Tumor-Infiltrating Lymphocytes and PD-L1 Expression in Pleomorphic Lobular Breast Carcinoma.

BACKGROUND: The prognostic and predictive role of stromal tumor-infiltrating lymphocytes (sTILs) is undetermined in pleomorphic invasive lobular cancer (pILC). The same applies for the expression of PD-1/PD-L1 in this rare breast cancer subtype. Here, we aimed to investigate the expression of sTILs and analyze the PD-L1 expression levels in pILC. METHODS: Archival tissues from sixty-six patients with pILC were collected. The sTIL density was scored as a percentage of tumor area using the following cut-offs: 0%; <5%; 5-9%; and 10-50%. The PD-L1 expression was analyzed using IHC on formalin-fixed, paraffin-embedded tissue sections using SP142 and 22C3 antibodies. RESULTS: A total of 82% of the sixty-six patients were hormone receptor positive and 8% of cases were triple negative (TN), while 10% showed human epidermal growth factor receptor 2 (HER2) amplification. sTILs (≥1%) were present in 64% of the study population. Using the SP142 antibody, 36% of tumors demonstrated a positive PD-L1 score of ≥1%, and using the 22C3 antibody, 28% had a positive PD-L1 score of ≥1. There was no correlation between sTILs or PD-L1 expression and tumor size, tumor grade, nodal status, expression of estrogen receptor (ER), or amplification of HER2. Our data did not show any difference in survival between the three molecular subtypes of pILC with respect to sTILs and PD-L1 expression. CONCLUSION: This study shows that pILCs show some degree of sTILs and PD-L1 expression; however, this was not associated with a survival improvement. Additional large trials are needed to understand immune infiltration in lobular cancer, especially in the pleomorphic subtype.

Safety of pre- or postoperative accelerated radiotherapy in 5 fractions: A randomized pilot trial.

OBJECTIVE: Neo-adjuvant radiotherapy (NART) for breast cancer has shown promising survival results in retrospective trials. However, there are some obstacles such as a chemotherapy delay, an increased overall treatment time (OTT) and the risk of increasing surgical morbidity. Accelerated radiotherapy (RT) in 5 fractions allows to deliver NART in a very short time span and minimizes the delay of surgery and chemotherapy. This trial investigates this NART schedule for safety, feasibility and OTT. MATERIAL AND METHODS: Twenty patients eligible for neo-adjuvant chemotherapy (NACT) and breast conserving surgery, were randomized between NART before NACT or NACT and postoperative RT. In both arms, RT treatment was given in 5 fractions to the whole breast with a simultaneously integrated boost (SIB) on the tumor(bed). Lymph node irradiation was given concomitantly in case of lymph node involvement. OTT was defined as the time from diagnosis to last surgery in the intervention group, while in the control group the time between diagnosis and last RT-fraction was used. In the intervention group NACT-delay was defined as time between diagnosis and start of chemotherapy. RESULTS: 20 patients were included, and 19 patients completed treatment. OTT was significantly shorter in the intervention group (mean 218 days, range 196-253) compared to the control group (mean 237, range 211-268, p = 0.001). The difference in mean duration from diagnosis to the first treatment was a non-significant 4 days longer (31 vs 27 days, p = 0.28), but the start of NACT after diagnosis was delayed by 21 days (48 vs 27 days, p < 0.001). NART did not result in additional surgery complications. CONCLUSION: This pilot trial is the first to report on accelerated NART in 5 fractions with SIB. NART before NACT resulted in a shorter OTT with good safety results.

Breast Amyloidosis: A Case Report and Literature Review.

Amyloidosis is an uncommon disorder characterized by extracellular accumulation of misfolded proteins in tissues. We report a unique case of localized breast amyloidosis in an asymptomatic 56-year-old woman with systemic lupus erythematosus, presented as suspicious microcalcifications without mass on mammography. Vacuum biopsy confirmed amyloidosis, producing the typical apple-green birefringence under polarized light after staining with Congo-red. Further workup and follow-up to exclude development into systemic amyloidosis or hematologic malignancy is recommended. If negative, the prognosis is very good, thus no further treatment is needed. A brief review of the literature revealed more about the typical findings and recommended management.

B-Cell Acute Lymphoblastic Leukaemia of the Uterus.

Teaching Point: Uterine involvement of B-cell acute lymphoblastic leukaemia is exceedingly rare. Globular enlargement of the uterus with diffuse homogeneous signal intensity is suggestive for the diagnosis on magnetic resonance imaging (MRI) in clinically suspected cases.

18F-FDG micro-PET/CT for intra-operative margin assessment during breast-conserving surgery.

Rationale: Positive surgical margins for invasive breast cancer (BC) treated with breast-conserving surgery (BCS) are defined as ink on tumor. The rate of positive margins is approximately 20%, since a time- and cost-effective method for margin assessment is lacking. In this study, we investigated margin status by intra-operative imaging using high-resolution 18 F-fluoro-deoxyglucose (FDG) positron emission tomography (PET) and X-ray computed tomography (CT).Methods: Twenty patients were enrolled and received 4 MBq/kg of FDG prior to surgery. Intra-operative imaging of the specimens was performed by the MOLECUBES ß-CUBE (PET) and X-CUBE (CT). Margin status was assessed by three surgeons and compared with an algorithm. The sensitivity and specificity were calculated by using histopathological assessment as a gold standard.Results: A region with high FDG uptake was visualized in all specimens. Automated analysis showed a sensitivity of 90%, a specificity of 60%, and an area under the curve (AUC) of 0.86 after ROC analysis. Margin assessment by the surgeons resulted in a mean sensitivity and specificity of 79% and 72%, respectively.Conclusions: This proof-of-concept study demonstrates that high-resolution FDG-PET/CT can facilitate intra-operative margin assessment during BCS. This technique achieves good sensitivity and specificity and may therefore reduce re-operation rates in the future.

Heterogeneous Response of Chemotherapy-Related Cognitive Decline in Patients with Breast Cancer: A Prospective Study.

OBJECTIVE: A significant proportion of adjuvant-treated breast cancer patients experience cognitive decline, challenging the person's ability to return to normal activities after treatment. However, not every patient experiences cognitive problems, and even in patients with impairments, determining clinically important cognitive decline remains challenging. Our objective was to explore differences in neuropsychological performance following adjuvant chemotherapy (CT) in patients with breast cancer. METHOD: We conducted a prospective observational study in an Oncology Breast Clinic and assessed neuropsychological performance before and after adjuvant CT and in non-CT-treated women with breast cancer and healthy controls (HCs). Standardised between-group differences and regression-based change scores were calculated. RESULTS: CT-treated patients (n = 66) performed significantly different from non-CT-treated patients (n = 39) and HCs (n = 56). There was a significant effect on verbal fluency (p = .0013). CT performed significantly worse than non-CT and HC [effect size (ES) = .89, p < .001 and ES = .61, p ≤ .001, respectively] and from HCs with regard to proactive interference (ES = .62, p ≤ .001). Regression-based scores revealed more severe cognitive decline in the CT-treated group [24.24% (16/66)] than in the non-CT-treated group [15.20% (6/39)] and HC group [7.14% (4/56)]. Patients who underwent CT and showed cognitive decline were less educated and older, with significantly lower baseline scores. CONCLUSIONS: CT-treated patients showed more vulnerability on cognitive control and monitoring than non-CT-treated breast cancer patients and HCs. Older patients with less education and lower baseline cognitive performance represent a group at risk for cognitive decline following CT. Identification of patients at risk for decline could improve targeted support and rehabilitation.

DNA double strand breaks induced by low dose mammography X-rays in breast tissue: A pilot study.

Breast tissue is very sensitive to ionizing radiation due to the presence of reproductive hormones, including estrogen. In the present pilot study, the efficiency of mammography X-rays to induce DNA double strand breaks (DSB) in mammary epithelial cells was investigated. For this, freshly resected healthy breast tissue was irradiated with 30 kV mammography X-rays in the dose range 0-500 mGy (2, 4, 10, 20, 40, 100 and 500 mGy). Breast specimens were also irradiated with identical doses of 60Co γ-rays as a radiation quality standard. With the γH2AX-foci assay, the number of DNA DSB induced by radiation were quantified in the mammary epithelial cells present in breast tissue. Results indicated that foci induced by 30 kV X-rays and γ-rays followed a biphasic linear dose-response. For 30 kV X-rays, the slope in the low dose region (0-20 mGy) was 8.71 times steeper compared with the slope in the higher dose region (20-500 mGy). Furthermore, compared with γ-rays, 30 kV X-rays were also more effective in inducing γH2AX-foci. This resulted in a relative biological effectiveness (RBE) value of 1.82 in the low dose range. In the higher dose range, an RBE close to 1 was obtained. In conclusion, the results indicated the existence of a low dose hypersensitive response for DSB induction in the dose range representative for mammography screening, which is probably caused by the bystander effect. This could affect the radiation risk calculations for women participating in mammography screening.

External partial breast irradiation in prone position: how to improve accuracy?

INTRODUCTION: In view of the limited incremental benefit between whole breast irradiation (WBI), accelerated partial breast irradiation (APBI) and omission of radiotherapy in favorable early-stage breast cancer (ESBC), APBI can only be justified if it combines adequate target coverage with the lowest achievable toxicity. Interobserver exercises demonstrated the difficulty of precise target delineation, especially in prone position; information on accuracy is even scarcer. We tested the impact of inserting an additional indicator clip, marking the depth of the tumor in the breast, and the added value of a preoperative CT in treatment position on precision and accuracy. MATERIAL AND METHODS: In 12 patients, tumor bed delineation was performed by four radiation oncologists, with CTVstandard (clinical target volume) based on standard delineation guidelines, CTVclip resulting from a 1-2-cm symmetrical expansion with the indicator clip as center and CTVclip_CT expanding from the midpoint between the indicator clip and preoperative gross tumor volume (GTV) as center. Precision was measured as the mean pairwise Jaccard index (JIpairs) between observers, accuracy as the mean overlap between GTV and respective CTVs. RESULTS: JIpairs was 0.38 for CTVstandard, 0.75 for CTVclip and 0.59 for CTVclip_CT. Overlap rate of GTV with CTVs was respectively 0.48, 0.67 and improved further to 0.88 for CTVclip_CT. High-dose coverage of GTV (D95 and D90) improved with an indicator clip, but the most optimal result was reached when preoperative CT was added. CONCLUSIONS: If EB-APBI in prone position is aimed for, an indicator clip intended to mark the depth of the tumor increases the probability of accurate target coverage, but cannot entirely replace the added value of a preoperative CT in treatment position. Avoiding the cost and effort of such CT implies a risk of missing the target, especially when small volumes are aimed for. Increasing target volumes to reduces this risk, questions the concept of APBI.

Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

Comparison of HER2 amplification status among breast cancer subgroups offers new insights in pathways of breast cancer progression.

Although the prognostic and predictive significance of human epidermal growth factor receptor 2 (HER2) in invasive breast cancer is well established, its role in ductal carcinoma in situ (DCIS) remains unclear. Reports on combined evaluation of both HER2 protein expression and HER2 amplification status in pure DCIS and DCIS adjacent to invasive ductal carcinoma (i.e., admixed DCIS) are scarce. In this study, immunohistochemistry and fluorescence in situ hybridization (FISH) were used to assess HER2 status in 72 cases of pure DCIS, 73 cases of DCIS admixed with invasive ductal carcinoma (IDC), and 60 cases of pure IDC. HER2 copy number-based amplification was present in 49% of pure DCIS, 16% of admixed DCIS, 18% of admixed IDC, and 8% of pure IDC. Amplified pure DCIS with clusters of HER2 signals showed a significantly lower HER2 copy number than amplified admixed DCIS with clusters. Whereas pure DCIS and admixed DCIS presented significant differences, the in situ and invasive component of admixed tumors showed striking similarities regarding mean HER2 and chromosome 17 centromere (CEP17) copy number, grade, and estrogen and progesterone receptor expression. The discrepant prevalence of HER2 amplification among breast cancer subgroups indirectly suggests that HER2 may not play a crucial role in the transition of in situ to invasive breast cancer. The similarities in HER2 amplification status between the in situ and invasive component of admixed tumors hint at a common biological pathway for both components. Our data support the theory that pure DCIS, pure IDC, and admixed lesions have a common progenitor, but can progress as separate lineages.

Secretome analysis of breast cancer-associated adipose tissue to identify paracrine regulators of breast cancer growth.

Adipose tissue secretes a plethora of adipokines as evidenced by characterization of subcutaneous and visceral adipose tissue secretomes. However, adipose tissue composition and secretion pattern is depot and disease dependent, influencing the adipose tissue secretome. We investigated the secretome of cancer-associated adipose tissue (CAAT) explants from breast cancer patients and explored its role in breast cancer proliferation. CAAT proteins were identified by LC-MS/MS and human protein antibody arrays and stimulated proliferation of three breast cancer cell lines. Kinomics and transcriptomics of MCF-7 breast cancer cells treated with the secretome of CAAT revealed activation of Akt-, ERK- and JNK-pathways and differential expression of activator protein 1 (AP-1) and cAMP responsive element-binding protein (CREB) target genes. The cyclin-dependent kinase (CDK)4/6-inhibitor palbociclib significantly abrogated CAAT-enhanced breast cancer cell proliferation. Our work characterizes the specific breast CAAT protein secretome and reveals its pro-proliferative potency in breast cancer.

Highly Accelerated Irradiation in 5 Fractions (HAI-5): Feasibility in Elderly Women With Early or Locally Advanced Breast Cancer.

PURPOSE: To investigate, in a prospective phase 1 to 2 trial, the safety and feasibility of delivering external beam radiation therapy in 5 fractions to the breast or thoracic wall, including boost and/or lymph nodes if needed, to women aged ≥65 years with breast cancer. METHODS AND MATERIALS: Ninety-five patients aged ≥65 years, referred for adjuvant radiation therapy, were treated in 5 fractions over 12 days with a total dose of 28.5 Gy/5.7 Gy to the breast or thoracic wall and, if indicated, 27 Gy/5.4 Gy to the lymph node regions and 32.5 Gy/6.5 Gy to 34.5 Gy/6.9 Gy to the tumor bed. The primary endpoint was clinically relevant dermatitis (grade ≥2). RESULTS: Mean follow-up time was 5.6 months, and mean age was 73.6 years. Clinically relevant dermatitis was observed in 11.6% of patients and only occurred in breast irradiation with boost (17.5% grade 2-3 vs 0% in the no-boost group). Although doses were high, treatment delivery with intensity modulated radiation therapy was swift, except for complex treatments, including lymph nodes for which single-arc volumetric modulated arc therapy was needed to reduce beam-on time. CONCLUSION: Accelerated radiation therapy in 5 fractions was technically feasible and resulted in low acute toxicity. Clinically relevant erythema was only observed in patients receiving a boost, but still at an acceptable rate. Although the follow-up is still short, the results on acute toxicity after accelerated radiation therapy were encouraging. A 5-fraction schedule is well tolerated in the elderly and may lower the threshold for radiation therapy in this population.

The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue.

Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076-1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active.

Intratissular expansion-mediated, serial fat grafting: A step-by-step working algorithm to achieve 3D biological harmony in autologous breast reconstruction.

BACKGROUND: Breast reconstruction involves the use of autologous tissues or implants. Occasionally, microsurgical reconstruction is not an option because of insufficient donor tissues. Fat grafting has become increasingly popular in breast surgery. The challenge with this technique is how to reconstruct a stable and living "scaffold" that resembles a breast. METHODS: Breast reconstruction (n = 7) was performed using intratissular expansion with serial deflation-lipofilling sessions. Mean age of the patients was 41 years (22-53). The expander generated a vascularized capsule at 8 weeks, which demarcated a recipient site between the skin and the capsule itself, and functioned as a vascular source for angiogenesis. Serial sessions of deflation and lipofilling were initiated at 8 weeks with removal of the expander at the completion of the treatment. An average of 644 ml (range, 415 ml-950 ml) of lipoaspirate material was injected to reconstruct the breast mound. An average of 4 (range, 3 to 5) fat-grafting sessions with a 3-month interval was needed to achieve symmetry with the contralateral breast. The average follow-up was 14 months (range, 9-29 months). MRI examination was performed at 8 months to analyze tissue survival and the residual volume. RESULTS: MRI examination retained tissue survival and the mean reconstructed breast volume was 386 ml (range, 231 ml-557 ml). An aesthetically pleasant breast mound was created, with a high satisfaction rate. CONCLUSION: We could reconstruct an aesthetically pleasant and stable breast mound in a selected group of patients by using intratissular expansion and fat grafting.

Reproducibility of deep inspiration breath hold for prone left-sided whole breast irradiation.

BACKGROUND: Investigating reproducibility and instability of deep inspiration breath hold (DIBH) in the prone position to reduce heart dose for left-sided whole breast irradiation. METHODS: Thirty patients were included and underwent 2 prone DIBH CT-scans during simulation. Overlap indices were calculated for the ipsilateral breast, heart and lungs to evaluate the anatomical reproducibility of the DIBH maneuver. The breathing motion of 21 patients treated with prone DIBH were registered using magnetic probes. These breathing curves were investigated to gain data on intra-fraction reproducibility and instability of the different DIBH cycles during treatment. RESULTS: Overlap index was 0.98 for the ipsilateral breast and 0.96 for heart and both lungs between the 2 prone DIBH-scans. The magnetic sensors reported population amplitudes of 2.8 ± 1.3 mm for shallow breathing and 11.7 ± 4.7 mm for DIBH, an intra-fraction standard deviation of 1.0 ± 0.4 mm for DIBH, an intra-breath hold instability of 1.0 ± 0.6 mm and a treatment time of 300 ± 69 s. CONCLUSION: Prone DIBH can be accurately clinically implemented with acceptable reproducibility and instability.

Heart dose reduction by prone deep inspiration breath hold in left-sided breast irradiation.

BACKGROUND AND PURPOSE: Cardiac disease has been related to heart dose after left-sided breast radiotherapy. This trial evaluates the heart sparing ability and feasibility of deep inspiration breath hold (DIBH) in the prone position for left-sided whole breast irradiation (WBI). MATERIALS AND METHODS: Twelve patients underwent CT-simulation in supine shallow breathing (SB), supine DIBH, prone SB and prone DIBH. A validation cohort of 38 patients received prone SB and prone DIBH CT-scans; the last 30 patients were accepted for prone DIBH treatment. WBI was planned with a prescription dose of 40.05 Gy. RESULTS: DIBH was able to reduce (p<0.001) heart dose in both positions, with results for prone DIBH at least as favorable as for supine DIBH. Mean heart dose was lowered from 2.2 Gy for prone SB to 1.3 Gy for prone DIBH (p<0.001), while preserving the lung sparing ability of prone positioning. Moreover prone DIBH nearly consistently reduced mean heart dose to less then 2 Gy, regardless of breast volume. All patients were able to perform the simulation procedure, 28/30 patients were treated with prone DIBH. CONCLUSIONS: This trial demonstrates the ability and feasibility of prone DIBH to acquire optimal heart and lung sparing for left-sided WBI.

Cancer-associated adipose tissue promotes breast cancer progression by paracrine oncostatin M and Jak/STAT3 signaling.

Increasing evidence supports the critical roles played by adipose tissue in breast cancer progression. Yet, the mediators and mechanisms are poorly understood. Here, we show that breast cancer-associated adipose tissue from freshly isolated tumors promotes F-actin remodeling, cellular scattering, invasiveness, and spheroid reorganization of cultured breast cancer cells. A combination of techniques, including transcriptomics, proteomics, and kinomics enabled us to identify paracrine secretion of oncostatin M (OSM) by cancer-associated adipose tissue. Specifically, OSM, expressed by CD45(+) leucocytes in the stromal vascular fraction, induced phosphorylation of STAT3 (pSTAT3-) Y705 and S727 in breast cancer cells and transcription of several STAT3-dependent genes, including S100 family members S100A7, S100A8, and S100A9. Autocrine activation of STAT3 in MCF-7 cells ectopically expressing OSM-induced cellular scattering and peritumoral neovascularization of orthotopic xenografts. Conversely, selective inhibition of OSM by neutralizing antibody and Jak family kinases by tofacitinib inhibited STAT3 signaling, peritumoral angiogenesis, and cellular scattering. Importantly, nuclear staining of pSTAT3-Y705 identified at the tumor invasion front in ductal breast carcinomas correlates with increased lymphovascular invasion. Our work reveals the potential of novel therapeutic strategies targeting the OSM and STAT3 axis in patients with breast cancer harboring nuclear pSTAT3-Y705.

Histopathological characterization of ductal carcinoma in situ (DCIS) of the breast according to HER2 amplification status and molecular subtype.

This study aimed to characterize ductal carcinoma in situ (DCIS) according to human epidermal growth factor receptor 2 (HER2) amplification status and molecular subtype. In addition, we performed a detailed HER2 and CEP17 copy number analysis and we assessed the impact of recent changes in the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on HER2 immunohistochemical (IHC) scores in DCIS. Nuclear grade, extensive comedonecrosis, stromal architecture, stromal inflammation, and progesterone receptor (PR) expression were significantly associated with HER2 amplification status. In multivariate analysis, stromal inflammation and extensive comedonecrosis were the only two features that remained significantly related to HER2 amplification status. The recent changes in ASCO/CAP guidelines resulted in significant upgrading of HER2 IHC score. Remarkably, about one in five non-amplified DCIS presented a 3+ IHC score, regardless of the scoring method. The biological significance of this phenomenon is presently unknown. After categorization according to molecular subtype, luminal A DCIS mainly presented histopathological features associated with good prognosis, whereas luminal B/HER2+ and HER2+ categories displayed a more aggressive phenotype. Overall, our results demonstrate that HER2-amplified DCIS constitute a clearly distinct subgroup which is characterized by histopathological features associated with poor prognosis. Further studies are required to elucidate the biological significance of a 3+ IHC score in non-amplified DCIS, as well as its mechanism.

Differential regulation of extracellular matrix protein expression in carcinoma-associated fibroblasts by TGF-ß1 regulates cancer cell spreading but not adhesion.

Cancer progression is characterized by a complex reciprocity between neoplastic epithelium and adjacent stromal cells. In ductal carcinoma in situ (DCIS) of the breast, both reduced stromal decorin expression and myxoid stroma are correlated with increased recurrence risk. In this study, we aimed to investigate paracrine regulation of expression of decorin and related extracellular matrix (ECM) proteins in cancer-associated fibroblasts (CAFs). Transforming growth factor-ß1 (TGF-ß1) was identified as a competent ECM modulator, as it reduced decorin and strongly enhanced versican, biglycan and type I collagen expression. Similar but less pronounced effects were observed when fibroblasts were treated with basic fibroblast growth factor (bFGF). Despite this concerted ECM modulation, TGF-ß1 and bFGF differentially regulated alpha-smooth muscle actin (α-SMA) expression, which is often proposed as a CAF-marker. Cancer cell-derived secretomes induced versican and biglycan expression in fibroblasts. Immunohistochemistry on twenty DCIS specimens showed a trend toward periductal versican overexpression in DCIS with myxoid stroma. Cancer cell adhesion was inhibited by decorin, but not by CAF-derived matrices. Cancer cells presented significantly enhanced spreading when seeded on matrices derived from TGF-ß1-treated CAF. Altogether these data indicate that preinvasive cancerous lesions might modulate the composition of surrounding stroma through TGF-ß1 release to obtain an invasion-permissive microenvironment.

On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids.

Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics. Here, a microfluidic chip with integrated light sheet illumination is reported, and accurate fluorescence single particle tracking size and concentration measurements of membrane vesicles in cell culture medium and in interstitial fluid collected from primary human breast tumours are demonstrated.