Circulating cancer stem cells: the importance to select.

It has been demonstrated that even localized tumors without clinically apparent metastasis give rise to circulating tumor cells (CTCs). A growing number of technically diverse platforms are being developed for detecting/isolating CTCs in the circulating blood. Despite the technical challenges of isolating rare CTCs from blood, recent studies have already shown the predictive value of CTCs enumeration. Thus, it is becoming increasingly accepted that CTC numbers are linked to patients' outcome and may also be used to monitor treatment response and disease relapse, respectively. Further CTCs provide a non-invasive source for tumor material, 'liquid biopsy', which is particularly important for patients, where no biopsy material can be obtained or where serial biopsies of the tumor, e.g., following treatment, are practically impossible. On the other hand the molecular and biological characterization of CTCs has still remained at a rather experimental stage. Future studies are necessary to define CTC heterogeneity to establish the crucial role of circulating cancer stem cells for driving metastasis, which represent a distinct subpopulation of CTCs that bear metastasis-initiating capabilities based on their stemness properties and invasiveness and thus are critical for the patients' clinical outcome. As compared to non-tumorigenic/metastatic bulk CTCs, circulating cancer stem cells may not only be capable of evading from the primary tumor, but also escape from immune surveillance, survive in the circulating blood and subsequently form metastases in distant organs. Thus, circulating cancer stem cells represent a subset of exclusively tumorigenic cancer stem cells characterized by their invasive characteristics and are potential therapeutic targets for preventing disease progression. To date, only a few original reports and reviews have been published focusing on circulating cancer stem cells. This review discusses the potential importance of isolating and characterizing these circulating cancer stem cells, but also highlights current technological limitations.

Differentiating Ductal Adenocarcinoma of the Pancreas from Benign Conditions Using Routine Health Records: A Prospective Case-Control Study.

The study aimed to develop a prediction model for differentiating suspected PDAC from benign conditions. We used a prospective cohort of patients with pancreatic disease (n = 762) enrolled at the Barts Pancreas Tissue Bank (2008-2021) and performed a case-control study examining the association of PDAC (n = 340) with predictor variables including demographics, comorbidities, lifestyle factors, presenting symptoms and commonly performed blood tests. Age (over 55), weight loss in hypertensive patients, recent symptoms of jaundice, high serum bilirubin, low serum creatinine, high serum alkaline phosphatase, low red blood cell count and low serum sodium were identified as the most important features. These predictors were then used for training several machine-learning-based risk-prediction models on 75% of the cohort. Models were assessed on the remaining 25%. A logistic regression-based model had the best overall performance in the validation cohort (area-under-the-curve = 0.90; Spiegelhalter's z = -1·82, p = 0.07). Setting a probability threshold of 0.15 guided by the maximum F2-score of 0.855, 96.8% sensitivity was reached in the full cohort, which could lead to earlier detection of 84.7% of the PDAC patients. The prediction model has the potential to be applied in primary, secondary and emergency care settings for the early distinction of suspected PDAC patients and expedited referral to specialist hepato-pancreatico-biliary services.

Pentraxin 3 is a stromally-derived biomarker for detection of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), characterized by dense desmoplastic stroma laid down by pancreatic stellate cells (PSC), has no reliable diagnostic biomarkers for timely detection. A multi-center cohort of PDAC patients and controls (chronic pancreatitis, intra-ductal papillary neoplasms, gallstones and otherwise healthy) donated serum in an ethically approved manner. Serum PTX3 above 4.34 ng/mL has a higher sensitivity (86%, 95% confidence interval (CI): 65-97%) and specificity (86%, 95% CI: 79-91%), positive predictive value (97%) and likelihood ratio (6.05), and is superior when compared to serum CA19-9 and CEA for detection of PDAC. In vitro and ex vivo analyses of PTX3, in human PDAC samples, PSCs, cell lines and transgenic mouse model for PDAC, suggest that PTX3 originates from stromal cells, mainly PSC. In activated PSC, PTX3 secretion could be downregulated by rendering PSC quiescent using all-trans-retinoic acid (ATRA). PTX3 organizes hyaluronan in conjunction with tumor necrosis factor-stimulated gene 6 (TSG-6) and facilitates stellate and cancer cell invasion. In SCALOP clinical trial (ISRCTN96169987) testing chemo-radiotherapy without stromal targeting, PTX3 had no prognostic or predictive role. However, in STARPAC clinical trial (NCT03307148), stromal modulation by ATRA even at first dose is accompanied with serum PTX3 response in patients who later go on to demonstrate disease control but not those in whom the disease progresses. PTX3 is a putative stromally-derived biomarker for PDAC which warrants further testing in prospective, larger, multi-center cohorts and within clinical trials targeting stroma.

Validation of a Novel, Flash-Freezing Method: Aluminum Platform.

Stored biological materials should have minimal pre-analytical variations in order to provide researchers with high-quality samples that will give reliable and reproducible results, yet methods of storage should be easy to implement, with minimal cost and health hazard. Frozen tissue samples are a valuable biological resource. Here we compare different methods, such as liquid nitrogen (LN) or dry ice (DI), to a cheap and safe alternative using an aluminum platform (AP). Murine fresh liver and pancreas tissues were used with varying lengths of warm ischemia time. Quality assessment was based on histological evaluation, DNA and RNA extraction and quantification, and RNA degradation analysis, as well preservation of antigens for immunofluorescence, in a blinded manner. Both in superficial and deep tissue sections, based on histological assessment, AP is superior to DI, or as good as LN techniques in terms of presence of ice crystals, cutting artifacts, and overall quality/structural preservation. DNA and RNA were successfully extracted in reasonable quantities from all freezing techniques, but RNA degradation was seen for pancreas samples across all techniques. Immunofluorescence with cytokeratin8 (CK-8), alpha smooth muscle actin (αSMA), CD3, and B220 shows equally good outcomes for AP and LN, which are better than DI. The aluminum platform is a cheap, yet reliable method to freeze samples, rapidly preserving histological, antigenic, and DNA/RNA quality. Wider testing is required across different sample types. © 2020 The Authors. Basic Protocol: Flash-freezing fresh tissue with aluminum platform Alternate Protocol 1: Freezing fresh tissue with liquid nitrogen Alternate Protocol 2: Freezing fresh tissue with dry ice.

Phase I clinical trial repurposing all-trans retinoic acid as a stromal targeting agent for pancreatic cancer.

Pre-clinical models have shown that targeting pancreatic stellate cells with all-trans-retinoic-acid (ATRA) reprograms pancreatic stroma to suppress pancreatic ductal adenocarcinoma (PDAC) growth. Here, in a phase Ib, dose escalation and expansion, trial for patients with advanced, unresectable PDAC (n = 27), ATRA is re-purposed as a stromal-targeting agent in combination with gemcitabine-nab-paclitaxel chemotherapy using a two-step adaptive continual re-assessment method trial design. The maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D, primary outcome) is the FDA/EMEA approved dose of gemcitabine-nab-paclitaxel along-with ATRA (45 mg/m2 orally, days 1-15/cycle). Dose limiting toxicity (DLT) is grade 4 thrombocytopenia (n = 2). Secondary outcomes show no detriment to ATRA pharmacokinetics.. Median overall survival for RP2D treated evaluable population, is 11.7 months (95%CI 8.6-15.7 m, n = 15, locally advanced (2) and metastatic (13)). Exploratory pharmacodynamics studies including changes in diffusion-weighted (DW)-MRI measured apparent diffusion coefficient after one cycle, and, modulation of cycle-specific serum pentraxin 3 levels over various cycles indicate stromal modulation. Baseline stromal-specific retinoid transport protein (FABP5, CRABP2) expression may be predicitve of response. Re-purposing ATRA as a stromal-targeting agent with gemcitabine-nab-paclitaxel is safe and tolerable. This combination will be evaluated in a phase II randomized controlled trial for locally advanced PDAC. Clinical trial numbers: EudraCT: 2015-002662-23; NCT03307148. Trial acronym: STARPAC.

Rapamycin inhibits prostate cancer cell growth through cyclin D1 and enhances the cytotoxic efficacy of cisplatin.

Prostate cancer is the most common malignancy in Western men and hormone refractory cancer (HRPC) kills most of the patients. Chemo-resistance is a major obstacle for the treatment of prostate cancer. Platinum-complexes have been used to treat a number of malignancies including prostate cancer. However, it has limited effect to prostate cancer and with significant toxicity at higher doses. In recent years, increasing numbers of new agents targeting cancer specific pathways have become available and with low toxic side-effects. Rapamycin (Sirolimus) is an mTORC1 inhibitor, which inhibits the PI3K/Akt/mTOR signaling pathway, which is commonly altered in prostate cancer. We determined the expression of cyclin D1 and phosphorylated-mTOR proteins in association with the response to rapamycin in two androgen sensitive (22RV1 and LNCaP) and two androgen independent (DU145 and PC3) prostate cancer cell lines and found that the base-line and changes of cyclin D1 level, but not the expression level of p-mTOR, correlated with rapamycin sensitivity. We evaluated the cell killing effect of combined rapamycin and cisplatin treatment and showed that the combination had a more than additive effect in both androgen dependent and independent prostate cancer cells, which may be partially explained by the reduction of cyclin D1 expression by rapamycin. We also evaluated a range of combined treatment schedules, simultaneously or sequentially and found that continuous rapamycin treatment after a short cisplatin exposure was effective. The clinical application of these findings for prostate cancer treatment should be further investigated.

Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System.

Isolation of circulating tumor cells (CTCs) from peripheral blood has the potential to provide a far easier "liquid biopsy" than tumor tissue biopsies, to monitor tumor cell populations during disease progression and in response to therapies. Many CTC isolation technologies have been developed. We optimized the Parsortix system, an epitope independent, size and compressibility-based platform for CTCs isolation, making it possible to harvest CTCs at the speed and sample volume comparable to standard CellSearch system. We captured more than half of cancer cells from different cancer cell lines spiked in blood samples from healthy donors using this system. Cell loss during immunostaining of cells transferred and fixed on the slides is a major problem for analyzing rare cell samples. We developed a novel cell transfer and fixation method to retain >90% of cells on the slide after the immunofluorescence process without affecting signal strength and specificity. Using this optimized method, we evaluated the Parsortix system for CTC harvest in prostate cancer patients in comparison to immunobead based CTC isolation systems IsoFlux and CellSearch. We harvested a similar number (p = 0.33) of cytokeratin (CK) positive CTCs using Parsortix and IsoFlux from 7.5 mL blood samples of 10 prostate cancer patients (an average of 33.8 and 37.6 respectively). The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02). Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively). We also captured CTC clusters using Parsortix. Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers. Interestingly, 95.6% of PC3 cells expressed Vimentin, including those cells that lacked both epithelial marker expression, indicating epithelial-to-mesenchymal transition. CK-positive/Vimentin-positive/CD45-negative, and CK-negative/Vimentin-positive/CD45-negative cells were also observed in four of five prostate cancer patients but rarely in three healthy controls, indicating that Parsortix harvests CTCs with both epithelial and mesenchymal features. We also demonstrated using PC3 and DU145 spiking experiment that Parsortix harvested cells were viable for cell culture.