Summary The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D “organoid” system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression and CHD1 loss. Whole exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as of DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.

Cancer cells display acoustic properties enabling acoustophoretic separation from white blood cells (WBCs) with 2-3 log suppression of the WBC background. However, a subset of WBCs has overlapping acoustic properties with cancer cells, which is why label-free acoustophoretic cancer cell isolation needs additional purification prior to analysis. This paper reports for the first time a proof of concept for continuous flow acoustophoretic negative selection of WBCs from cancer cells using negative acoustic contrast elastomeric particles (EPs) activated with CD45-antibodies that specifically bind to WBCs. The EP/WBC complexes align at the acoustic pressure anti-nodes along the channel walls while unbound cancer cells focus to the pressure node in the channel center, enabling continuous flow based depletion of WBC background in a cancer cell product. The method does not provide a single process solution for the CTC separation challenge, but provides an elegant part to a multi-step process by further reducing the WBC background in cancer cell separation products derived from an initial step of label-free acoustophoresis. We report the recorded performance of the negative selection immuno-acoustophoretic WBC depletion and cancer cell recovery. To eliminate the negative impact of the separation due to the known problems of aggregation of negative acoustic contrast particles along the sidewalls of the acoustophoresis channel and to enable continuous separation of EP/WBC complexes from cancer cells, a new acoustic actuation method has been implemented where the ultrasound frequency is scanned (1.991MHz ± 100 kHz, scan rate 200 kHz ms). Using this frequency scanning strategy EP/WBC complexes were acoustophoretically separated from mixtures of WBCs spiked with breast and prostate cancer cells (DU145 and MCF-7). An 86-fold (MCF-7) and 52-fold (DU145) reduction of WBCs in the cancer cell fractions were recorded with separation efficiencies of 98.6% (MCF-7) and 99.7% (DU145) and cancer cell recoveries of 89.8% (MCF-7) and 85.0% (DU145).

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Introduction: Circulating tumor cells (CTCs) are a promising tool for disease monitoring and for better-targeted therapies for patients with disseminating tumors. However, isolation of CTCs is challenging, due to their scarcity, heterogeneous size, and lack of universal expression markers. We employ a novel ultrasonic standing wave-based microfluidic technique (acoustophoresis) to enable unbiased, label-free isolation of circulating tumor cells (CTCs). The cell separation is based on intrinsic cell properties, such as size, morphology, density, and compressibility. During acoustophoresis cells/particles get positioned to nodes or antinodes depending on their acoustic contrast factor. The contrast factor depends on the density and compressibility ratios between the particles and the surrounding medium. Both cancer cells and subpopulations of white blood cells (WBCs) partly possess positive acoustic factors and are therefore positioned to pressure nodes during acoustophoresis. By employing negative contrast CD45-conjugated elastomeric particles (EPs) to capture the contaminating WBCs, a negative acoustic complex can be formed that positions the remaining WBCs to the antinodes. This cell separation technique is gentle towards the target cells and tunable to fit various situations, depending on whether throughput, cancer cell recovery, or purity is of major importance. Both paraformaldehyde (PFA) fixed cells and fresh blood can be processed through acoustophoresis. However, acoustophoretic cancer cell separation from fresh blood generates higher WBC contamination than processing of PFA fixed cells, due to overlapping acoustic properties. Therefore, an additional purification step is needed to remove excess contaminating WBCs. Hence, we have added an additional purging step after acoustophoresis, employing CD45-conjugated negative selection of WBCs using negative acoustic contrast EPs. Material and Methods: The acoustic separation device is a glass/silicon microchip connected to a pressure-driven system. For optimal separation performance, the system is temperature controlled and a prefocusing step is included before the main separation in the first cell separation step. The setup can process samples up to 10 mL, with a processing speed of 1 mL sample in 13 minutes without compromising the cell separation capacity. To evaluate the system performance, 1 mL model samples were processed, consisting of 10,000 DU145 prostate cancer cells spiked in 0.5 mL red blood cell (RBC) lysed blood from healthy donors. Results and Discussion: In the primary acoustic separation step, an average of 93% of the DU145 cancer cells were collected at the central outlet, whereas 7% were found in the side fraction. A 20-fold WBC depletion was achieved after the primary acoustophoresis-based tumor cell separation step. The central fraction output was subjected to a subsequent one-hour incubation with CD45-conjugated EPs in room temperature. The incubated samples were processed by acoustophoresis to negatively select EP/WBC complexes from cancer cells into separate outlet fractions, with subsequent flow cytometry analysis. Result showed a 98% cancer cell separation efficiency and a total 250-fold WBC depletion from the starting WBC concentration. Although the separation efficiency in the two steps was high, there was some cell loss in the total system, which needs to be further addressed. We propose a two-step acoustic-based microfluidic technology, consisting of an upstream primary label-free acoustophoretic separation of cancer cells from blood and a subsequent negative WBC depletion. This may lay the foundation for future analytical and clinical studies aimed to validate novel biomarkers for effective treatment management of cancer patients. Citation Format: Cecilia Magnusson, Eva Undvall, Thomas Laurell, Hans Lilja. A novel two-step tumor cell isolation system, combining acoustophoresis and negative selection of white blood cells using CD45-conjugated elastomeric particles [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A027.

33 Background: The inability to propagate patient-derived prostate cancer cells in vitro is a major impediment in the mechanistic understanding of tumorigenesis and therapeutic response. In order to generate accurate in vitro models that represent the diversity of in situ prostate cancer, we have developed a three-dimensional “organoid” system to culture metastasis samples and integrated it into our precision medicine workflow of attaining and characterizing pre-treatment biopsies. Methods: Biopsy samples of prostate cancer metastases, both soft tissue and bone, acquired at the time of therapeutic or diagnostic interventions following informed consent and institutional review board approval were obtained from two institutions. Samples were digested in Type II Collagenase (Gibco) and re-suspended in growth factor reduced Matrigel (BD), plated on plastic, and overlaid with prostate culture media (PCM). PCM consists of serum free Advanced DMEM/F12 (Gibco) with multiple growth factors optimized to propagate benign primary prostate cells. Cultures were maintained at 37°C in 5% CO2. Results: In the initial 51 samples, 15 continuous organoid cultures (29%) were established from distinct sites (9 of 32 bone, 6 of 19 soft). Tumor content of the biopsy represents a major determinant of organoid growth. Once established, organoids propagate indefinitely with different kinetics (approximately 48 hours to 1 week doubling time), and can be cryopreserved. Histological analysis shows that the organoids recapitulate the structure of the in situ cancer and genomic analysis using array CGH and whole-exome sequencing (WES) shows the presence of typical copy number alterations including TMPRSS2-ERG interstitial deletion, PTEN loss, CHD1 loss, and AR amplification. WES of two organoid/metastasis pairs shows that the growth conditions do not generate additional mutations. Conclusions: This novel tissue culture technique enables the development of new cell lines derived from metastatic deposits. This advance will facilitate research by availing new and varied cell lines, which will hopefully be more closely aligned to the spectrum of behavior of the clinical disease in comparison to the limited and problematic cell line models currently available.