Unbiased in vivo preclinical evaluation of anticancer drugs identifies effective therapy for the treatment of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.

Label-free proteomics reveals serum proteins whose levels differ between pancreatic ductal adenocarcinoma patients with short or long survival.

Pancreatic ductal adenocarcinoma is the most common and aggressive type of pancreatic cancer, with a 5-year survival rate that is less than 10%. New biomarkers to aid in predicting the prognosis of pancreatic ductal adenocarcinoma patients are needed. Previous proteomic studies have to a great extent focused on finding proteins of value for the diagnosis of pancreatic ductal adenocarcinoma. There is a lack of studies that have profiled the serum or plasma proteome in order to discover candidates for new prognostic biomarkers. In this study, we have used ultra-performance liquid chromatography-ultra-definition mass spectrometry to analyze the serum samples of 21 pancreatic ductal adenocarcinoma patients with short or long survival. Statistical analysis discovered 31 proteins whose expression differed significantly between pancreatic ductal adenocarcinoma patients with short or long survival. Pathway analysis discovered multiple canonical pathways enriched in this data set, with several pathways having roles in inflammation and lipid metabolism. The serum proteins identified here, which include complement components and several enzymes, could be of value as candidates for new noninvasive prognostic markers.

Preoperative Radiotherapy Leads to Significant Differences in the Plasma Protein Profile of Rectal Cancer Patients.

INTRODUCTION: Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for 10% of the global cancer burden. Rectal cancer accounts for around 30% of CRC cases, and patients with resectable rectal cancer are often given preoperative radiotherapy (PRT) to reduce the rate of local recurrence. The human plasma proteome is an exceptionally complex proteome and ideal to study due to its ability to reflect the presence of diseases such as cancer and the ease of obtaining blood samples. Previous proteomic studies involving rectal cancer patients have mostly focused on the identification of proteins involved in resistance to radiotherapy. OBJECTIVE: The aim of this study was to investigate the overall effects of PRT on plasma protein expression in rectal cancer patients, as there is a lack of such studies. METHODS: Here, we have used mass spectrometry and subsequent statistical analyses to analyze the plasma samples of 30 rectal cancer patients according to PRT status (positive or negative) and tumor stage (II or III). RESULTS AND CONCLUSIONS: We discovered 42 proteins whose levels differed significantly between stage II and III rectal cancer patients who did or did not receive PRT. This study shows that PRT, although localized to the pelvis, leads to measurable, tumor stage-specific changes in plasma protein expression. Future studies of plasma proteins should, when relevant, take this into account and be aware of the widespread effects that PRT has on the plasma proteome.

N-Glycomic Profiling of Microsatellite Unstable Colorectal Cancer.

Aberrant glycosylation affects cancer progression and immune evasion. Approximately 15% of colorectal cancers (CRCs) demonstrate microsatellite instability (MSI) and display major differences in outcomes and therapeutic responses, as compared to corresponding microsatellite stable (MSS) tumors. We compared the N-glycan profiles of stage II and IV MSI CRC tumors, further subdivided into BRAFV600E wild-type and mutated subgroups (n = 10 in each subgroup), with each other and with those of paired non-neoplastic mucosal samples using mass spectrometry. Further, the N-glycans of BRAFV600E wild-type stage II MSI tumors were compared to corresponding MSS tumors (n = 9). Multiple differences in N-glycan profiles were identified between the MSI CRCs and control tissues, as well as between the stage II MSI and MSS samples. The MSI CRC tumors showed a lower relative abundance of high-mannose N-glycans than did the control tissues or the MSS CRCs. Among MSI CRC subgroups, acidic N-glycans showed tumor stage and BRAF mutation status-dependent variation. Specifically, the large, sulfated/phosphorylated, and putative terminal N-acetylhexosamine-containing acidic N-glycans differed between the MSI CRC subgroups, showing opposite changes in stages II and IV, when comparing BRAF mutated and wild-type tumors. Our results show that molecular subgroups of CRC exhibit characteristic glycan profiles that may explain certain carcinogenic properties of MSI tumors.

Solid tumor growth depends on an intricate equilibrium of malignant cell states.

Control of cell identity and number is central to tissue function, yet principles governing organization of malignant cells in tumor tissues remain poorly understood. Using mathematical modeling and candidate-based analysis, we discover primary and metastatic pancreatic ductal adenocarcinoma (PDAC) organize in a stereotypic pattern whereby PDAC cells responding to WNT signals (WNT-R) neighbor WNT-secreting cancer cells (WNT-S). Leveraging lineage-tracing, we reveal the WNT-R state is transient and gives rise to the WNT-S state that is highly stable and committed to organizing malignant tissue. We further show that a subset of WNT-S cells expressing the Notch ligand DLL1 form a functional niche for WNT-R cells. Genetic inactivation of WNT secretion or Notch pathway components, or cytoablation of the WNT-S state disrupts PDAC tissue organization, suppressing tumor growth and metastasis. This work indicates PDAC growth depends on an intricately controlled equilibrium of functionally distinct cancer cell states, uncovering a fundamental principle governing solid tumor growth and revealing new opportunities for therapeutic intervention.

Quantitative glycoproteomics of human milk and association with atopic disease.

The prevalence of allergic diseases and asthma is increasing rapidly worldwide, with environmental and lifestyle behaviors implicated as a reason. Epidemiological studies have shown that children who grow up on farms are at lower risk of developing childhood atopic disease, indicating the presence of a protective "farm effect". The Old Order Mennonite (OOM) community in Upstate New York have traditional, agrarian lifestyles, a low rate of atopic disease, and long periods of exclusive breastfeeding. Human milk proteins are heavily glycosylated, although there is a paucity of studies investigating the milk glycoproteome. In this study, we have used quantitative glycoproteomics to compare the N-glycoprotein profiles of 54 milk samples from Rochester urban/suburban and OOM mothers, two populations with different lifestyles, exposures, and risk of atopic disease. We also compared N-glycoprotein profiles according to the presence or absence of atopic disease in the mothers and, separately, the children. We identified 79 N-glycopeptides from 15 different proteins and found that proteins including immunoglobulin A1, polymeric immunoglobulin receptor, and lactotransferrin displayed significant glycan heterogeneity. We found that the abundances of 38 glycopeptides differed significantly between Rochester and OOM mothers and also identified four glycopeptides with significantly different abundances between all comparisons. These four glycopeptides may be associated with the development of atopic disease. The findings of this study suggest that the differential glycosylation of milk proteins could be linked to atopic disease.

Quantitative bile and serum proteomics for the screening and differential diagnosis of primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by biliary strictures, cholestasis, and a markedly increased risk of cholangiocarcinoma. New markers for the screening and differential diagnosis of PSC are needed. In this pilot study, we have analyzed both the bile and serum proteomic profiles of 80 PSC patients and non-PSC controls (n = 6 for bile and n = 18 for serum). AIM: The aim of this study was to discover candidates for new biomarkers for the differential diagnosis of PSC. METHODS: Bile and serum samples were processed and subsequently analyzed using ultra performance liquid chromatography-ultra definition mass spectrometry (UPLC-UDMSE). Further analysis included statistical analyses such as receiver operating characteristic curve analysis as well as pathway analysis using Ingenuity Pathway Analysis. RESULTS AND CONCLUSIONS: In bile, we discovered 64 proteins with significantly different levels between the groups, with fold changes of up to 129. In serum, we discovered 112 proteins with significantly different levels. Receiver operating characteristic curve analysis found multiple proteins with high area under the curve values, up to 0.942, indicating that these serum proteins are of value as new non-invasive classifiers of PSC. Pathway analysis revealed multiple canonical pathways that were enriched in the dataset, which have roles in bile homeostasis and metabolism. We present several serum proteins that could serve as new blood-based markers for the diagnosis of PSC after further validation. The measurement of serum levels of these proteins could be of use in the screening of patients with suspected PSC.

Systematic Comparison of Pancreatic Ductal Adenocarcinoma Models Identifies a Conserved Highly Plastic Basal Cell State.

Intratumoral heterogeneity and cellular plasticity have emerged as hallmarks of cancer, including pancreatic ductal adenocarcinoma (PDAC). As PDAC portends a dire prognosis, a better understanding of the mechanisms underpinning cellular diversity in PDAC is crucial. Here, we investigated the cellular heterogeneity of PDAC cancer cells across a range of in vitro and in vivo growth conditions using single-cell genomics. Heterogeneity contracted significantly in two-dimensional and three-dimensional cell culture models but was restored upon orthotopic transplantation. Orthotopic transplants reproducibly acquired cell states identified in autochthonous PDAC tumors, including a basal state exhibiting coexpression and coaccessibility of epithelial and mesenchymal genes. Lineage tracing combined with single-cell transcriptomics revealed that basal cells display high plasticity in situ. This work defines the impact of cellular growth conditions on phenotypic diversity and uncovers a highly plastic cell state with the capacity to facilitate state transitions and promote intratumoral heterogeneity in PDAC. SIGNIFICANCE: This work provides important insights into how different model systems of pancreatic ductal adenocarcinoma mold the phenotypic space of cancer cells, highlighting the power of in vivo models.

Plasma protein expression differs between colorectal cancer patients depending on primary tumor location.

Colorectal cancer (CRC) includes tumors in the right colon, left colon, and rectum, although they differ significantly from each other in aspects such as prognosis and treatment. Few previous mass spectrometry-based studies have analyzed differences in protein expression depending on the tumor location. In this study, we have used mass spectrometry-based proteomics to analyze plasma samples from 83 CRC patients to study if differences in plasma protein expression can be seen depending on primary tumor location (right colon, left colon, or rectum). Differences were studied between the groups both regardless of and according to tumor stage (II or III). Large differences in plasma protein expression were seen, and we found that plasma samples from patients with rectal cancer separated from samples from patients with colon cancer when analyzed by principal component analysis and hierarchical clustering. Samples from patients with cancer in the right and left colon also tended to separate from each other. Pathway analysis discovered canonical pathways involved in lipid metabolism and inflammation to be enriched. This study will help to further define CRC as distinct entities depending on tumor location, as shown by the widespread differences in plasma protein profile and dysregulated pathways.

N-glycomic profiling of colorectal cancer according to tumor stage and location.

Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer and can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways regardless of tumor location.

Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing.

Circulating tumor DNA (ctDNA) is released from cancer cells and oncogenic mutations in ctDNA can be measured from plasma samples. Droplet digital PCR (ddPCR) is a sensitive and specific method for the detection of mutations in ctDNA. We analyzed serial plasma samples (n = 80) from ten metastatic colorectal cancer (mCRC) patients with a known KRAS mutation in their primary tumor. The patients were undergoing oncological treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan. Baseline ddPCR KRAS mutation allele frequency (MAF) values ranged from 0% to 63%. The first radiologic response evaluation criteria in solid tumors (RECIST) evaluation was performed 45-63 days after the initiation of treatment, and by this time three patients had an undetectable level of KRAS mutation, one had a MAF value of 0.5%, and one had a MAF value of 3% that had been reduced by 95% from the baseline value. In three of these patients the RECIST assessment was stable disease and in two partial response. In seven patients, ddPCR MAF values increased before radiological disease progression or death, while one patient remained disease-free with an undetectable KRAS mutation level. Next, we analyzed all available plasma samples with the Idylla ctKRAS system (n = 60), and found that the overall degree of agreement between ddPCR and Idylla was almost perfect (kappa value = 0.860). We used next-generation sequencing (NGS) to detect treatment-induced mutations in the last serial plasma sample of each patient, but were unable to find any new mutations when compared to the primary tumor. This study shows that ddPCR and Idylla are equally efficient for the detection of KRAS mutations in the liquid biopsies from mCRC patients and that ctDNA may indicate the disappearance of treatment responsive KRAS positive mCRC clones and serve as an early sign of disease progression.

Identification of several plasma proteins whose levels in colorectal cancer patients differ depending on outcome.

Colorectal cancer (CRC) stands for 10% of the worldwide cancer burden and has recently become the second most common cause of cancer death. The 5-year survival rate depends mainly on stage at diagnosis. Mass spectrometric proteomic analysis is widely used to study the plasma proteome, which is complex and contains multitudes of proteins. In this study, we have used Ultra Performance Liquid Chromatography-Ultra Definition Mass Spectrometry (UPLC-UDMSE)-based proteomics to analyze plasma samples from 76 CRC patients. We identified several plasma proteins, such as CP, TVP23C, FETUB, and IGFBP3, of which altered levels led to significant differences in survival, as seen by Cox regression and Kaplan-Meier analysis. Additionally, during Cox regression analysis, samples were adjusted for age and/or tumor stage, enabling stringent analysis. These proteins, although in need of further validation, could be of use during patient follow-up, as their levels can non-invasively be measured from blood samples, and could be of use in predicting patient outcome. Several of these proteins additionally have roles in metabolism and inflammation, two processes central to the development and progression of cancer, further indicating their importance in cancer.

Colorectal cancer patients with different C-reactive protein levels and 5-year survival times can be differentiated with quantitative serum proteomics.

Over 1.4 million people are diagnosed with colorectal cancer (CRC) each year, making it the third most common cancer in the world. Increased screening and therapeutic modalities including improved combination treatments have reduced CRC mortality, although incidence and mortality rates are still increasing in some areas. Serum-based biomarkers are mainly used for follow-up of cancer, and are ideal due to the ease and minimally invasive nature of sample collection. Unfortunately, CEA and other serum markers have too low sensitivity for screening and preoperative diagnostic purposes. Increasing interest is focused on the possible use of biomarkers for predicting treatment response and prognosis in cancer. In this study, we have performed mass spectrometry analysis (UPLC-UDMSE) of serum samples from 19 CRC patients. Increased levels of C-reactive protein (CRP), which occur during local inflammation and the presence of a systemic inflammatory response, have been linked to poor prognosis in CRC patients. We chose to analyze samples according to CRP values by dividing them into the categories CRP <30 and >30, and, separately, according to short and long 5-year survival. The aim was to discover differentially expressed proteins associated with poor prognosis and shorter survival. We quantified 256 proteins and performed detailed statistical analyses and pathway analysis. We discovered multiple proteins that are up- or downregulated in patients with CRP >30 as compared to CRP <30 and in patients with short as compared to long 5-year survival. Pathways that were enriched include LXR/RXR activation, FXR/RXR activation, complement and coagulation cascades and acute phase signaling response, with some of the proteins we identified having roles in these pathways. In this study, we have identified multiple proteins, of which a few have been previously identified as potential biomarkers, and others that have been identified as potential biomarkers for CRC for the first time, to the best of our knowledge. While these proteins still need to be validated in larger patient series, this pilot study will pave the way for future studies aiming to provide better biomarkers for patients with CRC.