Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma.

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFß, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

Spontaneous Fusion with Transformed Mesenchymal Stromal Cells Results in Complete Heterogeneity in Prostate Cancer Cells.

Tumor cells gain advantages in growth and survival by acquiring genotypic and phenotypic heterogeneity. Interactions with bystander cells in the tumor microenvironment contribute to the progression of heterogeneity. We have shown that fusion between tumor and bystander cells is one form of interaction, and that tumor-bystander cell fusion has contrasting effects. By trapping fusion hybrids in the heterokaryon or synkaryon state, tumor-bystander cell fusion prevents the progression of heterogeneity. However, if trapping fails, fusion hybrids will resume replication to form derivative clones with diverse genomic makeups and behavioral phenotypes. To determine the characteristics of bystander cells that influence the fate of fusion hybrids, we co-cultured prostate mesenchymal stromal cell lines and their spontaneously transformed sublines with LNCaP as well as HPE-15 prostate cancer cells. Subclones derived from cancer-stromal fusion hybrids were examined for genotypic and phenotypic diversifications. Both stromal cell lines were capable of fusing with cancer cells, but only fusion hybrids with the transformed stromal subline generated large numbers of derivative subclones. Each subclone had distinct cell morphologies and growth behaviors and was detected with complete genomic hybridization. The health conditions of the bystander cell compartment play a crucial role in the progression of tumor cell heterogeneity.

Overcoming Resistance in Prostate Cancer Therapy Using a DZ-Simvastatin Conjugate.

Prostate cancer (PC), particularly its metastatic castration-resistant form (mCRPC), is a leading cause of cancer-related deaths among men in the Western world. Traditional systemic treatments, including hormonal therapy and chemotherapy, offer limited effectiveness due to tumors' inherent resistance to these therapies. Moreover, they often come with significant side effects. We have developed a delivery method using a tumor-cell-specific heptamethine carbocyanine dye (DZ) designed to transport therapeutic agents directly to tumor cells. This research evaluated simvastatin (SIM) as the antitumor payload because of its demonstrated chemopreventive effects on human cancers and its well-documented safety profile. We designed and synthesized a DZ-SIM conjugate for tumor cell targeting. PC cell lines and xenograft tumor models were used to assess tumor-cell targeting specificity and killing activity and to investigate the corresponding mechanisms. DZ-SIM treatment effectively killed PC cells regardless of their androgen receptor status or inherent therapeutic resistance. The conjugate targeted and suppressed xenograft tumor formation without harming normal cells of the host. In cancer cells, DZ-SIM was enriched in subcellular organelles, including mitochondria, where the conjugate formed adducts with multiple proteins and caused the loss of transmembrane potential, promoting cell death. The DZ-SIM specifically targets PC cells and their mitochondria, resulting in a loss of mitochondrial function and cell death. With a unique subcellular targeting strategy, the conjugate holds the potential to outperform existing chemotherapeutic drugs. It presents a novel strategy to circumvent therapeutic resistance, offering a more potent treatment for mCRPC.

A cisplatin conjugate with tumor cell specificity exhibits antitumor effects in renal cancer models.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer and is notorious for its resistance to both chemotherapy and small-molecule inhibitor targeted therapies. Subcellular targeted cancer therapy may thwart the resistance to produce a substantial effect. METHODS: We tested whether the resistance can be circumvented by subcellular targeted cancer therapy with DZ-CIS, which is a chemical conjugate of the tumor-cell specific heptamethine carbocyanine dye (HMCD) with cisplatin (CIS), a chemotherapeutic drug with limited use in ccRCC treatment because of frequent renal toxicity. RESULTS: DZ-CIS displayed cytocidal effects on Caki-1, 786-O, ACHN, and SN12C human ccRCC cell lines and mouse Renca cells in a dose-dependent manner and inhibited ACHN and Renca tumor formation in experimental mouse models. Noticeably, in tumor-bearing mice, repeated DZ-CIS use did not cause renal toxicity, in contrast to the CIS-treated control animals. In ccRCC tumors, DZ-CIS treatment inhibited proliferation markers but induced cell death marker levels. In addition, DZ-CIS at half maximal inhibitory concentration (IC50) sensitized Caki-1 cells to small-molecule mTOR inhibitors. Mechanistically, DZ-CIS selectively accumulated in ccRCC cells' subcellular organelles, where it damages the structure and function of mitochondria, leading to cytochrome C release, caspase activation, and apoptotic cancer cell death. CONCLUSIONS: Results from this study strongly suggest DZ-CIS be tested as a safe and effective subcellular targeted cancer therapy.

Extracellular vesicles in fatty liver promote a metastatic tumor microenvironment.

Liver metastasis is a major cause of death in patients with colorectal cancer (CRC). Fatty liver promotes liver metastasis, but the underlying mechanism remains unclear. We demonstrated that hepatocyte-derived extracellular vesicles (EVs) in fatty liver enhanced the progression of CRC liver metastasis by promoting oncogenic Yes-associated protein (YAP) signaling and an immunosuppressive microenvironment. Fatty liver upregulated Rab27a expression, which facilitated EV production from hepatocytes. In the liver, these EVs transferred YAP signaling-regulating microRNAs to cancer cells to augment YAP activity by suppressing LATS2. Increased YAP activity in CRC liver metastasis with fatty liver promoted cancer cell growth and an immunosuppressive microenvironment by M2 macrophage infiltration through CYR61 production. Patients with CRC liver metastasis and fatty liver had elevated nuclear YAP expression, CYR61 expression, and M2 macrophage infiltration. Our data indicate that fatty liver-induced EV-microRNAs, YAP signaling, and an immunosuppressive microenvironment promote the growth of CRC liver metastasis.

Breast Cancer MCF-7 Cells Acquire Heterogeneity during Successive Co-Culture with Hematopoietic and Bone Marrow-Derived Mesenchymal Stem/Stromal Cells.

During disease progression and bone metastasis, breast tumor cells interact with various types of bystander cells residing in the tumor microenvironment. Such interactions prompt tumor cell heterogeneity. We used successive co-culture as an experimental model to examine cancer-bystander cell interaction. RMCF7-2, a clone of the human breast cancer MCF-7 cells tagged with a red fluorescent protein, was tracked for morphologic, behavioral, and gene expression changes. Co-cultured with various types of hematopoietic cells, RMCF7-2 adopted stable changes to a rounded shape in suspension growth of red fluorescent cells, from which derivative clones displayed marked expressional changes of marker proteins, including reduced E-cadherin and estrogen receptor α, and loss of progesterone receptor. In a successive co-culture with bone marrow-derived mesenchymal stem/stromal cells, the red fluorescent clones in suspension growth changed once more, adopting an attachment growth, but in diversified shapes. Red fluorescent clones recovered from the second-round co-culture were heterogeneous in morphology, but retained the altered marker protein expression while displaying increased proliferation, migration, and xenograft tumor formation. Interaction with bystander cells caused permanent morphologic, growth behavioral, and gene expressional changes under successive co-culture, which is a powerful model for studying cancer cell heterogeneity during breast cancer progression and metastasis.

A Multicenter Evaluation of the Seraph 100 Microbind Affinity Blood Filter for the Treatment of Severe COVID-19.

The Seraph100 Microbind Affinity Blood Filter (Seraph 100) (ExThera Medical, Martinez, CA) is an extracorporeal therapy that can remove pathogens from blood, including severe acute respiratory syndrome coronavirus 2. The aim of this study was to evaluate safety and efficacy of Seraph 100 treatment for COVID-19. DESIGN: Retrospective cohort study. SETTING: Nine participating ICUs. PATIENTS: COVID-19 patients treated with Seraph 100 (n = 53) and control patients matched by study site (n = 53). INTERVENTION: Treatment with Seraph 100. MEASUREMENTS AND MAIN RESULTS: At baseline, there were no differences between the groups in terms of sex, race/ethnicity, body mass index, and need for mechanical ventilation. However, patients in the Seraph 100 group were younger (median age, 54 yr; interquartile range [IQR], 41-65) compared with controls (median age, 64 yr; IQR, 56-69; p = 0.009). Charlson comorbidity index scores were lower in the Seraph 100 group (2; IQR, 0-3) compared with the control group (3; IQR, 2-4; p = 0.006). Acute Physiology and Chronic Health Evaluation II scores were also lower in Seraph 100 subjects (12; IQR, 9-17) compared with controls (16; IQR, 12-21; p = 0.011). The Seraph 100 group had higher vasopressor-free days with an incidence rate ratio of 1.30 on univariate analysis. This difference was not significant after adjustment. Seraph 100-treated subjects were less likely to die compared with controls (32.1% vs 64.2%; p = 0.001), a difference that remained significant after adjustment. However, no difference in mortality was observed in a post hoc analysis utilizing an external control group. In the full cohort of 86 treated patients, there were 177 total treatments, in which only three serious adverse events were recorded. CONCLUSIONS: Although this study did not demonstrate consistently significant clinical benefit across all endpoints and comparisons, the findings suggest that broad spectrum, pathogen agnostic, blood purification can be safely deployed to meet new pathogen threats while awaiting targeted therapies and vaccines.

Circulating Fatty Objects and Their Preferential Presence in Pancreatic Cancer Patient Blood Samples.

Human cancers are often complicated with increased incidences of blood vessel occlusion, which are mostly insensitive to anticoagulation therapy. We searched for causal factors of cancer-associated embolism. A total of 2,017 blood samples was examined for visible abnormalities. Examined were peripheral blood samples from cancer patients who were about to undergo surgical treatment for genitourinary, breast, gastrointestinal or abdominal tumors. Samples from ambulatory patients being treated for recurrent or castration-resistant prostate cancers were included in the study. The lipid-rich nature was studied with lipophilic stains and lipid panel analysis, while surface membrane was assessed with specific staining and antibody detection. We identified a new entity, lipid droplet-like objects or circulating fatty objects (CFOs), visible in the blood samples of many cancer patients, with the potential of causing embolism. CFOs were defined as lipid-rich objects with a membrane, capable of gaining in volume through interaction with peripheral blood mononuclear cells in ex vivo culture. Blood samples from pancreatic cancer patients were found to have the highest CFO incidence and largest CFO numbers. Most noticeably, CFOs from many pancreatic cancer samples presented as large clusters entangled in insoluble fiber networks, suggestive of intravascular clotting. This study identifies CFO as an abnormal entity in cancer patient blood, and a contributory factor to intravascular embolism during cancer development and progression.

The Movember Global Action Plan 1 (GAP1): Unique Prostate Cancer Tissue Microarray Resource.

BACKGROUND: The need to better understand the molecular underpinnings of the heterogeneous outcomes of patients with prostate cancer is a pressing global problem and a key research priority for Movember. To address this, the Movember Global Action Plan 1 Unique tissue microarray (GAP1-UTMA) project constructed a set of unique and richly annotated tissue microarrays (TMA) from prostate cancer samples obtained from multiple institutions across several global locations. METHODS: Three separate TMA sets were built that differ by purpose and disease state. RESULTS: The intended use of TMA1 (Primary Matched LN) is to validate biomarkers that help determine which clinically localized prostate cancers with associated lymph node metastasis have a high risk of progression to lethal castration-resistant metastatic disease, and to compare molecular properties of high-risk index lesions within the prostate to regional lymph node metastases resected at the time of prostatectomy. TMA2 (Pre vs. Post ADT) was designed to address questions regarding risk of castration-resistant prostate cancer (CRPC) and response to suppression of the androgen receptor/androgen axis, and characterization of the castration-resistant phenotype. TMA3 (CRPC Met Heterogeneity)'s intended use is to assess the heterogeneity of molecular markers across different anatomic sites in lethal prostate cancer metastases. CONCLUSIONS: The GAP1-UTMA project has succeeded in combining a large set of tissue specimens from 501 patients with prostate cancer with rich clinical annotation. IMPACT: This resource is now available to the prostate cancer community as a tool for biomarker validation to address important unanswered clinical questions around disease progression and response to treatment.

KRT13 promotes stemness and drives metastasis in breast cancer through a plakoglobin/c-Myc signaling pathway.

BACKGROUND: Keratins (KRTs) are intermediate filament proteins that interact with multiple regulatory proteins to initiate signaling cascades. Keratin 13 (KRT13) plays an important role in breast cancer progression and metastasis. The objective of this study is to elucidate the mechanism by which KRT13 promotes breast cancer growth and metastasis. METHODS: The function and mechanisms of KRT13 in breast cancer progression and metastasis were assessed by overexpression and knockdown followed by examination of altered behaviors in breast cancer cells and in xenograft tumor formation in mouse mammary fat pad. Human breast cancer specimens were examined by immunohistochemistry and multiplexed quantum dot labeling analysis to correlate KRT13 expression to breast cancer progression and metastasis. RESULTS: KRT13-overexpressing MCF7 cells displayed increased proliferation, invasion, migration and in vivo tumor growth and metastasis to bone and lung. Conversely, KRT13 knockdown inhibited the aggressive behaviors of HCC1954 cells. At the molecular level, KRT13 directly interacted with plakoglobin (PG, γ-catenin) to form complexes with desmoplakin (DSP). This complex interfered with PG expression and nuclear translocation and abrogated PG-mediated suppression of c-Myc expression, while the KRT13/PG/c-Myc signaling pathway increased epithelial to mesenchymal transition and stem cell-like phenotype. KRT13 expression in 58 human breast cancer tissues was up-regulated especially at the invasive front and in metastatic specimens (12/18) (p < 0.05). KRT13 up-regulation in primary breast cancer was associated with decreased overall patient survival. CONCLUSIONS: This study reveals that KRT13 promotes breast cancer cell growth and metastasis via a plakoglobin/c-Myc pathway. Our findings reveal a potential novel pathway for therapeutic targeting of breast cancer progression and metastasis.

The Clinical Cell-Cycle Risk (CCR) Score Is Associated With Metastasis After Radiation Therapy and Provides Guidance on When to Forgo Combined Androgen Deprivation Therapy With Dose-Escalated Radiation.

PURPOSE: The clinical cell-cycle risk (CCR) score, which combines the University of California, San Francisco's Cancer of the Prostate Risk Assessment (CAPRA) and the cell cycle progression (CCP) molecular score, has been validated to be prognostic of disease progression for men with prostate cancer. This study evaluated the ability of the CCR score to prognosticate the risk of metastasis in men receiving dose-escalated radiation therapy (RT) with or without androgen deprivation therapy (ADT). METHODS AND MATERIALS: This retrospective, multi-institutional cohort study included men with localized National Comprehensive Cancer Network (NCCN) intermediate-, high-, and very high-risk prostate cancer (N = 741). Patients were treated with dose-escalated RT with or without ADT. The primary outcome was time to metastasis. RESULTS: The CCR score prognosticated metastasis with a hazard ratio (HR) per unit score of 2.22 (95% confidence interval [CI], 1.71-2.89; P < .001). The CCR score better prognosticated metastasis than NCCN risk group (CCR, P < .001; NCCN, P = .46), CAPRA score (CCR, P = .002; CAPRA, P = .59), or CCP score (CCR, P < .001; CCP, P = .59) alone. In bivariable analyses, CCR score remained highly prognostic when accounting for ADT versus no ADT (HR, 2.18; 95% CI, 1.61-2.96; P < .001), ADT duration as a continuous variable (HR, 2.11; 95% CI, 1.59-2.79; P < .001), or ADT given at or below the recommended duration for each NCCN risk group (HR, 2.19; 95% CI, 1.69-2.86; P < .001). Men with CCR scores below or above the multimodality threshold (CCR score, 2.112) had a 10-year risk of metastasis of 3.7% and 21.24%, respectively. Men with below-threshold scores receiving RT alone had a 10-year risk of metastasis of 3.7%, and for men receiving RT plus ADT, the 10-year risk of metastasis was also 3.7%. CONCLUSIONS: The CCR score accurately and precisely prognosticates metastasis and adds clinically actionable information relative to guideline-recommended therapies based on NCCN risk in men undergoing dose-escalated RT with or without ADT. For men with scores below the multimodality threshold, adding ADT may not significantly reduce their 10-year risk of metastasis.

Two Patients With Severe COVID Pneumonia Treated With the Seraph-100 Microbind Affinity Blood Filter.

We present 2 patients with rapidly escalating oxygen requirements from severe acute respiratory syndrome coronavirus 2 infection (COVID-19) treated with the Seraph100 Microbind Affinity Blood Filter under Emergency Use Authorization from the US Federal Drug Administration. The Seraph100 is an extracorporeal hemoperfusion filter previously demonstrated to remove viral particles and pro-inflammatory cytokines from the blood. Treatment with the Seraph100 filter was associated with a rapid improvement in oxygenation and both patients were discharged from the hospital without supplemental oxygen.

Novel Mitochondria-Based Targeting Restores Responsiveness in Therapeutically Resistant Human Lung Cancer Cells.

Cisplatin and tyrosine kinase inhibitors (TKI) are recommended to treat non-small cell lung cancer (NSCLC). However, ubiquitously acquired drug resistance in patients with NSCLC diminishes their therapeutic efficacy. Strategies for overcoming cisplatin and TKI resistance are an unmet medical need. We previously described a group of near-infrared heptamethine carbocyanine fluorescent dyes, referred to as DZ, with tumor-homing properties via differentially expressed organic anion-transporting polypeptides on cancer cells. This group of organic dyes can deliver therapeutic payloads specifically to tumor cells in the form of a chemical conjugate. We synthesized DZ-simvastatin (DZ-SIM) initially to target cholesterol biosynthesis in lung cancer cells. DZ-SIM killed both cisplatin-sensitive and cisplatin-resistant as well as EGFR-TKI-sensitive and EGFR-TKI-resistant lung cancer cells. This conjugate specifically accumulated in and effectively inhibited the growth of xenograft tumors formed by NSCLC cells resistant to first-generation (H1650) and third-generation (PC9AR) EGFR TKIs. DZ-SIM induced cell death by targeting mitochondrial structure and function. We concluded that DZ-SIM could be a promising novel therapy for overcoming drug resistance in patients with NSCLC.

An asymptomatic anterior vaginal wall endometrioma, a rare manifestation of endometriosis: A case report.

A 23-year-old patient with an asymptomatic anterior vaginal wall cyst was referred to gynecology for evaluation and treatment. Preoperative assessment with physical examination and magnetic resonance (MR) imaging of the pelvis was most consistent with Gartner's cyst. Following resection of the cyst wall, histologic evaluation demonstrated endometrial glands, hemosiderin-laden macrophages and inflammation, consistent with vaginal wall endometrioma. This case highlights challenges in the diagnosis of endometriosis in the vagina and in other rare locations, possible mechanisms of development, and proposed treatments.

Focal 18F-NaF PET Prostate Activity in the Setting of Prostate Adenocarcinoma.

A 72-year-old man with a family history of prostate cancer and initial diagnosis of favorable intermediate risk prostate cancer via biopsy in 2017 elected for active surveillance. Two years later, he underwent prostate biopsy showing intermediate-risk cT1c Nx Mx lesion with Gleason score 3 + 4 = 7 (5 core positive). Transrectal ultrasound showed a prostate volume 28 mL, and the prostate-specific antigen was 8.1. Patient elected to proceed with combination radiation therapy and androgen deprivation therapy.

Chromosomal instability in untreated primary prostate cancer as an indicator of metastatic potential.

BACKGROUND: Metastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome. METHODS: PNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121). RESULTS: The CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene "metastasis" signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression. CONCLUSIONS: Measuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models.

Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1's role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.

Cancer cell's neuroendocrine feature can be acquired through cell-cell fusion during cancer-neural stem cell interaction.

Advanced and therapy-resistant prostate tumors often display neural or neuroendocrine behavior. We assessed the consequences of prostate cancer cell interaction with neural cells, which are rich in the human prostate and resident of the prostate tumor. In 3-dimensional co-culture with neurospheres, red fluorescent human LNCaP cells formed agglomerates on the neurosphere surface. Upon induced neural differentiation, some red fluorescent cells showed morphology of fully differentiated neural cells, indicating fusion between the cancer and neural stem cells. These fusion hybrids survived for extended times in a quiescent state. A few eventually restarted cell division and propagated to form derivative hybrid progenies. Clones of the hybrid progenies were highly heterogeneous; most had lost prostatic and epithelial markers while some had acquired neural marker expression. These results indicate that cancer cells can fuse with bystander neural cells in the tumor microenvironment; and cancer cell fusion is a direct route to tumor cell heterogeneity.

Cancer-stromal cell fusion as revealed by fluorescence protein tracking.

PURPOSE: We previously determined that cancer-stromal interaction was a direct route to tumor cell heterogeneity progression, since cancer-stromal cell fusion in coculture resulted in the creation of heterogeneous clones of fusion hybrid progeny. In this report, we modified the cancer-stromal coculture system to establish optimal experimental conditions for investigating cell fusion machinery and the mechanism of heterogeneity progression. EXPERIMENTAL DESIGN: Red fluorescence protein-tagged LNCaP cells were cocultured with green fluorescence protein-labeled prostate stromal cells for cancer-stromal cell fusion, which was tracked as dual fluorescent cells by fluorescence microscopy. RESULTS: We identified the most efficient strategy to isolate clones of fusion hybrid progenies. From the coculture, mixed cells including fusion hybrids were subjected to low-density replating for colony formation by fusion hybrid progeny. These colonies could propagate into derivative cell populations. Compared to the parental LNCaP cells, clones of the fusion hybrid progeny displayed divergent behaviors and exhibited permanent genomic hybridization. CONCLUSIONS: Cancer-stromal cell fusion leads to cancer cell heterogeneity. The cancer-stromal coculture system characterized in this study can be used as a model for molecular characterization of cancer cell fusion as the mechanism behind the progression of heterogeneity observed in clinical prostate cancers.