). Here we report the application of an allysine-targeted molecular magnetic resonance imaging (MRI) probe, MnL3, to provide an early, noninvasive assessment of treatment response in PDAC. Allysine increased 2- to 3-fold after one dose of NAT with FOLFIRINOX in sensitive human PDAC xenografts in mice. Molecular MRI with MnL3 could specifically detect and quantify fibrogenesis in PDAC xenografts. Comparing the MnL3 signal before and 3 days after one dose of FOLFIRINOX predicted subsequent treatment response. The MnL3 tumor signal increased by 70% from day 0 to day 3 in mice that responded to subsequent doses of FOLFIRINOX, while no signal increase was observed in FOLFIRINOX-resistant tumors. This study indicates the promise of allysine-targeted molecular MRI as a noninvasive tool to predict chemotherapy outcomes.

Absence of a pancreatic microbiome in intraductal papillary mucinous neoplasm.

OBJECTIVE: This study aims to validate the existence of a microbiome within intraductal papillary mucinous neoplasm (IPMN) that can be differentiated from the taxonomically diverse DNA background of next-generation sequencing procedures. DESIGN: We generated 16S rRNA amplicon sequencing data to analyse 338 cyst fluid samples from 190 patients and 19 negative controls, the latter collected directly from sterile syringes in the operating room. A subset of samples (n=20) and blanks (n=5) were spiked with known concentrations of bacterial cells alien to the human microbiome to infer absolute abundances of microbial traces. All cyst fluid samples were obtained intraoperatively and included IPMNs with various degrees of dysplasia as well as other cystic neoplasms. Follow-up culturing experiments were conducted to assess bacterial growth for microbiologically significant signals. RESULTS: Microbiome signatures of cyst fluid samples were inseparable from those of negative controls, with no difference in taxonomic diversity, and microbial community composition. In a patient subgroup that had recently undergone invasive procedures, a bacterial signal was evident. This outlier signal was not characterised by higher taxonomic diversity but by an increased dominance index of a gut-associated microbe, leading to lower taxonomic evenness compared with the background signal. CONCLUSION: The 'microbiome' of IPMNs and other pancreatic cystic neoplasms does not deviate from the background signature of negative controls, supporting the concept of a sterile environment. Outlier signals may appear in a small fraction of patients following recent invasive endoscopic procedures. No associations between microbial patterns and clinical or cyst parameters were apparent.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Extracellular vesicle analysis of plasma allows differential diagnosis of atypical pancreatic serous cystadenoma.

Increased use of cross-sectional imaging has resulted in frequent detection of incidental cystic pancreatic lesions. Serous cystadenomas (SCAs) are benign cysts that do not require surgical intervention unless symptomatic. Unfortunately, up to half of SCAs do not have typical imaging findings ("atypical SCAs"), overlap with potentially malignant precursor lesions, and thus pose a diagnostic challenge. We tested whether the analysis of circulating extracellular vesicle (EV) biomarkers using a digital EV screening technology (DEST) could enhance the discrimination of cystic pancreatic lesions and avoid unnecessary surgical intervention in these atypical SCAs. Analysis of 25 different protein biomarkers in plasma EV from 68 patients identified a putative biomarker signature of Das-1, Vimentin, Chromogranin A, and CAIX with high discriminatory power (AUC of 0.99). Analysis of plasma EV for multiplexed markers may thus be helpful in clinical decision-making.

Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts.

Digital PCR (dPCR) allows for highly sensitive quantification of low-frequency mutations and facilitates early detection of cancer. However, low-throughput targeting of single hotspots in dPCR hinders variant specification when multiple probes are used. We developed a dPCR method to simultaneously identify major variants related to pancreatic carcinogenesis. Using a two-dimensional plot of droplet fluorescence under the optimized concentration of two fluorescent probe pools, the absolute quantification of different KRAS and GNAS variants was determined. Successful detection of the multiple driver mutations was verified in 24 surgically resected tumor samples from 19 patients and 22 fine-needle aspiration samples from patients with pancreatic ductal adenocarcinoma. Precise quantification of the variant allele frequency was optimized by using template DNA at a concentration as low as 1 to 10 ng. Furthermore, amplicons targeting multiple hotspots were successfully enriched with fewer false-positive findings using high-fidelity polymerase, allowing for the detection of various KRAS and GNAS mutations with high probability in small amount of cell/tissue specimens. Using this target enrichment, mutations at a rate of 90% in small residual tissues, such as the fine-needle aspiration needle flush and microscopic lesions in resected specimens, were successfully identified. The proposed method allows for low-cost, accurate detection of driver mutations to diagnose cancers, even with minimal tissue collection.

Epigenetic regulation of pancreatic adenocarcinoma in the era of cancer immunotherapy.

Pancreatic adenocarcinoma is a lethal cancer with poor response to chemotherapy and immune checkpoint inhibitors. Recent studies suggest that epigenetic alterations contribute to its aggressive biology and the tumor microenvironment which render it unresponsive to immune checkpoint blockade. Here, we review our current understandings of epigenetic dysregulation in pancreatic adenocarcinoma, its effect on the tumor immune microenvironment, and the potential for epigenetic therapy to be combined with immune checkpoint inhibitors.

Single-EV analysis (sEVA) of mutated proteins allows detection of stage 1 pancreatic cancer.

Tumor cell-derived extracellular vesicles (EVs) are being explored as circulating biomarkers, but it is unclear whether bulk measurements will allow early cancer detection. We hypothesized that a single-EV analysis (sEVA) technique could potentially improve diagnostic accuracy. Using pancreatic cancer (PDAC), we analyzed the composition of putative cancer markers in 11 model lines. In parental PDAC cells positive for KRASmut and/or P53mut proteins, only ~40% of EVs were also positive. In a blinded study involving 16 patients with surgically proven stage 1 PDAC, KRASmut and P53mut protein was detectable at much lower levels, generally in <0.1% of vesicles. These vesicles were detectable by the new sEVA approach in 15 of the 16 patients. Using a modeling approach, we estimate that the current PDAC detection limit is at ~0.1-cm3 tumor volume, below clinical imaging capabilities. These findings establish the potential for sEVA for early cancer detection.

Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway.

BACKGROUND: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. METHODS: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. RESULTS: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. CONCLUSIONS: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

Case Report: A Rare Case of Esophagogastric Junctional Squamous Cell Carcinoma After the Successful Treatment of Neuroendocrine Carcinoma: Clonal Tumor Evolution Revealed by Genetic Analysis.

Neuroendocrine carcinoma (NEC) of the esophagogastric junction (EGJ) is a rare disease with no established treatments. Herein, we describe a case of recurrent squamous cell carcinoma (SCC) after achieving complete response to chemotherapy against NEC of the EGJ. A 67-year-old man was referred to our hospital because of epigastric discomfort. Computed tomography imaging and esophagogastroduodenoscopy revealed ulcerated tumors at the EGJ. Endoscopic biopsy revealed small tumor cells with a high nuclear/cytoplasmic ratio, suggesting small-cell NEC. Immunohistochemistry (IHC) analysis showed tumor cells with an MIB-1 index of 80%. The patient achieved complete response after 10 cycles of chemotherapy. Follow-up endoscopic examination revealed small red-colored mucosal lesions in the center of the cicatrized primary lesion. Re-biopsy detected cancer cells harboring large eosinophilic cytoplasm with keratinization and no evidence of NEC components. IHC of the cells were cytokeratin 5/6-positive and p53-negative. The tumor persisted without evidence of metastases after chemoradiotherapy, and total gastrectomy with lymph node dissection was performed. Pathological assessment of the resected specimens revealed SCC, without evidence of NEC. The patient survived without a recurrence for >3 years after the initial presentation. Somatic mutation profiles of the primary NEC and recurrent SCC were analyzed by targeted amplicon sequencing covering common cancer-related mutations. Both tumors possessed TP53 Q192X mutation, whereas SMAD4 S517T was found only in SCC, suggesting that both tumor components originated from a founder clone with a stop-gain mutation in TP53. The somatic mutation profile of the tumors indicated that that loss of heterozygosity (LOH) at the TP53 gene might have occurred during the differentiation of the founder clone into NEC, while a SMAD4 mutation might have contributed to SCC development, indicating branching and subclonal evolution from common founder clone to both NEC and SCC. The mutation assessments provided valuable information to better understand the clonal evolution of metachronous cancers.

Screening and Validation of Molecular Targeted Radiosensitizers.

The development of molecular targeted drugs with radiation and chemotherapy is critically important for improving the outcomes of patients with hard-to-treat, potentially curable cancers. However, too many preclinical studies have not translated into successful radiation oncology trials. Major contributing factors to this insufficiency include poor reproducibility of preclinical data, inadequate preclinical modeling of intertumoral genomic heterogeneity that influences treatment sensitivity in the clinic, and a reliance on tumor growth delay instead of local control (TCD50) endpoints. There exists an urgent need to overcome these barriers to facilitate successful clinical translation of targeted radiosensitizers. To this end, we have used 3-dimensional (3D) cell culture assays to better model tumor behavior in vivo. Examples of successful prediction of in vivo effects with these 3D assays include radiosensitization of head and neck cancers by inhibiting epidermal growth factor receptor or focal adhesion kinase signaling, and radioresistance associated with oncogenic mutation of KRAS. To address the issue of tumor heterogeneity, we leveraged institutional resources that allow high-throughput 3D screening of radiation combinations with small-molecule inhibitors across genomically characterized cell lines from lung, head and neck, and pancreatic cancers. This high-throughput screen is expected to uncover genomic biomarkers that will inform the successful clinical translation of targeted agents from the National Cancer Institute Cancer Therapy Evaluation Program portfolio and other sources. Screening "hits" need to be subjected to refinement studies that include clonogenic assays, addition of disease-specific chemotherapeutics, target/biomarker validation, and integration of patient-derived tumor models. The chemoradiosensitizing activities of the most promising drugs should be confirmed in TCD50 assays in xenograft models with or without relevant biomarker and using clinically relevant radiation fractionation. We predict that appropriately validated and biomarker-directed targeted therapies will have a higher likelihood than past efforts of being successfully incorporated into the standard management of hard-to-treat tumors.

Large-duct pattern invasive adenocarcinoma of the pancreas-a variant mimicking pancreatic cystic neoplasms: A minireview.

Pancreatic cancer currently has no subtypes that inform clinical decisions; hence, there exists an opportunity to rearrange the morphological and molecular taxonomy that guides a better understanding of tumor characteristics. Nonetheless, accumulating studies to date have revealed the large-duct type variant, a unique subtype of pancreatic ductal adenocarcinoma (PDA) with cystic features. This subtype often radiographically mimics intraductal papillary mucinous neoplasms (IPMNs) and involves multiple small cysts occasionally associated with solid masses. The "bunch-of-grapes" sign, an imaging characteristic of IPMNs, is absent in large-duct PDA. Large-duct PDA defines the mucin profile, and genetic alterations are useful in distinguishing large-duct PDA from IPMNs. Histologically, neoplastic ducts measure over 0.5 mm, forming large ductal elements. Similar to classic PDAs, this subtype is frequently accompanied by perineural invasion and abundant desmoplastic reactions, and KRAS mutations in codon 12 are nearly ubiquitous. Despite such morphological similarities with IPMNs, the prognosis of large-duct PDA is equivalent to that of classic PDA. Differential diagnosis is therefore essential.

Transcriptomic Analysis of Laser Capture Microdissected Tumors Reveals Cancer- and Stromal-Specific Molecular Subtypes of Pancreatic Ductal Adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) lethality is multifactorial; although studies have identified transcriptional and genetic subsets of tumors with different prognostic significance, there is limited understanding of features associated with the minority of patients who have durable remission after surgical resection. In this study, we performed laser capture microdissection (LCM) of PDAC samples to define their cancer- and stroma-specific molecular subtypes and identify a prognostic gene expression signature for short-term and long-term survival. EXPERIMENTAL DESIGN: LCM and RNA sequencing (RNA-seq) analysis of cancer and adjacent stroma of 19 treatment-naïve PDAC tumors was performed. Gene expression signatures were tested for their robustness in a large independent validation set. An RNA-ISH assay with pooled probes for genes associated with disease-free survival (DFS) was developed to probe 111 PDAC tumor samples. RESULTS: Gene expression profiling identified four subtypes of cancer cells (C1-C4) and three subtypes of cancer-adjacent stroma (S1-S3). These stroma-specific subtypes were associated with DFS (P = 5.55E-07), with S1 associated with better prognoses when paired with C1 and C2. Thirteen genes were found to be predominantly expressed in cancer cells and corresponded with DFS in a validation using existing RNA-seq datasets. A second validation on an independent cohort of patients using RNA-ISH probes to six of these prognostic genes demonstrated significant association with overall survival (median 17 vs. 25 months; P < 0.02). CONCLUSIONS: Our results identified specific signatures from the epithelial and the stroma components of PDAC, which add clarity to the nature of PDAC molecular subtypes and may help predict survival.

Simultaneous targeting of primary tumor, draining lymph node, and distant metastases through high endothelial venule-targeted delivery.

Cancer patients with malignant involvement of tumor-draining lymph nodes (TDLNs) and distant metastases have the poorest prognosis. A drug delivery platform that targets the primary tumor, TDLNs, and metastatic niches simultaneously, remains to be developed. Here, we generated a novel monoclonal antibody (MHA112) against peripheral node addressin (PNAd), a family of glycoproteins expressed on high endothelial venules (HEVs), which are present constitutively in the lymph nodes (LNs) and formed ectopically in the tumor stroma. MHA112 was endocytosed by PNAd-expressing cells, where it passed through the lysosomes. MHA112 conjugated antineoplastic drug Paclitaxel (Taxol) (MHA112-Taxol) delivered Taxol effectively to the HEV-containing tumors, TDLNs, and metastatic lesions. MHA112-Taxol treatment significantly reduced primary tumor size as well as metastatic lesions in a number of mouse and human tumor xenografts tested. These data, for the first time, indicate that human metastatic lesions contain HEVs and provide a platform that permits simultaneous targeted delivery of antineoplastic drugs to the three key sites of primary tumor, TDLNs, and metastases.

Extracellular Vesicle Analysis Allows for Identification of Invasive IPMN.

BACKGROUND AND AIMS: Advances in cross-sectional imaging have resulted in increased detection of intraductal papillary mucinous neoplasms (IPMNs), and their management remains controversial. At present, there is no reliable noninvasive method to distinguish between indolent and high risk IPMNs. We performed extracellular vesicle (EV) analysis to identify markers of malignancy in an attempt to better stratify these lesions. METHODS: Using a novel ultrasensitive digital extracellular vesicle screening technique (DEST), we measured putative biomarkers of malignancy (MUC1, MUC2, MUC4, MUC5AC, MUC6, Das-1, STMN1, TSP1, TSP2, EGFR, EpCAM, GPC1, WNT-2, EphA2, S100A4, PSCA, MUC13, ZEB1, PLEC1, HOOK1, PTPN6, and FBN1) in EV from patient-derived cell lines and then on circulating EV obtained from peripheral blood drawn from patients with IPMNs. We enrolled a total of 133 patients in two separate cohorts: a clinical discovery cohort (n = 86) and a validation cohort (n = 47). RESULTS: From 16 validated EV proteins in plasma samples collected from the discovery cohort, only MUC5AC showed significantly higher levels in high-grade lesions. Of the 11 patients with invasive IPMN (inv/HG), 9 had high MUC5AC expression in plasma EV of the 11 patients with high-grade dysplasia alone, only 1 had high MUC5AC expression (sensitivity of 82%, specificity of 100%). These findings were corroborated in a separate validation cohort. The addition of MUC5AC as a biomarker to imaging and high-riskstigmata allowed detection of all cases requiring surgery, whereas imaging and high-risk stigmata alone would have missed 5 of 14 cases (36%). CONCLUSIONS: MUC5AC in circulating EV can predict the presence of invasive carcinoma within IPMN. This approach has the potential to improve the management and follow-up of patients with IPMN including avoiding unnecessary surgery.

Neoplastic-Stromal Cell Cross-talk Regulates Matrisome Expression in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic reaction, warranting intense cancer-stroma communication. In this study, we interrogated the contribution of the BET family of chromatin adaptors to the cross-talk between PDAC cells and the tumor stroma. Short-term treatment of orthotopic xenograft tumors with CPI203, a small-molecule inhibitor of BET proteins, resulted in broad changes in the expression of genes encoding components of the extracellular matrix (matrisome) in both cancer and stromal cells. Remarkably, more than half of matrisome genes were expressed by cancer cells. In vitro cocultures of PDAC cells and cancer-associated fibroblasts (CAF) demonstrated that matrisome expression was regulated by BET-dependent cancer-CAF cross-talk. Disrupting this cross-talk in vivo resulted in diminished growth of orthotopic patient-derived xenograft tumors, reduced proliferation of cancer cells, and changes in collagen structure consistent with that of patients who experienced better survival. Examination of matrisome gene expression in publicly available data sets of 573 PDAC tumors identified a 65-gene signature that was able to distinguish long- and short-term PDAC survivors. Importantly, the expression of genes predictive of short-term survival was diminished in the cancer cells of orthotopic xenograft tumors of mice treated with CPI203. Taken together, these results demonstrate that inhibiting the activity BET proteins results in transcriptional and structural differences in the matrisome are associated with better patient survival. IMPLICATIONS: These studies highlight the biological relevance of the matrisome program in PDAC and suggest targeting of epigenetically driven tumor-stroma cross-talk as a potential therapeutic avenue.

FOLFIRINOX Versus Gemcitabine-based Therapy for Pancreatic Ductal Adenocarcinoma: Lessons from Patient-derived Cell Lines.

BACKGROUND/AIM: FOLFIRINOX [fluorouracil (5-FU), irinotecan, oxaliplatin] and gemcitabine plus nab-paclitaxel are standard treatments for patients with pancreatic ductal adenocarcinoma (PDAC). Despite efficacy rates of less than 32%, evidence is lacking to guide the use of one drug over the other. Herein, we compared the sensitivity of patient-derived PDAC cell lines to each of these regimens. MATERIALS AND METHODS: Changes in the growth of 19 low-passage patient-derived PDAC cell lines were evaluated in response to treatment with FOLFIRINOX and gemcitabine plus paclitaxel (Gem-Pac). RESULTS: Six cell lines exhibited optimal sensitivity (high EMax and low GI50) to FOLFIRINOX and three cell lines exhibited optimal sensitivity to Gem-Pac. Several cell lines that were optimally sensitive to one drug regimen exhibited very poor response to the other. CONCLUSION: Further characterization of cancer cells exhibiting preferential sensitivity to each of these regimens may allow the identification of biomarkers to guide the selection of appropriate chemotherapy for a given patient.

Pancreatic circulating tumor cell profiling identifies LIN28B as a metastasis driver and drug target.

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B-an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets-in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the "seeds" of metastasis.